Antibodies that bind to thyroid-stimulating hormone receptors and uses thereof

Abstract

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

Description

[Technical Field]

[0001] The present invention relates to antibodies or antigen-binding fragments thereof that specifically bind to TSHR, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof and host cells containing the same, and related uses. The present invention also relates to therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof. [Background technology]

[0002] Graves' disease (GD) is an intractable autoimmune disorder characterized by an enlarged and overactive thyroid gland, elevated heart rate, and eye abnormalities. The abnormally protruding eyes are called Graves' ophthalmopathy (GO) or hyperthyroid ophthalmopathy. Based on what is known about the disease, it is believed to be the result of a complex interaction between genetic and environmental factors. GO not only severely impacts quality of life, but also mental and professional performance. It also stimulates thyroid hormone secretion through overactivation of the associated thyroid hormone receptors, leading to an increased risk of mortality. Currently, clinical treatments for GO mainly involve antithyroid drug (ATD) therapy, radioactive iodine (RAI) therapy, and surgical treatment. However, the use of antithyroid drugs has low therapeutic efficacy and often results in side effects such as bone marrow suppression and liver toxicity. Surgical resection or radioactive iodine ablation treatment also carries significant risks, including recurrent neurological damage and hypoparathyroidism after surgery. Furthermore, while controlling thyroid function, glucocorticoid therapy, local orbital radiation therapy, or surgical treatment is required. However, these treatments are symptomatic and do not target the underlying mechanism of the disease. Currently, there is no effective clinical treatment for GO disease.

[0003] Thyroid-stimulating hormone receptor (TSHR) is mainly expressed on the basolateral surface of thyroid follicular cells and induces thyroid cell growth, hormone synthesis, and hormone secretion. It is also a major autoantigen in autoimmune thyroid diseases. In particular, in the case of GD disease, approximately 25% of GD patients develop GO disease as the disease course progresses.

[0004] It has been reported that some patients with autoimmune thyroid disease (AITD) develop autoantibodies that react with TSHR (Rees Smith B. et al. 1988. Endocrine Reviews 9:106-121). There are two main types of TSHR autoantibodies (TRAbs): stimulatory and blocking. Thyroid-stimulating autoantibodies bind to TSHR and mimic the action of TSH, stimulating the thyroid gland to produce high levels of T4 and T3. These autoantibodies have also been described as TRAbs with agonist activity (Rees Smith B et al. 2007. Thyroid 17:923-938). In the presence of such antibodies, the feedback control mechanism of thyroid function becomes ineffective, and patients develop clinical symptoms of an overactive thyroid gland characterized by excess thyroid hormones in the serum and their metabolic effects. This condition is called Graves' disease (GD). TRAb, which has stimulatory activity, may also interact with TSHR in retroorbital tissues to promote the development of Graves' ophthalmopathy (GO). Based on current research into the pathogenesis of GO disease, increased expression of TSHR in orbital fibroblasts of GO patients and activation of signaling pathways may lead to increased production of hyaluronic acid and adipose tissue in the orbit, resulting in increased intraocular pressure and extrusion of the eyeball, which are thought to be important causes of GO disease. Furthermore, TSHR expression is high in the thyroid and testes, but low in other tissues, potentially offering advantages in terms of drug targeting and side effects. Therefore, TSHR is expected to be a potential target for the treatment of GO disease.

[0005] Furthermore, the insulin-like growth factor 1 receptor (IGF1R) is thought to play another important role in the pathogenesis of GO. IGF1R blockers can improve GO disease. For example, the commercially available drug teprotumumab can competitively inhibit the activation of downstream signaling pathways by the IGF1R receptor, demonstrating remarkable therapeutic effects, particularly in improving exophthalmos and reducing inflammation. Its therapeutic efficiency reaches 78% in the treatment of moderate to severe thyroid eye disease (TED). Increased expression of TSHR and IGF1R is observed in orbital fibroblasts from patients with GO disease, and there is a certain interaction between TSHR and IGF1R. Therefore, direct blockade of TSHR (TSAb / antagonist) or combination therapy using simultaneous antagonism of TSHR and IGF1R receptors may be a fundamental treatment for GD or GO disease.

[0006] Currently, there are no commercially available drugs that target TSHR to treat GO disease, and there is a need in the art to develop therapeutic drugs that target TSHR. Summary of the Invention

[0007] Through extensive research, the inventors of the present application have obtained a mouse-derived antibody that specifically binds to TSHR and has significant TSHR inhibitory activity and cross-linking activity with human, mouse, and monkey TSHR. Furthermore, the inventors of the present application have also obtained a humanized antibody based on the mouse-derived antibody that has TSHR binding activity and TSHR inhibitory activity.

[0008] Based on these, the present application further provides pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof and host cells containing the same, and related uses.

[0009] Antibody or antigen-binding fragment thereof mAb001 and its humanized antibody Thus, in a first aspect, the present application provides an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) represented by any one of SEQ ID NOs: 1, 27, 29, and 30, which comprises VH CDR1 or a mutant thereof, VH CDR2 or a mutant thereof, and VH CDR3 or a mutant thereof; and / or a light chain variable region (VL) represented by any one of SEQ ID NOs: 5, 28, and 31, which comprises VL CDR1 or a mutant thereof, VL CDR2 or a mutant thereof, and VL CDR3 or a mutant thereof; However, a variant has one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In some embodiments, the substitutions are conservative substitutions.

[0010] In some embodiments, the antibody or antigen-binding fragment thereof It comprises three CDRs contained in a heavy chain variable region (VH) shown in any one of SEQ ID NOs: 1, 27, 29, and 30, and / or three CDRs contained in a light chain variable region (VL) shown in any one of SEQ ID NOs: 5, 28, and 31.

[0011] In some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat, IMGT, or Chothia numbering system, hi some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat numbering system.

[0012] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 2, VH CDR2 having the sequence of SEQ ID NO: 3 or 39, and VH CDR3 having the sequence of SEQ ID NO: 4; and / or a light chain variable region (VL) comprising three complementarity determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 6, VL CDR2 having the sequence of SEQ ID NO: 7, and VL CDR3 having the sequence of SEQ ID NO: 8; However, the CDRs are defined by the Kabat numbering system.

[0013] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a murine immunoglobulin.

[0014] In some 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.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof 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, However, a variant is a sequence which 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 which has 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.

[0016] In some embodiments, the substitutions are conservative substitutions.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:1 and a VL comprising the sequence set forth in SEQ ID NO:5.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a human immunoglobulin.

[0019] In some 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, ie, a heavy chain framework region from a human heavy chain germline sequence and / or a light chain framework region from a human light chain germline sequence.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NOs: 27, 29, and 30, or a variant thereof, and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 28 or 31, or a variant thereof; However, a variant is a sequence which 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 which has 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.

[0021] In some embodiments, the substitutions are conservative substitutions.

[0022] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) set forth in any one of SEQ ID NOs: 27, 29, and 30, and / or a light chain variable region (VL) set forth in SEQ ID NO: 28 or 31.

[0023] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:27 and a VL comprising the sequence set forth in SEQ ID NO:28.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:29 and a VL comprising the sequence set forth in SEQ ID NO:28.

[0025] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:30 and a VL comprising the sequence set forth in SEQ ID NO:28.

[0026] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:27 and a VL comprising the sequence set forth in SEQ ID NO:31.

[0027] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:29 and a VL comprising the sequence set forth in SEQ ID NO:31.

[0028] mAb012 and its humanized antibody Thus, in a second aspect, the present application provides an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) represented by any one of SEQ ID NOs: 9, 32, and 34, which comprises VH CDR1 or a mutant thereof, VH CDR2 or a mutant thereof, and VH CDR3 or a mutant thereof; and / or a light chain variable region (VL) represented by SEQ ID NO: 13 or 33, which comprises VL CDR1 or a mutant thereof, VL CDR2 or a mutant thereof, and VL CDR3 or a mutant thereof; However, a variant has one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In some embodiments, the substitutions are conservative substitutions.

[0029] In some embodiments, the antibody or antigen-binding fragment thereof It comprises three CDRs contained in the heavy chain variable region (VH) shown in any one of SEQ ID NOs: 9, 32, and 34, and / or three CDRs contained in the light chain variable region (VL) shown in SEQ ID NO: 13 or 33.

[0030] In some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat, IMGT, or Chothia numbering system, hi some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat numbering system.

[0031] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 10, VH CDR2 having the sequence of SEQ ID NO: 11 or 40, and VH CDR3 having the sequence of SEQ ID NO: 12 or 41; and / or a light chain variable region (VL) comprising three complementarity determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 14, VL CDR2 having the sequence of SEQ ID NO: 15, and VL CDR3 having the sequence of SEQ ID NO: 16; However, the CDRs are defined by the Kabat numbering system.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 10, VH CDR2 having the sequence of SEQ ID NO: 11 or 40, and VH CDR3 having the sequence of SEQ ID NO: 12; and / or a light chain variable region (VL) comprising three complementarity determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 14, VL CDR2 having the sequence of SEQ ID NO: 15, and VL CDR3 having the sequence of SEQ ID NO: 16; However, the CDRs are defined by the Kabat numbering system.

[0033] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 10, VH CDR2 having the sequence of SEQ ID NO: 11 or 40, and VH CDR3 having the sequence of SEQ ID NO: 41; and / or a light chain variable region (VL) comprising three complementarity determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 14, VL CDR2 having the sequence of SEQ ID NO: 15, and VL CDR3 having the sequence of SEQ ID NO: 16; However, the CDRs are defined by the Kabat numbering system.

[0034] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a murine immunoglobulin.

[0035] In some 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.

[0036] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 9 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 13 or a variant thereof, However, a variant is a sequence which 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 which has 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.

[0037] In some embodiments, the substitutions are conservative substitutions.

[0038] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:9 and a VL comprising the sequence set forth in SEQ ID NO:13.

[0039] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a human immunoglobulin.

[0040] In some 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, ie, a heavy chain framework region from a human heavy chain germline sequence and / or a light chain framework region from a human light chain germline sequence.

[0041] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 32 or 34 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 33 or a variant thereof; However, a variant is a sequence which 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 which has 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.

[0042] In some embodiments, the substitutions are conservative substitutions.

[0043] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) set forth in SEQ ID NO: 32 or 34 and / or a light chain variable region (VL) set forth in SEQ ID NO: 33.

[0044] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:32 and a VL comprising the sequence set forth in SEQ ID NO:33.

[0045] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:34 and a VL comprising the sequence set forth in SEQ ID NO:33.

[0046] mAb021 and its humanized antibody Accordingly, in a third aspect, the present application provides an antibody or antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or antigen-binding fragment thereof comprising: a heavy chain variable region (VH) represented by any one of SEQ ID NOs: 17, 35, and 37, which comprises VH CDR1 or a mutant thereof, VH CDR2 or a mutant thereof, and VH CDR3 or a mutant thereof; and / or a light chain variable region (VL) represented by any one of SEQ ID NOs: 21, 36, and 38, which comprises VL CDR1 or a mutant thereof, VL CDR2 or a mutant thereof, and VL CDR3 or a mutant thereof; However, a variant has one or more amino acid substitutions, deletions, or additions (e.g., one, two, or three amino acid substitutions, deletions, or additions, e.g., conservative substitutions) compared to the sequence from which it is derived. In some embodiments, the substitutions are conservative substitutions.

[0047] In some embodiments, the antibody or antigen-binding fragment thereof It comprises three CDRs contained in a heavy chain variable region (VH) shown in any one of SEQ ID NOs: 17, 35, and 37, and / or three CDRs contained in a light chain variable region (VL) shown in any one of SEQ ID NOs: 21, 36, and 38.

[0048] In some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat, IMGT, or Chothia numbering system, hi some embodiments, the three CDRs in the VH and / or the three CDRs in the VL are defined according to the Kabat numbering system.

[0049] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising three complementarity determining regions (CDRs): VH CDR1 having the sequence of SEQ ID NO: 18, VH CDR2 having the sequence of SEQ ID NO: 19, and VH CDR3 having the sequence of SEQ ID NO: 20; and / or a light chain variable region (VL) comprising three complementarity determining regions (CDRs): VL CDR1 having the sequence of SEQ ID NO: 22, VL CDR2 having the sequence of SEQ ID NO: 23, and VL CDR3 having the sequence of SEQ ID NO: 24; However, the CDRs are defined by the Kabat numbering system.

[0050] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a murine immunoglobulin.

[0051] In some 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.

[0052] In some embodiments, the antibody or antigen-binding fragment thereof a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 17 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 21 or a variant thereof, However, a variant is a sequence which 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 which has 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.

[0053] In some embodiments, the substitutions are conservative substitutions.

[0054] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:17 and a VL comprising the sequence set forth in SEQ ID NO:21.

[0055] In some embodiments, the antibody or antigen-binding fragment thereof comprises framework region sequences derived from a human immunoglobulin.

[0056] In some 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, ie, a heavy chain framework region from a human heavy chain germline sequence and / or a light chain framework region from a human light chain germline sequence.

[0057] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 35 or 37 or a variant thereof, and / or a light chain variable region (VL) comprising the sequence set forth in SEQ ID NO: 36 or 38 or a variant thereof; However, a variant is a sequence which 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 which has 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.

[0058] In some embodiments, the substitutions are conservative substitutions.

[0059] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) set forth in SEQ ID NO: 35 or 37, and / or a light chain variable region (VL) set forth in SEQ ID NO: 36 or 38.

[0060] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:35 and a VL comprising the sequence set forth in SEQ ID NO:36.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:37 and a VL comprising the sequence set forth in SEQ ID NO:38.

[0062] In some embodiments, the antibody or antigen-binding fragment thereof of the first, second or third aspect further comprises a constant region derived from a mammalian (e.g., human or murine) immunoglobulin.

[0063] In some 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).

[0064] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a human immunoglobulin heavy chain constant region (CH) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived (e.g., at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions, e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions); and / or The light chain of the antibody or antigen-binding fragment thereof comprises a human immunoglobulin light chain constant region (CL) or a variant thereof, wherein the variant has one or more amino acid substitutions, deletions, or additions compared to the sequence from which it is derived (e.g., at most 20, at most 15, at most 10, or at most 5 amino acid substitutions, deletions, or additions, e.g., 1, 2, 3, 4, or 5 amino acid substitutions, deletions, or additions). The substitutions may be conservative or non-conservative.

[0065] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) of a wild-type human immunoglobulin.

[0066] In some other embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a heavy chain constant region (CH) of a human immunoglobulin, which variant of the heavy chain constant region (CH) may have the same or essentially the same properties as the wild-type sequence from which it is derived. In some embodiments, the variant of the heavy chain constant region (CH) may have one or more conservative amino acid substitutions compared to the sequence from which it is derived.

[0067] In some other embodiments, the antibody or antigen-binding fragment thereof comprises a variant of a human immunoglobulin heavy chain constant region (CH), which may contain one or more amino acid mutations or chemical modifications to alter one or more properties of the antibody of the invention, such as Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function. Substitution of at least one amino acid residue in the antibody constant region with a different residue or chemical modification can result in altered function. For example, effector function can be altered (e.g., reduced or enhanced) by altering the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q). The Fc region of an antibody mediates several important effector functions, such as ADCC, phagocytosis, and CDC.

[0068] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a variant of a human immunoglobulin heavy chain constant region (CH), which variant may have reduced or eliminated effector function compared to the wild-type sequence from which it is derived.

[0069] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a variant of a human immunoglobulin heavy chain constant region (CH), which variant may have enhanced effector function compared to the wild-type sequence from which it is derived.

[0070] In some 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 variant may have the same or essentially the same effector function as compared to the wild-type sequence from which it is derived.

[0071] In some embodiments, the antibodies or antigen-binding fragments thereof of the present invention comprise a variant of a human immunoglobulin heavy chain constant region (CH), which variant has (i) an increased half-life, (ii) enhanced efficacy, and / or (iii) decreased ADCC activity compared to the wild-type sequence from which it is derived.

[0072] In some 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.

[0073] In some embodiments, the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.

[0074] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a mouse 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 mouse immunoglobulin (e.g., κ or λ).

[0075] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the first, second, or third aspect 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 human immunoglobulin (e.g., kappa or lambda).

[0076] In some 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.

[0077] In some embodiments, compared to 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 some embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations M252Y, S254T, and T256E; and in some embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises the substitution mutations L235E, M252Y, S254T, and T256E.

[0078] In some embodiments, the amino acid sites of substitution mutations are defined by the EU numbering system.

[0079] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth in any one of SEQ ID NOs: 25 and 42-43, and / or the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region (CL) set forth in SEQ ID NO: 26.

[0080] In some embodiments, the antibody or antigen-binding fragment thereof (1) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 27 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (2) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 29 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (3) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 30 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (4) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 27 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 31 and a CL of the sequence shown in SEQ ID NO: 26; (5) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 29 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 31 and a CL of the sequence shown in SEQ ID NO: 26; (6) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 32 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 33 and a CL of the sequence shown in SEQ ID NO: 26; (7) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 34 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 33 and a CL of the sequence shown in SEQ ID NO: 26; (8) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 35 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 36 and a CL of the sequence shown in SEQ ID NO: 26, or (9) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 37 and a CH of the sequence shown in any one of SEQ ID NOs: 25 and 42 to 43, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 38 and a CL of the sequence shown in SEQ ID NO: 26.

[0081] In some embodiments, the antibody or antigen-binding fragment thereof of the first aspect comprises: (1) a heavy chain comprising the sequence set forth in SEQ ID NO: 46 and / or a light chain comprising the sequence set forth in SEQ ID NO: 51; (2) a heavy chain comprising the sequence set forth in SEQ ID NO: 47 and / or a light chain comprising the sequence set forth in SEQ ID NO: 51; or (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.

[0082] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the first, second, or third aspect 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., kappa or lambda).

[0083] In some 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.

[0084] In some embodiments, the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A and L235A, and in some embodiments, the heavy chain constant region derived from human immunoglobulin IgG1 further comprises substitution mutations M252Y, S254T, and T256E, compared to the heavy chain constant region of wild-type human immunoglobulin IgG1.

[0085] In some embodiments, the amino acid sites of substitution mutations are defined by the EU numbering system.

[0086] In some embodiments, the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) set forth 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) set forth in SEQ ID NO: 26.

[0087] In some embodiments, the antibody or antigen-binding fragment thereof (1) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 27 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (2) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 29 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (3) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 30 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 28 and a CL of the sequence shown in SEQ ID NO: 26; (4) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 27 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 31 and a CL of the sequence shown in SEQ ID NO: 26; (5) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 29 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 31 and a CL of the sequence shown in SEQ ID NO: 26; (6) a heavy chain comprising a VH of the sequence shown in SEQ ID NO: 32 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 33 and a CL of the sequence shown in SEQ ID NO: 26; (7) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 34 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 33 and a CL of the sequence shown in SEQ ID NO: 26; (8) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 35 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 36 and a CL of the sequence shown in SEQ ID NO: 26, or (9) A heavy chain comprising a VH of the sequence shown in SEQ ID NO: 37 and a CH of the sequence shown in SEQ ID NO: 44 or 45, and / or a light chain comprising a VL of the sequence shown in SEQ ID NO: 38 and a CL of the sequence shown in SEQ ID NO: 26.

[0088] In some embodiments, the antibody or antigen-binding fragment thereof of the first aspect comprises: (1) a heavy chain comprising the sequence set forth in SEQ ID NO: 49 and / or a light chain comprising the sequence set forth in SEQ ID NO: 51; or (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.

[0089] In some embodiments, in the first, second or 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, humanized antibody, chimeric antibody, bispecific antibody or multispecific antibody.

[0090] According to a fourth aspect, the present application further provides an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to the first, second or third aspect, or a heavy chain variable region and / or a light chain variable region thereof, or a heavy and / or light chain thereof.

[0091] In some embodiments, an 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 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 in the same or different isolated nucleic acid molecules. When the first nucleotide sequence and the second nucleotide sequence are present in different isolated nucleic acid molecules, the isolated nucleic acid molecule according to the invention comprises a first nucleic acid molecule comprising the first nucleotide sequence and a second nucleic acid molecule comprising the second nucleotide sequence.

[0092] According to a fifth aspect, the present application further provides a vector comprising the isolated nucleic acid molecule described above. In some embodiments, the vector is a cloning vector or an expression vector.

[0093] In some 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 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 in the same or different vectors. When the first nucleotide sequence and the second nucleotide sequence are present in different vectors, the vector described in the present invention comprises a first vector comprising the first nucleotide sequence and a second vector comprising the second nucleotide sequence.

[0094] According to a sixth aspect, the present application further provides a host cell comprising the isolated nucleic acid molecule or vector described above.

[0095] 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, such as mouse cells, human cells, etc.). In some embodiments, the host cell is a microorganism.

[0096] The antibodies of the present invention can be produced by various methods known in the art, for example, by recombinant genetic engineering techniques. For example, DNA molecules encoding the heavy and light chain genes of the antibodies of the present invention are obtained by chemical synthesis or PCR amplification. The obtained DNA molecules are inserted into an expression vector and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the antibodies of the present invention.

[0097] Antigen-binding fragments of the present invention can be obtained by hydrolysis of intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)). Furthermore, these antigen-binding fragments can also be produced directly by recombinant host cells (see Hudson, Curr. Opin. Immunol. 11:548-557 (1999) and Little et al., Immunol. Today 21:364-370 (2000)). For example, Fab' fragments can be obtained directly from host cells, and Fab' fragments can be chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Furthermore, Fv, Fab or F(ab')2 fragments can be isolated directly from recombinant host cell culture. Those skilled in the art are familiar with other techniques for producing these antigen-binding fragments.

[0098] According to a seventh aspect, the present application further provides a method of producing an antibody or antigen-binding fragment thereof according to the first, second or third aspect, the method comprising culturing a host cell as described above 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 culture. therapeutic use

[0099] According to an eighth aspect, the present application further provides a bispecific or multispecific molecule comprising an antibody or antigen-binding fragment thereof according to the first, second or third aspect.

[0100] In some embodiments, the bispecific or multispecific molecule specifically binds to the TSHR and additionally specifically binds to one or more other targets.

[0101] In some embodiments, 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).

[0102] In some embodiments, the bispecific or multispecific molecule further comprises at least one secondary antibody that specifically binds to IGF1R. In some embodiments, the secondary antibody has an antagonistic effect on IGF1R signaling.

[0103] According to a ninth aspect, the present application further provides an immunoconjugate comprising an antibody or antigen-binding fragment thereof according to the first, second or third aspect and a therapeutic agent linked to the antibody or antigen-binding fragment thereof.

[0104] In some embodiments, the therapeutic agent is selected from an IGF1R antagonist or inhibitor.

[0105] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC).

[0106] In a tenth aspect, the present application further provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to the first, second or third aspect, an isolated nucleic acid molecule according to the fourth aspect, a vector according to the fifth aspect, a host cell according to the sixth aspect, a bispecific or multispecific molecule according to the eighth aspect or an immunoconjugate according to the ninth aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0107] In some embodiments, the pharmaceutical composition further comprises an additional pharmaceutically active agent, for example, an additional drug for treating an autoimmune thyroid disease (e.g., Graves' disease or Graves' ophthalmopathy) or thyroid cancer.

[0108] In some embodiments, the pharmaceutical composition further comprises an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor.

[0109] In some 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 some exemplary embodiments, such a 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 buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0110] According to an eleventh aspect, the present application further provides the use of an antibody or antigen-binding fragment thereof according to the first, second or third aspect, an isolated nucleic acid molecule according to the fourth aspect, a vector according to the fifth aspect, a host cell according to the sixth aspect, a bispecific or multispecific molecule according to the eighth aspect, an immunoconjugate according to the ninth aspect or a pharmaceutical composition according to the tenth aspect for use in the manufacture of a medicament for preventing and / or treating a disease associated with TSHR in a subject.

[0111] In some embodiments, the disease associated with TSHR would benefit from antagonism of TSHR signaling.

[0112] In some embodiments, the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0113] In some embodiments, the subject is a mammal, such as a human, mouse, or monkey.

[0114] In some 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, e.g., administered simultaneously or sequentially, with an additional pharmaceutically active agent (e.g., an additional drug for treating an autoimmune thyroid disease (e.g., Graves' disease or Graves' ophthalmopathy) or thyroid cancer).

[0115] In some 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.

[0116] According to a twelfth aspect, the present application further provides a method for preventing and / or treating a disease associated with TSHR in a subject, the method comprising administering to a subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof according to the first, second or third aspect, the isolated nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, the host cell according to the sixth aspect, the bispecific or multispecific molecule according to the eighth aspect, the immunoconjugate according to the ninth aspect or the pharmaceutical composition according to the tenth aspect.

[0117] In some embodiments, the disease associated with TSHR would benefit from antagonism of TSHR signaling.

[0118] In some embodiments, the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0119] In some embodiments, the subject is a mammal, such as a human, mouse, or monkey.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof according to the first, second or third aspect, the isolated nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, the host cell according to the sixth aspect, the bispecific or multispecific molecule according to the eighth aspect, the immunoconjugate according to the ninth aspect or the pharmaceutical composition according to the tenth aspect may be administered in combination, e.g. simultaneously or sequentially, with an additional pharmaceutically active agent (e.g. an additional drug for treating an autoimmune thyroid disease (e.g. Graves' disease or Graves' ophthalmopathy) or thyroid cancer).

[0121] In some embodiments, the antibody or antigen-binding fragment thereof according to the first, second or third aspect, the isolated nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, the host cell according to the sixth aspect, the bispecific or multispecific molecule according to the eighth aspect, the immunoconjugate according to the ninth aspect or the pharmaceutical composition according to the tenth aspect may be used in combination, e.g. administered simultaneously or sequentially, with an IGF1R antagonist or inhibitor and / or an additional TSHR antagonist or inhibitor.

[0122] The antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application may be prepared into any dosage form known in the medical arts, such as tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules, elixirs, topical applications, suppositories, injections (including injection solutions, sterile powders for injection, and concentrated solutions for injection), inhalants, sprays, etc. The preferred dosage form depends on the expected 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 the conditions of production and storage. A preferred dosage form is an injection. Such an injection may be a sterile injectable solution. For example, a sterile injectable solution can be prepared by incorporating the required amount of the antibodies or antigen-binding fragments thereof in an appropriate solvent and, optionally, co-incorporating other desired ingredients (including, but not limited to, pH adjusting agents, surfactants, adjuvants, ionic strength enhancing agents, isotonicity agents, preservatives, diluents, or any combination thereof), followed by filtration and sterilization. Alternatively, the sterile injectable solution can be prepared as a sterile lyophilized powder (e.g., by vacuum drying or freeze-drying) for ease of storage and use. Such sterile lyophilized powder 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, or any combination thereof.

[0123] The antibodies or antigen-binding fragments thereof, or pharmaceutical compositions of the present application may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, ocular, topical, parenteral, rectal, intrathecal, intravesical, intragastrointestinal, topical (e.g., powders, ointments, or drops), or nasal routes. However, for many therapeutic uses, the preferred route / mode of administration is parenteral administration (e.g., intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection). One of skill in the art should understand that the route and / or mode of administration will vary depending on the anticipated purpose. In some embodiments, the antibodies or antigen-binding fragments thereof, or pharmaceutical compositions of the present invention are administered by intravenous injection or bolus injection. Detective Use

[0124] According to a thirteenth aspect, the present application further provides a conjugate comprising an antibody or antigen-binding fragment thereof according to the first, second or third aspect, and a detectable marker linked to the antibody or antigen-binding fragment thereof.

[0125] In some embodiments, the detectable marker is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., luciferin or a fluorescent protein), a radionuclide, or biotin.

[0126] According to a 14th aspect, the present application further provides a reagent kit comprising an antibody or antigen-binding fragment thereof according to the first, second or third aspect, or a conjugate according to the 13th aspect.

[0127] In some embodiments, the reagent kit includes a detection buffer.

[0128] In some embodiments, the reagent kit comprises a conjugate according to the thirteenth aspect.

[0129] In some embodiments, the reagent kit comprises an antibody or antigen-binding fragment thereof according to the first, second or third aspect, and a secondary antibody that specifically recognizes the antibody or antigen-binding fragment thereof, and optionally the secondary antibody further comprises a detectable marker, such as an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., luciferin or a fluorescent protein), a radionuclide or biotin.

[0130] According to a 15th aspect, the present application further provides a method for detecting the presence or level of TSHR in a sample, the method comprising using an antibody or antigen-binding fragment thereof according to the first, second or third aspect, or a conjugate according to the 13th aspect.

[0131] In some embodiments, the methods are used for therapeutic, diagnostic, or non-therapeutic non-diagnostic purposes.

[0132] In some embodiments, the method is immunological detection, such as immunoblotting, enzyme-linked immunosorbent assay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.

[0133] In some embodiments, the method comprises using a conjugate according to the thirteenth aspect.

[0134] In some embodiments, the method comprises using an antibody or antigen-binding fragment thereof according to the first, second or third aspect, and the method further comprises detecting the antibody or antigen-binding fragment thereof using a secondary antibody bearing a detectable marker (e.g., an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., luciferin or a fluorescent protein), a radionuclide, or biotin).

[0135] In some embodiments, the method comprises: (1) contacting a sample with an antibody or antigen-binding fragment thereof, or conjugate of the invention; 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 the TSHR or a cell expressing the TSHR.

[0136] The present application further 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 some embodiments, the presence of TSHR or an increased level of TSHR compared to a reference level (e.g., compared to a healthy control) indicates that the subject is suffering from a disease associated with the given TSHR.

[0137] In some embodiments, the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0138] According to a 16th aspect, the present application further provides the use of an antibody or antigen-binding fragment thereof according to the first, second or third aspect, or a conjugate according to the 13th aspect, in the manufacture of a detection reagent for use in detecting the presence or level of TSHR in a sample and / or in diagnosing a disease associated with TSHR.

[0139] In some embodiments, the detection reagent detects the presence or level of TSHR in a sample by a method according to the fifteenth aspect.

[0140] In some embodiments, the detection reagent detects the presence or level of TSHR in a sample, thereby diagnosing a TSHR-associated disease, by the method described in aspect 15. In some embodiments, the presence of TSHR or an increased level of TSHR compared to a reference level (e.g., compared to a healthy control) indicates that the subject is afflicted with a TSHR-associated disease.

[0141] In some embodiments, the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.

[0142] In some embodiments, the sample is a tissue sample from a subject (eg, a mammal, preferably a human, mouse, or monkey). DETAILED DESCRIPTION OF THE INVENTION

[0143] Definition of Terms In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the laboratory operation steps of virology, biochemistry, and immunology used herein are all common steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the following provides definitions and interpretations of relevant terms.

[0144] When the terms "for example," "such as," "such as," "include," "containing," or variations thereof are used herein, these terms are not to be considered limiting terms and should be interpreted as "but not limited to" or "without being limited to."

[0145] Unless otherwise indicated herein or clearly contradicted by context, the terms "a," "an," and "the," and similar referents, in the context of describing the invention (particularly in the context of the claims that follow), should be construed to cover both the singular and the plural.

[0146] As used herein, the term "antibody" refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen, and the immunoglobulin-derived molecule binds to the target antigen via at least one antigen-binding site located in its variable region. References to the term "antibody" include not only intact antibodies, but also antigen-binding fragments capable of specifically binding to a target antigen, unless the context clearly indicates otherwise. An "intact antibody" typically consists of two pairs of polypeptide chains (each pair having a light chain (LC) and a heavy chain (HC)). Antibody light chains can be classified as kappa (κ) and lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and define antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant domains are not directly involved in binding of the antibody to an antigen but exhibit various effector functions. For example, they can mediate the binding of immunoglobulins to various cells of the immune system (e.g., effector cells) and host tissues or factors, including the first component of the classical complement system (C1q). The VH and VL regions can be further subdivided into regions of high variability (called complementarity-determining regions (CDRs)) interspersed with relatively conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of each heavy / light chain pair (VH and VL) form the respective antigen-binding sites.The amino acid assignments in each region or domain 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.

[0147] As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in an antibody variable region that are involved in antigen binding. Each heavy and light chain variable region 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. For example, they can be defined according to 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, those skilled in the art will easily identify the CDRs defined by each numbering system. The correspondence between various numbering systems is also well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003).

[0148] In the present invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art. In some embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention are preferably determined according to the Kabat, Chothia, or IMGT numbering system.

[0149] As used herein, the term "framework region" or "FR" residues refers to amino acid residues in an antibody variable region other than the CDR residues defined above.

[0150] The term "antibody" is not limited to any particular method of producing an antibody. It includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be of a different isotype, for example, an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.

[0151] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody, also called an "antigen-binding portion," 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 an antigen. 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 other such polypeptides which contain at least a sufficient portion of an antibody to confer specific antigen-binding ability on the polypeptide. Engineered antibody variants are summarized in Holliger et al., 2005, Nat Biotechnol, 23:1126-1136.

[0152] 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 in the hinge region; and the term "Fab' fragment" refers to a fragment obtained after reduction bonding of the disulfide bonds of the two heavy chain fragments in the F(ab')2 fragment, consisting of an intact light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

[0153] 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. An Fv fragment is generally considered to be the smallest antibody fragment capable of forming a complete antigen-binding site. In general, the six CDRs are considered to confer antigen-binding specificity to the antibody. However, even a single variable region (e.g., an Fd fragment containing only three antigen-specific CDRs) can recognize and bind to an antigen, although the affinity may be lower than that of the complete binding site.

[0154] 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 and the second and third constant regions of the second heavy chain of the antibody. The Fc fragment of an antibody has many different functions, but is not involved in antigen binding.

[0155] As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains connected via 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 may be used, although variants thereof may also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). Other linkers for use in the present invention are described in 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 be present between the VH and VL of the scFv. In some embodiments of the invention, scFv can form a di-scFv, which refers to an antibody formed by connecting two or more single scFvs in series. In some embodiments of the invention, scFv can form (scFv)2, which refers to an antibody formed by connecting two or more single scFvs in parallel.

[0156] As used herein, the term "diabody" means an antibody whose VH and VL domains are expressed on a single polypeptide chain, but which uses a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing pairing with complementary domains on another chain to produce 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)).

[0157] 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 consisting of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), which retains the ability to specifically bind to the same antigen bound by the full-length antibody. Single-domain antibodies are also called nanobodies.

[0158] As used herein, the term "bispecific antibody" refers to an antibody that has binding specificities for two different antigens (or epitopes). The term "multispecific antibody" refers to an antibody that has binding specificities for at least two or more (e.g., three or four) different antigens (or epitopes). A bispecific or multispecific antibody contains multiple antigen-binding domains that have binding specificities for different antigens (or epitopes) and can thereby bind to at least two different binding sites and / or target molecules. Each antigen-binding domain contained in a bispecific or multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, Fab fragment, F(ab')2 fragment, or scFv). In some cases, each antigen-binding domain is linked by a peptide linker.

[0159] Each of the above antibody fragments retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen.

[0160] Antigen-binding fragments of antibodies (e.g., the antibody fragments described above) can be obtained from a given antibody (e.g., an antibody provided by the present invention) by conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody can be specifically screened by the same methods used for intact antibodies.

[0161] As used herein, the term "humanized antibody" refers to a genetically engineered non-human antibody whose amino acid sequence has been modified to improve sequence homology with a human antibody. Generally, all or a portion of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). In some embodiments, the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or a portion of the non-CDR regions (e.g., variable region FRs and / or constant regions) are derived from a human immunoglobulin (acceptor antibody). Humanized antibodies generally retain the expected properties of the donor antibody, including, but not limited to, antigen specificity, affinity, reactivity, etc. In this application, the donor antibody may be a murine antibody with the expected properties (e.g., antigen specificity, affinity, reactivity, etc.). To produce a humanized antibody, the CDR regions of the donor antibody can be inserted into human framework sequences using methods known in the art. In some cases, the human framework sequences may include amino acid mutations substituted with corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) nor in the human framework sequences to further improve or optimize performance of the humanized antibody.

[0162] As used herein, the term "chimeric antibody" refers to an antibody in which a portion of its light and / or heavy chain is derived from one antibody (from a particular species or belonging to a particular antibody class or subclass) and another portion of its light and / or heavy chain is derived from another antibody (from the same or a different species or belonging to the same or a different antibody class or subclass), but which retains binding activity for a target antigen (Cabilly et al., U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In some embodiments, the term "chimeric antibody" may include antibodies in which the heavy and light chain variable regions of the antibody are derived from a first antibody, but the heavy and light chain constant regions of the antibody are derived from a second antibody.

[0163] As used herein, the term "identity" refers to the matching of sequences between two polypeptides or two nucleic acids. 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., gaps are introduced into a first amino acid or nucleic acid sequence to optimally align it with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. If 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 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 some embodiments, the two sequences are the same length.

[0164] The determination of percent identity between two sequences can also be carried out using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877, as modified. Such an algorithm is integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

[0165] As used herein, the term "variant" in the context of a polypeptide (including a polypeptide) also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently attaching any type of molecule to the polypeptide or peptide). For example, but not limited to, a polypeptide may be modified by, for example, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation 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. A variant also possesses similar, identical, or improved function as the polypeptide or peptide from which it is derived.

[0166] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and an antigen to which it is directed. The strength or affinity of a specific binding interaction is determined by the equilibrium dissociation constant (K D In the present invention, "K DThe term "antibody-antigen affinity" refers to the dissociation equilibrium constant of a particular antibody-antigen interaction and is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

[0167] The specific binding properties between two molecules can be measured using methods known in the art. One method involves measuring the rates of formation and dissociation of the antigen-binding site / antigen complex. Both the "association rate constant" (ka or k) and the "dissociation rate constant" (k or k) can be calculated from the concentration and the actual binding and dissociation rates (see Malmqvist M., Nature, 1993, 361:186-187). The ratio of k / k is the dissociation constant K D (See Davies et al., Annual Rev Biochem, 1990, 59:439-473). K can be calculated by any valid method. D The values ​​of k, k, and k can be measured. In some embodiments, the dissociation constant can be measured using surface plasmon resonance (SPR) on a Biacore. Alternatively, the dissociation constant can be measured using bioluminescence interferometry or Kinexa.

[0168] As used herein, a detectable marker according to the present invention may be any substance that can be detected by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means. Such markers are well known in the art and examples include 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 32P), 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 reagents such as acridinium ester compounds, luminol and its derivatives, and ruthenium derivatives such as ruthenium terpyridine), magnetic beads (e.g., Dynabeads®), colorimetric markers such as colloidal gold or colored glass or plastic (e.g., polystyrene, polyacrylic, latex, etc.), and biotin for binding to avidin (e.g., streptavidin) modified with the above markers. In some embodiments, the detectable marker is selected from an enzyme (e.g., horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (e.g., an acridinium ester compound, luminol and its derivatives, or a ruthenium derivative), a fluorescent dye (e.g., luciferin or a fluorescent protein), a radionuclide, or biotin. In some embodiments, the detectable marker can be linked to the antibody or antigen-binding fragment thereof of the present invention via a linker of various lengths to reduce potential steric hindrance.

[0169] As used herein, the term "vector" refers to a nucleic acid delivery tool into which a polynucleotide can be inserted. If the vector is capable of expressing a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the carried genetic material element to be expressed within 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), phages 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 viruses), poxviruses, baculoviruses, papilloma viruses, and papilloma vacuolating viruses (such as SV40). Vectors can contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Vectors may also contain an origin of replication site.

[0170] As used herein, the term "host cell" refers to cells that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as E. 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, HEK293 cells or human cells.

[0171] As used herein, the term "conservative substitution" refers to an amino acid substitution that does not adversely affect or alter the expected properties of a protein / polypeptide containing 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 that replace an amino acid residue with an amino acid residue having a similar side chain, such as substitution with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar chemical properties, including size, shape, charge, ability to form covalent 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). Accordingly, it is preferred to replace a 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).

[0172] The notation of the 20 conventional amino acids herein follows 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. And, in the present invention, amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.

[0173] 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 active ingredient, and is well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19th ed., Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, preservatives, and stabilizers. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Agents for maintaining osmotic pressure include, but are not limited to, sugars, NaCl, and analogs thereof. Agents for delaying absorption include, but are not limited to, monostearates and gelatin. Diluents include, but are not limited to, water, aqueous buffer solutions (e.g., buffered saline), alcohols, and polyols (e.g., glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, trichlorotert-butyl alcohol, phenol, and sorbic acid. Stabilizers have the meaning commonly understood by those skilled in the art and are capable of stabilizing the desired activity of the active ingredient in a drug, including, but not limited to, sodium glutamate, gelatin, SPGA, sugars (e.g., sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (e.g., glutamic acid, glycine), proteins (e.g., dried whey, albumin, or casein), or their degradation products (e.g., lactalbumin hydrolysate), etc. In some exemplary embodiments, the pharmaceutically acceptable carrier or excipient comprises a sterile injectable liquid (e.g., an aqueous or non-aqueous suspension or solution).In some exemplary embodiments, such sterile injectable fluids 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 buffer solution (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.

[0174] As used herein, the term "prevention" refers to a method performed to prevent or delay the onset of a disease or condition or symptom in a subject. As used herein, the term "treatment" refers to a method performed to obtain beneficial or desired clinical results. For purposes of the present invention, beneficial or desired clinical results include (but are not limited to) alleviation of symptoms, reduction in disease extent, stabilization of the disease state (i.e., not worsening), delay or mitigation of disease progression, improvement or alleviation of the disease state, and alleviation of symptoms (whether partial or total), whether detectable or undetectable. "Treatment" can also refer to prolonging survival compared to expected survival (e.g., survival without treatment).

[0175] As used herein, the term "subject" refers to a mammal, such as a human, mouse, or monkey. In some embodiments, the subject (e.g., a human, mouse, or monkey) has or is at risk for a TSHR-associated disease (e.g., Graves' disease, Graves' ophthalmopathy).

[0176] As used herein, the term "effective amount" refers to an amount sufficient to achieve, or at least partially achieve, a desired effect. For example, an amount effective for preventing a disease (e.g., Graves' disease, Graves' ophthalmopathy) refers to an amount sufficient to prevent, inhibit, or delay the onset of the disease, and an amount effective for treating a disease refers to an amount sufficient to cure or at least partially inhibit the disease and its complications in a patient suffering from the disease. Determining such effective amounts is entirely within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use depends on the severity of the disease being treated, the overall state of the patient's own immune system, the patient's general condition, such as age, weight, and sex, the method of drug administration, and other treatments administered simultaneously. Beneficial effects of the invention

[0177] The antibody provided herein has high affinity for human TSHR and cross-links to human, mouse, and monkey TSHR. Furthermore, the antibody has significant TSHR activity inhibitory activity, blocking the binding of TSHR to activating antibodies and thereby inhibiting downstream signaling pathways mediated by TSHR binding to activating antibodies. Verification has shown that the antibody significantly inhibits the secretion of hyaluronic acid, interleukin-6, and interleukin-8 in primary orbital fibroblasts from GO patients in in vitro functional experiments.

[0178]

[0023] The following detailed description of the preferred embodiments of the present invention will be made in conjunction with the accompanying drawings and examples. Those skilled in the art will appreciate that the following drawings and examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art based on the following detailed description of the preferred embodiments and the drawings. [Brief explanation of the drawings]

[0179] [Figure 1] The engineered antibodies inhibit the induction of TPO production in thyroid cells by serum from GD patients.

[0180] Sequence information A description of the sequences according to the present application is given in the table below.

[0181] [Table 1] TIFF2025529812000002.tif138138 [Example]

[0182] The present invention will now be described with reference to the following examples which are intended to illustrate, but not limit, the invention.

[0183] Unless otherwise specified, the molecular biology experimental methods and immunodetection methods used in the present invention are basically performed with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Reisen Port Laboratory Publishing, 1989, and FMA Usubel et al., Refined Molecular Biology Experimental Guidelines, 3rd Edition, John Wiley & Sons, Inc., 1995, and restriction enzymes are used in accordance with the conditions recommended by the product manufacturer. Those skilled in the art will recognize that the examples are illustrative of the present invention and are not intended to limit the scope claimed by the present invention.

[0184] Example 1: Antibody production 1. Hybridoma antibody production: 1.1 After immunizing mice with human TSHR antigen, mice with relatively high titers were selected for cell fusion and hybridoma production.

[0185] 1.2 Myeloma cells were prepared and counted using a counter before fusion to detect activity. SP2 / 0 cells were collected and centrifuged at 400 g for 5 minutes at room temperature. The culture medium was discarded, and the cell pellet was resuspended in 20 ml of fusion medium.

[0186] 1.3 Splenocytes were collected and cultured, and mice showing a relatively strong response to the immunogen were selected for hybridoma cell production. Following IACUC-approved methods, the mice were euthanized using carbon dioxide, pre-fusion plasma was collected, and serum (FB) was isolated and collected as a positive control for screening hybridoma supernatants.

[0187] 1.4 After the mice were disinfected by immersion in 70% alcohol, they were immediately transferred to a biological safety cabinet for handling. The spleens were collected and placed in a sterile culture dish. The spleens were placed on a filter and squeezed repeatedly with a sterile syringe plug until no obvious clumps remained. The filter was then rinsed with fusion medium to obtain a splenocyte suspension, which was then transferred to a 50 ml centrifuge tube.

[0188] 1.5 The cells were centrifuged at 400g for 5 minutes, the supernatant discarded, and electrofusion was used to prepare fused cells. The splenocyte pellet was resuspended in 5ml of red blood cell lysis solution, then incubated at 4°C for 5 minutes. The reaction was stopped with 50ml of DEME medium supplemented with 10% FBS. The splenocyte pellet was collected by centrifugation, and the cells were resuspended in fusion medium. The centrifuge tube was inverted 3-5 times and then counted. The cells were centrifuged at 400g for 5 minutes, the supernatant discarded, and the cell pellet was retained. The cells were then resuspended in 40ml of fusion medium. The splenocytes were incubated at room temperature for 2-3 minutes and then transferred to a new 50ml centrifuge tube. SP2 / 0 cells were added to the splenocytes at a 4:1 ratio, and the volume was adjusted to 50ml with fusion medium. The cell mixture was centrifuged, the supernatant discarded, and the cell pellet was loosened by repeatedly tapping the bottom of the tube. In the case of fusion and electrofusion, the cell mixture was placed in an electroporation tank and electroporation fusion was carried out according to an optimized program.

[0189] 1.6 After electrofusion, the fused cells were allowed to rest in the electroporation tank for an additional 10 minutes. The cells were resuspended in DMEM medium supplemented with 20% FBS and HAT and seeded into a 96-well microplate at a volume of 100 μl per well. After 24 hours, 100 μl of DMEM medium supplemented with 20% FBS and HAT was added to the wells of the plate. All fusion plates were returned to a 37°C, 5% CO2 incubator. Cell growth rate and the presence of microbial contamination were monitored daily. When hybridoma clones grew to a diameter of 1–2 mm (typically 10–14 days after fusion), hybridoma supernatants were screened using Acumen.

[0190] 1.7 Subcloning and amplification. Positive clones were transferred to 24-well plates and cultured in amplification medium. Hybridoma culture supernatants were collected for FACS detection. Positive mother clones were subcloned by limiting dilution to obtain monoclonals. The subcloned 96-well plates were placed in a carbon dioxide incubator and cultured for 7 days, after which the supernatants were screened for activity. One to two rounds of subcloning were required to secure monoclonals. Based on the screening results, four monoclonals with the best activity were selected from each subcloning plate and transferred to 24-well plates for amplification.

[0191] 1.8 Cell Cryopreservation. The amplified subclonal cell lines were cryopreserved and maintained in DMEM medium supplemented with 20% FBS and 10% DMSO, and placed in a liquid nitrogen tank for long-term storage.

[0192] The CDR and variable region sequences of the mouse monoclonal antibodies mAb001, mAb012, and mAb021 produced by the above method are shown in Table 2.

[0193] [Table 2]

[0194] 2. Humanization of mouse antibodies: Humanized antibodies were designed using CDR grafting, replacing the murine framework regions with the sequences closest to the murine sequences in BLAST human antibodies. The CDRs of the parent antibody were grafted onto the human receptor, and the risks of post-translational modifications (PTMs) of all sequences, such as isomerization, deamidation, and glycosylation, were analyzed. Appropriate PTMs were designed to remove mutations, ultimately obtaining the humanized light and heavy chains of 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 linked 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 humanized antibody heavy and light chain sequences.

[0195] 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.

[0196] Humanized antibodies of each mouse monoclonal antibody were obtained by the above method, and the amino acid sequences of the light chain and heavy chain variable regions of each humanized antibody are shown in SEQ ID NOs: 27 to 38.

[0197] Example 2: Mouse antibody-cell binding experiment Experimental procedure steps: Flow cytometry (FACS) was used to detect the binding strength of mouse antibodies to cell lines overexpressing 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR (293 cells overexpressing human, monkey, or mouse TSHR, respectively; see Uniprot for the amino acid sequences of human, monkey, and mouse TSHR). Experimental method: Cells in the logarithmic growth phase were harvested and counted using a cell counter (Countstar, IC1000). The cells were resuspended in cold FACS buffer. The buffer was 500 ml of PBS (HyClone, SH30256.01B) plus 50 ml of FBS (BI, 04-002-1A). The cell density was 5 × 10 6 The cell suspension was adjusted to 10 cells / ml. After incubating at room temperature for 10–20 minutes, 100 μL / well of the suspension was added to a FACS plate (Corning, 3799) and centrifuged to discard the supernatant. Antibodies to be tested were diluted in FACS buffer at a starting concentration of 200 nM, followed by a 1:3 gradient. 100 μL of the diluted antibody was added to the cells at 100 μL / well and incubated at 4°C for 1 hour. After incubation, the cells were washed three times with 250 μL / well of FACS buffer, centrifuged at 300×g (4°C) for 5 minutes, and then washed with FACS buffer. The secondary antibody, anti-mouse IgG-Alexa488 antibody (Invitrogen, A21202) (1:1k dilution) was prepared and incubated at 4°C for 1 hour in the dark at 100 μL / well. Thereafter, the cells were washed again three times with FACS buffer, and the cell pellet was resuspended in 100 μL of FACS buffer at 4° C., and the fluorescence intensity of the cells in each well, i.e., the binding signal, was read using a flow cytometer (BECKMAN COULTER, B53013).

[0198] Data processing (method of data processing) and experimental data: The binding signal values ​​corresponding to each antibody concentration were nonlinearly fitted using graphpad. The data in Table 3 are the maximum binding signals of the antibodies and the EC50 calculated by curve fitting.

[0199] [Table 3]

[0200] Testing conclusion: The selected antibodies and the 293 cell line overexpressing human TSHR both had relatively good binding activity, and both had monkey and mouse cross-binding activity.

[0201] Example 3: Humanized antibody cell binding experiment Experimental procedure steps: The binding activity of the humanized antibodies to the 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR-overexpressing cell lines was detected using the FACS method described in Example 2. However, the secondary antibody used was an anti-human IgG-Alexa488 antibody (Invitrogen, A-11013) (diluted 1:1k).

[0202] Data processing (method of data processing) and experimental data: The binding signal values ​​corresponding to each antibody concentration were nonlinearly fitted using Graphpad, and the data in Tables 4 and 5 were the maximum binding signals of the antibodies and the EC50 calculated by curve fitting.

[0203] [Table 4]

[0204] [Table 5]

[0205] Testing conclusion: Both the above-mentioned humanized antibodies and 293-human TSHR cells had relatively good binding activity, and at the same time, had the ability to bind to monkey TSHR and mouse TSHR.

[0206] Example 4: Humanized antibody cAMP experiments Experimental procedure steps: Antibody preparation: Different concentrations of antibodies were prepared in a 384-well plate (3824 Corning) using a Bravo (Agilent) instrument. The preparation solution was DPBS, the starting concentration was 200 nM, and diluted 1:3, for a total of 11 points.

[0207] In an experiment in which a humanized antibody blocked cAMP activation by an agonist antibody, antibody-induced cAMP was detected according to the instructions for the cAMP-Gs DYNAMIC Kit (62AM4PEB, Cisbio). The cell line used was the 293-human TSHR cell line, and the competing antibodies were Tab01 and Tab02, both agonist antibodies, at concentrations of 30 nM and 6.5 nM, respectively. The sampler was a MultiDrop Combi (Thermo) set to slow speed. The data reader was an Envision (PerkinElmer) and signals were read at wavelengths of 665 nM and 615 nM. A standard curve was plotted using the standard concentration versus the 665 nM / 615 nM signal ratio to determine the relative activity of the antibody.

[0208] Tab01 is a TSHR agonist antibody M22, and its sequences were derived from the antibody heavy chain variable region of SEQ ID NO: 1 and light chain variable region of SEQ ID NO: 6 in patent WO2004 / 050708A2. The heavy chain variable region and light chain variable region were linked to the human IgG1 heavy chain constant region and human lambda light chain constant region, respectively, to obtain the full-length Tab01 heavy chain and light chain sequences, and the human IgG1 heavy chain constant region and human lambda light chain constant region sequences were referenced to the positive control Tab03_hIgG1 heavy chain constant region and light chain constant region sequences.

[0209] Tab02 is the TSHR agonist antibody K1-18, the sequence of which was derived from the heavy chain sequence of SEQ ID NO: 15 and the light chain sequence of SEQ ID NO: 33 in the TSHR antibody patent with patent number US10428153.

[0210] The positive control was Tab03_hIgG1, the sequence of which was derived from the heavy chain sequence of SEQ ID NO:51 and the light chain sequence of SEQ ID NO:59 in the TSHR antibody patent of US Patent Application No. 10,428,153.

[0211] Data processing (method of data processing) and experimental data: The ratio of the signals at wavelengths 665 nM and 615 nM was calculated, and this ratio was directly proportional to the relative cAMP content, indicating the relative activity of TSHR. A nonlinear regression curve was plotted between the antibody concentration and the corresponding 665 nM / 615 nM ratio, and the IC50 and maximum inhibition rate were calculated. The results are shown in Tables 6 and 7.

[0212] [Table 6]

[0213] [Table 7]

[0214] Testing conclusion: All antibodies showed significant inhibition of TSHR activity (in the presence of agonist antibodies Tab01 and Tab02).

[0215] Example 5: Humanized antibody hyaluronic acid (HA) secretion experiment Experimental procedure steps: 1. Fibroblast Acquisition The experiment was divided into three groups, with cells derived from patients with acute GO and patients with stable GO, respectively. The cell acquisition method was as follows: Orbital connective tissue samples were obtained from two patients in each group. The specimens were quickly placed in sterile centrifuge tubes containing DMEM / F12 medium (a mixture containing penicillin and streptomycin). The tubes were then quickly transported to the cell culture laboratory and collected in a clean bench. After repeated washing with sterile PBS to remove blood stains, the specimens were transferred to a sterile culture dish containing DMEM / F12 medium (a mixture containing penicillin and streptomycin). To maintain tissue moistness and vitality, the specimens were completely immersed in the medium. Adipose tissue and large blood vessels were carefully removed using ophthalmic scissors and tweezers for microscopy. The isolated connective tissue was then washed three times with PBS and placed in a sterile culture dish containing a small amount of DMEM / F12 medium. The tissue was then cut into approximately 1 mm pieces using scissors and tweezers. 3 The tissue was cut into approximately 100 mm tissue chunks. The cell culture plate was washed once with complete medium (DMEM / F12 medium supplemented with 20% fetal bovine serum and 1% by volume of penicillin and streptomycin). After the tissue had dried slightly, it was carefully attached to the cell culture plate. The cell culture plate with the attached tissue was inverted in a 37°C, 5% CO2 cell incubator for 30 minutes to allow the tissue to adhere to the wall as quickly as possible. After 30 minutes, the cell culture plate was inverted and placed flat. Complete medium was added to just cover the tissue, and the plate was cultured for several days. After 3 days, half of the medium was replaced, and floating tissue chunks and residual red blood cells were carefully removed. Cells were observed for emergence and medium was added. The plate was digested with 0.25% trypsin-EDTA and subcultured at a ratio of 1:3 to 1:4 every 5–7 days.

[0216] 2. Obtaining GO patient serum immunoglobulin (GO-Ig) Serum samples from 40 GO patients were collected and centrifuged at 12,000 rpm / min for 10 minutes at 4°C using a 3K15 low-temperature high-speed centrifuge. The serum was then filtered through a 0.45 μm filter and collected for use. A 2 mL gravity-flow column (29920, Thermo Fisher Scientific) was packed with thiophillic-Superflow resin (635617, Takara) and allowed to settle overnight at 4°C. The affinity chromatography gravity-flow column was then equilibrated with 10 column volumes of equilibration buffer (50 mM sodium phosphate and 0.5 M sodium sulfate, pH 7.0). The collected serum samples were diluted 1:10 (volume ratio) with sample buffer (50 mM sodium phosphate and 0.55 M sodium sulfate, pH 7.0). The diluted serum samples were applied to an affinity chromatography gravity-flow column at a flow rate of 2 ml / min. Unbound proteins were then washed away with equilibration buffer (50 mM sodium phosphate and 0.5 M sodium sulfate, pH 7.0). Finally, GO-Ig was 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 then placed in Tube-O-Dialyzer dialysis tubing (786-619, G-Biosciences, 50K molecular weight cutoff) and dialyzed overnight at 4°C against Hanks' balanced salt solution (HBSS) containing 10 mM HEPES (pH 7.4). Finally, the dialyzed sample was concentrated using a Spin-X UF concentrator (CLS431480, Sigma). The protein concentration of the concentrated product was measured using the Pierce BCA protein assay.

[0217] 3. HA Secretion Experiment 3.1 Cell culture The patient fibroblasts from each group were counted using a Countess3 automated cell counter (Thermo Fisher Scientific) and adjusted for cell density. Then, 5,000 or 10,000 cells were seeded into each 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. Cells were then starved for 24 hours, the supernatant removed, and different concentrations of the target antibody added according to the experimental group. The GO-Ig antibody was then added and incubated for 96 hours. The supernatant was carefully collected and centrifuged at 1,000 × g in a 3K15 low-temperature high-speed centrifuge for 15 minutes. The cell debris was discarded, and the supernatant was removed for detection.

[0218] 3.2 HA detection This experiment used a human hyaluronic acid (HA) reagent kit (ELISA) (ml557801, Shanghai Enzyme Biotechnology Co., Ltd.). The specific procedure was as follows: After equilibrating at room temperature for 60 minutes, the required slats were removed from the aluminum foil bag. Standard and sample wells were placed in the standard wells, and 50 μL of standard samples at different concentrations (200 ng / mL, 100 ng / mL, 50 ng / mL, 25 ng / mL, 12.5 ng / mL, and 6.25 ng / mL) were added, respectively. 50 μL of the supernatant from the above treatment was added to the sample wells, but not to the blank wells. The wells were then incubated in a 37°C incubator for 1 hour. The wells were then washed three times with washing buffer. Except for the blank well, 100 μL of horseradish peroxidase (HRP)-conjugated detection antibody was added to each of the standard and sample wells, and the wells were incubated in a 37°C incubator for 60 minutes. The wells were then washed five times with washing buffer. 50 μL of substrate A and B were added to each well and incubated for 15 minutes at 37°C in the dark. 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.

[0219] Data processing (method of data processing) and experimental data: A standard curve was plotted with the OD values ​​of the standard as the abscissa and the concentration of the standard as the ordinate to obtain a linear regression equation. The OD values ​​of the sample wells were substituted into the equation to calculate the HA concentration in each well. Nonlinear fitting was performed using Graphpad Prism 9 to calculate the IC50 concentration for the HA and antibody concentrations. The results are shown in Table 8.

[0220] [Table 8]

[0221] Testing conclusion: As can be seen from the above results, all of the selected antibodies were able to inhibit GO-Ig-induced stimulation of orbital fibroblasts from patients to produce HA. 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, and cAb12-VH3-QG&S-VL3 had relatively good inhibitory effects on cells in the acute and stable phases, and the maximum inhibitory rate in the stable phase was superior to that of the control antibody Tab03_hIgG1, while cAb01-VH2-DA-VL3 and cAb12-VH3-QG&S-VL3 The IC50 of cAb21-VH2-VL2 and cAb21-VH1-VL3 was superior to that of the positive control Tab03_hIgG1, and cAb21-VH2-VL2 and cAb21-VH1-VL3 had relatively good inhibitory effects on stationary cells, with maximum inhibitory rates superior to that of Tab03_hIgG1.

[0222] Example 6: Humanized antibody IL6 secretion experiment Experimental procedure steps: 1. Cell Culture Following the method described in Example 5, fibroblasts and GO-Ig were harvested and counted using a Countess3 automated cell counter (Thermo Fisher Scientific). Then, 5,000 or 10,000 cells were seeded into each 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. Cells were then observed under a microscope to confirm adhesion to the plate wall. The cells were then starved and cultured for 24 hours, after which the supernatant was removed. The antibodies to be tested were added at different concentrations according to the experimental group, and the incubation was continued for 24 hours. GO-Ig was added and the incubation continued for 24 hours. The cell supernatant was carefully collected and centrifuged at 1,000 × g in a 3K15 low-temperature, high-speed centrifuge for 15 minutes. Cell debris was discarded, and the supernatant was removed for detection.

[0223] 2. Detection of IL6 This experiment used a Human IL-6 ELISA reagent kit (CSB-E04638h, Cusabio). The specific process was as follows: Before the experiment, the corresponding reagent was left at room temperature for 60 minutes. 100 μl of standard and sample were added to each well. No addition was made to blank wells. Standard concentrations 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 wells were sealed and incubated at 37°C for 2 hours. After adding liquid to each well, 100 μl of 1x Biotin antibody was added to each well and incubated in a 37°C incubator for 1 hour. The wells were washed three times with washing solution. 100 μl of 1x HRP-avidin was added to each well. The wells were sealed with a new slit and incubated at 37°C for 1 hour. After washing five times, 90 μl of TMB solution was added to each well and incubated for 15 minutes at 37°C in the dark. 50 μl of Stop Solution was added to each well and gently tapped to ensure thorough mixing. 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.

[0224] Data processing (method of data processing) and experimental data: A standard curve was plotted with the OD values ​​of the standard as the abscissa and the concentration of the standard as the ordinate to obtain a linear regression equation. The OD values ​​of the samples were then substituted into the equation to calculate the IL6 concentration of each well. Graphpad Prism 9 was used to perform nonlinear fitting of the IL6 concentration and antibody concentration to calculate the IC50 concentration. The results are shown in Table 9.

[0225] [Table 9]

[0226] Testing conclusion: All of the selected antibodies were able to inhibit GO-Ig-induced stimulation of orbital fibroblasts from patients to produce IL6, and all antibodies had relatively good inhibitory effects on acute- and stable-phase cells, with cAb01-VH1-DA-VL2, cAb01-VH1-DA-VL3, cAb01-VH2-DA-VL3, and cAb21-VH2-VL2 demonstrating superior maximum inhibitory rates and IC50 values ​​against acute- and stable-phase cells compared with the control antibody Tab03_hIgG1.

[0227] Example 7: Humanized antibody IL8 secretion experiment Experimental procedure steps: 1. Cell Culture The cells were cultured according to the method described in Example 6, and the supernatant was collected for detection.

[0228] 2. Detection of IL8 In this experiment, the concentration of IL-8 in the supernatants awaiting measurement was detected using a Human IL-8 ELISA reagent kit (D8000C, R&D).

[0229] Data processing (method of data processing) and experimental data: A standard curve was plotted with the OD values ​​of the standard as the abscissa and the concentration of the standard as the ordinate, and a linear regression equation was obtained. The OD values ​​of the samples were then substituted into the equation to calculate the IL8 concentration of the supernatant in each well. Graphpad Prism 9 was used to perform nonlinear fitting of the IL8 concentration and antibody concentration to calculate the IC50 concentration.

[0230] The data analysis results are shown in Table 10.

[0231] [Table 10]

[0232] Testing conclusion: All of the selected antibodies were able to inhibit GO-Ig-induced stimulation of orbital fibroblasts from patients to produce IL-8. All antibodies had relatively good inhibitory effects on cells in the acute and stable phases, and the maximum inhibitory rates of all antibodies were almost superior to those of the positive control, Tab03_hIgG1.

[0233] Example 8: Modification of humanized antibodies In this example, antibody cAb01-VH2-DA-VL3 (designated hAb01_G4P, in which the constant region is an IgG4 subtype containing the S228P mutation) was selected and subjected to Fc modifications (modifications included modifications based on IgG4-S228P and modifications based on IgG1). The modifications were aimed at extending half-life, increasing efficacy, and reducing ADCC activity. The positions of the modifications are shown in the table below.

[0234] [Table 11]

[0235] Example 9: Binding activity of modified antibodies The binding activity of the modified antibodies to the 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR-overexpressing cell lines was detected using the FACS method described in Example 2. However, the secondary antibody used was an anti-human IgG-Alexa488 antibody (Invitrogen, A-11013) (diluted 1:1k).

[0236] Here, hIgG1 was used as a negative control (purchased from Hyakuei B117901), and hIgG4 was used as a negative control (purchased from Hyakuei B107804).

[0237] The detection results were as follows:

[0238] [Table 12]

[0239] Testing conclusion: The modified antibody showed good binding to human TSHR and cross-linking activity to mouse and monkey TSHR.

[0240] Example 10: cAMP activity experiment of modified antibodies With reference to the cAMP detection method and data processing method described in Example 4, the results of activity detection of the modified antibodies were as follows.

[0241] [Table 13]

[0242] Testing conclusion: In the cAMP activity experiment, all of the modified antibodies exhibited the ability to inhibit the activation of TSHR activity by agonist antibodies (Tab01 and Tab02), and the inhibitory effect was greater than that of the unmodified molecule hAb01_G4P.

[0243] Example 11: ADCC activity of modified antibodies Experimental Method: The target cells for the experiment were 293-human TSHR cells (the amino acid sequence of overexpressed human TSHR is P16473, see Uniprot), and the effector cells were human PBMCs (obtained from Myojun, 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 the experimental buffer (RPMI 1640 + 10% FBS, RPMI Medium 1640, Gibco, A10491-01, FBS, BI, 040021A). 6 The solution was adjusted to 1 × 10 / mL and prepared for use. 6Target cells were labeled and incubated at 37°C for 20 minutes in the dark. Afterwards, the cells were washed four times with PBS and resuspended in experimental buffer for use. Antibodies were diluted in experimental buffer starting at 800 nM, with an 11-point gradient dilution at 1:5. 50 μL of target cells, 100 μL of antibody dilution, and 50 μL of effector cells were added to an experimental plate. Simultaneously, several control wells were set up for target cell maximum (50 μL target cells + 150 μL experimental buffer), target cell autofluorescence (50 μL target cells + 150 μL experimental buffer), and target and effector cell autofluorescence (50 μL target cells + 50 μL effector cells + 100 μL experimental buffer). The experimental plate was incubated in a carbon dioxide incubator at 37°C for 2 hours. Ten microliters of lysate was added to the target cell maximum, effector cell maximum, and target and effector cell maximum control wells. The experimental plates were centrifuged at 400 g for 5 minutes, and 25 μL of the supernatant was added to the flat-bottom detection plate. 200 μL / well of europium solution (Perkin Elmer, C136-100) was added to the detection plate. The detection plate was shaken at 250 rpm for 15 minutes. The plate was read using a multifunction microplate reader.

[0244] Here, the hAb01_G1 light chain variable region and heavy chain variable region sequences were identical to those of hAb01_G4P, and the constant region subtype was hIgG1 (the full-length heavy chain amino acid sequence is shown in SEQ ID NO: 49, and the full-length light chain amino acid sequence is shown in SEQ ID NO: 51).

[0245] Data processing (method of data processing) and experimental data: Calculation method: Inhibition rate % = (sample value - target cell and effector cell autofluorescence value) / (maximum target cell value - target cell autofluorescence value) × 100%.

[0246] The sample concentration value was taken as the abscissa and the inhibition rate as the ordinate. Nonlinear fitting was performed using Graphpad Prism 9 for the inhibition rate and antibody concentration to calculate the IC50 concentration. The results are shown in Table 14.

[0247] [Table 14]

[0248] Testing conclusion: Compared to hAb01_G1, the hAb01_G1_V4 molecule had a relatively weak ADCC effect. hAb01_G4P, hAb01_G4P_V1, and hAb01_G4P_V2 did not exhibit ADCC effect.

[0249] Example 12: FcRn-binding activity of modified antibodies Experimental Method: In this experiment, hFcRn-Avi (ACROBiosystems, FCM-H82W7) was captured using an SA chip (cytiva, BR100531). Different concentrations of antibody were flowed through the chip. Steady-state fitting analysis was performed based on the collected data. The experimental equipment used was a Biacore molecular interaction system (cytiva, Biacore8K+). The buffer solution used was HBS-EP buffer (cytiva, BR100669), with a pH of 6.0, an association time of 240 seconds, a dissociation time of 240 seconds, a flow rate of 30 μL / min, and detection conditions at 25°C. Steady-state fitting analysis of the obtained experimental data was performed using Biacore Insight Evaluation Software 3.0.12. The experimental results are shown in Table 15.

[0250] [Table 15]

[0251] Testing conclusion: When antibodies of the same Fc subtype were compared, the affinity of the modified antibody for FcRn was significantly improved, demonstrating that the modified antibody could have a longer half-life.

[0252] Example 13: In vitro testing of engineered antibodies for GD indications Experimental Method: 1. Extraction and culture of primary thyroid cells from patients Thyroid tissue samples were collected from normal thyroid tissue of patients who underwent total thyroidectomy for thyroid cancer, and for subculture amplification, the tissue was resuspended in a PBS solution containing 3 mg / mL type IV collagenase (Life Technologies) to obtain dispersed cells.

[0253] 2. Detection of thyroid catalase (TPO) gene levels Cells from each group were collected and the density was adjusted, and 6 x 10 cells were placed in each well of a 24-well plate. 4 Cells were seeded and cultured overnight. After attachment to the wall, the cells were starved and cultured for 24 hours. The supernatant was discarded, and different concentrations of the antibody to be tested and medium containing or without GD serum (final concentration 1:100, volume ratio) were added according to the experimental group, followed by 48 hours of incubation. Cells were harvested by trypsin digestion, and total RNA samples were extracted. The reaction system was reverse transcribed according to the flow configuration in the reagent kit instruction manual (iScript cDNA, Bio-Rad, 1708891EDU), and cDNA was obtained and stored at -70°C.

[0254] Real-time quantitative PCR reaction The reaction system was assembled on ice according to the instructions in the reagent instruction manual (Takara, RR420A), and amplification was performed. Data were processed using the 2-ΔΔCt formula to obtain the expression level of the target mRNA, using the internal reference gene GAPDH as a reference. The primers used are listed in Table 16.

[0255] [Table 16]

[0256] Data processing and experimental data: Data were analyzed and plotted using Graphpad Prism 9 (Version 9.4.0), organized and combined in Adobe Illustrator 2022 (Version 2022), and statistical differences between groups were verified using one-way ANOVA.

[0257] The experimental results are shown in Figure 1.

[0258] Testing conclusion: All of the modified antibodies were able to inhibit TPO induced by serum from GD patients, demonstrating that the antibodies of the present invention have the potential to treat GD disease.

[0259] Example 14: Modified antibody hyaluronic acid (HA) secretion experiment The experimental method and data processing method were as described in Example 5.

[0260] [Table 17]

[0261] Testing conclusion: As can be seen from the above results, all of the modified antibodies can inhibit GO-Ig-mediated stimulation of patients' orbital fibroblasts to produce HA, and have good inhibitory effects on cells in both the acute and stable phases.

[0262] Although specific embodiments of the present invention have been described in detail, it should be understood that those skilled in the art can, based on all the teachings disclosed, make various modifications and changes to the details, and all of these modifications are within the scope of the claims of the present invention, the full scope of which is given by the appended claims and any equivalents thereof.

Claims

1. An antithyroid-stimulating hormone receptor (TSHR) antibody or antigen-binding domain comprising a VH including one heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, and VH CDR3 of any one of SEQ ID NOs. 29, 1, 9, 17, 27, 30, 32, 34, 35, or 37 as defined by the Kabat numbering system, and a VL including one light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of any one of SEQ ID NOs. 31, 5, 13, 21, 28, 33, 36, or 38 as defined by the Kabat numbering system.

2. The anti-TSHR antibody or antigen-binding domain according to claim 1, wherein the VH comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO:

29.

3. The anti-TSHR antibody or antigen-binding domain according to claim 2, wherein the VL comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO:

31.

4. The anti-TSHR antibody or antigen-binding domain according to claim 1, wherein VH CDR1 comprises SEQ ID NO: 2, VH CDR2 comprises SEQ ID NO: 39, and VH CDR3 comprises SEQ ID NO:

4.

5. The anti-TSHR antibody or antigen-binding domain according to claim 4, wherein VLCDR1 comprises SEQ ID NO: 6, VLCDR2 comprises SEQ ID NO: 7, and VLCDR3 comprises SEQ ID NO:

8.

6. The anti-TSHR antibody or antigen-binding domain according to claim 1, comprising a heavy chain containing the aforementioned VH, wherein the heavy chain is at least 90% identical to SEQ ID NO:

46.

7. The anti-TSHR antibody or antigen-binding domain according to claim 6, wherein the heavy chain comprises the sequence of SEQ ID NO:

46.

8. The anti-TSHR antibody or antigen-binding domain according to claim 6, comprising a light chain containing the VL, wherein the light chain is at least 90% identical to SEQ ID NO:

51.

9. The anti-TSHR antibody or antigen-binding domain according to claim 8, wherein the light chain comprises the sequence of Sequence ID No.

51.

10. The anti-TSHR antibody or antigen-binding domain according to claim 1, comprising a framework region of human immunoglobulin.

11. The anti-TSHR antibody or antigen-binding domain according to claim 1, comprising a heavy chain framework region of a human heavy chain germline sequence and a light chain framework region of a human light chain germline sequence.

12. The anti-TSHR antibody or antigen-binding domain according to claim 1, comprising a constant region of human immunoglobulin.

13. An anti-TSHR antibody or antigen-binding domain according to any one of claims 1 to 12, comprising an IgG4 heavy chain constant region.

14. The anti-TSHR antibody or antigen-binding domain according to claim 13, wherein the IgG4 heavy chain constant region comprises substitutional mutations S228P, L235E, M252Y, S254T, and / or T256E, compared to wild-type human immunoglobulin IgG4, according to the Kabat numbering system.

15. The anti-TSHR antibody or antigen-binding domain according to claim 1, comprising the κ light chain constant region of SEQ ID NO:

26.

16. The antigen-binding domain is Fab, Fab', (Fab') ２ Fd, Fv, Fv linked to a disulfide bond, scFv, di-scFv, (scFv) ２ An anti-TSHR antibody or antigen-binding domain according to claim 1, selected from a biantibody and a single-domain antibody (sdAb).

17. An anti-TSHR antibody or antigen-binding domain comprising the heavy chain variable region (VH) sequence of SEQ ID NO: 29 and the light chain variable region (VL) sequence of SEQ ID NO:

31.

18. The anti-TSHR antibody or antigen-binding domain according to claim 17, comprising a heavy chain containing the VH, wherein the heavy chain comprises the sequence of SEQ ID NO: 46, and a light chain containing the VL, wherein the light chain comprises the sequence of SEQ ID NO:

51.

19. Use of an antithyroid-stimulating hormone receptor (TSHR) antibody or antigen-binding domain in the manufacture of a drug for the treatment of a disease, wherein the anti-TSHR antibody or antigen-binding domain comprises VH, which includes one heavy chain variable region (VH) complementarity-determining region (CDR) 1, VH CDR2, and VH CDR3 of any one of SEQ ID NOs: 29, 1, 9, 17, 27, 30, 32, 34, 35, or 37 as defined by the Kabat numbering system, and VL, which includes one light chain variable region (VL) CDR1, VL CDR2, and VL CDR3 of any one of SEQ ID NOs: 31, 5, 13, 21, 28, 33, 36, or 38 as defined by the Kabat numbering system.

20. The use according to claim 19, wherein the disease includes autoimmune thyroid disease, Graves' disease, Graves' ophthalmopathy, or thyroid cancer.

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