Group of fully human TSH receptor blocking monoclonal antibodies belonging to SH1 homocluster, preparation method therefor and use thereof

Abstract

Provided are a group of fully human thyrotropin receptor (TSHR) (TSH receptor, TSHR) blocking monoclonal antibodies belonging to a SH1 homocluster, a preparation method therefor, and a use thereof. The method comprises the following steps: using flow cytometry to sort plasma cells and individual memory B cells, which specifically recognize TSHR, from peripheral blood of a patient having a high TSH receptor blocking antibody (TBAb) titer, cloning antibody light and heavy chains in vitro, followed by recombinant expression, and performing antibody characteristic screening and verification by using hTSHR-CHO cells, thereby obtaining blocking monoclonal antibodies that specifically target human TSHR. The fully human TSH receptor blocking monoclonal antibodies can specifically bind to TSHR, thereby enabling effective blockage of signal transduction after TSH binds to the receptor, inhibiting the synthesis and secretion of thyroid hormones, and significantly reducing and suppressing the expression of TSHR and fibrosis in orbital fibroblasts, the effector cells in thyroid-related eye diseases. The present invention has broad application prospects in the treatment of Graves' diseases and diseases caused by hyperthyroidism, such as thyroid eye diseases.

Description

A group of SH1 homologous cluster full human TSH receptor blocking monoclonal antibodies and preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of monoclonal antibody technology and human antibody drugs, and relates to a group of full human TSH receptor blocking monoclonal antibodies and a preparation method and application thereof. BACKGROUND

[0002] The thyroid stimulating hormone receptor (TSHR) belongs to a seven-transmembrane G protein-coupled receptor and mainly exists on the cell membrane of thyroid follicular epithelial cells. The TSHR has a large extracellular domain, which is composed of a leucine-rich domain and a "hinge" region. Abnormalities in the function of the TSHR can lead to the occurrence of thyroid diseases. After the thyroid stimulating hormone (TSH) binds to and activates the TSHR, downstream signal transduction can be mediated by the Gs and Gq / 11 pathways, which regulates the transport and absorption of iodine, the organicization of iodine, and participates in the synthesis and release of thyroid hormones, as well as the growth and differentiation of thyroid cells. On the other hand, the large extracellular segment of the TSHR can be divided into an alpha-subunit and a beta-subunit, and the alpha-subunit is prone to falling off. In the background of genetic susceptibility, under the action of external environmental factors such as infection and mental trauma, the TSHR peptide segment can act as an exposed autoantigen, inducing the production of autoantibodies in the body, i.e., thyroid stimulating hormone receptor antibodies (TRAb), and participating in the occurrence and development of autoimmune thyroid diseases (AITD), mainly including Graves' disease (GD) and Hashimoto's thyroiditis.

[0003] Graves' disease is an organ-specific autoimmune disease with increased secretion of thyroid hormone, which is caused by the combined action of genetics and environment, and is the most common cause of hyperthyroidism. The number of GD patients is large, and the incidence of the population is about 0.2-2%. Like other autoimmune diseases, GD is more common in women of childbearing age, and the incidence of women is about 5-10 times that of men. In recent years, with the change of living environment, the incidence of hyperthyroidism has been increasing, and if the symptoms of hyperthyroidism are not controlled in time, it can involve the heart, leading to arrhythmia or heart failure, etc., and can lead to menstrual disorders, difficulty in pregnancy, abortion, etc. in women of childbearing age. In addition, hyperthyroidism can mostly cause mental abnormalities such as tension, anxiety, restlessness, etc., affecting the learning and life of patients, and even causing mental illness in severe cases. At present, the treatment of GD mainly includes drugs, radioactive iodine and surgical treatment, the former has a long course, and patients are difficult to adhere to, and the recurrence rate is high, about 60-70% of patients will relapse. The treatment of GD in clinical practice has not changed substantially for many years, and it is still a choice between antithyroid drugs, radioactive iodine or surgery. Among them, the treatment of Graves' disease with antithyroid drugs has been nearly 70 years. Except for the United States, doctors all over the world regard ATD as the first choice for the treatment of Graves' disease. However, after regular and systematic treatment of hyperthyroidism patients, only part of the patients can achieve cure, and the side effects are large, and a considerable part of the patients will relapse after a certain period of time. When the disease relapses, the condition worsens, and generally radioactive iodine or surgical treatment is needed, greatly increasing the economic and psychological burden of patients. In addition, radioactive iodine 131 radionuclide treatment can easily lead to permanent hypothyroidism; the complications of surgical treatment cannot be ignored. The current treatment dilemma of GD means that it is urgent to find better alternative drugs in clinical practice.

[0004] TRAb is the general term for antibodies produced by the body against TSHR, and is a group of polyclonal antibodies, which can be divided into TSH receptor stimulating antibodies (TSH-stimulating antibody, TSAb), TSH receptor blocking antibodies (TSH-stimulating blocking antibody, TBAb) and neutral antibodies. Their recognition epitopes on TSHR are not the same, and the recognition epitopes of TSAb, TBAb and neutral antibodies are concentrated in the amino terminal (N terminal), carboxy terminal (C terminal) and hinge region of the extracellular region of TSHR. Among them, the thyroid stimulating antibody TSAb binds to the TSHR on the membrane of the thyroid follicular epithelial cells, produces a biological effect similar to TSH, causes hyperthyroidism, and is the direct cause of Graves' disease. TSAb stimulates G protein coupling and further activates adenylate cyclase (AC) to stimulate the production of cAMP, and the AC-cAMP pathway is in a state of continuous activity, stimulating thyroid cell proliferation, and the thyroid gland synthesizes and secretes excessive thyroid hormones, i.e. triiodothyronine (T3) and thyroxine (T4). TSH cannot normally play a feedback regulatory role on T3 and T4 due to competitive inhibition, resulting in continuous increase of T3 and T4, causing a series of reactions in the body, and further causing hyperfunction of thyroid cells. From the histological level, the thyroid of GD patients shows thickened and enlarged follicular cells. The gland shows typical T cell and B cell lymphocyte infiltration, and there is a characteristic of thyroiditis, with a small amount of cell apoptosis and a certain degree of follicular destruction. TBAb blocks the binding of TSH to the receptor and inhibits thyroid hyperplasia and thyroid hormone production. Stimulating and blocking antibodies coexist in GD patients, and the final result of thyroid function depends on which antibody is dominant. This makes it possible to supplement a certain dose of blocking antibodies to TSHR from the outside, thereby improving the pathophysiological effects of autoantibodies, and becomes a new effective scheme for treating GD.

[0005] Furthermore, TSH receptor blocking monoclonal antibodies can inhibit the synthesis and secretion of thyroid hormones by blocking the signal transduction after TSH binds to the receptor, thereby treating a series of diseases caused by hyperthyroidism, such as thyroid eye disease (thyroid eye disease, TED), also known as thyroid associated ophthalmopathy (Thyroid associated ophthalmopathy, TAO), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis and pretibial myxedema, etc.

[0006] Thyroid eye disease (TED) is a common orbital disease in adults, an autoimmune disease closely related to Graves' disease (GD), which can manifest as hyperemia and edema of the eyelid and conjunctiva, fibrosis and adiposis of the intraorbital tissue, causing eye movement disorders, strabismus and diplopia, which can cause blindness and disability, and is difficult to diagnose and treat, seriously affecting the quality of life of patients. China is one of the countries with a high incidence of GD in the world, and the prevalence of TED in GD patients of Asian descent is as high as 45%. The TED patient population is large, and their visual health and quality of life have been severely affected. As a difficult problem that has plagued the global medical scientific community for two centuries, TED currently lacks effective treatment options. Although there are various treatment options for TED, many patients do not respond well to existing drugs or cannot tolerate side effects. High-dose glucocorticoid pulse therapy can significantly reduce inflammation, but it has little effect on exophthalmos and diplopia, and long-term use has significant side effects, such as hypertension, diabetes, osteoporosis, and gastric ulcers. The effectiveness of glucocorticoids for some patients is only 50-75%. In addition, there is the drawback of short-term effectiveness, but relapse after discontinuation. Immunosuppressants can reduce eye inflammation by suppressing the overreaction of the immune system, but can cause serious side effects, such as increased risk of infection and liver and kidney function damage. Therefore, there is an urgent need for new, safe and effective drugs on the market.

[0007] Orbital fibroblasts (OF) are effector cells of TED autoimmune response, which have thyroid stimulating hormone receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) on their surface. TSHR is ectopically highly expressed in CD34+ fibroblasts in the retrobulbar tissue of TED patients. TED immune response begins with abnormal recognition of autoantigen TSHR, and activated B cells produce TRAb. IGF-1R is another possible TED autoantigen, but it is not clear whether there are autoantibodies that directly stimulate IGF-1R. Studies have shown that IGF-1R and TSHR-mediated signal transduction pathways combine to cause the secretion of large amounts of hyaluronic acid (HAS). The antibody drug targeting IGF-1R (Tepezza) was approved by the FDA in 2020 for the treatment of TED. After treatment, exophthalmos and diplopia can be significantly improved, and nearly 40% of exophthalmos patients can basically return to normal. Although IGF-1R antibody drugs have significant efficacy, since IGF-1R is widely expressed in human organs, many significant side effects have been reported after large-scale clinical application, especially irreversible deafness and effects on the reproductive system. Because stimulating and blocking antibodies coexist in GD patients, the final result of thyroid function depends on which antibody is dominant. This makes it a new effective solution for treating thyroid eye disease to exogenously supplement a certain dose of blocking antibodies to inhibit TSHR.

[0008] Humanized monoclonal antibodies can be divided into murine humanized monoclonal antibodies and fully human monoclonal antibodies. Although the proportion of other non-human components is reduced to a certain extent in murine humanized monoclonal antibodies, all non-human components cannot be completely excluded, and the affinity and original biological activity of the antibodies are also reduced to a certain extent during the modification process. While fully human monoclonal antibodies directly amplify antibody genes from human single B cells, the antibody genes of single plasma cells or memory B cells isolated by amplification are obtained in large quantities, and then the paired antibody light and heavy chain genes are expressed to ultimately screen antibodies with antigen specificity and neutralizing activity. This method has the advantages of rapidity, high throughput, and small amount of required cells, and the fully human antibodies prepared by this method have the advantages of rich genetic diversity and natural pairing of light and heavy chain variable regions. At present, the preparation of fully human antibodies against influenza, anthrax virus and pneumococcus is based on this technology. SUMMARY

[0009] In view of the above prior art, the present application provides a group of fully human TSH receptor (TSHR) blocking monoclonal antibodies or antigen binding fragments thereof for treating hyperthyroidism, and also provides the coding sequence of the antibodies and the vectors containing the coding sequence.

[0010] In the present application, the term "TSHR" refers to a full-length human thyroid-stimulating hormone receptor having an amino acid sequence as shown in SEQ ID NO: 29, or a variant or fragment highly homologous to the thyroid-stimulating hormone receptor. Preferably, such variant and fragment have 70-99.9% homology with the amino acid sequence as shown in SEQ ID NO: 29.

[0011] The present application provides a group of fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof, which bind to TSH receptor and block the binding of TSH to TSH receptor; the fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof comprise a heavy chain variable region and a light chain variable region.

[0012] The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, the amino acid sequence of H-CDR1 is selected from one of SEQ ID NOs: 30-33; the amino acid sequence of H-CDR2 is selected from one of SEQ ID NOs: 34-36; and the amino acid sequence of H-CDR3 is selected from one of SEQ ID NOs: 37-40.

[0013] Preferably, the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:46; or,

[0014] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO:30, SEQ ID NO:34 and SEQ ID NO:37; or,

[0015] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO:31, SEQ ID NO:35 and SEQ ID NO:38; or,

[0016] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO:32, SEQ ID NO:35 and SEQ ID NO:39; or,

[0017] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 in the heavy chain variable region are respectively as shown in SEQ ID NO:33, SEQ ID NO:36 and SEQ ID NO:40.

[0018] Preferably, the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO:41, SEQ ID NO:43 and SEQ ID NO:46; or,

[0019] Preferably, the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO:42, SEQ ID NO:44 and SEQ ID NO:47; or,

[0020] Preferably, the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 in the light chain variable region are respectively as shown in SEQ ID NO:41, SEQ ID NO:45 and SEQ ID NO:48.

[0021] Further preferably, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region are as set forth in SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37, respectively; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region are as set forth in SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 46, respectively.

[0022] Further preferably, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region are as set forth in SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 37, respectively; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region are as set forth in SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 47, respectively.

[0023] Further preferably, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region are as set forth in SEQ ID NO: 31, SEQ ID NO: 35, SEQ ID NO: 38, respectively; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region are as set forth in SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 46, respectively.

[0024] Further preferably, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region are as set forth in SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 39, respectively; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region are as set forth in SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 46, respectively.

[0025] Further preferably, the amino acid sequences of H-CDR1, H-CDR2, and H-CDR3 of the heavy chain variable region are as set forth in SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 39, respectively; and the amino acid sequences of L-CDR1, L-CDR2, and L-CDR3 of the light chain variable region are as set forth in SEQ ID NO: 41, SEQ ID NO: 45, SEQ ID NO: 48, respectively.

[0026] Further preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are respectively as shown in SEQ ID NO:33, SEQ ID NO:36, SEQ ID NO:40; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are respectively as shown in SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:46.

[0027] wherein the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to one of SEQ ID NO:1, 3, 5, 7, 9, 11, 13;

[0028] wherein the amino acid sequence of the light chain variable region is selected from one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14 or has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14.

[0029] The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), Fav, dsFv, sc(Fv)2, single domain antibody (dAb).

[0030] In the present application, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof are as shown in SEQ ID NO:15-SEQ ID NO:28.

[0031] Preferably, the nucleotide sequences encoding the heavy chain variable region and the light chain variable region of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof have at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to SEQ ID NO:15-SEQ ID NO:28.

[0032] SEQ ID NO: 1 (SH1_1 amino acid sequence)

[0033] SEQ ID NO: 2 (SH1_2 amino acid sequence)

[0034] SEQ ID NO: 15 (SH1_3 nucleotide sequence)

[0035] SEQ ID NO: 16 (SH1_4 nucleotide sequence)

[0036] SEQ ID NO: 3 (SH2_1 amino acid sequence)

[0037] SEQ ID NO: 4 (SH2_2 amino acid sequence)

[0038] SEQ ID NO: 17 (SH2_3 nucleotide sequence)

[0039] SEQ ID NO: 18 (SH2_4 nucleotide sequence)

[0040] SEQ ID NO: 5 (SH3_1 amino acid sequence)

[0041] SEQ ID NO: 6 (SH3_2 amino acid sequence)

[0042] SEQ ID NO: 19 (SH3_3 nucleotide sequence)

[0043] SEQ ID NO: 20 (SH3_4 nucleotide sequence)

[0044] SEQ ID NO: 7 (SH7_1 amino acid sequence)

[0045] SEQ ID NO: 8 (SH7_2 amino acid sequence)

[0046] SEQ ID NO: 21 (SH7_3 nucleotide sequence)

[0047] SEQ ID NO: 22 (SH7_4 nucleotide sequence)

[0048] SEQ ID NO: 9 (SH18_1 amino acid sequence)

[0049] SEQ ID NO: 10 (SH18_2 amino acid sequence)

[0050] SEQ ID NO: 23 (SH18_3 nucleotide sequence)

[0051] SEQ ID NO: 24 (SH18_4 nucleotide sequence)

[0052] SEQ ID NO: 11 (SH19_1 amino acid sequence)

[0053] SEQ ID NO: 12 (SH19_2 amino acid sequence)

[0054] SEQ ID NO: 25 (SH19_3 nucleotide sequence)

[0055] SEQ ID NO: 26 (SH19_4 nucleotide sequence)

[0056] SEQ ID NO: 13 (SH63_1 amino acid sequence)

[0057] SEQ ID NO: 14 (SH63_2 amino acid sequence)

[0058] SEQ ID NO: 27 (SH63_3 nucleotide sequence)

[0059] SEQ ID NO: 28 (SH63_4 nucleotide sequence)

[0060] SEQ ID NO: 29 (human TSHR amino acid sequence)

[0061] Table 1: CDR regions of heavy chain (H) and light chain (L) of fully human TSH receptor blocking monoclonal antibodies with SH1 homology clusters

[0062] This invention comprises a group of fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments, namely SH1, SH3, SH2, SH7, SH18, SH19, and SH63. These antibodies share high homology with SH1, hence the name "Group of SH1 Homologous Cluster Fully Human TSH Receptor Blocking Monoclonal Antibodies or Their Antigen-Binding Fragments." In the table above, K1-70 represents a previously reported TSH receptor blocking monoclonal antibody with blocking activity. In contrast, the sequences provided in this invention are significantly different and show no homology whatsoever.

[0063] Preferably, the present invention proposes a set of fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments, named SH1 homology cluster fully human TSH receptor blocking monoclonal antibodies or their antigen-binding fragments.

[0064] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment is a TSH antagonist.

[0065] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment is an antagonist of thyroid-stimulating antibodies.

[0066] In this invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment each contains a V H Region (heavy chain variable region), the V H The region contains the complementarity-determining region (CDR) of the amino acid sequences as shown in SEQ ID NO: 30, 34, 37 (see Table 1), or the complementarity-determining region CDR of the amino acid sequences as shown in SEQ ID NO: 31, 35, 38 (see Table 1), or the complementarity-determining region CDR of the amino acid sequences as shown in SEQ ID NO: 32, 35, 39 (see Table 1), or the complementarity-determining region CDR of the amino acid sequences as shown in SEQ ID NO: 33, 36, 40 (see Table 1).

[0067] In this invention, each antibody contains a V L Region (light chain variable region), the V L The region contains CDRs of amino acid sequences as shown in SEQ ID NO: 41, 43, 46 (see Table 1) or CDRs of complementarity-determining regions of amino acid sequences as shown in SEQ ID NO: 42, 44, 47 (see Table 1) or CDRs of complementarity-determining regions of amino acid sequences as shown in SEQ ID NO: 41, 45, 48 (see Table 1).

[0068] In the present application, the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof comprises one or more amino acid sequences substantially homologous to the CDRs.

[0069] The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof provided by the present application inhibits the signal transduction of TSH receptor when binding to TSH receptor; inhibits the synthesis and secretion of thyroid hormone, reduces hyperthyroidism caused by various reasons; can significantly reduce thyroid enlargement caused by hyperthyroidism and the like after binding to TSH receptor; and reduces inflammation, edema and hyperplasia and the like of thyroid associated ophthalmopathy.

[0070] The thyroid associated ophthalmopathy (TAO) in the present application is also called thyroid eye disease (TED).

[0071] The present application also provides a preparation, a drug or a pharmaceutical composition comprising the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof described above.

[0072] The present application also provides a reagent or kit containing the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0073] The present application also provides a nucleotide encoding the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, and the nucleotide sequence is one of the following sequences:

[0074] (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NO: 15, 17, 19, 21, 23, 25, 27; and the nucleotide sequence encoding the light chain variable region is shown in one of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28;

[0075] (b) a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 more sequence identity to SEQ ID NO: 15-SEQ ID NO: 28;

[0076] (c) a nucleotide sequence obtained by adding, substituting, deleting or inserting one or several nucleotides to the nucleotide sequence shown in SEQ ID NO: 15-SEQ ID NO: 28;

[0077] (d) a nucleotide sequence hybridizing under stringent conditions to the nucleotide sequence of any of (a), (b), or (c) above, or the full-length complement thereof; or,

[0078] (e) a nucleotide sequence that differs from the nucleotide sequence of any of (a), (b), (c), or (d) above due to the degeneracy of the genetic code;

[0079] wherein the nucleotide sequence of the nucleotide or a portion thereof encodes an antibody V H domain; and an antibody V L domain or a CDR selected from the group consisting of SEQ ID NO: 30-SEQ ID NO: 48 (see Table 1).

[0080] The present application also provides a vector comprising the nucleotide as described above.

[0081] The present application also provides a host cell comprising the nucleotide as described above, and / or the vector as described above.

[0082] The present application also provides a cell which is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above, and / or the nucleotide as described above, and / or the vector as described above.

[0083] The present application also provides a cell which is an isolated cell expressing the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above.

[0084] The present application also provides a cell which is an isolated cell secreting the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above.

[0085] The present application also provides a composition comprising a determined concentration of TSH receptor autoantibodies and comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above.

[0086] The present application also provides a pharmaceutical composition for administration to a mammalian subject for the treatment of a thyroid-related condition, comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier;

[0087] wherein the thyroid-related condition is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

[0088] Further, the formulation, medicament or pharmaceutical composition can further comprise a physiologically compatible excipient, including a buffer, diluent, excipient, filler, binder, humectant, disintegrant, absorption enhancer, surfactant, adsorption carrier, lubricant, etc.

[0089] Further, the formulation, medicament or pharmaceutical composition can be prepared as an injection, sterile powder for injection, tablet, pill, capsule, lozenge, elixir, powder, granule, syrup, solution, tincture, aerosol, powder mist, or suppository, etc. The formulation, medicament or pharmaceutical composition in the above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0090] Further, the formulation, medicament or pharmaceutical composition can be introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, mucosal tissue, by injection, spraying, nasal instillation, ocular instillation, penetration, absorption, physical or chemical mediated methods; or mixed or encapsulated with other substances before being introduced into the body.

[0091] Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye disease comprises the full human TSH receptor blocking monoclonal antibody.

[0092] Further, the nucleotide sequence or at least part of the sequence can be expressed by a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include, but are not limited to, bacterial, insect cell and mammalian cell expression systems.

[0093] Further, the vector can be a plasmid, virus or their fragments, as well as various types of vectors known to those skilled in the art.

[0094] Preferably, the host cell is a CHO-K1 cell or the like.

[0095] In particular, the composition can comprise a determined concentration of TSHR autoantibodies having TSH antagonist activity, and comprises a full human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to the present application.

[0096] Further, the pharmaceutical composition is suitable for administration to a human; preferably, the pharmaceutical composition according to the present application has no significant adverse effect on the immune system of the subject.

[0097] Further, the pharmaceutical composition comprises one or more additional thyroid-stimulating hormone receptor antagonists.

[0098] Further, the pharmaceutical composition is used for treating a thyroid-related condition in an injectable form.

[0099] Preferably, said pharmaceutical composition for use in the treatment of Graves' ophthalmopathy is in the form of an intravenous injection formulation or an eye drop.

[0100] Preferably, said pharmaceutical composition for use in the treatment of pretibial myxedema is in the form of a topical administration.

[0101] In particular, said pharmaceutical composition comprises any antibody according to the present application and any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical compositions of this application include, but are not limited to, buffers such as phosphate, glycine, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), sorbitan, potatium sorbate, water, sodium chloride, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, partially

[0102] The pharmaceutical composition can be formulated in a wide variety of dosage forms. Such formulations include, but are not limited to, capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, inhalers. Preferably, the formulation is a solution or an ointment.

[0103] In particular, the pharmaceutical composition can be in the form of a sterile injectable preparation, such as a sterile injectable oil or water suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, mannitol, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables. The sterile oil solutions or suspensions can also contain long chain alcohol diluents or dispersants.

[0104] In particular, the pharmaceutical composition can be administered topically, by spray inhalation, orally, parenterally, by eye drop or eye ointment, buccal, vaginal, rectal, nasal administration or via an implanted reservoir, and the like. Preferably, the administration is oral or by injection. The term "parenterally" as used herein includes subcutaneous, intracutaneous, intra-articular, intrasynovial, intrasternal, intravenous, intramuscular, intralesional, intracranial, intraarterial, and intrathecal injection or infusion techniques.

[0105] In particular, the pharmaceutical compositions can be administered in a form suitable for rectal administration. Such compositions will contain the active ingredient in the form of a remedy which is not irritating to the rectal mucosa and which is liquefied at body temperature. Such materials include, but are not limited to, beeswax, cocoa butter, and polyethylene glycol.

[0106] In particular, the pharmaceutical compositions can be orally administered in any orally acceptable dosage form including, but not limited to, tablets, capsules, aqueous suspensions and solutions. In the case of tablets, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening and / or flavoring and / or coloring agents can be added.

[0107] In particular, the pharmaceutical compositions can be administered in the form of a nose spray or inhalant. These compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art.

[0108] In particular, topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For treatment of the eye or other external tissues, for example, the pharmaceutical composition can be applied as a topical ointment. Topical administration can also provide patterns of administration useful for the systemic treatment of systemic diseases in which localized effects are desirable, e.g., affecting the affected area. Topical application for transdermal absorption is a contemplated administration route. Topical application to the skin or mucosa of a pharmaceutically effective amount of the compounds of the present application, or salts or derivatives thereof, is a contemplated administration route. Topical administration of the pharmaceutical compositions of the present application is especially useful when the desired treatment involves areas or organs readily accessible by topical application. For treatment of the eye or other external tissues, for example, the pharmaceutical composition can be applied as a topical ointment. Topical administration can also provide patterns of administration useful for the systemic treatment of systemic diseases in which localized effects are desirable, e.g., affecting the affected area. Topical application for transdermal absorption is a contemplated administration route. Topical application to the skin or mucosa of a pharmaceutically effective amount of the compounds of the present application, or salts or derivatives thereof, is a contemplated administration route.

[0109] The present application also provides a method for preparing a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof, which method specifically comprises the following steps:

[0110] The first step is to sort the TSHR-aimed plasma cells and memory B cells in the peripheral blood of patients with high TBAb activity, to carry out single cell RNA extraction and cDNA synthesis, and to verify the sorted single cells by heavy chain H, light chain lambda and light chain kappa amplification through nested PCR, and to select single cells with positive heavy chain and light chain for subsequent cloning;

[0111] The second step is to amplify and clone the BCR heavy chain and light chain of all single B cells into the expression heavy chain vector AbVec-IGHG1, the lambda light chain expression vector AbVec-hIgKappa or the lambda light chain expression vector AbVec-hIgLambda through nested PCR in vitro.

[0112] The third step is to sequence and compare the obtained candidate clones after successfully obtaining the heavy chain and light chain recombinant plasmids, to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies, to transfect the heavy chain and light chain expression plasmids, to express the monoclonal antibodies in vitro, to further verify the antigen binding capacity and antibody blocking activity, and to finally obtain an antibody combination that can be specifically aimed at a target antigen, i.e., the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof.

[0113] The application also provides a method for treating a thyroid-related disease in a mammalian subject or in a cell derived from the subject, which comprises contacting the subject or the cell with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0114] The thyroid-related disease is selected from hyperthyroidism, thyroid-related eye disease (Graves' eye disease), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis and pretibial myxedema, etc.

[0115] The application also provides a method for inhibiting the stimulation of TSH receptors by thyroid stimulating antibodies in the thyroid of a mammalian subject, which comprises contacting the subject with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0116] Preferably, the binding of thyroid stimulating antibodies to TSH receptors is prevented.

[0117] The application also provides a method for inhibiting the binding of thyroid stimulating autoantibodies to TSH receptors outside the thyroid in a mammalian subject, which comprises contacting the subject with the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0118] In particular, the ectopic TSH receptor is located in retro-orbital tissue and / or pre-tibial tissue of the subject;

[0119] Preferably, the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof can block the binding of TSH receptor autoantibodies to ectopic TSH receptors.

[0120] The application also provides a method for treating thyroid cancer or metastatic thyroid cancer in the thyroid of a subject or in a thyroid cell derived from a subject, said method comprising contacting said cancer cell with a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, with the aim of inhibiting constitutive thyroid stimulating hormone receptor activity in said cell;

[0121] Preferably, the regrowth of said thyroid cancer cell is prevented or delayed.

[0122] The application also provides a method for treating hyperthyroidism caused by constitutive thyroid activity in a subject or in a thyroid cell derived from a subject, said method comprising contacting said subject or cell with a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above, with the aim of inhibiting such hyperthyroidism.

[0123] The application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid stimulating antibodies to TSH receptors, said method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above as a reference;

[0124] Preferably, a molecule is selected which prevents the binding of thyroid stimulating antibodies to TSH receptors.

[0125] The application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid blocking antibodies to TSH receptors, said method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above as a reference;

[0126] Preferably, a molecule is selected which prevents the binding of thyroid blocking antibodies to TSH receptors.

[0127] In particular, the thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis and pre-tibial myxedema.

[0128] Preferably, the subject treated in the above method is a human.

[0129] In the present application, the preparation method comprises the following steps: using flow cytometry to sort the plasma cells and memory single B cells specifically recognizing TSHR in peripheral blood of patients with high TSH-stimulating blocking antibody (TBAb) titer, cloning the antibody light and heavy chains in vitro and recombinantly expressing, and using hTSHR-CHO cells to screen and verify the antibody properties, so as to obtain blocking monoclonal antibodies specifically targeting human TSHR. Through the preparation method of the blocking monoclonal antibodies of human TSHR provided in the present application, the target antibody sequence can be obtained in 3 weeks to 1 month.

[0130] Further, the present application verifies and evaluates the antibody properties and effects in vitro by using hTSHR-CHO cells.

[0131] The present application also provides the use of the human TSHR blocking monoclonal antibody or the antigen binding fragment thereof as described above for treating a thyroid-related disease.

[0132] The present application also provides the use of the human TSHR blocking monoclonal antibody or the antigen binding fragment thereof as described above for preparing a medicament for treating a thyroid-related disease.

[0133] The present application also provides the use of the human TSHR blocking monoclonal antibody or the antigen binding fragment thereof as described above for preparing a reagent or a kit for detecting TSHR antibodies.

[0134] The present application also provides the use of the human TSHR blocking monoclonal antibody or the antigen binding fragment thereof as described above for preparing a reagent or a kit or a product for detecting hyperthyroidism and thyroid-related eye diseases.

[0135] The present application also provides the use of the human TSHR blocking monoclonal antibody or the antigen binding fragment thereof as described above, or the substance as described above, or the method as described above for preparing a preparation, a medicament or a pharmaceutical composition for detecting TSHR antibodies, a medicament for treating hyperthyroidism and thyroid-related eye diseases, a medicament for inhibiting thyroid hyperplasia and / or thyroid hormone production, a TSHR blocking medicament, and a medicament for antagonizing the activation of TSH on TSHR.

[0136] Specifically, the thyroid-related disease in the above method is selected from hyperthyroidism, thyroid-related eye disease (Graves' eye disease), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0137] The method of the present application does not need to immunize animals, and a large number of naturally paired antibody light and heavy chain genes are obtained by directly amplifying antibody genes from human single B cells, by amplifying antibody genes of isolated single plasma cells or memory B cells, and then by expressing paired antibody light and heavy chain genes, and finally screening to obtain antibodies with antigen specificity and neutralizing activity. The method of the present application has the advantages of rapidity, high throughput, and small amount of required cells, and the prepared fully human antibodies retain rich genetic diversity and natural pairing of light and heavy chain variable regions, and have great advantages. Compared with the traditional hybridoma antibody preparation technology, the experimental period can be significantly shortened, and the traditional hybridoma antibody preparation technology generally needs about 3 months to obtain antibody sequences, while the present application only needs 3 weeks to 1 month to obtain antibody sequences, greatly reducing the workload and cost of antibody preparation.

[0138] The present application also has the advantages that the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof can effectively block the signal transduction after TSH binds to the receptor, inhibit the synthesis and secretion of thyroid hormones, and significantly inhibit the expression of TSHR of effector cells of thyroid-associated eye disease and fibrosis. It can be used for treating a series of diseases caused by hyperthyroidism, such as thyroid eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid hyperactivity, thyroid cancer, thyroiditis, and pretibial myxedema, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0139] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor.

[0140] Figure 1 is a flow cytometry technology for sorting TSHR-specific plasma cells and memory single B cells from peripheral blood. Biotin-AF647 is used to label TSHR protein as a decoy, and other antibodies used for sorting are: CD19-Pacific Blue, IgM-PE, CD27-BV605, CD38-PE-Cy7, and finally TSHR-specific plasma cells and memory single B cells are sorted as CD19 + IgM - CD27 + CD38 - TSHR + .

[0141] Figure 2 is a graph showing the screening results of the anti-human TSHR monoclonal antibodies of the present application. The results shown are the mean ± standard deviation (n = 3). The dotted line value is 30%.

[0142] Figure 3 is a graph showing the results of the phylogenetic tree analysis of the anti-human TSHR monoclonal antibodies of the present application. The percentage of replicate trees in which a given clade was found is shown below the branches on the minimum loss of support test (1000 replicates).

[0143] Figure 4 is a graph showing the evaluation of the inhibitory effect of the SH1 antibody of the present application on orbital fibroblasts. The horizontal line numbers indicate the statistical differences (P value) between the antibody group and the control group (Control), and a value of 0.05 or less is considered to be a significant difference. DETAILED DESCRIPTION

[0144] The present application will be further described in conjunction with the following specific examples and drawings. The process, conditions, experimental methods, etc. for implementing the present application are generally known in the art and are not particularly limited, except for the following specifically mentioned content.

[0145] Example 1

[0146] Single cell sorting: Peripheral blood was collected from a volunteer with high TBAb titer, and a human B cell enrichment cocktail (STEMCELL, Cat. No. 15024) was added to the blood sample by immunodensity gradient centrifugation. The B cells in the peripheral blood were obtained by density gradient centrifugation using Ficoll-Paque Plus (Sigma, Cat. No. 10771). The CD19 + IgM - CD27 + CD38 - TSHR + plasmacytes and memory single B cells (Figure 1).

[0147] Light and heavy chain variable region cloning: The RNA bound to the single cells was captured using the SPRlselect nucleic acid fragment screening kit (Beckman Coulter, Cat. No. B23317), and the variable region sequences of the light and heavy chains were cloned according to the SuperScript III One-Step RT-PCR System (Invitrogen, Cat. No. 12574- 019) and the manufacturer's instructions. TM ​IV-step RT-PCR system (Invitrogen, Cat: 12594100) was used to synthesize cDNA. DreamTaq Green PCR 2X Master Mix (ThermoFisher, Cat: K1081) was used to amplify the variable region of light and heavy chain of antibody respectively. The PCR products were detected by agarose gel electrophoresis, and the bands with expected size were cut and recovered. The DNA fragments were purified by QIAquick Gel Extraction Kit (QIAGEN, Cat: 28704) and sent for sequencing. The sequencing results were analyzed by IgBLAST function of NCBI or IMGT database, and the corresponding V and J gene cloning primer was selected for cloning PCR of light and heavy chain. Then, the HiFi DNA Assembly Master Mix (NEB, Cat: E2621L) was used for fragment ligation, and the obtained light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors (NCBI GenBank number: FJ475055, FJ475056, FJ517647) containing the constant region of light and heavy chain. The transformed plates were picked and the final sequence was determined by sequencing. HiFi DNA Assembly Master Mix (NEB, Cat: E2621L) was used for fragment ligation, and the obtained light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors (NCBI GenBank number: FJ475055, FJ475056, FJ517647) containing the constant region of light and heavy chain. The transformed plates were picked and the final sequence was determined by sequencing.

[0148] Blocking antibody screening:

[0149] 1. Antigen binding capacity screening: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After incubation at 37℃, 5% CO2 for 3 days, the culture supernatant was collected by centrifugation, and the titer of TRAb antibody in the supernatant was detected according to the instructions of human anti-thyrotropin receptor antibody enzyme-linked immunosorbent assay kit (KeLu, Cat: ELK9540). The light and heavy chain combinations with binding capacity lower than the blank control group were removed, and the remaining combinations were screened for blocking activity.

[0150] 2. Blocking activity screening: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After incubation at 37℃, 5% CO2 for 3 days, the culture supernatant was collected by centrifugation, and 100 μL of supernatant was added with 1 IU / L bTSH (Sigma), and then hTSHR-CHO cells were incubated for 2 hours. The cell lysate was collected, and the change of cAMP level was detected (R&D, Cat: KGE002B).

[0151] Antibody expression and purification: the antibody light and heavy chain expression vectors were co-transfected into 293F cells at a ratio of 1:1; after 5 days of culture at 37°C, 8% CO2, 130 rpm shaking, the culture supernatant was collected by centrifugation, 0.45 μm filtered, and purified by Protein A (Genscript, Catalog No: L00210) affinity chromatography to obtain antibody proteins with high purity; the antibody concentration was determined by Bradford protein concentration method (Bi Yun Tian, Catalog No: P0006) and NanoDrop A280 method.

[0152] In vitro TSHR inhibition effect evaluation: the purified monoclonal antibodies were diluted according to different concentration gradients, bTSH 5 ng / ml was added, and hTSHR-CHO cells were incubated for 2 hours, then the cell lysate was collected, and the change of cAMP level was detected. If the inhibition of cAMP generation is greater than 30%, the monoclonal antibody is considered to have inhibitory activity. It was found that the monoclonal antibody concentration of 1 μg / mL can effectively antagonize the activation of TSH on TSH receptor, and show a concentration-dependent manner (Figure 2). The inhibition of cAMP generation percentage of SH1 at 2 μg / mL can reach 94%. SH1 is the strongest human TSHR inhibitory antibody in this group of inhibitory antibodies.

[0153] Phylogenetic tree analysis: MEGA 11 analysis software was used for phylogenetic analysis of antibodies, and a neighbor-joining method was used to construct a phylogenetic tree. The reliability of the phylogenetic tree was evaluated by Bootstrap value. The percentage of repeated trees in which the relevant taxa were clustered together in the bootstrap test (1000 repeats) is shown below the branch. The evolutionary distance was calculated using the Poisson correction method. Figure 3 is a rootless optimal tree combined with the results of in vitro inhibition activity determination. Antibody K1-70 is a reported TSHR inhibitory antibody, and the rest are antibodies obtained by screening this time. The closer the evolutionary distance of the antibodies, the closer the inhibitory activity. The results show that K1-70 and the strongest antibody SH1 in this application have the farthest evolutionary distance, indicating that the sequence difference between the two antibodies is the largest, and they are two completely different TSHR inhibitory monoclonal antibodies. The TSHR inhibitory monoclonal antibody screened in this application with the closest evolutionary distance to K1-70 is SH63, and its inhibitory activity is only 87% at an antibody concentration of 2 μg / mL, indicating that the inhibitory efficiency of K1-70 on TSHR is lower than that of the strongest antibody SH1 screened this time.

[0154] Orbital fibroblast (OF cell) inhibition effect evaluation: OF cells were cultured in 10 cm dishes in DMEM containing 10% FBS until they reached 70% confluence, then they were passaged to 12-well plates and cultured in DMEM containing 1% FBS for 16 hours. After adding 50 ng / ml of inhibitory antibody SH1, they were cultured for another 24 hours. The cells were collected to extract RNA, and the expression levels of fibrosis-related gene mRNA were detected by RT-qPCR. The results showed that TSHR inhibitory monoclonal antibody SH1 could significantly inhibit the expression of TSHR mRNA and fibrosis marker collagen 1A1 (COL1A1) mRNA (Figure 4).

[0155] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used in the description of the application herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0156] As used herein, the terms "comprises", "comprising", "includes", "including" or "contains", "containing" are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements or method steps.

[0157] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0158] The protection scope of the present application is not limited to the above examples. Variations and advantages that can be thought of by those skilled in the art without departing from the spirit and scope of the present application are included in the present application, and are protected by the appended claims.

Claims

1. A panel of fully human TSH receptor blocking monoclonal antibodies or antigen binding fragments thereof, characterized in that, The full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region comprises H-CDR1, H-CDR2 and H-CDR3, the amino acid sequence of H-CDR1 is selected from one of SEQ ID NO: 30-33; the amino acid sequence of H-CDR2 is selected from one of SEQ ID NO: 34-36; the amino acid sequence of H-CDR3 is selected from one of SEQ ID NO: 37-40; The light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, the amino acid sequence of L-CDR1 is selected from one of SEQ ID NO: 41-42; the amino acid sequence of L-CDR2 is selected from one of SEQ ID NO: 43-45; the amino acid sequence of L-CDR3 is selected from one of SEQ ID NO: 46-48.

2. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or has at least 85% sequence identity to one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13; The amino acid sequence of the light chain variable region is selected from one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14 or has at least 85% sequence identity to one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14.

3. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, characterized in that, The antigen binding fragment is selected from Fab, Fab', F(ab')2, Fv, single chain Fv (scFv), Fav, dsFv, sc(Fv)2, single domain antibody (dAb).

4. The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, characterized in that, The full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof is an antagonist of TSH; and / or is an antagonist of thyroid stimulating antibody.

5. The substance as claimed in any of the preceding claims, characterized in that The substance comprises: (1) a preparation, a drug or a pharmaceutical composition comprising the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2; (2) a reagent or kit containing the full human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (3) a nucleotide encoding the full human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, the nucleotide sequence of which comprises: (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NO: 15, 17, 19, 21, 23, 25, 27; the nucleotide sequence encoding the light chain variable region is shown in one of SEQ ID NO: 16, 18, 20, 22, 24, 26, 28; (b) a nucleotide sequence having at least 85% sequence identity to one of SEQ ID NO: 15-SEQ ID NO: 28; (c) a nucleotide sequence that is the addition, substitution, deletion or insertion of one or several nucleotides to the nucleotide sequence as shown in SEQ ID NO: 15-SEQ ID NO: 28; (d) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a), (b) or (c) above or the full-length complement thereof; or, (e) a nucleotide sequence that differs from the nucleotide sequence of (a), (b), (c), (d) above due to the degeneracy of the genetic code; wherein the nucleotide sequence of the nucleic acid or a portion thereof encodes an antibody V H domain and an antibody V L domain or is selected from the CDRs set forth as SEQ ID NO: 30-SEQ ID NO: 48; (4) a vector comprising the nucleotide of (3); (5) a host cell comprising the nucleotide of (3) and / or the vector of (4); (6) a cell that is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2, and / or the nucleotide of (3), and / or the vector of (4); and / or, an isolated cell expressing the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; and / or, an isolated cell secreting the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; (7) a composition comprising a determined concentration of TSH receptor autoantibodies and comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; (8) a pharmaceutical composition for administration to a mammalian subject for the treatment of a thyroid-related condition, said pharmaceutical composition comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and a pharmaceutically acceptable carrier; wherein said thyroid-related condition is selected from the group consisting of hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

6. The substance of claim 5, wherein, The pharmaceutical composition is suitable for administration to a human.

7. The substance of claim 5, wherein, The pharmaceutical composition comprises one or more additional thyroid-stimulating hormone receptor antagonists.

8. The substance of claim 5, wherein, The pharmaceutical composition comprises a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 and any pharmaceutically acceptable carrier, adjuvant or vehicle.

9. The substance of claim 5, wherein, The pharmaceutical composition is administered by oral administration, parenteral administration, administration by spray inhalation, topical administration, administration by eye drops or eye ointment, rectal administration, nasal administration, buccal administration, vaginal administration, administration via an implanted reservoir.

10. The substance of claim 5, wherein, The pharmaceutical composition is formulated in the form of a capsule, tablet, aqueous suspension, solution, rectal suppository, enema, ointment, lotion, cream, nasal spray, inhalant.

11. The substance of claim 5, wherein, The pharmaceutical composition is used to treat a thyroid-related condition in an injectable form. The pharmaceutical composition is used to treat a thyroid-related condition in an injectable form.

12. The substance of claim 5, wherein, The pharmaceutical composition is used for treating pretibial myxedema in a local administration form; and / or, The pharmaceutical composition is used for treating Graves' ophthalmopathy in the form of intravenous injection preparation or eye drop.

13. Any of the following methods, characterized in that, The method comprises: (1) A preparation method of a fully human TSH receptor blocking monoclonal antibody or an antigen binding fragment thereof, and the preparation method specifically comprises the following steps: Step 1: Sorting TSHR-targeting plasma cells and memory B cells in peripheral blood of patients with high TBAb activity, performing single cell RNA extraction and cDNA synthesis, and verifying the sorted single cells by nest-PCR amplification of heavy chain H, light chain lambda and light chain kappa, and selecting single cells with positive heavy chain and light chain for subsequent cloning; Step 2: Cloning the BCR heavy chain and light chain of all single B cells into an expression heavy chain vector AbVec-IGHG1, a lambda light chain expression vector AbVec-hIgKappa or a lambda light chain expression vector AbVec-hIgLambda by nest-PCR in vitro amplification; Step 3: After successfully obtaining the heavy chain and light chain recombinant plasmid, sequencing and alignment analysis are performed on the obtained candidate clones to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies; the heavy chain and light chain expression plasmid is transfected to express the monoclonal antibody in vitro; the antigen binding capacity and antibody blocking activity are further verified, and finally the antibody combination specific to the target antigen, i.e., the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof, is obtained; (2) A method for treating a thyroid-related disorder in a mammalian subject or in a cell derived from the subject, the method comprising contacting the subject or the cell with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2; (3) A method for inhibiting thyroid stimulating antibodies from stimulating TSH receptors in a thyroid of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal or the antigen binding fragment thereof according to claim 1 or 2; (4) A method for inhibiting thyroid stimulating autoantibodies from binding to TSH receptors outside the thyroid in a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2; (5) A method for treating thyroid cancer or metastatic thyroid cancer in a thyroid of a subject or in a thyroid cell derived from the subject, the method comprising contacting the cancer cell with the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof according to claim 1 or 2, with the purpose of inhibiting constitutive thyroid stimulating hormone receptor activity in the cell; (6) A method for treating hyperthyroidism caused by constitutive thyroid activity in a subject or in a thyroid cell derived from a subject, characterized in that said method comprises contacting said subject or cell with a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, with the aim of inhibiting such hyperthyroidism; (7) A method for identifying a molecule capable of inhibiting the binding of thyroid stimulating antibodies to the TSH receptor, said method comprising providing as a reference at least one fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; (8) A method for identifying a molecule capable of inhibiting the binding of thyroid blocking antibodies to the TSH receptor, said method comprising providing as a reference at least one fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

14. The method of claim 13, wherein, The thyroid-related condition is selected from the group consisting of hyperthyroidism, thyroid-associated ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema.

15. The method of claim 13, wherein, The subject is a human.

16. The method of claim 13, wherein, In method (3), the method prevents the binding of thyroid stimulating antibodies to the TSHR.

17. The method of claim 13, wherein, In method (4), wherein the ectopic TSH receptors are located in the retro-orbital tissue and / or pretibial tissue of the subject; and / or, The fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof blocks the binding of TSHR autoantibodies to ectopic TSHR.

18. The method of claim 13, wherein, In method (5), the method prevents or delays the regrowth of thyroid cancer cells.

19. The method of claim 13, wherein, In method (7), the method selects molecules to be tested that prevent the binding of thyroid stimulating antibodies to the TSHR.

20. The method of claim 13, wherein, In method (8), the method selects molecules that prevent the binding of thyroid blocking antibodies to the TSHR.

21. Use of any one of the preceding claims, characterized in that, The uses include: (1) Use of a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for the treatment of a thyroid-related condition; (2) Use of a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for the manufacture of a medicament for the treatment of a thyroid-related condition; (3) Use of a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for the manufacture of a reagent or kit for the detection of TSH receptor antibodies; (4) Use of a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2 for the manufacture of a reagent or kit or product for the detection of hyperthyroidism, thyroid-associated ophthalmopathy; (5) The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2, or the substance according to claim 5, or the method according to claim 13 for use in the manufacture of a preparation, medicament or pharmaceutical composition for detecting TSH receptor antibodies, for treating hyperthyroidism, thyroid-associated ophthalmopathy, for inhibiting thyroid hyperplasia and / or thyroid hormone production, for TSH receptor blockade, for antagonizing the activating effect of TSH on the TSH receptor.

22. The use according to claim 21, characterized in that, The thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-associated ophthalmopathy, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, thyroid overactivity, thyroid cancer, thyroiditis, and pretibial myxedema.

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