Group of SH23 homologous cluster fully-human TSH receptor-blocking monoclonal antibodies, preparation method therefor and use thereof

Abstract

Provided are a group of SH23 homologous cluster fully-human thyrotropin receptor (TSH receptor, TSHR)-blocking monoclonal antibodies, a preparation method therefor and the use thereof. The method comprises the following steps: by means of flow cytometry, sorting plasma cells and memory single B cells that specifically recognize TSHR from the peripheral blood of a patient with a high TSH receptor-blocking antibody (TSH-stimulating blocking antibody, TBAb) titer, performing in-vitro cloning of light and heavy chains of the antibody, performing recombination and expression, and carrying out antibody property screening and verification by using TSHR-CHO cells to obtain a blocking monoclonal antibody that specifically targets human TSHR. The fully-human TSH receptor blocking monoclonal antibodies can specifically bind to TSHR, can effectively block the signal transduction after TSH binds to a receptor, and have good inhibitory activity in in vitro and in vivo experiments, and therefore have broad application prospects in the treatment of diseases, such as Graves' disease (GD) and hyperthyroidism-induced diseases, such as thyroid eye disease (TED).

Description

A group of SH23 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. Abnormal function of the TSHR can cause the occurrence of thyroid diseases. After thyroid stimulating hormone (TSH) binds to and activates the TSHR, downstream signal transduction can be mediated by the Gs and Gq / 11 pathways, which can regulate iodine transport absorption, iodine organicization, and the like, and participate 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. Under the background of genetic susceptibility, the TSHR peptide segment can act as an exposed autoantigen to induce the production of autoantibodies, i.e., thyroid stimulating hormone receptor antibodies (TRAb), in the body under the action of external environmental factors such as infection and mental trauma, and participate 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, which greatly increases 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 is inhibited by competition, and cannot normally play a feedback regulation role on T3 and T4, leading to 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 has characteristics 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, 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 separated 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 retain rich genetic diversity and natural pairing of light and heavy chain variable regions, and have great advantages. 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: 53, 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: 53.

[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, and the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are respectively as shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56.

[0013] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 58, SEQ ID NO: 63, SEQ ID NO: 68.

[0014] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 58, SEQ ID NO: 63, SEQ ID NO: 68.

[0015] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 58, SEQ ID NO: 63, SEQ ID NO: 68.

[0016] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 58, SEQ ID NO: 63, SEQ ID NO: 68.

[0017] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 58, SEQ ID NO: 63, SEQ ID NO: 68.

[0018] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56, 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: 59, SEQ ID NO: 64, SEQ ID NO: 70, respectively.

[0019] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56, 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: 60, SEQ ID NO: 65, SEQ ID NO: 71, respectively.

[0020] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56, 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: 61, SEQ ID NO: 66, SEQ ID NO: 72, respectively.

[0021] 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: 54, SEQ ID NO: 55, SEQ ID NO: 56, 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: 62, SEQ ID NO: 67, SEQ ID NO: 73, respectively.

[0022] wherein,

[0023] the amino acid sequence of the heavy chain variable region is selected from one of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or has at least 85%, 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 NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25;

[0024] 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, 16, 18, 20, 22, 24, 26 or has at least 85%, 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%, or at least 99% sequence identity with one of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26.

[0025] The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fv, single-chain Fv(scFv), Fav, dsFv, sc(Fv)2, and single-domain antibody (dAb).

[0026] In this invention, the nucleotide sequences encoding the heavy chain variable region and light chain variable region of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment are shown in SEQ ID NO: 27-SEQ ID NO: 52.

[0027] Preferably, the nucleotide sequences encoding the heavy chain variable region and light chain variable region of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment 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%, and at least 99% sequence identity with SEQ ID NO: 27-SEQ ID NO: 52.

[0028] SEQ ID NO: 1 (SH23_1 amino acid sequence)

[0029] SEQ ID NO: 2 (SH23_2 amino acid sequence)

[0030] SEQ ID NO: 27 (SH23_3 nucleotide sequence)

[0031] SEQ ID NO: 28 (SH23_4 nucleotide sequence)

[0032] SEQ ID NO: 3 (CHL34_1 amino acid sequence)

[0033] SEQ ID NO: 4 (CHL34_2 amino acid sequence)

[0034] SEQ ID NO: 29 (CHL34_3 nucleotide sequence)

[0035] SEQ ID NO: 30 (CHL34_4 nucleotide sequence)

[0036] SEQ ID NO: 5 (SH33_1 amino acid sequence)

[0037] SEQ ID NO: 6 (SH33_2 amino acid sequence)

[0038] SEQ ID NO: 31 (SH33_3 nucleotide sequence)

[0039] SEQ ID NO: 32 (SH33_4 nucleotide sequence)

[0040] SEQ ID NO: 7 (CHL31_1 amino acid sequence)

[0041] SEQ ID NO: 8 (CHL31_2 amino acid sequence)

[0042] SEQ ID NO: 33 (CHL31_3 nucleotide sequence)

[0043] SEQ ID NO: 34 (CHL31_4 nucleotide sequence)

[0044] SEQ ID NO: 9 (CHL23_1 amino acid sequence)

[0045] SEQ ID NO: 10 (CHL23_2 amino acid sequence)

[0046] SEQ ID NO: 35 (CHL23_3 nucleotide sequence)

[0047] SEQ ID NO: 36 (CHL23_4 nucleotide sequence)

[0048] SEQ ID NO: 11 (CHL24_1 amino acid sequence)

[0049] SEQ ID NO: 12 (CHL24_2 amino acid sequence)

[0050] SEQ ID NO: 37 (CHL24_3 nucleotide sequence)

[0051] SEQ ID NO: 38 (CHL24_4 nucleotide sequence)

[0052] SEQ ID NO: 13 (CHL46_1 amino acid sequence)

[0053] SEQ ID NO: 14 (CHL46_2 amino acid sequence)

[0054] SEQ ID NO: 39 (CHL46_3 nucleotide sequence)

[0055] SEQ ID NO: 40 (CHL46_4 nucleotide sequence)

[0056] SEQ ID NO: 15 (CHL47_1 amino acid sequence)

[0057] SEQ ID NO: 16 (CHL47_2 amino acid sequence)

[0058] SEQ ID NO: 41 (CHL47_3 nucleotide sequence)

[0059] SEQ ID NO: 42 (CHL47_4 nucleotide sequence)

[0060] SEQ ID NO: 17 (CHL33_1 amino acid sequence)

[0061] SEQ ID NO: 18 (CHL33_2 amino acid sequence)

[0062] SEQ ID NO: 43 (CHL33_3 nucleotide sequence)

[0063] SEQ ID NO: 44 (CHL33_4 nucleotide sequence)

[0064] SEQ ID NO: 19 (CHL32_1 amino acid sequence)

[0065] SEQ ID NO: 20 (CHL32_2 amino acid sequence)

[0066] SEQ ID NO: 45 (CHL32_3 nucleotide sequence)

[0067] SEQ ID NO: 46 (CHL32_4 nucleotide sequence)

[0068] SEQ ID NO: 21 (SH24_1 amino acid sequence)

[0069] SEQ ID NO: 22 (SH24_2 amino acid sequence)

[0070] SEQ ID NO: 47 (SH24_3 nucleotide sequence)

[0071] SEQ ID NO: 48 (SH24_4 nucleotide sequence)

[0072] SEQ ID NO: 23 (CHL26_1 amino acid sequence)

[0073] SEQ ID NO: 24 (CHL26_2 amino acid sequence)

[0074] SEQ ID NO: 49 (CHL26_3 nucleotide sequence)

[0075] SEQ ID NO: 50 (CHL26_4 nucleotide sequence)

[0076] SEQ ID NO: 25 (SH31_1 amino acid sequence)

[0077] SEQ ID NO: 26 (SH31_2 amino acid sequence)

[0078] SEQ ID NO: 51 (SH31_3 nucleotide sequence)

[0079] SEQ ID NO: 52 (SH31_4 nucleotide sequence)

[0080] SEQ ID NO: 53 (human TSHR amino acid sequence)

[0081] Table 1: CDR regions of heavy chain (H) and light chain (L) of SH23 homologous cluster fully human TSH receptor blocking monoclonal antibodies

[0082] The present application comprises a group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, respectively SH23, CHL32, SH31, SH24, SH33, CHL26, CHL31, CHL23, CHL24, CHL46, CHL47, CHL33, CHL34, which have a high homology with SH23, so they are named as a group of SH23 homologous cluster fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof. K1-70 in the above table is a reported TSH receptor blocking monoclonal antibody with blocking activity, in comparison, the sequences provided by the present application are quite different and have no homology.

[0083] Preferably, the present application proposes a group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, which are named as SH23 homologous cluster fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof.

[0084] In the present application, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is a TSH antagonist.

[0085] In the present application, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is an antagonist of thyroid stimulating antibody.

[0086] In the present application, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof respectively comprises a V H region (heavy chain variable region), the V HThe CDRs comprise amino acid sequences as shown in SEQ ID NO: 54-SEQ ID NO: 56 (see Table 1).

[0087] In the present application, the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof comprises one V L region (light chain variable region), the V L region comprises a CDR with an amino acid sequence as shown in SEQ ID NO: 57, 63, 68 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 58, 63, 69 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 58, 64, 69 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 58, 63, 68 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 59, 64, 70 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 60, 65, 71 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 61, 66, 72 (see Table 1) or a CDR with an amino acid sequence as shown in SEQ ID NO: 62, 67, 73 (see Table 1).

[0088] 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.

[0089] The fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof provided by the present application can bind to the TSH receptor to inhibit the signal transduction of the TSH receptor, inhibit the synthesis and secretion of thyroid hormones, reduce hyperthyroidism caused by various reasons, significantly reduce the thyroid enlargement caused by hyperthyroidism and the like after binding to the TSH receptor, and reduce inflammation, edema and hyperplasia and the like of thyroid associated ophthalmopathy.

[0090] The thyroid associated ophthalmopathy (TAO) of the present application is also referred to as thyroid eye disease (TED).

[0091] 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.

[0092] 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.

[0093] 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 thereof is one of the following sequences:

[0094] (a) the nucleotide sequence encoding the heavy chain variable region is as shown in one of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51; and the nucleotide sequence encoding the light chain variable region is as shown in one of SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52;

[0095] (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% sequence identity to SEQ ID NO: 27-SEQ ID NO: 52;

[0096] (c) a nucleotide sequence which is the nucleotide sequence as shown in SEQ ID NO: 27-SEQ ID NO: 52 with addition, substitution, deletion or insertion of one or several nucleotides;

[0097] (d) a nucleotide sequence which hybridizes to the nucleotide sequence of the preceding (a), (b) or (c) or the full-length complement thereof under stringent conditions; or,

[0098] (e) a nucleotide sequence which is different from the nucleotide sequence of the preceding (a), (b), (c), (d) due to the degeneracy of the genetic code;

[0099] wherein the nucleotide sequence of the nucleotide or a part thereof encodes the antibody V H domain; and the antibody V L domain or is selected from the CDRs as shown in SEQ ID NO: 54-SEQ ID NO: 73 (see Table 1).

[0100] The present application also provides a vector containing the nucleotide as described above.

[0101] The present application also provides a host cell comprising the nucleotide as described above, and / or the vector as described above.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] The present application also provides a pharmaceutical composition for administration to a mammalian subject for treating a thyroid-related disorder, comprising a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier;

[0107] 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 pretibial myxedema.

[0108] Further, the preparation, medicament or pharmaceutical composition can further comprise a physiologically compatible adjuvant, including a buffer, a diluent, an excipient, a filler, a binder, a humectant, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, etc.

[0109] Further, the preparation, medicament or pharmaceutical composition can be prepared into an injection, a sterile powder for injection, a tablet, a pill, a capsule, a lozenge, a liquor, a powder, a granule, a syrup, a solution, a tincture, an aerosol, a powder mist, or a suppository, etc. The preparation, medicament or pharmaceutical composition in the above-mentioned various dosage forms can be prepared according to the conventional methods in the field of pharmacy.

[0110] Further, the preparation, 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 be introduced into the body after being mixed or wrapped with other substances.

[0111] Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye disease contains the fully human TSH receptor blocking monoclonal antibody.

[0112] 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.

[0113] Further, the vector can be a plasmid, a virus or fragments thereof and various types of vectors known to those skilled in the art.

[0114] Preferably, the host cell is a CHO-K1 cell or the like.

[0115] In particular, the composition can comprise a determined concentration of TSHR autoantibodies having TSH antagonist activity and comprises a fully human TSH receptor blocking monoclonal antibody or an antigen binding fragment thereof according to the present application.

[0116] Further, the pharmaceutical composition is suitable for administration to humans; preferably, the pharmaceutical composition according to the present application has no significant adverse effects on the immune system of the subject.

[0117] Further, the pharmaceutical composition comprises one or more additional thyroid stimulating hormone receptor antagonists.

[0118] Further, the pharmaceutical composition is for treating a thyroid-related condition in an injectable form.

[0119] Preferably, the pharmaceutical composition for treating Graves' ophthalmopathy is in the form of an intravenous injection preparation or eye drops.

[0120] Preferably, the pharmaceutical composition for treating pretibial myxedema is in a form for topical administration.

[0121] In particular, the pharmaceutical composition comprises any of the antibodies 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 the present application include, but are not limited to, buffers such as phosphate, glycine, ion exchangers, alumina, aluminium stearate, lecithin, serum proteins (such as human serum albumin), sorbitan, potassium sorbate, water, sodium chloride, salt or electrolytes (such as protamine sulphate), disodium hydrogen phosphate, potassium hydrogen phosphate, sodium- cellulose gum, polyethylene glycol, sodium carboxymethylcellulose, polypropylene

[0122] The pharmaceutical composition can be formulated in a variety of dosage forms including capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, inhalers. Preferably, the pharmaceutical composition is in the form of a solution and an ointment.

[0123] 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.

[0124] In particular, the pharmaceutical composition can be administered by topical administration, by administration by spray inhalation, oral administration, parenteral administration, by administration by eye drops or eye ointment, oral administration, vaginal administration, rectal administration, nasal administration or via an implanted reservoir, etc. Preferably, it is administered orally or by injection. The term "parenteral" as used herein includes subcutaneous, intracutaneous, intrasynovial, intrasternal, intravenous, intramuscular, intralesional, intracranial, intraarticular, and intrathecal injection or infusion techniques.

[0125] In particular, the pharmaceutical composition can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compound of the application with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and therefore will melt in the rectum to release the active components. Such materials include, but are not limited to, cocoa butter, beeswax and polyethylene glycols.

[0126] In particular, the pharmaceutical composition can be administered orally in any orally acceptable dosage form including, but not limited to, tablets, capsules, aqueous suspensions and solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For tablets for oral use, diluents which have been found to be used 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.

[0127] In particular, the pharmaceutical composition can be administered in the form of a nasal spray or an inhaler. 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.

[0128] In particular, topical administration of the pharmaceutical compositions of the present application is especially useful when the treatment desired involves areas or organs which are readily accessible by topical application. For topical administration in the treatment of skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active component suspended or dissolved in a carrier. Carriers for topical administration of the compounds of this application include, but are not limited to, mineral oil, white petrolatum, propylene glycol, liquid petrolatum, emulsifying wax and water. Alternatively, the pharmaceutical composition can be formulated with a suitable cream or lotion containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, and water. The pharmaceutical compositions of the present application can also be formulated for rectal administration as suppositories or enemas or for administration to the lower intestinal tract by using a suitable retention enema. The present application also includes topical transdermal patches.

[0129] The present application also provides a method for preparing a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof, which specifically comprises the following steps:

[0130] In the first step, the TSHR-directed plasma cells and memory B cells with high TBAb activity in the peripheral blood of patients are sorted, single-cell RNA is extracted and cDNA is synthesized, and the sorted single cells are amplified by nested PCR to verify the heavy chain H, light chain λ and light chain κ, and the single cells with positive heavy chain and light chain are selected for subsequent cloning;

[0131] In the second step, the BCR heavy chain and light chain of all single B cells are cloned into the expression heavy chain vector AbVec-IGHG1, the λ light chain expression vector AbVec-hIgKappa or the λ light chain expression vector AbVec-hIgLambda by in vitro amplification by nested PCR;

[0132] In the third step, after the successful heavy chain and light chain recombinant plasmids are obtained, the obtained candidate clones are sequenced and analyzed by comparison to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies; the heavy chain and light chain expression plasmids are 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 antigen binding fragment thereof, is obtained.

[0133] The present application also provides a method for treating a thyroid-related disorder in a mammalian subject or in a cell derived from the subject, which comprises contacting the subject or the cell with a fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof as described above.

[0134] Among them, 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.

[0135] The present application also provides a method for inhibiting the stimulation of TSH receptor by thyroid stimulating antibodies in the thyroid of a mammalian subject, said method comprising contacting said subject with a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described herein;

[0136] Preferably, the binding of thyroid stimulating antibodies to TSH receptor is prevented.

[0137] The present application also provides a method for inhibiting the binding of thyroid stimulating autoantibodies to ectopic TSH receptors in a mammalian subject, said method comprising contacting said subject with a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above;

[0138] In particular, said ectopic TSH receptors are located in retro-orbital tissue and / or pretibial tissue of said subject.

[0139] Preferably, said fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof blocks the binding of TSH receptor autoantibodies to ectopic TSH receptors.

[0140] The present application also provides a method for treating thyroid cancer or metastatic thyroid cancer in a subject or in thyroid cells derived from a subject, said method comprising contacting said cancer cells with a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above, with the aim of inhibiting constitutive thyrotropin receptor activity in said cells;

[0141] Preferably, the regrowth of said thyroid cancer cells is prevented or delayed.

[0142] The present application also provides a method for treating thyroid hyperactivity caused by constitutive thyroid activity in a subject or in thyroid cells derived from a subject, said method comprising contacting said subject or cells with a fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above, with the aim of inhibiting such thyroid hyperactivity.

[0143] The present application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid stimulating antibodies to TSH receptor, 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;

[0144] Preferably, the molecule is selected so as to prevent the binding of thyroid stimulating antibodies to the TSH receptor.

[0145] The present application also provides a method for identifying a molecule capable of inhibiting the binding of thyroid blocking antibodies to the TSH receptor, 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;

[0146] Preferably, the molecule is selected so as to prevent the binding of thyroid blocking antibodies to the TSH receptor.

[0147] In particular, the thyroid-related disorder is selected from hyperthyroidism, thyroid-associated ophthalmopathy (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, hyperthyroidism, thyroid cancer, thyroiditis, and pretibial myxedema, etc.

[0148] Preferably, the subject treated in the above method is a human.

[0149] In the present application, the preparation method comprises the following steps: sorting the plasma cells and memory single B cells that specifically recognize TSHR in the peripheral blood of patients with high TSH receptor blocking antibody (TBAb) titers using flow cytometry, cloning the antibody light and heavy chains in vitro and recombinantly expressing them, and using hTSHR-CHO cells to screen and verify the properties of the antibodies, thereby obtaining blocking monoclonal antibodies that specifically target human TSHR. Through the preparation method of the fully human TSH receptor blocking monoclonal antibody for treating hyperthyroidism provided by the present application, the target antibody sequence can be obtained in 3 weeks to 1 month.

[0150] Further, the present application verifies the properties of the antibodies and evaluates the effects in vitro by using hTSHR-CHO cells.

[0151] The present application also provides the use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above for the treatment of a thyroid-related disorder.

[0152] The present application also provides the use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above for the preparation of a medicament for the treatment of a thyroid-related disorder.

[0153] The present application also provides the use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above for the preparation of a reagent or kit for detecting TSH receptor antibodies.

[0154] The application also provides the use of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof as described above in the preparation of a reagent or a kit or a product for detecting hyperthyroidism, thyroid-related eye disease.

[0155] The application also provides the use of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof as described above, or the material as described above, or the method as described above in the preparation of a preparation, a medicine or a pharmaceutical composition for detecting TSH receptor antibody, a medicine for treating hyperthyroidism, thyroid-related eye disease, a medicine for inhibiting thyroid hyperplasia and / or thyroid hormone production, a medicine for blocking TSH receptor, a medicine for antagonizing the activation of TSH on TSH receptor.

[0156] 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.

[0157] The method of the 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 the antibody genes of the separated single plasma cells or memory B cells, and by finally screening the antibodies with antigen specificity and neutralizing activity by expressing the paired antibody light and heavy chain genes. The method of the application has the advantages of rapidity, high throughput, and small amount of required cells, etc. 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 application only needs 3 weeks to 1 month to obtain antibody sequences, greatly reducing the workload and cost of antibody preparation.

[0158] The application also provides the use of the fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof as described above in the preparation of a reagent or a kit or a product for detecting hyperthyroidism, thyroid-related eye disease. BRIEF DESCRIPTION OF DRAWINGS

[0159] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed in the embodiments or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0160] Figure 1 is a flow cytometry technique for sorting TSHR-specific plasma cells and memory single B cells from peripheral blood according to the present application. Biotin-AF647-labeled TSHR protein is used as a decoy, and the other antibodies used for sorting are: CD19-Pacific Blue, IgM-PE, CD27-BV605, CD38-PE-Cy7. The final sorting of TSHR-specific plasma cells and memory single B cells is CD19 + IgM - CD27 + CD38 - TSHR + .

[0161] Figure 2 is a graph showing the screening results of the anti-human TSHR monoclonal antibody according to the present application. The results shown are the mean ± standard deviation (n = 3). The dotted line value is 30%.

[0162] Figure 3 is a phylogenetic tree analysis result of the anti-human TSHR monoclonal antibody according to the present application. The percentage of repeated trees in which the relevant taxa are clustered together in the bootstrap test (1000 repetitions) is shown below the branches.

[0163] Figure 4 is an evaluation of the inhibitory effect of SH23 antibody on orbital fibroblasts according to the present application. The horizontal line number indicates the statistical difference (P value) between the antibody group and the control group (Control). A value less than or equal to 0.05 is considered to have a significant difference.

[0164] Figure 5 is a graph showing the changes in T4 and TSH content in vivo at different time points after intramuscular injection of SH23 antibody in rats according to the present application. The results shown are the mean ± standard deviation (n = 5), and **p < 0.01. DETAILED DESCRIPTION

[0165] 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 specifically limited by the present application, except for the following specifically mentioned content.

[0166] Example 1

[0167] Single cell sorting: Peripheral blood from volunteers with high TBAb titers was collected, and B cell-enriched mixture (STEMCELL, Cat# 15024) was added to the blood sample, and centrifuged using Ficoll-Paque Plus (Sigma, Cat# 10771) to obtain B cells from peripheral blood. CD19 + IgM - CD27 + CD38 - TSHR + plasma cells and memory single B cells (Figure 1).

[0168] Light and heavy chain variable region cloning: RNA bound to single cells was captured using SPRlselect Nucleic Acid Fragment Screening Kit (Beckman Coulter, Cat# B23317), and cDNA was synthesized according to the instruction of Superscript TM IV One-Step RT-PCR System (Invitrogen, Cat# 12594100). Using cDNA as template, DreamTaq Green PCR 2X Master Mix (ThermoFisher, Cat# K1081) was used to amplify the antibody light and heavy chain variable regions, respectively. The PCR products were then detected by agarose gel electrophoresis, and the bands with expected size were cut and recovered, and the DNA fragments were purified using 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 primers were selected to clone the light and heavy chains by PCR. Then, the light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors (NCBI GenBank No. FJ475055, FJ475056, FJ517647) containing light and heavy chain constant regions using HiFi DNA Assembly Master Mix (NEB, Cat# E2621L), and the obtained clones were sequenced to determine the final sequence.

[0169] Blocking antibody screening:

[0170] ​​1. Screening of antigen binding ability: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After 3 days of culture at 37°C and 5% CO2, the culture supernatant was collected by centrifugation, and the titer of TRAb antibody in the supernatant was detected according to the instructions of the human anti-thyrotropin receptor antibody enzyme-linked immunosorbent assay kit (Kelu, item number: ELK9540). The light and heavy chain combinations with a binding ability lower than the blank control group were removed, and the remaining combinations were subjected to blocking activity screening.

[0171] 2. Screening of blocking activity: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a ratio of 1:1. After 3 days of culture at 37°C and 5% CO2, the culture supernatant was collected by centrifugation, and 100 μL of the supernatant was used to incubate hTSHR-CHO cells for 2 hours with the addition of 1 IU / L bTSH (Sigma). Then, the cell lysate was collected, and the change in cAMP level was detected (R&D, item number: KGE002B).

[0172] 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 and 8% CO2 with 130 rpm shaking, the culture supernatant was collected by centrifugation, filtered with 0.45 μm, and purified by Protein A (Jinse, item number: L00210) affinity chromatography to obtain antibody proteins with high purity. The antibody concentration was determined by Bradford protein concentration method (Biyun Tian, item number: P0006) and NanoDrop A280 method.

[0173] Evaluation of in vitro TSHR inhibition effect: The purified monoclonal antibodies were diluted according to different concentration gradients, and 5 ng / ml of bTSH was added for co-incubation of hTSHR-CHO cells for 2 hours. Then, the cell lysate was collected, and the change in cAMP level was detected. If the inhibition percentage of cAMP generation was greater than 30%, the monoclonal antibody was considered to have inhibitory activity. It was found that the monoclonal antibody at a concentration of 1 μg / mL could effectively antagonize the activation of TSH on TSH receptor and showed a concentration-dependent effect (Figure 2). The inhibition percentage of cAMP generation of SH23 at 1 μg / mL was 94%, and the inhibition activity at a concentration of 2 μg / mL was as high as 98%.

[0174] Phylogenetic analysis: The phylogenetic analysis of antibodies was performed using MEGA 11 analysis software. The phylogenetic tree was constructed using the neighbor-joining method, and the reliability of the phylogenetic tree was evaluated using Bootstrap values. The percentage of replicate trees under which the relevant taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches. The evolutionary distances were calculated using the Poisson correction method. Figure 3 is a rootless optimal tree combined with the results of the in vitro inhibition activity assay. Antibody K1-70 is a reported inhibitory antibody, and the rest are antibodies obtained by screening. The closer the evolutionary distance of the antibodies, the closer the inhibitory activity. The results show that K1-70 and the antibody SH23 with the strongest inhibitory activity in the application have the farthest evolutionary distance, indicating that the two antibodies have the largest sequence difference and are two TSHR inhibitory monoclonal antibodies with completely different sequences. The two TSHR inhibitory monoclonal antibodies CHL34 and CHL33 screened in the application have the closest evolutionary distance to K1-70, and their inhibitory activities are only 79% and 75% at an antibody concentration of 1 μg / mL, and the inhibitory activities are both 88% at a concentration of 2 μg / mL, indicating that the inhibitory efficiency of K1-70 on TSHR is lower than that of the antibody SH23 with the strongest inhibitory activity screened in this screening.

[0175] Orbital fibroblast (OF cell) inhibition effect evaluation: OF cells were cultured in 10% FBS-containing DMEM in a 10 cm dish, and when they reached 70%, they were subcultured into a 12-well plate and cultured with 1% FBS-containing DMEM for 16 hours. After adding 50 ng / ml of inhibitory antibody SH23, continue to culture for 24 hours. Collect cells to extract RNA and perform RT-qPCR to detect the expression level of fibrosis-related gene mRNA. The results show that TSHR inhibitory monoclonal antibody SH23 can significantly inhibit the expression of TSHR mRNA, as well as the expression of fibrosis markers α-smooth muscle actin (α-SMA) and hyaluronic acid (HAS) mRNA (Figure 4).

[0176] In vivo effect evaluation: 6-8 weeks old male SD rats were selected, and each rat was injected with 0.1 mL of SH23 monoclonal antibody solution (200 μg of SH23 monoclonal antibody, buffer used: PBS; 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 8.1 mmol / L disodium hydrogen phosphate, 1.5 mmol / L potassium dihydrogen phosphate, pH 7.4; containing 3% mannitol and 1% Tween-80) by intramuscular injection. Tail blood was collected at 0 hour before injection, 4 hours, 24 hours, 48 hours and 72 hours after injection. The serum was separated by centrifugation at 4000 rpm for 5 minutes at 4°C and properly stored at -80°C for determination of the concentrations of thyroid hormone T4 (Yunke Lunn, Catalog No: CEA452Ge) and thyroid stimulating hormone TSH (Yunke Lunn, Catalog No: CEA463Ra). As shown in Figure 5, compared with 0 hour before injection, it was found that the concentration of T4 in the peripheral blood of rats began to decrease significantly at 4 hours after injection, and reached the highest peak of inhibition at 24 hours, indicating that SH23 had a strong effect on inhibiting the synthesis of thyroid hormone in vivo. After 24 hours, the T4 level recovered due to the compensatory increase of TSH, and the TSH level basically returned to normal at 72 hours.

[0177] 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.

[0178] 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.

[0179] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0180] The protection scope of the present application is not limited to the above embodiments. Changes 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, and the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 are respectively as shown in SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56; 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: 57-62; the amino acid sequence of L-CDR2 is selected from one of SEQ ID NO: 63-67; and the amino acid sequence of L-CDR3 is selected from one of SEQ ID NO: 68-73.

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, 15, 17, 19, 21, 23, 25 or has at least 85% sequence identity with one of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25; 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, 16, 18, 20, 22, 24, 26 or has at least 85% sequence identity with one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26.

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 or antigen binding fragment thereof 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, and the nucleotide sequence thereof comprises: (a) a nucleotide sequence encoding a heavy chain variable region as shown in one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51; and a nucleotide sequence encoding a light chain variable region as shown in one of SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52; (b) a nucleotide sequence having at least 85% sequence identity to one of SEQ ID NOs: 27-52; (c) a nucleotide sequence that is the addition, substitution, deletion or insertion of one or several nucleotides to a nucleotide sequence as shown in SEQ ID NOs: 27-52; (d) a nucleotide sequence that hybridizes under stringent conditions to a 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) or (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: 54-SEQ ID NO: 73; (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 that expresses the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; and / or, an isolated cell that secretes the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of claim 1 or 2; (7) a composition comprising a TSH receptor autoantibody in a defined concentration 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, the 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 the 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 administration of the pharmaceutical composition includes oral administration, parenteral administration, administration by spray inhalation, topical administration, administration by eye drop or eye ointment, rectal administration, nasal administration, oral administration, vaginal administration, administration via implantable reservoir.

10. The substance of claim 5, wherein, The preparation form of the pharmaceutical composition includes 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 for treating 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 topical 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, which specifically comprises the following steps: Step 1: Sorting the plasma cells and memory B cells against TSHR in the peripheral blood of patients with high TBAb activity, performing single-cell RNA extraction and cDNA synthesis, and verifying the sorted single cells by nested PCR amplification of heavy chain H, light chain λ and light chain κ, 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 the expression heavy chain vector AbVec-IGHG1, the λ light chain expression vector AbVec-hIgKappa or the λ light chain expression vector AbVec-hIgLambda by in vitro amplification through nested PCR; 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 condition in a mammalian subject or in a cell derived from the subject, which comprises 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 stimulating TSH receptors in the thyroid of a mammalian subject, which comprises contacting the subject with the fully human TSH receptor blocking monoclonal antibody 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, which comprises 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 of treating thyroid cancer or metastatic thyroid cancer in the thyroid in 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 according to claim 1 or 2, with the aim of inhibiting constitutive thyroid stimulating hormone receptor activity in said cell; (6) A method of 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 according to claim 1 or 2, with the aim of inhibiting such hyperthyroidism; (7) A method of 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 of 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-related eye disease, 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 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 a molecule under test that prevents the binding of thyroid stimulating antibodies to the TSHR.

20. The method of claim 13, wherein, In method (8), the method selects a molecule that prevents 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) The use of the fully human TSH receptor blocking monoclonal antibody or antigen binding fragment thereof according to claim 1 or 2 in the preparation of a reagent or kit or product for detecting hyperthyroidism, thyroid associated ophthalmopathy; (5) The use of 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 in the preparation of a preparation, medicament or pharmaceutical composition for detecting TSH receptor antibodies, in the preparation of a medicament for treating hyperthyroidism, thyroid associated ophthalmopathy, in the preparation of a medicament for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of a TSH receptor blocking medicament, in the preparation of a medicament for antagonizing the activating effect of TSH on the TSH receptor.

22. The use according to claim 21, characterized in that, The thyroid associated condition 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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