Fully human TSH receptor blocking monoclonal antibody and preparation therefor and use thereof

Abstract

Provided are a fully human TSH receptor (TSHR) blocking monoclonal antibody for treating hyperthyroidism, and a preparation method therefor and a use thereof. The method comprises the following steps: using flow cytometry to sort plasma cells and memory single B cells specifically recognizing a TSHR in the peripheral blood of patients having high titers of TSHR blocking antibody (TSH-stimulating blocking antibody, TBAb), performing in vitro cloning of antibody light and heavy chains and performing recombinant expression, and using hTSHR-CHO cells for antibody property screening and validation, to obtain a blocking monoclonal antibody specifically targeting human TSHR. The fully human TSHR blocking monoclonal antibody can specifically bind to the TSHR, has good inhibitory activity in vitro and in vivo experiments, and has broad application prospects in the development of treatment of Graves' diseases.

Description

A fully human TSH receptor blocking monoclonal antibody and its preparation and application Technical Field

[0001] The present invention belongs to the field of monoclonal antibody technology and human antibody drugs, and relates to a fully human TSH receptor blocking monoclonal antibody and its preparation and application. Background Art

[0002] Graves' disease (GD) is an organ-specific autoimmune disease caused by a combination of genetics and the environment, characterized by increased thyroid hormone secretion. It is the most common cause of hyperthyroidism. GD affects a large number of people, with a population prevalence of approximately 0.2-2%. Like other autoimmune diseases, GD is more common in women of childbearing age, with the incidence in women being approximately 5-10 times higher than in men. In recent years, the incidence of hyperthyroidism has been increasing with changes in living environments. If uncontrolled, hyperthyroidism can affect the heart, leading to arrhythmias or heart failure. In women of childbearing age, it can cause menstrual irregularities, difficulty conceiving, and miscarriage. Furthermore, hyperthyroidism can often cause psychiatric disturbances such as anxiety, restlessness, and restlessness, impacting patients' academic performance and personal life. In severe cases, it can even lead to psychiatric disorders. Current treatments for GD primarily include medication, radioactive iodine, and surgery. The former requires a long course of treatment, making it difficult for patients to adhere to it, and has a high recurrence rate, with approximately 60-70% of patients experiencing relapses. Clinically, the treatment of GD has remained largely unchanged for many years, remaining a choice between antithyroid drugs (ATDs), radioactive iodine, or surgery. Antithyroid drugs have been used to treat Graves' disease for nearly 70 years. With the exception of the United States, physicians worldwide consider ATDs the preferred treatment for Graves' disease. However, only a subset of patients with hyperthyroidism can achieve a cure after formal, systematic treatment, and the condition often suffers from significant side effects. A significant proportion of patients experience relapses after a period of time. Relapses can worsen the condition, often requiring further radioactive iodine or surgery, significantly increasing the patient's financial and psychological burden. Furthermore, radioactive iodine-131 therapy can easily lead to permanent hypothyroidism, and the complications of surgical treatment are also significant. The current challenges in GD treatment suggest an urgent need for better alternatives.

[0003] Antibodies to the thyroid stimulating hormone receptor (TSH receptor antibodies, TRAbs) are characteristic of Graves' disease (GD). TRAbs are a group of polyclonal antibodies that act on different binding sites of the TSH receptor and can be divided into TSH-stimulating antibodies (TSAbs), TSH-stimulating blocking antibodies (TBAbs), and neutral TSH receptor antibodies. TSAbs bind to the TSHR, producing biological effects similar to those of TSH and are the direct cause of Graves' disease. TBAbs bind to TSH, blocking TSH from binding to the receptor and inhibiting thyroid hyperplasia and thyroid hormone production. Both stimulating and blocking antibodies coexist in GD patients, and the ultimate outcome of thyroid function depends on which antibody predominates. This makes exogenous supplementation of a certain dose of blocking antibodies that bind to the TSHR, thereby ameliorating the pathophysiological effects of autoantibodies, a new and effective treatment option for GD.

[0004] Humanized monoclonal antibodies can be divided into mouse humanized monoclonal antibodies and fully human monoclonal antibodies. Although mouse humanized monoclonal antibodies reduce the proportion of other non-human components to a certain extent, they cannot completely eliminate all non-human components. Moreover, the modification process can also reduce the antibody's affinity and original biological activity to a certain extent. Fully human monoclonal antibodies, on the other hand, directly amplify the antibody gene from a single human B cell. By amplifying the antibody gene from a single isolated plasma cell or memory B cell, a large number of naturally paired antibody light and heavy chain genes are obtained. Then, by expressing these paired antibody light and heavy chain genes, antibodies with antigen specificity and neutralizing activity are finally screened. This method has the advantages of rapidity, high throughput, and low cell count requirements. The fully human antibodies produced retain rich genetic diversity and naturally paired light and heavy chain variable regions, which is a significant advantage. Currently, the preparation of fully human antibodies against influenza, anthrax, and pneumococcus is based on this technology.

[0005] Summary of the Invention

[0006] In response to the above-mentioned prior art, the present invention provides a fully human TSH receptor (TSHR) blocking monoclonal antibody for the treatment of hyperthyroidism, and also provides the coding sequence of the antibody and a vector containing the coding sequence.

[0007] As used herein, the term "TSHR" refers to the full-length human thyroid stimulating hormone receptor having the amino acid sequence set forth in SEQ ID NO: 5, or variants or fragments thereof that are highly homologous to the thyroid stimulating hormone receptor. Preferably, such variants or fragments have 70-99.9% homology to the amino acid sequence set forth in SEQ ID NO: 5.

[0008] The present invention provides a fully human TSH receptor blocking monoclonal antibody, which binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the amino acid sequence of the antibody is shown in SEQ ID NOs: 1 and 2; the nucleotide sequence of the antibody is shown in SEQ ID NOs: 3 and 4.

[0009] Preferably, the amino acid sequence of the antibody has a sequence identity of 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 to SEQ ID NO: 1, 2; and / or the nucleotide sequence of the antibody has a sequence identity of 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 to SEQ ID NO: 3, 4.

[0010] Preferably, the amino acid sequence of the antibody is as shown in SEQ ID NO: 1, or has a sequence identity of 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% or more to SEQ ID NO: 2.

[0011] Preferably, the nucleotide sequence of the antibody is as shown in SEQ ID NO: 3, or has a sequence identity of 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% or more to SEQ ID NO: 4.

[0012] The present invention includes a fully human TSH receptor blocking monoclonal antibody named TBI80. In Table 1, K1-70 is a reported TSH receptor blocking monoclonal antibody with blocking activity. In comparison, the sequence provided by the present invention is very different and has no homology at all.

[0013] Preferably, the present invention provides a fully human TSH receptor blocking monoclonal antibody, named TBI80.

[0014] In the present invention, the antibody is a TSH antagonist.

[0015] In the present invention, the antibody is an antagonist of thyroid stimulating antibodies.

[0016] In the present invention, the antibodies each comprise a V H Area, the V H The region comprises one or more complementarity determining regions (CDRs) selected from the group consisting of SEQ ID NO: 6 to SEQ ID NO: 8 (see Table 1).

[0017] In the present invention, the antibodies each comprise a V L Area, the V L The region comprises one or more CDRs selected from the group consisting of SEQ ID NO: 9 to SEQ ID NO: 11 (see Table 1).

[0018] In the present invention, the antibody comprises one or more amino acid sequences substantially homologous to these CDRs.

[0019] The antibody provided by the present invention binds to the TSH receptor to inhibit TSH receptor signal transduction; inhibits the synthesis and secretion of thyroid hormones, and reduces hyperthyroidism caused by various reasons; after binding to the TSH receptor, it can significantly reduce goiter caused by hyperthyroidism and other reasons; and alleviates inflammation, edema and hyperplasia of thyroid-related eye diseases.

[0020] The present invention also provides use of the fully human TSH receptor blocking monoclonal antibody described above in the preparation of a medicament for treating a thyroid-related disease.

[0021] The present invention also provides use of the fully human TSH receptor blocking monoclonal antibody described above in the preparation of a reagent or kit for detecting TSH receptor antibodies.

[0022] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody described above in the preparation of a reagent, kit or product for detecting hyperthyroidism and thyroid-related eye diseases.

[0023] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody in the preparation of drugs for treating hyperthyroidism, thyroid-related eye diseases and the like, in the preparation of drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, in the preparation of drugs for TSH receptor blocking, and in the preparation of drugs for antagonizing the activation effect of TSH on TSH receptors.

[0024] The present invention also provides a preparation, a medicine or a pharmaceutical composition, wherein the preparation, the medicine or the pharmaceutical composition comprises the above-mentioned fully human TSH receptor blocking monoclonal antibody.

[0025] The present invention also provides the use of the above-mentioned preparation, medicine or pharmaceutical composition in preparing a preparation, medicine or pharmaceutical composition for detecting TSH receptor antibodies, in preparing a medicine for treating hyperthyroidism and thyroid-related eye diseases, in preparing a medicine for inhibiting thyroid hyperplasia and / or thyroid hormone production, in preparing a medicine for blocking TSH receptors, and in preparing a medicine for antagonizing the activation effect of TSH on TSH receptors.

[0026] The present invention also provides the use of the above-mentioned fully human TSH receptor blocking monoclonal antibody in the preparation of a preparation, a medicine or a pharmaceutical composition for detecting TSH receptor antibodies.

[0027] Furthermore, the preparation, medicine or pharmaceutical composition may further comprise physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc.;

[0028] Furthermore, the preparation, medicine or pharmaceutical composition can be prepared into injection, sterile powder for injection, tablet, pill, capsule, lozenge, elixir, powder, granule, syrup, solution, tincture, aerosol, powder spray, or suppository, etc. The preparations, medicines or pharmaceutical compositions in various dosage forms can be prepared according to conventional methods in the pharmaceutical field.

[0029] Furthermore, the preparation, drug or pharmaceutical composition can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous, or mucosal tissue by injection, spraying, nasal drops, eye drops, penetration, absorption, physical or chemical mediation; or can be introduced into the body after being mixed or encapsulated with other substances.

[0030] The present invention also provides a reagent, a kit, or a product comprising the fully human TSH receptor blocking monoclonal antibody. Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye diseases comprises the fully human TSH receptor blocking monoclonal antibody.

[0031] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody in the preparation of a reagent, a kit or a product for detecting hyperthyroidism and thyroid-related eye diseases.

[0032] The present invention also provides the use of the reagent, kit or product in preparing drugs for treating hyperthyroidism and thyroid-related eye diseases, in preparing drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, in preparing drugs for blocking TSH receptors, and in preparing drugs for antagonizing the activation effect of TSH on TSH receptors.

[0033] The present invention also provides a method for preparing a fully human TSH receptor blocking monoclonal antibody, which specifically comprises the following steps:

[0034] (1) Plasma cells and memory B cells targeting TSHR in the peripheral blood of patients with high TBAb activity were sorted, and single-cell RNA was extracted and cDNA was synthesized. The sorted single cells were amplified and verified by nested PCR for heavy chain (H), light chain (λ), and light chain (κ). Single-cell clones that were positive for both heavy and light chains were selected for subsequent cloning.

[0035] (2) The BCR heavy and light chains of all single B cells were amplified in vitro by nested PCR and cloned into the heavy chain expression vector (AbVec-IGHG1), light chain expression vector (AbVec-hIgKappa), or light chain expression vector (AbVec-hIgLambda).

[0036] (3) After successfully obtaining the heavy chain and light chain recombinant plasmids, the obtained candidate clones are sequenced and compared 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 monoclonal antibodies in vitro; the antigen binding ability and antibody blocking activity are further verified, and finally an antibody combination that can specifically target the target antigen, namely a fully human TSH receptor blocking monoclonal antibody, is obtained.

[0037] The present invention also provides 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 described above.

[0038] The thyroid-related disease is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

[0039] The present invention also provides a method of inhibiting stimulation of TSH receptors by thyroid stimulating antibodies in the thyroid gland of a mammalian subject, the method comprising contacting the subject with an antibody of the present invention;

[0040] Preferably, the binding of thyroid stimulating antibodies to the TSH receptor is prevented.

[0041] The present invention also provides a method for inhibiting the binding of thyroid stimulating autoantibodies to extrathyroidal TSH receptors in a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody as described above;

[0042] Specifically, the extrathyroidal TSH receptor is located in the retroorbital tissue and / or anterior tibial tissue of the subject;

[0043] Preferably, the antibody can block the binding of TSH receptor autoantibodies to extrathyroidal TSH receptors.

[0044] The present invention also provides a method for treating thyroid cancer or metastatic thyroid cancer in a subject or in thyroid cells derived from a subject, the method comprising contacting the cancer cells with a fully human TSH receptor blocking monoclonal antibody as described above, in order to inhibit constitutive thyroid stimulating hormone receptor activity in the cells;

[0045] Preferably, the regrowth of the thyroid cancer cells is prevented or delayed.

[0046] The present invention also provides a method for treating thyroid hyperactivity caused by constitutive thyroid activity in a subject or in thyroid cells derived from a subject, the method comprising contacting the subject or cells with a fully human TSH receptor blocking monoclonal antibody as described above, in order to inhibit such thyroid hyperactivity.

[0047] The present invention also provides a method for identifying a molecule that can inhibit the binding of thyroid stimulating antibodies to TSH receptors, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody as described above as a reference;

[0048] Preferably, molecules are selected that prevent thyroid stimulating antibodies from binding to the TSH receptor.

[0049] The present invention also provides a method for identifying a molecule that can inhibit the binding of a thyroid blocking antibody to a TSH receptor, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody as described above as a reference;

[0050] Preferably, molecules are selected that prevent thyroid blocking antibodies from binding to the TSH receptor.

[0051] Specifically, the thyroid-related disease is selected from hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

[0052] Preferably, the subject treated in the above methods is a human.

[0053] The preparation method of the present invention includes the following steps: using flow cytometry to sort plasma cells and memory single B cells that specifically recognize TSHR from the peripheral blood of patients with high titers of TSH-stimulating blocking antibodies (TBAbs), cloning and recombinantly expressing the antibody light and heavy chains in vitro, and screening and verifying the antibody properties using hTSHR-CHO cells to obtain a blocking monoclonal antibody that specifically targets the human TSHR. The method for preparing a fully human TSH receptor-blocking monoclonal antibody for the treatment of hyperthyroidism proposed in this invention can obtain the target antibody sequence within 3 weeks to 1 month.

[0054] Furthermore, the present invention verifies the properties of the antibody and evaluates its effect in vitro using hTSHR-CHO cells; and confirms the blocking effect of the antibody through animal experiments.

[0055] The present invention also provides a nucleotide encoding the fully human TSH receptor blocking monoclonal antibody, the nucleotide sequence of which is one of the following sequences:

[0056] (1) the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0057] (2) 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%, or at least 99% sequence identity to SEQ ID NO: 3 or SEQ ID NO: 4;

[0058] (3) The nucleotide sequence after one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0059] (4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (1), (2) or (3) above or its full-length complement; or

[0060] (5) A nucleotide sequence that is different from the nucleotide sequences of (1), (2), (3), and (4) above due to the degeneracy of the genetic code.

[0061] Wherein, the nucleotide sequence of the nucleotide or a part thereof encodes the antibody V H domain; and antibody V LThe structural domain may be selected from the CDRs shown in SEQ ID NO: 6 to SEQ ID NO: 11 (see Table 1).

[0062] SEQ ID NO: 1 amino acid sequence

[0063] SEQ ID NO: 2 amino acid sequence

[0064] SEQ ID NO: 3 base sequence

[0065] SEQ ID NO: 4 base sequence

[0066] SEQ ID NO: 5 (human TSHR amino acid sequence)

[0067] Table 1: CDR regions of the heavy (H) and light (L) chains of the fully human TSH receptor TBI80 blocking monoclonal antibody

[0068] The nucleotide sequence of the present invention, or at least a portion thereof, can be expressed in a suitable expression system to obtain the corresponding protein or polypeptide, including but not limited to bacterial, insect cell, and mammalian cell expression systems.

[0069] The present invention also provides an expression vector comprising the above-mentioned nucleotide encoding the fully human TSH receptor blocking monoclonal antibody.

[0070] The present invention also provides a host cell comprising the above nucleotide and the above expression vector. Preferably, the host cell is a CHO-K1 cell or the like.

[0071] The present invention also provides a cell, which is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody described above, and / or the nucleotide described above, and / or the vector described above.

[0072] The present invention also provides a cell, which is an isolated cell expressing the fully human TSH receptor blocking monoclonal antibody as described above.

[0073] The present invention also provides a cell, which is an isolated cell that secretes the fully human TSH receptor blocking monoclonal antibody as described above.

[0074] The present invention also provides a composition comprising a determined concentration of TSH receptor autoantibodies and a fully human TSH receptor blocking monoclonal antibody as described above.

[0075] The present invention also provides a pharmaceutical composition for administering to a mammalian subject to treat a thyroid-related disorder, comprising the fully human TSH receptor blocking monoclonal antibody as described above and a pharmaceutically acceptable carrier;

[0076] The thyroid-related disease is selected from the group consisting of hyperthyroidism, thyroid-related eye disease (Graves' ophthalmopathy), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

[0077] Furthermore, the vector can be a plasmid, a virus, or a fragment thereof, as well as various different types of vectors known to those skilled in the art.

[0078] In particular, the composition may comprise a defined concentration of a TSHR autoantibody having TSH antagonist activity and an antibody according to the invention.

[0079] Furthermore, the pharmaceutical composition is suitable for administration to humans; preferably, the pharmaceutical composition according to the present invention has no significant adverse effects on the immune system of the subject.

[0080] Furthermore, the pharmaceutical composition comprises one or more additional thyroid stimulating hormone receptor antagonists.

[0081] Furthermore, the pharmaceutical composition is used to treat a thyroid-related disorder in an injectable form.

[0082] Preferably, the pharmaceutical composition for treating Graves' ophthalmopathy is in the form of an intravenous injection preparation or eye drops.

[0083] Preferably, the pharmaceutical composition for treating pretibial myxedema is in a topical administration form.

[0084] Specifically, the pharmaceutical composition comprises any antibody according to the present invention and any pharmaceutically acceptable carrier, adjuvant or vehicle. Pharmaceutically acceptable carriers, adjuvants and vehicles that can be used in the pharmaceutical composition of the present invention include, but are not limited to, buffer substances (such as phosphates), glycine, ion exchangers, aluminum oxide, aluminum stearate, lecithin, serum proteins (such as human serum albumin), sorbic acid, potassium sorbate, water, sodium chloride, salts or electrolytes (such as protamine sulfate), disodium hydrogen phosphate, potassium hydrogen phosphate, partial glyceride mixtures of saturated vegetable fatty acids, zinc salts, silica sol, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, magnesium trisilicate, polyvinyl pyrrolidone, polyethylene glycol and lanolin.

[0085] The pharmaceutical composition may be in the form of capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, and inhalants, preferably solutions and ointments.

[0086] Specifically, the pharmaceutical composition may be in the form of a sterile injectable preparation, such as a sterile injectable oil suspension or aqueous suspension. Such suspensions can be prepared using suitable dispersants or wetting agents (such as Tween 80) and suspending agents according to techniques known in the art. Sterile injectable agents may also be sterile injectable solutions or suspensions prepared with a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used are water, mannitol, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are commonly used as solvents or suspending media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids (such as oleic acid and its glyceride derivatives) can be used to prepare injectable preparations. The above-mentioned oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants.

[0087] Specifically, the pharmaceutical composition can be administered topically, by spray inhalation, orally, parenterally, by eye drops or eye ointment, orally, vaginally, rectally, nasally, or via an implanted reservoir. Preferably, it is administered orally or by injection. The term "parenteral" as used herein includes subcutaneous, intradermal, intrasynovial, intrasternal, intravenous, intramuscular, intralesional, intracranial, intraarticular, and intrathecal injection or infusion techniques.

[0088] Specifically, the pharmaceutical compositions can also be administered in the form of suppositories for rectal administration. These compositions can be prepared by mixing the compounds of the invention with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and thus dissolves in the rectum to release the active ingredient. Such materials include, but are not limited to, beeswax, cocoa butter, and polyethylene glycol.

[0089] Specifically, the pharmaceutical composition can be orally administered in any orally acceptable dosage form, including but not limited to tablets, capsules, aqueous suspensions, and solutions. For oral tablets, commonly used carriers include corn starch and lactose. Lubricants such as magnesium stearate are also typically added. For oral capsule forms, useful diluents include lactose and dry corn starch. When aqueous suspensions are administered orally, the active ingredient is combined with an emulsifier and a suspending agent. If desired, certain flavorings and / or sweeteners and / or coloring agents may also be added.

[0090] Specifically, the pharmaceutical composition can be administered in the form of a nasal spray or inhaler. These compositions are prepared according to techniques well known in the art of pharmaceutical formulation and can be prepared as saline solutions using benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and / or other dispersants or cosolvents known in the art.

[0091] Specifically, topical administration of the pharmaceutical compositions of the present invention is particularly useful when the desired treatment involves areas or organs readily accessible by topical application. For topical skin administration, the pharmaceutical compositions should be formulated with a suitable ointment containing the active ingredient suspended or dissolved in a carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, white petroleum, propylene glycol, liquid petroleum, emulsifying wax, polyoxyethylene polyoxypropylene compounds, and water. Alternatively, the pharmaceutical compositions 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, polysorbate 60, cetyl esters wax, cetyl alcohol, sorbitan monostearate, benzyl alcohol, 2-octyldodecanol, and water. The pharmaceutical compositions of the present invention can also be administered to the lower intestinal tract via rectal suppository formulations or in the form of suitable enema formulations. The present invention also includes topical transdermal patches.

[0092] The present invention also provides the use of the composition or pharmaceutical composition in preparing drugs for treating hyperthyroidism, thyroid-related eye diseases and the like, in preparing drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, in preparing drugs for blocking TSH receptors, and in preparing drugs for antagonizing the activation effect of TSH on TSH receptors.

[0093] The method of the present invention, because it does not require immunized animals, directly amplifies antibody genes from single human B cells, and amplifies the antibody genes of isolated single plasma cells or memory B cells to obtain a large number of naturally paired antibody light and heavy chain genes, and then expresses the paired antibody light and heavy chain genes to finally screen and obtain antibodies with antigen specificity and neutralizing activity. The method of the present invention has the advantages of being fast, high throughput, and requiring a small amount of cells. The fully human antibodies prepared retain rich genetic diversity and natural pairing of light and heavy chain variable regions, which has huge advantages. Compared with traditional hybridoma antibody preparation technology, the method of the present invention can significantly shorten the experimental cycle. Traditional hybridoma antibody preparation technology requires about 3 months to obtain antibody sequences, while the present invention only takes 3 weeks to 1 month to obtain antibody sequences, greatly reducing the workload and cost of antibody preparation.

[0094] The present invention also offers the following beneficial effects: The fully human TSH receptor-blocking monoclonal antibody described herein can specifically bind to the TSHR and exhibits excellent inhibitory activity in both in vitro and in vivo experiments, offering broad application prospects in the development of treatments for Graves' disease. GD is a common disease worldwide, and current treatment options for GD suffer from numerous drawbacks. The fully human TSHR-blocking monoclonal antibody prepared by the present invention not only effectively blocks TSH binding to the receptor, inhibiting thyroid hyperplasia and / or thyroid hormone production, but also does not produce a human anti-mouse antibody reaction, exhibits minimal side effects, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0096] Figure 1 shows the separation of plasma cells and single memory B cells that specifically recognize TSHR from peripheral blood using flow cytometry. Biotin-AF647 was used to label the TSHR protein as bait. Other antibodies used for separation were: CD19-Pacific Blue, IgM-PE, CD27-BV605, and CD38-PE-Cy7. TSHR-specific plasma cells and single memory B cells were ultimately isolated as CD19+IgM-CD27+CD38-TSHR+.

[0097] FIG2 is a diagram showing the antigen binding ability screening of the anti-human TSHR monoclonal antibody of the present invention.

[0098] Figure 3 is a diagram showing the blocking activity screening of the anti-human TSHR monoclonal antibody of the present invention. The results are shown as mean ± standard deviation (n=3).

[0099] Figure 4 is an SDS-PAGE gel electrophoresis diagram of the in vitro expression of the TBI80 antibody of the present invention. R: reduced state; NR: non-reduced state.

[0100] Figure 5 is a graph showing the in vitro dilution blocking activity test of the TBI80 antibody of the present invention, wherein the amount of bTSH added is 5 ng / ml and the amount of hTSH added is 100 ng / ml.

[0101] Figure 6 shows the changes in T4 and TSH levels in rats at different time points after intramuscular injection of the TBI80 antibody of the present invention. The results are mean ± standard deviation (n = 5), *p < 0.05.

[0102] Figure 7 shows the effects of three treatments with the TBI80 antibody of the present invention in a mouse model of Graves' disease. A shows the gross morphology of the thyroid gland after treatment; B shows the weight of the thyroid gland; C shows the changes in T4 levels in vivo; D shows the gross morphology of the thyroid gland in situ; E shows the morphology of the thyroid gland stained with HE. Scale bar: 100 μm. Results are mean ± SD (n = 5-6). *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0103] The present invention is further described in detail with reference to the following specific examples and accompanying drawings. The processes, conditions, experimental methods, etc. for implementing the present invention, except for those specifically mentioned below, are common knowledge and common common sense in the art and are not particularly limited by the present invention.

[0104] Example 1

[0105] Single cell sorting: Peripheral blood of volunteers with high TBAb titers was collected, and immune density gradient centrifugation human B cell enrichment mixture (STEMCELL, product number: 15024) was added to the blood sample. (Sigma, Catalog No. 10771) were used for density gradient centrifugation to obtain B cells from peripheral blood. CD19 cells were collected by flow cytometry. + IgM - CD27 + CD38 - TSHR + plasma cells and memory single B cells (Figure 1).

[0106] Light and heavy chain variable region cloning: RNA bound to single cells was captured using the SPR1select nucleic acid fragment screening kit (Beckman Coulter, Cat. No. B23317) and cloned according to SuperScript TMIV one-step RT-PCR system (Invitrogen, catalog number: 12594100) was used to synthesize cDNA. Using cDNA as a template, DreamTaq Green PCR 2X Master Mix (ThermoFisher, catalog number: K1081) was used to perform PCR amplification on the variable regions of the light and heavy chains of the antibody, respectively. The amplified PCR products were then subjected to agarose gel electrophoresis, and the bands with expected fragment sizes were cut and recovered. The DNA fragments were purified using the QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704) and sent for testing. The sequencing results were analyzed using the IgBLAST function of NCBI or the IMGT database, and the corresponding V and J gene cloning primers were selected for cloning PCR on the light and heavy chains. Subsequently, the PCR products were detected by agarose gel electrophoresis, and the bands with expected fragment sizes were cut and recovered. The DNA fragments were purified using the QIAquick Gel Extraction Kit (QIAGEN, catalog number: 28704) and sent for testing. The sequencing results were analyzed using the IgBLAST function of NCBI or the IMGT database, and the corresponding V and J gene cloning primers were selected for cloning PCR on the light and heavy chains. HiFi DNA Assembly Master Mix (NEB, Catalog No.: E2621L) was used for fragment ligation, and the resulting light and heavy chain variable region sequences were cloned into the corresponding light and heavy chain expression vectors containing the light and heavy chain constant regions (NCBI GenBank No.: FJ475055, FJ475056, FJ517647), respectively. Clones were selected after transformation plating, and the final sequences were determined by sequencing.

[0107] Blocking antibody screening:

[0108] 1. Antigen Binding Ability Screening: Antibody light and heavy chain expression vectors were co-transfected into 293T cells at a 1:1 ratio and cultured at 37°C, 5% CO2 for 3 days. The culture supernatant was collected by centrifugation and the TRAb antibody titer was determined according to the instructions of the Human Anti-Thyrotropin Receptor Antibody ELISA Kit (Kelu, Catalog No. ELK9540). Light and heavy chain combinations with binding abilities lower than the blank control were removed, and the remaining combinations were screened for blocking activity (Figure 2).

[0109] 2. Blocking activity screening: The antibody light and heavy chain expression vectors were co-transfected into 293T cells at a 1:1 ratio and cultured at 37°C, 5% CO2 for 3 days. The culture supernatant was collected by centrifugation and 100 μL of the supernatant was added with 1 IU / L bTSH (Sigma) to incubate hTSHR-CHO cells for 2 hours. Cell lysate was then collected and changes in cellular cAMP levels were assayed (R&D, Catalog No.: KGE002B). Antibody combinations with strong blocking activity were retained for further verification (Figure 3). Among them, the K12 combination showed the strongest inhibitory activity, and the corresponding monoclonal antibody was named TBI80, which was subsequently expressed and purified.

[0110] Antibody expression and purification: The antibody light and heavy chain expression vectors were co-transfected into 293F cells at a 1:1 ratio. After culturing at 37°C, 8% CO2, and shaking at 130 rpm for 5 days, the culture supernatant was collected by centrifugation, filtered through 0.45 μm, and purified by Protein A affinity chromatography (GenScript, Catalog No.: L00210) to obtain highly pure antibody protein. Antibody concentration was determined by the Bradford protein concentration method (Beyotime, Catalog No.: P0006) and the NanoDrop A280 method. Antibody expression was detected by SDS-PAGE gel electrophoresis and Coomassie Brilliant Blue staining (Beyotime, Catalog No.: P0017F) (Figure 4).

[0111] In vitro efficacy evaluation: The purified monoclonal antibody was diluted according to different concentration gradients and incubated with bTSH 5 ng / ml or human TSH 100 ng / ml. After incubation for 2 hours, the cell lysates were collected and the changes in cellular cAMP levels were measured (Figure 5). It was found that TBI80 monoclonal antibody at a concentration of 1 μg / mL could effectively antagonize the activation of TSH on the TSH receptor in a concentration-dependent manner.

[0112] In vivo efficacy evaluation: Male Sprague-Dawley rats aged 6-8 weeks were injected intramuscularly with 0.1 mL of TBI80 solution (200 μg TBI80, buffer: PBS; 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 8.1 mmol / L sodium hydrogen phosphate, 1.5 mmol / L potassium dihydrogen phosphate, pH 7.4; containing 3% mannitol and 1% Tween-80). Blood was collected from the rat tail at 0 hour before injection, and 4, 24, 48, and 72 hours after injection. Serum was separated by centrifugation at 4000 rpm for 5 minutes at 4°C and stored at -80°C. Thyroxine (T4) (Cloud Clone, Catalog No. CEA452Ge) and thyrotropin (TSH) (Cloud Clone, Catalog No. CEA463Ra) concentrations were measured (Figure 6). Compared to 0 hour before injection, T4 levels were significantly decreased 24 hours after injection, indicating that TBI80 maintains its inhibitory activity in vivo. Later, due to the feedback increase of TSH, the T4 level recovered.

[0113] A mouse model of Graves' disease was established. BALB / c mice were injected intramuscularly with an adenovirus overexpressing the α subunit of the human thyroid-stimulating hormone receptor (TSHR) (Ad-TSHR 289) three times, three weeks apart. Three weeks after the final immunization, the mice were evaluated for GD and divided into groups (control, virus-untreated, and TBI80-treated) for corresponding treatments. Three weeks after the final treatment, blood samples were collected (for thyroid function, liver and kidney function tests), and changes in thyroid size, weight, and morphology were observed (Figure 7). After three treatments with 50 μg of TBI80, T4 levels significantly decreased compared to the untreated group, thyroid size essentially recovered, and weight and morphology improved. Thyroid follicles showed regular morphology, with only a few epithelial cells remaining in a tall columnar shape, further demonstrating the in vivo inhibitory activity of the TSH receptor-blocking monoclonal antibody TBI80.

[0114] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. A fully human TSH receptor blocking monoclonal antibody, characterized in that: The antibody binds to the TSH receptor and blocks the binding of TSH to the TSH receptor; the amino acid sequence of the antibody is shown in SEQ ID NOs: 1 and 2; the nucleotide sequence of the antibody is shown in SEQ ID NOs: 3 and 4.

2. The antibody according to claim 1, characterized in that The amino acid sequence of the antibody 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% or more sequence identity with SEQ ID NO: 1, 2; and / or the nucleotide sequence of the antibody 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% or more sequence identity with SEQ ID NO: 3, 4.

3. The fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, characterized in that: The antibody is an antagonist of TSH; and / or, is an antagonist of thyroid stimulating antibody.

4. The fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, characterized in that: The antibodies each comprise a V H Area, the V H The region comprises one or more complementarity determining regions (CDRs) selected from SEQ ID NO: 6 to SEQ ID NO: 8; and / or, The antibodies each comprise a V L Area, the V L The region comprises one or more CDRs selected from SEQ ID NO: 9 to SEQ ID NO: 11; and / or, The antibody comprises one or more amino acid sequences that are substantially homologous to these CDRs.

5. A substance as described in any one of the following, characterized in that The substances include: (1) A preparation, a drug or a pharmaceutical composition, characterized in that the preparation, the drug or the pharmaceutical composition comprises the fully human TSH receptor blocking monoclonal antibody according to any one of claims 1 to 2; (2) A reagent or a kit, comprising the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (3) A nucleotide encoding the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, wherein the nucleotide sequence is one of the following sequences: (a) the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4; (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: 3 or SEQ ID NO: 4; (c) the nucleotide sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4 is supplemented, substituted, deleted or inserted with one or a nucleotide sequence after a number of nucleotides; (d) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (a), (b) or (c) above or its full-length complement; or, (e) a nucleotide sequence that is different from the nucleotide sequences of (a), (b), (c), and (d) above due to the degeneracy of the genetic code; Wherein, the nucleotide sequence of the nucleotide or a part thereof encodes antibody V H Domain and antibody V L A domain or a CDR selected from SEQ ID NO: 6 to SEQ ID NO: 11; (4) An expression vector comprising the nucleotide described in (3); (5) A host cell, comprising the nucleotide described in (3) and / or the expression vector described in (4); (6) A cell, wherein the cell is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, and / or the nucleotide according to (3), and / or the vector according to (4); and / or, An isolated cell expressing the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; and / or an isolated cell secreting the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (7) A composition comprising a determined concentration of TSH receptor autoantibodies and a fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (8) A pharmaceutical composition for administration to a mammalian subject to treat a thyroid-related disorder, the pharmaceutical composition comprising the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 and a pharmaceutically acceptable carrier; Wherein, the thyroid-related disease is selected from: hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis and pretibial myxedema.

6. The substance according to claim 5, characterized in that The pharmaceutical composition is suitable for administration to humans.

7. The substance according to claim 5, characterized in that The pharmaceutical composition includes one or more additional thyroid stimulating hormone receptor antagonists.

8. The substance according to claim 5, characterized in that The pharmaceutical composition comprises the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 and any pharmaceutically acceptable carrier, adjuvant or vehicle.

9. The substance according to claim 5, characterized in that The administration of the pharmaceutical composition includes oral administration, parenteral administration, administration by spray inhalation, topical administration, administration by eye drops or eye ointment, rectal administration, nasal administration, oral administration, vaginal administration, and administration via an implanted reservoir.

10. The substance according to claim 5, characterized in that The pharmaceutical composition may be in the form of capsules, tablets, aqueous suspensions, solutions, rectal suppositories, enemas, ointments, lotions, creams, nasal sprays, and inhalants.

11. The substance according to claim 5, characterized in that The pharmaceutical composition is used to treat a thyroid-related disorder in an injectable form.

12. The substance according to claim 5, characterized in that 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 drops.

13. Any of the following methods, characterized in that: The method comprises: (1) A method for preparing a fully human TSH receptor blocking monoclonal antibody, characterized in that it specifically comprises the following steps: In the first step, plasma cells and memory B cells targeting TSHR in the peripheral blood of patients with high TBAb activity were sorted, and single-cell RNA was extracted and cDNA was synthesized. The sorted single cells were amplified and verified by nested PCR for heavy chain H, light chain λ and light chain κ, and single-cell clones that were positive for both heavy and light chains were selected for subsequent cloning; In the second step, the BCR heavy and light chains of all single B cells were cloned into the heavy chain expression vector AbVec-IGHG1, the λ light chain expression vector AbVec-hIgKappa or the λ light chain expression vector AbVec-hIgLambda by nested PCR in vitro amplification; The third step is to sequence and compare the obtained candidate clones after the heavy chain and light chain recombinant plasmids are successfully obtained to determine the number of nucleotide and amino acid sequences of the obtained candidate antibodies; heavy chain and light chain expression plasmids are transfected to express monoclonal antibodies in vitro; the antigen binding ability and antibody blocking activity are further verified to finally obtain an antibody combination that can specifically target the target antigen, namely the fully human TSH receptor blocking monoclonal antibody; (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 a fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (3) A method for inhibiting stimulation of TSH receptors by thyroid stimulating antibodies in the thyroid gland of a mammalian subject, the method comprising contacting the subject with the antibody of the present invention; (4) A method for inhibiting the binding of thyroid stimulating autoantibodies to extrathyroidal TSH receptors in a mammalian subject, the method comprising contacting the subject with a fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2; (5) A method for treating thyroid cancer or metastatic thyroid cancer in a subject or in thyroid cells derived from a subject, the method comprising: treating the cancer cells with the fully human TSH receptor according to claim 1 or 2 contacting with a monoclonal antibody blocking the thyroxine receptor in order to inhibit constitutive thyroxine receptor activity in the cells; (6) A method for treating thyroid hyperactivity caused by constitutive thyroid activity in a subject or in thyroid cells derived from a subject, characterized in that the method comprises contacting the subject or cells with a fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, in order to inhibit such thyroid hyperactivity; (7) A method for identifying a molecule that can inhibit the binding of thyroid stimulating antibodies to TSH receptors, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 as a reference; (8) A method for identifying a molecule that can inhibit the binding of a thyroid blocking antibody to a TSH receptor, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 as a reference.

14. The method according to claim 13, characterized in that The thyroid-related disorder is selected from the group consisting of hyperthyroidism, thyroid-related eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

15. The method according to claim 13, characterized in that The subject is a human.

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

17. The method according to claim 13, characterized in that Method (4), wherein the extrathyroidal TSH receptor is located in the retro-orbital tissue and / or anterior tibial tissue of the subject; and / or, The antibodies block the binding of TSHR autoantibodies to the extrathyroidal TSHR.

18. The method according to claim 13, characterized in that In method (5), the method prevents or delays the regrowth of thyroid cancer cells.

19. The method according to claim 13, characterized in that In the method (7), the method selects a molecule to be tested that can prevent the binding of thyroid stimulating antibodies to TSHR.

20. The method according to claim 13, characterized in that In method (8), the method selects a molecule that can prevent thyroid blocking antibodies from binding to TSHR.

21. Any of the following applications, characterized in that: The applications include: (1) Use of the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 for treating a thyroid-related disease; (2) Use of the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 in the preparation of a medicament for treating a thyroid-related disease; (3) The fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 is used in the preparation of a TSH receptor detection Application of antibodies in reagents or kits; (4) Use of the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2 in the preparation of a reagent, kit or product for detecting hyperthyroidism or thyroid-related eye diseases; (5) Use of the fully human TSH receptor blocking monoclonal antibody according to claim 1 or 2, or the substance according to claim 5, or the method according to claim 13 in the preparation of preparations, drugs or pharmaceutical compositions for detecting TSH receptor antibodies, the preparation of drugs for treating hyperthyroidism and thyroid-related eye diseases, the preparation of drugs for inhibiting thyroid hyperplasia and / or thyroid hormone production, the preparation of drugs for TSH receptor blocking, and the preparation of drugs for antagonizing the activation effect of TSH on TSH receptors.

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

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