SH23 homologous cluster fully human TSH receptor blocking monoclonal antibody group as well as preparation method and application of SH23 homologous cluster fully human TSH receptor blocking monoclonal antibody group

Abstract

The invention discloses a group of blocking monoclonal antibodies of an SH23 homologous cluster fully human thyrotropin receptor (TSHR) (a TSH receptor, TSHR), a preparation method of the blocking monoclonal antibodies, and an application of the blocking monoclonal antibodies of the SH23 homologous cluster fully human thyrotropin receptor (TSHR), the SH23 homologous cluster fully human thyrotropin receptor (TSHR) and the preparation method of the blocking monoclonal antibodies. The method comprises the following steps: sorting plasma cells and memory single B cells for specifically recognizing TSHR in peripheral blood of a patient with high TSH-blocking antibody (TBAb) titer by using flow cytometry, cloning antibody light and heavy chains in vitro and performing recombinant expression, and performing antibody property screening verification by using hTSHR-CHO cells, so as to obtain the TSHR-TBAb-TSHR-TBAb. The blocking monoclonal antibody specifically targeting the human TSHR is obtained. The fully human TSH receptor blocking monoclonal antibody disclosed by the invention can be specifically combined with TSHR, and can effectively block signal transduction after the TSH is combined with the receptor; the compound disclosed by the invention has good inhibitory activity in vitro and in vivo experiments, and has a wide application prospect in the aspect of treating Graves' diseases (Graves' disease, GD) and diseases caused by hyperthyroidism, such as thyroid eye diseases (thyroid eye disease, TED) and the like.

Description

Technical Field

[0001] The present invention belongs to the field of monoclonal antibody technology and humanized antibody drugs, and relates to a group of fully humanized TSH receptor blocking monoclonal antibodies and their preparation methods and applications. Background Art

[0002] The thyroid-stimulating hormone receptor (TSHR) is a seven-transmembrane G protein-coupled receptor primarily found on the cell membrane of thyroid follicular epithelial cells. The TSHR has a large extracellular domain composed of a leucine-rich domain and a hinge region. Dysfunction of the TSHR may contribute to thyroid disease. Thyroid-stimulating hormone (TSH) binds to and activates the TSHR, initiating downstream signaling through the Gs and Gq / 11 pathways. These pathways regulate iodine transport and uptake, as well as iodine organoleptic properties, and contribute to thyroid hormone synthesis and release, as well as thyroid cell growth and differentiation. Furthermore, the large extracellular segment of the TSHR can be segmented into α- and β-subunits, with the α-subunit being susceptible to shedding. Against the backdrop of genetic susceptibility, under the influence of external environmental factors such as infection and trauma, TSHR peptides can act as exposed autoantigens, inducing the production of autoantibodies, namely thyrotropin receptor antibodies (TRAb), which participate in the occurrence and development of autoimmune thyroid diseases (AITD), mainly including Graves' disease (GD) and Hashimoto's thyroiditis.

[0003] Graves' disease (GD) is an organ-specific autoimmune disease caused by a combination of genetics and the environment, resulting in 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 a female incidence rate approximately 5-10 times higher than that of men. In recent years, the incidence of hyperthyroidism has been increasing with changes in living environments. If symptoms of hyperthyroidism are not promptly controlled, it 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 tension, anxiety, and irritability, impacting patients' academic performance and personal life. In severe cases, it can even lead to psychiatric disorders. Current treatments for GD mainly include medications, 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, with options remaining between antithyroid medications, radioactive iodine, and surgery. Among them, antithyroid drugs have been used to treat Graves' disease for nearly 70 years. Except for the United States, doctors around the world use ATD as the first choice for treating Graves' disease. However, after regular and systematic treatment, only some patients with hyperthyroidism can achieve a cure, and the side effects are significant. A considerable number of patients will relapse after a certain period of time. When the disease relapses, the condition worsens and generally requires radioactive iodine or surgical treatment, which greatly increases the financial and psychological burden on patients. In addition, radioactive iodine-131 nuclide treatment can easily lead to permanent hypothyroidism; the complications of surgical treatment cannot be ignored. The current dilemma in the treatment of GD means that there is an urgent need to find better alternative drugs in clinical practice.

[0004] TRAb is a general term for antibodies produced by the body against the thyroid stromal remodeling (TSHR). It is a group of polyclonal antibodies that can be divided into TSH receptor stimulating antibodies (TSAb), TSH receptor blocking antibodies (TBAb), and neutralizing antibodies. They recognize different epitopes on the TSHR. The recognition epitopes of TSAb, TBAb, and neutralizing antibodies are concentrated in the amino terminus (N-terminus), carboxyl terminus (C-terminus), and hinge region of the TSHR extracellular domain, respectively. Among them, thyroid stimulating antibodies (TSAb) bind to the TSHR on the membrane of thyroid follicular epithelial cells, producing a biological effect similar to that of TSH, causing hyperthyroidism and being the direct cause of Graves' disease. TSAbs, through stimulatory G protein coupling, further activate adenylate cyclase (AC), stimulating cAMP production. The AC-cAMP pathway remains continuously active, stimulating thyroid cell proliferation and the production and secretion of excessive thyroid hormones, triiodothyronine (T3) and thyroxine (T4). Competitive inhibition of TSH prevents its normal feedback regulation of T3 and T4, leading to a continuous increase in these two hormones, triggering a series of bodily responses and ultimately causing thyroid cell hyperfunction. Histologically, the thyroid glands of patients with GD display thickened and hypertrophic follicular cells. The glands show typical T and B cell lymphocytic infiltration, characteristic of thyroiditis, with occasional apoptosis and some follicular destruction. TBAbs bind to TSH, blocking its binding to receptors and suppressing thyroid proliferation and thyroid hormone production. Both stimulatory 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 to bind to TSHR, thereby improving the pathophysiological effects of autoantibodies and becoming a new effective option for the treatment of GD.

[0005] In addition, 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 (TED), also known as thyroid-associated ophthalmopathy (TAO), neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema.

[0006] Thyroid eye disease (TED) is a common orbital disease in adults and an autoimmune disease closely related to Graves' disease. It manifests as congestion and edema of the eyelids and conjunctiva, as well as fibrosis and fatty changes in the orbital tissues. This leads to ocular motility disorders, strabismus, and diplopia, which can cause blindness and disability. Diagnosis and treatment are challenging, significantly impacting patients' quality of life. my country has one of the highest rates of GD globally, with the prevalence of TED among Asian GD patients reaching as high as 45%. This large population of patients with TED severely impacts their visual health and quality of life. TED, a problem that has plagued the global medical community for two centuries, currently lacks effective treatment. While various treatment options are available, many patients respond poorly to existing medications or cannot tolerate their side effects. High-dose glucocorticoid pulse therapy, while significantly effective in reducing inflammation, is ineffective for proptosis and diplopia, and long-term use carries significant side effects, such as hypertension, diabetes, osteoporosis, and gastric ulcers. Some patients are insensitive to glucocorticoids, resulting in an efficacy rate of only 50-75%. Furthermore, while TED offers significant short-term benefits, it is prone to relapse after discontinuation. Immunosuppressants can alleviate ocular inflammation by suppressing the immune system's overreaction, but they can also cause serious side effects, such as increased risk of infection and damage to liver and kidney function. Therefore, the market urgently needs new, safe, and effective drugs.

[0007] Orbital fibroblasts (OF) are the effector cells of the TED autoimmune response, expressing thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) on their surface. TSHR is ectopically overexpressed in CD34+ fibroblasts in the retrobulbar tissue of TED patients. The immune response in TED fluid begins with abnormal recognition of the autoantigen thyroid-stimulating hormone receptor (TSHR), followed by the production of TRA antibodies by activated B cells. IGF-1R is another potential TED autoantigen, but the existence of autoantibodies that directly activate IGF-1R remains unclear. Studies have demonstrated that the signaling pathways mediated by IGF-1R and TSHR interact, leading to the secretion of hyaluronic acid (HAS). Tepezza, an antibody drug targeting IGF-1R, received FDA approval in 2020 for the treatment of TED. Treatment can significantly improve bulging eyes and diplopia, with nearly 40% of patients with bulging eyes achieving near-normal bulging eyes. Despite the remarkable efficacy of IGF-1R antibody drugs, due to the widespread expression of IGF-1R in human organs, significant side effects have been reported following large-scale clinical use, particularly irreversible deafness and reproductive system effects. Because both stimulating and blocking antibodies coexist in GD patients, the ultimate outcome for thyroid function depends on which antibody predominates. This makes exogenous supplementation with a dose of blocking antibodies to inhibit the TSHR a new and effective treatment option for thyroid eye disease.

[0008] 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 remodeling 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. Summary of the Invention

[0009] In response to the above-mentioned prior art, the present invention provides a group of fully human TSH receptor (TSHR) blocking monoclonal antibodies or their antigen-binding fragments for the treatment of hyperthyroidism, and also provides the coding sequence of the antibody and a vector containing the coding sequence.

[0010] 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: 53, 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: 53.

[0011] The present invention provides a group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, wherein the fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof bind to the TSH receptor and block the binding of TSH to the 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] Wherein, 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 shown in SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, respectively;

[0013] Wherein, 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 NOs: 57 to 62; the amino acid sequence of L-CDR2 is selected from one of SEQ ID NOs: 63 to 67; and the amino acid sequence of L-CDR3 is selected from one of SEQ ID NOs: 68 to 73.

[0014] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 57, SEQ ID NO: 63 and SEQ ID NO: 68, respectively.

[0015] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 58, SEQ ID NO: 63 and SEQ ID NO: 69, respectively.

[0016] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown in SEQ ID NO: 58, SEQ ID NO: 64 and SEQ ID NO: 69, respectively.

[0017] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 58, SEQ ID NO: 63 and SEQ ID NO: 68, respectively.

[0018] Preferably, the amino acid sequences of H-CDR1, H-CDR2 and H-CDR3 of the heavy chain variable region are shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 59, SEQ ID NO: 64 and 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 shown in SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown in SEQ ID NO: 60, SEQ ID NO: 65 and 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 shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 61, SEQ ID NO: 66 and 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 shown as SEQ ID NO: 54, SEQ ID NO: 55 and SEQ ID NO: 56, respectively; the amino acid sequences of L-CDR1, L-CDR2 and L-CDR3 of the light chain variable region are shown as SEQ ID NO: 62, SEQ ID NO: 67 and SEQ ID NO: 73, respectively.

[0022] in,

[0023] 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 a sequence identity of 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% to one of SEQ ID NO: 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 a sequence identity of 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% 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 the present 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 antigen-binding fragment thereof are shown in SEQ ID NO: 27 to SEQ ID NO: 52.

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

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

[0029] Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Th S Tyr Alar Val Ser Asp Alar Ser Gln GlyArg Leu Thr Ile SerArg Asp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAspThr Ala Val Tyr Tyr Cys Val Arg Glu Ala Val Ile Val Asp Gly Met Pro Phe GluTyr Trp GlyGln Gly Ala Leu Val Ser Val Ser

[0030] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISS SAATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQ GALVTVSS)

[0031] SEQ ID NO:2(SH23_2

[0032] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser ProGly Gln Ser Val Thr Val Thr IleSer Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr AsnTyr Val Ser Trp His Gln Gln His Pro GlyLys Ala Pro Lys Leu Met Ile Tyr AspVal Ser Arg Ser Gly Ser Asp Val Ser A Gly SerLys Ser Gly AsnThr Ala Ser Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr TyrCysCys Ser Tyr Ala Gly Ser Tyr Thr Leu Val Phe Gly Gly Gly Thr Lys Leu ThrVal Leu Gly Gln ProLys Ala Ala Pro Ser Val Pro Leu Pro

[0033] (SWAQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWHQQHPGKAPKLMIYDV SKRPSGVPDRFSGSSKSGNTASLTISGLQAEDADYYCCSYTLVFGGGTKLTVLGQPKA APSWTLFP)

[0034] SEQ ID NO:27(SH23_3

[0035] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0036] SEQ ID NO: 28 (Nucleotide sequence of SH23_4)

[0037] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCCCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0038] SEQ ID NO: 3 (Amino acid sequence of CHL34_1)

[0039] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0040] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0041] SEQ ID NO:4(CHL34_2

[0042] Ser Trp Ala Gln Ser Val Leu Thr Gln Pro Ser Val Ser Ala Ala ProGly Gln Lys Val Thr IleSer Cys Ser Gly Gly Ser Ser Asn Ile GlyAsn Asn Tyr IleSer Trp Tyr Gln Gln Leu Pro Gly Pro Gly Pro Lys Leu Leu Ile Tyr Aspn Ser A Arg Phe Ser Gly Ser Lys SerGly Thr SerAla Thr LeuGly Ile Thr Gly Leu Gln Thr GlyAsp GluAlaAsp Tyr Tyr Cys Gly Thr TrpAsp SerSer Leu SerAla Gly Val Phe Gly Gly Gly Thr Pro Lys Leu ThrVal Leu Gly Gln Pro Thal Leu PV

[0043] (SWAQSVLTQPPSVSAAPGQKVTISCSGGSSNIGNNYISWYQQLPGTAPKLLIYDNNKR PSGIPDRFSGSKSGTCATLGITGLQTGDEADYYCGTWDSSLSAGVFGGGTKLTVLGQPKAAP SVTLFP)

[0044] SEQ ID NO:29(CHL34_3

[0045] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0046] SEQ ID NO: 30 (CHL34_4 nucleotide sequence)

[0047] TCCTGGGCCCAGTCTGTGCTGACTCAGCCGCCCTCAGTGTCTGCGGCCCCCGGACAGAAGGTCACCATCTCCTGCTCTGGAGGCAGCTCCAACATTGGGAATAATTATATATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATTTATGACAATAATAAGAGACCCTCAGGGATTCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCACCCTGGGCATCACCGGACTCCAGACTGGGGACGAGGCCGATTATTACTGCGGAACATGGGATAGCAGCCTGAGTGCTGGGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCG

[0048] SEQ ID NO: 5 (SH33_1 amino acid sequence)

[0049] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0050] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0051] SEQ ID NO:6(SH33_2

[0052] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Tyr Thr TyrVal Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile TyrAg Arsp Val Serg Serg Arsp Val Serg P Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Trp Val Ser Gly Gly Gly Thr Lys Leu Pro Val Leu Gly Glyn Pro LysAla Thla Leu Leu Ser

[0053] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVSGGGTKLTVLGQPKA APSATLFP)

[0054] SEQ ID NO:31(SH33_3

[0055] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0056] SEQ ID NO: 32 (Nucleotide sequence of SH33_4)

[0057] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTCCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0058] SEQ ID NO: 7 (Amino acid sequence of CHL31_1)

[0059] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0060] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0061] SEQ ID NO:8(CHL31_2

[0062] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly TyrAsn TyrVal Ser Trp Tyr Gln Gln His Pro Gly LysAla Pro Lys Leu Met Ile Tyr P Asp Val Serg Arg Pro Arg Arg Val Ser Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Pro Val Leu Gly Glyn Pro

[0063] (SWAQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDYYCCSYTLVFGGGTKLTVLGQPKAA PSVTLFP)

[0064] SEQ ID NO:33(CHL31_3

[0065] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0066] SEQ ID NO: 34 (Nucleotide sequence of CHL31_4)

[0067] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGTACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCA

[0068] SEQ ID NO: 9 (Amino acid sequence of CHL23_1)

[0069] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0070] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0071] SEQ ID NO:10(CHL23_2

[0072] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Gly TyrAsn TyrVal Ser Trp His Gln Gln His Pro Gly LysAla Pro Lys Leu Met Ile Tyr Asp LyS Pros Val Gly Arg Pro Gly A Phe Ser Gly Ser LysSer GlyAsn ThrAlaSer Leu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAlaGly Ser Tyr Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glyn ProLys ProLys Pro Leu Thr

[0073] (SWAQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYNYVSWHQQHPGKAPKLMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDYYCCSYTLVFGGGTKLTVLGQPKAA PSVTLFP)

[0074] SEQ ID NO:35(CHL23_3

[0075] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0076] SEQ ID NO: 36 (CHL23_4 nucleotide sequence)

[0077] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATAACTATGTCTCCTGGCACCAACAGCACCCAGGCAAAGCCCCCAAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGACGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCA

[0078] SEQ ID NO: 11 (CHL24_1 amino acid sequence)

[0079] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0080] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0081] SEQ ID NO:12(CHL24_2

[0082] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Tyr Thr TyrVal Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile TyrAg Arsp Glys Val Serg P He Serg Val Ser Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glyn Pro LysAla ThrA Pro Leu Pro

[0083] (SWAQSALTQPRSVSGSPGQSITISCTGTSADVGDYDYVSWYQQHPGKAPKLIIYDVTR RPSGVPDRFSGSSKSGNTASLTISGLQADDEANYCCSYAGSDWLFGGGTQLTVLGQPKA APSVTLFP)

[0084] SEQ ID NO:37(CHL24_3

[0085] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0086] SEQ ID NO: 38 (CHL24_4 nucleotide sequence)

[0087] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAATCACCATCTCCTGCACTGGAACCAGCGCTGATGTTGGAGATTATGATTATGTCTCCTGGTACCAACAACACCCAGGCAAAGCCCCCAAACTCATAATTTATGATGTCACTAGGCGACCCTCAGGGGTCCCGGATCGCTTCTCTGGCTCCAAGTCTGGCAATACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGACGATGAGGCTAATTATTATTGCTGCTCATATGCAGGCAGCGACACTGATTGGCTGTTCGGCGGAGGGACCCAGCTGACCGTCCTGGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCA

[0088] SEQ ID NO: 13 (CHL46_1 amino acid sequence)

[0089] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0090] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0091] SEQ ID NO:14(CHL46_2

[0092] Ser Leu Ser Gln Pro Val Ser ProGly Thr Gln Pro Pro Ser Val Ser Val Ser ProGly Gln Thr Ala Arg IleThr Cys Ser Gly GlyArg Leu GlyAsp Thr PheAla Ser TrpTyr Gln GlnArg Pro Gly GlnAla Pro ValLeu Val Ile Tyr Asp Thr Met Pro Gly Arg Gly Arg I P Ser Asn Ser Gly HisAlaAla Thr Leu Thr IleSer Gly Thr Gln Thr MetAsp GluAlaAsp Tyr Tyr Cys Gln Val TrpAsp ThrSerAlaSerAla His Val Phe Gly Thr Gly Thr Thr Val Thr Pro Val Leu Gly Gln Pro Lys Alas Thr Pro Leu

[0093] (SLSQPVLTQPPSVSVSPGQTARITCSGGRLGDTFASWYQQRPGQAPVLVIYRDTMRPS GIPERFSGSNSGHAATLTISGTQTMDEADYYCQVWDTSASAHVFGTGTTVTVLGQPKANPT VTLFP)

[0094] SEQ ID NO:39(CHL46_3

[0095] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0096] SEQ ID NO: 40 (CHL46_4 nucleotide sequence)

[0097] TCTCTCTCGCAGCCTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGAATCACCTGCTCTGGGGGAAGATTGGGGGATACATTTGCTTCCTGGTATCAACAGAGGCCAGGCCAGGCCCCTGTGTTGGTCATCTATCGTGATACGATGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGCTCCAACTCTGGGCACGCAGCCACTCTGACCATCAGCGGGACGCAGACTATGGATGAGGCTGACTACTACTGTCAGGTGTGGGACACCAGCGCCAGCGCACATGTCTTCGGAACTGGGACCACGGTCACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTCCCG

[0098] SEQ ID NO: 15 (CHL47_1 amino acid sequence)

[0099] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0100] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0101] SEQ ID NO:16(CHL47_2

[0102] Ser Leu Ser Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser ProGly Gln ThrAlaArg IleThr Cys Ser Gly GlyArg Leu GlyAsp Thr PheAla Ser Trp TyrGln GlnArg Pro Gly GlnAla Pro ValLeu Val Ile Tyr Arg Arg Thr Arg Arg Pro Glu Ser I Phe Ser Gly Ser Asn Ser Gly HisAlaAla Thr Leu Thr Ile SerGly Thr Gln Thr MetAsp GluAlaAsp Tyr Tyr Cys Gln Val TrpAsp ThrSerAla SerAlaHis Val Phe Gly Thr Gly Thr Thr Val Thr Val Leu Gly Glyn Pro Lys Alar ProTh ProTh

[0103] (SLSQLVLTQPPSVSVSPGQTARITCSGGRLGDTFASWYQQRPGQAPVLVIYRDTMRPS GIPERFSGSNSGHAATLTISGTQTMDEADYYCQVWDTSASAHVFGTGTTVTVLGQPKANPT VTLFP)

[0104] SEQ ID NO:41(CHL47_3

[0105] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0106] SEQ ID NO: 42 (Nucleotide sequence of CHL47_4)

[0107] TCTCTCTCCCAGCTTGTGCTGACTCAGCCACCCTCAGTGTCCGTGTCCCCAGGACAGACAGCCAGAATCACCTGCTCTGGGGGAAGATTGGGGGATACATTTGCTTCCTGGTATCAACAGAGGCCAGGCCAGGCCCCTGTGTTGGTCATCTATCGTGATACGATGCGGCCCTCAGGGATCCCTGAGCGGTTCTCTGGCTCCAACTCTGGGCACGCAGCCACTCTGACCATCAGCGGGACGCAGACTATGGATGAGGCTGACTACTACTGTCAGGTGTGGGACACCAGCGCCAGCGCACATGTCTTCGGAACTGGGACCACGGTCACCGTCCTAGGTCAGCCCAAGGCCAACCCCACTGTCACTCTGTTCCCG

[0108] SEQ ID NO: 17 (Amino acid sequence of CHL33_1)

[0109] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0110] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0111] SEQ ID NO:18(CHL33_2

[0112] Ser Trp Ala Gln Ser Val Leu Thr Gln Pro SerAla Ser Gly Thr ProGly Gln Arg Val Thr IleSer Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn TyrVal Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Pro AlaPro Lys Leu Leu Ile TyrArgAs Gln Pro Glyn Pro Glyn Arg Phe Ser Gly Ser Lys SerGly Thr SerAla SerLeuAla Ile Ser Gly LeuArg Ser GluAsp Glu AlaAsp Tyr Tyr Cys AlaAla TrpAsp AlaSer Leu Ser Ala Leu Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro

[0113] (SWAQSVLTQPPSASGTPGQRVTISCSGSSNIGSNYVYWYQQLPGTAPKLLIYRNNQR PSGVPDRFSGSSKSGTSLAISGLRSEDEADYCAAWDASLSALFGGGTKLTVLGQPKAAPS VTLFP)

[0114] SEQ ID NO:43(CHL33_3

[0115] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0116] SEQ ID NO: 44 (Nucleotide sequence of CHL33_4)

[0117] TCCTGGGCCCAGTCTGTGCTGACGCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGCCAGCCTGAGTGCCTTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCG

[0118] SEQ ID NO: 19 (Amino acid sequence of CHL32_1)

[0119] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0120] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0121] SEQ ID NO:20(CHL32_2

[0122] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Tyr Thr TyrVal Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile TyrAg Arsp Val Serg Serg Arsp Val Serg Arg P Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Pro Val Leu Gly Gln Pro LysAla Thla Leu Pro Leu

[0123] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKA APSATLFP)

[0124] SEQ ID NO:45(CHL32_3

[0125] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0126] SEQ ID NO: 46 (Nucleotide sequence of CHL32_4)

[0127] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCA

[0128] SEQ ID NO: 21 (Amino acid sequence of SH24_1)

[0129] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0130] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0131] SEQ ID NO:22(SH24_2

[0132] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser ProGly Gln Ser Val Thr Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Tyr Thr TyrVal Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile TyrAg Arsp Glys Val Serg P He Serg Val Ser Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Glyn Pro LysAla ThrA Pro Leu Pro

[0133] (SWAQSALTQPASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRHSGVLDRFSGSKNGNTASLTISGLQAEDYYCCYAGSYTWVFGGGTKLTVLGQPKA APSATLFP)

[0134] SEQ ID NO:47(SH24_3

[0135] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0136] SEQ ID NO: 48 (Nucleotide sequence of SH24_4)

[0137] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCACTCAGGGGTCCTTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCG

[0138] SEQ ID NO: 23 (Amino acid sequence of CHL26_1)

[0139] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0140] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0141] SEQ ID NO:24(CHL26_2

[0142] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly Tyr Thr TyrVal Ser Trp Tyr Gln Gln His Pro Gly GluAla Pro Glu Leu Met Ile TyrAsp Arg Serg Val Serg Arg Serg Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Pro Val Leu Gly Glyn Pro LysAla Thla Leu Phe Pro

[0143] (SWAQSALTQPRSVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDADYCCSYAGSYTLVFGGGTKLTVLGQPKAA PSVTLFP)

[0144] SEQ ID NO:49(CHL26_3

[0145] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0146] SEQ ID NO: 50 (CHL26_4 nucleotide sequence)

[0147] TCCTGGGCCCAGTCTGCCCTGACTCAGCCTCGCTCAGTGTCCGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTTGGTATTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCG

[0148] SEQ ID NO: 25 (SH31_1 amino acid sequence)

[0149] Val His Ser Glu Val Gln Leu Val Gl Gly Ser Gly Gly Gly Leu Val Gln ProGly Gly Ser Leu ArgLeu Ser Cys Val Ala Ser Gly Phe Asp Phe Asn Gln Tyr SerMet Asn Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Leu Ser Tyr Ile Ser Tyr Alas Val Ser Alasp A Ser Gln GlyArg Leu Thr Ile Ser ArgAsp Asn Gly Lys Asn Ser Leu Phe Leu Gln Met Asn Ser Leu Arg Val GluAsp ThrAlaVal Tyr Tyr Cys Val Arg GluAla Val Ile Val Asp Gly Met Pro Phe Glu Tyr TrpGly GlnGlyAla Leu ThrVal Ser

[0150] (VHSEVQLVESGGGLVQPGGSLRLSCVASGFDFNQYSMNWVRQAPGKGLEWLSYISSS AATVFYADSVQGRLTISRDNGKNSLFLQMNSLRVEDTAVYYCVREAVIVDGMPFEYWGQGA LVTVSS)

[0151] SEQ ID NO:26(SH31_2

[0152] Ser Trp Ala Gln Ser Ala Leu Thr Gln Pro Arg Ser Val Ser Gly Ser ProGly Gln Ser Val Thr IleSer Cys Thr Gly Thr Ser SerAsp Val Gly Gly TyrAsn TyrVal Ser Trp Tyr Gln Gln His Pro Gly LysAla Pro Lys Leu Met Ile Tyr P Asp Val Serg Arg Pro Arg Arg Val Ser Gly Ser LysSer GlyAsn ThrAla SerLeu Thr Ile Ser Gly Leu GlnAla GluAsp GluAlaAsp Tyr Tyr Cys Cys SerTyrAla GlySer Tyr Thr Leu Val Phe Gly Gly Gly Thr Lys Leu Pro Val Leu Gly Glyn Pro

[0153] (SWAQSALTQHASVSGSPGQSVTISCTGTSSDVGGYTYVSWYQQHPGEAPELMIYDVS KRPSGVPDRFSGSSKSGNTASLTISGLQAEDEADYYCCSYAGSYTWVFGGGTKLTVLGQPKA APSATLFP)

[0154] SEQ ID NO:51(SH31_3

[0155] GTACATTCTGAGGTGCAGCTGGTGGAGTCCGGTGGAGGCTTGGTTCAACCGGGGGGGTCCCTGAGACTCTCCTGTGTAGCCTCTGGATTCGACTTCAACCAATATAGCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGCTTTCATACATCAGTAGCAGCGCTGCAACCGTGTTCTACGCAGACTCTGTGCAGGGCCGACTCACCATCTCCAGAGACAATGGCAAGAATTCACTCTTTCTGCAGATGAACAGCCTGAGAGTCGAGGACACGGCTGTCTATTACTGTGTCAGAGAAGCTGTTATAGTGGATGGCATGCCGTTTGAATACTGGGGCCAGGGAGCCCTGGTCACCGTCTCCTCAGC

[0156] SEQ ID NO: 52 (Nucleotide sequence of SH31_4)

[0157] TCCTGGGCCCAGTCTGCCCTGACTCAGCATGCCTCCGTGTCTGGGTCTCCTGGACAGTCAGTCACCATCTCCTGCACTGGAACCAGCAGTGATGTTGGTGGTTATACCTATGTCTCCTGGTACCAACAGCACCCAGGCGAAGCCCCCGAACTCATGATTTATGATGTCAGTAAGCGGCCCTCAGGGGTCCCTGATCGCTTCTCTGGCTCCAAGTCTGGCAACACGGCCTCCCTGACCATCTCTGGGCTCCAGGCTGAGGATGAGGCTGATTATTACTGCTGCTCATATGCAGGCAGCTACACTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTCAGCCCAAGGCTGCCCCCTCGGCCACTCTGTTCCCG

[0158] SEQ ID NO: 53 (Amino acid sequence of human TSHR)

[0159] MRPADLLQLVLLLDLPRDLGGMGCSSPPCECHQEEDFRVTCKDIQRIPSLPPSTQTLKLIETHLRTIPSHAFSNLPNISRIYVSIDVTLQQLESHSFYNLSKVTHIEIRNTRNLTYIDPDALKELPLLKFLGIFNTGLKMFPDLTKVYSTDIFFILEITDNPYMTSIPVNAFQGLCNETLTLKLYNNGFTSVQGYAFNGTKLDAVYLNKNKYLTVIDKDAFGGVYSGPSLLDVSQTSVTALPSKGLEHLKELIARNTWTLKKLPLSLSFLHLTRADLSYPSHCCAFKNQKKIRGILESLMCNESSMQSLRQRKSVNALNSPLHQEYEENLGDSIVGYKEKSKFQDTHNNAHYYVFFEEQEDEIIGFGQELKNPQEETLQAFDSHYDYTICGDSEDMVCTPKSDEFNPCEDIMGYKFLRIVVWFVSLLALLGNVFVLLILLTSHYKLNVPRFLMCNLAFADFCMGMYLLLIASVDLYTHSEYYNHAIDWQTGPGCNTAGFFTVFASELSVYTLTVITLERWYAITFAMRLDRKIRLRHACAIMVGGWVCCFLLALLPLVGISSYAKVSICLPMDTETPLALAYIVFVLTLNIVAFVIVCCCYVKIYITVRNPQYNPGDKDTKIAKRMAVLIFTDFICMAPISFYALSAILNKPLITVSNSKILLVLFYPLNSCANPFLYAIFTKAFQRDVFILLSKFGICKRQAQAYRGQRVPPKNSTDIQVQKVTHEMRQGLHNMEDVYELIENSHLTPKKQGQISEEYMQTVL

[0160] Table 1: CDR regions of the heavy chain (H) and light chain (L) of the SH23 homologous cluster fully human TSH receptor blocking monoclonal antibody

[0161]

[0162]

[0163] The present invention comprises a group of fully human TSH receptor-blocking monoclonal antibodies or antigen-binding fragments thereof, namely SH23, CHL32, SH31, SH24, SH33, CHL26, CHL31, CHL23, CHL24, CHL46, CHL47, CHL33, and CHL34. This group of antibodies shares high homology with SH23 and is therefore designated a group of SH23 homology cluster fully human TSH receptor-blocking monoclonal antibodies or antigen-binding fragments thereof. K1-70 in the table above is a reported TSH receptor-blocking monoclonal antibody with blocking activity. In contrast, the sequence provided by the present invention differs significantly and has no homology at all.

[0164] Preferably, the present invention proposes a group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, named SH23 homology cluster fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof.

[0165] In the present invention, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is a TSH antagonist.

[0166] In the present invention, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is an antagonist of thyroid stimulating antibodies.

[0167] In the present invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment respectively comprises a V H Region (heavy chain variable region), the V H The region comprises a complementarity determining region (CDR) having an amino acid sequence as shown in SEQ ID NO: 54 to SEQ ID NO: 56 (see Table 1).

[0168] In the present invention, the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment respectively comprises a V L Region (light chain variable region), the V LThe region comprises CDRs having amino acid sequences as set forth in SEQ ID NOs: 57, 63, and 68 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 58, 63, and 69 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 58, 64, and 69 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 58, 63, and 68 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 59, 64, and 70 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 60, 65, and 71 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 61, 66, and 72 (see Table 1), or CDRs having amino acid sequences as set forth in SEQ ID NOs: 62, 67, and 73 (see Table 1).

[0169] In the present invention, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof comprises one or more amino acid sequences substantially homologous to these CDRs.

[0170] The fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment 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 causes; after binding to the TSH receptor, it can significantly reduce goiter caused by hyperthyroidism and other reasons; and alleviate inflammation, edema and hyperplasia of thyroid-related eye diseases.

[0171] The thyroid associated ophthalmopathy (TAO) described in the present invention is also called thyroid eye disease (TED).

[0172] 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 or an antigen-binding fragment thereof.

[0173] The present invention also provides a reagent or a kit, which contains the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof as described above.

[0174] The present invention also provides a nucleotide encoding the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above, the nucleotide sequence of which is one of the following sequences:

[0175] (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, or 51; the nucleotide sequence encoding the light chain variable region is shown in one of SEQ ID NOs: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, or 52;

[0176] (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%, or at least 99% sequence identity to SEQ ID NO: 27 to SEQ ID NO: 52;

[0177] (c) a nucleotide sequence after one or more nucleotides are added, substituted, deleted, or inserted into the nucleotide sequence shown in SEQ ID NO: 27 to SEQ ID NO: 52;

[0178] (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

[0179] (e) a nucleotide sequence that differs from the nucleotide sequences of (a), (b), (c), and (d) above due to the degeneracy of the genetic code;

[0180] Wherein, the nucleotide sequence of the nucleotide or a part thereof encodes the antibody V H domain; and antibody V L The structural domain may be selected from the CDRs shown in SEQ ID NO: 54 to SEQ ID NO: 73 (see Table 1).

[0181] The present invention also provides a vector comprising the nucleotide described above.

[0182] The present invention also provides a host cell, comprising the nucleotide described above, and / or the vector described above.

[0183] The present invention also provides a cell, which is an isolated cell comprising the above-described fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof, and / or the above-described nucleotide, and / or the above-described vector.

[0184] The present invention 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.

[0185] The present invention also provides a cell, which is an isolated cell that secretes the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above.

[0186] The present invention also provides a composition comprising a determined concentration of TSH receptor autoantibodies and a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof as described above.

[0187] 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 or antigen-binding fragment thereof as described above and a pharmaceutically acceptable carrier;

[0188] 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.

[0189] Furthermore, the preparation, medicine or pharmaceutical composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc.

[0190] 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.

[0191] 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.

[0192] Preferably, the reagent or kit for detecting hyperthyroidism and thyroid-related eye diseases contains the fully human TSH receptor blocking monoclonal antibody.

[0193] Furthermore, the nucleotide sequence or at least a portion of the sequence 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.

[0194] 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.

[0195] Preferably, the host cell is a CHO-K1 cell or the like.

[0196] Specifically, the composition may comprise a TSHR autoantibody having TSH antagonist activity at a determined concentration and include a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to the present invention.

[0197] 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.

[0198] Furthermore, the pharmaceutical composition comprises one or more additional thyroid stimulating hormone receptor antagonists.

[0199] Furthermore, the pharmaceutical composition is used to treat a thyroid-related disorder in an injectable form.

[0200] Preferably, the pharmaceutical composition for treating Graves' ophthalmopathy is in the form of an intravenous injection preparation or eye drops.

[0201] Preferably, the pharmaceutical composition for treating pretibial myxedema is in a topical administration form.

[0202] 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.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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.

[0208] 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.

[0209] 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.

[0210] The present invention also provides a method for preparing a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof, the preparation method specifically comprising the following steps:

[0211] In the first step, plasma cells and memory B cells targeting the TSHR were isolated from the peripheral blood of patients with high TBAb activity. Single-cell RNA was extracted and cDNA synthesized. The sorted single cells were then amplified and verified by nested PCR for heavy chain H, light chain λ, and light chain κ. Single-cell clones positive for both heavy and light chains were selected for subsequent cloning.

[0212] In the second step, 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, the lambda light chain expression vector AbVec-hIgKappa, or the lambda light chain expression vector AbVec-hIgLambda;

[0213] In the third step, after successfully obtaining the heavy chain and light chain recombinant plasmids, the obtained candidate clones are sequenced and compared and analyzed 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 is obtained, namely the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment.

[0214] The present invention also provides a method for treating a thyroid-related disorder in a mammalian subject or in cells derived from the subject, the method comprising contacting the subject or the cell with the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above.

[0215] 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.

[0216] The present invention also provides a method for inhibiting thyroid stimulating antibodies from stimulating TSH receptors in the thyroid gland of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof of the present invention;

[0217] Preferably, the binding of thyroid stimulating antibodies to the TSH receptor is prevented.

[0218] 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 or antigen-binding fragment thereof as described above;

[0219] Specifically, the extrathyroidal TSH receptor is located in the retroorbital tissue and / or anterior tibial tissue of the subject;

[0220] Preferably, the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof can block the binding of TSH receptor autoantibodies to extrathyroidal TSH receptors.

[0221] 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 or antigen-binding fragment thereof as described above, for the purpose of inhibiting constitutive thyroid stimulating hormone receptor activity in the cells;

[0222] Preferably, the regrowth of the thyroid cancer cells is prevented or delayed.

[0223] 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 the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above, in order to inhibit such thyroid hyperactivity.

[0224] 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 or antigen-binding fragment thereof as described above as a reference;

[0225] Preferably, molecules are selected that prevent thyroid stimulating antibodies from binding to the TSH receptor.

[0226] 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 or an antigen-binding fragment thereof as described above as a reference;

[0227] Preferably, molecules are selected that prevent thyroid blocking antibodies from binding to the TSH receptor.

[0228] 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.

[0229] Preferably, the subject treated in the above methods is a human.

[0230] In the present invention, the preparation method includes the following steps: using flow cytometry to sort plasma cells and memory single B cells that specifically recognize TSHR in the peripheral blood of patients with high titers of TSH receptor blocking antibodies (TBAb), cloning the antibody light and heavy chains in vitro and recombinantly expressing them, and using hTSHR-CHO cells to screen and verify the antibody properties to obtain a blocking monoclonal antibody that specifically targets human TSHR. Through the preparation method of the fully human TSH receptor blocking monoclonal antibody for the treatment of hyperthyroidism proposed in the present invention, the target antibody sequence can be obtained in 3 weeks to 1 month.

[0231] Furthermore, the present invention uses hTSHR-CHO cells in vitro to verify the properties of the antibody and evaluate its effects.

[0232] The present invention also provides use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above for treating a thyroid-related disease.

[0233] The present invention also provides use of the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as described above in the preparation of a medicament for treating a thyroid-related disease.

[0234] The present invention also provides use of the above-mentioned fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof in the preparation of a reagent or kit for detecting TSH receptor antibodies.

[0235] The present invention also provides the use of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment as described above in the preparation of a reagent, kit or product for detecting hyperthyroidism and thyroid-related eye diseases.

[0236] The present invention also provides the use of the above-mentioned fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment, or the above-mentioned substance, or the above-mentioned method in the preparation of a preparation, a drug or a pharmaceutical composition for detecting TSH receptor antibodies, the preparation of a drug for treating hyperthyroidism and thyroid-related eye diseases, the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, the preparation of a TSH receptor blocking drug, and the preparation of a drug for antagonizing the activation effect of TSH on TSH receptors.

[0237] Specifically, the thyroid-related disease in the above method 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.

[0238] 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.

[0239] The present invention also has the following beneficial effects: the fully human TSH receptor-blocking monoclonal antibody or antigen-binding fragment thereof can effectively block signal transduction following TSH receptor binding; inhibit the synthesis and secretion of thyroid hormones; and significantly suppress TSHR expression and fibrosis in orbital fibroblasts, the effector cells of thyroid-associated eye disease. It can be used to treat a range of diseases caused by hyperthyroidism, such as thyroid eye disease, neonatal hyperthyroidism, human chorionic gonadotropin-induced hyperthyroidism, overactive thyroid, thyroid cancer, thyroiditis, and pretibial myxedema, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0240] 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.

[0241] Figure 1 The flow cytometry technology of the present invention is used to sort plasma cells and memory single B cells that specifically recognize TSHR from peripheral blood. Biotin-AF647 is used to label TSHR protein as bait. Other antibodies used for sorting are: CD19-PacificBlue, IgM-PE, CD27-BV605, CD38-PE-Cy7. Finally, TSHR-specific plasma cells and memory single B cells are sorted as CD19 + IgM - CD27 + CD38 - TSHR + .

[0242] Figure 2 This is a screening graph for the blocking activity of the anti-human TSHR monoclonal antibody of the present invention. The results shown are mean ± standard deviation (n=3). The dashed line represents 30%.

[0243] Figure 3 The results of phylogenetic tree analysis of the anti-human TSHR monoclonal antibody sequences of the present invention are shown below the branches. The percentage of repeated trees in which related taxa clustered together in the bootstrap test (1000 replicates) is shown.

[0244] Figure 4 The horizontal line represents the statistical difference (P value) between the antibody group and the control group (Control), and a value less than or equal to 0.05 is considered to be significant.

[0245] Figure 5 The graph shows the changes in T4 and TSH levels in rats at different time points after intramuscular injection of the SH23 antibody of the present invention. The results shown are mean ± standard deviation (n = 5), **p < 0.01. DETAILED DESCRIPTION

[0246] 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.

[0247] Example 1

[0248] 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. -1077 (Sigma, catalog number: 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 ).

[0249] 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 TM IV one-step RT-PCR system (Invitrogen, catalog number: 12594100) was used to synthesize cDNA. Using cDNA as a template, DreamTaq Green PCR 2X MasterMix (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 QIAquickGel 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 QIAquickGel 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.

[0250] Blocking antibody screening:

[0251] 1. Antigen Binding Ability Screening: Co-transfect 293T cells with the antibody light and heavy chain expression vectors at a 1:1 ratio and culture at 37°C, 5% CO2 for 3 days. Collect the culture supernatant by centrifugation and assay the TRAb antibody titer according to the instructions of the Human Anti-Thyrotropin Receptor Antibody ELISA Kit (Kelu, Catalog No. ELK9540). Remove light and heavy chain combinations with binding abilities lower than the blank control group, and screen the remaining combinations for blocking activity.

[0252] 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. 100 μL of the supernatant was added with 1 IU / L bTSH (Sigma) and incubated with hTSHR-CHO cells for 2 hours. The cell lysate was then collected and the changes in cellular cAMP levels were detected (R&D, Catalog No. KGE002B).

[0253] Antibody expression and purification: The antibody light and heavy chain expression vectors were co-transfected into 293F cells at a 1:1 ratio. After 5 days of incubation at 37°C, 8% CO2, and shaking at 130 rpm, 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.

[0254] In vitro TSHR inhibitory effect evaluation: The purified monoclonal antibody was diluted according to different concentration gradients, and bTSH 5ng / ml was added. After incubation with hTSHR-CHO cells for 2 hours, the cell lysate was collected and the changes in cellular cAMP levels were detected. If the percentage of inhibition of cAMP production was greater than 30%, the monoclonal antibody was considered to have inhibitory activity. It was found that the monoclonal antibody concentration of 1μg / mL could effectively antagonize the activation of TSH on TSH receptors, and showed a concentration-dependent effect ( Figure 2 SH23 inhibited cAMP production by 94% at a concentration of 1 μg / mL, and by 98% at a concentration of 2 μg / mL.

[0255] Phylogenetic tree analysis: Phylogenetic analysis of antibodies was performed using MEGA 11 analysis software. Phylogenetic trees were constructed using the neighbor-joining method, and bootstrap values ​​were used to assess the reliability of the trees. The percentage of replicate trees in which related taxa clustered together in the bootstrap test (1000 replicates) is shown below the branches. Evolutionary distances were calculated using the Poisson correction method. Figure 3The results are displayed by combining the unrooted optimal tree with the in vitro inhibitory activity assay. Antibody K1-70 is a reported inhibitory antibody, and the rest are antibodies obtained in this screening. The closer the evolutionary distance of the antibodies, the closer the inhibitory activity. The results show that K1-70 has the farthest evolutionary distance from SH23, the antibody with the strongest inhibitory activity in the present invention, indicating that the sequence difference between the two antibodies is the largest, and they are two TSHR inhibitory monoclonal antibodies with completely different sequences. The two TSHR inhibitory monoclonal antibodies screened by the present invention that are closest to K1-70 in evolutionary distance are CHL34 and CHL33. Their inhibitory activity is only 79% and 75% when the antibody concentration is 1 μg / mL, and the inhibitory activity is 88% when the concentration is 2 μg / mL. This shows that the inhibitory efficiency of K1-70 against TSHR is lower than that of SH23, the antibody with the strongest inhibitory activity screened in this screening.

[0256] Evaluation of the inhibitory effect on orbital fibroblasts (OF cells): OF cells were cultured in a 10 cm dish of DMEM containing 10% FBS, and when they grew to 70%, they were passaged to a 12-well plate and cultured for 16 hours with DMEM containing 1% FBS. After adding 50 ng / ml of the inhibitory antibody SH23, the cells were cultured for another 24 hours. The cells were collected to extract RNA, and RT-qPCR was performed to detect the expression level of fibrosis-related gene mRNA. The results showed that the TSHR inhibitory monoclonal antibody SH23 could 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 ).

[0257] In vivo effect evaluation: Male SD rats aged 6-8 weeks were selected, and each rat was intramuscularly injected with 0.1 mL of SH23 monoclonal antibody solution (200 μg SH23 monoclonal antibody, buffer used: PBS; 137 mmol / L sodium chloride, 2.7 mmol / L potassium chloride, 8.1 mmol / L sodium dihydrogen 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, 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 the determination of thyroxine T4 (Cloud Clone, Product No.: CEA452Ge) and thyrotropin TSH (Cloud Clone, Product No.: CEA463Ra). Figure 5 As shown, compared to the 0-hour period before injection, T4 concentrations in the rats' peripheral blood began to decline significantly 4 hours after injection, reaching their peak inhibition at 24 hours, demonstrating that SH23 has a strong inhibitory effect on thyroid hormone synthesis in vivo. After 24 hours, T4 levels recovered somewhat due to a compensatory increase in TSH, and TSH levels returned to normal by 72 hours.

[0258] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0259] As used in the present invention, the terms "include" and "comprising" are open expressions, that is, including the contents specified in the present invention, but not excluding other aspects.

[0260] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0261] 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 group of fully human TSH receptor blocking monoclonal antibodies or antigen-binding fragments thereof, characterized in that: The fully human TSH receptor blocking monoclonal antibody or its antigen binding fragment binds to the TSH receptor to block the binding of TSH to the TSH receptor; the fully human TSH receptor blocking monoclonal antibody or its antigen binding fragment comprises a heavy chain variable region and a light chain variable region; Wherein, 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 shown in SEQ ID NO:54, SEQ ID NO:55 and SEQ ID NO:56, respectively; The light chain variable region comprises L-CDR1, L-CDR2 and L-CDR3, the amino acid sequence of the L-CDR1 is selected from one of SEQ ID NOs: 57 to 62; the amino acid sequence of the L-CDR2 is selected from one of SEQ ID NOs: 63 to 67; the amino acid sequence of the L-CDR3 is selected from one of SEQ ID NOs: 68 to 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, and 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 fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof is a TSH antagonist; and / or is an antagonist of thyroid stimulating antibodies.

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, comprising the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof as claimed in claim 1 or 2; (2) A reagent or a kit, comprising the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2; (3) A nucleotide encoding the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2, wherein the nucleotide sequence comprises: (a) the nucleotide sequence encoding the heavy chain variable region is shown in one of SEQ ID NOs: 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51; the nucleotide sequence encoding the light chain variable region is 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 NO: 27 to SEQ ID NO: 52; (c) a nucleotide sequence after one or more nucleotides are added, substituted, deleted or inserted into the nucleotide sequence shown in SEQ ID NO: 27 to SEQ ID NO: 52; (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: 54 to SEQ ID NO: 73; (4) A vector comprising the nucleotide described in (3); (5) A host cell, comprising the nucleotide described in (3) and / or the vector described in (4); (6) A cell, wherein the cell is an isolated cell comprising the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof 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 or antigen-binding fragment thereof as claimed in claim 1 or 2; and / or, An isolated cell secreting the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2; (7) A composition comprising a determined concentration of TSH receptor autoantibodies and a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof 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 or antigen-binding fragment thereof 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 or antigen-binding fragment thereof 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 or an antigen-binding fragment thereof, the preparation method specifically comprising 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; In the third step, after the heavy chain and light chain recombinant plasmids are successfully obtained, the obtained candidate clones are sequenced and compared and analyzed 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, and finally an antibody combination that can specifically target the target antigen, namely the fully human TSH receptor blocking monoclonal antibody or its antigen binding fragment, is obtained; (2) A method for treating a thyroid-related disorder in a mammalian subject or in a cell derived from the subject, the method comprising contacting the subject or the cell with the fully human TSH receptor blocking monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2; (3) A method for inhibiting thyroid stimulating antibodies from stimulating TSH receptors in the thyroid gland of a mammalian subject, the method comprising contacting the subject with the fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2; (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 or an antigen-binding fragment thereof 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 contacting the cancer cells with a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof according to claim 1 or 2, with the purpose of inhibiting constitutive thyroid stimulating hormone 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 the subject, characterized in that the method comprises contacting the subject or the cell with a fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof 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 a thyroid stimulating antibody to a TSH receptor, the method comprising providing at least one fully human TSH receptor blocking monoclonal antibody or an antigen-binding fragment thereof 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 or an antigen-binding fragment thereof 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 fully human TSH receptor blocking monoclonal antibody or the antigen binding fragment thereof blocks 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 or antigen-binding fragment thereof according to claim 1 or 2 for treating a thyroid-related disease; (2) 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 medicament for treating a thyroid-related disease; (3) 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 for detecting TSH receptor antibodies; (4) 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, kit or product for detecting hyperthyroidism or thyroid-related eye diseases; (5) Use of the fully human TSH receptor blocking monoclonal antibody or its antigen-binding fragment 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, a drug or a pharmaceutical composition for detecting TSH receptor antibodies, the preparation of a drug for treating hyperthyroidism and thyroid-related eye diseases, the preparation of a drug for inhibiting thyroid hyperplasia and / or thyroid hormone production, the preparation of a TSH receptor blocking drug, and the preparation of a drug 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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