WO2024046384A1 - 结合促甲状腺激素受体的抗体及其用途 - Google Patents

结合促甲状腺激素受体的抗体及其用途 Download PDF

Info

Publication number
WO2024046384A1
WO2024046384A1 PCT/CN2023/115886 CN2023115886W WO2024046384A1 WO 2024046384 A1 WO2024046384 A1 WO 2024046384A1 CN 2023115886 W CN2023115886 W CN 2023115886W WO 2024046384 A1 WO2024046384 A1 WO 2024046384A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
sequence shown
antibody
antigen
heavy chain
Prior art date
Application number
PCT/CN2023/115886
Other languages
English (en)
French (fr)
Inventor
马彦彬
卫培培
陈达锴
孙杲
康立山
骆天红
王思勤
金磊
Original Assignee
长春金赛药业有限责任公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 长春金赛药业有限责任公司 filed Critical 长春金赛药业有限责任公司
Publication of WO2024046384A1 publication Critical patent/WO2024046384A1/zh

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P5/00—Drugs for disorders of the endocrine system
    • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
    • A61P5/08—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH for decreasing, blocking or antagonising the activity of the anterior pituitary hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/166—Animal cells resulting from interspecies fusion
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9

Definitions

  • Graves' disease is a difficult-to-treat autoimmune disease. Its symptoms include enlargement and overactivity of the thyroid gland, accelerated heart rate, and eye abnormalities. The disease with abnormally protruding eyes is also called Graves' disease. Graves' ophthalmopathy (GO) or hyperthyroidism. Based on what is known about the disease, it is thought to be the result of a complex interaction of genetic and environmental factors. GO disease not only has a serious impact on the quality of life, but also affects the mind and work, and the related overactivation of thyroid hormone receptors stimulates the secretion of thyroid hormone, which will lead to an increased risk of death.
  • GO Graves' ophthalmopathy
  • the current clinical treatments for GO disease mainly include antithyroid drug (ATD) treatment, radioactive iodine (RAI) treatment and surgical treatment.
  • ATD antithyroid drug
  • RAI radioactive iodine
  • surgical treatment mainly includes side effects such as bone marrow suppression and liver toxicity.
  • Treatment with surgical resection or radioactive iodine ablation also has obvious risks, and can lead to recurrent nerve damage and parathyroid gland damage after surgery. Decreased function.
  • the above therapies are all symptomatic treatments and do not target the pathogenesis of the disease.
  • GO disease currently lacks effective clinical treatments.
  • Thyroid-stimulating hormone receptor (TSHR) is mainly expressed on the basolateral surface of thyroid follicular cells, inducing thyroid cell growth, hormone synthesis and hormone secretion. It is also the main autoantigen in autoimmune thyroid diseases, especially GD disease. As the disease progresses, Over time, approximately 25% of GD patients will develop GO disease.
  • TSHR autoantibodies There are two main types of TSHR autoantibodies (TRAb): one is the stimulatory type and the other is the blocking type. Thyroid-stimulating autoantibodies bind to TSHR and mimic the effects of TSH, thereby stimulating the thyroid to produce high levels of T4 and T3. These autoantibodies have also been described as TRAbs with agonist activity (Rees Smith et al. 2007. Thyroid 17:923-938).
  • TRAb with stimulatory activity may also interact with TSHR in retroorbital tissue to promote the occurrence of Graves' disease ophthalmopathy (GO).
  • GO Graves' disease ophthalmopathy
  • Elevated exophthalmos is an important cause of GO disease.
  • the expression of TSHR is high in the thyroid and testis but low in other tissues, so it may have advantages in terms of drug targeting and side effects. Therefore, TSHR is expected to become a potential target for the treatment of GO diseases.
  • IGF1R insulin-like growth factor 1 receptor
  • Teprotumumab can competitively inhibit IGF1R receptor activates downstream signaling pathways, especially in improving eye proptosis and reducing inflammation. Its therapeutic efficiency reaches 78% in the treatment of moderate to severe thyroid eye disease (TED). Increased expression of TSHR and IGF1R was found in orbital fibroblasts of patients with GO disease, and there was a certain interaction between TSHR and IGF1R. Therefore, combined therapy that directly blocks TSHR (TSAb/antagonist) or simultaneously antagonizes TSHR and IGF1R receptors may be a way to cure GD or GO diseases.
  • TSAb/antagonist TSAb/antagonist
  • the present invention relates to antibodies that specifically bind to TSHR or antigen-binding fragments thereof, pharmaceutical compositions containing the antibodies or antigen-binding fragments thereof, nucleic acid molecules encoding the antibodies or antigen-binding fragments thereof, host cells containing the same, and related use. Furthermore, the invention relates to the therapeutic and diagnostic uses of these antibodies or antigen-binding fragments thereof.
  • the inventors of the present application have obtained murine antibodies that specifically bind to TSHR, which have significant inhibitory effects on TSHR activity and have cross-binding activity against human/mouse/monkey TSHR.
  • the inventor of the present application further obtained a humanized antibody with TSHR binding activity and TSHR activity inhibitory effect based on the mouse-derived antibody.
  • the present application also provides pharmaceutical compositions containing the antibody or antigen-binding fragment thereof, nucleic acid molecules encoding the antibody or antigen-binding fragment thereof, host cells containing the same, and related uses.
  • the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or an antigen-binding fragment thereof comprising:
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • the 3 CDRs contained in the heavy chain variable region (VH) as shown in any one of SEQ ID NO: 1, 27, 29, and 30; and/or, as any one of SEQ ID NO: 5, 28, and 31 Three CDRs contained in the light chain variable region (VL) are shown.
  • the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system. In certain embodiments, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region containing the following three complementarity determining regions (CDRs): VH CDR1 with sequence SEQ ID NO:2, VH CDR2 with sequence SEQ ID NO:3 or 39, VH CDR2 with sequence SEQ ID NO :4 VH CDR3; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:6, VL CDR1 with a sequence of SEQ ID NO:7 VL CDR2, VL CDR3 with sequence SEQ ID NO:8;
  • the CDR is defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a murine immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and/or a light chain framework region of a murine light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region which includes the sequence shown in SEQ ID NO: 1 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 5 or its variations;
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 1 and a VL comprising the sequence set forth in SEQ ID NO: 5.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and/or a light chain framework region of a human light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in any one of SEQ ID NO: 27, 29, 30, or a variant thereof; and /or, a light chain variable region (VL) comprising the sequence shown in SEQ ID NO: 28 or 31 or a variant thereof;
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as set forth in any one of SEQ ID NO: 27, 29, 30; and/or a heavy chain variable region (VH) as set forth in any one of SEQ ID NO: 27, 29, 30; The light chain variable region (VL) shown in ID NO:28 or 31.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:27 and a VL comprising the sequence set forth in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:29 and a VL comprising the sequence set forth in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:30 and a VL comprising the sequence set forth in SEQ ID NO:28.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:27 and a VL comprising the sequence set forth in SEQ ID NO:31.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:29 and a VL comprising the sequence set forth in SEQ ID NO:31.
  • the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or an antigen-binding fragment thereof comprising:
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region 3 CDRs contained in the area (VL).
  • the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system. In certain embodiments, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region contains the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:10, VH CDR2 with the sequence SEQ ID NO:11 or 40, and VH CDR2 with the sequence SEQ ID NO: : VH CDR3 of 12 or 41; and/or, a light chain variable region (VL) containing the following 3 complementarity determining regions (CDRs): VL CDR1 with the sequence SEQ ID NO: 14, SEQ ID NO: VL CDR2 of 15, VL CDR3 with sequence SEQ ID NO:16;
  • the CDR is defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • the heavy chain variable region contains the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:10, VH CDR2 with the sequence SEQ ID NO:11 or 40, and VH CDR2 with the sequence SEQ ID NO: : VH CDR3 of SEQ ID NO: 12; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO: 14, VL CDR1 with a sequence of SEQ ID NO: 15 VL CDR2, VL CDR3 with sequence SEQ ID NO:16;
  • the CDR is defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region containing the following three complementarity determining regions (CDRs): VH CDR1 with the sequence SEQ ID NO:10, VH CDR2 with the sequence SEQ ID NO:11 or 40, and VH CDR2 with the sequence SEQ ID NO :41 VH CDR3; and/or, a light chain variable region (VL) containing the following three complementarity determining regions (CDRs): VL CDR1 with a sequence of SEQ ID NO:14, VL CDR1 with a sequence of SEQ ID NO:15 VL CDR2, VL CDR3 whose sequence is SEQ ID NO:16;
  • the CDR is defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a murine immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and/or a light chain framework region of a murine light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region which includes the sequence shown in SEQ ID NO: 9 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 13 or its variations;
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 9 and a VL comprising the sequence set forth in SEQ ID NO: 13.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and/or a light chain framework region of a human light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 32 or 34, or a variant thereof; and/or comprising as The light chain variable region (VL) of the sequence shown in SEQ ID NO:33 or a variant thereof;
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) as set forth in SEQ ID NO: 32 or 34; and/or a heavy chain variable region (VH) as set forth in SEQ ID NO: 33
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:32 and a VL comprising the sequence set forth in SEQ ID NO:33.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:34 and a VL comprising the sequence set forth in SEQ ID NO:33.
  • the application provides an antibody or an antigen-binding fragment thereof capable of specifically binding to TSHR, the antibody or an antigen-binding fragment thereof comprising:
  • the variant has one or several amino acid substitutions, deletions or additions (eg 1, 2 or 3 amino acid substitutions, deletions or additions, eg conservative substitutions) compared to the sequence from which it is derived.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH heavy chain variable region
  • VL light chain variable region
  • the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat, IMGT or Chothia numbering system. In certain embodiments, the three CDRs contained in the VH and/or the three CDRs contained in the VL are defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises:
  • VH The heavy chain variable region
  • CDRs complementarity determining regions
  • VH CDR1 with the sequence SEQ ID NO:18
  • VH CDR2 with the sequence SEQ ID NO:19
  • SEQ ID NO:20 The VH CDR3 of , VL CDR3 whose sequence is SEQ ID NO:24;
  • the CDR is defined by the Kabat numbering system.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a murine immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a murine heavy chain germline sequence, and/or a light chain framework region of a murine light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises:
  • Heavy chain variable region which includes the sequence shown in SEQ ID NO: 17 or a variant thereof; and/or, light chain variable region (VL), which includes the sequence shown in SEQ ID NO: 21 or its variations;
  • the variant has one or several amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least Sequences with 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • amino acid substitutions, deletions or additions compared to the sequence from which it is derived (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least Sequences with 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO: 17 and a VL comprising the sequence set forth in SEQ ID NO: 21.
  • the antibody or antigen-binding fragment thereof comprises framework sequence derived from a human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a framework region contained in the amino acid sequence encoded by a human germline antibody gene. In certain embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain framework region of a human heavy chain germline sequence, and/or a light chain framework region of a human light chain germline sequence.
  • the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) comprising the sequence set forth in SEQ ID NO: 35 or 37 or a variant thereof; and/or comprising as The light chain variable region (VL) of the sequence shown in SEQ ID NO: 36 or 38 or a variant thereof;
  • VH heavy chain variable region
  • VL light chain variable region
  • the variant has one or several amino acid substitutions, deletions or additions (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the sequence from which it is derived. , or have at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% , or a sequence with 100% sequence identity.
  • the substitutions are conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises: comprising a heavy chain variable region (VH) as set forth in SEQ ID NO: 35 or 37; and/or comprising as set forth in SEQ ID NO: 36 or The light chain variable region (VL) shown in 38.
  • VH heavy chain variable region
  • VL light chain variable region
  • the antibody or antigen-binding fragment thereof comprises: a VH comprising the sequence set forth in SEQ ID NO:35 and a VL comprising the sequence set forth in SEQ ID NO:36.
  • the antibody or antigen-binding fragment thereof comprises a VH comprising the sequence set forth in SEQ ID NO:37 and a VL comprising the sequence set forth in SEQ ID NO:38.
  • the antibody or antigen-binding fragment thereof further comprises a constant region derived from a mammalian (eg, human or murine) immunoglobulin.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a human immunoglobulin (e.g., IgG1, IgG2, IgG3, or IgG4), and/or the antibody or antigen-binding fragment thereof
  • the light chain of the antigen-binding fragment comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) of a human immunoglobulin or a variant thereof that has a or multiple amino acid substitutions or deletions Loss or addition (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, substitution, deletion of 1, 2, 3, 4, or 5 amino acids or add); and/or,
  • CH heavy chain constant region
  • the light chain of the antibody or antigen-binding fragment thereof comprises the light chain constant region (CL) of a human immunoglobulin or a variant thereof having one or more amino acid substitutions compared to the sequence from which it is derived, Deletion or addition (e.g., substitution, deletion, or addition of up to 20, up to 15, up to 10, or up to 5 amino acids; for example, substitution, deletion of 1, 2, 3, 4, or 5 amino acids or add).
  • the substitutions may be conservative substitutions or non-conservative substitutions.
  • the antibody or antigen-binding fragment thereof comprises the heavy chain constant region (CH) of a wild-type human immunoglobulin.
  • the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin that is identical to the wild-type variant from which it is derived.
  • Type sequences may have the same or substantially the same characteristics.
  • the variant of the heavy chain constant region (CH) may have one or more conservative amino acid substitutions compared to the sequence from which it is derived.
  • the antibody or antigen-binding fragment thereof comprises a variant of the heavy chain constant region (CH) of a human immunoglobulin, which variant of the heavy chain constant region (CH) may comprise one or more Amino acid mutations or chemical modifications to alter one or more of the following properties of the antibodies of the invention: Fc receptor binding, antibody glycosylation, number of cysteine residues, effector cell function or complement function, etc.
  • Effector function can be altered by replacing at least one amino acid residue in the constant region of the antibody with a different residue or by chemical modification to produce a functional change, e.g., changing the affinity of the antibody for an effector ligand such as FcR or complement C1q. (e.g. lower or boost).
  • the Fc region of antibodies mediates several important effector functions, such as ADCC, phagocytosis, CDC, etc.
  • the antibodies of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH) that may have reduced or Eliminated effector functions.
  • CH human immunoglobulin heavy chain constant region
  • the antibodies of the invention comprise variants of the human immunoglobulin heavy chain constant region (CH) that may have enhanced potency compared to the wild-type sequence from which they are derived. Effector functions.
  • CH human immunoglobulin heavy chain constant region
  • the antibodies of the invention, or antigen-binding fragments thereof comprise a human immunoglobulin heavy chain constant region (CH) or a variant thereof, which variant may have the same sequence as the wild-type sequence from which it is derived. or essentially the same effector function.
  • CH human immunoglobulin heavy chain constant region
  • the antibodies of the invention, or antigen-binding fragments thereof comprise a variant of the human immunoglobulin heavy chain constant region (CH) that, compared to the wild-type sequence from which it is derived, has: (i ) extended half-life, (ii) enhanced efficacy, and/or, (iii) reduced ADCC activity.
  • CH human immunoglobulin heavy chain constant region
  • the heavy chain constant region (CH) is an IgG heavy chain constant region, such as an IgGl, IgG2, IgG3 or IgG4 heavy chain constant region.
  • the light chain constant region is a kappa light chain constant region or a lambda light chain constant region.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from a murine immunoglobulin (e.g., IgGl, IgG2, IgG3, or IgG4), the antibody or antigen-binding fragment thereof
  • the light chain includes a light chain constant region derived from a murine immunoglobulin (eg, kappa or lambda).
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG4, and/or,
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the heavy chain constant region derived from human immunoglobulin IgG4 comprises substitution mutations S228P, L235E, M252Y, S254T and/or compared to the heavy chain constant region of wild-type human immunoglobulin IgG4.
  • T256E e.g., (i) S228P, (ii) L235E, and/or (iii) M252Y, S254T, and T256E).
  • the heavy chain constant region derived from human immunoglobulin IgG4 comprises the substitution mutation S228P compared to the heavy chain constant region of wild-type human immunoglobulin IgG4; in certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 The heavy chain constant region derived from human immunoglobulin IgG4 further comprises substitution mutations M252Y, S254T and T256E; in certain embodiments, the heavy chain constant region derived from human immunoglobulin IgG4 further comprises substitution mutations L235E, M252Y, S254T and T256E.
  • amino acid positions of the substitution mutations are defined by the EU numbering system.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as shown in any one of SEQ ID NO: 25, 42-43, and/or, the antibody Or the light chain of the antigen-binding fragment thereof includes the light chain constant region (CL) as shown in SEQ ID NO:26.
  • CH heavy chain constant region
  • CL light chain constant region
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain containing the VH of the sequence shown in SEQ ID NO:27 and the CH of the sequence shown in any one of SEQ ID NO:25 and 42-43, and/or, containing the VH of the sequence shown in SEQ ID NO:28 The VL of the sequence shown and the light chain of CL of the sequence shown in SEQ ID NO:26;
  • a heavy chain comprising VH of the sequence shown in SEQ ID NO: 27 and CH of the sequence shown in any one of SEQ ID NO: 25 and 42-43, and/or, including SEQ ID NO: 31
  • the VL of the sequence shown and SEQ ID NO:26 are The light chain of CL of the sequence shown;
  • a heavy chain containing the VH of the sequence shown in SEQ ID NO:29 and the CH of the sequence shown in any one of SEQ ID NO:25 and 42-43, and/or, containing the VH of the sequence shown in SEQ ID NO:31 The VL of the sequence shown and the light chain of CL of the sequence shown in SEQ ID NO:26;
  • a heavy chain containing the VH of the sequence shown in SEQ ID NO:34 and the CH of the sequence shown in any one of SEQ ID NO:25 and 42-43, and/or, containing the VH of the sequence shown in SEQ ID NO:33 The VL of the sequence shown and the light chain of CL of the sequence shown in SEQ ID NO:26;
  • the antibody of the first aspect, or antigen-binding fragment thereof comprises:
  • a heavy chain comprising the sequence shown in SEQ ID NO:46, and/or a light chain comprising the sequence shown in SEQ ID NO:51;
  • a heavy chain comprising the sequence shown in SEQ ID NO:48, and/or a light chain comprising the sequence shown in SEQ ID NO:51.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region derived from human immunoglobulin IgG1, and/or,
  • the light chain of the antibody or antigen-binding fragment thereof comprises a light chain constant region derived from a human immunoglobulin (eg, kappa or lambda).
  • the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A, L235A, M252Y, S254T and/or compared to the heavy chain constant region of wild-type human immunoglobulin IgG1 T256E (e.g., (i) L234A and L235A, and/or (ii) M252Y, S254T, and T256E).
  • the heavy chain constant region derived from human immunoglobulin IgG1 comprises substitution mutations L234A and L235A compared to the heavy chain constant region of wild-type human immunoglobulin IgG1; in certain embodiments , described
  • the heavy chain constant region derived from human immunoglobulin IgG1 further contains substitution mutations M252Y, S254T and T256E.
  • amino acid positions of the substitution mutations are defined by the EU numbering system.
  • the heavy chain of the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 44 or 45, and/or, the antibody or antigen-binding fragment thereof
  • the light chain includes a light chain constant region (CL) as shown in SEQ ID NO:26.
  • the antibody or antigen-binding fragment thereof comprises:
  • a heavy chain comprising VH of the sequence shown in SEQ ID NO:37 and CH of the sequence shown in SEQ ID NO:44 or 45, and/or, containing the sequence shown in SEQ ID NO:38 VL and the sequence shown in SEQ ID NO:26 Column light chain of CL.
  • the antibody of the first aspect, or antigen-binding fragment thereof comprises:
  • a heavy chain comprising the sequence shown in SEQ ID NO:49, and/or, a light chain comprising the sequence shown in SEQ ID NO:51; or,
  • a heavy chain comprising the sequence shown in SEQ ID NO:50, and/or a light chain comprising the sequence shown in SEQ ID NO:51.
  • the antigen-binding fragment is selected from the group consisting of Fab, Fab', (Fab') 2 , Fd, Fv, disulfide-linked Fv, scFv, di-scFv, (scFv) 2 , diabody and single domain antibody (sdAb); and/or, the antibody is a murine antibody, a humanized antibody, a chimeric antibody, a bispecific antibody or Multispecific antibodies.
  • the application also provides an isolated nucleic acid molecule encoding an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, or a heavy chain variable region and/or light chain thereof. variable region, or its heavy and/or light chain.
  • the isolated nucleic acid molecule comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a first nucleotide sequence encoding said antibody or antigen-binding fragment thereof A second nucleotide sequence of a light chain or light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different separate nucleic acid molecules.
  • the isolated nucleic acid molecule of the present invention includes a third nucleotide sequence containing the first nucleotide sequence. a nucleic acid molecule and a second nucleic acid molecule containing said second nucleotide sequence.
  • the present application also provides a vector comprising the isolated nucleic acid molecule as described above.
  • the vector is a cloning vector or an expression vector.
  • the vector comprises a first nucleotide sequence encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof of the invention and a light chain encoding said antibody or antigen-binding fragment thereof. or a second nucleotide sequence of the light chain variable region, wherein said first nucleotide sequence and said second nucleotide sequence are present on the same or different vectors.
  • the vector of the present invention includes a first vector containing the first nucleotide sequence and a vector containing the A second vector for a second nucleotide sequence.
  • the present application also provides a host cell comprising an isolated nucleic acid molecule or vector as described above.
  • host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., E. coli cells), and eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, For example, mouse cells, human cells, etc.).
  • prokaryotic cells such as bacterial cells (e.g., E. coli cells)
  • eukaryotic cells such as fungal cells (e.g., yeast cells), insect cells, plant cells, and animal cells (e.g., mammalian cells, For example, mouse cells, human cells, etc.).
  • the host cell is a microorganism.
  • the antibodies of the present invention can be prepared by various methods known in the art, such as by genetic engineering and recombinant technology.
  • DNA molecules encoding the heavy chain and light chain genes of the antibody of the present invention are obtained by chemical synthesis or PCR amplification.
  • the resulting DNA molecule is inserted into an expression vector and then transfected into host cells. Then, the transfected host cells are cultured under specific conditions and express the antibody of the invention.
  • the antigen-binding fragments of the present invention can be obtained by hydrolyzing intact antibody molecules (see Morimoto et al., J. Biochem. Biophys. Methods 24:107-117 (1992) and Brennan et al., Science 229:81 (1985)) .
  • these antigen-binding fragments can also be produced directly from recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11:548-557 (1999); Little et al., Immunol. Today, 21:364-370 (2000) )).
  • Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F(ab′) 2 fragments (Carter et al., Bio/Technology, 10:163-167 (1992)).
  • Fv, Fab or F(ab') 2 fragments can also be directly isolated from the recombinant host cell culture medium. Those of ordinary skill in the art are well aware of other techniques for preparing such antigen-binding fragments.
  • the present application also provides a method for preparing an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, which includes, under conditions that allow the expression of the antibody or antigen-binding fragment thereof.
  • the host cells as described above are cultured, and the antibody or antigen-binding fragment thereof is recovered from the cultured host cell culture.
  • the present application also provides bispecific or multispecific molecules, which comprise the antibodies or antigen-binding fragments thereof as described in the first aspect, the second aspect or the third aspect.
  • the bispecific or multispecific molecule specifically binds TSHR and additionally specifically binds one or more other targets.
  • the bispecific or multispecific molecule further comprises at least one molecule (eg, a second antibody) having a second binding specificity for a second target.
  • the bispecific or multispecific molecule further comprises at least one second antibody that specifically binds IGF1R.
  • the second antibody has an antagonistic effect on IGF1R signaling.
  • the present application also provides an immunoconjugate comprising an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, and a treatment connected to the antibody or antigen-binding fragment thereof. agent.
  • the therapeutic agent is selected from IGF1R antagonists or inhibitors.
  • the immunoconjugate is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the present application also provides a pharmaceutical composition, which includes the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, the isolated nucleic acid molecule as described in the fourth aspect, such as The vector according to the fifth aspect, the host cell according to the sixth aspect, the bispecific or multispecific molecule according to the eighth aspect or the immunoconjugate according to the ninth aspect, and pharmaceutically acceptable Acceptable carriers and/or excipients.
  • the pharmaceutical composition further includes an additional pharmaceutically active agent, such as an additional drug for treating autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer.
  • an additional pharmaceutically active agent such as an additional drug for treating autoimmune thyroid disease (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer.
  • the pharmaceutical composition further comprises an IGF1R antagonist or inhibitor, and/or, an additional TSHR antagonist or inhibitor.
  • the pharmaceutically acceptable carrier and/or excipient comprises a sterile injectable liquid (such as an aqueous or non-aqueous suspension or solution).
  • a sterile injectable liquid such as an aqueous or non-aqueous suspension or solution.
  • sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% Glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.
  • the application also provides the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the fifth aspect
  • the vector, the host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect, the immunoconjugate as described in the ninth aspect or the pharmaceutical combination as described in the tenth aspect Use of the substance for preparing a medicament for preventing and/or treating TSHR-related diseases in a subject.
  • the TSHR-related diseases will benefit from antagonism of TSHR signaling.
  • the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.
  • an autoimmune thyroid disease eg, Graves' disease, Graves' ophthalmopathy
  • the subject is a mammal, such as a human, mouse, or monkey.
  • the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is used alone or with additional Pharmaceutically active agents (eg, additional drugs used to treat autoimmune thyroid disease (eg, Graves' disease or Graves' ophthalmopathy) or thyroid cancer) are administered in combination, eg, simultaneously or sequentially.
  • additional Pharmaceutically active agents eg, additional drugs used to treat autoimmune thyroid disease (eg, Graves' disease or Graves' ophthalmopathy) or thyroid cancer
  • the antibody or antigen-binding fragment thereof, isolated nucleic acid molecule, vector, host cell, bispecific or multispecific molecule, immunoconjugate or pharmaceutical composition is combined with an IGF1R antagonist or inhibitor and/or additionally
  • the TSHR antagonists or inhibitors are used in combination, for example, administered simultaneously or sequentially.
  • the present application also provides a method for preventing and/or treating a TSHR-related disease in a subject, comprising: administering to a subject in need an effective amount of, e.g. The antibody or antigen-binding fragment thereof according to the first, second or third aspect, the isolated nucleic acid molecule according to the fourth aspect, the vector according to the fifth aspect, the host according to the sixth aspect Cells, bispecific or multispecific molecules as described in the eighth aspect, immunoconjugates as described in the ninth aspect, or pharmaceutical compositions as described in the tenth aspect.
  • the TSHR-related diseases will benefit from antagonism of TSHR signaling.
  • the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.
  • an autoimmune thyroid disease eg, Graves' disease, Graves' ophthalmopathy
  • the subject is a mammal, such as a human, mouse, or monkey.
  • the host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect, the immunoconjugate as described in the ninth aspect or the pharmaceutical composition as described in the tenth aspect can be combined with Additional pharmaceutically active agents, such as additional drugs for the treatment of autoimmune thyroid diseases (such as Graves' disease or Graves' ophthalmopathy) or thyroid cancer, are administered in combination, for example simultaneously or sequentially.
  • autoimmune thyroid diseases such as Graves' disease or Graves' ophthalmopathy
  • the antibody or antigen-binding fragment thereof as described in the first, second or third aspect, the isolated nucleic acid molecule as described in the fourth aspect, the vector as described in the fifth aspect, The host cell as described in the sixth aspect, the bispecific or multispecific molecule as described in the eighth aspect, the immunoconjugate as described in the ninth aspect or the pharmaceutical composition as described in the tenth aspect can be combined with The IGF1R antagonist or inhibitor and/or an additional TSHR antagonist or inhibitor is administered in combination, for example simultaneously or sequentially.
  • the antibody or antigen-binding fragment thereof or pharmaceutical composition of the present application can be formulated into any dosage form known in the medical field, for example, tablets, pills, suspensions, emulsions, solutions, gels, capsules, powders, granules , elixirs, tablets, suppositories, injections (including injections, sterile powders for injection and concentrated solutions for injection), inhalants, sprays, etc.
  • the preferred dosage form depends on the intended mode of administration and therapeutic use.
  • the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention should be sterile and stable under the conditions of production and storage.
  • One preferred dosage form is an injection. Such injections may be sterile injectable solutions.
  • sterile injectable solutions may be prepared by incorporating in an appropriate solvent the requisite dose of an antibody of the invention, or antigen-binding fragment thereof, and, optionally, other desired ingredients including, but not limited to, pH adjuster, surfactant, adjuvant, ionic strength enhancer, isotonic agent, preservative, diluent, or any combination thereof) followed by filter sterilization.
  • sterile injectable solutions may be prepared as sterile Lyophilize the powder (eg, by vacuum drying or freeze drying) for ease of storage and use.
  • Such sterile lyophilized powder can be dispersed in a suitable carrier before use, such as water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (for example, 0.9% NaCl), glucose solution (for example, 5 % glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution for example, 0.9% NaCl
  • glucose solution for example, 5 % glucose
  • surfactant-containing solutions e.g., 0.01% polysorbate 20
  • pH buffer solutions e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any combination thereof.
  • the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the present application may be administered by any suitable method known in the art, including, but not limited to, oral, buccal, sublingual, eyeball, topical, parenteral, rectal, intrathecal , intracytoplasmic reticulum, groin, bladder, topically (eg, powder, ointment, or drops), or nasal route.
  • the preferred route/mode of administration is parenteral (eg intravenous or bolus injection, subcutaneous injection, intraperitoneal injection, intramuscular injection).
  • the antibodies or antigen-binding fragments thereof or pharmaceutical compositions of the invention are administered by intravenous injection or bolus injection.
  • the present application also provides a conjugate, which comprises the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, and the antibody or antigen-binding fragment thereof connected to the Detectable markers.
  • the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • enzymes e.g., horseradish peroxidase or alkaline phosphatase
  • chemiluminescent reagents e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the present application also provides a kit, which includes the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, or the conjugate as described in the thirteenth aspect.
  • the kit includes a detection buffer.
  • the kit includes the conjugate of the thirteenth aspect.
  • the kit comprises an antibody or an antigen-binding fragment thereof as described in the first, second or third aspect, and a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof.
  • the second antibody also includes a detectable label, such as an enzyme (such as horseradish peroxidase or alkaline phosphatase), a chemiluminescent reagent (such as acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • an enzyme such as horseradish peroxidase or alkaline phosphatase
  • a chemiluminescent reagent such as acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the present application also provides a method for detecting the presence of TSHR in a sample or its level, which includes using the antibody or antigen-binding fragment thereof as described in the first aspect, the second aspect or the third aspect, or As mentioned in the thirteenth aspect of conjugates.
  • the methods are used for therapeutic purposes, diagnostic purposes, or non-therapeutic non-diagnostic purposes.
  • the method is an immunological assay, such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • immunological assay such as a western blot, enzyme immunoassay (eg, ELISA), chemiluminescent immunoassay, fluorescent immunoassay, or radioimmunoassay.
  • the method includes using the conjugate of the thirteenth aspect.
  • the method includes using an antibody or an antigen-binding fragment thereof as described in the first aspect, the second aspect, or the third aspect, and the method further includes using an antibody carrying a detectable label (e.g., an enzyme (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclide or biotin) to detect the antibody or antigen-binding fragment thereof.
  • a detectable label e.g., an enzyme (such as horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (such as acridinium ester compounds, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent protein), radionuclide or biotin) to detect the antibody or
  • the method includes: (1) contacting the sample with an antibody of the invention, or an antigen-binding fragment or conjugate thereof; (2) detecting the formation of an antigen-antibody immune complex or detecting the resulting The amount of immune complexes.
  • the formation of the immune complex indicates the presence of TSHR or cells expressing TSHR.
  • the present application also provides a method of diagnosing a disease associated with TSHR, which includes detecting the presence of TSHR or the level thereof in a sample from a subject using the method described in the fifteenth aspect.
  • a reference level eg, compared to healthy controls
  • the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.
  • an autoimmune thyroid disease eg, Graves' disease, Graves' ophthalmopathy
  • the application also provides the use of the antibody or antigen-binding fragment thereof as described in the first, second or third aspect or the conjugate as described in the thirteenth aspect in preparing a detection reagent,
  • the detection reagent is used to detect the presence of TSHR in a sample or its level and/or to diagnose TSHR-related diseases.
  • the detection reagent detects the presence or level of TSHR in the sample by the method described in the fifteenth aspect.
  • the detection reagent detects the presence of TSHR in a sample or its level through the method described in the fifteenth aspect to diagnose TSHR-related diseases. In certain embodiments, when TSHR is present or the level of TSHR is increased compared to a reference level (eg, compared to healthy controls), the subject has a disease associated with TSHR.
  • a reference level eg, compared to healthy controls
  • the TSHR-associated disease is an autoimmune thyroid disease (eg, Graves' disease, Graves' ophthalmopathy), thyroid cancer, or a combination thereof.
  • an autoimmune thyroid disease eg, Graves' disease, Graves' ophthalmopathy
  • the sample is from a group of subjects (e.g., mammals, preferably humans, mice, or monkeys) Textile samples.
  • antibody refers to an immunoglobulin-derived molecule capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region.
  • the target antigen When referring to the term “antibody”, it includes not only intact antibodies but also antigen-binding fragments capable of specifically binding a target antigen, unless the context clearly indicates otherwise.
  • An “intact antibody” typically consists of two pairs of polypeptide chains, each pair having a light chain (LC) and a heavy chain (HC). Antibody light chains can be classified into kappa (kappa) and lambda (lambda) light chains.
  • Heavy chains can be classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, and the heavy chain also contains a "D" region of approximately 3 or more amino acids.
  • Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH).
  • the heavy chain constant region consists of 3 domains (CH1, CH2 and CH3).
  • Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL).
  • the light chain constant region consists of one domain, CL.
  • the constant domain is not directly involved in the binding of antibodies to antigens, but exhibits a variety of effector functions, such as mediating the interaction of immunoglobulins with host tissues or factors, including various cells of the immune system (e.g., effector cells) and classical complement. Binding of the first component of the system (C1q).
  • the VH and VL regions can also be subdivided into regions of high variability called complementarity determining regions (CDRs), interspersed with more conservative regions called framework regions (FRs).
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions (VH and VL) of each heavy chain/light chain pair respectively form the antigen-binding site.
  • the assignment of amino acids to each region or domain can follow Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196:901- 917; Definition of Chothia et al. (1989) Nature 342:878-883.
  • CDR complementarity determining region
  • the variable regions of the heavy chain and light chain each contain three CDRs, named CDR1, CDR2 and CDR3.
  • CDR1, CDR2 and CDR3 The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, such as the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al.
  • the CDRs contained in the antibody or antigen-binding fragment thereof of the present invention can be determined according to various numbering systems known in the art.
  • the CDRs contained in the antibodies of the invention, or antigen-binding fragments thereof are preferably determined by the Kabat, Chothia, or IMGT numbering systems.
  • framework region or "FR” residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.
  • antibody is not limited to any particular method of producing the antibody. This includes, for example, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies.
  • the antibodies may be of different isotypes, for example, IgG (eg, IgGl, IgG2, IgG3 or IgG4 subtypes), IgA1, IgA2, IgD, IgE or IgM antibodies.
  • the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody Specific binding to an antigen, which is also called an "antigen-binding moiety.”
  • an antigen-binding moiety which is also called an "antigen-binding moiety.
  • Non-limiting examples of antigen-binding fragments include Fab, Fab', F(ab') 2 , Fd, Fv, disulfide-linked Fv, scFv , di-scFv, diabodies, single domain antibodies, and polypeptides containing at least a portion of the antibody sufficient to confer specific antigen-binding ability to the polypeptide.
  • Engineered antibody variants are reviewed in Holliger et al. , 2005; Nat Biotechnol, 23:1126-1136.
  • Fd means an antibody fragment consisting of VH and CH1 domains
  • Fab fragment means an antibody fragment consisting of VL, VH, CL and CH1 domains
  • F( "ab') 2 fragment means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region
  • Fab'fragment means two heavy chain fragments in a reduced-linked F(ab') 2 fragment The fragment obtained after the disulfide bond consists of a complete light chain and the Fd fragment of the heavy chain (consisting of VH and CH1 domains).
  • Fv means an antibody fragment consisting of the VL and VH domains of a single arm of an antibody. Fv fragments are generally considered to be the smallest antibody fragments that can form a complete antigen-binding site. It is generally believed that six CDRs confer the antigen-binding specificity of an antibody. However, even a variable region (such as an Fd fragment, which contains only three CDRs specific for the antigen) can recognize and bind the antigen, although its affinity may be lower than that of the intact binding site.
  • Fc means a region formed by disulfide bonding of the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain.
  • Antibody fragments The Fc fragment of an antibody has many different functions but does not participate in antigen binding.
  • scFv refers to a single polypeptide chain comprising VL and VH domains connected by a linker (see, e.g., Bird et al., Science 242:423 -426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Roseburg and Moore, eds., Springer-Verlag, New York, pp. 269-315 (1994)).
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof.
  • a linker having the amino acid sequence (GGGGS) 4 can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linkers useful in the present invention are provided by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol.
  • scFv can form di-scFv, which refers to two or more individual scFvs connected in series to form an antibody.
  • scFv can form (scFv) 2 , which refers to two or more individual scFvs joining in parallel to form an antibody.
  • the term "diabody” means one whose VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains of the same chain, This forces the domain to pair with the complementary domain of the other chain and creates two antigen binding sites (see, e.g., Holliger P. et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993), and Poljak R.J. et al., Structure 2:1121-1123 (1994)).
  • single-domain antibody has the meaning commonly understood by those skilled in the art, which refers to an antibody composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable An antibody fragment consisting of a region) that retains the ability to specifically bind to the same antigen that the full-length antibody binds.
  • Single domain antibodies are also called nanobodies.
  • bispecific antibody refers to an antibody that has binding specificities for two different antigens (or epitopes).
  • multispecific antibody refers to an antibody that has binding specificity for at least two (eg, three or four) different antigens (or epitopes).
  • Bispecific antibodies or multispecific antibodies contain multiple antigen-binding domains with binding specificities for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and/or target molecules.
  • Each antigen-binding domain comprised by a bispecific antibody or a multispecific antibody can be independently selected from a full-length antibody (e.g., IgG antibody) or an antigen-binding fragment thereof (e.g., Fv fragment, Fab fragment, F(ab') 2 fragment or scFv).
  • the individual antigen binding domains are linked by a peptide linker.
  • Each of the above antibody fragments retains the ability to specifically bind to the same antigen that the full-length antibody binds, and/or competes with the full-length antibody for specific binding to the antigen.
  • Antigen-binding fragments of an antibody can be obtained from a given antibody (e.g., the antibodies provided by the invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods) ), and the antigen-binding fragments of the antibody are screened for specificity in the same manner as for intact antibodies.
  • humanized antibody refers to a non-human antibody that has been genetically engineered and whose amino acid sequence has been modified to increase sequence homology to that of a human antibody.
  • all or part of the CDR region of a humanized antibody comes from a non-human antibody (donor antibody), and all or part of the non-CDR region (for example, variable region FR and/or constant region) comes from a human source.
  • Immunoglobulins receptor antibodies.
  • the CDR regions of the humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., variable region FR and/or constant region) are derived from a human source.
  • Humanized antibodies generally retain the expected properties of the donor antibody, including but not limited to, antigen specificity, affinity, reactivity, etc.
  • the donor antibody may be a murine antibody with desired properties (eg, antigen specificity, affinity, reactivity, etc.).
  • the CDR regions of the donor antibody can be inserted into human framework sequences using methods known in the art.
  • the human framework sequences may contain amino acid mutations that are replaced by corresponding non-human residues.
  • humanized antibodies can also contain residues that are not found in either the original donor antibody variable region (e.g., light chain variable region or heavy chain variable region) or human framework sequences to further improve or optimize Performance of this humanized antibody.
  • chimeric antibody refers to an antibody in which part of the light chain and/or heavy chain is derived from an antibody (which may be derived from a specific species or belonging to a specific species). a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), but regardless of However, it still retains binding activity to the target antigen (USP 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).
  • the term “chimeric antibody” may include antibodies in which the heavy chain variable region and light chain variable region of the antibody are derived from the first antibody, and the heavy chain constant region and light chain constant region of the antibody are from secondary antibodies.
  • the term "identity" is used to refer to the match of sequences between two polypeptides or between two nucleic acids.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the first amino acid sequence or nucleic acid sequence to best match the second amino acid or nucleic acid sequence). Good comparison).
  • the amino acid residues or nucleotides at the corresponding amino acid positions or nucleotide positions are then compared. Molecules are identical when a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence.
  • Determination of percent identity between two sequences can also be accomplished using mathematical algorithms.
  • One non-limiting example of a mathematical algorithm for comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Improved in .Acad.Sci.U.S.A.90:5873-5877.
  • Such algorithms were integrated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.
  • variant in the context of polypeptides (including polypeptides), also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing substitutions, deletions, or additions of amino acid residues. In some cases, the term “variant” also refers to a polypeptide or peptide that has been modified (ie, by covalently linking any type of molecule to the polypeptide or peptide).
  • polypeptides may be modified, e.g., by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, Attached to cellular ligands or other proteins, etc.
  • Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like.
  • a variant has a similar, identical or improved function to the polypeptide or peptide from which it is derived.
  • the term “specific binding” refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen against which it is directed.
  • the strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant (K D ) of the interaction.
  • K D refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen.
  • the specific binding properties between two molecules can be determined using methods known in the art.
  • One approach involves measuring the rate at which antigen binding site/antigen complexes form and dissociate.
  • Both the "association rate constant” (ka or kon) and the “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361 :186-187).
  • the ratio kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59:439-473). Any valid method can be used to measure K D , kon and kdis values.
  • dissociation constants can be measured in Biacore using surface plasmon resonance (SPR).
  • bioluminescence interferometry or Kinexa can be used to measure dissociation constants.
  • a detectable label of the invention may be any substance detectable by fluorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical or chemical means.
  • labels are well known in the art and examples include, but are not limited to, enzymes (e.g., horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides fluorescein (e.g., 3H , 125I , 35S , 14C , or 32P ), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethylrhodamine isothiocyanate (TRITC) , fluorescent proteins (such as phycoerythrin (PE)), Texas red, rhodamine, quantum dots or cyanine dye derivatives (such as Cy7, Alexa750)), luminescent substances (such as chemilum
  • the detectable label is selected from the group consisting of enzymes (e.g., horseradish peroxidase or alkaline phosphatase), chemiluminescent reagents (e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives), fluorescent dyes (such as fluorescein or fluorescent proteins), radionuclides or biotin.
  • enzymes e.g., horseradish peroxidase or alkaline phosphatase
  • chemiluminescent reagents e.g., acridinium esters, luminol and its derivatives, or ruthenium derivatives
  • fluorescent dyes such as fluorescein or fluorescent proteins
  • the term "vector” refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • papillomaviruses papillomaviruses
  • Polyomavacuolating viruses such as SV40.
  • a vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
  • conservative substitution means an amino acid substitution that does not adversely affect or alter the expected properties of the protein/polypeptide comprising the amino acid sequence.
  • conservative amino acid substitutions include those in which an amino acid residue is replaced with an amino acid residue having a similar side chain, e.g., one that is physically or functionally similar to the corresponding amino acid residue (e.g., has similar size, shape, charge, chemical properties, including ability to form covalent bonds or hydrogen bonds, etc.). Families of amino acid residues with similar side chains have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine , asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isoleucine amino acids, proline, phenylalanine, methionine), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, Phenylalanine, tryptophan, histidine) amino acids.
  • basic side chains e.g., lysine, arginine, and histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • amino acids involved in this article have been prepared following conventional usage. See, e.g., Immunology-A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
  • the term "pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, They are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but are not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers Agents, diluents, agents to maintain osmotic pressure, agents to delay absorption, preservatives, stabilizers.
  • pH adjusting agents include, but are not limited to, phosphate buffer.
  • Surfactants include, but are not limited to, cationic, anionic or nonionic surfactants such as Tween-80.
  • Ionic strength enhancers include, but are not limited to, sodium chloride.
  • Agents that maintain osmotic pressure include, but are not limited to, sugar, NaCl, and the like.
  • Agents that delay absorption include, but are not limited to, monostearate and gelatin.
  • Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols and polyols (such as glycerol), and the like.
  • Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, sorbic acid, etc.
  • Stabilizers have the meaning commonly understood by those skilled in the art, which can stabilize the desired activity of active ingredients in medicines, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose) , lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dry whey, albumin, or casein) or their degradation products (such as milk Albumin hydrolyzate), etc.
  • the pharmaceutically acceptable carrier or excipient includes sterile injectable liquids (such as aqueous or non-aqueous suspensions or solutions).
  • sterile injectable liquids are selected from water for injection (WFI), bacteriostatic water for injection (BWFI), sodium chloride solution (e.g., 0.9% NaCl), glucose solution (e.g., 5% Glucose), surfactant-containing solutions (e.g., 0.01% polysorbate 20), pH buffer solutions (e.g., phosphate buffer solution), Ringer's solution, and any combination thereof.
  • WFI water for injection
  • BWFI bacteriostatic water for injection
  • sodium chloride solution e.g. 0.9% NaCl
  • glucose solution e.g., 5% Glucose
  • surfactant-containing solutions e.g., 0.01% polysorbate 20
  • pH buffer solutions e.g., phosphate buffer solution
  • Ringer's solution e.g., Ringer's solution, and any
  • prevention refers to a method performed to prevent or delay the occurrence of a disease or condition or symptom in a subject.
  • treatment refers to a method performed to obtain a beneficial or desired clinical result.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, reduction of the extent of the disease, stabilization (i.e., no worsening) of the state of the disease, delaying or slowing the progression of the disease, ameliorating or alleviating the disease. status, and relief of symptoms (whether partial or complete), whether detectable or undetectable.
  • treatment may also refer to prolonging survival compared with expected survival (eg, survival without treatment).
  • the term "subject” refers to a mammal, such as a human, rat, or monkey.
  • the subject eg, human, mouse, or monkey
  • TSHR eg, Graves' disease, Graves' ophthalmopathy
  • an effective amount refers to an amount sufficient to obtain, at least in part, the desired effect.
  • an effective amount to prevent a disease e.g., Graves' disease, Graves' ophthalmopathy
  • an effective amount to treat a disease refers to an amount sufficient to cure or at least partially prevent the disease.
  • the amount of disease and its complications in patients with pre-existing disease. Determining such effective amounts is well within the capabilities of those skilled in the art.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.
  • the antibodies provided in this application have high affinity to human TSHR and have cross-binding activity against human/mouse/monkey TSHR.
  • the antibody of the present application has a significant inhibitory effect on TSHR activity. It can inhibit the downstream signaling pathway mediated by the combination of TSHR and activating antibodies by blocking the binding between TSHR and activating antibodies. It has been verified that the antibody of the present application has the function of significantly inhibiting the secretion of hyaluronic acid, interleukin 6 and interleukin 8 in primary orbital fibroblasts of GO patients in in vitro functional experiments.
  • Figure 1 Engineered antibodies inhibit GD patient serum from inducing TPO production by thyroid cells.
  • the molecular biology experimental methods and immunoassay methods used in the present invention basically refer to J. Sambrook et al., Molecular Cloning: Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and The method was carried out as described in F.M. Ausubel et al., Compiled Experimental Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes was in accordance with the conditions recommended by the product manufacturer.
  • restriction enzymes was in accordance with the conditions recommended by the product manufacturer.
  • mice with human TSHR antigen After immunizing mice with human TSHR antigen, select mice with higher titers for cell fusion and hybridoma preparation.
  • mice with strong response to the immunogen for preparation of hybridoma cells use carbon dioxide to euthanize mice according to the method approved by IACUC, collect pre-fusion plasma, separate and collect serum (FB) as Positive control for hybridoma supernatant screening.
  • FB serum
  • splenocytes stand at room temperature for 2-3 minutes, then transfer to a new 50 ml centrifuge tube.
  • ratio of splenocytes to SP2/0 cells 4:1, add SP2/0 cells to splenocytes, and adjust the volume to 50 ml with fusion culture medium. Centrifuge the cell mixture, discard the supernatant, and loosen the cell pellet by repeatedly tapping the bottom of the tube. Fusion, for electrofusion, place the cell mixture in the electroporation tank and perform electroporation fusion according to the optimized procedure.
  • the fused cells need to rest in the electroporation tank for an additional 10 minutes. Resuspend in DMEM culture medium supplemented with 20% FBS and HT, and inoculate into a 96-well microplate at a volume of 100 ⁇ l per well. After 24 hours, DMEM culture medium supplemented with 20% FBS and HAT was added to the well plate, 100 ⁇ l per well. Transfer all confluent plates back to the CO2 incubator, 37°C, 5% CO2 . Monitor cell growth rate and presence of microbial contamination daily. When the hybridoma clone grows to a size of 1-2 mm in diameter (generally 10-14 days after fusion), use Acumen to screen the hybridoma supernatant.
  • Subcloning and expansion culture The positive clones were transferred to a 24-well plate and cultured with amplification medium. The hybridoma culture supernatant was collected for FACS detection. By limiting dilution method, the positive mother clone was subcloned to obtain a single clone. Place the subclone 96-well plate in a carbon dioxide incubator and culture it for 7 days, and then detect and screen the activity of the supernatant. To ensure a single clone, 1-2 rounds of subcloning are required. Based on the screening results, select the four most active single clones from each subclone plate and transfer them to a 24-well plate for amplification and culture.
  • the amplified subcloned cell lines were cryopreserved, stored in DMEM culture medium supplemented with 20% FBS and 10% DMSO, and placed in a liquid nitrogen tank for long-term storage.
  • mice monoclonal antibodies mAb001, mAb012 and mAb021 were prepared by the above method, and their CDR and variable region sequences are shown in Table 2.
  • CDR transplantation to design humanized antibodies.
  • BLAST the sequence closest to the mouse sequence in the human antibody as the variable region skeleton, replace the mouse framework region, transplant the CDRs of the parent antibody into the human recipient, and analyze all sequences Risks of post-translational modifications (PTM), such as isomerization, deamidation and glycosylation, were designed to remove mutations and ultimately obtain humanized light chains and humanized heavy chains of each parent antibody.
  • PTM post-translational modifications
  • the humanized light and heavy chain variable regions of each parent antibody were paired with each other for affinity ranking experiments.
  • the heavy chain variable region and the light chain variable region were connected to the human IgG4-S228P heavy chain constant region (SEQ ID NO:25) and the human kappa light chain constant region (SEQ ID NO:26) respectively to obtain humanized antibodies.
  • Full-length heavy and light chain sequences were connected to the human IgG4-S228P heavy chain constant region (SEQ ID NO:25) and the human kappa light chain constant region (SEQ ID NO:26) respectively to obtain humanized antibodies.
  • the DNA sequences encoding the humanized antibody heavy chain and light chain were synthesized and inserted into the pcDNA3.4 vector to construct a full-length antibody expression plasmid.
  • Humanized antibodies were expressed in Expi293F cell cultures, and the supernatant was purified using a protein A affinity column. Buffer exchange the purified antibody into PBS using a PD-10 desalting column. The concentration and purity of the purified protein were determined by OD280 and SDS-PAGE, respectively.
  • Humanized antibodies of each mouse-derived monoclonal antibody were obtained by the above method.
  • the amino acid sequences of the light and heavy chain variable regions of each humanized antibody are shown in SEQ ID NO: 27-38.
  • the flow cytometry (FACS) method was used to detect mouse-derived antibodies and 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpression cell lines (the cells used were overexpressed human, monkey or mouse-derived TSHR. 293 cells, wherein the amino acid sequences of the TSHRs derived from humans, monkeys and mice can be found in P16473, A0A2K5X226, P47750 (Uniprot) respectively.
  • the experimental method is as follows: collect cells in the logarithmic growth phase and count them with a cell counter (Countstar; IC1000); resuspend the cells in cold FACS buffer, and the buffer is 500 ml PBS (HyClone, SH30256.01B) plus 50 ml FBS.
  • the cell density was adjusted to 5 ⁇ 10 6 cells per ml; the cell suspension was left to stand at room temperature for 10 to 20 minutes before being added to the FACS plate (Corning, 3799) , 100 ⁇ L per well, centrifuge and discard the supernatant; dilute the antibody to be tested with FACS buffer, with a starting concentration of 200 nM, and a 1:3 gradient dilution; add the diluted antibody to the above cells, 100 ⁇ L per well, and incubate at 4 degrees for 1 hour; After the incubation is completed, wash the cells with FACS buffer, 250 ⁇ L per well, centrifuge at 300g (4 degrees) for 5 minutes, and wash three times; add FACS buffer to prepare a secondary anti-mouse IgG-Alexa488 antibody (invitrogen; A21202) (1: 1k dilution), 100 ⁇ L per well, incubate in the dark at 4 degrees for 1 hour, then wash the cells three times with FACS buffer
  • the selected antibodies all have good binding activity to the 293 cell line overexpressing human TSHR, and all have monkey and mouse cross-binding activity.
  • Example 2 Refer to the FACS method described in Example 2 to detect the binding effect of the humanized antibody to 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpression cell lines.
  • anti-human IgG-Alexa488 antibody invitrogen; A-11013 (dilution of 1:1k) was used as the secondary antibody.
  • the above humanized antibodies have good binding activity to 293-human TSHR cells, and have the ability to bind to monkey TSHR and mouse TSHR.
  • Antibody preparation Use Bravo (Agilent) instrument to prepare antibodies of different concentrations in a 384-well plate (3824Corning).
  • the preparation solution is DPBS, with a starting concentration of 200nM, diluted 1:3, and a total of 11 points.
  • Humanized antibody blocks agonistic antibody activation of cAMP experiment According to the instruction manual of cAMP-Gs DYNAMIC Kit (62AM4PEB, Cisbio), complete the detection of antibody cAMP.
  • the cell line used is the 293-human TSHR cell line;
  • the competing antibodies are Tab01 and Tab02, both of which are agonistic antibodies, and the concentrations used are 30nM and 6.5nM respectively;
  • the sampling instrument is MultiDrop Combi (Thermo), set to low speed ;
  • the data reading instrument is Envision (PerkinElmer) plate reading to detect the signals at 665nM and 615nM wavelengths.
  • the standard curve is made with the concentration of the standard and the signal ratio of the wavelength 665nM/615nM to detect the relative activity of the antibody.
  • Tab01 is TSHR agonistic antibody M22, and its sequence is derived from the antibody heavy chain variable region with sequence number No. 1 and the light chain variable region with sequence number No. 6 in patent WO 2004/050708A2.
  • the heavy chain variable region and the light chain variable region were respectively connected to the human IgG1 heavy chain constant region and the human lambda light chain constant region to obtain the full-length sequences of Tab01 heavy chain and light chain, and the human IgG1 heavy chain constant region and The human lambda light chain constant region sequence is referred to the positive control Tab03_hlgG1 heavy chain constant region and light chain constant region sequences.
  • Tab02 is TSHR agonistic antibody K1-18, and its sequence is derived from the heavy chain sequence No. 15 and the light chain sequence No. 33 in the TSHR antibody patent patent No. US10428153.
  • the positive control is Tab03_hIgG1, and its sequence is derived from the heavy chain sequence No. 51 and the light chain sequence No. 59 in the TSHR antibody patent patent No. US10428153.
  • the ratio of the signals at wavelengths 665nM and 615nM is calculated.
  • the ratio is proportional to the relative content of cAMP and represents the relative activity of TSHR.
  • the experiment was divided into three groups, and the cell sources were GO patients in the acute phase and GO patients in the stable phase.
  • the cell acquisition method is as follows. Obtain tissue specimens from the patients' orbital connective parts. There are 2 patients in each group. The specimens are quickly placed into sterile centrifuge tubes filled with DMEM/F12 culture medium (containing penicillin-streptomycin mixture), and then centrifuged with the tissue.
  • DMEM/F12 culture medium containing penicillin-streptomycin mixture
  • the tube is quickly transferred to the cell culture laboratory; the tissue in the centrifuge tube is picked up in a clean bench and washed repeatedly with sterile PBS to remove blood stains, then transferred to DMEM/F12 culture medium (containing penicillin-streptomycin mixture)
  • DMEM/F12 culture medium containing penicillin-streptomycin mixture
  • DMEM/F12 culture medium containing penicillin-streptomycin mixture
  • equilibration buffer 50mM sodium phosphate and 0.5M sodium sulfate , pH7.0
  • sample buffer 50mM sodium phosphate and 0.55M sodium sulfate, pH 7.0
  • concentration ratio 50mM sodium phosphate and 0.5M sodium sulfate, pH7.0
  • elution buffer 20mM phosphate Sodium and 20% glycerol, pH 7.0
  • the fibroblasts obtained from each group of patients were counted using a Countess 3 automatic cell counter (Thermo Fisher Scientific). After adjusting the density, 5,000 or 10,000 cells were seeded into each well of a 96-well plate. The plate was placed in a Forma 3111 water-jacketed CO 2 incubator (Thermo Electron company) and cultured overnight. All cells were observed under the microscope for adhesion and then starved for 24 hours. The supernatant was removed, and different concentrations of the antibodies to be tested were added according to experimental groups and incubated. After 24 hours, add GO-Igs and continue to incubate for 96 hours. Carefully collect the cell supernatant. Use a 3K15 low-temperature high-speed centrifuge to centrifuge the supernatant at 1000 ⁇ g for 15 minutes. Discard the cell debris and take the supernatant for detection.
  • This experiment used the human hyaluronic acid (HA) kit (ELISA) (ml557801, Shanghai Enzyme Biotechnology Co., Ltd.).
  • the specific process is as follows: Take out the required slats from the aluminum foil bag that has been equilibrated at room temperature for 60 minutes. Set up standard wells and sample wells, and add 50 ⁇ L of standards of different concentrations to each standard well; 200ng/mL, 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL. Add 50 ⁇ L of the supernatant after the above treatment to the sample well; do not add it to the blank well, and incubate in a 37°C incubator for 1 hour.
  • HA human hyaluronic acid
  • HRP horseradish peroxidase
  • the selected antibodies can inhibit GO-Igs from stimulating the patient's orbital fibroblasts to produce HA, among which cAb01-VH1-DA-VL2, cAb01-VH2-DA-VL2, cAb01-VH3-DA-VL2, cAb01- VH1-DA-VL3, cAb01-VH2-DA-VL3, cAb12-VH1-QG&S-VL3, cAb12-VH3-QG&S-VL3 have good inhibitory effects on cells in the acute phase and stable phase, with the maximum inhibition rate in the stable phase Better than the control antibody Tab03_hIgG1; cAb01-VH2-DA-VL3 and cAb12-VH3-QG&S-VL3IC50 are better than the positive control Tab03_hIgG1; cAb21-VH2-VL2 and cAb21-VH1-VL3 have good inhibitory effects on cells in the acute phase and
  • Fibroblasts and GO-Igs were collected according to the method described in Example 5, and the cells were counted using a Countess 3 automatic cell counter (Thermo Fisher Scientific). After adjusting the density, 5,000 or 10,000 cells were seeded into each well of a 96-well plate. cells, and place the 96-well plate in a Forma 3111 water-jacketed CO 2 incubator (Thermo Electron company) overnight. Observe all cells under the microscope and then starve them for 24 hours. Remove the supernatant. Add different concentrations of antibodies to be tested according to experimental groups and incubate for 24 hours. Add GO-Igs and continue incubating for 24 hours. Carefully collect the cell supernatant. , centrifuge the supernatant at 1000 ⁇ g for 15 minutes using a 3K15 low-temperature high-speed centrifuge, discard the cell debris, and take the supernatant for detection.
  • This experiment used the Human IL-6 ELISA kit (CSB-E04638h, Cusabio Company).
  • the specific process is as follows: before the experiment, place the corresponding reagents at room temperature for 60 minutes. Add 100 ⁇ l of standard and sample to each well. Do not add the standard to the blank well. The concentrations are 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.2pg/ml, 15.6pg/ml, 7.8pg/ml, seal the plate, and incubate at 37°C for 2 hours. Discard the liquid from each well, add 100 ⁇ l 1 ⁇ Biotin-antibody to each well, and incubate in a 37°C incubator for 1 hour.
  • the selected antibodies can all inhibit GO-Igs from stimulating the patient's orbital fibroblasts to produce IL6. All antibodies have good inhibitory effects on cells in the acute phase and stable phase, among which cAb01-VH1-DA-VL2, cAb01-VH1-DA- The maximum inhibition rate and IC50 of VL3, cAb01-VH2-DA-VL3, and cAb21-VH2-VL2 in the acute phase and stationary phase were better than those of the control antibody Tab03_hIgG1.
  • Human IL-8 ELISA kit (D8000C, R&D Company) was used to detect the concentration of IL8 in the supernatant to be tested.
  • All the selected antibodies can inhibit GO-Igs from stimulating the production of IL8 by orbital fibroblasts in patients. All antibodies have good inhibitory effects on cells in the acute phase and stable phase, and the maximum inhibition rate is almost better than the positive control Tab03_hIgG1.
  • the antibody cAb01-VH2-DA-VL3 (named hAb01_G4P, in which the constant region is of IgG4 subtype and contains the S228P mutation) was selected for Fc transformation (the transformation includes transformation based on IgG4-S228P and transformation based on IgG1) , the purpose of the modification is to extend the half-life, increase the efficacy and reduce the ADCC effect.
  • the modification sites are shown in the table below:
  • Example 2 Refer to the FACS method described in Example 2 to detect the binding effect of the modified antibody to 293-human TSHR, 293-mouse TSHR, and 293-monkey TSHR overexpression cell lines.
  • anti-human IgG-Alexa488 antibody invitrogen; A-11013 (dilution of 1:1k) was used as the secondary antibody.
  • hIgG1 is a negative control (purchased from Baiying B117901); hIgG4 is a negative control (purchased from Baiying B107804).
  • the modified antibody binds well to human TSHR and has cross-binding activity in mice and monkeys.
  • the activity detection results of the modified antibodies are as follows:
  • the target cells of the experiment were 293-human TSHR cells (for the amino acid sequence of overexpressed human TSHR, please refer to P16473, Uniprot), and the effector cells were human PBMCs (from Miaoshun, P122070903C). Resuscitate effector cell PBMC the day before the experiment and harvest on the day of the experiment. Adjust the cell concentration to 5 ⁇ 10 6 / mL, reserve. Use fluorescent dye (Perkin Elmer, C136-100) to label 1 ⁇ 10 6 target cells, incubate at 37°C in the dark for 20 minutes, wash the cells 4 times with PBS, and resuspend the cells in experimental buffer for later use.
  • fluorescent dye Perkin Elmer, C136-100
  • sequences of the light chain variable region and heavy chain variable region of hAb01_G1 are consistent with hAb01_G4P, and the constant region subtype is hIgG1 (the full-length amino acid sequence of the heavy chain is shown in SEQ ID NO: 49, and the full-length amino acid sequence of the light chain is shown in SEQ ID NO :51 shown).
  • Inhibition rate % (sample value - autofluorescence value of target cells and effector cells) / (maximum value of target cells - autofluorescence value of target cells) ⁇ 100%.
  • hAb01_G1_V4 molecule Compared with hAb01_G1, hAb01_G1_V4 molecule has a weaker ADCC effect. hAb01_G4P, hAb01_G4P_V1, and hAb01_G4P_V2 did not exhibit ADCC effects.
  • This experiment uses SA chip (cytiva, BR100531) to capture hFcRn-Avi (Bipsis, FCM-H82W7), flow different concentrations of antibodies through the chip, and perform steady-state fitting analysis based on the collected data.
  • the experimental instrument is Biacore molecular interaction instrument (cytiva, Biacore 8K+), the buffer solution used is HBS-EP Buffer (cytiva, BR100669), pH 6.0, binding time 240s, dissociation time 240s, flow rate 30 ⁇ L/min, 25°C conditions Detection was carried out under the conditions, and the experimental data obtained were subjected to steady-state fitting analysis through Biacore Insight Evaluation Software 3.0.12 software. The experimental results are shown in Table 15.
  • Example 13 In vitro experiments of engineered antibodies against GD indications
  • Thyroid tissue samples were taken from normal thyroid tissue of patients who underwent total thyroidectomy for thyroid cancer. The tissue was resuspended in PBS solution containing 3 mg/mL type IV collagenase (Life Technologies) to obtain dispersed cells for passage and amplification.
  • TPO Thyroid catalase
  • Cells from each group were collected, and after adjusting the density, 6 ⁇ 10 4 cells were seeded into each well of a 24-well plate and cultured overnight. After adhesion, starve culture for 24 hours, remove the supernatant, add different concentrations of the antibody to be tested and culture medium with or without GD serum (final concentration is 1:100, volume ratio) according to the experimental group, continue to incubate for 48 hours, and digest with trypsin. Cells were collected and total RNA samples were extracted. Configure the reverse transcription reaction system according to the procedure in the kit instructions (iScript cDNA, Bio-Rad, 1708891EDU), obtain cDNA, and store it at -70°C.
  • kit instructions iScript cDNA, Bio-Rad, 1708891EDU
  • the reaction system was configured on the ice box and amplified.
  • the data was processed through the 2- ⁇ Ct formula, and the expression level of the target mRNA was obtained using the internal reference gene GAPDH as a reference.
  • the primers used are shown in Table 16.
  • All the modified antibodies can inhibit TPO induced by GD patient serum, indicating that the antibody of the present invention has the potential to treat GD disease.
  • the modified antibodies can inhibit GO-Igs from stimulating the patient's orbital fibroblasts to produce HA, and have a good inhibitory effect on cells in the acute and stable phases.

Abstract

本发明涉及特异性结合TSHR的抗体或其抗原结合片段、含有所述抗体或其抗原结合片段的药物组合物、编码所述抗体或其抗原结合片段的核酸分子及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段的治疗和诊断用途。

Description

结合促甲状腺激素受体的抗体及其用途 背景技术
格雷夫斯病(Graves'disease,GD)是一种难治的自身免疫性疾病，其症状表现为甲状腺肿大和过度活跃、心率加速和眼部异常，其中眼部异常突出的疾病又称为格雷夫斯眼病(Graves'ophthalmopathy,GO)或者甲亢突眼病。根据对该疾病的了解，认为这是遗传和环境因素复杂相互作用的结果。GO疾病不仅对生活质量有严重影响，同样会影响精神和工作，并且相关的过度激活甲状腺激素受体刺激甲状腺激素的分泌将导致死亡风险的增加。目前临床上针对GO疾病的治疗主要是抗甲状腺药物(ATD)治疗、放射性碘(RAI)治疗及外科手术治疗。但是使用抗甲状腺药物往往治疗效率不高且会产生如骨髓抑制和肝脏毒性等副作用，而通过手术切除或者放射性碘消融术治疗也存在明显的风险，并且术后导致复发性神经损伤和甲状旁腺功能减退。此外，在控制甲状腺功能的同时还需要选择糖皮质激素治疗、眼眶局部放射治疗或手术治疗，然而上述疗法均为对症治疗，并非针对疾病发病机制进行治疗。GO疾病目前临床缺乏有效的治疗手段。
促甲状腺激素受体(TSHR)主要表达于甲状腺滤泡细胞基底外侧表面，诱导甲状腺细胞生长、激素合成和激素分泌，同时也是自身免疫性甲状腺疾病的主要自身抗原，特别是GD疾病，随着病程的延长，大约25％的GD患者会发展成GO疾病。
文献记载，一些自身免疫性甲状腺疾病(AITD)患者会产生与TSHR反应的自身抗体(Rees Smith B,et al 1988.Endocrine Reviews 9:106-121)。TSHR自身抗体(TRAb)主要有两种类型：一种是刺激型，一种是阻断型。促甲状腺型自身抗体与TSHR结合，模仿TSH的作用，从而刺激甲状腺产生高水平的T4和T3。这些自身抗体也被描述为具有激动剂活性的TRAb(Rees Smith B et al.2007.Thyroid 17:923-938)。当存在该抗体时，甲状腺功能的反馈控制机制不再有效，且患者出现甲状腺过度活跃的临床症状，其特征是血清中甲状腺激素过多及其代谢后果，这种情况被称为格雷夫斯病(Graves’，GD)。具有刺激活性的TRAb也可能与眼眶后组织的TSHR相互作用，促进Graves病眼病(GO)的发生。基于目前对GO疾病的发病机制的研究认为，在GO患者眼眶成纤维细胞中，TSHR的表达量增加，并且当信号通路激活时将增加眼眶内透明质酸和脂肪组织的产生，导致眼内压升高眼球突出，是导致GO疾病的重要原因。此外，TSHR的表达在甲状腺和睾丸中表达较多，而在其他组织中的表达量低，因此在药物靶向性和副作用方面可能具有优势。因此，TSHR有望成为治疗GO疾病的潜在靶点。
另外，胰岛素样生长因子1受体(IGF1R)被认为是另一个在GO发病机制中的重要角色。IGF1R阻断剂能够改善GO疾病，例如，上市的药物Teprotumumab能够竞争性抑制 IGF1R受体激活下游的信号通路，特别是在改善眼睛突出和炎症减少方面具有显著疗效，其在治疗中度至重度甲状腺眼病(TED)中治疗效率达到78％。在GO病患者的眼眶成纤维细胞中发现TSHR和IGF1R的表达上升，并且TSHR和IGF1R存在一定的相互作用。因此，采用直接阻断TSHR(TSAb/antagonist)或者同时拮抗TSHR和IGF1R受体的联合治疗可能是根治GD或者GO疾病的方法。
目前没有以TSHR为靶点治疗GO疾病的药物上市，本领域存在开发以TSHR为靶点的治疗药物的需求。
技术领域
本发明涉及特异性结合TSHR的抗体或其抗原结合片段、含有所述抗体或其抗原结合片段的药物组合物、编码所述抗体或其抗原结合片段的核酸分子及包含其的宿主细胞，以及相关用途。此外，此发明涉及这些抗体或其抗原结合片段的治疗和诊断用途。
发明内容
本申请发明人通过大量的研究，获得了特异性结合TSHR的鼠源抗体，其具备显著的TSHR活性抑制作用并且具有针对人/鼠/猴TSHR的交叉结合活性。此外，本申请发明人还基于所述鼠源抗体进一步获得了具有TSHR结合活性和TSHR活性抑制作用的人源化抗体。
基于此，本申请还提供了含有所述抗体或其抗原结合片段的药物组合物，编码所述抗体或其抗原结合片段的核酸分子及包含其的宿主细胞，以及相关用途。
抗体或其抗原结合片段
mAb001及其人源化抗体
因此，在第一方面，本申请提供了能够特异性结合TSHR的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
如SEQ ID NO:1、27、29、30任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:5、28、31任一项所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
如SEQ ID NO:1、27、29、30任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:5、28、31任一项所示的轻链可变区(VL)中含有的3个CDR。
在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:2的VH CDR1、序列为SEQ ID NO:3或39的VH CDR2、序列为SEQ ID NO:4的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:6的VL CDR1、序列为SEQ ID NO:7的VL CDR2、序列为SEQ ID NO:8的VL CDR3；
其中，所述CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于鼠免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：鼠重链胚系序列的重链框架区，和/或，鼠轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含SEQ ID NO：1所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：5所示的序列或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:1所示的序列的VH和包含如SEQ ID NO:5所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含人胚系抗体基因所编码的氨基酸序列中所包含的框架区。在某些实施方案中，所述抗体或其抗原结合片段包含：人重链胚系序列的重链框架区，和/或，人轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:27、29、30任一项所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:28或31所示的序列或其变体的轻链可变区(VL)；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:27、29、30任一项所示的重链可变区(VH)；和/或，包含如SEQ ID NO:28或31所示的轻链可变区(VL)。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:27所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:29所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:30所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:27所示的序列的VH和包含如SEQ ID NO:31所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:29所示的序列的VH和包含如SEQ ID NO:31所示的序列的VL。
mAb012及其人源化抗体
因此，在第二方面，本申请提供了能够特异性结合TSHR的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
如SEQ ID NO:9、32、34任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:13或33所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
如SEQ ID NO:9、32、34任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:13或33所示的轻链可变区(VL)中含有的3个CDR。
在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:10的VH CDR1、序列为SEQ ID NO:11或40的VH CDR2、序列为SEQ ID NO:12或41的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:14的VL CDR1、序列为SEQ ID NO:15的VL CDR2、序列为SEQ ID NO:16的VL CDR3；
其中，所述CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:10的VH CDR1、序列为SEQ ID NO:11或40的VH CDR2、序列为SEQ ID NO:12的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:14的VL CDR1、序列为SEQ ID NO:15的VL CDR2、序列为SEQ ID NO:16的VL CDR3；
其中，所述CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:10的VH CDR1、序列为SEQ ID NO:11或40的VH CDR2、序列为SEQ ID NO:41的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:14的VL CDR1、序列为SEQ ID NO:15的VL CDR2、序列为SEQ ID NO:16的VL CDR3；
其中，所述CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于鼠免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：鼠重链胚系序列的重链框架区，和/或，鼠轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含SEQ ID NO：9所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：13所示的序列或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:9所示的序列的VH和包含如SEQ ID NO:13所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含人胚系抗体基因所编码的氨基酸序列中所包含的框架区。在某些实施方案中，所述抗体或其抗原结合片段包含：人重链胚系序列的重链框架区，和/或，人轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:32或34所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:33所示的序列或其变体的轻链可变区(VL)；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:32或34所示的重链可变区(VH)；和/或，包含如SEQ ID NO:33所示的轻链可变区(VL)。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:32所示的序列的VH和包含如SEQ ID NO:33所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:34所示的序列的VH和包含如SEQ ID NO:33所示的序列的VL。
mAb021及其人源化抗体
因此，在第三方面，本申请提供了能够特异性结合TSHR的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
如SEQ ID NO:17、35、37任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:21、36、38任一项所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)。在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：
如SEQ ID NO:17、35、37任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:21、36、38任一项所示的轻链可变区(VL)中含有的3个CDR。
在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。在某些实施方案中，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含：
包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:18的VH CDR1、序列为SEQ ID NO:19的VH CDR2、序列为SEQ ID NO:20的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:22的VL CDR1、序列为SEQ ID NO:23的VL CDR2、序列为SEQ ID NO:24的VL CDR3；
其中，所述CDR由Kabat编号系统定义。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于鼠免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含：鼠重链胚系序列的重链框架区，和/或，鼠轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：
重链可变区(VH)，其包含SEQ ID NO：17所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：21所示的序列或其变体；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少 96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:17所示的序列的VH和包含如SEQ ID NO:21所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列。
在某些实施方案中，所述抗体或其抗原结合片段包含人胚系抗体基因所编码的氨基酸序列中所包含的框架区。在某些实施方案中，所述抗体或其抗原结合片段包含：人重链胚系序列的重链框架区，和/或，人轻链胚系序列的轻链框架区。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:35或37所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:36或38所示的序列或其变体的轻链可变区(VL)；
其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列。
在某些实施方案中，所述的置换是保守置换。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:35或37所示的重链可变区(VH)；和/或，包含如SEQ ID NO:36或38所示的轻链可变区(VL)。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:35所示的序列的VH和包含如SEQ ID NO:36所示的序列的VL。
在某些实施方案中，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:37所示的序列的VH和包含如SEQ ID NO:38所示的序列的VL。
在某些实施方案中，在第一方面、第二方面或第三方面中，所述抗体或其抗原结合片段进一步包含来源于哺乳动物(例如人或鼠)免疫球蛋白的恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含人免疫球蛋白的重链恒定区(CH)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺 失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)；和/或，
所述抗体或其抗原结合片段的轻链包含人免疫球蛋白的轻链恒定区(CL)或其变体，所述变体与其所源自的序列相比具有一个或多个氨基酸的置换、缺失或添加(例如，至多20个、至多15个、至多10个、或至多5个氨基酸的置换、缺失或添加；例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)。所述置换可以是保守置换或非保守置换。
在一些实施方案中，所述抗体或其抗原结合片段包含野生型人免疫球蛋白的重链恒定区(CH)。
在另一些实施方案中，所述抗体或其抗原结合片段包含人免疫球蛋白的重链恒定区(CH)的变体，所述重链恒定区(CH)的变体与其所源自的野生型序列相比可以具有相同或基本相同的特性。在某些实施方案中，所述重链恒定区(CH)的变体与其所源自的序列相比可以具有一个或多个氨基酸的保守置换。
在另一些实施方案中，所述抗体或其抗原结合片段包含人免疫球蛋白的重链恒定区(CH)的变体，所述重链恒定区(CH)的变体可以包含一个或多个氨基酸突变或化学修饰以改变本发明抗体的下列中的一个或更多个特性：Fc受体结合、抗体糖基化、半胱氨酸残基的数目、效应细胞功能或补体功能等。可以通过将抗体恒定区中的至少一个氨基酸残基替换为不同残基或化学修饰，产生功能改变，例如，改变抗体对效应子配体(如FcR或补体C1q)的亲和力，从而改变效应子功能(例如降低或增强)。抗体的Fc区介导几种重要的效应子功能，例如ADCC、吞噬作用、CDC等。
在某些实施方案中，本发明的抗体或其抗原结合片段包含人免疫球蛋白重链恒定区(CH)的变体，所述变体与其所源自的野生型序列相比可以具有降低或消除的效应子功能。
在某些实施方案中，本发明的抗体或其抗原结合片段包含人免疫球蛋白重链恒定区(CH)的变体，所述变体与其所源自的野生型序列相比可以具有增强的效应子功能。
在某些实施方案中，本发明的抗体或其抗原结合片段包含人免疫球蛋白重链恒定区(CH)或其变体，所述变体与其所源自的野生型序列相比可以具有相同或基本相同的效应子功能。
在某些实施方案中，本发明的抗体或其抗原结合片段包含人免疫球蛋白重链恒定区(CH)的变体，所述变体与其所源自的野生型序列相比具有：(i)延长的半衰期，(ii)增强的药效，和/或，(iii)降低的ADCC活性。
在某些实施方案中，所述重链恒定区(CH)是IgG重链恒定区，例如IgG1、IgG2、IgG3或IgG4重链恒定区。
在某些实施方案中，所述轻链恒定区是κ轻链恒定区或λ轻链恒定区。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含来源于鼠免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，所述抗体或其抗原结合片段的轻链包含来源于鼠免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，在第一方面、第二方面或第三方面中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白IgG4的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，与野生型人免疫球蛋白IgG4的重链恒定区相比，所述来源于人免疫球蛋白IgG4的重链恒定区包含置换突变S228P、L235E、M252Y、S254T和/或T256E(例如，(i)S228P，(ii)L235E，和/或(iii)M252Y、S254T和T256E)。
在某些实施方案中，与野生型人免疫球蛋白IgG4的重链恒定区相比，所述来源于人免疫球蛋白IgG4的重链恒定区包含置换突变S228P；在某些实施方案中，所述来源于人免疫球蛋白IgG4的重链恒定区进一步包含置换突变M252Y、S254T和T256E；在某些实施方案中，所述来源于人免疫球蛋白IgG4的重链恒定区进一步包含置换突变L235E、M252Y、S254T和T256E。
在某些实施方案中，所述置换突变的氨基酸位置由EU编号系统所定义。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含如SEQ ID NO:25、42-43任一项所示的重链恒定区(CH)，和/或，所述抗体或其抗原结合片段的轻链包含如SEQ ID NO:26所示的轻链恒定区(CL)。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(1)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(2)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(3)包含如SEQ ID NO:30所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(4)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所 示的序列的CL的轻链；
(5)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(6)包含如SEQ ID NO:32所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(7)包含如SEQ ID NO:34所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(8)包含如SEQ ID NO:35所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:36所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；或，
(9)包含如SEQ ID NO:37所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:38所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链。
在某些实施方案中，第一方面的所述抗体或其抗原结合片段包含：
(1)包含如SEQ ID NO:46所示的序列的重链，和/或，包含如SEQ ID NO:51所示的序列的轻链；
(2)包含如SEQ ID NO:47所示的序列的重链，和/或，包含如SEQ ID NO:51所示的序列的轻链；或，
(3)包含如SEQ ID NO:48所示的序列的重链，和/或，包含如SEQ ID NO:51所示的序列的轻链。
在某些实施方案中，在第一方面、第二方面或第三方面中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白IgG1的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区。
在某些实施方案中，与野生型人免疫球蛋白IgG1的重链恒定区相比，所述来源于人免疫球蛋白IgG1的重链恒定区包含置换突变L234A、L235A、M252Y、S254T和/或T256E(例如，(i)L234A和L235A，和/或(ii)M252Y、S254T和T256E)。
在某些实施方案中，与野生型人免疫球蛋白IgG1的重链恒定区相比，所述来源于人免疫球蛋白IgG1的重链恒定区包含置换突变L234A和L235A；在某些实施方案中，所述 来源于人免疫球蛋白IgG1的重链恒定区进一步包含置换突变M252Y、S254T和T256E。
在某些实施方案中，所述置换突变的氨基酸位置由EU编号系统所定义。
在某些实施方案中，所述抗体或其抗原结合片段的重链包含如SEQ ID NO:44或45所示的重链恒定区(CH)，和/或，所述抗体或其抗原结合片段的轻链包含如SEQ ID NO:26所示的轻链恒定区(CL)。
在某些实施方案中，所述抗体或其抗原结合片段包含：
(1)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(2)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(3)包含如SEQ ID NO:30所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(4)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(5)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(6)包含如SEQ ID NO:32所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(7)包含如SEQ ID NO:34所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
(8)包含如SEQ ID NO:35所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:36所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；或，
(9)包含如SEQ ID NO:37所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:38所示的序列的VL和SEQ ID NO:26所示的序 列的CL的轻链。
在某些实施方案中，第一方面的所述抗体或其抗原结合片段包含：
(1)包含如SEQ ID NO:49所示的序列的重链，和/或，包含如SEQ ID NO:51所示的序列的轻链；或者，
(2)包含如SEQ ID NO:50所示的序列的重链，和/或，包含如SEQ ID NO:51所示的序列的轻链。
在某些实施方案中，在第一方面、第二方面或第三方面中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fd、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv)2、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、人源化抗体、嵌合抗体、双特异性抗体或多特异性抗体。
第四方面，本申请还提供了分离的核酸分子，其编码如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区，或其重链和/或轻链。
在某些实施方案中，所述分离的核酸分子包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的分离的核酸分子上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的分离的核酸分子上时，本发明所述的分离的核酸分子包含含有所述第一核苷酸序列的第一核酸分子以及含有所述第二核苷酸序列的第二核酸分子。
第五方面，本申请还提供了载体，其包含如上所述的分离的核酸分子。在某些实施方案中，所述载体为克隆载体或表达载体。
在某些实施方案中，所述载体包含编码本发明的抗体或其抗原结合片段的重链或重链可变区的第一核苷酸序列和编码所述抗体或其抗原结合片段的轻链或轻链可变区的第二核苷酸序列，其中所述第一核苷酸序列和所述第二核苷酸序列存在于相同或不同的载体上。当所述第一核苷酸序列和所述第二核苷酸序列存在于不同的载体上时，本发明所述的载体包含含有所述第一核苷酸序列的第一载体以及含有所述第二核苷酸序列的第二载体。
第六方面，本申请还提供了宿主细胞，其包含如上所述的分离的核酸分子或载体。
此类宿主细胞包括但不限于，原核细胞例如细菌细胞(如大肠杆菌细胞)，以及真核细胞例如真菌细胞(例如酵母细胞)，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞， 例如小鼠细胞、人细胞等)。在某些实施方案中，所述宿主细胞是微生物。
本发明的抗体可以本领域已知的各种方法来制备，例如通过基因工程重组技术来获得。例如，通过化学合成或PCR扩增获得编码本发明抗体的重链和轻链基因的DNA分子。将所得DNA分子插入表达载体内，然后转染宿主细胞。然后，在特定条件下培养转染后的宿主细胞，并表达本发明的抗体。
本发明的抗原结合片段可以通过水解完整的抗体分子获得(参见Morimoto et al.,J.Biochem.Biophys.Methods 24:107-117(1992)and Brennan et al.,Science 229:81(1985))。另外，这些抗原结合片段也可以直接由重组宿主细胞产生(reviewed in Hudson,Curr.Opin.Immunol.11:548-557(1999)；Little et al.,Immunol.Today,21:364-370(2000))。比如，Fab’片段可以直接从宿主细胞中获得；可以将Fab’片段化学偶联形成F(ab’)2片段(Carter et al.,Bio/Technology,10:163-167(1992))。另外，Fv、Fab或F(ab’)2片段也可以直接从重组宿主细胞培养液中直接分离得到。本领域的普通技术人员完全知晓制备这些抗原结合片段的其它技术。
第七方面，本申请还提供了制备如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养如上所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
治疗用途
第八方面，本申请还提供了双特异性或多特异性分子，其包含如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段。
在某些实施方案中，所述双特异性或多特异性分子特异性结合TSHR，并且额外地特异性结合一个或多个其他靶标。
在某些实施方案中，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)。
在某些实施方案中，所述双特异性或多特异性分子还包含至少一种特异性结合IGF1R的第二抗体。在某些实施方案中，所述第二抗体具有对IGF1R信号传导的拮抗作用。
第九方面，本申请还提供了免疫缀合物，其包含如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂。
在某些实施方案中，所述治疗剂选自IGF1R拮抗剂或抑制剂。
在某些实施方案中，所述免疫缀合物是抗体-药物偶联物(ADC)。
第十方面，本申请还提供了药物组合物，其包含如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段、如第四方面所述的分离的核酸分子、如第五方面所述的载体、如第六方面所述的宿主细胞、如第八方面所述的双特异性或多特异性分子或如第九方面所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂。
在某些实施方案中，所述药物组合物还包含另外的药学活性剂，例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物。
在某些实施方案中，所述药物组合物还包含IGF1R拮抗剂或抑制剂，和/或，另外的TSHR拮抗剂或抑制剂。
在某些示例性实施方案中，所述药学上可接受的载体和/或赋形剂包含无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
第十一方面，本申请还提供了如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段、如第四方面所述的分离的核酸分子、如第五方面所述的载体、如第六方面所述的宿主细胞、如第八方面所述的双特异性或多特异性分子、如第九方面所述的免疫缀合物或如第十方面所述的药物组合物用于制备药物的用途，所述药物用于预防和/或治疗受试者的与TSHR相关的疾病。
在某些实施方案中，所述与TSHR相关的疾病将得益于对TSHR信号传导的拮抗作用。
在某些实施方案中，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合。
在某些实施方案中，所述受试者为哺乳动物，例如人、鼠或猴。
在某些实施方案中，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物单独使用，或与另外的药学活性剂(例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物)联合使用，例如同时或相继施用。
在某些实施方案中，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物与IGF1R拮抗剂或抑制剂和/或另外 的TSHR拮抗剂或抑制剂联合使用，例如，同时或相继施用。
第十二方面，本申请还提供了用于在受试者中预防和/或治疗受试者的与TSHR相关的疾病的方法，其包括：给有此需要的受试者施用有效量的如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段、如第四方面所述的分离的核酸分子、如第五方面所述的载体、如第六方面所述的宿主细胞、如第八方面所述的双特异性或多特异性分子、如第九方面所述的免疫缀合物或如第十方面所述的药物组合物。
在某些实施方案中，所述与TSHR相关的疾病将得益于对TSHR信号传导的拮抗作用。
在某些实施方案中，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合。
在某些实施方案中，所述受试者为哺乳动物，例如人、鼠或猴。
在某些实施方案中，如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段、如第四方面所述的分离的核酸分子、如第五方面所述的载体、如第六方面所述的宿主细胞、如第八方面所述的双特异性或多特异性分子、如第九方面所述的免疫缀合物或如第十方面所述的药物组合物可以与另外的药学活性剂(例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物)联合施用，例如同时或相继施用。
在某些实施方案中，如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段、如第四方面所述的分离的核酸分子、如第五方面所述的载体、如第六方面所述的宿主细胞、如第八方面所述的双特异性或多特异性分子、如第九方面所述的免疫缀合物或如第十方面所述的药物组合物可以与IGF1R拮抗剂或抑制剂和/或另外的TSHR拮抗剂或抑制剂联合施用，例如同时或相继施用。
本申请的抗体或其抗原结合片段或药物组合物可以配制成医学领域已知的任何剂型，例如，片剂、丸剂、混悬剂、乳剂、溶液、凝胶剂、胶囊剂、粉剂、颗粒剂、酏剂、锭剂、栓剂、注射剂(包括注射液、注射用无菌粉末与注射用浓溶液)、吸入剂、喷雾剂等。优选剂型取决于预期的给药方式和治疗用途。本发明的抗体或其抗原结合片段或药物组合物应当是无菌的并在生产和储存条件下稳定。一种优选的剂型是注射剂。此类注射剂可以是无菌注射溶液。例如，可通过下述方法来制备无菌注射溶液：在适当的溶剂中掺入必需剂量的本发明的抗体或其抗原结合片段，以及任选地，同时掺入其他期望的成分(包括但不限于，pH调节剂，表面活性剂，佐剂，离子强度增强剂，等渗剂，防腐剂，稀释剂，或其任何组合)，随后过滤除菌。此外，可以将无菌注射溶液制备为无菌 冻干粉剂(例如，通过真空干燥或冷冻干燥)以便于储存和使用。此类无菌冻干粉剂可在使用前分散于合适的载体中，例如注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
本申请的抗体或其抗原结合片段或药物组合物可以通过本领域已知的任何合适的方法来施用，包括但不限于，口服、口腔、舌下、眼球、局部、肠胃外、直肠、叶鞘内、内胞浆网槽内、腹股沟、膀胱内、局部(如，粉剂、药膏或滴剂)，或鼻腔途径。但是，对于许多治疗用途而言，优选的给药途径/方式是胃肠外给药(例如静脉注射或推注，皮下注射，腹膜内注射，肌内注射)。技术人员应理解，给药途径和/或方式将根据预期目的而发生变化。在某些实施方案中，本发明的抗体或其抗原结合片段或药物组合物通过静脉注射或推注给予。
检测用途
第十三方面，本申请还提供了缀合物，其包含如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段，以及与所述抗体或其抗原结合片段连接的可检测的标记。
在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
第十四方面，本申请还提供了试剂盒，其包括如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段或第十三方面所述的缀合物。
在某些实施方案中，所述试剂盒包含检测用缓冲液。
在某些实施方案中，所述试剂盒包含第十三方面所述的缀合物。
在某些实施方案中，所述试剂盒包含如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段，以及特异性识别所述抗体或其抗原结合片段的第二抗体；任选地，所述第二抗体还包括可检测的标记，例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
第十五方面，本申请还提供了用于检测TSHR在样品中的存在或其水平的方法，其包括使用如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段或第十三方面所述 的缀合物。
在某些实施方案中，所述方法用于治疗目的，诊断目的，或者非治疗非诊断目的。
在某些实施方案中，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法。
在某些实施方案中，所述方法包括使用第十三方面所述的缀合物。
在某些实施方案中，所述方法包括使用如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述抗体或其抗原结合片段。
在某些实施方案中，所述方法包括：(1)将所述样品与本发明的抗体或其抗原结合片段或缀合物接触；(2)检测抗原-抗体免疫复合物的形成或检测所述免疫复合物的量。所述免疫复合物的形成表明存在TSHR或表达TSHR的细胞。
本申请还提供了诊断与TSHR相关的疾病的方法，其包括使用第十五方面所述的方法检测来自受试者的样品中TSHR的存在或其水平。在某些实施方案中，当存在TSHR或与参照水平相比(例如与健康对照相比)TSHR的水平增加时，表明所述受试者患有与TSHR相关的疾病。
在某些实施方案中，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合。
第十六方面，本申请还提供了如第一方面、第二方面或第三方面所述的抗体或其抗原结合片段或第十三方面所述的缀合物在制备检测试剂中的用途，所述检测试剂用于检测TSHR在样品中的存在或其水平和/或诊断与TSHR相关的疾病。
在某些实施方案中，所述检测试剂通过第十五方面所述的方法来检测TSHR在样品中的存在或其水平。
在某些实施方案中，所述检测试剂通过第十五方面所述的方法来检测TSHR在样品中的存在或其水平从而诊断与TSHR相关的疾病。在某些实施方案中，当存在TSHR或与参照水平相比(例如与健康对照相比)TSHR的水平增加时，表明所述受试者患有与TSHR相关的疾病。
在某些实施方案中，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合。
在某些实施方案中，所述样品为来自受试者(例如哺乳动物，优选人、鼠或猴)的组 织样品。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的病毒学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
当本文使用术语“例如”、“如”、“诸如”、“包括”、“包含”或其变体时，这些术语将不被认为是限制性术语，而将被解释为表示“但不限于”或“不限于”。
除非本文另外指明或根据上下文明显矛盾，否则术语“一个”和“一种”以及“该”和类似指称物在描述本发明的上下文中(尤其在以下权利要求的上下文中)应被解释成覆盖单数和复数。
本文所用的术语“抗体”是指能够特异性结合靶抗原的源自免疫球蛋白的分子，所述源自免疫球蛋白的分子通过位于其可变区中的至少一个抗原结合位点来结合所述靶抗原。当提及术语“抗体”时，除非上下文明确指出，其不仅包括完整抗体，而且包括能够特异性结合靶抗原的抗原结合片段。“完整抗体”典型地由两对多肽链(每对具有一条轻链(LC)和一条重链(HC))组成。抗体轻链可分类为κ(kappa)和λ(lambda)轻链。重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE。在轻链和重链内，可变区和恒定区通过大约12或更多个氨基酸的“J”区连接，重链还包含大约3个或更多个氨基酸的“D”区。各重链由重链可变区(VH)和重链恒定区(CH)组成。重链恒定区由3个结构域(CH1、CH2和CH3)组成。各轻链由轻链可变区(VL)和轻链恒定区(CL)组成。轻链恒定区由一个结构域CL组成。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如可介导免疫球蛋白与宿主组织或因子，包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(C1q)的结合。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的称为框架区(FR)的区域。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。氨基酸在各区域或结构域的分配可遵循Kabat,Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda,Md.(1987and 1991))，或Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883的定义。
如本文中所使用的，术语“互补决定区”或“CDR”是指抗体可变区中负责抗原结合的氨基酸残基。在重链和轻链的可变区中各含有三个CDR，命名为CDR1、CDR2和CDR3。这些CDR的精确边界可根据本领域已知的各种编号系统进行定义，例如可按照Kabat编号系统(Kabat et al.,Sequences of Proteins of Immunological Interest,5th Ed.Public Health Service,National Institutes of Health,Bethesda,Md.,1991)、Chothia编号系统(Chothia&Lesk(1987)J.Mol.Biol.196:901-917；Chothia等人(1989)Nature 342:878-883)或IMGT编号系统(Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)中的定义。对于给定的抗体，本领域技术人员将容易地鉴别各编号系统所定义的CDR。并且，不同编号系统之间的对应关系是本领域技术人员熟知的(例如，可参见Lefranc et al.,Dev.Comparat.Immunol.27:55-77,2003)。
在本发明中，本发明的抗体或其抗原结合片段含有的CDR可根据本领域已知的各种编号系统确定。在某些实施方案中，本发明的抗体或其抗原结合片段含有的CDR优选地通过Kabat、Chothia或IMGT编号系统确定。
如本文中所使用的，术语“框架区”或“FR”残基是指，抗体可变区中除了如上定义的CDR残基以外的那些氨基酸残基。
术语“抗体”不受任何特定的产生抗体的方法限制。例如，其包括，重组抗体、单克隆抗体和多克隆抗体。抗体可以是不同同种型的抗体，例如，IgG(例如，IgG1，IgG2，IgG3或IgG4亚型)，IgA1，IgA2，IgD，IgE或IgM抗体。
如本文中所使用的，术语抗体的“抗原结合片段”是指包含全长抗体的片段的多肽，其保持特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合，其也被称为“抗原结合部分”。通常参见，Fundamental Immunology,Ch.7(Paul,W.,ed.,第2版，Raven Press,N.Y.(1989)，其以其全文通过引用合并入本文，用于所有目的。可通过重组DNA技术或通过完整抗体的酶促或化学断裂产生抗体的抗原结合片段。抗原结合片段的非限制性实例包括Fab、Fab’、F(ab’)2、Fd、Fv、二硫键连接的Fv、scFv、di-scFv、双抗体(diabody)、单域抗体(single domain antibody)和这样的多肽，其包含足以赋予多肽特异性抗原结合能力的抗体的至少一部分。工程改造的抗体变体综述于Holliger等,2005；Nat Biotechnol,23:1126-1136中。
如本文中所使用的，术语“Fd”意指由VH和CH1结构域组成的抗体片段；术语“Fab片段”意指由VL、VH、CL和CH1结构域组成的抗体片段；术语“F(ab’)2片段”意指包含通过铰链区上的二硫桥连接的两个Fab片段的抗体片段；术语“Fab’片段”意指还原连接F(ab’)2片段中两个重链片段的二硫键后所获片段，由一条完整的轻链和重链的Fd片段(由VH和CH1结构域组成)组成。
如本文中所使用的，术语“Fv”意指由抗体的单臂的VL和VH结构域组成的抗体片段。Fv片段通常被认为是，能形成完整的抗原结合位点的最小抗体片段。一般认为，六个CDR赋予抗体的抗原结合特异性。然而，即便是一个可变区(例如Fd片段，其仅仅含有三个对抗原特异的CDR)也能够识别并结合抗原，尽管其亲和力可能低于完整的结合位点。
如本文中所使用的，术语“Fc”意指，由抗体的第一重链的第二、第三恒定区与第二重链的第二、第三恒定区经二硫键结合而形成的抗体片段。抗体的Fc片段具有多种不同的功能，但不参与抗原的结合。
如本文中所使用的，术语“scFv”是指，包含VL和VH结构域的单个多肽链，其中所述VL和VH通过接头(linker)相连(参见，例如,Bird等人,Science 242:423-426(1988)；Huston等人,Proc.Natl.Acad.Sci.USA 85:5879-5883(1988)；和Pluckthun，The Pharmacology of Monoclonal Antibodies，第113卷，Roseburg和Moore编，Springer-Verlag，纽约，第269-315页(1994))。此类scFv分子可具有一般结构：NH2-VL-接头-VH-COOH或NH2-VH-接头-VL-COOH。合适的现有技术接头由重复的GGGGS氨基酸序列或其变体组成。例如，可使用具有氨基酸序列(GGGGS)4的接头，但也可使用其变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。在一些情况下，scFv的VH与VL之间还可以存在二硫键。在本发明的某些实施方案中，scFv可形成di-scFv，其指的是两个或两个以上单个scFv串联而形成抗体。在本发明的某些实施方案中，scFv可形成(scFv)2，其指的是两个或两个以上单个scFv并联而形成抗体。
如本文中所使用的，术语“双抗体”意指，其VH和VL结构域在单个多肽链上表达，但使用太短的连接体以致不允许在相同链的两个结构域之间配对，从而迫使结构域与另一条链的互补结构域配对并且产生两个抗原结合部位(参见，例如,Holliger P.等人,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993)，和Poljak R.J.等人,Structure 2:1121-1123(1994))。
如本文中所使用的，术语“单域抗体(single-domain antibody,sdAb)”具有本领域技术人员通常理解的含义，其是指由单个单体可变抗体结构域(例如单个重链可变区)所组成的抗体片段，其保持特异性结合全长抗体所结合的相同抗原的能力。单域抗体也称为纳米抗体(nanobody)。
如本文中所使用的，术语“双特异性抗体”是指对两种不同抗原(或表位)具有结合特异性的抗体。术语“多特异性抗体”是指对至少两种以上(例如三种或四种)不同抗原(或表位)具有结合特异性的抗体。双特异性抗体或多特异性抗体包含对不同抗原(或表位)具有结合特异性的多个抗原结合结构域，从而能够结合至少两个不同的结合位点和/或靶分子。双特异性抗体或多特异性抗体所包含的各个抗原结合结构域可以各自独立地选自全长抗体(例如IgG抗体)或其抗原结合片段(例如Fv片段、Fab片段、F(ab’)2片段或scFv)。在一些情况下，各个抗原结合结构域通过肽接头连接。
上述各个抗体片段均保持了特异性结合全长抗体所结合的相同抗原的能力，和/或与全长抗体竞争对抗原的特异性结合。
可使用本领域技术人员已知的常规技术(例如，重组DNA技术或酶促或化学断裂法)从给定的抗体(例如本发明提供的抗体)获得抗体的抗原结合片段(例如，上述抗体片段)，并且以与用于完整抗体的方式相同的方式就特异性筛选抗体的抗原结合片段。
如本文中所使用的，术语“人源化抗体”是指，经基因工程改造的非人源抗体，其氨基酸序列经修饰以提高与人源抗体的序列的同源性。通常而言，人源化抗体的全部或部分CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。在某些实施方案中，人源化抗体的CDR区来自于非人源抗体(供体抗体)，全部或部分的非CDR区(例如，可变区FR和/或恒定区)来自于人源免疫球蛋白(受体抗体)。人源化抗体通常保留了供体抗体的预期性质，包括但不限于，抗原特异性、亲和性、反应性等。在本申请中，供体抗体可以是有预期性质(例如，抗原特异性、亲和性、反应性等)的鼠源抗体。为制备人源化抗体，可以使用本领域已知的方法将供体抗体的CDR区插入人源框架序列。在一些情形中，所述人源框架序列可以包含由相应非人类残基替换的氨基酸突变。此外，人源化抗体还可包含在初始供体抗体可变区(例如，轻链可变区或重链可变区)或人源框架序列中均未发现的残基，以进一步改进或优化该人源化抗体的性能。
如本文中所使用的，术语“嵌合抗体(Chimeric antibody)”是指，这样的抗体，其轻链或/和重链的一部分源自一个抗体(其可以源自某一特定物种或属于某一特定抗体类或亚类)，且轻链或/和重链的另一部分源自另一个抗体(其可以源自相同或不同的物种或属于相同或不同的抗体类或亚类)，但无论如何，其仍保留对目标抗原的结合活性(U.S.P 4,816,567 to Cabilly et al.；Morrison et al.,Proc.Natl.Acad.Sci.USA,81:6851-6855(1984))。在某些实施方案中，术语“嵌合抗体”可包括这样的抗体，其中抗体的重链可变区和轻链可变区来自第一抗体，而抗体的重链恒定区和轻链恒定区来自第二抗体。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。为了测定两个氨基酸序列或两个核酸序列的百分比同一性，为了最佳比较目的将序列进行比对(例如，可在第一氨基酸序列或核酸序列中引入缺口以与第二氨基酸或核酸序列最佳比对)。然后比较对应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的对应位置相同的氨基酸残基或核苷酸占据时，则分子在该位置上是同一的。两个序列之间的百分比同一性是由序列所共享的同一性位置的数目的函数(即，百分比同一性＝同一重叠位置的数目/位置的总数×100％)。在某些实施方案中，两个序列长度相同。
两个序列之间的百分比同一性的测定还可使用数学算法来实现。用于两个序列的比较的数学算法的一个非限制性实例是Karlin和Altschul的算法，1990,Proc.Natl.Acad.Sci.U.S.A.87:2264-2268，如同Karlin和Altschul,1993,Proc.Natl.Acad.Sci.U.S.A.90:5873-5877中改进的。将这样的算法整合至Altschul等人,1990,J.Mol.Biol.215:403的NBLAST和XBLAST程序中。
如本文中所使用的，术语“变体”，在多肽的情境中(包括多肽)也指包含已通过引入氨基酸残基置换、缺失或添加改变的氨基酸序列的多肽或肽。在某些情况下，术语“变体”还指已被修饰(即，通过将任何类型的分子共价连接至多肽或肽)的多肽或肽。例如，但非限制性地，多肽可以被修饰，例如通过糖基化、乙酰化、聚乙二醇化、磷酸化、酰胺化、通过已知保护/封闭基团进行的衍生化、蛋白水解切割、连接至细胞配体或其它蛋白质等。衍生多肽或肽可使用本领域技术人员已知的技术通过化学修饰来产生，所述技术包括但不限于特异性化学切割、乙酰化、甲酰化、衣霉素的代谢合成等。此外，变体具有与其所源自的多肽或肽相似、相同或改善的功能。
如本文中所使用的，术语“特异性结合”是指，两分子间的非随机的结合反应，如抗体和其所针对的抗原之间的反应。特异性结合相互作用的强度或亲和力可以该相互作用的平衡解离常数(KD)表示。在本发明中，术语“KD”是指特定抗体-抗原相互作用的解离平衡常数，其用于描述抗体与抗原之间的结合亲和力。平衡解离常数越小，抗体-抗原结合越紧密，抗体与抗原之间的亲和力越高。
两分子间的特异性结合性质可使用本领域公知的方法进行测定。一种方法涉及测量抗原结合位点/抗原复合物形成和解离的速度。“结合速率常数”(ka或kon)和“解离速率常数”(kdis或koff)两者都可通过浓度及缔合和解离的实际速率而计算得出(参见Malmqvist M,Nature,1993,361:186-187)。kdis/kon的比率等于解离常数KD(参见Davies等人,Annual Rev Biochem,1990；59:439-473)。可用任何有效的方法测量KD、 kon和kdis值。在某些实施方案中，可以使用表面等离子体共振术(SPR)在Biacore中来测量解离常数。除此以外还可用生物发光干涉测量法或Kinexa来测量解离常数。
如本文中所使用的，本发明所述的可检测的标记可以是可通过荧光、光谱、光化学、生物化学、免疫学、电学、光学或化学手段检测的任何物质。这类标记是本领域熟知的，其实例包括但不限于，酶(例如，辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、脲酶、葡萄糖氧化酶，等)、放射性核素(例如，3H、125I、35S、14C或32P)、荧光染料(例如，异硫氰酸荧光素(FITC)、荧光素、异硫氰酸四甲基罗丹明(TRITC)、荧光蛋白(如藻红蛋白(PE))、德克萨斯红、罗丹明、量子点或花菁染料衍生物(例如Cy7、Alexa750))、发光物质(例如化学发光试剂，如吖啶酯类化合物、鲁米诺及其衍生物、钌衍生物如三联吡啶钌)、磁珠(例如，)、测热标记物例如胶体金或有色玻璃或塑料(例如，聚苯乙烯、聚丙烯、乳胶，等)珠、以及用于结合上述标记物修饰的亲和素(例如，链霉亲和素)的生物素。在某些实施方案中，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。在某些实施方案中，可通过不同长度的接头(linker)将如上所述的可检测标记连接至本发明的抗体或其抗原结合片段，以降低潜在的位阻。
如本文中所使用的，术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多聚核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的预期性质的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点 诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997)，其通过引用并入本文)。
本文涉及的二十个常规氨基酸的编写遵循常规用法。参见例如，Immunology-A Synthesis(2nd Edition,E.S.Golub and D.R.Gren,Eds.,Sinauer Associates,Sunderland,Mass.(1991))，其以引用的方式并入本文中。在本发明中，术语“多肽”和“蛋白质”具有相同的含义且可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂，表面活性剂，佐剂，离子强度增强剂，稀释剂，维持渗透压的试剂，延迟吸收的试剂，防腐剂，稳定剂。例如，pH调节剂包括但不限于磷酸盐缓冲液。表面活性剂包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80。离子强度增强剂包括但不限于氯化钠。维持渗透压的试剂包括但不限于糖、NaCl及其类似物。延迟吸收的试剂包括但不限于单硬脂酸盐和明胶。稀释剂包括但不限于水，水性缓冲液(如缓冲盐水)，醇和多元醇(如甘油)等。防腐剂包括但不限于各种抗细菌试剂和抗真菌试剂，例如硫柳汞，2-苯氧乙醇，对羟苯甲酸酯，三氯叔丁醇，苯酚，山梨酸等。稳定剂具有本领域技术人员通常理解的含义，其能够稳定药物中的活性成分的期望活性，包括但不限于谷氨酸钠，明胶，SPGA，糖类(如山梨醇，甘露醇，淀粉，蔗糖，乳糖，葡聚糖，或葡萄糖)，氨基酸(如谷氨酸，甘氨酸)，蛋白质(如干燥乳清，白蛋白或酪蛋白)或其降解产物(如乳 白蛋白水解物)等。在某些示例性实施方案中，所述药学上可接受的载体或赋形剂包括无菌可注射液体(如水性或非水性悬浮液或溶液)。在某些示例性实施方案中，此类无菌可注射液体选自注射用水(WFI)、抑菌性注射用水(BWFI)、氯化钠溶液(例如0.9％NaCl)、葡萄糖溶液(例如5％葡萄糖)、含有表面活性剂的溶液(例如0.01％聚山梨醇20)、pH缓冲溶液(例如磷酸盐缓冲溶液)、Ringer氏溶液及其任意组合。
如本文中所使用的，术语“预防”是指，为了阻止或延迟疾病或病症或症状在受试者体内的发生而实施的方法。如本文中所使用的，术语“治疗”是指，为了获得有益或所需临床结果而实施的方法。为了本发明的目的，有益或所需的临床结果包括(但不限于)减轻症状、缩小疾病的范围、稳定(即，不再恶化)疾病的状态，延迟或减缓疾病的发展、改善或减轻疾病的状态、和缓解症状(无论部分或全部)，无论是可检测或是不可检测的。此外，“治疗”还可以指，与预期的存活期(如，未接受治疗的存活期)相比，延长存活期。
如本文中所使用的，术语“受试者”是指哺乳动物，例如人、鼠或猴。在某些实施方案中，所述受试者(例如人、鼠或猴)患有与TSHR相关的疾病(例如，格雷夫斯病、格雷夫斯眼病)，或者，具有患有上述疾病的风险。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病(例如，格雷夫斯病、格雷夫斯眼病)有效量是指，足以预防，阻止，或延迟所述疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
发明的有益效果
本申请提供的抗体与人TSHR具有高亲和力，并且具有针对人/鼠/猴TSHR的交叉结合活性。此外，本申请的抗体具备显著的TSHR活性抑制作用，其可通过阻断TSHR与激活型抗体的结合作用，抑制由TSHR与激活型抗体的结合介导的下游信号通路。经验证，本申请的抗体在体外功能性实验中具备显著抑制GO病人原代眼眶纤维原细胞透明质酸、白介素6及白介素8分泌的功能。
下面将结合附图和实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本发明，而不是对本发明的范围的限定。根据附图和 优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
附图说明
图1：改造抗体抑制GD病人血清诱导甲状腺细胞产生TPO。
序列信息
本申请涉及的序列的描述提供于下表中。
表1：序列信息

具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
实施例1：抗体制备
1、杂交瘤抗体的制备：
1.1用人TSHR抗原免疫小鼠后选择具有较高效价的小鼠用于细胞融合和杂交瘤制备。
1.2准备骨髓瘤细胞，融合前，用计数仪计数并检测活力，收集SP2/0细胞，室温下400g，离心5分钟，弃掉培养液，用20ml融合培养液重悬细胞沉淀。
1.3收集和培养脾细胞，选择对免疫原反应较强的小鼠用于制备杂交瘤细胞，按照IACUC批准的方法，使用二氧化碳安乐死小鼠，采集融合前血浆，分离和收集血清(FB)，作为杂交瘤上清筛选的阳性对照。
1.4将小鼠浸泡在70％酒精中进行消毒处理，然后立即转移到生物安全柜进行操作，收集脾脏，置于无菌培养皿，将脾脏置于滤网上，用无菌注射器的栓塞反复挤压脾脏直至无明显团块，然后用融合培养液冲洗滤网，以获得脾细胞悬液，将其全部转移至50毫升离心管。
1.5在400g下离心5分钟，弃掉上清，采用电融合方法制备融合细胞，用5ml红细胞裂解液重悬脾细胞沉淀，然后置于4摄氏度静置5分钟，用添加10％FBS的50毫升DEME培养液终止反应。离心收集脾细胞沉淀，然后加入融合培养液重悬细胞，颠倒离心管3-5次，然后进行计数。400g下离心5分钟，弃掉上清，保留细胞沉淀，然后用40ml融合培养液重悬细胞。将脾细胞于室温静置2-3分钟，然后转移到新50ml离心管中。按照脾细胞对SP2/0细胞4:1的比例，将SP2/0细胞加到脾细胞中，用融合培养液定容到50ml。将细胞混合液离心，弃上清，通过反复敲打管子底部使细胞沉淀松散。融合，对于电融合，将细胞混合物置于电击槽内，按照优化好的程序进行电击融合。
1.6电融合后，融合细胞需在电击槽内额外静置10分钟。用添加20％FBS和HT的DMEM培养液重悬，按照100μl每孔的体积接种至96孔微孔板。24小时后，将添加20％FBS和HAT的DMEM培养液加到孔板中，100微升每孔。将全部融合板转移回二氧化碳培养箱，37℃,5％CO2。每天监测细胞生长速度和是否有微生物污染。当杂交瘤克隆生长到1-2mm直径(一般融合后10-14天)大小时，用Acumen筛选杂交瘤上清。
1.7亚克隆和扩增培养。将阳性克隆转移到24孔板，用扩增培养液进行培养。收集杂交瘤培养上清进行FACS检测。通过有限稀释法，对阳性的母克隆进行亚克隆以获得单克隆。将亚克隆96孔板置于二氧化碳培养箱，培养7天，然后检测筛选上清活性。为了确保单克隆，需要进行1-2轮亚克隆。根据筛选结果，选择每块亚克隆板选择4个活性最好的单克隆，转移至24孔板进行扩增培养。
1.8细胞冻存。将扩增起来的亚克隆细胞株进行冻存，保存于添加20％FBS，10％DMSO的DMEM培养液中，放置于液氮罐进行长期保存。
通过上述方法制备获得鼠源单克隆抗体mAb001、mAb012和mAb021，其CDR及可变区序列如表2所示。
表2鼠源抗体可变区及CDR序列信息
2.鼠源抗体人源化：
使用CDR移植设计人源化抗体，BLAST人抗体中与鼠源序列最接近的序列作为可变区骨架，替换鼠源框架区，将亲本抗体的CDRs移植到人受体中，并分析了所有序列的翻译后修饰(PTM)，比如异构化，脱酰胺基和糖基化等风险，设计了合适的PTM去除突变，最终获得每个亲本抗体的各人源化轻链和人源化重链。将每个亲本抗体的各人源化轻重链可变区相互配对进行亲和力排序实验。然后将重链可变区与轻链可变区分别与人IgG4-S228P重链恒定区(SEQ ID NO:25)和人kappa轻链恒定区(SEQ ID NO:26)连接获得人源化抗体重链和轻链全长序列。
合成编码人源化抗体重链和轻链的DNA序列，插入pcDNA3.4载体，构建全长抗体表达质粒。在Expi293F细胞培养物中进行人源化抗体的表达，并用蛋白A亲和柱纯化上清液。使用PD-10脱盐柱将纯化的抗体缓冲液交换为PBS。纯化蛋白的浓度和纯度分别通过OD280和SDS-PAGE测定。
通过上述方法获得各鼠源单抗的人源化抗体，各人源化抗体的轻重链可变区氨基酸序列如SEQ ID NO:27-38所示。
实施例2：鼠源抗体细胞结合实验
实验操作步骤：
通过流式细胞术(FACS)的方法检测鼠源抗体与293-人TSHR，293-小鼠TSHR，293-猴TSHR过表达细胞系(所用细胞为过表达人、猴或小鼠来源的TSHR的293细胞，其中，所述人、猴和小鼠来源的TSHR的氨基酸序列分别参见P16473,A0A2K5X226,P47750 Uniprot)的结合强度。实验方法如下：收集对数生长期的细胞，用细胞计数仪(Countstar；IC1000)计数；将细胞重悬至冷的FACS缓冲液，缓冲液为500ml PBS(HyClone，SH30256.01B)中加入50ml FBS(BI；04-002-1A)中，细胞密度调整到5×106个细胞每毫升；细胞悬液置于室温静置10到20分钟后，将其加到FACS板中(Corning，3799)，每孔100μL，离心弃上清；用FACS缓冲液稀释待测抗体，起始浓度200nM，1:3梯度稀释；将稀释好的抗体加入上述细胞中，每孔100μL，4度孵育1小时；孵育完成后，用FACS缓冲液清洗细胞，每孔250μL，300g(4度)离心5分钟，清洗三遍；加入FACS缓冲液配制二抗抗小鼠IgG-Alexa488抗体(invitrogen；A21202)(1:1k的稀释度)，每孔100μL，4度避光孵育1小时后，再次用FACS缓冲液洗细胞三次；细胞沉淀用100μL 4度FACS 缓冲液重悬，通过流式细胞仪(BECKMAN COULTER；B53013)读取每孔细胞的荧光强度，即结合信号。
数据处理(数据处理的方法)和实验数据：
将抗体每个浓度对应的结合信号值用graphpad做非线性拟合，表3中的数据为抗体的最大结合信号和曲线拟合计算出的EC50。
表3各抗原最大结合信号及EC50
实验结论：
所选抗体与过表达人TSHR的293细胞系均具有较好的结合活性，且均具有猴、鼠交叉结合活性。
实施例3：人源化抗体细胞结合实验
实验操作步骤：
参照实施例2所述FACS方法检测人源化抗体与293-人TSHR，293-小鼠TSHR，293-猴TSHR过表达细胞系的结合作用。其中，二抗使用抗人IgG-Alexa488抗体(invitrogen；A-11013)(1:1k的稀释度)。
数据处理(数据处理的方法)和实验数据：
将抗体每个浓度对应的结合信号值用Graphpad做非线性拟合，表4和表5中的数据为抗体的最大结合信号和曲线拟合计算出的EC50。
表4各抗原最大结合信号及EC50
表5各抗原最大结合信号及EC50
实验结论：
以上人源化抗体与293-人TSHR细胞均有较好的结合活性，且同时具备结合猴TSHR和鼠TSHR的能力。
实施例4：人源化抗体cAMP实验
实验操作步骤：
抗体配制：使用Bravo(Agilent)仪器在384孔板(3824Corning)配制不同浓度的抗体，配制溶液为DPBS，起始浓度200nM，1:3稀释，共11个点。
人源化抗体阻断激动型抗体激活cAMP实验：根据cAMP-Gs DYNAMIC Kit(62AM4PEB,Cisbio)使用说明书，完成抗体cAMP的检测。其中，使用的细胞系为293-人TSHR细胞系；竞争抗体分别为Tab01，Tab02，均为激动型抗体，使用浓度分别为30nM，6.5nM；加样仪器为MultiDrop Combi(Thermo)，设置为低速；数据读取仪器为Envision(PerkinElmer)读板检测665nM和615nM波长的信号，以标准品浓度和波长665nM/615nM的信号比值做标准曲线，检测抗体的相对活性。
Tab01为TSHR激动型抗体M22,序列来源于专利WO 2004/050708A2中序列编号为No.1的抗体重链可变区及序列编号为No.6的轻链可变区。将所述重链可变区与所述轻链可变区分别与人IgG1重链恒定区和人lambda轻链恒定区连接获得Tab01重链和轻链全长序列，人IgG1重链恒定区和人lambda轻链恒定区序列参照阳性对照Tab03_hIgG1重链恒定区和轻链恒定区序列。
Tab02为TSHR激动型抗体K1-18，序列来源于专利号为US10428153的TSHR抗体专利中序列编号为No.15的重链序列和序列编号为No.33的轻链序列。
阳性对照为Tab03_hIgG1，序列来源于专利号为US10428153的TSHR抗体专利中序列编号为No.51的重链序列和No.59的轻链序列。
数据处理(数据处理的方法)和实验数据：
将波长665nM和615nM的信号计算比值，比值与cAMP的相对含量成正比，表示TSHR的相对活性。将抗体浓度和相应665nM/615nM比值做非线性回归曲线，计算IC50 和最大抑制率，结果如表6和表7所示。
表6各抗体IC50和最大抑制率
表7各抗体IC50和最大抑制率
实验结论：
所有抗体都表现出对TSHR活性的显著抑制(在激动型抗体Tab01和Tab02存在的情况下)。
实施例5：人源化抗体透明质酸(HA)分泌实验
实验操作步骤：
1成纤维细胞的获取
实验分为三组，细胞来源分别为急性期GO病人和稳定期GO病人。细胞获取方法如下。获取病人眼眶结缔部位组织标本，每组2个病人，将标本迅速放入装有DMEM/F12培养基(含青霉素-链霉素混合液)的无菌离心管中，随后将装有组织的离心管迅速转移至细胞培养实验室；在超净台中夹取离心管中的组织使用无菌PBS反复洗涤去除血渍后，转移至加有DMEM/F12培养基(含青霉素-链霉素混合液)的无菌培养皿中，保证标本完全浸润在培养基中，以维持组织湿润状态以及活性；使用显微眼科剪及镊子小心剔除脂肪组织和较大的血管，将分离出来的结缔组织使用PBS清洗3遍后，放置于含有少许DMEM/F12培养基的无菌培养皿中，用剪刀和镊子剪成约1mm3左右的组织块。用完全培养基(DMEM/F12培养基加20％胎牛血清，加1％体积分数青霉素和链霉素)将细胞培养板润洗一遍，待组织稍干后小心贴于细胞培养板上。将贴有组织的细胞培养板置于37℃，5％CO2细胞培养箱中倒置30min，使组织尽快贴壁。30min后将细胞培养板翻转平放，加入完全培养基，以刚好没过组织即可，培养数天。3d后半量换液，小心除去漂 浮组织块和残留的红细胞，观察细胞爬出情况并添加培养基，每5～7天使用0.25％胰酶-EDTA消化，按照1:3-1:4传代培养。
2 GO病人血清免疫球蛋白(GO-Igs)获取
收集40个GO病人的血清样本使用3K15型低温高速离心机，4℃，12000rpm/min，离心10min，使用0.45μm滤膜孔径的过滤器进行过滤，收集血清备用，使用亲硫超流树脂(Thiophillic-Superflow，635617，Takara公司)填充容积为2mL的重力流柱(29920，Thermo Fisher Scientific公司)，于4℃沉淀过夜；然后使用10倍柱体积的平衡缓冲液(50mM磷酸钠和0.5M硫酸钠，pH7.0)冲洗亲和层析重力流柱进行平衡。把收集的血清样品按1:10(体积比)的比例稀释于样品缓冲液(50mM磷酸钠和0.55M硫酸钠，pH7.0)中，将稀释后的血清样本加入亲和层析重力流柱中，控制样品流速为2ml/min，然后使用平衡缓冲液(50mM磷酸钠和0.5M硫酸钠，pH7.0)洗涤未结合蛋白，最后使用2-3倍柱体积的洗脱缓冲液(20mM磷酸钠和20％甘油，pH7.0)来洗脱GO-Igs，收集并合并洗脱产物，将洗脱产物至于Tube-O-Dialyzer透析管中(786-619，G-Biosciences公司；截留分子量为50K)在含10mM HEPES(pH 7.4)的Hanks平衡盐溶液(HBSS)中4℃透析过夜，最后使用Spin-X UF浓缩器(CLS431480，Sigma公司)对透析后的样品进行浓缩，浓缩产物使用Pierce BCA蛋白测定法来测定蛋白浓度。
3 HA分泌实验
3.1细胞培养
将获取的各组病人的成纤维细胞，分别用Countess 3自动细胞计数仪(Thermo Fisher Scientific公司)进行细胞计数，调整密度后，向96孔板中每孔接种5000或10000个细胞，将96孔板置于Forma 3111型水套式CO2培养箱(Thermo Electron company)中培养过夜，在镜下观察所有细胞贴壁后饥饿培养24h，去上清，按照实验分组加入不同浓度的待测抗体孵育24h后，加入GO-Igs继续孵育96h，小心收集细胞上清，将上清使用3K15型低温高速离心机1000×g离心15分钟，弃去细胞碎片，取上清待检测。
3.2 HA检测
本次实验使用人透明质酸(HA)试剂盒(ELISA)(ml557801，上海酶联生物科技有限公司)，具体过程如下：从室温平衡60min后的铝箔袋中取出所需板条。设置标准品孔和样本孔，标准品孔各加不同浓度的标准品50μL；200ng/mL、100ng/mL、50ng/mL、25ng/mL、12.5ng/mL、6.25ng/mL。样本孔中加入经上述处理后的上清50μL；空白孔不加，37℃恒温箱孵育1小时。用洗涤液重复洗涤三次。除空白孔外，标准品孔和样本孔中每孔加入辣根过氧化物酶(HRP)标记的检测抗体100μL，37℃恒温箱温育60min，洗涤液洗5次。每孔加入底物A、B各50μL，37℃避光孵育15min。每孔加入终止液50μL，15min 内，使用酶标仪(Tecan Infinite 200 PRO酶标仪，Tecan公司)在450nm波长处测定各孔的OD值。
数据处理(数据处理的方法)和实验数据：
以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘制标准曲线，并得到直线回归方程，将样本孔的OD值代入方程，计算出每孔HA的浓度。通过Graphpad prism9对HA浓度及抗体浓度进行非线性拟合，计算出IC50浓度，结果如表8所示。
表8各抗体最大抑制率及IC50
实验结论：
根据以上结果可知，所选抗体均能抑制GO-Igs刺激病人眼眶成纤维细胞产生HA，其中cAb01-VH1-DA-VL2，cAb01-VH2-DA-VL2,cAb01-VH3-DA-VL2，cAb01-VH1-DA-VL3，cAb01-VH2-DA-VL3,cAb12-VH1-QG&S-VL3，cAb12-VH3-QG&S-VL3对于急性期和稳定期的细胞具有较好的抑制作用，在稳定期最大抑制率优于对照抗体Tab03_hIgG1；cAb01-VH2-DA-VL3和cAb12-VH3-QG&S-VL3IC50优于阳性对照Tab03_hIgG1；cAb21-VH2-VL2和cAb21-VH1-VL3对稳定期细胞有较好的抑制作用，最大抑制率优于Tab03_hIgG1。
实施例6：人源化抗体IL6分泌实验
实验操作步骤：
1细胞培养
根据实施例5所述的方法收集成纤维细胞及GO-Igs，采用Countess 3自动细胞计数仪(Thermo Fisher Scientific公司)对细胞进行计数，调整密度后，向96孔板中每孔接种5000或10000个细胞，将96孔板置于Forma 3111型水套式CO2培养箱(Thermo Electron  company)中培养过夜，在镜下观察所有细胞贴壁后饥饿培养24h，去上清，按照实验分组加入不同浓度的待测抗体孵育24h后，加入GO-Igs继续孵育24h，小心收集细胞上清，将上清使用3K15型低温高速离心机1000×g离心15分钟，弃去细胞碎片，取上清待检测。
2 IL6检测
本次实验使用Human IL-6 ELISA试剂盒(CSB-E04638h，Cusabio公司)，具体过程如下：实验前将相应试剂放置室温下60min，每孔加标准品和样品100μl，空白孔不加，标准品浓度为500pg/ml、250pg/ml、125pg/ml、62.5pg/ml、31.2pg/ml、15.6pg/ml、7.8pg/ml，封板，37℃孵育2小时。弃去每孔液体，每孔加100μl 1×Biotin-antibody，37℃恒温箱孵育1小时。用洗涤液重复洗涤三次。每孔加1×HRP-avidin 100μl，用新的胶条封板，37℃孵育1小时。清洗5次，每孔加入90μl TMB溶液，37℃避光孵育15分钟。每孔加入50μl的Stop Solution，轻轻敲板，以确保充分混合，15min内，使用酶标仪(Tecan Infinite 200 PRO酶标仪，Tecan公司)在450nm波长处测定各孔的OD值。
数据处理(数据处理的方法)和实验数据：
以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出每孔上清的IL6浓度。通过Graphpad prism9对IL6浓度及抗体浓度进行非线性拟合，计算出IC50浓度，结果如表9所示。
表9各抗体最大抑制率及IC50
实验结论：
所选抗体均能抑制GO-Igs刺激病人眼眶成纤维细胞产生IL6，所有抗体对于急性期和稳定期的细胞具有较好的抑制作用，其中cAb01-VH1-DA-VL2，cAb01-VH1-DA-VL3,cAb01-VH2-DA-VL3,cAb21-VH2-VL2对于急性期和稳定期的细胞最大抑制率和IC50优于对照抗体Tab03_hIgG1。
实施例7：人源化抗体IL8分泌实验
实验操作步骤：
1细胞培养
根据实施例6所述方法进行细胞培养，收集上清待检测。
2 IL8检测
本实验使用Human IL-8 ELISA试剂盒(D8000C，R&D公司)检测待测上清中IL8的浓度。
数据处理(数据处理的方法)和实验数据：
以所测标准品的OD值为横坐标，标准品的浓度值为纵坐标，绘制标准曲线，并得到直线回归方程，将样品的OD值代入方程，计算出每孔上清的IL8浓度。通过Graphpad prism9对IL8浓度及抗体浓度进行非线性拟合，计算出IC50浓度。
数据分析结果如表10。
表10各抗体最大抑制率及IC50
实验结论：
所选抗体均能抑制GO-Igs刺激病人眼眶成纤维细胞产生IL8，所有抗体对于急性期和稳定期的细胞具有较好的抑制作用，最大抑制率几乎均优于阳性对照Tab03_hIgG1。
实施例8：人源化抗体的改造
本实施例选取抗体cAb01-VH2-DA-VL3(命名为hAb01_G4P，其中恒定区为IgG4亚型，包含S228P突变)进行Fc改造(所述改造包括基于IgG4-S228P的改造，以及基于IgG1的改造)，改造目的为延长半衰期，增加药效和减少ADCC作用。改造位点如下表所示：
表11 hAb01 Fc改造位点
实施例9：改造抗体的结合活性
参照实施例2所述FACS方法检测改造抗体与293-人TSHR，293-小鼠TSHR，293-猴TSHR过表达细胞系的结合作用。其中，二抗使用抗人IgG-Alexa488抗体(invitrogen；A-11013)(1:1k的稀释度)。
其中hIgG1为阴性对照(购自百英B117901)；hIgG4为阴性对照(购自百英B107804)。
检测结果如下：
表12改造抗体最大结合信号
注释：“/”代表拟合很差。
实验结论：
改造抗体与人TSHR结合良好，且有鼠、猴交叉结合活性。
实施例10：改造抗体的cAMP活性实验
参照实施例4所述cAMP的检测方法及数据处理方法，改造抗体的活性检测结果如下：
表13改造抗体cAMP活性的最大抑制率及IC50
注释：“/”代表拟合很差。
实验结论：
在cAMP活性实验中，所有改造抗体都表现出抑制激动型抗体(Tab01和Tab02)激活TSHR活性的作用，且抑制效果不弱于未改造分子hAb01_G4P。
实施例11：改造抗体的ADCC活性
实验方法：
实验的靶细胞为293-人TSHR细胞(过表达的人TSHR的氨基酸序列参见P16473，Uniprot)，效应细胞为人的PBMC(来源于妙顺，P122070903C)。实验前一天复苏效应细胞PBMC，实验当天收获，在实验缓冲液(RPMI 1640+10％FBS；RPMI Medium 1640，gibco，A10491-01；FBS，BI，040021A)中调整细胞浓度至5×106/mL，备用。使用荧光染料(Perkin Elmer，C136-100)标记1×106个靶细胞，37℃避光孵育20分钟后用PBS清洗细胞4次，将细胞重悬至实验缓冲液备用。使用实验缓冲液稀释抗体800nM起始，1：5梯度稀释，11个点，备用。在实验板中加50μL靶细胞，100μL抗体稀释液，50μL效应细胞。同时设定以下几个对照孔：靶细胞最大值(靶细胞50μL+实验缓冲液150μL)；靶细胞自发荧光值(靶细胞50μL+实验缓冲液150μL)；靶细胞和效应细胞自发荧光值(靶细胞50μL+效应细胞50μL+实验缓冲液100μL)。将上述实验板在37℃二氧化碳培养箱中孵育2h。加10uL裂解液至以下对照孔：靶细胞最大值，效应细胞最大值，靶细胞和效应细胞最大值。将实验板离心，400g 5min，取25μL上清至平底检测板，每孔加200uL Europium Solution(Perkin Elmer，C136-100)至待测板，以250rpm的速度震荡检测板，震荡15min。在多功能酶标仪中读取。
其中hAb01_G1轻链可变区与重链可变区序列与hAb01_G4P一致，恒定区亚型为hIgG1(重链全长氨基酸序列如SEQ ID NO：49所示，轻链全长氨基酸序列如SEQ ID NO：51所示)。
数据处理(数据处理的方法)和实验数据：
计算方法：抑制率％＝(样品值-靶细胞和效应细胞自发荧光值)/(靶细胞最大值-靶细胞自发荧光值)×100％。
以样品的浓度值为横坐标，抑制率为纵坐标，通过Graphpad prism9对抑制率及抗体浓度进行非线性拟合，计算出IC50浓度，结果如表14所示。
表14抗体ADCC抑制率及IC50
注释：“无”代表无ADCC作用。
实验结论：
与hAb01_G1相比，hAb01_G1_V4分子具有较弱ADCC作用。hAb01_G4P，hAb01_G4P_V1，hAb01_G4P_V2未展现出ADCC作用。
实施例12：改造抗体的FcRn结合活性
实验方法：本实验使用SA芯片(cytiva，BR100531)捕获hFcRn-Avi(百普赛斯，FCM-H82W7)，将不同浓度的抗体流过芯片，根据采集数据进行稳态拟合分析。实验仪器为Biacore分子互作仪(cytiva，Biacore 8K+)，所用缓冲溶液为HBS-EP Buffer(cytiva,BR100669)，pH6.0，结合时间240s，解离时间240s，流速30μL/min，25℃条件下进行检测，所得实验数据通过Biacore Insight Evaluation Software 3.0.12软件进行稳态拟合分析，实验结果如表15所示。
表15改造抗体的FcRn的KD值
实验结论：
相同Fc亚型的抗体对比，改造后抗体与FcRn的亲和力显著升高，说明改造后抗体可以延长半衰期。
实施例13：改造抗体针对GD适应症的体外实验
实验方法：
1病人原代甲状腺细胞提取及培养
甲状腺组织样本取自甲状腺癌全甲状腺切除术患者的正常甲状腺组织，用含3mg/mL IV型胶原酶(Life Technologies)的PBS溶液重悬组织，获得分散的细胞，进行传代扩增。
2.甲状腺过氧化氢酶(TPO)基因水平检测
收集各组的细胞，调整密度后，向24孔板中每孔接种6×104个细胞，培养过夜。贴壁后饥饿培养24h，去上清，按照实验分组加入不同浓度的待测抗体和含或不含GD血清(终浓度为1:100，体积比)的培养基后继续孵育48h，胰酶消化收集细胞，提取总RNA样品。按照试剂盒说明书上(iScript cDNA，Bio-Rad，1708891EDU)的流程配置逆转录反应体系，获得cDNA，-70℃保存。
实时定量PCR反应
按照试剂说明书(Takara，RR420A)的流程在冰盒上进行反应体系的配置，进行扩增。通过2-ΔΔCt公式对数据进行处理，以内参基因GAPDH为参照，得到目的mRNA的表达水平。所用引物见表16。
表16-实时定量PCR使用的引物
数据处理和实验数据：
数据采用Graphpad Prism 9(Version 9.4.0)进行分析与作图，Adobe Illustrator 2022 (Version 2022)进行整理合图，组间统计学差异采用one-way ANOVA检验。
实验结果如图1所示。
实验结论：
所有的改造抗体均可以抑制GD病人血清诱导的TPO，说明本发明抗体具有治疗GD疾病的潜力。
实施例14：改造抗体透明质酸(HA)分泌实验
实验方法及数据处理方法参照实施例5所述。
数据处理和实验数据：
注释：“/”代表拟合很差。
实验结论：
根据以上结果可知，改造抗体均能抑制GO-Igs刺激病人眼眶成纤维细胞产生HA，且对于急性期和稳定期的细胞具有较好的抑制作用。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (22)

  1. 能够特异性结合TSHR的抗体或其抗原结合片段，所述抗体或其抗原结合片段包含：
    (a)如SEQ ID NO:1、27、29、30任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:5、28、31任一项所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
    (b)如SEQ ID NO:9、32、34任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:13或33所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；或，
    (c)如SEQ ID NO:17、35、37任一项所示的重链可变区(VH)中含有的VH CDR1或其变体、VH CDR2或其变体以及VH CDR3或其变体；和/或，如SEQ ID NO:21、36、38任一项所示的轻链可变区(VL)中含有的VL CDR1或其变体、VL CDR2或其变体以及VL CDR3或其变体；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个或3个氨基酸的置换、缺失或添加，例如保守置换)；优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    (a)如SEQ ID NO:1、27、29、30任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:5、28、31任一项所示的轻链可变区(VL)中含有的3个CDR；
    (b)如SEQ ID NO:9、32、34任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:13或33所示的轻链可变区(VL)中含有的3个CDR；或，
    (c)如SEQ ID NO:17、35、37任一项所示的重链可变区(VH)中含有的3个CDR；和/或，如SEQ ID NO:21、36、38任一项所示的轻链可变区(VL)中含有的3个CDR；
    优选地，所述VH中含有的3个CDR和/或所述VL中含有的3个CDR由Kabat、IMGT或Chothia编号系统定义。
  2. 权利要求1所述的抗体或其抗原结合片段，其包含：
    (a)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:2的VH CDR1、序列为SEQ ID NO:3或39的VH CDR2、序列为SEQ ID NO:4的VH  CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:6的VL CDR1、序列为SEQ ID NO:7的VL CDR2、序列为SEQ ID NO:8的VL CDR3；
    (b)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:10的VH CDR1、序列为SEQ ID NO:11或40的VH CDR2、序列为SEQ ID NO:12或41的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:14的VL CDR1、序列为SEQ ID NO:15的VL CDR2、序列为SEQ ID NO:16的VL CDR3；或，
    (c)包含下述3个互补决定区(CDRs)的重链可变区(VH)：序列为SEQ ID NO:18的VH CDR1、序列为SEQ ID NO:19的VH CDR2、序列为SEQ ID NO:20的VH CDR3；和/或，包含下述3个互补决定区(CDRs)的轻链可变区(VL)：序列为SEQ ID NO:22的VL CDR1、序列为SEQ ID NO:23的VL CDR2、序列为SEQ ID NO:24的VL CDR3；
    其中，所述CDR由Kabat编号系统定义。
  3. 权利要求1或2所述的抗体或其抗原结合片段，其包含：
    (a)重链可变区(VH)，其包含SEQ ID NO：1所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：5所示的序列或其变体；
    (b)重链可变区(VH)，其包含SEQ ID NO：9所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：13所示的序列或其变体；或，
    (c)重链可变区(VH)，其包含SEQ ID NO：17所示的序列或其变体；和/或，轻链可变区(VL)，其包含SEQ ID NO：21所示的序列或其变体；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:1所示的序列的VH和包含如SEQ ID NO:5所示的序列的VL；
    优选地，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:9所示的序列的VH和包含如SEQ ID NO:13所示的序列的VL；
    优选地，所述抗体或其抗原结合片段包含：包含如SEQ ID NO:17所示的序列的VH和包含如SEQ ID NO:21所示的序列的VL。
  4. 权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含来源于人免疫球蛋白的框架区序列；
    优选地，所述抗体或其抗原结合片段包含：人重链胚系序列的重链框架区，和/或，人轻链胚系序列的轻链框架区。
  5. 权利要求4所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包含：
    (a)包含如SEQ ID NO:27、29、30任一项所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:28或31所示的序列或其变体的轻链可变区(VL)；
    (b)包含如SEQ ID NO:32或34所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:33所示的序列或其变体的轻链可变区(VL)；或，
    (c)包含如SEQ ID NO:35或37所示的序列或其变体的重链可变区(VH)；和/或，包含如SEQ ID NO:36或38所示的序列或其变体的轻链可变区(VL)；
    其中，所述变体与其所源自的序列相比具有一个或几个氨基酸的置换、缺失或添加(例如1个，2个，3个，4个或5个氨基酸的置换、缺失或添加)，或具有至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％、或100％的序列同一性的序列；
    优选地，所述的置换是保守置换；
    优选地，所述抗体或其抗原结合片段包含：
    (a)包含如SEQ ID NO:27、29、30任一项所示的重链可变区(VH)；和/或，包含如SEQ ID NO:28或31所示的轻链可变区(VL)；
    (b)包含如SEQ ID NO:32或34所示的重链可变区(VH)；和/或，包含如SEQ ID NO:33所示的轻链可变区(VL)；或，
    (c)包含如SEQ ID NO:35或37所示的重链可变区(VH)；和/或，包含如SEQ ID NO:36或38所示的轻链可变区(VL)；
    优选地，所述抗体或其抗原结合片段包含：
    (1)包含如SEQ ID NO:27所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL；
    (2)包含如SEQ ID NO:29所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL；
    (3)包含如SEQ ID NO:30所示的序列的VH和包含如SEQ ID NO:28所示的序列的VL；
    (4)包含如SEQ ID NO:27所示的序列的VH和包含如SEQ ID NO:31所示的序列的VL；
    (5)包含如SEQ ID NO:29所示的序列的VH和包含如SEQ ID NO:31所示的序列的VL；
    (6)包含如SEQ ID NO:32所示的序列的VH和包含如SEQ ID NO:33所示的序列的VL；
    (7)包含如SEQ ID NO:34所示的序列的VH和包含如SEQ ID NO:33所示的序列的VL；
    (8)包含如SEQ ID NO:35所示的序列的VH和包含如SEQ ID NO:36所示的序列的VL；或，
    (9)包含如SEQ ID NO:37所示的序列的VH和包含如SEQ ID NO:38所示的序列的VL。
  6. 权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段进一步包含来源于哺乳动物(例如人或鼠)免疫球蛋白的恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，所述抗体或其抗原结合片段的重链包含来源于鼠免疫球蛋白(例如IgG1、IgG2、IgG3或IgG4)的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于鼠免疫球蛋白(例如κ或λ)的轻链恒定区。
  7. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白IgG4的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，与野生型人免疫球蛋白IgG4的重链恒定区相比，所述来源于人免疫球蛋白IgG4的重链恒定区包含置换突变S228P、L235E、M252Y、S254T和/或T256E(例如，(i)S228P，(ii)L235E，和/或(iii)M252Y、S254T和T256E)；
    优选地，与野生型人免疫球蛋白IgG4的重链恒定区相比，所述来源于人免疫球蛋白IgG4的重链恒定区包含置换突变S228P；优选地，所述来源于人免疫球蛋白IgG4的重链恒定区进一步包含置换突变M252Y、S254T和T256E；更优选地，所述来源于人免疫球蛋白IgG4的重链恒定区进一步包含置换突变L235E、M252Y、S254T和T256E；
    优选地，所述抗体或其抗原结合片段的重链包含如SEQ ID NO:25、42-43任一项所示的重链恒定区(CH)，和/或，所述抗体或其抗原结合片段的轻链包含如SEQ ID NO:26所示的轻链恒定区(CL)；
    优选地，所述抗体或其抗原结合片段包含：
    (1)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (2)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (3)包含如SEQ ID NO:30所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (4)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (5)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (6)包含如SEQ ID NO:32所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (7)包含如SEQ ID NO:34所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (8)包含如SEQ ID NO:35所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:36所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；或，
    (9)包含如SEQ ID NO:37所示的序列的VH和SEQ ID NO:25、42-43任一项所示的序列的CH的重链，和/或，包含如SEQ ID NO:38所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链。
  8. 权利要求1-6任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段的重链包含来源于人免疫球蛋白IgG1的重链恒定区，和/或，所述抗体或其抗原结合片段的轻链包含来源于人免疫球蛋白(例如κ或λ)的轻链恒定区；
    优选地，与野生型人免疫球蛋白IgG1的重链恒定区相比，所述来源于人免疫球蛋白IgG1的重链恒定区包含置换突变L234A、L235A、M252Y、S254T和/或T256E(例如，(i)L234A和L235A，和/或(ii)M252Y、S254T和T256E)；
    优选地，与野生型人免疫球蛋白IgG1的重链恒定区相比，所述来源于人免疫球蛋白IgG1的重链恒定区包含置换突变L234A和L235A；优选地，所述来源于人免疫球蛋白IgG1的重链恒定区进一步包含置换突变M252Y、S254T和T256E；
    优选地，所述抗体或其抗原结合片段的重链包含如SEQ ID NO:44或45所示的重链恒定区(CH)，和/或，所述抗体或其抗原结合片段的轻链包含如SEQ ID NO:26所示的轻链恒定区(CL)；
    优选地，所述抗体或其抗原结合片段包含：
    (1)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (2)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (3)包含如SEQ ID NO:30所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:28所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (4)包含如SEQ ID NO:27所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (5)包含如SEQ ID NO:29所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:31所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (6)包含如SEQ ID NO:32所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (7)包含如SEQ ID NO:34所示的序列的VH和SEQ ID NO:44或45所示的序列的 CH的重链，和/或，包含如SEQ ID NO:33所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；
    (8)包含如SEQ ID NO:35所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:36所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链；或，
    (9)包含如SEQ ID NO:37所示的序列的VH和SEQ ID NO:44或45所示的序列的CH的重链，和/或，包含如SEQ ID NO:38所示的序列的VL和SEQ ID NO:26所示的序列的CL的轻链。
  9. 权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗原结合片段选自Fab、Fab’、(Fab’)2、Fd、Fv、二硫键连接的Fv、scFv、di-scFv、(scFv)2、双抗体(diabody)和单域抗体(sdAb)；和/或，所述抗体为鼠源抗体、人源化抗体、嵌合抗体、双特异性抗体或多特异性抗体。
  10. 分离的核酸分子，其编码权利要求1-9任一项所述的抗体或其抗原结合片段，或其重链可变区和/或轻链可变区，或其重链和/或轻链。
  11. 载体，其包含权利要求10所述的分离的核酸分子；优选地，所述载体为克隆载体或表达载体。
  12. 宿主细胞，其包含权利要求10所述的分离的核酸分子或权利要求11所述的载体。
  13. 制备权利要求1-9任一项所述的抗体或其抗原结合片段的方法，其包括，在允许所述抗体或其抗原结合片段表达的条件下，培养权利要求12所述的宿主细胞，和从培养的宿主细胞培养物中回收所述抗体或其抗原结合片段。
  14. 双特异性或多特异性分子，其包含权利要求1-9任一项所述的抗体或其抗原结合片段；
    优选地，所述双特异性或多特异性分子特异性结合TSHR，并且额外地特异性结合一个或多个其他靶标；
    优选地，所述双特异性或多特异性分子还包含至少一种具有针对第二靶标的第二结合特异性的分子(例如第二抗体)；
    优选地，所述双特异性或多特异性分子还包含至少一种特异性结合IGF1R的第二抗体；优选地，所述第二抗体具有对IGF1R信号传导的拮抗作用。
  15. 免疫缀合物，其包含权利要求1-9任一项所述的抗体或其抗原结合片段以及连接于所述抗体或其抗原结合片段的治疗剂；
    优选地，所述治疗剂选自IGF1R拮抗剂或抑制剂；
    优选地，所述免疫缀合物是抗体-药物偶联物(ADC)。
  16. 药物组合物，其包含权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求10所述的分离的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求14所述的双特异性或多特异性分子或权利要求15所述的免疫缀合物，以及药学上可接受的载体和/或赋形剂；
    优选地，所述药物组合物还包含另外的药学活性剂，例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物；
    优选地，所述药物组合物还包含IGF1R拮抗剂或抑制剂，和/或，另外的TSHR拮抗剂或抑制剂。
  17. 权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求10所述的分离的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求14所述的双特异性或多特异性分子、权利要求15所述的免疫缀合物或权利要求16所述的药物组合物用于制备药物的用途，所述药物用于预防和/或治疗受试者的与TSHR相关的疾病；
    优选地，所述与TSHR相关的疾病将得益于对TSHR信号传导的拮抗作用；
    优选地，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合；
    优选地，所述受试者为哺乳动物，例如人、鼠或猴；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物单独使用，或与另外的药学活性剂(例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物)联合使用；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物与IGF1R拮抗剂或抑制剂和/或另外的TSHR拮抗剂或抑制剂联合使用。
  18. 用于在受试者中预防和/或治疗受试者的与TSHR相关的疾病的方法，其包括：给有此需要的受试者施用有效量的权利要求1-9任一项所述的抗体或其抗原结合片段、权利要求10所述的分离的核酸分子、权利要求11所述的载体、权利要求12所述的宿主细胞、权利要求14所述的双特异性或多特异性分子、权利要求15所述的免疫缀合物或权利要求16所述的药物组合物；
    优选地，所述与TSHR相关的疾病将得益于对TSHR信号传导的拮抗作用；
    优选地，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合；
    优选地，所述受试者为哺乳动物，例如人、鼠或猴；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物单独使用，或与另外的药学活性剂(例如另外的用于治疗自身免疫性甲状腺疾病(如格雷夫斯病或格雷夫斯眼病)或甲状腺癌的药物)联合使用；
    优选地，所述抗体或其抗原结合片段、分离的核酸分子、载体、宿主细胞、双特异性或多特异性分子、免疫缀合物或药物组合物与IGF1R拮抗剂或抑制剂和/或另外的TSHR拮抗剂或抑制剂联合使用。
  19. 缀合物，其包含权利要求1-9任一项所述的抗体或其抗原结合片段，以及与所述抗体或其抗原结合片段连接的可检测的标记；
    优选地，所述可检测的标记选自酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素。
  20. 试剂盒，其包含权利要求1-9任一项所述的抗体或其抗原结合片段或权利要求19所述的缀合物。
  21. 用于检测TSHR在样品中的存在或其水平的方法，其包括使用权利要求1-9任一项所述的抗体或其抗原结合片段或权利要求19所述的缀合物；
    优选地，所述方法是免疫学检测，例如免疫印迹法、酶免疫测定法(例如ELISA)、化学发光免疫分析法、荧光免疫分析法或放射免疫测定法；
    优选地，所述方法包括使用权利要求19所述的缀合物；
    优选地，所述方法包括使用权利要求1-9任一项所述的抗体或其抗原结合片段，并且所述方法还包括使用携带可检测的标记(例如酶(例如辣根过氧化物酶或碱性磷酸酶)、化学发光试剂(例如吖啶酯类化合物、鲁米诺及其衍生物、或钌衍生物)、荧光染料(例如荧光素或荧光蛋白)、放射性核素或生物素)的第二抗体来检测所述抗体或其抗原结合片段；
    优选地，所述方法包括：(1)将所述样品与权利要求1-9任一项所述的抗体或其抗原结合片段或权利要求19所述的缀合物接触；(2)检测抗原-抗体免疫复合物的形成或检测所述免疫复合物的量，所述免疫复合物的形成表明存在TSHR或表达TSHR的细胞；
    优选地，所述方法为诊断与TSHR相关的疾病的方法，其中，所述样品为来自受试者的样品；
    优选地，当存在TSHR或与参照水平相比(例如与健康对照相比)TSHR的水平增加时，表明所述受试者患有与TSHR相关的疾病；
    优选地，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合。
  22. 权利要求1-9任一项所述的抗体或其抗原结合片段或权利要求19所述的缀合物在制备检测试剂中的用途，所述检测试剂用于检测TSHR在样品中的存在或其水平和/或诊断与TSHR相关的疾病；
    优选地，所述检测试剂通过权利要求21所述的方法来检测TSHR在样品中的存在或其水平或诊断与TSHR相关的疾病；
    优选地，所述与TSHR相关的疾病为自身免疫性甲状腺疾病(例如格雷夫斯病、格雷夫斯眼病)、甲状腺癌或其组合；
    优选地，所述样品为来自受试者(例如哺乳动物，优选人、鼠或猴)的组织样品。
PCT/CN2023/115886 2022-08-31 2023-08-30 结合促甲状腺激素受体的抗体及其用途 WO2024046384A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202211054215.2 2022-08-31
CN202211054215 2022-08-31

Publications (1)

Publication Number Publication Date
WO2024046384A1 true WO2024046384A1 (zh) 2024-03-07

Family

ID=90100389

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2023/115886 WO2024046384A1 (zh) 2022-08-31 2023-08-30 结合促甲状腺激素受体的抗体及其用途

Country Status (1)

Country Link
WO (1) WO2024046384A1 (zh)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1717418A (zh) * 2002-11-29 2006-01-04 Rsr有限公司 促甲状腺素受体的抗体及其用途
CN101657468A (zh) * 2007-02-15 2010-02-24 Rsr有限公司 用作拮抗剂的抗促甲状腺激素受体的人单克隆抗体
CN102264764A (zh) * 2008-12-24 2011-11-30 Rsr有限公司 抗体
CN103224562A (zh) * 2001-08-23 2013-07-31 Rsr有限公司 促甲状腺素受体的表位区域和针对该区域的抗体
CN110579593A (zh) * 2019-09-17 2019-12-17 郑州安图生物工程股份有限公司 一种刺激性促甲状腺激素受体抗体浓度的检测试剂盒
CN114113637A (zh) * 2021-12-07 2022-03-01 郑州安图生物工程股份有限公司 一种促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103224562A (zh) * 2001-08-23 2013-07-31 Rsr有限公司 促甲状腺素受体的表位区域和针对该区域的抗体
CN1717418A (zh) * 2002-11-29 2006-01-04 Rsr有限公司 促甲状腺素受体的抗体及其用途
CN101657468A (zh) * 2007-02-15 2010-02-24 Rsr有限公司 用作拮抗剂的抗促甲状腺激素受体的人单克隆抗体
CN102264764A (zh) * 2008-12-24 2011-11-30 Rsr有限公司 抗体
CN110579593A (zh) * 2019-09-17 2019-12-17 郑州安图生物工程股份有限公司 一种刺激性促甲状腺激素受体抗体浓度的检测试剂盒
CN114113637A (zh) * 2021-12-07 2022-03-01 郑州安图生物工程股份有限公司 一种促甲状腺激素受体抗原试剂及促甲状腺激素受体抗体定量检测试剂盒

Similar Documents

Publication Publication Date Title
US10906978B2 (en) Anti-CD3 antibodies, anti-CD123 antibodies and bispecific antibodies specifically binding to CD3 and/or CD123
US10273307B2 (en) Humanized anti-CD134 (OX40) antibodies and uses thereof
RU2739610C1 (ru) Антитело против PD-1 и его применение
JP2022130393A (ja) 抗ｃｔｌａ４－抗ｐｄ－１二機能性抗体、その医薬組成物および使用
CN113544156B (zh) 抗Claudin18.2抗体及其应用
WO2020168554A1 (zh) 改造的Fc片段，包含其的抗体及其应用
KR20170081699A (ko) Cd47 항체, 방법 및 용도
CN114507284B (zh) 抗tigit的抗体、其制备方法和应用
KR20100067682A (ko) 항-헵시딘 항체 및 그의 용도
US11639388B2 (en) CD3 antigen binding fragment and application thereof
JP2024026260A (ja) カリクレイン関連ペプチダーゼ２抗原結合ドメインを含むタンパク質及びその使用
WO2018219327A1 (zh) 抗cd40抗体、其抗原结合片段及其医药用途
WO2017043466A1 (ja) 抗ＥｐｈＡ４抗体
WO2022143794A1 (zh) 抗cldn18.2抗体及其制备方法和应用
WO2022174813A1 (zh) 抗GPRC5DxBCMAxCD3三特异性抗体及其用途
WO2023001155A1 (zh) 一种磷脂酰肌醇蛋白聚糖3抗体及其应用
CA3235697A1 (en) Anti-mesothelin nanobodies and use thereof
WO2024046384A1 (zh) 结合促甲状腺激素受体的抗体及其用途
JP2023539524A (ja) エンゲージャを含む薬物治療剤の開発及び応用
JP2023505123A (ja) 医薬組成物、その製造方法及び用途
JP2022550121A (ja) Ｌｉｆに特異的な結合分子及びその使用
JP2012528812A (ja) ヒトｃｃｎ１に対する抗体とその用途
TW202411255A (zh) 結合促甲狀腺激素受體的抗體及其用途
WO2024022478A1 (zh) 结合大麻素受体cb1的抗体及其用途
WO2022117050A1 (zh) 一种新型肿瘤衔接器治疗药物的开发和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 23859413

Country of ref document: EP

Kind code of ref document: A1