WO2020125744A1 - 双特异性蛋白 - Google Patents

双特异性蛋白 Download PDF

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WO2020125744A1
WO2020125744A1 PCT/CN2019/126903 CN2019126903W WO2020125744A1 WO 2020125744 A1 WO2020125744 A1 WO 2020125744A1 CN 2019126903 W CN2019126903 W CN 2019126903W WO 2020125744 A1 WO2020125744 A1 WO 2020125744A1
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seq
variable region
chain variable
antibody
heavy chain
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PCT/CN2019/126903
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English (en)
French (fr)
Inventor
曹卓晓
罗肖
何宁
胡齐悦
张连山
陶维康
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江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to MX2021007444A priority Critical patent/MX2021007444A/es
Priority to KR1020217021692A priority patent/KR20210109552A/ko
Priority to EP19898966.7A priority patent/EP3901173A4/en
Priority to AU2019410643A priority patent/AU2019410643A1/en
Priority to JP2021535930A priority patent/JP2022516439A/ja
Priority to CA3123979A priority patent/CA3123979A1/en
Priority to US17/416,237 priority patent/US20220153853A1/en
Priority to CN202211312212.4A priority patent/CN115947849A/zh
Priority to CN201980071999.XA priority patent/CN112996811B/zh
Publication of WO2020125744A1 publication Critical patent/WO2020125744A1/zh

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    • 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
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • 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
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/626—Diabody or triabody
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

  • the present disclosure relates to human GCGR antibody, GLP-1 peptide and mutants thereof, as well as bispecific protein formed by fusion of GCGR antibody and GLP-1 peptide, and preparation method and application thereof.
  • Diabetes is a metabolic disease characterized by defects in insulin secretion and/or dysfunction of insulin, characterized by hyperglycemia, and its onset is mainly the result of the joint action of insulin and glucagon.
  • GLP-1 is one of the most important hormones that affect the secretion of insulin, together with glucagon (Glucagon) is derived from proinsulin.
  • Proproinsulin is composed of about 158 amino acids and is cleaved into different peptide chains at different sites.
  • the biologically active GLP-1 in the human body mainly includes GLP-1 (7-36) amide and GLP-1 (7-37) two forms.
  • GLP-1 is secreted by L cells in the small intestine, mainly promotes insulin secretion in a glucose concentration-dependent manner, protects islet ⁇ cells, and inhibits glucagon secretion to reduce the body's blood glucose level.
  • GLP-1 also suppresses gastric emptying and reduces appetite. It can be used clinically to treat type 2 diabetes and obesity.
  • the half-life of natural active GLP-1 in the body is very short (less than 2 minutes), it is easily degraded by DPPIV enzyme in the body, and has no clinical value.
  • GLP-1 GLP-1 proliferative protein
  • Glucagon has the opposite effect of insulin and mainly plays a role in raising the body's blood sugar.
  • Glucagon is a 29-amino acid peptide secreted by pancreatic islet alpha cells. After binding to the GCGR receptor on the hepatocyte membrane, it mainly accelerates glycogenolysis, lipolysis and gluconeogenesis by activating downstream cAMP/PKA pathways to make blood glucose Rise.
  • GCGR knockout mice exhibited a series of phenotypes such as increased GLP-1, decreased glycogen output, increased lipid metabolism, and decreased appetite.
  • GCGR is one of the most popular targets for diabetes treatment, but the development of antagonist drugs for GCGR is slow.
  • REMD-477 of REMD Biotherapeutics is currently the most advanced GCGR monoclonal antibody drug and is in clinical phase two.
  • GCGR antibodies in patents such as CN101589062A, CN101983208A, CN102482350A, CN103314011A, CN105189560A, CN107614695A, US20180273629A1, WO2013059531A1, etc., but still needs to provide new and efficient GCGR antibodies and diabetes treatment methods.
  • the present disclosure provides an anti-GCGR monoclonal antibody or antigen-binding fragment thereof.
  • the antibody or antigen-binding fragment thereof has the ability to bind to human GCGR (or the epitope contained therein).
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a combination of a heavy chain variable region and a light chain variable region selected from the following a) or b):
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 regions as shown in SEQ ID NO: 48, 49, and 50, and the light chain variable region contains SEQ ID NO: 51, 52, and 50, respectively.
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 38, 39 and 54 respectively, and the light chain variable region contains as SEQ ID NO: 55, 56 and 57 shows the LCDR1, LCDR2, and LCDR3 areas.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises any combination of a heavy chain variable region and a light chain variable region selected from the following i) to vi):
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 14, 15 and 16, respectively, and the light chain variable region contains SEQ ID NO: 17, 18 and LCDR1, LCDR2 and LCDR3 areas shown in 19;
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 20, 21 and 22, and the light chain variable region contains SEQ ID NO: 23, 24 and LCDR1, LCDR2 and LCDR3 areas shown in 25;
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 regions shown in SEQ ID NOs: 26, 27, and 28, and the light chain variable region contains the SEQ ID NOs: 29, 30, and 28, respectively.
  • the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 32, 33 and 34, and the light chain variable region comprises SEQ ID NO: 35, 36 and LCDR1, LCDR2 and LCDR3 areas shown in 37;
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 38, 39 and 40, and the light chain variable region contains SEQ ID NO: 41, 42 and LCDR1, LCDR2 and LCDR3 areas shown in 43; or
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions as shown in SEQ ID NO: 38, 39 and 44, respectively, and the light chain variable region contains SEQ ID NO: 45, 46 and 47 shows the LCDR1, LCDR2 and LCDR3 areas.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof is a murine antibody, chimeric antibody or humanized antibody or antigen-binding fragment thereof.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a framework region derived from a human antibody or a framework region variant thereof, the framework region variant is in human There are at most 10 back mutations on the light chain framework region of the antibody, and/or, the framework region variant has at most 10, at most 9, at most 8, at most 7, based on the heavy chain framework region of the human antibody , At most 6, at most 5, at most 4, at most 3, at most 2, at most 1 amino acid back mutation.
  • the framework region variant comprises a member selected from:
  • the light chain variable region contains one or more amino acid back mutations in 42G, 44V, 71Y and 87F, and/or the heavy chain variable region contains 38K, 48I, 67A, 69F, 71A, 73P, 78A and One or more amino acids in 93S are back-mutated; or
  • the light chain variable region contains one or more amino acid back mutations in 38L, 44V, 59S, 70E and 71Y, and/or the heavy chain variable region contains 38K, 48I, 66K, 67A, 69L, 73R, One or more amino acids in 78M and 94S are back-mutated. Further, the position of the back mutation site is determined according to the Kabat numbering rule.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a sequence as shown in any one of SEQ ID NO: 2, 61, 62, 63, and 64 or is identical to SEQ ID NO: 2, 61 , 62, 63, and 64 show variable heavy chains with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity District; and/or
  • sequence is as shown in any of SEQ ID NO: 3, 58, 59 and 60 or has at least 90%, 91%, 92%, 93%, 94% as shown in any of SEQ ID NO: 3, 58, 59 and 60 , 95%, 96%, 97%, 98%, or 99% light chain variable regions with sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a sequence as described in SEQ ID NO: 63 and a light chain having a sequence as shown in SEQ ID NO: 58 Variable area.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 4 or has at least 90%, 91%, 92% as shown in SEQ ID NO: 4 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable regions and/or sequences are shown in SEQ ID NO: 5 or with SEQ ID NO: 5
  • the light chain variable regions shown have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises the sequence shown in SEQ ID NO: 6 or at least 90%, 91%, 92% as shown in SEQ ID NO: 6 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable regions and/or sequences are shown in SEQ ID NO: 7 or with SEQ ID NO: 7
  • the light chain variable regions shown have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises the sequence shown in any of SEQ ID NO: 8, 68, 69, 70, and 71 or is in accordance with SEQ ID NO: 8, 68 , 69, 70, and 71 show heavy chain variable with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • the regions and/or sequences are shown in any of SEQ ID NO: 9, 65, 66 and 67 or have at least 90%, 91%, 92%, 93 as shown in SEQ ID NO: 9, 65, 66 and 67 %, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity light chain variable regions.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region whose sequence is as described in SEQ ID NO: 71 and a light chain whose sequence is as shown in SEQ ID NO: 67 Variable area.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a sequence shown in SEQ ID NO: 10 or at least 90%, 91%, 92% as shown in SEQ ID NO: 10 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable regions and/or sequences are shown in SEQ ID NO: 11 or with SEQ ID NO: 11
  • the light chain variable regions shown have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a sequence shown in SEQ ID NO: 12 or at least 90%, 91%, 92% as shown in SEQ ID NO: 12 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity heavy chain variable regions and/or sequences are shown in SEQ ID NO: 13 or with SEQ ID NO: 13
  • the light chain variable regions shown have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof wherein the antibody is a full-length antibody, further including an antibody constant region, specifically, the heavy chain constant region of the antibody constant region is selected from Human IgG1, IgG2, IgG3 and IgG4 constant regions and their conventional variants, the light chain constant regions of the antibody constant regions are selected from human antibody ⁇ and ⁇ chain constant regions and their conventional variants, more preferably comprising SEQ ID NO: 72 The human antibody heavy chain constant region shown and the human light chain constant region shown in SEQ ID NO: 73 are shown.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 75 or has at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 77 or has at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 74, 76 or 78 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 79 or has at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 80 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 81 or has at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 82 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 83 or has at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 84 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO:85 or has at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 86 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 87 or has at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof includes a heavy chain and a light chain, wherein:
  • the heavy chain is shown in SEQ ID NO: 88 or has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, or 99% sequence identity
  • the light chain is shown in SEQ ID NO: 89 or has at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 78 and a light chain as shown in SEQ ID NO: 79.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain as shown in SEQ ID NO: 84 and a light chain as shown in SEQ ID NO: 85.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof comprises any combination of a heavy chain variable region and a light chain variable region selected from the following ac) to ah):
  • the heavy chain variable region contains the HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 2, and the light chain variable region contains the SEQ ID NO: 3
  • the light chain variable region has the same sequence of LCDR1, LCDR2 and LCDR3 regions;
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 4, and the light chain variable region contains the same as SEQ ID NO: 5.
  • the light chain variable region has the same sequence of LCDR1, LCDR2 and LCDR3 regions;
  • the heavy chain variable region comprises the HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 6, and the light chain variable region comprises the SEQ ID NO: 7
  • the light chain variable region has the same sequence of LCDR1, LCDR2 and LCDR3 regions;
  • the heavy chain variable region contains HCDR1, HCDR2 and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 8, and the light chain variable region contains the same as SEQ ID NO: 9.
  • the light chain variable region has the same sequence of LCDR1, LCDR2 and LCDR3 regions;
  • the heavy chain variable region contains HCDR1, HCDR2, and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 10, and the light chain variable region contains SEQ ID NO: 11
  • the light chain variable region has the same sequence of LCDR1, LCDR2, and LCDR3 regions; or
  • the heavy chain variable region contains the HCDR1, HCDR2, and HCDR3 regions having the same sequence as the heavy chain variable region shown in SEQ ID NO: 12, and the light chain variable region contains the SEQ ID NO: 13
  • the light chain variable region has the same sequence of LCDR1, LCDR2, and LCDR3 regions.
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab') 2, single-chain antibody, and dimerized V Region (diabodies) and disulfide-stabilized V region (dsFv).
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof is characterized by competing with the aforementioned monoclonal antibody or antigen-binding fragment thereof for binding to human GCGR (or its epitope).
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof has at least one of the following characteristics:
  • the anti-GCGR monoclonal antibody or antigen-binding fragment thereof wherein the monoclonal antibody or antigen-binding fragment thereof binds to the same antigen table as the monoclonal antibody or antigen-binding fragment thereof as described above Bit.
  • the present disclosure provides a bispecific protein.
  • a bispecific protein comprising a GLP-1 peptide and a GCGR antibody, the GLP-1 peptide and the polypeptide chain of the GCGR antibody by a peptide bond Or the connector is covalently connected.
  • the bispecific protein wherein the carboxy terminus of the GLP-1 peptide is connected to the amino terminus of the heavy chain variable region of the GCGR antibody by a peptide bond or linker, or the GLP-1 peptide
  • the carboxyl terminus of the GCGR antibody is connected to the amino terminus of the light chain variable region of the GCGR antibody by a peptide bond or linker.
  • the bispecific protein wherein the carboxy terminus of the GLP-1 peptide and the amino terminus of the heavy chain variable region of the GCGR antibody are connected by a peptide bond or linker.
  • the bispecific protein wherein the carboxy terminus of the GLP-1 peptide and the amino terminus of the light chain variable region of the GCGR antibody are connected by a peptide bond or linker.
  • the bispecific protein wherein the carboxy terminus of the GLP-1 peptide is connected to the amino terminus of the heavy chain of the GCGR full-length antibody by a peptide bond or linker.
  • the bispecific protein wherein the carboxy terminus of the GLP-1 peptide and the light chain amino terminus of the full-length GCGR antibody are connected by a peptide bond or linker.
  • the GCGR antibody is selected from the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above.
  • the bispecific protein, the GLP-1 peptide is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide has Q17E, GLP-1A peptide variants with one or more amino acid substitutions in I23V, K28R and G30R.
  • the bispecific protein, the GLP-1 peptide is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide has Q17E based on GLP-1A GLP-1A peptide variants.
  • the bispecific protein, the GLP-1 peptide is GLP-1A shown in SEQ ID NO: 91, or the GLP-1 peptide has a Q17E substitution based on GLP-1A , And also has one or more amino acids in I23V, K28R and G30R instead of GLP-1A peptide variants.
  • the bispecific protein, the GLP-1 peptide is GLP-1A shown in SEQ ID NO: 91, or the GLP-1 peptide has Q17E or GLP-1A on the basis of GLP-1A GLP-1A peptide variant with Q17E and I23V.
  • the bispecific protein, the GLP-1A peptide variant comprises the sequence shown in SEQ ID NO: 92, 93, 94, 95, 96, 97, 98 or 99 or by composition.
  • the bispecific protein, the GCGR antibody is selected from the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described in any one of the preceding items and the GLP-1A peptide as described in any one of the preceding items Or GLP-1A peptide variants.
  • the bispecific protein has a first polypeptide chain and a second polypeptide chain, wherein: the first polypeptide chain is selected from SEQ ID NO: 100 , 101, 102, 103, 104, 105, 106, 107, and 108, and the second polypeptide chain is selected from the polypeptides shown in SEQ ID NO: 79.
  • the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first A polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 109, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 81;
  • the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first One polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 110, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 83;
  • the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first One polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 111, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 85;
  • the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first A polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 112, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 87; or
  • the bispecific protein has a first polypeptide chain comprising a GCGR antibody heavy chain and a second polypeptide chain comprising a GCGR antibody light chain, wherein: the first A polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 113, and the second polypeptide chain is selected from the polypeptide shown in SEQ ID NO: 89.
  • the present disclosure also provides a GLP-1 peptide variant.
  • the GLP-1 peptide variant is a mutant having one or more amino acid mutations in Q17E, I23V, K28R, and G30R based on GLP-1A shown in SEQ ID NO: 91.
  • the GLP-1 peptide is GLP-1A as shown in SEQ ID NO: 91, or the GLP-1 peptide has Q17E or GLP-1A with Q17E and I23V based on GLP-1A Peptide variants.
  • the GLP-1 peptide variant has the sequence shown in SEQ ID NO: 92, 93, 94, 95, 96, 97, 98 or 99.
  • the present disclosure also provides a pharmaceutical composition containing a therapeutically effective amount of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above, or the bispecific protein as described above, or as described above GLP-1 peptide variants, and one or more pharmaceutically acceptable carriers, diluents, buffers or excipients.
  • the present disclosure also provides an isolated nucleic acid molecule encoding the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above, or the bispecific protein as described above, or the GLP-1 as described above Peptide variants.
  • the present disclosure provides a recombinant vector comprising the isolated nucleic acid molecule as described above.
  • the present disclosure provides a host cell transformed with the recombinant vector as described above, the host cell being selected from prokaryotic cells and eukaryotic cells, preferably eukaryotic cells, more preferably mammalian cells or insect cells.
  • the present disclosure provides methods for producing anti-GCGR monoclonal antibodies or antigen-binding fragments thereof as previously described, or bispecific proteins as previously described, or GLP-1 peptide variants as previously described,
  • the method includes culturing the host cell as described above in a medium to form and accumulate the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above, or the bispecific protein as described above, or GLP-1 peptide variants as previously described, and recovering the monoclonal antibody or antigen-binding fragment or bispecific protein or GLP-1 peptide variant from the culture.
  • the present disclosure provides a method for in vitro detection or determination of human GCGR, which method includes the use of a monoclonal antibody or antigen-binding fragment thereof as previously described.
  • a kit for detecting human GCGR which contains the monoclonal antibody or antigen-binding fragment thereof as described above.
  • kits eg kits, arrays, test papers, multi-well plates, magnetic beads, coated microparticles
  • a kit includes a multi-well plate coated with the aforementioned anti-GCGR monoclonal antibody or antigen-binding fragment thereof.
  • the present disclosure provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof in the preparation of reagents for detecting or measuring human GCGR.
  • the present disclosure provides a method of reducing the blood glucose concentration of a subject, the method comprising administering to the subject a therapeutically effective amount of an anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above, or as previously described Bispecific protein, or the GLP-1 peptide variant as described above, or the pharmaceutical composition as described above.
  • the therapeutically effective amount is a unit dose of the composition containing 0.1-3000 mg of the anti-GCGR monoclonal antibody or antigen-binding fragment thereof as described above, or the bispecific protein as described above, or according to GLP-1 peptide variants as previously described.
  • the present disclosure provides a method of treating a metabolic disorder, the method comprising administering to the subject an anti-GCGR monoclonal antibody or antigen-binding fragment thereof, or a bispecific protein as described above, or as The aforementioned GLP-1 peptide variant, or the pharmaceutical composition as described above; preferably, the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, high Glucose, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting blood glucose, dyslipidemia, atherosclerosis, or prediabetes.
  • the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, high Glucose, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia, insulin resistance syndrome, impaired fasting blood glucose, dyslipidemia, atherosclerosis, or prediabetes.
  • the present disclosure also provides anti-GCGR monoclonal antibodies or antigen-binding fragments thereof as described above, or bispecific proteins as described above, or GLP-1 peptide variants as described above, or as previously described Use of the pharmaceutical composition described in the preparation of a medicament for treating metabolic disorders or reducing the blood glucose concentration of a subject
  • the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia , Insulin resistance syndrome, impaired fasting blood glucose, dyslipidemia, atherosclerosis, or prediabetes.
  • the present disclosure also provides the use of anti-GCGR monoclonal antibodies or antigen-binding fragments thereof as described above, or bispecific proteins as described above, or GLP-1 peptide variants as described above, or as previously described
  • the pharmaceutical composition described above is used as a medicine, preferably as a medicine for treating metabolic disorders or lowering the blood glucose concentration of a subject.
  • the metabolic disorder is metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia Syndrome, insulin resistance syndrome, impaired fasting blood glucose, dyslipidemia, atherosclerosis, or prediabetes.
  • FIG. 1 Schematic diagram of the bispecific protein (GLP-1/GCGR anti) of the present disclosure.
  • Figure 2 Antagonistic activity of bispecific protein and GCGR antibody against GCGR.
  • Figure 3 Activating activity of GLP-1R by bispecific protein and duraglutide.
  • Figure 4 Effect of long-term medication on random blood glucose in ob/ob mice.
  • Vehicle empty carrier
  • PBS phosphate buffered saline
  • hu1803-9D-3mpk hu1803-9-2.84mpk
  • dora Glycopeptide-1.16mpk is the amount concentration of the same substance.
  • Figure 5 Effect of long-term medication on hungry blood glucose in ob/ob mice. All experimental groups can significantly reduce the fasting blood glucose concentration of mice, of which hu1803-9D-3mpk and hu1803-9-2.84mpk have the strongest hypoglycemic concentration ability, and hu1803-9D-3mpk has better hypoglycemic activity than hu1803-9- 2.84mpk.
  • bispecific protein refers to a protein molecule capable of binding two target proteins or target antigens.
  • the bispecific protein refers to a protein that can bind GCGR and GLP-1R (GLP-1 receptor) and is formed by fusion of the polypeptide chain of the GLP-1 peptide and the GCGR antibody (or antigen-binding fragment thereof).
  • GLP-1 peptide refers to a peptide that can bind to and activate the GLP-1 receptor.
  • Prior art peptides are described in patent applications WO2008/071972, WO2008/101017, WO2009/155258, WO2010/096052, WO2010/096142, WO2011/075393, WO2008/152403, WO2010/070251, WO2010/070252, WO2010/070253, WO2010 /070255, WO2011/160630, WO2011/006497, WO2011/117415, WO2011/117416, WO2006/134340, WO1997046584, WO2007124461, WO2017100107, WO2007039140, CN1935261A, CN1935846A, WO2006036834, WO2005058958, WO2002046227, WO1999043705, WO1999043708, WO1999043341, WO1999043341 ,
  • GLP-1 Including GLP-1, GLP-1 analogs and GLP-1 receptor peptide agonists, some specific GLP-1 peptides such as: Lixisenatide/AVE0010/ZP10/Lyxumia, Exenatide /Exendin-4/Byetta/Bydureon/ITCA650/AC-2993, Liraglutide/Victoza, Somalutide (Semaglutide), Taspoglutide, Syncria/Abilutide ( Albiglutide), Dulaglutide, rExendin-4, CJC-1134-PC, PB-1023, TTP-054, Langlenatide/HM-11260C, CM-3, GLP-1Eligen, ORMD-0901, NN-9924 , NN-9926, NN-9927, Nodexen, Viador-GLP-1, CVX-096, ZYOG-1, ZYD-1, GSK-2374697, DA-3091, MAR-701, MAR709
  • GPCR G protein-coupled Receptor
  • GPCR G protein-coupled receptor
  • GPCR consists of more than 800 members and is currently the largest family of membrane proteins in the mammalian genome. In the human body, GPCR proteins are widely distributed in the central nervous system, immune system, cardiovascular, retina and other organs and tissues, and participate in the development of the body and normal functioning.
  • the main body of the GPCR protein is composed of 7-segment ⁇ -helix structures across the plasma membrane.
  • the N-terminus and three loops are located outside the cell and are involved in the interaction between the protein and its receptor; the C-terminus and 3 loops are located inside the cell, where the C-terminus and the third loop are mediated by the interaction between the GPCR protein and the downstream G protein It plays an important role in the intracellular signaling process.
  • GCGR is a glucagon (Glucagon) receptor and is one of the members of the GPCR family. After combining glucagon with GCGR, it mainly accelerates glycogenolysis, lipolysis and/or gluconeogenesis by activating downstream pathways, Increase blood sugar.
  • antibody (Ab) includes at least one antigen-binding molecule (or molecule that specifically binds to or interacts with (eg recognizes and/or binds to) a specific antigen (or its epitope) (eg GCGR) (eg, recognition and/or binding) Complex).
  • a specific antigen or its epitope
  • eg GCGR eg, recognition and/or binding
  • antibody includes: immunoglobulin molecules including four polypeptide chains interconnected by disulfide bonds, two heavy (H) chains and two light (L) chains, and multimers thereof (eg, IgM).
  • Each heavy chain includes a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH).
  • This heavy chain constant region contains three regions (domains), CH1, CH2 and CH3.
  • Each light chain includes a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region (CL).
  • VH and VL regions can be further subdivided into hypervariable regions, called complementarity-determining regions (CDR), with more conservative regions interspersed between them, called framework regions (FR, also known as framework regions, framework regions).
  • CDR complementarity-determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs, arranged from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the FR of the anti-GCGR antibody may be the same as the human germline sequence, or may be modified naturally or artificially.
  • the antibodies may be antibodies of different subclasses, for example, IgG (eg, IgG1, IgG2, IgG3, or IgG4 subclass), IgA1, IgA2, IgD, IgE, or IgM antibodies.
  • antibody also includes antigen-binding fragments of complete antibody molecules.
  • antigen-binding portion include any naturally occurring, enzymatically produced, synthetic or genetically engineered complex that specifically binds to an antigen to form a complex Polypeptide or glycoprotein.
  • Antigen-binding fragments of antibodies can be derived from, for example, whole antibody molecules using any suitable standard techniques, such as proteolytic digestion or recombinant genetic engineering techniques involving DNA manipulation and expression of variable regions and (optionally) constant regions of antibodies. This DNA is known and/or can be easily obtained from, for example, commercially available sources, DNA databases (including, for example, phage-antibody databases) or can be synthesized.
  • This DNA can be sequenced and manipulated chemically or through the use of molecular biotechnology, such as arranging one or more variable and/or constant regions into a suitable configuration, or introducing codons to produce cysteine residues, modifications , Add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab′) 2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; vi) dAb fragment.
  • Other engineered molecules such as region-specific antibodies, single-domain antibodies, region-deleting antibodies, chimeric antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (such as monovalent Nanobodies, bivalent Nanobodies, etc.), Small module immunopharmaceutical (SMIP) and shark variable IgNAR region are also covered by the term "antigen-binding fragment" used in the text.
  • the antigen-binding fragment will typically contain at least one variable region.
  • the variable region may be a region of any size or amino acid composition and will generally contain CDRs adjacent to or within one or more framework sequences.
  • the antigen-binding fragment of the antibody is in any configuration of a variable region and a constant region, the variable region and the constant region may be directly connected to each other or may be connected by a complete or partial hinge or linker region .
  • the hinge region may be composed of at least 2 (e.g. 5, 10, 15, 20, 40, 60 or more) amino acids, so that flexibility is created between adjacent variable and/or constant regions in a single polypeptide molecule And semi-flexible connection.
  • the "murine antibody” in this disclosure is a monoclonal antibody derived from a mouse or a rat prepared according to the knowledge and skills in the art. When preparing, inject the test subjects with antigen, and then isolate the hybridoma expressing the antibody with the desired sequence or functional characteristics. When the injected test subject is a mouse, the antibody produced is a mouse-derived antibody. When the injected test subject is In the case of rats, the antibodies produced are those derived from rats.
  • a “chimeric antibody” is an antibody obtained by fusing a variable region of an antibody of a first species (such as a mouse) with a constant region of an antibody of a second species (such as a human).
  • a first species such as a mouse
  • a second species such as a human
  • To create a chimeric antibody first create a hybridoma that secretes monoclonal antibodies of the first species (such as a mouse), then clone the variable region genes from the hybridoma cells, and then clone the constant region genes of the second species (such as human) antibodies as needed
  • the gene of the variable region of the first species and the gene of the constant region of the second species are connected into a chimeric gene and inserted into an expression vector, and finally the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.
  • the antibody light chain of the chimeric antibody further comprises a light chain constant region of human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or a variant thereof, preferably a human heavy chain constant region of IgG1, IgG2 or IgG4, or amino acid mutation ( IgG1, IgG2 or IgG4 heavy chain constant region variants such as YTE mutation or back mutation, L234A and/or L235A mutation, or S228P mutation).
  • humanized antibody includes CDR-grafted antibodies, which refers to the grafting of the CDR sequences of animal-derived antibodies, such as murine antibodies, into the framework region (or framework of human antibody variable regions) Region, framework) region.
  • Humanized antibodies can overcome the heterogeneous reactions induced by chimeric antibodies due to the large number of heterologous protein components.
  • the sequences of such framework regions can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of human heavy chain and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at http://www.vbase2.org/), and in Kabat, EA, etc. 1991 Sequences of Proteins of Immunological Interest, found in 5th edition.
  • the framework sequence of the variable region of the human antibody may be subjected to minimal reverse mutation or back mutation to maintain activity.
  • the humanized antibodies of the present disclosure also include humanized antibodies after further affinity affinity maturation of CDRs by phage display.
  • the grafting of the CDR may result in the reduced affinity of the produced antibody or antigen-binding fragment thereof for the antigen due to the framework residues in contact with the antigen. Such interactions may be the result of somatic cell mutations. Therefore, it may still be necessary to graft amino acids of such donor frameworks to the framework of humanized antibodies.
  • Amino acid residues from non-human antibodies or antigen-binding fragments involved in antigen binding can be identified by examining the sequence and structure of the variable regions of animal monoclonal antibodies. Each residue in the CDR donor framework that is different from the germline can be considered to be related.
  • the sequence can be compared to the consensus sequence of a subclass consensus sequence or animal antibody sequence with a high percentage of similarity. Rare framework residues are thought to be the result of highly somatic mutations, and thus play an important role in binding.
  • the antibody or antigen-binding fragment thereof may further comprise a light chain constant region of human or murine ⁇ , ⁇ chain or a variant thereof, or further comprise human or murine IgG1 , IgG2, IgG3, IgG4 or its heavy chain constant region.
  • “Conventional variants" of human antibody heavy chain constant regions and human antibody light chain constant regions refer to human-derived heavy chain constant regions or light chain constant regions derived from humans that do not change the structure and function of antibody variable regions Variants
  • exemplary variants include IgG1, IgG2, IgG3, or IgG4 heavy chain constant region variants that undergo site-directed modification and amino acid replacement of the heavy chain constant region, specifically replacing YTE mutations known in the prior art, L234A and/ Or L235A mutation, or S228P mutation, or mutations that obtain knob-into-hole structure (making the antibody heavy chain have a combination of knob-Fc and hole-Fc), these mutations have been proven to make the antibody have new properties, but do not change the antibody Variable area function.
  • Human antibody and “human antibody” are used interchangeably, and can be an antibody derived from human or an antibody obtained from a transgenic organism that has been "modified” to produce in response to antigen stimulation
  • Specific human antibodies can be produced by any method known in the art. In some techniques, elements of human heavy and light chain loci are introduced into cell lines of organisms derived from embryonic stem cell lines, and endogenous heavy and light chain loci in these cell lines are targeted damage. Transgenic organisms can synthesize human antibodies specific for human antigens, and the organisms can be used to produce human antibody-secreting hybridomas.
  • the human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more human DNA sources. Fully human antibodies can also be constructed by gene or chromosome transfection methods and phage display technology, or by B cells activated in vitro, all of which are known in the art.
  • “Monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous antibodies, that is, except for possible variant antibodies (eg, containing naturally occurring mutations or mutations that were produced during the manufacture of monoclonal antibody preparations, these variants are usually Except in small amounts), the individual antibodies that make up the population recognize the same and/or bind the same epitope. Unlike polyclonal antibody preparations that usually contain different antibodies against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation (preparation) is directed against a single determinant on the antigen.
  • monoclonal indicates the characteristics of antibodies as obtained from a substantially homogeneous population of antibodies, and should not be interpreted as requiring the production of antibodies by any particular method.
  • monoclonal antibodies used in accordance with the present disclosure can be prepared by various techniques including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of transgenic animals containing all or part of human immunoglobulin loci Methods, such methods, and other exemplary methods for preparing monoclonal antibodies are described herein.
  • the term "monoclonal antibody or antigen-binding fragment thereof" refers to a full-length antibody.
  • full-length antibody intact antibody
  • complete antibody completely antibody
  • whole antibody in which the heavy chain sequentially includes the VH region, CH1 region, hinge region, and Fc region from the amino terminal to the carboxy terminal
  • the light chain sequentially includes the VL region and CL region from the amino terminal to the carboxy terminal.
  • single-chain Fv single-chain Fv
  • scFv single-chain Fv
  • Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody.
  • Such antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as for intact antibodies.
  • the antigen-binding portion can be produced by recombinant DNA technology or by enzymatic or chemical fragmentation of intact immunoglobulins.
  • Antigen-binding fragments can also be incorporated into single-chain molecules containing a pair of tandem Fv fragments (VH-CH1-VH-CH1), which together with complementary light chain polypeptides form a pair of antigen-binding regions (Zapata et al. , 1995 Protein Eng. 8 (10): 1057-1062; and US Patent US5641870).
  • Fab is an antibody fragment having a molecular weight of about 50,000 Da obtained by treating an IgG antibody molecule with papain (cutting the amino acid residue at position 224 of the H chain), which has antigen-binding activity, in which about half of the N-terminal side of the H chain and The entire L chain is joined together by disulfide bonds.
  • F(ab')2 is an antibody fragment with a molecular weight of about 100,000 Da obtained by digesting the lower part of the two disulfide bonds in the hinge region of IgG with pepsin, and has antigen-binding activity and contains two connected at the hinge position Fab area.
  • Fab' is an antibody fragment having a molecular weight of about 50,000 Da and having antigen-binding activity obtained by cleaving the disulfide bond of the hinge region of F(ab') 2 described above.
  • Fab' can be produced by treating F(ab')2 that specifically recognizes and binds an antigen with a reducing agent such as dithiothreitol.
  • the Fab' can be produced by inserting DNA encoding the Fab' fragment of the antibody into a prokaryotic expression vector or a eukaryotic expression vector and introducing the vector into a prokaryotic or eukaryotic organism to express the Fab'.
  • single-chain antibody single-chain Fv
  • scFv single-chain Fv
  • Such scFv molecules may have a general structure: NH 2 -VL-linker-VH-COOH or NH 2 -VH-linker-VL-COOH.
  • Suitable prior art linkers consist of repeating GGGGS amino acid sequences or variants thereof, for example using 1-4 (including 1, 2, 3 or 4) repeating variants (Holliger et al. (1993), Proc Natl Acad Sci USA. 90:6444-6448).
  • linkers that can be used in the present disclosure are Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur J Immuno. 31:94-106, Hu et al. (1996), Cancer Res .56:3055-3061, described by Kipriyanov et al. (1999), J Mol Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol Immunother. 50:51-59.
  • Linker or “linker” or “linker” refers to a connecting polypeptide sequence used to connect protein domains, and usually has a certain flexibility. The use of linkers will not cause the original function of the protein domain to be lost.
  • Diabody refers to an antibody fragment in which scFv is dimerized, and is an antibody fragment having bivalent antigen binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
  • dsFv is obtained by connecting a polypeptide in which one amino acid residue in each VH and VL is replaced by a cysteine residue via a disulfide bond between the cysteine residues.
  • Amino acid residues substituted with cysteine residues can be selected according to a known method (Protein Engineering. 7:697 (1994)) based on the prediction of the three-dimensional structure of the antibody.
  • the antigen-binding fragment can be produced by obtaining the coding cDNA of the VH and/or VL of the monoclonal antibody of the present disclosure that specifically recognizes and binds the antigen and other required domains, and constructs the coding antigen Binding fragment DNA, inserting the DNA into a prokaryote expression vector or eukaryote expression vector, and then introducing the expression vector into a prokaryote or eukaryote to express the antigen-binding fragment.
  • the "Fc region” may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of the immunoglobulin heavy chain may vary, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl terminus. The numbering of the residues in the Fc region is the numbering of the EU index as in Kabat. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of immunoglobulins usually has two constant region domains CH2 and CH3.
  • Knob-Fc refers to a point mutation containing T366W in the Fc region of an antibody to form a spatial structure similar to a knob.
  • hole-Fc refers to a point mutation containing T366S, L368A, and Y407V in the Fc region of an antibody to form a hole-like spatial structure.
  • Knob-Fc and hole-Fc are more likely to form heterodimers due to steric hindrance.
  • point mutations of S354C and Y349C can also be introduced in knock-Fc and hole-Fc, respectively, to further promote the formation of heterodimers through disulfide bonds.
  • knob-Fc or hole-Fc can be used either as the Fc region of the first polypeptide chain or as the Fc region of the second polypeptide chain.
  • the first polypeptide The Fc region of the chain and the second polypeptide chain are not knob-Fc or hole-Fc at the same time.
  • amino acid difference or “amino acid mutation” means that the variant protein or polypeptide has an amino acid change or mutation compared to the original protein or polypeptide, including the insertion of one or several amino acids on the basis of the original protein or polypeptide, Missing or replacing.
  • variable region of an antibody refers to the variable region (VL) of the antibody light chain or the variable region (VH) of the antibody heavy chain, alone or in combination.
  • VL variable region
  • VH variable region
  • the variable regions of the heavy and light chains each consist of 4 framework regions (FR) connected by 3 complementarity determining regions (CDRs) (also called hypervariable regions).
  • FR framework regions
  • CDRs complementarity determining regions
  • the CDRs in each chain are held tightly by the FR and together with the CDRs from the other chain contribute to the formation of the antigen-binding site of the antibody.
  • There are at least 2 techniques for determining CDRs (1) Methods based on cross-species sequence variability (ie, Kabat et al.
  • CDR may refer to a CDR determined by either method or a combination of both methods.
  • antibody framework or "FR region” refers to a part of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDR.
  • CDR complementarity determining region
  • HCDR1, HCDR2, HCDR3 three CDRs in each heavy chain variable region and three CDRs (LCDR1, LCDR2, LCDR3) in each light chain variable region.
  • Any of a variety of well-known schemes can be used to determine the amino acid sequence boundaries of the CDR, including "Kabat” numbering rules (see Kabat et al.
  • the CDR amino acid residue numbers in the heavy chain variable domain (VH) are 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); light
  • the CDR amino acid residues in the chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid numbers in VH are 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residue numbers in VL are 26-32 (LCDR1), 50- 52 (LCDR2) and 91-96 (LCDR3).
  • the CDR is composed of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24- in human VL 34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3).
  • the CDR amino acid residue numbers in VH are approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3)
  • the CDR amino acid residue numbers in VL are approximately 27-32 (CDR1 ), 50-52 (CDR2) and 89-97 (CDR3).
  • the CDR region of an antibody can be determined using the program IMGT/DomainGap Align.
  • Any CDR variant thereof in “HCDR1, HCDR2, and HCDR3 regions or any CDR variants thereof” refers to a variant obtained by amino acid mutation of any one, or two, or three HCDRs in HCDR1, HCDR2, and HCDR3 regions body.
  • Antibody constant region domains refer to the constant region domains derived from the light and heavy chains of antibodies, including CL and the CH1, CH2, CH3, and CH4 domains derived from different classes of antibodies.
  • Epitope or “antigenic determinant” refers to a site on an antigen where immunoglobulins or antibodies specifically bind. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols Methods Molecular Biology, Volume 66, G.E. Morris, Ed. (1996).
  • antibodies bind with an affinity (KD) of about less than 10 -8 M, such as about less than 10 -9 M, 10 -10 M, 10 -11 M or less.
  • the term “competition” in the case of competition for antigen-binding proteins (eg, neutralizing antigen-binding proteins or neutralizing antibodies) of the same epitope, it means competition between antigen-binding proteins, which is determined by the following assay:
  • the antigen-binding protein to be detected eg, antibody or antigen-binding fragment thereof
  • prevents or inhibits eg, reduces
  • the specific binding of the reference antigen-binding protein eg, ligand or reference antibody
  • Many types of competitive binding assays can be used to determine whether one antigen-binding protein competes with another.
  • These assays include, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), Sandwich competition assay (see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253); solid phase direct biotin-avidin EIA (see for example Kirkland et al., 1986, J. Immunol. 137:3614-3619), solid Phase direct labeling assay, solid phase direct labeling sandwich assay (see for example Harlow and Lane, 1988, Antibodies, A Laboratory (Manual), Cold Spring Harbor Press); solid phase direct labeling with I-125 marker RIA (see for example Morel et al., 1988, Molec.
  • RIA solid-phase direct or indirect radioimmunoassay
  • EIA enzyme immunoassay
  • Sandwich competition assay see, for example, Stahli et al., 1983, Methods in Enzymology 9: 242-253
  • the assay involves the use of purified antigen (the antigen is on a solid surface or cell surface) that can bind to an unlabeled detection antigen binding protein and a labeled reference antigen binding protein. In the presence of the antigen-binding protein to be tested, the amount of label bound to the solid surface or cell is measured to measure competitive inhibition. Usually, the antigen-binding protein to be tested is present in excess.
  • the antigen binding protein identified by the competitive assay includes: an antigen binding protein that binds to the same epitope as the reference antigen binding protein; and an antigen binding that binds to an adjacent epitope sufficiently close to the binding epitope of the reference antigen binding protein Protein, the two epitopes prevent each other from binding spatially. Additional details regarding methods for determining competitive binding are provided in the embodiments of the present disclosure. Usually when the antigen binding protein in competition is present in excess, it will inhibit (eg, reduce) at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70 -75% or 75% or more of the specific binding of the reference antigen binding protein to the common antigen. In some cases, binding is inhibited by at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.
  • affinity refers to the strength of the interaction between antibody and antigen at a single epitope. Within each antigenic site, the variable region of the "arm" of the antibody interacts with the antigen at multiple amino acid sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.
  • an antibody or antigen-binding fragment thereof e.g. Fab fragments
  • the term “high affinity” generally refers to having a K D 1E -9 M in K D or less (e.g.
  • KD refers to a particular antibody - antigen interaction dissociation equilibrium constant.
  • antibodies bind antigen with a dissociation equilibrium constant (KD) of less than about 1E -8 M, such as less than about 1E -9 M, 1E -10 M, or 1E -11 M or less, for example, if using surface plasmon resonance (SPR) technology is measured in the BIACORE instrument.
  • KD dissociation equilibrium constant
  • SPR surface plasmon resonance
  • nucleic acid molecule refers to DNA molecules and RNA molecules.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, then the promoter or enhancer is effectively linked to the coding sequence.
  • vector means a construct capable of delivering one or more genes or sequences of interest and preferably expressing it in a host cell.
  • vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA encapsulated in liposomes or RNA expression vectors and certain eukaryotic cells such as producer cells.
  • mice can be immunized with antigens or fragments thereof, the obtained antibodies can be renatured and purified, and amino acid sequencing can be performed by conventional methods.
  • Antigen-binding fragments can also be prepared by conventional methods.
  • the antibody or antigen-binding fragment described in the present disclosure is genetically engineered to add one or more human FR regions to a non-human CDR region.
  • the human FR germline sequence can be obtained from the website http://www.imgt.org/ by aligning the IMGT human antibody variable region germline gene database and MOE software, or from the Journal of Immunoglobulin, 2001ISBN012441351.
  • host cell refers to a cell into which an expression vector has been introduced.
  • Host cells may include bacterial, microbial, plant or animal cells.
  • Bacteria that are easily transformed include members of the Enterobacteriaceae family, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae.
  • Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris.
  • Suitable animal host cell lines include CHO (Chinese Hamster Ovary Cell Line), HEK293 cells (non-limiting examples such as HEK293E cells) and NS0 cells.
  • Engineered antibodies or antigen-binding fragments can be prepared and purified by conventional methods. For example, cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors. The recombinant immunoglobulin expression vector can stably transfect CHO cells. As an alternative prior art, mammalian expression systems can lead to glycosylation of antibodies, especially at the highly conserved N-terminal sites in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to the antigen. Positive clones were expanded in serum-free medium in the bioreactor to produce antibodies. The antibody-secreted culture fluid can be purified by conventional techniques.
  • a protein A or protein G Sepharose FF column containing adjusted buffer is used for purification. Non-specifically bound components are washed away. Then, the bound antibody was eluted by pH gradient method, and antibody fragments were detected by SDS-PAGE and collected. Antibodies can be filtered and concentrated by conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The resulting product should be frozen immediately, such as -70 °C, or lyophilized.
  • administering when applied to animals, humans, experimental subjects, cells, tissues, organs or biological fluids, refers to the application of exogenous drugs, therapeutic agents, Diagnostic agents, compositions, or human operations (such as “euthanasia” in the examples) are provided to animals, humans, subjects, cells, tissues, organs, or biological fluids.
  • administering and “treatment” may refer to, for example, treatment, pharmacokinetics, diagnosis, research, and experimental methods.
  • the treatment of cells includes contact of reagents with cells and contact of reagents with fluids, wherein the fluids contact cells.
  • administering and “treating” also mean in vitro and ex vivo treatment of, for example, cells by an agent, diagnosis, binding composition, or by another cell.
  • Treatment when applied to human, veterinary or research subjects refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
  • Treatment means administration of a therapeutic agent for internal or external use to a subject, such as a composition comprising any of the compounds of the embodiments of the present disclosure, the subject has (or is suspected of having, or is susceptible to) Or a variety of disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms.
  • the therapeutic agent is administered to the treated subject or population in an amount effective to relieve the symptoms of one or more diseases to induce the regression of such symptoms or inhibit the development of such symptoms to any clinically measurable extent.
  • the amount of therapeutic agent effective to relieve the symptoms of any specific disease may vary according to various factors, such as the disease state, age, and weight of the subject, and the effect of the drug on the subject. ability.
  • embodiments of the present disclosure may not be effective in relieving the symptoms of each target disease, according to any statistical test methods known in the art such as Student's test, chi-square test, according to Mann and Whitney's U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that it should alleviate the target disease symptoms in a statistically significant number of subjects.
  • Constant amino acid modification or “conservative amino acid substitution” refers to the substitution of amino acids in a protein or polypeptide by other amino acids with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.), thereby This allows frequent changes without changing the biological activity of the protein or polypeptide or other desired characteristics (such as antigen affinity and/or specificity).
  • Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially change biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co ., page 224 (4th edition)).
  • substitution of structurally or functionally similar amino acids is unlikely to destroy biological activity.
  • Exemplary conservative amino acid substitutions are as follows:
  • Effective amount and “effective dose” refer to the amount of drug, compound or pharmaceutical composition necessary to obtain any one or more beneficial or desired therapeutic results.
  • beneficial or desired results include elimination or reduction of risk, reduction of severity or delay of the onset of the disorder, including the disorder, its complications, and the biochemical, tissue, and intermediate pathological phenotypes exhibited during the development of the disorder Academic and/or behavioral symptoms.
  • beneficial or desired results include clinical results, such as reducing the incidence or improving one or more symptoms of the various target antigen-related disorders of the present disclosure, reducing the dose of other agents required to treat the disorder , Enhance the efficacy of another agent, and/or delay the progression of the subject's target antigen-related disorders of the present disclosure.
  • Exogenous refers to substances produced outside the body of organisms, cells or humans as the case may be.
  • Endogenous refers to substances produced in cells, organisms, or humans according to circumstances.
  • Homology and “identity” are interchangeable herein and refer to the sequence similarity between two polynucleotide sequences or between two polypeptides. When the positions in the two compared sequences are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is occupied by adenine, then the molecules are homologous at that position .
  • the percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared ⁇ 100.
  • the two sequences are 60% homologous; if there are 95 matches at 100 positions in the two sequences Or homology, then the two sequences are 95% homologous.
  • a comparison is made when aligning two sequences to give the maximum percentage of homology.
  • the comparison can be performed by the BLAST algorithm, where the parameters of the algorithm are selected to give the maximum match between the individual sequences over the entire length of the individual reference sequences.
  • BLAST ALGORITHMS Altschul, SF et al., (1990) J. Mol. Biol. 215:403-410; Gish, W. et al., (1993 ) Nature Genet. 3: 266-272; Madden, TL et al. (1996) Meth. Enzymol. 266: 131-141; Altschul, SF et al. (1997) Nucleic Acids Res. 25: 3389-3402; Zhang, J. et al. (1997) Genome Res. 7:649-656.
  • Other conventional BLAST algorithms such as those provided by NCBI BLAST are also well known to those skilled in the art.
  • isolated means changed to "depart from its original state of existence", and in this case means that the designated molecule is substantially free of other non-target biomolecules.
  • isolated is not intended to refer to the complete absence of these materials or the absence of water, buffers, or salts unless they are present in amounts that significantly interfere with the experimental or therapeutic use of the compounds as described herein.
  • “Pharmaceutical composition” means a mixture containing one or more compounds of the present disclosure or a physiological/pharmaceutically acceptable salt or prodrug thereof with other chemical components, such as physiological/pharmaceutically acceptable Carriers and excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the living body, facilitate the absorption of the active ingredient and thereby exert the biological activity.
  • pharmaceutically acceptable carrier refers to any inactive substance suitable for use in formulations for delivery of antibodies or antigen-binding fragments.
  • Carriers can be anti-adherents, binders, coatings, disintegrating agents, fillers or diluents, preservatives (such as antioxidants, antibacterial or antifungal agents), sweeteners, absorption delaying agents, wetting agents Agent, emulsifier, buffer, etc.
  • suitable pharmaceutically acceptable carriers include water, ethanol, polyol (e.g. glycerin, propylene glycol, polyethylene glycol, etc.) dextrose, vegetable oil (e.g. olive oil), saline, buffer, buffered saline and the like Osmotic agents such as sugar, polyols, sorbitol and sodium chloride.
  • metabolic disorders are metabolic syndrome, obesity, impaired glucose tolerance, diabetes, diabetic ketoacidosis, hyperglycemia, hyperglycemic hyperosmolar syndrome, perioperative hyperglycemia, hyperinsulinemia Syndrome, insulin resistance syndrome, impaired fasting blood glucose, dyslipidemia, atherosclerosis, or prediabetes.
  • another aspect of the present disclosure relates to a method for immunodetection or determination of a target antigen, a reagent for immunodetection or determination of a target antigen, a method for immunodetection or determination of cells expressing the target antigen, and a method for diagnosis and target antigen
  • a diagnostic agent for a disease related to positive cells which contains a monoclonal antibody or antibody fragment of the present disclosure that specifically recognizes and binds a target antigen as an active ingredient.
  • the method for detecting or measuring the amount of target antigen may be any known method.
  • it includes immunodetection or assay methods.
  • the immunodetection or measurement method is a method of detecting or measuring the amount of antibody or antigen using labeled antigen or antibody.
  • immunodetection or measurement methods include radioactive substance-labeled immunoantibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescence immunoassay (FIA), luminescence immunoassay, Western blot, physical-chemical method Wait.
  • the above-mentioned diseases related to target antigen-positive cells can be diagnosed by detecting or measuring cells expressing the target antigen with the monoclonal antibodies or antibody fragments of the present disclosure.
  • the living sample used for detecting or measuring the target antigen there is no particular limitation on the living sample used for detecting or measuring the target antigen, as long as it has the possibility of including cells expressing the target antigen, such as tissue cells, blood, plasma, serum, pancreatic juice, urine, Feces, tissue fluid or culture fluid.
  • cells expressing the target antigen such as tissue cells, blood, plasma, serum, pancreatic juice, urine, Feces, tissue fluid or culture fluid.
  • the diagnostic agent containing the monoclonal antibody or antibody fragment of the present disclosure may also contain a reagent for performing an antigen-antibody reaction or a reagent for detecting the reaction.
  • Reagents for performing antigen-antibody reactions include buffers, salts, and the like.
  • the reagents used for detection include reagents commonly used in immunodetection or measurement methods, such as a labeled second antibody that recognizes the monoclonal antibody, its antibody fragment, or a conjugate thereof, a substrate corresponding to the label, and the like.
  • Human GCGR cDNA encoding a glucagon receptor with a total length of 477 amino acids was subcloned into an expression vector (such as pcDNA3.1) and transfected into CHO-K1 cells. After screening and single-cell cloning, monoclonals were selected for antigen expression and receptor-based cell surface expression characterization studies. The following GCGR antigens refer to human GCGR unless otherwise specified.
  • Full-length GCGR used to construct GCGR overexpressing cell lines, or used for immunization antigen and subsequent detection:
  • ProteinG is preferred for affinity chromatography.
  • the supernatant of the cultured hybridoma is centrifuged to take 10-15% volume of 1M Tris-HCl (pH8.0-8.5) according to the volume of the supernatant. Clear pH.
  • the ProteinG column is washed 3-5 times the column volume with 6M guanidine hydrochloride, and then washed 3-5 times the column volume with pure water; use a buffer system such as 1 ⁇ PBS (pH7.4) as the equilibrium buffer to equilibrate the column 3-5 Double the column volume; combine the cell supernatant with a low flow rate and control the flow rate so that the retention time is about 1min or longer; wash the column 3-5 times the column volume with 1 ⁇ PBS (pH7.4) until the UV absorption drops to Baseline; use 0.1M acetic acid/sodium acetate (pH3.0) buffer for sample elution, collect elution peaks according to UV detection, and use 1M Tris-HCl (pH8.0) to quickly adjust pH to 5-6.
  • a buffer system such as 1 ⁇ PBS (pH7.4) as the equilibrium buffer to equilibrate the column 3-5 Double the column volume; combine the cell supernatant with a low flow rate and control the flow
  • the solution can be replaced by methods well known to those skilled in the art, such as ultrafiltration concentration using ultrafiltration tubes and solution replacement to the desired buffer system, or the use of molecular exclusion such as G-25 desalting to replace the desired Buffer system, or use high-resolution size exclusion columns such as Superdex 200 to remove polymer components in the eluted product to improve sample purity.
  • methods well known to those skilled in the art such as ultrafiltration concentration using ultrafiltration tubes and solution replacement to the desired buffer system, or the use of molecular exclusion such as G-25 desalting to replace the desired Buffer system, or use high-resolution size exclusion columns such as Superdex 200 to remove polymer components in the eluted product to improve sample purity.
  • the cell culture supernatant expressing Fc bispecific protein or antibody is subjected to high-speed centrifugation to collect the supernatant.
  • the ProteinA affinity column was washed 3-5 times the column volume with 6M guanidine hydrochloride and then 3-5 times the column volume with pure water.
  • the chromatographic column is equilibrated 3-5 times the column volume using a buffer system such as 1 ⁇ PBS (pH7.4) as the equilibration buffer.
  • the supernatant of the cells was loaded with a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the solution can be replaced by methods well known to those skilled in the art, such as ultrafiltration concentration using ultrafiltration tubes and solution replacement to the desired buffer system, or the use of molecular exclusion such as G-25 desalting to replace the desired Buffer system, or use high-resolution size exclusion columns such as Superdex 200 to remove polymer components in the eluted product to improve sample purity.
  • Mouse immunity Anti-human GCGR monoclonal antibodies are produced by immunizing mice. Experimental SJL white mice, female, 6-8 weeks of age (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001). Feeding environment: SPF level. After the mice were purchased, they were kept in the laboratory environment for 1 week, adjusted for a 12/12 hour light/dark cycle, and the temperature was 20-25°C; The mice that have adapted to the environment are immunized according to the following protocol. The immune antigen is GCGRCHO-K1 stable cells.
  • IP intraperitoneal
  • GCGR CHO-K1 intraperitoneally
  • the inoculation time is 0, 14, 28, 42, 56, 70, 84, 98, 112 days.
  • Blood was collected on days 35, 63, and 91, and the antibody titer in the serum of mice was determined by ELISA.
  • mice with high antibody titers in the serum and titers tending to the platform were selected for fusion of spleen cells.
  • booster immunization was performed, and 1E7 cells/GCGC CHO-K1 cells were injected intraperitoneally (IP) to stabilize the cells.
  • Rat immunization 6-8 week old SD rats were immunized with DNA (encoding full length hGCGR, see Genbank accession number: NM_000160), and the antibody titer in rat serum was determined by FACS method. After the 3-4th immunization, rats with high antibody titers in the serum and titers tending to plateau were selected for splenocyte fusion. Immunization was boosted 3 days before splenocyte fusion.
  • hybridoma cells spleen lymphocytes and myeloma cells Sp2/0 cells ( CRL-8287 TM ) to obtain hybridoma cells.
  • the fused hybridoma cells were resuspended in a complete medium (IMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI) at a density of 0.5-1E6/ml, and 100 ⁇ l/well was seeded in a 96-well plate at 37°C After incubating for 3-4 days with 5% CO 2 , supplement 100 ⁇ l/well of HAT complete medium and continue culturing for 3-4 days until colonies are formed.
  • IMEM medium containing 20% FBS, 1 ⁇ HAT, 1 ⁇ OPI
  • HT complete medium IMDM medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI
  • ELISA test was performed after incubation at 37° C. and 5% CO 2 for 3 days.
  • the hybridoma culture supernatant was detected by cell binding ELISA method.
  • the well cells that tested positive were expanded, frozen, and subcloned two to three times in time until a single cell clone was obtained.
  • Each subcloned cell also needs to be tested by GCGR cell combined with ELISA test.
  • the hybridoma clones were obtained through experimental screening, and the antibody was further prepared by serum-free cell culture method, and the antibody was purified according to the purification example for use in the test example.
  • the process of cloning sequences from positive hybridomas is as follows.
  • the logarithmic growth phase hybridoma cells were collected, RNA was extracted using Trizol (Invitrogen, Cat No. 15596-018) according to the kit instructions, and reverse transcription was performed using PrimeScript TM Reverse Transcriptase kit (Takara, Cat No. 2680A).
  • the cDNA obtained by reverse transcription was amplified by PCR using mouse Ig-Primer Set (Novagen, TB326 Rev. B 0503) and then sequenced.
  • Screened positive clones were obtained from the obtained DNA sequence, antibodies were prepared by serum-free cell culture method, the antibodies were purified according to the purification example, and cell-based GCGR binding blocking experiments were used in the test examples after multiple rounds of screening to obtain Mouse hybridoma clones 1803, 1805, 1808 and 1810, and rat hybridoma clones 1817 and 1822.
  • the light and heavy chain CDR sequences of the above mouse-derived antibodies m1803, m1805, m1808 and m1810 have high homology
  • the light and heavy chain CDR sequences of the rat-derived antibodies rat1817 and rat1822 have high The homology, the consensus sequence is shown in the following table:
  • IMGT human antibody heavy and light chain variable region germline gene databases were compared, and heavy chain and light chain variable region germline genes with high homology to murine antibodies were selected as templates.
  • the CDRs of murine antibodies were transplanted into the corresponding human templates to form variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. If necessary, part of the amino acids in the backbone sequence are back-mutated to the amino acids corresponding to the murine antibody to obtain the humanized anti-GCGR monoclonal antibody.
  • the determination of amino acid residues in the CDR region is determined and annotated by the Kabat numbering system.
  • the light and heavy chain variable regions of the mouse antibody and the light and heavy chain constant regions of the human antibody are connected to form a chimeric antibody.
  • the chimeric antibody corresponding to clone 1803 is named ch1803, and other antibodies can be deduced by analogy.
  • the humanized light chain templates of the murine antibody m1803 are IGKV1-39*01 and hjk4.1, and the humanized heavy chain templates are IGHV1-3*01 and hjh6.1.
  • the sequence of the humanized variable region is as follows:
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR sequence.
  • hybridoma clone 1803 The humanized template selection and back mutation design of hybridoma clone 1803 are as follows:
  • P44V means to mutate Kabat number 44 P back to V according to Kabat numbering system.
  • the grafting represents that the mouse antibody CDR is directly implanted into the human germline FR region sequence.
  • the humanized antibody light chain variable region and heavy chain variable region sequence combinations of antibody derived from hybridoma clone 1803 are as follows:
  • the antibody light and heavy chain variable regions indicated by the antibody names in the above table may be connected to the antibody light and heavy chain constant regions to form a full-length antibody.
  • the light chain variable region is linked to the Kappa chain constant region shown in SEQ ID NO: 73 to form the antibody light chain
  • the heavy chain variable region is linked to SEQ ID NO: 72
  • the IgG4-AA shown are joined to form the antibody heavy chain.
  • the humanized light chain templates of the murine antibody m1810 are IGKV1-39*01 and hJK4.1, and the humanized heavy chain templates are IGHV1-69*02 and hJH4.1.
  • the sequence of the humanized variable region is as follows:
  • Hu1810VH-CDR grafting (SEQ ID NO: 68)
  • Hu1810VL-CDR grafting (SEQ ID NO: 65)
  • the order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italic in the sequence is the FR sequence, and the underline is the CDR sequence.
  • hybridoma clone 1810 The humanized template selection and back mutation design of hybridoma clone 1810 are as follows:
  • P44V means to mutate Kabat number 44 P back to V according to Kabat numbering system.
  • the grafting represents that the mouse antibody CDR is directly implanted into the human germline FR region sequence.
  • the antibody light and heavy chain variable regions referred to by the antibody names in the above table can be connected to the antibody light and heavy chain constant regions respectively to form full-length antibodies.
  • the light chain variable regions and The Kappa chain constant region shown in SEQ ID NO: 73 is connected to form an antibody light chain
  • the heavy chain variable region is connected to the IgG4-AA shown in SEQ ID NO: 72 to form an antibody heavy chain.
  • Example 4 Construction and expression of IgG4-AA form of GCGR chimera/humanized antibody
  • IgG4-AA antibody form can be obtained by simple point mutation of the IgG4 antibody form, and IgG4-AA represents F234A, L235A and S228P mutations. Mutations of F234A and L235A can reduce the binding ability of IgG4-Fc and Fc ⁇ R, and further reduce ADCC/CDC.
  • S228P represents the mutation of amino acid S at position 228 of the hinge region of wild-type IgG4 to P. This site mutation can avoid natural IgG4 antibody in Mismatches caused by Fab-exchanges occur in the body.
  • ch1803, ch1805, ch1808, ch1810, ch1817, and ch1822 are chimeric antibodies formed by linking the light and heavy chain variable regions of animal origin shown in Table 1 to the human antibody kappa chain and the human antibody IgG4-AA heavy chain constant region, respectively.
  • the sequence of the heavy chain constant region of IgG4-AA is as follows: (SEQ ID NO: 72)
  • the sequence of the light chain (Kappa chain) constant region of the antibody is as follows: (SEQ ID NO: 73)
  • hu1803-1 Antibody form IgG4AA
  • hu1803-1 heavy chain sequence (SEQ ID NO: 76)
  • ch1808 heavy chain sequence (SEQ ID NO: 82)
  • hu1810-12 heavy chain sequence (SEQ ID NO: 84)
  • Duramose peptide is a double-stranded form, the single-stranded is GLP-1/hIgG4Fc (SEQ ID NO: 90)
  • the human GLP-1 peptide is used as the GLP-1 receptor agonist part in the bispecific protein, and the GCGR antibody is used as the GCGR antagonist part of the bispecific protein to form the GLP-1/GCGR antibody bispecific protein.
  • GLP-1 amino acid mutations at specific positions of GLP-1 (such as Q17E, I23V, K28R or G30R)
  • the new GLP-1/GCGR antibody bispecific protein formed was more stable in vitro, and the GLP-1A
  • the Q mutation at position 17 is E
  • the I mutation at position 23 is V form (GLP-1C), with the highest stability.
  • Non-limiting example sequences of GLP-1 and its mutant forms of the present disclosure are as follows:
  • the C-terminal amino acid of the GLP-1 peptide of the present disclosure is connected to the N-terminal amino acid of the GCGR antibody heavy chain through a peptide bond or linker.
  • Regular expression through the 293 expression system yields the model structure of the bispecific protein shown in Table 9:
  • the GLP-1 peptide can be attached to the amino terminus of the variable region of the GCGR antibody heavy chain or to the amino terminus of the variable region of the GCGR antibody light chain. It has been verified by experiments that the bispecific protein linked to the amino terminus of the heavy chain variable region of the GCGR antibody by the GLP-1 peptide is more stable than the amino terminus linked to the variable region of the light chain of the GCGR antibody.
  • the structure of the bispecific protein in some embodiments of the GLP-1 peptide of the present disclosure linked to the variable region of the heavy chain of the GCGR full-length antibody is shown in FIG.
  • the different GLP-1 peptides are connected to the heavy chain amino acids of different antibodies with a linker (eg (GGGGS) 3 ) to form the following proteins:
  • the linker linker of the bispecific protein can be (GGGGS) 3 , and in other embodiments, it can also be a peptide bond or other linkers conventionally used for polypeptide connection.
  • the use of (GGGGS) 3 is not a bispecific protein of the present disclosure. Restrictions on connectors.
  • the nucleotide sequence encoding GLP-1, the nucleotide sequence encoding the GCGR antibody, and the nucleotide sequence of the linker protein fragment ((GGGGS) 3 ) are obtained by conventional technical means in the art.
  • the C-terminal nucleotide of GLP-1 was connected to the N-terminal nucleotide of the GCGR antibody via a linker protein, and cloned into the Phr-BsmbI vector.
  • the recombinant GLP-1/GCGR antibody bispecific protein was expressed in 293 cells and purified by the method of Example 6. The purified protein can be used in the following examples.
  • the supernatant was collected after centrifugation of the cell culture fluid at high speed, and the first step of purification was performed by affinity chromatography.
  • the chromatographic medium is Protein A that interacts with Fc or a derivative filler, such as GE's Mabselect.
  • the equilibration buffer is 1 ⁇ PBS (137 mmol/L NaCl, 2.7 mmol/L KCl, 10 mmol/L Na 2 HPO 4 , 2 mmol/L KH 2 PO 4 , pH7.4), after equilibrating 5 times the column volume, place the cells on The combination of clearing and loading, the flow rate is controlled to the sample retention time on the column ⁇ 1min.
  • the column was rinsed with 1 ⁇ PBS (pH 7.4) until the UV absorption of A280 dropped to the baseline.
  • the column was then washed with 0.1 M glycine (pH 3.0) elution buffer, the elution peak was collected based on the A280 ultraviolet absorption peak, and the collected elution sample was neutralized with 1 M Tris (pH 8.5).
  • the neutralized eluted sample was ultrafiltered and concentrated, and then subjected to size exclusion chromatography.
  • the buffer solution was 1 ⁇ PBS
  • the chromatography column was XK26/60Superdex200 (GE)
  • the flow rate was controlled at 4 ml/min
  • the sample loading volume was less than 5 ml.
  • the collected protein was identified by SEC-HPLC with a purity of greater than 95%, and was identified as correct by LC-MS and then distributed. GLP-1/GCGR antibody bispecific protein was obtained.
  • Test Example 1 ELISA experiment of GCGR chimeric antibody binding to GCGR of human, mouse and cynomolgus monkey
  • the binding capacity of the anti-GCGR antibody was tested by the antibody binding experiment with CHO cells overexpressing GCGR. After transfection, the GCGR full-length plasmids of human, mouse and cynomolgus monkeys were transferred into CHO cells for two weeks under pressure and the expression of GCGR was detected. After the overexpression cells were fixed to the bottom of the 96-well plate, the strength of the signal after the addition of the antibody was used to determine the binding activity of the antibody and GCGR overexpressing CHO cells.
  • the GCGR method is used as an example. The specific experimental methods are as follows:
  • the cells were seeded in 96-well plates at a density of 0.9-1.0 ⁇ 10 6 /ml, 100 ⁇ l/well, and cultured overnight. After discarding the supernatant and washing it three times with PBS, 100 ⁇ l/well of cell immunofixation solution (Beyotime, Cat Wo. P0098) was added for fixation at room temperature for 1 hour, and PBS was washed four times. After discarding the liquid, 5% skimmed milk (BD skim milk, Cat Wo.232100) blocking solution diluted with PBS was added 200 ⁇ l/well, and incubated at 37° C. incubator for 3 hours for blocking.
  • 5% skimmed milk BD skim milk, Cat Wo.232100
  • the plate was washed 3 times with PBST buffer (pH7.4PBS containing 0.05% tweeeW-20), and 50 ⁇ l/well of different concentrations of the test antibody (hybridized antibody) diluted with the sample diluent were added , Chimeric antibody or humanized antibody), placed in an incubator at 37 °C for 2 hours. After the incubation, the plate was washed 3 times with PBST, and 50 ⁇ l/well of HRP-labeled goat anti-mouse secondary antibody (JacksoW ImmuWo Research, Cat Wo. 115-035-003) or goat anti-human secondary antibody (JacksoW) ImmuWo Research, Cat Wo.
  • HRP-labeled goat anti-mouse secondary antibody JacksoW ImmuWo Research, Cat Wo. 115-035-003
  • goat anti-human secondary antibody JacksoW
  • Test Example 2 GCGR chimeric antibody blocks the binding of GCGR ligand to GCGR
  • the antagonistic activity of GCGR chimeric antibodies was evaluated by the GCGR chimeric antibody blocking the binding of GCGR ligand glucagon to GCGR.
  • the combination of cAMP and CRE can start the expression of the luciferase gene (luciferase) downstream of the CRE.
  • the luciferase binds to its substrate and emits fluorescence, which reflects the inhibition efficiency through changes in the fluorescence signal.
  • the CRE was cloned upstream of the luciferase gene, and the CHO-K1 cells were co-transfected with the plasmid containing the GCGR gene to select monoclonal cells that highly expressed both CRE and GCGR.
  • the GLP-1/GCGR antibody bispecific protein and glucagon can competitively bind to GCGR, blocking the downstream signal transmission of GCGR, affecting the downstream cAMP expression, and evaluating the GLP-1/GCGR antibody by measuring the change in fluorescence signal Antagonistic activity of bispecific protein against GCGR.
  • Test Example 3 Blocking test of GCGR ligand binding to GCGR by GCGR humanized antibody
  • Test Example 4 Stability of GLP-1/GCGR antibody bispecific protein in PBS
  • test antibody bispecific protein dissolved in 1ml 1 ⁇ PBS (pH7.4), store in a 37°C incubator; take samples on days 0 and 14, respectively, and use Agilent 6530Q-TOF for LC-MS to detect the complete heavy chain retention , The results are shown in Table 14 below.
  • the bispecific proteins containing the modified GLP-1 peptides are much more stable than the bispecific proteins containing GLP-1A (SEQ ID NO: 91), and hu1803-9B, hu1803-9D and hu1803-9G show Better stability.
  • Figure 2 and Table 15 show that both hu1803-9B and hu1803-9D can completely inhibit the antagonistic activity of GCGR, and have comparable efficacy and IC50 (concentration required to inhibit 50% of the maximum activity) with GCGR monoclonal antibody, indicating that hu1803-9B Both hu1803-9D and GCGR antibody retain partial biological activity.
  • IC50 concentration required to inhibit 50% of the maximum activity
  • the GLP-1/GCGR antibody bispecific protein GLP-1 portion was evaluated for GLP-1R activation activity.
  • cAMP cyclopentase
  • CRE cyclopentase binds to its substrate, it emits fluorescence, which reflects the inhibition efficiency through the change of fluorescence signal.
  • CRE was cloned upstream of the luciferase gene, and CHO-K1 cells were co-transfected with the plasmid containing the GLP-1R gene to select monoclonal cells that simultaneously expressed CRE and GLP-1R.
  • the GLP-1/GCGR antibody bispecific protein and the positive control Doxyl glycopeptide can bind to GLP-1R, activate downstream signal transmission of GLP-1R, stimulate downstream cAMP expression, and evaluate GLP-1/ by measuring changes in fluorescence signal Activating activity of GCGR antibody bispecific protein on GLP-1R.
  • a Prepare a cell suspension with fresh cell culture medium, add 25000 cells/well to a 96-well cell culture plate of 90 ⁇ l culture system, and incubate at 37°C for 16 hours with 5% carbon dioxide.
  • Figure 3 and Table 17 show that both hu1803-9B and hu1803-9D can fully activate GLP-1R, and have comparable efficacy and EC50 (concentration required to activate 50% of the maximum activity) with the positive control duraglutide, indicating hu1803 Both -9B and hu1803-9D retained the full biological activity of GLP-1.
  • Table 18 shows that the ch1805-D, ch1808-D, and ch1817-D chimeric antibodies can fully activate GLP-1R by linking with GLP1 peptides, and they have comparable efficacy and EC50 (50% of the maximum activation activity) as the positive control degree of glycopyrrolate The desired concentration) indicates that the bispecific protein formed by linking different GCGR antibodies to the GLP1 peptide in the manner disclosed herein does not affect the biological activity of the GLP1 peptide.
  • C57 mice Four C57 mice were used in the experiment, female, with light/dark adjustment for 12/12 hours, constant temperature of 24 ⁇ 3°C, humidity of 50-60%, and free access to drinking water. Purchased from Jie Jie Jie Experimental Animal Co., Ltd. On the day of the experiment, C57 mice were injected with equimolar hu1803-9D and the positive control drug dula glycopeptide into the tail vein, respectively, the dosage was 6 mg/kg and 2.35 mg/kg, and the injection volume was 10 ml/kg.
  • the drug metabolism time in mice is shorter, and the selected blood sampling time point is: Day 0h, 1h, 24h (day 2), on day 3, blood was taken from the fundus vein of mice, each time 150 ⁇ l (before blood collection, 1.5 ⁇ l DPP-4 inhibitor was added to blood collection tube in advance); The collected blood samples were placed at 4°C for half an hour to agglomerate, and then centrifuged at 14000 ⁇ g for 5 minutes at 4°C. Collect the supernatant (approximately 80 ⁇ l) and store at -80°C immediately.
  • the detection process is described as follows:
  • the PK analysis result shows that the half-life of the bispecific protein molecule hu1803-9D of the present disclosure in mice is about 23.4h, which is twice that of the positive control drug.
  • Test Example 8 In vivo drug efficacy experiment of ob/ob mice
  • mice diabetic ob/ob mice and wild-type mice of the same age (Nanjing Institute of Model Animals, Nanjing University), the purpose is to observe the continuous multiple administration of GLP-1/GCGR antibody bispecific protein Therapeutic effect on diabetes-related indicators such as blood glucose, glycated hemoglobin, weight, and food intake of ob/ob mice.
  • mice in the model group were divided into 6 groups according to the random and fasting body weight and random and fasting blood glucose on the day of the experiment, respectively:
  • Model control group 2.84mg/kg, 1.42mg/kg GCGR monoclonal antibody group (hu1803-9), 1.16mg/kg positive control group (duratopeptide) and 3mg/kg, 1.5mg/kghu1803-9D
  • model control Group mice were given subcutaneous injection (SC) with phosphate buffer solution, and mice in each group were given subcutaneous injection (9:00AM) once a week for a total of 4 times (Table 20).
  • Group deal with dose Dosing frequency Mode of administration 1 ob/ob mouse model control group PBS Once a week x 4 weeks S.C. 2 GCGR monoclonal antibody low dose group 1.42mpk Once a week x 4 weeks S.C. 3 GCGR monoclonal antibody high-dose group 2.84mpk Once a week x 4 weeks S.C. 4 Positive control high-dose group 1.16mpk Once a week x 4 weeks S.C. 4 hu1803-9D low-dose group 1.5mpk Once a week x 4 weeks S.C. 5 hu1803-9D high-dose group 3mpk Once a week x 4 weeks S.C.
  • Random blood glucose was measured before the first administration and on days 1, 2, 3, and 7 after the administration, and random blood glucose was measured once a week thereafter.
  • the fasting blood glucose was measured at 6h before the first administration and on the 3rd and 7th days after the administration, and the fasting blood glucose was measured once a week thereafter.
  • mice were euthanized after fasting for 6 hours (8:00-14:00). After the heart was drawn, the whole blood was divided into two parts, and a part of about 30 ⁇ l was injected into the centrifuge tube with the added anticoagulant and stored. It is used for measuring glycated hemoglobin in wet ice, and the other part is centrifuged and the serum is centrifuged to determine the levels of TG, FFA, CHOL, HDL and LDL.
  • the data is analyzed by graphpad Prism 6 software, and Student-t test is used for statistical analysis of the data.
  • the random blood glucose of ob/ob mice in the model control group was maintained at a high level throughout the test period.
  • the random blood glucose of mice in each group had different degrees Decreased, showing a good dose effect and significantly lower than the model control group.
  • 2.84mg/kg administration of hu1803-9 and 3mg/kg administration of hu1803-9D were significantly better than other test groups, while 3mg/kg administration of hu1803-9D administration on days 3, 6, 14 and 30 Random blood sugar is better.
  • Test Example 9 Competitive ELISA experiment of GCGR antibody
  • the biotinylated antibody competes with standard antibodies at different concentrations for binding to CHO cells overexpressing GCGR to detect and classify the epitope that GCGR antibody binds to GCGR.
  • the preparation method of the cell plate is the same as that of Test Example 1.
  • the specific experimental methods are as follows:
  • the antibody was labeled according to the instructions of the biotin labeling kit (Dojindo Molecular Technologies, Inc. LK03).
  • a biotin labeling kit (Dojindo Molecular Technologies, Inc. LK03).
  • 50 ⁇ l/well of different concentrations of unbiotin-labeled test antibody diluted with the sample diluent were placed and incubated at 37°C for 2 hours. After incubation, wash the plate 3 times with PBST, add 50 ⁇ l/well of biotinylated antibody diluted with sample diluent to 0.1 ⁇ g/ml, wash the plate 3 times with PBST after incubating for 2 hours at 37°C, add HRP-labeled goat anti-human Secondary antibody (JacksoW ImmuWo Research, Cat Wo.
  • IC% (test antibody highest OD 450nm - test antibody Minimum OD 450nm) / (tested up to OD 450nm - labeled antibody Minimum OD 450nm) calculated competition efficiency, 22 results in the table below.

Abstract

提供一种人GCGR抗体、GLP-1肽及其突变体，以及GCGR抗体与GLP-1肽融合形成的双特异性蛋白及其制备方法，可用于减肥、治疗糖尿病。

Description

双特异性蛋白
本申请要求2018年12月21日提交的专利申请201811573634.0和2018年12月27日提交的专利申请201811606887.3的优先权；两者通过引用并入本文。
技术领域
本公开涉及人GCGR抗体、GLP-1肽及其突变体，以及GCGR抗体与GLP-1肽融合形成的双特异性蛋白及其制备方法和应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然地构成现有技术。
糖尿病(diabetes mellitus，DM)是一种表现为胰岛素分泌缺陷和/或胰岛素作用障碍，以高血糖为特征的代谢性疾病，其发病主要是胰岛素与胰高血糖素共同作用的结果。
GLP-1是影响胰岛素分泌最主要的激素之一，与胰高血糖素(Glucagon)一起均来源于前胰岛素原。前胰岛素原由约158个氨基酸组成，在不同的部位被切割成不同的肽链。人体内具有生物活性的GLP-1主要包括GLP-1(7-36)酰胺和GLP-1(7-37)两种形式。GLP-1通过小肠L细胞分泌，主要以葡萄糖浓度依赖性方式促进胰岛素分泌，保护胰岛β细胞，抑制胰高血糖素分泌来降低机体血糖水平。同时GLP-1还有抑制胃排空，降低食欲的作用。临床可用于II型糖尿病和肥胖症的治疗。机体内天然活性GLP-1的半衰期很短(不足2分钟)，很容易被机体内DPPIV酶降解，不具有临床使用价值。
延长半衰期一直是GLP-1类药物研发的主要方向，目前已有多款GLP-1激动剂上市，如度拉糖肽(Dulaglutide)和索马鲁肽(Semaglutide)等。虽然GLP-1在疗效上已经获得人们的充分肯定，但也存在诸多副作用，主要表现为胃肠道症状，低血糖，胰腺炎及肾损伤等。
胰高血糖素与胰岛素作用相反，主要起升高机体血糖的作用。胰高血糖素是由胰岛α细胞分泌的具有29个氨基酸的肽，与肝细胞膜上受体GCGR结合后主要通过激活下游cAMP/PKA途径，加速糖原分解、脂肪分解和糖异生，使血糖升高。
研究发现，GCGR基因敲除鼠呈现出GLP-1升高，肝糖输出减少，脂代谢增加，食欲减退等一系列表型。GCGR是糖尿病治疗最热门的靶点之一，但目前针对GCGR的拮抗类药物研发进展缓慢。REMD Biotherapeutics公司的REMD-477，目前是最前沿的GCGR单克隆抗体药物，处于临床二期。
现有技术已在专利CN101589062A、CN101983208A、CN102482350A、CN103314011A、CN105189560A、CN107614695A、US20180273629A1、WO2013059531A1等专利中公开GCGR抗体，但仍有待于提供新的高效的GCGR 抗体及糖尿病治疗方法。
发明内容
本公开提供一种抗GCGR的单克隆抗体或其抗原结合片段。该抗体或其抗原结合片段具有与人GCGR(或其所包含的抗原表位)结合的能力。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，包含选自如下a)或b)的重链可变区和轻链可变区的组合：
a)所述重链可变区包含分别如SEQ ID NO：48、49和50所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：51、52和53所示的LCDR1、LCDR2和LCDR3区；或
b)所述重链可变区包含分别如SEQ ID NO：38、39和54所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：55、56和57所示的LCDR1、LCDR2和LCDR3区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，包含选自如下i)至vi)的重链可变区和轻链可变区的任一组合：
i)所述重链可变区包含分别如SEQ ID NO：14、15和16所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：17、18和19所示的LCDR1、LCDR2和LCDR3区；
ii)所述重链可变区包含分别如SEQ ID NO：20、21和22所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：23、24和25所示的LCDR1、LCDR2和LCDR3区；
iii)所述重链可变区包含分别如SEQ ID NO：26、27和28所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：29、30和31所示的LCDR1、LCDR2和LCDR3区；
iv)所述重链可变区包含分别如SEQ ID NO：32、33和34所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：35、36和37所示的LCDR1、LCDR2和LCDR3区；
v)所述重链可变区包含分别如SEQ ID NO：38、39和40所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：41、42和43所示的LCDR1、LCDR2和LCDR3区；或
vi)所述重链可变区包含分别如SEQ ID NO：38、39和44所示的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含分别如SEQ ID NO：45、46和47所示的LCDR1、LCDR2和LCDR3区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段是鼠源抗体、嵌合抗体或人源化抗体或其抗原结合片段。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，所述人 源化抗体包含源自人抗体的框架区或其框架区变体，所述框架区变体为在人抗体的轻链框架区上具有至多10个回复突变，和/或，所述框架区变体在人抗体的重链框架区基础上具有至多10个、至多9个、至多8个、至多7个、至多6个、至多5个、至多4个、至多3个、至多2个、至多1个氨基酸的回复突变。
在一些实施方式中，所述框架区变体包含选自：
aa)轻链可变区中包含42G、44V、71Y和87F中的一个或多个氨基酸回复突变，和/或重链可变区中包含38K、48I、67A、69F、71A、73P、78A和93S中的一个或多个氨基酸回复突变；或者
ab)轻链可变区中包含38L、44V、59S、70E和71Y中的一个或多个氨基酸回复突变，和/或重链可变区中包含38K、48I、66K、67A、69L、73R、78M和94S中的一个或多个氨基酸回复突变。进一步地，所述回复突变位点的位置依据Kabat编号规则确定。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列如SEQ ID NO:2、61、62、63和64任一所示或与SEQ ID NO：2、61、62、63和64任一所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区；和/或
序列如SEQ ID NO：3、58、59和60任一所示或与SEQ ID NO：3、58、59和60任一所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方案中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列如SEQ ID NO：63所述的重链可变区和序列如SEQ ID NO：58所示的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列为SEQ ID NO:4所示或与SEQ ID NO：4所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区和/或序列为SEQ ID NO：5所示或与SEQ ID NO：5所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列为SEQ ID NO:6所示或与SEQ ID NO：6所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区和/或序列为SEQ ID NO：7所示或与SEQ ID NO：7所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列为SEQ ID NO:8、68、69、70和71任一所示或与SEQ ID NO：8、68、69、70和71任一所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区和/或序列为SEQ ID NO：9、65、66和67 任一所示或与SEQ ID NO：9、65、66和67任一所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方案中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列如SEQ ID NO：71所述的重链可变区和序列如SEQ ID NO：67所示的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列为SEQ ID NO:10所示或与SEQ ID NO：10所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区和/或序列为SEQ ID NO：11所示或与SEQ ID NO：11所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其包含序列为SEQ ID NO:12所示或与SEQ ID NO：12所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的重链可变区和/或序列为SEQ ID NO：13所示或与SEQ ID NO：13所示具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％序列同一性的轻链可变区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括抗体恒定区，具体地，所述抗体恒定区的重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述抗体恒定区的轻链恒定区选自人抗体κ和λ链恒定区及其常规变体，更优选包含SEQ ID NO:72所示的人抗体重链恒定区和SEQ ID NO:73所示的人轻链恒定区。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:74、76或78所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:75所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:74、76或78所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:77所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:74、76或78所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:79所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:80所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:81所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:82所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:83所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:84所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:85所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:86所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:87所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方式中，所述的抗GCGR的单克隆抗体或其抗原结合片段，包括重链和轻链，其中：
所述重链为SEQ ID NO:88所示或与其具有至少85％、86％、87％、88％、89％、 90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性，且所述轻链为SEQ ID NO:89所示或与其具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、或99％的序列同一性。
在一些实施方案中，所述的抗GCGR的单克隆抗体或其抗原结合片段，其包含如序列如SEQ ID NO：78所示的重链和序列如SEQ ID NO：79所示的轻链。
在一些实施方案中，所述的抗GCGR的单克隆抗体或其抗原结合片段，其包含如序列如SEQ ID NO：84所示的重链和序列如SEQ ID NO：85所示的轻链。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，包含选自如下ac)至ah)的重链可变区和轻链可变区的任一组合：
ac)所述重链可变区包含与SEQ ID NO：2所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：3所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
ad)所述重链可变区包含与SEQ ID NO：4所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：5所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
ae)所述重链可变区包含与SEQ ID NO：6所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：7所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
af)所述重链可变区包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：9所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
ag)所述重链可变区包含与SEQ ID NO：10所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：11所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；或
ah)所述重链可变区包含与SEQ ID NO：12所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和所述轻链可变区包含与SEQ ID NO：13所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其中所述抗原结合片段是选自Fab、Fab'、F(ab')2、单链抗体、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其特征在于与如前所述的单克隆抗体或其抗原结合片段竞争结合人GCGR(或其表位)。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，具有以下特征中的至少一种：
i.以小于500nM、小于450nM、小于400nM、小于350nM或小于300nM(优 选小于300nM)IC50值的水平阻断人GCGR与人胰高血糖素结合的拮抗活性；
ii.阻断食蟹猴或恒河猴GCGR与食蟹猴或恒河猴胰高血糖素结合的拮抗活性；
iii.抑制人血糖浓度的升高；
iv.阻断小鼠GCGR与小鼠胰高血糖素结合的拮抗活性。
在一些实施方式中，所述抗GCGR的单克隆抗体或其抗原结合片段，其中所述单克隆抗体或其抗原结合片段与如前所述的单克隆抗体或其抗原结合片段结合相同的抗原表位。
另一方面，本公开提供一种双特异性蛋白。在一些实施方式中，所提供的一种双特异性蛋白，所述双特异性蛋白包含GLP-1肽和GCGR抗体，所述GLP-1肽与所述GCGR抗体的多肽链之间由肽键或接头共价连接。
在一些实施方式中，所述的双特异性蛋白，其中所述GLP-1肽的羧基端与GCGR抗体的重链可变区的氨基端由肽键或接头连接，或者所述GLP-1肽的羧基端与GCGR抗体的轻链可变区的氨基端由肽键或接头连接。
在一些实施方式中，所述的双特异性蛋白，其中所述GLP-1肽的羧基端与GCGR抗体的重链可变区的氨基端由肽键或接头连接。
在一些实施方式中，所述的双特异性蛋白，其中所述GLP-1肽的羧基端与GCGR抗体的轻链可变区的氨基端由肽键或接头连接。
在一些实施方式中，所述的双特异性蛋白，其中所述GLP-1肽的羧基端与GCGR全长抗体的重链氨基端由肽键或接头连接。
在一些实施方式中，所述的双特异性蛋白，其中所述GLP-1肽的羧基端与全长GCGR抗体的轻链氨基端由肽键或接头连接。
在一些实施方式中，所述的双特异性蛋白，所述GCGR抗体选自如前所述的抗GCGR的单克隆抗体或其抗原结合片段。
在一些实施方式中，所述的双特异性蛋白，所述GLP-1肽是如SEQ ID NO：91所示的GLP-1A，或所述GLP-1肽在GLP-1A基础上具有Q17E、I23V、K28R和G30R中一个或多个氨基酸替代的GLP-1A肽变体。
在一些实施方式中，所述的双特异性蛋白，所述GLP-1肽是如SEQ ID NO：91所示的GLP-1A，或所述GLP-1肽在GLP-1A基础上具有Q17E的GLP-1A肽变体。
在一些实施方式中，所述的双特异性蛋白，所述GLP-1肽是如SEQ ID NO：91所示的GLP-1A，或所述GLP-1肽在GLP-1A基础上具有Q17E替代，且同时还具有I23V、K28R和G30R中一个或多个氨基酸替代GLP-1A肽变体。
在一些实施方式中，所述的双特异性蛋白，所述GLP-1肽是如SEQ ID NO：91所示的GLP-1A，或所述GLP-1肽在GLP-1A基础上具有Q17E或具有Q17E和I23V的GLP-1A肽变体。
在一些实施方式中，所述的双特异性蛋白，所述GLP-1A肽变体包含如SEQ ID NO：92、93、94、95、96、97、98或99所示的序列或由其组成。
在一些实施方式中，所述的双特异性蛋白，所述GCGR抗体是选自如前任一项所述的抗GCGR的单克隆抗体或其抗原结合片段和如前任一项所述的GLP-1A肽或GLP-1A肽变体。
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含第一多肽链和第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：100、101、102、103、104、105、106、107和108任一所示的多肽，和所述第二多肽链选自如SEQ ID NO：79所示的多肽。
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含GCGR抗体重链的第一多肽链和包含GCGR抗体轻链的第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：109所示的多肽，和所述第二多肽链选自如SEQ ID NO：81所示的多肽；
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含GCGR抗体重链的第一多肽链和包含GCGR抗体轻链的第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：110所示的多肽，和所述第二多肽链选自如SEQ ID NO：83所示的多肽；
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含GCGR抗体重链的第一多肽链和包含GCGR抗体轻链的第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：111所示的多肽，和所述第二多肽链选自如SEQ ID NO：85所示的多肽；
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含GCGR抗体重链的第一多肽链和包含GCGR抗体轻链的第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：112所示的多肽，和所述第二多肽链选自如SEQ ID NO：87所示的多肽；或者
在一些实施方式中，所述的双特异性蛋白，所述双特异性蛋白具有包含GCGR抗体重链的第一多肽链和包含GCGR抗体轻链的第二多肽链，其中：所述第一多肽链选自如SEQ ID NO：113所示的多肽，和所述第二多肽链选自如SEQ ID NO：89所示的多肽。
另一方面，本公开还提供一种GLP-1肽变体。在一些实施方式中，所述GLP-1肽变体，其是在SEQ ID NO：91所示的GLP-1A基础上具有Q17E、I23V、K28R和G30R中一个或多个氨基酸突变的突变体。
在一些实施方式中，所述GLP-1肽是如SEQ ID NO：91所示的GLP-1A，或所述GLP-1肽在GLP-1A基础上具有Q17E或具有Q17E和I23V的GLP-1A肽变体。
在一些实施方式中，所述的GLP-1肽变体，其中具有如SEQ ID NO：92、93、 94、95、96、97、98或99所示的序列。
本公开还提供一种药物组合物，其含有治疗有效量的如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，以及一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
本公开还提供一种分离的核酸分子，其编码如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体。
本公开提供一种重组载体，其包含如前所述的分离的核酸分子。
本公开提供一种用如前所述的重组载体转化的宿主细胞，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞或昆虫细胞。
本公开提供用于生产如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或根据如前所述的GLP-1肽变体的方法，所述方法包括将如前所述宿主细胞在培养基中进行培养以形成并积累如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，以及从培养物回收所述单克隆抗体或其抗原结合片段或双特异性蛋白或GLP-1肽变体。
本公开提供用于体外检测或测定人GCGR的方法，所述方法包括使用如前所述的单克隆抗体或其抗原结合片段。
一种检测人GCGR的试剂盒，其包含如前所述的单克隆抗体或其抗原结合片段。
根据一些实施方式，还提供了前述抗GCGR单克隆抗体或其抗原结合片段在制备医疗器械(例如试剂盒、阵列、试纸、多孔板、磁珠、包被微粒)中的用途，所述医疗器械包含前所述的单克隆抗体或其抗原结合片段。作为一个示例，试剂盒中包含包被有前述抗GCGR单克隆抗体或其抗原结合片段的多孔板。
本公开提供如前所述的单克隆抗体或其抗原结合片段在制备用于检测或测定人GCGR的试剂中的用途。
本公开提供一种降低受试者血糖浓度的方法，所述方法包括向受试者施用治疗有效量的如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，或如前所述的药物组合物。
优选地，所述治疗有效量为单位剂量的组合物中含有0.1-3000mg的如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或根据如前所述的GLP-1肽变体。
本公开提供一种治疗代谢障碍的方法，所述方法包括向受试者施用如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，或如前所述的药物组合物；优选地，所述代谢障碍为代谢综合征、肥胖症、葡萄糖耐量受损、糖尿病、糖尿病酮症酸中毒、高血糖症、高血糖高渗综合征、围术期高血糖症、高胰岛素血症、胰岛素抵抗综合症、空腹 血糖受损、血脂异常、动脉粥样硬化或糖尿病前期状态。
本公开还提供根据如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，或如前所述的药物组合物在制备治疗代谢障碍或降低受试者血糖浓度的药物中的用途；
优选地，所述代谢障碍为代谢综合征、肥胖症、葡萄糖耐量受损、糖尿病、糖尿病酮症酸中毒、高血糖症、高血糖高渗综合征、围术期高血糖症、高胰岛素血症、胰岛素抵抗综合症、空腹血糖受损、血脂异常、动脉粥样硬化或糖尿病前期状态。
本公开还提供用如前所述的抗GCGR的单克隆抗体或其抗原结合片段，或如前所述的双特异性蛋白，或如前所述的GLP-1肽变体，或如前所述的药物组合物用作药物，优选的用作治疗代谢障碍或降低受试者血糖浓度的药物。
更优选地，所述代谢障碍为代谢综合征、肥胖症、葡萄糖耐量受损、糖尿病、糖尿病酮症酸中毒、高血糖症、高血糖高渗综合征、围术期高血糖症、高胰岛素血症、胰岛素抵抗综合症、空腹血糖受损、血脂异常、动脉粥样硬化或糖尿病前期状态。
附图说明
图1：本公开的双特异性蛋白(GLP-1/GCGR抗)结构示意图。
图2：双特异性蛋白及GCGR抗体对GCGR的拮抗活性。
图3：双特异性蛋白及度拉糖肽对GLP-1R的激活活性。
图4：长期用药对ob/ob小鼠随机血糖的影响，Vehicle(空载体)为注射磷酸盐缓冲液(PBS)的模型对照组，hu1803-9D-3mpk与hu1803-9-2.84mpk和度拉糖肽-1.16mpk是相同物质的量浓度。
图5：长期用药对ob/ob小鼠饥饿血糖的影响。所有实验组均能显著降低小鼠空腹血糖浓度，其中hu1803-9D-3mpk与hu1803-9-2.84mpk的降血糖浓度能力最强，且hu1803-9D-3mpk的降血糖活性优于hu1803-9-2.84mpk。
具体实施方式
术语
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
术语“双特异性蛋白”指能够与两个目标蛋白或目标抗原结合的蛋白分子。在本公开双特异性蛋白特指能够结合GCGR和GLP-1R(GLP-1受体)，由GLP-1肽和GCGR抗体(或其抗原结合片段)的多肽链融合后形成的蛋白。
“GLP-1肽”指能结合并激活GLP-1受体的肽。现有技术的肽描述于专利申请WO2008/071972、WO2008/101017、WO2009/155258、WO2010/096052、 WO2010/096142、WO2011/075393、WO2008/152403、WO2010/070251、WO2010/070252、WO2010/070253、WO2010/070255、WO2011/160630、WO2011/006497、WO2011/117415、WO2011/117416、WO2006/134340、WO1997046584、WO2007124461、WO2017100107、WO2007039140、CN1935261A、CN1935846A、WO2006036834、WO2005058958、WO2002046227、WO1999043705、WO1999043708、WO1999043341、CN102949730、CN104293834、CN104327187、WO2015067716、WO2015049651、WO2014096145、WO2014096148、WO2014096150、WO2014096149中，其内容通过引用并入本文。包括GLP-1、GLP-1类似物和GLP-1受体肽激动剂，部分具体的GLP-1肽例如：利西拉来(Lixisenatide)/AVE0010/ZP10/Lyxumia、艾塞那肽(Exenatide)/毒蜥外泌肽-4/Byetta/Bydureon/ITCA 650/AC-2993、利拉鲁肽/Victoza、索马鲁肽(Semaglutide)、他司鲁肽(Taspoglutide)、Syncria/阿必鲁肽(Albiglutide)、度拉糖肽(Dulaglutide)、rExendin-4、CJC-1134-PC、PB-1023、TTP-054、Langlenatide/HM-11260C、CM-3、GLP-1Eligen、ORMD-0901、NN-9924、NN-9926、NN-9927、Nodexen、Viador-GLP-1、CVX-096、ZYOG-1、ZYD-1、GSK-2374697、DA-3091、MAR-701、MAR709、ZP-2929、ZP-3022、TT-401、BHM-034、MOD-6030、CAM-2036、DA-15864、ARI-2651、ARI-2255、艾塞那肽-XTEN和胰高血糖素-Xten，除上述GLP-1肽外，还包括本公开基于SEQ ID NO：91所示GLP-1A及其突变体(如SEQ ID NO：92-99所示)。
GPCR(G Protein-Coupled Receptor)，即G蛋白偶联受体，是表达在细胞质膜上的一类跨膜蛋白。GPCR由超过800个成员组成，是目前已知的哺乳动物基因组中最大的膜蛋白家族。在人体内，GPCR蛋白广泛分布于中枢神经系统、免疫系统、心血管、视网膜等器官和组织，参与机体的发育和正常的功能行使。
GPCR蛋白主体由7段跨细胞质膜的α螺旋结构构成。N端和三个环位于胞外，参与蛋白与其受体的相互作用；C端和3个环位于胞内，其中C端和第3个环在GPCR蛋白与下游G蛋白的相互作用从而介导胞内的信号传导过程中发挥重要的作用。
“GCGR”是胰高血糖素(Glucagon)受体，是GPCR家族的成员之一，胰高血糖素与GCGR结合后主要通过激活下游途径，加速糖原分解、脂肪分解和/或糖异生，使血糖升高。
术语“抗体(antibody，Ab)”包含至少一个与具体抗原(或其表位)(例如GCGR)特异性结合或相互作用(例如识别和/或结合)的互补决定区的抗原结合分子(或分子复合物)。
术语“抗体”包含：包括通过双硫键相互连接的四条多肽链，两条重(H)链和二条轻(L)链的免疫球蛋白分子以及其多聚体(例如IgM)。各重链包含重链可变区(文中缩写为HCVR或VH)和重链恒定区(CH)。这一重链恒定区包含三个 区(结构域)，CH1、CH2和CH3。各轻链包含轻链可变区(文中缩写为LCVR或VL)和轻链恒定区(CL)。VH和VL区可进一步细分为高变区，称为互补决定区(CDR)，其间散布着较保守性区域，称为框架区(framework region,FR，也称骨架区、构架区)。各VH和VL是由三个CDR和四个FR所组成，以下列顺序由氨基端排列到羧基端：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。
在本公开的不同实施例中，抗GCGR抗体(或其抗原结合片段)的FR可与人种系序列相同，或可经自然或人工修饰。抗体可以是不同亚类(subclass)的抗体，例如，IgG(例如，IgG1、IgG2、IgG3、或IgG4亚类)、IgA1、IgA2、IgD、IgE或IgM抗体。
术语“抗体”还包含完全抗体分子的抗原结合片段。
术语抗体的“抗原结合部分”、“抗原结合结构域”、“抗原结合片段”等，如文中所用，包含任何与抗原特异性结合形成复合物的天然生成、酶制得、合成或基因工程改造的多肽或糖蛋白。抗体的抗原结合片段可使用任何适合的标准技术，例如蛋白质水解消化作用或涉及编码抗体可变区和(视需要)恒定区的DNA操作和表达的重组基因工程技术，来源于例如全抗体分子。这一DNA是已知的和/或可容易地从例如市售来源、DNA资料库(包含，例如噬菌体-抗体资料库)取得或可经合成。这一DNA可用化学或通过使用分子生物技术来定序和操作，例如将一个或多个可变和/或恒定区排列成适合的配置，或导入密码子，产生半胱氨酸残基、修饰、增添或删除氨基酸等。
抗原结合片段的非限定示例包含：(i)Fab片段；(ii)F(ab′)2片段；(iii)Fd片段；(iv)Fv片段；(v)单链Fv(scFv)分子；(vi)dAb片段。其它工程改造分子，例如区域特异性抗体、单域抗体、区域删除抗体、嵌合抗体、双抗体、三抗体、四抗体、微抗体、纳米抗体(例如单价纳米抗体、双价纳米抗体等)、小模块免疫医药(SMIP)和鲨可变IgNAR区，也涵盖在文中所用的“抗原结合片段”的词语内。
抗原结合片段典型地将包含至少一个可变区。可变区可以是任何大小或氨基酸组成的区域且一般将包含与一个或多个框架序列相邻或在其框架内的CDR。
在某些实施例中，抗体的抗原结合片段在任何可变区和恒定区的配置中，可变区和恒定区可直接彼此相连接或可通过完整或部分的绞链或连接子区相连接。绞链区可由至少2个(例如5、10、15、20、40、60或更多个)氨基酸所组成，使得在单一多肽分子中在相邻的可变和/或恒定区之间产生柔性和半柔性连结。
“鼠源抗体”在本公开中为根据本领域知识和技能制备的来源于小鼠或大鼠的单克隆抗体。制备时用抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤，当所注射的试验对象为小鼠时，所产生的抗体为小鼠来源抗体，当所注射的试验对象为大鼠时，所产生的抗体为大鼠来源抗体。
“嵌合抗体(chimeric antibody)”，是将第一物种(如鼠)抗体的可变区与第 二物种(如人)抗体的恒定区融合而成的抗体。建立嵌合抗体，要先建立分泌第一物种(如鼠)单抗的杂交瘤，然后从杂交瘤细胞中克隆可变区基因，再根据需要克隆第二物种(如人)抗体的恒定区基因，将第一物种可变区基因与第二物种恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
在本公开一个优选的实施方案中，所述的嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区，优选包含人源IgG1、IgG2或IgG4重链恒定区，或者使用氨基酸突变(如YTE突变或回复突变，L234A和/或L235A突变，或S228P突变)的IgG1、IgG2或IgG4重链恒定区变体。
术语“人源化抗体(humanized antibody)”，包括CDR移植抗体(CDR-grafted antibody)，是指将动物来源抗体，例如鼠源抗体的CDR序列移植到人的抗体可变区框架区(或构架区，framework region)中产生的抗体。人源化抗体可以克服嵌合抗体由于携带大量异源蛋白成分，从而诱导的异源性反应。此类框架区的序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网http://www.vbase2.org/获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。
为避免免疫原性下降引起的活性下降，可对所述的人抗体可变区框架序列进行最少的反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
由于抗原的接触残基，CDR的移植可由于与抗原接触的构架残基而导致产生的抗体或其抗原结合片段对抗原的亲和力减弱。此类相互作用可以可能是体细胞高度突变的结果。因此，可能仍然需要将此类供体构架氨基酸移植至人源化抗体的构架。来自非人抗体或其抗原结合片段的参与抗原结合的氨基酸残基可通过检查动物单克隆抗体可变区序列和结构来鉴定。CDR供体构架中与种系不同的各残基可被认为是相关的。如果不能确定最接近的种系，那么可将序列与亚类共有序列或具有高相似性百分数的动物抗体序列的共有序列相比较。稀有构架残基被认为可能是体细胞高度突变的结果，从而在结合中起着重要作用。
在本公开一个的实施方案中，所述的抗体或其抗原结合片段，可进一步包含人源或鼠源κ、λ链或其变体的轻链恒定区，或进一步包含人源或鼠源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。
人抗体重链恒定区和人抗体轻链恒定区的“常规变体”是指现有技术已公开的来源于人的不改变抗体可变区结构和功能的重链恒定区或轻链恒定区的变体，示例性变体包括对重链恒定区进行定点改造和氨基酸替换的IgG1、IgG2、IgG3或IgG4重链恒定区变体，具体替换如现有技术已知的YTE突变，L234A和/或L235A 突变，或S228P突变，或获得knob-into-hole结构的突变(使得抗体重链具有knob-Fc和hole-Fc组合)，这些突变已被证实使得抗体具有新的性能，但不改变抗体可变区的功能。
“人抗体”与“人源抗体”可以互换使用，可以是源于人的抗体或者是从一种转基因生物体中获得的抗体，该转基因生物体经“改造”以响应于抗原刺激而产生特异性人抗体并且可以通过本领域已知的任何方法产生。在某些技术中，将人重链和轻链基因座的元件引入到源于胚胎干细胞系的生物体的细胞株中，这些细胞株中的内源性重链和轻链基因座被靶向破坏。转基因生物可以合成对人抗原特异的人抗体，并且该生物可以用于产生人抗体-分泌杂交瘤。人抗体还可以是一种抗体，其中重链和轻链是由源于一个或多个人DNA来源的核苷酸序列编码的。完全人抗体还可以通过基因或染色体转染方法以及噬菌体展示技术来构建，或者由体外活化的B细胞构建，所有的这些都是本领域已知的。
“单克隆抗体”是指从基本上均质抗体的群体获得的抗体，即除可能的变体抗体(例如含有天然存在的突变或在制造单克隆抗体制剂的期间产生的突变，这些变体通常以少量存在)之外，构成所述群体的个别抗体识别相同和/或结合相同表位。与通常包含针对不同决定簇(表位)的不同抗体的多克隆抗体制备物不同，单克隆抗体制备物(制剂)的每个单克隆抗体是针对抗原上的单一决定簇的。因此，修饰语“单克隆”指示如从基本上均质抗体群体获得的抗体的特性，且不应解释为需要通过任何特定方法来制造抗体。例如，根据本公开使用的单克隆抗体可通过各种技术制备，所述技术包括但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法以及利用含有全部或部分人免疫球蛋白基因座的转基因动物的方法，此类方法以及用于制备单克隆抗体的其他示例性方法在本文中进行描述。术语“单克隆抗体或其抗原结合片段”中单克隆抗体指全长抗体。
术语“全长抗体”、“完整抗体”、“完全抗体”和“全抗体”在本文中可互换使用，指基本上完整形式的抗体，与下文定义的抗原结合片段相区分。该术语特别指重链由氨基端至羧基端依次包含VH区、CH1区、铰链区和Fc区，轻链由氨基端至羧基端依次包含VL区和CL区的抗体。
此外，虽然Fv片段的两个结构域VL和VH由分开的基因编码，但可使用重组方法，通过合成的接头连接它们，从而使得其能够产生为其中VL和VH区配对形成单价分子的单个蛋白质链(称为单链Fv(scFv)；参见，例如，Bird等人(1988)Science242:423-426；和Huston等人(1988)Proc.Natl.Acad.Sci USA85:5879-5883)。此类单链抗体也意欲包括在术语抗体的“抗原结合片段”中。使用本领域技术人员已知的常规技术获得此类抗体片段，并且以与对于完整抗体的方式相同的方式就功用性筛选的片段。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。
抗原结合片段还可并入至包含一对串联Fv片段(VH-CH1-VH-CH1)的单链 分子中，该对串联Fv片段连同互补轻链多肽一起形成一对抗原结合区(Zapata等人,1995Protein Eng.8(10):1057-1062；及美国专利US5641870)。
Fab是通过用木瓜蛋白酶(切割H链的224位的氨基酸残基)处理IgG抗体分子所获得的具有约50,000Da分子量的抗体片段，其具有抗原结合活性，其中H链N端侧的约一半和整个L链通过二硫键结合在一起。
F(ab')2是通过用胃蛋白酶消化IgG铰链区中两个二硫键的下方部分而获得的分子量为约100,000Da的抗体片段，并具有抗原结合活性并包含在铰链位置相连的两个Fab区。
Fab'是通过切割上述F(ab')2的铰链区的二硫键而获得的分子量为约50,000Da并具有抗原结合活性的抗体片段。Fab'可以通过用还原剂例如二硫苏糖醇处理特异性识别并结合抗原的F(ab')2来生产。
此外，可以通过将编码抗体的Fab'片段的DNA插入到原核生物表达载体或真核生物表达载体中并将载体导入到原核生物或真核生物中以表达Fab'来生产所述Fab'。
术语“单链抗体”、“单链Fv”或“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-接头-VH-COOH或NH 2-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，例如使用1-4个(包括1个、2个、3个或4个)重复的变体(Holliger等人(1993)，Proc Natl Acad Sci USA.90:6444-6448)。可用于本公开的其他接头由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur J Immuno.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 Immunother.50:51-59描述。
“Linker”或“接头”或“连接子”指用于连接蛋白质结构域的连接性多肽序列，通常具有一定的柔性，接头的使用不会使蛋白质结构域原有的功能丧失。
双抗体(diabody)是指scFv被二聚体化的抗体片段，是具有二价抗原结合活性的抗体片段。在二价抗原结合活性中，两个抗原可以是相同或不同的。
dsFv是通过将其中每个VH和VL中的一个氨基酸残基被半胱氨酸残基取代的多肽经由半胱氨酸残基之间的二硫键相连而获得的。可以按照已知方法(Protein Engineering.7:697(1994))基于抗体的三维结构预测来选择被半胱氨酸残基取代的氨基酸残基。
本公开一些实施例中抗原结合片段可以通过以下步骤来生产：获得本公开的特异性识别并结合抗原的单克隆抗体的VH和/或VL及所需的其他结构域的编码cDNA，构建编码抗原结合片段的DNA，将所述DNA插入到原核生物表达载体或真核生物表达载体中，然后将所述表达载体导入到原核生物或真核生物中以表达抗原结合片段。
"Fc区"可以是天然序列Fc区或变体Fc区。虽然免疫球蛋白重链的Fc区的边界可能变化，但人IgG重链Fc区通常被定义成从位置Cys226上的氨基酸残基或从Pro230延伸至其羧基端。Fc区中的残基的编号为如Kabat中的EU索引的编号。Kabat等，Sequences of Proteins of Immunological Interest，第5版Public Health Service,National Institutes of Health,Bethesda,Md.,1991。免疫球蛋白的Fc区通常具有两个恒定区结构域CH2和CH3。
“knob-Fc”指在抗体Fc区包含T366W的点突变，以形成类似knob的空间结构。相对应地，“hole-Fc”指在抗体Fc区包含T366S，L368A，Y407V的点突变，以形成类似hole的空间结构。Knob-Fc和hole-Fc由于空间位阻的原因，更易形成异二聚体。为进一步地促进异二聚体的形成，还可在knob-Fc和hole-Fc分别引入S354C和Y349C的点突变，通过二硫键进一步促进异二聚体的形成。同时，为消除或减弱抗体Fc引起的ADCC效应，还可向Fc引入的234A和235A的取代突变。在双特异性抗体中，knob-Fc或hole-Fc既可以作为第一多肽链的Fc区域，也可以作为第二多肽链的Fc区域，在同一双特异性抗体中，第一多肽链和第二多肽链的Fc区不同时为knob-Fc或hole-Fc。
术语“氨基酸差异”或“氨基酸突变”是指相较于原蛋白质或多肽，变体蛋白质或多肽存在氨基酸的改变或突变，包括在原蛋白质或多肽的基础上发生1个或数个氨基酸的插入、缺失或替换。
抗体的“可变区”是指单独的或组合的抗体轻链的可变区(VL)或抗体重链的可变区(VH)。如在本领域中已知的，重链和轻链的可变区各自由通过3个互补决定区(CDR)(也称为高变区)连接的4个框架区(FR)组成。每一条链中的CDR通过FR紧密地保持在一起并且与来自另一条链的CDR一起促成抗体的抗原结合部位的形成。存在至少2个用于确定CDR的技术：(1)基于跨种序列变异性的方法(即，Kabat等Sequences of Proteins of Immunological Interest,(第5版，1991,National Institutes of Health,Bethesda MD))；和(2)基于抗原-抗体复合物的晶体学研究的方法(Al-Lazikani等，J.Molec.Biol.273:927-948(1997))。如本文中所用，CDR可指由任一方法或由两种方法的组合确定的CDR。
术语“抗体框架”或“FR区”，是指可变结构域VL或VH的一部分，其用作该可变结构域的抗原结合环(CDR)的支架。从本质上讲，其是不具有CDR的可变结构域。
术语“互补决定区”和“CDR”是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。通常，每个重链可变区中存在三个CDR(HCDR1、HCDR2、HCDR3)和每个轻链可变区中存在三个CDR(LCDR1、LCDR2、LCDR3)。可以使用各种公知方案中的任何一种来确定CDR的氨基酸序列边界，包括“Kabat”编号规则(参见Kabat等(1 991)，“Sequences of Proteins of Immunological Interest”，第5版，Public Health Service，National Institutes of Health，Bethesda，MD)、“Chothia” 编号规则(Al-Lazikani等人，(1997)JMB 273：927-948)和ImMunoGenTics(IMGT)编号规则(Lefranc M.P.，Immunologist,7,132-136(1999)；Lefranc,M.P.等，Dev.Comp.Immunol.,27,55-77(2003))等。例如，对于经典格式，遵循Kabat规则，所述重链可变域(VH)中的CDR氨基酸残基编号为31-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)；轻链可变域(VL)中的CDR氨基酸残基编号为24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)。遵循Chothia规则，VH中的CDR氨基酸编号为26-32(HCDR1)、52-56(HCDR2)和95-102(HCDR3)；并且VL中的氨基酸残基编号为26-32(LCDR1)、50-52(LCDR2)和91-96(LCDR3)。通过组合Kabat和Chothia两者的CDR定义，CDR由人VH中的氨基酸残基26-35(HCDR1)、50-65(HCDR2)和95-102(HCDR3)和人VL中的氨基酸残基24-34(LCDR1)、50-56(LCDR2)和89-97(LCDR3)构成。遵循IMGT规则，VH中的CDR氨基酸残基编号大致为26-35(CDR1)、51-57(CDR2)和93-102(CDR3)，VL中的CDR氨基酸残基编号大致为27-32(CDR1)、50-52(CDR2)和89-97(CDR3)。遵循IMGT规则，抗体的CDR区可以使用程序IMGT/DomainGap Align确定。
“HCDR1、HCDR2和HCDR3区或其任意CDR变体”中的“其任意CDR变体”是指对HCDR1、HCDR2和HCDR3区中任意一个、或两个、或三个HCDR进行氨基酸突变获得的变体。
“抗体恒定区结构域”指来源于抗体的轻链和重链的恒定区的结构域，包括CL和来源于不同类抗体的CH1、CH2、CH3和CH4结构域。
“表位”或“抗原决定簇”是指抗原上免疫球蛋白或抗体特异性结合的部位。表位通常以独特的空间构象包括至少3、4、5、6、7、8、9、10、11、12、13、14或15个连续或非连续的氨基酸。参见，例如，Epitope Mapping Protocols in Methods in Molecular Biology，第66卷，G.E.Morris，Ed.(1996)。
术语“特异性结合”、“选择性结合”、“选择性地结合”和“特异性地结合”是指抗体对预先确定的抗原上的表位的结合。通常，抗体以大约小于10 -8M，例如大约小于10 -9M、10 -10M、10 -11M或更小的亲和力(KD)结合。
当术语“竞争”用于竞争相同表位的抗原结合蛋白(例如中和抗原结合蛋白或中和抗体)的情况中时，意指在抗原结合蛋白之间竞争，其通过以下测定法来测定：在所述测定法中，待检测的抗原结合蛋白(例如抗体或其抗原结合片段)防止或抑制(例如降低)参考抗原结合蛋白(例如配体或参考抗体)与共同抗原的特异性结合。众多类型的竞争性结合测定可用于确定一种抗原结合蛋白是否与另一种竞争，这些测定例如：固相直接或间接放射免疫测定(RIA)、固相直接或间接酶免疫测定(EIA)、夹心竞争测定(参见例如Stahli等，1983，Methodsin Enzymology 9：242-253)；固相直接生物素-亲和素EIA(参见例如Kirkland等，1986，J.Immunol.137：3614-3619)、固相直接标记测定、固相直接标记夹心测定(参见例 如Harlow和Lane，1988，Antibodies，A Laboratory Manual(抗体，实验室手册)，Cold Spring Harbor Press)；用I-125标记物的固相直接标记RIA(参见例如Morel等，1988，Molec.Immunol.25：7-15)；固相直接生物素-亲和素EIA(参见例如Cheung，等，1990，Virology176：546-552)；和直接标记的RIA(Moldenhauer等，1990，Scand.J.Immunol.32：77-82)。通常所述测定法涉及使用能与带有未标记的检测抗原结合蛋白及标记的参考抗原结合蛋白结合的纯化抗原(所述抗原在固态表面或细胞表面)。在待测抗原结合蛋白存在下，测量结合于固态表面或细胞的标记的量，来测量竞争性抑制。通常，待测抗原结合蛋白是过量存在的。由竞争性测定(竞争抗原结合蛋白)鉴定的抗原结合蛋白包括：结合与参考抗原结合蛋白同一表位的抗原结合蛋白；和结合充分接近参考抗原结合蛋白的结合表位的邻近表位的抗原结合蛋白，所述两个表位在空间上互相妨碍发生结合。在本公开实施例中提供关于用于测定竞争性结合的方法的其它详细资料。通常当竞争的抗原结合蛋白过量存在时，其将抑制(例如降低)至少40-45％、45-50％、50-55％、55-60％、60-65％、65-70％、70-75％或75％或更多参考抗原结合蛋白与共同抗原的特异性结合。在某些情况下，结合被抑制至少80-85％、85-90％、90-95％、95-97％或97％或更多。
术语“亲和力”是指在单一表位处，抗体与抗原之间相互作用的强度。在各抗原位点内，抗体“臂”的可变区通过弱非共价力与抗原在多个氨基酸位点处相互作用；相互作用愈大，亲和力愈强。如本文所用，抗体或其抗原结合片段(例如Fab片段)的术语“高亲和力”通常是指具有1E -9M或更小的K D(例如1E -10M或更小的K D、1E -11M或更小的K D、1E -12M或更小的K D、1E -13M或更小的K D、1E -14M或更小的K D等)的抗体或抗原结合片段。
术语"KD"或“K D”是指特定抗体-抗原相互作用的解离平衡常数。通常，抗体以小于大约1E -8M，例如小于大约1E -9M、1E -10M或1E -11M或更小的解离平衡常数(KD)结合抗原，例如，如使用表面等离子体共振(SPR)技术在BIACORE仪中测定的。KD值越小，亲和力越大。
术语“核酸分子”是指DNA分子和RNA分子。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。
术语"载体"意指能够递送一个或多个目标基因或序列并且优选在宿主细胞中表达其的构建体。载体的示例包括，但不限于，病毒载体、裸露DNA或RNA表达载体、质粒、粘粒或噬菌体载体、与阳离子凝聚剂缔合的DNA或RNA表达载体、包封在脂质体中的DNA或RNA表达载体和某些真核生物细胞诸如生产细胞。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。例如，鼠可以用抗原或其片段免疫，所得到的抗体 能被复性、纯化，并且可以用常规的方法进行氨基酸测序。抗原结合片段同样可以用常规方法制备。本公开所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从网站http://www.imgt.org/得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
术语“宿主细胞”是指已向其中引入了表达载体的细胞。宿主细胞可包括细菌、微生物、植物或动物细胞。易于转化的细菌包括肠杆菌科(enterobacteriaceae)的成员，例如大肠杆菌(Escherichia coli)或沙门氏菌(Salmonella)的菌株；芽孢杆菌科(Bacillaceae)例如枯草芽孢杆菌(Bacillus subtilis)；肺炎球菌(Pneumococcus)；链球菌(Streptococcus)和流感嗜血菌(Haemophilus influenzae)。适当的微生物包括酿酒酵母(Saccharomyces cerevisiae)和毕赤酵母(Pichia pastoris)。适当的动物宿主细胞系包括CHO(中国仓鼠卵巢细胞系)、HEK293细胞(非限制性实施例如HEK293E细胞)和NS0细胞。
工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种可选的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与抗原特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的蛋白A或蛋白G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“施用”、“给药”、“给予”、“处理”，当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指将外源性药物、治疗剂、诊断剂、组合物或者人为操作(比如实施例中的“安乐死”)提供给与动物、人、受试者、细胞、组织、器官或生物流体。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予受试者内用或外用治疗剂，例如包含本公开实施例的任一种化合物的组合物，所述受试者具(或疑似患有、或易感于)有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗受试者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床有测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也 称作“治疗有效量”)可根据多种因素变化，例如受试者的疾病状态、年龄和体重，以及药物在受试者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本公开的实施方案(例如治疗方法或制品)可能无法在缓解每个目标疾病症状方面都有效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的受试者中应当减轻目标疾病症状。
“氨基酸保守修饰”或“氨基酸保守取代”指蛋白质或多肽中的氨基酸被具有相似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其他氨基酸取代，从而使得在不改变蛋白质或多肽的生物活性或其他所需特性(例如抗原亲和力和/或特异性)的情况下，可以经常进行改变。本领域技术人员认识到，通常，多肽的非必需区域中的单个氨基酸取代基本上不改变生物活性(参见，例如，Watson等人，(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页(第4版))。此外，结构上或功能上相似的氨基酸的取代不太可能破坏生物活性。示例性氨基酸保守取代如下：
Figure PCTCN2019126903-appb-000001
“有效量”、“有效剂量”是指获得任一种或多种有益的或所需的治疗结果所必需的药物、化合物或药物组合物的量。对于预防用途，有益的或所需的结果包括消除或降低风险、减轻严重性或延迟病症的发作，包括病症、其并发症和在病症的 发展过程中呈现的中间病理表型的生物化学、组织学和/或行为症状。对于治疗应用，有益的或所需的结果包括临床结果，如减少各种本公开靶抗原相关病症的发病率或改善所述病症的一个或多个症状，减少治疗病症所需的其它药剂的剂量，增强另一种药剂的疗效，和/或延缓受试者的本公开靶抗原相关病症的进展。
“外源性”指根据情况在生物、细胞或人体外产生的物质。
“内源性”指根据情况在细胞、生物或人体内产生的物质。
“同源性”、“同一性”在本文中可以互换，是指两个多核苷酸序列之间或两个多肽之间的序列相似性。当两个比较序列中的位置均被相同碱基或氨基酸单体亚基占据时，例如如果两个DNA分子的每一个位置都被腺嘌呤占据时，那么所述分子在该位置是同源的。两个序列之间的同源性百分率是两个序列共有的匹配或同源位置数除以比较的位置数×100的函数。例如，在序列最佳比对时，如果两个序列中的10个位置有6个匹配或同源，那么两个序列为60％同源；如果两个序列中的100个位置有95个匹配或同源，那么两个序列为95％同源。通常，当比对两个序列时进行比较以给出最大百分比同源性。例如，可以通过BLAST算法执行比较，其中选择算法的参数以在各个参考序列的整个长度上给出各个序列之间的最大匹配。
以下参考文献涉及经常用于序列分析的BLAST算法：BLAST算法(BLAST ALGORITHMS):Altschul,S.F.等人,(1990)J.Mol.Biol.215:403-410；Gish,W.等人,(1993)Nature Genet.3:266-272；Madden,T.L.等人,(1996)Meth.Enzymol.266:131-141；Altschul,S.F.等人,(1997)Nucleic Acids Res.25:3389-3402；Zhang,J.等人,(1997)Genome Res.7:649-656。其他如NCBI BLAST提供的常规BLAST算法也为本领域技术人员所熟知。
“分离的”指加以改为“脱离其原始存在环境状态”，并且在这种情况下意味着，指定的分子基本上不含其他非目标生物分子。通常，术语“分离的”并不意图指完全不存在这些材料或不存在水、缓冲液或盐，除非它们以显著干扰如本文所述的化合物的实验或治疗用途的量存在。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生的场合。
“药物组合物”表示含有一种或多种本公开所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
术语“药学上可接受的载体”指适合用于制剂中用于递送抗体或抗原结合片段的任何无活性物质。载体可以是抗粘附剂、粘合剂、包衣、崩解剂、充填剂或稀释剂、防腐剂(如抗氧化剂、抗菌剂或抗真菌剂)、增甜剂、吸收延迟剂、润湿剂、乳化剂、缓冲剂等。合适的药学上可接受的载体的示例包括水、乙醇、多元 醇(例如甘油、丙二醇、聚乙二醇等)右旋糖、植物油(例如橄榄油)、盐水、缓冲液、缓冲的盐水和等渗剂例如糖、多元醇、山梨糖醇和氯化钠。
术语“代谢障碍”的具体示例为代谢综合征、肥胖症、葡萄糖耐量受损、糖尿病、糖尿病酮症酸中毒、高血糖症、高血糖高渗综合征、围术期高血糖症、高胰岛素血症、胰岛素抵抗综合症、空腹血糖受损、血脂异常、动脉粥样硬化或糖尿病前期状态。
此外，本公开另一方面涉及用于免疫检测或测定目标抗原的方法、用于免疫检测或测定目标抗原的试剂、用于免疫检测或测定表达目标抗原的细胞的方法和用于诊断与目标抗原阳性细胞相关的疾病的诊断剂，其包含本公开的特异性识别并结合目标抗原的单克隆抗体或抗体片段作为活性成分。
在本公开中，用于检测或测定目标抗原的量的方法可以是任何已知方法。例如，它包括免疫检测或测定方法。
免疫检测或测定方法是使用标记的抗原或抗体检测或测定抗体量或抗原量的方法。免疫检测或测定方法的示例包括放射性物质标记的免疫抗体方法(RIA)、酶免疫测定法(EIA或ELISA)、荧光免疫测定法(FIA)、发光免疫测定法、蛋白质免疫印迹法、物理化学方法等。
上述与目标抗原阳性细胞相关的疾病可以通过用本公开的单克隆抗体或抗体片段检测或测定表达目标抗原的细胞来诊断。
为了检测表达多肽的细胞，可以使用已知的免疫检测方法，并优选使用免疫沉淀法、荧光细胞染色法、免疫组织染色法等。此外，可以使用利用FMAT8100HTS系统(Applied Biosystem)的荧光抗体染色法等。
在本公开中，对用于检测或测定目标抗原的活体样本没有特别限制，只要它具有包含表达目标抗原的细胞的可能性即可，例如组织细胞、血液、血浆、血清、胰液、尿液、粪便、组织液或培养液。
根据所需的诊断方法，含有本公开的单克隆抗体或其抗体片段的诊断剂还可以含有用于执行抗原-抗体反应的试剂或用于检测反应的试剂。用于执行抗原-抗体反应的试剂包括缓冲剂、盐等。用于检测的试剂包括通常用于免疫检测或测定方法的试剂，例如识别所述单克隆抗体、其抗体片段或其结合物的标记的第二抗体和与所述标记对应的底物等。
在以上说明书中提出了本发明一种或多种实施方式的细节。虽然可使用与本文所述类似或相同的任何方法和材料来实施或测试本发明，但是以下描述优选的方法和材料。通过说明书和权利要求书，本发明的其他特点、目的和优点将是显而易见的。在说明书和权利要求书中，除非上下文中有清楚的另外指明，单数形式包括复数指代物的情况。除非另有定义，本文使用的所有技术和科学术语都具有本发明所属领域普通技术人员所理解的一般含义。说明书中引用的所有专利和出版物都通过引用纳入本文。提出以下实施例是为了更全面地说明本发明的优选 实施方式。这些实施例不应以任何方式理解为限制本发明的范围，本发明的范围由权利要求书限定。
实施例
实施例1：GCGR抗原抗体的制备
1.1抗原构建和筛选
将编码全长477个氨基酸的胰高血糖素受体的人类GCGR cDNA亚克隆到表达载体(如pcDNA3.1)中，并转染CHO-K1细胞。在筛选和单细胞克隆后，选择单克隆用于抗原的表达、及基于受体的细胞表面表达的特性研究。以下GCGR抗原未特殊说明的均指人GCGR。
全长GCGR：用于构建GCGR过表达细胞株，或用于免疫用抗原及后续检测：
Figure PCTCN2019126903-appb-000002
1.2GCGR杂交瘤、重组抗体的纯化
(1)杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化首选ProteinG进行亲和层析，将培养所得杂交瘤离心取上清，根据上清体积加入10-15％体积的1M Tris-HCl(pH8.0-8.5)调节上清pH。ProteinG柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M醋酸/醋酸钠(pH3.0)缓冲液进行样本洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样本纯度。
(2)Protein A亲和层析提取带Fc标签的双特异性蛋白或者抗体：
首先将表达Fc双特异性蛋白或者抗体的细胞培养上清进行高速离心收取上清。ProteinA亲和柱利用6M盐酸胍洗3-5倍柱体积，然后利用纯水清洗3-5倍柱 体积。利用如1×PBS(pH7.4)缓冲体系作为平衡缓冲液对层析柱平衡3-5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线。利用0.1M醋酸/醋酸钠(pH3.0-3.5)缓冲液进行样本洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)快速调节pH至5-6暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系，或者利用如Superdex 200等高分辨率分子排阻柱去除洗脱产物中的聚体成分以提高样本纯度。
实施例2：抗人GCGR单克隆抗体的制备
2.1免疫
(1)小鼠免疫：抗人GCGR单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6-8周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按以下方案免疫。免疫抗原为GCGR CHO-K1稳转细胞。
免疫方案：用佐剂
Figure PCTCN2019126903-appb-000003
Gold Adjuvant(Sigma Cat No.T2684)预先免疫小鼠，腹膜内(IP)注射0.1ml/只(首次免疫)。15min后，腹膜内(IP)注射GCGR CHO-K1稳转细胞，1E7细胞/只。接种时间为第0、14、28、42、56、70、84、98、112天。于第35，63，91天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第6-9次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫，腹膜内(IP)注射1E7细胞/只的GCGR CHO-K1稳转细胞。
(2)大鼠免疫：使用DNA(编码全长hGCGR，编码序列参见Genbank登录号：NM_000160)免疫6-8周龄SD大鼠，用FACS方法确定大鼠血清中的抗体滴度。在第3-4次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的大鼠进行脾细胞融合。在进行脾细胞融合前3天加强免疫。
2.2脾细胞融合
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2019126903-appb-000004
CRL-8287 TM)进行融合得到杂交瘤细胞。融合的杂交瘤细胞以0.5-1E6/ml的密度用完全培养基(含20％FBS、1×HAT、1×OPI的IMEM培养基)重悬，100μl/孔种于96孔板中，37℃，5％CO 2孵育3-4天后，补充HAT完全培养基100μl/孔，继续培养3-4天至形成克隆。去除上清，加入200μl/孔的HT完全培养基(含20％FBS、1×HT和1×OPI的IMDM培养基)，37℃，5％CO 2培养3天后进行ELISA检测。
2.3杂交瘤细胞筛选
根据杂交瘤细胞生长密度，用细胞结合ELISA方法进行杂交瘤培养上清检测。将测试呈为阳性的孔细胞及时进行扩增、冻存、和二到三次亚克隆直至获得单细胞克隆。
每次亚克隆细胞也均需进行GCGR细胞结合ELISA实验检测。通过实验筛选得到杂交瘤克隆，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在测试例中使用。
2.4杂交瘤阳性克隆序列测定
从阳性杂交瘤中克隆序列过程如下。收集对数生长期杂交瘤细胞，用Trizol(Invitrogen,Cat No.15596-018)按照试剂盒说明书步骤提取RNA，用PrimeScript TM Reverse Transcriptase试剂盒反转录(Takara,Cat No.2680A)。将反转录得到的cDNA采用小鼠Ig-Primer Set(Novagen,TB326Rev.B 0503)进行PCR扩增后测序。
从得到的DNA序列获得所筛选到的阳性克隆，用无血清细胞培养法制备抗体，按纯化实例纯化抗体，在测试例中使用基于细胞的GCGR结合阻断实验经过多轮筛选，得到来源于小鼠的杂交瘤克隆1803，1805，1808和1810，来源于大鼠的杂交瘤克隆1817和1822。
表1.GCGR抗体轻链可变区和重链可变区序列
Figure PCTCN2019126903-appb-000005
Figure PCTCN2019126903-appb-000006
表2.Kabat编号的CDR区序列
Figure PCTCN2019126903-appb-000007
Figure PCTCN2019126903-appb-000008
经序列比对及计算机模拟发现，上述小鼠来源抗体m1803、m1805、m1808和m1810的轻重链CDR序列具有较高的同源性，大鼠来源抗体rat1817和rat1822的轻重链CDR序列具有较高的同源性，其共有序列如下表所示：
表3.重链及轻链CDR区序列的共有序列
Figure PCTCN2019126903-appb-000009
实施例3：鼠源抗人GCGR抗体的人源化
使用MOE软件，比对IMGT人类抗体重轻链可变区种系基因数据库，分别挑 选与鼠源抗体同源性高的重链和轻链可变区种系基因作为模板。将鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。根据需要，将骨架序列中部分氨基酸回复突变为鼠源抗体对应的氨基酸，即得到人源化抗GCGR单克隆抗体。其中CDR区氨基酸残基的确定由Kabat编号系统确定并注释。
上述鼠源抗体的轻重链可变区与人源抗体的轻重链恒定区连接后形成嵌合抗体，1803号克隆对应的嵌合抗体命名为ch1803，其他抗体类推。
3.1杂交瘤克隆1803的人源化
(1)杂交瘤克隆1803人源化构架选择：
鼠源抗体m1803的人源化轻链模板为IGKV1-39*01和hjk4.1，人源化重链模板为IGHV1-3*01和hjh6.1，人源化可变区序列如下：
hu1803VH-CDR嫁接：(SEQ ID NO：61)
Figure PCTCN2019126903-appb-000010
hu1803VL-CDR嫁接：(SEQ ID NO：58)
Figure PCTCN2019126903-appb-000011
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)杂交瘤克隆1803号的人源化模板选择和回复突变设计如下表：
表4.模板选择和回复突变
Figure PCTCN2019126903-appb-000012
注：如P44V表示依照Kabat编号系统，将Kabat编号的第44位P突变回V。嫁接代表鼠抗体CDR直接植入人种系FR区序列。
(3)杂交瘤克隆1803的人源化具体序列如下：
>hu1803_VL.1(同Hu1803VL-CDR嫁接)：(SEQ ID NO：58)
Figure PCTCN2019126903-appb-000013
>hu1803_VL.1A：(SEQ ID NO：59)
Figure PCTCN2019126903-appb-000014
>hu1803_VL.1B：(SEQ ID NO：60)
Figure PCTCN2019126903-appb-000015
>hu1803_VH.1(同Hu1803VH-CDR嫁接)：(SEQ ID NO：61)
Figure PCTCN2019126903-appb-000016
>hu1803_VH.1A：(SEQ ID NO：62)
Figure PCTCN2019126903-appb-000017
>hu1803_VH.1B：(SEQ ID NO：63)
Figure PCTCN2019126903-appb-000018
>hu1803_VH.1C：(SEQ ID NO：64)
Figure PCTCN2019126903-appb-000019
(4)杂交瘤克隆1803来源抗体的人源化抗体轻链可变区和重链可变区序列组合如下：
表5.不同人源化抗体轻重链可变区组合
  hu1803_VL.1 hu1803_VL.1A hu1803_VL.1B
hu1803_VH.1 hu1803-1 hu1803-5 hu1803-9
hu1803_VH.1A hu1803-2 hu1803-6 hu1803-10
hu1803_VH.1B hu1803-3 hu1803-7 hu1803-11
hu1803_VH.1C hu1803-4 hu1803-8 hu1803-12
上表中抗体名称所指代的抗体轻重链可变区可以分别与抗体轻重链恒定区连接形成全长抗体。在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA连接形成抗体重链。
3.2杂交瘤克隆1810的人源化
(1)杂交瘤克隆1810人源化构架选择：
鼠源抗体m1810的人源化轻链模板为IGKV1-39*01和hJK4.1，人源化重链模板为IGHV1-69*02和hJH4.1，人源化可变区序列如下：
Hu1810VH-CDR嫁接：(SEQ ID NO：68)
Figure PCTCN2019126903-appb-000020
Hu1810VL-CDR嫁接：(SEQ ID NO：65)
Figure PCTCN2019126903-appb-000021
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
(2)杂交瘤克隆1810号的人源化模板选择和回复突变设计如下表：
表6.模板选择和回复突变
Figure PCTCN2019126903-appb-000022
注：如P44V表示依照Kabat编号系统，将Kabat编号的第44位P突变回V。嫁接代表鼠抗体CDR直接植入人种系FR区序列。
(3)杂交瘤克隆1810的人源化具体序列如下：
>hu1810_VL.1(同Hu1810VL-CDR嫁接)：(SEQ ID NO：65)
Figure PCTCN2019126903-appb-000023
>hu1810_VL.1A：(SEQ ID NO：66)
Figure PCTCN2019126903-appb-000024
>hu1810_VL.1B：(SEQ ID NO：67)
Figure PCTCN2019126903-appb-000025
>hu1810_VH.1(同Hu1810VH-CDR嫁接)：(SEQ ID NO：68)
Figure PCTCN2019126903-appb-000026
>hu1810_VH.1A：(SEQ ID NO：69)
Figure PCTCN2019126903-appb-000027
>hu1810_VH.1B：(SEQ ID NO：70)
Figure PCTCN2019126903-appb-000028
>hu1810_VH.1C：(SEQ ID NO：71)
Figure PCTCN2019126903-appb-000029
(4)杂交瘤克隆1810的人源化抗体轻重链可变区序列组合如下：
表7.不同人源化抗体轻重链可变区的组合
  hu1810_VL.1 hu1810_VL.1A hu1810_VL.1B
hu1810_VH.1 hu1810-1 hu1810-5 hu1810-9
hu1810_VH.1A hu1810-2 hu1810-6 hu1810-10
hu1810_VH.1B hu1810-3 hu1810-7 hu1810-11
hu1810_VH.1C hu1810-4 hu1810-8 hu1810-12
上表中抗体名称所指代的抗体轻重链可变区可以分别与抗体轻重链恒定区连接形成全长抗体，在本公开中如无明确说明时，形成全长抗体时轻链可变区与SEQ ID NO：73所示的Kappa链恒定区连接形成抗体轻链，重链可变区与SEQ ID NO：72所示的IgG4-AA连接形成抗体重链。
实施例4：构建和表达GCGR嵌合体/人源化抗体的IgG4-AA形式
设计引物，PCR搭建各嵌合体/人源化抗体VH/VK基因片段，再与表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)进行同源重组，构建抗体全长表达载体VH-CH1-FC-pHr/VK-CL-pHr。IgG4-AA抗体形式可以通过IgG4抗体形式简单点突变获得，IgG4-AA代表F234A、L235A和S228P突变。F234A和L235A的突变可降低IgG4-Fc与FcγR的结合能力，进一步降低ADCC/CDC，S228P代表野生型IgG4铰链区第228位的氨基酸S突变成P，该位点突变能够避免天然IgG4抗体在体内发生Fab-交换(exchange)导致的错配。
ch1803、ch1805、ch1808、ch1810、ch1817和ch1822为表1所示动物来源的轻重链可变区分别与人抗体kappa链和人抗体IgG4-AA重链恒定区连接后形成的嵌合抗体。
IgG4-AA的重链恒定区序列如下：(SEQ ID NO：72)
Figure PCTCN2019126903-appb-000030
抗体的轻链(Kappa链)恒定区序列如下：(SEQ ID NO：73)
Figure PCTCN2019126903-appb-000031
构建后的抗体序列示例性列举如下：
ch1803：抗体形式IgG4AA
ch1803重链序列：(SEQ ID NO：74)
Figure PCTCN2019126903-appb-000032
ch1803轻链序列：(SEQ ID NO：75)
Figure PCTCN2019126903-appb-000033
hu1803-1：抗体形式IgG4AA
hu1803-1重链序列：：(SEQ ID NO：76)
Figure PCTCN2019126903-appb-000034
hu1803-1轻链序列：(SEQ ID NO：77)
Figure PCTCN2019126903-appb-000035
hu1803-9重链序列：(SEQ ID NO：78)
Figure PCTCN2019126903-appb-000036
Figure PCTCN2019126903-appb-000037
hu1803-9轻链序列：(SEQ ID NO：79)
Figure PCTCN2019126903-appb-000038
ch1805重链序列：(SEQ ID NO：80)
Figure PCTCN2019126903-appb-000039
ch1805轻链序列：(SEQ ID NO：81)
Figure PCTCN2019126903-appb-000040
ch1808重链序列：(SEQ ID NO：82)
Figure PCTCN2019126903-appb-000041
ch1808轻链序列：(SEQ ID NO：83)
Figure PCTCN2019126903-appb-000042
hu1810-12重链序列：(SEQ ID NO：84)
Figure PCTCN2019126903-appb-000043
hu1810-12轻链序列：(SEQ ID NO：85)
Figure PCTCN2019126903-appb-000044
ch1817重链序列：(SEQ ID NO：86)
Figure PCTCN2019126903-appb-000045
ch1817轻链序列：(SEQ ID NO：87)
Figure PCTCN2019126903-appb-000046
ch1822重链序列：(SEQ ID NO：88)
Figure PCTCN2019126903-appb-000047
Figure PCTCN2019126903-appb-000048
ch1822轻链序列：(SEQ ID NO：89)
Figure PCTCN2019126903-appb-000049
阳性对照：度拉糖肽是双链形式，单链为GLP-1/hIgG4Fc(SEQ ID NO：90)
Figure PCTCN2019126903-appb-000050
实施例5：抗体双特异性蛋白的克隆和表达
采用人GLP-1肽作为双特异性蛋白中GLP-1受体激动剂部分，将GCGR抗体作为双特异性蛋白的GCGR拮抗部分，形成GLP-1/GCGR抗体双特异性蛋白。
研究发现，GLP-1特定位点氨基酸突变(如Q17E、I23V、K28R或G30R)后，形成的新GLP-1/GCGR抗体双特异性蛋白，其体外稳定性更高，其中将GLP-1A第17位的Q突变为E和第23位的I突变为V形式(GLP-1C)，稳定性最高。本公开GLP-1及其突变形式的非限制性实施例序列如下：
表8.GLP-1A多肽及其变体序列
Figure PCTCN2019126903-appb-000051
Figure PCTCN2019126903-appb-000052
利用同源重组技术，将本公开GLP-1肽的C末端氨基酸通过肽键或接头(linker)连接GCGR抗体重链N末端氨基酸。通过293表达系统进行常规表达，得到如表9所示的双特异性蛋白的模式结构：
表9.GLP-1/GCGR双特异性蛋白的结构模式
Figure PCTCN2019126903-appb-000053
*注：Ab为本公开所述GCGR抗体。GLP-1肽可以连接至GCGR抗体重链可变区的氨基端或连接至GCGR抗体轻链可变区的氨基端。经试验验证，GLP-1肽连接至GCGR抗体中重链可变区的氨基端的双特异性蛋白较连接至GCGR抗体轻 链可变区的氨基端稳定性更佳。本公开GLP-1肽连接至GCGR全长抗体重链可变区的部分实施方式中双特异性蛋白结构示意如图1所示。
将不同GLP-1肽与不同抗体的重链氨基酸用接头(例如(GGGGS) 3)相连接，形成以下蛋白：
表10.双特异性蛋白序列
Figure PCTCN2019126903-appb-000054
Figure PCTCN2019126903-appb-000055
Figure PCTCN2019126903-appb-000056
Figure PCTCN2019126903-appb-000057
Figure PCTCN2019126903-appb-000058
Figure PCTCN2019126903-appb-000059
此处双特异性蛋白中接头接头可选用(GGGGS) 3，在其他实施方式中也可以是肽键或其他常规用于多肽连接的接头，(GGGGS) 3的使用并不是对本公开双特异性蛋白接头的限制。编码GLP-1的核苷酸序列、编码GCGR抗体的核苷酸序列、接头蛋白片段((GGGGS) 3)的核苷酸序列通过所属领域常规技术手段获得。利用同源重组技术将GLP-1的C末端核苷酸通过接头蛋白连接GCGR抗体的N末端核苷酸，克隆到Phr-BsmbI载体上。重组的GLP-1/GCGR抗体双特异性蛋白在293细胞表达，通过实施例6的方法进行纯化。纯化的蛋白可用于下述各实施例中。
实施例6：抗体双特异性蛋白的纯化
细胞培养液高速离心后收集上清，利用亲合层析进行第一步纯化。层析介质为与Fc相互作用的Protein A或者衍生填料，如GE的Mabselect。平衡缓冲液为1×PBS(137mmol/L NaCl，2.7mmol/L KCl，10mmol/L Na 2HPO 4，2mmol/L KH 2PO 4，pH7.4)，平衡5倍柱体积后，将细胞上清上样结合，流速控制为样本在柱上保留时间≧1min。上样结束后，用1×PBS(pH7.4)冲洗柱子，直至A280紫外吸收降至基线。然后用0.1M甘氨酸(pH3.0)的洗脱缓冲液冲洗层析柱，根据A280紫外吸收峰收集洗脱峰，收集的洗脱样本用1M Tris(pH8.5)中和。
将上述中和后的洗脱样本超滤浓缩后进行体积排阻层析，缓冲液为1×PBS，层析柱为XK26/60Superdex200(GE)，流速控制在4ml/min，上样体积小于5ml，根据A280紫外吸收合并目的蛋白峰。收集的蛋白经SEC-HPLC鉴定纯度大于95％，经 LC-MS鉴定为正确后分装备用。得到GLP-1/GCGR抗体双特异性蛋白。
测试例
测试例1：GCGR嵌合抗体结合人、小鼠和食蟹猴GCGR的ELISA实验
抗GCGR抗体的结合力通过抗体与过表达GCGR的CHO细胞的结合实验来检测。通过转染的方法分别将人、小鼠和食蟹猴GCGR全长质粒转进CHO细胞中加压筛选两周后，检测GCGR的表达量。将过表达细胞固定于96孔板底后，抗体加入后信号的强弱被用于判断抗体和GCGR过表达CHO细胞的结合活性，三种种属GCGR抗体结合实验方法相同，以检测GCGR抗体结合人GCGR方法为例，具体实验方法如下：
将细胞以0.9-1.0×10 6/ml密度，100μl/孔接种于96孔板中过夜培养。弃上清，用PBS洗三遍后，加入100μl/孔的细胞免疫固定液(Beyotime，Cat Wo.P0098)室温固定1小时，PBS洗四遍。弃去液体后，加入用PBS稀释的5％脱脂牛奶(BD skim milk,Cat Wo.232100)封闭液200μl/孔，37℃孵育箱孵育3小时进行封闭。封闭结束后，弃去封闭液，并用PBST缓冲液(pH7.4PBS含0.05％tweeeW-20)洗板3次后，加入50μl/孔用样本稀释液稀释的不同浓度待测抗体(杂交瘤纯化抗体、嵌合抗体或人源化抗体)，放于37℃孵育箱孵育2小时。孵育结束后用PBST洗板3次，加入50μl/孔用样本稀释液稀释的HRP标记的羊抗鼠二抗(JacksoW ImmuWo Research，Cat Wo.115-035-003)或羊抗人二抗(JacksoW ImmuWo Research，Cat Wo.109-035-003)，37℃孵育1小时。用PBST洗板3次后，加入50μl/孔TMB显色底物(KPL，Cat Wo.52-00-03)，于室温孵育10min，加入50μl/孔1M H 2SO4终止反应，用酶标仪(Thermo scientific Multiskan MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据，计算GCGR嵌合抗体对GCGR过表达CHO细胞的结合EC50值，结果见下表。
表11.嵌合抗体结合活性
抗体EC50(nM) ch1803 ch1805 ch1808 ch1810 ch1817 ch1822
人GCGR 0.1167 0.2969 0.6769 0.1168 0.1926 0.3834
小鼠GCGR 0.7183 0.3428 1.019 0.4415 0.1638 0.5681
食蟹猴GCGR 0.2128 0.3585 0.5356 0.2480 0.2968 0.4280
结果显示各嵌合抗体ch1803、ch1805、ch1808、ch1810、ch1817和ch1822均与人GCGR具有良好的细胞表面结合活性，并且与小鼠GCGR和食蟹猴GCGR也有较好的交叉亲和活性。
测试例2：GCGR嵌合抗体阻断GCGR配体与GCGR结合试验
1、测试目的：
通过GCGR嵌合抗体阻断GCGR配体胰高血糖素与GCGR结合试验来评估 GCGR嵌合抗体的拮抗活性。
2、测试原理：
cAMP与CRE结合可启动CRE下游荧光素酶基因(luciferase)的表达，荧光素酶与其底物结合后发出荧光，通过荧光信号变化反映抑制效率。将CRE克隆至荧光素酶基因的上游，通过与含GCGR基因的质粒共转染CHO-K1细胞，挑选出同时高表达CRE及GCGR的单克隆细胞。GLP-1/GCGR抗体双特异性蛋白和胰高血糖素可竞争性的与GCGR结合，阻断GCGR的下游信号传递，影响下游cAMP的表达，通过测定荧光信号变化可评估GLP-1/GCGR抗体双特异性蛋白对GCGR的拮抗活性。
3、测试样本：
嵌合抗体ch1803，ch1805，ch1808，ch1810，ch1817，ch1822。
4、实验步骤：
a.用新鲜细胞培养基制取细胞悬液，以20000个细胞/孔加入80μl培养体系的96孔细胞培养板中，5％二氧化碳，37℃培养16小时。
b.每孔分别加入10μl配制好的待测蛋白，再加入10μl配好的胰高血糖素，5％二氧化碳，37℃培养5小时。
c.每孔加入加100μl检测液ONE Glo(Promega)，室温避光放置7分钟。
d.酶标仪Victor3上检测荧光，计算GCGR嵌合抗体融合对人、小鼠和食蟹猴GCGR配体胰高血糖素的结合阻断的IC50值及阻断效率Imax。
表12.嵌合抗体拮抗活性
Figure PCTCN2019126903-appb-000060
测试例3：GCGR人源化抗体对GCGR配体与GCGR结合的阻断试验
通过抗GCGR抗体阻断GCGR配体胰高血糖素与GCGR的结合试验来评估GCGR抗体的拮抗活性。实验原理和步骤同测试例2，结果见下表：
表13.人源化抗体拮的抗活性
Figure PCTCN2019126903-appb-000061
Figure PCTCN2019126903-appb-000062
体外活性生物学评价
测试例4：GLP-1/GCGR抗体双特异性蛋白在PBS中的稳定性
取200μg待测抗体双特异性蛋白溶于1ml 1×PBS(pH7.4)中，37℃恒温箱存放；分别于0和14天取样，Agilent 6530Q-TOF进行LC-MS检测完整重链保留情况，结果如下表14所示。含各突变改造的GLP-1肽的双特异性蛋白均较含GLP-1A(SEQ ID NO：91)的双特异性蛋白稳定性大大提高，并且hu1803-9B、hu1803-9D和hu1803-9G显示出更良好的稳定性。
表14.GLP-1/GCGR抗体双特异性蛋白的稳定性检测
蛋白 第14天
hu1803-9A 70.12％
hu1803-9B ≥97％
hu1803-9C 93.98％
hu1803-9D ≥97％
hu1803-9E 94.15％
hu1803-9F 94.65％
hu1803-9G ≥97％
测试例5：基于细胞的GCGR结合阻断实验
实验原理和步骤同测试例2。
测试样本：
①GCGR抗体(hu1803-9，hu1810-12)
②双特异性蛋白hu1803-9B，hu1803-9D。
表15.双特异性蛋白对GCGR的拮抗活性
Figure PCTCN2019126903-appb-000063
Figure PCTCN2019126903-appb-000064
表16.人源化抗体对小鼠和食蟹猴GCGR的拮抗活性
Figure PCTCN2019126903-appb-000065
图2和表15显示hu1803-9B和hu1803-9D均能完全抑制GCGR的拮抗活性，且与GCGR单抗具有相当的功效和IC50(抑制最大活性的50％所需的浓度)，表明hu1803-9B和hu1803-9D均保留了GCGR抗体部分完全的生物活性。除验证hu1803-9及含有hu1803-9的双特异性蛋白具有抑制人GCGR的拮抗活性外，进一步地还证明(见表16)hu1803-9和hu1810-12具有抑制小鼠GCGR和食蟹猴GCGR的拮抗活性。
测试例6：基于细胞的GLP-1R结合激活实验
1、测试目的：
评估GLP-1/GCGR抗体双特异性蛋白的GLP-1部分对GLP-1R的激活活性。
2、测试原理：
cAMP与CRE结合可启动CRE下游荧光素酶基因的表达，荧光素酶与其底物结合后发出荧光，通过荧光信号变化反映抑制效率。将CRE克隆至荧光素酶基因的上游，通过与含GLP-1R基因的质粒共转染CHO-K1细胞，挑选出同时高表达CRE及GLP-1R的单克隆细胞。GLP-1/GCGR抗体双特异性蛋白和阳性对照度拉糖肽可与GLP-1R结合，激活GLP-1R的下游信号传递，刺激下游cAMP的表达，通过测定荧光信号变化可评估GLP-1/GCGR抗体双特异性蛋白对GLP-1R的激活活性。
3、测试样本：
①阳性对照：度拉糖肽
②hu1803-9B，hu1803-9D。
4、实验步骤：
a.用新鲜细胞培养基制取细胞悬液，以25000个细胞/孔加入90μl培养体系的96孔细胞培养板中，5％二氧化碳，37℃培养16小时。
b.每孔分别加入10μl配制好的待测蛋白，5％二氧化碳，37℃培养5小时。
c.每孔加入加100μl检测液ONE Glo(Promega)，室温避光放置7分钟。
d.酶标仪Victor3上检测荧光，计算GLP-1/GCGR抗体双特异性蛋白对GLP-1R的结合激活的EC50值。
表17.双特异性蛋白对GLP-1R的激活活性
蛋白 EC50(nM) Emax(％)
度拉糖肽 0.21 100
hu1803-9B 0.22 100
hu1803-9D 0.32 100
图3和表17显示hu1803-9B和hu1803-9D均能完全激活GLP-1R，且与阳性对照度拉糖肽具有相当的功效和EC50(激活最大活性的50％所需的浓度)，表明hu1803-9B和hu1803-9D均保留了GLP-1部分完全的生物活性。
表18.双特异性蛋白对人GLP-1R的激活活性
样本 EC50(nM) Emax％
ch1805-D 0.11 108
ch1808-D 0.14 107
ch1817-D 0.30 106
表18显示ch1805-D、ch1808-D、ch1817-D嵌合抗体与GLP1肽连接均能完全激活GLP-1R，且与阳性对照度拉糖肽具有相当的功效和EC50(激活最大活性的50％所需的浓度)，表明不同GCGR抗体与GLP1肽以本公开的方式连接所形成的双特异性蛋白不影响GLP1肽的生物活性。
药代动力学评价
测试例7：C57小鼠体内药代动力学检测
实验用C57小鼠4只，雌性，12/12小时光/暗调节，温度24±3℃恒温，湿度50-60％，自由进食饮水。购自杰思捷实验动物有限公司。实验当天对C57小鼠分别尾静脉注射等摩尔hu1803-9D和阳性对照药物度拉糖肽，给药剂量分别为6mg/kg和2.35mg/kg，注射体积为10ml/kg。
因本公开的GLP-1/GCGR抗体双特异性蛋白和阳性对照度拉糖肽均与小鼠有交叉活性，因此在小鼠体内药物代谢时间较短，选择的取血时间点为：第1天给药后0h、1h、24h(第2天)，第3天，于小鼠眼底静脉取血，每次150μl(采血前，提前向采血管中加入1.5μl的DPP-4抑制剂)；收集的血样在4℃下置放半小时至凝集，然后4℃下14000×g离心5分钟。收集上清(约80μl)，立即放置-80℃贮存。
检测流程描述如下：
a.1μg/mL抗-GLP1(Novus,NBP1-05180)抗体铺板，4℃过夜。
b.250μl 1×PBST洗涤4次，加入200μl含5％脱脂牛奶的PBS,37℃封闭3小时。
c.250μl 1×PBST洗涤4次，加入100μl小鼠空白血清梯度稀释的待测药物，37℃孵育2小时。
d.250μl 1×PBST洗涤3次。
e.每孔加入100μl辣根过氧化物酶标记的二抗抗人IgG Fc，37℃孵育1小时。
f.250μl 1×PBST洗涤3次。
g.每孔加入100μl TMB，室温孵育10分钟后加入100μl 1M H 2SO 4终止反应。
h.酶标仪上检测450nm测吸收值，Graphpad Prism5分析数据。
表19.双特异性蛋白在小鼠中的T1/2
受试药物 给药方式 T1/2(h)
hu1803-9D IV(6mg/kg) 23.4
度拉糖肽 IV(2.35mg/kg) 10
PK分析结果表明，本公开双特异性蛋白分子hu1803-9D在小鼠体内的半衰期约为23.4h，为阳性对照药物半衰期的2倍。
体内活性生物学评价
测试例8：ob/ob小鼠体内药效实验
1、本实验应用的小鼠品系为糖尿病ob/ob小鼠和同龄野生型小鼠(南京大学南京模式动物研究所)，目的为观察GLP-1/GCGR抗体双特异性蛋白连续多次给药后对ob/ob小鼠血糖、糖化血红蛋白、体重、摄食量等糖尿病相关指标的治疗作用。
实验进行前对模型组动物按照实验当天的随机和空腹体重、随机和空腹血糖将其分成6组，分别为：
模型对照组、2.84mg/kg、1.42mg/kg GCGR单抗组(hu1803-9)、1.16mg/kg阳性对照组(度拉糖肽)和3mg/kg、1.5mg/kghu1803-9D，模型对照组小鼠皮下注射(S.C.)给予磷酸盐缓冲液，各组小鼠每周1次皮下注射(9:00AM)，一共4次(表20)。
表20.试验分组及给药情况
组别 处理 剂量 给药频率 给药方式
1 ob/ob小鼠模型对照组 PBS 每周一次x 4周 S.C.
2 GCGR单抗低剂量组 1.42mpk 每周一次x 4周 S.C.
3 GCGR单抗高剂量组 2.84mpk 每周一次x 4周 S.C.
4 阳性对照高剂量组 1.16mpk 每周一次x 4周 S.C.
4 hu1803-9D低剂量组 1.5mpk 每周一次x 4周 S.C.
5 hu1803-9D高剂量组 3mpk 每周一次x 4周 S.C.
2、实验步骤：
a.每周测定一次空腹和随机体重，每天测定一次摄食量和饮水量。
b.首次给药前及给药后第1、2、3、7天测随机血糖，后每周测一次随机血糖。首次给药前及给药后第3、7天测6h空腹血糖，后每周测一次空腹血糖。
c.给药第26天，动物禁食6h(8:00-14:00)后，单次腹腔给予葡萄糖水溶液2g/kg，并将给糖时间记为0点，在给糖前0min，给糖后15，30，60，90，120min分别对动物进行血糖检测，根据时间对血糖数据绘制糖耐量曲线，计算曲线下面积(AUC)。
d.实验结束后，小鼠禁食6小时(8:00-14:00)后给予安乐死，心脏取血后全血分为两部分，一部分约30μl注入已加好抗凝剂离心管并存放于湿冰中用于糖化血红蛋白测定，另一部分静置后离心取血清用于TG、FFA、CHOL、HDL、LDL水平测定。
e.数据采用graphpad Prism 6软件分析，采用Student-t test对数据进行统计学分析。
3、实验结果：
1)长期用药对ob/ob小鼠随机血糖的影响：
如图4所示，整个试验期间，模型对照组ob/ob小鼠随机血糖维持在较高的水平，一周一次皮下注射不同剂量各组药物后，各组小鼠的随机血糖均有不同程度的下降，呈现良好的剂量效应且显著低于模型对照组。2.84mg/kg的hu1803-9给药和3mg/kg hu1803-9D给药的降糖效果显著优于其他各试验组，而3mg/kg hu1803-9D给药第3、6、14和30天的随机血糖效果更佳。
2)长期用药对ob/ob小鼠饥饿血糖的影响:如图5所示，整个试验期间，模型对照组ob/ob小鼠随机血糖维持在较高的水平，一周一次皮下注射不同剂量各组药物后，各组小鼠的饥饿血糖均有不同程度的下降，呈现良好的剂量效应且显著低于模型对照组。与随机血糖浓度试验相似，2.84mg/kg的hu1803-9给药和3mg/kg hu1803-9D给药的降糖效果显著优于其他各试验组，而3mg/kg hu1803-9D给药第3、6、13和30天的饥饿血糖效果更佳。
3)长期用药对ob/ob小鼠糖化血红蛋白(HbA1c)的影响：
结果如表21所示，ob/ob小鼠一周一次皮下注射不同剂量各组药物30天后，糖化血红蛋白水平均有不同程度的下降，且显著低于模型对照组(P<0.05)，3mg/kg和1.5mg/kg的hu1803-9D组糖化血红蛋白水平分别为5.5±0.2％和4.7±0.1％，呈明显的剂量效应关系；其中3mg/kg的hu1803-9D组低于等摩尔的1.16mg/kg阳性对照度拉糖肽及2.84mg/kg的GCGR单抗hu1803-9(P<0.05)。
表21.长期用药对ob/ob小鼠HbA1c％的影响
Figure PCTCN2019126903-appb-000066
Figure PCTCN2019126903-appb-000067
测试例9：GCGR抗体的竞争性ELISA实验
通过生物素标记的抗体与不同浓度的标准抗体竞争结合过表达GCGR的CHO细胞的ELISA实验来检测，对GCGR抗体与GCGR结合的表位分类。细胞板准备方法与测试例1相同，后续具体实验方法如下：
按生物素标记试剂盒说明书标记抗体(Dojindo Molecular Technologies,Inc.LK03)。在96孔细胞板中，按50μl/孔用样本稀释液稀释的不同浓度未生物素标记待测抗体，放于37℃孵育2小时。孵育结束后用PBST洗板3次，加入50μl/孔用样本稀释液稀释至0.1μg/ml的生物素标记抗体，37℃孵育2小时后用PBST洗板3次，加入HRP标记的羊抗人二抗(JacksoW ImmuWo Research，Cat Wo.109-035-003)，37℃孵育1小时。用PBST洗板3次后，加入50μl/孔TMB显色底物(KPL，Cat Wo.52-00-03)，于室温孵育10min，加入50μl/孔1M H 2SO 4终止反应，用酶标仪(Thermo scientific Multiskan MK3)在波长450nm处读取吸收值，用GraphPad Prism 5分析数据。
当生物素标记抗体竞争结合至细胞板上的浓度越低时，OD值越低，反之信号越高。按公式IC％＝(待测抗体最高OD 450nm-待测抗体最低OD 450nm)/(待测最高OD 450nm-标记抗体最低OD 450nm)计算竞争效率，结果见下表22。
表22.各抗体间竞争结合关系
Figure PCTCN2019126903-appb-000068
结果显示，ch1817与ch1822具有非常相近的表位，ch1808、hu1803-9和hu1810-12具有非常相近的表位；而ch1808、hu1803-9和hu1810-12的表位推测在 ch1805的表位范围内。

Claims (25)

  1. 一种抗GCGR的单克隆抗体或其抗原结合片段，其包含选自如下所示的重链可变区和轻链可变区的组合：
    a)重链可变区，其包含分别如SEQ ID NO：48、49和50所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：51、52和53所示的LCDR1、LCDR2和LCDR3区；或
    b)重链可变区，其包含分别如SEQ ID NO：38、39和54所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：55、56和57所示的LCDR1、LCDR2和LCDR3区。
  2. 根据权利要求1所述的抗GCGR的单克隆抗体或其抗原结合片段，其包含选自如下所示的重链可变区和轻链可变区的组合：
    i)重链可变区，其包含分别如SEQ ID NO：14、15和16所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：17、18和19所示的LCDR1、LCDR2和LCDR3区；
    ii)重链可变区，其包含分别如SEQ ID NO：20、21和22所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：23、24和25所示的LCDR1、LCDR2和LCDR3区；
    iii)重链可变区，其包含分别如SEQ ID NO：26、27和28所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：29、30和31所示的LCDR1、LCDR2和LCDR3区；
    iv)重链可变区，其包含分别如SEQ ID NO：32、33和34所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：35、36和37所示的LCDR1、LCDR2和LCDR3区；
    v)重链可变区，其包含分别如SEQ ID NO：38、39和40所示的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：41、42和43所示的LCDR1、LCDR2和LCDR3区；和
    vi)重链可变区，其包含分别如SEQ ID NO：38、39和44所示的HCDR1、 HCDR2和HCDR3区，和
    轻链可变区，其包含分别如SEQ ID NO：45、46和47所示的LCDR1、LCDR2和LCDR3区。
  3. 根据权利要求1或2所述的抗GCGR的单克隆抗体或其抗原结合片段，其是鼠源抗体或其抗原结合片段、嵌合抗体或其抗原结合片段、或人源化抗体或其抗原结合片段。
  4. 根据权利要求3所述的抗GCGR的单克隆抗体或其抗原结合片段，所述人源化抗体包含源自人抗体的框架区或其框架区变体，其中：
    所述框架区变体在人抗体的轻链框架区和/或重链框架区上分别具有至多10个氨基酸的回复突变，
    优选地，所述氨基酸回复突变选自：
    aa)轻链可变区中包含42G、44V、71Y和87F中的一个或更多个回复突变，和/或
    重链可变区中包含38K、48I、67A、69F、71A、73P、78A和93S中的一个或更多个回复突变；或者
    ab)轻链可变区中包含38L、44V、59S、70E和71Y中的一个或更多个回复突变，和/或
    重链可变区中包含38K、48I、66K、67A、69L、73R、78M和94S中的一个或更多个回复突变。
  5. 根据权利要求3所述的抗GCGR的单克隆抗体或其抗原结合片段，其包含选自如下所示的轻链可变区和重链可变区组合：
    c)重链可变区，其序列如SEQ ID NO：2、61、62、63和64任一所示或与SEQ ID NO：2、61、62、63和64任一所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：3、58、59和60任一所示或与SEQ ID NO：3、58、59和60任一所示具有至少90％序列同一性；
    d)重链可变区，其序列如SEQ ID NO:4所示或与SEQ ID NO：4所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：5所示或与SEQ ID NO：5所示具有至少90％序列同一性；
    e)重链可变区，其序列如SEQ ID NO：6所示或与SEQ ID NO：6所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：7所示或与SEQ ID NO：7所示具有至少90％序列同一性；
    f)重链可变区，其序列如SEQ ID NO：8、68、69、70和71任一所示或与SEQ ID NO：8、68、69、70和71任一所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：9、65、66和67任一所示或与SEQ ID NO：9、65、66和67任一所示具有至少90％序列同一性；
    g)重链可变区，其序列如SEQ ID NO:10所示或与SEQ ID NO：10所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：11所示或与SEQ ID NO：11所示具有至少90％序列同一性；和
    h)重链可变区，其序列如SEQ ID NO:12所示或与SEQ ID NO：12所示具有至少90％序列同一性；和
    轻链可变区，其序列如SEQ ID NO：13所示或与SEQ ID NO：13所示具有至少90％序列同一性。
  6. 根据权利要求1至5任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，其中所述抗体为全长抗体，进一步包括抗体恒定区，
    优选地，所述重链恒定区选自人IgG1、IgG2、IgG3和IgG4恒定区及其常规变体，所述轻链恒定区选自人抗体κ和λ链恒定区及其常规变体，
    更优选包含SEQ ID NO：72所示的人抗体重链恒定区和SEQ ID NO：73所示的人抗体轻链恒定区。
  7. 根据权利要求6所述的抗GCGR的单克隆抗体或其抗原结合片段，包括选自如下任一项所示的重链和轻链的组合：
    j)所述重链为SEQ ID NO：74、76或78所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：75、77或79所示或与其具有至少85％序列同一性；
    k)所述重链为SEQ ID NO：80所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：81所示或与其具有至少85％序列同一性；
    l)所述重链为SEQ ID NO：82所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：83所示或与其具有至少85％序列同一性；
    m)所述重链为SEQ ID NO：84所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：85所示或与其具有至少85％序列同一性；
    n)所述重链为SEQ ID NO：86所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：87所示或与其具有至少85％序列同一性；和
    o)所述重链为SEQ ID NO：88所示或与其具有至少85％序列同一性，且所述轻链为SEQ ID NO：89所示或与其具有至少85％序列同一性。
  8. 一种抗GCGR的单克隆抗体或其抗原结合片段，其中包含选自如下所示的重链可变区和轻链可变区组合：
    ac)重链可变区，其包含与SEQ ID NO：2所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：3所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
    ad)重链可变区，其包含与SEQ ID NO：4所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：5所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
    ae)重链可变区，其包含与SEQ ID NO：6所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：7所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
    af)重链可变区，其包含与SEQ ID NO：8所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：9所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；
    ag)重链可变区，其包含与SEQ ID NO：10所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：11所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区；和
    ah)重链可变区，其包含与SEQ ID NO：12所示重链可变区具有相同序列的HCDR1、HCDR2和HCDR3区，和
    轻链可变区，其包含与SEQ ID NO：13所示轻链可变区具有相同序列的LCDR1、LCDR2和LCDR3区。
  9. 根据权利要求1至8任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，其中所述抗原结合片段选自Fab、Fab'、F(ab')2、单链抗体、二聚化的V区(双抗体)和二硫键稳定化的V区(dsFv)。
  10. 一种双特异性蛋白，其包含GLP-1肽和抗GCGR抗体或其抗原结合片段，所述GLP-1肽通过肽键或接头共价连接至所述的抗GCGR抗体或其抗原结合片段。
  11. 根据权利要求10所述的双特异性蛋白，其中：
    所述GLP-1肽的羧基端通过肽键或接头连接至所述抗GCGR的单克隆抗体或其抗原结合片段的重链可变区的氨基端；或者
    所述GLP-1肽的羧基端通过肽键或接头连接至所述抗GCGR抗体或其抗原结合片段的轻链可变区的氨基端。
  12. 根据权利要求11所述的双特异性蛋白，其中所述的抗GCGR抗体或其抗原结合片段是权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段。
  13. 根据权利要求10至12任一项所述的双特异性蛋白，其中：
    所述GLP-1肽是SEQ ID NO：91所示的GLP-1肽或其变体，
    所述GLP-1肽变体为SEQ ID NO：91所示GLP-1肽中包含Q17E、I23V、K28R和G30R中一个或更多个氨基酸突变，
    优选地，所述GLP-1肽变体包含Q17E突变、或包含Q17E和I23V突变。
  14. 根据权利要求13所述的双特异性蛋白，其中所述GLP-1肽变体的序列如SEQ ID NO：92、93、94、95、96、97、98或99所示。
  15. 根据权利要求14所述的双特异性蛋白，所述双特异性蛋白包含第一多肽链和第二多肽链，所述第一多肽链包含权利要求1至9任一项所述抗GCGR的单克隆抗体的重链；所述第二多肽链包含权利要求1至9任一项所述抗GCGR的单克隆抗体的轻链；其中：
    (ai)所述第一多肽链包含：选自SEQ ID NO：100、101、102、103、104、105、106、107和108任一所示的多肽，和
    所述第二多肽链包含SEQ ID NO：79所示的多肽；
    (aj)所述第一多肽链包含SEQ ID NO：109所示的多肽，和
    所述第二多肽链包含SEQ ID NO：81所示的多肽；
    (ak)所述第一多肽链包含SEQ ID NO：110所示的多肽，和所述第二多肽链包含SEQ ID NO：83所示的多肽；
    (al)所述第一多肽链包含SEQ ID NO：111所示的多肽，和所述第二多肽链包含SEQ ID NO：85所示的多肽；
    (am)所述第一多肽链包含SEQ ID NO：112所示的多肽，和所述第二多肽链包含SEQ ID NO：87所示的多肽；或者
    (an)所述第一多肽链包含SEQ ID NO：113所示的多肽，和所述第二多肽链包含SEQ ID NO：89所示的多肽。
  16. 一种GLP-1肽变体，所述GLP-1肽变体是在SEQ ID NO：91所示的GLP-1 肽基础上包含Q17E、I23V、K28R和G30R中一个或更多个氨基酸突变，优选地，所述GLP-1肽变体包含Q17E突变、或包含Q17E和I23V突变。
  17. 根据权利要求16所述的GLP-1肽变体，所述GLP-1肽变体的序列如SEQ ID NO：92、93、94、95、96、97、98或99所示。
  18. 一种药物组合物，其含有：
    治疗有效量的根据权利要求1至9任一项所述抗GCGR的单克隆抗体或其抗原结合片段，或根据权利要求10至15任一项所述的双特异性蛋白，或根据权利要求16或17所述的GLP-1肽变体，以及
    一种或多种药学上可接受的载体、稀释剂、缓冲剂或赋形剂。
  19. 一种分离的核酸分子，其编码权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或根据权利要求10至15任一项所述的双特异性蛋白，或根据权利要求16或17所述的GLP-1肽变体。
  20. 一种重组载体，其包含权利要求19所述的分离的核酸分子。
  21. 一种宿主细胞，其转化有权利要求20所述的重组载体，所述宿主细胞选自原核细胞和真核细胞，优选为真核细胞，更优选哺乳动物细胞或昆虫细胞。
  22. 一种制备权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或权利要求10至15任一项所述的双特异性蛋白，或权利要求16或17所述的GLP-1肽变体的方法，所述方法包括：
    培养权利要求21所述的宿主细胞形成权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或权利要求10至15任一项所述的双特异性蛋白，或权利要求16或17所述的GLP-1肽变体，以及
    从培养物回收所述单克隆抗体或其抗原结合片段、或双特异性蛋白、或GLP-1肽变体。
  23. 一种检测样本中人GCGR的试剂，其包含权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段。
  24. 一种降低受试者血糖浓度的方法，所述方法包括：
    向受试者施用治疗有效量的权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或权利要求10至15任一项所述的双特异性蛋白，或权利要求16或17所述的GLP-1肽变体，或权利要求18所述的药物组合物，
    优选地，所述治疗有效量为单位剂量中含有0.1至3000mg的权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或权利要求10至15任一项所述的双特异性蛋白，或权利要求16或17所述的GLP-1肽变体。
  25. 一种治疗代谢障碍的方法，所述方法包括：
    向受试者施用治疗有效量的权利要求1至9任一项所述的抗GCGR的单克隆抗体或其抗原结合片段，或权利要求10至15任一项所述的双特异性蛋白，或权利要求16或17所述的GLP-1肽变体，或权利要求18所述的药物组合物；
    优选地，所述代谢障碍选自：代谢综合征、肥胖症、葡萄糖耐量受损、糖尿病、糖尿病酮症酸中毒、高血糖症、高血糖高渗综合征、围术期高血糖症、高胰岛素血症、胰岛素抵抗综合症、空腹血糖受损、血脂异常、动脉粥样硬化和糖尿病前期状态。
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WO2022083720A1 (zh) * 2020-10-23 2022-04-28 江苏恒瑞医药股份有限公司 Glp1-gcgr抗体融合蛋白变体及包含其的组合物
WO2022143516A1 (zh) * 2020-12-29 2022-07-07 苏州康宁杰瑞生物科技有限公司 一种人glp-1多肽变体及其应用

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US20220153853A1 (en) 2022-05-19
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