WO2019196603A1 - Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用 - Google Patents

Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用 Download PDF

Info

Publication number
WO2019196603A1
WO2019196603A1 PCT/CN2019/078674 CN2019078674W WO2019196603A1 WO 2019196603 A1 WO2019196603 A1 WO 2019196603A1 CN 2019078674 W CN2019078674 W CN 2019078674W WO 2019196603 A1 WO2019196603 A1 WO 2019196603A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
amino acid
glp
fusion protein
Prior art date
Application number
PCT/CN2019/078674
Other languages
English (en)
French (fr)
Inventor
张�成
章华
汪笑峰
药晨江
姜艳
毕良亮
景书谦
Original Assignee
鸿运华宁（杭州）生物医药有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 鸿运华宁（杭州）生物医药有限公司 filed Critical 鸿运华宁（杭州）生物医药有限公司
Priority to US17/046,686 priority Critical patent/US11542336B2/en
Priority to EP19785909.3A priority patent/EP3778634A4/en
Priority to CA3096556A priority patent/CA3096556A1/en
Publication of WO2019196603A1 publication Critical patent/WO2019196603A1/zh

Links

Images

Classifications

    • 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
    • A61P3/06—Antihyperlipidemics
    • 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
    • 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
    • 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
    • C07K2317/50—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
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • 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
    • 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
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • GCGR antibodies and fusion proteins thereof with GLP-1 are provided herein.
  • GCGR antibodies and fusion proteins thereof with GLP-1 for treating, preventing or ameliorating one or more symptomatic methods of hyperglycemia, type 2 diabetes, metabolic syndrome, or dyslipidemia.
  • Human glucagon is an important hormonal substance that acts together with insulin and participates in the regulation of glucose levels in human blood. Both glucagon and insulin are peptide hormones. Glucagon is produced in alpha pancreatic islet cells of the pancreas, and insulin is produced in beta islet cells. When blood sugar levels are lowered, glucagon mainly releases glucose by stimulating some target cells (mainly liver cells). Glucagon and insulin regulate blood sugar. The insulin stimulates cells to take up and store glucose when blood sugar rises. , lower blood sugar concentration.
  • Natural human glucagon is composed of 29 amino acid residues: His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg- Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr.
  • Glucagon binds to its receptor and activates downstream signaling pathways.
  • the glucagon receptor belongs to the seven-pass transmembrane G protein-coupled receptor family secretin subtype, after ligand-receptor binding.
  • GCGR glucagon receptor
  • Diabetes is a common form of glucose metabolism, characterized by hyperglycemia, which is divided into type 1 diabetes with absolute insulin deficiency and type 2 diabetes with relatively insufficient insulin.
  • Type 1 diabetic patients exhibit hyperglycemia and hypoinsulinemia, and conventional treatment for the disease provides insulin.
  • absolute or relatively high glucagon causes hyperglycemia.
  • the removal of glucagon from the blood circulation with selective or specific antibodies can reduce blood glucose levels (Brand et al., 1996, Diabetes 45: 1076).
  • Blocking the binding of glucagon and glucagon receptors by antibodies that target the glucagon receptor can also be used as a means of controlling or lowering blood sugar, making it a new treatment for diabetes (US 2008/ 036341 A2 and US 2012/0128679 A1).
  • US 2008/ 036341 A2 and US 2012/0128679 A1 a new treatment for diabetes
  • Prime transiently elevated liver transaminases, and significant proliferation of islet alpha cells (Kostic et al, 2018, Diabetes Obes Metab. 20: 283-91; Gu et al, 2009, JPET 331: 871-881).
  • a fusion protein of GCGR antibody and GLP-1 (GLP-1 fusion protein).
  • the GCGR antibody partially reduces intracellular cAMP levels and lowers blood glucose by inhibiting the binding of glucagon to its receptor;
  • GLP- Part 1 also has the effect of lowering blood sugar.
  • two parts of the GLP-1 fusion protein the antibody of GCGR and the part of GLP-1 simultaneously reduce blood sugar, and the two have a synergistic effect, which can lower blood sugar more greatly, thereby increasing blood sugar lowering. Drug effect.
  • the clinical dose of anti-glucagon antibodies does not need to be particularly high, that is, at low doses, together with GLP-1 agonists, can significantly lower blood sugar, which is better than alone.
  • the use of high-dose GCGR antibodies can lower blood glucose more significantly, thereby avoiding the side effects caused by antibodies using high-dose glucagon receptors, such as the proliferation of islet alpha cells.
  • GLP-1 agonists promote insulin secretion, which in turn antagonizes glucagon production (Mari et al., 2016, Diabetes Obes Metab. 18:834-9), giving GLP-1 fusion proteins,
  • the GLP-1 moiety can inhibit the increase of glucagon, and also reduces the negative feedback effect of GCGR antibody on glucagon, which can significantly reduce the side effects of the drug.
  • hypoglycemic effect of the GCGR antibody portion of the GLP-1 fusion protein and the GLP-1 partial hypoglycemic effect are superimposed to treat hyperglycemia, type 2 diabetes, metabolic syndrome, and other related symptoms including dyslipidemia by a dual action. This is greater than the GLP-1 agonist alone, and it is also less toxic than the glucagon receptor alone.
  • the GLP-1 fusion protein has a more pronounced hypoglycemic effect and is safer.
  • antibodies that specifically bind to GCGR which are antagonists of GCGR.
  • an antibody that specifically binds to GCGR comprising one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the following Amino acid sequence:
  • heavy chain CDR1 amino acid sequence SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28, SEQ ID NO: 31, and SEQ ID NO: 34;
  • heavy chain CDR2 amino acid sequence SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 35;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36.
  • GLP-1 fusion protein comprising an antibody that specifically binds to GCGR, and one, two, three, four, five, six, seven, or eight GLP-1 Fragment; the fusion protein ligates the carboxy terminus of a GLP-1 fragment to the amino terminus of a GCGR antibody light or heavy chain via a linker sequence (Linker) or the amino terminus of a GLP-1 fragment to a GCGR antibody The carboxy terminus of the chain or heavy chain is attached.
  • Linker linker sequence
  • GLP-1 fusion protein comprising an antibody that specifically binds to GCGR, and one, two, three, four, five, six, seven, or eight reverse GLPs a -1 fragment; the fusion protein ligates the carboxy terminus of a reverse GLP-1 fragment to the amino terminus of a GCGR antibody light or heavy chain via a peptide linker sequence (Linker) or an amino group of a reverse GLP-1 fragment The terminus is linked to the carboxy terminus of a GCGR antibody light or heavy chain.
  • Linker peptide linker sequence
  • a GLP-1 fusion protein comprising a GCGR antibody and two GLP-1 fragments; the fusion protein carboxy terminus of a GLP-1 fragment to a GCGR antibody light chain via a peptide linker (Linker) Amino-terminal linkage: N'-GLP-1-Linker-R-C'; or the carboxy terminus of a GLP-1 fragment and the amino terminus of a GCGR antibody heavy chain: N'-GLP-1-Linker-R- C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxy terminus of the fusion protein polypeptide chain, GLP-1 represents the GLP-1 fragment, and R is the amino acid sequence of the light or heavy chain of the GCGR antibody, And Linker stands for peptide linker sequence.
  • Linker stands for peptide linker sequence.
  • a GLP-1 fusion protein comprising a GCGR antibody and two inverted GLP-1 fragments; the fusion protein is linked to the amino terminus of a reverse GLP-1 fragment by a peptide linker (Linker)
  • the carboxy-terminal linkage of the antibody light chain N'-R-Linker-reverse GLP-1-C'; or the amino terminus of a reverse GLP-1 fragment and the carboxy terminus of a GCGR antibody heavy chain: N'- R-Linker-reverse GLP-1-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxy terminus of the fusion protein polypeptide chain, and reverse GLP-1 represents the reverse GLP-1 fragment, R is the amino acid sequence of the light or heavy chain of the GCGR antibody, and Linker represents the peptide linker sequence.
  • Linker represents the peptide linker sequence.
  • a polynucleotide encoding a GCGR antibody described herein is provided herein.
  • a polynucleotide encoding a fusion protein of a GCGR antibody and GLP-1 described herein.
  • a vector comprising a polynucleotide encoding a GCGR antibody described herein.
  • a vector comprising a polynucleotide encoding a fusion protein of a GCGR antibody and GLP-1 described herein.
  • a host cell comprising a vector described herein.
  • composition comprising a GCGR antibody described herein and a pharmaceutically acceptable carrier.
  • composition comprising a fusion protein of a GCGR antibody and GLP-1 described herein and a pharmaceutically acceptable carrier.
  • a GCGR antibody described herein in the manufacture of a medicament for the treatment, prevention or amelioration of type 2 diabetes.
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the manufacture of a medicament for the treatment, prevention or amelioration of type 2 diabetes.
  • a GCGR antibody described herein for the manufacture of a medicament for the treatment, prevention or amelioration of complications of type 2 diabetes.
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the manufacture of a medicament for the treatment, prevention or amelioration of complications of type 2 diabetes.
  • a GCGR antibody described herein for the manufacture of a medicament for the treatment, prevention or amelioration of hyperglycemia.
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the manufacture of a medicament for the treatment, prevention or amelioration of hyperglycemia.
  • a GCGR antibody described herein in the manufacture of a medicament for the treatment, prevention or amelioration of metabolic syndrome.
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the manufacture of a medicament for the treatment, prevention or amelioration of metabolic syndrome.
  • GCGR antibody described herein for the preparation of other related drugs for the treatment, prevention or amelioration of dyslipidemia.
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the preparation of other related drugs for the treatment, prevention or amelioration of dyslipidemia.
  • a GCGR antibody described herein for the manufacture of a medicament for the simultaneous treatment, prevention or amelioration of two or more related conditions of hyperglycemia, type 2 diabetes, metabolic syndrome, or dyslipidemia .
  • a fusion protein of a GCGR antibody and GLP-1 described herein for the preparation of two or more other related treatments for the simultaneous treatment, prevention or amelioration of hyperglycemia, type 2 diabetes, metabolic syndrome, or dyslipidemia Use in drugs for the above conditions.
  • GCGR antibody described herein.
  • a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 described herein.
  • GCGR antibody described herein.
  • a method of treating, preventing, or ameliorating type 2 diabetes comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 described herein.
  • GCGR antibody described herein.
  • a method of treating, preventing, or ameliorating metabolic syndrome comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 described herein.
  • GCGR antibody described herein.
  • a method of treating, preventing, or ameliorating dyslipidemia comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 described herein.
  • GCGR antibody described herein.
  • a GCGR antibody and GLP-1 described herein Fusion protein are provided herein.
  • Figure 1 shows the results of flow cytometry (FACS) detection of mouse ascites antibody L5H5 (which comprises SEQ ID NO: 85 and SEQ ID NO: 95) specifically binding to hGCGR, gray peak is 500 nM mouse ascites antibody L5H5 Negative control combined with blank cell CHO-DHFR-, solid line peaks were binding curves of mouse ascites antibody L5H5 and CHO-DHFR-hGCGR of 500 nM (1a), 50 nM (1b) and 5 nM (1c), respectively, relative to gray The peak negative control showed a significant right shift, demonstrating the specific binding of L5H5 and CHO-DHFR-hGCGR.
  • FACS flow cytometry
  • Figure 2 shows the results of flow cytometry (FACS) detection of mouse ascites antibody L4H4 (which comprises SEQ ID NO: 84 and SEQ ID NO: 94) specifically binding to hGCGR, gray peak is 500 nM mouse ascites antibody L4H4 Negative control combined with blank cell CHO-DHFR-, solid line peaks were binding curves of mouse ascites antibody L4H4 and CHO-DHFR-hGCGR of 500 nM (2a), 50 nM (2b) and 5 nM (2c), respectively, relative to gray The peak negative control showed a significant right shift, demonstrating the specific binding of L4H4 and CHO-DHFR-hGCGR.
  • FACS flow cytometry
  • Figure 4 shows the direct cAMP assay for detection of mouse ascites GCGR antibodies (L4H4 and L5H5) antagonizing the gradient dilution of the glucagon-activated hGCGR signaling pathway. As the concentration of anti-GCGR antibody increased, the S-curve of glucagon-activated hGCGR showed a significant right shift.
  • Figure 6 shows the results of a single subcutaneous injection of L4H4, GMA102 (every two days) in C57BL/6 obese mice induced by high fat diet, and the results of oral glucose tolerance on day 14 after combination therapy.
  • Figure 7 shows the pharmacokinetic (PK) time curve of hGCGR antibody and GLP-1 fusion protein GLP-1-Linker-L7H7 in the healthy rhesus monkey experimental cycle.
  • Figure 8 shows insulin changes before a single subcutaneous injection of a positive control drug and GLP-1-Linker-L7H7 in a healthy rhesus monkey intravenous glucose tolerance test (IVGTT).
  • Figure 9 shows insulin changes 48 hours after a single subcutaneous injection of a positive control drug and GLP-1-Linker-L7H7 in a healthy rhesus monkey intravenous glucose tolerance test (IVGTT).
  • Figure 10 shows the changes in blood glucose before a single subcutaneous injection of a positive control drug and GLP-1-Linker-L7H7 in a healthy rhesus monkey intravenous glucose tolerance test (IVGTT).
  • Figure 11 shows blood glucose changes 48 hours after a single subcutaneous injection of a positive control drug and GLP-1-Linker-L7H7 in a healthy rhesus monkey intravenous glucose tolerance test (IVGTT).
  • IVGTT intravenous glucose tolerance test
  • Polynucleotide and polypeptide sequences are indicated herein using standard one-letter or three-letter abbreviations.
  • the first amino acid residue (N') carrying an amino group is at the far left and the last amino acid residue (C') having a carboxyl group is at the far right, such as the GLP-1 fragment sequence referred to herein.
  • SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123 are amino acid residue carrying an amino group carrying an amino group.
  • the reverse polypeptide sequence refers to a sequence formed by reversely arranging the amino acid appearance sequences of the polypeptide sequence, for example, the reverse GLP-1 fragment sequence formed by the above GLP-1 fragment sequence: SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131.
  • the 5' end of the upstream strand of the single-stranded nucleic acid sequence and the double-stranded nucleic acid sequence is to the left and their 3' end is to the right.
  • Particular portions of a polypeptide may be represented by amino acid residue numbers, such as amino acids 80 to 130, or by actual residues of the site, such as Lys80 to Lys130.
  • a particular polypeptide or polynucleotide sequence can also be described by interpreting its differences from the reference sequence.
  • peptide refers to a molecule comprising two or more amino acids joined together by a peptide. These terms encompass polypeptide analogs (eg, muteins, variants, and fusion proteins) such as native and artificial protein and protein sequences, as well as proteins that are post-transcriptionally or otherwise covalently or non-covalently modified.
  • polypeptide analogs eg, muteins, variants, and fusion proteins
  • the peptide, polypeptide or protein can be a monomer or a multimer.
  • polypeptide fragment refers to a polypeptide having an amino terminus and/or a carboxy terminus deletion compared to the corresponding full length protein.
  • the fragment length can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids.
  • the segment length can be, for example, up to 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids.
  • the fragment may further comprise one or more additional amino acids at one or both ends thereof, for example, an amino acid sequence (eg, an Fc or leucine zipper domain) or an artificial amino acid sequence (eg, an artificial linker sequence) from a different native protein.
  • Polypeptides herein include polypeptides modified for any reason and by any method, for example, to: (1) reduce proteolytic sensitivity, (2) reduce oxidation sensitivity, and (3) alter the affinity of protein-forming complexes, ( 4) altering the binding affinity, and (5) imparting or modifying other physicochemical or functional properties.
  • Analogs comprise a mutein of a polypeptide. For example, single or multiple amino acid substitutions (eg, conservative amino acid substitutions) can be made in a native sequence (eg, a portion of a polypeptide that is outside of the domain that forms the intramolecular contact).
  • a “conservative amino acid substitution” is one that does not significantly alter the structural properties of the parent sequence (eg, a replacement amino acid should not disrupt the helix present in the parent sequence or interfere with other secondary structure types that confer a maternal sequence property or is essential for its function).
  • a "variant" of a polypeptide comprises an amino acid sequence in which one or more amino acid residues have been inserted, deleted and/or replaced in the amino acid sequence relative to another polypeptide sequence. Variants herein include fusion proteins.
  • a “derivative" of a polypeptide is a chemically modified polypeptide, for example by binding to other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.
  • albumin e.g., human serum albumin
  • phosphorylation e.g., phosphorylation
  • glycosylation e.g., glycosylation
  • antibody includes two full-length heavy chains and two full-length light chain antibodies, as well as derivatives, variants, fragments, and muteins thereof, examples of which are described below.
  • antibody is a protein comprising a scaffold or framework portion that binds to an antigen and optionally allows the antigen binding portion to adopt a conformation that promotes binding of the antibody to the antigen.
  • antibodies include intact antibodies, antibody fragments (e.g., antigen binding portions of antibodies), antibody derivatives, and antibody analogs.
  • the antibody may comprise, for example, a selectable protein scaffold or a human scaffold with grafted CDRs or CDRs derivatives.
  • Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising, for example, a three-dimensional structure that stabilizes the antibody, and fully synthetic scaffolds comprising, for example, biocompatible multimers.
  • the antibody may be a peptidomimetic antibody ("PAMs") or a scaffold comprising a mock antibody that utilizes fibrin lignin as a scaffold.
  • PAMs peptidomimetic antibody
  • the antibody may have a structure such as a native immunoglobulin.
  • Immunoglobulin is a tetrameric molecule. In natural immunoglobulins, each tetramer is composed of two identical pairs of polypeptide chains, each pair having a "light” chain (about 25 kDa) and a "heavy” chain (about 50-70 kDa). The amino terminus of each chain includes a variable domain of about 100 to 110 amino acids, primarily associated with antigen recognition. The carboxy terminal portion of each chain defines a constant region that is primarily associated with effector action. Human antibody light chains are classified into kappa and lambda light chains.
  • the heavy chain is divided into ⁇ , ⁇ , ⁇ or ⁇ and the isoforms of the antigen are determined, for example IgM, IgD, IgG, IgA, and IgE, respectively.
  • the variable and constant regions are joined by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D” region of about 10 amino acids. See, Fundamental Immunology Ch. 7 (Paul Editor, 2nd ed., Raven Press, 1989).
  • the variable regions of each light/heavy chain pair form an antibody binding site such that one intact immunoglobulin has two binding sites.
  • Native immunoglobulin chains display the same basic structure of relatively conserved framework regions (FR) joined by three highly variable regions, also referred to as complementarity determining regions or CDRs. From the N-terminus to the C-terminus, the light and heavy chains each comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The assignment of amino acids for each domain is consistent with the definition of Kabat et al. in Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242,1991.
  • antibody refers to an intact immunoglobulin or an antigen binding portion thereof that can compete for specific binding to an intact antibody.
  • the antigen binding portion can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding portions include, inter alia, Fab, Fab', F(ab') 2 , Fv, domain antibodies (dAbs), including fragments of complementarity determining regions (CDRs), single-chain antibodies (scFv), chimeric antibodies, Diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient to confer specific antigen binding to the polypeptide.
  • F (ab ') 2 fragment is a bivalent fragment having two Fab fragments linked by the hinge region disulfide bonds; an Fv fragment V H and V L, having a domain; a dAb fragment having a V H domain, V L domain, or a V H or V L domain antigen binding fragment (U.S. Patent No. US6,846,634 and US 6,696,245; U.S. Patent application Publication No. US 2005/0202512, US 2004/0202995, US 2004/0038291, US 2004/0009507, and US 2003/0039958; Ward et al, 1989, Nature 341: 544-546).
  • Single-chain antibody is a fusion protein in which the V L and V H region by a linker (e.g., a synthetic amino acid residue sequences) linked to form antibodies continuous protein, wherein the linker is long enough to allow the protein chain folds It returns to itself and forms a monovalent antigen binding site (see, for example, Bird et al, 1988, Science 242: 423-26; and Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-83).
  • a linker e.g., a synthetic amino acid residue sequences
  • Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V H domains and V L, connected by a linker, the linker is short so as to allow pairing the two domains on the same chain, Thus each domain is allowed to pair with a complementary domain on another polypeptide chain (see, for example, Holliger et al, 1993, Proc. Natl. Acad. Sci. USA 90:6444-48; and Poljak et al, 1994, Structure 2: 1121-23). If the two polypeptide chains of the diabody are identical, the diabody obtained by their pairing will have the same antigen binding site. Polypeptide chains with different sequences can be used to prepare diabodies with different antigen binding sites. Similarly, the triple-chain antibody and the four-chain antibody are antibodies comprising three and four polypeptide chains, respectively, and form three and four antigen-binding sites, respectively, which may be the same or different.
  • CDRs complementarity determining region of a given antibody using the method described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, USDept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991 (CDRs) and framework regions (FR).
  • CDRs can be incorporated into the molecule either covalently or non-covalently into an antibody.
  • Antibodies can incorporate CDR(s) into larger polypeptide chains.
  • the CDR(s) can be covalently linked to another native peptide chain, or non-covalently incorporated into the CDR(s).
  • CDRs allow antibodies to specifically bind to specific antigens of interest.
  • An antibody can have one or more binding sites. If there is more than one binding site, the binding site can be the same or different from the other. For example, a native human immunoglobulin typically has two identical binding sites, while a "bispecific" or "bifunctional” antibody has two different binding sites.
  • murine antibody includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.
  • humanized antibody is an antibody produced by grafting the complementarity determining region sequence of a mouse antibody molecule into a human antibody variable region framework.
  • antigen binding domain is part of an antibody comprising an amino acid residue that interacts with an antigen and which contributes to the specificity and affinity of the antibody for the antigen.
  • antigen binding domain for an antibody that specifically binds to its antigen, this will include at least a portion of at least one of its CDR domains.
  • epitope is a portion of a molecule that binds to an antibody (eg, by an antibody).
  • An epitope may comprise a non-contiguous portion of a molecule (eg, in a polypeptide, amino acid residues that are not contiguous in the primary sequence of the polypeptide are sufficiently close together in the tertiary and quaternary structure of the polypeptide that they are bound by one antibody) .
  • the "same percentage" of two polynucleotides or two polypeptide sequences was determined using the GAP computer program (GCG Wisconsin Package; version 10.3 (accelrys, San Diego, CA)) using its default parameter comparison sequence.
  • nucleic acid refers to DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), and nucleotides used. DNA or RNA analogs and hybrids thereof produced by analogs (eg, peptide nucleic acids and non-natural nucleotide analogs).
  • the nucleic acid molecule can be single stranded or double stranded.
  • a nucleic acid molecule herein comprises an open reading frame encoding a continuous antibody, or a fragment, derivative, mutein or variant thereof, provided herein.
  • Two single-stranded polynucleotides are "complementary" to each other if their sequences are arranged in anti-parallel, such that each nucleotide in one polynucleotide is complementary to another polynucleotide In contrast to nucleotides, no gaps are introduced and there are no unpaired nucleotides at the 5' or 3' end of each sequence.
  • a polynucleotide is "complementary" to another polynucleotide if it can hybridize to each other under moderately stringent conditions. Thus, one polynucleotide may be complementary to another polynucleotide, but not its complement.
  • vector is a nucleic acid that can be used to introduce another nucleic acid to which it is linked into a cell.
  • plasmid which refers to a linear or circular double stranded DNA molecule to which additional nucleic acid segments can be ligated.
  • viral vector e.g., replication defective retrovirus, adenovirus, and adenovirus companion virus
  • certain vectors can be autonomously replicated in the host cell into which they are introduced (e.g., bacterial vectors comprising bacterial origins of replication as well as episomal mammalian vectors).
  • vectors when introduced into a host cell, integrate into the genome of the host cell and are therefore replicated along with the host genome.
  • An "expression vector” is a type of vector that directs expression of a selected polynucleotide.
  • a nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression of the nucleotide sequence (eg, expression level, time or site).
  • a "regulatory sequence” is a nucleic acid that can affect the expression (eg, expression level, time or locus) of a nucleic acid to which it is operably linked.
  • a regulatory gene for example, acts directly on a regulated nucleic acid or through one or more other molecules (eg, a polynucleotide that binds to a regulatory sequence and/or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (eg, polyadenylation signals).
  • the term "host cell” is a cell used to express a nucleic acid, such as a nucleic acid provided herein.
  • the host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a single cell eukaryote (eg, yeast or other fungus), a plant cell (eg, a tobacco or tomato plant cell), an animal cell (eg, Human cells, monkey cells, hamster cells, rat cells, mouse cells or insect cells) or hybridomas.
  • the host cell is a cultured cell transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in a host cell.
  • the phrase "recombinant host cell” can be used to describe a host cell transformed or transfected with a nucleic acid that is expected to be expressed.
  • a host cell can also be a cell that comprises the nucleic acid but is not expressed at a desired level unless a regulatory sequence is introduced into the host cell such that it is operably linked to the nucleic acid. It is to be understood that the term host cell refers not only to a particular subject cell but also to the progeny or progeny of that cell. Since certain modifications may occur to subsequent generations, such as mutations or environmental influences, the progeny may in fact be different from the parent cell but still fall within the scope of the term as used herein.
  • the glucagon receptor belongs to the seven-transmembrane G protein-coupled receptor family of type B, which is linked to one or more intracellular signaling via a heterotrimeric guanine nucleotide binding protein (G protein).
  • G protein heterotrimeric guanine nucleotide binding protein
  • an antibody provided herein is an antibody that specifically binds to human GCGR. In another embodiment, an antibody provided herein is an antibody that specifically binds to GCGR on a cell membrane and that inhibits or blocks the transmission of glucagon signaling within such cells. In another embodiment, an antibody provided herein is an antibody that specifically binds to human GCGR, and the antibody binds to GCGR of other species (eg, monkey or mouse) and blocks glucagon in these species Signaling. In a further embodiment, the antibody provided herein is a murine antibody that binds to human GCGR and is capable of binding to GCGR of other species (eg, monkey).
  • amino acid and polynucleotide sequences of the GCGR are listed below, and the sequence data is derived from the GeneBank database of the National Center for Biotechnology Information and the Uniprot database of the European Bioinformatics Institute:
  • Rhesus macaque polynucleotide (SEQ ID NO: 78); accession number: XM_015120592;
  • Rhesus macaque amino acid (SEQ ID NO: 74); accession number: A0A1D5QZY8;
  • Rat (Rattus norvegicus) polynucleotide (SEQ ID NO: 79); accession number: X68692;
  • Rat (Rattus norvegicus) amino acid SEQ ID NO: 75; accession number: P30082;
  • Glucagon receptor antibody (GCGR antibody)
  • a GCGR antibody In one embodiment, provided herein is a GCGR antibody. In another embodiment, the GCGR antibody provided herein is a complete GCGR antibody. In another embodiment, a GCGR antibody provided herein is a GCGR antibody fragment. In another embodiment, the GCGR antibody provided herein is a GCGR antibody derivative. In another embodiment, the GCGR antibody provided herein is a GCGR antibody mutant protein. In a further embodiment, the GCGR antibody provided herein is a GCGR antibody variant.
  • a GCGR antibody provided herein comprises one, two, three, four, five, or six amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • heavy chain CDR1 amino acid sequence SEQ ID NO: 22, SEQ ID NO: 25, and SEQ ID NO: 28, SEQ ID NO: 31, and SEQ ID NO: 34;
  • heavy chain CDR2 amino acid sequence SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, and SEQ ID NO: 35;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36.
  • Table 1 lists the amino acid sequences of the light chain CDRs of the GCGR antibodies provided herein, as well as their corresponding polynucleotide coding sequences.
  • Table 2 lists the amino acid sequences of the heavy chain CDRs of the GCGR antibodies provided herein, as well as their corresponding polynucleotide coding sequences.
  • the antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences set forth in Tables 1 and 2 by 5, 4, 3, 2, or 1 single amino acid additions, substitutions, and/or deletions. .
  • an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences set forth in Tables 1 and 2 by 4, 3, 2, or 1 single amino acid additions, substitutions, and/or deletions.
  • the antibodies provided herein comprise sequences that differ by one, two, or one single amino acid additions, substitutions, and/or deletions from one of the CDR amino acid sequences set forth in Tables 1 and 2.
  • an antibody provided herein comprises a sequence that differs from one of the CDR amino acid sequences set forth in Tables 1 and 2 by two or one single amino acid additions, substitutions, and/or deletions.
  • the antibodies provided herein comprise a sequence that differs from one of the CDR amino acid sequences set forth in Tables 1 and 2 by a single amino acid addition, substitution, and/or deletion.
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Heavy chain CDR2 amino acid sequences SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, and SEQ ID NO:35.
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Heavy chain CDR3 amino acid sequence SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36.
  • a GCGR antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Heavy chain CDR2 amino acid sequences SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, SEQ ID NO:32, and SEQ ID NO:35.
  • a GCGR antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Heavy chain CDR3 amino acid sequences SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36.
  • a GCGR antibody provided herein comprises one, two, three, or four amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Heavy chain CDR3 amino acid sequences SEQ ID NO:24, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:33, and SEQ ID NO:36.
  • a GCGR antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below: SEQ ID NO: 1, SEQ ID NO: 2. SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.
  • a GCGR antibody provided herein comprises one, two, or three amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below: SEQ ID NO: 22, SEQ ID NO :23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31 SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36.
  • a GCGR antibody provided herein comprises a combination of a light chain and a heavy chain CDR1 amino acid sequence independently selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 22, SEQ ID NO: 4 And SEQ ID NO: 25, SEQ ID NO: 7 and SEQ ID NO: 28, SEQ ID NO: 10 and SEQ ID NO: 31, SEQ ID NO: 13 and SEQ ID NO: 34, SEQ ID NO: 16 and SEQ ID NO: 34, SEQ ID NO: 18 and SEQ ID NO: 34, and SEQ ID NO: 20 and SEQ ID NO: 34.
  • a GCGR antibody provided herein comprises a combination of a light chain and a heavy chain CDR2 amino acid sequence independently selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 23, SEQ ID NO: 5 and SEQ ID NO: 26, SEQ ID NO: 8 and SEQ ID NO: 29, SEQ ID NO: 11 and SEQ ID NO: 32, and SEQ ID NO: 14 and SEQ ID NO: 35.
  • a GCGR antibody provided herein comprises a combination of a light chain and a heavy chain CDR3 amino acid sequence independently selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 24, SEQ ID NO: 6 with SEQ ID NO:27, SEQ ID NO:9 and SEQ ID NO:30, SEQ ID NO:12 and SEQ ID NO:33, SEQ ID NO:15 and SEQ ID NO:36, SEQ ID NO:17 SEQ ID NO: 36, SEQ ID NO: 19 and SEQ ID NO: 36, and SEQ ID NO: 21 and SEQ ID NO: 36.
  • GCGR antibodies provided herein comprise:
  • GCGR antibodies provided herein comprise:
  • GCGR antibodies provided herein comprise:
  • GCGR antibodies provided herein comprise:
  • a combination of the light chain and heavy chain CDR3 amino acid sequences independently selected from the group consisting of SEQ ID NO: 3 and SEQ ID NO: 24, SEQ ID NO: 6 and SEQ ID NO: 27, SEQ ID NO :9 and SEQ ID NO:30, SEQ ID NO:12 and SEQ ID NO:33, SEQ ID NO:15 and SEQ ID NO:36, SEQ ID NO:17 and SEQ ID NO:36, SEQ ID NO:19 And SEQ ID NO: 36, and SEQ ID NO: 21 and SEQ ID NO: 36.
  • GCGR antibodies provided herein comprise:
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences set forth below:
  • Light chain variable domain amino acid sequence SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO : 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90; and an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to any of its sequences;
  • Heavy chain variable domain amino acid sequence SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, and SEQ ID NO: 97; and an amino acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to any of its sequences.
  • a polynucleotide coding sequence for a GCGR antibody comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the following Column polynucleotide sequence:
  • Light chain variable domain polynucleotide coding sequence SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO :103, and SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, and SEQ ID NO: 107; and at least 80%, at least 85%, at least 90%, or at least 95 with any of its sequences % identical polynucleotide coding sequences; and
  • a GCGR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, and SEQ ID NO: 90.
  • a GCGR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO:95, SEQ ID NO:96, and SEQ ID NO:97.
  • a GCGR antibody provided herein comprises a combination of a light chain and heavy chain variable domain amino acid sequence independently selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 91, SEQ ID NO: 82 and SEQ ID NO: 92, SEQ ID NO: 83 and SEQ ID NO: 93, SEQ ID NO: 84 and SEQ ID NO: 94, SEQ ID NO: 85 and SEQ ID NO: 95, SEQ ID NO: 86 with SEQ ID NO: 96, SEQ ID NO: 87 and SEQ ID NO: 97, SEQ ID NO: 88 and SEQ ID NO: 97, SEQ ID NO: 89 and SEQ ID NO: 97, and SEQ ID NO: 90 And SEQ ID NO:97.
  • a GCGR antibody provided herein comprises an amino acid sequence independently selected from the group consisting of SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO :96, and SEQ ID NO:97.
  • a GCGR antibody provided herein comprises a combination of a light chain and heavy chain variable domain amino acid sequence independently selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 91 (L1H1) ), SEQ ID NO: 82 and SEQ ID NO: 92 (L2H2), SEQ ID NO: 84 and SEQ ID NO: 94 (L4H4), SEQ ID NO: 85 and SEQ ID NO: 95 (L5H5), SEQ ID NO :86 with SEQ ID NO:96 (L6H6), SEQ ID NO:87 and SEQ ID NO:97 (L7H7), SEQ ID NO:88 and SEQ ID NO:97 (L8H7), and SEQ ID NO:89 and SEQ ID NO: 97 (L9H7).
  • L2H2 refers to an intact antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 82 (L2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 92 (H2).
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of the light chain and heavy chain constant amino acid sequences set forth below: SEQ ID NO: 115 and SEQ ID NO:117, SEQ ID NO:115 and SEQ ID NO:118, SEQ ID NO:116 and SEQ ID NO:117, and SEQ ID NO:116 and SEQ ID NO:118.
  • a GCGR antibody provided herein comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from the group consisting of the light chain and heavy chain constant amino acid sequences set forth below: SEQ ID NO: 115 And SEQ ID NO: 117, SEQ ID NO: 115 and SEQ ID NO: 118, SEQ ID NO: 116 and SEQ ID NO: 117, SEQ ID NO: 116 and SEQ ID NO: 118, SEQ ID NO: 132 and SEQ ID NO: 117, SEQ ID NO: 132 and SEQ ID NO: 118, SEQ ID NO: 133 and SEQ ID NO: 117, and SEQ ID NO: 133 and SEQ ID NO: 118.
  • a GCGR antibody provided herein comprises the light and heavy chain CDRs set forth herein, as well as the amino acid sequence of the FRs (framework).
  • the amino acid sequence of the FRs is contained in the light chain or heavy chain variable domain amino acid sequence and is not separately displayed.
  • the antibody comprises a light chain CDR1 sequence set forth herein.
  • the antibody comprises a light chain CDR2 sequence set forth herein.
  • the antibody comprises a light chain CDR3 sequence set forth herein.
  • the antibody comprises a heavy chain CDR1 sequence set forth herein.
  • the antibody comprises a heavy chain CDR2 sequence set forth herein.
  • the antibody comprises a heavy chain CDR3 sequence set forth herein.
  • the antibody comprises a light chain FR1 sequence herein. In another embodiment, the antibody comprises a light chain FR2 sequence herein. In another embodiment, the antibody comprises a light chain FR3 sequence herein. In another embodiment, the antibody comprises a light chain FR4 sequence herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence herein.
  • the light chain CDR3 sequence of the antibody and the light chain CDR3 amino acid sequences set forth herein are SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID NO One of 21: the difference must not exceed 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution and/or deletion.
  • the heavy chain CDR3 sequence of the antibody differs from the heavy chain CDR3 amino acid sequence set forth herein as SEQ ID NO: 33 or SEQ ID NO: 36 by no more than 6, 5, 4, 3, 2, or 1 Add, replace, and/or delete single amino acids.
  • the light chain CDR3 sequence of the antibody and the light chain CDR3 amino acid sequences set forth herein are SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, and SEQ ID One of the NO:21 phases must not differ by more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and/or deletion
  • the heavy chain CDR3 sequence of the antibody and the heavy chain CDR3 amino acid sequence set forth herein are SEQ ID NO: 33 or SEQ ID NO: 36 differs by no more than 6, 5, 4, 3, 2, or 1 single amino acid addition, substitution, and/or deletion.
  • the antibody further comprises 1, 2, 3, 4, 5, or 6 light heavy chain CDR light heavy chain sequence combinations as set forth herein.
  • a GCGR antibody provided herein comprises a light chain variable domain amino acid sequence selected from L1 (SEQ ID NO: 81), L2 (SEQ ID NO: 82), L4 (listed herein).
  • SEQ ID NO: 84 L5 (SEQ ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • L1 SEQ ID NO: 81
  • L2 SEQ ID NO: 82
  • L4 listed herein.
  • SEQ ID NO: 84 L5 (SEQ ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • L1 SEQ ID NO: 81
  • L2 SEQ ID NO: 82
  • L4 listed herein.
  • SEQ ID NO: 84 L5 (SEQ ID NO
  • the light chain variable domain amino acid sequence of the GCGR antibody is linked to L1 (SEQ ID NO:81), L2 (SEQ ID NO:82), L4 (SEQ ID NO:84), L5 (SEQ ID Light chain variable domain of one of NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • the amino acid sequence differs by 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, wherein the difference in each sequence is independently an amino acid residue. Missing, inserting or replacing.
  • the light chain variable domain amino acid sequence of the GCGR antibody is linked to L1 (SEQ ID NO:81), L2 (SEQ ID NO:82), L4 (SEQ ID NO:84), L5 (SEQ Light chain variable structure of one of ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • the domain amino acid sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the light chain variable domain polynucleotide coding sequence of the GCGR antibody is linked to L1 (SEQ ID NO:81), L2 (SEQ ID NO:82), L4 (SEQ ID NO:84) , L5 (SEQ ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • the polynucleotide coding sequence is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the light chain variable domain polynucleotide coding sequence of the GCGR antibody comprises under moderate conditions with L1 (SEQ ID NO: 81), L2 (SEQ ID NO: 82), L4 ( SEQ ID NO: 84), L5 (SEQ ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • L1 SEQ ID NO: 81
  • L2 SEQ ID NO: 82
  • L4 SEQ ID NO: 84
  • L5 SEQ ID NO: 85
  • L6 SEQ ID NO: 86
  • L7 SEQ ID NO: 87
  • L8 SEQ ID NO: 88
  • L9 SEQ ID NO: 89
  • the light chain variable domain polynucleotide coding sequence of the GCGR antibody comprises under stringent conditions with L1 (SEQ ID NO: 81), L2 (SEQ ID NO: 82), L4 ( SEQ ID NO: 84), L5 (SEQ ID NO: 85), L6 (SEQ ID NO: 86), L7 (SEQ ID NO: 87), L8 (SEQ ID NO: 88), and L9 (SEQ ID NO: 89)
  • L1 SEQ ID NO: 81
  • L2 SEQ ID NO: 82
  • L4 SEQ ID NO: 84
  • L5 SEQ ID NO: 85
  • L6 SEQ ID NO: 86
  • L7 SEQ ID NO: 87
  • L8 SEQ ID NO: 88
  • L9 SEQ ID NO: 89
  • a GCGR antibody provided herein comprises a heavy chain variable domain amino acid sequence selected from H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 (SEQ) ID NO: 94), H5 (SEQ ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97) heavy chain variable domain sequences.
  • the heavy chain variable domain amino acid sequence of the GCGR antibody is H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 (SEQ ID NO: 94), H5 (SEQ.
  • the heavy chain variable domain sequences of one of ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97) are present at 15, 14, 13, 12, 11, 10, 9, and 8. , 7, 6, 5, 4, 3, 2, or 1 amino acid difference, wherein the difference in each sequence is independently the deletion, insertion or substitution of one amino acid residue.
  • the heavy chain variable domain amino acid sequence of the GCGR antibody is H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 (SEQ ID NO: 94), H5 (SEQ.
  • the heavy chain variable domain sequence of one of ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97) has at least 70%, at least 75%, at least 80%, at least 85% At least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the heavy chain variable domain polynucleotide coding sequence of the GCGR antibody is H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 (SEQ ID NO: 94) a heavy chain variable domain polynucleotide coding sequence of at least 70% of H5 (SEQ ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97), At least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical.
  • the heavy chain variable domain polynucleotide coding sequence of the GCGR antibody comprises H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 under moderate stringency conditions Polynucleotide of the heavy chain variable domain of one of (SEQ ID NO: 94), H5 (SEQ ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97) A polynucleotide sequence that hybridizes to a sequence complementary sequence.
  • the heavy chain variable domain polynucleotide coding sequence of the GCGR antibody comprises H1 (SEQ ID NO: 91), H2 (SEQ ID NO: 92), H4 (SEQ) under stringent conditions
  • Polynucleotide coding sequence of heavy chain variable domain of ID NO: 94), H5 (SEQ ID NO: 95), H6 (SEQ ID NO: 96), and H7 (SEQ ID NO: 97) A polynucleotide sequence in which a complementary sequence hybridizes.
  • the antibodies provided herein are one comprising L1H1 (SEQ ID NO: 81 and SEQ ID NO: 91), L2H2 (SEQ ID NO: 82 and SEQ ID NO: 92), L3H3 (SEQ ID NO: 83) And SEQ ID NO: 93), L4H4 (SEQ ID NO: 84 and SEQ ID NO: 94), L5H5 (SEQ ID NO: 85 and SEQ ID NO: 95), L6H6 (SEQ ID NO: 86 and SEQ ID NO: 96), L7H7 (SEQ ID NO: 87 and SEQ ID NO: 97), L8H7 (SEQ ID NO: 88 and SEQ ID NO: 97), L9H7 (SEQ ID NO: 89 and SEQ ID NO: 97), or L10H7 An antibody (SEQ ID NO: 90 and SEQ ID NO: 97), or a desired phenotype thereof (eg, IgA, IgG1, IgG2a, IgG2
  • the antibodies provided herein are one comprising L1H1 (SEQ ID NO: 81 and SEQ ID NO: 91), L2H2 (SEQ ID NO: 82 and SEQ ID NO: 92), L4H4 (SEQ ID NO: 84) And SEQ ID NO: 94), L5H5 (SEQ ID NO: 85 and SEQ ID NO: 95), L6H6 (SEQ ID NO: 86 and SEQ ID NO: 96), L7H7 (SEQ ID NO: 87 and SEQ ID NO: 97), an antibody that binds L8H6 (SEQ ID NO: 88 to SEQ ID NO: 97) or L9H7 (SEQ ID NO: 89 to SEQ ID NO: 97), or a class of such converted antibodies (eg, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, and IgD), or a Fab or F(ab')2 fragment thereof.
  • L1H1
  • the antibodies provided herein can comprise any of the constant regions known in the art.
  • the light chain constant region can be, for example, a kappa or lambda type light chain constant region, such as a mouse kappa or lambda light chain constant region.
  • the heavy chain constant region can be, for example, an alpha, delta, epsilon, gamma, or mu-type heavy chain constant region, such as a mouse alpha, delta, epsilon, gamma, or mu-type heavy chain constant region.
  • the light or heavy chain constant region is a fragment, derivative, variant, or mutein of the native constant region.
  • an antibody provided herein further comprises a human constant light chain kappa or lambda domain or a fragment thereof.
  • the amino acid sequence of the constant region of the light chain is as follows:
  • an antibody provided herein further comprises a human constant light chain kappa or lambda domain or a fragment thereof.
  • the amino acid sequence of the constant region of the light chain is as follows:
  • an antibody provided herein further comprises a human heavy chain constant domain or a fragment thereof.
  • the amino acid sequence sequence of the heavy chain constant region is as follows:
  • Human heavy chain constant region amino acid sequence (hIgG2): (SEQ ID NO: 117);
  • Human heavy chain constant region amino acid sequence (hIgG4): (SEQ ID NO: 118).
  • the GCGR antibody provided herein is selected from the group consisting of a murine antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a single-chain antibody, a double-chain antibody , a triple-chain antibody, a four-chain antibody, a Fab fragment, an F(ab')x fragment, a domain antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody.
  • the GCGR antibody provided herein is a GCGR monoclonal antibody.
  • a GCGR antibody provided herein is a monoclonal antibody comprising a combination of amino acid sequences selected from the group consisting of SEQ ID NO: 81 and SEQ ID NO: 91, SEQ ID NO: 82 and SEQ ID NO: 92, SEQ ID NO: 83 and SEQ ID NO: 93, SEQ ID NO: 84 and SEQ ID NO: 94, SEQ ID NO: 85 and SEQ ID NO: 95, SEQ ID NO: 86 with SEQ ID NO: 96, SEQ ID NO: 87 and SEQ ID NO: 97, SEQ ID NO: 88 and SEQ ID NO: 97, SEQ ID NO: 89 and SEQ ID NO: 97, and SEQ ID NO: 90 And SEQ ID NO:97.
  • the GCGR antibody provided herein is a murine GCGR antibody. In another embodiment, the GCGR antibody provided herein is a humanized GCGR antibody.
  • GCGR human antibodies provided herein reduce signaling IC 50 value of about 1nM to about 300nM or from about 1nM to about 150nM.
  • the antibodies provided herein are intact antibodies (including polyclonal, monoclonal, chimeric, humanized, or human antibodies having full length heavy and/or light chains).
  • the antibodies provided herein are antibody fragments, such as F(ab') 2 , Fab, Fab', Fv, Fc, or Fd fragments, single domain antibodies, single chain antibodies, maximal antibodies (maxibodies) ), minibodies, intrabodies, di-chain antibodies, tri-chain antibodies, tetra-chain antibodies, v-NAR, or bis-scFv (see, eg, Hollinger and Hudson, 2005, Nature Biotechnology 23: 1126- 1136).
  • the antibodies provided herein include antibody polypeptides as disclosed in U.S. Patent No. 6,703,199, including fibronectin polypeptide single antibodies.
  • the antibodies provided herein include single-chain polypeptides as disclosed in U.S. Patent Publication No. 2005/0238646.
  • variable region of the gene of the monoclonal antibody is expressed in a hybridoma using nucleotide primer amplification.
  • These primers can be synthesized by one of ordinary skill in the art or purchased from commercial sources.
  • Murine and human variable region primer comprises a V Ha, V Hb, V Hc , V Hd, primer C H1, V L, and C L regions may be purchased from commercial sources.
  • These primers can be used to amplify heavy or light chain variable regions are then inserted into vectors such IMMUNOZAP TM H or IMMUNOZAP TM L (Stratagene) in. These vectors are then introduced into an E. coli, yeast or mammalian based expression system. These methods can be used for producing large quantities comprising V H and V L domains of a single chain fusion protein (see Bird et al., 1988, Science 242: 423-426) .
  • proteins may undergo a variety of post-transcriptional modifications.
  • the type and extent of these modifications will depend on the host cell line used to express the protein as well as the culture conditions.
  • modifications include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartic acid isomerization, and asparagine deamidation.
  • the carboxy terminal basic residue of the antibody e.g., lysine or arginine
  • Murine monoclonal antibodies can be produced using conventional hybridoma cell methods.
  • the monoclonal antibody can be isolated and purified by a variety of established techniques. Such separation techniques include affinity chromatography of Protein A-Sepharose, size exclusion chromatography, and ion exchange chromatography (see, for example, Coligan, pages 2.7.1-2.7.12 and 2.9.1-2.9. 3 pages; Baines et al., "Purification of Immunoglobulin G (IgG),” Methods in Molecular Biology, Vol. 10, pp. 79-104 (The Humana Press, Inc., 1992)).
  • the monoclonal antibody can be purified by affinity chromatography using a suitable ligand selected based on the specific properties of the antibody (eg, heavy or light chain isoform, binding specificity, etc.).
  • suitable ligands for affinity chromatography include protein A, protein G, anti-constant (light or heavy chain) antibodies, anti-idiotypic antibodies, and TGF-beta binding proteins or fragments or variants thereof.
  • Modification of affinity maturation of the molecule can be performed using complementarity determining regions (CDRs) at the center of the antibody binding site to obtain antibodies with increased affinity, such as antibodies with increased affinity for c-erbB-2 (Schier et al, 1996, J. Mol. Biol. 263: 551-567). Therefore, this type of technology can be used to prepare antibodies to human GCGR.
  • CDRs complementarity determining regions
  • antibodies against human GCGR can be used in an in vitro or in vivo assay to detect the presence or absence of human GCGR.
  • Antibodies can also be prepared by any conventional technique. For example, cells that naturally express these antibodies can be purified (e.g., can be purified from antibody producing hybridomas) or produced in recombinant expression systems using any technique known in the art. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, edited by Kennet et al, Plenum Press (1980); and Antibodies: A Laboratory Manual, edited by Harlow and Land, Cold Spring Harbor Laboratory Press (1988). This is discussed in the Nucleic Acids section below.
  • Antibodies can be prepared by any known technique and screened for the desired properties. Some techniques involve isolating a nucleic acid encoding a polypeptide chain (or a portion thereof) of a related antibody (eg, an anti-GCGR antibody) and operating the nucleic acid by recombinant DNA techniques.
  • the nucleic acid can be fused or modified (eg, by mutagenesis or other conventional techniques) with another related nucleic acid to add, delete or replace one or more amino acid residues.
  • CDRs When it is desired to increase the affinity of an antibody according to one or more of the above CDRs herein, maintenance of the CDRs can be accomplished by a variety of affinity maturation protocols (Yang et al, 1995, J. Mol. Biol. 254: 392-403), Strand replacement (Marks et al, 1992, Bio/Technology 10: 779-783), DNA rearrangement using a mutant strain of E. coli (Low et al, 1996, J. Mol. Biol. 250: 350-368) (Patten et al, 1997) , Curr. Opin. Biotechnol. 8: 724-733), phage display (Thompson et al, 1996, J. Mol. Biol.
  • the antibodies provided herein are anti-GCGR fragments.
  • the fragment may consist entirely of antibody-derived sequences or may comprise additional sequences.
  • antigen-binding fragments include Fab, F(ab')2, single-chain antibodies, di-chain antibodies, tri-chain antibodies, tetra-chain antibodies, and domain antibodies, and other examples are provided in Lunde et al., 2002, Biochem. Soc. Trans. 30:500-06.
  • a single-chain antibody can be formed by linking an amino acid bridge (short peptide linker) to a heavy chain and a light chain variable domain (Fv region) to obtain a single polypeptide chain.
  • V L and V H two coding variable domain polypeptides
  • the resulting polypeptides can be folded back into themselves to form antigen binding monomers, or they can form multimers (eg, dimers, trimers, or tetramers), depending on the length of the flexible linker between the two variable domains.
  • scFvs binding member may be formed with a plurality of different phenotypes (Kriangkum et, 2001, Biomol.Eng.18: 31-40).
  • Techniques developed for the production of single chain antibodies include U.S. Patent No. 4,946,778; Bird, 1988, Science 242: 423; Huston et al, 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883; Ward et al, 1989 , Nature 334: 544-546; de Graaf et al, 2002, Methods Mol Biol. 178: 379-87.
  • Single-chain antibodies derived from the antibodies provided herein include, but are not limited to, scFvs comprising the variable domain combination L1H1, all of which are encompassed herein.
  • Antibody-derived antigen-binding fragments can also be obtained by proteolysis of antibodies, for example, by digestion of intact antibodies with pepsin or papain according to conventional methods.
  • an antibody fragment called F(ab')2 can be produced by a pepsinase cleavage antibody to produce an antibody fragment. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment.
  • the cleavage reaction can be carried out using a thiol protecting group to obtain cleavage of a disulfide bond; in addition, enzymatic cleavage of papain can be used to directly produce two monovalent Fab fragments and one Fc fragment.
  • lysing antibodies such as preparing heavy chains to form monovalent heavy, light chain fragments (Fd), further cleavage fragments or other enzyme, chemical or genetic techniques can be used as long as the fragments bind to antigens that are recognized by the intact antibody.
  • Fd light chain fragments
  • Other methods of lysing antibodies such as preparing heavy chains to form monovalent heavy, light chain fragments (Fd), further cleavage fragments or other enzyme, chemical or genetic techniques can be used as long as the fragments bind to antigens that are recognized by the intact antibody.
  • CDRs can be obtained by constructing a polypeptide encoding a related CDR.
  • Such polypeptides can be prepared, for example, by polymerase chain reaction using mRNA of antibody-producing cells as a template to synthesize variable regions, see, for example, Larrick et al., 1991, Methods: A Companion to Methods in Enzymology 2:106; Courtenay-Luck , "Genetic Manipulation of Monoclonal Antibodies,” Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al., ed., 166 (Cambridge University Press, 1995); and Ward et al, "Genetic Manipulation and Expression of Antibodies,” Monoclonal Antibodies: Principles And Applications, Birch et al., ed., 137 (Wiley-Liss, Inc., 1995).
  • the antibody fragment can further comprise at least one variable domain of an antibody described herein.
  • V region domain may be monomeric and a V H or V L domain, which may be as follows at least equal to 1x 10 -7 M or higher affinity binding independent of GCGR hereinbefore described.
  • variable region domain can be any naturally variable domain or a genetically engineered form thereof.
  • Genetically engineered forms refer to variable region domains produced using recombinant DNA engineering techniques.
  • the genetically engineered form includes, for example, production from a specific antibody variable region by insertion, deletion or alteration of an amino acid sequence of a specific antibody.
  • Specific examples include variable region domains comprising one or more framework amino acids comprising only one CDR and optionally from one antibody and the remainder of the variable region domain from another antibody, and assembled by genetic engineering.
  • variable region domain can be covalently linked to at least one other antibody domain or fragment thereof at the C-terminal amino acid.
  • V H region domain may be linked to an immunoglobulin C H1 domain or fragment thereof.
  • V L domain may be attached to C K domain or fragment thereof.
  • the antibody may be a Fab fragment wherein the antigen binding domain comprises a V H and V L, United domains thereof are connected to the C-terminus and C K C Hl domain covalently.
  • Other amino acids can be used to extend the C H1 domain, for example to provide a hinge region or as Fab 'portions of the hinge domain fragment or provide other domains, such as an antibody C H2 and C H3 domains.
  • the nucleotide sequence encoding the amino acid sequences L1 and H1 can be altered by random mutagenesis or by site-directed mutagenesis (eg, oligonucleotide-induced site-directed mutagenesis) to produce an unmutated polynucleotide compared to an unmutated polynucleotide.
  • site-directed mutagenesis eg, oligonucleotide-induced site-directed mutagenesis
  • An altered polynucleotide comprising one or more specific nucleotide substitutions, deletions or insertions.
  • anti-GCGR antibody derivatives in the art include covalent or aggregated bindings of an anti-GCGR antibody or fragment thereof to its protein or polypeptide, for example by expression of a heterologous polypeptide comprising a fusion to the N-terminus or C-terminus of an anti-GCGR antibody polypeptide.
  • Recombinant fusion protein can be a heterologous signal (or leader) polypeptide, such as a yeast alpha factor leader peptide or a peptide such as an epitope tag.
  • An antibody comprising a fusion protein can comprise a peptide (eg, a polyhistidine) that is added to aid in the purification or identification of the antibody.
  • Antibodies can also be linked to FLAG peptides as described in Hopp et al, 1988, Bio/Technology 6: 1204 and U.S. Patent 5,011,912.
  • the FLAG peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing rapid detection and convenient purification of the expressed recombinant protein.
  • mAb monoclonal antibody
  • oligomers comprising one or more antibodies are available As a GCGR antagonist or with a higher order oligomer.
  • the oligomer may be in the form of a dimer, trimer or higher oligomer that is covalently linked or non-covalently linked. Oligospecifics comprising two or more antibodies can be used, one of which is a homodimer. Other oligomers include heterodimers, homotrimers, heterotrimers, homotetramers, heterotetramers, and the like.
  • One embodiment is directed to oligomers comprising a plurality of antibodies linked by covalent or non-covalent interactions between peptide moieties fused to the antibody.
  • Such peptides may be peptide spacers or peptides having properties that promote oligomerization.
  • Leucine zippers and certain antibody-derived polypeptides are peptides that promote oligomerization of antibodies, as described in detail below.
  • the oligomer comprises two to four antibodies.
  • the antibody to the oligomer can be in any form, such as any of the forms described above, such as variants or fragments.
  • the oligomer comprises an antibody having GCGR binding activity.
  • the oligomer is prepared using a polypeptide derived from an immunoglobulin.
  • a polypeptide derived from an immunoglobulin Preparation of heterologous polypeptides comprising fusions to different portions of antibody-derived polypeptides, including Fc domains, has been described, for example, in Ashkenazi et al, 1991, PNAS USA 88: 10535; Byrn et al, 1990, Nature 344: 677; and Hollenbaugh et al. Construction of Immunoglobulin Fusion Proteins, Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11.
  • One embodiment herein is directed to a dimer comprising a fusion protein produced by fusion of a glucagon binding fragment of an anti-GCGR antibody with an Fc region of an antibody.
  • a dimer can be prepared by, for example, inserting a gene fusion encoding a fusion protein into a suitable expression vector, expressing the fusion gene in a host cell transformed with the recombinant expression vector, and allowing the expressed fusion protein to be assembled like an antibody molecule, The interchain disulfide bond between the Fc moieties forms a dimer.
  • Fc polypeptide includes polypeptides derived from the native and mutein forms of the Fc region of an antibody. Also included are truncated forms of such polypeptides comprising a hinge region that promotes dimerization. Fusion proteins comprising an Fc portion (and oligomers formed therefrom) provide the advantage of convenient purification by affinity chromatography on a Protein A or Protein G column.
  • Fc polypeptide in PCT application WO 93/10151 is a native C-terminal single-chain polypeptide that extends from the N-terminal hinge region to the Fc region of a human IgGl antibody.
  • Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,545, 705 and Baum et al., 1994, EMBO J. 13: 3992-4001.
  • the amino acid sequence of the mutein is identical to the amino acid sequence of the native Fc sequence shown in WO 93/10151 except that amino acid 19 is changed from leucine to alanine, amino acid 20 is changed from leucine to glutamine, and amino acid 22 is derived from Glycine becomes alanine.
  • This mutant protein exhibits a decrease in affinity for the Fc receptor.
  • the heavy and/or light chains of an anti-GCGR antibody can be substituted with a variable portion of an antibody heavy and/or light chain.
  • the oligomer is a fusion protein comprising a plurality of antibodies, with or without spacer peptides.
  • linker peptides are described in U.S. Patents 4,751,180 and 4,935,233.
  • Leucine zipper domains are peptides that promote oligomerization of proteins in which they are present. Leucine zippers were originally found to be present in several DNA binding proteins (Landschulz et al, 1988, Science 240: 1759) and have since been found to be present in a variety of different proteins. A natural peptide or a derivative thereof which is dimerizable or trimerizable in a known leucine zipper.
  • leucine zipper domain suitable for the production of soluble oligomeric proteins is described in PCT application WO 94/10308, a leucine zipper derived from pulmonary surfactant protein D (SPD) as described in Hoppe Zheng, 1994, FEBS Letters 344:191, in the form of a reference.
  • SPD pulmonary surfactant protein D
  • the use of a modified leucine zipper that permits heterotrimeric stabilization of a heterologous protein fused thereto is described in Fanslow et al, 1994, Semin. Immunol. 6:267-78.
  • a recombinant fusion protein comprising an anti-GCGR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and a soluble oligomeric anti-GCGR antibody fragment is collected from the culture supernatant or Its derivatives.
  • the antibody derivative can comprise at least one of the CDRs disclosed herein.
  • one or more CDRs can be integrated into a known antibody framework region (IgGl, IgG2, etc.) or combined with a suitable vector to enhance its half-life.
  • Suitable carriers include, but are not limited to, Fc, albumin, transfer iron, and the like. These and other suitable carriers are known in the art.
  • the binding CDR peptide can be monomeric, dimeric, tetrameric or other forms.
  • one or more water soluble polymers bind at one or more specific sites of the binding agent, for example at the amino terminus.
  • the antibody derivative comprises one or more water soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol.
  • the derivatives comprise one or more monomethoxy.polyethylene glycol, dextran, cellulose or other Carbohydrate-based polymers, poly(N-vinylpyrrolidone).
  • one or more water soluble polymers are randomly associated with one or more side chains.
  • PEG can increase the therapeutic effect of a binding agent such as an antibody.
  • the antibodies provided herein may have at least one amino acid substitution as long as the antibody retains binding specificity. Therefore, modifications of the antibody structure are included in the context of this document. These may include amino acid substitutions that do not disrupt the ability of the antibody to bind to GCGR, which may be conservative or non-conservative. Conservative amino acid substitutions can include non-natural amino acid residues, which are typically synthesized by chemical peptide synthesis rather than biological systems. These include peptidomimetics and other amino acid moieties in reverse or inverted form. Conservative amino acid substitutions may also involve replacing a natural amino acid residue with a non-native residue such that the polarity or charge effect of the amino acid residue at the site has little or no effect. Non-conservative substitutions may involve the exchange of one member of a class of amino acids or amino acid analogs with members of another class of amino acids having different physical properties (eg, volume, polarity, hydrophobicity, charge).
  • variants to be tested comprising an amino acid substitution at each desired amino acid residue.
  • Such variants can be screened using activity assays known to those skilled in the art.
  • Such variants can be used to gather information about appropriate variants. For example, variants having such changes can be avoided if an amino acid residue is found to cause activity disruption, undesired decrease or inappropriate activity.
  • one skilled in the art can readily determine that amino acids should be avoided by further substitution (alone or in combination with other mutations).
  • polypeptides can be determined suitable variants of the polypeptides as listed herein using known techniques.
  • one skilled in the art can identify suitable regions of the molecule that do not disrupt activity upon alteration by targeting regions that are not critical to activity.
  • residues or molecular moieties that are conserved in similar polypeptides can be identified.
  • one skilled in the art can examine structural. Functional studies to identify residues in similar polypeptides that are important for activity or structure. In view of this comparison, the importance of amino acid residues in proteins corresponding to amino acid residues important for activity or structure in similar proteins can be predicted.
  • One skilled in the art can select chemically similar amino acid substitutions for these predicted important amino acid residues.
  • One of skill in the art can also analyze three dimensional structures and amino acid sequences associated with the structure of similar polypeptides. In view of this type of information, one skilled in the art can predict amino acid residue alignments of antibodies in terms of three dimensional structure. In some embodiments, one skilled in the art can choose not to make significant changes to amino acid residues predicted to be on the surface of the protein, as such residues may be involved in important interactions with other molecules. Many scientific publications are dedicated to the prediction of secondary structures. See, Moult, 1996, Curr. Op. Biotech. 7: 422-427, Chou et al, 1974, Biochemistry 13: 222-245; Chou et al, 1974, Biochemistry 113: 211-222; Chou et al, 1978, Adv. Enzymol.
  • antibody variants include glycosylation variants, wherein the parent polypeptide The amino acid sequence changes the number and/or type of glycosylation sites compared to the native protein. In some embodiments, the variant has a greater or lesser number of N-linked glycosylation sites than the native protein. Substitution of this sequence removes existing N-linked sugar chains. It also provides rearrangement of N-linked sugar chains in which one or more N-linked sugar chain sites (usually those that occur naturally) are removed and created One or more new N-ligation sites.
  • Other preferred antibody variants include cysteine variants, The parent amino acid sequence is one or more cysteine residues replaced by another amino acid (eg, serine).
  • cyst When the antibody must be folded into a biologically active conformation (eg, after isolation of soluble inclusion bodies), the cyst can be used.
  • Cysteine variants typically have fewer cysteine residues than native proteins and usually have an even number of cysteines to minimize the interaction caused by unpaired cysteines.
  • amino acid substitutions can be used to identify important residues of a human GCGR antibody or to increase or decrease the affinity of a human GCGR antibody described herein.
  • preferred amino acid substitutions are as follows: (1) reduced proteolysis sensitivity, (2) reduced oxidation sensitivity, (3) altered binding affinity to form a protein complex, (4) altered binding affinity and/or (4) imparting or modifying other physicochemical or functional properties on such polypeptides.
  • single or multiple amino acid substitutions in some embodiments, conservative amino acid substitutions can be made in a naturally occurring sequence (in some embodiments, a portion of the polypeptide other than the domain that forms the intermolecular contacts) .
  • conservative amino acid substitutions generally do not substantially alter the structural properties of the parent sequence (eg, the replacement amino acid should not cleave the helix present in the parent sequence or interfere with other types of secondary structures that characterize the parent sequence).
  • Examples of secondary and tertiary structures of peptides recognized in the art are described in Proteins, Structures and Molecular Principles, edited by Creighton, WH Freeman and Company (1984); Introduction to Protein Structure, edited by Branden and Tooze, Garland Publishing (1991); Thornton et al, 1991, Nature 354: 105, which is incorporated herein by reference.
  • the antibodies provided herein can be chemically bonded to a polymer, lipid, or other moieties.
  • the antigen binding reagent can comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure.
  • the biocompatible backbone structure comprises a polypeptide or portion thereof sufficient to form a conformationally stable structural support or backbone or scaffold that can display one or more amino acid sequences that can bind to an antigen at a localized surface region (eg, , CDRs, variable regions, etc.).
  • Such structures may be "folded" (structural motifs) of a naturally occurring polypeptide or polypeptide, or may have one or more modifications, such as amino acid additions, deletions or substitutions, relative to a native polypeptide or fold.
  • These scaffolds can be derived from polypeptides of any species (or more than one species), for example, humans, other mammals, other vertebrates, invertebrates, bacteria or viruses.
  • Biosoluble backbone structures are typically based on protein scaffolds or backbones rather than immunoglobulin domains. For example, based on fibronectin, ankyrin, lipocalin, neo-oncoprotein, cytochrome b, CP1 zinc finger protein, PST1, coiled coil, LACI-D1, Z domain and starch Enzyme aprotinin domain (see, for example, Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7: 463-469).
  • suitable binding agents include portions of such antibodies, such as one or more heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3, as specifically disclosed herein.
  • the at least one heavy chain CDR1, CDR2, CDR3, CDR1, CDR2 and CDR3 regions have at least one amino acid substitution as long as the antibody retains the binding specificity of the non-replacement CDR.
  • the non-CDR portion of the antibody can be a non-protein molecule, wherein the binding agent cross-blocks binding of the antibodies disclosed herein to human GCGR and/or inhibits glucagon signaling via the receptor.
  • the non-CDR portion of the antibody can be a non-protein molecule, wherein the antibody exhibits a binding type to a human glucagon peptide similar to that exhibited by at least one of the antibodies L4H4/L5H5 in a competitive binding assay, and/or And the activity of glucagon.
  • the non-CDR portion of the antibody may be composed of an amino acid, wherein the antibody is a recombinant binding protein or a synthetic peptide, and the recombinant binding protein cross-blocks binding of the antibody disclosed herein to human GCGR and/or neutralizes glucagon activity in vivo or in vitro .
  • the non-CDR portion of the antibody may consist of an amino acid, wherein the antibody is a recombinant antibody, and the recombinant antibody exhibits a binding type to a human GCGR peptide similar to that exhibited by at least one of the antibodies L4H4/L5H5 in a competition binding assay, and / Or neutralize glucagon signaling.
  • a fusion protein of a GCGR antibody and GLP-1 comprising an antibody that specifically binds to GCGR, and one, two, three, four, five, six, Seven, or eight GLP-1 fragments or a reverse GLP-1 fragment; the fusion protein carboxy terminus of a GLP-1 fragment to the amino terminus of a GCGR antibody light chain or heavy chain via a peptide linker sequence (Linker) Ligation or ligation of the amino terminus of a reverse GLP-1 fragment to the carboxy terminus of a GCGR antibody light or heavy chain.
  • Linker peptide linker sequence
  • a fusion protein of a GCGR antibody and GLP-1 comprising an antibody that specifically binds to GCGR, and one, two, three, or four GLP-1 fragments;
  • the fusion protein ligates the carboxy terminus of a GLP-1 fragment to the amino terminus of a GCGR antibody light or heavy chain via a linker sequence (Linker).
  • a fusion protein of a GCGR antibody to GLP-1 comprising an antibody that specifically binds to GCGR, and one, two, three, or four reverse GLP-1 Fragment; the fusion protein ligates the amino terminus of a reverse GLP-1 fragment to the carboxy terminus of a GCGR antibody light or heavy chain via a linker sequence (Linker).
  • Linker linker sequence
  • a fusion protein of a GCGR antibody and GLP-1 comprising an antibody that specifically binds to GCGR, and two GLP-1 fragments; the fusion protein is passed through a peptide linker sequence ( Linker) ligates the carboxy terminus of a GLP-1 fragment to the amino terminus of a GCGR antibody light chain or heavy chain.
  • Linker peptide linker sequence
  • a fusion protein of a GCGR antibody and GLP-1 comprising an antibody that specifically binds to GCGR, and two inverted GLP-1 fragments; the fusion protein is passed through a peptide linker
  • the Linker links the amino terminus of a reverse GLP-1 fragment to the carboxy terminus of a GCGR antibody light chain or heavy chain.
  • a GLP-1 fusion protein comprising a GCGR antibody and two GLP-1 fragments; the fusion protein carboxy terminus of a GLP-1 fragment by a peptide linker (Linker) Linking to the amino terminus of a GCGR antibody light chain: N'-GLP-1-Linker-R-C'; or linking the carboxy terminus of a GLP-1 fragment to the amino terminus of a GCGR antibody heavy chain: N'-GLP -1-Linker-R-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxy terminus of the fusion protein polypeptide chain, GLP-1 represents a GLP-1 fragment, and R is a GCGR antibody The amino acid sequence of the light or heavy chain, and Linker represents a peptide linker sequence.
  • Linker represents a peptide linker sequence.
  • a GLP-1 fusion protein comprising a GCGR antibody and two inverted GLP-1 fragments; the fusion protein is a reverse GLP-1 fragment by a peptide linker (Linker)
  • the amino terminus is linked to the carboxy terminus of a GCGR antibody light chain: N'-R-Linker-reverse GLP-1-C'; or the amino terminus of a reverse GLP-1 fragment and the carboxyl group of a GCGR antibody heavy chain Terminal ligation: N'-R-Linker-reverse GLP-1-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, C' represents the carboxy terminus of the fusion protein polypeptide chain, and reverse GLP-1 represents a In the reverse GLP-1 fragment, R is the amino acid sequence of the light or heavy chain of a GCGR antibody, and Linker represents a peptide linker sequence.
  • a GLP-1 fusion protein comprising a GCGR antibody and two GLP-1 fragments; the fusion protein carboxy terminus of a GLP-1 fragment by a peptide linker (Linker) Linking to the amino terminus of the light chain of the GCGR antibody: N'-GLP-1-Linker-R-C'; wherein: N' represents the amino terminus of the fusion protein polypeptide chain, and C' represents the carboxy terminus of the fusion protein polypeptide chain, GLP- 1 represents a GLP-1 fragment, R is the amino acid sequence of the light chain of a GCGR antibody, and Linker represents a peptide linker sequence.
  • Linker represents a peptide linker sequence.
  • the GLP-1 fragments are each independently selected from the amino acid sequence of one of: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.
  • the inverted GLP-1 fragments are each independently selected from the amino acid sequence of one of: SEQ ID NO: 127, SEQ ID NO: 128 SEQ ID NO: 129, SEQ ID NO: 130, and SEQ ID NO: 131.
  • the sequence of the linker consists independently of from 1 to 200 amino acid amines, from 2 to 100 amino acid amines, from 5 From 50 to amino acid amines, from 6 to 25 amino acid amines, or from 10 to 20 amino acid amines.
  • the sequence of the linker comprises a full length, partial, or repeat amino acid sequence independently selected from one of the following: SEQ ID NO: 124, SEQ ID NO: 125, and SEQ ID NO: 126.
  • isolated nucleic acid molecules are provided herein.
  • the nucleic acid molecule comprises, for example, a polynucleotide encoding all or part of an antibody, such as one or both strands of an antibody or GLP-1 fusion protein herein, or a fragment, derivative, mutein or variant thereof; a polynucleotide of a hybridization probe; a PCR primer or a sequencing primer for identifying, analyzing, mutating or amplifying a polynucleotide encoding the polypeptide; an antisense nucleic acid for inhibiting expression of the polynucleotide and Complementary sequence.
  • the nucleic acid can be of any length.
  • nucleic acids may be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides, and/or comprising one or more additional sequences, such as regulatory sequences, and/or a larger nucleic acid such as a portion of a vector.
  • the nucleic acid can be single or double stranded and comprise RNA and/or DNA nucleotides as well as artificial variants thereof (eg, peptide nucleic acids).
  • a nucleic acid encoding an antibody polypeptide (eg, a heavy or light chain, a variable domain only, or a full length) can be isolated from mouse B cells immunized with a GCGR antigen.
  • the nucleic acid of the antibody or GLP-1 fusion protein can be isolated by a conventional method such as polymerase chain reaction (PCR).
  • Nucleic acid sequences encoding the heavy and light chain variable regions are as indicated above. The skilled artisan will appreciate that due to the degeneracy of the genetic code, each polypeptide sequence disclosed herein can be encoded by a greater number of other nucleic acid sequences. Provided herein are each degenerate nucleotide sequence encoding an antibody or GLP-1 fusion protein provided herein.
  • nucleic acids that hybridize to other nucleic acids (eg, nucleic acids comprising any of the nucleotide sequences of L4H4/L5H5) under specific hybridization conditions.
  • Methods for hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Son (1989), 6.3.1-6.3.6.
  • moderately stringent conditions use a prewash solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6x
  • SSC sodium chloride/sodium citrate
  • 1.0 mM EDTA pH 8.0
  • hybridization buffer of about 50% formamide
  • 6x The SSC is hybridized to a temperature of 55 ° C (or other similar hybridization solution, for example, containing 50% formamide, hybridized at 42 ° C), and eluted at 60 ° C using 0.5 x SSC, 0.1% SDS.
  • Stringent hybridization conditions were hybridized in 6x SSC at 45 °C and then washed one or more times in 0.1x SSC, 0.2% SDS at 68 °C.
  • nucleosides comprising at least 65, 70, 75, 80, 85, 90, 95, 98 or 99% homology to each other are nucleosides.
  • the nucleic acids of the acid sequence can usually still hybridize to each other.
  • Mutations can be introduced using any technique known in the art.
  • one or more specific amino acid residues are altered using, for example, a site-directed mutagenesis protocol.
  • one or more randomly selected residues are altered using, for example, a random mutagenesis protocol. Regardless of how it is produced, the mutant polypeptide can be expressed and screened for the desired properties.
  • nucleic acid without significantly altering the biological activity of the polypeptide encoded thereby.
  • nucleotide substitutions that result in amino acid substitutions at non-essential amino acid residues can be made.
  • the nucleotide sequence provided herein is a L1 to L10 and H1 to H7 or GLP-1 fusion protein, or a fragment, variant or derivative thereof, such that its encoding comprises L1 to L10 and H1 as indicated herein.
  • One or more deletions or substitutions of the amino acid residues of H7 become two or more residues that differ in sequence.
  • the mutagenesis inserts an amino acid into one or more amino acid residues adjacent to one or more amino acid residues of the L1 to L10 and H1 to H7 or GLP-1 fusion proteins shown herein. base.
  • one or more mutations can be introduced into the nucleic acid to selectively alter the biological activity of the polypeptide encoded thereby (eg, binding to GCGR).
  • the mutation can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing or eliminating this activity. Examples of qualitative changes include altering the antigen specificity of an antibody or GLP-1 fusion protein.
  • nucleic acid molecules suitable for use as primers or hybridization probes for detecting nucleic acid sequences herein may comprise only a portion of a nucleic acid sequence encoding a full length polypeptide herein, for example, a fragment useful as a probe or primer or a fragment encoding an active portion of a polypeptide herein (eg, a GCGR binding portion).
  • Probes based on the nucleic acid sequences herein can be used to detect the nucleic acid or a similar nucleic acid, such as a transcript encoding a polypeptide herein.
  • the probe may comprise a labeling group such as a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.
  • a vector comprising a nucleic acid encoding a polypeptide herein or a portion thereof.
  • vectors include, but are not limited to, plasmids, viral vectors, non-free gene mammalian vectors, and expression vectors, such as recombinant expression vectors.
  • a recombinant expression vector herein can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell.
  • the recombinant expression vector comprises one or more regulatory sequences that are screened based on the host cell for expression operably linked to the pre-expressed nucleic acid sequence.
  • Regulatory sequences include constitutive expression of a guide nucleotide sequence in a plurality of species of host cells (eg, SV40 early gene enhancer, Rous sarcoma virus promoter, and cytomegalovirus promoter), directing only in certain hosts Expression of nucleotide sequences in cells (eg, tissue-specific regulatory sequences, see Voss et al, 1986, Trends Biochem. Sci.
  • the host cell can be any prokaryotic or eukaryotic cell.
  • Prokaryotic host cells include Gram-negative or Gram-positive organisms such as E. coli or Bacillus. More advanced eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines grown in serum-free medium (see Rasmussen et al, 1998, Cytotechnology 28: 31) or CHO DXB-11, which lacks DHFR (see Urlaub et al, 1980, Proc. Natl. Acad, Sci.
  • CHO Chinese hamster ovary
  • CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC#CRL-1861), and UV20 (ATCC#CRL-1862), and other host cells include the COS-7 line of monkey kidney cells (ATCC#).
  • CRL-1651) see Gluzman et al, 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL-163), AM-1/D cells (described in US Patent Serial No.
  • HeLa cells derived from African green monkey kidney cell line CV1 (see McMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney Cells such as 293, 293EBNA or MSR 293, human epithelial A431 cells, human C010205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissues, primary transplants, HL- 60, U937, HaK or Jurkat cells. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, 1985).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells by conventional transformation or transfection techniques.
  • a gene encoding a screening marker eg, antibiotic resistance
  • Preferred screening markers include those that confer resistance to drugs such as G418, hygromycin, and methotrexate.
  • stable transfected cells containing the introduced nucleic acid can be identified by drug screening (e.g., cells incorporating the screened gene can survive while other cells die).
  • the transformed cells can be cultured under conditions that increase the expression of the polypeptide, and the polypeptide can be recovered by conventional protein purification methods.
  • One such purification method is described in the examples below.
  • Polypeptides pre-administered herein include substantially homologous recombinant mammalian anti-GCGR antibodies or GLP-1 fusion protein polypeptides that are substantially free of contaminating endogenous materials.
  • the activity of a GCGR antibody means that the antibody provided herein has a therapeutic effect that specifically binds to GCGR and inhibits or blocks glucagon signaling, such as treatment of hyperglycemia, type 2 diabetes, metabolic syndrome, and Dyslipidemia.
  • the term "reducing the biological activity of glucagon signaling” or “inhibiting or blocking the biological activity of glucagon signaling” means that the GCGR antibody or its binding to the GLP-1 fusion protein in vivo binds to GCGR and Blocking glucagon causes a cellular emergency response downstream of the receptor. Responses include, but are not limited to, reducing hepatic glycogen output, lowering blood glucose, and associated changes in fat metabolism.
  • provided herein are murine or humanized antibodies that specifically bind to human GCGR. Such antibodies include antagonistic or neutralizing antibodies that reduce or neutralize glucagon signaling.
  • K antibody when binding to human GCGR d provided herein is from about 0.01nM to about 1000nM, from about 0.1nM to about 500nM, from about 0.5nM to about 200nM, from about 1nM to about 200nM, or about 10nM to About 100nM. In another embodiment, K antibody when binding to human GCGR d provided herein is from about 1nM to about 200nM. In another embodiment, K antibody when binding to human GCGR d provided herein is from about 1nM to about 100nM.
  • K antibody when binding to human GCGR d provided herein is from about 1nM, about 2nM, about 5nM, about 10 nM, about 20 nM, about 30 nM, about 40nM, about 50 nM, about 60 nM, about 70nM, Approximately 80 nM, approximately 90 nM, or approximately 100 nM.
  • K antibody when binding to human GCGR d provided herein is about 100nM, about 110nM, about 120nM, about 130nM, about 140nM, about 150nM, about 160nM, about 170nM, about 180nM, about 190nM, Or about 200nM.
  • the antibody provided herein in reducing human glucagon signaling IC 50 value from about 0.01nM to about 500 nM, from about 0.1nM to about 200nM, from about 0.5nM to about 200nM, from about 1nM to about 200nM , or about 10 nM to about 100 nM. In another embodiment, the antibody provided herein in reducing human glucagon signaling IC 50 value of about 1nM to about 200nM. In another embodiment, the antibody provided herein in reducing human glucagon signaling IC 50 value of about 10nM to about 100nM.
  • the antibodies provided herein have an IC50 value of about 1 nM, about 2 nM, about 5 nM, about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about reducing human glucagon signaling. 60 nM, approximately 70 nM, approximately 80 nM, approximately 90 nM, or approximately 100 nM.
  • the GCGR antibodies provided herein, when combined with human GCGR, have one or more of the properties listed below:
  • the reference antibody comprises a combination of the light chain variable domain amino acid sequence SEQ ID NO:87 and the heavy chain variable domain amino acid sequence SEQ ID NO:97.
  • the reference antibody is monoclonal antibody L4H4, L5H5, or L7H7.
  • a reference antibody comprises, for example, an antibody having a combination of light chain SEQ ID NO:87 and heavy chain SEQ ID NO:97.
  • the reference antibody comprises the GCGR antibody L4H4, L5H5, or L7H7.
  • the biological activity of the fusion protein of the GCGR antibody and GLP-1 includes both the biological activity of GLP-1 and the activity of GCGR antibody.
  • the activity of the GCGR antibody is as described above.
  • GLP-1 biological activity means that a fusion protein of a GCGR antibody and GLP-1 binds in vivo and activates the GLP-1 receptor and causes a cellular stress response, and exhibits a therapeutic biological activity such as hyperglycemia, Type 2 diabetes, metabolic syndrome, and other related symptoms including dyslipidemia.
  • the aforementioned cellular stress responses include, but are not limited to, reducing hepatic glycogen output, lowering blood glucose, and associated changes in fat metabolism.
  • the GLP-1 fusion proteins described herein can be used to treat a variety of diseases and conditions associated with GLP-1R and GCGR.
  • the fusion protein exerts its biological effects by acting on GLP-1R and/or GCGR, and thus can be advantageously treated with the GLP-1 fusion protein described herein for "increasing GLP-1R stimulation” or "reducing GCGR stimulation”.
  • Subjects who respond to diseases and conditions. These subjects were referred to as subjects who required "GLP-1R stimulation therapy" or “need to reduce GCGR stimulation.” Includes hyperglycemia, type 2 diabetes, metabolic syndrome, and other related symptoms of dyslipidemia.
  • the biological activity of the GCGR antibody or GLP-1 fusion protein is detected by direct cAMP detection, and the GCGR antibody or GLP-1 fusion protein is quantified to inhibit the function of GCGR in vitro.
  • a pharmaceutical composition comprising a GCGR antibody provided herein and one or more pharmaceutically acceptable carriers.
  • a pharmaceutical composition comprising a fusion protein of a GCGR antibody and GLP-1 provided herein, together with one or more pharmaceutically acceptable carriers.
  • carrier includes carriers, pharmaceutical excipients, or stabilizers which are not harmful to the exposure of cells or mammals at the dosages and concentrations employed.
  • provided herein is a method of treating, preventing, or ameliorating type 2 diabetes comprising administering to a subject a therapeutically effective amount of a GCGR antibody or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating, preventing, or ameliorating type 2 diabetes comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 provided herein, or a pharmaceutical combination thereof Things.
  • provided herein is a method of treating, preventing, or ameliorating a complication of type 2 diabetes comprising administering to a subject a therapeutically effective amount of a GCGR antibody or a pharmaceutical composition thereof provided herein.
  • provided herein is a method of treating, preventing, or ameliorating a complication of type 2 diabetes comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 provided herein, or a medicament thereof Use the composition.
  • provided herein is a method of treating, preventing, or ameliorating hyperglycemia comprising administering to a subject a therapeutically effective amount of a GCGR antibody provided herein, or a pharmaceutical composition thereof.
  • provided herein is a method of treating, preventing, or ameliorating hyperglycemia comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 provided herein, or a pharmaceutical combination thereof Things.
  • provided herein is a method of treating, preventing, or ameliorating a metabolic syndrome comprising administering to a subject a therapeutically effective amount of a GCGR antibody provided herein, or a pharmaceutical composition thereof.
  • provided herein is a method of treating, preventing, or ameliorating a metabolic syndrome comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 provided herein, or a pharmaceutical combination thereof Things.
  • provided herein is a method of treating, preventing, or ameliorating dyslipidemia comprising administering to a subject a therapeutically effective amount of a GCGR antibody provided herein, or a pharmaceutical composition thereof.
  • a method of treating, preventing, or ameliorating dyslipidemia comprising administering to a subject a therapeutically effective amount of a fusion protein of a GCGR antibody and GLP-1 provided herein, or a pharmaceutical composition thereof .
  • the pharmaceutical composition is for intravenous or subcutaneous injection.
  • the fusion protein of GLP-1R antibody and GLP-1 described herein comprises
  • heavy chain CDR1 amino acid sequence SEQ ID NO: 137;
  • heavy chain CDR2 amino acid sequence SEQ ID NO: 138;
  • Heavy chain CDR3 amino acid sequence SEQ ID NO: 139.
  • the fusion protein of a GLP-1R antibody and GLP-1 described herein comprises a combination of the light chain and heavy chain variable region amino acid sequences set forth below: SEQ ID NO: 140 and SEQ ID NO: 141.
  • subject refers to mammals, including humans, and can be used interchangeably with the term “patient.”
  • treating includes alleviating or preventing other aspects of at least one symptom or condition, or reducing the severity of the disease.
  • the fusion protein of the GCGR antibody or GCGR antibody and GLP-1 provided herein does not need to produce a complete healing effect, or eradicate all symptoms or manifestations of the disease, and constitute an effective therapeutic agent.
  • a drug as a therapeutic agent can reduce the severity of a given disease state, but can be considered as an effective therapeutic agent without eliminating all manifestations of the disease.
  • prophylactic administration forms an effective preventive agent without being completely effective in preventing the onset of symptoms.
  • One embodiment herein relates to a method of administering to a patient a GCGR antibody or a GCGR antibody and a GLP-1 fusion protein in an amount and for a time sufficient to induce a sustained improvement in the severity of the response to a particular condition.
  • the fusion protein pharmaceutical composition of the GCGR antibody or GCGR antibody and GLP-1 can be administered by any suitable technique including, but not limited to, parenteral, topical or inhalation.
  • the pharmaceutical composition can be administered by rapid injection or continuous infusion by, for example, intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes. Administration may be considered, for example, for topical administration at the site of the disease or injury, such as transdermal administration and sustained release of the implant.
  • Administration by inhalation includes, for example, nasal or oral inhalation, administration with a spray, inhalation of the antibody in the form of an aerosol, and the like. Other options include oral formulations including tablets, syrups or lozenges.
  • composition comprising one or more additional components, such as a physiologically acceptable carrier, adjuvant or diluent.
  • the composition may optionally additionally comprise one or more physiologically active agents as described below.
  • the composition comprises one, two, three, four, in addition to one or more of the antibodies (eg, murine or humanized antibodies) or GLP-1 fusion proteins provided herein. , five or six physiologically active agents.
  • the pharmaceutical composition comprises a murine or humanized antibody or GLP-1 fusion protein provided herein and one or more substances selected from the group consisting of pH suitable for buffering of antibodies or GLP-1 fusion proteins Liquids, antioxidants such as ascorbic acid, low molecular weight polypeptides (eg, polypeptides containing less than 10 amino acids), proteins, amino acids, sugars such as dextrin, complexes such as EDTA, glutathione, stabilizers, and adjuvants. Preservatives may also be added according to appropriate industry standards.
  • the composition can be formulated as a lyophilized powder using a suitable adjuvant solution as a diluent. The appropriate components are not toxic to the recipient at the dosages and concentrations employed.
  • kits for use by medical practitioners, including one or more of the antibodies provided herein. Or a GLP-1 fusion protein and a label or other description for treating any of the conditions discussed herein.
  • the kit comprises a sterile preparation of one or more antibodies or GLP-1 fusion proteins in one or more vials in the form of the above compositions.
  • the dosage and frequency of administration can vary depending on the route of administration, the particular antibody or GLP-1 fusion protein used, the nature and severity of the condition being treated, whether the condition is acute or chronic, and the volume and overall symptoms of the patient. Suitable dosages can be determined by methods well known in the art, such as including dose escalation studies in clinical trials.
  • the antibody or GLP-1 fusion protein provided herein can be administered one or more times at regular intervals, for example, over a period of time.
  • the murine or humanized antibody or GLP-1 fusion protein is administered once every at least one month or longer, for example one, two or three months or even uncertain.
  • long-term treatment is usually the most effective.
  • short-term administration is sufficient, for example, from one week to six weeks.
  • human antibodies are administered until the patient exhibits a selected medical condition or a medically relevant improvement in the indicator above baseline levels.
  • An example of a treatment regimen provided herein includes subcutaneous injection of an antibody or a GLP-1 fusion protein at a suitable dose once a week or longer for the treatment of hyperglycemia, type 2 diabetes, type 2 diabetes complications, metabolic syndrome, and dyslipidemia, and the like.
  • the antibody or GLP-1 fusion protein can be administered weekly or monthly until the desired result is achieved, for example, the patient's symptoms resolve.
  • the treatment can be re-treated as needed or, alternatively, a maintenance dose can be administered.
  • the patient's blood glucose concentration, body weight can be monitored before, during, and/or after treatment with an antibody or GLP-1 fusion protein, such as a human antibody or GLP-1 fusion protein, to detect any change in its pressure.
  • an antibody or GLP-1 fusion protein such as a human antibody or GLP-1 fusion protein
  • changes in blood glucose can vary with factors such as the course of the disease.
  • the blood glucose concentration can be determined by known techniques.
  • an antibody or GLP-1 fusion protein and one or more glucagon antagonists two or more of the antibodies or GLP-1 fusion proteins provided herein, or An antibody or GLP-1 fusion protein of the invention and one or more additional glucagon antagonists.
  • the antibody or GLP-1 fusion protein is administered alone or in combination with other agents for treating the symptoms that are afflicting the patient. Examples of such agents include proteins as well as non-protein drugs. When multiple drugs are administered in combination, the dosages as known in the art should be adjusted accordingly.
  • “Co-administered" combination therapies are not limited to simultaneous administration, but also include treatment regimens that administer at least one antigen and protein in a course of treatment involving administration of at least one other therapeutic agent to the patient.
  • provided herein is a method of preparing an agent for treating hyperglycemia, type 2 diabetes, type 2 diabetes complications, metabolic syndrome, and dyslipidemia and related disorders, comprising an antibody or GLP-1 fusion protein provided herein and a pharmaceutical Mixtures in excipients are acceptable for the treatment of conditions associated with the above conditions.
  • the preparation method of the medicament is as described above.
  • nucleic acid molecules, and derivatives and fragments thereof, comprising a polynucleotide encoding all or part of a polypeptide that binds to GCGR, for example, encoding all or part of an anti-GCGR antibody, antibody fragment, antibody derivative or GLP-1 A nucleic acid that fuses a protein.
  • vectors and plasmids comprising such nucleic acids, as well as cells and cell lines comprising such nucleic acids and/or vectors and plasmids.
  • the method provided includes, for example, a method of preparing, identifying or isolating an antibody or a GLP-1 fusion protein, such as an anti-GCGR antibody or a GLP-1 fusion protein, which binds to human GCGR, and determining whether the antibody or GLP-1 fusion protein binds to GCGR.
  • CHO-DHFR- cells were seeded into 6-well plates. After 24 hours of culture (hr), the pTM15 plasmid having the hGCGR gene (see SEQ ID NO: 77 for nucleotide sequence and SEQ ID NO: 73 for amino acid sequence) was transfected into the cells. Transfection was performed according to Invitrogen's recommended transfection conditions for Lipofectamine 2000. After 48 hr, switch to complete medium containing 10 nM MTX, change the solution every 3 days (d), wait for about two weeks, and grow stable clones, digest and disperse cell colonies, and grow to 50% healing, gradually The concentration of MTX was increased for pressure screening until the concentration of MTX was 300 nM.
  • a fusion protein of the extracellular region of hGCGR and hIgG Fc can also be used as an immunogen for the preparation of antibodies, which is prepared as follows: subcloning the extracellular region of hGCGR, a fusion protein of hIgG2Fc and a peptide linker (Linker) The sequence gene was in the pTM5 plasmid. The cell supernatant was obtained by suspension of HEK293 cells for a large amount of transient expression, and then purified by affinity chromatography to obtain the hGCGR extracellular domain fusion protein.
  • Linker peptide linker
  • mice The immunogen and the aluminum hydroxide adjuvant were mixed, and BALB/c mice (6-8 weeks old) were injected subcutaneously, and then the mice were boosted once a week. After a total of 6 immunizations, blood was collected by tail-cutting. Serum was separated by centrifugation and serum titer was measured by FACS. When the antibody titer is reached, the mice are sacrificed by cervical dislocation, and spleen cells are obtained under aseptic conditions. In addition, SP2/0 cells in a logarithmic growth phase were collected, and the cells were centrifuged, and the pelleted cells were cultured to serum-free to resuspend, centrifuged again - resuspended, and counted.
  • the spleen cells and SP2/0 cells were mixed to ensure that the SP2/0 and spleen cells were close in number, and then "washed-centrifuged" three times after mixing.
  • the fused hybridoma cells were incubated with feeder cells in a 96-well plate and subjected to HAT (hypoxanthine, methotrexate, and thymidine) screening to remove non-fused cells. After 10 days, the supernatant of the hybridoma cells in the culture plate was collected for ELISA detection.
  • HAT hypoxanthine, methotrexate, and thymidine
  • CHO-DHFR-hGCGR cells overexpressing hGCGR and CHO-DHFR-cells not expressing hGCGR were inoculated into 96-well plates, respectively. After the cells were grown to 90% healing, the cell culture supernatant was removed, washed twice with PBS, fixed with 100% methanol at 4 ° C, and then added with 100 ⁇ L of freshly prepared 0.6% H 2 O 2 -PBS, and treated at room temperature for 20 minutes (min). Wash PBS twice. After blocking with 1% BSA (dissolved in PBS), the supernatant of the hybridoma cells was added and incubated at 4 ° C for 90 min.
  • BSA 1% BSA
  • a 96-well plate is coated with a fusion protein comprising an amino acid sequence of the N-terminal domain of hGCGR and hFc as a coating antigen.
  • the supernatant of the hybridoma cells was added and incubated at 4 ° C for 90 min. Thereafter, the anti-hGCGR monoclonal antibody was screened by the above ELISA method.
  • the positive control was the serum of the immunized mice; the negative control was the cell culture supernatant.
  • several positive hybridoma cell lines secreting anti-hGCGR antibodies were screened. These hybridoma strains secreting anti-hGCGR antibodies were selected and cloned to obtain cell lines stably secreting anti-hGCGR antibodies. Finally, the ascites antibody prepared by the positive hybridoma cells was selected for FACS verification (refer to Example 10).
  • the antibody-secreting hybridoma cells are collected and the mRNA of the hybridoma cells is extracted according to QIAGEN's mRNA extraction kit protocol.
  • the extracted mRNA is then reverse transcribed into cDNA, the reverse transcription primer is a specific primer for the mouse light and heavy chain constant region, and the heavy chain reverse transcription primer is (5'-TTTGGRGGGAAGATGAAGAC-3'), light chain reverse transcription primer.
  • the reverse transcription primer is a specific primer for the mouse light and heavy chain constant region
  • the heavy chain reverse transcription primer is (5'-TTTGGRGGGAAGATGAAGAC-3'), light chain reverse transcription primer.
  • 5'-TTAACACTCTCCCCTGTTGAA-3' and 5'-TTAACACTCATTCCTGTTGAA-3'.
  • the reaction conditions of RT-PCR were: 25 ° C for 5 min; 50 ° C for 60 min; and 70 ° C for 15 min.
  • the reverse transcribed cDNA was diluted to 500 ⁇ L with 0.1 mM TE, added to an ultrafiltration centrifuge tube (Amicon Ultra-0.5), centrifuged at 2000 g for 10 min; the filtrate was discarded, 500 ⁇ L of 0.1 mM TE was added, and 2000 g was centrifuged for 10 min; The filtrate was inverted, and the preparation tube was inverted into a new centrifuge tube, and centrifuged at 2000 g for 10 min to obtain purified cDNA; 10 ⁇ L of the purified cDNA was used as a template, and 4 ⁇ L of 5 ⁇ tailing buffer (Promega), 4 ⁇ L of dATP (1 mM) and 4 ⁇ L were added.
  • 10U end-transferase (Promega) was mixed, incubated at 37 ° C for 5 min and then incubated at 65 ° C for 5 min; then the cDNA of the PolyA tail was used as a template to PCR-amplify the light and heavy chain variable region genes of the antibody.
  • the upstream primers were all OligodT, the heavy chain downstream primers were (5'-TGGACAGGGATCCAGAGTTCC-3') and (5'-TGGACAGGGCTCCATAGTTCC-3'), and the light chain downstream primer was (5'-ACTCGTCCTTGGTCAACGTG-3').
  • PCR reaction conditions 95 ° C for 5 min; 95 ° C for 30 s, 56 ° C for 30 s, 72 ° C for 1 min 40 cycles; 72 ° C for 7 min; PCR products were ligated into the PMD 18-T vector (Takara Bio) and sequenced. PCR primers were designed based on the DNA sequence of the sequenced antibody, thereby ligating the entire light chain, heavy chain signal peptide and variable domain as well as the mouse IgGl constant region to the expression vector pTM5. 5, humanization and optimization of antibodies
  • the NCBI database was used to search for the human antibody germ cell line gene sequence homologous to the murine antibody variable region sequence (Ig Germline Gene Sequence).
  • the human gene sequence with the highest homology is used as a template sequence for CDR grafting to obtain a humanized antibody variable region sequence.
  • the light and heavy chain genes of the humanized antibody are synthesized and ligated with the human IgG2 or IgG4 constant region sequence to obtain a complete recombinant humanized antibody sequence.
  • Recombinant antibodies were expressed as in Example 8, and their affinity for GCGR was verified according to the FACS technique in Step 10, and the antibodies with the best affinity were selected.
  • the variable region sequence of the humanized antibody was modified by site-directed mutagenesis to further increase its affinity for GCGR.
  • the optimized humanized antibody heavy chain and light chain variable region sequences were over-synthesized.
  • the 5' end of the heavy chain variable region is introduced into the Nhe1 cleavage site, and the 3' end is introduced into the Sal1 cleavage site, thereby integrating the entire heavy chain variable region sequence with the expression vector loaded into the heavy chain constant region.
  • pTM5 is connected.
  • the 5' end of the light chain variable region is introduced into the Nhe1 cleavage site, and the 3' end is introduced into the Bsiw1 cleavage site, thereby ligating the entire light chain variable region sequence into the light chain constant region.
  • the expression vector pTM5 is ligated.
  • the optimized humanized antibody is fused to the GLP-1 and its derivative sequences at the N-terminus or C-terminus of the light chain to form a GLP-1 fusion protein.
  • the sequences of the two are linked by a linker sequence (Linker).
  • the nucleotide sequence of the signal peptide -GLP-1-Linker was synthesized by Kingsray Biotech Co., Ltd.
  • the sequence of the "signal peptide-GLP-1-Linker" portion was PCR amplified using the synthetic gene as a template. Further, the nucleotide sequence of the humanized antibody is used as a template to amplify the sequence of the antibody portion of the fusion protein.
  • the "signal peptide-GLP-1-Linker" portion of the fusion protein nucleic acid sequence was ligated to the antibody portion by overlapping PCR, and the restriction sites of Nhe1 and Not1 were added to both ends of the primer to thereby integrate the entire fusion protein sequence with the expression vector pTM5. Connected.
  • the collected cell supernatant of Example 8 was subjected to high-speed (8000 rpm) centrifugation to remove cells and cell debris, and then clarified by filtration through a 0.22 ⁇ m filter. The clarified supernatant was used for purification.
  • the purification process is done by a chromatograph.
  • the supernatant was first passed through a protein A/G affinity column.
  • the antibody contained in the supernatant was retained in the column during this period in combination with the ligand of the protein A/G affinity chromatography column.
  • the column is then lysed with a low pH (less than or equal to 3.0) elution buffer to dissociate the antibody bound to the column.
  • the collected antibody eluate was rapidly neutralized with 1 M Tris-HCl.
  • the resulting antibody eluate is dialyzed and replaced with PBS or other buffer system.
  • the positive treatment group was treated with 200 ⁇ L of specific concentration of hGCGR antibody per tube, and incubated at room temperature; after centrifugation, centrifuge at 1500 rpm, discard the supernatant, wash the cell pellet with flow-type loading buffer, centrifuge again, and resuspend the cells; The suspension was added with a 1:50 dilution of FITC-labeled goat anti-mouse fluorescent secondary antibody, 200 ⁇ L/well, and incubated at room temperature for 30 min in the dark; centrifuge, discard the supernatant, wash once with flow-loading buffer, centrifuge, and finally flow. The cell suspension was resuspended in the loading buffer and detected on the machine.
  • the functional antibody against recombinant hGCGR specifically binds to CHO-DHFR-CHO-GCGR cells expressing GCGR.
  • the gray peak is a negative control of 500 nM mouse ascites antibody L5H5 and blank cell CHO-DHFR-binding, and the solid peaks are 500 nM (1a), 50 nM (1b) and 5 nM (1c), respectively.
  • the binding curves of the murine ascites antibody L5H5 and CHO-DHFR-hGCGR showed a significant right shift relative to the gray peak negative control, demonstrating the specific binding of L5H5 and CHO-DHFR-hGCGR.
  • the gray peak is a negative control of 500 nM mouse ascites antibody L4H4 and blank cell CHO-DHFR-binding
  • the solid peaks are 500 nM (2a), 50 nM (2b) and 5 nM (2c), respectively.
  • the binding curves of the murine ascites antibody L4H4 and CHO-DHFR-hGCGR showed a significant right shift relative to the gray peak negative control, demonstrating the specific binding of L4H4 and CHO-DHFR-hGCGR.
  • HGCGR-expressing CHO-DHFR- cells were seeded at 30,000 per well into 96-well cell culture plates and placed in a 37 ° C, 5% CO 2 incubator overnight. The cell supernatant was removed the next day, and hybridoma cell culture supernatant or gradient-diluted antibody 45 ⁇ L/well was added. After standing for 30 min at room temperature, 45 ⁇ L/well of glucagon polypeptide (Phoenix Pharmaceuticals, 50 pM) was added. The 96-well cell culture plate was then placed in a 37 ° C, 5% CO 2 incubator for 30 min, and 10 ⁇ L/well of 10% Triton X-100 was added, lysed at room temperature, and uniformly mixed with a lance.
  • Reporter gene assay to detect the fusion protein of GCGR antibody and GLP-1 activates GLP-1R in vitro.
  • CHO-DHFR-cells co-expressing hGLP-1R-CRE-Luciferase were seeded at 40,000 per well into 96-well cell culture plates, and cultured overnight at 37 °C. On the next day, the culture supernatant was removed, the cell surface was washed twice with serum-free medium, and the residue was aspirated, and 100 ⁇ L of the purified antibody or GMA102 was diluted with serum-free medium, and incubated at 37 ° C for 4 hours.
  • FIG. 5 shows the reporter gene assay for the GCGR antibody/GLP-1 fusion protein GLP-1-Linker-L7H7, and the activation profile of GMA102 for activation of the hGLP-1R signaling pathway (EC 50 of 106 pM and 16 pM, respectively).
  • GMA102 is a long-acting GLP-1 agonist developed by our company (Li et al., Biochem Pharmacol. 2018, 150: 46-53; CN 201410349725), which activates intracellular cAMP signaling pathway and significantly reduces blood sugar and body weight in mice. Is in the second phase of clinical development.
  • the animals in the tails were bled, and the blood glucose level (0 min) was measured by Roche's excellent blood glucose meter (the same below), followed by subcutaneous injection (0.1 mL/10 g body weight, and the normal control group was given PBS).
  • administration group L4H4 alone used 2.5, 5, 10mg/kg, or GMA102 dose 2.5, 5mg/kg
  • GMA102 is a humanized antibody fusion protein, there are anti-drug antibodies in mice, so every two One dose per day, the mouse L4H4 was given only once), and the glucose solution of each group was intragastrically administered (2g/kg, 0.1 mL/10g body weight) 30 minutes after subcutaneous injection, and 30, 60 and 120 min after the sugar load Blood was collected and blood glucose levels were measured.
  • On the second day of administration in order to reduce the irritation to the animals, only the blood glucose levels of each group of animals after fasting for 5 hr (free drinking water) (0 min) and 30 min after glucose load were measured. Thereafter, the blood glucose level after the sugar load was continuously measured for four days, six days, eight days, ten days, and twelve days after administration.
  • Table 3 shows that from day 2 to day 8, GMA102, L4H4 alone, and GMA102 and L4H4 in combination significantly reduced oral glucose tolerance in mice, and combined The effect is more obvious than the single use effect, and has the synergistic effect.
  • mice 60% high fat diet induced C57BL/6 mice to establish a model of obesity (DIO mice).
  • DIO mice 60% high fat diet induced C57BL/6 mice to establish a model of obesity
  • mice One week after the mice were fed a normal diet, a certain number of mice were randomly selected as normal control groups to feed the normal mice, and the remaining animals were given high fat diet.
  • the animals were fed for 8 weeks (wk) and the body weight and food intake were weighed once a week.
  • the mice in the high fat diet group were then randomized by body weight. After fasting overnight (free drinking water), the animals in the tails were bled, and the blood glucose level (0 min) was measured by Roche's excellent blood glucose meter (the same below), followed by subcutaneous injection (0.1 mL/10 g body weight, and the normal control group was given PBS).
  • a total of 6 cynomolgus monkeys were given a single injection of a fusion protein of GCGR antibody and GLP-1 at a dose of 2 mg/kg, before administration (0 min), 2 hr, 4 hr, 8 hr, 12 hr after administration. 24hr, 2d, 4d, 6d, 8d, 10d, 12d, 18d, 28d were administered to the side limbs to take whole blood 0.6mL into a centrifuge tube, and after natural coagulation on ice, the serum was extracted by centrifugation and cryopreserved (-80 °C) until the test.
  • the GLP-1 portion of the fusion protein of the GCGR antibody and GLP-1 in the serum sample and the hGCGR antibody portion were separately quantified by ELISA, and the half-life of both in the cynomolgus monkey was determined by software analysis.
  • GLP-1-Linker-L7H7 Two healthy and healthy rhesus monkeys were given a single single GCGR antibody and GLP-1 fusion protein (GLP-1-Linker-L7H7) at a dose of 2 mg/kg, and before administration (0 min), 2h, 4h, 8h, 12h, 24h, 2d, 4d, 6d, 8d, 10d, 12d, 16d, 20th, 25th, 30th, 40th, 50th, and 60th after drug Tianjing non-administered limb veins Take 0.6 mL of whole blood and place it in a centrifuge tube with 8 ⁇ L of DDP-IV enzyme inhibitor (Millipore).
  • PK studies showed that the half-life T 1/2 of the GLP-1 moiety was approximately 38 hours, while the antibody partial half-life T 1/2 of GLP-1-Linker-L7H7 was approximately 131 hours. PK curves and parameters are shown in Figure 7 and Table 4.
  • GLP-1-Linker-L7H7 and positive control GMA102 a fusion protein of our company's GLP-1R antibody and GLP-1
  • All animals were fasted for 14-16 hours overnight before the test.
  • the animals were anesthetized with a 50% glucose solution (0.25 g/kg) via the lower extremities, 5 min, 3 min before the sugar supply and 3 min and 5 min after the sugar.
  • Blood samples were collected from the forearm vein at 8 min, 10 min, 20 min and 30 min.
  • the EDTA2K was anticoagulated and separated for centrifugation for insulin and blood glucose testing.
  • Insulin ( ⁇ U/mL) was detected using a Roche cobas 6000analyzer series E601. The results are shown in Figures 8 and 9. Before the injection, the two groups of animals secreted the same amount of insulin, and 48 hours after the injection, the GLP-1-Linker-L7H7 group secreted more insulin than the positive control group. Glucose (mmol/L) was measured by Roche cobas 6000analyzer series C501. The results are shown in Figure 10 and Figure 11. Before the injection, the blood glucose of the two groups of monkeys was comparable, and 48 hours after the injection, GLP-1-Linker-L7H7 The group had a greater rate of hypoglycemia than the positive control group.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Endocrinology (AREA)
  • Diabetes (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • General Chemical & Material Sciences (AREA)
  • Obesity (AREA)
  • Veterinary Medicine (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Hematology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Public Health (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Emergency Medicine (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

本文提供了GCGR抗体及其与GLP-1的融合蛋白质，以及其药物组合物。本文还提供了GCGR抗体及其与GLP-1的融合蛋白质用于治疗、预防或改善高血糖症、二型糖尿病、代谢综合症、或血脂异常的一种或多种症状方法。

Description

GCGR抗体及其与GLP-1的融合蛋白质，以及其药物组合物和应用 技术领域
本文提供了能与GCGR特异性结合的抗体及其与GLP-1的融合蛋白质，以及其药物组合物。本文还提供了GCGR抗体及其与GLP-1的融合蛋白质用于治疗、预防或改善高血糖症、二型糖尿病、代谢综合症、或血脂异常的一种或多种症状方法。
发明背景
人胰高血糖素是与胰岛素一起作用的重要激素类物质，参与人体血液中葡萄糖含量的调控。胰高血糖素和胰岛素都是多肽类激素。胰高血糖素是在胰腺的α胰岛细胞产生的，胰岛素是在β胰岛细胞产生的。当血糖水平降低时，胰高血糖素主要通过刺激一些靶细胞(主要是肝细胞)释放葡萄糖，胰高血糖素和胰岛素调节血糖的作用正好相反，胰岛素在血糖升高时刺激细胞摄取并储存葡萄糖，降低血糖浓度。
天然人胰高血糖素是由29个氨基酸残基：His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr。
胰高血糖素通过结合其受体并激活下游信号通路，胰高血糖素受体(简称GCGR)属于七次跨膜G蛋白偶联受体家族分泌素亚型，配体-受体结合后，通过激活第二信使腺苷酸环化酶来行使功能，升高肝细胞内cAMP水平，继而启动葡萄糖的从头合成途径和肝糖原降解途径，使得血糖浓度升高(Wakelam等，1986，Nature 323:68-71；Pittner和Fain，1991，Biochem J.277:371-8)。
糖尿病是常见的葡萄糖代谢症，其特征是高血糖，分为胰岛素绝对不足的一型糖尿病和胰岛素相对不足的二型糖尿病。一型糖尿病患者表现出高血糖和低胰岛素血症，并且对该病的常规治疗措施为提供胰岛素。但是，在某些一型或二型糖尿病中，绝对或相对高的胰高血糖素导致高血糖。在健康的动物或糖尿病模型动物中，用选择性或特异性的抗体清除血液循环中的胰高血糖素可以使血糖水平下降(Brand等，1996，Diabetes45:1076)。这些研究表明，抑制胰高血糖素或拮抗胰高血糖素受体可以作为糖尿病常规高血糖治疗的辅助手段。
通过靶向胰高血糖素受体的抗体阻断胰高血糖素和胰高血糖素受体的结合，也可以作为控制或降低血糖的一种手段，从而成为治疗糖尿病的新方法(US 2008/036341 A2和US 2012/0128679 A1)。但在实际的动物实验及临床试验中却发现，直接使用胰高血糖素受体的抗体，阻断胰高血糖素信号通路，会引起显著的负反馈，血液中生成浓度特别高的胰高血糖素，短暂升高的肝转氨酶，以及胰岛α细胞的显著增生(Kostic等，2018，Diabetes Obes Metab.20:283-91；Gu等，2009，JPET 331:871-881)。
GCGR抗体与GLP-1的融合蛋白质(GLP-1融合蛋白质)，一方面，GCGR抗体部分通过抑制胰高血糖素与其受体的结合，降低细胞内cAMP水平，降低血糖；另外一 方面，GLP-1部分，同样具有降低血糖的作用。这样GLP-1融合蛋白质中的两个部分：GCGR的抗体以及GLP-1部分同时起到降低血糖的作用，二者具有协同增效的作用，可以更大幅度地降低血糖，从而提高降血糖的药效。
另外，在协同增效的背景下，抗胰高血糖素抗体的临床剂量也不需要用到特别高，即在低剂量下，配合GLP-1激动剂一起，就可以显著降低血糖，这比单独使用高剂量的GCGR的抗体，能更大幅度地降低血糖，从而避免了使用高剂量胰高血糖素受体的抗体引起的副作用，比如胰岛α细胞的增生。更进一步来说，GLP-1激动剂能促进胰岛素分泌，而胰岛素又会拮抗胰高血糖素的生成(Mari等，2016，Diabetes Obes Metab.18:834-9)，给予GLP-1融合蛋白质，其中的GLP-1部分能够抑制胰高血糖素的升高，同样也降低了GCGR抗体显著升高胰高血糖素的负反馈作用，这样会显著降低药物的毒副作用。
GLP-1融合蛋白质中的GCGR抗体部分的降糖效果和GLP-1部分降糖效果都会叠加，通过双重作用来治疗高血糖症、二型糖尿病、代谢综合症和包括血脂异常的其它相关症状。这比单独使用GLP-1激动剂降糖幅度更大，也比单独使用胰高血糖素受体的抗体毒副作用更低，GLP-1融合蛋白质具有更显著的降血糖效果，又更安全。
发明内容
本文提供了能与GCGR特异性结合的抗体，该抗体是GCGR的拮抗剂。
本文还提供了一个能与GCGR特异性结合的抗体，其抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；
b.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；
d.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34；
e.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35；及
f.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
本文还提供了一个GLP-1融合蛋白质，其包含一个能与GCGR特异性结合的抗体，和一个，二个，三个，四个，五个，六个，七个，或八个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接或者将一GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
本文进一步提供了一个GLP-1融合蛋白质，其包含一个能与GCGR特异性结合的抗体，和一个，二个，三个，四个，五个，六个，七个，或八个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接或者将一反向GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
本文提供了一个GLP-1融合蛋白质，其包含一个GCGR抗体和二个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链的氨基端连接：N'-GLP-1-Linker-R-C'；或者将一GLP-1片段的羧基端和一GCGR抗体重链的氨基端连接：N'-GLP-1-Linker-R-C'；其中:N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,GLP-1代表GLP-1片段，R为GCGR抗体的轻链或者重链的氨基酸序列,及Linker代表肽接头序列。
本文提供了一个GLP-1融合蛋白质，其包含一个GCGR抗体和二个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链的羧基端连接：N'-R-Linker-反向GLP-1-C'；或者将一反向GLP-1片段的氨基端和一GCGR抗体重链的羧基端连接：N'-R-Linker-反向GLP-1-C'；其中:N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,反向GLP-1代表反向GLP-1片段，R为GCGR抗体的轻链或者重链的氨基酸序列,及Linker代表肽接头序列。
本文提供了一个多核苷酸，其编码本文中所述的一个GCGR抗体。
本文提供了一个多核苷酸，其编码本文中所述的一个GCGR抗体与GLP-1的融合蛋白质。
本文提供了一个载体，其包含编码本文中所述的一个GCGR抗体的多核苷酸。
本文提供了一个载体，其包含编码本文中所述的一个GCGR抗体与GLP-1的融合蛋白质的多核苷酸。
本文提供了一个宿主细胞，其包含本文中所述的一个载体。
本文提供了一个药用组合物，其包含本文所述的一个GCGR抗体和一个药用可接受载体。
本文提供了一个药用组合物，其包含本文所述的一个GCGR抗体与GLP-1的融合蛋白质和一个药用可接受载体。
本文提供了本文所述的一个GCGR抗体在制备用于治疗、预防或改善二型糖尿病的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于治疗、预防或改善二型糖尿病的药物中的用途。
本文提供了本文所述的一个GCGR抗体在制备用于治疗、预防或改善二型糖尿病并发症的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于治疗、 预防或改善二型糖尿病并发症的药物中的用途。
本文提供了本文所述的一个GCGR抗体在制备用于治疗、预防或改善高血糖症的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于治疗、预防或改善高血糖症的药物中的用途。
本文提供了本文所述的一个GCGR抗体在制备用于治疗、预防或改善代谢综合症的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于治疗、预防或改善代谢综合症的药物中的用途。
本文提供了本文所述的一个GCGR抗体在制备用于治疗、预防或改善血脂异常的其它相关的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于治疗、预防或改善血脂异常的其它相关的药物中的用途。
本文提供了本文所述的一个GCGR抗体在制备用于同时治疗、预防或改善高血糖症、二型糖尿病、代谢综合症、或血脂异常的其它相关二种及二种以上病症的药物中的用途。
本文提供了本文所述的一个GCGR抗体与GLP-1的融合蛋白质在制备用于同时治疗、预防或改善高血糖症、二型糖尿病、代谢综合症、或血脂异常的其它相关二种及二种以上病症的药物中的用途。
本文提供了治疗、预防或改善高血糖症的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体。
本文提供了治疗、预防或改善高血糖症的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体与GLP-1的融合蛋白质。
本文提供了治疗、预防或改善二型糖尿病的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体。
本文提供了治疗、预防或改善二型糖尿病的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体与GLP-1的融合蛋白质。
本文提供了治疗、预防或改善代谢综合症的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体。
本文提供了治疗、预防或改善代谢综合症的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体与GLP-1的融合蛋白质。
本文提供了治疗、预防或改善血脂异常的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体。
本文提供了治疗、预防或改善血脂异常的方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体与GLP-1的融合蛋白质。
本文提供了治疗、预防或改善高血糖症、代谢综合症和包括血脂异常的一种或多种 症状方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体。
本文提供了治疗、预防或改善高血糖症、代谢综合症、或血脂异常的一种或多种症状方法，其包括给予受试者治疗有效量的本文所述的一个GCGR抗体与GLP-1的融合蛋白质。
附图简述
图一：显示了流式细胞术(FACS)检测小鼠腹水抗体L5H5(其包含SEQ ID NO:85与SEQ ID NO:95)与hGCGR特异性结合的结果，灰色峰为500nM小鼠腹水抗体L5H5和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(1a)、50nM(1b)和5nM(1c)的小鼠腹水抗体L5H5和CHO-DHFR-hGCGR的结合曲线，相对于灰色峰阴性对照有明显右移，证明了L5H5和CHO-DHFR-hGCGR的特异性结合。
图二：显示了流式细胞术(FACS)检测小鼠腹水抗体L4H4(其包含SEQ ID NO:84与SEQ ID NO:94)与hGCGR特异性结合的结果，灰色峰为500nM小鼠腹水抗体L4H4和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(2a)、50nM(2b)和5nM(2c)的小鼠腹水抗体L4H4和CHO-DHFR-hGCGR的结合曲线，相对于灰色峰阴性对照有明显右移，证明了L4H4和CHO-DHFR-hGCGR的特异性结合。
图三：显示了直接cAMP实验检测hGCGR抗体L7H7(其包含SEQ ID NO:87与SEQ ID NO:97)拮抗胰高血糖素激活hGCGR信号通路的浓度抑制曲线(IC 50＝139nM,R 2＝0.99)。
图四：显示了直接cAMP实验检测小鼠腹水GCGR抗体(L4H4和L5H5)拮抗梯度稀释的胰高血糖素激活hGCGR信号通路的Schild曲线。随着抗GCGR抗体浓度的提高，胰高血糖素激活hGCGR的S型曲线有明显的右移。
图五：显示了报告基因实验检测GCGR抗体与GLP-1的融合蛋白质GLP-1-Linker-L7H7，以及GMA102激活hGLP-1R信号通路的激活性曲线(EC 50分别为106pM和16pM,R 2＝0.99)。
图六：显示了高脂饲料诱导的C57BL/6肥胖小鼠中单次皮下注射L4H4、GMA102(每两天一次)，以及二者联合用药后，第14天检测口服糖耐量的结果。
图七：显示了健康恒河猴药代实验周期内hGCGR抗体与GLP-1融合蛋白质GLP-1-Linker-L7H7的药代(PK)时间曲线。
图八：显示了在健康恒河猴静脉葡萄糖耐量试验(IVGTT)中，单次皮下注射阳性对照药和GLP-1-Linker-L7H7前的胰岛素变化。
图九：显示了在健康恒河猴静脉葡萄糖耐量试验(IVGTT)中，单次皮下注射阳性对照药和GLP-1-Linker-L7H7后48小时的胰岛素变化。
图十：显示了在健康恒河猴静脉葡萄糖耐量试验(IVGTT)中，单次皮下注射阳性对照药和GLP-1-Linker-L7H7前的血糖变化。
图十一：显示了在健康恒河猴静脉葡萄糖耐量试验(IVGTT)中，单次皮下注射阳性对照药和GLP-1-Linker-L7H7后48小时的血糖变化。
具体实施方式
定义
除非本文另外定义，与本文相关的科学和技术术语应具有本领域普通技术人员所理解的含义。通常，与本文所述药物学、生物学、生物化学、细胞和组织培养学、生物学、分子生物学、免疫学、微生物学、遗传学和蛋白质核酸化学以及杂交相关的命名法和技术为本领域熟知和经常使用的。
本文使用标准的单字母或三字母缩写表明多聚核苷酸和多肽序列。多肽序列在书写时，带有氨基的首个氨基酸残基(N')在最左而带有羧基的最末氨基酸残基(C')在最右,例如本文所涉及的GLP-1片段序列：SEQ ID NO:119,SEQ ID NO:120,SEQ ID NO:121,SEQ ID NO:122,及SEQ ID NO:123。反向多肽序列指将多肽序列的氨基酸出现顺序逆向排列后形成的序列，例如上述GLP-1片段序列所形成的反向GLP-1片段序列：SEQ ID NO:127,SEQ ID NO:128,SEQ ID NO:129,SEQ ID NO:130,及SEQ ID NO:131。单链核酸序列和双链核酸序列的上游链的5’端在左而它们的3’端在右。多肽的具体部分可由氨基酸残基编号表示，例如氨基酸80至130，或由该位点的实际残基表示例如Lys80至Lys130。也可通过解释其与参比序列的差异描述具体的多肽或多聚核苷酸序列。
术语“肽”、“多肽”、和“蛋白”均指包含两个或多个通过肽健相互连接的氨基酸的分子。这些术语涵盖例如天然和人工蛋白和蛋白序列的多肽类似物(例如突变蛋白、变异体和融合蛋白)以及转录后或否则为共价或非共价修饰的蛋白。肽、多肽或蛋白可为单体或多聚体。
术语“多肽片段”指与对应的全长蛋白相比具有氨基端和/或羧基端缺失的多肽。片段长度可为，例如，至少5、6、7、8、9、10、11、12、13、14、15、20、50、70、80、90、100、150、或200个氨基酸。片段长度可为，例如，最多1000、750、500、250、200、175、150、125、100、90、80、70、60、50、40、30、20、15、14、13、12、11、或10个氨基酸。片段可在其一端或两端进一步包含一个或多个附加氨基酸，例如，来自不同天然蛋白质的氨基酸序列(例如，Fc或亮氨酸拉链结构域)或人工氨基酸序列(例如，人工接头序列)。
本文的多肽包括以任何原因和经任何方法修饰的多肽，例如，以：(1)降低蛋白水解敏感性，(2)降低氧化敏感性，(3)改变形成蛋白复合物的亲和性，(4)改变结合亲和性，以及(5)赋予或修饰其它物理化学或功能性质。类似物包含多肽的突变蛋白。例如，可在天然序列(例如在形成分子内接触的结构域之外的多肽部分)中进行单个或多个氨基酸替换(例如，保守氨基酸替换)。“保守氨基酸替换”为不显著改变母体序列结构特性者(例如，替换氨基酸不应破坏母体序列中出现的螺旋或干扰其它赋予母体序列特性或对其功能是必须的二级结构类型)。
多肽的“变异体”包含相对于另一多肽序列在氨基酸序列中插入、缺失和/或替换了一个或多个氨基酸残基的氨基酸序列。本文的变异体包括融合蛋白。
多肽的“衍生物”为经化学修饰的多肽，例如通过与其它化学部分例如聚乙二醇、白蛋白(例如人血清白蛋白)结合、磷酸化和糖基化。
除非另外说明，术语“抗体”包括两个全长重链和两个全长轻链的抗体，以及其衍生物，变异体、片段、和突变蛋白，其实例见下文。
术语“抗体”为包含与抗原结合部分并任选为允许抗原结合部分采取促进该抗体与该抗原结合的构象的支架或框架部分的蛋白。抗体的实例包括完整抗体、抗体片段(例如抗体的抗原结合部分)、抗体衍生物、和抗体类似物。该抗体可包含例如可选择的蛋白支架或具有移植CDRs或CDRs衍生物的人工支架。该支架包括但不限于包含被引入的例如以稳定化该抗体的三维结构的抗体衍生支架以及包含例如生物相容性多聚体的全合成支架。参见，例如，Korndorfer等，2003，Proteins 53:121-129；Roque等，2004,Biotechnol.Prog.20:639-654。此外，该抗体可以是模拟肽抗体(“PAMs”)或包含模拟抗体的支架，其如支架一样利用纤维蛋白连接素。
抗体可具有例如天然免疫球蛋白的结构。“免疫球蛋白”为四聚体分子。在天然的免疫球蛋白中，各四聚体由两个相同的多肽链对组成，各对具有一个“轻”链(约25kDa)和一个“重”链(约50-70kDa)。各链的氨基端包括约100至110氨基酸的可变结构域，主要与抗原识别相关。各链的羧基端部分确定了主要与效应器作用相关的恒定区。人的抗体轻链分为κ和λ轻链。重链分为μ、δ、α或ε，并确定了抗原的同种型，例如分别为IgM、IgD、IgG、IgA、和IgE。在轻链和重链中，可变和恒定区由约12或更多个氨基酸的“J”区连接，重链也包括约10多个氨基酸的“D”区。参见，Fundamental Immunology Ch.7(Paul编辑，第2版，Raven Press，1989)。各轻/重链对的可变区形成抗体结合位点，这样一个完整的免疫球蛋白具有两个结合位点。
天然免疫球蛋白链显示出由三个高度可变区连接的相对保守骨架区(FR)的相同基本结构，也被称作互补决定区或CDRs。从N端到C端，轻和重链均包含结构域FR1、CDR1、FR2、CDR2、FR3、CDR3、和FR4。各结构域氨基酸的分配与Kabat等在Sequences of Proteins of Immunological Interest，第5版，U.S.Dept.of Health and Human Services,PHS，NIH，NIH Publication No.91-3242，1991中的定义一致。
除非另外指明，“抗体”指完整的免疫球蛋白或其可与完整抗体竞争特异性结合的抗原结合部分。可由重组DNA技术或通过酶或化学裂解完整抗体生产抗原结合部分。抗原结合部分包括，尤其是，Fab、Fab’、F(ab’) 2、Fv、结构域抗体(dAbs)，包括互补决定区(CDRs)的片段、单链抗体(scFv)、嵌合抗体、双链抗体(diabodies)、三链抗体(triabodies)、四链抗体(tetrabodies)和至少包含足以赋予多肽特异抗原结合的免疫球蛋白的一部分的多肽。
Fab片段为具有V L、V H、C L、和C H1结构域的单价片段；F(ab’) 2片段为具有两个在铰链区由二硫键连接的Fab片段的二价片段；Fv片段具有V H和V L结构域；dAb片段具有V H结构域、V L结构域，或V H或V L结构域的抗原结合片段(美国专利号US6,846,634及US 6,696,245；美国专利申请公开号US 2005/0202512、US 2004/0202995、 US 2004/0038291、US 2004/0009507、及US 2003/0039958；Ward等，1989，Nature341:544-546)。
单链抗体(scFv)为一融合蛋白，其中的V L和V H区由接头(例如，合成的氨基酸残基序列)连接以形成连续蛋白质的抗体，其中该接头足够长以允许该蛋白链折叠回自身并形成单价抗原结合位点(参见，例如，Bird等，1988，Science 242:423-26；和Huston等，1988,Proc.Natl.Acad.Sci.U.S.A.85:5879-83)。
双链抗体为包含两个多肽链的二价抗体，其中各多肽链包含由接头连接的V H和V L结构域，该接头很短以致于不允许两个结构域在相同链上的配对，因此允许各结构域与另一多肽链上的互补结构域配对(参见，例如，Holliger等，1993，Proc.Natl.Acad.Sci.U.S.A.90:6444-48；和Poljak等，1994，Structure 2:1121-23)。如果双链抗体的两个多肽链是相同的，那么由它们配对得到的双链抗体将具有相同的抗原结合位点。具有不同序列的多肽链可用于制备具有不同抗原结合位点的双链抗体。相似地，三链抗体和四链抗体分别为包含三个和四个多肽链的抗体并分别形成三个和四个抗原结合位点，其可相同或不同。
本文使用Kabat等在Sequences of Proteins of Immunological Interest，第5版,U.S.Dept.of Health and Human Services,PHS,NIH,NIH Publication No.91-3242，1991中描述的方法鉴定给定抗体的互补决定区(CDRs)和框架区(FR)。可向分子中共价或非共价并入一个或多个CDRs使其成为抗体。抗体可以较大多肽链并入CDR(s)。可将CDR(s)共价连接至另一乡肽链，或非共价并入CDR(s)。CDRs允许抗体与具体的相关抗原特异性结合。
抗体可有一个或多个结合位点。如果多于一个结合位点，该结合位点可与另一个相同或不同。例如，天然人免疫球蛋白通常具有两个相同的结合位点，而“双特异性”或“双功能”抗体具有两个不同的结合位点。
术语“鼠源抗体”包括具有一个或多个来源于小鼠免疫球蛋白序列的可变区和恒定区的抗体。
术语“人源化抗体”是将小鼠抗体分子的互补决定区序列移植到人抗体可变区框架中而制成的抗体。
术语“抗原结合结构域”、“抗原结合区”或“抗原结合位点”为包含与抗原相互作用的氨基酸残基并有助于抗体对抗原的特异性和亲和力的抗体的部分。对与其抗原特异性结合的抗体而言，这将包括至少部分的至少一个其CDR结构域。
术语“表位”为与抗体(例如，通过抗体)结合的分子部分。表位可包含分子的非连续部分(例如，在多肽中，在多肽的一级序列中不连续的氨基酸残基在该多肽的三级和四级结构中相互足够接近以致于被一个抗体结合)。
两个多聚核苷酸或两个多肽序列的“相同百分比”由使用GAP计算机程序(GCG Wisconsin Package；version 10.3(Accelrys，San Diego，CA)的一部分)使用其默认参数比较序列测定。
术语“多聚核苷酸”、“寡聚核苷酸”和“核酸”可在全文中交替使用并包括DNA分子(例如，cDNA或基因组DNA)、RNA分子(例如mRNA)、使用核苷酸类似物(例如，肽核酸和非天然核苷酸类似物)生成的DNA或RNA类似物及其杂交体。核酸分子可为单链或双链。在一个实施方案中，本文的核酸分子包含编码本文提供抗体或其片段、衍生物、突变蛋白或变异体连续的开放阅读框。
如果它们的序列可反向平行排列则两个单链多聚核苷酸是相互“互补的”，这样一个多聚核苷酸中的各核苷酸与另一多聚核苷酸中的互补核苷酸相反，不会引入空隙并且各序列的5’或3’端没有未配对的核苷酸。如果两个多聚核苷酸可在中等严格条件下相互杂交那么一个多聚核苷酸与另一多聚核苷酸“互补”。因此，一个多聚核苷酸可与另一多聚核苷酸互补，但并不是它的互补序列。
术语“载体”为可用于将与其相连的另一核酸引入细胞的核酸。载体的一种类型为“质粒”，其指可连接附加核酸区段的线性或环状双链DNA分子。载体的另一类型为病毒载体(例如，复制缺陷逆转录病毒、腺病毒和腺病毒伴随病毒)，其中可将附加DNA区段引入病毒基因组。某些载体可在它们被引入的宿主细胞中自主复制(例如，包含细菌复制起点的细菌载体以及游离型哺乳动物载体)。其它载体(例如，非游离型哺乳动物载体)在引入宿主细胞时整合入宿主细胞的基因组中并因此与宿主基因组一起复制。“表达载体”为可引导所选多聚核苷酸表达的载体类型。
如果调控序列影响核苷酸序列的表达(例如，表达水平、时间或位点)那么核苷酸序列与调控序列“可操作地相连”。“调控序列”为可影响与其可操作相连的核酸的表达(例如，表达水平、时间或位点)的核酸。调控基因，例如，直接对受调控核酸发挥作用或通过一个或多个其它分子(例如，与调控序列和/或核酸结合的多聚核苷酸)的作用。调控序列的实例包括启动子、增强子和其它表达控制元件(例如，多腺苷酸化信号)。调控序列的进一步实例描述于例如Goeddel，1990,Gene Expression Technology:Methods in Enzymology,Volume 185,Academic Press,San Diego,CA；和Baron等,1995,Nucleic Acids Res.23:3605-06。
术语“宿主细胞”为用于表达核酸例如本文提供核酸的细胞。宿主细胞可为原核生物，例如大肠杆菌，或者其可为真核生物，例如单细胞真核生物(例如，酵母或其它真菌)、植物细胞(例如烟草或番茄植物细胞)、动物细胞(例如，人细胞、猴细胞、仓鼠细胞、大鼠细胞、小鼠细胞或昆虫细胞)或杂交瘤。通常，宿主细胞为可用多肽编码核酸转化或转染的培养细胞，其可接着在宿主细胞中表达。短语“重组宿主细胞”可用于表述用预期表达的核酸转化或转染的宿主细胞。宿主细胞也可为包含该核酸但是不以期望水平表达的细胞，除非向该宿主细胞引入了调控序列这样其与核酸可操作地相连。应理解的是术语宿主细胞不仅指具体的受试者细胞也指该细胞的子代或可能的子代。由于例如突变或环境影响后续世代会出现某些修饰，该子代事实上可能与母体细胞不同但是仍然属于本文使用的术语范围。
胰高血糖素受体
胰高血糖素受体属于七次跨膜的G蛋白偶联受体家族的B型，其通过异源三聚体鸟嘌呤核苷酸结合蛋白(G蛋白)与一个或多个胞内信号传导途径偶联(Jelinek等，1993，Science 259:1614-16)。截止目前的研究发现，GCGR主要表达在肝脏、肾、大脑、脂肪组织、胰腺和心脏(Ahren等，2015，Peptides 67:74-81；Jazayeri等，2016，Nature533:274-7)，主要参与人体内的糖代谢过程，因此也与糖尿病、血脂异常相关病症紧密关联(Lee等，2016，Diabetologia 59:1372-5)，本文中所使用的“人GCGR”和“hGCGR”均指代人源的胰高血糖素受体，可交替使用。本文中所使用的“鼠GCGR”和“mGCGR”均指代鼠源的胰高血糖素受体，也可交替使用。
在一个实施方案中，本文提供的抗体是与人GCGR特异性结合的抗体。在另一个实施方案中，本文提供的抗体是与细胞膜上的GCGR特异性结合的抗体，并且该抗体能抑制或者阻断胰高血糖素信号在这些细胞内的传导。在另一个实施方案中，本文提供的抗体是与人GCGR特异性结合的抗体，并且该抗体能与其它物种的GCGR结合(例如猴子或小鼠)，并阻断在这些物种中胰高血糖素的信号传导。在进一步的实施例中，本文提供的抗体是与人GCGR结合的鼠源抗体，并且该抗体能与其它物种的GCGR结合(例如猴子)。
在一个实施方案中，GCGR的氨基酸和多聚核苷酸序列如下所列，序列数据来源于美国国立生物技术信息中心的GeneBank数据库以及欧洲生物信息研究所的Uniprot数据库：
人(Homo sapiens)多聚核苷酸(SEQ ID NO：77)；登录号：BC104854；
人(Homo sapiens)氨基酸(SEQ ID NO：73)；登录号：P47871；
猴子(Rhesus macaque)多聚核苷酸(SEQ ID NO：78)；登录号：XM_015120592；
猴子(Rhesus macaque)氨基酸(SEQ ID NO：74)；登录号：A0A1D5QZY8；
大鼠(Rattus norvegicus)多聚核苷酸(SEQ ID NO:79)；登录号：X68692；
大鼠(Rattus norvegicus)氨基酸(SEQ ID NO:75)；登录号：P30082；
小鼠(Mus musculus)多聚核苷酸(SEQ ID NO：80)；登录号：BC031885；及
小鼠(Mus musculus)氨基酸(SEQ ID NO：76)；登录号：Q61606。
胰高血糖素受体抗体(GCGR抗体)
在一个实施方案中，本文提供的是GCGR抗体。在另一个实施方案中，本文提供的GCGR抗体是完整GCGR抗体。在另一个实施方案中，本文提供的GCGR抗体是GCGR抗体片段。在另一个实施方案中，本文提供的GCGR抗体是GCGR抗体衍生物。在另一个实施方案中，本文提供的GCGR抗体是GCGR抗体突变蛋白质。在进一步的实施方案中，本文提供的GCGR抗体是GCGR抗体变异体。
在一个实施方式中，本文提供的GCGR抗体包含一个、两个、三个、四个、五个、或六个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；
b.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；
d.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34；
e.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35；及
f.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
表1列出本文提供的GCGR抗体的轻链CDRs的氨基酸序列，以及其相应的多聚核苷酸编码序列。表2列出本文提供的GCGR抗体的重链CDRs的氨基酸序列，以及其相应的多聚核苷酸编码序列。
表1：轻链CDRs的氨基酸序列及其多聚核苷酸编码序列
Figure PCTCN2019078674-appb-000001
Figure PCTCN2019078674-appb-000002
表2：重链CDRs的氨基酸序列及其多聚核苷酸编码序列
Figure PCTCN2019078674-appb-000003
Figure PCTCN2019078674-appb-000004
在一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差5、4、3、2、或1个单氨基酸添加、替换和/或缺失的序列。在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差4、3、2、或1个单氨基酸添加、替换和/或缺失的序列。
在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差3、2、或1个单氨基酸添加、替换和/或缺失的序列。
在另一个实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差2或1个单氨基酸添加、替换和/或缺失的序列。
在进一步的实施方案中，本文提供的抗体包含与表1和表2中所列的CDR氨基酸序列中之一相差1个单氨基酸添加、替换和/或缺失的序列。
在一个实施方案中，本文提供的GCGR抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；及
b.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34。
在另一个实施方案中，本文提供的GCGR抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；及
b.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35。
在另一个实施方案中，本文提供的GCGR抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；及
b.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
在另一个实施方案中，本文提供的GCGR抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；
b.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34；
c.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；及
d.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35。
在另一个实施方案中，本文提供的GCGR抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；
b.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；及
d.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
在进一步的实施方案中，本文提供的GCGR抗体包含一个，两个，三个，或四个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；
b.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35；
c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；及
d.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
在一个实施方案中，本文提供的GCGR抗体包含一个，两个，或三个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、及SEQ ID NO:21。
在另一个实施方案中，本文提供的GCGR抗体包含一个，两个，或三个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36。
在一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:22、SEQ ID NO:4与SEQ ID NO:25、SEQ ID NO:7与SEQ ID NO:28、SEQ ID NO:10与SEQ ID NO:31、SEQ ID NO:13与SEQ ID NO:34、SEQ ID NO:16与SEQ ID NO:34、SEQ ID NO:18与SEQ ID NO:34、及SEQ ID NO:20与SEQ ID NO:34。
在另一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:23、SEQ ID NO:5与SEQ ID NO:26、SEQ ID NO:8与SEQ ID NO:29、SEQ ID NO:11与SEQ ID NO:32、及SEQ ID NO:14与SEQ ID NO:35。
在进一步的实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:24、SEQ ID NO:6与SEQ ID NO:27、SEQ ID NO:9与SEQ ID NO:30、SEQ ID NO:12与SEQ ID NO:33、SEQ ID NO:15与SEQ ID NO:36、SEQ ID NO:17与SEQ ID NO:36、SEQ ID NO:19与SEQ ID NO:36、及SEQ ID NO:21与SEQ ID NO:36。
在一个实施方案中，本文提供的GCGR抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:22、SEQ ID NO:4与SEQ ID NO:25、SEQ ID NO:7与SEQ ID NO:28、SEQ ID NO:10与SEQ ID NO:31、SEQ ID NO:13与SEQ ID NO:34、SEQ ID NO:16与SEQ ID NO:34、SEQ ID NO:18与SEQ ID NO:34、及SEQ ID NO:20与SEQ ID NO:34；和
b.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:23、SEQ ID NO:5与SEQ ID NO:26、SEQ ID NO:8与SEQ ID NO:29、SEQ ID NO:11与SEQ ID NO:32、及SEQ ID NO:14与SEQ ID NO:35。
在另一个实施方案中，本文提供的GCGR抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:22、SEQ ID NO:4与SEQ ID NO:25、SEQ ID NO:7与SEQ ID NO:28、SEQ ID NO:10与SEQ ID NO:31、SEQ ID NO:13与SEQ ID NO:34、SEQ ID NO:16与SEQ ID NO:34、SEQ ID NO:18与SEQ ID NO:34、及SEQ ID NO:20与SEQ ID NO:34；和
b.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:24、SEQ ID NO:6与SEQ ID NO:27、SEQ ID NO:9与SEQ ID NO:30、SEQ ID NO:12与SEQ ID NO:33、SEQ ID NO:15与SEQ ID NO:36、SEQ ID NO:17与SEQ ID NO:36、SEQ ID NO:19与SEQ ID NO:36、及SEQ ID NO:21与SEQ ID NO:36。
在另一个实施方案中，本文提供的GCGR抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:23、SEQ ID NO:5与SEQ ID NO:26、SEQ ID NO:8与SEQ ID NO:29、SEQ ID NO:11与SEQ ID NO:32、及SEQ ID NO:14与SEQ ID NO:35；和
b.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:24、SEQ ID NO:6与SEQ ID NO:27、SEQ ID NO:9与SEQ ID NO:30、SEQ ID NO:12与SEQ ID NO:33、SEQ ID NO:15与SEQ ID NO:36、SEQ ID NO:17与SEQ ID NO:36、SEQ ID NO:19与SEQ ID NO:36、及SEQ ID NO:21与SEQ ID NO:36。
在进一步实施方案中，本文提供的GCGR抗体包含：
a.一个独立地选自于以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:22、SEQ ID NO:4与SEQ ID NO:25、SEQ ID NO:7与SEQ ID NO:28、SEQ ID NO:10与SEQ ID NO:31、SEQ ID NO:13与SEQ ID NO:34、SEQ ID NO:16与SEQ ID NO:34、SEQ ID NO:18与SEQ ID NO:34、及SEQ ID NO:20与SEQ ID NO:34；
b.一个独立地选自于以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:23、SEQ ID NO:5与SEQ ID NO:26、SEQ ID NO:8与SEQ ID NO:29、 SEQ ID NO:11与SEQ ID NO:32、及SEQ ID NO:14与SEQ ID NO:35；和
c.一个独立地选自于以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:24、SEQ ID NO:6与SEQ ID NO:27、SEQ ID NO:9与SEQ ID NO:30、SEQ ID NO:12与SEQ ID NO:33、SEQ ID NO:15与SEQ ID NO:36、SEQ ID NO:17与SEQ ID NO:36、SEQ ID NO:19与SEQ ID NO:36、及SEQ ID NO:21与SEQ ID NO:36。
在一个实施方案中，本文提供的GCGR抗体包含：
a.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:22、SEQ ID NO:23、及SEQ ID NO:24；
b.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:25、SEQ ID NO:26、及SEQ ID NO:27；
c.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:28、SEQ ID NO:29、及SEQ ID NO:30；
d.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:31、SEQ ID NO:32、及SEQ ID NO:33；
e.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36；
f.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:16、SEQ ID NO:14、SEQ ID NO:17、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36；
g.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:18、SEQ ID NO:14、SEQ ID NO:19、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36；及
h.轻链和重链CDR1、CDR2、及CDR3氨基酸序列的组合：SEQ ID NO:20、SEQ ID NO:14、SEQ ID NO:21、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36。
在一个实施方案中，本文提供的GCGR抗体包含一个或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列氨基酸序列：
a.轻链可变结构域氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、及SEQ ID NO:90；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列；及
b.重链可变结构域氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列。
在另一个实施方案中，本文提供的GCGR抗体的多聚核苷酸编码序列包含一个或两个多聚核苷酸编码序列，其中每个多聚核苷酸编码序列独立地选自于以下所列多聚核苷酸序列：
a.轻链可变结构域多聚核苷酸编码序列：SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO: 100、及SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、及SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、及SEQ ID NO:107；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列；及
b.重链可变结构域多聚核苷酸编码序列：SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、及SEQ ID NO:114；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸编码序列。
在一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、及SEQ ID NO:90。
在另一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97。
在一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的轻链与重链可变结构域氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91、SEQ ID NO:82与SEQ ID NO:92、SEQ ID NO:83与SEQ ID NO:93、SEQ ID NO:84与SEQ ID NO:94、SEQ ID NO:85与SEQ ID NO:95、SEQ ID NO:86与SEQ ID NO:96、SEQ ID NO:87与SEQ ID NO:97、SEQ ID NO:88与SEQ ID NO:97、SEQ ID NO:89与SEQ ID NO:97、及SEQ ID NO:90与SEQ ID NO:97。
在一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97。
在另一个实施方案中，本文提供的GCGR抗体包含一个独立地选自于以下所列的轻链与重链可变结构域氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91(L1H1)、SEQ ID NO:82与SEQ ID NO:92(L2H2)、SEQ ID NO:84与SEQ ID NO:94(L4H4)、SEQ ID NO:85与SEQ ID NO:95(L5H5)、SEQ ID NO:86与SEQ ID NO:96(L6H6)、SEQ ID NO:87与SEQ ID NO:97(L7H7)、SEQ ID NO:88与SEQ ID NO:97(L8H7)、及SEQ ID NO:89与SEQ ID NO:97(L9H7)。
本文也可用“LxHy”符号来指代所提供的GCGR抗体，其中“x”对应于轻链可变区序列代号；“y”对应于重链可变区序列代号。例如，L2H2指具有包含SEQ ID NO:82(L2)氨基酸序列的轻链可变区和包含SEQ ID NO:92(H2)氨基酸序列的重链可变区的完整抗体。
在一个实施方案中，本文提供的GCGR抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链恒定氨基酸序列：SEQ ID NO:115及SEQ ID NO:116；及
b.重链恒定氨基酸序列：SEQ ID NO:117及SEQ ID NO:118。
在一个实施方案中，本文提供的GCGR抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
a.轻链恒定氨基酸序列：SEQ ID NO:115、SEQ ID NO:116、SEQ ID NO:132、及SEQ ID NO:133；及
b.重链恒定氨基酸序列：SEQ ID NO:117及SEQ ID NO:118。
在一个实施方案中，本文提供的GCGR抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的轻链和重链恒定氨基酸序列的组合：SEQ ID NO:115和SEQ ID NO:117、SEQ ID NO:115和SEQ ID NO:118、SEQ ID NO:116和SEQ ID NO:117、和SEQ ID NO:116和SEQ ID NO:118。
在另一个实施方案中，本文提供的GCGR抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的轻链和重链恒定氨基酸序列的组合：SEQ ID NO:115和SEQ ID NO:117、SEQ ID NO:115和SEQ ID NO:118、SEQ ID NO:116和SEQ ID NO:117、SEQ ID NO:116和SEQ ID NO:118、SEQ ID NO:132和SEQ ID NO:117、SEQ ID NO:132和SEQ ID NO:118、SEQ ID NO:133和SEQ ID NO:117、及SEQ ID NO:133和SEQ ID NO:118。
在一个实施方案中，本文提供的GCGR抗体包含本文所列轻链及重链CDRs，以及FRs(框架)的氨基酸序列。FRs的氨基酸序列包含于轻链或者重链可变结构域氨基酸序列中，未单独陈列。在一个实施方案中，该抗体包含一个本文所列的轻链CDR1序列。在另一个实施方案中，该抗体包含一个本文所列的轻链CDR2序列。在另一个实施方案中，该抗体包含一个本文所列的轻链CDR3序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR1序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR2序列。在另一个实施方案中，该抗体包含一个本文所列的重链CDR3序列。在另一个实施方案中，该抗体包含一个本文的轻链FR1序列。在另一个实施方案中，该抗体包含一个本文的轻链FR2序列。在另一个实施方案中，该抗体包含一个本文的轻链FR3序列。在另一个实施方案中，该抗体包含一个本文的轻链FR4序列。在另一个实施方案中，该抗体包含一个本文的重链FR1序列。在另一个实施方案中，该抗体包含一个本文的重链FR2序列。在另一个实施方案中，该抗体包含一个本文的重链FR3序列。在进一步的实施方案中，该抗体包含一个本文的重链FR4序列。
在一个实施方案中，该抗体的轻链CDR3序列与本文所列轻链CDR3氨基酸序列SEQ ID NO:12、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21之一相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。在另一个实施方案中，该抗体的重链CDR3序列与本文所列重链CDR3氨基酸序列SEQ ID NO:33或SEQ ID NO:36相差最多不超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。在进一步的实施方案中，该抗体的轻链CDR3序列与本文所列轻链CDR3氨基酸 序列SEQ ID NO:12、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21之一相差不得超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失，并且该抗体的重链CDR3序列与本文所列重链CDR3氨基酸序列SEQ ID NO:33或SEQ ID NO:36相差最多不超过6、5、4、3、2、或1个单氨基酸添加、替换和/或缺失。在另一个实施方案中，该抗体进一步包含1、2、3、4、5、或6个本文所列轻重链CDR轻重链序列组合。
在一个实施方案中，本文提供的GCGR抗体包含一个轻链可变结构域氨基酸序列，该序列选择于本文所列的L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)轻链可变结构域序列。在一个实施方案中，该GCGR抗体的轻链可变结构域氨基酸序列与L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)之一的轻链可变结构域氨基酸序列存在15、14、13、12、11、10、9、8、7、6、5、4、3、2、或1个氨基酸的差异的，其中各序列的差异独立为一个氨基酸残基的缺失、插入或替换。在另一个实施方案中，该GCGR抗体的轻链可变结构域氨基酸序列与L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)之一的轻链可变结构域氨基酸序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该GCGR抗体的轻链可变结构域多聚核苷酸编码序列与L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)之一的多聚核苷酸编码序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该GCGR抗体的轻链可变结构域多聚核苷酸编码序列包含在中等条件下与L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)之一的轻链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。在进一步的实施方案中，该GCGR抗体的轻链可变结构域多聚核苷酸编码序列包含在严格条件下与L1(SEQ ID NO:81)、L2(SEQ ID NO:82)、L4(SEQ ID NO:84)、L5(SEQ ID NO:85)、L6(SEQ ID NO:86)、L7(SEQ ID NO:87)、L8(SEQ ID NO:88)、及L9(SEQ ID NO:89)之一的轻链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。
在一个实施方案中，本文提供的GCGR抗体包含一个重链可变结构域氨基酸序列，该序列选择于本文所列H1(SEQ ID NO:91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)重链可变结构域序列。在另一个实施方案中，该GCGR抗体的重链可变结构域氨基酸序列与H1(SEQ ID NO: 91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)之一的重链可变结构域序列存在15、14、13、12、11、10、9、8、7、6、5、4、3、2、或1个氨基酸差异，其中各序列的差异独立为一个氨基酸残基的缺失、插入或替换。在另一个实施方案中，该GCGR抗体的重链可变结构域氨基酸序列与H1(SEQ ID NO:91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)之一的重链可变结构域序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该GCGR抗体的重链可变结构域多聚核苷酸编码序列与H1(SEQ ID NO:91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)之一的重链可变结构域多聚核苷酸编码序列有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少97％、或至少99％相同。在另一个实施方案中，该GCGR抗体的重链可变结构域多聚核苷酸编码序列包含在中等严格条件下与H1(SEQ ID NO:91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)之一的重链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。在一个实施方案中，该GCGR抗体的重链可变结构域多聚核苷酸编码序列包含在严格条件下与H1(SEQ ID NO:91)、H2(SEQ ID NO:92)、H4(SEQ ID NO:94)、H5(SEQ ID NO:95)、H6(SEQ ID NO:96)、及H7(SEQ ID NO:97)之一的重链可变结构域的多聚核苷酸编码序列互补序列杂交的多聚核苷酸序列。
在一个实施方案中，本文提供的抗体是一包含L1H1(SEQ ID NO:81与SEQ ID NO:91)、L2H2(SEQ ID NO:82与SEQ ID NO:92)、L3H3(SEQ ID NO:83与SEQ ID NO:93)、L4H4(SEQ ID NO:84与SEQ ID NO:94)、L5H5(SEQ ID NO:85与SEQ ID NO:95)、L6H6(SEQ ID NO:86与SEQ ID NO:96)、L7H7(SEQ ID NO:87与SEQ ID NO:97)、L8H7(SEQ ID NO:88与SEQ ID NO:97)、L9H7(SEQ ID NO:89与SEQ ID NO:97)、或L10H7(SEQ ID NO:90与SEQ ID NO:97)组合的抗体，或其一的期望表型(例如，IgA、IgG1、IgG2a、IgG2b、IgG3、IgM、IgE、或IgD)，或其一Fab或F(ab')2片段。
在一个实施方案中，本文提供的抗体是一包含L1H1(SEQ ID NO:81与SEQ ID NO:91)、L2H2(SEQ ID NO:82与SEQ ID NO:92)、L4H4(SEQ ID NO:84与SEQ ID NO:94)、L5H5(SEQ ID NO:85与SEQ ID NO:95)、L6H6(SEQ ID NO:86与SEQ ID NO:96)、L7H7(SEQ ID NO:87与SEQ ID NO:97)、L8H6(SEQ ID NO:88与SEQ ID NO:97)或L9H7(SEQ ID NO:89与SEQ ID NO:97)组合的抗体，或其一类转换的抗体(例如，IgA、IgG1、IgG2a、IgG2b、IgG3、IgM、IgE、和IgD)，或其一Fab或F(ab')2片段。
本文提供的抗体可包含本领域已知的恒定区中任何一个。轻链恒定区可为例如κ或λ型轻链恒定区，例如小鼠κ或λ型轻链恒定区。重链恒定区可为例如α、δ、ε、γ、或μ型重链恒定区，例如小鼠α、δ、ε、γ、或μ型重链恒定区。在一个实施方案中，该轻链或重链恒定区为天然恒定区的片段、衍生物、变异体、或突变蛋白。
在一个实施方案中，本文提供的抗体进一步包含人恒定轻链κ或λ结构域或其片段。轻链恒定区的氨基酸序列如下：
人恒定轻链κ结构域氨基酸序列：(SEQ ID NO:115)；及
人恒定轻链λ结构域氨基酸序列：(SEQ ID NO:116)。
在一个实施方案中，本文提供的抗体进一步包含人恒定轻链κ或λ结构域或其片段。轻链恒定区的氨基酸序列如下：
人恒定轻链κ结构域氨基酸序列：(SEQ ID NO:132)；及
人恒定轻链λ结构域氨基酸序列：(SEQ ID NO:133)。
在另一个实施方案中，本文提供的抗体进一步包含人重链恒定结构域或其片段。重链恒定区氨基酸序列序列如下：
人重链恒定区氨基酸序列(hIgG2)：(SEQ ID NO:117)；及
人重链恒定区氨基酸序列(hIgG4)：(SEQ ID NO:118)。
在一个实施方式中，本文所提供的GCGR抗体选自鼠源抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、抗原结合抗体片段、单链抗体、双链抗体、三链抗体、四链抗体、Fab片段、F(ab’)x片段、结构域抗体、IgD抗体、IgE抗体、IgM抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
在一个实施方式中，本文提供的GCGR抗体为GCGR单克隆抗体。
在另一个实施方式中，本文提供的GCGR抗体为一单克隆抗体,该单克隆抗体包含一个选自于以下所列的氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91、SEQ ID NO:82与SEQ ID NO:92、SEQ ID NO:83与SEQ ID NO:93、SEQ ID NO:84与SEQ ID NO:94、SEQ ID NO:85与SEQ ID NO:95、SEQ ID NO:86与SEQ ID NO:96、SEQ ID NO:87与SEQ ID NO:97、SEQ ID NO:88与SEQ ID NO:97、SEQ ID NO:89与SEQ ID NO:97、及SEQ ID NO:90与SEQ ID NO:97。
在一个实施方式中，本文提供的GCGR抗体为鼠源GCGR抗体。在另一个实施方式中，本文提供的GCGR抗体为人源化GCGR抗体。
在一个实施方式中，本文提供的GCGR抗体降低人信号传导的IC 50值为大约1nM至大约300nM或大约1nM至大约150nM。
抗体和抗体片段
在一个实施方案中，本文提供的抗体为完整抗体(包括具有全长重和/或轻链的多克隆、单克隆、嵌合、人源化、或人类抗体)。在另一个实施方案中，本文提供的抗体为抗体片段，列如F(ab’) 2、Fab、Fab’、Fv、Fc、或Fd片段，单结构域抗体、单链抗体、最大抗体(maxibodies)、微抗体(minibodies)、内抗体(intrabodies)、二链抗体、三链抗体、四链抗体、v-NAR、或bis-scFv(参见，例如Hollinger and Hudson，2005，Nature Biotechnology 23:1126-1136)。在另一个实施方案中，本文提供的抗体包括如美国专利号6703199中所公开的抗体多肽，包括纤维结合素多肽单抗体。在进一步的实施方案中，本文提供的抗体包括如美国专利出版物2005/0238646中所公开的单链多肽。
在一个实施方案中，单克隆抗体的基因的可变区是使用核苷酸引物扩增在杂交瘤中表达。这些引物可由本领域普通技术人员合成或从商业来源购买。鼠和人可变区引物包括V Ha、V Hb、V Hc、V Hd、C H1、V L、和C L区的引物可从商业来源购买。这些引物可用于扩增重链或轻链可变区，然后将其分别插入载体例如IMMUNOZAP TMH或IMMUNOZAP TML(Stratagene)中。然后将这些载体引入大肠杆菌、酵母或哺乳动物为基础的表达系统。这些方法可使用于生产大量包含V H和V L结构域融合的单链蛋白(参见Bird等，1988，Science 242:423-426)。
本领域技术人员应理解的是一些蛋白质，例如抗体，可能进行可多种转录后修饰。这些修饰的类型和程度取决于用于表达该蛋白的宿主细胞系以及培养条件。该类修饰包括糖基化作用、甲硫氨酸氧化、二酮哌嗪形成、天冬氨酸异构化和天冬酰胺脱酰胺作用的变化。抗体的羧端碱性残基(例如赖氨酸或精氨酸)因羧肽酶的频繁修饰作用而可能丢失(参见，Harris，1995，Journal of Chromatography 705:129-134)。
鼠单克隆抗体可使用常用的杂交瘤细胞方法来生产。该单克隆抗体可通过多种已确立的技术分离和纯化。该类分离技术包括蛋白A-琼脂糖的亲和色谱法、分子排阻色谱法、和离子交换色谱法(参见，例如，Coligan第2.7.1-2.7.12页和第2.9.1-2.9.3页；Baines等，“Purification of Immunoglobulin G(IgG),”Methods in Molecular Biology，第10卷，第79-104页(The Humana Press，Inc.，1992))。该单克隆抗体可使用基于抗体的特殊性质(例如，重链或轻链同种型、结合特异性等)筛选的适当配基通过亲和色谱法来纯化。亲和色谱的适当配基的实例包括蛋白A、蛋白G、抗恒定区(轻链或重链)抗体、抗独特型抗体以及TGF-β结合蛋白或其片段或变异体。
可使用抗体结合位点中央的互补决定区(CDRs)对分子进行亲和力成熟化的改造，来得到亲和性增加的抗体，例如针对c-erbB-2的亲和性增加的抗体(Schier等，1996，J.Mol.Biol.263:551-567)。因此，该类技术可用于制备人GCGR的抗体。
例如可在检测是否存在人GCGR的体外或体内测定法中使用针对人GCGR的抗体。
也可通过任何传统技术制备抗体。例如，可从天然表达这些抗体的细胞将其纯化(例如，可从生产抗体的杂交瘤将其纯化)或使用本领域任何已知的技术在重组表达系统中生产。参见，例如，Monoclonal Antibodies,Hybridomas:A New Dimension in Biological Analyses,Kennet等编辑，Plenum Press(1980)；和Antibodies:A Laboratory Manual,Harlow and Land编辑,Cold Spring Harbor Laboratory Press(1988)。这在下文的核酸部分讨论。
可通过任何已知技术制备抗体并筛选期望性质。一些技术涉及分离编码相关抗体(例如，抗GCGR抗体)的多肽链(或其部分)的核酸，并通过重组DNA技术操作核酸。该核酸可与另一相关核酸融合或经修饰(例如通过诱变或其它传统技术)以添加、缺失或替换一个或多个氨基酸残基。
当需要提高根据本文包含一个或多个上述CDRs的抗体的亲和性时，可通过多种亲和成熟方案包括维持CDRs(Yang等，1995，J.Mol.Biol.254:392-403)、链替换(Marks 等，1992，Bio/Technology 10:779-783)、使用大肠杆菌的突变株(Low等，1996，J.Mol.Biol.250:350-368)DNA重排(Patten等，1997，Curr.Opin.Biotechnol.8:724-733)、噬菌体展示(Thompson等，1996，J.Mol.Biol.256:7-88)以及其它PCR技术(Crameri等，1998，Nature 391：288-291)。所有这些亲和力成熟方法讨论于Vaughan等，1998，Nature Biotechnology 16:535-539中。
在一个实施方案中，本文提供的抗体为抗GCGR片段。该片段可完全由抗体衍生序列组成或可包含附加序列。抗原结合片段的实例包括Fab、F(ab’)2、单链抗体、双链抗体、三链抗体、四链抗体和结构域抗体，其它实例提供于Lunde等，2002，Biochem.Soc.Trans.30:500-06。
可经氨基酸桥(短肽接头)连接重链和轻链可变结构域(Fv区)形成单链抗体，从而得到单多肽链。已通过将编码肽接头的DNA融合在编码两个可变结构域多肽(V L和V H)的DNAs之间制备该单链Fvs(scFvs)。所得多肽可折叠回自身形成抗原结合单体，或它们可形成多聚体(例如，二聚体、三聚体或四聚体)，取决于两个可变结构域之间的柔性接头的长度(Kortt等，1997，Prot.Eng.10:423；Kortt等2001，Biomol.Eng.18:95-108)。通过组合包含多肽的不同V L和V H，可形成与不同表型结合的多体scFvs(Kriangkum等，2001，Biomol.Eng.18:31-40)。已研发的用于生产单链抗体的技术包括美国专利号4946778；Bird，1988，Science 242:423；Huston等，1988,Proc.Natl.Acad.Sci.USA 85:5879-5883；Ward等，1989，Nature 334:544-546；de Graaf等，2002，Methods Mol Biol.178:379-87申描述的那些。来源于本文提供的抗体的单链抗体包括但不限于包含可变结构域组合L1H1的scFvs，均涵盖于本文。
也可通过抗体的蛋白水解作用例如根据传统方法用胃蛋白酶或木瓜蛋白酶消化完整的抗体获得来源于抗体的抗原结合片段。举例而言，可用胃蛋白酶酶裂解抗体提供称作F(ab’)2的SS片段生产抗体片段。可使用巯基还原剂进一步裂解这一片段产生3.5S Fab’单价片段。可选择的方案有，使用巯基保护基团进行该裂解反应得到二硫键的裂解；另外还可以使用木瓜蛋白酶的酶裂解直接产生两个单价Fab片段和一个Fc片段。这些方法描述于例如Goldenberg，美国专利号4,331,647，Nisonoff等，1960，Arch.Biochem.Biophys.89:230；Porter，1959，Biochem.J.73:119；Edelman等，Methods in Enzymology l:422(Academic Press，1967)；以及Andrews和Titus,J.A.Current Protocols in Immunology(Coligan等编辑，John Wiley&Sons,2003)，第2.8,1-2.8.10页和第2.10A.1-2.10A.5页。其它裂解抗体的方法，例如制备重链以形成单价重、轻链片段(Fd)，进一步裂解片段或也可使用其它酶、化学或基因技术，只要片段与可被该完整抗体识别的抗原结合。
另一种形式的抗体片段为包含一个或多个抗体互补决定区(CDRs)的肽。可通过构建编码相关CDR的多肽获得CDRs。例如可通过使用聚合酶链式反应用抗体生成细胞的mRNA作为模板合成可变区制备该类多肽，参见，例如，Larrick等，1991，Methods:A Companion to Methods in Enzymology 2:106；Courtenay-Luck，“Genetic Manipulation of Monoclonal Antibodies,”Monoclonal Antibodies:Production,Engineering and Clinical  Application，Ritter等编辑，166页(Cambridge University Press，1995)；和Ward等，“Genetic Manipulation and Expression of Antibodies,”Monoclonal Antibodies:Principles and Applications，Birch等编辑，137页(Wiley-Liss，Inc.，1995)。该抗体片段可进一步包含本文所述抗体的至少一个可变结构域。因此，例如，V区结构域可为单体并且是V H或V L结构域，其可以如下文所述的至少等于1x 10 -7M或更高的亲和力独立与GCGR结合。
该可变区结构域可为任何天然可变结构域或其基因工程形式。基因工程形式指使用重组DNA工程技术生产的可变区结构域。该基因工程形式包括例如通过向特异抗体的氨基酸序列插入、缺失或改变从特异抗体可变区产生的。具体实例包括包含只含一个CDR以及任选来自一个抗体的一个或多个框架氨基酸和来自另一抗体的可变区结构域剩余部分，并由基因工程组装成的可变区结构域。
可变区结构域可与至少一个其它抗体结构域或其片段在C端氨基酸共价连接。因此，举例而言，存在于可变区结构域的V H结构域可与免疫球蛋白C H1结构域或其片段相连。相似地，V L结构域可与C K结构域或其片段相连。以这种方式，例如，该抗体可为Fab片段，其中抗原结合结构域包含它们的C端分别与C H1和C K结构域共价连接的联合V H和V L结构域。可用其它氨基酸延长C H1结构域，例如以提供铰链区或如Fab’片段中的部分铰链结构域或提供其它结构域，例如抗体C H2和C H3结构域。
抗体的衍生物和变异体
例如可通过随机诱变或通过定点诱变(例如寡聚核苷酸诱导的定点诱变)改变编码对应于氨基酸序列L1和H1的核苷酸序列以产生与未突变多聚核苷酸相比包含一个或多个具体核苷酸替换、缺失或插入的经改变的多聚核苷酸。用于产生该类改变的技术实例描述于Walder等，1986，Gene 42:133；Bauer等，1985，Gene 37:73；Craik,1985,BioTechniques,3:12-19；Smith等，1981,Genetic Engineering:Principles and Methods，Plenum Press；以及美国专利号4518584和4737462。这些和其它方法可用于产生例如与未衍生化抗体相比具有期望性质例如亲和性、亲和力或对GCGR的特异性增强、体内或体外活性或稳定性增强或体内副作用降低的抗GCGR抗体的衍生物。
本文领域的其它抗GCGR抗体衍生物包括抗GCGR抗体或其片段与它蛋白或多肽的共价或聚集结合物，例如通过表达包含与抗GCGR抗体多肽的N端或C端融合的异源多肽的重组融合蛋白。例如，该结合肽可为异源信号(或引导)多肽，例如酵母α因子前导肽或例如表位标签的肽。包含融合蛋白的抗体可包含被添加以辅助抗体的纯化或鉴定的肽(例如多聚组氨酸)。抗体也可与FLAG肽连接，如Hopp等，1988,Bio/Technology 6:1204和美国专利5011912所述。FLAG肽具有高抗原性并提供被特异单克隆抗体(mAb)可逆结合的表位，允许已表达重组蛋白的快速检测和方便纯化。可商业购买(Sigma-Aldrich，St.Louis，MO)用于制备其中FLAG肽与给定多肽融合的融合蛋白的试剂，在另一个实施方案中，包含一个或多个抗体的寡聚体可用作GCGR拮抗剂或用更高级的寡聚体。寡聚体可以是共价连接的或非共价连接的二聚体、三聚体或 更高的寡聚体形式。可使用包含两个或更多个抗体的寡具体，其中一个实例为同型二聚体。其它寡聚体包括异二聚体、同型三聚体、异三聚体、同型四聚体、杂四聚体等。
一个实施方案是针对包含多个抗体的寡聚体，它们通过与抗体融合的肽部分之间的共价或非共价相互作用连接。该类肽可为肽接头(spacers)或具有促进寡聚化作用性质的肽。亮氨酸拉链和某些来源于抗体的多肽为可促进抗体寡聚化的肽，如下文详细描述。
在具体的实施方案中，寡聚体包含两个至四个抗体。寡聚体的抗体可为任何形式，如上文所述任何形式，例如变异体或片段。优选地，该寡聚体包含具有GCGR结合活性的抗体。
在一个实施方案中，使用来源于免疫球蛋白的多肽制备寡聚体。制备包含一些与抗体衍生多肽(包括Fc结构域)的不同部位融合的异源多肽已描述于例如Ashkenazi等，1991，PNAS USA 88:10535；Byrn等，1990,Nature 344:677；和Hollenbaugh等，Construction of Immunoglobulin Fusion Proteins,Current Protocols in Immunology,Suppl.4，第10.19.1-10.19.11页。本文的一个实施方案是针对包含两个由融合抗GCGR抗体的胰高血糖素结合片段与抗体的Fc区产生的融合蛋白的二聚体。可通过以下方式制备二聚体：例如在适当的表达载体中插入编码融合蛋白的基因融合，在用重组表达载体转化的宿主细胞中表达该融合基因并允许已表达融合蛋白像抗体分子一样组装，其中Fc部分之间的链间二硫键形成二聚体。
如本文所使用术语“Fc多肽”包括来源于抗体Fc区的天然和突变蛋白形式的多肽。也包括包含促进二聚体化的铰链区的该类多肽的截短形式。包含Fc部分(以及由其形成的寡聚体)的融合蛋白提供了在蛋白A或蛋白G柱子上进行亲和层析法方便纯化的优势。
PCT申请W0 93/10151(以参考形式并于本文)中一种适当的Fc多肽为从N端铰链区延伸至人IgG1抗体的Fc区的天然C端的单链多肽。另一种可用的Fc多肽为美国专利5457035和Baum等，1994,EMBO J.13:3992-4001中描述的Fc突变蛋白。该突变蛋白的氨基酸序列与W0 93/10151中所示天然Fc序列的氨基酸序列相同，除了氨基酸19从亮氨酸变为丙氨酸，氨基酸20从亮氨酸变为谷氨酰胺以及氨基酸22从甘氨酸变为丙氨酸。该突变蛋白表现出对Fc受体的亲和力降低。在其它实施方案中，抗GCGR抗体的重链和/或轻链可被取代为抗体重链和/或轻链的可变部分。
或者，该寡聚体为包含多个抗体的融合蛋白，包含或不包含接头肽(spacer peptides)。这些适当的接头肽描述于美国专利4751180和4935233。
制备寡聚抗体的另一种方法涉及使用亮氨酸拉链。亮氨酸拉链结构域为促进它们所存在的蛋白寡聚化作用的肽。最初发现亮氨酸拉链存在于数种DNA结合蛋白中(Landschulz等，1988，Science 240:1759)，此后发现存在于各种不同蛋白中。在已知的亮氨酸拉链中为可二聚体化或三聚体化的天然肽或其衍生物。适用于生产可溶寡聚蛋白的亮氨酸拉链结构域的实例描述于PCT申请W0 94/10308，来源于肺表面活性蛋白D(SPD)的亮氨酸拉链描述于Hoppe筝，1994，FEBS Letters 344:191，以参考形式并于 本文。允许与其融合的异源蛋白稳定三聚体化的经修饰的亮氨酸拉链的使用描述于Fanslow等，1994，Semin.Immunol.6:267-78。在一种方法中，在适当的宿主细胞中表达包含与亮氨酸拉链肽融合的抗GCGR抗体片段或衍生物的重组融合蛋白，从培养物上清中收集可溶寡聚抗GCGR抗体片段或其衍生物。
在另一个实施方案中，该抗体衍生物可包含至少本文公开的CDRs之一。举例而言，可将一个或多个CDR整合入已知的抗体骨架区(IgG1，IgG2等)或与适当的载体结合以增强其半衰期。适当载体包括但不限于Fc、白蛋白、转铁蛋及类似物质。这些和其它适当的载体为本领域已知。该结合CDR肽可为单体、二聚体、四聚体或其它形式。在一个实施方案中，一个或多个水溶性多聚体在结合剂的一个或多个特异位点结合，例如在氨基端。在一个实例中，抗体衍生物包含一个或多个水溶性多聚体附着物包括但不限于聚乙二醇、聚氧乙烯二醇或聚丙二醇。参见，例如，美国专利号4640835、4496689、4301144、4670417、4791192和4179337，在一些实施方案中，衍生物包含一个或多个一甲氧基.聚乙二醇、葡聚糖、纤维素或其它基于碳水化合物的聚合物，聚(N-乙烯基吡咯酮).聚乙二醇、聚氧乙烯多元醇(例如甘油)和聚乙烯醇，以及该类聚合物的混合物。在一些实施方案中，一个或多个水溶性聚合物与一个或多个侧链随机结合。在一些实施方案中。PEG可提高结合剂例如抗体的治疗作用。一些该类方法描述于例如美国专利号6133426，其以参考形式以任何目的并于本文。
应当理解的是本文提供的抗体可具有至少一个氨基酸替换，只要该抗体保留了结合特异性。因此，抗体结构的修饰包含于本文范畴。这些可包括不破坏抗体GCGR结合能力的氨基酸替换，其可为保守或非保守的。保守氨基酸替换可包括非天然氨基酸残基，其通常经化学肽合成整合而不是生物系统合成。这些包括拟肽和其它反向或倒转形式的氨基酸部分。保守氨基酸替换也可涉及用非天然残基替换天然氨基酸残基这样对该位点氨基酸残基的极性或电荷作用很小或没有作用。非保守替换可涉及一类氨基酸或氨基酸类似物的一个成员与具有不同物理性质(例如，体积、极性、疏水性、电荷)的另一类氨基酸的成员交换。
而且，本领域技术人员可生成在各期望氨基酸残基上包含氨基酸替换的待测变异体。可使用本领域技术人员已知的活性测定法筛选该类变异体。该类变异体可用于收集关于适当变异体的信息。举例而言，如果发现某一氨基酸残基可引起活性破坏、非期望的降低或不当的活性，可避免具有该类变化的变异体。换言之，基于从这些常规试验收集的信息，本领域技术人员可轻松确定应避免进一步替换(单独或与其它突变组合)的氨基酸。
技术人员可使用已知技术确定如本文所列的多肽的适当变异体。在一些实施方案中，本领域技术人员可通过靶向对于活性不重要的区域鉴定经改变后不会破坏活性的分子适当区域。在一些实施方案中，可鉴定在相似多肽中保守的残基或分子部分。在一些实施方案中，甚至可保守替换对于生物活性或结构重要的区域而不破坏生物活性或不会有不利作用于多肽结构。此外，本领域技术人员可考察结构.功能研究鉴定对活性或结 构重要的相似多肽中的残基。鉴于这一对比，可预测对应于在相似蛋白中对活性或结构重要的氨基酸残基的蛋白质中氨基酸残基的重要性。本领域技术人员可为这些经预测重要的氨基酸残基选择化学相似氨基酸替换。
本领域技术人员也可分析与相似多肽的结构相关的三维结构和氨基酸序列。鉴于该类信息，本领域技术人员可预测就三维结构而言抗体的氨基酸残基比对。在一些实施方案中，本领域技术人员可选择不对经预测在蛋白质表面的氨基酸残基进行显著改变，因为该类残基可能参与与其它分子的重要相互作用。许多科学出版物致力于二级结构的预测。参见，Moult，1996，Curr.Op.Biotech.7:422-427，Chou等，1974，Biochemistry13:222-245；Chou等，1974，Biochemistry 113:211-222；Chou等，1978，Adv.Enzymol.Relat.Areas Mol.Biol.47:45-148；Chou等，1979，Ann.Rev.Biochem.47:251-276和Chou等，Biophys.J.26:367-384。此外，目前可使用计算机程序辅助预测二级结构。举例而言，序列同一性大于30％或相似性大于40％的两个多肽或蛋白质通常具有相似的高级结构。近期蛋白结构数据库(PDB)的增长增强了二级结构的可预测性，包括多肽或蛋白结构中潜在的折叠数量。参见，Holm等，1999，Nucl.Acid.Res.27:244-247。已表明(Brenner等，1997，Curr.Op.Struct.Biol.7:369-376)在给定多肽或蛋白质中存在有限数量的折叠并且一旦确定了临界数量的结构，结构预测将变得显著更加精确。
预测二级结构的其它方法包括“穿接(threading)”(Jones，1997，Curr.Opin.Struct.Biol.,7:377-87；Sippl等，1996，Structure 4:15-19；“图谱分析(profile analysis)”(Bowie等，1991，Science 253:164-170；Gribskov等，1990，Meth.Enzym.183:146-159；Gribskov等，1987，Proc.Nat.Acad.Sci.USA 84:4355-4358和“进化联系(evolutionary linkage)”(参见Holm,supra(1999),and Brenner,supra(1997))。在一些实施方案中，抗体变异体包括糖基化变异体，其中与母体多肽的氨基酸序列相比改变了糖基化位点的数量和/或类型。在一些实施方案中，变异体与天然蛋白质相比具有更多或更少数量的N连接糖基化位点。或者，去除该序列的替换可移除现有的N连接糖链。也提供了N连接糖链的重排，其中去除了一个或多个N连接糖链位点(通常为天然存在的那些)并创造了一个或多个新的N连接位点。其它优选抗体变异体包括半胱氨酸变异体，与母体氨基酸序列相比其中缺失或由另一氨基酸(例如丝氨酸)替换一个或多个半胱氨酸残基。当抗体必须折叠成生物活性构象时(例如在分离可溶包涵体之后)可用半胱氨酸变异体。半胱氨酸变异体通常比天然蛋白质具有较少的半胱氨酸残基，并通常具有偶数个半胱氨酸以最小化未配对半胱氨酸引起的相互作用。
本领域技术人员可在需要该类替换时确定期望的氨基酸替换(保守或非保守)。在一些实施方案中，氨基酸替换可用于鉴定人GCGR抗体的重要残基或者增加或降低本文所述人GCGR抗体的亲和力。
根据一些实施方案，优选的氨基酸替换为以下：(1)降低蛋白质水解敏感性，(2)降低氧化敏感性，(3)改变形成蛋白质复合物的结合亲和力，(4)改变结合亲和力和/或(4)赋予或修饰该类多肽上的其它物理化学或功能性质。根据一些实施方案，可在 天然存在序列中(在一些实施方案中，在形成分子间接触的结构域之外的多肽部分)进行单个或多个氨基酸替换(在一些实施方案中为保守氨基酸替换)。在一些实施方案中，保守氨基酸替换通常不会本质上改变母体序列的结构特性(例如，替换氨基酸不应破解存在于母体序列中的螺旋或干扰特征化母体序列的其它类型二级结构)。本领域认可的多肽二级和三级结构的实例描述于Proteins，Structures and Molecular Principles，Creighton编辑，W.H.Freeman and Company(1984)；Introduction to Protein Structure，Branden and Tooze编辑,Garland Publishing(1991)；以及Thornton等，1991,Nature354:105，其以参考形式并于本文。
在一些实施方案中，本文提供的抗体可与多聚体、脂类或其它部分(moieties)化学键合。
抗原结合试剂可包含至少一个本文描述的CDRs，其掺入生物相容性骨架结构中。在一个实例中，该生物相容性骨架结构包含足以形成构象稳定结构支持或骨架或支架的多肽或其部分，其可在局限的表面区域展示可与抗原结合的一个或多个氨基酸序列(例如，CDRs、可变区等)。该类结构可为天然存在多肽或多肽“折叠”(结构基序)，或相对与天然多肽或折叠可具有一个或多个修饰，例如氨基酸添加、缺失或替换。这些支架可来源于任何物种(或多于一个物种)的多肽，例如，人类、其它哺乳动物、其它脊椎动物、无脊椎动物、细菌或病毒。
生物可溶性骨架结构通常是基于蛋白质支架或骨架而不是免疫球蛋白结构域。举例而言，可使用基于纤维结合素、锚蛋白、脂质运载蛋白(lipocalin)、新抑癌蛋白、细胞色素b、CP1锌指蛋白、PST1、卷曲螺旋、LACI-D1、Z结构域和淀粉酶抑肽结构域(参见，例如，Nygren和Uhlen,1997,Current Opinion in Structural Biology 7:463-469)。
此外，本领域技术人员可认识到适当的结合剂包括这些抗体的部分，例如一个或多个重链CDR1、CDR2、CDR3，轻链CDR1、CDR2和CDR3，如本文所具体公开。至少一个重链CDR1、CDR2、CDR3、CDR1，CDR2和CDR3区具有至少一个氨基酸替换，只要该抗体保留了非替换CDR的结合特异性。该抗体的非CDR部分可为非蛋白分子，其中该结合剂交叉阻断本文公开的抗体与人GCGR的结合和/或抑制经该受体的胰高血糖素的信号传导。该抗体的非CDR部分可为非蛋白质分子，其中该抗体在竞争结合测定法中显示出与至少抗体L4H4/L5H5之一所显示相似的与人胰高血糖素肽的结合类型，和/或中和胰高血糖素的活性。抗体的非CDR部分可由氨基酸组成，其中该抗体为重组结合蛋白或合成肽，并且该重组结合蛋白交叉阻断本文公开的抗体与人GCGR的结合和/或中和体内或体外胰高血糖素活性。抗体的非CDR部分可由氨基酸组成，其中该抗体为重组抗体，并且该重组抗体在竞争结合测定法中显示出与至少抗体L4H4/L5H5之一所显示相似的与人GCGR肽的结合类型，和/或中和胰高血糖素信号传导。
GCGR抗体与GLP-1的融合蛋白质
在一个实施方案中，本文提供了一个GCGR抗体与GLP-1的融合蛋白质，其包含 一个能与GCGR特异性结合的抗体，和一个，二个，三个，四个，五个，六个，七个，或八个GLP-1片段或反向GLP-1片段；该融合蛋白质通过一肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接，或者将一反向GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
在另一个实施方案中，本文提供了一个GCGR抗体与GLP-1的融合蛋白质，其包含一个与GCGR特异性结合的抗体，和一个，二个，三个，或四个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接。
在另一个实施方案中，本文提供了一个GCGR抗体与GLP-1的融合蛋白质，其包含一个能与GCGR特异性结合的抗体，和一个，二个，三个，或四个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
在另一个实施方案中，本文提供了一个GCGR抗体与GLP-1的融合蛋白质，其包含一个能与GCGR特异性结合的抗体，和二个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或者重链的氨基端连接。
在另一个实施方案中，本文提供了一个GCGR抗体与GLP-1的融合蛋白质，其包含一个能与GCGR特异性结合的抗体，和二个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链或者重链的羧基端连接。
在另一个实施方案中，本文提供了一个GLP-1融合蛋白质，其包含一个GCGR抗体和二个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链的氨基端连接：N'-GLP-1-Linker-R-C'；或者将一GLP-1片段的羧基端和一GCGR抗体重链的氨基端连接：N'-GLP-1-Linker-R-C'；其中:N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,GLP-1代表一GLP-1片段，R为一GCGR抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头序列。
另一个实施方案中，本文提供了一个GLP-1融合蛋白质，其包含一个GCGR抗体和二个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链的羧基端连接：N'-R-Linker-反向GLP-1-C'；或者将一反向GLP-1片段的氨基端和一GCGR抗体重链的羧基端连接：N'-R-Linker-反向GLP-1-C'；其中:N'代表融合蛋白质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,反向GLP-1代表一反向GLP-1片段，R为一GCGR抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头序列。
在进一步的实施方案中，本文提供了一个GLP-1融合蛋白质，其包含一个GCGR抗体和二个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与GCGR抗体轻链的氨基端连接：N'-GLP-1-Linker-R-C'；其中:N'代表融合蛋白 质多肽链的氨基端,C'代表融合蛋白质多肽链的羧基端,GLP-1代表一GLP-1片段，R为一GCGR抗体的轻链的氨基酸序列,及Linker代表一肽接头序列。
在一实施方案中，在本文提供的GLP-1融合蛋白质中，所述的GLP-1片段各自独立地选自于以下之一的氨基酸序列：SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO:121、SEQ ID NO:122、及SEQ ID NO:123。在一实施方案中，在本文提供的GLP-1融合蛋白质中，所述的反向GLP-1片段各自独立地选自于以下之一的氨基酸序列：SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、及SEQ ID NO:131。
在一实施方案中，在本文提供的GLP-1融合蛋白质中，所述的肽接头(Linker)的序列自独立包含从1个至200个氨基酸胺，从2个至100个氨基酸胺，从5个至50个氨基酸胺，从6个至25个氨基酸胺，或从10个至20个氨基酸胺。
在另一个实施方案中，在本文提供的GLP-1融合蛋白质中，所述的肽接头(Linker)的序列包含全长的、部分的、或者重复的独立选自以下之一的氨基酸序列：SEQ ID NO:124、SEQ ID NO:125、及SEQ ID NO:126。
核酸
一方面，本文提供分离的核酸分子。该核酸分子包含例如编码全部或部分抗体的多聚核苷酸，例如本文抗体或GLP-1融合蛋白质的一条链或两条链，或其片段、衍生物、突变蛋白或变异体；足以用作杂交探针的多聚核苷酸；PCR引物或用于鉴定、分析、突变或扩增编码多肽的多聚核苷酸的测序引物；用于抑制多聚核苷酸表达的反义核酸以及其互补序列。该核酸可为任何长度。例如它们的长度可为5、10、15、20、25、30、35、40、45、50、75、100、125、150、175、200、250、300、350、400、450、500、750、1000、1500、3000、5000或更多个核苷酸，和/或包含一个或多个附加序列，例如调控序列，和/或是较大核酸例如载体的一部分。该核酸可为单链或双链并包含RNA和/或DNA核苷酸以及其人工变异体(例如，肽核酸)。
可从经GCGR抗原免疫的小鼠B细胞中分离编码抗体多肽(例如，重链或轻链、仅可变结构域或全长)的核酸。可通过常规方法例如聚合酶链式反应(PCR)分离抗体或GLP-1融合蛋白质的核酸。
编码重链和轻链可变区的核酸序列如上文所示。熟练的技术人员可理解由于遗传密码的简并性，本文公开的各多肽序列可由更多数量的其他核酸序列编码。本文提供编码本文提供的抗体或GLP-1融合蛋白质的各简并核苷酸序列。
本文进一步提供在具体杂交条件下与其他核酸(例如，包含任何L4H4/L5H5的核苷酸序列的核酸)杂交的核酸。杂交核酸的方法为本领域熟知。参见，例如，Current Protocols in Molecular Biology，John Wiley&Son(1989)，6.3.1-6.3.6。如本文定义，例如，中等严格条件使用包含5x氯化钠/柠檬酸钠(SSC)的预洗溶液、0.5％SDS、1.0mM EDTA(pH 8.0)、约50％甲酰胺的杂交缓冲液、6x SSC和55℃的杂交温度(或其他相似的杂交溶液，例如包含绚50％甲酰胺的，以42℃杂交)，并且洗脱条件为60℃，使用0.5x SSC、0.1％SDS。严格杂交条件在6x SSC中于45℃杂交，然后于68℃在0.1x SSC、 0.2％SDS中洗涤一次或多次。此外，本领域技术人员可操作杂交和/或洗涤条件以增加或降低杂交严格度这样包含相互之间至少65、70、75、80、85、90、95、98或99％同源的核苷酸序列的核酸通常仍可以相互杂交。影响杂交条件选择的基本参数和设计适当条件的指导列于例如Sambrook，Fritsch和Maniatis，1989，Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory Press，第9和11章；Current Protocols in Molecular Biology，1995，Ausubel等编辑，John Wiley&Sons，Inc.，第2.10和6.3-6.4节)并可由具有本领域普通技术的人员基于例如DNA的长度和/或碱基组成轻松确定。可通过突变在核酸中引入变化，藉此导致其编码的多肽(例如，抗原结合蛋白)氨基酸序列的变化。可使用本领域已知的任何技术引入突变。在一个实施方案中，使用例如定点诱变方案改变一个或多个具体氨基酸残基。在另一个实施方案中，使用例如随机诱变方案改变一个或多个随机选择的残基。无论其如何生成，可表达突变多肽并筛选期望性质。
可将突变引入核酸而不显著改变其编码多肽的生物学活性。例如，可进行引起非必需氨基酸残基处氨基酸替换的核苷酸替换。在一个实施方案中，突变本文为L1至L10和H1至H7或GLP-1融合蛋白质提供的核苷酸序列或其片段、变异体或衍生物这样其编码包含本文所示L1至L10和H1至H7的氨基酸残基的一个或多个缺失或替换，成为两个或多个序列相异的残基。在另一个实施方案中，诱变作用在本文所示L1至L10和H1至H7或GLP-1融合蛋白质的一个或多个氨基酸残基附近插入一个氨基酸成为两个或多个序列相异的残基。或者，可将一个或多个突变引入核酸以选择性改变其编码多肽的生物学活性(例如，与GCGR结合)。例如，该突变可在数量上或性质上改变生物学活性。量变的实例包括增加、降低或消除该活性。质变的实例包括改变抗体或GLP-1融合蛋白质的抗原特异性。
在另一方面，本文提供适于用做引物或检测本文核酸序列的杂交探针的核酸分子。本文的核酸分子可仅包含编码本文全长多肽的核酸序列的一部分，例如，可用作探针或引物或编码本文多肽活性部分的片段(例如，GCGR结合部分)的片段。
基于本文核酸序列的探针可用于检测该核酸或相似核酸，例如编码本文多肽的转录物。该探针可包含标记基团，例如放射性同位素、荧光化合物、酶或酶辅因子。该类探针可用于鉴定表达该多肽的细胞。
在另一方面本文提供包含编码本文多肽或其部分的核酸的载体。载体的实例包括但是不限于质粒、病毒载体、非游离基因哺乳动物载体和表达载体，例如重组表达载体。
本文的重组表达载体可包含适于该核酸在宿主细胞中表达的形式的本文核酸。该重组表达载体包括一个或多个调控序列，基于用于表达的宿主细胞进行筛选，其与该预表达的核酸序列可操作性相连。调控序列包括引导核苷酸序列在多个种类宿主细胞中组成型表达的(例如，SV40早期基因增强剂、劳斯氏肉瘤病毒启动子和细胞巨化病毒启动子)，引导仅在某些宿主细胞中核苷酸序列的表达的(例如，组织特异调控序列，参见Voss等，1986，Trends Biochem.Sci.11:287，Maniatis等，1987，Science 236:1237， 其完整内容以参考形式并于本文)以及引导核苷酸序列响应具体处理或条件的诱导型表达的(例如，哺乳动物细胞中的金属硫堇启动子和原核和真核系统二者中的四环霉素反应(tet-sesponsive)启动子和/或链霉素反应启动子(同前))。本领域技术人员应理解表达载体的设计取决于例如用于转化的宿主细胞的选择、所需蛋白表达水平等因素。本文的表达载体可引入宿主细胞，藉此生产由本文所述核酸编码的蛋白或肽，包括融合蛋白或肽。
另一方面，本文提供可引入本文表达载体的宿主细胞。宿主细胞可为任何原核或真核细胞。原核宿主细胞包括革兰氏阴性或革兰氏阳性生物体，例如大肠杆菌或杆菌。更高级的真核细胞包括昆虫细胞、酵母细胞以及哺乳动物源的确立细胞系。适当哺乳动物宿主细胞系的实例包括中国仓鼠卵巢(CHO)细胞或它们的衍生物例如Veggie CHO和在无血清培养基中生长的相关细胞系(参见Rasmussen等，1998，Cytotechnology 28:31)或CHO株DXB-11，其缺失DHFR(参见Urlaub等，1980，Proc.Natl.Acad,Sci.USA77:4216-20)。其它CHO细胞系包括CHO-K1(ATCC#CCL-61)、EM9(ATCC#CRL-1861)，和UV20(ATCC#CRL-1862)，其它宿主细胞包括猴肾细胞的COS-7系(ATCC#CRL-1651)(参见Gluzman等，1981，Cell 23:175)、L细胞、C127细胞、3T3细胞(ATCC CCL-163)，AM-1/D细胞(描述于美国专利序列号6210924)、HeLa细胞、BHK(ATCC CRL-10)细胞系、来源于非洲绿猴肾细胞系CV1的CV1/EBNA细胞系(ATCC CCL-70)(参见McMahan等，1991，EMBO J.10:2821)、人胚肾细胞例如293，293EBNA或MSR 293、人上皮A431细胞、人C010205细胞、其它经转化灵长动物细胞系、正常二倍体细胞、来源于初生组织体外培养物的细胞株、初移植体、HL-60、U937、HaK或Jurkat细胞。用于细菌、真菌、酵母和哺乳细胞宿主的适当克隆和表达载体描述于Pouwels等(Cloning Vectors:A Laboratory Manual,Elsevier,1985)。
可通过传统转化或转染技术将载体DNA引入原核或真核细胞中。对于稳定的哺乳动物转染而言，取决于使用的表达载体和转染技术，已知只有一小部分细胞可将外源DNA鏊合入它们的基因组中。为了鉴定和筛选这些整合子，通常将编码筛选标记(例如抗生素抗性)的基因与所关注基因一起引入宿主细胞。优选的筛选标记包括可赋予药物(如G418、潮霉素和甲氨喋呤)抗性的那些。在其它方法中可通过药物筛选鉴别包含被引入核酸的稳定转染细胞(例如，整合了筛选基因的细胞可存活，而其它细胞则死亡)。
可在提高多肽表达的条件下培养已转化细胞，可通过常规蛋白纯化方法回收多肽。一种该纯化方法描述于下文实施例。预用于本文的多肽包括基本同源的重组哺乳动物抗GCGR抗体或GLP-1融合蛋白质多肽，其基本不含污染性内源材料。
GCGR抗体的活性
GCGR抗体的活性是指本文提供的抗体具有与GCGR特异性结合，抑制或者阻断胰高血糖素信号传导后显示出的治疗性作用，例如治疗高血糖症、二型糖尿病、代谢综合症、和血脂异常。术语“降低胰高血糖素信号传导的生物学活性”或者“抑制或阻断胰 高血糖素信号传导的生物学活性”指GCGR抗体或其与GLP-1融合蛋白质在体内与GCGR结合并抑制或者阻断胰高血糖素引起该受体下游的细胞应急反应。反应包括但是不限于降低肝糖原输出、降低血糖、以及相关的脂肪代谢变化。在一个实施方案中，本文提供了能与人GCGR特异性结合的鼠源抗体或人源化抗体。该类抗体包括可减少或中和胰高血糖素信号传导的拮抗或中和抗体。
在一个实施方案中，本文提供的抗体与人GCGR结合时的K d为大约0.01nM至大约1000nM、大约0.1nM至大约500nM、大约0.5nM至大约200nM、大约1nM至大约200nM、或大约10nM至大约100nM。在另一个实施方案中，本文提供的抗体与人GCGR结合时的K d为大约1nM至大约200nM。在另一个实施方案中，本文提供的抗体与人GCGR结合时的K d为大约1nM至大约100nM。在另一个实施方案中，本文提供的抗体与人GCGR结合时的K d为大约1nM、大约2nM、大约5nM、大约10nM、大约20nM、大约30nM、大约40nM、大约50nM、大约60nM、大约70nM、大约80nM、大约90nM、或大约100nM。在另一个实施方案中，本文提供的抗体与人GCGR结合时的K d为大约100nM、大约110nM、大约120nM、大约130nM、大约140nM、大约150nM、大约160nM、大约170nM、大约180nM、大约190nM、或大约200nM。
在一个实施方案中，本文提供的抗体在降低人胰高血糖素信号传导的IC 50值为大约0.01nM至大约500nM、大约0.1nM至大约200nM、大约0.5nM至大约200nM、大约1nM至大约200nM、或大约10nM至大约100nM。在另一个实施方案中，本文提供的抗体在降低人胰高血糖素信号传导的IC 50值为大约1nM至大约200nM。在另一个实施方案中，本文提供的抗体在降低人胰高血糖素信号传导的IC 50值为大约10nM至大约100nM。在另一个实施方案中，本文提供的抗体在降低人胰高血糖素信号传导的IC 50值为大约1nM、大约2nM、大约5nM、大约10nM、大约20nM、大约30nM、大约40nM、大约50nM、大约60nM、大约70nM、大约80nM、大约90nM、或大约100nM。在另一个实施方案中，本文提供的抗体在降低人胰高血糖素信号传导的IC 50值为大约100nM、大约110nM、大约120nM、大约130nM、大约140nM、大约150nM、大约160nM、大约170nM、大约180nM、大约190nM、或大约200nM。
在一个实施方式中，本文提供的GCGR抗体与人GCGR结合时，具有一个或多个以下所列的性质：
a.当与人GCGR结合时以与所述参比抗体基本类似的K d；
b.当抑制人GCGR的胰高血糖素激活时以与所述参比抗体基本类似的IC 50；和
c.在人GCGR上与所述参比抗体交叉竞争结合。
在一个实施方式中,所述参比抗体包含轻链可变结构域氨基酸序列SEQ ID NO:87和重链可变结构域氨基酸序列SEQ ID NO:97的组合。在另一实施方式中,所述参比抗体为单克隆抗体L4H4、L5H5、或L7H7。
在本文中，术语“基本相似”意为与参比抗体的IC 50或K d可比或是参比抗体的IC 50或K d值的大约200％、大约180％、大约160％、大约150％、大约140％、大约120％、 大约110％、大约100％、大约99％、大约98％、大约97％、大约95％、大约90％、大约85％、大约80％、大约75％、大约70％、大约65％、或大约50％。在一个实施方案中，参比抗体包括，例如，具有轻链SEQ ID NO:87和重链SEQ ID NO:97组合的抗体。在另一个实施方案中，参比抗体包括GCGR抗体L4H4、L5H5、或L7H7。
GCGR抗体与GLP-1的融合蛋白质(GLP-1融合蛋白质)的生物学活性
GCGR抗体与GLP-1的融合蛋白质的生物学活性包含GLP-1的生物学活性和GCGR抗体活性两个方面。GCGR抗体的活性如前文所述。“GLP-1生物学活性”指GCGR抗体与GLP-1的融合蛋白质在体内结合并激活GLP-1受体并引起细胞应激反应，并显示出治疗作用的生物学活性，例如高血糖症、二型糖尿病、代谢综合症和包括血脂异常的其它相关症状。前述细胞应激反应包括但是不限于降低肝糖原输出、降低血糖，以及相关的脂肪代谢变化。综合了GLP-1和GCGR抗体的生物学活性，本文所述的GLP-1融合蛋白质可以用于治疗多种与GLP-1R和GCGR相关联的疾病和病症。该融合蛋白质通过作用于GLP-1R和/或GCGR发挥其生物学作用，因此可以用本文所述的GLP-1融合蛋白质治疗对“增加GLP-1R刺激”或对“降低GCGR刺激”做出有利应答的疾病和病症的受试者。这些受试者称为“需要GLP-1R刺激治疗”或“需要降低GCGR刺激”的受试者。包括高血糖症、二型糖尿病、代谢综合症、和血脂异常的其它相关症状。
在一个实施方案中，GCGR抗体或GLP-1融合蛋白质的生物学活性变化是用直接cAMP检测方法来检测的，量化GCGR抗体或GLP-1融合蛋白质在体外抑制GCGR的功能。
药物组合物
在一个实施方案中，本文提供了一药物组合物，其中包含本文提供的一个GCGR抗体及一种或多种可药用载体。
在另一个实施方案中，本文提供了一药物组合物，其包括本文提供的一个GCGR抗体与GLP-1的融合蛋白质，与一种或多种可药用载体。
本文所使用的术语“载体”包括载体、药用辅料或者在使用的剂量和浓度下将细胞或哺乳动物暴露于其中无害的稳定剂。
治疗方法
在一个实施方案中，本文提供了治疗、预防、或改善二型糖尿病的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善二型糖尿病的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体与GLP-1的融合蛋白质,或其药用组合物。
在一个实施方案中，本文提供了治疗、预防、或改善二型糖尿病并发症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善二型糖尿病并发症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体与GLP-1的融合蛋白质,或其 药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善高血糖症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体，或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善高血糖症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体与GLP-1的融合蛋白质,或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善代谢综合症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体,或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善代谢综合症的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体与GLP-1的融合蛋白质,或其药用组合物。
在另一个实施方案中，本文提供了治疗、预防、或改善血脂异常的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体，或其药用组合物。
在进一步的实施方案中，本文提供了治疗、预防、或改善血脂异常的方法，其包括给予受试者治疗有效量的本文提供的GCGR抗体与GLP-1的融合蛋白质,或其药用组合物。
在本文提供的任意一项用途，其所述的药用组合物是用于静脉或皮下注射。
在本文提供的任意一项用途，进一步包含给予受试者治疗有效量的一种GLP-1R抗体与GLP-1的融合蛋白质。
在一个实施方案中，本文所述的GLP-1R抗体与GLP-1的融合蛋白质包含
a.轻链CDR1氨基酸序列：SEQ ID NO:134；
b.轻链CDR2氨基酸序列：SEQ ID NO:135；
c.轻链CDR3氨基酸序列：SEQ ID NO:136；
d.重链CDR1氨基酸序列：SEQ ID NO:137；
e.重链CDR2氨基酸序列：SEQ ID NO:138；及
f.重链CDR3氨基酸序列：SEQ ID NO:139。
在另一个实施方案中，本文所述的GLP-1R抗体与GLP-1的融合蛋白质包含以下所列的轻链与重链可变区氨基酸序列的组合：SEQ ID NO:140与SEQ ID NO:141。
其它GLP-1R抗体与GLP-1的融合蛋白质的实例公开于US 10,059,773and US2018/0000934，其公开内容通过参考并入本文。
本文中，术语“受试者”指哺乳动物，包括人类，可与术语“患者”交替使用。
术语“治疗”包括减轻或预防至少一种症状或病症的其它方面，或者减轻疾病严重性。本文提供的GCGR抗体或GCGR抗体与GLP-1的融合蛋白质不需要产生完全治愈的效果，或根除疾病的所有症状或表现，即可构成有效治疗剂。如相关领域所公认，作为治疗剂的药物可减少给定疾病状态的严重程度，但不需消除疾病的所有表现即可被认为是有效的治疗剂。相似地，预防给药治疗不需在预防症状出现上完全有效即可构成有 效预防剂。只减少疾病的影响(例如，通过减少其症状的数量或严重度，或通过提高另一治疗效果，或通过产生另一有效作用)，或者减少受试者中疾病发生或加重的可能性就已经足够。本文的一个实施方案涉及包含以足以诱导反应具体病症严重性的指示剂高于基线水平的持续改善的量和时间给予患者GCGR抗体或GCGR抗体与GLP-1融合蛋白质的方法。
GCGR抗体或GCGR抗体与GLP-1的融合蛋白质药物组合物可采用任意适当技术包括但不限于肠道外、局部或吸入给药。如果是注射，可通过例如关节内、静脉内、肌肉内、损伤区内、腹膜内或皮下途径，以快速注射或连续输注给予药用组合物。可考虑例如在疾病或损伤部位局部给药，如透皮给药和埋植剂持续释放给药。吸入给药包括例如鼻腔或口腔吸入、采用喷雾剂、以气雾剂形式吸入抗体等等。其它选择包括口腔制剂包括片剂、糖浆剂或锭剂。
以包含一个或更多其它组分例如生理学可接受载体、辅料或稀释剂的组合物形式给予本文提供的GCGR抗体或GLP-1融合蛋白质是有利的。组合物可任选额外包含一个或更多如下所述的生理学活性剂。在多个具体实施方案中，组合物包含除一个或更多本文提供的抗体(例如鼠源抗体或人源化抗体)或GLP-1融合蛋白质之外的一个、两个、三个、四个、五个或六个生理学活性剂。
在一个实施方案中，药物组合物包含本文提供的鼠源抗体或人源化抗体或GLP-1融合蛋白质以及一个或更多选自以下的物质：pH适合于抗体或GLP-1融合蛋白质的缓冲液、抗氧化剂例如抗坏血酸、低分子量多肽(例如含少于10个氨基酸的多肽)、蛋白质、氨基酸、糖例如糊精、络合物例如EDTA、谷胱甘肽、稳定剂和辅料。根据适当工业标准，也可加入防腐剂。可使用适当辅料溶液作为稀释剂将组合物配制成冻干粉末。适当组分在所用剂量和浓度下对受者无毒。可用于药物处方组分的进一步实例见Remington's Pharmaceutical Sciences，第16版(1980)和20版(2000)，Mack Publishing Company提供医学从业者使用的试剂盒，其包括一种或更多本文提供的抗体或GLP-1融合蛋白质以及治疗本文讨论任何病症的标签或其它说明。在一个实施方案中，试剂盒包括以上述组合物形式装在一个或多个管型瓶中的一种或多种抗体或GLP-1融合蛋白质的无菌制剂。
给药剂量和频率可根据以下因素而改变：给药途径、所用具体抗体或GLP-1融合蛋白质、所治疾病的性质和严重度、症状为急性还是慢性以及患者的体积和总体症状。可通过本领域熟知的方法确定适当剂量，例如在临床试验中包括剂量放大研究。
本文提供的抗体或GLP-1融合蛋白质可在例如一段时间内按规律间隔给药一次或多次。在具体实施方案中，在至少一个月或更长时间给药一次给予鼠源抗体或人源化抗体或GLP-1融合蛋白质，例如一个、两个或三个月或者甚至不确定。对于治疗慢性症状，长期治疗通常最有效。但是，对于治疗急性症状，短期给药例如从一周至六周就已足够。通常，给予人类抗体直至患者表现出所选体征或指示剂高于基线水平的医学相关改善度为止。
本文提供的治疗方案的一个实例包括以适当剂量一周一次或者更长的皮下注射抗体或GLP-1融合蛋白质治疗高血糖症、二型糖尿病、二型糖尿病并发症、代谢综合症和包括血脂异常等引起的症状。可持续每周或每月给予抗体或GLP-1融合蛋白质直到达到所需结果例如病人症状消退。可按需要重新治疗，或者，可选择地，给予维持剂量。
可在使用抗体或GLP-1融合蛋白质例如人类抗体或GLP-1融合蛋白质治疗之前、进行中和/或之后监测病人的血糖浓度、体重，以检测其压力的任何变化。对于某些病症，血糖的变化可随例如疾病进程等因素而变化。可用已知技术测定其血糖浓度。
本文的方法和组合物的具体实施方案涉及使用例如抗体或GLP-1融合蛋白质和一个或多个胰高血糖素拮抗剂、两个或更多本文提供的抗体或GLP-1融合蛋白质，或者本发明抗体或GLP-1融合蛋白质和一个或更多其它胰高血糖素拮抗剂。在进一步的实施方案中，单独或与其它用于治疗使患者痛苦的症状的药剂组合给予抗体或GLP-1融合蛋白质。这些药剂的实例包括蛋白质以及非蛋白质药物。当联合给予多种药物时，如本领域所熟知其剂量应相应调整。“联合给药”组合疗法不限于同时给药，也包括在涉及给予患者至少一种其它治疗剂的疗程中至少给予一次抗原和蛋白的治疗方案。
另一方面，本文提供制备治疗高血糖症、二型糖尿病、二型糖尿病并发症、代谢综合症、和血脂异常及相关病症药剂的方法，其包含本文提供的抗体或GLP-1融合蛋白质与药学可接受辅料中的混合物，用于治疗上述疾病的相关病症。药剂制备方法如上所述。
本文进一步提供可特异性结合至人GCGR的抗体或GLP-1融合蛋白质相关的组合物、试剂盒和方法。也提供了核酸分子及其衍生物和片段，其包含编码与GCGR结合的多肽的全部或部分的多聚核苷酸，例如编码全部或部分抗GCGR抗体、抗体片段、抗体衍生物或GLP-1融合蛋白质的核酸。本文进一步提供包含该类核酸的载体和质粒以及包含该类核酸和/或载体和质粒的细胞和细胞系。所提供方法包括，例如，制备、鉴定或分离与人GCGR结合的抗体或GLP-1融合蛋白质例如抗GCGR抗体或GLP-1融合蛋白质的方法，测定该抗体或GLP-1融合蛋白质是否与GCGR结合的方法、以及将与GCGR结合的抗体或GLP-1融合蛋白质给予动物模型的方法。
下面通过具体实例，对本文的技术方案作进一步的说明。
本文中，若非特指，所采用的原料和设备等均可从市场购得或是本领域常用的。下述实例中的方法，如无特别说明，均为本领域的常规方法。
1、免疫用抗原的制备
接种CHO-DHFR-细胞至6孔板中。培养24小时(hr)后，细胞中转染克隆有hGCGR基因(核苷酸序列见SEQ ID NO:77及氨基酸序列见SEQ ID NO:73)的pTM15质粒。转染是按照Invitrogen公司推荐Lipofectamine 2000的转染条件进行。48hr后，再换为含有10nM MTX的完全培养基，每隔3天(d)换液，待两周左右，出现稳定生长的克隆，消化分散细胞集落，待长至50％愈合度时，逐渐提高MTX的浓度进行加压筛选，直至MTX的浓度为300nM。待两周左右，出现稳定生长的克隆。消化分散细胞集落，将细 胞传代，继续培养细胞,待传代细胞长至100％愈合度。利用V5标签的抗体(Life Technologies)对构建的稳定细胞株分别进行FACS检测，根据FACS检测结果鉴定加压后的细胞群体。筛选后的CHO-DHFR-hGCGR细胞膜上有大量hGCGR表达。最后经过亚克隆和进一步鉴定后，选出2株hGCGR细胞为高表达的稳定细胞株。这些高表达hGCGR的细胞株可被作为制备抗体的免疫原(参考实施例2)。此外，在某些实施方案中，hGCGR胞外区和hIgG Fc的融合蛋白也可以作为制备抗体的免疫原，其制备方法如下：亚克隆hGCGR胞外区,hIgG2Fc和肽接头(Linker)的融合蛋白序列基因于pTM5质粒。通过悬浮HEK293细胞进行大量瞬时表达，获得细胞上清液，然后通过亲和层析纯化得到hGCGR胞外区融合蛋白。
2、抗体的制备
将免疫原和氢氧化铝佐剂混匀，皮下注射BALB/c小鼠(6-8周龄)，此后每周加强免疫小鼠一次。经过总共6次免疫后，通过剪尾的方式采血。离心分离血清，用FACS检测血清效价。达到适合抗体滴度时，断颈处死小鼠，无菌状态下获取脾脏细胞。另外收集生长状态处于对数生长期的SP2/0细胞，离心细胞，将沉淀细胞用无血清培养至重悬，再次离心-重悬，计数。混合脾脏细胞和SP2/0细胞，保证SP2/0和脾脏细胞数量接近，混合以后再“洗涤-离心”3次。弹散最后一次离心后的细胞沉淀，逐滴加入预温的PEG-1500，上下吹吸后，缓慢加入30mL预热的无血清培养基以终止PEG的融合作用。再次离心后弹散细胞沉淀，加入融合培养基，将脾细胞和饲养层细胞铺于96孔板中,每孔加入100μL培养基。融合后的杂交瘤细胞和饲养层细胞一起在96孔板中进行培养，并进行HAT(次黄嘌呤、氨甲喋呤和胸苷)筛选，以除去非融合的细胞。10d后收取培养板中的杂交瘤细胞上清进行ELISA检测。
3、ELISA筛选抗体
将过表达hGCGR的CHO-DHFR-hGCGR细胞和不表达hGCGR的CHO-DHFR-细胞分别接种至96孔板。待细胞长至90％愈合度，除去细胞培养上清，PBS洗两遍，加入100％甲醇4℃固定，然后加入100μL新鲜配制的0.6％H 2O 2-PBS，室温处理20分钟(min)，PBS洗两遍。经过1％BSA(溶于PBS中)封闭后，加入杂交瘤细胞上清4℃孵育90min。多次洗涤后，每孔加入100μL稀释的羊抗鼠Fc-HRP二抗(Sigma-Aldrich)，37℃孵育30min。洗涤5次后,每孔加入100μL TMB显色底物，37℃反应15min，加入50μL 2M H 2SO 4终止显色，读取OD 450值。此外，在某些实施方案中，使用包含hGCGR的N端结构域的氨基酸序列与hFc的融合蛋白作为包被抗原，包被96孔板。经过1％BSA(溶于PBS中)封闭后，加入杂交瘤细胞上清4℃孵育90min。之后步骤同上述ELISA方法筛选抗hGCGR单克隆抗体。阳性对照为免疫小鼠的血清；阴性对照为细胞培养基上清。经过ELISA的初步检测，筛选到了数个分泌抗hGCGR抗体的阳性杂交瘤细胞株。选取这些分泌抗hGCGR抗体的杂交瘤株，进行克隆化以获得能稳定分泌抗hGCGR抗体的细胞株。最后选取阳性杂交瘤细胞制备的腹水抗体进行FACS验证(参考实例10)。
4、抗体基因的克隆及亚克隆
收集分泌抗体的杂交瘤细胞，按照QIAGEN的mRNA抽提试剂盒操作规程，提取杂交瘤细胞的mRNA。然后将提取后的mRNA反转录成cDNA，逆转录引物为小鼠轻、重链恒定区的特异性引物，重链逆转录引物为(5’-TTTGGRGGGAAGATGAAGAC-3’)，轻链逆转录引物为(5’-TTAACACTCTCCCCTGTTGAA-3’)和(5’-TTAACACTCATTCCTGTTGAA-3’)。RT-PCR的反应条件为：25℃5min；50℃60min；70℃15min。将反转录的cDNA用0.1mM的TE稀释至500μL，加入到超滤离心管(Amicon Ultra-0.5)中，2000g离心10min；弃滤液，再加500μL的0.1mM的TE，2000g离心10min；弃滤液，将制备管倒置到新的离心管中，2000g离心10min，得到纯化后的cDNA；取10μL的纯化后的cDNA作为模板，加入4μL的5x tailing buffer(Promega)，4μL的dATP(1mM)和10U的末端转移酶(Promega)后混匀，37℃孵育5min后再65℃孵育5min；然后以加上PolyA尾的cDNA为模板，PCR扩增抗体的轻、重链可变区基因。上游引物均为OligodT，重链下游引物为(5’-TGGACAGGGATCCAGAGTTCC-3’)和(5’-TGGACAGGGCTCCATAGTTCC-3’)，轻链下游引物为(5’-ACTCGTCCTTGGTCAACGTG-3’)。PCR反应条件：95℃5min；95℃30s，56℃30s，72℃1min 40cycles；72℃7min；PCR产物连接到PMD 18-T载体(Takara Bio)后进行测序。基于已测序得到的抗体的DNA序列设计PCR引物，从而将完整轻链、重链信号肽和可变域以及小鼠IgG1恒定区与表达载体pTM5相连。5、抗体的人源化及优化
首先，根据筛选所得的鼠源抗体轻、重链可变区序列，使用NCBI数据库搜索与筛选所得的鼠源的抗体可变区序列同源的人源抗体生殖细胞系基因序列(Ig Germline Gene Sequence)，并将除CDR序列外，同源性最高的人源基因序列做为模板序列进行CDR嫁接，得到人源化的抗体可变区序列。合成人源化抗体轻、重链的基因，与人IgG2或者IgG4恒定区序列拼接后得到完整的重组人源化抗体序列。重组抗体按照实例8进行表达，并按照步骤10中的FACS技术验证其针对GCGR的亲和力，遴选出亲和力表现最优秀的抗体。最后通过定点突变，对人源化抗体的可变区序列进行改造，进一步提高其对GCGR的亲和力。
6、人源化hGCGR抗体的基因克隆与亚克隆
优化后的人源化抗体重链及轻链可变区序列外包合成。合成时重链可变区5’端带入Nhe1酶切位点，3’端带入Sal1酶切位点，从而将完整的重链可变区序列与已装入重链恒定区的表达载体pTM5相连。同样，合成时轻链可变区5’端带入Nhe1酶切位点，3’端带入Bsiw1酶切位点，从而将完整的轻链可变区序列与已装入轻链恒定区的表达载体pTM5相连。
7、人源化hGCGR抗体与GLP-1的融合蛋白质的构建
优化后的人源化抗体在轻链的N端或者C端与GLP-1及其衍生物序列进行融合组成GLP-1融合蛋白质。两者的序列由肽接头序列(Linker)做为桥梁进行连接。信号肽 -GLP-1-Linker的核苷酸序列由金斯瑞生物科技有限公司合成。以合成基因为模板，PCR扩增“信号肽-GLP-1-Linker”部分的序列。另以人源化抗体的核苷酸序列为模板，扩增融合蛋白质的抗体部分的序列。然后通过overlapping PCR将融合蛋白质核酸序列的“信号肽-GLP-1-Linker”部分与抗体部分连接，引物两端添加Nhe1和Not1的酶切位点，从而将完整的融合蛋白质序列与表达载体pTM5相连。
8、GCGR抗体和GLP-1的融合蛋白质的瞬时表达
接种5×10 5/mL的悬浮HEK293或者CHO表达细胞株至转瓶中。经过37℃，5％CO 2旋转培养24hr后，密度达到1×10 6/mL后被用于转染。转染过程中使用polyethylenimine(PEI)作为转染介质，将其与的DNA混合。两者混合物在静置孵育15min后被加入到细胞培养中。细胞在接受PEI与DNA混合物后继续37℃，5％CO 2旋转培养24hr后，向细胞培养液中加入胰蛋白胨作为表达需要的添加物。最后在表达完成后(96hr以上)收集细胞上清用于抗体的纯化分离。
9、GCGR抗体和GLP-1的融合蛋白质的纯化分离
收集的实例8中的细胞上清经过高速(8000rpm)离心去除细胞以及细胞碎片后，再用0.22μm滤膜过滤澄清。澄清后的上清被用于纯化。纯化过程由层析仪完成。上清首先流过蛋白A/G亲和层析柱。上清中包含的抗体在此期间与蛋白A/G亲和层析柱的配基相结合后被滞留于柱内。然后用低pH值(小于等于3.0)的洗脱缓冲液灌洗层析柱解离与层析柱结合的抗体。收集到的抗体洗脱液用1M的Tris-HCl迅速中和。得到的抗体洗脱液经过透析后置换成PBS或者其他缓冲体系。
10、流式细胞仪验证功能性GCGR抗体的结合活性
用含10mM EDTA的PBS消化、收集10 5个CHO-DHFR-hGCGR细胞，分别加入1.5mL EP管，离心后弃上清。阴性对照样本用流式上样缓冲液(PBS，2％FBS)重悬。阳性处理组细胞每管加200μL特定浓度的hGCGR抗体，室温孵育；孵育完成后1500rpm离心，弃上清，用流式上样缓冲液洗一次细胞沉淀，再离心，将细胞重悬；向细胞重悬液加入1:50稀释的FITC标记的羊抗鼠荧光二抗，200μL/孔，室温避光孵育30min；离心，弃上清，再用流式上样缓冲液洗一次，离心，最后用流式上样缓冲液将细胞沉淀重悬，上机检测。重组抗hGCGR的功能性抗体和表达GCGR的CHO-DHFR-CHO-GCGR细胞有特异性结合。图一显示的实验结果中，灰色峰为500nM小鼠腹水抗体L5H5和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(1a)、50nM(1b)和5nM(1c)的小鼠腹水抗体L5H5和CHO-DHFR-hGCGR的结合曲线，相对于灰色峰阴性对照有明显右移，证明了L5H5和CHO-DHFR-hGCGR的特异性结合。图二显示的实验结果中，灰色峰为500nM小鼠腹水抗体L4H4和空白细胞CHO-DHFR-结合的阴性对照，实线峰分别为500nM(2a)、50nM(2b)和5nM(2c)的小鼠腹水抗体L4H4和CHO-DHFR-hGCGR的结合曲线，相对于灰色峰阴性对照有明显右移，证明了L4H4和CHO-DHFR-hGCGR的特异性结合。
11、cAMP实验检测GCGR抗体和GCGR抗体与GLP-1的融合蛋白质在体外拮抗GCGR 的生物学活性
以每孔30000个接种表达hGCGR的CHO-DHFR-细胞至96孔细胞培养板，置于37℃，5％CO 2培养箱中过夜。第二天除去细胞上清，加入杂交瘤细胞培养上清或梯度稀释的抗体45μL/孔。室温放置30min，再加入胰高血糖素多肽(Phoenix Pharmaceuticals，50pM)45μL/孔。然后将96孔细胞培养板置于37℃，5％CO 2培养箱中继续孵育30min后，加10μL/孔的10％Triton X-100，室温裂解，用排枪混合均匀。采用cAMP试剂盒(CisBio)检测实验中产生的cAMP。取上述10μL/孔细胞裂解液于白色384孔板中，加入5μL/孔1:20稀释的cAMP-d2，最后加5μL/孔1:20稀释的Anti-cAMP-Eu3±cryptate，室温孵育1hr。在Envision 2103酶标仪上读取时间分辨荧光665nm/620nm信号比值，然后采用Prism5.0计算IC 50值。图三显示了直接cAMP实验检测hGCGR抗体L7H7拮抗胰高血糖素激活hGCGR信号通路的浓度抑制曲线(IC 50＝139nM,R2＝0.99)。
Schild作图分析：采用以上直接cAMP方法，分别固定抗GCGR抗体(L4H4和L5H5)浓度为3160nM、1000nM、316nM、0nM，连续稀释胰高血糖素(100nM至1fM)，检测存在GCGR抗体的情况下，采用Prism5.0计算使得胰高血糖素剂量响应曲线2倍右移所需抗体浓度的负对数，即抗体的pA2。图四显示直接cAMP实验检测小鼠腹水GCGR抗体，拮抗梯度稀释的胰高血糖素激活hGCGR信号通路的Schild曲线。随着抗GCGR抗体浓度的提高，胰高血糖素激活其受体的S型曲线有明显的右移。
12、报告基因实验检测GCGR抗体与GLP-1的融合蛋白质在体外激活GLP-1R
以每孔40000个接种共表达hGLP-1R-CRE-Luciferase的CHO-DHFR-细胞至96孔细胞培养板，37℃培养过夜。第二天除去培养基上清，用无血清培养基清洗细胞表面两次，吸去残液，再加入100μL用无血清培养基稀释纯化抗体或GMA102，37℃孵育4小时。刺激结束后，加入100μL Bright Glo化学发光底物(Promega)，最后将细胞裂解物转移至白色96孔板，在SpectraMax L酶标仪(Molecular Devices)上读取相对荧光强度。图五显示了报告基因实验检测GCGR抗体/GLP-1融合蛋白质GLP-1-Linker-L7H7，以及GMA102激活hGLP-1R信号通路的激活性曲线(EC 50分别为106pM和16pM)。其中，GMA102是本公司开发的长效GLP-1激动剂(Li等，Biochem Pharmacol.2018，150:46-53；CN 201410349725)，具有激活细胞内cAMP信号通路，显著降低小鼠的血糖和体重，正处于临床二期开发阶段。
13、普通ICR小鼠药效试验评估GCGR抗体L4H4单独或联合GMA102(本公司的GLP-1R抗体与GLP-1的融合蛋白质)的体内药效
各组动物禁食过夜(自由饮水)后，尾尖采血，以罗氏卓越型血糖仪(下同)测定血糖水平(0min)，之后皮下注射给药(0.1mL/10g体重，正常对照组给予PBS，给药组：L4H4单独使用2.5、5、10mg/kg，或者分别联用GMA102剂量2.5、5mg/kg，GMA102由于是人源化抗体融合蛋白，在小鼠内存在抗药抗体，因而每两天给一次药，鼠源L4H4仅给一次药)，在皮下注射后30min各组动物葡萄糖溶液灌胃(2g/kg，0.1 mL/10g体重)，并于糖负荷后30、60和120min尾尖采血，测定血糖水平。给药第2天，为了减少对动物的刺激，仅测定各组动物禁食5hr(自由饮水)后(0min)及葡萄糖负荷后30min的血糖水平。之后连续测定给药四天、六天、八天、十天、十二天后糖负荷后血糖值。
将GMA102和抗GCGR抗体L4H4联用后，表3显示了从第二天到第八天，单用GMA102、L4H4，以及联用GMA102和L4H4都明显降低小鼠的口服糖耐量，而且联用的效果比单用效果更明显，具有协同增效的作用。以血糖浓度变化的线下面积AUC表示(mean±SD),N＝6。
Figure PCTCN2019078674-appb-000005
14、高脂饲料诱导的C57BL/6肥胖小鼠药效实验评价L4H4单独或联合GMA102(本公司的GLP-1R抗体与GLP-1的融合蛋白质)的体内药效
60％高脂饲料诱导C57BL/6小鼠建立肥胖模型(DIO mice)。小鼠购入后正常饮食饲养一周后，随机挑选一定数量的老鼠作为正常对照组给予普通小鼠饲料喂养，剩余动物给予高脂饲料。连续饲喂8周(wk)，每周称量一次体重和进食量。随后将高脂饲料组老鼠按体重随机分组。各组动物禁食过夜(自由饮水)后，尾尖采血，以罗氏卓越型血糖仪(下同)测定血糖水平(0min)，之后皮下注射给药(0.1mL/10g体重，正常对照组给予PBS，给药组：L4H4单独使用2.5、5mg/kg，GMA102单独使用2.5mg/kg，抗L4H4和低剂量、高剂量的GMA102联用，L4H4每周一次，GMA102每两天一次给药)。监测动物的体重、耗食量、随机血糖水平、空腹血糖、OGTT和糖化血红蛋白。图六显示了高脂饲料诱导的C57BL/6肥胖小鼠中单次皮下注射L4H4、GMA102，以及联用GMA102和L4H4都显著降低肥胖小鼠的口服糖耐量，而且联用的效果比单用效果更明显，也具有协同增效的作用。单次用药至两周后，仍然具有明显的降血糖效果，表明L4H4抗体具有明显长效的作用。注：与模型组组相比，*表示P<0.05，**表示P<0.01；与L4H4-5mg/kg组相比，△表示P<0.05，△△表示P<0.01；与GMA102-2.5mg/kg组相比，☆表示P<0.05，☆☆表示P<0.01。
15、GCGR抗体与GLP-1的融合蛋白质在食蟹猴上的药代动力学实验
给予雌雄各半共6只食蟹猴皮下单次GCGR抗体与GLP-1的融合蛋白质注射，剂量为2mg/kg，并在给药前(0min)，给药后2hr，4hr，8hr，12hr，24hr，2d，4d，6d，8d，10d，12d，18d，28d进行给药侧肢静脉取全血0.6mL置于离心管中在冰上待其自然凝固后离心提取血清，超低温保存(-80℃)至检测为止。血清样品中的GCGR抗体与GLP-1的融合蛋白质的GLP-1部分以及hGCGR抗体部分是用ELISA方法对其进行分别定量，并通过软件分析确定两者在食蟹猴体内的半衰期。
16、GCGR抗体与GLP-1的融合蛋白质在恒河猴上的药代动力学实验
给予雌雄各半共2只健康恒河猴皮下单次GCGR抗体与GLP-1的融合蛋白质(GLP-1-Linker-L7H7)注射，剂量为2mg/kg，并在给药前(0min)，给药后2h、4h、8h、12h、24h、2天、4天、6天、8天、10天、12天、16天、20天、25天、30天、40天、50天、及60天经非给药肢静脉取全血0.6mL置于已有8μL DDP-IV酶抑制剂(Millipore)的离心管中，放在冰上待其自然凝固后离心提取血清，超低温保存(-80℃)至检测为止。血清样品中的GCGR抗体与GLP-1的融合蛋白质的GLP-1部分以及hGCGR抗体部分是用ELISA方法对其进行分别定量，并通过软件分析确定两者在恒河猴体内的半衰期。
PK研究显示，GLP-1部分的半衰期T 1/2约为38小时，而GLP-1-Linker-L7H7的抗体部分半衰期T 1/2约为131小时。PK曲线及参数见图七及表四。
表四：GCGR抗体与GLP-1的融合蛋白质(GLP-1-Linker-L7H7)药代动力学参数
    GLP-1部分 抗体部分
T 1/2 小时 38±6 131±40
Tmax 小时 8 12-48
Cmax 纳克/毫升 25585±5537 43743±503
17、实验研究GCGR抗体与GLP-1的融合蛋白质单次给药对健康恒河猴静脉葡萄糖耐量(IVGTT)影响
给予雌雄各半共4只健康恒河猴皮下单次注射给药：GLP-1-Linker-L7H7和阳性对照GMA102(本公司的GLP-1R抗体与GLP-1的融合蛋白质)，剂量为2mg/kg。所有动物试验前一天晚上禁食14-16小时过夜，试验当天动物麻醉后经下肢静脉注射50％葡萄糖溶液(0.25g/kg)，分别于给糖前5min、3min及给糖后3min、5min、7min，10min、20min和30min经前臂静脉采集血样0.8mL，EDTA2K抗凝，离心后分离血浆，用于胰岛素和血糖的检测。
采用Roche cobas 6000analyzer series E601检测胰岛素(μU/mL)。结果如图八及图九所示，注射前，二组动物分泌等量的胰岛素，而注射后48小时后，GLP-1-Linker-L7H7组比阳性对照组分泌更多的胰岛素。采用Roche cobas 6000analyzer series C501检测血糖Glucose(mmol/L)，结果如图十及图十一所示，注射前，二组猴子的血糖相当，而注射后48小时后，GLP-1-Linker-L7H7组比阳性对照组的降糖幅度更大。
提供以上例用于向本领域普通技术人员充分公开和说明如何制造和使用要求保护的实施方式，而不意味着限制本文披露的范围。对本领域技术人员而言显而易见的修饰都在本文权利要求书的范围内。将本说明书中引用的所有出版物、专利和专利申请都通过参考并入本文，就如同每个出版物、专利或专利申请均被具体和单独地通过参考并入本文一样。

Claims (44)

  1. 一个能与人GCGR特异性结合的抗体，所述抗体包含一，两，三，四，五，或六个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：
    a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；
    b.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；
    c.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；
    d.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34；
    e.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35；及
    f.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
  2. 根据权利要求1所述的抗体，其中所述抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：
    a.轻链CDR1氨基酸序列：SEQ ID NO:1、SEQ ID NO:4、SEQ ID NO:7、SEQ ID NO:10、SEQ ID NO:13、SEQ ID NO:16、SEQ ID NO:18、及SEQ ID NO:20；及
    b.重链CDR1氨基酸序列：SEQ ID NO:22、SEQ ID NO:25、及SEQ ID NO:28、SEQ ID NO:31、及SEQ ID NO:34。
  3. 根据权利要求1或2所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：
    a.轻链CDR2氨基酸序列：SEQ ID NO:2、SEQ ID NO:5、SEQ ID NO:8、SEQ ID NO:11、及SEQ ID NO:14；及
    b.重链CDR2氨基酸序列：SEQ ID NO:23、SEQ ID NO:26、SEQ ID NO:29、SEQ ID NO:32、及SEQ ID NO:35。
  4. 根据权利要求1至3中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：
    a.轻链CDR3氨基酸序列：SEQ ID NO:3、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:12、及SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、及SEQ ID NO:21；及
    b.重链CDR3氨基酸序列：SEQ ID NO:24、SEQ ID NO:27、SEQ ID NO:30、SEQ ID NO:33、及SEQ ID NO:36。
  5. 根据权利要求1至4中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、及SEQ ID NO:21。
  6. 根据权利要求1至5中任一项所述的抗体，其中所述抗体包含或还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自以下所列的氨基酸序列：SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、及SEQ ID NO:36。
  7. 根据权利要求1至6中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自以下所列的轻链和重链CDR1氨基酸序列的组合：SEQ ID NO:1与SEQ ID NO:22、SEQ ID NO:4与SEQ ID NO:25、SEQ ID NO:7与SEQ ID NO:28、SEQ ID NO:10与SEQ ID NO:31、SEQ ID NO:13与SEQ ID NO:34、SEQ ID NO:16与SEQ ID NO:34、SEQ ID NO:18与SEQ ID NO:34、及SEQ ID NO:20与SEQ ID NO:34。
  8. 根据权利要求1至7中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自以下所列的轻链和重链CDR2氨基酸序列的组合：SEQ ID NO:2与SEQ ID NO:23、SEQ ID NO:5与SEQ ID NO:26、SEQ ID NO:8与SEQ ID NO:29、SEQ ID NO:11与SEQ ID NO:32、及SEQ ID NO:14与SEQ ID NO:35。
  9. 根据权利要求1至8中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自以下所列的轻链和重链CDR3氨基酸序列的组合：SEQ ID NO:3与SEQ ID NO:24、SEQ ID NO:6与SEQ ID NO:27、SEQ ID NO:9与SEQ ID NO:30、SEQ ID NO:12与SEQ ID NO:33、SEQ ID NO:15与SEQ ID NO:36、SEQ ID NO:17与SEQ ID NO:36、SEQ ID NO:19与SEQ ID NO:36、及SEQ ID NO:21与SEQ ID NO:36。
  10. 根据权利要求1至9中任一项所述的抗体，其中所述抗体包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链可变结构域氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、及SEQ ID NO:90；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列；及
    b.重链可变结构域氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的氨基酸序列。
  11. 根据权利要求1至10中任一项所述的抗体，其中所述抗体的多聚核苷酸编码序列包含一或两个多聚核苷酸序列，其中每个多聚核苷酸列独立地选自于以下所列多聚核苷酸序列：
    a.轻链可变结构域多聚核苷酸编码序列：SEQ ID NO:98、SEQ ID NO:99、SEQ ID NO:100、及SEQ ID NO:101、SEQ ID NO:102、SEQ ID NO:103、及SEQ ID NO:104、SEQ ID NO:105、SEQ ID NO:106、及SEQ ID NO:107；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸序列；及
    b.重链可变结构域多聚核苷酸编码序列：SEQ ID NO:108、SEQ ID NO:109、SEQ ID NO:110、SEQ ID NO:111、SEQ ID NO:112、SEQ ID NO:113、及SEQ ID NO:114；及与其任一序列有至少80％、至少85％、至少90％、或至少95％相同的多聚核苷酸序列。
  12. 根据权利要求1至11中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:83、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、及SEQ ID NO:90。
  13. 根据权利要求1至12中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:93、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97。
  14. 根据权利要求1至13中任一项所述的抗体，其中所述抗体包含一个独立地选自于以下所列的轻链与重链可变区氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91、SEQ ID NO:82与SEQ ID NO:92、SEQ ID NO:83与SEQ ID NO:93、SEQ ID NO:84与SEQ ID NO:94、SEQ ID NO:85与SEQ ID NO:95、SEQ ID NO:86与SEQ ID NO:96、SEQ ID NO:87与SEQ ID NO:97、SEQ ID NO:88与SEQ ID NO:97、SEQ ID NO:89与SEQ ID NO:97、及SEQ ID NO:90与SEQ ID NO:97。
  15. 根据权利要求1至14中任一项所述的抗体，其中所述抗体包含或还包含一个独立地选自于以下所列的氨基酸序列：SEQ ID NO:81、SEQ ID NO:82、SEQ ID NO:84、SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、SEQ ID NO:91、SEQ ID NO:92、SEQ ID NO:94、SEQ ID NO:95、SEQ ID NO:96、及SEQ ID NO:97。
  16. 根据权利要求1至15中任一项所述的抗体，其中所述抗体包含一个独立地选自于以下所列的轻链与重链可变区氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91、SEQ ID NO:82与SEQ ID NO:92、SEQ ID NO:84与SEQ ID NO:94、SEQ ID NO:85与SEQ ID NO:95、SEQ ID NO:86与SEQ ID NO:96、SEQ ID NO:87与SEQ ID NO:97、SEQ ID NO:88与SEQ ID NO:97、及SEQ ID NO:89与SEQ ID NO:97。
  17. 根据权利要求1至16中任一项所述的抗体，其中所述抗体还包含一或两个氨基酸序列，其中每个氨基酸序列独立地选自于以下所列的氨基酸序列：
    a.轻链恒定氨基酸序列：SEQ ID NO:115及SEQ ID NO:116；
    轻链恒定氨基酸序列：SEQ ID NO:132及SEQ ID NO:133；及
    b.重链恒定氨基酸序列：SEQ ID NO:117及SEQ ID NO:118。
  18. 根据权利要求1至17中任一项所述的抗体，其中所述抗体为鼠源GCGR抗体或人源化GCGR抗体。
  19. 根据权利要求1至18中任一项所述的抗体，其中所述抗体为GCGR单克隆抗体。
  20. 根据权利要求1至19中任一项所述的抗体，其中所述抗体为一单克隆抗体,该单克隆抗体包含一个选自于以下所列的氨基酸序列的组合：SEQ ID NO:81与SEQ ID NO:91、SEQ ID NO:82与SEQ ID NO:92、SEQ ID NO:83与SEQ ID NO:93、SEQ ID NO:84与SEQ ID NO:94、SEQ ID NO:85与SEQ ID NO:95、SEQ ID NO:86与SEQ ID NO:96、SEQ ID NO:87与SEQ ID NO:97、SEQ ID NO:88与SEQ ID NO:97、SEQ ID NO:89与SEQ ID NO:97、及SEQ ID NO:90与SEQ ID NO:97。
  21. 根据权利要求1至20中任一项所述的抗体，其特征在于：所述抗体为鼠源抗体、人 类抗体、人源化抗体、嵌合抗体、单克隆抗体、多克隆抗体、重组抗体、抗原结合抗体片段、单链抗体、双链抗体、三链抗体、四链抗体、Fab片段、F(ab’)x片段、结构域抗体、IgD抗体、IgE抗体、IgM抗体、IgGl抗体、IgG2抗体、IgG3抗体、或IgG4抗体。
  22. 根据权利要求1至21中任一项所述的抗体，其中所述抗体的降低人Glucagon信号传导的IC50值为大约1nM至大约300nM或大约1nM至大约150nM。
  23. 一个GCGR抗体与GLP-1的融合蛋白质(GLP-1融合蛋白质)，其结构特征在于：所述的融合蛋白质包含权利要求1至22中任一项所述的一个GCGR抗体、和一个，二个，三个，四个，五个，六个，七个，或八个GLP-1片段或反向GLP-1片段；该融合蛋白质通过一肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接，或者将一反向GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
  24. 根据权利要求23所述的融合蛋白质，其所述的融合蛋白质包含一个GCGR抗体，和一个，二个，三个，或四个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或重链的氨基端连接。
  25. 权利要求23所述的融合蛋白质，其所述的融合蛋白质包含一个GCGR抗体，和一个，二个，三个，或四个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链或重链的羧基端连接。
  26. 权利要求23所述的融合蛋白质，其所述的融合蛋白质包含一个GCGR抗体，和二个GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一GLP-1片段的羧基端与一GCGR抗体轻链或者重链的氨基端连接。
  27. 权利要求23所述的融合蛋白质，其所述的融合蛋白质包含一个GCGR抗体，和二个反向GLP-1片段；该融合蛋白质通过一个肽接头序列(Linker)将一反向GLP-1片段的氨基端与一GCGR抗体轻链或者重链的羧基端连接。
  28. 根据权利要求23所述的融合蛋白质，其中所述的GCGR抗体、GLP-1片段、反向GLP-1片段、和肽接头序列(Linker)通过以下所述中一方式融合形成所述的融合蛋白质：
    通过肽接头序列(Linker)将一GLP-1片段的羧基端和一GCGR抗体轻链或者重链的氨基端连接：N'-GLP-1-Linker-R-C'；
    通过肽接头序列(Linker)将一反向GLP-1片段的氨基端和一GCGR抗体轻链或者重链的羧基端连接：N'-R-Linker-反向GLP-1-C'；
    其中:N'代表多肽链的氨基端,C'代表多肽链的羧基端,GLP-1代表一GLP-1片段，反向GLP-1代表一反向GLP-1片段，R为权利要求1至22所述的一GCGR抗体的轻链或者重链的氨基酸序列,及Linker代表一肽接头。
  29. 根据权力要求23至28中任一项所述的GLP-1融合蛋白质，所述的肽接头(Linker)的序列包含全长的、部分的、或者重复的独立选自以下之一的氨基酸序列：SEQ ID NO:124、SEQ ID NO:125、及SEQ ID NO:126。
  30. 根据权力要求23至29中任一项所述的GLP-1融合蛋白质，其中所述的GLP-1片段包含独立选自以下之一的氨基酸序列：SEQ ID NO:119、SEQ ID NO:120、SEQ ID NO: 121、SEQ ID NO:122、及SEQ ID NO:123；或者其中所述的反向GLP-1片段包含独立选自以下之一的氨基酸序列：SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ ID NO:130、及SEQ ID NO:131。
  31. 一种多核苷酸，其编码权利要求1至22中任一项所述的GCGR抗体或权利要求23至30中任一项所述的GLP-1融合蛋白质。
  32. 一种载体，其包含权利要求31中所述的多核苷酸。
  33. 一种宿主细胞，其包含权利要求32中所述的载体。
  34. 一种药用组合物，其包含与药用可接受载体混合的权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质。
  35. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于预防或治疗高血糖症的药物中的用途。
  36. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于预防或治疗二型糖尿病的药物中的用途。
  37. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于预防或治疗二型糖尿病并发症的药物中的用途。
  38. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于预防或治疗代谢综合症的药物中的用途。
  39. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于预防或治疗血脂异常的药物中的用途。
  40. 一种包含权利要求1至22中任一项所述的GCGR抗体或者权利要求23至30中任一项所述的GLP-1融合蛋白质或者权利要求34所述的药用组合物在制备用于同时治疗高血糖症、二型糖尿病、二型糖尿病并发症、代谢综合症、或血脂异常二种及二种以上病症的药物中的用途。
  41. 根据权利要求35至40中任一项所述用途，其所述的药用组合物是用于静脉或皮下注射。
  42. 根据权利要求35至41中任一项所述用途，进一步包含注射一种GLP-1R抗体与GLP-1的融合蛋白质。
  43. 根据权利要求42所述用途，其所述的GLP-1R抗体与GLP-1的融合蛋白质包含:
    a.轻链CDR1氨基酸序列：SEQ ID NO:134；
    b.轻链CDR2氨基酸序列：SEQ ID NO:135；
    c.轻链CDR3氨基酸序列：SEQ ID NO:136；
    d.重链CDR1氨基酸序列：SEQ ID NO:137；
    e.重链CDR2氨基酸序列：SEQ ID NO:138；及
    f.重链CDR3氨基酸序列：SEQ ID NO:139。
  44. 根据权利要求43所述用途，其所述的GLP-1R抗体与GLP-1的融合蛋白质包含以下所列的轻链与重链可变区氨基酸序列的组合：SEQ ID NO:140与SEQ ID NO:141。
PCT/CN2019/078674 2018-04-10 2019-03-19 Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用 WO2019196603A1 (zh)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US17/046,686 US11542336B2 (en) 2018-04-10 2019-03-19 GCGR antibody and GLP-1 fusion protein thereof, pharmaceutical composition thereof and application thereof
EP19785909.3A EP3778634A4 (en) 2018-04-10 2019-03-19 GCGR ANTIBODY AND GLP-1 FUSION PROTEIN THEREOF, PHARMACEUTICAL COMPOSITION THEREOF AND USES THEREOF
CA3096556A CA3096556A1 (en) 2018-04-10 2019-03-19 Gcgr antibody and its fusion protein with glp-1, and pharmaceutical composition and application thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201810316473.0A CN110357959B (zh) 2018-04-10 2018-04-10 Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
CN201810316473.0 2018-04-10

Publications (1)

Publication Number Publication Date
WO2019196603A1 true WO2019196603A1 (zh) 2019-10-17

Family

ID=68163932

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2019/078674 WO2019196603A1 (zh) 2018-04-10 2019-03-19 Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用

Country Status (5)

Country Link
US (1) US11542336B2 (zh)
EP (1) EP3778634A4 (zh)
CN (1) CN110357959B (zh)
CA (1) CA3096556A1 (zh)
WO (1) WO2019196603A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3901173A4 (en) * 2018-12-21 2022-11-23 Jiangsu Hengrui Medicine Co., Ltd. BISPECIFIC PROTEIN

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
US5011912A (en) 1986-12-19 1991-04-30 Immunex Corporation Hybridoma and monoclonal antibody for use in an immunoaffinity purification system
WO1993010151A1 (en) 1991-11-22 1993-05-27 Immunex Corporation Receptor for oncostatin m and leukemia inhibitory factor
WO1994010308A1 (en) 1992-10-23 1994-05-11 Immunex Corporation Methods of preparing soluble, oligomeric proteins
US5457035A (en) 1993-07-23 1995-10-10 Immunex Corporation Cytokine which is a ligand for OX40
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
US6210924B1 (en) 1998-08-11 2001-04-03 Amgen Inc. Overexpressing cyclin D 1 in a eukaryotic cell line
US20030039958A1 (en) 1999-12-03 2003-02-27 Domantis Limited Direct screening method
US20040009507A1 (en) 2000-10-13 2004-01-15 Domantis, Ltd. Concatenated nucleic acid sequence
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US20040202995A1 (en) 2003-04-09 2004-10-14 Domantis Nucleic acids, proteins, and screening methods
US20050238646A1 (en) 2001-01-17 2005-10-27 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
WO2007124463A1 (en) * 2006-04-20 2007-11-01 Amgen Inc. Glp-1 compound/glucagon antibody compositions
US20080036341A1 (en) 2006-08-11 2008-02-14 Nilsen John Ceiling storage container mounting system
US20120128679A1 (en) 2010-11-23 2012-05-24 Regeneron Pharmaceuticals, Inc. Human Antibodies to the Glucagon Receptor

Family Cites Families (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CL2007002668A1 (es) * 2006-09-20 2008-05-09 Amgen Inc Proteina de union a antigeno que se une al receptor de glucagon humano; acido nucleico que la codifica; metodo de produccion; composicion farmaceutica que la comprende; y su uso para tratar o prevenir la diabetes tipo 2.
CL2009000586A1 (es) * 2008-03-27 2010-06-04 Lilly Co Eli Fragmento fab o anticuerpo monoclonal humanizado que lo comprende que se une a receptor de glucagon humano/glucr, de rata, de raton, y mono cinomolgus; vector que comprende polinuccleotido codificante; celula huesped que lo comprende; composicion farmaceutica; uso para tratar diabetes tipo 1 o 2, o para la perdida de peso
US9102732B2 (en) * 2009-09-08 2015-08-11 Neopharm Co., Ltd. Antibodies against glucagon receptor and their use
WO2013059531A1 (en) 2011-10-20 2013-04-25 Genentech, Inc. Anti-gcgr antibodies and uses thereof
CN105189560A (zh) 2013-05-07 2015-12-23 瑞纳神经科学公司 抗胰高血糖素受体抗体和其使用方法
CN104231083A (zh) 2013-06-09 2014-12-24 杭州鸿运华宁生物医药工程有限公司 抗胰高血糖素受体的抗体及其用途
CN104371019B (zh) 2013-08-13 2019-09-10 鸿运华宁（杭州）生物医药有限公司 一种能与glp-1r特异性结合的抗体及其与glp-1的融合蛋白质
CN104926934B (zh) * 2014-09-23 2016-11-09 蒋先兴 胃泌酸调节素类似物
CN107614695B (zh) 2015-04-02 2022-01-25 瑞美德生物医药科技有限公司 用胰高血糖素受体拮抗性抗体治疗肥胖症和非酒精性脂肪肝病或非酒精性脂肪性肝炎的方法
PL3464318T3 (pl) * 2016-06-02 2021-11-08 Abbvie Inc. Agonista receptora glukokortykoidowego i jego immunokoniugaty
CN106084031B (zh) * 2016-06-02 2020-03-31 中国药科大学 一类glp-1r/gcgr双重激动剂在用于降糖和减肥药物中的运用
EP3506940A1 (en) 2016-08-30 2019-07-10 Regeneron Pharmaceuticals, Inc. Methods of treating severe insulin resistance by interfering with glucagon receptor signaling

Patent Citations (31)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4179337A (en) 1973-07-20 1979-12-18 Davis Frank F Non-immunogenic polypeptides
US4301144A (en) 1979-07-11 1981-11-17 Ajinomoto Company, Incorporated Blood substitute containing modified hemoglobin
US4331647A (en) 1980-03-03 1982-05-25 Goldenberg Milton David Tumor localization and therapy with labeled antibody fragments specific to tumor-associated markers
US4640835A (en) 1981-10-30 1987-02-03 Nippon Chemiphar Company, Ltd. Plasminogen activator derivatives
US4737462A (en) 1982-10-19 1988-04-12 Cetus Corporation Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β
US4518584A (en) 1983-04-15 1985-05-21 Cetus Corporation Human recombinant interleukin-2 muteins
US4496689A (en) 1983-12-27 1985-01-29 Miles Laboratories, Inc. Covalently attached complex of alpha-1-proteinase inhibitor with a water soluble polymer
US4751180A (en) 1985-03-28 1988-06-14 Chiron Corporation Expression using fused genes providing for protein product
US4670417A (en) 1985-06-19 1987-06-02 Ajinomoto Co., Inc. Hemoglobin combined with a poly(alkylene oxide)
US4935233A (en) 1985-12-02 1990-06-19 G. D. Searle And Company Covalently linked polypeptide cell modulators
US4791192A (en) 1986-06-26 1988-12-13 Takeda Chemical Industries, Ltd. Chemically modified protein with polyethyleneglycol
US5011912A (en) 1986-12-19 1991-04-30 Immunex Corporation Hybridoma and monoclonal antibody for use in an immunoaffinity purification system
US4946778A (en) 1987-09-21 1990-08-07 Genex Corporation Single polypeptide chain binding molecules
WO1993010151A1 (en) 1991-11-22 1993-05-27 Immunex Corporation Receptor for oncostatin m and leukemia inhibitory factor
WO1994010308A1 (en) 1992-10-23 1994-05-11 Immunex Corporation Methods of preparing soluble, oligomeric proteins
US5457035A (en) 1993-07-23 1995-10-10 Immunex Corporation Cytokine which is a ligand for OX40
US6133426A (en) 1997-02-21 2000-10-17 Genentech, Inc. Humanized anti-IL-8 monoclonal antibodies
US6703199B1 (en) 1997-06-12 2004-03-09 Research Corporation Technologies, Inc. Artificial antibody polypeptides
US6696245B2 (en) 1997-10-20 2004-02-24 Domantis Limited Methods for selecting functional polypeptides
US20040038291A2 (en) 1997-10-20 2004-02-26 Domantis Limited Method to screen phage display libraries with different ligands
US6846634B1 (en) 1997-10-20 2005-01-25 Domantis Limited Method to screen phage display libraries with different ligands
US20050202512A1 (en) 1997-10-20 2005-09-15 Domantis Limited Method to screen phage display libraries with different ligands
US6210924B1 (en) 1998-08-11 2001-04-03 Amgen Inc. Overexpressing cyclin D 1 in a eukaryotic cell line
US20030039958A1 (en) 1999-12-03 2003-02-27 Domantis Limited Direct screening method
US20040009507A1 (en) 2000-10-13 2004-01-15 Domantis, Ltd. Concatenated nucleic acid sequence
US20050238646A1 (en) 2001-01-17 2005-10-27 Trubion Pharmaceuticals, Inc. Binding domain-immunoglobulin fusion proteins
US20040202995A1 (en) 2003-04-09 2004-10-14 Domantis Nucleic acids, proteins, and screening methods
WO2007124463A1 (en) * 2006-04-20 2007-11-01 Amgen Inc. Glp-1 compound/glucagon antibody compositions
US20080036341A1 (en) 2006-08-11 2008-02-14 Nilsen John Ceiling storage container mounting system
US20120128679A1 (en) 2010-11-23 2012-05-24 Regeneron Pharmaceuticals, Inc. Human Antibodies to the Glucagon Receptor
CN103314011A (zh) * 2010-11-23 2013-09-18 瑞泽恩制药公司 胰高血糖素受体的人抗体

Non-Patent Citations (75)

* Cited by examiner, † Cited by third party
Title
"Proteins, Structures and Molecular Principles", 1984, W. H. FREEMAN AND COMPANY
"Remington's Pharmaceutical Sciences", 1980, MACK PUBLISHING COMPANY
AHREN ET AL., PEPTIDES, vol. 67, 2015, pages 74 - 81
ANDREWS, S.M.TITUS, J. A. ET AL.: "Current Protocols in Immunology", 2003, JOHN WILEY & SONS, pages: 2.8.1 - 2.8.10,2.10A.1-2.10A.5
ASHKENAZI ET AL., PNAS USA, vol. 88, 1991, pages 10535
BAINES ET AL.: "Methods in Molecular Biology", vol. 10, 1992, THE HUMANA PRESS, INC., article "Purification of Immunoglobulin G (IgG", pages: 79 - 104
BARON ET AL., NUCLEIC ACIDS RES., vol. 23, pages 3605 - 06
BAUER ET AL., GENE, vol. 37, 1985, pages 73
BAUM ET AL., EMBO J., vol. 13, 1994, pages 3992 - 4001
BOWIE ET AL., SCIENCE, vol. 253, 1991, pages 164 - 170
BRAND ET AL., DIABETES, vol. 45, 1996, pages 1076
BRENNER ET AL., CURR. OP. STRUCT. BIOL., vol. 7, 1997, pages 369 - 376
BYRN ET AL., NATURE, vol. 344, 1990, pages 677
CHOU ET AL., ADV. ENZYMOL. RELAT. AREAS MOL. BIOL., vol. 47, 1978, pages 45 - 148
CHOU ET AL., ANN. REV. BIOCHEM., vol. 47, 1979, pages 251 - 276
CHOU ET AL., BIOCHEMISTRY, vol. 113, 1974, pages 211 - 222
CHOU ET AL., BIOPHYS. J., vol. 26, pages 367 - 384
COURTENAY-LUCK ET AL.: "Monoclonal Antibodies: Production, Engineering and Clinical Application", 1995, CAMBRIDGE UNIVERSITY PRESS, article "Genetic Manipulation of Monoclonal Antibodies", pages: 166
CRAIK, BIOTECHNIQUES, vol. 3, 1985, pages 12 - 19
DE GRAAF ET AL., METHODS MOL. BIOL., vol. 178, 2002, pages 379 - 87
EDELMAN ET AL.: "Methods in Enzymology", vol. 1, 1967, ACADEMIC PRESS, pages: 422
FANSLOW ET AL., SEMIN. IMMUNOL., vol. 6, 1994, pages 267 - 78
GRIBSKOV ET AL., METH. ENZYM., vol. 183, 1990, pages 146 - 159
GRIBSKOV ET AL., PROC. NAT. ACAD. SCI., vol. 84, 1987, pages 4355 - 4358
GU ET AL., JPET, vol. 331, 2009, pages 871 - 881
HARRIS, JOURNAL OF CHROMATOGRAPHY, vol. 705, 1995, pages 129 - 134
HOLLENBAUGH ET AL.: "Construction of Immunoglobulin Fusion Proteins", CURRENT PROTOCOLS IN IMMUNOLOGY, 1992, pages 10.19.1 - 10.19.11
HOLLIGER ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 90, 1993, pages 6444 - 48
HOLLINGERHUDSON, NATURE BIOTECHNOLOGY, vol. 23, 2005, pages 1126 - 1136
HOLM ET AL., NUCL. ACID. RES., vol. 27, 1999, pages 244 - 247
HOPP ET AL., BIO/TECHNOLOGY, vol. 6, 1988, pages 1204
HOPPE ET AL., FEBS LETTERS, vol. 344, 1994, pages 191
HUSTON ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 85, 1988, pages 5879 - 83
HUSTON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 5879
JELINEK ET AL., SCIENCE, vol. 259, 1993, pages 1614 - 16
JONES, CURR. OPIN. STRUCT. BIOL., vol. 7, 1997, pages 377 - 87
KABAT ET AL.: "Sequences of Proteins of Immunological Interest", 1991, U.S. DEPT. OF HEALTH AND HUMAN SERVICES, PHS, NIH, NIH, article "Publication No. 91-3242"
KORNDORFER ET AL., PROTEINS, vol. 53, 2003, pages 121 - 129
KORTT ET AL., PROT. ENG., vol. 10, 1997, pages 423
KOSTIC ET AL., DIABETES OBES METAB, vol. 20, 2018, pages 283 - 91
KRIANGKUM ET AL., BIOMOL. ENG., vol. 18, 2001, pages 31 - 108
LANDSCHULZ ET AL., SCIENCE, vol. 240, 1988, pages 1759 - 426
LARRICK ET AL., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, vol. 2, 1991, pages 106
LEE ET AL., DIABETOLOGIA, vol. 59, pages 1372 - 5
LI ET AL., BIOCHEM PHARMACOL., vol. 150, 2018, pages 46 - 53
LI, C ET AL.: "Glutazumab, a novel long-lasting GLP-1/anti-GLP-1R antibo- dy fusion protein, exerts anti-diabetic effects through targeting dual re- ceptor binding sites", BIOCHEM. PHARMACOL., 3 February 2018 (2018-02-03), XP055642385, Retrieved from the Internet <URL:https://api.elsevier.com/content/article/PII:S0006295218300297?httpAccept=text/plain> *
LUNDE ET AL., BIOCHEM. SOC. TRANS., vol. 30, 2002, pages 500 - 06
MANIATIS ET AL., SCIENCE, vol. 236, 1987, pages 1237
MARI, DIABETES OBES METAB, vol. 18, 2016, pages 834 - 9
MARKS ET AL., BIOLTECHNOLOGY, vol. 10, 1992, pages 779 - 783
MCMAHAN ET AL., EMBO J., vol. 10, 1991, pages 2821
MOULT, CURR. OP. BIOTECH., vol. 7, 1996, pages 422 - 427
NISONOFFET ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 89, 1960, pages 230
NYGRENUHLEN, CURRENT OPINION IN STRUCTURAL BIOLOGY, vol. 7, 1997, pages 463 - 469
PATTEN ET AL., CURR. OPIN. BIOTECHNOL., vol. 8, 1997, pages 724 - 733
PITTNERFAIN, BIOCHEM J., vol. 277, 1991, pages 371 - 8
POLJAK ET AL., STRUCTURE, vol. 2, 1994, pages 1121 - 23
PORTER, BIOCHEM. J., vol. 73, 1959, pages 119
POUWELS ET AL.: "Cloning Vectors: A Laboratory Manual", 1985, ELSEVIER
RASMUSSEN ET AL., CYTOTECHNOLOGY, vol. 28, 1998, pages 31
ROQUE ET AL., BIOTECHNOL. PROG., vol. 20, 2004, pages 639 - 654
SAMBROOKFRITSCHMANIATIS: "Current Protocols in Molecular Biology", 1995, COLD SPRING HARBOR LABORATORY PRESS, pages: 6.3.1 - 6.3.6
See also references of EP3778634A4
SIPPL ET AL., STRUCTURE, vol. 4, 1996, pages 15 - 19
SMITH ET AL.: "Genetic Engineering: Principles and Methods", 1981, PLENUM PRESS
THOMPSON ET AL., J. MOL. BIOL., vol. 256, 1996, pages 350 - 368
THORNTON ET AL., NATURE, vol. 354, 1991, pages 105 - 291
URLAUB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 77, 1980, pages 4216 - 20
VAUGHAN ET AL., NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 535 - 539
VOSS ET AL., TRENDS BIOCHEM. SCI., vol. 11, 1986, pages 287
WAKELAM ET AL., NATURE, vol. 323, 1986, pages 68 - 71
WALDER ET AL., GENE, vol. 42, 1986, pages 133
WARD ET AL., NATURE, vol. 334, 1989, pages 544 - 546
WARD ET AL.: "Monoclonal Antibodies: Principles and Applications", 1995, WILEY-LISS, INC., article "Genetic Manipulation and Expression or Antibodies"
YANG ET AL., J. MOL. BIOL., vol. 254, 1995, pages 392 - 403

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3901173A4 (en) * 2018-12-21 2022-11-23 Jiangsu Hengrui Medicine Co., Ltd. BISPECIFIC PROTEIN

Also Published As

Publication number Publication date
CN110357959B (zh) 2023-02-28
EP3778634A1 (en) 2021-02-17
CA3096556A1 (en) 2019-10-17
TW201945392A (zh) 2019-12-01
CN110357959A (zh) 2019-10-22
EP3778634A4 (en) 2022-04-06
US11542336B2 (en) 2023-01-03
US20210122827A1 (en) 2021-04-29

Similar Documents

Publication Publication Date Title
WO2021052349A1 (zh) Gipr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
TWI832847B (zh) Gipr抗體及其與glp-1的融合蛋白質，以及其藥物組合物和應用
WO2021008519A1 (zh) ETA抗体与TGF-βTrap的融合蛋白质，以及其药物组合物和应用
WO2019196603A1 (zh) Gcgr抗体及其与glp-1的融合蛋白质，以及其药物组合物和应用
TWI826351B (zh) Ｒ抗體,其藥物組合物及其應用
WO2020248967A1 (zh) Eta抗体与bnp的融合蛋白质，以及其药物组合物和应用
EP3808847A1 (en) Apj antibody, fusion protein thereof with elabela, and pharmaceutical compositions and use thereof
TWI842699B (zh) Gcgr抗體及其與glp-1的融合蛋白質，以及其藥物組合物和應用
WO2024051802A1 (zh) Gipr抗体及其与fgf21的融合蛋白，以及其药物组合物和应用
RU2800370C2 (ru) Антитело к gipr и его слитый с glp-1 белок, а также фармацевтическая композиция на его основе и его применение
EP4317187A1 (en) Antibody capable of specifically binding to human endothelin receptor, and use thereof in treatment of diabetic nephropathy and chronic nephropathy

Legal Events

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

Ref document number: 19785909

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 3096556

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2019785909

Country of ref document: EP

Effective date: 20201110