Classifications

• • 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
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • 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
  • A61P3/04—Anorexiants; Antiobesity agents
• • 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
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
• • 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
  • C07K2317/56—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
  • C07K2317/70—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
  • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/90—Fusion polypeptide containing a motif for post-translational modification
  • C07K2319/92—Fusion polypeptide containing a motif for post-translational modification containing an intein ("protein splicing")domain

Definitions

• Obesity is a complex medical disorder of appetite regulation and/or metabolism resulting in excessive accumulation of adipose tissue mass.
• Obesity is an important clinical problem and is becoming an epidemic disease in western cultures, affecting more than one-third of the US adult population. It is estimated that about 97 million adults in the United States are overweight or obese. Obesity is further associated with premature death and with a significant increase in mortality and morbidity from stroke, myocardial infarction, congestive heart failure, coronary heart disease, and sudden death. Obesity also exacerbates many health problems, both independently and in association with other diseases. The primary goals of obesity therapy are to reduce excess body weight, improve or prevent obesity-related morbidity and mortality, and maintain long-term weight loss.
• Nonalcoholic fatty liver disease including its more aggressive form nonalcoholic steatohepatitis (NASH) is also increasing in epidemic proportions concurrent with the obesity epidemic (Sowers JR et al., Cardiorenal Med, 1 :5-12, 201 1 ).
• the dramatic rise in obesity and NAFLD appears to be due, in part, to consumption of a western diet (WD) containing high amounts of fat and sugar (e.g., sucrose or fructose), as fructose consumption in the US has more than doubled in the last three decades (Barrera et al, Clin Liver Dis, 18:91 - 1 12, 2014).
• NAFLD is characterized by macrovesicular steatosis of the liver occurring in individuals who consume little or no alcohol.
• the histological spectrum of NAFLD includes the presence of steatosis alone, fatty liver and inflammation.
• NASH is a more serious chronic liver disease characterized by excessive fat accumulation in the liver that, for reasons that are still incompletely understood, induces chronic inflammation which leads to progressive fibrosis (scarring) that can lead to cirrhosis, hepatocellular carcinoma (HCC), eventual liver failure and death (Brunt et al., Am. J. Gastroenterol. 94:2467-2474, 1999; Brunt, Semin. Liver Dis. 21 :3-16, 2001 ; Takahashi Y et al., World J Gastroenterol, 18:2300-2308, 2012).
• the present disclosure is based in part on the inventors' unique insight that isolated antigen binding and antagonizing proteins that specifically bind to the human glucagon receptor may provide for improved, effective therapies for treatment of diet induced obesity (DIO) and treatment of NAFLD/NASH in diabetic and non-diabetic subjects.
• DIO diet induced obesity
• NAFLD/NASH NAFLD/NASH in diabetic and non-diabetic subjects.
• the beneficial therapeutic effects provided by regulating glucose output in DIO and/or NAFLD/NASH subjects may include: reducing insulin resistance; reducing or preventing hyperinsulinemia, reducing or preventing fat deposits in the liver; reducing or preventing inflammation in the liver; reducing or preventing the accumulation of lipid, e.g., hepatic triacylglycerol, hepatic diacylglycerol, and ceramides; and preventing injury in the liver.
• lipid e.g., hepatic triacylglycerol, hepatic diacylglycerol, and ceramides
• the present disclosure comprises a method for treating or preventing NAFLD/NASH in a subject, comprising administering to a subject diagnosed with NAFLD/NASH, or a subject at risk of contracting NAFLD/NASH, a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor.
• the isolated antagonistic antigen binding protein comprises an antibody selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen- binding antibody fragment, a Fab, a Fab', a Fab 2 , a Fab' 2 , a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody.
• the antibody is a fully human monoclonal antibody.
• the present disclosure comprises a method for treating NAFLD.
• the present disclosure comprises a method for treating NASH.
• the present disclosure comprises a method for treating or preventing NAFLD/NASH in a subject, comprising (a) administering to a subject diagnosed with NAFLD/NASH, or a subject at risk of contracting NAFLD/NASH, a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor, and (b) an anti-obesity agent.
• the isolated antagonistic antigen binding protein comprises an antibody selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab 2 , a Fab' 2 , a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody.
• the antibody is a fully human monoclonal antibody.
• the present disclosure comprises a method for treating NAFLD. In various embodiments, the present disclosure comprises a method for treating NASH.
• the anti-obesity agent is selected from gut-selective MTP inhibitors, CCKa agonists, 5HT2c agonists, MCR4 agonists, lipase inhibitors, opioid antagonists, oleoyl-estrone, obinepitide, pramlintide (SYMLIN®), tesofensine, leptin, bromocriptine, orlistat, AOD-9604, and sibutramine.
• the present disclosure relates to methods of treating a subject classified as obese (e.g., having a body mass index (BMI) of 30 kg/m 2 or more) comprising administering to the subject a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor.
• BMI body mass index
• the isolated antagonistic antigen binding protein comprises an antibody selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen-binding antibody fragment, a Fab, a Fab', a Fab 2 , a Fab' 2 , a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody.
• the antibody is a fully human monoclonal antibody.
• the present disclosure relates to methods of treating a subject classified as obese (e.g., having a body mass index (BMI) of 30 kg/m 2 or more) comprising : (a) administering to the subject a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor, and (b) an anti- obesity agent.
• BMI body mass index
• the isolated antagonistic antigen binding protein comprises an antibody selected from a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigen- binding antibody fragment, a Fab, a Fab', a Fab 2 , a Fab' 2 , a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, or a diabody.
• the antibody is a fully human monoclonal antibody.
• the anti-obesity agent is selected from gut-selective MTP inhibitors, CCKa agonists, 5HT2c agonists, MCR4 agonists, lipase inhibitors, opioid antagonists, oleoyl-estrone, obinepitide, pramlintide (SYMLIN®), tesofensine, leptin, bromocriptine, orlistat, AOD-9604, and sibutramine.
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment, prophylaxis and/or prevention of nonalcoholic steatohepatitis (NASH) in a subject in need thereof.
• NASH nonalcoholic steatohepatitis
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment, prophylaxis and/or prevention of nonalcoholic fatty liver disease (NAFLD) in a subject in need thereof.
• NAFLD nonalcoholic fatty liver disease
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment a subject classified as obese (e.g., having a body mass index (BMI) of 30 kg/m 2 or more).
• BMI body mass index
• the isolated antagonistic binding protein binds to a human glucagon receptor with a dissociation constant (K D ) of at least about 1 x1 0 ⁇ 7 M, at least about 1 x10 8 M, at least about 1 x1 0 9 M, at least about 1 x10 10 M, at least about 1 x1 0 1 1 M, or at least about 1 x1 0 12 M.
• K D dissociation constant
• the isolated antagonistic binding protein is a fully human antibody which comprises the amino acid sequence encoding the heavy chain variable region of SEQ ID NO: 2 and the amino acid sequence encoding the light chain variable region of SEQ ID NO: 3.
• the isolated antagonistic protein is a fully human antibody which comprises the amino acid sequence encoding the heavy chain variable region of SEQ ID NO: 4 and the amino acid sequence encoding the light chain variable region of SEQ ID NO: 5.
• the isolated antagonistic protein is a fully human antibody which comprises the amino acid sequence encoding the heavy chain variable region of SEQ ID NO: 6 and the amino acid sequence encoding the light chain variable region of SEQ ID NO: 7.
• the isolated antagonistic protein is a fully human antibody which comprises a heavy chain variable region having the amino acid sequence selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 1 2, SEQ ID NO: 1 3, SEQ ID NO: 14, SEQ ID NO: 1 5, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.
• the isolated antagonistic protein is a fully human antibody which comprises a light chain variable region having the amino acid sequence selected from the group consisting of 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, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.
• the isolated antagonistic protein is a fully human antibody which comprises the amino acid sequence encoding the heavy chain variable region of SEQ ID NO: 28 and the amino acid sequence encoding the light chain variable region of SEQ ID NO: 47.
• the isolated antagonistic protein is a fully human antibody which comprises the amino acid sequence encoding the heavy chain of SEQ ID NO: 51 and the amino acid sequence encoding the light chain of SEQ ID NO: 52.
• the isolated antagonistic antigen binding protein that specifically binds the human glucagon receptor will be admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition that can be systemically administered to the subject via intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal injection, intra-arterial injection, intrasternal injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.
• a pharmaceutical composition that can be systemically administered to the subject via intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, transdermal injection, intra-arterial injection, intrasternal injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or via infusions.
• Figure 1 is a line plot depicting the in vivo effects on body weight (g) for the
• Vehicle group REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated for up to twenty weeks. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 2 is a line plot depicting the in vivo effects on food consumption
• Figure 3 is a line plot depicting the in vivo effects on blood glucose (mmol/L) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated for up to 19 weeks. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 4 is a line plot depicting the in vivo effects of repeat dosing of REMD 2.59 on blood glucose (mmol/L) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the
• Figure 5 is a bar graph depicting the AUC levels (mmol/L min blood glucose) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 6 is a bar graph depicting the in vivo effects on triglyceride (TG) levels
• Figure 7 is a bar graph depicting the in vivo effects on total cholesterol (TCHO) levels (mmol/L) in serum for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated for 20 weeks. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the
• REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 8 is a bar graph depicting the in vivo effects on alanine aminotransferase
• ALT aspartate aminotransferase
• AST aspartate aminotransferase
• GTT transpeptidase
• ALP alkaline phosphatase
• Figure 9 is a bar graph depicting the in vivo effects on triglyceride (TG) levels
• REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 10 is a bar graph depicting the in vivo effects on total cholesterol (TCHO) levels (mmol/L), high density lipoprotein cholesterol (HDL-C) levels (mg/g), and low density lipoprotein cholesterol (LDL-C) levels (mg/g) in the liver for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• TCHO total cholesterol
• HDL-C high density lipoprotein cholesterol
• LDL-C low density lipoprotein cholesterol
• Figure 1 1 is a bar graph depicting the in vivo effects on insulin levels (ng/mL) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated for 20 weeks. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding. The insulin levels on Day 57, 85, and 1 13 were tested after 6 hours of fasting, and on Day 141 after fating for 16 hours (OGTT study was conducted on that day).
• Figure 12 is a bar graph depicting the in vivo effects on leptin levels (ng/mL) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding. The insulin levels on Day 57, 85, and 1 13 were tested after 6 hours of fasting, and on Day 141 after fating for 16 hours (OGTT study was conducted on that day).
• Figure 13 is a bar graph depicting the in vivo effects on active glucagon-like peptide-1 (GLP-1 ) levels (pM) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• GLP-1 active glucagon-like peptide-1
• Figure 14 is a bar graph depicting the wet weight (g) for white adipose tissue
• Figure 15 is a bar graph depicting the IHC results (% insulin area/islet area and
• % glucagon are/islet area) for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the
• REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• Figure 16 depicts the results of histological H&E staining (20 X 10) of various liver sections for the Vehicle group, REMD2.59 group, Pair feeding group, Normal diet group, and Prevention group as described herein, in a HFD DIO mouse study, evaluated at week 20. Treatment commenced on Day 57 (i.e., start of week 9) for all groups but the Prevention group. For the Prevention group, REMD2.59C antibody was administered weekly starting from day 1 of HFD feeding.
• peptide refers to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. These terms encompass, e.g., native and artificial proteins, protein fragments and polypeptide analogs (such as muteins, variants, and fusion proteins) of a protein sequence as well as post- translationally, or otherwise covalently or non-covalently, modified proteins.
• a peptide, polypeptide, or protein may be monomeric or polymeric.
• “peptides”, “polypeptides”, and “proteins” are chains of amino acids whose alpha carbons are linked through peptide bonds.
• amino terminus refers to the free a-amino group on an amino acid at the amino terminal of a peptide or to the a-amino group (imino group when participating in a peptide bond) of an amino acid at any other location within the peptide.
• carboxy terminus refers to the free carboxyl group on the carboxy terminus of a peptide or the carboxyl group of an amino acid at any other location within the peptide.
• Peptides also include essentially any polyamino acid including, but not limited to, peptide mimetics such as amino acids joined by an ether as opposed to an amide bond.
• polypeptide sequences are indicated using standard one- or three-letter abbreviations. Unless otherwise indicated, polypeptide sequences have their amino termini at the left and their carboxy termini at the right, and single-stranded nucleic acid sequences, and the top strand of double-stranded nucleic acid sequences, have their 5' termini at the left and their 3' termini at the right.
• a particular section of a polypeptide can be designated by amino acid residue number such as amino acids 80 to 1 19, or by the actual residue at that site such as Ser80 to Ser1 19.
• a particular polypeptide or polynucleotide sequence also can be described by explaining how it differs from a reference sequence.
• Polypeptides of the disclosure include polypeptides that have been modified in any way and for any reason, for example, to: (1 ) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinities, and (5) confer or modify other physicochemical or functional properties.
• single or multiple amino acid substitutions e.g., conservative amino acid substitutions
• a "conservative amino acid substitution” refers to the substitution in a polypeptide of an amino acid with a functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another:
• I Isoleucine
• L Leucine
• M Methionine
• V Valine
• Phenylalanine (F), Tyrosine (Y), and Tryptophan (W) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)
• a "non-conservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class.
• the hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
• the substitution of like amino acids can be made effectively on the basis of hydrophilicity, particularly where the biologically functional protein or peptide thereby created is intended for use in immunological embodiments, as disclosed herein.
• the greatest local average hydrophilicity of a protein as governed by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with a biological property of the protein.
• hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1 ); glutamate (+3.0.+-.1 ); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1 ); alanine (-0.5); histidine (-0.5); cysteine (-1 .0); methionine (-1 .3); valine (-1 .5); leucine (-1 .8); isoleucine (-1 .8); tyrosine (-2.3); phenylalanine (-2.5) and tryptophan (-3.4).
• the substitution of amino acids whose hydrophilicity values are within + 2 is included, in certain embodiments, those that are within + 1 are included, and in certain embodiments, those within + 0.5 are included.
• Exemplary amino acid substitutions are set forth in Table 1 .
• a skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques.
• one skilled in the art may identify suitable areas of the molecule that may be changed without destroying activity by targeting regions not believed to be important for activity.
• the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides.
• even areas that may be important for biological activity or for structure may be subject to conservative amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
• One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of a polypeptide with respect to its three-dimensional structure. In certain embodiments, one skilled in the art may choose to not make radical changes to amino acid residues predicted to be on the surface of the polypeptide, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those skilled in the art. Such variants could be used to gather information about suitable variants.
• polypeptide fragment and "truncated polypeptide” as used herein refers to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion as compared to a corresponding full-length protein.
• fragments can be, e.g., at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 amino acids in length.
• fragments can also be, e.g., at most 1000, at most 900, at most 800, at most 700, at most 600, at most 500, at most 450, at most 400, at most 350, at most 300, at most 250, at most 200, at most 150, at most 100, at most 50, at most 25, at most 10, or at most 5 amino acids in length.
• a fragment can further comprise, at either or both of its ends, one or more additional amino acids, for example, a sequence of amino acids from a different naturally-occurring protein ⁇ e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence ⁇ e.g., an artificial linker sequence).
• polypeptide variant and “polypeptide mutant” as used herein refers to a polypeptide that comprises an amino acid sequence wherein one or more amino acid residues are inserted into, deleted from and/or substituted into the amino acid sequence relative to another polypeptide sequence.
• the number of amino acid residues to be inserted, deleted, or substituted can be, e.g., at least 1 , at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length.
• Variants of the present disclosure include fusion proteins.
• a "derivative" of a polypeptide is a polypeptide that has been chemically modified, e.g., conjugation to another chemical moiety such as, for example, polyethylene glycol, albumin ⁇ e.g., human serum albumin), phosphorylation, and glycosylation.
• % sequence identity is used interchangeably herein with the term “% identity” and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences, when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same thing as 80% sequence identity determined by a defined algorithm, and means that a given sequence is at least 80% identical to another length of another sequence.
• the % identity is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence identity to a given sequence. In certain embodiments, the % identity is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
• % homology refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences, when aligned using a sequence alignment program.
• 80% homology means the same thing as 80% sequence homology determined by a defined algorithm, and accordingly a homologue of a given sequence has greater than 80% sequence homology over a length of the given sequence.
• the % homology is selected from, e.g., at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% or more sequence homology to a given sequence.
• the % homology is in the range of, e.g., about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.
• Sequence searches are typically carried out using the BLASTP program when evaluating a given amino acid sequence relative to amino acid sequences in the GenBank Protein Sequences and other public databases.
• the BLASTX program is preferred for searching nucleic acid sequences that have been translated in all reading frames against amino acid sequences in the GenBank Protein Sequences and other public databases. Both BLASTP and BLASTX are run using default parameters of an open gap penalty of 1 1 .0, and an extended gap penalty of 1 .0, and utilize the BLOSUM-62 matrix. See id.
• the BLAST algorithm In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787 (1993)).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is, e.g., less than about 0.1 , less than about 0.01 , or less than about 0.001 .
• isolated molecule is a molecule that by virtue of its origin or source of derivation
• a protein or polypeptide is “substantially pure,” “substantially homogeneous,” or
• substantially purified when at least about 60% to 75% of a sample exhibits a single species of polypeptide.
• a substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80% or 90% W W of a protein sample, more usually about 95%, and e.g., will be over 99% pure.
• Protein purity or homogeneity may be indicated by a number of means well known in the art, such as polyacrylamide gel electrophoresis of a protein sample, followed by visualizing a single polypeptide band upon staining the gel with a stain well known in the art. For certain purposes, higher resolution may be provided by using HPLC or other means well known in the art for purification.
• an "antigen binding and antagonizing protein” is a protein comprising a portion that binds to an antigen and, optionally, a scaffold or framework portion that allows the antigen binding portion to adopt a conformation that promotes binding of the isolated antagonistic antigen binding protein to the antigen.
• antigen binding and antagonizing proteins include antibodies, antibody fragments (e.g., an antigen binding portion of an antibody), antibody derivatives, and antibody analogs.
• the isolated antagonistic antigen binding protein can comprise, for example, an alternative protein scaffold or artificial scaffold with grafted CDRs or CDR derivatives.
• Such scaffolds include, but are not limited to, antibody-derived scaffolds comprising mutations introduced to, for example, stabilize the three-dimensional structure of the isolated antagonistic antigen binding protein as well as wholly synthetic scaffolds comprising, for example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins:
• PAMs peptide antibody mimetics
• An isolated antagonistic antigen binding protein can have, for example, the structure of a naturally occurring immunoglobulin.
• An "immunoglobulin” is a tetrameric molecule. In a naturally occurring immunoglobulin, each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
• the amino-terminal portion of each chain includes a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
• the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. Human light chains are classified as kappa and lambda light chains.
• Heavy chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.
• the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D” region of about 10 more amino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in its entirety for all purposes).
• the variable regions of each light/heavy chain pair form the antibody binding site such that an intact immunoglobulin has two binding sites.
• an “antibody” refers to a protein comprising one or more polypeptides
• immunoglobulin genes substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes and having specificity to a tumor antigen or specificity to a molecule overexpressed in a pathological state.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as subtypes of these genes and myriad of immunoglobulin variable region genes.
• Light chains (LC) are classified as either kappa or lambda.
• Heavy chains (HC) are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• a typical immunoglobulin (e.g., antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy" chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
• each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or V H ) and a heavy chain constant region.
• the heavy chain constant region is comprised of three domains, Cm , CH2 and CH3 (and in some instances, CH 4 ).
• Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or Vi_) and a light chain constant region.
• the light chain constant region is comprised of one domain, Ci_.
• the V H and V L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
• CDR complementarity determining regions
• Each V H and V L is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRi , CDRi , FR 2 , CDR 2 , FR 3 , CDR 3 , FR 4 .
• the extent of the framework region and CDRs has been defined.
• the sequences of the framework regions of different light or heavy chains are relatively conserved within a species, such as humans.
• the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
• Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass.
• type e.g., IgG, IgE, IgM, IgD, IgA and IgY
• class e.g., lgG1 , lgG2, IgG 3, lgG4, lgA1 and lgA2 or subclass.
• Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab 2 , F(ab)' 2 fragments, single chain Fv proteins ("scFv”) and disulfide stabilized Fv proteins ("dsFv”), that bind to the target antigen.
• a scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
• antibody encompasses e.g., monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab') 2 fragments, antibody fragments that exhibit the desired biological activity, disulfide-linked Fvs (sdFv), intrabodies, and epitope-binding fragments or antigen binding fragments of any of the above.
• monoclonal antibodies including full-length monoclonal antibodies
• polyclonal antibodies multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab'
• a Fab fragment is a monovalent fragment having the V L , V H , Ci_ and Cm domains; a F(ab') 2 fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the V H and C.sub.HI domains; an Fv fragment has the Vi_ and V H domains of a single arm of an antibody; and a dAb fragment has a V H domain, a V L domain, or an antigen-binding fragment of a V H or V L domain (U.S. Pat. Nos. 6,846,634, 6,696,245, US App. Pub. No. 05/0202512, 04/0202995, 04/0038291 , 04/0009507, 03/0039958, Ward et al., Nature 341 :544-546 (1989)).
• a single-chain antibody is an antibody in which a V L and a V H region are joined via a linker (e.g., a synthetic sequence of amino acid residues) to form a continuous protein chain wherein the linker is long enough to allow the protein chain to fold back on itself and form a monovalent antigen binding site (see, e.g., Bird et al., Science 242:423-26 (1988) and Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83 (1988)).
• a linker e.g., a synthetic sequence of amino acid residues
• Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises V H and Vi_ domains joined by a linker that is too short to allow for pairing between two domains on the same chain, thus allowing each domain to pair with a complementary domain on another polypeptide chain (see, e.g., Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48 (1993), and Poljak et al., Structure 2:1 121 -23 (1994)). If the two polypeptide chains of a diabody are identical, then a diabody resulting from their pairing will have two identical antigen binding sites.
• Polypeptide chains having different sequences can be used to make a diabody with two different antigen binding sites.
• tribodies and tetrabodies are antibodies comprising three and four polypeptide chains, respectively, and forming three and four antigen binding sites, respectively, which can be the same or different.
• An isolated antagonistic antigen binding protein may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For example, a naturally occurring human immunoglobulin typically has two identical binding sites, while a "bispecific" or "Afunctional" antibody has two different binding sites.
• human antibody includes all antibodies that have one or more variable and constant regions derived from human immunoglobulin sequences. In one embodiment, all of the variable and constant domains are derived from human immunoglobulin sequences (a fully human antibody). These antibodies may be prepared in a variety of ways, examples of which are described below, including through the immunization with an antigen of interest of a mouse that is genetically modified to express antibodies derived from human heavy and/or light chain-encoding genes.
• a "humanized antibody” has a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject.
• certain amino acids in the framework and constant domains of the heavy and/or light chains of the non-human species antibody are mutated to produce the humanized antibody.
• the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species.
• one or more amino acid residues in one or more CDR sequences of a non-human antibody are changed to reduce the likely immunogenicity of the non-human antibody when it is administered to a human subject, wherein the changed amino acid residues either are not critical for immunospecific binding of the antibody to its antigen, or the changes to the amino acid sequence that are made are conservative changes, such that the binding of the humanized antibody to the antigen is not significantly worse than the binding of the non-human antibody to the antigen. Examples of how to make humanized antibodies may be found in U.S. Pat. Nos. 6,054,297, 5,886,152 and 5,877,293.
• An isolated antagonistic antigen binding protein of the present disclosure including an antibody, "specifically binds" to an antigen, such as the human glucagon receptor if it binds to the antigen with a high binding affinity as determined by a dissociation constant (Kd, or corresponding Kb, as defined below) value of 10 ⁇ 7 M or less.
• Kd dissociation constant
• An isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor may be able to bind to glucagon receptors from other species as well with the same or different affinities.
• An “epitope” is the portion of a molecule that is bound by an isolated antagonistic antigen binding protein (e.g., by an antibody).
• An epitope can comprise non-contiguous portions of the molecule (e.g., in a polypeptide, amino acid residues that are not contiguous in the polypeptide's primary sequence but that, in the context of the polypeptide's tertiary and quaternary structure, are near enough to each other to be bound by an antigen binding and antagonizing protein).
• blood glucose level or "level of blood glucose” shall mean blood glucose concentration.
• a blood glucose level is a plasma glucose level.
• Plasma glucose may be determined in accordance with, e.g., Etgen et al., Metabolism, 49(5): 684-688, 2000) or calculated from a conversion of whole blood glucose concentration in accordance with D'Orazio et al., Clin. Chem. Lab. Med., 44(12): 1486- 1490, 2006.
• normal glucose levels refers to mean plasma glucose values in humans of less than about 100 mg/dL for fasting levels, and less than about 145 mg/dL for 2- hour post-prandial levels or 125 mg/dL for a random glucose.
• elevated blood glucose level or “elevated levels of blood glucose” shall mean an elevated blood glucose level such as that found in a subject demonstrating clinically inappropriate basal and postprandial
• hyperglycemia or such as that found in a subject in oral glucose tolerance test (oGTT), with "elevated levels of blood glucose” being greater than about 100 mg/dL when tested under fasting conditions, and greater than about 200 mg/dL when tested at 1 hour.
• OGTT oral glucose tolerance test
• glucagon inhibitor a molecule that detectably inhibits glucagon signaling.
• the inhibition caused by an inhibitor need not be complete so long as the inhibition is detectable using an assay that is recognized and understood in the art as being determinative of glucagon signaling inhibition.
• a “pharmaceutical composition” refers to a composition suitable for oral delivery.
• a pharmaceutical composition comprises a pharmacologically and/or therapeutically effective amount of an active agent and a pharmaceutically acceptable carrier.
• “Pharmaceutically acceptable carrier” refers to compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
• pharmaceutically acceptable carrier refers to any of the standard pharmaceutical carriers, vehicles, buffers, and carriers, such as a phosphate buffered saline solution, 5% aqueous solution of dextrose, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants.
• a "pharmaceutically acceptable salt” is a salt that can be formulated into a compound for pharmaceutical use including, e.g., metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.
• a "therapeutically effective amount" of an isolated antagonistic antigen binding protein that specifically binds the human glucagon receptor refers to an amount of such protein that, when provided to a subject in accordance with the disclosed and claimed methods effects one of the following biological activities: treats obesity; treats NAFLD; treats NASH; or reduces, suppresses, attenuates, or inhibits one or more symptoms of NASH.
• beneficial or desired clinical results include, but are not limited to, one or more of the following: improvement in blood glucose to within about 80-180 mg/dL, or to within about 80-170 mg/dL, or to within about 80-160 mg/dL, or to within about 80-150 mg/dL, or to within about 80-140 mg/dL, or an improvement in any one or more conditions, diseases, or symptoms associated with, or resulting from, elevated levels of blood glucose including, but not limited to, hyperglycemia, hyperglucanemia, and hyperinsulinemia.
• Obesity is a medical condition in which excess body fat has accumulated in multiple tissues, including the liver, to the extent that it may have an adverse effect on health.
• BMI body mass index
• Obesity is most commonly caused by a combination of excessive food energy intake, lack of physical activity, and genetic susceptibility, although a few cases are caused primarily by genes, endocrine disorders, medications, or psychiatric illness. Obesity increases the likelihood of various diseases, particularly heart disease, type 2
• diabetes NAFLD/NASH
• obstructive sleep apnea certain types of cancer
• osteoarthritis and asthma.
• Complications are either directly caused by obesity or indirectly related through mechanisms sharing a common cause such as a poor diet or a sedentary lifestyle.
• the strength of the link between obesity and specific conditions varies. One of the strongest is the link with type 2 diabetes. Excess body fat underlies 64% of cases of diabetes in men and 77% of cases in women.
• Bariatric surgery may be considered as a weight loss intervention for subjects at or exceeding a BMI of 40 kg/m 2 .
• Subjects with a BMI of ⁇ 35 kg/m 2 and with an associated serious medical condition are also candidates for this treatment option.
• postoperative complications commonly result from bariatric surgical procedures, including bleeding, embolism or thrombosis, wound complications, deep infections, pulmonary infections
• Rates of reoperation or conversion surgery beyond the postoperative period depend upon the type of bariatric procedure, and in one study ranged from 17% to 31 %. Intestinal absorptive abnormalities, such as micronutrient deficiency and protein-calorie malnutrition, also are typically seen with bypass procedures, requiring lifelong nutrient supplementation. Major and serious adverse outcomes associated with bariatric surgery are common, observed in approximately 4 percent of procedures performed (including death in 0.3 to 2 percent of all subjects receiving laparoscopic banding or bypass surgeries, respectively).
• DIO diet induced obesity
• the present disclosure provides antigen binding and antagonizing proteins that specifically bind to the human glucagon receptor that may provide for improved, effective therapies for treatment of DIO in diabetic and non- diabetic subjects.
• NAFLD Nonalcoholic Fatty Liver Disease
• NASH Nonalcoholic Steatohepatitis
• Nonalcoholic fatty liver disease is highly prevalent in the Western population. Recent studies suggest that this disease may occur at a frequency of 70% in obese individuals and 35% in lean individuals (Wanless IR, et al., Hepatology, 12:1 106-1 1 10, 1990).
• NAFLD is characterized by macrovesicular steatosis of the liver occurring in individuals who consume little or no alcohol. The histological spectrum of NAFLD is classified as simple steatosis alone, or nonalcoholic steatohepatitis (NASH).
• NASH nonalcoholic steatohepatitis
• NAFLD is also strongly associated with the ingestion of fructose, especially from sweetened beverages (Ouyang X, et al., J Hepatol., 48:993-999, 2008).
• the classic Western diet is high in both saturated fats and in sugar.
• hyperinsulinemia that develops with consumption of high-fat and high-sugar diets (e.g., simple sugar such as glucose, fructose) is closely linked to NAFLD.
• high-fat and high-sugar diets e.g., simple sugar such as glucose, fructose
• NAFLD simple sugar such as glucose, fructose
• hepatic insulin resistance is caused by dysfunction in three pathways of energy metabolism (Samuel et al, Cell, 148:852-871 , 2012).
• excess carbohydrate flux e.g., glucose, fructose
• Id de novo lipogenesis
• triacylglycerols which are inert but often track with increased levels of bioactive lipid intermediates diacylglycerols (DAG) and ceramides that putatively lead to hepatic insulin resistance
• DAG bioactive lipid intermediates diacylglycerols
• ceramides that putatively lead to hepatic insulin resistance
• the hepatic steatosis linked to insulin resistance is associated with mitochondrial dysfunction and altered hepatic fatty acid oxidation (Rector RS et al., J Hepatol, 52:727-736, 2010).
• NAFLD nonalcoholic steatohepatitis
• HCC hepatocellular carcinoma
• Glucagon is a 29 amino acid hormone processed from its pre-pro-form in the pancreatic alpha cells by cell specific expression of prohormone convertase 2 (PC2), a neuroendocrine-specific protease involved in the intracellular maturation of prohormones and proneuropeptides (Furuta et al., J. Biol. Chem. 276: 27197-27202 (2001 )).
• PC2 prohormone convertase 2
• glucagon is a major counter-regulatory hormone for insulin actions. During fasting, glucagon secretion increases in response to falling glucose levels.
• glucagon secretion stimulates glucose production by promoting hepatic glycogenolysis and gluconeogenesis (Dunning and Gerich, Endocrine Reviews, 28:253-283 (2007)). Thus glucagon counterbalances the effects of insulin in maintaining normal levels of glucose in animals.
• the biological effects of glucagon are mediated through the binding and subsequent activation of a specific cell surface receptor, the glucagon receptor.
• the glucagon receptor (GCGR) is a member of the secretin subfamily (family B) of G-protein-coupled receptors.
• the human GCGR is a 477 amino acid sequence GPCR and the amino acid sequence of GCGR is highly conserved across species (Mayo et al, Pharmacological Rev., 55:167-194, (2003)).
• the glucagon receptor is predominantly expressed in the liver, where it regulates hepatic glucose output, on the kidney, and on islet ⁇ -cells, reflecting its role in gluconeogenesis.
• glucagon receptors The activation of the glucagon receptors in the liver stimulates the activity of adenyl cyclase and phosphoinositol turnover which subsequently results in increased expression of gluconeogenic enzymes including phosphoenolpyruvate carboxykinase (PEPCK), fructose- 1 ,6-bisphosphatase (FBPase-1 ), and glucose-6-phosphatase (G-6-Pase).
• PEPCK phosphoenolpyruvate carboxykinase
• FBPase-1 fructose- 1 ,6-bisphosphatase
• G-6-Pase glucose-6-phosphatase
• glucagon signaling activates glycogen phosphorylase and inhibits glycogen synthase.
• the antigen binding and antagonizing proteins of the present disclosure may be selected to bind to membrane-bound glucagon receptors as expressed on cells, and inhibit or block glucagon signaling through the glucagon receptor.
• the antigen binding and antagonizing proteins of the present disclosure specifically bind to the human glucagon receptor.
• the antigen binding and antagonizing proteins binding to the human glucagon receptor may also bind to the glucagon receptors of other species.
• the polynucleotide and polypeptide sequences for several species of glucagon receptor are known (see, e.g., U.S. Pat. No.
• the antigen binding and antagonizing proteins specifically bind the human glucagon receptor having the amino acid sequence set forth in SEQ ID NO: 1 :
• the antigen binding and antagonizing proteins of the present disclosure specifically bind glucagon receptors which have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (as calculated using methods known in the art and described herein) to the glucagon receptors described in the cited references are also included in the present disclosure.
• the antigen binding and antagonizing proteins of the present disclosure function to block the interaction between glucagon and its receptor, thereby inhibiting the glucose elevating effects of glucagon.
• the use of the antigen binding and antagonizing proteins of the present disclosure are an effective means of achieving normal levels of glucose, thereby ameliorating, or preventing one or more symptoms of, or long term complications caused by diabetes including, but not limited to, hyperglycemia, hyperglucanemia, and hyperinsulinemia.
• the use of the antigen binding and antagonizing proteins of the present disclosure are also an effective means of achieving normal levels of glucose in non-diabetic patients, thereby lowering the risk of hyperglycemia, hyperglucanemia, and hyperinsulinemia in subjects having disorders including, but not limited to, obesity or NAFLDs, and for treating such non-diabetic disorders.
• Methods of generating antibodies that bind to antigens such as the human glucagon receptor are known to those skilled in the art.
• a method for generating a monoclonal antibody that binds specifically to a targeted antigen polypeptide may comprise administering to a mouse an amount of an immunogenic composition comprising the targeted antigen polypeptide effective to stimulate a detectable immune response, obtaining antibody- producing cells (e.g., cells from the spleen) from the mouse and fusing the antibody-producing cells with myeloma cells to obtain antibody-producing hybridomas, and testing the antibody- producing hybridomas to identify a hybridoma that produces a monocolonal antibody that binds specifically to the targeted antigen polypeptide.
• antibody- producing cells e.g., cells from the spleen
• a hybridoma can be propagated in a cell culture, optionally in culture conditions where the hybridoma-derived cells produce the monoclonal antibody that binds specifically to targeted antigen polypeptide.
• the monoclonal antibody may be purified from the cell culture. A variety of different techniques are then available for testing an antigen/antibody interaction to identify particularly desirable antibodies.
• Antibodies can be engineered in numerous ways. They can be made as single- chain antibodies (including small modular immunopharmaceuticals or SMIPsTM), Fab and F(ab') 2 fragments, etc. Antibodies can be humanized, chimerized, deimmunized, or fully human.
• Chimeric antibodies can be produced by recombinant DNA techniques known in the art. For example, a gene encoding the Fc constant region of a murine (or other species) monoclonal antibody molecule is digested with restriction enzymes to remove the region encoding the murine Fc, and the equivalent portion of a gene encoding a human Fc constant region is substituted (see Robinson et al., International Patent Publication PCT/US86/02269; Akira, et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171 ,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO 86/01533; Cabilly et al.
• a humanized antibody has one or more amino acid residues introduced from a source that is nonhuman, in addition to the nonhuman CDRs.
• Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525, 1986; Riechmann et al., Nature, 332:323-327, 1988; Verhoeyen et al., Science, 239:1534-1536, 1988), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S.
• humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some framework region residues are substituted by residues from analogous sites in rodent antibodies.
• U.S. Patent No. 5,693,761 to Queen et al discloses a refinement on Winter et al. for humanizing antibodies, and is based on the premise that ascribes avidity loss to problems in the structural motifs in the humanized framework which, because of steric or other chemical incompatibility, interfere with the folding of the CDRs into the binding-capable conformation found in the mouse antibody.
• Queen teaches using human framework sequences closely homologous in linear peptide sequence to framework sequences of the mouse antibody to be humanized. Accordingly, the methods of Queen focus on comparing framework sequences between species. Typically, all available human variable region sequences are compared to a particular mouse sequence and the percentage identity between correspondent framework residues is calculated.
• the human variable region with the highest percentage is selected to provide the framework sequences for the humanizing project. Queen also teaches that it is important to retain in the humanized framework, certain amino acid residues from the mouse framework critical for supporting the CDRs in a binding-capable conformation. Potential criticality is assessed from molecular models. Candidate residues for retention are typically those adjacent in linear sequence to a CDR or physically within 6A of any CDR residue.
• framework shuffling Another method of humanizing antibodies, referred to as “framework shuffling", relies on generating a combinatorial library with nonhuman CDR variable regions fused in frame into a pool of individual human germline frameworks (Dall'Acqua et al., Methods, 36:43, 2005). The libraries are then screened to identify clones that encode humanized antibodies which retain good binding.
• variable regions both light and heavy
• sequence of the variable region of a rodent antibody is screened against the entire library of known human variable-domain sequences.
• the human sequence that is closest to that of the rodent is then accepted as the human framework region (framework region) for the humanized antibody (Sims et al., J. Immunol., 151 :2296, 1993; Chothia et al., J. Mol. Biol., 196:901 , 1987).
• Another method uses a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions.
• the choice of nonhuman residues to substitute into the human variable region can be influenced by a variety of factors. These factors include, for example, the rarity of the amino acid in a particular position, the probability of interaction with either the CDRs or the antigen, and the probability of participating in the interface between the light and heavy chain variable domain interface. (See, for example, U.S. Patent Nos. 5,693,761 , 6,632,927, and 6,639,055).
• One method to analyze these factors is through the use of three-dimensional models of the nonhuman and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences.
• a method for producing an anti-GCGR antibody or antigen binding antibody fragment thereof comprises the steps of synthesizing a library of human antibodies on phage, screening the library with GCGR or an antibody binding portion thereof, isolating phage that bind GCGR, and obtaining the antibody from the phage.
• one method for preparing the library of antibodies for use in phage display techniques comprises the steps of immunizing a non-human animal comprising human immunoglobulin loci with GCGR or an antigenic portion thereof to create an immune response, extracting antibody-producing cells from the immunized animal; isolating RNA encoding heavy and light chains of antibodies of the disclosure from the extracted cells, reverse transcribing the RNA to produce cDNA, amplifying the cDNA using primers, and inserting the cDNA into a phage display vector such that antibodies are expressed on the phage.
• Recombinant anti-GCGR antibodies of the disclosure may be obtained in this way.
• recombinant human anti-GCGR antibodies of the disclosure can also be isolated by screening a recombinant combinatorial antibody library.
• the library is a scFv phage display library, generated using human V L and V H cDNAs prepared from mRNA isolated from B cells.
• Methods for preparing and screening such libraries are known in the art.
• Kits for generating phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAPTM phage display kit, catalog no. 240612).
• There also are other methods and reagents that can be used in generating and screening antibody display libraries see, e.g., U.S. Pat. No. 5,223,409; PCT Publication Nos.
• WO 92/18619 WO 91 /17271 , WO 92/20791 , WO 92/15679, WO 93/01288, WO 92/01047, WO 92/09690; Fuchs et al., Bio/Technology, 9:1370-1372 (1991 ); Hay et al., Hum. Antibod. Hybridomas, 3:81 -85, 1992; Huse et al., Science, 246:1275-1281 , 1989; McCafferty et al., Nature, 348:552-554, 1990; Griffiths et al., EMBO J., 12:725-734, 1993; Hawkins et al., J. Mol.
• Human antibodies are also produced by immunizing a non-human, transgenic animal comprising within its genome some or all of human immunoglobulin heavy chain and light chain loci with a human IgE antigen, e.g., a XenoMouseTM animal (Abgenix, Inc./Amgen, Inc.-Fremont, Calif.).
• XenoMouseTM mice are engineered mouse strains that comprise large fragments of human immunoglobulin heavy chain and light chain loci and are deficient in mouse antibody production. See, e.g., Green et al., Nature Genetics, 7:13-21 , 1994 and U.S. Pat. Nos.
• XenoMouseTM mice produce an adult-like human repertoire of fully human antibodies and generate antigen-specific human antibodies.
• the XenoMouseTM mice contain approximately 80% of the human antibody V gene repertoire through introduction of megabase sized, germline configuration fragments of the human heavy chain loci and kappa light chain loci in yeast artificial chromosome (YAC).
• YAC yeast artificial chromosome
• XenoMouseTM mice further contain approximately all of the human lambda light chain locus. See Mendez et al., Nature Genetics, 15:146-156, 1997; Green and Jakobovits, J. Exp. Med., 188:483-495, 1998; and WO 98/24893.
• the isolated antagonistic antigen binding protein of the present disclosure utilize an antibody or antigen binding antibody fragment thereof is a polyclonal antibody, a monoclonal antibody or antigen-binding fragment thereof, a recombinant antibody, a diabody, a chimerized or chimeric antibody or antigen-binding fragment thereof, a humanized antibody or antigen-binding fragment thereof, a fully human antibody or antigen- binding fragment thereof, a CDR-grafted antibody or antigen-binding fragment thereof, a single chain antibody, an Fv, an Fd, an Fab, an Fab', or an F(ab') 2 , and synthetic or semi-synthetic antibodies.
• the isolated antagonistic antigen binding protein of the present disclosure utilize an antibody or antigen-binding fragment that binds to a glucagon receptor antigen with a dissociation constant (K D ) of, e.g., at least about 1 x10 ⁇ 7 M, at least about 1 x10 8 M, at least about 1 x10 9 M, at least about 1 x10 10 M, at least about 1 x10 11 M, or at least about 1 x10 12 M.
• K D dissociation constant
• the isolated antagonistic antigen binding protein of the present disclosure utilize an antibody or antigen-binding fragment that binds to a glucagon receptor antigen with a dissociation constant (K D ) in the range of, e.g., at least about 1 x10 ⁇ 7 M to at least about 1 x10 8 M, at least about 1 x10 8 M to at least about 1 x10 9 M, at least about 1 x10 9 M to at least about 1 x10 _10 M, at least about 1 x10 10 M to at least about 1 x10 11 M, or at least about 1 x10 11 M to at least about 1 x10 12 M.
• K D dissociation constant
• Antibodies to the glucagon receptor have been described in, e.g., U.S. Pat. Nos.
• the isolated antagonistic antigen binding protein is an anti-GCGR ("antagonistic") antibody or antigen-binding fragment which comprises the polynucleotide and polypeptide sequences set forth in, e.g., U.S. Pat. No. 7,947,809, and 8,158,759, each herein incorporated by reference in its entirety for its specific teaching of polynucleotide and polypeptide sequences of various anti-GCGR antibodies or antigen-binding fragments.
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 2.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 2.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 2.
• the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 2. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 2:
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 3.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 3.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 3.
• the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 3. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 3:
• the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 2 or 3.
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 4.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 4.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 4.
• the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 4. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 4:
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 5.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 5.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 5.
• the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 5. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 5:
• the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 4 or 5.
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 6.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the heavy chain variable region sequence as set forth in SEQ ID NO: 6.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the heavy chain variable region sequence as set forth in SEQ ID NO: 6.
• the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 6. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the heavy chain variable region sequence as set forth in SEQ ID NO: 6:
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 7.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the light chain variable region sequence as set forth in SEQ ID NO: 7.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the light chain variable region sequence as set forth in SEQ ID NO: 7.
• the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 7. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the light chain variable region sequence as set forth in SEQ ID NO: 7:
• the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 6 or 7.
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the heavy chain sequence as set forth in SEQ ID NO: 8.
• the antibody is an anti-GCGR antibody which competes with the antibody comprising the heavy chain sequence as set forth in SEQ ID NO: 8.
• the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the heavy chain sequence as set forth in SEQ ID NO: 8.
• the antibody may be an anti-GCGR antibody which comprises the heavy chain sequence as set forth in SEQ ID NO: 8.
• the antibody may be an anti- GCGR antibody which comprises the heavy chain sequence as set forth in SEQ ID NO: 8: MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWV
• EKSLSHSPGK (SEQ ID NO: 8)
• the antibody may be an anti-
• the antibody may be an anti-GCGR antibody which binds to the same epitope as the antibody comprising the light chain sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody is an anti-GCGR antibody which competes with the antibody comprising the light chain sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-GCGR antibody which comprises at least one (such as two or three) CDRs of the light chain sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the light chain sequence as set forth in SEQ ID NO: 9. In various embodiments, the antibody may be an anti-GCGR antibody which comprises the light chain sequence as set forth in SEQ ID NO: 9:
• the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 8 or 9.
• the antibody may be an anti-
• GCGR antibody which comprises a heavy chain variable region sequence selected from SEQ ID NO: 10, SEQ ID NO: 1 1 , 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, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28, and a light chain variable region sequence selected from, 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, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 , SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID
• the antibody contains an amino acid sequence that shares an observed homology of, e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% with the sequences of SEQ ID NOS: 10-28 or SEQ ID NOS: 29-47.
• the isolated antagonistic antibody is a fully human antibody which comprises the amino acid sequence encoding the heavy chain variable region of SEQ ID NO: 28 and the amino acid sequence encoding the light chain variable region of SEQ ID NO: 47.
• An isolated anti-GCGR antibody, antibody fragment, or antibody derivative of the present disclosure can comprise any constant region known in the art.
• the light chain constant region can be, for example, a kappa- or lambda-type light chain constant region, e.g., a human kappa- or lambda-type light chain constant region.
• the heavy chain constant region can be, for example, an alpha-, delta-, epsilon-, gamma-, or mu-type heavy chain constant regions, e.g., a human 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 a naturally occurring constant region.
• IgG antibodies may be derived from an IgM antibody, for example, and vice versa.
• Such techniques allow the preparation of new antibodies that possess the antigen-binding properties of a given antibody (the parent antibody), but also exhibit biological properties associated with an antibody isotype or subclass different from that of the parent antibody.
• Recombinant DNA techniques may be employed. Cloned DNA encoding particular antibody polypeptides may be employed in such procedures, e.g., DNA encoding the constant domain of an antibody of the desired isotype. See also Lanitto et al., Methods Mol. Biol. 178:303-16 (2002).
• an isolated antigen binding protein of the present disclosure comprises the constant light chain kappa region as set forth in SEQ ID NO: 48, or a fragment thereof. In various embodiments, an isolated antigen binding protein of the present disclosure comprises the constant light chain lambda region as set forth in SEQ ID NO: 49, or a fragment thereof. In various embodiments, an isolated antigen binding protein of the present disclosure comprises a lgG2 heavy chain constant region set forth in SEQ ID NO: 50, or a fragment thereof.
• an isolated antagonistic antigen binding protein of the present disclosure is a fully human anti-GCGR antibody that comprises a heavy chain sequence as set forth in SEQ ID NO: 51 and a light chain as set forth in SEQ ID NO: 52.
• the isolated antagonistic antigen binding protein is a hemibody.
• a "hemibody” is an immunologically-functional immunoglobulin construct comprising a complete heavy chain, a complete light chain and a second heavy chain Fc region paired with the Fc region of the complete heavy chain.
• a linker can, but need not, be employed to join the heavy chain Fc region and the second heavy chain Fc region.
• the hemibody is a monovalent antigen binding protein comprising (i) an intact light chain, and (ii) a heavy chain fused to an Fc region (e.g., an lgG2 Fc region).
• the present disclosure provides a pharmaceutical composition
• a pharmaceutical composition comprising an isolated antagonistic antigen binding protein as described herein, with one or more pharmaceutically acceptable carrier(s).
• the pharmaceutical compositions and methods of uses described herein also encompass embodiments of combinations (co-administration) with other active agents, as detailed below.
• the antagonistic antigen binding proteins of the present disclosure are suitable to be administered as a formulation in association with one or more pharmaceutically acceptable carrier(s).
• the term 'carrier' is used herein to describe any ingredient other than the compound(s) of the disclosure.
• carrier(s) will to a large extent depend on factors such as the particular mode of administration, the effect of the carrier on solubility and stability, and the nature of the dosage form.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• compositions of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.
• the composition will include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• additional examples of pharmaceutically acceptable substances are wetting agents or minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the antibody.
• Pharmaceutical compositions of the present disclosure and methods for their preparation will be readily apparent to those skilled in the art.
• compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995).
• the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all GMP regulations of the U.S. Food and Drug Administration.
• parenteral administration of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to,
• parenteral administration is contemplated to include, but is not limited to, subcutaneous injection, intraperitoneal injection, intramuscular injection, intrasternal injection, intravenous injection, intraarterial injection, intrathecal injection, intraventricular injection, intraurethral injection, intracranial injection, intrasynovial injection or infusions; or kidney dialytic infusion techniques.
• a pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe.
• a pen delivery device readily has applications in delivering a
• Such a pen delivery device can be reusable or disposable.
• a reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused.
• a disposable pen delivery device there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.
• Numerous reusable pen and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present disclosure including, but not limited to AUTOPENTM (Owen Mumford, Inc., Woodstock, UK), DISETRONICTM (Disetronic Medical Systems, Burghdorf, Switzerland), and HUMALOG MIX 75/25TM, HUMALOGTM pen, HUMALIN 70/30TM pen (Eli Lilly and Co., Indianapolis, Ind.).
• administration typically generally comprise the active ingredient combined with a
• formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration.
• injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or in multi-dose containers containing a preservative.
• Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and the like.
• Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents.
• the active ingredient is provided in dry (i.e.
• Parenteral formulations also include aqueous solutions which may contain carriers such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
• a suitable vehicle e.g. sterile pyrogen-free water
• parenteral formulations also include aqueous solutions which may contain carriers such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but, for some applications, they may be more suitably formulated as a sterile non-aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
• Exemplary parenteral administration forms include solutions or
• administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation.
• administration may be formulated to be immediate and/or modified release.
• Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
• sterile injectable solutions can be prepared by incorporating the isolated antagonistic antigen binding protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
• methods of preparation such as vacuum drying and freeze-drying yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable
• compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
• the injectable compositions will be administered using commercially available disposable injectable devices.
• the isolated antagonistic antigen binding protein of the present disclosure can be administered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture, or as a mixed component particle, for example, mixed with a suitable
• a dry powder inhaler as an aerosol spray from a pressurized container, pump, spray, atomiser (preferably an atomiser using
• electrohydrodynamics to produce a fine mist), or nebulizer with or without the use of a suitable propellant, or as nasal drops.
• the pressurized container, pump, spray, atomizer, or nebulizer generally contains a solution or suspension of an isolated antagonistic antigen binding protein of the disclosure comprising, for example, a suitable agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent.
• the drug product Prior to use in a dry powder or suspension formulation, the drug product is generally micronized to a size suitable for delivery by inhalation (typically less than 5 microns). This may be achieved by any appropriate comminuting method, such as spiral jet milling, fluid bed jet milling, supercritical fluid processing to form nanoparticles, high pressure
• Capsules, blisters and cartridges for use in an inhaler or insufflator may be formulated to contain a powder mix of the isolated antagonistic antigen binding protein of the disclosure, a suitable powder base and a performance modifier.
• Suitable flavours such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium, may be added to those formulations of the disclosure intended for inhaled/intranasal administration.
• Formulations for inhaled/intranasal administration may be formulated to be immediate and/or modified release.
• Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.
• the dosage unit is determined by means of a valve which delivers a metered amount.
• Units in accordance with the disclosure are typically arranged to administer a metered dose or "puff" of an antibody of the disclosure.
• the overall daily dose will typically be administered in a single dose or, more usually, as divided doses throughout the day.
• the isolated antagonistic antigen binding protein of the present disclosure may also be formulated for an oral administration.
• Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract, and/or buccal, lingual, or sublingual administration by which the compound enters the blood stream directly from the mouth.
• Formulations suitable for oral administration include solid, semi-solid and liquid systems such as tablets; soft or hard capsules containing multi- or nano-particulates, liquids, or powders;
• lozenges including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules;
• compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents in order to provide a pharmaceutically elegant and palatable preparation.
• the isolated antagonistic antigen binding protein is mixed with at least one pharmaceutical carrier, and the solid formulation is compressed to form a tablet according to known methods, for delivery to the gastrointestinal tract.
• the tablet composition is typically formulated with additives, e.g. a saccharide or cellulose carrier, a binder such as starch paste or methyl cellulose, a filler, a disintegrator, or other additives typically usually used in the manufacture of medical preparations.
• DHEA is mixed with at least one pharmaceutical carrier, and the solid formulation is placed in a capsular container suitable for delivery to the gastrointestinal tract.
• compositions comprising isolated antagonistic antigen binding protein may be prepared as described generally in Remington's Pharmaceutical Sciences, 18th Ed. 1990 (Mack Publishing Co. Easton Pa. 18042) at Chapter 89, which is herein incorporated by reference.
• the pharmaceutical compositions are formulated as orally deliverable tablets containing isolated antagonistic antigen binding protein in admixture with non-toxic pharmaceutically acceptable carriers which are suitable for manufacture of tablets.
• These carriers may be inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, gelatin or acacia, and lubricating agents, for example, magnesium stearate, stearic acid, or talc.
• the tablets may be uncoated or they may be coated with known techniques to delay disintegration and absorption in the gastrointestinal track and thereby provide a sustained action over a longer period of time.
• a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.
• the pharmaceutical compositions are formulated as hard gelatin capsules wherein the isolated antagonistic antigen binding protein is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate, or kaolin or as soft gelatin capsules wherein the isolated antagonistic antigen binding protein is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.
• an inert solid diluent for example, calcium carbonate, calcium phosphate, or kaolin
• an aqueous or an oil medium for example, arachis oil, peanut oil, liquid paraffin or olive oil.
• Liquid formulations include suspensions, solutions, syrups and elixirs. Such formulations may be employed as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and typically comprise a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspending agents. Liquid formulations may also be prepared by the reconstitution of a solid, for example, from a sachet.
• Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for administering the isolated antagonistic antigen binding protein of the disclosure.
• the present antigen binding and antagonizing proteins are useful for lowering blood glucose levels by regulating
• gluconeogenesis and glycogenlysis and also for the treatment of a wide range of conditions and disorders in which blocking the interaction of glucagon with its receptor is beneficial, while also reducing and or eliminating one or more of the unwanted side effects associated with the current treatments.
• an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor is provided.
• the antigen binding and antagonizing proteins are fully human monoclonal antibodies and the disorder is obesity.
• the antigen binding and antagonizing proteins are fully human monoclonal antibodies and the disorder is NAFLD.
• the antigen binding and antagonizing proteins are fully human monoclonal antibodies and the disorder is NASH.
• An antagonistic antigen binding protein in particular a human antibody according to the present disclosure, need not effect a complete cure, or eradicate every symptom or manifestation of a disease, to constitute a viable therapeutic agent.
• drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents.
• a prophylactically administered treatment need not be completely effective in preventing the onset of a condition in order to constitute a viable prophylactic agent.
• One embodiment of the disclosure is directed to a method comprising administering to a subject an isolated antagonistic antigen binding protein such as a human antibody in an amount and for a time sufficient to induce a sustained improvement over baseline of an indicator that reflects the severity of the particular disorder.
• obesity is defined as BMI of 30 kg/m 2 or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)).
• the present disclosure is also intended to include a disease, disorder, or condition that is
• BMI body mass index
• An isolated antagonistic antigen binding protein that specifically binds the human glucagon receptor in particular, the fully human antibodies of the disclosure, may be
• a fully human antibody is administered, e.g., once or more than once, at regular intervals over a period of time.
• a fully human antibody is administered over a period of at least once a month or more, e.g., for one, two, or three months or even indefinitely.
• long-term treatment is generally most effective.
• administration for shorter periods e.g. from one to six weeks, may be sufficient.
• the fully human antibody is administered until the subject manifests a medically relevant degree of improvement over baseline for the chosen indicator or indicators.
• One example of therapeutic regimens provided herein comprise subcutaneous injection of an isolated antagonistic antigen binding protein once a week, or once every two weeks, at an appropriate dosage, to treat a condition in which blood glucose levels play a role. Weekly or monthly administration of isolated antagonistic antigen binding protein would be continued until a desired result is achieved, e.g., the subject's symptoms subside. Treatment may resume as needed, or, alternatively, maintenance doses may be administered.
• a subject's levels of blood glucose may be monitored before, during and/or after treatment with an isolated antagonistic antigen binding protein such as a human antibody, to detect changes, if any, in their levels.
• an isolated antagonistic antigen binding protein such as a human antibody
• the incidence of elevated blood glucose may vary according to such factors as the stage of the disease.
• Known techniques may be employed for measuring glucose levels.
• Glucagon levels may also be measured in the subject's blood using known techniques, for example, ELISA.
• a therapeutically effective dose can be estimated initially from cell culture assays by determining an IC 5 o-
• a dose can then be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 5 o as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured by, e.g., HPLC or immunoassays using the anti-idiotypic antibodies specific to the therapeutic drug. The exact composition, route of administration and dosage can be chosen by the individual physician in view of the subject's condition.
• Dosage regimens can be adjusted to provide the optimum desired response
• Dosage unit form refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
• the specification for the dosage unit forms of the present disclosure will be dictated primarily by the unique characteristics of the antibody and the particular therapeutic or prophylactic effect to be achieved.
• the dose and dosing regimen is adjusted in accordance with methods well-known in the therapeutic arts. That is, the maximum tolerable dose can be readily established, and the effective amount providing a detectable therapeutic benefit to a subject may also be determined, as can the temporal requirements for administering each agent to provide a detectable therapeutic benefit to the subject. Accordingly, while certain dose and administration regimens are exemplified herein, these examples in no way limit the dose and administration regimen that may be provided to a subject in practicing the present disclosure.
• dosage values may vary with the type and severity of the condition to be ameliorated, and may include single or multiple doses. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. Further, the dosage regimen with the compositions of this disclosure may be based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the subject, the severity of the condition, the route of administration, and the particular antibody employed. Thus, the dosage regimen can vary widely, but can be determined routinely using standard methods.
• doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such as toxic effects and/or laboratory values.
• the present disclosure encompasses intra-subject dose-escalation as determined by the skilled artisan. Determining appropriate dosages and regimens are well-known in the relevant art and would be understood to be encompassed by the skilled artisan once provided the teachings disclosed herein.
• the total monthly dose of the isolated antagonistic antigen binding protein of the disclosure can be in the range of 0.5-1200 mg per patient, 0.5-1 100 mg per patient, 0.5-1000 mg per patient, 0.5-900 mg per patient, 0.5-800 mg per patient, 0.5-700 mg per patient, 0.5-600 mg per patient, 0.5-500 mg per patient, 0.5-400 mg per patient, 0.5-300 mg per patient, 0.5-200 mg per patient, 0.5-100 mg per patient, 0.5-50 mg per patient, 1 -1200 mg per patient, 1 -1 100 mg per patient, 1 -1000 mg per patient, 1 -900 mg per patient, 1 -800 mg per patient, 1 -700 mg per patient, 1 -600 mg per patient, 1 -500 mg per patient, 1 -400 mg per patient, 1 -300 mg per patient, 1 -200 mg per patient, 1 -100 mg per patient, or 1 -50 mg per patient depending, of course, on the mode of administration.
• an intravenous monthly dose can require about 1 -1000 mg/patient.
• the isolated antagonistic antigen binding protein of the disclosure can be administered at an intravenous monthly dose of about 1 -500 mg per patient.
• the isolated antagonistic antigen binding protein of the disclosure can be administered at an intravenous monthly dose of about 1 -400 mg per patient.
• the isolated antagonistic antigen binding protein of the disclosure can be administered at an intravenous monthly dose of about 1 -300 mg per patient.
• the isolated antagonistic antigen binding protein of the disclosure can be administered at an intravenous monthly dose of about 1 -200 mg per patient.
• the isolated antagonistic antigen binding protein of the disclosure can be administered, at an intravenous monthly dose of about 1 -150 mg per patient. In various embodiments, the isolated antagonistic antigen binding protein of the disclosure can be administered or at an intravenous monthly dose of about 1 -100 mg/patient. In various embodiments, the isolated antagonistic antigen binding protein of the disclosure can be administered at an intravenous monthly dose of about 1 -50 mg per patient.
• the total monthly dose can be administered in single or divided doses and can, at the physician's discretion, fall outside of the typical ranges given herein.
• an isolated antagonistic antigen binding protein of the disclosure can be 0.001 to 100 mg/kg body weight, 0.001 to 90 mg/kg, 0.001 to 80 mg/kg, 0.001 to 70 mg/kg, 0.001 to 60 mg/kg, 0.001 to 50 mg/kg, 0.001 to 40 mg/kg, 0.001 to 30 mg/kg, 0.001 to 20 mg/kg, 0.001 to 10 mg/kg, 0.001 to 5 mg/kg, 0.001 to 4 mg/kg, 0.001 to 3 mg/kg, 0.001 to 2 mg/kg, 0.001 to 1 mg/kg, 0.010 to 50 mg/kg, 0.010 to 40 mg/kg, 0.010 to 30 mg/kg, 0.010 to 20 mg/kg, 0.010 to 10 mg/kg, 0.010 to 5 mg/kg, 0.010 to 4 mg/kg, 0.010 to 3 mg/kg, 0.010 to 2 mg/kg, 0.1 to 50 mg
• dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular patient, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.
• the total dose administered will achieve a plasma antibody concentration in the range of, e.g., about 1 to 1000 g/ml, about 1 to 750 g/ml, about 1 to 500 ⁇ / ⁇ , about 1 to 250 ⁇ / ⁇ , about 10 to 1000 ⁇ / ⁇ , about 10 to 750 ⁇ / ⁇ , about 10 to 500 ⁇ / ⁇ , about 10 to 250 ⁇ / ⁇ , about 20 to 1000 ⁇ / ⁇ , about 20 to 750 ⁇ / ⁇ , about 20 to 500 ⁇ / ⁇ , about 20 to 250 ⁇ / ⁇ , about 30 to 1000 ⁇ / ⁇ , about 30 to 750 ⁇ / ⁇ , about 30 to 500 ⁇ / ⁇ , about 30 to 250 ⁇ / ⁇ .
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.01 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.025 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.05 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.075 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.1 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.25 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 0.75 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 1 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 1 .5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 2 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 2.5 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 3 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 3.5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 4 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 4.5 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 5.5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 6 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 6.5 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 7 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 7.5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 8 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 8.5 mg/kg body weight.
• the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 9 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 9.5 mg/kg body weight. In various embodiments, the weekly or bi-weekly dose for a therapeutically effective amount of an isolated antagonistic antigen binding protein of the disclosure will be 10 mg/kg body weight.
• Toxicity and therapeutic index of the pharmaceutical compositions of the disclosure can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 5 o (the dose therapeutically effective in 50% of the population).
• the dose ratio between toxic and therapeutic effective dose is the therapeutic index and it can be expressed as the ratio LD 5 o/ED 5 o.
• Compositions that exhibit large therapeutic indices are generally preferred.
• compositions are administered depending on the dosage and frequency as required and tolerated by the subject.
• the composition should provide a sufficient quantity of at least one of the isolated antagonistic antigen binding protein disclosed herein to effectively treat the subject.
• the dosage can be administered once but may be applied periodically until either a therapeutic result is achieved or until side effects warrant discontinuation of therapy.
• the dosing frequency of the administration of the isolated antagonistic antigen binding protein pharmaceutical composition depends on the nature of the therapy and the particular disease being treated.
• the subject can be treated at regular intervals, such as weekly or monthly, until a desired therapeutic result is achieved.
• Exemplary dosing frequencies include, but are not limited to: once weekly without break; once weekly, every other week; once every 2 weeks; once every 3 weeks; weakly without break for 2 weeks, then monthly; weakly without break for 3 weeks, then monthly; monthly; once every other month; once every three months; once every four months; once every five months; or once every six months, or yearly.
• the terms "co-administration”, “co-administered” and “in combination with”, referring to the isolated antagonistic antigen binding protein of the present disclosure and one or more other therapeutic agent(s), is intended to mean, and does refer to and include the following: simultaneous administration of such combination of isolated antagonistic antigen binding protein of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated together into a single dosage form which releases said components at substantially the same time to said subject; substantially simultaneous administration of such combination of isolated antagonistic antigen binding protein of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at substantially the same time by said subject, whereupon said components are released at substantially the same time to said subject; sequential administration of such combination of isolated antagonistic antigen binding protein of the disclosure and therapeutic agent(s) to a subject in need of treatment, when such components are formulated apart from each other into separate dosage forms which are taken at consecutive times by said subject
• Suitable pharmaceutical agents that may be used in combination with the compounds of the present invention include anti-obesity agents (including appetite
• anti-diabetic agents anti-diabetic agents
• anti-hyperglycemic agents anti-hyperglycemic agents
• lipid lowering agents antihypertensive agents
• Suitable anti-obesity agents include 1 1 ⁇ -hydroxy steroid dehydrogenase- 1 (1 1 ⁇ -HSD type 1 ) inhibitors, stearoyl- CoA desaturase-1 (SCD-1 ) inhibitor, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (such as sibutramine), sympathomimetic agents, ⁇ 3 adrenergic agonists, dopamine agonists (such as bromocriptine), melanocyte-stimulating hormone analogs, 5HT2c agonists, melanin concentrating hormone antagonists, leptin (the OB protein), leptin analogs, leptin agonists, galanin antagonists, lipase inhibitors (such as tetrahydrolipstatin, i.e.
• anorectic agents such as a bombesin agonist
• neuropeptide-Y antagonists e.g., NPY Y5 antagonists such as velneperit
• PYY3-36 including analogs thereof
• BRS3 modulator mixed antagonists of opiod receptor subtypes, thyromimetic agents, dehydroepiandrosterone or an analog thereof, glucocorticoid agonists or antagonists, orexin antagonists, glucagon-like peptide-1 agonists, ciliary neurotrophic factors (such as AXOKINETM available from Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.
• AXOKINETM available from Regeneron Pharmaceuticals, Inc., Tarrytown, N.Y.
• GTP/ApoB inhibitors e.g., gut-selective MTP inhibitors, such as dirlotapide, JTT130, Usistapide, SLx4090
• opioid antagonist e.g., mu opioid receptor modulators, including but not limited to GSK1521498, MetAp2 inhibitors, including but not limited to ZGN- 433, agents with mixed modulatory activity at 2 or more of glucagon, GIP and GLP1 receptors, such as MAR-701 or ZP2929, norepinephrine transporter inhibitors, cannabinoid-1 -receptor antagonist/inverse agonists, ghrelin agonists/antagonists, oxyntomodulin and analogs, monoamine uptake inhibitors, such as but not limited to tesofensine, an orexin antagonist, combination agents (such as
• the anti-obesity agent is selected from gut-selective
• MTP inhibitors e.g., dirlotapide, mitratapide and implitapide, R56918 (CAS No. 403987) and CAS No. 913541 -47-6
• CCKa agonists e.g., N-benzyl-2-[4-(1 H-indol-3-ylmethyl)-5-oxo-1 - phenyl-4,5-dihydro-2,3,6,1 Ob- tetraaza-benzo[e]azulen-6-yl]-N-isopropyl-acetamide (described in PCT Publication No. WO 2005/1 16034 or US Publication No. 2005-0267100 A1 ), 5HT2c agonists (e.g., lorcaserin), MCR4 agonist (e.g., compounds described in U.S. Pat. No.
• PYY3-36 includes analogs, such as peglated PYY3-36 e.g., those described in US Publication 2006/0178501 ), opioid antagonists (e.g., naltrexone), oleoyl-estrone (CAS No. 180003-17-2), obinepitide (TM30338), pramlintide (SYMLINTM), tesofensine (NS2330), leptin, bromocriptine, orlistat, AOD-9604 (CAS No. 221231 -10-3) and sibutramine.
• opioid antagonists e.g., naltrexone
• oleoyl-estrone CAS No. 180003-17-2
• TM30338 obinepitide
• pramlintide SYMLINTM
• tesofensine tesofensine
• N2330 leptin, bromocriptine, orlistat
• AOD-9604 CAS No. 221231
• the combination therapy comprises administering the isolated antagonistic antigen binding protein composition and the second agent composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions.
• isolated antagonistic antigen binding protein comprises administering the isolated antagonistic antigen binding protein
• compositions and the second agent composition are administered sequentially, i.e., the isolated antagonistic antigen binding protein composition is administered either prior to or after the administration of the second agent composition.
• the administrations of the isolated antagonistic antigen binding protein composition and the second agent composition are concurrent, i.e., the administration period of the isolated antagonistic antigen binding protein composition and the second agent composition overlap with each other.
• the administrations of the isolated antagonistic antigen binding protein composition and the second agent composition are non-concurrent.
• the administration of the isolated antagonistic antigen binding protein composition is terminated before the second agent composition is administered.
• the administration second agent composition is terminated before the isolated antagonistic antigen binding protein composition is administered.
• the present disclosure comprises a method for treating an overweight or obese subject comprising administering to the subject a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor.
• the present disclosure comprises a method for treating an overweight or obese subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor; and (b) an anti-obesity agent.
• the present disclosure comprises a method for treating or preventing NAFLD/NASH in a subject, comprising administering to a subject diagnosed with NAFLD/NASH, or a subject at risk of contracting NAFLD/NASH, a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor.
• the antibody is a fully human monoclonal antibody.
• the present disclosure comprises a method for treating NAFLD.
• the present disclosure comprises a method for treating NASH.
• the present disclosure comprises a method for treating or preventing NAFLD/NASH in a subject, comprising administering to a subject diagnosed with NAFLD/NASH, or a subject at risk of contracting NAFLD/NASH, (a) a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor; and (b) an anti-obesity agent.
• the antibody is a fully human monoclonal antibody.
• the present disclosure comprises a method for treating NAFLD.
• the present disclosure comprises a method for treating NASH.
• the present disclosure provides methods for treating a subject who is at risk of developing NASH (e.g., subjects who are overweight or obese or subjects with NAFLD) comprising administering to the subject a therapeutically effective amount of an isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor.
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment, prophylaxis and/or prevention of nonalcoholic steatohepatitis (NASH) in a subject in need thereof.
• NASH nonalcoholic steatohepatitis
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment, prophylaxis and/or prevention of nonalcoholic fatty liver disease (NAFLD) in a subject in need thereof.
• NAFLD nonalcoholic fatty liver disease
• the present disclosure relates to the use of a non-naturally occurring isolated antagonistic antigen binding protein that specifically binds to the human glucagon receptor for the preparation of a medicament for treatment a subject classified as obese (e.g., having a body mass index (BMI) of 30 kg/m 2 or more).
• BMI body mass index
• Wild-type C57BL/6 mice are commonly used for obesity research, because they show increasing body fat mass, hyperglycemia, and hyperinsulinemia when they are fed a high fat diet (“HFD”)(REbuffe-Scrive, M et al., Metabolism, 42:1405-1409, 1993; Surwit, RS., Metabolism 44:645-651 , 1995).
• HFD high fat diet
• One group of 8 wild-type C57BL/6J male, age 4-6 weeks, 20-22 g are fed with a HFD and dosed weekly with 7.5 mg/kg REMD2.59 antibody for 8 weeks (starting on day 1 ) (hereinafter "Prevention Group”).
• the mice are kept in laminar flow rooms at constant temperature and humidity with one animal in each cage. Animals are housed in polycarbonate cages and in an environmentally monitored, well-ventilated room maintained at a temperature of (22 ⁇ 2°C) and a relative humidity of 40%-70%. Fluorescent lighting provided illumination approximately 12 hours per day.
• the bedding material is soft wood, which is changed once per week. All procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals.
• NASH National Institutes of Health
• Feeding Group remain on HFD and the "Normal Diet Group” remains on chow and are all dosed weekly (starting on day 57 and up thru week 20) via subcutaneous injection with vehicle (PBS).
• the HFD "REMD2.59 Group” is dosed weekly (starting on day 57 and up thru week 20) via subcutaneous injection with 7.5 mg/kg (10 mL/kg) REMD2.59C antibody.
• Prevention Group continues to be dosed weekly up thru week 20 via subcutaneous injection with 7.5 mg/kg (10 mL/kg) REMD2.59C antibody.
• the Study group assignments are outlined in Table 2 below.
• Body weight is measured weekly throughout the study.
• Food consumption (food in/food out) is recorded weekly throughout the study.
• Food consumption is monitored daily for the first week post treatment for the "REMD2.59 Group” and the amount on post-treatment day 7 is then used to feed the "Pair Feeding Group” during week 10.
• food consumption of the "REMD2.59 Group” is monitored weekly and the food consumption amount at the end of each week is used to feed the "Pair Feeding Group” the following week.
• the chow fed "Normal diet group” is fed the same amount of chow throughout the entire 20 week study.
• the blood glucose levels were measured at different time points (30, 60, 120 min) by using Accu-Chek Performa System; iii) the lipid profile in serum and blood bio-chemistry parameters (ALT, AST, GGT, ALP, TG and TCHO) were tested throughout the study. Blood samples were obtained on week 8, 12, 16 and 20, the samples were immediately processed by centrifugation at 4°C, 4000 g for 15 minutes, and then they were transferred into new test tubes.
• Lipid profile and blood bio-chemistry parameters were measured by using TOSHIBA TBA-40FR automated biochemical analyzer; iv) the lipid profiles (TG, TCHO, HDL-C and LDL-C) were extracted from the liver of the animal according to the protocol, and then lipid profile were measured by using TOSHIBA TBA-40FR automated biochemical analyzer; v) the insulin level of all study animals were measured on week 8, 12, 16 and 20. GLP-1 and leptin were measured at the end of the study with ELISA method. The blood serum was used for the analysis; and vi) on the termination day, after OGTT study necropsy was conducted.
• tissue or organs were collected and the wet weights of the pancreas, white adipose tissue (WAT), muscle (gastrocnemius muscle) and liver were measured. Half of these tissue samples were fixed and brought up to paraffin block for H&E (liver and WAT) or IHC (pancreas) analysis. Hypothalamus, brain, heart and remaining part of pancreas, WAT, muscle, liver were stored at -80 °C or future analysis. The various measurements and/or analysis described above is made as described in the Additional
• the Pair Feeding Group which was given the same amount of daily food as that of the REMD2.59 Group from Week 9 to Week 20, showed a similar, but slightly greater weight gain, than the REMD2.59 Group. This observation suggests that the effects of REMD2.59 are not limited to the reduction in food intake.
• the Prevention Group which received REMD2.59 weekly injections concurrently with HFD from Week 1 through Week 20, showed the lowest weight gain compared with all other groups, despite the HFD feeding.
• the average body weight of the Prevention Group (35.3 ⁇ 3.0 g) was 34% lower (P ⁇ 0.01 ) than the Vehicle Group (53.3 ⁇ 2.4 g), and 9% lower even than the Normal Diet Group (38.6 ⁇ 3.1 g), at the end of the study (on Day 140, or end of Week-20).
• the Prevention Group consumed the same amount of calories per gram of body weight as the Normal Diet Group.
• the HFD fed groups (Vehicle Group, REMD2.59 Group and Pair Feeding Group) all consumed nearly the same amount of calories when adjusted by gram of body weight. Nevertheless, taking into consideration of the body weight differences, the Vehicle Group still consumes greater amount of calories per animal, than the REMD2.59 Group and Pair Feeding Groups, since the Vehicle Group has the highest average body weight.
• TG TG
• REMD2.59 Treatment did not significantly affect TG levels, and although the Prevention Group showed reduced TG levels at Week 16, the changes were not sustained by the end of study. Thus, overall REMD2.59 did not cause any major change in circulating TG levels.
• TCHO total cholesterol
• hyperinsulinemia as insulin levels were brought back to, or even lower than, the levels seen in the Normal Diet Group.
• leptin were reduced in the Prevention Group compared to the Vehicle Group, by as much as 92%, and the reduced leptin level was even lower than that of the Normal Diet Group. This is significant in that leptin levels in circulation signifies the extent of adiposity.
• the REMD2.59 Group reduced leptin levels slightly, although statistically insignificantly.
• the REMD2.59 Group but not the
• the Prevention Group exhibited a significantly reduced the white adipose tissue weight, suggesting that blockade of glucagon receptor is associated with lower adiposity.
• the Prevention Group also exhibited reduced liver wet weight, which may be related to the reduced fat (TG) and glycogen contents.
• the REMD2.59 Group exhibited reduced wet liver weight, similarly to the Prevention Group, but slightly increased the pancreatic wet tissue weight, which might be due to the reactive increase in the islet tissue mass.
• the REMD 2.59 Group exhibited reduced insulin area/islet area, similar to the Prevention Group, collectively suggesting that blockade of glucagon receptor signaling attenuated the stimulation to islet beta-cells and to insulin synthesis/secretion.
• Such a histological finding is in line with the observation that the REMD2.59 Group and Prevention Group exhibited lower insulin levels in circulation ( Figure 1 1 , above).
• both the REMD2.59 Group and Prevention Group induced marked increases in glucagon area/islet area, suggesting a reactive feedback stimulation of glucagon synthesis/secretion from the islet alpha-cells.
• the liver tissues from the Vehicle Group show abundance of vacuoles due to fat droplets, which confirms the fatty liver diagnosis of these mice; 2) the liver samples from the REMD2.59 Group appear to show markedly reduced density and sizes of fat droplets, i.e., the areas occupied by overt fat droplets appear to be much smaller than those of the Vehicle Group; 3) the liver tissue samples from the Pair Feeding Group show very little, if any, reduction in the density and sizes of fat droplets from those observed in the Vehicle Group; 4) the liver tissue samples from the Normal Diet Group show very clean liver sections, with virtually no visible fat droplets, comparable to those of the Prevention Group; and 5) the liver tissue samples from the Prevention Group show very clean liver sections, with virtually no visible fat droplets, comparable to those of the Normal Diet Group.
• the above histological evidence clearly indicates the robust effects of the antagonistic glucagon receptor antibody in correcting, or preventing, the fatty liver changes in the diet-induced obese mice.
• the histological improvement in fatty liver is closely associated with the correction of hyperglycemia and hyperinsulinemia, and improvements in glucose and fat metabolism.
• Example 1 the relationship between regulating glucose output and the development of
• NAFLD/NASH in various murine models is evaluated.
• the in vivo activity of REMD2.59C is evaluated in the NASH-derived HCC murine model (STAMTM model, Fujii et al, Med Mol Morphol, 46:141 -152, 2013).
• STAMTM model C57BL/6J mice are injected with a single subcutaneous injection of 200 ⁇ g STZ at 2 days after birth and put on a HFD or chow after 4 weeks of age. In male mice, this combined STZ-HFD treatment results in the
• HCC hepatocellular carcinoma
• the STZ-Chow group is dosed weekly via subcutaneous injection with vehicle (PBS) up until age 24 weeks, and one STZ-HFD group is dosed weekly with 7.5 mg/kg REMD2.59 antibody up to age 24 weeks.
• one STZ-HFD group is dosed weekly with 2.5 mg/kg REMD2.59 antibody up to age 24 weeks
• one STZ-HFD group is dosed weekly with 5.0 mg/kg REMD2.59 antibody up to age 24 weeks
• one STZ-HFD group is dosed weekly with 7.5 mg/kg REMD2.59 antibody up to age 24 weeks.
• Various parameters are measured throughout the 24 week study, including, e.g., i) body weight (once a week); ii) fasting blood glucose determination (mice are fasted for 6 hours prior to the test and fasting blood glucose levels are measured via tail veins weekly using Accu-Chek Aviva System®; iii) serum hemoglobin-A1 c (HbA1 c) determination; iv) serum GLP-1 determination; v) serum insulin and leptin levels via radioimmunoassay (Linco, St.
• ALT serum alanine aminotransferase
• adioponectin serum lipids (e.g., total cholesterol, LDL, HDL and triglycerides) determination
• CGT gamma-glutamyl transpeptidase
• livers are rapidly excised, rinsed in ice-cold saline, and weighed. Aliquots of liver are snap frozen in liquid nitrogen and kept at -80 °C until being analyzed. A portion of each liver is fixed in 10% formalin for proper histological analysis of the liver. Liver triglyceride (TG) content, diacylglyceride (DG) content, and ceramide content measurements are made as described in the Additional Materials and Methods section below. Inflammation, central vein fibrosis, and portal tract fibrosis will be evaluated as described in the Additional Materials and Methods section below.
• TG Liver triglyceride
• DG diacylglyceride
• ceramide content measurements are made as described in the Additional Materials and Methods section below. Inflammation, central vein fibrosis, and portal tract fibrosis will be evaluated as described in the Additional Materials and Methods section below.
• Example 1 In view of the results demonstrated in Example 1 , it is expected that treatment of the wild-type mice using an anti-GCGR antibody will provide beneficial therapeutics effects which may include, e.g., reducing insulin resistance; reducing or preventing hyperinsulinemia, reducing or preventing fat deposits in the liver; reducing or preventing inflammation in the liver; reducing or preventing the accumulation of lipid, e.g., hepatic triacylglycerol, hepatic
• diacylglycerol, and ceramides and preventing injury in the liver, and that the development of NAFLD/NASH in such mice may be prevented or treated, thus reducing the risk of the diabetic subject from developing HCC.
• Example 1 the in vivo activity of REMD2.59C is evaluated in a murine model of NASH, using db/db mice.
• the study will be a 24 week study. An additional 48 week study may also be performed, db/db mice from the C57BL/6 background are purchased from Jackson Laboratory (Bar Harbor, ME), db/db mice are hyperleptinemic, obese and diabetic mice, db/db mice that are fed a methionine and choline deficient (MCD) diet spontaneously develop hepatic steatosis, which progresses to NASH (Wortham et al., Dig Dis Sci., 53(10): 2761-2774, 2008 October).
• MCD methionine and choline deficient
• mice each of db/db at age 10 -12 weeks are fed ad libitum with either a methionine and choline deficient (MCD) diet (MP Biomedicals Solon, OH, cat. no. 960439) or the same diet supplemented with methionine and choline (MCDS) diet (MP Biomedicals cat. no. 960441 ) for 4 weeks, or a HFD + fructose Western Diet (WD)(#58Y1 , TestDiet, St Louis, MO), or chow (Control Diet)(#58Y2, TestDiet, St Louis, MO) for 24 weeks.
• MCD methionine and choline deficient
• MCDS methionine and choline
• mice are housed individually in steel microisolator cages at 22 °C with a 12-h/12-h, light/dark cycle. All procedures were conducted in accordance with the National Institutes of Health (NIH) guidelines for the care and use of laboratory animals.
• the mice are dosed weekly or bi-weekly via subcutaneous injection with either vehicle (10 mM sodium acetate, 5% sorbitol, and 0.004% polysorbate 20), 2.5 mg/kg REMD2.59C antibody ("Low Dose”), or 5 mg/kg REMD2.59C antibody (“High Dose”) for 4 weeks or 24 weeks, as appropriate. The dose administered will not exceed 10mg/kg per month.
• Various parameters are measured throughout the 4 week or 24 week study, including, e.g., i) body weight (once a week); ii) fasting blood glucose determination (mice are fasted for 6 hours prior to the test and fasting blood glucose levels are measured via tail veins weekly using Accu-Chek Aviva System®; iii) serum hemoglobin-A1 c (HbA1 c) determination; iv) serum GLP-1 determination; v) serum insulin and leptin levels via radioimmunoassay (Linco, St.
• ALT serum alanine aminotransferase
• TG serum lipids
• CCT gamma-glutamyl transpeptidase
• livers are rapidly excised, rinsed in ice-cold saline, and weighed. Aliquots of liver are snap frozen in liquid nitrogen and kept at -80 °C until being analyzed. A portion of each liver is fixed in 10% formalin for proper histological analysis of the liver. Liver triglyceride (TG) content, diacylglyceride (DG) content, and ceramide content measurements are made as described in the Additional Materials and Methods section below. Inflammation, central vein fibrosis, and portal tract fibrosis will be evaluated as described in the Additional Materials and Methods section below.
• TG Liver triglyceride
• DG diacylglyceride
• ceramide content measurements are made as described in the Additional Materials and Methods section below. Inflammation, central vein fibrosis, and portal tract fibrosis will be evaluated as described in the Additional Materials and Methods section below.
• Example 1 In view of the results demonstrated in Example 1 , it is expected that treatment of the db/db mice using an anti-GCGR antibody will provide beneficial therapeutics effects which may include, e.g., reducing insulin resistance; reducing or preventing hyperinsulinemia, reducing or preventing fat deposits in the liver; reducing or preventing inflammation in the liver; reducing or preventing the accumulation of lipid, e.g., hepatic triacylglycerol, hepatic
• diacylglycerol, and ceramides preventing injury in the liver, and that the development of NAFLD/NASH in such mice may be prevented or treated.
• This Example describes a randomized, double-blind, placebo-controlled, parallel group, multiple dose study to evaluate the safety, pharmacokinetics and pharmacodynamic effects of weekly treatment using a fully human anti-GCGR antibody in subjects diagnosed with NASH.
• the treatment may last a period up to 6 or 12 months, long enough to observe and quantitate treatment efficacy and safety.
• Treatment groups include a placebo group and treatment groups to be treated with various dosages of a fully human anti-GCGR antibody which comprises the heavy chain sequence set forth in SEQ ID NO: 51 and the light chain sequence set forth in SEQ ID NO: 52 ("REMD-477").
• non-placebo treatment groups will include, e.g., subjects who receive injections of either 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 .0 mg/kg, 1 .5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, or 10 mg/kg REMD-477 per week, and subjects who receive injections of either 0.01 mg/kg, 0.025 mg/kg, 0.05 mg/kg, 0.075 mg/kg, 0.1 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 .0 mg/kg, 1 .5 mg/kg, 2.0 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, or 10 mg/kg REMD-477 weekly.
• Primary outcome measures will include, e.g., change in percentage of liver fat content by MRI at: baseline, at 4-week or 8-week intervals, until the end of study; change in the proportion of REMD-477 treated patients relative to placebo achieving improvement of liver fibrosis by at least one stage, at the end of study, in comparison to the baseline assessment; and change in liver enzyme and metabolic markers, including Aspartate Transaminase (AST), Alanine Transaminase (ALT), Bilirubin and Alkaline phosphatase (ALP), at: baseline, at 4-week intervals, until the end of study.
• AST Aspartate Transaminase
• ALT Alanine Transaminase
• ALP Alkaline phosphatase
• Secondary outcome measures will include, e.g., change in fasting plasma glucose levels, average of daily morning glucose after an overnight fast, at pre- treatment baseline, and at weekly intervals until the end of study; change in plasma insulin levels at pre-treatment baseline, and at weekly intervals until the end of study; change in hemoglobin A1 c levels (indicator of chronic glucose control) at pre-treatment baseline, and at 4- week or 8-week intervals until the end of study; change in glucose profiles at oral glucose tolerance tests (OGTT), measured at 0, 30, 60, 90 and 120 min after an oral glucose load, at pre-treatment baseline, and at 8-week intervals until the end of study; composite long term outcome measured by the number of patients with the onset of any adjudicated events, including cirrhosis, all-cause mortality, and liver-related clinical outcomes, at the Baseline and the end of study; and changes in scores of the Quality of Life (36-ltem Short-Form Health Survey [SF-36]) Questionnaire.
• OGTT oral glucose tolerance tests
• Body Weights Body weights of all animals are measured weekly throughout the duration of the various studies.
• Food Consumption Food consumption of all animals are measured daily and/or weekly throughout the duration of the various studies.
• OGTT was performed for all animals at the end of the study.
• the baseline (time 0) glucose level was measured after 16 hours fast and prior to glucose challenge.
• the blood glucose levels were measured at different time points (30, 60, 120 min) by using Accu-Chek Performa System.
• LDL-C LDL-C
• ELISA Kits Analysis The insulin level of all study animals were measured on week 8, 12, 16 and 20. GLP-1 and leptin were measured at the end of the study with ELISA method. The blood serum was used for the analysis.
• livers are rapidly excised, rinsed in ice- cold saline, and weighed. Aliquots of liver are snap frozen in liquid nitrogen and kept at - ⁇ ' ⁇ until being analyzed. A portion of each liver is fixed in 10% formalin for histology.
• Liver TG/DG/Ceramide Content Liver triglyceride (TG), diacylglyceride (DG), and ceramide content of all animals are measured at the end of the study. Liver samples are homogenized in 50 mM Tris- HCI buffer, pH 7.4, containing 150 mM NaCI, 1 mM EDTA, and 1 ⁇ PMSF and lipids isolated by extraction into chloroform with appropriate internal standards included for each protocol. Extracted lipids were resuspended and diluted in
• DG molecular species were quantified as sodiated adducts using selected reaction monitoring as previously described with intensity of each species normalized to that of the internal standard di- 20:0 DG (Demarco VG et al., Endocrinology, 154:159-171 , 2013).
• TG aliphatic groups were quantified byTG fingerprinting techniques with neutral loss scanning for the loss of each fatty acid from theTG species and comparisons to that of the neutral loss 268 which is derived from the internal standard Tri-17:1 TG (Han et al., Anal Biochem, 295:88-100, 2001 ).
• Individual ceramide molecular species were quantified in negative ion mode using neutral loss 256 by comparing the ion intensity of individual molecular species to that of the internal standard (17:0 ceramide) after corrections for type I and type II C isotope effects.
• H&E hematoxylin and eosin
• Masson's trichrome for histological analysis. Inflammation is evaluated on H&E-stained sections and is given a score from 0 to 3 as follows: 0, no inflammation; 1 , mild; 2, moderate; 3, severe. The degree of fibrosis is assessed by digital morphometry. Five discrete regions of a trichrome-stained sections from each mouse are randomly selected and within each region identified a portal track and central vein to be digitally photographed.
• Photographs are obtained with a X20 objective with the portal tracks or central veins of interest in the center of the field, thus obtaining five portal/periportal and five central/ pericentral fields of interest for each mouse.
• the pixels corresponding to fibrosis are measured based on a narrow band of the blue spectrum corresponding to the stain of clear-cut fibrosis in each specimen, carefully excluding the normal stromal collagen in those areas.
• the number of pixels corresponding to fibrosis is measured as a percentage of the total pixels of each image using the Image Processing Tool Kit, version 5.0 (Reindeer Graphics, Asheville, NC).
• the results of the five portal/periportal and five central/pericentral fields from each specimen are averaged, and fibrosis is expressed as a percentage of total cross-sectional area for each animal.
• MCD Methionine and Choline Deficient Diet: Of the dietary approaches discussed herein, MCD diets produce the most severe NASH phenotype in the shortest time frame. MCD diet is high in sucrose and fat, but lacks methionine and choline, which are essential for hepatic beta-oxidation and the production of very low density lipoprotein (VLDL). This results in the accumulation of intra-hepatic lipid and decreased VLDL synthesis (Anstee et al., Int J Exp Pathol, 87(1 ):1 -16, 2006).
• VLDL very low density lipoprotein
• MCD diets will quickly induce measurable hepatic steatosis (mainly macrovesicular) in rodents by 2-4 weeks and this progresses to inflammation and fibrosis shortly thereafter.
• Fat levels in MCD diets can vary, though typically they contain about 20% fat by energy.
• rodents fed MCD diets lose weight (due to a vastly lower caloric intake) and do not become insulin resistant. Since most humans with NASH are obese and insulin resistant, this represents an important difference in how MCD diets model human NASH.
• HFD High-fat diets
• HFD' encompasses a wide variety of diet formulas and diets of different composition can be expected to alter the liver phenotype in various ways.
• An exemplary HFD diet may consist of 36% fat derived-calories (9% corn oil and 27% butter) and 43.2% carbohydrate-derived calories without sugar. HFD (Research Diets, D12492, HFD) was used in these studies.
• HFD + fructose diet (Western diet ("WD"): An exemplary WD may consist of
• fat derived-calories (9% corn oil and 27% butter), which is the same as HFD, and 43.2% carbohydrate-derived calories with e.g., fructose (e.g., 30% sugar-derived calories).
• Murine Models of NASH The anti-GCGR antibodies of the present disclosure may be evaluated in any of the other various published murine models of NASH (see, e.g.
• SEQ ID NO: 1 is the amino acid sequence of a human glucagon receptor
• SEQ ID NO: 2 is the amino acid sequence encoding the heavy chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 3 is the amino acid sequence encoding the light chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 4 is the amino acid sequence encoding the heavy chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 5 is the amino acid sequence encoding the light chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 6 is the amino acid sequence encoding the heavy chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 7 is the amino acid sequence encoding the light chain variable region of a fully human anti-GCGR antibody.
• SEQ ID NO: 8 is the amino acid sequence encoding the heavy chain of a chimeric anti-GCGR antibody.
• SEQ ID NO: 9 is the amino acid sequence encoding the light chain of a chimeric anti-GCGR antibody.
• SEQ ID NOS: 10-28 are amino acid sequences encoding the heavy chain variable regions of various fully human anti-GCGR antibodies.
• SEQ ID NOS: 29-47 are amino acid sequences encoding the light chain variable regions of various fully human anti-GCGR antibodies.
• SEQ ID NO: 48 is the amino sequence encoding the kappa light chain constant region.
• SEQ ID NO: 49 is the amino sequence encoding the lambda light chain constant region.
• SEQ ID NO: 50 is the amino sequence encoding the lgG2 heavy chain constant region.
• SEQ ID NO: 51 is the amino acid sequence encoding the heavy chain of a human anti-GCGR antibody.
• SEQ ID NO: 52 is the amino acid sequence encoding the light chain of a human anti-GCGR antibody.
• SEQ ID NO: 1 Amino acid sequence of a human glucagon receptor (GCGR) molecule
• SEQ ID NO: 2 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 3 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 5 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 6 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 7 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 8 Amino acid sequence of a heavy chain of a chimeric antibody that binds GCGR
• SEQ ID NO: 9 Amino acid sequence of a light chain of a chimeric antibody that binds GCGR
• SEQ ID NO: 10 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 1 1 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 12 Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYY DYAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCTRDRSSGWNEGYYYYGMDVWGQG TTVTVSS
• SEQ ID NO: 13 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 14 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 15 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 16 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 17 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 18 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 19 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 20 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 21 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• VSS SEQ ID NO: 22 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 23 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 24 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 25 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 26 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 27 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 28 Amino acid sequence of a HCVR of a human antibody that binds GCGR
• SEQ ID NO: 29 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 30 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 31 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 33 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 34 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 35 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 36 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 37 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 38 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• KIVMTQTPLALPVIPG EPASISCRSSQSLVDSDDGDTYLDWYLQKPGQSPQVLIHRLSYRASGV PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMHRIEFPFTFGGGTKVEIKR
• SEQ ID NO: 39 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 40 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 41 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 42 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 43 Amino acid sequence of a LCVR of a human antibody that binds GCGR SYELTQPPSVSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQSTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVLG
• SEQ ID NO: 44 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 45 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 46 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 47 Amino acid sequence of a LCVR of a human antibody that binds GCGR
• SEQ ID NO: 48 Amino acid sequence of the constant light chain kappa region
• SEQ ID NO: 51 Amino acid sequence of a HC of a human antibody that binds GCGR
• SEQ ID NO: 52 Amino acid sequence of a LC of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGV PSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

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