Combination therapy for the treatment of obesity

Abstract

The present disclosure relates to methods for treating or preventing obesity and other metabolic disorders in diabetic and non-diabetic subjects using combination therapy comprising a therapeutically effective dose of an antagonistic antigen binding molecule that specifically binds to the human glucagon receptor (GCGR) co-administered with a therapeutically effective low dose of glucagon like peptide- 1 receptor (GLP-1 R) agonist peptide approved for treating T2DM or using a bifunctional fusion molecule comprising a glucagon like peptide- 1 receptor (GLP-1 R) agonist peptide fused directly to an antagonistic antigen binding molecule that specifically binds to the human glucagon receptor (GCGR).

Description

PCT Patent Application Docket No: CACRE1.0026WOCOMBINATION THERAPY FOR THE TREATMENT OF OBESITYRELATED PATENT APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Application No. 63 / 557,044, filed on February 23, 2024, and U.S. Provisional Application No. 63 / 689992, filed September 3, 2024, each incorporated in its entirety by reference herein.SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (REMD GCGR-GLP-1.xml; Size: 66 Kilobytes; Date of Initial Creation: February 11, 2025; Production Date: February 12, 2025, is herein incorporated by reference in its entirety.TECHNICAL FIELD

[0003] Glucose homeostasis is mainly maintained by the pancreatic hormones, insulin and glucagon. Under normal circumstances, the body is able to balance the amount of glucose, or sugar, in the blood with the amount of glucose that the cells need for fuel (glucose homeostasis). The hormone insulin, which the pancreas produces, facilitates the transport of glucose into the cells while glucagon, which is also produced by pancreas, increases the glucose level in the body through gluconeogenesis and glycogenolysis. Loss of glucose homeostasis as a result of imbalanced faulty insulin secretion and glucagon actions typically results in metabolic disorders such as obesity, diabetes and hyperglycemia.

[0004] 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 promotes the emergence of associated complications, such as type 2 diabetes, cardiovascular diseases, metabolic dysfunction-associated steatohepatitis (MASH), and non-alcoholic liver steatohepatitis (NASH), as well as certain cancers, imposing a considerable burden on healthcare systems. 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 exacerbatesPCT Patent Application Docket No: CACRE1.0026WOmany 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. Despite major efforts, pharmacotherapies for obesity have historically faced continued challenges associated with limited efficacy and, more importantly, clinically relevant safety liabilities.

[0005] More recent efforts in the field of obesity pharmacotherapy have focused on incretin-based therapy, e.g., glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP) and glucagon (Muller et al, 2022, Nature Reviews Drug Discovery, 21:201 -223). These incretin-based therapies were initially developed for treatment of type 2 diabetes (T2D). The discovery that GLP-1 receptor agonists (GLP-1 R agonists, also referred to herein as GLP-1 RAs and GLP-1 agonists) reduce food intake / calorie intake alongside increasing insulin secretion and inhibiting gastric emptying led to the development and approval of the long-acting GLP-1 RAs liraglutide, dulaglutide and semaglutide for obesity treatment.

[0006] Importantly, past and current studies have shown that the GLP-1 agonist dosages required for the treatment of T2D vs. obesity are quite different. Studies show that higher dose incretin-based therapy is needed to achieve significant weight loss. For example, current approved step-up doses of semaglutide (Ozempic®) for treatment of T2D are 0.25 mg weekly for 4 weeks, 0.5 mg weekly for 4 weeks, 1.0 mg weekly for 4 weeks, and then 2.0 mg weekly for 4 weeks if tolerable. Major benefits observed with the T2D treatment are the reduction in glycated HbA1c with limited effect on weight loss. But as relates to the treatment of obesity, current approved step-up doses of semaglutide (Wegovy®) are 0.25 mg weekly for 4 weeks, 0.5 mg weekly for 4 weeks, 1.0 mg weekly for 4 weeks, 1.7 mg weekly for 4 weeks, and then 2.4 mg weekly for 4 weeks if tolerable. Current approved step-up doses of tirzepatide (Mounjaro®) for T2D is 2.5 mg injected subcutaneously once weekly for 4 weeks, and then 5 mg injected subcutaneously once weekly. If additional glycemic control is needed, the dose is increased in 2.5 mg increments after at least 4 weeks on the current dose. As relates to treatment of obesity the approved step-up doses of tirzepatide (Zepbound®) is 12.5 or 15 mg per 0.5 mL in a single-does pen or single dose vial.

[0007] Unfortunately, the higher dose incretin-based therapy required to achieve significant weight loss (i.e., desired fat mass loss) also results in a problematic loss of lean muscle mass, and there is often a rapid regain of fat, not muscle mass, observed after treatment stops (“rebound effect”). Another common observation from all incretin-based therapies is that they have dose-limiting gastrointestinal side effects. There thus also remains a need to developPCT Patent Application Docket No: CACRE1.0026WOtherapies that address both the muscle loss issue and high Gl effects associated with higher dose incretin-based therapy.

[0008] There have also been extensive studies evaluating incretin-based poly-agonists that simultaneously target the GLP-1 receptor (GLP-1 R), GIP receptor (GIPR) and / or glucagon receptor (GCGR) (Muller et al, 2022, Nature Reviews Drug Discovery, 21:201-223). Importantly, these studies are focused on the basic and acceptable concept that there is additional benefit for treating obesity by agonizing two or three of these receptors simultaneously.

[0009] However, in human and preclinical studies, in the case of targeting GIP receptor (GIPR) and glucagon-like peptide-1 receptor (GLP-1 R), both antagonism and agonism of the GIPR prevent weight gain and lead to dramatic weight loss when combined with GLP-1 RAs (Coskun et al, 2018, Molecular Metabolism, 18:3-14; Murielle et al, 2024, Nature Metabolism, Feb 2024 online publication). A recent study reconciled the paradoxical observations that GIPR agonism and antagonism can both reduce body weight; it is postulated that the chronic GIPR agonism (that is with long-acting GIP peptide) produced desensitization resulting in the loss of GIPR activity, which mimicked antagonism of the receptor by the GIPR blocking antibody (Killion et al, 2020, Nature Communications 11:4981 ).

[0010] Retatrutide, a triple agonist of GLP-1, GIP, and glucagon receptors, has been studied in a phase 2 human trial for the treatment of obesity. It showed a dose-dependent body weight reduction, but also caused gastrointestinal side effects and increased heart rate (Jastreboff et al, 2023, N Engl J Med, 389:514-26). Similar to GIPR, the effect of chronic GCGR agonism on GCGR activity remains to be investigated.

[0011] While there have been continued advancements in the treatment of obesity, there remains a pressing need for more research on the molecular mechanisms that underlie obesity and its medical consequences, as well as new approaches for developing safe and effective anti-obesity agents, addressing the issues of current therapies.DISCLOSURE OF THE INVENTION

[0012] The present disclosure is based in part on the inventors' unique insight that combining human glucagon receptor (GCGR) antagonism with low-dose glucagon like peptide-1 receptor (GLP-1 R) agonism may provide for improved, effective therapies for treatment of obesity and other metabolic disorders in diabetic and non-diabetic subjects. This combination therapy is expected to show enhanced glycemic control compared to single agent treatment by dual actions of GLP-1 R agonism and GCGR antagonism, without the gastrointestinal sidePCT Patent Application Docket No: CACRE1.0026WOeffects and unwanted lean mass loss observed in high dose incretins or dual or triple agonist currently used for obese T2D and obesity treatment.

[0013] More specifically, in order to address the muscle loss issue associated with higher dose incretin-based therapy, the present inventors propose to use therapeutically effective low doses of GLP-1 agonists (e.g., doses approved and commonly used for T2D treatment) in combination with a glucagon receptor (GCGR) antagonist to prevent catabolism and sarcopenia while continuing to drive preferential fat loss. This unique approach is expected to achieve robust weight loss without muscle loss in the first place, show limited gastrointestinal side effects, and attenuate the rebound effects associated with discontinuation of treatment with higher dose incretin-based therapy. Importantly, such co-targeting may be accomplished using co-administration of single agents that target each receptor (formulated separately or coformulated as a single product) or using recombinant bifunctional fusion proteins comprising each targeting agent.

[0014] In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is an FDA-approved dose for the treatment of T2D. In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is the FDA-approved T2D dose of semaglutide. In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is an FDA-approved T2D dose of tirzepatide.

[0015] In one aspect, the present disclosure relates to methods of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GCGR antagonist in combination with a therapeutically effective T2D low dose of GLP-1 R agonist. In various embodiments, the agents are formulated separately. In various embodiments, the agents are co-formulated as a single product. In various embodiments, the combination therapy has a synergistic effect for the treatment of obesity.

[0016] In another aspect, the present disclosure relates to methods of treating a metabolic disorder selected from the group consisting of obese T2D, obesity with prediabetes, insulin resistance, metabolic syndrome, fatty liver diseases, metabolic dysfunction-associated steatohepatitis (MASH), and Nonalcoholic steatohepatitis (NASH) and other indications that obesity is an important contributing factor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GCGR antagonist in combination with a therapeutically effective low dose of GLP-1 R agonist. In various embodiments, the agents are formulated separately. In various embodiments, the agents are co-formulated as a single product. In various embodiments, the combination therapy has a synergistic effect for the treatment of the metabolic disorder.PCT Patent Application Docket No: CACRE1.0026WO

[0017] There have been extensive studies evaluating the use of GCGR small molecules for therapeutic use and such studies have been reported in the literature. In various embodiments, the isolated antagonistic antigen binding molecule to human GCGR is a small molecule. In various embodiments, the GCGR small molecule antagonist is selected from the GCGR antagonists disclosed in Sammons and Lee, Bioorg Med Chem Lett. 2015 Oct1;25(19):4057-64; Chen et al, Expert Opinion on Therapeutic Patents, Vol 30, 2020, Issue 7; Filipski, Expert Opinion on Therapeutic Patents, Vol 25, 2015, Issue 7; Shen et al, Expert Opinion on Therapeutic Patents, Vol 21, 2011, Issue 8; Sheen et al, Expert Opinion on Investigational Drugs, Volume 26, 2017, Issue 12; Yang et al, Acta Pharmacologica Sinica (2015) 36: 1033-1042; US 12208071; US 8609892; US 20230104956; US 20210121422; US 20190194143.

[0018] In various embodiments, the isolated antagonistic antigen binding molecule to human GCGR is selected from the group consisting of 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 Fab2, a Fab'2, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, a diabody, and a hemibody. In various embodiments, the antibody is a fully human monoclonal antibody. In various embodiments, the isolated antibody or antigen-binding antibody fragment specifically binds to a human GCGR with a dissociation constant (KD) of at least about 1x10-7M, at least about 1x10-8M, at least about 1x10-9M, at least about 1x10-10M, at least about 1x10-11M, or at least about 1x10-12M.

[0019] In various embodiments, the isolated GCGR antagonistic antigen binding molecule comprises an 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. In various embodiments, the isolated antagonistic antigen binding molecule comprises an 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. In various embodiments, the isolated antagonistic antigen binding molecule comprises an 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.

[0020] In various embodiments, the isolated GCGR antagonistic antigen binding molecule is a chimeric antibody which comprises the amino acid sequence encoding the heavy chain of SEQ ID NO: 8 and the amino acid sequence encoding the light chain of SEQ ID NO: 9.PCT Patent Application Docket No: CACRE1.0026WO

[0021] In various embodiments, an isolated GCGR antagonistic antigen binding molecule of the present disclosure is a fully human 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.

[0022] In various embodiments, the GLP-1 agonist is selected from the group consisting of a GLP-1 agonist that is a peptide, a GLP-1 agonist that is a modified peptide, a GLP-1 agonist that is a small molecule, a GLP-1 agonist that is an antisense or siRNA molecule, a GLP-1 agonist that is an antibody, or a fragment thereof, or an artificial construct comprising an antibody or fragment thereof, or an artificial construct designed to mimic the binding of an antibody or fragment thereof to its antigen, a GLP-1 agonist that is a peptide-Ab fusion, or a GLP-1 agonist that is a peptide-Fc fusion. In various embodiments, the GLP-1 agonist is in the form of an incretin-based poly-agonist. In various embodiments, the GLP-1 agonist of the present invention is GLP-1 (7-37) (SEQ ID NO: 53). In various embodiments, the GLP-1 agonist of the present invention is a GLP-1 (7-37) analog selected from the group consisting of exenatide (tradename Byetta®, Amylin / Astrazeneca); liraglutide (tradename Victoza®, Novo Nordisk A / S); lixisenatide (tradename Lyxumia®, Sanofi); albiglutide (tradename Tanzeum®, GlaxoSmithKline); dulaglutide (tradename Trulicity®, Eli Lilly); semaglutide (tradename Ozempic® and Wegovy®, Novo Nordisk A / S) and taspoglutide. In various embodiments, the GLP-1 agonist is selected from the group consisting of exendin-3 and derivatives thereof and exendin-4 and derivatives thereof. In various embodiments, the GLP-1 agonist comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 54-58.

[0023] In various embodiments, the GLP-1 agonist is a peptidic compound described in, e.g., US 9181305, US 20140206608; US 20140206609; 20140221281; US 20140213513; and US 20150164997, of which the contents of each is incorporated herein by reference. An illustrative, but not limiting, list of suitable GLP-1 agonist peptides to be used for coadministration of single agents that target each receptor (formulated separately or co-formulated as a single product) is provided in Table 2 and Table 3.

[0024] In another aspect, the present disclosure relates to the preparation of bifunctional fusion proteins comprising a GLP-1 R agonist peptide fused to an GCGR antagonistic antigen binding molecule (the bifunctional fusion protein hereinafter referred to as GCGR antagonist / GLP-1 R agonist bifunctional fusion protein). In various embodiments, the GCGR antagonist will be directly fused to the GLP-1 R agonist. In various embodiments, the GCGR antagonist will be directly fused to the GLP-1 R agonist using a peptide linker. In various embodiments, the bifunctional fusion protein is a recombinant protein containing glucagon receptor antagonistic antibody fused to GLP-1 peptide via a peptide linker at the GCGRPCT Patent Application Docket No: CACRE1.0026WOantibody heavy chain N-terminal. In various embodiments, the GCGR antagonist / GLP-1 R agonist bifunctional fusion protein has the structure depicted in FIG. 1. Importantly, the bifunctional fusion proteins of the present invention advance the state of art by providing bifunctional fusion molecules having a greater concentration therapeutic window than those previously described. An illustrative, but not limiting, list of suitable GLP-1 agonist peptides to be used for GCGR antagonist / GLP-1 R agonist bifunctional fusion proteins of the present disclosure is provided in Table 2 and Table 3.

[0025] In another aspect, the present disclosure relates to methods of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein.

[0026] In another aspect, the present disclosure relates to methods of treating a metabolic disorder selected from the group consisting of obese T2D, obesity with prediabetes, insulin resistance, metabolic syndrome, fatty liver diseases, metabolic dysfunction-associated steatohepatitis (MASH), and Nonalcoholic steatohepatitis (NASH) and other indications that obesity is an important contributing factor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein.

[0027] In various embodiments, an isolated GCGR antagonist, or GLP-1 R agonist, or GCGR antagonist / GLP-1 R agonist bifunctional fusion protein 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. In various embodiments, the isolated GCGR antagonist and GLP-1 R agonist will be combined and admixed with a pharmaceutically acceptable carrier as a single product.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] FIG. 1 is a schematic depicting an exemplary recombinant GCGR antagonist / GLP-1 R agonist bifunctional fusion protein of the present invention.

[0029] FIG. 2 is a line graph depicting the body weight change rate (%) vs. day 1 over 39 days with 28 days treatment with Vehicle, Dulag lutide (“Dula”) at 0.6 mg / kg (“mpk”), REMD-514 (bifunctional antibody containing glucagon receptor antagonist mAb (“GCGR mAb”) andPCT Patent Application Docket No: CACRE1.0026WOGLP-1 peptide) at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + standard deviation (SD).

[0030] FIG. 3 is a line graph depicting food consumption (g / day) over 41 days with 28 days treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SD.

[0031] FIG. 4 is a line graph depicting fed glucose (mM) over 42 days with 28 days treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SD.

[0032] FIG. 5 is a line graph depicting fasting glucose (mM) after a 6h fast over 42 days with 28 days treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SD.

[0033] FIG. 6 is a bar graph depicting the area under the curve (AUC) (determined by glucose levels measured at t=0, 15min, 30min, 60min, and 120min) after oral glucose tolerance test (OGTT) was performed on day 29 after a 16h fast, after 4 weeks treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.

[0034] FIG. 7 is a bar graph depicting AUC (determined by glucose levels measured at t=0, 15min, 30min, 60min, and 120min) after OGTT was performed on day 43 following 2 weeks of no treatment after a 16h fast, in animals previously treated for 4 weeks with Vehicle, Dula at 0.6 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.

[0035] FIG. 8A is a bar graph depicting lean weight (g) on day 27 following treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. FIG. 8B is a bar graph depicting lean percent (%) following 4 weeks treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.PCT Patent Application Docket No: CACRE1.0026WO

[0036] FIG. 9A is a bar graph depicting fat weight (g) on day 27 following treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. FIG. 9B is a bar graph depicting fat percent (%) following 4 weeks treatment with Vehicle, Dula at 0.6 mpk, REMD-514 at 2.47 mpk, GCGR mAb at 2.34 mpk, Dula at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.

[0037] FIG. 10A is a bar graph depicting lean weight (g) on day 41 following 2 weeks of no treatment in animals previously treated for 4 weeks with Vehicle, Dula at 0.6 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. FIG. 10B is a bar graph depicting lean percent (%) on day 41 in animals previously treated with Vehicle, Dula at 0.6 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.

[0038] FIG. 11 A is a bar graph depicting fat weight (g) on day 41 following 2 weeks of no treatment in animals previously treated for 4 weeks with Vehicle, Dula at 0.6 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. FIG. 11 B is a bar graph depicting fat percent (%) on day 41 in animals previously treated with Vehicle, Dula at 0.6 mpk, GCGR mAb at 5 mpk, or Dula at 0.6 mpk plus GCGR mAb at 5 mpk. Shown are mean + SEM.

[0039] FIG. 12 is a line graph depicting the body weight change rate (%) vs. day 1 over 41 days with 28 days treatment with Vehicle, Semaglutide (“Serna”) at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SD.

[0040] FIG. 13 is a line graph depicting food consumption (g / day) (%) over 41 days with 28 days treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SD.

[0041] FIG. 14 is a line graph depicting fed glucose (mmol / L) over 42 days with 28 days treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SD.

[0042] FIG. 15 is a line graph depicting fasting glucose (mM) after a 6h fast over 42 days with 28 days treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SD.PCT Patent Application Docket No: CACRE1.0026WO

[0043] FIG. 16 is a bar graph depicting AUC (determined by glucose levels measured at t=0, 15min, 30min, 60min, and 120min) after OGTT was performed on day 30 after a 16h fast, after 4 weeks treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0044] FIG. 17 is a bar graph depicting AUC (determined by glucose levels measured at t=0, 15min, 30min, 60min, and 120min) after OGTT was performed on day 43 following 2 weeks of no treatment after a 16h fast in animals previously treated for 4 weeks with Vehicle, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0045] FIG.18A is a bar graph depicting lean weight (g) on day 13 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 18B is a bar graph depicting lean percent (%) on day 13 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0046] FIG. 19A is a bar graph depicting fat weight (g) on day 13 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 19B is a bar graph depicting fat percent (%) on day 13 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0047] FIG.20A is a bar graph depicting lean weight (g) on day 28 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 20B is a bar graph depicting lean percent (%) on day 28 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0048] FIG. 21 A is a bar graph depicting fat weight (g) on day 28 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 21 B is a bar graph depicting fat percent (%) on day 28 following treatment with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.PCT Patent Application Docket No: CACRE1.0026WO

[0049] FIG. 22A is a bar graph depicting lean weight (g) on day 41 following 12 days of no treatment in animals previously treated for 4 weeks with Vehicle, Sema at 12 nmol / kg, GCGR mAb at 5 mpk, or Sema at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 22B is a bar graph depicting lean percent (%) on day 41 following 12 days of no treatment in animals previously treated for 4 weeks with Vehicle, Sema at 12 nmol / kg, GCGR mAb at 5 mpk, or Sema at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.

[0050] FIG. 23A is a bar graph depicting fat weight (g) on day 41 following 12 days of no treatment in animals previously treated for 4 weeks with Vehicle, Sema at 12 nmol / kg, GCGR mAb at 5 mpk, or Sema at 6 nmol / kg + GCGR mAb at 5 mpk. FIG. 23B is a bar graph depicting fat percent (%) on day 41 following 12 days of no treatment in animals previously treated for 4 weeks with Vehicle, Sema at 12 nmol / kg, GCGR mAb at 5 mpk, or Sema at 6 nmol / kg + GCGR mAb at 5 mpk. Shown are mean + SEM.MODE(S) FOR CARRYING OUT THE INVENTION

[0051] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (1990), incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used andPCT Patent Application Docket No: CACRE1.0026WOwell known in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of subjects.Definitions

[0052] The terms "peptide" "polypeptide" and "protein" each 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. In certain embodiments, "peptides", "polypeptides", and "proteins" are chains of amino acids whose alpha carbons are linked through peptide bonds. The terminal amino acid at one end of the chain (amino terminal) therefore has a free amino group, while the terminal amino acid at the other end of the chain (carboxy terminal) has a free carboxyl group. As used herein, the term "amino terminus" (abbreviated N-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. Similarly, the term "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.

[0053] Polynucleotide and 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 119, or by the actual residue at that site such as Ser80 to Ser119. A particular polypeptide or polynucleotide sequence also can be described by explaining how it differs from a reference sequence.

[0054] 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) alterPCT Patent Application Docket No: CACRE1.0026WObinding affinities, and (5) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the naturally occurring sequence (e.g., in the portion of the polypeptide outside the domain(s) forming intermolecular contacts). 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:Alanine (A), Serine (S), and Threonine (T)Aspartic acid (D) and Glutamic acid (E)Asparagine (N) and Glutamine (Q)Arginine (R) and Lysine (K)Isoleucine (I), Leucine (L), Methionine (M), and Valine (V)Phenylalanine (F), Tyrosine (Y), and Tryptophan (W)

[0055] A “non-conservative amino acid substitution” refers to the substitution of a member of one of these classes for a member from another class. In making such changes, according to certain embodiments, 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. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0056] The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (see, for example, Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, in certain embodiments, the substitution of amino acids whose hydropathic indices 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.

[0057] It is also understood in the art that 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, asPCT Patent Application Docket No: CACRE1.0026WOdisclosed herein. In certain embodiments, 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.

[0058] The following 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). In making changes based upon similar hydrophilicity values, in certain embodiments, 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.Table 1Original Residues Exemplary Substitutions Preferred Substitutions Ala Val, Leu, Ile ValArg Lys, Gin, Asn LysAsn GinAsp GluCys Ser, Ala SerGin Asn AsnGlu Asp AspGly Pro, Ala AlaHis Asn, Gin, Lys, Arg ArgIle Leu, Val, Met, Ala, LeuPhe, NorleucineLeu Norleucine, Ile, IleVal, Met, Ala, PheLys Arg, 1,4 Diamino-butyric ArgAcid, Gin, AsnMet Leu, Phe, Ile LeuPhe Leu, Val, Ile, Ala, Tyr LeuPro Ala GlyPCT Patent Application Docket No: CACRE1.0026WOSer Thr, Ala, Cys ThrThr SerTrp Tyr, Phe TyrTyr Trp, Phe, Thr, Ser PheVal Ile, Met, Leu, Phe, LeuAla, Norleucine

[0059] A skilled artisan will be able to determine suitable variants of polypeptides as set forth herein using well-known techniques. In certain embodiments, 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. In other embodiments, the skilled artisan can identify residues and portions of the molecules that are conserved among similar polypeptides. In further embodiments, 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.

[0060] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides that are important for activity or structure. In view of such a comparison, the skilled artisan can predict the importance of amino acid residues in a polypeptide that correspond to amino acid residues important for activity or structure in similar polypeptides. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.

[0061] 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. For example, if one discovered that a change to a particular amino acid residue resulted in destroyed, undesirably reduced, or unsuitable activity, variants with such a change can be avoided. In other words, based on information gathered from suchPCT Patent Application Docket No: CACRE1.0026WOroutine experiments, one skilled in the art can readily determine the amino acids where further substitutions should be avoided either alone or in combination with other mutations.

[0062] The term "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. In certain embodiments, 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. In certain embodiments, 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).

[0063] The terms "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. In certain embodiments, 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.

[0064] 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.

[0065] The term "% 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. In certain embodiments, the % identity is selected from, e.g., at least 60%, atPCT Patent Application Docket No: CACRE1.0026WOleast 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%.

[0066] The term "% sequence homology" is used interchangeably herein with the term "% homology" and 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. For example, as used herein, 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. In certain embodiments, 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. In certain embodiments, 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%.

[0067] Exemplary computer programs which can be used to determine identity between two sequences include, but are not limited to, the suite of BLAST programs, e.g., BLASTN, BLASTX, and TBLASTX, BLASTP and TBLASTN, publicly available on the Internet at the NCBI website. See also Altschul et al., 1990, J. Mol. Biol. 215:403-10 (with special reference to the published default setting, i.e., parameters w=4, t=17) and Altschul et al., 1997, Nucleic Acids Res., 25:3389-3402. 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 11.0, and an extended gap penalty of 1.0, and utilize the BLOSUM-62 matrix. See id.

[0068] 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. For example, a nucleic acid is considered similar to a reference sequence if thePCT Patent Application Docket No: CACRE1.0026WOsmallest 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.

[0069] The term "isolated molecule" (where the molecule is, for example, a polypeptide, a polynucleotide, or an antibody) is a molecule that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a molecule that is chemically synthesized, or expressed in a cellular system different from the cell from which it naturally originates, will be "isolated" from its naturally associated components. A molecule also may be rendered substantially free of naturally associated components by isolation, using purification techniques well known in the art. Molecule purity or homogeneity may be assayed by a number of means well known in the art. For example, the purity of a polypeptide sample may be assayed using polyacrylamide gel electrophoresis and staining of the gel to visualize the polypeptide using techniques 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.

[0070] 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.

[0071] 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 molecule to the antigen. Examples of 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 molecule 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 molecule as well as wholly synthetic scaffolds comprising,PCT Patent Application Docket No: CACRE1.0026WOfor example, a biocompatible polymer. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, Volume 53, Issue 1:121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimetics (" PAMs") can be used, as well as scaffolds based on antibody mimetics utilizing fibronection components as a scaffold.

[0072] An isolated antagonistic antigen binding molecule 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 110 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. Within light and heavy chains, 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.

[0073] An "antibody" refers to a protein comprising one or more polypeptides 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 110 or more amino acids primarily responsible for antigen recognition.

[0074] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavyPCT Patent Application Docket No: CACRE1.0026WOchain constant region is comprised of three domains, CH1, CH2and CH3(and in some instances, CH4). Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VHand VLregions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FRi, CDRi, FR2, CDR2, FR3, CDR3, FR4. 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., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0075] Antibodies exist as intact immunoglobulins or as a number of well characterized fragments. Such fragments include Fab fragments, Fab' fragments, Fab2, F(ab')2fragments, 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. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, as used herein, the term 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')2fragments, 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.

[0076] A Fab fragment is a monovalent fragment having the VL, V, CL and Cm domains; a F(ab')2fragment is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment has the VHand CH1domains; an Fv fragment has the VLand VHdomains of a single arm of an antibody; and a dAb fragment has a VHdomain, a VLPCT Patent Application Docket No: CACRE1.0026WOdomain, or an antigen-binding fragment of a VHor VLdomain (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)).

[0077] A single-chain antibody (scFv) is an antibody in which a VL and a VH 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)). Diabodies are bivalent antibodies comprising two polypeptide chains, wherein each polypeptide chain comprises VHand VL 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 2:1121 -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. Similarly, 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.

[0078] An isolated antagonistic antigen binding molecule 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 "bifunctional" antibody has two different binding sites.

[0079] The term "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.

[0080] 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 speciesPCT Patent Application Docket No: CACRE1.0026WOantibody, when it is administered to a human subject. In one embodiment, 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. In another embodiment, the constant domain(s) from a human antibody are fused to the variable domain(s) of a non-human species. In another embodiment, 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.

[0081] An isolated antagonistic antigen binding molecule 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 10-7M or less. An isolated antagonistic antigen binding molecule 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.

[0082] An "epitope" is the portion of a molecule that is bound by an isolated antagonistic antigen binding molecule (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).

[0083] A "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal or human. 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. As used herein "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.Suitable pharmaceutical carriers and formulations are described in Remington's PharmaceuticalPCT Patent Application Docket No: CACRE1.0026WOSciences, 21st Ed. 2005, Mack Publishing Co, Easton. 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.

[0084] As used herein, a "therapeutically effective amount" of an isolated antagonistic antigen binding molecule 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 MASH; or reduces, suppresses, attenuates, or inhibits one or more symptoms of MASH; treats NASH; or reduces, suppresses, attenuates, or inhibits one or more symptoms of NASH.

[0085] The terms "treat", "treating" and "treatment" refer refers to an approach for obtaining beneficial or desired clinical results. Further, references herein to "treatment" include references to curative, palliative and prophylactic treatment. For purposes of this disclosure, 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.

[0086] The phrase "synergistic effect" refers to the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the active ingredients separately.

[0087] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the disclosure described herein include "consisting" and / or "consisting essentially of" aspects and variation.

[0088] Reference to "about" a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to "about X" includes description of " X".Glucagon Receptor and Antigen binding and antagonizing proteins

[0089] 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 andPCT Patent Application Docket No: CACRE1.0026WOproneuropeptides (Furuta et al., J. Biol. Chem. 276: 27197-27202 (2001 )). In vivo, glucagon is a major counter- regulatory hormone for insulin actions. During fasting, glucagon secretion increases in response to falling glucose levels. Increased 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.

[0090] 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. 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). In addition, glucagon signaling activates glycogen phosphorylase and inhibits glycogen synthase. Studies have shown that higher basal glucagon levels and lack of suppression of postprandial glucagon secretion contribute to diabetic conditions in humans (Muller et al., N Eng J Med 283: 109-115 (1970)). As such, methods of controlling and lowering blood glucose by targeting glucagon production or function using a GCGR antagonist have been explored.

[0091] In various embodiments, 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. In various embodiments, the antigen binding and antagonizing proteins of the present disclosure specifically bind to the human glucagon receptor. In various embodiments, 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. 7,947,809, herein incorporated by reference in its entirety for its specific teaching of polynucleotide and polypeptide sequences of a human, rat, mouse and cynomolgus glucagon receptor). In various embodiments of the present disclosure, the antigen binding and antagonizing proteins specifically bind the human glucagon receptor having the amino acid sequence set forth in SEQ ID NO: 1:PCT Patent Application Docket No: CACRE1.0026WOGlucagon Receptor Human (Homo sapiens) amino acid sequence (Accession Number AAI04855)MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFD KYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQ MDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLK ASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLH NLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFL AILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSA KLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHG PPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPF (SEQ ID NO: 1)In various embodiments, 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.

[0092] 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. As such, 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, T1 D, T2D, obesity, NAFLD, MASH, NASH, and for treating such non-diabetic disorders.

[0093] In various embodiments, the isolated antagonistic antigen binding molecule to human GCGR is a small molecule. In various embodiments, the GCGR small molecule antagonist is selected from the GCGR antagonists disclosed in Sammons and Lee, Bioorg Med Chem Lett. 2015 Oct 1;25(19):4057-64; Chen et al, Expert Opinion on Therapeutic Patents, Vol 30, 2020, Issue 7; Filipski, Expert Opinion on Therapeutic Patents, Vol 25, 2015, Issue 7; Shen et al, Expert Opinion on Therapeutic Patents, Vol 21, 2011, Issue 8; Sheen et al, Expert Opinion on Investigational Drugs, Volume 26, 2017, Issue 12; Yang et al, Acta Pharmacologica SinicaPCT Patent Application Docket No: CACRE1.0026WO(2015) 36: 1033-1042; US 12208071; US 8609892; US 20230104956; US 20210121422; US 20190194143.

[0094] Methods of generating antibodies that bind to antigens such as the human glucagon receptor are known to those skilled in the art. For example, 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 antibodyproducing 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 antibodyproducing hybridomas to identify a hybridoma that produces a monoclonal antibody that binds specifically to the targeted antigen polypeptide. Once obtained, 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.

[0095] In various embodiments, the isolated antagonistic antigen binding molecule to human GCGR is selected from the group consisting of 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 Fab2, a Fab'2, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, a diabody, and a hemibody.

[0096] In various embodiments, the isolated antagonistic antigen binding molecule of the present disclosure utilize an antibody or antigen-binding fragment that binds to a glucagon receptor antigen with a dissociation constant (KD) of, e.g., at least about 1x10-7M, at least about 1x10-8M, at least about 1x10-9M, at least about 1x10-10M, at least about 1x10-11M, or at least about 1x10-12M. In various embodiments, the isolated antagonistic antigen binding molecule of the present disclosure utilize an antibody or antigen-binding fragment that binds to a glucagon receptor antigen with a dissociation constant (KD) in the range of, e.g., at least about 1x10-7M to at least about 1x10-8M, at least about 1x10-8M to at least about 1x10-9M, at least about 1x10-9M to at least about 1x10-10M, at least about 1x10-10M to at least about 1x10-11M, or at least about 1x10-11M to at least about 1x10-12M.

[0097] Antibodies to the glucagon receptor have been described in, e.g., U. S. Pat. Nos.5,770,445; 7,947,809; 7,968,686; 8,545,847; 8,771,696; 9,102,732; 9,248,189; European patent application EP2074149A2; EP patent EP0658200B1; U. S. patent publications 2009 / 0041784; 2009 / 0252727; 2013 / 0344538; 2014 / 0335091; and 20160075778 and PCT publicationPCT Patent Application Docket No: CACRE1.0026WOW02008 / 036341. In various embodiments of the present invention, the isolated antagonistic antigen binding molecule 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.

[0098] In various embodiments, the isolated GCGR antagonistic antigen binding molecule comprises an 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. In various embodiments, the isolated antagonistic antigen binding molecule comprises an 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. In various embodiments, the isolated antagonistic antigen binding molecule comprises an 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.

[0099] In various embodiments of the present disclosure 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: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, 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 NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47. In various embodiments, 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.Examples of Anti-GCGR AntibodiesHCVR LCVRT1PCT Patent Application Docket No: CACRE1.0026WOSEQ ID NO 2 SEQ ID NO 3SEQ ID NO 4 SEQ ID NO 5SEQ ID NO 6 SEQ ID NO 7SEQ ID NO 10 SEQ ID NO 29SEQ ID NO 11 SEQ ID NO 30SEQ ID NO 12 SEQ ID NO 31SEQ ID NO 13 SEQ ID NO 32SEQ ID NO 14 SEQ ID NO 33SEQ ID NO 15 SEQ ID NO 34SEQ ID NO 16 SEQ ID NO 35SEQ ID NO 17 SEQ ID NO 36SEQ ID NO 18 SEQ ID NO 37SEQ ID NO 19 SEQ ID NO 38SEQ ID NO 20 SEQ ID NO 39SEQ ID NO 21 SEQ ID NO 40SEQ ID NO 22 SEQ ID NO 41SEQ ID NO 23 SEQ ID NO 42SEQ ID NO 24 SEQ ID NO 43SEQ ID NO 25 SEQ ID NO 44SEQ ID NO 26 SEQ ID NO 45SEQ ID NO 27 SEQ ID NO 46SEQ ID NO: 28 SEQ ID NO: 4728

[0100] In various embodiments, 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.

[0101] 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. In various embodiments, the light or heavy chain constant region is a fragment, derivative, variant, or mutein of a naturally occurring constant region.

[0102] Techniques are known for deriving an antibody of a different subclass or isotype from an antibody of interest, i.e., subclass switching. Thus, IgG antibodies may be derived from an IgM antibody, for example, and vice versa. Such techniques allow the preparation of newPCT Patent Application Docket No: CACRE1.0026WOantibodies 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).

[0103] In various embodiments, 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 IgG2 heavy chain constant region set forth in SEQ ID NO: 50, or a fragment thereof.

[0104] In various embodiments, the isolated GCGR antagonistic antigen binding molecule is an chimeric antibody which comprises the amino acid sequence encoding the heavy chain of SEQ ID NO: 8 and the amino acid sequence encoding the light chain of SEQ ID NO: 9 (also hereinafter “REMD2.59C”).

[0105] In various embodiments, an isolated GCGR antagonistic antigen binding molecule of the present disclosure is a fully human 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

[0106] In various embodiments of the present disclosure, the isolated antagonistic antigen binding molecule 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. In various embodiments, 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 IgG2 Fc region). Methods for preparing hemibodies are described in, e.g., U. S. patent application 2012 / 0195879, herein incorporated by reference in its entirety herein for purposes of teaching the preparation of such hemibodies.IncretinsPCT Patent Application Docket No: CACRE1.0026WO

[0107] Incretins belong to a class of gastrointestinal hormones that increase insulin release from beta cells of the islets of Langerhans after eating, even before blood glucose levels are elevated. Incretins also slow the rate of absorption of nutrients into the blood stream by reducing gastric emptying and may reduce food intake. Incretins inhibit glucagon release from the alpha cells of the Islets of Langerhans. The two main members of incretins that exert these activities are glucagon-like peptide-1 (GLP-1) and gastric inhibitory peptide (GIP). Both GLP-1 and GIP are rapidly inactivated by the enzyme dipeptidyl peptidase 4 (DPP-4).

[0108] The incretins or incretin mimetic peptides that can be used in the present disclosure may be naturally occurring incretins or incretin mimetic peptides or modified naturally occurring incretins or incretin mimetic peptides. The peptides may be chemically synthesized using standard techniques for peptide synthesis such as solid-phase peptide synthesis or may be prepared using DNA techniques known in the art (see, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2. sup. nd Ed., Cold Spring Harbor, 1989). The peptides thus produced may or may not be identical to the naturally occurring peptides. Analogs, fragments and conjugates of the naturally occurring incretins or incretin mimetic peptides are encompassed in the present invention so long as they retain one or more of the biological activities of the naturally occurring incretins or incretin mimetic peptides.

[0109] GLP-1 is a physiological incretin hormone that is synthesized in intestinal L-cells by tissue-specific post-translational processing of the glucagon precursor preproglucagon and implicated in the control of appetite and satiety. GLP-1 acts through GLP-1 receptor (GLP-1 R), a 463 amino-acid member of the G protein-coupled receptor (GPCR) superfamily (Drucker DJ et al., Mol Endocrinol, 17(2):161 — 171, 2003). Bioactive GLP-1 exists in two equipotent molecular forms: GLP-17-37and GLP-17-36 amide. GLP-1 is rapidly cleaved by diaminopeptidyl peptidase-4 (DPP-4), which results in the generation of largely GLP-17-37and inactive molecular GLP-19-36 amideforms. The majority of GLP-1 leaving the intestinal venous circulation has already been cleaved by DPP-4 expressed in capillary surrounding gut L cells, which provides an estimated half-life of 1-2 minutes for intact GLP-1 in vivo (Drucker DJ, Gastroenterology, 122(2):531 -544, 2002).

[0110] The GLP-1 R is widely distributed in tissues, including brain, pancreas, intestine, lung, stomach, and kidney. The effects of GLP-1 appear to be both insulinotropic and insulinomimetic, depending on the ambient glucose concentration. Due to their ability to increase insulin secretion from the pancreas, increase insulin-sensitivity in both alpha cells and beta cells, and decrease glucagon secretion from the pancreas, GLP-1 and its analogs havePCT Patent Application Docket No: CACRE1.0026WOattracted considerable attention as a therapeutic strategy for diabetes, obesity and hyperglycemia.

[0111] GLP-1 agonists (also known as GLP-1 receptor agonists (GLP-1 RAs), incretin mimetics, or GLP-1 analogs) represent a class of medications used to treat type 2 diabetes mellitus and, in some cases, obesity. Examples of drugs in this class include exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and semaglutide. (Burcelin R et al., Obes Rev.2017 Jan;18(1):86-98). Several GLP-1 agonists have been approved for treatment of T2D, including, e.g., exenatide (tradename Byetta®, Amylin / Astrazeneca); liraglutide (tradename Victoza®, Novo Nordisk A / S); lixisenatide (tradename Lyxumia®, Sanofi); albiglutide (tradename Tanzeum®, GlaxoSmithKline); dulaglutide (tradename Trulicity®, Eli Lilly); semaglutide (tradename Ozempic®, Novo Nordisk A / S); tirzepatide (tradename Mounjaro®, Eli Lilly).

[0112] Many formulations of GLP-1 agonists, all of which historically were injectable and administered subcutaneously due to poor oral bioavailability, can be prescribed in the United States. Lixisenatide and liraglutide dosing are once-daily, albiglutide, dulaglutide, semaglutide dosing is once weekly, and exenatide can be dosed either as a twice-daily or a once-weekly injection. Recently, the FDA approved an oral formulation of semaglutide. Researchers have suspended trials investigating taspoglutide as a novel GLP-1 analog due to gastrointestinal side effects and hypersensitivity reactions (Gupta V. Glucagon-like peptide-1 analogues: An overview. Indian J Endocrinol Metab. 2013 May;17(3):413-21 ).

[0113] While proven efficacious, the major drawback associated with the clinical use of GLP-1 agonists is the short biological half-life, necessitating continuous administration intravenously or by frequent subcutaneous injections, and all GLP-1 drugs approved to date are subcutaneous administered on a twice daily or once weekly basis. Moreover, there are safety concerns associated with the use of these GLP-1 agonists, namely, pancreatitis and pancreatic neoplasia, hypoglycemia, and renal impairment. In addition, GLP-1 drugs cause gastrointestinal side effects such as vomiting and diarrhea when given at efficacious doses for obesity. Due to this poor tolerability, patients are required to start with low dose and gradually increase up to higher doses over time to limit the Gl toxicity.

[0114] As used herein, the term GLP-1 agonist refers to a protein or peptide having the activity of native GLP-1, or a peptide comprising the amino acid sequence set forth in of SEQ ID NO. 53. Also included is GLP-17-36 amide. GLP-1 refers to GLP-1 from any source which has the sequence of SEQ ID NO: 53, including isolated, purified and / or recombinant GLP-1 produced from any source or chemically synthesizes, for example using solid phase synthesis. AlsoPCT Patent Application Docket No: CACRE1.0026WOincluded herein are conserved amino acid substitutions of native GLP-1. For example, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function. Conservative amino acid substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In certain embodiments, the GLP-1 molecule has 80% homology to native GLP-1; 85% homology; 90% homology; 92% homology; 95% homology; 96% homology; 97% homology; 98% homology; or 99% homology to native GLP-1 while retaining at least one biological activity of native GLP-1. Many molecular derivatives of GLP-1 have been disclosed, including some of which are reported to have agonist activity, see e.g., U. S. Pat. Nos. 6,358,924; 6,344,180; 6,284,725; 6,277,819; 6,271,241; 6,268,343; and 6,191,102, the contents of which are incorporated herein by reference.

[0115] In various embodiments, the GLP-1 R agonist of the present invention is a GLP-1 R (7-37) analog selected from the group consisting of exenatide (tradename Byetta®, Amylin / Astrazeneca); liraglutide (tradename Victoza®, Novo Nordisk A / S); lixisenatide (tradename Lyxumia®, Sanofi); albiglutide (tradename Tanzeum®, GlaxoSmithKline); dulaglutide (tradename Trulicity®, Eli Lilly), semaglutide (tradename Ozempic®, Novo Nordisk A / S) and taspoglutide. In various embodiments, the GLP-1 agonist comprises the amino acid sequence selected from the group consisting of SEQ ID NOS: 54-58.

[0116] Additional GLP-1 related molecules, e.g., a proteins called exendin-4 and exendin-3, have also been disclosed and reported to be capable of inducing pancreatic endocrine differentiation, islet proliferation and an increase in [3-cell mass (Parkes et aL, Metabolism 50:583, 2001). Exendin 4 and exendin 3 are 39 amino acid peptides (differing at residues 2 and 3) which are approximately 53% homologous to GLP-1 and have insulinotropic activity. Many molecular derivatives of exendin-3 and exendin-4 have been disclosed, including some of which are reported to have agonist activity, see, e.g., U. S. Pat. Nos. 5,424,286;6,268,343; 6,384,016; 6,458,924; 6,858,576; 6,989,366; 7,115,569; 7,153,825; 7,223,725;7,235,627; 7,297,761; 7,419,952; 7,521,423; 7,696,161; 7,700,549; 8,097,698; 8,853,160;8,889,619; 9,012,398; US 20120283179; US 20140206608; US 20140206609; 20140221281; US 20140213513; US 20150164997; and U. S. Pat. No. 9,181,305, the contents of which are incorporated herein by reference.

[0117] In various embodiments, the GLP-1 agonist is selected from the group consisting of exendin-3 and derivatives thereof and exendin-4 and derivatives thereof. In certain embodiments, analogs and derivatives of exendin-3 or / and exendin-4 may contain aPCT Patent Application Docket No: CACRE1.0026WOmodified amino acid sequence comprising a single amino acid deletion (e.g. desPro36, desPro37, desPro37, desAsp28, desMet(O)14in exendin-4 and the corresponding positions in exendin-3). In certain embodiments, single amino acid positions can be substituted (e.g. Met(O)14Trp(O2)25, IsoAsp28, Asp28, Pro38in exendin-4 and the corresponding positions in exendin-3), in which case unnatural amino acids such as Met(0)(methionine sulfoxide or methionine sulfone), Trp(O2) (N-formylkynurenine), or / and IsoAsp (-aspartate or isoaspartate) can also be used. Unnatural amino acids can be readily inserted, in the form of corresponding amino acid building blocks, into the sequence. In certain embodiments, the C-terminus or / and the N-terminus can be modified, for example, by an additional sequence such as -(Lys)-, -(Lys)2-, -(Lys)3-, -(Lys)4-, -(Lys)5-, -(Lys)6-, and -Asn-(Glu)5-.

[0118] In certain embodiments, the GLP-1 agonist is an exendin-4 analog selected from the group consisting of: desPro36-exendin-4(1-39)-Lys6NH2; H-des(Pro36'37)-exendin-4-Lys4-NH2; H-des(Pro36’37)-exendin-4-Lys5-NH2; desPro36[Asp28]exendin-4 (1 -39); desPro36[lsoAsp28]exendin-4 (1-39); desPro36[Met(O)14, Asp28]exendin-4 (1-39); desPro36[Met(O)14, lsoAsp28]exendin-4 (1-39); desPro36[Trp(O2)26, Asp28]exendin-4 (1-39); desPro36[Trp(O2)25, lsoAsp28]exendin-4 (1-39); desPro36[Met(O)14Trp(O2)25, Asp28]exendin-4 (1-39); and desPro36[Met(O)14Trp(O2)25, lsoAsp28]exendin-4 (1-39). In certain embodiments, the C-terminus or / and the N-terminus can be modified by an additional sequence selected from the group consisting of -(Lys)-, -(Lys)2-, -(Lys)3-, -(Lys)4-, -(Lys)5-, -(Lys)6-, and -Asn-(Glu)5-. In certain embodiments, the exendin-4 analog is desPro36-exendin-4(1-39)-Lys6NH2.

[0119] In certain embodiments, the GLP-1 agonist is an exendin-4 peptide analog or other peptidic compounds described in, e.g., US 9181305, US 20140206608; US 20140206609; 20140221281; US 20140213513; and US 20150164997, of which the contents of each is incorporated herein by reference.

[0120] An illustrative, but not limiting, list of suitable GLP-1 agonist peptides to be used in the GCGR antagonist / GLP-1 agonist combination therapy regimens of the present invention and / or in the GCGR antagonist / GLP-1 R agonist bispecific fusion proteins of the present disclosure is provided in Table 2 and Table 3.Table 2PCT Patent Application Docket No: CACRE1.0026WOGlucose metabolism-related RefSeq (NCBI / Uniprot) proteinsGlucagon proprotein NP_002045.1Glucagon peptide NP_002045.1 (aa 53-81)Glucagon-like peptide 1 NP_002045.1 (aa 98-127)Glucagon-like peptide 2 NP_002045.1 (aa 146-178) Glicentin P01275 (aa 21-89)Glicentin-related polypeptide P01275 (aa 21-50)Gastric inhibitory polypeptide NP_004114.1preproteinGastric inhibitory polypeptide NP_004114.1 (aa 52-93)Dipeptidyl peptidase 4 P27487Glucose transporter member 4 NP_001033.1Preproglucagon AAA52567.1Insulin receptor substrate 1 NP_005535.1Human Insulin Preprotein NP_000198.1Apolipoprotein A-ll P02652Solute carrier family 2, facilitated P11166glucose transporter member 1Glycogen synthase 1 P13807Glycogen synthase 2 P54840Tyrosin-protein phosphatase nonP18031receptor type 1RAC-alpha serine threonine-protein P31749kinasePeroxisome proliferator-activated P37231receptor gammaHexokinase 3 P52790Phosphatidylinositol-3,4,5- P60484triphosphate 3-phosphatase and dualspecificity proteinPyruvate dehydrogenase kinase 1 Q15118Calcium-binding and coiled-coil Q9P1Z2domain-containing protein 1Max-like protein X Q9UH92Fructose-bisphosphate aldolase A P04075Glucagon-like peptide 1 receptor P43220Glucagon-like peptide 2 receptor 095838Gastric inhibitory polypeptide receptor P48546Insulin-like growth factor 1 receptor P08069.1Insulin-like growth factor 2 receptor P11717.3Insulin Receptor P06213GLP-1 agonist-Exenatide DB01276GLP-1 agonist-Liraglutide DB06655GLP-1 agonist-Lixisenatide DB09265GLP-1 agonist-Albiglutide DB09043GLP-1 agonist-Dulaglutide DB09045PCT Patent Application Docket No: CACRE1.0026WOGLP-1 agonist-Semaglutide DB13928GIPR agonist (Des-Ala2-GIPi-3o) NP_004114.1 (aa 52-81)GIPR agonist-Truncated GIP130 NP_004114.1 (aa 52-81)GLP-1 R agonist (aa 1 -37 of GIP) NP_004114.1 (aa 52-88)GLP-1 R agonist (aa 7-36 of GIP) NP_004114.1 (aa 58-87)Table 3ReceptorsType Drug Name Company TargetedDanuglipron GLP-1 R PfizerMono Orforglipron GLP-1 R Eli LillyAgonists CarmotCT-996 GLP-1 RTherapeutics Tirzepatide GLP-1 R,Eli Lilly(Mounjaro) GIPRSurvodutide (Bl GLP-1 R, Boehringer 456906) GCGR IngelheimGLP-1 R,Cagrilintide + Sema Novo Nordisk AmylinGLP-1 R,Cotadutide AstraZeneca GCGR GLP-1 R, Innovent MazdutideGCGRA BiologiesGLP-1 R, Hanmi EfinopegdutideGCGRA Pharmaceutical GLP-1 R,Pemvidutide Altimmune GCGRADual GLP-1 R,NNC0165+Sema Novo Nordisk Agonists PYYRAGLP-1 R,Bimagrumab+Sema Versanis Bio Activin R IIGLP-1 R, Regeneron Trevogrumab+SemaGDF8 Pharmaceuticals GLP-1 R,MK-8521 MerckGCGR GLP-1 R, GIP VikingVK2735RA, Therapeutics GLP-1 R, GIP ScohiaSCO-094RA, Therapeutics GLP-1 R, GIP CarmotCT-388RA, Therapeutics Amycretin GLP-1 R,Novo Nordisk(NNC0482) Amylin RA,PCT Patent Application Docket No: CACRE1.0026WOGLP-1 R, HanmiHM12525AGIPR, GCGR Pharmaceutical LY3437943 GLP-1 R,Eli Lilly(Retatrutide) GIPR, GCGRTri-AgonistGLP-1 R,SAR441255 SanofiGIPR, GCGRBl 456906 + GIPR GLP-1 R, Boehringeragonist GCGR, GIPR IngelheimAntisense and siRNA Molecules

[0121] In certain embodiments, the glucose-lowering agent is a GLP-1 agonist that is an antisense or siRNA molecule to a designated GLP-1 polypeptide or one of the defined polypeptides in its pathway(s). Nucleotide sequences of the designated GLP-1 polypeptides and the defined polypeptides in their pathways are known and are readily available from publicly available databases. Exemplary sites of targeting include, but are not limited to, the initiation codon, the 5' regulatory regions, the coding sequence and the 3' untranslated region. In some embodiments, the oligonucleotides are about 10 to 100 nucleotides in length, about 15 to 50 nucleotides in length, about 18 to 25 nucleotides in length, or more. In some embodiments, the oligonucleotides are about 10, 15, 20, 25, 20, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 500, 1000 nucleotides in length, or more. The oligonucleotides can comprise backbone modifications such as, for example, phosphorothioate linkages, and 2'-0 sugar modifications well known in the art.

[0122] Included among the GLP-1 agonists of the invention are antisense oligonucleotides that act to directly block the translation of mRNA by hybridizing to targeted mRNA and preventing polypeptide translation. Antisense oligonucleotides are suitable for use in treating any of the medical disorders disclosed herein, either alone or in combination with other GLP-1 agonists or in combination with other agents for treating the same condition. Antisense molecules of the invention may interfere with the translation of the GLP-1, the GLP-1 receptor, or an enzyme in the metabolic pathways for the synthesis of the GLP-1 polypeptide. Antisense nucleic acids are typically at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides. In some embodiments, the oligonucleotide is between 18-21 nucleotides, 10-30 nucleotides, 6-35 nucleotides, or 15-25 nucleotides. In some embodiments, the oligonucleotide is between 18-21 nucleotides.PCT Patent Application Docket No: CACRE1.0026WO

[0123] The oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded. The oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc. The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al., 1989, Proc, Natl. Acad. Sci. U. S. A. 86:6553-6556; Lemaitre et. al., 1987, Proc. Natl. Acad. Sci. 84:648-652; PCT Publication No. W088 / 09810, published Dec. 15, 1988), or hybridization-triggered cleavage agents or intercalating agents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). The antisense molecules should be delivered to cells which express the targeted transcript.

[0124] Ribozyme molecules designed to catalytically cleave mRNA transcripts can also be used to prevent the translation of mRNAs encoding a GLP-1, GLP-1 receptors, or enzymes involved in synthesis of a GLP-1 polypeptide or GLP-1 Rs (see. e.g., PCT WO90 / 11, 364; U. S. Pat. No. 5,824,519). Ribozymes useful for this purpose include hammerhead ribozymes (Haseloff and Gerlach, 1988, Nature, 334:585-591), RNA endoribonucleases (hereinafter " Cech-type ribozymes") such as the one that occurs naturally in Tetrahymena thermophila (known as the IVS, or L-19 IVS RNA) (see, for example, WO 88 / 04300; Been and Cech, 1986, Cell, 47:207-216). Ribozymes can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express the target peptide in vivo. A preferred method of delivery involves using a DNA construct encoding the ribozyme under the control of a strong constitutive pol II or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous target mRNA, thereby inhibiting its translation.

[0125] Antisense RNA and DNA, ribozyme, triple helix molecules, etc. of the invention may be prepared by any method known in the art for the synthesis of DNA and RNA molecules, including, for example, solid phase phosphoramidite chemical synthesis. Oligonucleotides can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be synthesized by the method of Stein et al., 1988, Nucl. Acids Res. 16:3209, and methylphosphonate oligonucleotides can be prepared as described by Sarin et al., 1988, Proc. Natl. Acad. Sci. U. S. A. 85:7448-7451. Alternatively, RNA molecules may generated by in vitro and in vivo transcription of DNA sequences encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerasePCT Patent Application Docket No: CACRE1.0026WOpromoters. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.SMIPs

[0126] In certain embodiments, the glucose-lowering agent is a GLP-1 agonist that is a Small Modular Immuno-Pharmaceuticals (SMIP). SMIPs are single-chain polypeptides that are engineered to retain full binding and activity function of a monoclonal Ab (mAb); are approximately one-third to one-half the size of conventional therapeutic mAbs; and retain Remediated effector functions. In various embodiments, the GLP-1 agonist is selected from the group consisting of Danuglipron (Pfizer), Orforglipron (Eli Lilly), and CT-996 (Carmot Therapeutics).Biogenerics, Biosimilars, Follow On Biologies, and Follow-On Proteins

[0127] The glucose-lowering agent may be a GLP-1 agonist that is a biosimilar, biogeneric, follow-on biologic, or follow-on protein version of a currently contemplated GLP-1 agonist, including a direct GLP-1 agonist. For example, once the patents covering one of the approved GLP-1 agonists expire, other manufacturers will likely produce molecules similar or identical to that GLP-1 agonist, by manufacturing processes that are substantially similar or the same, or different from, those used to manufacture the GLP-1 agonist. Their objective would be to make, offer to sell, and sell therapeutics similar or identical in structure and activity to the approved GLP-1 agonist. Such molecules are generally referred to as biogenerics, generic biologies, biosimilars, follow on biologies, and follow on proteins, depending on details of the molecule, the manufacturing process and the regulatory pathway. In certain instances, the new product might differ by one or a few amino acids, which might be purported to improve the manufacturing efficiency or the therapeutic efficacy. In all such instances, these molecules are viewed as substantially the same as, or the same as currently contemplated GLP-1 agonists.Peptide Linkers

[0128] In various embodiments, the bifunctional fusion protein is a recombinantly expressed fusion molecule. In various embodiments, the bifunctional fusion protein comprises aPCT Patent Application Docket No: CACRE1.0026WOGLP-1 agonist that is directly attached to the GCGR antagonist. In various embodiments, the bifunctional fusion protein comprises a GLP-1 agonist that is attached to the GCGR antagonist via a peptide linker. In various embodiments, the linker may be an artificial sequence of between 5, 10, 15, 20, 30, 40 or more amino acids that are relatively free of secondary structure. In various embodiments, the linker is a rigid linker peptide of between 10, 15, 20, 30, 40 or more amino acids that display a-helical conformation and may act as rigid spacers between protein domains. In various embodiments, the peptide linker is a G / S rich linker. In various embodiments, the peptide linker is an alpha-helical linker. In various embodiments, the peptide linker is a glycine linker. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 62. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 63. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 64. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 65. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 66. In various embodiments, the peptide linker has the sequence set forth in SEQ ID NO: 67.

[0129] In various embodiments, the bifunctional fusion protein is a recombinant protein containing GCGR antagonistic antibody fused to GLP-1 R agonist via a peptide linker at the GCGR antibody heavy chain (e.g., SEQ ID NO: 51) N-terminal In various embodiments, the GCGR antagonist / GLP-1 R agonist bifunctional fusion protein has the structure depicted in Figure 1.

[0130] In various embodiments, the fusion molecules of the present disclosure will comprise the antibody and GLP-1 R agonist combinations recited in Table 4.Table 4Examples of GCGR antagonist / GLP-1 R agonist bifunctional fusion proteinsName GCGR Ab (HC / LC) Linker GLP-1 Agonist peptide from the GLP-1 R Agonist Drug BelowREMD-514 SEQ ID NOS: 51 / 52 GGGGS DulaglutideREMD-524 SEQ ID NOS: 51 / 52 (G4S)3DulaglutideN / A SEQ ID NOS: 51 / 52 GGGGS ExenatideN / A SEQ ID NOS: 51 / 52 GGGGS LiraglutideN / A SEQ ID NOS: 51 / 52 GGGGS AlbiglutideN / A SEQ ID NOS: 51 / 52 GGGGS Lixisenatide

[0131] In various embodiments, the GCGR antagonist / GLP-1 R agonist bifunctional fusion protein comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 59 and a light chain having the amino acid sequence set forth in SEQ ID NO: 61 (alsoPCT Patent Application Docket No: CACRE1.0026WOhereinafter “REMD-514”). In various embodiments, the GCGR antagonist / GLP-1 R agonist bifunctional fusion protein comprises a heavy chain having the amino acid sequence set forth in SEQ ID NO: 60 and a light chain having the amino acid sequence set forth in SEQ ID NO: 61 (also hereinafter “REMD-524”).Pharmaceutical Compositions

[0132] In another aspect, the present disclosure provides a pharmaceutical composition comprising an isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion 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 (coadministration) with other active agents, as detailed below. In various embodiments, the isolated GCGR antagonist and GLP-1 R agonist will be combined and admixed with a pharmaceutically acceptable carrier as a single product.

[0133] Generally, the isolated GCGR antagonistic antigen binding proteins and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion protein 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. The choice of 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. As used herein, "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. Some examples of pharmaceutically acceptable carriers are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, 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. Such compositions and methods for their preparation may be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). ThePCT Patent Application Docket No: CACRE1.0026WOpharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all GMP regulations of the U. S. Food and Drug Administration.

[0134] The pharmaceutical compositions of the present disclosure are typically suitable for parenteral administration. As used herein, "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, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissuepenetrating non-surgical wound, and the like. In particular, 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.

[0135] A pharmaceutical composition of the present disclosure can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present disclosure. 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. In 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 AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ (Disetronic Medical Systems, Burghdorf, Switzerland), and HUMALOG MIX 75 / 25™, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Ind.).

[0136] Formulations of a pharmaceutical composition suitable for parenteral administration typically generally comprise the active ingredient combined with aPCT Patent Application Docket No: CACRE1.0026WOpharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such 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. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition. 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 suspensions in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, or in a liposomal preparation. Formulations for parenteral administration may be formulated to be immediate and / or modified release. Modified release formulations include delayed-, sustained-, pulsed-, controlled-, targeted and programmed release.

[0137] For example, in one aspect, sterile injectable solutions can be prepared by incorporating the isolated antagonistic antigen binding molecule in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, 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. In the case of sterile powders for the preparation of sterile injectable solutions, 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 delaysPCT Patent Application Docket No: CACRE1.0026WOabsorption, for example, monostearate salts and gelatin. In various embodiments, the injectable compositions will be administered using commercially available disposable injectable devices.

[0138] The isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion 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 pharmaceutically acceptable carrier) from 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.

[0139] The pressurized container, pump, spray, atomizer, or nebulizer generally contains a solution or suspension of an isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion protein of the disclosure comprising, for example, a suitable agent for dispersing, solubilizing, or extending release of the active, a propellant(s) as solvent.

[0140] 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 homogenization, or spray drying.

[0141] 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 molecule of the disclosure, a suitable powder base and a performance modifier.

[0142] 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.

[0143] 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.

[0144] In the case of dry powder inhalers and aerosols, 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.PCT Patent Application Docket No: CACRE1.0026WO

[0145] The isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion 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; sprays; and buccal / mucoadhesive patches.

[0146] Pharmaceutical 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. For example, to prepare orally deliverable tablets, the isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion 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. To prepare orally deliverable capsules, 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 molecule 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.

[0147] In various embodiments, the pharmaceutical compositions are formulated as orally deliverable tablets containing isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist 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. ForPCT Patent Application Docket No: CACRE1.0026WOexample, a time delay material such as glyceryl monostearate or glyceryl distearate alone or with a wax may be employed.

[0148] In various embodiments, the pharmaceutical compositions are formulated as hard gelatin capsules wherein the isolated GCGR antagonistic antigen binding molecule and / or GLP-1 agonist and / or GCGR antagonist / GLP-1 R agonist bifunctional fusion 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 molecule is mixed with an aqueous or an oil medium, for example, arachis oil, peanut oil, liquid paraffin or olive oil.

[0149] 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.

[0150] Any method for administering peptides, proteins or antibodies accepted in the art may suitably be employed for administering the isolated antagonistic antigen binding molecule of the disclosure.

[0151] Exemplary pharmaceutical formulations of GLP-1 agonist compounds are described in US Patent Nos. 7,521, 423, 7,456,254; US Publication No. 2004 / 0106547, WO 2006 / 068910, WO 2006 / 125763, the disclosures of which are incorporated by reference herein.Methods of Treatment

[0152] In various embodiments, the present disclosure comprises a method for treating obesity in diabetic and non-diabetic subjects comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a glucagon like peptide-1 receptor (GLP-1 R) agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human glucagon receptor (GCGR).

[0153] In various embodiments, the present disclosure comprises a method for treating obesity in diabetic and non-diabetic subjects comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0154] In various embodiments, the present disclosure comprises a method for treatingPCT Patent Application Docket No: CACRE1.0026WOobese Type-2 Diabetes in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0155] In various embodiments, the present disclosure comprises a method for treating obese Type-2 Diabetes in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0156] In various embodiments, the present disclosure comprises a method for treating obesity with prediabetes in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0157] In various embodiments, the present disclosure comprises a method for treating obesity with prediabetes in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0158] In various embodiments, the present disclosure comprises a method for treating insulin resistance in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0159] In various embodiments, the present disclosure comprises a method for treating insulin resistance in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0160] In various embodiments, the present disclosure comprises a method for treating metabolic syndrome in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0161] In various embodiments, the present disclosure comprises a method for treating metabolic syndrome in a subject comprising administering to the subject: (a) a therapeuticallyPCT Patent Application Docket No: CACRE1.0026WOeffective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0162] In various embodiments, the present disclosure comprises a method for treating fatty liver diseases in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0163] In various embodiments, the present disclosure comprises a method for treating fatty liver diseases in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0164] In various embodiments, the present disclosure comprises a method for treating metabolic dysfunction-associated steatohepatitis (MASH) in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0165] In various embodiments, the present disclosure comprises a method for treating MASH in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0166] In various embodiments, the present disclosure comprises a method for treating Nonalcoholic steatohepatitis (NASH) in a subject comprising administering to the subject a therapeutically effective amount of a bifunctional fusion molecule comprising a GLP-1 agonist peptide fused directly into an antagonistic antigen binding molecule that specifically binds to the human GCGR.

[0167] In various embodiments, the present disclosure comprises a method for treating NASH in a subject comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human GCGR; and (b) a therapeutically effective low dose of GLP-1 R agonist.

[0168] In various embodiments of the present disclosure, obesity is defined as BMI of 30 kg / m2or more (National Institute of Health, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults (1998)). In various other embodiments, the present disclosure is also intended to include a disease, disorder, or condition that is characterized by a body mass index (BMI) of 25 kg / m2or more, 26 kg / m2or more, 27 kg / m2orPCT Patent Application Docket No: CACRE1.0026WOmore, 28 kg / m2or more, 29 kg / m2or more, 29.5 kg / m2or more, or 29.9 kg / m2or more, all of which are typically referred to as overweight.

[0169] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus can be administered, several divided doses (multiple or repeat or maintenance) can be administered over time and the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein 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.

[0170] Thus, the skilled artisan would appreciate, based upon the disclosure provided herein, that 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.

[0171] It is to be noted that 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. For example, doses may be adjusted based on pharmacokinetic or pharmacodynamic parameters, which may include clinical effects such asPCT Patent Application Docket No: CACRE1.0026WOtoxic effects and / or laboratory values. Thus, 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.

[0172] For administration to human patients, the total monthly dose of the isolated GCGR antagonistic antigen binding molecule of the disclosure can be in the range of 0.5-1200 mg per patient, 0.5-1100 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, 1-1100 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. For example, an intravenous monthly dose can require about 1-1000 mg / patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered at an intravenous monthly dose of about 1-500 mg per patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered at an intravenous monthly dose of about 1-400 mg per patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered at an intravenous monthly dose of about 1-300 mg per patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered at an intravenous monthly dose of about 1-200 mg per patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered, at an intravenous monthly dose of about 1-150 mg per patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule of the disclosure can be administered or at an intravenous monthly dose of about 1-100 mg / patient. In various embodiments, the isolated GCGR antagonistic antigen binding molecule 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.

[0173] An exemplary, non-limiting weekly, or bi-weekly dosing range for a therapeutically or prophylactically effective amount of an isolated GCGR antagonistic antigen binding molecule 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,PCT Patent Application Docket No: CACRE1.0026WO0.001 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, 0.010 to 1 mg / kg, 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.1 to 4 mg / kg, 0.1 to 3 mg / kg, 0.1 to 2 mg / kg, 0.1 to 1 mg / kg, 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30 mg / kg, 1 to 25 mg / kg, 1 to 20 mg / kg, 1 to 15 mg / kg, 1 to 10 mg / kg, 1 to 7.5 mg / kg, 1 to 5 mg / kg, 1 to 4 mg / kg, 1 to 3 mg / kg, 1 to 2 mg / kg, or 1 mg / kg body weight. It is to be noted that 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.

[0174] In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is an FDA-approved dose for the treatment of T2D. In various embodiments, the therapeutically effective amount of an isolated GLP-1 agonist described herein for use in the methods described herein will typically be from about 0.01 pg to about 5 mg; about 0.1 pg to about 2.5 mg; about 1 pg to about 1 mg; about 1 pg to about 50 pg; or about 1 pg to about 25 pg. Alternatively, the therapeutically effective amount of the GLP-1 receptor agonist compounds may be from about 0.001 pg to about 100 pg based on the weight of a 70 kg patient; or from about 0.01 pg to about 50 pg based on the weight of a 70 kg patient. These therapeutically effective doses may be administered once / day, twice / day, thrice / day, once / week, biweekly, or once / month, depending on the formulation. In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is a dose of up to 1.0 mg weekly. In various embodiments, the therapeutically effective low dose of GLP-1 R agonist is selected from the group consisting of 0.25 mg, 0.50 mg, 0.75 mg, 1.0 mg, and 2.0 mg.

[0175] In various embodiments, an exemplary summary of dosing frequency for some commonly prescribed GLP-1 agonists can be Dulaglutide - once weekly; Albiglutide - once weekly; Liraglutide - once daily; Semaglutide - one weekly subcutaneously, daily orally;Exenatide BID - twice daily; Exenatide QW - once weekly; Lixisenatide - once daily;Tirzepatide - once weekly.

[0176] 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 LD50(the dose lethal to 50% of the population)PCT Patent Application Docket No: CACRE1.0026WOand the ED50(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 LD50 / ED50. Compositions that exhibit large therapeutic indices are generally preferred.

[0177] In various embodiments, single or multiple administrations of the pharmaceutical compositions are administered depending on the dosage and frequency as required and tolerated by the subject. In any event, the composition should provide a sufficient quantity of at least one of the isolated antagonistic antigen binding molecule 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.

[0178] The dosing frequency of the administration of the isolated GCGR antagonistic antigen binding molecule 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.

[0179] As used herein, the terms "co-administration", "co-administered" and "in combination with", referring to the isolated GCGR antagonistic antigen binding molecule 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 molecule 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 molecule 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 molecule 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 with a significant time interval between each administration, whereupon said components are released at substantially different times to said subject; and sequential administration of such combinationPCT Patent Application Docket No: CACRE1.0026WOof isolated antagonistic antigen binding molecule 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 in a controlled manner whereupon they are concurrently, consecutively, and / or overlappingly released at the same and / or different times to said subject, where each part may be administered by either the same or a different route.

[0180] In various embodiments, the combination therapy has a synergistic effect for the treatment of obesity.

[0181] In various embodiments, the combination therapy comprises administering the isolated GCGR antagonistic antigen binding molecule composition and the second agent composition simultaneously, either in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, isolated GCGR antagonistic antigen binding molecule composition and the second agent composition are administered sequentially, i.e., the isolated GCGR antagonistic antigen binding molecule composition is administered either prior to or after the administration of the second agent composition.

[0182] In various embodiments, the administrations of the isolated GCGR antagonistic antigen binding molecule composition and the second agent composition are concurrent, i.e., the administration period of the isolated GCGR antagonistic antigen binding molecule composition and the second agent composition overlap with each other.

[0183] In various embodiments, the administrations of the isolated GCGR antagonistic antigen binding molecule composition and the second agent composition are non-concurrent. For example, in various embodiments, the administration of the isolated GCGR antagonistic antigen binding molecule composition is terminated before the second agent composition is administered. In various embodiments, the administration second agent composition is terminated before the isolated antagonistic antigen binding molecule composition is administered.

[0184] The invention having been described, the following examples are offered by way of illustration, and not limitation.Example 1

[0185] In this example, C57BL / 6 mice, which have been fed on high fat diet for 14 weeks already (DIO mice, average body weight 42g; n=8 / group) were treated twice / week subcutaneously for 4 weeks with Vehicle, Dulaglutide (“Dula”) at 0.6 mg / kg (“mpk”), REMD-514 (bifunctional antibody containing glucagon receptor antagonist mAb and GLP-1 agonist peptide)PCT Patent Application Docket No: CACRE1.0026WOat 2.47 mpk, REMD-2.59c (chimeric glucagon receptor antagonist mAb; also referred to as “GCGR mAb”) at 2.34 mpk, Dulaglutide at 0.6 mpk plus GCGR mAb at 2.34 mpk, GCGR mAb at 5 mpk, or Dulaglutide at 0.6 mpk plus GCGR mAb at 5 mpk. After 4 weeks of treatment, a 2-week period of no treatment followed to assess any rebound effects for selected treatment groups. Body weight and food weight were measured twice / week on dosing days. Fed and fasting (6h) blood glucose was measured once / week. Oral glucose tolerance test (OGTT) was performed on the final day of dosing after 16h fast. Body composition was assessed on days 27 and 41 using the UltraFocus DXA Specimen Radiography System. Blood and tissues were collected at the end of the study for further evaluation.ResultsBody Weight Change

[0186] Treatment with Vehicle resulted in a 7% body weight gain by day 25, vs. day 1. Treatment with the test articles resulted in body weight loss of 6.4-19.5%, vs day 1 (Table 5, FIG. 2). Unexpectedly, the combination of Dula + GCGR mAb resulted in additional body weight loss above single agent treatment. Also unexpectedly, REMD-514 treatment resulted in less body weight loss than the combination of Dula + GCGR mAb and the single agent Dula alone.Table 5Day 25 Vehicle REMD- Dula GCGR mAb Dula + GCGR Dula +514 2.34 mg / kgAlone GCGR mAb GCGR (mpk) mAb 5 mg / kg mAb 2.34 mg / kg (mpk) 5 mg / kg (mpk) (mpk) Body WeightChange Rate 7.0 ± 4.0 -8.8 ± 3.3 -12.6 ± 2.7 -7.7 ± 2.8 -16.5 ± 7.5 -6.4 ± 4.1 -19.5 ± 4.5 % (vs. day 1)MeaniSD

[0187] On day 39, 10 days following the last treatment dose, Vehicle resulted in a 6.8% body weight gain vs. day 1. Treatment with the test articles resulted in body weight loss of 2.7-14.2% vs. day 1 (Table 6, FIG. 2). Importantly and unexpectedly, the combination of Dula + GCGR mAb resulted in better maintained body weight loss above Dula single agent treatment.Table 6PCT Patent Application Docket No: CACRE1.0026WODay 39 Vehicle Dula GCGR mAb Dula +Alone 5 mg / kg GCGR mAb(mpk) 5 mg / kg(mpk)Body WeightChange Rate 6.8 ± 5.2 -5.0 ± 5.9 -2.7 ± 3.4 -14.2 ± 3.3% (vs. day 1)Mean+SDFood Consumption

[0188] Treatment with the test articles resulted in lower food consumption vs. Vehicle treated animals. The combination of Dula plus GCGR mAb resulted in even lower consumption vs. single agent alone (FIG. 3).Fed Glucose

[0189] Fed glucose was measured weekly throughout the study. On day 28 after 4 weeks of treatment, animals treated with test articles had lower fed glucose than Vehicle treated animals (Table 7, FIG. 4). GCGR mAb treatment at either dose resulted in lower fed glucose than Dula treatment, and the combination of Dula + GCGR mAb resulted in even lower fed glucose. REMD-514 treatment resulted in similar fed glucose to Dula alone. On day 42, 2 weeks after treatment stopped, animals who had previously been treated with GCGR mAb or the combination of Dula + GCGR mAb still had lower fed glucose than Vehicle, while animals previously treated with Dula alone had higher fed glucose than Vehicle (Table 8, FIG. 4).Table 7Day 28 Vehicle REMD-514 Dula Alone GCGR Dula + GCGR Dula + mAb GCGR mAb GCGR 2.34 mg / kg mAb 5 mg / kg mAb 2.34 mg / kg 5 mg / kg FedGlucose 10.1 ± 1.0 7.1 ± 0.7 7.5 ± 0.8 6.7 ± 1.3 6.6 ±0.6 6.5 ± 0.3 6.2 ± 0.4 (mM)Mean±SDTable 8PCT Patent Application Docket No: CACRE1.0026WODay 42 Vehicle Dula Alone GCGR Dula +mAb GCGR5 mg / kg mAb5 mg / kgFedGlucose 7.9 ± 0.5 8.4 ± 0.6 6.8 ± 0.3 6.6 ± 0.4(mM)Mean±SDFasting Glucose

[0190] Fasting glucose was measured weekly throughout the study. On day 28 after 4 weeks of treatment, animals treated with test articles had lower fasting glucose than Vehicle treated animals (Table 9, FIG. 5). GCGR mAb treatment at either dose with or without Dula cotreatment resulted in lower fasting glucose than Dula treatment alone. Fasting glucose was slightly lower in animals treated with REMD-514 vs. Dula alone. On day 42, 2 weeks after treatment stopped, animals who had previously been treated with GCGR mAb or the combination of Dula + GCGR mAb still had lower fasting glucose vs. Vehicle (Table 10, FIG. 5). The fasting glucose levels of animals previously treated with Dula were higher than Vehicle, suggesting a robust rebound effect on fasting glucose.Table 9Day 28 Vehicle REMD-514 Dula Alone GCGR Dula + GCGR Dula + mAb GCGR mAb GCGR 2.34 mg / kg mAb 5 mg / kg mAb 2.34 mq / kq 5 mq / kq FastingGlucose 8.8 ± 0.6 6.5 ± 0.3 6.9 ± 0.2 5.7 ± 0.4 5.6 ±0.2 6.1 ± 0.5 6.0 ± 0.6 (mM)Mean+SDTable 10Day 42 Vehicle Dula Alone GCGR Dula +mAb GCGR5 mg / kg mAb5 mg / kgFastingGlucose 8.9 ± 0.2 9.3 ± 0.8 6.6 ± 0.3 6.2 ± 0.5(mM)Mean±SDOGTTPCT Patent Application Docket No: CACRE1.0026WO

[0191] OGTT was performed on days 29 and 43 after a 16h fast, and glucose levels were measured at t=0, 15min, 30min, 60min, and 120min and the AUG determined. On day 29, following 4 weeks of treatment, all test articles resulted in lower AUG than Vehicle treatment (Table 11, FIG. 6). Dula or Dula + GCGR mAb resulted in lower AUG than GCGR mAb alone and REMD-514 treatments. On day 43, after 2 weeks of no treatment, only GCGR mAb with or without Dula showed an improvement compared to Vehicle (Table 12, FIG. 7). Dula treated animals had similar AUC compared to Vehicle, suggesting a loss in glycemic control after the treatment stopped.Table 11Day 29 Vehicle REMD- Dula Alone GCGR mAb Dula + GCGR Dula + OTGG 514 2.34 mg / kg GCGR mAb GCGR mAb mAb 5 mg / kg 5 mg / kg 2.34 mg / kgAUC(mM*min) 1,532.8 ± 1,034 ± 838.5 ± 982.8 ± 85.1 879.9 ± 1,024.8 ± 828.5 ± 50.6 MeaniSD 178.7 63 43.8 63.9 68.3Table 12Day 42 Vehicle Dula Alone GCGR Dula + OTGG mAb GCGR5 mg / kg mAb5 mq / kqAUC(mM*min) 1736 ± 128 1720 ± 350 1304 ± 167 1241 ± 77Mean+SDBody Composition

[0192] Lean weight and fat weight were measured using the UltraFocus DXA Specimen Radiography System following 4 weeks of treatment (day 27) and following 2 weeks off treatment (day 41). Treatment with the test articles did not significantly affect lean weight, as expected, due to the low dose of dulag lutide and known effects of GCGR mAb. However, treatment with the test articles significantly reduced fat weight vs. Vehicle, with additional fat weight loss observed in the Dula + GCGR mAb combination groups compared to the singlePCT Patent Application Docket No: CACRE1.0026WOagent treated groups. (Table 13, FIGS. 8A-8B). Dula alone and REMD-514 treatment resulted in more fat weight loss vs. Vehicle compared to GCGR mAb treatment alone. Due to the selective loss of fat vs. lean weight, the percentage of lean mass increased and percentage of fat decreased for the treatment groups vs. Vehicle (FIGS. 9A-9B). On day 41 after 2 weeks of no treatment, lean weight was slightly higher for the animals previously treated with Dula, GCGR mAb or the combination of the two. Fat weight was significantly lower for the combination group vs. Dula alone, demonstrating a rapid re-gain of fat mass after ending Dula treatment. These data suggest that the fat loss provided by Dula + GCGR mAb is better maintained after stopping treatment (Table 14, FIGS. 10A-10B and FIGS. 11A-11 B).Table 13Day 27 Vehicle REMD- Dula GCGR Dula + GCGR Dula + Body Composition 514 Alone mAb GCGR mAb GCGR 2.34 mpk mAb 5 mpk mAb 2.34 mpk 5 mpk Soft weight (g) 45.1+5.6 38.613.9 36.513.6 39.6+4.8 34.813.8 39.513.1 33.9+2.5 Mean±SDLean weight (g) 29.312.2 30.012.4 28.010.8 29.812.2 28.512.4 28.812.6 28.011.9 MeaniSDFat weight (g) 15.815.0 8.612.1 8.513.8 9.913.2 6.312.5 10.712.0 5.811.8 MeaniSDLean Percent (%) 65.5+7.1 77.813.5 77.317.6 75.6+5.2 82.415.4 73.114.2 83.0+4.4 MeaniSDFat Percent (%) 34.517.1 22.213.5 22.717.6 24.415.2 17.615.4 26.914.2 17.014.4MeaniSDTable 14Day 41 Vehicle Dula GCGR mAb Dula + GCGR mAb Body Composition Alone 5 mpk 5 mpkSoft weight (g) 44.918.6 43.217.2 41.513.7 35.913.0MeaniSDLean weight (g) 26.814.8 28.9+2.1 30.911.9 28.911.5MeaniSDFat weight (g) 18.117.2 14.216.0 10.612.1 7.112.2MeaniSDLean Percent (%) 60.5111.3 67.918.4 74.613.4 80.614.4MeaniSDFat Percent (%) 39.5111.3 32.118.4 25.4+3.4 19.414.4MeaniSDExample 2PCT Patent Application Docket No: CACRE1.0026WO

[0193] In this example, C57BL / 6 mice, which have been fed on high fat diet for 19 weeks already (DIO mice, average weight 44g; n=10 / group) were treated twice / week (GCGR mAb) or daily (Semaglutide; also referred to as “Serna”) subcutaneously for 4 weeks with Vehicle, Serna at 6 nmol / kg, Serna at 12 nmol / kg, GCGR mAb at 5 mpk, Serna at 6 nmol / kg + GCGR mAb at 2.34 mpk, or Serna at 6 nmol / kg + GCGR mAb at 5 mpk. After 4 weeks of treatment, a 2-week period of no treatment followed to assess any rebound effects for selected treatment groups. Body weight and food weight were measured twice / week on dosing days. Fed and fasting (6h) blood glucose were measured once / week. Oral glucose tolerance test (OGTT) was performed on the final day of dosing and two weeks after stopping treatment after 16h fast. Body composition was assessed using the UltraFocus DXA Specimen Radiography System on days 13, 28, and 41. Blood and tissues were collected at the end of the study for further evaluation.ResultsBody Weight Change

[0194] Treatment with Vehicle for 4 weeks resulted in a 3.9% body weight gain on day 29, vs. day 1. Treatment with the test articles resulted in body weight loss of 10.6-24%, vs day 1 (Table 15, FIG. 12). The combination of Serna + GCGR mAb resulted in additional body weight loss above single agent treatment. There was no dose response observed for the Serna alone groups as animals treated with the two different doses achieved comparable weight loss, likely due to the two doses being similar.Table 15Day 29 Vehicle Serna Sema GCGR mAb Sema + Sema +6 nmol / kg 12 nmol / kg 5 mpk GCGR mAb GCGR mAb 2.34 mpk 5 mpk Body Weight 3.9+3.9 -17.9+4.6 -17.3+3.8 -10.6+3.6 -21.0+5.4 -24.0+2.8 Change Rate% (vs. day 1)Mean±SDPCT Patent Application Docket No: CACRE1.0026WO

[0195] On day 41, 11 days following the last treatment dose, Vehicle resulted in a 5.1% body weight gain vs. day 1. Treatment with GCGR mAb and the combination of GCGR mAb + Serna resulted in body weight loss of 8.5 and 13.5% vs. day 1, respectively (Table 16, FIG. 12). Animals treated with Serna alone had gained 1.4% body weight vs. day 1, suggesting that the combination of Serna + GCGR mAb resulted in better maintained body weight loss compared to Serna alone and stopping treatment with Serna alone results in a rapid re-gain of weight (i.e. rebound effect).Table 16Day 41 Vehicle Sema GCGR mAb Sema + GCGR mAb12 nmol / kg 5 mpk 5 mpkBody Weight 5.1+4.2 1.4+7.5 -8.5+4.8 -13.5+2.9Change Rate% (vs. day 1)Mean+SDFood Consumption

[0196] Treatment with the test articles resulted in lower food consumption vs. Vehicle treated animals during the 4-week treatment period (FIG. 13). Stopping treatment resulted in increased food consumption for all treatment groups.Fed Glucose

[0197] Fed glucose was measured weekly throughout the study. On day 29 after 4 weeks of treatment, animals treated with test articles had lower fed glucose than Vehicle treated animals (Table 17, FIG. 14). GCGR mAb treatment at either dose with or without Serna resulted in lower fed glucose than Serna treatment alone. On day 42, 2 weeks after treatment stopped, animals who had previously been treated with GCGR mAb or the combination of Serna and GCGR mAb still had lower fed glucose than Vehicle, and also lower than animals previously treated with Serna alone (Table 18, FIG. 14).Table 17Day 29 Vehicle Sema Sema GCGR mAb Sema + Sema +6 nmol / kg 12 nmol / kg 5 mpk GCGR mAb GCGR mAbPCT Patent Application Docket No: CACRE1.0026WO2.34 mpk 5 mpkFed Glucose 8.9+0.6 7.7+0.4 7.0+0.6 5.9+0.4 6.3+0.4 6.1+0.5(mM)MeaniSDTable 18Day 42 Vehicle Serna GCGR mAb Serna + GCGR mAb12 nmol / kg 5 mpk 5 mpkFed Glucose 8.6+0.3 8.0+0.5 6.5+0.3 6.7+0.6(mM)Mean±SDFasting Glucose

[0198] Fasting glucose was measured weekly throughout the study. On day 29 after 4 weeks of treatment, animals treated with test articles had lower fasting glucose than Vehicle treated animals (Table 19, FIG. 15). Single agent treatment with Serna or GCGR mAb resulted in similar fasting glucose levels on day 29. Combination treatment resulted in slightly lower fasting glucose levels than single agent treatment on day 29. On day 42, 2 weeks after treatment stopped, animals who had previously been treated with GCGR mAb or the combination of Serna and GCGR mAb still had significantly lower fasting glucose levels than Vehicle (Table 20, FIG. 15). The fasting glucose levels of animals previously treated with Serna were higher than Vehicle, suggesting a robust rebound effect on fasting glucose.Table 19Day 29 Vehicle Serna Serna GCGR Sema + Sema +6 nmol / kg 12 nmol / kg mAb 5 mpk GCGR mAb GCGR mAb 2.34 mpk 5 mpk Fasting Glucose 9.2+0.8 6.3+0.4 6.2+0.4 6.2+0.3 6.0+0.3 6.0+0.5(mM)Mean±SDTable 20Day 42 Vehicle Sema GCGR mAb Sema + GCGR mAb12 nmol / kg 5 mpk 5 mpkFasting Glucose 8.5±0.4 9.4±0.6 6.4±0.3 6.6±0.2(mM)Mean+SDPCT Patent Application Docket No: CACRE1.0026WOOGTT

[0199] OGTT was performed on days 30 and 43 after a 16h fast, and glucose levels were measured at t=0, 15min, 30min, 60min, and 120min and AUC determined. On day 30, following 4 weeks of treatment, all test articles resulted in lower AUC than Vehicle treatment (Table 21, FIG. 16). Sema or Sema + GCGR mAb resulted in lower AUC than GCGR mAb alone. On day 43, after 2 weeks of no treatment, GCGR mAb with or without Sema showed an improvement compared to Vehicle (Table 22, FIG. 17). Sema treated animals had an equivalent AUC compared to Vehicle. These results suggest that the beneficial effects of Sema on glycemic control are not maintained as well as with GCGR mAb or the combination of GCGR mAb + Sema after stopping treatment.Table 21Day 30 Vehicle Sema Sema GCGR mAb Sema + Sema + OGTT 6 nmol / kg 12 nmol / kg 5 mpk GCGR mAb GCGR mAb 2.34 mpk 5 mpk AUC 1192.6±127.5 826.2±84.5 810.2±143.3 895.6±92.8 792.8±56.6 814.1±94.7 (mM*min)Mean±SDTable 22Day 43 Vehicle Sema GCGR mAb Sema + GCGR mAb OGTT Alone 5 mg / kg 5 mg / kgAUC 1362±79 1385±68 1137±97 1109±90(mM*min)Mean±SDBody Composition

[0200] Lean weight and fat weight were measured using the UltraFocus DXA Specimen Radiography System following 2 weeks treatment (day 13), 4 weeks treatment (day 28) and following 2 weeks after treatment ended (day 41). Treatment with the test articles for 2 weeks did not significantly affect lean weight, as expected due to the low dose of Semaglutide used and known effects of GCGR mAb. However, treatment with the test articles significantly reducedPCT Patent Application Docket No: CACRE1.0026WOfat weight, with additional fat weight loss observed in the Sema + GCGR mAb combination groups compared to the single agent treated groups (Table 23, FIGS. 18A-18B and FIGS. 19A-19B). After 4 weeks of treatment a similar trend was observed, with single agent treated animals showing reduced fat weight and maintained lean weight and combination groups having additional fat loss (Table 24, FIGS. 20A-20B and FIGS. 21A-21B). On day 41, 2 weeks after treatment stopped, lean weight was comparable for all treatment groups (Table 25, FIGS. 22A-22B and FIGS. 23A-23B). Fat weight was much lower for the combination group vs. Sema alone, suggesting that the fat loss provided by Sema + GCGR mAb is better maintained after stopping treatment.Table 23Day 13 Vehicle Sema Sema GCGR mAb Sema + Sema + Body 6 nmol / kg 12 nmol / kg 5 mpk GCGR mAb GCGR mAb Composition 2.34 mpk 5 mpk Soft weight (g) 44.8±2.6 38.6±3.5 38.0±2.0 41.7±2.3 36.7±1.9 36.4±1.8 Mean±SDLean weight (g) 28.3±1.8 27.7±1.8 27.1±1.4 28.5±1.5 27.8±1.2 27.3±1.6 Mean±SDFat weight (g) 16.5±2.3 10.9±2.2 10.9±1.9 13.2±1.6 8.9±1.7 9.1±1.0 Mean±SDLean Percent (%) 63.4±4.0 72.1±3.6 71.4±4.0 68.3±2.7 75.9±3.6 75.1±2.5 Mean±SDFat Percent (%) 36.6±4.0 27.9±3.6 28.6±4.0 31.7±2.7 24.1±3.6 24.9±2.5Mean±SDTable 24Day 28 Vehicle Sema Sema GCGR Sema + Sema + Body 6 nmol / kg 12 nmol / kg mAb 5 mpk GCGR mAb GCGR mAb Composition 2.34 mpk 5 mpkSoft weight (g) 45.2±2.5 36.7±3.8 36.6±1.8 39.6±2.0 35.6±2.2 34.9±1.7 Mean±SDLean weight (g) 26.8±1.8 26.8±1.5 26.5±1.5 27.1±1.8 27.8±1.7 27.2±1.6 Mean±SDFat weight (g) 18.5±2.5 9.9±3.5 10.2±2.2 12.4±2.7 7.7±1.3 7.6±1.2 Mean±SDLean Percent (%) 59.3±4.2 73.5±6.9 72.4±4.9 68.8±5.6 78.4±3.1 78.1±3.1 Mean±SDFat Percent (%) 40.7±4.2 26.5±6.9 27.6±4.9 31.2±5.6 21.6±3.1 21.9±3.1Mean±SDTable 25PCT Patent Application Docket No: CACRE1.0026WODay 41 Vehicle Sema GCGR mAb Sema + GCGR mAb Body 12 nmol / kg 5 mpk 5 mpkCompositionSoft weight (g) 46.3±3.6 44.9±2.3 40.5±2.6 38.6±1.9 Mean±SDLean weight (g) 29.3±1.5 29.6±1.4 29.2±1.7 29.8±1.7 Mean±SDFat weight (g) 17.0±3.5 15.2±2.2 11.2±1.6 8.8±1.0MeaniSDLean Percent (%) 63.6±5.1 66.1±3.9 72.3±2.8 77.1±2.2 Mean±SDFat Percent (%) 36.4±5.1 33.9±3.9 27.7±2.8 22.9±2.2MeaniSD

[0201] The collective data from the Examples above demonstrates the surprising and unexpected finding that GCGR mAb + low dose GLP-1 prevents muscle loss yet still achieves robust weight loss in the Diet Induced Obesity mouse model. Moreover, GCGR mAb combined with low dose Dulaglutide or Semaglutide resulted in robust body weight loss while preserving muscle mass and enhanced glycemic control with less rebound in fat mass and hyperglycemia after the treatment stopped. The finding that a GCGR antagonist can also have beneficial effects on obesity when combined with a GLP-1 agonist, similar to GCGR agonist, is new and unexpected. The collective data in Example 1 also demonstrates the surprising and unexpected finding that the combination of GCGR antagonist with GLP-1 agonist is superior to a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein. One skilled in the art would expect a bifunctional fusion protein acting on both the GCGR and GLP-1 R to have more potent activity in an obese subject. Surprisingly in our rodent obesity study the bifunctional fusion protein had much weaker effect on body weight loss and glucose lowering than the combination of GCGR antagonist mAb and GLP-1 agonist.Example 3

[0202] In this example, we propose a Phase 2 clinical study in human subjects to test the combination of low dose Semaglutide (dose approved for T2D) plus Volagidemab (GCGR antagonist mAb comprising SEQ ID NOs: 51 and 52) at 2 different doses, versus Semaglutide alone in obese or overweight T2D patients (see Table 26 below). An accelerated dose escalation scheme is proposed to shorten the time taken to reach the target GLP-1 dose of 1 mg weekly. Subjects will initiate SC weekly dosing on day 0 with Semaglutide with or without 35 or 70 mg Volagidemab and after the dose escalation reaches the target dose of 1 mg the subjects will be treated for 12 weeks. Men and women between 18 and 80 years old with bodyPCT Patent Application Docket No: CACRE1.0026WOmass index (BMI) > 30 or BMI > 27 with one or more obesity-associated comorbidities (e.g., hypertension, insulin resistance, sleep apnea, or dyslipidemia) will be eligible. The primary outcome measure will be change from baseline in body weight after 12 weeks of treatment at the target dose of Semaglutide. Secondary outcome measures will include change from baseline in waist circumference, change from baseline in total body fat mass, change from baseline in percent body fat, proportion of participants with change in waist circumference ≥5 cm, proportion of participants with change in body weight ≥5%, ≥10%, and ≥15%, percentage of weight loss due to fat mass or lean mass by dual-energy x-ray absorptiometry (DXA), change from baseline in fat mass by bioelectrical impedance analysis (BIA), change from baseline in lean mass and appendicular lean mass by DXA, change from baseline in lean mass by BIA, safety and tolerability measurements throughout the study by treatment-emergent adverse events (TEAEs), proportion of participants with change from baseline in BMI categories, change from baseline in HbA1c, change from baseline in Quality of Life Short Form 36 survey (SF-36), and change from baseline in Impact of Weight on Quality of Life-Lite for Clinical Trials (IWQOL-Lite).Table 26

[0203] Based on the data and observations from Example 1 and 2, the present inventors believe this unique approach will achieve robust weight loss without muscle loss in the firstPCT Patent Application Docket No: CACRE1.0026WOplace, show limited gastrointestinal side effects, and attenuate the rebound effects associated with higher dose incretin-based therapy.

[0204] All of the articles and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the articles and methods without departing from the spirit and scope of the disclosure. All such variations and equivalents apparent to those skilled in the art, whether now existing or later developed, are deemed to be within the spirit and scope of the disclosure as defined by the appended claims. All patents, patent applications, and publications mentioned in the specification are indicative of the levels of those of ordinary skill in the art to which the disclosure pertains. All patents, patent applications, and publications are herein incorporated by reference in their entirety for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for any and all purposes. The disclosure illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising”, “consisting essentially of”, and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. Thus, it should be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure as defined by the appended claims.Sequence Listings

[0205] The amino acid sequences listed in the accompanying sequence listing are shown using standard three letter code for amino acids, as defined in 37 C. F. R. 1.822. This disclosure includes a Sequence Listing in computer readable form (ST25 format text file) prepared through the use of software program Patentin and is identical to the accompanyingPCT Patent Application Docket No: CACRE1.0026WOsequence listings.

[0206] SEQ ID NO: 1 is the amino acid sequence of a human glucagon receptor (GCGR) molecule (Accession Number AAI04855).

[0207] 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.

[0208] 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.

[0209] 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.

[0210] 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.

[0211] SEQ ID NOS: 10-28 are amino acid sequences encoding the heavy chain variable regions of various fully human anti-GCGR antibodies.

[0212] SEQ ID NOS: 29-47 are amino acid sequences encoding the light chain variable regions of various fully human anti-GCGR antibodies.

[0213] 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.

[0214] SEQ ID NO: 50 is the amino sequence encoding the IgG2 heavy chain constant region.

[0215] 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.

[0216] SEQ ID NOs: 53-58 are the amino acid sequences of GLP-1 agonists.

[0217] SEQ ID NOs: 59 and 60 are amino acid sequence of a heavy chain of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein.

[0218] SEQ ID NO: 61 is the amino acid sequence of a light chain of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein.

[0219] SEQ ID NOs: 62-67 are the amino acid sequences of peptide linkers and a peptide leader sequence.PCT Patent Application Docket No: CACRE1.0026WOSEQUENCE LISTINGSSEQ ID NO: 1 - Amino acid sequence of a human glucagon receptor (GCGR) molecule MPPCQPQRPLLLLLLLLACQPQVPSAQVMDFLFEKWKLYGDQCHHNLSLLPPPTELVCNRTFD KYSCWPDTPANTTANISCPWYLPWHHKVQHRFVFKRCGPDGQWVRGPRGQPWRDASQCQ MDGEEIEVQKEVAKMYSSFQVMYTVGYSLSLGALLLALAILGGLSKLHCTRNAIHANLFASFVLK ASSVLVIDGLLRTRYSQKIGDDLSVSTWLSDGAVAGCRVAAVFMQYGIVANYCWLLVEGLYLH NLLGLATLPERSFFSLYLGIGWGAPMLFVVPWAVVKCLFENVQCWTSNDNMGFWWILRFPVFL AILINFFIFVRIVQLLVAKLRARQMHHTDYKFRLAKSTLTLIPLLGVHEVVFAFVTDEHAQGTLRSA KLFFDLFLSSFQGLLVAVLYCFLNKEVQSELRRRWHRWRLGKVLWEERNTSNHRASSSPGHG PPSKELQFGRGGGSQDSSAETPLAGGLPRLAESPFSEQ ID NO: 2 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYD GSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNY YYGLDVWGQGTTVTVSS SEQ ID NO: 3 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGV PSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIK SEQ ID NO: 4 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV AVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREKDHYDI LTGYNYYYGLDVWGQGTTVTVSS SEQ ID NO: 5 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGV PSRFSGSGSGTEFTLTISSLQPEDFVTYYCLQHNSNPLTFGGGTKVEIK SEQ ID NO: 6 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYD GSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNY YYGLDVWGQGTTVTVSS SEQ ID NO: 7 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGV PSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIK SEQ ID NO: 8 - Amino acid sequence of a heavy chain of a chimeric antibody that binds GCGR MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPG KGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHY DILTGYNYYYGLDVWGQGTTVTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTPCT Patent Application Docket No: CACRE1.0026WOVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD CGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQT QPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPP PKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKS NWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 9 - Amino acid sequence of a light chain of a chimeric antibody that binds GCGR MDMRVPAQLLGLLLLWFPGARCDIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP GKAPKRLIYAASSLESGVPSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKV EIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDS KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 10 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVILSDGRNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SS SEQ ID NO: 11 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVILNDGRNKYYA DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDDYEILTGYGYYGMDVWGQGTTVTV SS 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 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYDIHWVRQAPGKGLEWVAVLSSDGNNKYCA DSVKGRFTISRDNSKNTLYLQMNSLRTEDTAVYYCAREEVYYDILTGYYDYYGMDVWGQGTTV TVSS SEQ ID NO: 14 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGKGLEWIGYIFYSGSTNYNPSLK SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGMDVWGQGTTVTVSS SEQ ID NO: 15 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLQQSGPGLVKPSQILSLTCAISGDRVSSNGAAWNWIRQSPSRGLEWLGRTYYRSKWYYD YAGSVKSRININPDTSKNQFSLQVNSVTPEDTAVYYCARDRSSGWNEGYYYYGMDVWGQGT TVTVSS SEQ ID NO: 16 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLQESGPGLVKPSETLSLTCTVSGGSISTYFWTWIRQFPGEGLEWIGYIFYSGNTNYNPSLT SRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGYYDILTGEDYSYGIDVWGQGTTVTVSS SEQ ID NO: 17 - Amino acid sequence of a HCVR of a human antibody that binds GCGRPCT Patent Application Docket No: CACRE1.0026WOQVQLVESGGGVVQPGRSLRLSCAASGFIFSSYGMHWVRQAPGKGLEWVAVISNDGSNKYYA DFVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREDYDILTGNGVYGMDVWGQGTTVTV SS SEQ ID NO: 18 - Amino acid sequence of a HCVR of a human antibody that binds GCGR EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTMNWVRQAPGKGLEWVSYISGSSSLIYYAD SVKGRFTISRDNAKNSLYLHMNSLRDEDTAVYYCARARYNWNDYYGMDVWGQGTTVTVSS SEQ ID NO: 19 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFAFSSYGIHWVRQAPGKGLEWVAGIWYDGSNKYYA DSVKGRFTVSRDNSKNTLYLQMNSLRAEDTAVYYCARLFDAFDIWGQGTMVTVSS SEQ ID NO: 20 - Amino acid sequence of a HCVR of a human antibody that binds GCGR EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYTMNWVRQAPGKGLEWVSYISSSSSLIYYADS VKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARSDYYGSGSYYKGNYYGMDVWGQGTTV TVSS SEQ ID NO: 21 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYAD SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSS SEQ ID NO: 22 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVTIIWSDGINKYYAD SVKGRFTISRDNSKNTLNLQMNSLRAEDTAVYYCARERGLYDILTGYYDYYGIDVWGQGTTVT VSS SEQ ID NO: 23 - Amino acid sequence of a HCVR of a human antibody that binds GCGR EVQLVESGGGLVKPGGSLRLSCAASGITFRSYSMNWVRQAPGKGLEWVSAISSSSSYIYYADS VKGRFTISRDNAKNSVYLQMNSLRAEDTAVYYCARGRYGMDVWGQGTTVTVSS SEQ ID NO: 24 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGSTFRSYDMHWVRQAPGKGLEWVAVISYDGSNKYYG DSVKGRLTISRDNSKNTLYLQMNSLRAEDTAVYYCARDQYDILTGYSSDAFDIWGQGTMVTV SS SEQ ID NO: 25 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWVAVIWYDGSHKYY EDSVKGRFTISRDNSKNTLYLQMNSLRADDTGVYYCARVGYGSGWYEYYYHYGMDVWGQGT TVTVSS SEQ ID NO: 26 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDY VDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSPCT Patent Application Docket No: CACRE1.0026WOSEQ ID NO: 27 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDY VDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTT VTVSSSEQ ID NO: 28 - Amino acid sequence of a HCVR of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGITFSSYGMHWVRQAPGKGLEWVASIWYDGSNKYYV DSVKGRFTIFRDNSKKTLYLQMNRLRAEDTAVYYCARLGGGFDYWGQGTLVTVSS SEQ ID NO: 29 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQKKPGKAPKSLIYVVSSLQSGVPSRFSG SGSGTDFTLTINNLQPEDFATYYCQQYNHYPLTFGGGTRVEIKR SEQ ID NO: 30 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQDISNYLAWFQQRPGKAPKSLIYVVSSLQSGVPSRFSG SGSGTDFTLTISNLQPEDFATYFCQQYNHYPLTFGGGTKVEIKR SEQ ID NO: 31 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGS GAGTDFTFTISSLQPEDIATYFCQQYDDLPLTFGGGTRVDIKR SEQ ID NO: 32 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFS GSGSGTEFTLTISSLQPEDFATYYCLQHNSNPLTFGGGTKVEIKR SEQ ID NO: 33 - Amino acid sequence of a LCVR of a human antibody that binds GCGR QNVLTQSPGTLSLSPGERVTLSCRASQSVSSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRF SGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKR SEQ ID NO: 34 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQFPSSLSASIGDRVTITCQASQDISNFLNWFQQKPGKAPKLLIYDASDLETGVPSRFSGS GAGTDFTFTISSLQPEDVATYFCQQYDNLPLTFGGGTKVDIKR SEQ ID NO: 35 - Amino acid sequence of a LCVR of a human antibody that binds GCGR ENVLTQSPGTLSLSPGERATLSCRASQSVTSSYLAWYQQKPGQAPRLLIFGVSSRATGIPDRF SGSGSGTDFSLTISRLEPEDFAVYYCQQYGNSPFTFGPGTKVDIKR SEQ ID NO: 36 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIDMYLAWFQQKPGKAPKSLIYAASSLQSGVPSKFS GSGFGTDFTLTISSLQPEDFATYYCQQYNIFPFTFGPGTKVDVKR SEQ ID NO: 37 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFS GSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKRPCT Patent Application Docket No: CACRE1.0026WOSEQ ID NO: 38 - Amino acid sequence of a LCVR of a human antibody that binds GCGR KIVMTQTPLALPVIPGEPASISCRSSQSLVDSDDGDTYLDWYLQKPGQSPQVLIHRLSYRASGV PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMHRIEFPFTFGGGTKVEIKR SEQ ID NO: 39 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQRPGKAPKRLIYAASSLQTGVPSRFS GSGSGTEFTLTISSLQPEDFATYYCLQHNSYPWTFGQGTKVEIKR SEQ ID NO: 40 - Amino acid sequence of a LCVR of a human antibody that binds GCGR GIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMCSFGQGTKLEIKR SEQ ID NO: 41 - Amino acid sequence of a LCVR of a human antibody that binds GCGR GIVLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVP DRFSGSGSGTDFTLKISRVEAEDVGVYYCMEALQTMSSFGQGTKLEIKR SEQ ID NO: 42 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIVMTQTPLFLPVTPGEPASISCRSSQTLLDSDDGNTYLDWYLQKPGQSPQRLIYTLSYRASGV PDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQHIEFPSTFGQGTRLEIKR SEQ ID NO: 43 - Amino acid sequence of a LCVR of a human antibody that binds GCGR SYELTQPPSVSVSPGQTASITCSGDKLGDKYASWYQQKPGQSPVLVIYQSTKRPSGIPERFSG SNSGNTATLTISGTQAMDEADYYCQAWDSSTVVFGGGTKLTVLG SEQ ID NO: 44 - Amino acid sequence of a LCVR of a human antibody that binds GCGR NIVMTQTPLSLSVTPGQPASISCKSSQSLLHSDGKNYLFWYLQKPGQSPQLLIYEVSYRFSGVP DRFSGSGSGTDFSLKISRVEAEDVGVYYCMQNIQPPLTFGQGTRLEIKR SEQ ID NO: 45 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFS GSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKR SEQ ID NO: 46 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLESGVPSRFS GSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKR SEQ ID NO: 47 - Amino acid sequence of a LCVR of a human antibody that binds GCGR DIVLTQTPLSLPVTPGEPASISCRSSQSLLDRDDGDTYLDWYLQKPGQSPQLLIYTLSYRASGV PDRFSGSGSGTDFSLKISRVEAEDVGVYYCMQRIEFPFTFGPGTKVDIKR SEQ ID NO: 48 - Amino acid sequence of the constant light chain kappa region RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKD STYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 49 - Amino acid sequence of the constant light chain lambda regionPCT Patent Application Docket No: CACRE1.0026WOGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN N KYAASSYLSLTP EQWKSHRSYSCQVTH EGSTVE KTVAPTECS SEQ ID NO: 50 - Amino sequence of the IgG2 heavy chain constant region ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKP KDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVH QDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPGK SEQ ID NO: 51 - Amino acid sequence of a HC of a human antibody that binds GCGR QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYD GSNKDYVDSVKGRFTISRDNSKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNY YYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVEC PPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTK PREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 52 - Amino acid sequence of a LC of a human antibody that binds GCGR DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGV PSRFSGSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRTVAAPSVFIF PPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 53 - Amino acid sequence of a GLP-1 agonist (GLP-1 (7-37)) HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG SEQ ID NO: 54 - Amino acid sequence of a GLP-1 agonist (Exenatide) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO: 55 - Amino acid sequence of a GLP-1 agonist (Liraglutide) HAEGTFTSDVSSYLEGQAAKEEFIAWLVRGRG SEQ ID NO: 56 - Amino acid sequence of a GLP-1 agonist (Liraglutide) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPSKKKKKK SEQ ID NO: 57 - Amino acid sequence of a GLP-1 agonist (Albiglutide) HGEGTFTSDVSSYLEGQAAKEFIAWLVKGRHGEGTFTS SEQ ID NO: 58 - Amino acid sequence of a GLP-1 agonist (Dulaglutide) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGG SEQ ID NO: 59 - Heavy Chain amino acid sequence of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein (HC of REMD-514)PCT Patent Application Docket No: CACRE1.0026WOHGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSAQVQLVESGGGVVQPGRSLRLSCAA SGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDNSKNTLYLQMN RLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTS ESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYT CNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO: 60 - Heavy Chain amino acid sequence of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein (HC of REMD-524) HGEGTFTSDVSSYLEEQAAKEFIAWLVKGGGGGGGSGGGGSGGGGSAQVQLVESGGGVVQ PGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVMWYDGSNKDYVDSVKGRFTISRDN SKNTLYLQMNRLRAEDTAVYYCAREKDHYDILTGYNYYYGLDVWGQGTTVTVSSASTKGPSVF PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP SSSLGTQTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNG KEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVE WESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK SEQ ID NO: 61 - Light Chain amino acid sequence of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein (LC of REMD-514) DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQQKPGKAPKRLIYAASSLQSGVPSRFS GSGSGTEFTLTISSVQPEDFVTYYCLQHNSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 62 - Amino acid sequence of a peptide linkerGGGGS SEQ ID NO: 63 - Amino acid sequence of a peptide linkerAEAAAKEAAAKAGS SEQ ID NO: 64 - Amino acid sequence of a peptide linkerGGGGGGGG SEQ ID NO: 65 - Amino acid sequence of a peptide linkerGGGGSGGGGSGGGGS SEQ ID NO: 66 - Amino acid sequence of a peptide linkerSGGGGS SEQ ID NO: 67 - Amino acid sequence of a peptide leader sequenceMGWSWILLFLLSVTAGVHS

Claims

PCT Patent Application Docket No: CACRE1.0026WOWhat is claimed is:

1. A method of treating obesity in a subject in need thereof, comprising administering to the subject: (a) a therapeutically effective amount of an isolated antagonistic antigen binding molecule that specifically binds to the human glucagon receptor (“GCGR antagonist”); and (b) a therapeutically effective low dose of a glucagon-like peptide-1 (“GLP-1 ”) receptor agonist (“GLP-1 R agonist”).

2. A method of treating a metabolic disorder selected from the group consisting of obese Type-2 Diabetes (T2D), obesity with prediabetes, insulin resistance, metabolic syndrome, fatty liver diseases, metabolic dysfunction-associated steatohepatitis (MASH), Nonalcoholic steatohepatitis (NASH) and other indications that obesity is an important contributing factor in a subject in need thereof, comprising administering to the subject: (a) a therapeutically effective amount of a GCGR antagonist; and (b) a therapeutically effective low dose of a GLP-1 R agonist.

3. The method according to any one of claims 1 to 2, wherein the GCGR antagonist and GLP-1 R agonist are formulated separately.

4. The method according to any one of claims 1 to 2, wherein the GCGR antagonist and GLP-1 R agonist are co-formulated as a single product.

5. The method according to any one of claims 1 to 4, wherein the combination therapy has a synergistic effect for the treatment of the metabolic disorder.

6. A method of treating obesity in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bifunctional fusion protein, wherein the bifunctional fusion protein is a recombinant protein comprising a GLP-1 R agonist peptide fused to an GCGR antagonist (“GCGR antagonist / GLP-1 R agonist bifunctional fusion protein”).

7. A method of treating a metabolic disorder selected from the group consisting of obese Type-2 Diabetes, obesity with prediabetes, insulin resistance, metabolic syndrome, fatty liver diseases, metabolic dysfunction-associated steatohepatitis (MASH), and NonalcoholicPCT Patent Application Docket No: CACRE1.0026WOsteatohepatitis (NASH) and other indications that obesity is an important contributing factor in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a GCGR antagonist / GLP-1 R agonist bifunctional fusion protein.

8. The method according to any one of claims 6 to 7, wherein the GLP-1 R agonist will be directly fused to the GCGR antagonist.

9. The method according to any one of claims 6 to 7, wherein the GLP-1 R agonist will be fused to the GCGR antagonist using a peptide linker.

10. The method according to claim 9, wherein the GLP-1 R agonist will be fused to the GCGR antagonist using a peptide linker at the GCGR antibody heavy chain N-terminal.

11. The method according to any one of claims 9 to 10, wherein the peptide linker is a peptide linker comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 62-67.

12. The method according to any one of claims 1 to 11, wherein the GLP-1 R agonist is selected from is selected from the group consisting of a GLP-1 agonist that is a peptide, a GLP-1 agonist that is a modified peptide, a GLP-1 agonist that is a small molecule, a GLP-1 agonist that is an antisense or siRNA molecule, a GLP-1 agonist that is an antibody, or a fragment thereof, or an artificial construct comprising an antibody or fragment thereof, or an artificial construct designed to mimic the binding of an antibody or fragment thereof to its antigen, a GLP-1 agonist that is a peptide-Ab fusion, and a GLP-1 agonist that is a peptide-Fc fusion.

13. The method according to any one of claims 1 to 12, wherein the GLP-1 R agonist is GLP-1 (7-37) having the amino acid sequence of SEQ ID NO: 53.

14. The method according to any one of claims 1 to 12, wherein the GLP-1 R agonist is a peptide comprising the amino acid sequence selected from the group consisting of SEQ ID NOS: 54-58.PCT Patent Application Docket No: CACRE1.0026WO15. The method according to any one of claims 1 to 11, wherein the GLP-1 R agonist is a GLP-1 (7-37) analog selected from the group consisting of exenatide, liraglutide, lixisenatide, albiglutide, dulag lutide, semaglutide and taspoglutide.

16. The method according to claim 15, wherein the GLP-1 R agonist is dulaglutide.

17. The method according to claim 15, wherein the GLP-1 R agonist is semaglutide.

18. The method according to any one of claims 1 to 11, wherein the GLP-1 R agonist is in the form of an incretin-based poly-agonist.

19. The method according to any one of claims 1 to 18, wherein the GCGR antagonist is selected from the group consisting of a GCGR antagonist that is a small molecule.

20. The method according to any one of claims 1 to 18, wherein the GCGR antagonist is selected from the group consisting of a fully human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, an antigenbinding antibody fragment, a Fab, a Fab', a Fab2, a Fab'2, a IgG, a IgM, a IgA, a IgE, a scFv, a dsFv, a dAb, a nanobody, a unibody, a diabody, and a hemibody.

21. The method according to claim 20, wherein the isolated GCGR antagonistic antibody or antibody fragment specifically binds to a human glucagon receptor with a dissociation constant (KD) of at least about 1 x107M, at least about 1 x108M, at least about 1x109M, at least about 1x10-10M, at least about 1x10-11M, or at least about 1x10-12M.

22. The method according to any one of claims 20 to 21, wherein the isolated GCGR antagonistic antibody is a fully human antibody.

23. The method according to claim 22, wherein the fully human GCGR antagonist antibody is selected from an 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; an antibody which comprises an 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; and an antibody whichPCT Patent Application Docket No: CACRE1.0026WOcomprises 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.

24. The method according to claim 22, wherein the fully human GCGR antagonist antibody is selected from an antibody which comprises a heavy chain variable region sequence selected from SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, 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 NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47.

25. The method according to claim 22, wherein the fully human GCGR antagonist antibody 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.

26. The method according to claim 20, wherein the isolated GCGR antagonist antibody is a chimeric antibody which comprises the amino acid sequence encoding the heavy chain of SEQ ID NO: 8 and the amino acid sequence encoding the light chain of SEQ ID NO: 9.

27. The method according to any one of claims 1-26, wherein the therapeutically effective amount of the isolated antagonistic antigen binding molecule is selected from the group consisting of 0.001 to 100 mg / kg, 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, 0.010 to 1 mg / kg, 0.1 to 50 mg / kg, 0.1 to 40 mg / kg, 0.1 to 30 mg / kg, 0.1 to 20 mg / kg, 0.1 to 10 mg / kg, 0.1 to 5 mg / kg, 0.1 to 4 mg / kg, 0.1 to 3 mg / kg, 0.1 to 2 mg / kg, 0.1 to 1 mg / kg, 0.5 to 50 mg / kg, 0.5 to 40 mg / kg, 0.5 to 30 mg / kg, 0.5 to 20 mg / kg, 0.5 to 10 mg / kg, 0.5 to 5 mg / kg, 0.5 to 4 mg / kg, 0.5 to 3 mg / kg, 0.5 to 2 mg / kg, 0.5 to 1 mg / kg, 1 to 50 mg / kg, 1 to 40 mg / kg, 1 to 30PCT Patent Application Docket No: CACRE1.0026WOmg / kg, 1 to 20 mg / kg, 1 to 10 mg / kg, 1 to 5 mg / kg, 1 to 4 mg / kg, 1 to 3 mg / kg, 1 to 2 mg / kg, and 0.1 mg / kg to 1 mg / kg body weight per week.

28. The method according to any one of claims 1-27, wherein the therapeutically effective amount of the GLP-1 R agonist is selected from about 0.01 mg to about 0.10 mg, about 0.10 mg to about 0.25 mg, about 0.25 mg to about 0.50 mg, about 0.50 mg to about 0.75 mg, about 0.75 mg to about 1.0 mg.

29. The method according to any one of claims 1-27, wherein the therapeutically effective amount of the GLP-1 R agonist is up to about 1.0 mg.

30. The method according to any one of claims 1 -27, wherein the therapeutically effective amount of the GLP-1 R agonist is an FDA-approved dose for the treatment of T2D.

31. The method according to any one of claims 1 to 30 wherein the isolated GCGR antagonistic antigen binding molecule is admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for systemic administration to a subject.

32. The method according to any one of claims 1 to 30 wherein the GLP-1 R agonist is admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for systemic administration to a subject.

33. The method according to any one of claims 1 to 30 wherein the GCGR antagonist / GLP-1 R agonist bifunctional fusion protein is admixed with a pharmaceutically acceptable carrier to form a pharmaceutical composition for systemic administration to a subject.

34. The method according to any one of claims 31 to 33 wherein the systemic administration to a subject is selected from 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.

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