Anti-GLP1R antagonist antibody and method of using the same

Abstract

The present disclosure relates to an antibody or antigen-binding fragment thereof that specifically binds to the glucagon-like peptide 1 receptor (GLP1R) protein, and methods of using the same. In various embodiments, the antibody or antigen-binding fragment thereof is a fully human antibody that binds to GLP1R. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for attenuating GLP1R activity, thereby providing a means of treating, preventing, or alleviating GLP1R-related diseases, disorders, or conditions in humans. In certain embodiments, the antibody or antigen-binding fragment thereof treats hypoglycemia, such as post-bariatric hypoglycemia (PBH), by increasing glucose levels when administered to a subject, thereby attenuating insulin secretion from pancreatic beta cells and reducing insulin expression.

Description

Technical Field

[0001] The present disclosure relates to antagonist antibodies that specifically bind to the glucagon-like peptide 1 receptor (GLP1R) and antigen-binding fragments thereof, and methods of their therapeutic use including the treatment of hypoglycemia.

Background Art

[0002] Bariatric surgery is a typical and effective treatment option for patients with severe obesity. Each year, approximately 44,000 gastric bypass surgeries and 138,000 gastrectomies are performed in the United States alone. When successful weight loss occurs after bariatric surgery, insulin sensitivity is often improved. Improvement of insulin sensitivity accompanied by an increase in the level of glucagon-like peptide 1 (GLP1) induces postprandial hyperinsulinemia in some of the patients who have undergone the surgery. Hypoglycemia is generally characterized by a low plasma glucose level at the onset of symptoms, and the symptoms are alleviated when the glucose level rises. Post-bariatric hypoglycemia (PBH) is characterized by postprandial hypoglycemia that develops 1 to 3 hours after a meal. GLP1 is secreted in response to food intake, activates the GLP1 receptor (GLP1R) on pancreatic beta cells, and induces insulin secretion. Therefore, the increase in GLP1 plays an important role in the development of PBH. As the number of bariatric surgeries continues to increase worldwide, reports of severe complications - hyperinsulinemic hypoglycemia have been increasing. For example, symptomatic hypoglycemia has been reported to develop 0.5 to 10 years after surgery in 0.2 to 15% of gastric bypasses and 1 to 7% of patients who have undergone gastrectomy. Non-Patent Document 1; Non-Patent Document 2; Non-Patent Document 3; Non-Patent Document 4; Non-Patent Document 5; Non-Patent Document 6.

[0003] GLP-1 plays an important role in PBH because surgery causes an increase in GLP-1 due to the accelerated passage of food through the small intestine where GLP-1-producing L cells are located. Generally, the increase in GLP-1 aids in postoperative weight loss in obese patients, while symptoms of hypoglycemia, such as adrenergic effects (e.g., tremor, palpitations, anxiety), cholinergic effects (e.g., sweating, hunger, paresthesia), and neuroglycopenic effects (e.g., cognitive impairment, seizures, and loss of consciousness) can occur, particularly in patients who achieve significant weight loss postoperatively. Patients who experience recurrent hypoglycemia may undergo a sudden onset of neuroglycopenia, which can lead to severe falls, motor vehicle accidents, seizures, and loss of consciousness. Severe PBH is debilitating and significantly reduces QOL. Currently, treatment options for PBH include dietary improvement, acarbose (a starch digestion blocker), somatostatin analogs, nifedipine (a Ca channel blocker), diazoxide, and the placement of a gastrostomy tube. Non-Patent Document 7. The increasing prevalence of morbid obesity indicates an increase in PBH cases as a complication of bariatric surgery.

[0004] Other disorders, diseases, and conditions can also lead to low plasma glucose levels. For example, dumping syndrome is a frequent complication of surgery on the upper abdomen (e.g., bariatric, esophageal, or gastric), where GLP-1 is thought to play a major role. Dumping syndrome is estimated to occur in up to 40% of bariatric surgery patients (Roux-en-Y bypass or sleeve gastrectomy), up to 50% of patients who undergo esophagectomy for the treatment of esophageal cancer, and up to 75% of patients who undergo gastrectomy for the treatment of gastric cancer and peptic ulcer. The diagnosis of dumping syndrome may be made several months to years after surgery. The hyperinsulinemic response due to GLP-1 after carbohydrate ingestion causes hypoglycemia-related symptoms, such as neuroglycopenia (fatigue, weakness, confusion, hunger, and fainting) and autonomic / adrenergic reactivity (sweating, palpitations, tremor, and hypersensitivity).

Prior Art Documents

Non-Patent Documents

[0005] [Non-Patent Document 1] Kellogg et al., Surg Obes Relat Dis., 4(4):492-99 (2008) [Non-Patent Document 2] Marsk et al., Diabetologia, 53(11):2307-11 (2010) [Non-Patent Document 3] Nambron et al., WMJ, 112(3):136 (2013) [Non-Patent Document 4] Lee et al., Obesity (Silver Spring), 23(5):1079-84 (2015) [Non-Patent Document 5] Gribsholt et al., Ugeskr Laeger, 178(44) (2016) [Non-Patent Document 6] Sun et al., Surg Obes Relat Dis., 15(9):1439-1446 (2019). [Non-Patent Document 7] Eisenberg et al., Surg Obes Relat Dis., 13(3):371-378 (2017) [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] Hypoglycemia involving postprandial hypoglycemia (PBH) can occur over a wide range of severities. Quality of life (QOL) is greatly affected by severe hypoglycemia, such that affected patients may be incapacitated and unable to care for themselves or others, or to transport themselves or others. There is no approved or effective drug therapy for PBH or severe hypoglycemia that has a long duration of action and does not cause hyperglycemia, and surgical interventions have not been uniformly successful in restoring hypoglycemia. As a result, there is a great and unmet need for therapeutic agents that effectively raise blood glucose levels and thus act as useful treatments for hypoglycemia of any origin, including PBH. [Means for Solving the Problems]

[0007] The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the glucagon-like peptide 1 receptor (GLP1R) protein. In certain embodiments, the GLP1R antibody is a fully human antibody that binds to GLP1R with high affinity and blocks the binding and / or activity of GLP1R or destabilizes the active conformation. The antibodies or antigen-binding fragments thereof of the present disclosure are useful, inter alia, for inactivating or reducing the activity of the GLP1R protein. In certain embodiments, the antibody or antigen-binding fragment thereof is useful for preventing, treating, or ameliorating at least one symptom or sign of a GLP1R-related disease or disorder in a subject. In certain embodiments, the antibody or antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having or at risk of having a GLP1R-related disease or disorder, such as PBH or other types of hypoglycemia of any origin. In certain specific embodiments, the antibody or antigen-binding fragment thereof is used to increase glucose levels in a subject in need thereof. Such an antibody or antigen-binding fragment thereof can be used as a therapy for disorders such as hypoglycemia of any origin (e.g., postprandial hypoglycemia or hypoglycemia due to other upper abdominal surgeries such as esophagectomy, gastrectomy for gastric cancer and peptic ulcer, or hypoglycemia due to an underlying etiology such as a genetic abnormality) when administered to a subject in need thereof. In certain embodiments, the GLP1 antagonist antibody or antigen-binding fragment of the present disclosure has a longer half-life and reduced immunogenicity compared to other therapies, such as Exendin(9-39) (Avexitide, Eiger BioPharmaceuticals). In certain embodiments, the antibodies or antigen-binding fragments disclosed herein bind to GLP1R with high affinity and have improved pharmacokinetic properties compared to standard therapeutic agents.

[0008] The antibodies and antigen-binding fragments disclosed herein specifically bind to GLP1R in an antagonist-like manner, i.e., in a manner that attenuates, prevents or blocks the binding and / or activity of GLP1R. This antibody is effective in raising blood glucose levels and maintaining normal levels when administered to a subject in need thereof. A single administration of the antibody of the present disclosure resulted in persistently normalized blood glucose levels in mice for 46 days. Such antibodies can be used to provide excellent efficacy at a lower dosing frequency to subjects having a GLP1R-related disease or disorder (e.g., hypoglycemia).

[0009] The antibodies of the present disclosure may be full-length (e.g., IgG1 or IgG4 antibodies) or may contain only the antigen-binding portion (e.g., Fab, F(ab’)2, or scFv fragments) and may be modified to affect functionality, e.g., to increase persistence in the host or to remove residual effector functions (Reddy et al., 2000, J. Immunol. 164:1925-1933). In certain embodiments, the antibody may be bispecific.

[0010] In one aspect, the present disclosure provides an isolated recombinant monoclonal antibody or antigen-binding fragment thereof that specifically binds to GLP1R.

[0011] In certain embodiments, the antibody is a fully human monoclonal antibody.

[0012] The GLP1R antagonists of the present disclosure are useful, inter alia, for reducing the activation of GLP1R. In certain embodiments, the GLP1R antagonist functions by decreasing insulin secretion and lowering blood glucose levels. In certain embodiments, the GLP1R antagonist functions by attenuating glucose-induced insulin secretion from pancreatic beta cells and decreasing insulin expression. In certain embodiments, the GLP1R antagonist is useful in preventing, treating, or ameliorating at least one symptom of a hypoglycemia-related disease or disorder (e.g., PBH) in a subject.

[0013] In certain embodiments, the disclosed anti-GLP1R antagonist antibodies and antigen-binding fragments substantially or completely correct exendin-4-induced hypoglycemia over 45 days after a single administration, as shown in Example 5.

[0014] Exemplary GLP1R antagonist antibodies of the present disclosure are set forth in Tables 1 and 2 herein. Table 1 lists the amino acid sequence identification names of the heavy chain variable region (HCVR), light chain variable region (LCVR), heavy chain complementarity determining regions (HCDR) (HCDR1, HCDR2, and HCDR3), and light chain complementarity determining regions (LCDR) (LCDR1, LCDR2, and LCDR3) of representative antibodies. Table 2 lists the nucleic acid sequence identification names of the HCVR, LCVR, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of representative antibodies.

[0015] The present disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCVR comprising an amino acid sequence selected from any of the HCVR amino acid sequences listed in Table 1 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0016] The present disclosure also provides an antibody or an antigen-binding fragment thereof that comprises an LCVR comprising an amino acid sequence selected from any of the LCVR amino acid sequences listed in Table 1, or a substantially similar sequence thereto having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0017] The present disclosure also provides an antibody or an antigen-binding fragment thereof that comprises an HCVR / LCVR amino acid sequence pair (HCVR / LCVR) that includes any of the HCVR amino acid sequences listed in Table 1 that pair with any of the LCVR amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides an antibody, or an antigen-binding fragment thereof, that comprises the HCVR / LCVR amino acid sequence pair included in any of the exemplary anti-GLP1R antibodies described in Table 1. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from one of SEQ ID NO: 2 / 10 (e.g., mAb36986), 22 / 30 (e.g., mAb37639), and 40 / 48 (e.g., mAb37645).

[0018] The present disclosure also provides an antibody comprising an HCVR and an LCVR, or an antigen-binding fragment thereof, wherein the HCVR comprises the amino acid sequence set forth in Table 1 having 12 or fewer amino acid substitutions, and / or the LCVR comprises the amino acid sequence set forth in Table 1 having 10 or fewer amino acid substitutions. For example, the present disclosure provides an antibody comprising an HCVR and an LCVR, or an antigen-binding fragment thereof, wherein the HCVR comprises the amino acid sequence set forth in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid substitutions. In another example, the present disclosure provides an antibody comprising an HCVR and an LCVR, or an antigen-binding fragment thereof, wherein the LCVR comprises the amino acid sequence set forth in Table 1, and the amino acid sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In one embodiment, the present disclosure provides an anti-GLP1R antibody or an antigen-binding fragment thereof comprising an HCVR and an LCVR, wherein the HCVR comprises the amino acid sequence set forth in Table 1, the amino acid sequence has at least one amino acid substitution, and / or the LCVR comprises the amino acid sequence set forth in Table 1, and the amino acid sequence has at least one amino acid substitution.

[0019] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain complementarity determining region 1 (HCDR1) comprising an amino acid sequence selected from any of the HCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0020] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain complementarity-determining region 2 (HCDR2) comprising an amino acid sequence selected from any of the HCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0021] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a heavy-chain complementarity-determining region 3 (HCDR3) comprising an amino acid sequence selected from any of the HCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0022] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a light-chain complementarity-determining region 1 (LCDR1) comprising an amino acid sequence selected from any of the LCDR1 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0023] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a light-chain complementarity-determining region 2 (LCDR2) comprising an amino acid sequence selected from any of the LCDR2 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0024] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a light-chain complementarity-determining region 3 (LCDR3) comprising an amino acid sequence selected from any of the LCDR3 amino acid sequences listed in Table 1, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0025] The present disclosure also provides an antibody or an antigen-binding fragment thereof that includes an HCDR1 and LCDR1 amino acid sequence pair (HCDR1 / LCDR1) that pairs with any of the LCDR1 amino acid sequences set forth in Table 1 and any of the HCDR1 amino acid sequences set forth in Table 1. According to certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof that includes an HCDR1 / LCDR1 amino acid sequence pair included in any of the exemplary anti-GLP1R antibodies set forth in Table 1. In certain embodiments, the HCDR1 / LCDR1 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 4 / 12 (e.g., mAb36986), 24 / 32 (e.g., mAb37639), and 42 / 50 (e.g., mAb37645).

[0026] The present disclosure also provides an antibody or an antigen-binding fragment thereof that includes an HCDR2 and LCDR2 amino acid sequence pair (HCDR2 / LCDR2) that pairs with any of the LCDR2 amino acid sequences set forth in Table 1 and any of the HCDR2 amino acid sequences set forth in Table 1. According to certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof that includes an HCDR2 / LCDR2 amino acid sequence pair included in any of the exemplary anti-GLP1R antibodies set forth in Table 1. In certain embodiments, the HCDR2 / LCDR2 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 6 / 14 (e.g., mAb36986), 26 / 14 (e.g., mAb37639), and 44 / 52 (e.g., mAb37645).

[0027] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 and LCDR3 amino acid sequence pair (HCDR3 / LCDR3) comprising any of the HCDR3 amino acid sequences listed in Table 1 that pair with any of the LCDR3 amino acid sequences listed in Table 1. According to certain embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising an HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-GLP1R antibodies listed in Table 1. In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair is selected from the group consisting of SEQ ID NO: 8 / 16 (e.g., mAb36986), 28 / 34 (e.g., mAb37639), and 46 / 54 (e.g., mAb37645).

[0028] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising an HCVR and an LCV, wherein the HCVR comprises an HCDR1 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid, an HCDR2 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid, and an HCDR3 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid.

[0029] In certain embodiments, the present disclosure provides an antibody, or an antigen-binding fragment thereof, comprising an HCVR and an LCVR, wherein the LCVR comprises an LCDR1 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid, an LCDR2 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid, and an LCDR3 comprising an amino acid sequence that differs from the amino acid sequence set forth in Table 1 by one amino acid.

[0030] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a heavy chain (HC) comprising an amino acid selected from any of the HC amino acid sequences set forth in Table 3 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0031] The present disclosure also provides an antibody or an antigen-binding fragment thereof that includes a light chain (LC) comprising an amino acid sequence selected from any of the LC amino acid sequences set forth in Table 3, or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity thereto.

[0032] The present disclosure also provides an antibody, or an antigen-binding fragment thereof, that includes a heavy chain (HC) and light chain (LC) amino acid sequence pair (HC / LC) comprising any of the HC amino acid sequences set forth in Table 3 paired with any of the LC amino acid sequences set forth in Table 3. According to certain embodiments, the invention provides an antibody or an antigen-binding fragment thereof that includes the HC / LC amino acid sequence pair included in any of the exemplary anti-GLP1R antibodies set forth in Table 3. In certain embodiments, the HC / LC amino acid sequence pair is an HC / LC amino acid sequence pair selected from the group consisting of SEQ ID NOs: 18 / 20, 36 / 38, and 56 / 58.

[0033] The present disclosure also provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) contained in any of the exemplary antibodies listed in Table 1. In certain embodiments, the HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequence set is selected from the group consisting of SEQ ID NOs: 4 / 6 / 8 / 12 / 14 / 16 (e.g., mAb36986), 24 / 26 / 28 / 32 / 14 / 34 (e.g., mAb37639), and 42-44-46-50-52-54 (e.g., mAb37645). In related embodiments, the present disclosure provides an antibody or an antigen-binding fragment thereof comprising a set of six CDRs (i.e., HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) contained in an HCVR / LCVR amino acid sequence pair defined by any of the exemplary antibodies listed in Table 1. For example, the present disclosure includes an antibody or an antigen- / binding fragment thereof comprising an HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 amino acid sequence set contained in an HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10 (e.g., mAb36986), 22 / 30 (e.g., mAb37639), and 40 / 48 (e.g., mAb37645).

[0034] Methods and techniques for identifying CDRs within HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs within the specified HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions used to identify CDR boundaries include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence variability, the Chothia definition is based on the location of structurally defined loop regions, and the AbM definition is a compromise between the Kabat and Chothia approaches. See, e.g., Kabat, "Sequences of Proteins of Immunological Interest", National Institutes of Health, Bethesda, Md. (1991); Al-Lazikani et al., J. Mol. Biol. 273:927-948 (1997); and Martin et al., Proc. Natl. Acad. Sci. USA 86:9268-9272 (1989). Public databases are also available for identifying CDR sequences within antibodies.

[0035] In certain embodiments, the disclosure includes an antibody or antigen-binding fragment thereof that specifically binds to GLP1R, wherein the fragment contained within the heavy chain variable region (HCVR) thereof comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light chain variable region (LCVR), wherein the HCVR comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 40; (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 40; (iii) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 40; or (iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 22, and 40 and having 12 or fewer amino acid substitutions; and the LCVR comprises: (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, and 48; (ii) an amino acid sequence having at least 90% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, and 48; (iii) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, and 48; or (iv) an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 30, and 48 and having 12 or fewer amino acid substitutions.

[0036] The disclosure includes anti-GLP1R antibodies having an altered glycosylation pattern. In certain embodiments, modifications to remove undesired glycosylation sites may be useful, or antibodies lacking a fucose moiety, as shown on the oligosaccharide chain, for example, increase antibody-dependent cell cytotoxicity (ADCC) function (see Shield et al. (2002) JBC 277:26733). In other applications, modifications such as galactosylation are made to modify complement-dependent cytotoxicity (CDC).

[0037] In certain embodiments, the present disclosure includes antibodies and antigen-binding fragments thereof that exhibit pH-dependent binding to GLP1R. For example, the present disclosure includes antibodies and antigen-binding fragments thereof that bind to GLP1R with higher affinity at acidic pH than at neutral pH (i.e., binding is decreased at acidic pH).

[0038] The present disclosure also provides antibodies and antigen-binding fragments thereof that compete with an antibody or antigen-binding fragment thereof that includes the CDRs of HCVR and the CDRs of LCVR for specific binding to GLP1R, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0039] The present disclosure also provides antibodies and antigen-binding fragments thereof that cross-compete with a reference antibody or antigen-binding fragment thereof that includes the CDRs of HCVR and the CDRs of LCVR for binding to GLP1R, wherein each of HCVR and LCVR has an amino acid sequence selected from the HCVR and LCVR sequences listed in Table 1.

[0040] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to the same epitope as a reference antibody or antigen-binding fragment thereof that includes three CDRs of HCVR and three CDRs of LCVR, wherein HCVR and LCVR each have an amino acid sequence selected from the HCVR and LCVR sequences described in Table 1.

[0041] The present disclosure also provides antibodies and antigen-binding fragments thereof that decrease or destabilize the binding of GLP1R to GLP1. In certain embodiments, an antibody or antigen-binding fragment thereof that blocks GLP1R binding to GLP1 may bind to an epitope on GLP1R that is the same as or different from that of GLP1.

[0042] In certain embodiments, the antibodies or antigen-binding fragments of the present disclosure are bispecific, comprising a first binding specificity for a first epitope of GLP1R and a second binding specificity for a second epitope of GLP1R, wherein the first and second epitopes are distinct and non-overlapping.

[0043] In certain embodiments, the present disclosure provides an isolated antibody or antigen-binding fragment thereof that: (a) is a fully human monoclonal antibody; (b) binds to human GLP1R with a dissociation constant (K D ) of less than 6 nM at 25°C as measured by surface plasmon resonance (SPR) assay; (c) binds to human GLP1R with a K D of less than 20 nM at 37°C as measured by SPR assay; (d) binds to monomeric cynomolgus GLP1R with a K D of less than 20 nM at 25°C as measured by SPR assay; (e) binds to monomeric cynomolgus GLP1R with a K D of less than 60 nM at 37°C as measured by SPR assay; (f) binds to hdesA-GLP1_GLP1R-MMH with a K D of less than 18 nM at 25°C as measured by SPR assay; (g) binds to hdesA-GLP1_GLP1R-MMH with a K D of less than 75 nM at 37°C as measured by SPR assay; (h) binds to monomeric mouse GLP1R monomer with a K D of less than 22 nM at 25°C as measured by SPR assay; (i) binds to monomeric mouse GLP1R monomer with a K D of less than 75 nM at 37°C as measured by SPR assay; (j) blocks the interaction between GLP1 and GLP1R with an IC 50 value of less than about 10 nM at 37°C as measured by an in vitro receptor / ligand binding assay; (k) reduces GLP1-induced hypoglycemia without causing hyperglycemia; and (l) raises blood glucose to normal levels.

[0044] In other aspects, the present disclosure provides nucleic acid molecules encoding an anti-GLP1R antibody or a fragment thereof. For example, the present disclosure provides nucleic acid molecules encoding any of the HCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the HCVR nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. The present disclosure also provides nucleic acid molecules encoding any of the LCVR amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the LCVR nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0045] The present disclosure also provides nucleic acid molecules encoding any of the HCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the HCDR1 nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0046] The present disclosure also provides nucleic acid molecules encoding any of the HCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is selected from any of the HCDR2 nucleic acid sequences listed in Table 2, or a polynucleotide sequence of a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0047] The present disclosure also provides a nucleic acid molecule encoding any of the HCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is any of the HCDR3 nucleic acid sequences listed in Table 2, or a polynucleotide sequence selected from substantially similar sequences thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0048] The present disclosure also provides a nucleic acid molecule encoding any of the LCDR1 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is any of the LCDR1 nucleic acid sequences listed in Table 2, or a polynucleotide sequence selected from substantially similar sequences thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0049] The present disclosure also provides a nucleic acid molecule encoding any of the LCDR2 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is any of the LCDR2 nucleic acid sequences listed in Table 2, or a polynucleotide sequence selected from substantially similar sequences thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0050] The present disclosure also provides a nucleic acid molecule encoding any of the LCDR3 amino acid sequences listed in Table 1; in certain embodiments, the nucleic acid molecule is any of the LCDR3 nucleic acid sequences listed in Table 2, or a polynucleotide sequence selected from substantially similar sequences thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto.

[0051] The present disclosure also provides a nucleic acid molecule encoding an HCVR, wherein the HCVR comprises a set of three CDRs (i.e., HCDR1-HCDR2-HCDR3), and wherein the set of HCDR1-HCDR2-HCDR3 amino acid sequences is as defined by any of the exemplary antibodies listed in Table 1.

[0052] The present disclosure also provides a nucleic acid molecule encoding an LCVR, wherein the LCVR comprises a set of three CDRs (i.e., LCDR1-LCDR2-LCDR3), and wherein the set of LCDR1-LCDR2-LCDR3 amino acid sequences is as defined by any of the exemplary antibodies listed in Table 1.

[0053] The present disclosure also provides a nucleic acid molecule encoding both an HCVR and an LCVR, wherein the HCVR comprises an amino acid sequence of any of the HCVR amino acid sequences listed in Table 1, and wherein the LCVR comprises an amino acid sequence of any of the LCVR amino acid sequences listed in Table 1. In certain embodiments, the nucleic acid molecule comprises a polynucleotide sequence selected from any of the HCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto, and a polynucleotide sequence selected from any of the LCVR nucleic acid sequences listed in Table 2, or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity thereto. In certain embodiments according to this aspect, the nucleic acid molecule encodes an HCVR and an LCVR, wherein both the HCVR and the LCVR are derived from the same anti-GLP1R antibody listed in Table 1.

[0054] The present disclosure provides a nucleic acid molecule encoding any of the heavy chain amino acid sequences listed in Table 3. The present invention also provides a nucleic acid molecule encoding any of the light chain amino acid sequences listed in Table 3. The present invention also provides a nucleic acid molecule encoding both a heavy chain (HC) and a light chain (LC), where the HC comprises an amino acid sequence of any of the HC amino acid sequences described in Table 3, and the LC comprises an amino acid sequence of any of the LC amino acid sequences described in Table 3.

[0055] In related aspects, the present disclosure provides a recombinant expression vector having the ability to express a polypeptide comprising a heavy chain and / or a light chain variable region of an antibody. For example, the present disclosure includes a recombinant expression vector comprising any of the above-described nucleic acid molecules, i.e., any nucleic acid molecule encoding any of the HCVR, LCVR, and / or CDR sequences described in Table 2. In certain embodiments, the present disclosure provides: (a) a nucleic acid molecule comprising a nucleic acid sequence encoding an HCVR of an antibody that binds to GLP1R, wherein the HCVR comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 1; and / or (b) a nucleic acid molecule comprising a nucleic acid sequence encoding an LCVR of an antibody that binds to GLP1R, wherein the HCVR comprises an amino acid sequence selected from the group consisting of the sequences set forth in Table 1. Also included within the scope of the present disclosure is a method of producing an antibody or a portion thereof by culturing a host cell into which such a vector has been introduced under conditions that allow for the production of the antibody or antibody fragment and recovering the thus-produced antibody and antibody fragment. In certain embodiments, the host cell comprises a mammalian cell or a prokaryotic cell. In certain embodiments, the host cell is a Chinese hamster ovary (CHO) cell or an Escherichia coli (E. coli) cell. In certain embodiments, the present disclosure provides a method of producing an antibody or an antigen-binding fragment thereof. The method comprises introducing into a host cell an expression vector comprising a nucleic acid sequence encoding an HCVR and / or an LCVR of an antibody or an antigen-binding fragment thereof of the present disclosure operably linked to a promoter; culturing the host cell under conditions favorable for expression of the nucleic acid sequence; and isolating the antibody or an antigen-binding fragment thereof from the culture medium and / or the host cell.In certain embodiments, the method comprises introducing into a host cell (e.g., a CHO cell) (a) a first expression vector comprising a nucleic acid sequence encoding the HCVR of an antibody or an antigen-binding fragment thereof of the present disclosure operably linked to a promoter, and (b) a second expression vector comprising a nucleic acid sequence encoding the LCVR of an antibody or an antigen-binding fragment thereof of the present disclosure operably linked to a promoter; culturing the host cell under conditions favorable for expression of the nucleic acid sequences; and isolating the antibody or an antigen-binding fragment thereof from the culture medium and / or the host cell. The isolated antibody or an antigen-binding fragment thereof can be purified using any of the methods known in the prior art.

[0056] In other aspects, the present disclosure provides a pharmaceutical composition comprising a therapeutically effective amount of at least one recombinant monoclonal antibody or an antigen-binding fragment thereof that specifically binds to GLP1R and a pharmaceutically acceptable carrier. In related aspects, the present disclosure features a composition that is a combination of an anti-GLP1R antibody and a second therapeutic agent. In one embodiment, the second therapeutic agent is advantageously combined with an anti-GLP1R antibody - for example, insulin or an insulin receptor inhibitor, a starch digestion inhibitor (e.g., acarbose), a somatostatin analog (e.g., octreotide, lanreotide), a calcium channel inhibitor (e.g., nifedipine), diazoxide, glucagon, a somatostatin receptor type 5 agonist, or a combination thereof, and any agent that can be advantageously combined therewith. Exemplary agents that can be advantageously combined with an anti-GLP1R antibody include, but are not limited to, other agents that bind to GLP1R and / or inactivate GLP1R activity (including other antibodies or antigen-binding fragments thereof, etc.) and / or agents that do not directly bind to GLP1R but nevertheless treat or ameliorate at least one symptom or sign of a GLP1R-related disease or disorder (disclosed elsewhere herein). Additional combination therapies and co-formulations comprising the anti-GLP1R antagonist antibody of the present disclosure are disclosed elsewhere in this specification.

[0057] Also provided herein is a method of treating a disease or disorder associated with GLP1R in a subject using an anti-GLP1R antagonist antibody or an antigen-binding fragment thereof, the method comprising administering to a subject in need thereof a pharmaceutical composition comprising a therapeutically effective amount of the antibody or an antigen-binding fragment thereof. The disorder to be treated is any disease or condition (e.g., hypoglycemia) that is ameliorated, improved, inhibited, or prevented by attenuation of GLP1R activity. In certain embodiments, the present disclosure provides a method of preventing or treating a GLP1R-related disease or disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-GLP1R antibody or an antigen-binding fragment thereof disclosed herein. In certain embodiments, the antibody or an antigen-binding fragment thereof can be administered prophylactically or therapeutically to a subject having or at risk of having a GLP1R-related disease or disorder. In certain embodiments, the antibody or an antigen-binding fragment thereof is administered to a subject after upper abdominal surgery. In certain embodiments, the antibody or an antigen-binding fragment thereof is administered to a subject in need thereof in combination with a second therapeutic agent. In certain embodiments, the second therapeutic agent may be an agent that serves to counteract or mitigate any possible side effects associated with the antibody or an antigen-binding fragment thereof disclosed herein, if such side effects occur. The antibody or fragment thereof can be administered subcutaneously, intravenously, intradermally, intraperitoneally, orally, or intramuscularly. The antibody or fragment thereof can be administered at a dosage of from about 0.1 mg / kg to about 100 mg / kg of the subject's body weight. In certain embodiments, the antibody or fragment thereof of the present disclosure may be administered in one or more dosages comprising from 10 mg to 600 mg.

[0058] The present disclosure also includes the use of an anti-GLP1R antibody or an antigen-binding fragment thereof of the present disclosure in the manufacture of a medicament for treating a disease or disorder - hypoglycemia, such as PBH, that would benefit from blockade of GLP1R binding and / or activity.

[0059] Other embodiments will become apparent from consideration of the following detailed description.

Brief Description of the Drawings

[0060]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0061] It is to be understood that the present disclosure is not limited to the specific methods and experimental conditions described, and that such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. The scope of the present disclosure is limited only by the appended claims and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present disclosure, but the preferred methods and materials are described herein. All publications mentioned herein are incorporated herein by reference in their entirety.

[0062] Definitions The term "GLP1R" refers to the glucagon-like peptide 1 receptor and includes recombinant GLP1R protein or fragments thereof. GLP1R has a sequence of 463 residues. Donnelly, Br J Pharmacol, 166(1):27-41 (2011). Glucagon-like peptide 1 (GLP1) is a 31-amino acid peptide hormone that is released from intestinal L cells in response to nutrient intake. Binding of GLP1 to GLP1R promotes insulin secretion from glucose-responsive pancreatic beta cells, increases insulin expression, inhibits apoptosis of beta cells, promotes beta cell neogenesis, decreases glucagon secretion, delays gastric emptying, promotes satiety, and increases glucose disposal in the periphery.

[0063] As used herein, the term "antibody" is intended to refer to an immunoglobulin molecule (i.e., a "complete antibody molecule") that includes four polypeptide chains, two heavy (H) chains and two light (L) chains that are inter-connected by disulfide bonds, as well as multimers thereof (e.g., IgM) or antigen-binding fragments thereof. Each heavy chain includes a heavy chain variable region ("HCVR" or "V H "), as well as a heavy chain constant region (domains C H 1, C H 2, and C H 3). Each light chain includes a light chain variable region ("LCVR" or "V L "), and a light chain constant region (C L ). The V H and V L regions are further subdivided into regions of high-frequency variability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each V H as well as V Lcomprises three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In certain embodiments, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified. The amino acid contiguous sequences are defined based on the contiguous analysis of two or more adjacent CDRs.

[0064] Substitution of one or more CDR residues or exclusion of one or more CDRs is also possible. Antibodies that bind even after omitting one or two CDRs have been described in the scientific literature. Padlan et al. (FASEB J. 9: 133-139, 1995) analyzed the contact regions between an antibody and its antigen based on the published crystal structures and concluded that only about one-fifth to one-third of the CDR residues actually contact the antigen. Padlan also found many antibodies in which one or two CDRs do not have amino acids that contact the antigen (see also Vajdos et al., J Mol Biol 320: 415-428, 2002).

[0065] CDR residues that do not contact the antigen are identified by molecular modeling and / or experimentally from regions of Kabat CDRs outside the Chothia CDRs in previous studies. If a CDR or its residue is excluded, it is usually substituted with an amino acid occupying the corresponding position in another human antibody sequence or a common such sequence. The CDRs for substitution and the positions for substitution within the amino acids are also selected experimentally. The experimental substitutions are conservative or non-conservative substitutions.

[0066] The fully human anti-GLP1R monoclonal antibodies disclosed herein include one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains as compared to the corresponding germline sequences. Such mutations can be readily ascertained by comparing the amino acid sequences disclosed herein to, for example, germline sequences available from public antibody sequence databases. The present disclosure includes antibodies and antigen-binding fragments thereof derived from any of the amino acid sequences disclosed herein, where one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody is derived, or to the corresponding residues of another human germline sequence, or to conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). One of ordinary skill in the art can readily generate a number of antibodies and antigen-binding fragments that include one or more individual germline mutations or combinations thereof, starting from the heavy and light chain variable region sequences disclosed herein. In certain embodiments, V H and / or V LMutate all of the framework and / or CDR residues within the domain to return to the residues found in the original germline sequence from which the antibody is derived. In other embodiments, only certain residues are mutated, for example, mutated residues found within the first 8 amino acids of FR1, or within the last 8 amino acids of FR4, or only mutated residues found within CDR1, CDR2, or CDR3, to return to the original native germline sequence. In other embodiments, one or more of the framework and / or CDR residues are mutated to the corresponding residues of a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody originally derived). Further, the antibodies of the present disclosure contain any combination of two or more germline mutations within the framework and / or CDR regions, for example, where certain individual residues are mutated to the corresponding residues of a particular germline sequence, while certain other residues different from the original germline sequence are maintained or mutated to the corresponding residues of a different germline sequence. Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations are readily tested for one or more desired properties such as improvement in binding specificity, increase in binding affinity, improvement or enhancement of antagonist biological properties, reduction of immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general method are encompassed by the present disclosure.

[0067] The present disclosure also includes fully human anti-GLP1R monoclonal antibodies comprising variants of any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein having one or more conservative substitutions. For example, the present disclosure includes anti-GLP1R antibodies having HCVR, LCVR, and / or CDR amino acid sequences having, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc. conservative amino acid substitutions related to any of the HCVR, LCVR, and / or CDR amino acid sequences disclosed herein.

[0068] As used herein, the terms "human antibody" or "fully human antibody" are intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human mAbs disclosed herein may contain, for example, amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis, or somatic mutations in vivo) in the CDRs, particularly CDR3. However, the terms "human antibody" or "fully human antibody" as used herein are not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human FR sequences. The terms include antibodies recombinantly produced in non-human mammals or in cells of non-human mammals. The terms are not intended to include antibodies isolated from or occurring in a human subject.

[0069] As used herein, the term "recombinant" refers to an antibody or antigen-binding fragment thereof that is made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transfection expression. The term refers to antibodies expressed in a non-human mammalian (including transgenic non-human mammals, e.g., transgenic mice) or cell (e.g., CHO cell) expression system or isolated from a recombinant combinatorial human antibody library.

[0070] The terms "specifically binds" or "binds specifically to" mean that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is characterized by an equilibrium dissociation constant of at least about 1×10 -8 M or less (e.g., K DThe smaller it is, the stronger the binding (is shown). Methods for determining whether two molecules specifically bind are well-known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, etc. As described herein, antibodies are identified by surface plasmon resonance, for example, BIACORE™, and specifically bind to GLP1R. Further, a multispecific antibody that binds to one domain in GLP1R and one or more additional antigens, or a bispecific antibody that binds to two different regions of GLP1R, is nevertheless considered an antibody that "specifically binds" as used herein.

[0071] The term "high affinity" antibody refers to its mAb having a binding affinity for GLP1R, as measured by surface plasmon resonance, for example, BIACORE™ or solution affinity ELISA, of at least 10 -8 M; preferably, 10 -9 M; more preferably, 10 -10 M, even more preferably, 10 -11 M of K D as represented.

[0072] The terms "slow off-rate", "Koff", or "kd" refer to an antibody that dissociates from GLP1R with a rate constant of 1×10 -3 s -1 or less, preferably, 1×10 -4 s -1 or less.

[0073] As used herein, terms such as "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc. include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. As used herein, the term "antigen-binding fragment" of an antibody, or "antibody fragment" refers to one or more fragments of an antibody that retain the ability to bind to the GLP1R protein.

[0074] In certain embodiments, the antibodies or antibody fragments of the present disclosure bind to a moiety such as a ligand, or a second anti-GLP1R antibody, or any other therapeutic moiety such as any other therapeutic moiety useful for treating a disease or disorder associated with GLP1R (an "immune complex").

[0075] As used herein, an "isolated antibody" is intended to refer to an antibody substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody or fragment thereof that specifically binds to GLP1R is substantially free of Abs that specifically bind to antigens other than GLP1R).

[0076] As used herein, a "blocking antibody" or "antagonist antibody" (or "antibody that reduces or attenuates GLP1R activity" or "antibody that destabilizes the active conformation") is intended to refer to an antibody whose binding to GLP1R results in inhibition of at least one biological activity of GLP1R. For example, the antibodies of the present disclosure can increase glucose levels upon administration to a subject in need thereof.

[0077] As used herein, the term "surface plasmon resonance" refers to an optical phenomenon that enables real-time analysis of biomolecular interactions, for example, by detection of changes in protein concentration within a biosensor matrix using a BIACORE™ system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

[0078] As used herein, the term "K D " is intended to refer to the equilibrium dissociation constant of the interaction of a particular antibody with an antigen.

[0079] The term "epitope" refers to an antigenic determinant that interacts with the specific antigen-binding site in the variable region of an antibody molecule, also known as a paratope. A single antigen has more than one epitope. Thus, different antibodies bind to different regions on the antigen and have different biological effects. The term "epitope" also refers to the site on an antigen to which B and / or T cells respond. It also refers to the region of an antigen that is bound by an antibody. Epitopes are defined either structurally or functionally. Functional epitopes are generally a subset of structural epitopes and have those residues that are directly involved in the affinity of the interaction. Epitopes are also conformational, i.e., they contain non-linear amino acids. In certain embodiments, an epitope comprises determinants that are chemically active surface groups of a molecule such as an amino acid, sugar side chain, phosphate group, or sulfonyl group, and in certain embodiments, has specific three-dimensional structural features, and / or specific modifying features.

[0080] As used herein, the term "cross-competing" means that an antibody or an antigen-binding fragment thereof binds to an antigen and inhibits or blocks the binding of another antibody or an antigen-binding fragment thereof. The term also includes competition between two antibodies in both directions, i.e., a first antibody that binds to a second antibody and blocks the binding of the second antibody, and vice versa. In certain embodiments, the first and second antibodies bind to the same epitope. Alternatively, the first and second antibodies bind to different but overlapping epitopes such that one binding inhibits or blocks the binding of the second antibody, for example, through steric hindrance. Cross-competition between antibodies is measured by methods known in the art, for example, by real-time, label-free biolayer interferometry assay. Cross-competition between two antibodies is expressed as the binding of the second antibody, which is less than the background signal resulting from self-to-self binding (where the first and second antibodies are the same antibody). Cross-competition between two antibodies is expressed, for example, as the % binding of the second antibody that is less than the baseline self-to-self background binding (where the first and second antibodies are the same antibody).

[0081] When referring to a nucleic acid or a fragment thereof, the term "substantial identity" or "substantially identical" means that when optimally aligned with another nucleic acid (or its complementary strand) with appropriate nucleotide insertions or deletions, as discussed below, there is at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, or 99% nucleotide sequence identity of nucleotide bases, as measured by any well-known algorithm for sequence identity, such as FASTA, BLAST, or GAP. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule encodes a polypeptide having an amino acid sequence that is the same as or substantially similar to the polypeptide encoded by the reference nucleic acid molecule in certain examples.

[0082] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 90% sequence identity, more preferably at least 95%, 98%, or 99% sequence identity when optimally aligned, for example, by the programs GAP or BESTFIT using the default gap weights. Preferably, residue positions differ by conservative amino acid substitutions rather than being identical. A "conservative amino acid substitution" is one in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage or degree of similarity is adjusted upward to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, for example, Pearson (1994) Methods Mol. Biol. 24:307-331. Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine, and threonine; 3) amide-containing side chains: asparagine, and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate, and glutamate, and 7) sulfur-containing side chains: cysteine, and methionine. Preferred conservative amino acid substituents are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-45. A "moderately conservative" substitution is any change having a non-negative value in the PAM250 log-likelihood matrix.

[0083] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using similarity measurements assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG software includes programs such as GAP and BESTFIT that are used with default parameters for determining sequence homology or sequence identity between closely related polypeptides, such as between homologous polypeptides from different species of organisms or between a wild-type protein and its mutant protein. See, for example, GCG Version 6.1. Polypeptide sequences are also compared using FASTA with default or recommended parameters; the programs in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) yields alignment of the region of optimal overlap between the query and the search sequence and percent sequence identity (supra, Pearson (2000)). Another preferred algorithm for comparing the sequences of the present disclosure to a database containing numerous sequences from different organisms is the computer program BLAST, particularly BLASTP or TBLASTN, with default parameters. See, for example, Altschul et al. (1990) J. Mol. Biol. 215: 403-410, and (1997) Nucleic Acids Res. 25: 3389-3402.

[0084] As used herein, the phrase "therapeutically effective amount" means an amount that produces the desired effect on the thing being administered. The amount will depend on the purpose of the treatment and will be elucidated by one of ordinary skill in the art using known techniques (see, for example, Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding).

[0085] As used herein, the term "subject" refers to an animal, preferably a mammal, more preferably a human, in need of amelioration, prevention, and / or treatment of a GLP1R-related disease or disorder, such as hypoglycemia.

[0086] As used herein, the terms "treating," "treatment," or "treat" refer to reducing or ameliorating the severity of at least one symptom or manifestation of a GLP1R-related disease or disorder (e.g., hypoglycemia) by administering a therapeutic agent, such as an antibody or antigen-binding fragment thereof of the present disclosure, to a subject in need thereof. This term includes inhibiting the progression of the disease, or inhibiting the worsening of symptoms / manifestations. This term may also include a positive prognosis of the disease, i.e., the subject may be freed from the disease or the disease may be reduced upon administration of a therapeutic agent such as an antibody of the present disclosure. The therapeutic agent may be administered to the subject at a therapeutic dose.

[0087] The terms "preventing," "prevention," or "prevent" refer to inhibiting the development of a GLP1R-related disease or disorder (e.g., hypoglycemia), or any symptom or manifestation of such a disease or disorder (e.g., low glucose levels), upon administration of an antibody of the present disclosure.

[0088] Antigen-binding fragment of an antibody Unless otherwise specifically indicated, the term "antibody" as used herein is understood to encompass antibody molecules (i.e., "complete antibody molecules") comprising two immunoglobulin heavy chains and two immunoglobulin light chains, as well as antigen-binding fragments thereof. The terms "antigen-binding portion of an antibody", "antigen-binding fragment of an antibody", etc. as used herein include any naturally occurring, enzymatically obtained, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. The term "antigen-binding fragment of an antibody", or "antibody fragment" as used herein refers to one or more fragments of an antibody that retain the ability to specifically bind to the GLP1R protein. Antibody fragments include Fab fragments, F(ab')2 fragments, Fv fragments, dAb fragments, fragments containing CDRs, or isolated CDRs. In certain embodiments, the term "antigen-binding fragment" refers to a polypeptide fragment of a multispecific antigen-binding molecule. The antigen-binding fragment of an antibody can be obtained from a complete antibody molecule using any suitable standard techniques, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding the antibody variable and (optionally) constant domains. Such DNA is known and / or can be readily obtained, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or synthesized. The DNA is sequenced and manipulated chemically or using molecular biology techniques such that, for example, one or more variable and / or constant domains are arranged in an appropriate configuration, or codons are introduced, cysteine residues are created, amino acids are modified, added, or deleted.

[0089] Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab’)2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic the hypervariable regions of an antibody (e.g., isolated complementarity-determining regions (CDRs) such as CDR3 peptides), or limited FR3-CDR3-FR4 peptides. Domain-specific antibodies, single-domain antibodies, antibodies lacking domains, chimeric antibodies, antibodies with transplanted CDRs, bispecific antibodies, trispecific antibodies, tetravalent antibodies, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also encompassed by the term "antigen-binding fragment" as used herein.

[0090] An antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally includes at least one CDR and is adjacent to or in-frame with one or more framework sequences. V L domain-linked V H In an antigen-binding fragment having a V H and V L domain, the V H and V H domains are positioned relative to each other in any suitable arrangement. For example, the variable region is a dimer and contains V H -V L or V L -V L dimers. Alternatively, an antigen-binding fragment of an antibody contains a monomeric V H or V L domain.

[0091] In certain embodiments, an antigen-binding fragment of an antibody contains at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary arrangements of variable and constant domains found within the antigen-binding fragments of the antibodies of the present disclosure are: (i) V H -C H 1; (ii) V H -C H 2; (iii) V H -C H 3; (iv) V H -C H 1-C H 2; (v) V H -C H 1-C H 2-C H 3; (vi) V H -C H 2-C H 3; (vii) V H -C L ; (viii) V L -C H 1; (ix) V L -C H 2; (x) V L -C H 3; (xi) V L -C H 1-C H 2; (xii) V L -C H 1-C H 2-C H 3; (xiii) V L -C H 2-C H 3; and (xiv) V L -C LIt includes. In any arrangement of variable and constant domains, including any of the typical arrangements listed above, the variable and constant domains are either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region consists of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids and provides a mobile or semi-mobile linkage between adjacent variable and / or constant domains in a single polypeptide molecule. Further, the antigen-binding fragments of the antibodies of the present disclosure are non-covalently associated with each other and / or with one or more monomeric V H or V L domains and include homo-dimers or hetero-dimers (or other multimers) of any of the variable and constant domain arrangements listed above.

[0092] Similar to whole antibody molecules, the antigen-binding fragments are monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding fragments of antibodies typically include at least two different variable domains, where each variable domain has the ability to specifically bind to a different antigen or to different epitopes on the same antigen. Any multispecific antibody format, including the typical bispecific antibody formats disclosed herein, is adapted for use in the context of the antigen-binding fragments of the antibodies of the present disclosure using conventional techniques available in the art.

[0093] Preparation of Human Antibodies Methods for making human antibodies in transgenic mice are known in the art. Any such known method is used in the context of the present disclosure to make human antibodies that specifically bind to GLP1R.

[0094] An antibody against the GLP1R protein is prepared using an immunogen comprising any one of the following. In certain embodiments, the antibodies disclosed herein are obtained from mice immunized with the full-length native GLP1R protein, or DNA encoding the protein or a fragment thereof. Alternatively, the protein or a fragment thereof can be produced and modified using standard biochemical techniques and used as an immunogen. In certain embodiments, the immunogen may be a recombinant GLP1R protein or a fragment thereof expressed in other optional eukaryotic or mammalian cells such as Escherichia coli or Chinese hamster ovary (CHO) cells.

[0095] Using the VELOCIMMUNE® technology (e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®), or any other known method for making monoclonal antibodies, a high-affinity chimeric antibody against GLP1R having human variable regions and mouse constant regions is first isolated. The VELOCIMMUNE® technology involves the generation of transgenic mice having a genome comprising human heavy and light chain variable regions operably linked to the endogenous mouse constant region locus such that the mouse produces antibodies comprising human variable regions and mouse constant regions in response to antigen stimulation. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0096] Generally, VELOCIMMUNE® mice are loaded with the antigen of interest, and lymphocytes (e.g., B cells) are recovered from the mice that express the antibody. The lymphocytes are fused with a myeloma cell line to prepare an immortal hybridoma cell line, and such hybridoma cell lines are screened and selected to identify a hybridoma cell line that produces an antibody specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains is isolated and ligated to the constant regions of the desired isotype of the heavy and light chains. Such antibody proteins are produced in cells such as CHO cells. Alternatively, antigen-specific chimeric antibodies, or DNA encoding the variable domains of the light and heavy chains, are isolated directly from antigen-specific lymphocytes.

[0097] First, high-affinity chimeric antibodies having human variable regions and mouse constant regions are isolated. As in the experimental sections below, the antibodies are characterized and selected for desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are replaced with the desired human constant regions to generate the fully human antibodies of the present disclosure, such as wild-type, or modified IgG1 or IgG4. While the selected constant regions vary according to the particular use, the high-affinity antigen binding and target specificity characteristics reside in the variable regions.

[0098] Biological equivalents The anti-GLP1R antagonist antibodies and antibody fragments of the present disclosure include proteins that have amino acid sequences different from the described antibodies but maintain the ability to bind to the GLP1R protein. Such variant antibodies and antibody fragments contain one or more additions, deletions, or substitutions of amino acids when compared to the parental sequence, but exhibit biological activity that is substantially equivalent to that of the described antibodies. Similarly, antibodies encoding the DNA sequences of the present disclosure include sequences that contain one or more additions, deletions, or substitutions of nucleotides when compared to the disclosed sequences, but encode antibodies or antibody fragments that are substantially biological equivalents of the antibodies or antibody fragments disclosed herein.

[0099] Two antigen-binding proteins, or antibodies, are considered to be biological equivalents if, for example, they are pharmaceutical equivalents or pharmaceutical alternatives that do not show significant differences in the rate and extent of absorption when administered at the same molar dose, either as a single dose or multiple doses, under similar experimental conditions. If an antibody is equivalent in terms of the extent of its absorption but not in terms of the rate of its absorption, it is considered an equivalent or a pharmaceutical alternative, and such a difference in the rate of absorption is intentional and reflected in the labeling and is considered not to be pharmaceutically significant for the particular drug product being studied, as it is not essential for achieving the effective body drug concentration during chronic use, and is still considered to be a biological equivalent.

[0100] In one embodiment, two antigen-binding proteins are biological equivalents if there are no clinically significant differences in the safety, purity, or efficacy of the two antigen-binding proteins.

[0101] In one embodiment, two antigen-binding proteins are biological equivalents if a patient can be switched one or more times between a reference product and a biological product without expecting a clinically significant change in immunogenicity or an increased risk of side effects, including impaired efficacy, compared to a therapy that is continued without switching.

[0102] In one embodiment, two antigen-binding proteins are biological equivalents if both act by the normal mechanism or mechanisms of action for the conditions of use or multiple conditions, as long as such mechanisms are known.

[0103] Biological equivalents are demonstrated by in vivo and / or in vitro methods. Measurement of biological equivalents includes, for example, (a) in vivo tests in humans or other mammals where the concentration of an antibody or its metabolite is measured as a function of time in blood, plasma, serum, or other biological fluids; (b) in vitro tests that correlate with and adequately predict human in vivo bioavailability data; (c) in vivo tests in humans or other mammals where the appropriate acute pharmacological effect of an antibody (or its target) is measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, bioavailability, or biological equivalence of an antibody.

[0104] Biological equivalent variants of the antibodies disclosed herein are constructed, for example, by making various substitutions of residues or sequences, or deleting terminal or internal residues or sequences not required for biological activity. For example, cysteine residues that are not essential for biological activity are deleted or substituted with other amino acids to prevent the formation of unwanted or inaccurate intramolecular disulfide bridges upon regeneration. In other contexts, biological equivalent antibodies include antibody variants that contain amino acid changes that modify the glycosylation characteristics of the antibody, such as removing or modifying mutations that remove glycosylation.

[0105] Biological characteristics of the antibody Generally, the antibodies of the present disclosure function by binding to the GLP1R protein and reducing its activity. For example, the present disclosure uses an assay format as defined, for example, in Example 3 herein, and measures a K of less than 20 nM by surface plasmon resonance. DComprising antibodies and antigen-binding fragments of antibodies that bind to monomeric human GLP1R protein at (e.g., at 25°C or 37°C). In certain embodiments, these antibodies or their antigen-binding fragments bind to GLP1R with a K of less than about 20 nM, less than about 18 nM, less than about 10 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by surface plasmon resonance or a substantially similar assay using an assay format as defined in Example 3 herein. D and bind to GLP1R.

[0106] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to monomeric cynomolgus GLP1R at (e.g., at 25°C or 37°C) with a K of less than 60 nM, as measured by surface plasmon resonance using an assay format as defined in Example 3 herein. D Comprising antibodies and antigen-binding fragments of antibodies that bind to GLP1R with a K of less than about 60 nM, less than about 20 nM, less than about 2 nM, less than about 1 nM, or less than about 0.5 nM, as measured by surface plasmon resonance or a substantially similar assay using an assay format as defined in Example 3 herein. D and bind to GLP1R.

[0107] The present disclosure also provides antibodies and antigen-binding fragments thereof that bind to hdesA-GLP1_GLP1R-MMH at (e.g., at 25°C or 37°C) with a K of less than 75 nM, as measured by surface plasmon resonance using an assay format as defined in Example 3 herein. D Comprising antibodies and antigen-binding fragments of antibodies that bind to hdesA-GLP1_GLP1R-MMH with a K of less than about 75 nM, less than about 70 nM, less than about 60 nM, less than about 40 nM, less than about 20 nM, or less than about 5 nM at 25°C or 37°C, as measured by surface plasmon resonance or a substantially similar assay using an assay format as defined in Example 3 herein. Dand binds to GLP1R.

[0108] The present disclosure also provides an antibody that binds to monomeric mouse GLP1R (e.g., at 25° C. or 37° C.) with a K of less than about 75 nM, as measured by surface plasmon resonance using an assay format as defined in Example 3 herein. D and antigen-binding fragments thereof. In certain embodiments, these antibodies or antigen-binding fragments thereof bind to GLP1R with a K of less than about 75 nM, less than about 50 nM, less than about 30 nM, less than about 20 nM, or less than about 11 nM at 25° C. or 37° C., as measured by surface plasmon resonance using an assay format as defined in Example 3 herein or by a substantially similar assay. D and binds to GLP1R.

[0109] The present disclosure also provides an antibody and antigen-binding fragment that blocks the interaction between GLP1 and GLP1R with an IC value of less than about 10 nM in an in vitro receptor / ligand binding assay measurement at 37° C., as described in Example 4 herein. 50 In certain embodiments, these antibodies or antigen-binding fragments thereof block the interaction between GLP1 and GLP1R with an IC value of less than about 10 nM, less than about 7 nM, or less than about 2 nM when measured in an in vitro receptor / ligand binding assay at 37° C. or a substantially similar assay as described in Example 4 herein. 50 value.

[0110] The present disclosure also includes an antibody or an antigen-binding fragment thereof that binds to GLP1R, raises blood glucose levels, and reduces GLP1-induced hypoglycemia without causing hyperglycemia when administered to a subject in need thereof, as shown, for example, in Example 5 or 6 of this specification. In certain embodiments, the antibodies and antigen-binding fragments of antibodies disclosed herein raise blood glucose to a normalizing level. In certain embodiments, the antibodies and antigen-binding fragments of antibodies disclosed herein raise blood glucose from an initial level of less than 55 mg / dL to a normalizing level (e.g., greater than 70 mg / dL). In such embodiments, raising blood glucose to a normalizing level does not cause hyperglycemia.

[0111] The antibodies of the present disclosure can have one or more of the aforementioned biological properties, or any combination thereof. Other biological properties of the antibodies of the present disclosure will be apparent to those skilled in the art from consideration of the present disclosure, including the examples herein.

[0112] Epitope mapping and related techniques The present disclosure includes anti-GLP1R antagonist antibodies and antigen-binding fragments thereof that interact with one or more amino acids found within one or more regions of the GLP1R protein molecule. The epitope to which the antibody binds consists of a single continuous sequence of three or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids located in any of the aforementioned domains of the GLP1R protein molecule (e.g., a linear epitope in the domain). Alternatively, the epitope consists of a number of non-contiguous amino acids (or amino acid sequences) located in any or both of the aforementioned domains of the protein molecule (e.g., a conformational epitope).

[0113] Using various techniques known to those skilled in the art, it is determined whether an antibody "interacts with one or more amino acids within a polypeptide or protein". Typical techniques include, for example, conventional cross-blocking assays such as those described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY). Other methods include alanine scanning mutagenesis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide cleavage analysis, crystal studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of the antigen are utilized (Tomer (2000) Prot. Sci. 9:487-496). Another method used to identify the amino acids within a polypeptide with which an antibody interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling the protein of interest with deuterium, followed by binding the antibody to the deuterium-labeled protein. The protein / antibody complex is then transferred to water, and the exchangeable protons within the amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than the exchangeable protons within amino acids that are not part of the contact surface. As a result, the amino acids that form part of the protein / antibody contact surface retain deuterium and thus exhibit a relatively large mass compared to the amino acids not included in the contact surface. After dissociation of the antibody, the target protein is subjected to proteolytic enzyme cleavage and mass spectrometry, thereby revealing the deuterium-labeled residues corresponding to the specific amino acids with which the antibody interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0114] The term "epitope" refers to the site on an antigen to which B and / or T cells respond. B cell epitopes are formed from both contiguous and non-contiguous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes typically contain at least 3 amino acids, more usually at least 5 or 8 - 10 amino acids, in their native three-dimensional structure.

[0115] Modified-assisted profiling (MAP), also known as antigen-structure-based antibody profiling (ASAP), is a method of classifying multiple monoclonal antibodies (mAbs) directed against the same antigen based on the similarity of the binding profiles of each antibody to a chemically or enzymatically modified antigen surface (US2004 / 0101920, incorporated herein by reference in its entirety). Each category reflects a distinct epitope that is clearly different from or partially overlapping with the epitope represented by another category. This technique enables the rapid selection of genetically identical antibodies such that the focus is on antibodies that are genetically well-defined. When applied to hybridoma screening, MAP facilitates the identification of rare hybridoma clones that produce mAbs with desired characteristics. Using MAP, the antibodies of the present disclosure are classified into groups of antibodies that bind to different epitopes.

[0116] In certain embodiments, the present disclosure includes anti-GLP1R antibodies and antigen-binding fragments thereof that interact with one or more epitopes found within the extracellular domain of GLP1R. The epitope may consist of one or more contiguous sequences of 3 or more (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) amino acids found within the extracellular domain of GLP1R. Alternatively, the epitope may consist of a plurality of non-contiguous amino acids (or amino acid sequences) located within the extracellular domain of GLP1R.

[0117] The present disclosure includes anti-GLP1R antibodies that bind to the same epitope, or a portion of the epitope, as any of the specific exemplary antibodies listed in Table 1. Similarly, the present disclosure also includes anti-GLP1R antibodies that compete with any of the specific exemplary antibodies listed in Table 1 for binding to the GLP1R protein or a fragment thereof. For example, the present disclosure includes anti-GLP1R antibodies that cross-compete with one or more of the antibodies listed in Table 1 for binding to the GLP1R protein.

[0118] By using conventional methods known in the art, it can be readily determined whether an antibody binds to the same epitope as a reference anti-GLP1R antibody or competes with the reference anti-GLP1R antibody for binding. For example, to determine whether a test antibody binds to the same epitope as a reference anti-GLP1R antibody of the present disclosure, it is possible to bind the reference antibody to the GLP1R protein or peptide under saturating conditions. Next, the ability of the test antibody to bind to the GLP1R protein molecule is evaluated. If the test antibody can bind to GLP1R after saturating binding with the reference anti-GLP1R antibody, it is concluded that the test antibody binds to an epitope different from the reference anti-GLP1R antibody. On the other hand, if the test antibody cannot bind to the GLP1R protein after saturating binding with the reference anti-GLP1R antibody, then the test antibody binds to the same epitope as the epitope bound by the reference anti-GLP1R antibody of the present disclosure.

[0119] To determine whether an antibody competes with a reference anti-GLP1R antibody for binding, the binding methodology described above is performed in two ways. In the first approach, the reference antibody is allowed to bind to the GLP1R protein under saturation conditions, and subsequently, the binding of the test antibody to the GLP1R molecule is evaluated. In the second approach, the test antibody is allowed to bind to the GLP1R molecule under saturation conditions, and subsequently, the binding of the reference antibody to the GLP1R molecule is evaluated. In both approaches, if only the first (saturating) antibody has the ability to bind to the GLP1R molecule, then the test antibody and the reference antibody are concluded to compete for binding to GLP1R. As will be understood by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily bind to the same epitope as the reference antibody, but rather sterically blocks the binding of the reference antibody by binding to overlapping or adjacent epitopes.

[0120] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other to its antigen. That is, a 1-, 5-, 10-, 20-, or 100-fold excess of one antibody inhibits the binding of the other by at least 50%, but preferably 75%, 90%, or even 99%, as measured in a competitive binding assay (see, for example, Junghans et al., Cancer Res. 1990, 50:1495-1502). Alternatively, two antibodies have the same epitope if essentially all amino acid mutations in an antigen that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other. Two antibodies have overlapping epitopes if some amino acid mutations that reduce or eliminate the binding of one antibody also reduce or eliminate the binding of the other.

[0121] Next, further conventional experiments (e.g., peptide mutations, and binding assays) are performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same epitope as the reference antibody, or whether steric hindrance (or another phenomenon) is involved in the observed lack of binding. This screening experiment is performed using ELISA, RIA, surface plasmon resonance, flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0122] In certain embodiments, the disclosure provides an isolated antibody or antigen-binding fragment thereof that specifically binds to a GLP1R protein and that interacts with one or more amino acids contained within the extracellular domain of GLP1R as determined by hydrogen / deuterium exchange and that binds to and inactivates GLP1R.

[0123] Immunoconjugate The disclosure includes a human anti-GLP1R monoclonal antibody conjugated to a therapeutic moiety (an "immunoconjugate") for treating a GLP1R-related disease or disorder (e.g., hypoglycemia). As used herein, the term "immunoconjugate" refers to an antibody that is chemically or biologically conjugated to a radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antibody can be conjugated to the radioactive agent, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule so long as it can bind to its target. Examples of immunoconjugates include antibody-drug conjugates, and fusion proteins of antibodies and toxins. In one embodiment, the agent is a second, different antibody to the GLP1R protein. The type of therapeutic moiety conjugated to the anti-GLP1R antibody is considered in view of the condition to be treated and the desired therapeutic effect to be achieved. Examples of agents suitable for forming immunoconjugates are known in the art; see, e.g., WO05 / 103081.

[0124] Multispecific antibody The antibodies of the present disclosure are monospecific, bispecific, or multispecific. Multispecific antibodies are specific for different epitopes of one target polypeptide or contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004, Trends Biotechnol. 22:238-244.

[0125] Any of the multispecific antigen-binding molecules of the present disclosure, or variants thereof, are constructed using standard molecular biology techniques (e.g., recombinant DNA and protein expression techniques) known to those of skill in the art.

[0126] In certain embodiments, GLP1R-specific antagonist antibodies are made in a bispecific format ("bispecific") in which variable regions that bind to separate domains of the GLP1R protein are joined together to confer dual-domain specificity within a single binding molecule. Suitably, the designed bispecificity enhances the overall GLP1R protein inhibitory efficacy through both increased specificity and binding activity. Variable regions having specificity for an individual domain (e.g., a segment of the N-terminal domain) or binding to different regions within one domain are paired on a structural scaffold such that each region is capable of simultaneously binding to a distinct epitope or different regions within one domain. In one example of bispecificity, a heavy chain variable region (V H ) from a binder having specificity for one domain is recombined with a series of light chain variable regions (V L ) from binders having specificity for a second domain and paired with the original V H such that the original specificity for the V H is not disrupted and a non-cognate V L partner is identified. In this method, a single V L segment (e.g., V L 1) is paired with two different V H domains (e.g., VH 1, and V H 2) combined with two binding "arms" (V H 1-V L 1, and V H 2-V L 1) to produce bispecificity. The use of a single V L segment reduces the complexity of the system, thereby increasing the efficiency in the cloning, expression, and purification methods used to simplify and produce bispecificity. See, for example, US2011 / 0195454, and US2010 / 0331527.

[0127] Alternatively, more than one domain, and non-limitingly, for example, an antibody that binds to a second target such as a second different anti-GLP1R antibody, is prepared in a bispecific format using the techniques described herein or other techniques known to those skilled in the art. Antibody variable regions that bind to distinct regions are combined, for example, with a variable region that binds to a relevant site on the extracellular domain of GLP1R, to ensure bis-antigen specificity within a single binding molecule. Suitably, the designed bispecificity of this nature serves a dual function. The variable region having specificity for the extracellular domain pairs on a structural scaffold with a variable region having specificity for outside the extracellular domain, each variable region being capable of binding to a different antigen.

[0128] A typical bispecific antibody format used in the context of the present disclosure involves the use of a first immunoglobulin (Ig) C H 3 domain, and a second Ig C H 3 domain, where the first and second Ig C H 3 domains differ from each other by at least one amino acid, where at least one amino acid difference reduces the binding of the bispecific antibody to protein A compared to a bispecific antibody lacking the amino acid difference. In one embodiment, the first Ig C H 3 domain binds to protein A, and the second Ig C HThe 3 domains contain mutations that reduce or abolish protein A binding, such as the H95R modification (according to the IMGT exon numbering; H435R according to the EU numbering). The second C H 3 further includes the Y96F modification (according to IMGT; Y436F according to EU). The second C H Further modifications found within the second C3 include: for IgG1 antibodies, D16E, L18M, N44S, K52N, V57M, and V82I (according to IMGT; according to EU, D356E, L358M, N384S, K392N, V397M, and V422I); for IgG2 antibodies, N44S, K52N, and V82I (IMGT; according to EU, N384S, K392N, and V422I); and for IgG4 antibodies, Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (according to IMGT; according to EU, Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I). Variations in the bispecific antibody formats described above are considered to be within the scope of the present disclosure.

[0129] Other exemplary bispecific formats used in the context of the present disclosure include, without limitation, for example, scFv-based or bispecific antibody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-Ig, quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgG1 / IgG2, dual action Fab (DAF)-IgG, and Mab 2Including bispecific formats (see, for example, Klein et al., 2012, mAbs 4:6, 1-11, and references cited therein for descriptions of such formats). Bispecific antibodies can also be constructed using peptide / nucleic acid binding, for example, here using unnatural amino acids with orthogonal chemical reactivity to generate site-specific antibody-oligonucleotide binding, which then self-assembles into multimeric complexes with defined composition, valence, and geometry. See, for example, Kazane et al., J. Am. Chem. Soc. [Epub: December 4, 2012].

[0130] Therapeutic Administration and Formulations The present disclosure provides a therapeutic composition comprising a GLP1R antagonist antibody of the present disclosure or an antigen-binding fragment thereof. The therapeutic composition is administered with suitable carriers, excipients, and other agents incorporated into the formulation to effect transport, delivery, improvement of tolerance, etc., in accordance with the present disclosure. A number of suitable formulations can be found in the formulary known to all pharmaceutical chemists: Remington’s Pharmaceutical Sciences, Mack Publishing Company, Easton, PA. These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. See also Powell et al., “Compendium of excipients for parenteral formulations” PDA (1998) J Pharm Sci Technol 52:238-311.

[0131] The dosage of the antibody or its antigen-binding fragment may vary depending on the age and body size of the subject to be administered, the target disease, condition, route of administration, and the like. When the antibody or its antigen-binding fragment of the present disclosure is used to treat a disease or disorder or for the prevention of such a disease in an adult patient, it is generally advantageous to administer the antibody or its antigen-binding fragment of the present disclosure at a single dose of about 0.1 to about 100 mg per kg of body weight. Depending on the severity of the condition, the frequency and duration of treatment are adjusted. In certain embodiments, the antibody or its antigen-binding fragment of the present disclosure is administered as a starting dose of at least about 0.1 mg to about 800 mg, about 1 to about 600 mg, about 5 to about 500 mg, or about 10 to about 400 mg. In certain embodiments, following the starting dose, a second or multiple subsequent doses of the antibody or its antigen-binding fragment in an amount approximately the same as or less than that of the starting dose are administered, where the subsequent doses are separated by at least 1 day to 3 days; at least 1 week; at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0132] A variety of delivery systems are known and can be used to encapsulate the pharmaceutical compositions of the present disclosure, for example, in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis is administered (see, for example, Wu et al. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition can be administered by any convenient route, for example, by injection or bolus injection, or by absorption through epithelial or skin mucosa (such as oral mucosa, rectum, and intestinal mucosa), and can be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in vesicles, particularly liposomes (see, for example, Langer (1990) Science 249:1527-1533).

[0133] The use of nanoparticles for delivering the antibodies and antigen-binding fragments thereof of the present disclosure is also contemplated herein. Antibody-conjugated nanoparticles are used for both therapeutic and diagnostic applications. Antibody-conjugated nanoparticles, as well as methods of preparation and use, are described in more detail by Arruebo et al., 2009, "Antibody-conjugated nanoparticles for biomedical applications", J. Nanomat, 2009, Article No. 439389, page 24. Nanoparticles are developed and conjugated to antibodies contained in pharmaceutical compositions for targeting cells. Nanoparticles for drug delivery are also described, for example, in US8257740 or US8246995.

[0134] In certain situations, the pharmaceutical composition is delivered in a controlled-release system. In one embodiment, a pump is used. In another embodiment, a polymeric material is used. In yet another embodiment, the controlled-release system is placed near the target of the composition, and thus only a small systemic dose is required.

[0135] Injectable preparations include forms for intravenous, subcutaneous, intracranial, intraperitoneal, and intramuscular injection, forms for intravenous drip infusion, etc. These injectable preparations are prepared by publicly known methods.

[0136] The pharmaceutical compositions of the present disclosure are delivered subcutaneously or intravenously with standard needles and syringes. In addition, with respect to subcutaneous delivery, pen delivery devices are readily applicable in the delivery of the pharmaceutical compositions of the present disclosure. Such pen delivery devices are either reusable or disposable. Reusable pen delivery devices generally utilize replaceable cartridges containing the pharmaceutical composition. When all of the pharmaceutical composition in the cartridge has been administered, the cartridge becomes empty, the empty cartridge is easily discarded, and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device is then reused. In disposable pen delivery devices, there are no replaceable cartridges. Rather, disposable pen delivery devices are pre-filled with the pharmaceutical composition held in a reservoir within the device. When the pharmaceutical composition is empty, the entire device is discarded.

[0137] Advantageously, the pharmaceutical compositions for oral or parenteral use described above are prepared in dosage forms in unit doses suitable to match the dosage of the active ingredient. Such dosage forms in unit doses include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of antibody contained is generally, in a unit dose; in particular, in the form of injection, preferably about 5 to about 500 mg of antibody per dosage form, about 5 to about 300 mg for other dosage forms, and about 10 to about 300 mg.

[0138] Therapeutic use of the antibody The antibodies and antigen-binding fragments thereof of the present disclosure are useful for the treatment, and / or prevention, and / or amelioration of at least one symptom associated with a disease or disorder or condition related to GLP1R. In certain embodiments, the antibodies or antigen-binding fragments thereof of the present disclosure can be administered at a therapeutically effective dose to a patient having a disease, disorder, or condition related to GLP1R. Non-limiting examples of diseases, disorders, or conditions that can be treated, prevented, or alleviated using the antibodies and antigen-binding fragments thereof of the present disclosure include hypoglycemia of any origin, such as hypoglycemia resulting from PBH or other upper abdominal surgeries (e.g., esophagectomy, gastrectomy for gastric cancer and peptic ulcer), hyperinsulinemia (e.g., congenital hyperinsulinemia), recurrent hypoglycemia, postprandial hypoglycemia, excessive insulin secretion in patients with recurrent hypoglycemia after gastric bypass, and hypoglycemia as a symptom of late dumping syndrome.

[0139] Hypoglycemia (e.g., PBH) is a disorder characterized by hypoglycemia with or without an increase in insulin levels. In PBH, hypoglycemia typically occurs in the subject 1 to 3 hours after a meal. Mild to moderate hypoglycemia presents as hypoglycemic symptoms confirmed at a blood glucose concentration <70 mg / dL. Severe hypoglycemia presents as neuroglycopenia confirmed at a blood glucose concentration <55 mg / dL. Neuroglycopenic symptoms include confusion, decreased concentration, fatigue, ataxia, paralysis, seizures, or loss of consciousness. Other symptoms, such as vasomotor symptoms (e.g., sweating and tremor), and / or adrenal symptoms (e.g., palpitations), can also occur in subjects with hypoglycemia.

[0140] In certain embodiments, the antibodies and antigen-binding fragments thereof of the present disclosure are useful for the treatment, alleviation, or prevention of hypoglycemia, such as hypoglycemia resulting from esophagectomy, gastrectomy, or other upper abdominal surgeries for PBH or gastric cancer and peptic ulcer, or hypoglycemia resulting from intrinsic etiologies such as genetic abnormalities. Also contemplated herein is the prophylactic use of one or more antibodies of the present disclosure in a subject at risk of having low glucose levels or hypoglycemia.

[0141] In one embodiment, the antibodies and antigen-binding fragments thereof are used in the preparation of a pharmaceutical composition or medicament for treating a patient suffering from a disease, disorder, or condition (e.g., low glucose levels or hypoglycemia) disclosed herein. In other embodiments, the antibodies and antigen-binding fragments of the present invention are used as adjuvant therapy or any other therapy with any other agent known to those of skill in the art useful for treating or ameliorating a disease, disorder, or condition (e.g., low glucose levels or hypoglycemia) disclosed herein.

Examples

[0142] The following examples are set forth to provide a complete disclosure and description of how to make and use the methods and compositions of the present disclosure to those of skill in the art and are not intended to limit the scope of what the inventors regard as their invention. Similarly, the present disclosure is not limited to any particular preferred embodiment described herein. Indeed, modifications and variations of the embodiments may be apparent to those of skill in the art upon reading this specification and can be made without departing from the spirit and scope thereof. Although attempts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), some experimental error and deviation should, of course, be allowed for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric.

Examples

[0143] Example 1: Preparation of Human Antibodies Against Glucagon-like Peptide 1 Receptor (GLP1R) Human antibodies against the GLP1R protein, which contain DNA encoding the human immunoglobulin heavy and κ light chain variable regions, were prepared in VELOCIMMUNE® mice. These mice were immunized with a stabilized full-length GLP1R protein.

[0144] The antibody immune response was monitored by a GLP1R-specific immunoassay. Once the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to retain viability and form hybridoma cell lines. The hybridoma cell lines were screened and selected to identify cell lines that produce GLP1R-specific antibodies. Using the cell lines, several anti-GLP1R chimeric antibodies (for example, antibodies having human variable domains and mouse constant domains) were obtained.

[0145] Anti-GLP1R antibodies were also isolated directly from antigen-positive mouse B cells (described in US Patent Application Publication No. 2007 / 10280945). Using this method, several fully human anti-GLP1R antibodies (that is, antibodies having human variable domains and human constant domains) were obtained.

[0146] Exemplary antibodies prepared as disclosed above were designated mAb36986, mAb37639, and mAb37645. The biological properties of the exemplary antibodies prepared according to the method of this example are further described in the following examples.

Examples

[0147] Example 2: Amino Acid Sequences and Nucleotide Sequences of Heavy and Light Chain Variable Regions Table 1 lists the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of selected anti-GLP1R antagonist antibodies of the present disclosure.

[0148]

Table 1

[0149] The corresponding nucleic acid sequence identifiers are listed in Table 2.

[0150]

Table 2

[0151] The antibodies referred to in this specification typically have fully human variable regions, but may have human or mouse constant regions. As will be understood by those skilled in the art, antibodies having a particular Fc isotype can be converted to antibodies having a different Fc isotype (e.g., an antibody having a mouse IgG1 Fc can be converted to an antibody having a human IgG4, etc.). In any case, the variable domains (including CDRs) indicated by the identification names represented by the numbers shown in Table 2 remain the same, and it was predicted that the antigen-binding properties would be identical or substantially similar regardless of the nature of the Fc domain. In certain embodiments, a selected antibody having a mouse IgG1 Fc was converted to an antibody having a human IgG4 Fc.

[0152] Table 3 lists the amino acid sequence identifiers of the heavy and light chains of the selected anti-GLP1R antibodies having a human IgG4 Fc.

[0153]

Table 3

Examples

[0154] Example 3: GLP1R Monoclonal Antibodies That Bind to GLP1R as Determined by Surface Plasmon Resonance at 25°C and 37°C The equilibrium dissociation constant (K D) was determined using real-time surface plasmon resonance with a Biacore 4000 instrument. All binding tests were performed at 25 °C and 37 °C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET) running buffer. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a human Fc-specific mouse mAb to capture different GLP1R mAbs. Different concentrations (100, 25, and 6.25 nM) of the N-terminal region of human GLP1R-MMH (hGLP1R-MMH; SEQ ID NO: 59), human GLP1R (hGLP1R-mFc; SEQ ID NO: 63) expressed with mouse IgG2a Fc, an in-line fusion protein of human desA-GLP1 and GLP1R-MMH (hdesA-GLP1_GLP1R-MMH; SEQ ID NO: 62), cynomolgus monkey (Macaca fascicularis) GLP1R-MMH (mfGLP1R-MMH; SEQ ID NO: 60) or 100 nM mouse GLP1R-MMH (mGLP1R-MMH; SEQ ID NO: 61) prepared in HBS-ET running buffer were injected at a flow rate of 30 μL / min for 150 s onto the surface capturing the GLP1R mAb, and dissociation in HBS-ET running buffer was monitored for 10 min. At the end of each cycle, the GLP1R mAb capture surface was regenerated using a 12 s injection of 20 mM phosphoric acid.

[0155] Association rate (k a ) and dissociation rate (k d ) were determined by fitting the real-time binding sensorgram to a 1:1 binding model with mass transport limitation using Scrubber 2.0c curve fitting software. The binding dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

Equation

[0156] The kinetic parameters regarding the binding of GLP1R to the selected GLP1R mAbs at 25°C and 37°C are shown in Tables 4 to 13.

[0157]

Table 4

[0158]

Table 5

[0159]

Table 6

[0160]

Table 7

[0161]

Table 8

[0162]

Table 9

[0163]

Table 10

[0164]

Table 11

[0165]

Table 12

[0166]

Table 13

Example

[0167] Example 4: Functional inhibition of human GLP1R This example relates to the functional inhibition of human GLP1R in a cell-based bioassay using the following anti-GLP1R antagonist antibodies: mAb36986, mAb37639, and mAb37645, and HEK293 / FSC11 / pCDNA3.1+GLP1R+1nM GLP1 cells activated by hGLP1.

[0168] GLP1R is a member of the secretin family (class B) of G protein-coupled receptors (GPCRs). When the ligand GLP1 binds, GLP1R initiates a downstream signaling cascade via a G αS protein, which results in transcriptional regulation of genes (Donnelly, Br J Pharmacol, 166(1):27-41 (2011)). To evaluate the inhibition of GLP1R by anti-hGLP1R antibodies, a bioassay was established using HEK293 cells (human embryonic kidney 293, ATCC) transfected to stably express a reporter gene [cAMP response element (4X)-luciferase-lRES-GFP] together with full-length human GLP1R. The resulting cell line was named HEK293 / FSC11 / pCDNA3.1+GLP1R+1nM GLP (ACL#6822, Regeneron) and is hereafter referred to as HEK293 / CRE-luc / GLP1R.

[0169] For the bioassay, HEK293 / CRE-luc / GLP1R cells were seeded at 20,000 cells per well in assay medium (Opti-MEM medium containing 0.1% fetal bovine serum and 1X penicillin-streptomycin-glutamine) and incubated overnight at 37 °C and 5% CO2. The next day, the selected anti-GLP1R antibody or isotype control antibody was serially diluted 1:3 in assay medium (along with additional wells containing only assay medium without test molecule) from 300 nM to 5.08 pM and pre-incubated with HEK293 / CRE-luc / GLP1R cells for 30 minutes at 5% CO2 and 37 °C. Exendin-3(9-39) amide (Tocris, catalog number 2081), hereafter referred to as Exendin-9, was serially diluted 1:3 in assay medium (along with additional wells without test molecule) from 500 nM to 8.47 pM and pre-incubated with the cells for 30 minutes at 5% CO2 and 37 °C. After 30 minutes, GLP-1(7-36) amide (Phoenix Pharmaceuticals, catalog number 028-11), hereafter referred to as GLP-1, was added to the cells at a fixed concentration of 40 pM in assay medium. Also, GLP1 was serially diluted 1:3 in assay medium (along with additional wells without test molecule) from 100 nM to 1.69 pM and added to cells not treated with antibody or Exendin-9 for dose response of the ligand. After incubation at 5% CO2 and 37 °C for 5 hours, luciferase activity was evaluated by the addition of ONEGLO™ luciferase assay system reagent (Promega, catalog number E6130), and relative light units (RLU) were measured using an Envision Plate reader (Perkin Elmer). The results were analyzed using non-linear regression (4-parameter logistic) of PRISM® 7 software, and the EC 50 and IC 50 values were determined. The percent inhibition was calculated by the following formula:

Equation

[0170] In the above formula, "RLU GLP1" refers to the relative light unit (RLU) value from cells treated with 40 pM GLP1 without antibody. "RLU" 阻害 " refers to the lowest RLU value measured at the dose - response concentration of the antibody or Exendin - 9 with the maximum value together with 40 pM GLP1. "RLU" GLP1なし対照 " refers to the RLU value of cells measured in the absence of GLP1, Exendin - 9 or antibody.

[0171] Results: Three anti - GLP1R antibodies were tested for their inhibition of HEK293 / CRE - luc / GLP1R cells activated with 40 pM GLP1. As shown in Table 14, two anti - GLP1R antibodies, mAb36986 and mAb37639, showed 100% inhibition with IC 50 values of 1.69 nM and 6.55 nM respectively. mAb37645 inhibited GLP1R activation by 73% with an IC 50 value of 1.23 nM. The isotype - control mAb showed no inhibition. Exendin - 9 showed 100% inhibition with an IC 50 of 29.4 nM. GLP1 activated the cells with an EC 50 value of 44.6 pM.

[0172]

Table 14

Example

[0173] Example 5: Effect of GLP1R - antagonist antibodies on Exendin - 4 - induced hypoglycemia in GLP1R - humanized mice This example relates to in - vivo tests using the following anti - GLP1R antagonist antibodies: mAb36986, mAb37639, and mAb37645.

[0174] To determine the glucose-elevating effect of an anti-GLP1R antagonist antibody against GLP1-induced hypoglycemia, a single dose of the selected anti-GLP1R antibody was administered to 6-month-old male homozygous mice expressing human GLP1R instead of mouse GLP1R (GLP1R humanized mice), and they were challenged with the GLP1 analog Exendin-4 (Sigma, catalog number E7144) for the next 67 days periodically.

[0175] For the study, 35 mice were randomly divided into one group of 14 mice and three groups of 7 mice each. On day 0, the group of 14 (later divided into group 1 and group 2) received a subcutaneous (s.c.) injection of an mIgG2 isotype control antibody (referred to as the isotype control, dose 25 mg / kg), and the other three groups (n = 7 / group) received s.c. injections of mAb36986 (group 3), mAb37639 (group 4), or mAb37645 (group 5) (dose 25 mg / kg), respectively. The next day (day 1), the 14 mice that received the isotype control were randomly divided into two groups of 7 mice each (group 1, group 2). At the 0-minute time point on day 1, animals in group 1 received an intraperitoneal (i.p.) injection of saline, and animals in group 2 received an i.p. injection of Exendin-4 (dose 0.01 mg / kg). At the same time point, animals in groups 3, 4, and 5 received an i.p. injection of Exendin-4 (dose 0.01 mg / kg). For all animals, blood glucose levels were measured using a handheld glucometer immediately before and 15, 30, 60, and 120 minutes after the injection of saline or Exendin-4. All animals remained in their assigned groups until the end of the 67-day study. The Exendin-4 challenge performed on day 1 was repeated on days 8, 22, 46, and 67. The mean ± SEM of blood glucose levels at each time point was calculated for each group and shown in Table 15. The mean ± SEM of the percent differences in blood glucose levels from group 1 at each time point was calculated for each group and shown in Table 16. Statistical analysis was performed by two-way analysis of variance followed by Bonferroni post hoc test, comparing groups 2, 3, 4, and 5 with group 1.

[0176] In animals that received isotype control on day 0 and saline on the day of Exendin-4 challenge (Group 1), an increase in blood glucose level due to injection was shown at early time points (i.e., 15 minutes, 30 minutes, and 60 minutes), and it was consistent on day 1, day 8, day 22, day 46, and day 67. Animals that received isotype control on day 0 and Exendin-4 on the Exendin-4 challenge day (Group 2) showed a decrease in Exendin-4-induced blood glucose level on each day of the challenge. Animals that received mAb36986 on day 0 and Exendin-4 on day 1, day 8, day 22, day 46, and day 67 (Group 3) showed glucose levels similar to the measured levels of Group 1 at each time point on each day when Exendin-4 challenge was performed, except on the last day (day 67). This data indicated that a single high dose of mAb36986 in mice could improve GLP1-induced hypoglycemia for at least 46 days. There was no difference in blood glucose level between Group 1 and Group 3 at the 0-minute time point on each day. This data showed that mAb36986 did not cause hyperglycemia in mice under the tested conditions.

[0177] As shown in Figure 1, animals that received mAb37639 on day 0 and Exendin-4 on day 1, day 8, day 22, day 46, and day 67 (Group 4) showed glucose normalization only on day 1 and day 8, indicating that the action time of mAb37639 was shorter compared to mAb36986. Animals that received mAb37645 on day 0 and Exendin-4 on day 1, day 8, day 22, day 46, and day 67 (Group 5) did not show glucose normalization. Furthermore, it was shown that mAb36986 had a long-lasting action and improved GLP1-induced hypoglycemia without causing hyperglycemia in mice.

[0178]

Table 15

[0179]

Table 16

Example

[0180] Example 6: Effect of GLP1R antagonist antibody on GLP1-induced hypoglycemia in GLP1R humanized mice This example relates to a test conducted to determine the glucose-elevating efficacy and duration of action of the GLP1R antagonist antibody mAb36986 against GLP1-induced hypoglycemia. mAb36986 was administered to homozygous mice expressing human GLP1R instead of mouse GLP1R (hereinafter referred to as GLP1R humanized mice) and repeatedly challenged with Exendin-4 (Sigma, catalog number E7144), a GLP1 analog. In this example, the efficacy and duration of action of mAb36986 were compared with those of the peptide GLP1R antagonist Exendin(9-39) (Bachem, catalog number 4017799), hereinafter referred to as Exendin-9.

[0181] 2. Thirty-nine 5-month-old male GLP1R humanized mice were randomly divided into groups of 16 mice, two groups of 8 mice, and one group of 7 mice. On day 0, an isotype control antibody of hIgG4 (referred to as the isotype control, dosage 10 mg / kg) was administered subcutaneously (s.c.) to the group of 16 mice (Groups 1 and 2). Exendin-9 (dosage 10 mg / kg) was administered s.c. to one group of 8 mice (Group 3), and mAb36986 was administered s.c. (dosage 3 mg / kg) to the other group of 8 mice (Group 4). mAb36986 was administered s.c. (dosage 10 mg / kg) to the group of 7 mice (Group 5). The next day (day 1), the animals administered the isotype control were randomly divided into two groups of 8 mice (Groups 1 and 2). Twenty-four hours after the administration of the test substance (= 0 minute time point on day 1), physiological saline was administered intraperitoneally (i.p.) to the animals in Group 1, and Exendin-4 (dosage 0.01 mg / kg) was administered i.p. to the animals in Group 2. At the same time point, Exendin-4 (dosage 0.01 mg / kg) was administered i.p. to the animals in Groups 3, 4, and 5. For all animals, blood glucose levels were measured using a handheld glucometer immediately before the administration of physiological saline or Exendin-4, 15 minutes later, 30 minutes later, 60 minutes later, and 120 minutes later. All animals remained in the groups to which they were assigned until the completion of the test on day 80. The animals in Groups 1, 2, 4, and 5 were repeatedly challenged with Exendin-4 on days 3, 7, 15, 21, 29, 65, and 80. The animals in Group 3 were administered Exendin-9 once again on day 3, and the Exendin-4 challenge was performed 0.5 and 4.5 hours after the administration of Exendin-9.

[0182] For each Exending-4 challenge, the area under the curve (AUC) of blood glucose levels during the 120-minute challenge was calculated. The AUC value of each animal in each challenge was normalized to the mean AUC value of Group 1. The mean ± SEM of the normalized AUC was calculated for each group for each challenge and is shown in Table 17 and Figure 2. Statistical analysis was performed for each challenge by one-way analysis of variance followed by Bonferroni post hoc test to compare Group 1, Group 3, Group 4, or Group 5 with Group 2.

[0183] Isotype controls on Day 0 were compared with animals administered saline on the day of the Exendin-4 challenge (Group 1), and animals administered saline on the day of the Exendin-4 challenge (Group 2) induced a decrease in normalized glucose AUC on each day of the challenge. In animals administered Exendin-9 (Group 3), when the Exendin-4 challenge was performed 30 minutes after Exendin-9 administration, improvement in Exendin-4-induced hypoglycemia was shown, but no improvement was observed when the challenge was performed 4.5 hours or 24 hours after Exendin-9 administration. In animals administered mAb36986 at 3 mg / kg (Group 4), when the Exendin-4 challenge was performed 1, 3, 7, 15, 21, and 29 days after mAb36986 administration, improvement in Exendin-4-induced hypoglycemia was shown, but no improvement was observed when the challenge was performed on Day 65 and Day 80 after mAb36986 administration. Animals administered mAb36986 at 10 mg / kg (Group 5) showed improvement in Exendin-4-induced hypoglycemia when the Exendin-4 challenge was performed on Day 1, Day 3, Day 7, Day 15, Day 21, Day 29, and Day 65 after mAb36986 administration, but no improvement was observed when the challenge was performed on Day 80 after mAb36986 administration.

[0184] This data shows that a single administration of mAb36986 can improve GLP1-induced hypoglycemia in mice for 1 to 2 months, and that the duration of action is dose-dependent, while a single administration of Exendin-9 improves GLP-1-induced hypoglycemia within 4 hours. As a conclusion, mAb36986, a GLP1R antagonist antibody of the present disclosure, improved GLP1-induced hypoglycemia with a long-duration dose-dependent action.

[0185]

Table 17

[0186] The present disclosure should not be limited in scope by the specific embodiments described herein. Indeed, various modifications other than those described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

Claims

1. An isolated antibody or antigen-binding fragment thereof that specifically binds to a glucagon-like peptide 1 receptor (GLP1R) protein, wherein the antibody or antigen-binding fragment thereof attenuates GLP1R activity and increases blood glucose levels, the antibody or antigen-binding fragment thereof comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR); and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR), HCDR1 comprises the amino acid sequence of SEQ ID NO: 4, HCDR2 comprises the amino acid sequence of SEQ ID NO: 6, HCDR3 comprises the amino acid sequence of SEQ ID NO: 8, LCDR1 comprises the amino acid sequence of SEQ ID NO: 12, LCDR2 comprises the amino acid sequence of SEQ ID NO: 14, and LCDR3 comprises the amino acid sequence of SEQ ID NO: 16, the antibody or antigen-binding fragment thereof.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody is a fully human monoclonal antibody.

3. The antibody or antigen-binding fragment thereof according to claim 1 or 2, comprising an IgG1 or IgG4 isotype.

4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a heavy-chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:

2.

5. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, comprising a light-chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:

10.

6. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 2 / 10.

7. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising a heavy chain (HC) and a light chain (LC), wherein the HC comprises the amino acid sequence of SEQ ID NO:

18.

8. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, comprising an HC and an LC, wherein the LC comprises the amino acid sequence of SEQ ID NO:

20.

9. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, comprising an HC and an LC, wherein the HC comprises the amino acid sequence of SEQ ID NO: 18; and the LC comprises the amino acid sequence of SEQ ID NO:

20.

10. A pharmaceutical composition comprising an antibody that binds to GLP1R or an antigen-binding fragment thereof according to any one of claims 1 to 9, and a pharmaceutically acceptable carrier or diluent.

11. An isolated polynucleotide molecule comprising a polynucleotide sequence encoding the HCVVR and / or LCVR of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

12. An isolated vector comprising the polynucleotide molecule according to claim 11.

13. An isolated host cell expressing the vector according to claim 12.

14. A first vector comprising a polynucleotide sequence encoding the HCVVR of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and a second vector comprising a polynucleotide sequence encoding the LCVR of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and expressing the isolated host cell according to claim 13.

15. A method for producing an anti-GLP1R antagonist antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to claim 13 or 14 under conditions enabling the production of the antibody or fragment, and recovering the antibody or fragment thus produced.

16. The method according to claim 15, further comprising formulating the antibody or antigen-binding fragment as a pharmaceutical composition comprising an acceptable carrier.

17. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 for use in a method of treating, preventing or ameliorating at least one symptom or sign of a GLP1R-related disease or disorder in a subject in need thereof.

18. The pharmaceutical composition according to claim 17, wherein the GLP1R-related disease or disorder is hypoglycemia.

19. The pharmaceutical composition according to claim 18, wherein the hypoglycemia is post-bariatric surgery hypoglycemia.

20. The pharmaceutical composition according to any one of claims 17 to 19, wherein the pharmaceutical composition is administered to a subject after upper abdominal surgery.

21. The pharmaceutical composition according to any one of claims 17 to 20, wherein the pharmaceutical composition is administered prophylactically or therapeutically to a subject in need thereof.

22. The pharmaceutical composition according to any one of claims 17 to 21, wherein the pharmaceutical composition is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

23. A pharmaceutical composition comprising a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9 for use in a method of increasing blood glucose levels in a subject in need thereof.

24. The pharmaceutical composition according to claim 23, wherein the subject has a GLP1R-related disease or disorder.

25. The pharmaceutical composition according to claim 24, wherein the GLP1R-related disease or disorder is hypoglycemia.

26. The pharmaceutical composition according to claim 25, wherein the hypoglycemia is post-bariatric surgery hypoglycemia.

27. The pharmaceutical composition according to any one of claims 23 to 26, wherein the antibody or an antigen-binding fragment thereof is administered to the subject after upper abdominal surgery.

28. The pharmaceutical composition according to any one of claims 23 to 27, wherein the antibody or an antigen-binding fragment thereof is administered prophylactically or therapeutically to a subject in need thereof.

29. The pharmaceutical composition according to any one of claims 23 to 28, wherein the antibody or an antigen-binding fragment thereof is administered subcutaneously, intravenously, intradermally, intraperitoneally, or intramuscularly.

30. The pharmaceutical composition according to any one of claims 23 to 29, which increases the blood glucose level of the subject to a normal level.

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