Methods for treating severe insulin resistance by interfering with glucagon receptor signaling

Abstract

To provide methods for treating severe insulin resistance by interfering with glucagon receptor signaling.SOLUTION: A method for treating a patient with severe insulin resistance is provided. The method comprises administering a therapeutic amount of GCG / GCGR signaling pathway inhibitor to a patient in need thereof, thereby reducing blood glucose or beta-hydroxybutyrate levels, or mediating severe insulin resistance, or mediating condition or disease characterized by severe insulin resistance, or alleviating at least one symptom or complication associated with the condition or disease or severity thereof.SELECTED DRAWING: Figure 1A

Description

[Technical field]

[0001] The present invention relates to methods of treating or slowing the progression of severe insulin resistance and / or reducing the therapeutic dose of insulin in patients in need thereof using glucagon (GCG) inhibitors or glucagon receptor (GCGR) antagonists.

[0002] Sequence Listing Concurrently herewith, an official copy of the Sequence Listing is being submitted electronically via EFS-Web as an ASCII formatted Sequence Listing with filename 10282WO01_SEQ_LIST_ST25, creation date August 25, 2017, and size of approximately 116 kilobytes. The Sequence Listing contained in this ASCII formatted document is a part of this specification and is incorporated herein by reference in its entirety. [Background technology]

[0003] Glucagon is a 29-residue polypeptide hormone that, in cooperation with insulin, mediates homeostatic control of the amount of glucose in the blood. Glucagon acts primarily to maintain normal blood glucose levels by stimulating certain cells, such as liver cells, to release glucose when blood glucose levels fall. Glucagon's action is opposite to that of insulin, which stimulates cells to take up and store glucose whenever blood glucose levels rise. Glucagon is produced in the alpha cells of the pancreas, while insulin is secreted from nearby beta cells.

[0004] The imbalance of glucagon and insulin may play an important role in some diseases such as diabetes mellitus and diabetic ketoacidosis.In particular, studies have shown that higher basal glucagon levels and lack of suppression of postprandial glucagon secretion contribute to the diabetic state in humans (Muller et al. (1970), N Eng J Med, vol. 283: 109-115).

[0005] The effect of glucagon on increasing blood glucose levels is believed to be mediated in part by the activation of certain cellular pathways following the binding of glucagon (GCG) to its receptor (termed GCGR). GCGR is a member of the secretin subfamily (family B) of G protein-coupled receptors, which is expressed primarily in the liver. Binding of glucagon to its receptor triggers a G protein signal transduction cascade that activates intracellular cyclic AMP, leading to increased glucose production (gluconeogenesis) and glycogenolysis by de novo synthesis (Wakelam et al., 1986, Nature, 323:68-71; Unson et al., 1989, Peptides, 10:1171-1177; and Pittner and Fain, 1991, Biochem. J., 277:371-378).

[0006] The action of glucagon can be suppressed by providing an antagonist such as a small molecule inhibitor, a GCG antibody, or a GCGR antibody, as described herein.Anti-GCG antibody is mentioned in, for example, U.S. Patent No. 4,206,199, U.S. Patent No. 4,221,777, U.S. Patent No. 4,423,034, U.S. Patent No. 4,272,433, U.S. Patent No. 4,407,965, U.S. Patent No. 5,712,105, and PCT Publication No. WO2007 / 124463 and WO2013 / 081993. Anti-GCGR antibodies are described in U.S. Patent Nos. 5,770,445, 7,947,809, and 8,545,847, European Patent Application No. EP2074149(A2), European Patent No. EP0658200(B1), U.S. Patent Publication Nos. 2009 / 0041784, 2009 / 0252727, and 2011 / 0223160, and PCT Publication No. WO2008 / 036341. Small molecule inhibitors of GCG or GCGR are mentioned, for example, in WO07 / 47676, WO06 / 86488, WO05 / 123688, WO05 / 121097, WO06 / 14618, WO08 / 42223, WO08 / 98244, WO2010 / 98948, US20110306624, WO2010 / 98994, WO2010 / 88061, WO2010 / 71750, WO2010 / 30722, WO06 / 104826, WO05 / 65680, WO06 / 102067, WO06 / 17055, WO2011 / 07722, or WO09 / 140342. Severe insulin resistance syndrome is a rare metabolic disorder in which patients do not respond well to insulin. Current treatments available for severe insulin resistance syndrome include regular feeding and very high doses of insulin with the goal of achieving adequate glycemic control. Administration of IGF-I is effective in the short term but has failed to achieve long-term glycemic control in patients with severe insulin resistance. Vestergaard et al. (1997), European Journal of Endocrinology, 136:475-482. Administration of recombinant leptin has shown some success by lowering blood glucose levels over several months in patients with Rabson-Mendenhall syndrome (RMS). Cochran et al. (2004), Journal of Clinical Endocrinology and Metabolism, 89:1548-1554. Given the lack of effective therapies for treating severe insulin resistance diseases or slowing their progression, i.e., prolonging the life and / or improving the quality of life of patients with severe insulin resistance, there is a need to identify and explore the use of other agents to treat these diseases, such as the GCG / GCGR signaling pathway inhibitors and antagonists described herein. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Pat. No. 4,206,199 [Patent Document 2] U.S. Pat. No. 4,221,777 [Patent Document 3] U.S. Patent No. 5,770,445 [Patent Document 4] U.S. Pat. No. 7,947,809 [Non-patent literature]

[0008] [Non-Patent Document 1] Vestergaard et al., European Journal of Endocrinology (1997) 136:475-482 [Non-Patent Document 2] Cochran et al., Journal of Clinical Endocrinology and Metabolism (2004) 89:1548-1554 [Non-Patent Document 3] Wakelam et al. (1986), Nature 323:68-71 [Non-Patent Document 4] Unson et al. (1989), Peptides, 10:1171-1177 [Non-Patent Document 5] Pittner and Fain (1991), Biochem. J. 277:371-378. Summary of the Invention [Means for solving the problem]

[0009] Provided herein is a method for treating patients with conditions or diseases characterized by severe insulin resistance by administering a pharmaceutical composition that contains a GCG inhibitor or GCGR antagonist, for example.A GCG inhibitor or GCGR antagonist is a compound that can block or inhibit glucagon receptor signaling pathway.Antagonists can take the form of small molecule inhibitors, peptide inhibitors, CRISPR technology (clustered regularly interspaced short palindromic repeats; CRISPR technology can generate GCGR knockdown or deletion of regulatory sequences that affect GCGR activity), antisense inhibitors, DARPin, and GCG or GCGR neutralizing monoclonal antibodies. The GCG inhibitors or GCGR antagonists may be administered alone, in a pharmaceutical composition, or in combination with one or more therapeutic agents useful in treating a condition or disease associated with severe insulin resistance, or treating one or more symptoms associated with the condition or disease, or lowering blood glucose and / or ketones in a patient having a condition or disease associated with severe insulin resistance.

[0010] In some embodiments, methods are provided for lowering blood glucose levels and / or beta-hydroxybutyrate levels, or for reducing ketonemia and / or ketoacidosis, or for treating a condition or disease associated with or characterized in part by high blood glucose and / or ketonemia and / or ketoacidosis, or at least one symptom or complication associated with the condition or disease. In some aspects, the method comprises administering to a patient with severe insulin resistance a therapeutically effective amount of a composition comprising an inhibitor of GCG / GCGR signaling, thereby lowering blood glucose or beta-hydroxybutyrate levels, or mediating the condition or disease, or alleviating or reducing the severity of at least one symptom or complication associated with the condition or disease. In some embodiments, the inhibitor of GCGR signaling is a GCGR antagonist, such as an anti-GCGR antibody. In some embodiments, the anti-GCGR antibody has the HCVR / LCVR sequence pair of SEQ ID NO: 86 / 88. In some embodiments, the inhibitor of GCGR signaling is a GCG inhibitor, such as an anti-GCG antibody. In some embodiments, the anti-GCG antibody has the HCVR / LCVR sequence pair of SEQ ID NOs: 182 / 190. In some embodiments, the anti-GCG antibody has the HCVR / LCVR sequence pair of SEQ ID NOs: 166 / 174.

[0011] In some embodiments, a method is provided for treating a patient with severe insulin resistance, the patient exhibiting elevated blood glucose levels. The method comprises administering to the patient a therapeutically effective amount of a composition comprising a GCG inhibitor or a GCGR antagonist.

[0012] In some embodiments, a method is provided for treating a patient with severe insulin resistance, wherein the patient does not exhibit elevated blood glucose levels. The method comprises administering to the patient a therapeutically effective amount of a composition comprising a GCG inhibitor or a GCGR antagonist.

[0013] In some embodiments, a method is provided for reducing the amount and / or dosage of insulin required to treat a patient with severe insulin resistance, the patient exhibiting severe insulin resistance and / or elevated blood glucose levels. In some aspects, the method comprises administering to the patient a therapeutically effective amount of a composition comprising a GCG inhibitor or GCGR antagonist. In some aspects, the GCG inhibitor or GCGR antagonist is administered in combination with insulin. The amount and / or dosage of insulin may be reduced by about 30% to about 95%, or about 90%, when administered in combination with an isolated human monoclonal antibody that specifically binds to GCGR.

[0014] In some embodiments, the GCGR antagonist can be an anti-GCGR antibody. The anti-GCGR antibody can inhibit or antagonize GCGR. The anti-GCGR antibody can inhibit or block GCGR signal transduction pathway. In some embodiments, the GCG inhibitor can be an anti-GCG antibody. The anti-GCG antibody can inhibit GCG from binding to GCGR.

[0015] In certain embodiments, the antibody or antigen-binding fragment specifically binds to hGCGR and comprises heavy and light chain CDR domains contained in a heavy and light chain sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148.

[0016] In certain embodiments, the antibody or antigen-binding fragment comprises the heavy and light chain CDR domains contained in the HCVR / LCVR amino acid sequence pair of SEQ ID NO:86 / 88.

[0017] In certain embodiments, the antibody or antigen-binding fragment comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NOs:86 / 88.

[0018] In one embodiment, a human antibody or antigen-binding fragment of a human antibody that binds to hGCGR comprises a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0019] In one embodiment, a human antibody or antigen-binding fragment of a human antibody that binds to hGCGR comprises a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0020] In certain embodiments, a human antibody or fragment thereof that binds to hGCGR comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148. In certain embodiments, the HCVR / LCVR amino acid sequence pair is selected from the group consisting of SEQ ID NOs: 34 / 42, 70 / 78, 86 / 88, 110 / 118, and 126 / 128.

[0021] In certain embodiments, an isolated human antibody or antigen-binding fragment thereof that specifically binds to hGCGR comprises a HCVR comprising three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained in a HCVR sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or a LCVR comprising three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a LCVR sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148.

[0022] In certain embodiments, the methods provided herein contemplate the use of an isolated human antibody or antigen-binding fragment thereof that binds to hGCGR, comprising an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116, and 136, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and / or an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0023] In one embodiment, the method provided herein comprises: a) selecting an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112, and 132, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and b) selecting an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114, and 134, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; The use of an antibody or fragment thereof is contemplated, further comprising an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120, and 140, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122, and 142, or a substantially similar sequence thereof with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0024] In one embodiment, the antibody or antigen-binding fragment of the antibody comprises: (a) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116, and 136; (b) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144; Includes.

[0025] In a related embodiment, the antibody or antigen-binding fragment of the antibody comprises: (c) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112, and 132; (d) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114, and 134; (e) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120, and 140; (f) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122, and 142; Further includes:

[0026] In one embodiment, the antibody or antigen-binding fragment thereof comprises an HCDR1 domain having an amino acid sequence selected from one of SEQ ID NOs: 4, 20, 36, 52, 72, 92, 112, and 132; an HCDR2 domain having an amino acid sequence selected from one of SEQ ID NOs: 6, 22, 38, 54, 74, 94, 114, and 134; and an HCDR3 domain having an amino acid sequence selected from one of SEQ ID NOs: 8, 24, 40, 56, 76, 96, 116, and 136. and a LCVR comprising an LCDR1 domain having an amino acid sequence selected from one of SEQ ID NOs: 12, 28, 44, 60, 80, 100, 120, and 140, an LCDR2 domain having an amino acid sequence selected from one of SEQ ID NOs: 14, 30, 46, 62, 82, 102, 122, and 142, and an LCDR3 domain having an amino acid sequence selected from one of SEQ ID NOs: 16, 32, 48, 64, 84, 104, 124, and 144.

[0027] In certain embodiments, a human antibody or antigen-binding fragment of a human antibody that binds to human GCGR comprises an HCDR3 / LCDR3 amino acid sequence pair selected from the group consisting of SEQ ID NOs: 8 / 16, 24 / 32, 40 / 48, 56 / 64, 76 / 84, 86 / 88, 96 / 104, 116 / 124, and 136 / 144. Non-limiting examples of anti-GCGR antibodies having these HCDR3 / LCDR3 pairs are the antibodies designated H4H1345N, H4H1617N, H4H1765N, H4H1321B and H4H1321P, H4H1327B and H4H1327P, H4H1328B and H4H1328P, H4H1331B and H4H1331P, H4H1339B and H4H1339P, respectively.

[0028] In one embodiment, an isolated antibody or antigen-binding fragment thereof that specifically binds to GCG and neutralizes at least one activity associated with GCG, useful according to the methods provided herein, comprises (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0029] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to GCG and neutralizes at least one activity associated with GCG comprises a HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and a LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0030] In some embodiments, the isolated antibody or antigen-binding fragment thereof that specifically binds to GCG and neutralizes at least one activity associated with GCG comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302.

[0031] In some embodiments, the HCVR / LCVR amino acid sequence pair comprises SEQ ID NO: 166 / 174.

[0032] In some embodiments, the HCVR / LCVR amino acid sequence pair comprises SEQ ID NO: 182 / 190.

[0033] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 152, 168, 184, 200, 216, 232, 248, 264, 280, and 296; (b) an HCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 154, 170, 186, 202, 218, 234, 250, 266, 282, and 298; (c) an HCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 156, 172, 188, 204, 220, 236, 252, 268, 284, and 300; (d) an LCDR1 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 160, 176, 192, 208, 224, 240, 256, 272, 288, and 304; (e) an LCDR2 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 162, 178, 194, 210, 226, 242, 258, 274, 290, and 306; (f) an LCDR3 domain having an amino acid sequence selected from the group consisting of SEQ ID NOs: 164, 180, 196, 212, 228, 244, 260, 276, 292, and 308; Includes.

[0034] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 168; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 170; (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 172; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 176; and (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 178; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 180; Includes.

[0035] In one embodiment, an isolated antibody or antigen-binding fragment thereof useful in accordance with the methods provided herein comprises: (a) an HCDR1 domain comprising the amino acid sequence of SEQ ID NO: 184; (b) an HCDR2 domain comprising the amino acid sequence of SEQ ID NO: 186; and (c) an HCDR3 domain comprising the amino acid sequence of SEQ ID NO: 188; (d) an LCDR1 domain comprising the amino acid sequence of SEQ ID NO: 192; and (e) an LCDR2 domain comprising the amino acid sequence of SEQ ID NO: 194; and (f) an LCDR3 domain comprising the amino acid sequence of SEQ ID NO: 196; Includes.

[0036] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR1 (HCDR1) that comprises an amino acid sequence selected from any of the HCDR1 amino acid sequences provided herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0037] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR2 (HCDR2) that comprises an amino acid sequence selected from any of the HCDR2 amino acid sequences provided herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0038] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a heavy chain CDR3 (HCDR3) that comprises an amino acid sequence selected from any of the HCDR3 amino acid sequences provided herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0039] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR1 (LCDR1) that comprises an amino acid sequence selected from any of the LCDR1 amino acid sequences provided herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0040] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR2 (LCDR2) that comprises an amino acid sequence selected from any of the LCDR2 amino acid sequences provided herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0041] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a light chain CDR3 (LCDR3) that comprises an amino acid sequence selected from any of the LCDR3 amino acid sequences listed herein, or a substantially similar sequence thereof having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0042] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising HCDR3 and LCDR3 amino acid sequence pairs (HCDR3 / LCDR3) that comprise any of the HCDR3 amino acids provided herein paired with any of the LCDR3 amino acid sequences provided herein.According to certain embodiments, the antibodies or antigen-binding fragments thereof comprise the HCDR3 / LCDR3 amino acid sequence pair contained in any of the exemplary anti-GCG antibodies provided herein.In certain embodiments, the HCDR3 / LCDR3 amino acid sequence pair comprises SEQ ID NO: 172 / 180.

[0043] Also useful according to the methods provided herein are antibodies or antigen-binding fragments thereof that specifically bind to GCG, comprising a set of six CDRs (i.e., HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3) contained in any of the exemplary anti-GCG antibodies provided herein. In certain embodiments, the set of amino acid sequences of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 comprises SEQ ID NOs: 168 / 170 / 172 / 176 / 178 / 180. In certain embodiments, the set of amino acid sequences of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 comprises SEQ ID NOs: 184 / 186 / 188 / 192 / 194 / 196.

[0044] In a related embodiment, an antibody or antigen-binding fragment thereof that specifically binds to GCG comprises a set of six CDRs (i.e., HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3) contained in the HCVR / LCVR amino acid sequence pair defined by any of the exemplary anti-GCG antibodies provided herein. For example, an antibody or antigen-binding fragment thereof that specifically binds to GCG comprises a set of amino acid sequences of HCDR1 / HCDR2 / HCDR3 / LCDR1 / LCDR2 / LCDR3 contained in the HCVR / LCVR amino acid sequence pair selected from the group consisting of 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302.

[0045] Non-limiting examples of antibodies that specifically bind to GCG and comprise the CDR sequences provided above include HIH059P, H4H10223P, H4H10231P, H4H10232P, H4H10236P, H4H10237P, H4H10238P, H4H10250P, H4H10256P, and H4H10270P.

[0046] Methods and techniques for identifying CDRs in HCVR and LCVR amino acid sequences are well known in the art and can be used to identify CDRs in the particular HCVR and / or LCVR amino acid sequences disclosed herein. Exemplary conventions that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, and the AbM definition. In general terms, the Kabat definition is based on sequence diversity, the Chothia definition is based on the location of structural loop regions, and the AbM definition is a compromise between the Kabat and Chothia methods. See, for example, Kabat (1991) "Sequences of Proteins of Immunological See, "Interest Rate" National Institutes of Health, Bethesda, Md.; Al-Lazikani et al. (1997), J. Mol. Biol. 273:927-948; and Martin et al. (1989), Proc. Natl. Acad. Sci. USA 86:9268-9272. Public databases are also available to identify CDR sequences in antibodies.

[0047] In some embodiments, the patient with severe insulin resistance may suffer from one of the following conditions or diseases: Donohue syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolic disorders, Bardet-Biedl syndrome, and conditions or diseases associated with the presence of one or more genetic variants reported to result in severe insulin resistance. In some embodiments, insulin-degrading protease activity is detected in the serum of the patient. In some embodiments, neutralizing anti-insulin antibodies or anti-insulin receptor antibodies are detected in the serum of the patient. In some patients, severe insulin resistance occurs in the context of autoimmune destruction of fat cells resulting in lipodystrophy.

[0048] In some embodiments, the genetic variants associated with severe insulin resistance are selected from the following: INSR, PSMD6, ADRA2A, AGPAT2 (associated with lipodystrophy and insulin resistance), AKT2, APPL1, BBS1 (associated with Bardet-Biedl syndrome 1), BSCL2, CIDEC, GRB10, IRS2, KLF14, LEP, LEPR, LMNA (associated with lipodystrophy), MC4R, PCNT, PIK2CA, POLD1 (associated with lipodystrophy), PPARG, PTPRD, PTRF (associated with lipodystrophy), RASGRP1, TBC1D4, and TCF7L2.

[0049] In some aspects, the composition comprising glucagon / GCGR antagonist is administered to the patient in combination with at least one additional therapeutic agent.The additional therapeutic agent can be any agent that alleviates or reduces the symptoms and signs associated with severe insulin resistance.In some embodiments, the at least one additional therapeutic agent is selected from the following: insulin, biguanide, hIGF1, leptin, metraleptin, pioglitazone, vildagliptin, acarbose, alpha-glycosidase inhibitor, L-arginine, dipeptidyl-peptidase-4 inhibitor, insulin secretagogue, amylin receptor agonist, insulin sensitizer, FGF21, SGLT2 inhibitor, SGLT1 inhibitor, GLP-1 receptor agonist, GLP-1 receptor activator, second GCG inhibitor, and second GCGR antagonist. In some embodiments, the insulin secretagogue is selected from sulfonylureas, ATP-sensitive K channel antagonists, and meglitinides.In some embodiments, the insulin sensitizer is selected from thiazolidinediones and rosiglitazone.In some embodiments, the additional therapeutic agent can be an agent that increases energy expenditure and / or brown fat activity, such as β3 adrenergic agonists (e.g., miglitol), NPR1 agonists, NPR3 antagonists, triiodothyronine, thiazolidinediones, VEGF, irisin, meteorin-like, natriuretic peptides, orexin, norepinephrine, T4, bile acid, FGF-21, menthol, slit2-C BMP7, BMP8β, and FnIII domain-like / Tn3 scaffold (a binding molecule based on the third fibronectin type III domain of human tenascin-C), etc.

[0050] Other objects and advantages will become apparent from a consideration of the ensuing detailed description. [Brief description of the drawings]

[0051] [Figure 1A]1A-1E show blood glucose, insulin, glucagon, and B-hydroxybutyrate levels, as well as body weight, in a mouse model of severe insulin resistance. In FIG. 1A, mice treated with S961, an insulin receptor antagonist, and H4H1327P, an antibody against GCGR (open triangles), exhibited elevated blood glucose levels compared to those in mice treated with the insulin receptor antagonist and an isotype control antibody (closed squares). In FIG. 1B, treatment of mice with S961 demonstrated elevated insulin levels over time (closed squares), even in the presence of H4H1327P (open triangles). In FIG. 1C, mice treated with H4H1327P exhibited higher glucagon levels than isotype control-treated mice (closed circles) or S961-treated mice (closed squares) in the absence (open circles) or presence (open triangles) of S961. In Figure ID, mice treated with S961 and H4H1327P (open triangles) maintained beta-hydroxybutyrate levels similar to isotype control treatment (closed circles) and antibody alone control (open circles). Mice treated with insulin receptor antagonist in the absence of GCGR antibody exhibited higher beta-hydroxybutyrate levels (closed squares) compared to the other treatment groups. Body weight did not change among the four treatment groups. See Figure ID. [Figure 1B]1A-1E show blood glucose, insulin, glucagon, and B-hydroxybutyrate levels, as well as body weight, in a mouse model of severe insulin resistance. In FIG. 1A, mice treated with S961, an insulin receptor antagonist, and H4H1327P, an antibody against GCGR (open triangles), exhibited elevated blood glucose levels compared to those in mice treated with the insulin receptor antagonist and an isotype control antibody (closed squares). In FIG. 1B, treatment of mice with S961 demonstrated elevated insulin levels over time (closed squares), even in the presence of H4H1327P (open triangles). In FIG. 1C, mice treated with H4H1327P exhibited higher glucagon levels than isotype control-treated mice (closed circles) or S961-treated mice (closed squares) in the absence (open circles) or presence (open triangles) of S961. In Figure ID, mice treated with S961 and H4H1327P (open triangles) maintained beta-hydroxybutyrate levels similar to isotype control treatment (closed circles) and antibody alone control (open circles). Mice treated with insulin receptor antagonist in the absence of GCGR antibody exhibited higher beta-hydroxybutyrate levels (closed squares) compared to the other treatment groups. Body weight did not change among the four treatment groups. See Figure ID. [Figure 1C]1A-1E show blood glucose, insulin, glucagon, and B-hydroxybutyrate levels, as well as body weight, in a mouse model of severe insulin resistance. In FIG. 1A, mice treated with S961, an insulin receptor antagonist, and H4H1327P, an antibody against GCGR (open triangles), exhibited elevated blood glucose levels compared to those in mice treated with the insulin receptor antagonist and an isotype control antibody (closed squares). In FIG. 1B, treatment of mice with S961 demonstrated elevated insulin levels over time (closed squares), even in the presence of H4H1327P (open triangles). In FIG. 1C, mice treated with H4H1327P exhibited higher glucagon levels than isotype control-treated mice (closed circles) or S961-treated mice (closed squares) in the absence (open circles) or presence (open triangles) of S961. In Figure ID, mice treated with S961 and H4H1327P (open triangles) maintained beta-hydroxybutyrate levels similar to isotype control treatment (closed circles) and antibody alone control (open circles). Mice treated with insulin receptor antagonist in the absence of GCGR antibody exhibited higher beta-hydroxybutyrate levels (closed squares) compared to the other treatment groups. Body weight did not change among the four treatment groups. See Figure ID. [Figure 1D]1A-1E show blood glucose, insulin, glucagon, and B-hydroxybutyrate levels, as well as body weight, in a mouse model of severe insulin resistance. In FIG. 1A, mice treated with S961, an insulin receptor antagonist, and H4H1327P, an antibody against GCGR (open triangles), exhibited elevated blood glucose levels compared to those in mice treated with the insulin receptor antagonist and an isotype control antibody (closed squares). In FIG. 1B, treatment of mice with S961 demonstrated elevated insulin levels over time (closed squares), even in the presence of H4H1327P (open triangles). In FIG. 1C, mice treated with H4H1327P exhibited higher glucagon levels than isotype control-treated mice (closed circles) or S961-treated mice (closed squares) in the absence (open circles) or presence (open triangles) of S961. In Figure ID, mice treated with S961 and H4H1327P (open triangles) maintained beta-hydroxybutyrate levels similar to isotype control treatment (closed circles) and antibody alone control (open circles). Mice treated with insulin receptor antagonist in the absence of GCGR antibody exhibited higher beta-hydroxybutyrate levels (closed squares) compared to the other treatment groups. Body weight did not change among the four treatment groups. See Figure ID. [Figure 1E]1A-1E show blood glucose, insulin, glucagon, and B-hydroxybutyrate levels, as well as body weight, in a mouse model of severe insulin resistance. In FIG. 1A, mice treated with S961, an insulin receptor antagonist, and H4H1327P, an antibody against GCGR (open triangles), exhibited elevated blood glucose levels compared to those in mice treated with the insulin receptor antagonist and an isotype control antibody (closed squares). In FIG. 1B, treatment of mice with S961 demonstrated elevated insulin levels over time (closed squares), even in the presence of H4H1327P (open triangles). In FIG. 1C, mice treated with H4H1327P exhibited higher glucagon levels than isotype control-treated mice (closed circles) or S961-treated mice (closed squares) in the absence (open circles) or presence (open triangles) of S961. In Figure ID, mice treated with S961 and H4H1327P (open triangles) maintained beta-hydroxybutyrate levels similar to isotype control treatment (closed circles) and antibody alone control (open circles). Mice treated with insulin receptor antagonist in the absence of GCGR antibody exhibited higher beta-hydroxybutyrate levels (closed squares) compared to the other treatment groups. Body weight did not change among the four treatment groups. See Figure ID.

[0052] [Figure 2A]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F. [Figure 2B]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F. [Figure 2C]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F. [Figure 2D]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F. [Figure 2E]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F. [Figure 2F]Figures 2A-2F show blood glucose, insulin, glucagon, B-hydroxybutyrate, and amino acid levels, as well as body weight, in a mouse model of severe insulin resistance. Treatment with an insulin receptor antagonist (S961) prior to treatment with antibody H4H1327P elevated blood glucose levels, and the ability of the antibody to lower blood glucose levels was demonstrated within days of initiating antibody treatment (open triangles). See Figure 2A. In Figure 2B, treatment with S961 elevated insulin levels (closed squares), while subsequent treatment with GCGR antibody H4H1327P did not lower insulin levels (open triangles). As shown in Figure 2C, glucagon levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). Figure 2D shows that plasma beta-hydroxybutyrate levels were elevated in response to treatment with S961 (closed squares), but within a few days of treatment with H4H1327P, levels fell to those of untreated and antibody-only controls (open triangles). Figure 2E shows that amino acid levels were higher in mice treated with H4H1327P (open circles) and even higher in mice treated with both antibody and S961 (open triangles). No changes in body weight were observed. See Figure 2F.

[0053] [Diagram 3] Figures 3A and 3B provide the results of Western blot analysis of mouse liver samples obtained from mice treated with either or both S961 and H4H1327P. Treatment with H4H1327P reduced phosphoenolpyruvate carboxykinase (Pepck) in mouse liver by 70% compared to isotype-treated control, and treatment with S961 increased Pepck levels by 2.3-fold. Treatment with H4H1327P reversed the increase in levels caused by S961 to less than 30% of baseline. See Figures 3A and 3B.

[0054] [Figure 4A]Figures 4A-4D show the effect of the four treatments on pancreatic tissue: pancreas weight, Figure 4A; pancreatic alpha cell mass, Figure 4B; pancreatic beta cell mass, Figure 4C; and islet number relative to total pancreatic area, Figure 4D. Beta cell mass doubled in the presence of S961 and H4H1327P when compared to S961 alone, and was increased 5.8-fold compared to control mice. See Figure 4C. [Figure 4B] Figures 4A-4D show the effect of the four treatments on pancreatic tissue: pancreas weight, Figure 4A; pancreatic alpha cell mass, Figure 4B; pancreatic beta cell mass, Figure 4C; and islet number relative to total pancreatic area, Figure 4D. Beta cell mass doubled in the presence of S961 and H4H1327P when compared to S961 alone, and was increased 5.8-fold compared to control mice. See Figure 4C. [Figure 4C] Figures 4A-4D show the effect of the four treatments on pancreatic tissue: pancreas weight, Figure 4A; pancreatic alpha cell mass, Figure 4B; pancreatic beta cell mass, Figure 4C; and islet number relative to total pancreatic area, Figure 4D. Beta cell mass doubled in the presence of S961 and H4H1327P when compared to S961 alone, and was increased 5.8-fold compared to control mice. See Figure 4C. [Figure 4D] Figures 4A-4D show the effect of the four treatments on pancreatic tissue: pancreas weight, Figure 4A; pancreatic alpha cell mass, Figure 4B; pancreatic beta cell mass, Figure 4C; and islet number relative to total pancreatic area, Figure 4D. Beta cell mass doubled in the presence of S961 and H4H1327P when compared to S961 alone, and was increased 5.8-fold compared to control mice. See Figure 4C. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Before describing the method, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as 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, since the scope of the present invention will be limited only by the appended claims.

[0056] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs.In the practice or testing of this invention, any method and material similar or equivalent to those described herein can be used, but preferred methods and materials are described below.All patents, applications, and non-patent publications mentioned herein are incorporated herein in their entirety by reference.

[0058] Overview Severe insulin resistance occurs in association with a variety of physiological and pathophysiological conditions. Clinical findings include hyperinsulinemia, acanthosis nigricans, ovarian hyperandrogenism, polycystic ovaries, and eventual hyperglycemia; in rare cases, patients may develop ketoacidosis. Although there is no agreed-upon definition to distinguish severe insulin resistance from more general insulin resistance, symptomatic insulin resistance has been classified as either primary insulin signaling defects (insulin receptoropathy or partial disruption of the insulin signaling pathway) or insulin resistance secondary to adipose tissue abnormalities (severe obesity or lipodystrophy). See Semple et al. (2011), Genetic Syndromes of Severe Insulin Resistance, Endocrine Reviews, 32(4):498-514.

[0059] Evidence of severe insulin resistance is seen in patients who require exogenous insulin at doses greater than 100-200 units per day or who have chronically elevated circulating levels of endogenous insulin. Moller and Flier (1991), New England Journal of Medicine, 325:938-948. Fasting insulin levels greater than 50-70 μU / mL or peak (after an oral glucose tolerance test) insulin levels greater than 350 μU / mL suggest severe insulin resistance. 2×10 4 Insulin sensitivity index values ​​below μU / mL min typically occur in the presence of severe insulin resistance. Patients with severe insulin resistance also exhibit glucose disposal rates below 2 mg / kg min. See Tritos and Mantzoros (1998), Journal of Clinical Endocrinology and Metabolism, 83:3025-3030.

[0060] Insulin interacts with insulin receptors on the plasma membrane of target cells. The insulin receptor is a transmembrane tyrosine kinase receptor that functions to control glucose homeostasis. It consists of two α subunits that contain sites for insulin binding and two β subunits that contain tyrosine kinase domains, and the subunits are connected by disulfide bridges to form a 350 kDa β-α-α-β tetramer. There are two isoforms of the receptor, an isoform with exon 11 (IR-B) and an isoform without exon 11 (IR-A), and the expression levels of the isoforms differ in various tissues. The IR-B isoform exhibits higher and more efficient signaling activity than the IR-A isoform, and the IR-B isoform is mainly expressed in liver, adipose tissue, and muscle tissue. The IR-A isoform is expressed in CNS and hematopoietic cells and has a slightly higher insulin binding affinity.

[0061] The tyrosine kinase activity of the activated insulin receptor is involved in transmembrane signaling of glucose transport and the control of glucose homeostasis.

[0062] Severe insulin resistance is typically associated with insulin receptor mutations that result in reduced cell surface expression or reduced receptor signaling capacity.Other mutations include defective receptor binding affinity or mutations in proteins involved in the insulin signal transduction pathway, such as conserved regions of the tyrosine kinase domain of the insulin receptor.

[0063] Patients with severe insulin resistance may suffer from a condition or disease selected from the following: Donahue syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, disorders of arginine metabolism, or Bardet-Biedl syndrome.

[0064] Genetic and acquired severe insulin resistance conditions are rare disorders in which the body's tissues and organs do not respond appropriately to insulin. Clinical findings associated with severe insulin resistance include growth retardation, organomegaly, impaired skeletal and adipose tissue development, soft tissue hyperplasia, diabetes mellitus, hepatic steatosis, acanthosis nigricans, ovarian hyperandrogenism, and hirsutism. Laboratory findings include hyperinsulinemia, reduced insulin clearance, hyperglycemia, dyslipidemia, and elevated androgens. Each of the various syndromes associated with severe insulin resistance has unique features in addition to some or all of the common clinical and laboratory features.

[0065] Donohue syndrome (DS, also known as elfin syndrome) and Rabson-Mendenhall syndrome (RMS) are rare autosomal recessive conditions in which both alleles of the insulin receptor are abnormal and patients are unable to respond to endogenous and exogenous insulin. Individuals with DS and RMS are underdeveloped from prenatal onset and fail to develop as infants. Patients exhibit extremely high levels of circulating insulin, up to 1000 times higher than normal levels. The primary metabolic consequence of DS is fasting hypoglycemia and secondarily postprandial hyperglycemia. Individuals diagnosed with DS usually die before the age of 1 year and do not develop diabetic ketoacidosis. Individuals with RMS also experience fasting hypoglycemia and typically survive infancy, but eventually develop severe and refractory diabetic ketoacidosis and reduced insulin levels.

[0066] Ketonemia occurs when ketone bodies are formed and accumulate in the blood by the breakdown of fatty acids and the deamination of amino acids. If left untreated, the patient may then continue on to diabetic ketoacidosis. Beta-hydroxybutyrate and acetoacetate are two of the more common ketones, and elevated levels can be used to measure the severity of ketonemia and an indicator of ketoacidosis.

[0067] Type A insulin resistance syndrome is another rare disorder characterized by severe insulin resistance, with symptoms typically appearing during puberty in females or adulthood in males. Females present with primary amenorrhea or oligomenorrhea, ovarian cysts, hirsutism, and acanthosis nigricans, but are not typically overweight. Affected males become symptomatic when they develop diabetes mellitus. As in DS and RMS, insulin receptor gene mutations contribute to type A insulin resistance syndrome.

[0068] Lipodystrophies refer to a group of disorders characterized by abnormal fat distribution, utilization, and metabolism due to defects in the insulin receptor itself or in downstream components of the insulin signaling cascade. Lipodystrophies patients exhibit generalized or partial absence of adipose tissue, insulin resistance (with or without diabetes), marked dyslipidemia, and fatty liver. Some lipodystrophies syndromes, such as Berardinelli-Seip syndrome, are hereditary, while others, including Lawrence syndrome, are acquired, sometimes after an infectious prodrome. Additional lipodystrophies syndromes include Kobberling-Dunnigan syndrome, lipodystrophy with other dysmorphic features, and cephalothoracic lipodystrophy.

[0069] Type B insulin resistance syndrome differs from DS, RMS, and type A insulin resistance syndrome in that it is associated with the presence of serum autoantibodies against the insulin receptor and can occur in the context of autoimmune disease. Symptoms are similar to other insulin resistance syndromes and include nonketotic and severe insulin-resistant diabetes mellitus, acanthosis nigricans, and hirsutism, in addition to occasional paradoxal hypoglycemia.

[0070] HAIR-AN (Hyperandrogenism, Insulin Resistance, and Acanthosis Nigricans) syndrome manifests in typically obese young women with various forms of insulin resistance. Some individuals have high insulin concentrations but normal glucose levels, while others exhibit diabetic symptoms. Unlike other severe insulin resistance syndromes, which are rare, HAIR-AN syndrome is estimated to affect approximately 5% of adolescent girls worldwide. The syndrome has been associated with mutations in the tyrosine kinase domain of the insulin receptor gene.

[0071] Pseudoacromegaly shows severe insulin resistance in association with acromegaly and may result from defects in the insulin signaling pathway or from high insulin levels signaling through the IGF-1 receptor.

[0072] Other severe insulin resistance syndromes include Alström's syndrome, myotonic dystrophy, and Werner's syndrome, to name a few.

[0073] In some patients, condition or disease is associated with the presence of gene variants that have been reported to cause severe insulin resistance.Exemplary gene variants include INSR, PSMD6, ADRA2A, AGPAT2 (associated with lipodystrophy and insulin resistance), AKT2, APPL1, BBS1 (associated with Bardet-Biedl syndrome 1), BSCL2, CIDEC, GRB10, IRS2, KLF14, LEP, LEPR, LMNA (associated with lipodystrophy), MC4R, PCNT, PIK2CA, POLD1 (associated with lipodystrophy), PPARG, PTPRD, PTRF (associated with lipodystrophy), RASGRP1, TBC1D4 and TCF7L2.

[0074] In some patients, insulin-degrading protease activity is detectable in the patient's serum. In some patients, neutralizing anti-insulin antibodies or anti-insulin receptor antibodies are detectable in the patient's serum. In some patients, severe insulin resistance occurs in conjunction with autoimmune destruction of fat cells resulting in lipodystrophy.

[0075] Patients with severe insulin resistance eventually develop hyperglycemia and, in some syndromes, ketoacidosis. For example, in patients with RMS, insulin levels begin very high during childhood and even during periods of paradoxical fasting hypoglycemia. As the disease progresses, insulin levels remain high but decline. In addition, levels of partially oxidized fatty acids increase. This indicates that insulin is unable to inhibit the release of fatty acids from adipocytes, ultimately resulting in homeostatic ketoacidosis. Similarly, homeostatic hyperglycemia results in the inability of insulin levels to inhibit hepatic glucose production and release. However, continuous infusion of extremely high concentrations of insulin (9.5 U / kg·h) can reverse the increased fatty acid oxidation and prevent ketonuria. Longo et al. (1991), Journal of Clinical Endocrinology & Metabolism, 84:2623-2629. In addition, hypertriglyceridemia and low high density lipoprotein cholesterol levels are associated with severe insulin resistance.

[0076] Patients with severe insulin resistance syndrome have normal or even slightly elevated plasma glucagon levels despite hyperglycemia. West et al. (1975), Arch. Dis. Child., 50(9):703-708; Desbois-Mouthon et al. (1997), Pediatr. Res., 42(1):72-77. Hyperglycemia results from enhanced hepatic glucose production due to lack of insulin suppression and abnormally high glucagon signaling.

[0077] To date, there are no studies that have investigated the effect of antagonizing the GCG / GCGR signaling pathway on severe insulin resistance conditions or diseases.The study described in the Examples uses an antagonist of GCGR as an exemplary inhibitor of the GCG / GCGR signaling pathway in a mouse model of severe insulin resistance, and demonstrates the effect on blood glucose levels and ketonemia measured by plasma beta-hydroxybutyrate levels over several weeks of treatment.

[0078] definition The "glucagon receptor", also referred to herein as "GCGR", belongs to the G protein-coupled receptor class 2 family and consists of a long amino-terminal extracellular domain, seven transmembrane segments, and an intracellular C-terminal domain. Glucagon receptors are expressed, among others, on the surface of hepatocytes, where they bind glucagon and transmit the resulting signal into the cell. Thus, the term "glucagon receptor" also refers to one or more receptors that specifically interact with glucagon, resulting in a biological signal. In the art, DNA sequences encoding glucagon receptors of rat and human origin have been isolated and disclosed (EP0658200B1). Mouse and cynomolgus monkey homologs have also been isolated and sequenced (Burcelin et al. (1995), Gene, 164:305-310; McNally et al. (2004), Peptides, 25:1171-1178). As used herein, "glucagon receptor" and "GCGR" are used interchangeably. The terms "GCGR", "hGCGR", or fragments thereof, as used herein, refer to the human GCGR protein or fragments thereof, unless specified to be derived from a non-human species, e.g., "mouse GCGR", "rat GCGR", or "monkey GCGR".

[0079] The term "GCGR antagonist" refers to the inhibitor, antagonist or inverse agonist of GCGR signal transduction pathway. "GCG inhibitor" can prevent glucagon from binding to receptor. GCGR inhibitor can also prevent glucagon from binding to receptor. However, either can effectively block or attenuate the activation of receptor or interfere with the signal transduction cascade downstream of GCGR activation.

[0080] GCGR antagonists can bind to glucagon receptors, thereby antagonizing the activity of GCG mediated by GCGR. Inhibiting the activity of GCG by antagonizing the binding and activity of GCG at GCGR reduces the rate of gluconeogenesis and glycogenolysis, and the concentration of glucose in plasma. Methods for determining the binding of putative antagonists to glucagon receptors are known in the art, and means for determining interference with glucagon activity at glucagon receptors are published. See, for example, SE de Laszlo et al. (1999), Bioorg. Med. Chem. Lett., vol. 9:641-646. Contemplated as useful herein are GCGR antagonists or GCG inhibitors that have small molecule compounds, or in other words, low molecular weight organic compounds, as their functional components. Small molecules are typically less than 800 daltons. In addition, CRISPR technology can be used to knock down the expression of GCG or GCGR.

[0081] The term "inhibitor" or "antagonist" includes substances that retard or prevent chemical or physiological reactions or responses.Common inhibitors or antagonists include, but are not limited to, antisense molecules, antibodies, small molecule inhibitors, peptide inhibitors, DARPins, Spiegelmers, aptamers, engineered Fn type III domains, and their derivatives.

[0082] Examples of GCG inhibitors or GCGR signaling pathway antagonists include, but are not limited to, antibodies (human or humanized antibodies) against GCG or GCGR, or antigen-binding fragments thereof, that block the binding or inhibit the activity of GCGR signaling pathway.Exemplary GCGR antagonists that can be used in the methods described herein include isolated human monoclonal antibodies or antigen-binding fragments thereof, comprising (a) HCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) LCVR having an amino acid sequence selected from the group consisting of SEQ ID NO: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. Exemplary GCG inhibitors that may be used in the methods described herein include an isolated human monoclonal antibody or antigen-binding fragment thereof comprising: (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302.

[0083] A "therapeutically effective dose" is a dose that produces the desired effect upon administration. The exact dose will depend on the purpose of the treatment and can be ascertained by one skilled in the art using known techniques (see, for example, Lloyd (1999), The Art, Science and Technology of Pharmaceutical Compounding).

[0084] The phrase "substantially identical" refers to a protein sequence that has at least 95% identity to (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148, and is capable of binding to GCGR and inhibiting the biological activity of GCGR. The phrase "substantially identical" also refers to a protein sequence that has at least 95% identity to the amino acid sequences: (a) HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or (b) LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302, and is capable of binding to GCG and inhibiting the biological activity of GCG.

[0085] The term "identity" or "homology" is taken to mean the percentage of amino acid residues of a candidate sequence that are identical compared to the residues of the corresponding sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity throughout the sequence, without considering any conservative substitutions as part of the sequence identity. Neither N-terminal nor C-terminal extensions nor insertions are to be construed as reducing identity or homology. Methods and computer programs for alignment are well known in the art. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, Wis. 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

[0086] The term "treating" (or "treat" or "treatment") refers to a process that involves slowing, hindering, inhibiting, suppressing, controlling, halting, reducing, ameliorating, or reversing the progression, duration, or severity of an existing symptom, disorder, condition, or disease by use of a GCG inhibitor or GCGR antagonist described herein, but does not necessarily involve complete disappearance of all of the symptoms, condition, or disorder associated with the disease.Additionally, "treating," "treatment," or "treat" refers to an approach for obtaining beneficial or desired results, including clinical results, including, but not limited to, the following: inhibiting, slowing, or preventing the progression of severe insulin resistance; diseases associated with severe insulin resistance or characterized by elevated plasma insulin levels, elevated blood glucose levels, and / or ketonemia or ketoacidosis (as measured by elevated beta-hydroxybutyrate levels), such as Donahue's syndrome, Rabson-Mendenhall syndrome, Type A insulin resistance, Type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner's syndrome, lipodystrophy, cirrhosis, monoclonal antibody therapy, and the like; The present invention includes one or more of the following: inhibiting, delaying, or preventing the progression of a condition or disease associated with monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolism disorder, Bardet-Biedl syndrome, or the presence of a genetic variant reported to cause severe insulin resistance; inhibiting, preventing, or reversing at least one symptom associated with a disease associated with severe insulin resistance; or reducing blood glucose levels and / or beta-hydroxybutyrate levels (as an indicator of ketoacidosis) to mediate a condition or disease associated with high blood glucose levels and ketonemia, or alleviating or reducing the severity of at least one symptom or complication associated with a condition or disease. "Treatment" or "treating" as used herein also refers to improving the quality of life of those suffering from a disease, reducing the dose of other drugs required to treat the disease, and / or prolonging the survival of a patient. For example, "treatment" or "treating" can include reducing the amount and / or dosage of insulin required to treat a patient with severe insulin resistance.

[0087] The phrase "insulin resistance" refers to a situation in which greater than normal amounts of insulin are required to elicit a quantitatively normal response. The phrase "severe insulin resistance" generally refers to a clinical entity that typically exhibits near-normal or elevated blood glucose levels despite marked elevations in endogenous insulin secretion and / or plasma levels of insulin. Evidence of severe insulin resistance is seen in patients who require exogenous insulin at doses of more than 100-200 units per day or in patients with chronically elevated circulating levels of endogenous insulin. Moller and Flier (1991), New England Journal of Medicine, 325:938-948. Fasting insulin levels of more than 50-70 μU / mL or peak (after an oral glucose tolerance test) insulin levels of more than 350 μU / mL suggest severe insulin resistance. 2×10 4 Insulin sensitivity index values ​​below μU / mL min typically occur in the presence of severe insulin resistance. Patients with severe insulin resistance also exhibit glucose disposal rates below 2 mg / kg min. See Tritos and Mantzoros (1998), Journal of Clinical Endocrinology and Metabolism, 83:3025-3030.

[0088] GCG / GCGR signaling pathway inhibitors Provided herein is a GCG inhibitor and a GCGR antagonist for treating the condition or disease characterized by severe insulin resistance.In some embodiments, the antagonist is an inhibitor of glucagon.In some embodiments, the antagonist is an inhibitor of GCGR.In some embodiments, the GCGR antagonist is MK-0893, PF-06291874, LGD-6972 or LY2409021.

[0089] In some embodiments, the antagonist comprises an antibody or fragment thereof that can bind to GCG or GCGR.In some embodiments, the signal transduction pathway is inhibited by disrupting the expression of GCG or GCGR, for example by using CRISPR technology or antisense.

[0090] In some embodiments, the GCG inhibitor or GCGR antagonist is an antisense molecule, an antibody, a small molecule inhibitor, a peptide inhibitor, a DARPin, a Spiegelmer, an aptamer, an engineered Fn type III domain, or a derivative thereof.

[0091] Anti-GCGR antibodies, anti-GCG antibodies, and antibody fragments In some embodiments, the GCGR antagonist is an antibody or antibody fragment disclosed in U.S. Patent No. 8,545,847, the entirety of which is incorporated herein by reference. The antibodies disclosed therein are provided in Table 1. [Table 1]

[0092] Additional GCGR antibodies or antibody fragments contemplated as useful herein include those disclosed in U.S. Patent Nos. 5,770,445 and 7,947,809, European Patent Application No. EP2074149(A2), European Patent No. EP0658200(B1), U.S. Patent Publication Nos. 2009 / 0041784, 2009 / 0252727, and 2011 / 0223160, and PCT Publication No. WO2008 / 036341. These patents and publications are incorporated herein by reference in their entireties.

[0093] In some embodiments, the GCG inhibitor is an antibody or antibody fragment thereof disclosed in US2016 / 0075778, the entirety of which is incorporated herein by reference. The antibodies disclosed in this document are presented in Table 2. [Table 2]

[0094] Additional GCG antibodies or antibody fragments contemplated as useful herein include those disclosed in U.S. Pat. Nos. 4,206,199, 4,221,777, 4,423,034, 4,272,433, 4,407,965, 5,712,105, and PCT Publication Nos. WO2007 / 124463 and WO2013 / 081993.

[0095] Antibody fragments include any fragment having the required target specificity, for example, antibody fragments produced by modification (e.g., enzymatic digestion) of whole antibodies or those produced using recombinant DNA techniques. These include those synthesized novo (scFv, single domain antibodies, DVDs (dual variable domain immunoglobulins), or dAbs (single variable domain antibodies)) or identified using human phage display libraries or yeast display libraries (see, for example, McCafferty et al. (1990), Nature 348:552-554). Alternatively, antibodies may be isolated from mice producing human, human-mouse, human-rat, and human-rabbit chimeric antibodies using standard immunization and antibody isolation methods, including, but not limited to, the generation of hybridomas or the use of B-cell screening techniques such as SLAM. Immunoglobulin binding domains include those comprising the heavy chain (V) of an immunoglobulin, H ) or light chain (V L Alternatively, by immunizing people, isolating antigen-positive B cells, and cloning cDNAs that encode the heavy and light chains and co-express them in cells such as CHO.

[0096] The term "antibody" as used herein refers to a polypeptide comprising a framework region derived from an immunoglobulin gene or a fragment thereof, which specifically binds and recognizes an antigen. Recognized immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant regions, as well as a wide variety of immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Within each IgG class, there are different isotypes (e.g., IgG1, IgG2, IgG3, IgG4). Typically, the antigen-binding region of an antibody is most critical in determining the specificity and affinity of binding.

[0097] An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one light chain (about 25 kD) and one heavy chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition. The "variable light chain" (VLC) is L ) and variable heavy chain (V H ) refer to these light and heavy chains respectively.

[0098] Antibodies exist as intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases. For example, pepsin digests antibodies down the disulfide linkages in the hinge region to produce a dimer of Fab, F(ab)', itself separated by disulfide bonds. H -C H1. F(ab)'2 can be reduced under mild conditions that break the disulfide linkages in the hinge region, thereby converting the F(ab)'2 dimer into a Fab' monomer. A Fab' monomer is essentially a Fab with a portion of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill in the art will realize that such fragments can be synthesized chemically or de novo using recombinant DNA methodology.

[0099] Methods for preparing antibodies useful according to the methods herein are known in the art. See, for example, Kohler and Milstein (1975), Nature, vol. 256:495-497; Harlow and Lane (1988), Antibodies: a Laboratory Manual, Cold Spring Harbor Lab., Cold Spring Harbor, NY. Genes encoding the heavy and light chains of an antibody of interest can be cloned from cells. For example, genes encoding monoclonal antibodies can be cloned from hybridomas and used to produce recombinant monoclonal antibodies. Monoclonal antibodies can be humanized using standard cloning of CDR regions into human scaffolds. Gene libraries encoding human heavy and light chains of monoclonal antibodies can also be generated from hybridomas or plasma cells. Random combination of heavy and light chain gene products produces a large pool of antibodies with different antigen specificities. To produce antibodies for use in the methods disclosed herein, techniques for the production of single chain antibodies or recombinant antibodies (U.S. Patent No. 4,946,778, U.S. Patent No. 4,816,567) may be adapted. Also, transgenic mice, or other organisms such as other mammals, may be used to express human, human-mouse chimeric, human-rat chimeric, human-rabbit chimeric, or humanized antibodies. Alternatively, phage display or yeast display technology may be used to identify human antibodies and heteromeric Fab fragments that specifically bind to a selected antigen.

[0100] Immunoconjugates The present disclosure encompasses the treatment of severe insulin resistance with human anti-GCGR monoclonal antibodies ("immunoconjugates") conjugated to therapeutic moieties such as drugs that can lower blood glucose levels or address another symptom of severe insulin resistance. The type of therapeutic moiety that can be conjugated to the anti-GCGR antibody takes into account the condition to be treated and the desired therapeutic effect to be achieved. For example, in an effort to lower blood glucose and / or maintain normal blood glucose levels, drugs such as biguanides (e.g., metformin), sulfonylureas (e.g., glyburide, glipizide), PPAR gamma agonists (e.g., pioglitazone, rosiglitazone), alpha glucosidase inhibitors (e.g., acarbose, voglibose), inhibitors of advanced glycation end product formation (e.g., aminoguanidine), or a second GCGR inhibitor or GCG inhibitor can be conjugated to the GCGR antibody. Alternatively, when the desired therapeutic effect is to treat ketonemia or any other symptoms or conditions associated with severe insulin resistance, it may be advantageous to conjugate an appropriate agent to anti-GCGR antibody.The example of suitable agent for forming immunoconjugate is known in the art.See, for example, WO05 / 103081.

[0101] multispecific antibody Antibodies useful according to the methods provided herein can be monospecific, bispecific, or multispecific. Multispecific antibodies may be specific for different epitopes of one target polypeptide or may contain antigen-binding domains specific for more than one target polypeptide. See, for example, Tutt et al. (1991), J. Immunol., vol. 147:60-69; Kufer et al. (2004), Trends Biotechnol., vol. 22:238-244. The anti-GCGR antibody may be linked to or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or otherwise) to one or more other molecular entities, such as another antibody or antibody fragment, to produce a bispecific or multispecific antibody having a second binding specificity. For example, bispecific antibodies are contemplated, where one arm of the immunoglobulin is specific for human GCGR or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is conjugated to a therapeutic moiety.In certain embodiments, one arm of the immunoglobulin is specific for an epitope or a fragment thereof in the N-terminal domain of hGCGR, and the other arm of the immunoglobulin is specific for an epitope or a fragment thereof in one of the EC loops of hGCGR.In certain embodiments, one arm of the immunoglobulin is specific for an EC loop or a fragment thereof, and the second arm is specific for a second EC loop or a fragment thereof.In certain embodiments, one arm of the immunoglobulin is specific for an epitope in an EC loop of hGCGR, and the other arm is specific for a second epitope in the same EC loop of hGCGR.

[0102] An exemplary bispecific antibody format that can be used according to the methods described herein is a bispecific antibody format comprising a first immunoglobulin (Ig) C H 3 domain and the C of the second IgH 3 domain, where the C of the first and second Ig H The three domains differ from each other by at least one amino acid, and the at least one amino acid difference reduces binding of the bispecific antibody to Protein A compared to a bispecific antibody lacking the amino acid difference. H The 3 domain binds protein A and the C domain of the second Ig H The third domain contains a mutation that reduces or eliminates Protein A binding, such as the H95R modification (according to the IMGT exon numbering, H435R according to the EU numbering). H 3 may further comprise a Y96F modification (according to IMGT; Y436F according to EU). H Additional modifications that may be found in 3 include D16E, L18M, N44S, K52N, V57M, and V82I for IgG1 antibodies (according to IMGT; D356E, L358M, N384S, K392N, V397M, and V422I for EU); N44S, K52N, and V821 for IgG2 antibodies (according to IMGT; N384S, K392N, and V422I for EU); and Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I for IgG4 antibodies (according to IMGT; Q355R, N384S, K392N, V397M, R409K, E419Q, and V422I for EU). Variations in the above bispecific antibody formats are contemplated within the scope of this disclosure.

[0103] Screening and Selection of Antibodies Screening and selection of preferred antibodies useful according to the methods provided herein can be performed by various methods known in the art. Initial screening for the presence of monoclonal antibodies specific to the target antigen can be performed, for example, by using an ELISA-based method. Secondary screening is preferably performed to identify and select desired monoclonal antibodies for use in constructing antibody-drug conjugates. Secondary screening may be performed using any suitable method known in the art. One preferred method, called "Biosensor Modification-Assisted Profiling" ("BiaMAP"), is described in U.S. Patent Publication No. 2004 / 0101920, which is expressly incorporated herein by reference in its entirety. BiaMAP allows for the rapid identification of hybridoma clones that produce monoclonal antibodies with desired characteristics. More specifically, monoclonal antibodies are sorted into distinct epitope-related groups based on evaluation of antibody:antigen interactions. Antibodies capable of blocking either the ligand or the receptor can be identified by cell-based assays such as luciferase assays utilizing the luciferase gene under the control of an NFκB-driven promoter or a cAMP response-driven promoter. Stimulation of GCGR by glucagon results in a signal through NFκB / cAMP / CREB, thus increasing luciferase levels in the cell. Blocking antibodies are identified as those that blocked glucagon induction of luciferase activity.

[0104] Treatment population The therapeutic method provided herein is useful for treating individuals with severe insulin resistance or conditions or diseases associated with severe insulin resistance.Exemplary conditions or diseases include Donahue syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolic disorder, Bardet-Biedl syndrome, and conditions or diseases associated with the presence of genetic variants that are reported to cause severe insulin resistance.In some embodiments, insulin-degrading protease activity is detected in the serum of the patient.In some embodiments, neutralizing anti-insulin antibodies are detected in the serum of the patient.In some patients, severe insulin resistance occurs in association with autoimmune destruction of fat cells, which leads to lipodystrophy.

[0105] Therapeutic Administration and Formulations According to the methods provided herein, therapeutic compositions comprising glucagon / GCGR antagonists, such as anti-GCGR antibodies, are useful. Administration of therapeutic compositions according to the methods described herein is by any suitable route, including but not limited to, intravenous, subcutaneous, intramuscular, intrathecal, intracerebral, intraventricular, intranasal, or oral, with suitable carriers, excipients, and other agents incorporated into the formulation to improve entry, delivery, tolerance, etc. Numerous suitable formulations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., a formulary known to all pharmacists. These formulations include, for example, powders, pastes, ointments, jellies, 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, emulsions of carbowax (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowax. Powell et al., "Compendium of excipients for See also, "parenteral formulations," PDA (1998), J Pharm Sci Technol 52:238-311.

[0106] The dose of the antibody may vary depending on the age and size of the subject to be administered, the target disease, condition, route of administration, etc. When the antibody is used to lower blood glucose levels and / or reduce ketonemia (e.g., as measured by beta-hydroxybutyrate levels) associated with severe insulin resistance in various conditions and diseases of patients, such as type A insulin resistance syndrome, RMS, or DS, it is usually advantageous to administer the antibody intravenously at a dose of about 0.01 to about 30 mg / kg body weight, more preferably about 0.02 to about 7, about 0.03 to about 5, or about 0.05 to about 3 mg / kg body weight. The frequency and duration of the treatment may be adjusted depending on the severity of the condition and the response to treatment. In certain embodiments, the antibody or antigen-binding fragment thereof may be administered as an initial dose of at least about 0.1 mg to about 800 mg, about 1 to about 500 mg, about 5 to about 300 mg, or about 10 to about 200 mg, up to about 100 mg, or up to about 50 mg.

[0107] In certain embodiments, the initial dose may be followed by a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof, in an amount that may be about the same as the initial dose or less than the initial dose, where the subsequent doses are administered at least 1 to 3 days apart, 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 apart.

[0108] A variety of delivery systems, such as liposomal encapsulation, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, receptor-mediated endocytosis, are known and can be used to administer pharmaceutical compositions containing antibodies (see, for example, Wu et al. (1987), J. Biol. Chem., vol. 262:4429-4432). Methods of introduction include, but are not limited to, depot formulations, aerosol, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, intrathecal, intraventricular, and oral routes. The compositions may be administered by any convenient route, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration may be systemic or local.

[0109] The pharmaceutical composition can also be delivered in a vesicle, in particular a liposome (see, eg, Langer (1990) Science 249:1527-1533).

[0110] In certain circumstances, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used. In another embodiment, a polymeric material can be used. In yet another embodiment, the controlled release system can be placed close to the target of the composition, thus requiring only a fraction of the systemic dose.

[0111] The injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal and intramuscular injections, drip infusions, and the like. These injectable preparations may be prepared by publicly known methods. For example, the injectable preparations may be prepared by dissolving, suspending, or emulsifying the above-mentioned antibody or its salt in a sterile aqueous or oily medium conventionally used for injections. Aqueous solvents for injection include, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents, and the like, which may be used in combination with suitable solubilizers, such as alcohols (e.g., ethanol), polyhydric alcohols (e.g., propylene glycol, polyethylene glycol), nonionic surfactants [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], and the like. As oily media, for example, sesame oil, soybean oil, and the like may be used in combination with solubilizers, such as benzyl benzoate, benzyl alcohol, and the like. The injectable preparations thus prepared are preferably filled into suitable ampoules.

[0112] The pharmaceutical compositions useful herein can be delivered subcutaneously or intravenously using a standard needle and syringe. In addition, for subcutaneous delivery, a pen delivery device is easily applied in the delivery of pharmaceutical compositions useful in the methods described herein. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge containing the pharmaceutical composition. Once all the pharmaceutical composition in the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, a disposable pen delivery device is pre-filled with a pharmaceutical composition held in a reservoir in the device. Once the pharmaceutical composition in the reservoir is depleted, the entire device is discarded.

[0113] A number of reusable pen and autoinjector delivery devices are adapted for subcutaneous delivery of pharmaceutical compositions useful according to the methods described herein. Examples include the AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), the DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), the HUMALOG MIX 75 / 25™ pen, the HUMALOG™ pen, the HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, Inn.), the NOVOPEN™ I, II, and III (Novo Nordisk, Copenhagen, Denmark), the NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pens (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi-Aventis, Frankfurt, Germany), to name just a few, but certainly not limited to these. Examples of disposable pen delivery devices adapted for subcutaneous delivery of pharmaceutical compositions useful according to the methods described herein include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, Calif.), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ pen (Abbott Labs, Abbott Park, Ill.), to name just a few.

[0114] Advantageously, the pharmaceutical composition for oral or parenteral use described above is prepared in a unit dose dosage form suitable for containing a dose of active ingredient. Such unit dose dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 750 mg per unit dose dosage form. The antibody is preferably contained in an amount of about 5 to about 100 mg, particularly in the form of an injection, and about 10 to about 250 mg in the case of other dosage forms.

[0115] Combination therapy In many embodiments, the GCG inhibitors or GCGR antagonists useful herein may be administered in combination with one or more additional compounds or therapies. The combination therapy may be simultaneous or sequential.

[0116] In some embodiments, the GCG inhibitor or GCGR antagonist is administered with at least one additional therapeutic agent selected from the following: insulin, biguanides, hIGF1, leptin, pioglitazone, vildagliptin, acarbose, alpha-glycosidase inhibitor, L-arginine, dipeptidyl-peptidase-4 inhibitor, insulin secretagogue, amylin receptor agonist, insulin sensitizer, SGLT2 inhibitor, SGLT1 inhibitor, GLP-1 analog, GLP-1 receptor activator, second GCG inhibitor, and second GCGR antagonist. In some embodiments, the GCG inhibitor or GCGR antagonist is administered with at least one additional therapeutic agent selected from the following: vanadate or vanadium salt, phenytoin, benzafibrate. In some embodiments, the GCG inhibitor or GCGR antagonist is administered with a dietary supplement, such as fish oil rich in omega-3 fatty acids.

[0117] In some embodiments, the insulin sensitizer is a thiazolidinedione, such as troglitazone. In some embodiments, the insulin sensitizer is rosiglitazone.

[0118] In some embodiments, the insulin secretagogue is a sulfonylurea, an ATP-sensitive K channel antagonist, or a meglitinide.

[0119] An additional therapeutically active component may be administered prior to, simultaneously with, or after administration of the GCG inhibitor or GCGR antagonist, and for purposes of this disclosure, such an administration regimen is considered administration of the GCG inhibitor or GCGR antagonist "in combination with" the second therapeutically active component.

[0120] Dosing regimen According to certain embodiments described herein, multiple doses of glucagon / GCGR antagonist may be administered to a subject over a predefined time course. The method includes sequentially administering multiple doses of glucagon / GCGR antagonist to a subject. As used herein, "sequentially administering" means that each dose of antagonist is administered to a subject at different times, for example, on different days separated by a predefined interval (e.g., hours, days, weeks, or months). The method described herein includes sequentially administering a single initial dose of glucagon / GCGR antagonist, followed by one or more secondary doses of glucagon / GCGR antagonist, optionally followed by one or more tertiary doses of glucagon / GCGR antagonist to a patient.

[0121] The terms "initial dose", "secondary dose" and "tertiary dose" refer to the time sequence of administration of glucagon / GCGR antagonist useful herein. Thus, the "initial dose" is the dose administered at the beginning of a treatment regimen (also referred to as the "baseline dose"), the "secondary dose" is the dose administered after the initial dose, and the "tertiary dose" is the dose administered after the secondary dose. The initial dose, secondary dose and tertiary dose may all contain the same amount of glucagon / GCGR antagonist, but generally may differ from each other in terms of frequency of administration. However, in certain embodiments, the amount of glucagon / GCGR antagonist contained in the initial dose, secondary dose and / or tertiary dose differs from each other during the course of treatment (e.g., adjusted upwards or downwards as necessary). In certain embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered as a "loading dose" at the start of a treatment regimen, followed by subsequent doses (e.g., "maintenance doses") administered less frequently.

[0122] Pharmaceutical Compositions The methods disclosed herein contemplate the use of pharmaceutical compositions comprising at least a therapeutically effective amount of an active agent, such as a glucagon / GCGR antagonist, useful in the treatment of severe insulin resistance, and a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in animals, more particularly in humans, or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which a therapeutic agent is administered. Such pharmaceutical carriers can be sterile liquids, such as water, and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The compositions may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The compositions may be formulated as suppositories, with traditional binders and carriers, such as triglycerides. Oral formulations may include standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by EW Martin.

[0123] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration to humans.If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to reduce pain at the injection site.When the composition is administered by injection, the composition may be dispensed using an infusion bottle containing sterile pharmaceutical grade water or saline.When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the components can be mixed before administration.

[0124] The active agents useful according to the methods described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with free carboxyl groups, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0125] The amount of active agent that is effective in treating severe insulin resistance can be determined by standard clinical techniques based on the present specification. In addition, in vitro assays can be optionally used to help identify optimal dosage ranges. The exact dose to be used in the formulation will also depend on the route of administration and the seriousness of the condition, and should be determined according to the judgment of the practitioner and each subject's circumstances. However, suitable dosage ranges for intravenous administration are generally about 20 micrograms to 2 grams of active compound per kilogram of body weight. Suitable dosage ranges for intranasal administration are generally about 0.01 pg / kg to 1 mg / kg of body weight. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0126] For systemic administration, the therapeutically effective dose can be estimated initially from in vitro assays, e.g., IC determined in cell culture. 50Dosage can be formulated in animal models to achieve a circulating concentration range that includes. Such information can be used to more accurately determine useful doses in humans. Initial dosages can also be estimated from in vivo data, for example animal models, using techniques that are well known in the art. Those skilled in the art can easily optimize administration to humans based on animal data.

[0127] Dosage and interval can be adjusted individually to provide sufficient plasma level of compound to maintain therapeutic effect.In case of local administration or selective uptake, effective local concentration of compound may not be related to plasma concentration.Those skilled in the art can optimize therapeutically effective local dosage without undue experimentation.

[0128] The amount of compound administered will of course depend on the subject being treated, the subject's weight, the severity of the illness, the mode of administration, and the judgment of the prescribing physician.Therapy may be repeated intermittently while symptoms are detectable, or even when symptoms are not detectable.Therapy may be provided alone or in combination with other drugs.

[0129] kit Also provided herein is an article of manufacture comprising packaging material and a pharmaceutical agent contained within the packaging material, wherein the pharmaceutical agent comprises at least one GCG / GCGR antagonist useful according to the methods disclosed herein, and the packaging material comprises a label or insert indicating that the GCG / GCGR antagonist can be used to treat a condition or disease characterized by severe insulin resistance.

[0130] While the present invention has been particularly shown and described with reference to certain embodiments, it will be understood by those skilled in the art that changes in form and detail may be made in the various embodiments disclosed herein without departing from the spirit and scope of the present invention, and that the various embodiments disclosed herein are not intended to be limitations on the scope of the claims. EXAMPLES

[0131] The following examples are provided to allow those skilled in the art to have a complete disclosure and description of how the methods disclosed herein are carried out.Efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be taken into account.Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric pressure.

[0132] Example 1: Evaluation of GCGR antagonists in preventing hyperglycemia in a mouse model of severe insulin resistance The insulin receptor antagonist S961, when administered to mice via osmotic minipumps, leads to severe insulin resistance and hyperglycemia (Gusarova (V et al. (2014), Cell 159:691-696; Yi P et al. (2013), Cell 153:747-758; Schaffer L. (2008), Biochem. Biophys. Res. Commun. 376:380-383). This model of severe insulin resistance was used to determine the effect of anti-GCGR antibodies in preventing hyperglycemia, as well as on blood glucose levels and plasma beta-hydroxybutyrate levels (as a measure of ketonemia) resulting from severe insulin resistance. material: hIgG4 isotype control H4H1327P, anti-hGCGR hIgG4 S961, insulin receptor antagonist (custom synthesized by Celtek Peptides using published sequence (Schaffer L. (2008) Biochem. Biophys. Res. Commun. 376:380-383)

[0133] Animals and injections: Twenty-nine mice were divided into four groups of 6–8 mice. The first group was subcutaneously injected with 10 mg / kg hIgG4 isotype control on days 0, 6, and 14, and was subcutaneously infused with PBS via osmotic minipumps (Alzet 2002) from day 7 onwards. The second group was subcutaneously injected with 10 mg / kg H4H1327P on days 0, 6, and 14, and was subcutaneously infused with PBS via osmotic minipumps (Alzet 2002) from day 7 onwards. The third group was subcutaneously injected with 10 mg / kg hIgG4 isotype control on days 0, 6, and 14, and was subcutaneously infused with 20 nmol / week S961 via osmotic minipumps (Alzet 2002) from day 7 onwards. The fourth group was injected subcutaneously with 10 mg / kg H4H1327P on days 0, 6, and 14, and 20 nmol / week S961 via osmotic minipump (Alzet 2002) from day 7 onwards. Mice were bled on days 0, 3, 6, 10, 14, 17, and 21 for blood glucose measurements. Mean ± SEM of blood glucose levels at each time point was calculated for each group and is shown in Table 3. Plasma was collected at baseline and on days 6, 14, and 21 for determination of insulin and beta-hydroxybutyrate levels. Mean ± SEM of plasma beta-hydroxybutyrate and insulin levels at each time point was calculated for each group and is shown in Tables 4 and 5. [Table 3] [Table 4] [Table 5]

[0134] result: Statistical analysis was performed using Prism software (version 6). Two-way ANOVA with Bonferroni's multiple comparison test was used to assess significance versus the control group (group 1). a: p<0.05, b: p<0.01, c: p<0.001, d: p<0.0001.

[0135] H4H1327P-treated and PBS-injected animals (Group 2) showed reduced blood glucose after H4H1327P administration (between days 3 and 21) compared to isotype control-treated and PBS-injected animals (Group 1), confirming the glucose-lowering efficacy of H4H1327P. Isotype control-treated and S961-injected animals (Group 3) showed increased blood glucose after S961 injection (between days 10 and 21) compared to isotype control-treated and PBS-injected animals (Group 1), confirming the hyperglycemic effect of S961. H4H1327P-treated and S961-injected animals (Group 4) had blood glucose levels comparable to those of Group 1 mice between days 10 and 21 after S961 injection. See Figure 1A.

[0136] Plasma insulin levels were elevated in animals treated with isotype control and injected with S961 (Group 3) compared to animals treated with isotype control and injected with PBS (Group 1) on days 14 and 21, confirming the effect of S961 to inhibit the insulin receptor during the study. Insulin levels were also elevated in animals treated with H4H1327P and injected with S961 (Group 4) compared to animals treated with isotype control and injected with S961 (Group 3). See Figure 1B.

[0137] Consistent with previous studies (Okamoto et al., 2015, Endocrinology 156(8):2781-2794), H4H1327P demonstrated elevated plasma glucagon levels, an effect independent of S961 administration (see Figure 1C).

[0138] Plasma beta-hydroxybutyrate levels were elevated on days 14 and 21 in animals treated with isotype control and injected with S961 (Group 3) compared to animals treated with isotype control and injected with PBS (Group 1), but were unchanged in animals treated with H4H1327P and injected with S961 (Group 4). See Figure 1D. In addition, no differences in body weight were observed between treatment groups (see Figure 1E).

[0139] These data indicate that H4H1327P prevents insulin receptor antagonist-induced hyperglycemia and ketonemia and lowers blood glucose even in the presence of severe hyperinsulinemia.

[0140] Example 2: Evaluation of GCGR antagonists in reversing hyperglycemia in a mouse model of severe insulin resistance Using the same animal model and the same materials as mentioned in Example 1, except that an insulin receptor antagonist was administered 4 days before the injection of anti-GCGR antibody, the effect of anti-GCGR antibody in reversing established hyperglycemia induced by severe insulin resistance was determined. The effect on blood glucose and plasma beta-hydroxybutyrate levels was also determined.

[0141] Animals and injections: Thirty-two mice were divided into four groups of eight mice. The first group was subcutaneously infused with PBS via osmotic minipumps (Alzet 2002) from day 0 and subcutaneously injected with 10 mg / kg hIgG4 isotype control on days 4, 11, and 18. The second group was subcutaneously infused with PBS from day 0 and subcutaneously injected with 10 mg / kg H4H1327P on days 4, 11, and 18. The third group was subcutaneously infused with 20 nmol / week S961 from day 0 and subcutaneously injected with 10 mg / kg hIgG4 isotype control on days 4, 11, and 18. The fourth group was subcutaneously infused with 20 nmol / week S961 from day 0 and subcutaneously injected with 10 mg / kg H4H1327P on days 4, 11, and 18. Mice were bled on days 0, 4, 7, 11, 14, 18, and 21 for blood glucose measurements. The mean ± SEM of blood glucose levels at each time point was calculated for each group and is shown in Table 6. Plasma was collected at baseline and days 4, 11, and 21 for determination of insulin and beta-hydroxybutyrate levels. The mean ± SEM of plasma beta-hydroxybutyrate and insulin levels at each time point was calculated for each group and is shown in Tables 7 and 8. [Table 6] [Table 7] [Table 8]

[0142] result: Statistical analysis was performed using Prism software (version 6). Two-way ANOVA with Bonferroni's multiple comparison test was used to assess significance versus the control group (group 1). a: p<0.05, b: p<0.01, c: p<0.001, d: p<0.0001.

[0143] S961-injected and isotype control-treated animals (Group 3) showed increased blood glucose after S961 injection (between days 4 and 21) compared to PBS-injected and isotype control-treated animals (Group 1), confirming the hyperglycemic effect of S961. S961-injected and H4H1327P-treated animals (Group 4) showed blood glucose levels after H4H1327P injection that were nearly identical to those of PBS-injected and isotype control-treated animals (Group 1). PBS-injected and H4H1327P-treated animals (Group 2) maintained reduced blood glucose levels after H4H1327P injection (between days 4 and 21) compared to isotype control-injected and PBS-injected animals (Group 1), confirming the glucose-lowering efficacy of H4H1327P. See Figure 2A.

[0144] Plasma insulin levels were elevated on days 4, 11, and 21 in animals that received S961 and isotype control (Group 3) compared to animals that received PBS and isotype control (Group 1), confirming the effect of S961 in inhibiting the insulin receptor over the course of the study. See Figure 2B. Hyperinsulinemia (Table 8 and Figure 2B) and hyperglucagonemia (see Figure 2C) were more pronounced in mice that received both receptor antagonists.

[0145] Plasma beta-hydroxybutyrate levels were elevated in animals injected with S961 and treated with isotype control (Group 3) compared to animals injected with PBS and treated with isotype control (Group 1) on days 11 and 21, but levels were unchanged at these same time points in animals injected with S961 and treated with H4H1327P (Group 4) versus animals in Group 1. See FIG. 2D.

[0146] Consistent with previous findings (Okamoto et al., 2015), H4H1327P increased circulating amino acid levels. S961 also increased circulating amino acid levels, but to a lesser extent than the antibody (see Figure 2E). Inhibition of both insulin and glucagon receptors led to a further increase in plasma amino acid levels (see Figure 2E). No changes in body weight were observed (see Figure 2F).

[0147] These data indicate that H4H1327P reverses insulin receptor antagonist-induced hyperglycemia and ketonemia, lowering blood glucose even in the presence of severe hyperinsulinemia.

[0148] Example 3: Evaluation of GCGR antagonists in reversing hepatic Pepck expression induced by insulin receptor antagonists Liver samples obtained from mice treated according to each of the four groups in Example 1 were lysed in ice-cold RIPA buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 10 mM β-glycerophosphate, 5 mM dibasic sodium pyrophosphate, and 1% NP-40) in the presence of protease and phosphatase inhibitor cocktail (Thermo-Fisher), 1 mM DTT, and 2 mM Na3VO4. Total sample lysates were mixed with 6x SDS loading buffer (Alfa-Aesar) and boiled for 5 min. Protein samples (10-100 μg) were loaded onto 4-20% gradient SDS-PAGE gels (Bio-Rad), separated, and transferred to polyvinylidene difluoride membranes. Membranes were blocked for 1 h with 5% bovine serum albumin in 1× TBS supplemented with 0.1% Tween 20 (Bio-Rad) and incubated with an antibody against phosphoenolpyruvate carboxykinase (PEPCK) (1:250, Abcam). Bound antibodies were detected using anti-rabbit or anti-mouse secondary antibodies conjugated to horseradish peroxidase (1:10,000, Jackson ImmunoResearch) and enhanced chemiluminescence reagents (Thermo-Fisher). Band intensities were quantified in Image J software.

[0149] Western blot analysis revealed that the levels of phosphoenolpyruvate carboxykinase (Pepck), a rate-limiting gluconeogenic enzyme, were reduced by 70% in the livers of mice treated with H4H1327P (see Figures 3A and B). Conversely, Pepck levels in the livers of mice injected with S961 were elevated 2.3-fold, but this effect was reversed by H4H1327P to 30% below baseline. Thus, the relative levels of glucagon and insulin signaling control Pepck expression, as previously demonstrated (Lynedjian et al., 1995; Rucktaeschel et al., 2000; Chakravarty et al., 2005). These data indicate that blocking GCGR in mice with H4H1327P prevents hyperglycemia induced by severe insulin resistance by suppressing hepatic glucose production.

[0150] Example 4: Evaluation of GCGR and insulin receptor antagonism in alpha and beta cell mass Pancreata from mice treated according to each of the four groups in Example 2 were fixed in 10% neutral buffered formalin solution for 48 hours, embedded in paraffin, and sectioned on slides. Pancreatic tissues and cells were permeabilized and hybridized with a combination of mRNA probes for mouse Gcg and Ins2 according to the manufacturer's instructions (Advanced Cell Diagnostics). A chromogenic kit was used to amplify the mRNA signal (Advanced Cell Diagnostics). Halo digital imaging analysis software (Indica Labs) was used to measure the area of ​​glucagon and insulin positive cells. Percentage of glucagon and insulin positive areas were calculated as a percentage of the total pancreatic area. Alpha and beta cell mass was calculated by multiplying the area of ​​alpha and beta cells of each animal by the corresponding pancreatic weight. Islet number was measured by counting the number of insulin positive islets on the section using Halo digital imaging analysis software and normalized by the total pancreatic area of ​​the section.

[0151] H4H1327P increased pancreatic weight by 19%, an effect that was greater in the presence of both H4H1327P and S961 (33%) (see Figure 4A). RNA in situ hybridization (RNA ISH) using probes for Gcg and Ins2 was used for morphometric analysis of pancreatic sections. H4H1327P increased α-cell mass 5.7-fold (see Figure 4B), while S961 administration increased β-cell mass 3-fold (see Figure 4C). H4H1327P alone had no effect on β-cell mass, but unexpectedly, β-cell mass doubled in the simultaneous presence of S961 and H4H1327P when compared to S961 alone, and increased 5.8-fold compared to control mice (see Figure 4C). It is important to note that further expansion of β-cell mass occurred in the environment of normal blood glucose levels (Table 3). Alpha cell mass was slightly increased by S961 treatment (1.6-fold) and in the simultaneous presence of H4H1327P (1.4-fold compared to H4H1327P alone) (see FIG. 4B). S961 increased the number of islets per total pancreatic area by 49%, whereas combined treatment with S961 and H4H1327P increased the number of islets per area by 82% (see FIG. 4D). In summary, when glucagon and insulin signaling were inhibited, a compensatory increase in alpha and beta cell mass was produced. A novel finding is that beta cell mass doubled when glucagon signaling was blocked in insulin-resistant mice, and this effect occurred at normal blood glucose levels. The present invention provides, for example, the following items. (Item 1) 1. A method for reducing blood glucose levels and / or ketone body levels or for treating a condition or disease associated with or characterized in part by high blood glucose or elevated ketone bodies, or at least one symptom or complication associated with said condition or disease, comprising administering to a patient having severe insulin resistance a therapeutically effective amount of a composition comprising a glucagon (GCG) inhibitor or a glucagon receptor (GCGR) antagonist, thereby reducing blood glucose levels or ketone body levels or mediating said condition or disease, or alleviating or reducing the severity of at least one symptom or complication associated with said condition or disease. (Item 2) 2. The method according to item 1, wherein the patient with severe insulin resistance suffers from a condition or disease selected from the group consisting of Donohue syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolism disorders, Bardet-Biedl syndrome, and conditions or diseases associated with the presence of genetic variants reported to result in severe insulin resistance. (Item 3) 2. The method of claim 1, wherein insulin-degrading protease activity is detected in the patient's serum. (Item 4) 2. The method of claim 1, wherein neutralizing anti-insulin antibodies or anti-insulin receptor antibodies are detected in the patient's serum. (Item 5) 2. The method of claim 1, wherein the insulin resistance is associated with a genetic variant of one or more genes selected from the group consisting of INSR, PSMD6, ADRA2A, AGPAT2, AKT2, APPL1, BBS1, BSCL2, CIDEC, GRB10, IRS2, KLF14, LEP, LEPR, LMNA, MC4R, PCNT, PIK2CA, POLD1, PPARG, PTPRD, PTRF, RASGRP1, TBC1D4, and TCF7L2. (Item 6) 2. The method of claim 1, wherein the GCG inhibitor or the GCGR antagonist is administered in combination with insulin. (Item 7) 13. The method of claim 1, wherein the composition is administered to the patient in combination with at least one additional therapeutic agent. (Item 8) 8. The method of claim 7, wherein the at least one additional therapeutic agent is selected from the group consisting of insulin, a biguanide, hIGF1, leptin, pioglitazone, vildagliptin, acarbose, an alpha-glycosidase inhibitor, L-arginine, a dipeptidyl-peptidase-4 inhibitor, an insulin secretagogue, an amylin receptor agonist, an insulin sensitizer, FGF21, an SGLT2 inhibitor, an SGLT1 inhibitor, a GLP-1 agonist, a GLP-1 receptor activator, a beta3 adrenergic agonist, an NPR1 agonist, an NPR3 antagonist, triiodothyronine, a second GCG inhibitor, and a second GCGR antagonist. (Item 9) The method according to item 1, wherein the GCG inhibitor or the GCGR antagonist is an isolated human monoclonal antibody or an antigen-binding fragment thereof. (Item 10) The method of claim 1, wherein the GCGR antagonist is an isolated human monoclonal antibody or an antigen-binding fragment thereof, comprising a complementarity determining region (CDR) of a heavy chain variable region (HCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and a CDR of a light chain variable region (LCVR) having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 11) 11. The method of claim 10, wherein the isolated antibody or antigen-binding fragment thereof comprises (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 12) 11. The method of claim 10, wherein the isolated antibody or antigen-binding fragment thereof comprises a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148. (Item 13) The method of claim 10, wherein the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair set forth in SEQ ID NO: 86 / 88. (Item 14) The method of claim 1, wherein the GCG inhibitor is an isolated human monoclonal antibody or an antigen-binding fragment thereof, comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 15) 15. The method of claim 14, wherein the isolated antibody or antigen-binding fragment thereof comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 16) 15. The method of claim 14, wherein the isolated antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. (Item 17) 15. The method of claim 14, wherein the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 166 / 174 or SEQ ID NO: 182 / 190. (Item 18) 9. The method of claim 8, wherein the insulin secretagogue is selected from the group consisting of sulfonylureas, ATP-sensitive K channel antagonists, and meglitinides. (Item 19) 9. The method of claim 8, wherein the insulin sensitizer is selected from the group consisting of thiazolidinediones and rosiglitazone. (Item 20) 2. The method of claim 1, wherein the ketone body is beta-hydroxybutyrate. (Item 21) 1. A method of treating a patient having severe insulin resistance, the patient exhibiting elevated blood glucose levels, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a GCG inhibitor or GCGR antagonist. (Item 22) 22. The method of claim 21, wherein the patient with severe insulin resistance suffers from a condition or disease selected from the group consisting of Donohue syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolism disorders, Bardet-Biedl syndrome, and conditions or diseases associated with the presence of genetic variants reported to result in severe insulin resistance. (Item 23) 22. The method of claim 21, wherein insulin-degrading protease activity is detected in the patient's serum. (Item 24) 22. The method of claim 21, wherein neutralizing anti-insulin antibodies or anti-insulin receptor antibodies are detected in the patient's serum. (Item 25) 22. The method of claim 21, wherein the insulin resistance is associated with a genetic variant of one or more genes selected from the group consisting of INSR, PSMD6, ADRA2A, AGPAT2, AKT2, APPL1, BBS1, BSCL2, CIDEC, GRB10, IRS2, KLF14, LEP, LEPR, LMNA, MC4R, PCNT, PIK2CA, POLD1, PPARG, PTPRD, PTRF, RASGRP1, TBC1D4, and TCF7L2. (Item 26) 22. The method of claim 21, wherein the GCG inhibitor or the GCGR antagonist is administered in combination with insulin. (Item 27) 22. The method of claim 21, wherein the composition is administered to the patient in combination with at least one additional therapeutic agent. (Item 28) 28. The method of claim 27, wherein the at least one additional therapeutic agent is selected from the group consisting of insulin, a biguanide, hIGF1, leptin, pioglitazone, vildagliptin, acarbose, an alpha-glycosidase inhibitor, L-arginine, a dipeptidyl-peptidase-4 inhibitor, an insulin secretagogue, an amylin receptor agonist, an insulin sensitizer, FGF21, an SGLT2 inhibitor, an SGLT1 inhibitor, a GLP-1 agonist, a GLP-1 receptor activator, a beta3 adrenergic agonist, an NPR1 agonist, an NPR3 antagonist, triiodothyronine, a second GCG inhibitor, and a second GCGR antagonist. (Item 29) 22. The method of claim 21, wherein the GCG inhibitor or GCGR antagonist is an isolated human monoclonal antibody or an antigen-binding fragment thereof. (Item 30) The method of claim 21, wherein the GCGR antagonist is an isolated human monoclonal antibody comprising a CDR of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and a CDR of LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 31) 31. The method of claim 30, wherein the isolated antibody or antigen-binding fragment thereof comprises (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 32) 31. The method of claim 30, wherein the isolated antibody or antigen-binding fragment comprises a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148. (Item 33) 31. The method of claim 30, wherein the isolated human monoclonal antibody comprises the HCVR / LCVR amino acid sequence pair set forth in SEQ ID NO: 86 / 88. (Item 34) The method of claim 21, wherein the GCG inhibitor is an isolated human monoclonal antibody or an antigen-binding fragment thereof, comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 35) 35. The method of claim 34, wherein the isolated antibody or antigen-binding fragment thereof comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 36) 35. The method of claim 34, wherein the isolated antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. (Item 37) 35. The method of claim 34, wherein the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 166 / 174 or SEQ ID NO: 182 / 190. (Item 38) 29. The method of claim 28, wherein the insulin secretagogue is selected from the group consisting of sulfonylureas, ATP-sensitive K channel antagonists, and meglitinides. (Item 39) 29. The method of claim 28, wherein the insulin sensitizer is selected from the group consisting of thiazolidinediones and rosiglitazone. (Item 40) A method for reducing the amount and / or dosage of insulin required to treat a patient with severe insulin resistance, the patient exhibiting severe insulin resistance and elevated blood glucose levels, the method comprising administering to the patient a therapeutically effective amount of a composition comprising a GCG inhibitor or GCGR antagonist. (Item 41) 41. The method of item 40, wherein the GCG inhibitor or the GCGR antagonist is administered in combination with insulin. (Item 42) 41. The method of claim 40, wherein the amount and / or dosage of insulin may be reduced by about 30% to about 95% when co-administered with a GCGR antagonist, wherein the GCGR antagonist is an isolated human monoclonal antibody that specifically binds to GCGR. (Item 43) 41. The method of claim 40, wherein the amount and / or dosage of insulin can be reduced by about 90% when co-administered with a GCGR antagonist, wherein the antagonist is an isolated human monoclonal antibody that specifically binds to GCGR. (Item 44) 41. The method of item 40, wherein the patient with severe insulin resistance suffers from a condition or disease selected from the group consisting of Donahue's syndrome, Rabson-Mendenhall syndrome, type A insulin resistance, type B insulin resistance, HAIR-AN (hyperandrogenism, insulin resistance, and acanthosis nigricans) syndrome, pseudoacromegaly, Alström syndrome, myotonic dystrophy, Werner's syndrome, lipodystrophy, cirrhosis, monogenic morbid obesity, hyperproinsulinemia, carboxypeptidase E deficiency, arginine metabolism disorders, Bardet-Biedl syndrome, and conditions or diseases associated with the presence of genetic variants reported to result in severe insulin resistance. (Item 45) 41. The method of claim 40, wherein insulin-degrading protease activity is detected in the patient's serum. (Item 46) 41. The method of claim 40, wherein neutralizing anti-insulin antibodies or anti-insulin receptor antibodies are detected in the patient's serum. (Item 47) 41. The method of claim 40, wherein the insulin resistance is associated with a genetic variant of one or more genes selected from the group consisting of INSR, PSMD6, ADRA2A, AGPAT2, AKT2, APPL1, BBS1, BSCL2, CIDEC, GRB10, IRS2, KLF14, LEP, LEPR, LMNA, MC4R, PCNT, PIK2CA, POLD1, PPARG, PTPRD, PTRF, RASGRP1, TBC1D4, and TCF7L2. (Item 48) 41. The method of claim 40, wherein the composition is administered to the patient in combination with at least one additional therapeutic agent. (Item 49) 49. The method of item 48, wherein the at least one additional therapeutic agent is selected from the group consisting of a biguanide, hIGF1, leptin, pioglitazone, vildagliptin, acarbose, an alpha-glycosidase inhibitor, L-arginine, a dipeptidyl-peptidase-4 inhibitor, an insulin secretagogue, an amylin receptor agonist, an insulin sensitizer, FGF21, an SGLT2 inhibitor, an SGLT1 inhibitor, a GLP-1 agonist, a GLP-1 receptor activator, a beta3 adrenergic agonist, an NPR1 agonist, an NPR3 antagonist, triiodothyronine, a second GCG inhibitor, and a second GCGR antagonist. (Item 50) 41. The method of claim 40, wherein the GCG inhibitor or GCGR antagonist is an isolated human monoclonal antibody or an antigen-binding fragment thereof. (Item 51) The method of claim 40, wherein the GCGR antagonist is an isolated human monoclonal antibody comprising a CDR of HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and a CDR of LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 52) 52. The method of claim 51, wherein the isolated antibody or antigen-binding fragment thereof comprises (a) an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 18, 34, 50, 66, 70, 86, 90, 106, 110, 126, 130, and 146, and / or (b) an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 26, 42, 58, 68, 78, 88, 98, 108, 118, 128, 138, and 148. (Item 53) 52. The method of claim 51, wherein the isolated antibody or antigen-binding fragment comprises a HCVR / LCVR sequence pair selected from the group consisting of SEQ ID NOs: 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 68, 70 / 78, 86 / 88, 90 / 98, 106 / 108, 110 / 118, 126 / 128, 130 / 138, and 146 / 148. (Item 54) 52. The method of claim 51, wherein the isolated antibody comprises the HCVR / LCVR amino acid sequence pair set forth in SEQ ID NO: 86 / 88. (Item 55) The method of claim 40, wherein the GCG inhibitor is an isolated human monoclonal antibody or an antigen-binding fragment thereof, comprising: (a) three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) contained in a heavy chain variable region (HCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294; and (b) three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained in a light chain variable region (LCVR) amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 56) 56. The method of claim 55, wherein the isolated antibody or antigen-binding fragment thereof comprises an HCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 150, 166, 182, 198, 214, 230, 246, 262, 278, and 294, and / or an LCVR having an amino acid sequence selected from the group consisting of SEQ ID NOs: 158, 174, 190, 206, 222, 238, 254, 270, 286, and 302. (Item 57) 56. The method of claim 55, wherein the isolated antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair selected from the group consisting of SEQ ID NOs: 150 / 158, 166 / 174, 182 / 190, 198 / 206, 214 / 222, 230 / 238, 246 / 254, 262 / 270, 278 / 286, and 294 / 302. (Item 58) 56. The method of claim 55, wherein the isolated antibody or antigen-binding fragment thereof comprises the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 166 / 174 or SEQ ID NO: 182 / 190. (Item 59) 50. The method of claim 49, wherein the insulin secretagogue is selected from the group consisting of sulfonylureas, ATP-sensitive K channel antagonists, and meglitinides. (Item 60) 50. The method of claim 49, wherein the insulin sensitizer is selected from the group consisting of thiazolidinediones and rosiglitazone. (Item 61) A method for lowering blood glucose levels and / or beta-hydroxybutyrate levels or for treating a condition or disease associated with or characterized in part by high blood glucose or high levels of ketone bodies, or at least one symptom or complication associated with said condition or disease, comprising administering to a patient having severe insulin resistance a therapeutically effective amount of a composition comprising an inhibitor of GCGR signaling, thereby lowering blood glucose levels or beta-hydroxybutyrate levels, or mediating said condition or disease, or alleviating or reducing the severity of at least one symptom or complication associated with said condition or disease. (Item 62) 61. The method of item 60, wherein the inhibitor of GCGR signaling is selected from the group consisting of antisense molecules, GCGR antibodies, small molecule inhibitors, peptide inhibitors, DARPins, Spiegelmers, aptamers, engineered Fn type III domains, GCG antibodies, and derivatives thereof. (Item 63) A method for suppressing glucose production in the liver, comprising administering to a patient having severe insulin resistance a therapeutically effective amount of a composition comprising an inhibitor of GCGR signaling, thereby suppressing glucose production in the liver. (Item 64) 1. A method for increasing beta cell mass in a patient having severe insulin resistance, comprising administering to the patient a therapeutically effective amount of a composition comprising an inhibitor of GCGR signaling, thereby increasing beta cell mass compared to the beta cell mass before treatment.

Claims

[Claim 1] The invention as depicted in the drawings.

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