WO2019023056A1 - Insulin receptor antibodies and uses thereof - Google Patents

Abstract

Antibodies that modulate insulin receptor signaling are disclosed. As agonists, the antibodies may be developed as therapeutics. The antagonists may serve as a tool to induce hyperglycemia and insulin resistance in cellular and animal models.

Description

INSULIN RECEPTOR ANTIBODIES AND USES THEREOF

FIELD OF THE INVENTION

[0001] Provided herein are novel isolated antibodies that immunospecifically bind to insulin receptor (IR) and modulate its signaling, and methods of using the same.

BACKGROUND OF THE INVENTION

[0002] The peptide hormone insulin is a major regulator of glucose homeostasis and cell growth. The first step in insulin action is the binding of the hormone to the insulin receptor (IR), an integral membrane glycoprotein, also designated as CD220 or HHF5. The IR belongs to the tyrosine kinase growth factor receptor superfamily and is composed of two extracellular a subunits that bind insulin, and two transmembrane β subunits with intrinsic tyrosine kinase activity. The IR is expressed in two isoforms, IR-A and IR-B which may form heterodimers, IR-A/IR-B, and hybrid IR/IGF-IR receptors (Belfiore et al, Endocrine Rev., 2009, 30(6):586-623).

[0003] When insulin binds to the IR, the receptor is activated by tyrosine

autophosphorylation and the IR tyrosine kinase phosphorylates various effector molecules, including the insulin receptor substrate-1 (IRS-1), leading to hormone action (Ullrich et al, Nature 313 : 756-761 , 1985; Goldfine et al, Endocrine Reviews 8: 235-255, 1987; White and Kahn, Journal Biol. Chem. 269: 1-4, 1994). IRS-1 binding and phosphorylation eventually leads to an increase in the high affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue, and therefore to an increase in the uptake of glucose from blood into these tissues. Glut4 is transported from cellular vesicles to the cell surface, where it then can mediate the transport of glucose into the cell. A decrease in IR signaling, leads to a reduction in the uptake of glucose by cells, hyperglycemia (an increase in circulating glucose).

[0004] Reduction in glucose uptake can result in insulin resistance, which describes a condition in which physiological amounts of insulin are inadequate to produce a normal insulin response from cells or tissues. Severe insulin resistance is associated with diabetes, while less severe insulin resistance is also associated with a number of disease states and conditions present in approximately 30-40% of non-diabetic individuals (reviewed in Woods et al, End, Metab & Immune Disorders— Drug Targets 9: 187-198, 2009).

[0005] Current treatments for diabetes and insulin resistance are directed toward improving insulin secretion, reducing glucose production, and enhancing insulin action.

[0006] Several models have been used to induce and study insulin resistance in vitro and in vivo. However, many of these treatments act through a variety of mechanisms, either directly or indirectly affecting the IR. In addition, these models may exhibit off- target effects and may not translate between in vitro and in vivo systems (Lo et al, Cell Rep, 2013, 5(1): p. 259-70). Treating cultured cells with cytokines, high insulin levels, excessive glucose and/or lipid concentrations, and hypoxia exposure lead to decreased IR phosphorylation and insulin signaling (Kroder et al, J Clin Invest, 1996, 97(6): p. 1471 -7; Kawanaka et al, J Biol Chem, 2001 , 276(23): p. 20101 -7; Rotter et al., J Biol Chem, 2003, 278(46): p. 45777-84). In vivo models have relied on diet-induced and/or genetic models, which have provided some valuable understanding into glucose homeostasis. However, these methods are time consuming and costly to establish. The diet-induced obesity (DIO) mouse model is routinely used, but implicates numerous metabolic and inflammatory pathways, and consists of a number of dietary factors that may influence physiological responses (Thomson et al, J Biol Chem, 1997, 272(12): p. 7759-64; Storlien et al, Br J Nutr, 2000. 83 Suppl 1 : p. S85-90; Winzell et al, Diabetes, 2004, 53 Suppl 3 : p. S215-9; Nilsson et al., Acta Pharmacol Sin, 2012. 33(2): p. 173- 81). Genetic models of insulin resistance provide a more direct evaluation, yet these models must be made individually and are not easily transferable among species. One valuable example is the liver insulin receptor knockout mouse (LIRKO). These animals exhibit significant hyperinsulinemia and hyperglycemia, but do not account for IR function in peripheral tissues, such as skeletal muscle and adipose (Michael et al., Mol Cell, 2000, 6(1): p. 87-97; Escribano et al, Diabetes, 2009. 58(4): p. 820-8). In addition, LIRKO mice develop hepatic impairment by 6 months resulting in hypoglycemia (Barrett, J Clin Invest, 2003. 11 1(4): p. 434-5). While the LIRKO mouse is valuable, there are no higher order species with which this model can be directly compared. Genetic manipulations of other species have either not been performed or require long development times to create. Therefore, improved methods and tools that induce insulin resistance are needed for understanding the biology and treatment of this disorder. [0007] Insulin resistance has been induced consistently both in vivo and in vitro by a peptide that directly targets the IR. The biosynthetic peptide, S961, directly competes with insulin for the receptor orthosteric site (Schaffer et al, Biochem Biophys Res Commun, 2008, 376(2): p. 380-3; Knudsen et al, PLoS One, 2012, 7(12): p. e51972). In vivo, S961 efficiently induces insulin resistance in rats at high concentrations and has been used to generate rodent models of diabetes (Vikram and Jena, Biochem Biophys Res Commun, 2010, 398(2): p. 260-5; Rostoker et al, Endocrinology, 2013, 154(5): p. 1701-10). While S961 induces insulin resistance in vivo, it is not a pure IR antagonist as it exhibits agonist activity at low concentrations in vitro (Knudsen et al, PLoS One, 2012, 7(12): p. e51972). This may produce altered signaling depending on systemic distribution and elimination in vivo. Nevertheless, designing antagonists that specifically target the IR holds promise for generating insulin resistant cell lines and animal models using a consistent and efficient method.

SUMMARY OF THE INVENTION

[0008] The invention relates to novel monoclonal antibodes that immunospecifically bind to IR. In one embodiment, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR, comprises a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 3, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; wherein the CDRs are defined according to Kabat [BBBB138]. In another embodiment, the isolated antibody, or antigen-binding fragment thereof, has a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9. In another embodiment, the isolated antibody, or antigen- binding fragment thereof, is a human antibody or antigen-binding fragment. In another embodiment, the isolated antibody, or antigen-binding fragment thereof, is

recombinant. In another embodiment, the isolated antibody, or antigen-binding fragment thereof, is a Fab fragment, a F(ab')2 fragment, or a single chain antibody. In another embodiment, the invention relates to a nucleic acid molecule encoding the isolated antibody, or antigen-binding fragment thereof. In another embodiment, the invention relates to a vector comprising the nucleic acid molecule encoding the isolated antibody, or antigen-binding fragment thereof. In another embodiment, the invention relates to a cell expressing the isolated antibody, or antigen-binding fragment thereof.

[0009] The invention also relates to a method of antagonizing insulin receptor signaling, comprising administering to a subject an effective amount of the antibody, that immunospecifically binds to IR, or antigen-binding fragment thereof. In another embodiment, the method induces hyperglycemia, hyperinsulinemia, or insulin resistance. In another embodiment, the method induces hyperglycemia,

hyperinsulinemia, or insulin resistance, associated with type 2 diabetes.

[0010] The invention also relates to a method of screening compounds for the ability to modulate hyperglycemia, hyperinsulinemia , or insulin resistance, comprising: contacting a cell or a subject with the anti-IR antibody and with a test compound, and measuring the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the presence of the test compound relative to the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the absence of the test compound, wherein an increase in the levels of IR phosphorylation, Akt phosphorylation, or a decrease in blood glucose in the presence of the test compound indicates that the test compound is capable of reducing hyperglycemia, hyperinsulinemia , or insulin resistance; and wherein a decrease or no change in the levels of IR phosphorylation, Akt

phosphorylation, or increase or no change in blood glucose in the presence of the test compound indicates that the test compound is not capable of reducing hyperglycemia, hyperinsulinemia, or insulin resistance.

[0011] The invention also relates to an isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds IR, the antibody or antigen-binding fragment thereof comprising: a heavy chain CDRl comprising the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDRl comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; wherein the CDRs are defined according to Kabat [BBBB141]. In another embodiment, the isolated antibody, or antigen-binding fragment thereof has a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17. In another embodiment, the antibody or antigen-binding fragment is a human antibody or antigen- binding fragment. In another embodiment, the antibody or antigen-binding fragment is recombinant. In another embodiment, the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, or a single chain antibody. In another embodiment, the invention relates to a nucleic acid molecule encoding the isolated antibody, or antigen-binding fragment thereof. In another embodiment, the invention relates to a vector comprising the nucleic acid molecule.

[0012] The invention also relates to a method of agonizing insulin receptor signaling, comprising administering to a cell or a subject an effective amount of the agonist anti- IR antibody, or antigen-binding fragment thereof. In another embodiment, the method comprises an increase in insulin receptor tyrosine phosphorylation. In another embodiment, the method comprises an increase in insulin receptor tyrosine

phosphorylation. In another embodiment, the method comprises a reduction of hyperglycemia, hyperinsulinemia, or insulin resistance. In another embodiment, the hyperglycemia, hyperinsulinemia, or insulin resistance is associated with type 2 diabetes.

DESCRIPTION OF THE FIGURES

[0013] Figure 1 illustrates that BBBB138 binds allosterically to IR-A insulin receptor isoform at concentrations 1.56, 6.25, 25.0, 100 and 400 nM, determined by Surface Plasmon Resonance (SPR), as shown from bottom (lowest concentration) to top (highest concentration), with the black lines representing 1 : 1 Langmuir models of binding profiles.

[0014] Figure 2 illustrates that BBBB138 binds allosterically to IR-B insulin receptor isoform at concentrations 1.56, 6.25, 25.0, 100 and 400 nM, determined by SPR, as shown from bottom (lowest concentration) to top (highest concentration), with the black lines representing 1 : 1 Langmuir models of binding profiles.

[0015] Figure 3 illustrates insulin binding allosterically to the receptor in the presence of BBBB138 by SPR. BBBB138 was captured on an anti-human/anti-mouse Fc surface (box "BBBB138"); this was followed by the capture of the receptor through BBBB138 (box "Insulin Receptor"), and finally, insulin was titrated to test for binding to the receptor (box "Insulin"; insulin concentrations were 3.2, 16, 80, 400 and 2000 nM, shown from bottom (lowest concentration) to top (highest concentration)). The sensorgram shows the sequential injection of BBBB138, the receptor and the titration of insulin. Insulin binds to the receptor as can be observed. Insulin is of very low molecular weight compared to the receptor and generates low binding response;

therefore the binding profile of insulin to the receptor (box "Insulin") is zoomed in.

[0016] Figure 4 illustrates that BBBB138 decreases insulin-induced IR

phosphorylation in vitro in a dose-dependent manner. IR tyrosine phosphorylation levels were measured in HuH7 treated with increasing concentrations of BBBB138 for 30 minutes followed 1 nM insulin for 5 minutes. Data are presented as a percentage of the phospho-IR signal relative to the total IR levels and normalized to IgGl Isotype control (CNTO3930)-treated cells at equivalent concentrations. IC50 value was calculated using Nonlinear regression and Variable Slope model analyses (GraphPad Prism 6, GraphPad Software, Inc, La Jolla, CA).

[0017] Figure 5 illustrates that BBBB138 decreases insulin-induced IR tyrosine (Tyr) phosphorylation in vitro following treatments with different doses of insulin. Cells were treated for 30 minutes with either 30 nM IgGl Isotype control antibody

(CNTO3930) or BBBB138 at 3, 10 or 30 nM of BBBB138 followed by increasing concentrations insulin for 5 minutes. Data presented as mean ± SEM, n = 3 presented as a percentage of the phospho-IR signal relative to the total IR levels and normalized to the insulin dose-response curve. EC50 values were calculated using Nonlinear regression and Variable Slope model (GraphPad Prism 6).

[0018] Figure 6 illustrates the antagonistic effects of BBBB138 on in vitro IR phosphorylation over 120 min. Insulin time course of cells stimulated with 1 nM insulin that were treated for 30 mins with either 30 nM IgGl Isotype control

(CNTO3930) or 30 nM BBBB138. Data are presented as mean ± SEM, n = 3, with Phospho IR signal/Total IR levels normalized to % maximum of insulin curve.

[0019] Figure 7 illustrates insulin dose-response curves of IRS-1 tyrosine

phosphorylation was measured in HuH7 cells treated with either 30 nM IgGl Isotype control (CNTO3930) or BBBB138 for 30 minutes followed by insulin stimulation for an additional 5 minutes. Data were obtained using MSD analysis normalized to IgGl Isotype curve and are presented as mean ± SEM, n = 3.

[0020] Figure 8 illustrates that BBBB138 antagonizes downstream insulin signaling in vitro. Western blots from HuH7 cells that were treated for 30 minutes with either 10 nM of IgGl Isotype control (CNTO3930) or BBBB138. Cells were either unstimulated or stimulated with 1 nM insulin for an additional 15 minutes. Western blots were performed against Akt Ser473 phosphorylation and a Total Akt loading control.

[0021] Figure 9 illustrates that BBBB138 antagonizes glucose uptake in vitro. Glucose uptake was measured in 3T3 cells treated for 15 minutes with increasing concentrations of BBBB138 and stimulated with insulin for 15 minutes. Data are measured as counts per minute (CPM)^g and presented as mean ± SEM, n = 3.

[0022] Figure 10 illustrates that BBBB138 induces hyperglycemia in vivo. Blood glucose was measured in lean C57 mice treated with either BBBB138 or IgGl Isotype control (CNTO3930) using indicated concentrations. Body weight and fed blood glucose were measured one hour prior to sub-cutaneous injection of either PBS, IgG Isotype Control mAb, or BBBB138 at doses of 5, 10, and 25 mg/kg. Changes in blood glucose were monitored at 2, 4, 6, and 8 hours post dose.

[0023] Figure 11 illustrates that BBBB138 induces hyperglycemia and insulin resistance in vivo. Blood glucose was measured in lean C57 mice treated with either BBBB138 or IgGl Isotype control (CNTO3930). Body weight and fed blood glucose were measured one hour prior to sub-cutaneous injection of PBS, IgG Isotype Control mAb, or BBBB138 at doses of 5, 10, and 25 mg/kg. One day after treatment, a fasted 2 g/kg oral glucose tolerance test (OGTT) was performed. Glucose excursions were measured at 0, 30, 60, and 120 minutes post glucose challenge.

[0024] Figure 12 illustrates blood glucose levels 5 days post-treatment with either IgGl Isotype control antibody (CNTO3930) or BBBB138 at a dose of 5 mg/kg. A 4 hour food removal preceded intraperitoneal administration of saline or Exendin-4 (1 μg/kg). Blood glucose was measured at 0, 30, 60, 120, 180, and 240 minutes post- treatment. Data are presented as mean ± SEM, n = 8 for each experimental group.

[0025] Figure 13 illustrates changes in blood glucose values following the treatment with either BBBB138 or insulin receptor antagonist S961. Lean C57 mice were treated subcutaneously with BBBB138 (10 mg/kg), IgGl Isotype control (CNTO3930) (10 mg/kg), PBS or one of three doses of S691 (0.25, 0.5, and 1 mg/kg). Blood glucose was measured at peak agent exposure: 24 hours post-injection for mAbs or 1 hour post injection for the peptide antagonist. Data were presented as mean ± SEM, n = 8 for each experimental group except for 1 mg/kg S961 cohort where data is presented as mean ± SEM, n = 7. Statistical comparisons were performed with One-Way ANOVA and Dunnett's t Test using Prism Graphpad 6. PO.05*, PO.01 **, PO.001 ***, PO.0001 ****. Black bars represent the IgGl Isotype-treated animals and grey bars represent BBBB138-treated animals.

[0026] Figure 14 illustrates the effects of either BBBB138 or S961 on insulin levels in vivo. Data are presented as mean ± SEM, n = 8 for each experimental group except for 1 mg/kg S961 cohort where data is presented as mean ± SEM, n = 7. Statistical comparisons are performed with One-Way ANOVA and Dunnett's t Test using Prism Graphpad 6. PO.05*, PO.01 **, PO.001***, PO.0001****. Black bars represent the IgGl Isotype control (CNTO3930)-treated animals and grey bars represent BBBB138- treated animals.

[0027] Figure 15 illustrates phosphorylation of IR and Akt induced by varying concentrations of insulin in HuH7 cells treated with either BBBB138 or S961. Cells were pretreated with 100 nM BBBB138 or 1 nM S961 for 30 minutes followed by a 15 minute stimulation with increasing concentrations (1, 10 or 100 nM) of insulin.

[0028] Figure 16 illustrates the effects of pre-treatment with either IgGl Isotype control (CNTO3930) (30 nM) or BBBB141 (3 nM or 30 nM) on the levels of phospho- IR (total Tyr phosphorylation) relative to total IR insulin in HUH7 cells. Results are presented as mean ±SEM, n=3.

[0029] Figure 17 illustrates the induction of insulin receptor phosphorylation by BBBB141 and insulin. HUH7 cells were pretreated with either BBBB141 or IgGl isotype control (CNTO3930). Levels of phosphorylated insulin receptor were measured over 120 min time course. Results are presented as mean ±SEM, n=3.

[0030] Figure 18 illustrates the phosphorylation levels of insulin receptor site Tyrl328, relative to total IR, at various time points in HuH7 cells treated with indicated compounds. Results are presented as mean ±SEM, n=3. [0031] Figure 19 illustrates insulin receptor phosphorylation dose-response curves of HUH7 cells treated with either insulin, BBBB141, or a 1 : 1 co-stimulation of insulin and BBBB141. Results are presented as mean ±SEM, n=3.

[0032] Figure 20 illustrates insulin receptor phosphorylation dose-response curves of HUH7 cells treated with either bivalent, monovalent or the fab of BBBB141. Results are presented as mean ±SEM, n=3.

[0033] Figure 21 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor (IR)

phosphorylation at either Tyrl l50 or Tyrl l51 in the liver of C57BL/6 mice, as measured using 50 μg of total protein. **** indicates P<0.0001 using Students 's t-test.

[0034] Figure 22 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor (IR)

phosphorylation at either Tyrl 150 or Tyrl 151 in the gastrocnemius muscle of

C57BL/6 mice, as measured using 50 μg of total protein. **** indicates PO.0001 using Student's t-test.

[0035] Figure 23 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor signaling pathway (Thr308-Akt) phosphorylation in the liver of C57BL/6 mice, as measured using 20 μg of total protein.

[0036] Figure 24 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor signaling pathway (Thr308-Akt) phosphorylation in the gastrocnemius muscle of C57BL/6 mice, as measured using 20 μg of total protein. ** indicates PO.01, * indicates PO.05 using Student's t- test.

[0037] Figure 25 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor signaling pathway (Ser473-Akt) phosphorylation in the liver of C57BL/6 mice, as measured using 20 μg of total protein.

[0038] Figure 26 illustrates the effects of treatment with either IgGl isotype control (CNTO3930, 10 mg/kg) or BBBB141 (10 mg/kg) on insulin receptor signaling pathway (Ser473-Akt) phosphorylation in the gastrocnemius muscle of C57BL/6 mice, as measured using 20 μg of total protein. DETAILED DESCRIPTION OF THE INVENTION

[0039] The disclosed subject matter may be understood more readily by reference to the following detailed description taken in connection with the accompanying figures, which form a part of this disclosure. It is to be understood that the disclosed subject matter is not limited to those described and/or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting of the claimed subject matter.

[0040] Unless specifically stated otherwise, any description as to a possible mechanism or mode of action or reason for improvement is meant to be illustrative only, and the disclosed subject matter are not to be constrained by the correctness or incorrectness of any such suggested mechanism or mode of action or reason for improvement.

[0041] Throughout this text, the descriptions refer to antibodies and methods of using said antibodies. Where the disclosure describes or claims a feature or embodiment associated with an antibody, such a feature or embodiment is equally applicable to the methods of using the antibody. Likewise, where the disclosure describes or claims a feature or embodiment associated with a method of using an antibody, such a feature or embodiment is equally applicable to the antibody.

[0042] When a range of values is expressed, another embodiment includes from the one particular value and/or to the other particular value. Further, reference to values stated in ranges include each and every value within that range. All ranges are inclusive and may be combined. When values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value, unless the context clearly dictates otherwise.

[0043] It is to be appreciated that certain features of the disclosed subject matter which are, for clarity, described herein in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosed subject matter that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination. Definitions

[0044] As used herein, the singular forms "a," "an," and "the" include the plural.

[0045] Various terms relating to aspects of the description are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definitions provided herein.

[0046] The term "about" when used in reference to numerical ranges, cutoffs, or specific values is used to indicate that the recited values may vary by up to as much as 10% from the listed value. Thus, the term "about" is used to encompass variations of ± 10% or less, variations of ± 5% or less, variations of ± 1% or less, variations of ± 0.5% or less, or variations of ± 0.1% or less from the specified value.

[0047] "Antibodies" as used herein is meant in a broad sense and includes immunoglobulin molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies, antibody fragments, bispecific or multispecific antibodies formed from at least two intact antibodies or antibody fragments, dimeric, tetrameric or multimeric antibodies, single chain antibodies, domain antibodies and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity.

[0048] Immunoglobulins can be assigned to five major classes, IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgAl, IgA2, IgGl, IgG2, IgG3 and IgG4. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0049] "Antibody fragments" refers to a portion of an immunoglobulin molecule that retains the heavy chain and/or the light chain antigen binding site, such as heavy chain complementarity determining regions (HCDR) 1, 2 and 3, light chain complementarity determining regions (LCDR) 1, 2 and 3, a heavy chain variable region (VH), or a light chain variable region (VL). Antibody fragments include well known Fab, F(ab')2, Fd and Fv fragments as well as domain antibodies (dAb) consisting one VH domain. VH and VL domains may be linked together via a synthetic linker to form various types of single chain antibody designs where the VH/VL domains pair intramolecularly, or intermolecularly in those cases when the VH and VL domains are expressed by separate single chain antibody constructs, to form a monovalent antigen binding site, such as single chain Fv (scFv) or diabody; described for example in Int. Pat. Publ. No. WO1998/44001, Int. Pat. Publ. No. WO1988/01649; Int. Pat. Publ. No.

WO1994/13804; Int. Pat. Publ. No. WO 1992/01047.

[0050] An antibody variable region consists of a "framework" region interrupted by three "antigen binding sites". The antigen binding sites are defined using various terms: (i) Complementarity Determining Regions (CDRs), three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3) are based on sequence variability (Wu and Kabat, J Exp Med 132:211-50, 1970; Kabat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "Hypervariable regions", "HVR", or "HV", three in the VH (HI, H2, H3) and three in the VL (LI, L2, L3) refer to the regions of an antibody variable domains which are hypervariable in structure as defined by Chothia and Lesk (Chothia and Lesk, Mol Biol 196:901-17, 1987). Other terms include "IMGT-CDRs" (Lefranc et al, Dev Comparat Immunol 27:55-77, 2003) and "Specificity Determining Residue Usage" (SDRU) (Almagro, Mol Recognit 17: 132-43, 2004). The International ImMunoGeneTics (IMGT) database

(http://www_imgt_org) provides a standardized numbering and definition of antigen- binding sites. The correspondence between CDRs, HVs and IMGT delineations is described in Lefranc et al, Dev Comparat Immunol 27:55-77, 2003.

[0051] "Monoclonal antibody" as used herein refers to a homogenous antibody population with singular molecular composition. Monoclonal antibody may be monospecific or multispecific, or monovalent, bivalent or multivalent. A bispecific antibody is included in the term monoclonal antibody.

[0052] "Chothia residues" as used herein are the antibody VL and VH residues numbered according to Al-Lazikani (Al-Lazikani et al, J Mol Biol 273:927-48, 1997).

[0053] "Human antibody" refers to an antibody having heavy and light chain variable regions in which both the framework and the antigen binding site regions are derived from sequences of human origin. If the antibody contains a constant region, the constant region also is derived from sequences of human origin.

[0054] Human antibody comprises heavy or light chain variable regions that are "derived from" sequences of human origin if the variable regions of the antibody are obtained from a system that uses human germline immunoglobulin or rearranged immunoglobulin genes. Such exemplary systems are human immunoglobulin gene libraries displayed on phage, and transgenic non-human animals such as mice carrying human immunoglobulin loci as described herein. "Human antibody" may contain amino acid differences when compared to the human germline or rearranged immunoglobulin sequences due to for example naturally occurring somatic mutations or intentional introduction of substitutions. Typically, "human antibody" is at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical in amino acid sequence to an amino acid sequence encoded by a human germline or rearranged immunoglobulin gene. In some cases, "human antibody" may contain consensus framework sequences derived from human framework sequence analyses, for example as described in Knappik et al, J Mol Biol 296:57-86, 2000, or synthetic HCDR3 incorporated into human immunoglobulin gene libraries displayed on phage, for example as described in Shi et ctl, J Mol Biol 397:385-96, 2010 and Int. Pat. Publ. No. WO2009/085462.

[0055] Isolated humanized antibodies are synthetic. Human antibodies, while derived from human immunoglobulin sequences, may be generated using systems such as phage display incorporating synthetic CDRs and/or synthetic frameworks, or may be subjected to in vitro mutagenesis to improve antibody properties, resulting in antibodies that do not naturally exist within the human antibody germline repertoire in vivo.

[0056] "Heavy chain variable region" as used herein refers to the region of the antibody molecule comprising at least one complementarity determining region (CDR) of said antibody heavy chain variable domain. The heavy chain variable region may contain one, two, or three CDR of said antibody heavy chain.

[0057] "Light chain variable region" as used herein refers to the region of an antibody molecule, comprising at least one complementarity determining region (CDR) of said antibody light chain variable domain. The light chain variable region may contain one, two, or three CDR of said antibody light chain, which may be either a kappa or lambda light chain depending on the antibody.

[0058] The phrase "glucose tolerance", as used herein, refers to the ability of a subject to control the level of plasma glucose and/or plasma insulin when glucose intake fluctuates. For example, glucose tolerance encompasses the subject's ability to reduce, within about 120 minutes, the level of plasma glucose back to a level determined before the intake of glucose.

[0059] Broadly speaking, the terms "diabetes" and "diabetic" refer to a progressive disease of carbohydrate metabolism involving inadequate production or utilization of insulin, frequently characterized by hyperglycemia and glycosuria. The terms "prediabetes" and "pre-diabetic" refer to a state wherein a subject does not have the characteristics, symptoms and the like typically observed in diabetes, but does have characteristics, symptoms and the like that, if left untreated, may progress to diabetes. The presence of these conditions may be determined using, for example, either the fasting plasma glucose (FPG) test or the oral glucose tolerance test (OGTT). Both usually require a subject to fast for at least 8 hours prior to initiating the test. In the FPG test, a subject's blood glucose is measured after the conclusion of the fasting; generally, the subject fasts overnight and the blood glucose is measured in the morning before the subject eats. A healthy subject would generally have a FPG concentration between about 90 and about 100 mg/dl, a subject with "pre-diabetes" would generally have a FPG concentration between about 100 and about 125 mg/dl, and a subject with "diabetes" would generally have a FPG level above about 126 mg/dl. In the OGTT, a subject's blood glucose is measured after fasting and again two hours after drinking a glucose-rich beverage. Two hours after consumption of the glucose-rich beverage, a healthy subject generally has a blood glucose concentration below about 140 mg/dl, a pre-diabetic subject generally has a blood glucose concentration about 140 to about 199 mg/dl, and a diabetic subject generally has a blood glucose concentration about 200 mg/dl or above. While the aforementioned glycemic values pertain to human subjects, normoglycemia, moderate hyperglycemia and overt hyperglycemia are scaled differently in murine subjects. A healthy murine subject after a four-hour fast would generally have a FPG concentration between about 100 and about 150 mg/dl, a murine subject with "pre-diabetes" would generally have a FPG concentration between about 175 and about 250 mg/dl and a murine subject with "diabetes" would generally have a FPG concentration above about 250 mg/dl.

[0060] "Effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired result. An effective amount of an antibody that binds to IR may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the beneficial effects.

[0061] The term "hyperglycemia", as used herein, refers to a condition in which an elevated amount of glucose circulates in the blood plasma of a subject relative to a healthy individual. Hyperglycemia can be diagnosed using methods known in the art, including measurement of fasting blood glucose levels as described herein.

[0062] The term "hyperinsulinemia", as used herein, refers to a condition in which there are elevated levels of circulating insulin when, concomitantly, blood glucose levels are either elevated or normal. Hyperinsulinemia can be caused by insulin resistance which is associated with dyslipidemia, such as high triglycerides, high cholesterol, high low-density lipoprotein (LDL) and low high-density lipoprotein (HDL); high uric acids levels; polycystic ovary syndrome; type II diabetes and obesity. Hyperinsulinemia can be diagnosed as having a plasma insulin level higher than about 2 μυ/mL.

[0063] The term "insulin resistance" as used herein refers to a condition where a normal amount of insulin is unable to produce a normal physiological or molecular response. In some cases, a hyper-physiological amount of insulin, either endogenously produced or exogenously administered, is able to overcome the insulin resistance, in whole or in part, and produce a biologic response.

[0064] "Immunospecifically" when used in the context of antibodies, or antibody fragments, represents binding via domains encoded by immunoglobulin genes or fragments of immunoglobulin genes to one or more epitopes of a protein of interest, without preferentially binding other molecules in a sample containing a mixed population of molecules. Typically, an antibody binds to a cognate antigen with a Kd of less than about 1x10-8 M, as measured by a surface plasmon resonance assay or a cell binding assay. Phrases such as "anti-[antigen] antibody" (e.g., anti-insulin receptor antibody) are meant to convey that the recited antibody specifically binds the recited antigen.

[0065] "Isolated" means a biological component (such as an antibody) has been substantially separated, produced apart from, or purified away from other biological components of the organism in which the component naturally occurs, i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins. Antibodies that have been "isolated" thus include antibodies purified by standard purification methods. "Isolated antibodies" can be part of a composition and still be isolated if such composition is not part of the native environment of the antibody. The term also embraces antibodies prepared by recombinant expression in a host cell as well as chemically synthesized antibodies. An "isolated antibody or antigen-binding fragment thereof," as used herein, is intended to refer to an antibody or antigen-binding fragment thereof which is substantially free of other antibodies or antigen-binding fragments having different antigenic specificities (for instance, an isolated antibody that specifically binds to IR is substantially free of antibodies that specifically bind antigens other than IR). An isolated antibody that specifically binds to an epitope, isoform or variant of IR may, however, have cross-reactivity to other related antigens, for instance from other species (such as IR species homologs).

[0066] "Recombinant" as used herein, includes antibodies and other proteins that are prepared, expressed, created or isolated by recombinant means.

[0067] As used herein, an antibody that "specifically binds" is "antigen specific", is "specific for" antigen target or is "immunoreactive" with an antigen refers to an antibody or polypeptide binding agent of the invention that binds an antigen with greater affinity than other antigens of similar sequence. In one aspect, the polypeptide binding agents of the invention, or fragments, variants, or derivatives thereof, will bind with a greater affinity to human antigen as compared to its binding affinity to similar antigens of other, i.e., non-human, species, but polypeptide binding agents that recognize and bind orthologs of the target are within the scope of the invention.

[0068] "Subject" refers to human and non-human animals, including all vertebrates, e.g., mammals and non-mammals, such as non-human primates, mice, rabbits, sheep, dogs, cats, horses, cows, chickens, amphibians, and reptiles. In many embodiments of the described subject matter, the subject is a human. [0069] "Treating" or "treatment" refer to any success or indicia of success in the attenuation or amelioration of an injury, pathology, or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of degeneration less debilitating, improving a subject's physical or mental well-being, or prolonging the length of survival. The treatment may be assessed by objective or subjective parameters, including the results of a physical examination, neurological examination, or psychiatric evaluations.

[0070] "Vector" means a polynucleotide capable of being duplicated within a biological system or that can be moved between such systems. Vector polynucleotides typically contain elements, such as origins of replication, polyadenylation signal or selection markers, that function to facilitate the duplication or maintenance of these polynucleotides in a biological system. Examples of such biological systems may include a cell, virus, animal, plant, and reconstituted biological systems utilizing biological components capable of duplicating a vector. The polynucleotide comprising a vector may be DNA or RNA molecules or a hybrid of these.

IR-Specific Antibodies and Antigen-Binding Fragments

[0071] Disclosed herein are isolated antibodies, or antigen-binding fragments thereof, that immunospecifically bind to the IR.

[0072] The isolated antibody or antigen-binding fragment thereof can comprise: a. a heavy chain CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:2, b. a heavy chain CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:3, c. a heavy chain CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:4, d. a light chain CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:5, e. a light chain CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 6, and f. a light chain CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 7, wherein the CDRs are defined according to Kabat. [0073] The isolated antibody or antigen-binding fragment thereof can comprise a heavy chain variable domain comprising an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 8 and a light chain variable domain comprising an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO:9. In some embodiments, the heavy chain variable domain can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 8 and the light chain variable domain can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO:9.

[0074] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR is BBBB138 or an antigen-binding fragment thereof. In a related aspect, the BBBB138 antibody, or antigen-binding fragment thereof, binds to the IR allosterically.

[0075] The isolated antibody or antigen-binding fragment thereof can also comprise: a. a heavy chain CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 10, b. a heavy chain CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 11, c. a heavy chain CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 12, d. a light chain CDR1 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 13, e. a light chain CDR2 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 14, and f. a light chain CDR3 comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO: 15, wherein the CDRs are defined according to Kabat.

[0076] The isolated antibody or antigen-binding fragment thereof can comprise a heavy chain variable domain comprising an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain comprising an amino acid sequence that is at least 90%, 95%, or 99% identical to the amino acid sequence of SEQ ID NO: 17.

[0077] In some embodiments, the heavy chain variable domain can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 16 and the light chain variable domain can comprise, consist of, or consist essentially of the amino acid sequence of SEQ ID NO: 17. [0078] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR, is BBBB141 or an antigen-binding fragment thereof.

[0079] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR, is a human antibody or antigen-binding fragment.

[0080] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR, is recombinant.

[0081] In some embodiments, the isolated antibody, or antigen-binding fragment thereof, that immunospecifically binds to IR, is a Fab fragment, a F(ab')2 fragment, or a single chain antibody.

[0082] In addition to the described anti-IR antibodies and antigen-binding fragments thereof, also provided are polynucleotide sequences encoding the disclosed antibodies and antigen-binding fragments thereof.

[0083] Vectors comprising the polynucleotides are also provided. The vectors can be expression vectors. Recombinant expression vectors containing a sequence encoding the disclosed antibodies or antigen-binding fragments thereof are thus contemplated as within the scope of this disclosure. The expression vector may contain one or more additional sequences such as, but not limited, to regulatory sequences (e.g., promoter, enhancer), selection markers, and polyadenylation signals. Vectors for transforming a wide variety of host cells are well known and include, but are not limited to, plasmids, phagemids, cosmids, baculoviruses, bacmids, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), as well as other bacterial, yeast and viral vectors.

[0084] Also described are cells expressing, and capable of expressing, the disclosed vectors. These cells may be mammalian cells (such as 293F cells, CHO cells), insect cells (such as Sf7 cells), yeast cells, plant cells, or bacteria cells (such as E. coli). The disclosed antibodies may also be produced by hybridoma cells.

Methods of modulating IR signaling.

[0085] The IR agonist and antagonist antibodies provided by this invention are useful as lead compounds for identifying other more potent or selective therapeutics, assay reagents for identifying other useful ligands by, for example, competition screening assays, as research tools for further analysis of IR, and as potential therapeutics in pharmaceutical compositions. In one embodiment, one or more of the disclosed peptides can be provided as components in a kit for identifying other ligands (e.g., small, organic molecules) that bind to IR. Such kits may also comprise IR, or functional fragments thereof. The peptide and receptor components of the kit may be labeled (e.g., by radioisotopes, fluorescent molecules, chemiluminescent molecules, enzymes or other labels), or may be unlabeled and labeling reagents may be provided. The kits may also contain peripheral reagents such as buffers, stabilizers, etc. Instructions for use can also be provided.

[0086] In another embodiment, the peptide sequences provided by this invention can be used to design secondary peptide libraries, which are derived from the peptide sequences, and include members that bind to IR. Such libraries can be used to identify sequence variants that increase or modulate the binding and/or activity of IR, as described in the related applications of Beasley et al. International Application

PCT/USOO/08528, filed Mar. 29, 2000, and Beasley et al, U. S. application Ser. No. 09/538, 038, filed Mar. 29, 2000, in accordance with well-established techniques.

[0087] Also provided herein, a method of antagonizing insulin receptor signaling, comprising administering to the subject an effective amount of the BBBB138 antibody or antigen-binding fragment thereof.

[0088] In some embodiments, the administration to the subject an effective amount of the BBBB138 antibody, or antigen-binding fragment thereof, induces hyperglycemia, hyperinsulinemia, or insulin resistance.

[0089] In some embodiments, the hyperglycemia, hyperinsulinemia, or insulin resistance is associated with type 2 diabetes.

[0090] Also provided a method of screening compounds for the ability to modulate hyperglycemia, hyperinsulinemia, or insulin resistance, comprising: contacting a cell or a subject with the BBBB 138 antibody, and with a test compound, and measuring the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the presence of the test compound relative to the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the absence of the test compound, wherein an increase in the levels of IR phosphorylation, Akt phosphorylation, or a decrease in blood glucose levels in the presence of the test compound indicates that the test compound is capable of reducing hyperglycemia, hyperinsulinemia, or insulin resistance; and wherein a decrease or no change in the level of IR phosphorylation, Akt phosphorylation, or an increase or no change in blood glucose levels in the presence of the test compound indicates that the test compound is not capable of reducing hyperglycemia, hyperinsulinemia, or insulin resistance.

[0091] Also provided a method of agonizing insulin receptor signaling, comprising administering to the subject an effective amount of the BBBB141 antibody or antigen- binding fragment thereof.

[0092] In some embodiments, the agonizing insulin receptor signaling comprises an increase in insulin receptor tyrosine phosphorylation.

[0093] In some embodiments, the administration to the subject an effective amount of the BBBB141 antibody or antigen-binding fragment thereof, reduces hyperglycemia, hyperinsulinemia, or insulin resistance.

[0094] In some embodiments, the hyperglycemia, hyperinsulinemia , or insulin resistance is associated with type 2 diabetes.

Methods for screening for modulators of IR signaling.

[0095] Examples of compounds that can be screened include antibodies, antigen- binding proteins, polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates. Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described WO 95/12608, WO 93/06121 , WO 94/08051 , WO 95/35503 and Scripps, WO 95/30642. Peptide libraries can also be generated by phage display methods. See, e.g., US5,432,018.

[0096] Cellular assays generally involve contacting a cell (or more typically a culture of such cells) with a compound and determining whether a property of the cells changes. The change can be assessed from levels of the property before and after contacting the cell with the compound or by performing a control experiment performed on the same cell or population of cells without the compound. The property measured is often a level of insulin secreted by the cell or proinsulin within a cell. The screening may also include treating the cell with an agent inducing secretion of insulin, such as glucose, arginine or a secretagogue, and measuring insulin secreted in response. Optionally, the insulin secretion response can be measured in response to successive challenges of the inducing agent.

[0097] Analogous experiments can be performed on an animal. Suitable signs or symptoms that can be monitored include elevated blood glucose levels (e.g., fasting blood glucose levels or blood glucose levels following an oral glucose challenge), and insulin levels. Glucose tolerance refers to a state of proper functioning of the homeostatic mechanisms by which insulin is secreted in response to an elevation in serum glucose concentrations.

[0098] A normal level of glucose in human is in the range of from about 65 mg/dL to about 140 mg/dL. Impairment in this system results in transient hyperglycemia as the organism is unable to maintain normoglycemia following a glucose load (for example, a carbohydrate containing meal) because of insufficient secretion of insulin from the islet beta-cells or because of insensitivity of target tissues to circulating insulin.

Impaired glucose tolerance in humans can be defined as a plasma glucose concentration greater than or equal to 140 mg/dl (7.8 mmol/1) two hours after ingestion of a 75 g oral glucose load. Impaired insulin sensitivity can be determined by IV glucose tolerance test (FSIVGTT), insulin tolerance test (ITT), insulin sensitivity test (1ST), and continuous infusion of glucose with model assessment (CIGMA), or the glucose clamp. See, e.g., Krentz, Insulin Resistance (Wiley-Blackwell, 2002); de Paula Martins et al., Eur. J. Obst. Gynecol. Reprod. Biol. 133(2):203-207 (2007). Normal ranges of blood sugar in mice are 60-130 mg/ml, similar to those in humans.

[0099] In another embodiment of this invention, screening assays to identify pharmacologically active ligands for IR are provided. Ligands may encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Such ligands can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. Ligands often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Ligands can also comprise biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof. [0100] Ligands may include, for example, 1) peptides such as soluble peptides, including Ig-tailed fusion peptides and members of random peptide libraries (see, e.g., Lam et al, 1991 , Nature 354: 82-84; Houghten et al., 1991, Nature 354: 84-86) and combinatorial chemistry-derived molecular libraries made of D-and/or L-configuration amino acids; 2) phosphopeptides (e.g., members of random and partially degenerate, directed phosphopeptide libraries, see, e.g., Songyang et al, 1993, Cell 72:767-778); 3) antibodies (e.g., polyclonal, monoclonal, humanized, anti-idiotypic, chimeric, and single chain antibodies as well as Fab, F(ab')2, Fab expression library fragments, and epitope-binding fragments of antibodies); and 4) small organic and inorganic molecules.

[0101] Ligands can be obtained from a wide variety of sources including libraries of synthetic or natural compounds. Synthetic compound libraries are commercially available from, for example, Maybridge Chemical Co. (Trevillet, Cornwall, UK), Comgenex (Princeton, N.J.), Brandon Associates (Merrimack, N.H.), and Microsource (New Milford, Conn.). A rare chemical library is available from Aldrich Chemical Company, Inc. (Milwaukee, Wis.) . Natural compound libraries comprising bacterial, fungal, plant or animal extracts are available from, for example, Pan Laboratories (Bothell, Wash.). In addition, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides.

[0102] Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be readily produced. Methods for the synthesis of molecular libraries are readily available (see, e.g., DeWitt et al, 1993 , Proc. Natl. Acad. Sci. USA 90:6909; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91 : 1 1422;

Zuckermann et al, 1994, J. Med. Chem. 37:2678; Cho et al, 1993, Science 261 : 1303; Carell et al, 1994, Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al, 1994, Angew. Chem. Int. Ed. Engl. 33 :2061 ; and in Gallop et al, 1994, J. Med. Chem. 37: 1233). In addition, natural or synthetic compound libraries and compounds can be readily modified through conventional chemical, physical and biochemical means (see, e.g., Blondelle et al, 1996, Trends in Biotech. 14:60), and may be used to produce combinatorial libraries. In another approach, previously identified pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, and the analogs can be screened for IR-modulating activity.

[0103] Numerous methods for producing combinatorial libraries are known in the art, including those involving biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the One- bead one-compound' library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to polypeptide or peptide libraries, while the other four approaches are applicable to polypeptide, peptide, non-peptide oligomer, or small molecule libraries of compounds (K. S. Lam, 1997 , Anticancer Drug Des. 12: 145).

[0104] Libraries may be screened in solution by methods generally known in the art for determining whether ligands competitively bind at a common binding site. Such methods may including screening libraries in solution (e.g., Houghten, 1992 ,

Biotechniques 13:412-421), or on beads (Lam, 1991, Nature 354:82-84), chips (Fodor, 1993, Nature 364:555-556), bacteria or spores (Ladner U.S. Pat. No. 5,223,409), plasmids (Cull et al., 1992, Proc. Natl. Acad. Sci. USA 89: 1865-1869), or on phage (Scott and Smith, 1990, Science 249:386-390; Devlin, 1990, Science 249:404-406; Cwirla et al, 1990, Proc. Natl. Acad. Sci. USA 97:6378-6382; Felici, 1991, J. Mol. Biol. 222:301-310; Ladner, supra).

[0105] Where the screening assay is a binding assay, IR, or one of the IR-binding antibodies disclosed herein, may be joined to a label, where the label can directly or indirectly provide a detectable signal. Various labels include radioisotopes, fluorescent molecules, chemiluminescent molecules, enzymes, specific binding molecules, particles, e.g., magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.

[0106] A variety of other reagents may be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc., which are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., may be used. The components are added in any order that produces the requisite binding. Incubations are performed at any temperature that facilitates optimal activity, typically between 4° and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. Normally, between 0.1 and 1 hr will be sufficient. In general, a plurality of assay mixtures is run in parallel with different test agent concentrations to obtain a differential response to these concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0107] The screening assays provided in accordance with this invention are based on those disclosed in International application WO 96/04557, which is incorporated herein in its entirety. Briefly, WO 96/04557 discloses the use of reporter peptides that bind to active sites on targets and possess agonist or antagonist activity at the target. These reporters are identified from recombinant libraries and are either peptides with random amino acid sequences or variable antibody regions with at least one CDR region that has been randomized (rVab). The reporter peptides may be expressed in cell recombinant expression systems, such as for example in E coli, or by phage display (see WO 96/04557 and Kay et al. 1996 , Mol. Divers. 1 (2): 139-40, both of which are incorporated herein by reference). The reporters identified from the libraries may then be used in accordance with this invention either as therapeutics themselves, or in competition binding assays to screen for other molecules, preferably small, active molecules, which possess similar properties to the reporters and may be developed as drug candidates to provide agonist or antagonist activity. Preferably, these small organic molecules are orally active.

[0108] The basic format of an in vitro competitive receptor binding assay as the basis of a heterogeneous screen for small organic molecular replacements for insulin may be as follows: occupation of the active site of IR is quantified by time-resolved fluorometric detection (TRFD) with streptavidin-labeled europium (saEu) complexed to biotinylated peptides (bP). In this assay, saEu forms a ternary complex with bP and IR (i.e., IR:bP:saEu complex). The TRFD assay format is well established, sensitive, and quantitative (Tompkins et al, 1993 , J. Immunol. Methods 163:209-216). The assay can use a single-chain antibody or a biotinylated peptide. Furthermore, both assay formats faithfully report the competition of the biotinylated ligands binding to the active site of IR by insulin. [0109] In these assays, soluble IR is coated on the surface of microtiter wells, blocked by a solution of 0.5% bovine serum albumin (BSA) and 2% non-fat milk in PBS, and then incubated with biotinylated peptide or rVab. Unbound bP is then washed away and saEu is added to complex with receptor-bound bP. Upon addition of the acidic enhancement solution, the bound europium is released as free Eu ⁺ which rapidly forms a highly fluorescent and stable complex with components of the enhancement solution. The IR:bP bound saEu is then converted into its highly fluorescent state and detected by a detector such as Wallac Victor II (EG&G Wallac, Inc.).

[0110] Phage display libraries can also be screened for ligands that bind to IR, as described above. Details of the construction and analyses of these libraries, as well as the basic procedures for biopanning and selection of binders, have been published (see, e.g., WO 96/04557; Mandecki et al, 1997 , Display Technologies— Novel Targets and Strategies, P. Guttry (ed), International Business Communications, Inc. Southborogh, Mass., pp. 231-254; Ravera et al., 1998, Oncogene 16: 1993-1999; Scott and Smith, 1990, Science 249:386-390); Grihalde et al, 1995, Gene 166: 187-195; Chen et al, 1996, Proc. Natl. Acad. Sci. USA 93: 1997-2001; Kay et al, 1993, Ge«el28:59-65; Carcamo et al, 1998, Proc. Natl. Acad. Sci. USA 95: 11146-11151; Hoogenboom, 1997, Trends Biotechnol. 15:62-70; Rader and Barbas, 1997, Curr. Opin. Biotechnol. 8.503- 508; all of which are incorporated herein by reference).

[0111] The designing of mimetics to a known pharmaceutically active compound is a known approach to the development of pharmaceuticals based on a "lead" compound. This might be desirable where the active compound is difficult or expensive to synthesize or where it is unsuitable for a particular method of administration, e.g., peptides are generally unsuitable active agents for oral compositions as they tend to be quickly degraded by proteases in the alimentary canal. Mimetic design, synthesis, and testing are generally used to avoid large-scale screening of molecules for a target property.

[0112] There are several steps commonly taken in the design of a mimetic from a compound having a given target property. First, the particular parts of the compound that are critical and/or important in determining the target property are determined. In the case of a peptide, this can be done by systematically varying the amino acid residues in the peptide (e.g., by substituting each residue in turn). These parts or residues constituting the active region of the compound are known as its

"pharmacophore".

[0113] Once the pharmacophore has been found, its structure is modeled according to its physical properties (e.g., stereochemistry, bonding, size, and/or charge), using data from a range of sources (e.g., spectroscopic techniques, X-ray diffraction data, and NMR). Computational analysis, similarity mapping (which models the charge and/or volume of a pharmacophore, rather than the bonding between atoms), and other techniques can be used in this modeling process.

[0114] In a variant of this approach, the three dimensional structure of the ligand and its binding partner are modeled. This can be especially useful where the ligand and/or binding partner change conformation on binding, allowing the model to take account of this in the design of the mimetic.

[0115] A template molecule is then selected, and chemical groups that mimic the pharmacophore can be grafted onto the template. The template molecule and the chemical groups grafted on to it can conveniently be selected so that the mimetic is easy to synthesize, is likely to be pharmacologically acceptable, does not degrade in vivo, and retains the biological activity of the lead compound. The mimetics found are then screened to ascertain the extent they exhibit the target property, or to what extent they inhibit it. Further optimization or modification can then be carried out to arrive at one or more final mimetics for in vivo or clinical testing.

[0116] This invention provides specific amino acid sequences that function as either IR agonist or antagonist. Additional sequences may be obtained in accordance with the procedures described herein.

Modification of antibodies

[0117] The antibodies of the invention may be subjected to one or more modifications known in the art, which may be useful for manipulating storage stability,

pharmacokinetics, and/or any aspect of the bioactivity of the peptide, such as, e.g., potency, selectivity, and drug interaction. Chemical modification to which the peptides may be subjected includes, without limitation, the conjugation to a peptide of one or more of polyethylene glycol (PEG), monomethoxy-polyethylene glycol, dextran, poly- (N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polypropylene glycol, polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, colominic acids or other carbohydrate based polymers, polymers of amino acids, and biotin derivatives. PEG conjugation of proteins at Cys residues is disclosed, e.g., in Goodson, R. J. & Katre, N. V. (1990) Bio/Technology 8, 343 and Kogan, T. P. (1992) Synthetic Comm. 22, 2417.

[0118] Other useful modifications include, without limitation, acylation, using methods and compositions such as described in, e.g., U. S. Pat. No. 6,251 , 856, and WO

00/55119.

Therapeutic administration

[0119] The antibodies of the present invention may be administered individually or in combination with other pharmacologically active agents. It will be understood that such combination therapy encompasses different therapeutic regimens, including, without limitation, administration of multiple agents together in a single dosage form or in distinct, individual dosage forms. If the agents are present in different dosage forms, administration may be simultaneous or near-simultaneous or may follow any predetermined regimen that encompasses administration of the different agents.

[0120] For example, when used to treat diabetes or other diseases or syndromes associated with a decreased response or production of insulin, hyperlipidemia, obesity, appetite-related syndromes, and the like, the peptides of the invention may be advantageously administered in a combination treatment regimen with one or more agents, including, without limitation, insulin, insulin analogues, insulin derivatives, glucagon-like peptide- 1 or-2 (GLP-1, GLP-2), derivatives or analogues of GLP-1 or GLP-2 (such as are disclosed, e.g., in WO 00/551 19). It will be understood that an "analogue" of insulin, GLP-1, or GLP-2 as used herein refers to a peptide containing one or more amino acid substitutions relative to the native sequence of insulin, GLP-1, or GLP-2, as applicable; and "derivative" of insulin, GLP-1 , or GLP-2 as used herein refers to a native or analogue insulin, GLP-1 , or GLP-2 peptide that has undergone one or more additional chemical modifications of the amino acid sequence, in particular relative to the natural sequence. Insulin derivatives and analogues are disclosed, e.g., in U. S. Pat. Nos. 5,656,722, 5,750,497, 6,251 ,856, and 6,268,335. In some embodiments, the combination agent is one of Lys ^B29(-myristoyl)des(B30) human insulin, Lys^B29(- tetradecanoyl)des(B30) human insulin and B²⁹-N-(N-lithocolyl-glutamyl)-des(B30) human insulin. Also suitable for combination therapy are non-peptide

antihyperglycemic agents, antihyperlipidemic agents, and the like such as those well- known in the art.

[0121] In one embodiment, the invention encompasses methods of treating diabetes or related syndromes comprising administering a first amount of peptide S597 or peptide S557 and a second amount of a long-acting insulin analogue, such as, e.g., Lys ^B29(- myristoyl)des(B30) human insulin, Lys^B29(-tetradecanoyl)des(B30) human insulin or B²⁹-N-(N-lithocolyl-glutamyl)-des(B30) human insulin, wherein the first and second amounts together are effective for treating the syndrome. As used herein, a long-acting insulin analogue is one that exhibits a protracted profile of action relative to native human insulin, as disclosed, e.g., in U. S. Pat. No. 6,451 ,970.

Methods of Administration

[0122] The amino acid sequences of this invention may be administered as pharmaceutical compositions comprising standard carriers known in the art for delivering proteins and peptides and by gene therapy. Preferably, a pharmaceutical composition includes, in admixture, a pharmaceutically (i. e., physiologically) acceptable carrier, excipient, or diluent, and one or more of an IR agonist or antagonist peptide, as an active ingredient. The preparation of pharmaceutical compositions that contain peptides as active ingredients is well understood in the art. Typically, such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients that are pharmaceutically (i.e.,

physiologically) acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH-buffering agents, which enhance the effectiveness of the active ingredient.

[0123] An IR agonist or antagonist peptide can be formulated into a pharmaceutical composition as neutralized physiologically acceptable salt forms. Suitable salts include the acid addition salts (i.e., formed with the free amino groups of the peptide molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0124] The pharmaceutical compositions can be administered systemically by oral or parenteral routes. Non-limiting parenteral routes of administration include

subcutaneous, intramuscular, intraperitoneal, intravenous, transdermal, inhalation, intranasal, intra-arterial, intrathecal, enteral, sublingual, or rectal. Due to the labile nature of the amino acid sequences parenteral administration is preferred. Preferred modes of administration include aerosols for nasal or bronchial absorption; suspensions for intravenous, intramuscular, intrasternal or subcutaneous, injection; and compounds for oral administration.

[0125] Intravenous administration, for example, can be performed by injection of a unit dose. The term "unit dose" when used in reference to a pharmaceutical composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., liquid used to dilute a concentrated or pure substance (either liquid or solid), making that substance the correct (diluted) concentration for use. For injectable administration, the composition is in sterile solution or suspension or may be emulsified in

pharmaceutically-and physiologically-acceptable aqueous or oleaginous vehicles, which may contain preservatives, stabilizers, and material for rendering the solution or suspension isotonic with body fluids (i.e., blood) of the recipient.

[0126] Excipients suitable for use are water, phosphate buffered saline, pH 7.4, 0.15 M aqueous sodium chloride solution, dextrose, glycerol, dilute ethanol, and the like, and mixtures thereof. Illustrative stabilizers are polyethylene glycol, proteins, saccharides, amino acids, inorganic acids, and organic acids, which may be used either on their own or as admixtures. The amounts or quantities, as well as routes of administration, used are determined on an individual basis, and correspond to the amounts used in similar types of applications or indications known to those of skill in the art. [0127] Pharmaceutical compositions are administered in a manner compatible with the dosage formulation, and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, capacity of the subject's immune system to utilize the active ingredient, and degree of modulation of IR activity desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are specific for each individual.

[0128] Further guidance in preparing pharmaceutical formulations can be found in, e.g., Gilman et al. (eds), 1990 , Goodman and Gilman's: The Pharmacological Basis of Therapeutics, 8th ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed., 1990, Mack Publishing Co., Easton, Pa.; Avis et al. (eds), 1993, Pharmaceutical Dosage Forms: Parenteral Medications, Dekker, New York; Lieberman et al. (eds), 1990, Pharmaceutical Dosage Forms: Disperse Systems, Dekker, New York.

[0129] The present invention further contemplates compositions comprising an IR agonist or antagonist peptide, and a physiologically acceptable carrier, excipient, or diluent as described in detail herein.

[0130] The following examples illustrate the invention. These examples should not be construed as to limit the scope of this invention. The examples are included for purposes of illustration and the present invention is limited only by the claims.

Example 1. Isolation of antibodies which bind to insulin receptor from phage display libraries

[0131] BBBB138 and BBBB141 phage panning. Fab phage display panning was conducted to identify insulin receptor binding antibodies.

[0132] IR binding Fabs were selected from de novo pIX phage display libraries as described in Shi et al, J Mol Biol 397:385-96, 2010, Int. Patent Publ. No.

WO2009/085462 and U.S. Patent Publ. No. US2010/0021477. Briefly, the libraries were generated by diversifying human scaffolds where germline VH genes IGHVl- 69*01, IGHV3-23*01, and IGHV5-51 *01 were recombined with the human IGHJ-4 minigene via the H3 loop, and human germline VL kappa genes 012 (IGKV1-39*01), L6 (IGKV3-11 *01), A27 (IGKV3-20*01), and B3 (IGKV4-1*01) were recombined with the IGKJ-1 minigene to assemble complete VH and VL domains. The positions in the heavy and light chain variable regions around HI, H2, LI, L2 and L3 loops corresponding to positions identified to be frequently in contact with protein and peptide antigens were chosen for diversification. Sequence diversity at selected positions was limited to residues occurring at each position in the IGHV or IGLV germline gene families of the respective IGHV or IGLV genes. Diversity at the H3 loop was generated by utilizing short to mid-sized synthetic loops of lengths 7-14 amino acids. The amino acid distribution at H3 was designed to mimic the observed variation of amino acids in human antibodies. Library design is detailed in Shi et al, (2010) JMol Biol 397:385-96. The scaffolds utilized to generate libraries were named according to their human VH and VL germline gene origin. The three heavy chain libraries were combined with the four germline light chains or combined with the diversified light chain libraries to generate 12 unique VH:VL combinations. These libraries were later combined further based on library versions to generate additional libraries for panning experiments against IR.

[0133] The libraries were panned against human IR long isoform (SEQ ID No: 1):

HLYPGEVCPGMDIRNNLTRLHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDY

LLLFRVYGLESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVRIE

K ELCYLATIDWSRILDSVEDNYIVLNKDDNEECGDICPGTAKGKTNCPATVINGQF

VERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGNCSQPDDPTKCVACRNFYLDG

RCVETCPPPYYHFQDWRCVNFSFCQDLHHKCKNSRRQGCHQYVIHNNKCIPECPSGYT

MNSSNLLCTPCLGPCPKVCHLLEGEKTIDSVTSAQELRGCTVINGSLIINIRGG LAAE

LEANLGLIEEISGYLKIRRSYALVSLSFFRKLRLIRGETLEIGNYSFYALDNQNLRQLWD

WSKHNLTITQGKLFFHYNPKLCLSEIHKMEEVSGTKGRQERNDIALKTNGDQASCENE

LLKFSYIRTSFDKILLRWEPYWPPDFRDLLGFMLFYKEAPYQNVTEFDGQDACGSNSW

TVVDIDPPLRSNDPKSQNHPGWLMRGLKPWTQYAIFVKTLVTFSDERRTYGAKSDIIY

VQTD ATNP S VPLDPI S VSNS SSQIILKWKPP SDPNGNITHYL VF WERQ AED SELFELD YC

LKGLKLP SRT WSPPFE SED SQKHNQSEYED S AGECC SCPKTD SQILKELEES SFRKTFED

YLHNVVFVPRKTSSGTGAEDPRPSRKRRSLGDVGNVTVAVPTVAAFPNTSSTSVPTSPE

EHRPFEKVVNKESLVISGLRHFTGYRIELQACNQDTPEERCSVAAYVSARTMPEAKAD

DIVGPVTHEIFE VVHLMWQEPKEPNGLIVLYEVSYRRYGDEELHLCVSRKHFALER

GCRLRGLSPG YSVRIRATSLAGNGSWTEPTYFYVTDYLDVPSNIAKAHHHHHHHHH

H

The recombinant protein was biotinylated (bt) and captured on streptavidin magnetic beads (Dynal), then exposed to the de novo pIX Fab libraries at a final concentration up to and including 10 uM. Non-specific phages were washed away in PBS-Tween and bound phages were recovered by infection of MCI 06 IF' E. coli cells. Phages were amplified from these cells overnight and panning was repeated for a total of three rounds. Following the final round of biopanning, monoclonal Fabs were screened for binding to human IR using ELISA. Briefly, biotinylated IR antigen was captured on ELISA plates by Streptavidin and secreted Fab was added to the captured antigen, followed by detection of the Fab with streptavidin/HRP. Clones that demonstrated binding to the proteins were sequenced in the heavy and light chain variable regions.

[0134] Select Fabs were chosen for further characterization and were cloned onto a human IgGl Fc. The antibodies were evaluated for their ability to bind to cells endogenously expressing human IR (hepato-cellular carcinoma HuH-7 cells) in the presence or absence of insulin (Sigma- Aldrich, St. Louis, MO).

[0135] The antibodies that bound to the cells were then sorted by a competition binding experiment via MSD (Meso Scale Discovery). Antibodies were distinguished based on their ability to compete with each other for binding to the insulin receptor (Sino Biologies, Beijing, China).

[0136] Antibodies were confirmed for binding to rat L6 skeletal muscle cells (ATCC, Manassas, VA) as well as the absence of binding to insulin-like growth factor 1 receptor (Sino Biologicals). Representative molecules from each epitope were tested in insulin signaling pathway assays.

Example 2. BBBB138 allosteric binding to insulin receptor isoforms

[0137] BBBB138 was identified through phage panning against the human insulin receptor. The antibody sequences are shown in Table 1. Because the insulin receptor exists as a short isoform (IR-A) and a long isoform (IR-B), the ability of BBBB138 to bind to each of these extracellular constructs was tested by Surface Plasmon Resonance (SPR) using a ProteOn XPR36 protein interaction array system (BioRad). The biosensor surface was prepared by covalently coupling goat anti-human IgG (Fc) to the surface of a GLC chip (BioRad) using the manufacturer instructions for amine-coupling chemistry. Approximately 5500 RU (response units) of goat anti-human IgG (Fc) antibody (Jackson ImmunoResearch laboratories Prod # 109-005-098) was

immobilized. The goat anti-human IgG (Fc) surface also included a goat anti-mouse IgG (Fc). Since the Fc mixture was 1 : 1, about 50% of these RU immobilized are expected to be goat anti-human Fc. The sensor chip surface was also coated with 500- 700 RU of a non-specific IgG (IgGl Lambda, Sigma Aldrich, Prod# 15029- IMG) to generate a uniform surface for evaluating any non-specific binding. The kinetics experiments were performed at 25 °C in running buffer (PBS pH 7.4, 0.005% P20 surfactant, 3 mM EDTA). Four-fold (1 :3) serial dilutions of human IR-A or IR-B, starting at 400 nM were prepared in running buffer. Around 40-60 RU of the mAbs were captured on each channel of the sensor chip. Capture of the mAbs was followed by 4 min injection (association phase) of antigen at 50 μί/ηϋη, followed by 30 min of buffer flow (dissociation phase). The chip surface was regenerated by short injections of 0.85% phosphoric acid, 100 mM NaOH and running buffer at 100 μί/ηιίη. Data was processed on the instrument software. The reference spots (goat anti-human Fc) containing no candidate captured were used as for reference subtraction. Double referencing of the data was performed by subtracting the curves generated by buffer injection from the reference-subtracted curves for analyte injections. Kinetics analysis of the mAb binding to both insulin receptor isoforms was performed using 1 : 1 Langmuir binding model with group fitting option. The binding results were reported in the format of k_on (on-rate), k₀ff (off-rate), KD (Equilibrium dissociation constant) (Table 2). BBBB138 bound to each construct with similar nM affinity (Figures 1-2 and Table 2).

Table 1. Sequences of BBBB138 antibody.

ISTAYLQWSSLKASDTAMYYCARWALLNLDYWGQGT

LVTVSS

9 EIVLTQSPGTLSLSPGERATLSCRASQSVGKSGLAWYQ VL

QKPGQAPRLLIYNASSRATGIPDRFSGSGSGTDFTLTISR

LEPEDFAVYYCQQYNGPPLTFGQGTKVEIK

Table 2. BBBB138 Insulin Receptor Affinity

Example 3. Insulin binds allosterically to insulin receptor in the presence of BBBB138

[0138] To determine if insulin was able to bind to the receptor in the presence of BBBB138, binding studies were performed by SPR. BBBB138 was captured on the anti-Fc channels of a GLC sensor chip. This was followed by the injection of IR-A and/or IR-B receptors which were captured through the BBBB mAb on the anti-Fc surface. Finally, a series of insulin solutions (Humulin, Eli Lilly, Prod # NDC-0002- 8215-01, Indianapolis, IN) starting from 2000 nM at 5-fold dilutions were injected over the IR/mAb complex surface for 4 min (association phase) and was followed by 10 min of running buffer flow (dissociation phase) at 50 μΐνηηη. The interspots with nonspecific IgG and without any capture molecules were used for reference correction. Double referencing of the data was performed by subtracting the curves generated by buffer injection from the reference-subtracted curves for analyte injections. [0139] In the presence of BBBB138, insulin bound to the insulin receptor (Figure 3), suggesting that the BBBB138 and insulin binding sites do not overlap.

Example 4. BBBB138 antagonizes insulin-induced insulin receptor

phosphorylation in vitro

[0140] The activity of BBBB138 was tested in a dose titration experiment against a fixed, physiologically relevant concentration of 1 nM insulin in HuH7 cells. HuH7 cells were plated at 50,000 cells/well (lOOuL) in 96-well in DMEM medium, supplemented with GlutaMAX, 10% heat inactivated FBS and incubated at 37°C in 5% C02 air for 18-24 hours prior to use. Cells were prestimulated with increasing concentrations of bivalent BBBB138 in cell medium for 30 minutes at 37°C. Insulin was added to cells and samples for 5 minutes at 37°C. Assay was stopped by addition of ice-cold PBS followed by lysis buffer, supplemented with protease and phosphatase inhibitors. Samples were analyzed using Meso Scale Discovery (MSD) phospho Insulin Receptor Signaling Panel Kit (Rockville, MD).

[0141] BBBB138 decreased insulin-induced IR phosphorylation in a dose dependent manner with an IC50 of 6.5 nM (Figure 4). Next, full insulin dose-response curves were performed in the presence of three different concentrations of BBBB138. HuH7 cells were treated with 3, 10 or 30 nM of BBBB138 followed by stimulation with an insulin dose-titration. Treatment with BBBB138 decreased insulin potency with dose- dependent rightward shift of the insulin-response curves (Figure 5). The EC50 for insulin alone (or with isotype) was observed as 0.2 nM. This curve clearly shifted with the 3 nM concentration of BBBB138 and continued to shift approximately 10 fold with the 30 nM BBBB138. Interestingly, BBBB138 did not impact the Cmax values despite the effect on the EC50 values.

[0142] We next performed a time course experiment in HuH7 cells treated with insulin alone or in the presence of BBBB138. Insulin induced a sharp peak of IR

phosphorylation at approximately 5 minutes and gradually decreased but remained sustained above baseline phosphorylation over the 2 hour time period, which is consistent with previous reports in HuH7 cells (Figure 6). Pretreatment with BBBB138 clearly prevented insulin-induced IR tyrosine phosphorylation (Figure 5). [0143] These data suggested that BBBB138 is a potent insulin receptor antagonist. Treatment with this mAb blocks insulin induced IR phosphorylation in a dose- dependent manner in vitro.

Example 5. BBBB138 effects downstream insulin signaling cascade and functional glucose uptake in vitro.

[0144] Having observed the antagonist effects of BBBB138 on insulin receptor phosphorylation, we sought to confirm if downstream signaling and cellular function was inhibited. The IRS-1 protein is essential for insulin signaling propagation as it binds to and is phosphorylated by the active IR kinase. Because IRS-1 is a direct substrate of the IR we tested the effects of BBBB138 antagonist activity on insulin induced IRS-1 tyrosine phosphorylation (Figure 7). BBBB138 resulted in decreased phosphorylation levels and insulin potency for inducing IRS-1 phosphorylation. Next, we evaluated BBBB138 activity on a further downstream endpoint, Akt, which is a well-known target and master regulator of metabolic signaling (Gonzalez, E. and T.E. McGraw, Mol Biol Cell, 2006, 17(10): p. 4484-93). As expected, BBBB138 treatment decreased both basal and insulin induced levels of Akt phosphorylation on Ser473 (Figure 8). To further assess the activity of BBBB138 on functional output and ensure antagonism is observed in an additional cell line, glucose uptake was measured in 3T3 cells (Figure 9). BBBB138 effectively decreased insulin induced glucose uptake at concentrations of 1 nM and 10 nM in vitro.

Example 6. BBBB138 induces hyperglycemia in vivo

[0145] Because BBBB138 impaired insulin signaling in vitro by specifically targeting the IR, we next tested if this antagonist could induce a model of insulin resistance in vivo. Lean C57 mice were subcutaneously dosed with 5, 10 or 25 mg/kg of BBBB138. Changes in fed and fasted blood glucose levels were monitored. BBBB138 resulted in elevated fed blood glucose levels as rapidly as 4 hours post-dose and resulted in severe hyperglycemia (>400 mg/dL) at 6 hours post-dose in the 25 mg/kg group (Figure 10). These effects were observed with the 25 mg/kg dosage and no change in fed blood glucose was observed for lower concentrations at these early time points. Next, mice were fasted overnight and challenged with an oral glucose tolerance test (OGTT, 2g/kg) 24 hours post-dose with BBBB138. While fasted-blood glucose levels were dose- dependent increased on BBBB138 treatment, administration of OGTT resulted in blood glucose levels >500mg/dL within 30 minutes for each concentration of BBBB138 (Figure 11). Each dose of BBBB138 resulted in severe glucose intolerance as blood glucose levels remained above 400 mg/dL over a time course of 120 minutes.

[0146] To assess whether the hyperglycemia induced by BBBB138 is an appropriate model for evaluating anti-diabetic therapies, we tested the effects of Exendin-4, a well- established agonist for glucagon-like peptide-1 (GLP-1) receptor, used as a type 2 diabetes treatment (Bhavsar, S, Mudaliar, S and Cherrington A; Current Diabetes Reviews 9: 161-193, 2013), on blood glucose levels in BBBB138 treated mice. Mice were dosed with either BBBB138 or IgGl Isotype control (CNTO3930) at 5 mg/kg (single dose). On day 5 after dosing, blood glucose was measured and Exendin-4 or saline were administered (Figure 12). Blood glucose was measured over a time course of 240 minutes. BBBB138 treated mice had elevated blood glucose on day 5 compared to Isotype control. Exendin-4 lowered blood glucose to baseline levels in BBBB138 treated mice within 120 minutes on Day 5. This demonstrates that the BBBB138 model of hyperglycemia can be therapeutically treated.

Example 7. BBBB138 and S961 display distinct IR antagonism

[0147] Next we sought to compare BBBB138 activity with a known IR peptide antagonist, S961. Because S961 has been reported to induce hyperglycemia and hyperinsulinemia in mice (Yip, L. et al. Diabetes. February 2015 vol. 64 no. 2 p.604- 617), we compared the proficiency of BBBB138 and S961 to raise glucose and insulin levels in normal C57 mice. Because the BBBB138 mAb and S961 peptide display different pharmacokinetic properties, measurements were taken 24 hours post-dose for BBBB138 and 1 hour post-dose for S961 to achieve comparable blood glucose levels. We observed that 24 hour treatment with 10 mg/kg BBBB138 and 1 hour treatment with 1.0 mg/kg S961 resulted in blood glucose levels >450 mg/dL (Figure 13). When insulin levels were compared at these same conditions both antagonists induced hyperinsulinemia, but BBBB138 resulted in mean levels >50 ng/dL while S961 mean levels were < 30 ng/dL (Figure 14). At comparable blood glucose concentrations, BBBB138 induces more severe hyperinsulinemia than S961.

[0148] Aside from different pharmacokinetic properties, BBBB138 and S961 bind to different sites on the IR. We therefore hypothesized that these molecules may exhibit differential antagonism of the IR and downstream insulin signaling. To elucidate signaling differences, antagonist effects of 100 nM BBBB138 and 1 nM of S961 were challenged against an insulin titration (Figure 15). Antagonist concentrations of 100 nM BBBB138 and 1 nM S961 resulted in similar phosphorylation decreases on the IR and Akt when cells were stimulated with 1 nM or 10 nM of insulin. However, at 100 nM insulin, BBBB138 resulted in a greater degree of phosphorylation on the IR and Akt compared to S 961.

Example 8. BBBB141 agonized insulin-induced insulin receptor phosphorylation in vitro

[0149] BBBB141 was identified through phage panning against the human insulin receptor. The antibody sequences are shown in Table 3. Insulin dose-response curves from HUH7 cells pretreated with either different concentrations of bivalent BBBB141 (3nM or 30nM) or isot pe control (CNTO 3930) (Figure 16) indicated that

(1) BBBB141 was an insulin receptor agonist at 3nM and 30nM concentrations;

(2) BBBB141 increased insulin receptor tyrosine phosphorylation in the absence of insulin; (3) BBBB141 increased the Cmax of the insulin dose-response curve of insulin receptor total tyrosine phosphorylation.

Table 3. Sequences of BBBB141 antibody.

LVTVSS

17 EIVLTQSPATLSLSPGERATLSCRASQSVDKALAWYQQ VL

KPGQ APRLLI YYASNRATGIP ARF S GS GS GTDFTLTI SSL EPEDFAVYYCQQRYDWPYTFGQGTKVEIK

Example 9. BBBB141 induced insulin receptor phosphorylation differently from insulin

[0150] IR phosphorylation was compared over the 120 min time course in HuH7 cells stimulated with either BBBB141 (30 nM) or insulin (1 nM), and where the insulin- treated cells were pretreated with either BBBB141 (30nM) or isotype control

(CNTO3930, 30 nM), as indicated (Figure 17). The results suggested that (1)

BBBB141 alone stimulated insulin receptor phosphorylation over the entire 120 minute time course; (2) BBBB141 displayed a different trend in insulin receptor

phosphorylation over time, as compared to that in insulin-treated cells.

[0151] Phosphorylation levels on insulin receptor site Tyrl328 at various time points were also measured (Figure 18). BBBB141 led to different phosphorylation levels on insulin receptor tyrosine 1328.

Example 10. BBBB141 and insulin co-stimulation resulted in greater insulin receptor phosphorylation than either BBBB141 or insulin alone

[0152] Insulin receptor phosphorylation was compared using dose-response curves from HuH7 cells treated with either insulin alone, BBBB141 alone, or a 1 : 1 co- stimulation of insulin and BBBB141 (Figure 19). The results suggested that (1) BBBB141 stimulated insulin receptor phosphorylation in a dose-dependent manner; (2) BBBB141 and insulin dose-response curves for insulin receptor phosphorylation were different; and (3) co-stimulation with BBBB141 and insulin simultaneously resulted in greater insulin receptor phosphorylation than either BBBB141 or insulin alone (Figure 19). Example 11. Different constructs of BBBB141 activate the insulin receptor in the absence of insulin

[0153] Insulin receptor phosphorylation EC50 values were compared in HuH7 cells treated with increasing concentrations between 0 - 1000 nM of bivalent, monovalent or the Fab portion of BBBB141. The results suggested that the treatment with different constructs of BBBB141 resulted in insulin receptor phosphorylation in the absence of insulin (Figure 20). EC50 values for the BBBB141 constructs tested were calculated using Nonlinear regression curve fit and Variable Slope model analyses (GraphPad Prism 6, Table 4).

Table 4. Insulin receptor phosphorylation EC50 values of BBBB141 constructs.

*: P<0.05, compared with isotype-treated group (T-test, compared with vehicle treated group in the same animal model)

Example 12. BBBB141 single-dose on blood glucose and plasma insulin in C57BL/6 and Diet-induced obese (DIO) mice

[0154] Fed blood glucose levels were compared in C57BL/6 lean mice and DIO mice, treated with either isotype control (CNTO3930, 10 mg/kg), or BBBB141 (10 mg/kg). The results suggested that in C57BL/6 lean mice the treatment with BBBB141 statistically significantly reduced fed blood glucose levels starting at 2 hr post antibody injection (Table 5). This blood glucose lowering effect lasted up to 7 days. The treatment of DIO mice with BBBB141 (10 mg/kg) also resulted in a significant reduction of fed blood glucose levels starting at 4 hrs and lasting through 8 hrs following antibody administration at day 0, as well as at day 7. (Table 5) Table 5. Fed blood glucose levels before and after BBBB141 treatment in C57BL/6 lean mice and DIO mice (Mean±SE, n=8). *: P<0.05, compared with isotype-treated group (T-test, compared with vehicle treated group in the same animal model)

[0155] Fed plasma insulin levels were compared in C57BL/6 lean mice and DIO mice, treated with either isotype control (CNTO3930, 10 mg/kg), or BBBB141 (10 mg/kg). The results suggested that the treatment of C57BL/6 lean mice with BBBB141 had a trend of reduction of plasma insulin at day 5 and day 7 post-antibody injection, however the difference did not reach statistical significance. Fed plasma insulin levels in DIO mice treated with BBBB141, were statistically significantly reduced 7 days post-antibody injection, as compared with the isotype mAb control treatment (p < 0.05, as determined using t-test) (Table 6).

Table 6. Fed plasma insulin levels before and after the BBBB141 treatment in C57BL/6 lean mice and DIO mice (Mean±SE, n=8).

Example 13. BBBB141 single-dose on liver and skeletal muscle insulin signaling pathway in C57BL/6 mice

[0156] Phosphorylation of insulin receptor and its downstream signaling target Akt was analyzed in C57/BL6 mice treated with either BBBB141 (10 mg/kg) or isotype control (CNTO3930, 10 mg/kg). Briefly, male C57BL/6 mice (20-22 grams, fed with regular chow) were grouped such that basal fed blood glucose levels and body weights were evenly distributed among the groups. After grouping, mice were dosed with either isotype control or BBBB141 Insulin Receptor agonist mAb subcutaneously at 9am; food was removed at 5 pm in order to fast for 16 h prior to oral glucose tolerance test (OGTT).

[0157] Twenty four hours post antibody administration, a 2 g/kg oral glucose challenge was administered to the animals. Mice were sacrificed at baseline (no oral glucose), and 30, 60 and 120min post-glucose challenge; liver and gastrocnemius muscle samples were rapidly removed and snap frozen in liquid N2. Tissue samples were then homogenized (30s on ice) in 5mL IX Tris Lysis Buffer (MSD Rockville, Maryland) containing Complete Protease Inhibitors (Roche; Indianapolis, ID) and PhosStop (Roche; Indianapolis, ID) [1 tab/lOmL buffer each], and clarified by refrigerated centrifugation (14000 m/4C/10min). Lysates were used for protein concentration analysis (BCA assay; Pierce Rockford, IL), and phospho-Insulin Receptor beta (Tyrl 150/1151) Assay (Cell Signaling, Danvers, MA), pSer473/Total Akt Assay (MSD Rockville, Maryland) and pSer308 (MSD Rockville, Maryland).

[0158] BBBB141 treatment significantly elevated phospho-IR at 24h post- mAb injection, following 16h fast in both liver and gastrocnemius muscle (Figure 21-22) In liver, significantly elevated phospho-IR was sustained during OGTT; a small effect of the glucose challenge could be observed on phospho-IR levels in both control and BBBB141 tissue (Figure 21). In muscle, significantly elevated phospho-IR was sustained during OGTT; phospho-IR levels in BBBB141 tissue appeared to trend towards increasing out to 2 h in a linear dependency (Figure 22). No effect of the glucose challenge was observed in control mice.

[0159] In liver, no effect of BBBB141 was observed on the insulin receptor signaling pathway, including phospho-T308 or phospho-S473 Akt. A mild inverse trend in response to glucose challenge was noted. (Figures 21 - 26). In muscle, both (P)T308 or (P)S473 Akt were significantly elevated 24h post-mAb injection, following 16h fast. In muscle, (P)T308 or (P)S473 Akt were both significantly elevated at the 30 and 60min post-glucose time points relative to control animals; muscle (P)Akt appeared to increase in response to glucose in both control and BBBB141 groups. No effect was observed on (P)Erkl/2 in liver or muscle (data not shown).

Example 14. The effect of a single dose of BBBB141 on blood glucose and plasma insulin levels in Streptozotocin (STZ)-treated diabetic mice

[0160] Male DIO C57BL/6J mice at 9 weeks of age, treated with Streptozotocin (STZ), 50 mg/kg i.p. daily for 5 days, with fed blood glucose levels greater than 425 mg/dL, were used in this study, n == 8 per group (Table 7). Mice were grouped such that the mean fed blood glucose levels and body weights were comparable among the groups. Animals received either isotype control (CNTO3930, 10 mg/kg,) or BBBB141 (10 mg/kg) via subcutaneously injection at day 0. Fed blood glucose levels were measured daily from day 1 to day 10. Fed plasma insulin levels were determined at day 10.

[0161] In mice with STZ -induced diabetes, a single dose of BBBB141 was statistically significantly associated with reduced fed blood glucose levels, starting from day 2 and continuing through day 10, as compared with that in isotype mAb control treated mice (Table 7).

Table 7. Fed blood glucose levels before and after BBBB141 treatment in STZ- induced diabetic mice. *: PO.05, compared with isotype-treated group (T-test).

6 501.7±20.4 269.5±43.3*

7 562.5±23.5 191.8±36.0*

8 552.5±25.0 146.7±11.8*

9 585.2±12.3 262.3±31.0*

10 570.0±17.4 349.8±30.3*

[0162] Fed plasma insulin levels were determined at day 10 of the study, and were statistically significantly higher in BBBB141 treated mice (241.6 ± 35.8 pg/mL), as compared to that of the isotype control treated mice (120.5±13.6 pg/mL, P<0.05).

Example 15. The effect of a multiple-dose treatment with BBBB141 on blood glucose and plasma insulin levels in DIO mice

[0163] Blood glucose and plasma insulin were analyzed in male DIO C57BL/6J mice, 11-12 weeks of age. The mice have been maintained on a high-fat diet, D12492 (Research Diets, New Brunswick, NJ), for 5-6 weeks prior to the start of the experiment , and continued to receive the high-fat diet during the experiment. The mice were grouped such that the mean blood glucose levels and body weights were comparable among the groups. Next, mice were subcutaneously injected with either isotype control or BBBB141, 3 or 10 mg/kg, twice per week. Blood glucose and plasma insulin levels were determined using OneTouch Ultra gluco meter (LifeScan, Milpitas, CA) and MSD Insulin assay ( Rockvi!le. MD) at indicated time points (Tables 8-9), following food removal 6 hours prior to obtaining the samples.

[0164] The results suggested that blood glucose levels were significantly reduced in mice treated with either dose of BBBB141, as compared with that in isotype control- treated mice (Table 8). Table 8. Blood glucose levels (mg/dL) before and after BBBB141 treatment in DIO mice (Mean±SE, n=8). * : P<0.05, compared with isotype-treated group (One way ANOVA, followed by Dunnelt's multiple comparisons test)

[0165] The results also suggested that plasma insulin levels at 7, 14, and 24 days of treatment were statistically significantly reduced in mice treated with BBBB141 , either 3 mg/kg or 10 mg/kg, as compared with that in isotype control -treated mice (Table 9).

Table 9. Plasma insulin levels (mg/dL) before and after BBBB141 treatment in DIO mice (Mean±SE, n=8). * : P<0.05, compared with isotype-treated group (One way A OVA. followed by Dunnett's multiple comparisons test)

Claims

CLAIMS We claim:

1. An isolated antibody, or antigen-binding fragment thereof, that

immunospecifically binds to insulin receptor (IR), the antibody or antigen-binding fragment thereof comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO:2, a heaw chain CDR2 comprising the amino acid sequence of SEQ ID NO:3, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO:4, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5. a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7; wherein the CDRs are defined according to Kabat.

2. The isolated antibody, or antigen-binding fragment thereof of claim 1 wherein: the antibody, or antigen-binding fragment thereof, has a heavy chain variable domain comprising an amino acid se uence that is at least 90% identical to the amino acid sequence of SEQ I D NO: 8 and a light chain variable domain comprisin an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:9.

3. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-2 wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.

4. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 -3 wherein the antibody or antigen-binding fragment is recombinant.

5. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 -4 wherein the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, or a single chain antibody.

6. A nucleic acid molecule encoding the isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A cell expressing the isolated antibody, or antigen-binding fragment thereof, of any one of claims 1-5.

9. A method of antagonizing insulin receptor signaling, comprising

administering to a subject an effective amount of the antibody of any one of claims 1-5, or antigen-binding fragment thereof.

10. The method of claim 9 wherein the administration to the subject an effecti e amount of the antibody of claim 1, or antigen-binding fragment thereof, induces hyperglycemia, hypennsulinemia. or insulin resistance.

1 1 . The method of claim 10 wherein the hyperglycemia, hypennsulinemia. or insulin resistance is associated with type 2 diabetes.

12. A method of screening compounds for the ability to modulate

hyperglycemia, hyperinsulinemia, or insulin resistance, comprising: contacting a cell or a subject with the antibody of claim 1 and with a test compound, and measuring the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the presence of the test compound relative to the levels of IR phosphorylation, Akt phosphorylation, or blood glucose in the absence of the test compound, wherein an increase in the levels of IR phosphorylation, Akt phosphorylation, or a decrease in blood glucose in the presence of the test compound, relative to that in the absence of the test compound, indicates that the test compound is capable of reducing hyperglycemia,

hyperinsulinemia, or insulin resistance; and wherein a decrease or no change in the levels of IR phosphorylation, Akt phosphorylation, or increase or no change in blood glucose in the presence of the test compound, relative to that in the absence of the test compound, indicates that the test compound is not capable of reducing hyperglycemia, hyperinsulinemia, or insulin resistance.

13. An isolated antibody, or antigen-binding fragment thereof, that

immunospeci fically binds I R. the antibody or antigen-binding fragment thereof comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12, a light chain CDR1 comprising the amino acid sequence of SEQ I D NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15; wherein the CDRs are defined according to Kabat.

14. The isolated antibody, or antigen-bindin fragment thereof, o claim 13 wherein: the antibody, or antigen-binding fragment thereof, has a heavy chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 16 and a light chain variable domain comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 17.

15. The isolated antibody, or antigen-binding fragment thereof, o any one of claims 13-14 wherein the antibody or antigen-binding fragment is a human antibody or antigen-binding fragment.

16. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 1 3- 1 5 wherein the antibody or antigen-binding fragment is recombinant.

17. The isolated antibody, or antigen-binding fragment thereof, of any one of claims 13-16 wherein the antigen-binding fragment is a Fab fragment, a F(ab')2 fragment, or a single chain antibody.

18. A nucleic acid molecule encoding the isolated antibody, or antigen-binding fragment thereof, of any one of claims 13-17.

19. A vector comprising the nucleic acid molecule of claim 18.

20. A cell expressing the isolated antibody, or antigen-binding fragment thereof, of any one of claims 13-17.

21 . A method of agonizing insulin receptor signaling, comprising administerin to a cell or a subject an effective amount of the antibody of any one of claims 13- 1 7. or antigen-binding fragment thereof.

22. The method of claim 21 wherein agonizing insulin receptor signaling comprises an increase in insulin receptor tyrosine phosphorylation.

23. The method of claim 21 wherein the administration to the subject an effective amount of the antibody of claim 1, or antigen-bindin fragment thereof, reduces hyperglycemia, hyperinsulinemia. or insulin resistance.

24. The method of claim 23 wherein the hyperglycemia, hyperinsulinemia , or insulin resistance is associated with type 2 diabetes.