Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/22—Hormones
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/06—Antihyperlipidemics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
  • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/48—Drugs for disorders of the endocrine system of the pancreatic hormones
  • A61P5/50—Drugs for disorders of the endocrine system of the pancreatic hormones for increasing or potentiating the activity of insulin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL

Definitions

• CGRP CGRP
• fragments thereof including Fab fragments which specifically bind to CGRP and promote glucose uptake and utilization in peripheral tissue and/or inhibit hepatic glucose production.
• Exemplary embodiments of the subject methods may preserve functional pancreatic beta cells, thereby slowing the progression to overt diabetes.
• the invention also pertains to methods of screening for diseases and disorders associated with insulin resistance (including disorders of glucose, carbohydrate and fat metabolism), and methods of preventing or treating diseases and disorders associated with insulin resistance by administering said antibodies or fragments thereof.
• Calcitonin Gene Related Peptide is produced as a multifunctional neuropeptide of 37 amino acids in length.
• CGRP-alpha and CGRP-beta differ by three amino acids in humans, and are derived from different genes.
• the CGRP family of peptides also includes amylin, adrenomedullin, and calcitonin, although each has distinct receptors and biological activities. Doods, H., Curr. Op. Invest. Drugs, 2(9): 1261-68 (2001).
• CGRP protein family amino acid residues at putative receptor binding sites are conserved, although overall homology varies. For example, human CGRP and amylin share 46% amino acid sequence identity overall while human calcitonin and CGRP share 15% amino acid sequence identity. Wimalawansa, S.J., Endocrine Rev. 17(5):533-585 (1996).
• CGRP-R which consists of a seven-transmembrane component, in conjunction with receptor- associated membrane protein (RAMP).
• RAMP receptor-associated membrane protein
• CGRP is found throughout the peripheral and central nervous system and influences the cardiovascular, nervous and endocrine systems. When CGRP is released from tissues such as trigeminal nerves, it can result in a sequential activation and release of neuropeptides within the meninges, to mediate neurogenic inflammation that is characterized by vasodilation, vessel leakage, and mast-cell degradation. Durham, P.L., New Eng. J. Med , 350 (11): 1073-75 (2004). CGRP is thought to play a prominent role in the development of migraines. It has been shown that elevated levels of CGRP identified in plasma from jugular venous blood during the headache phase of migraines, to the exclusion of other neuropeptides.
• CGRP receptors In addition to nervous tissue, CGRP receptors have been identified in cardiovascular tissue, adrenal gland, pituitary gland, kidney, pancreas and bone. Wimalawansa, S.J., Endocrine Rev. 17(5):533-585 (1996). In in vitro studies, both CGRP and amylin were found to inhibit insulin secretion using isolated pancreatic tissue, counteract the insulin-stimulated rate of glycogen synthesis in a dose-dependent manner and block the effects of insulin in isolated hepatocytes (Gomez-Foix et al. Biochem. J. 276:607-610, 1991).
• Glucose homeostasis is maintained by balancing glycogen synthesis with glycogenolysis by the hormone glucagon and glucose utilization and uptake into tissue by the hormone insulin.
• the presence of glucose normally stimulates insulin production, which functions to increase the transport rate of glucose into skeletal muscle, myocytes, brain and adipocytes. Insulin also normally inhibits lipid degradation in adipocytes.
• pancreatic beta cells compensate by secreting increasing levels of insulin.
• pancreatic beta cell ability to compensate becomes exhausted and exogenous insulin is required.
• the present disclosure provides a method of increasing peripheral and/or hepatic glucose utilization in a subject in need thereof, comprising administering an effective amount of a composition comprising an anti-human CGRP antibody or antibody fragment to said subject.
• the present disclosure provides a method of decreasing insulin resistance in a subject in need thereof, comprising administering an effective amount of a composition comprising an anti-human CGRP antibody or antibody fragment to said subject.
• the present disclosure provides a method of treating, preventing or controlling obesity in a subject in need thereof comprising administering an effective amount of a composition comprising an anti-human CGRP antibody or antibody fragment to said subject.
• the present disclosure provides a method to achieve sustained normoglycemia in a subject in need thereof comprising administering an effective amount of a composition comprising an anti-human CGRP antibody or antibody fragment to said subject.
• the present disclosure provides a method for increasing the ratio of lean tissue to body fat in a subject in need thereof, comprising administering an effective amount of a composition comprising an anti-human CGRP antibody or antibody fragment to said subject.
• the subject methods may be effective to treat or delay the onset of type II diabetes and/or obesity. For example, the need for administering exogenous insulin may be delayed.
• the method may be effective to prevent or slow the loss of pancreatic beta cells. For example, without intent to be limited by theory, it is thought that the method may allow pancreatic beta cells of an insulin-resistant human or non-human animal to rest, thereby preventing loss of functional pancreatic beta cells.
• Said subject may have been diagnosed with pre-diabetes or may exhibit one or more risk factors for development of type II diabetes.
• the subject may be pre-menopausal, perimenopausal, menopausal or postmenopausal.
• the subject may exhibit one or more symptoms of pre-diabetes such as fasting blood glucose level of between 100 mg/dL and 125 mg/dl; blood sugar level of between 140 mg/dL and 199 mg/dL two hours after ingesting a 75 gram glucose solution or a glucose solution of 1.75 grams of glucose per kilogram of body weight, to a maximum dose of 75 grams; and/or glycated hemoglobin of between 5.7 percent and 6.4 percent.
• pre-diabetes such as fasting blood glucose level of between 100 mg/dL and 125 mg/dl; blood sugar level of between 140 mg/dL and 199 mg/dL two hours after ingesting a 75 gram glucose solution or a glucose solution of 1.75 grams of glucose per kilogram of body weight, to a maximum dose of 75 grams; and/or glycated hemoglobin of between 5.7 percent and 6.4 percent.
• the subject may exhibit one or more symptoms of diabetes, such as fasting blood glucose level greater than 125 mg/dl; blood sugar level of at least 200 mg/dL two hours after ingesting a 75 gram glucose solution or a glucose solution of 1.75 grams of glucose per kilogram of body weight, to a maximum dose of 75 grams; and/or glycated hemoglobin of at least 6.5 percent.
• diabetes such as fasting blood glucose level greater than 125 mg/dl; blood sugar level of at least 200 mg/dL two hours after ingesting a 75 gram glucose solution or a glucose solution of 1.75 grams of glucose per kilogram of body weight, to a maximum dose of 75 grams; and/or glycated hemoglobin of at least 6.5 percent.
• the subject may exhibit one or more risk factors for development of type II diabetes, such as a family history of type II diabetes; one or more parents or siblings previously diagnosed with type II diabetes; dyslipidemia; total blood triglyceride levels of at least 200 mg/dL; blood high density lipoprotein level less than 35 mg/dL; obesity; body mass index greater than 25 kg/m 2 ; history of gestational diabetes; previously gave birth to an infant with birth weight greater than 9 lbs.; hypertension; systolic blood pressure of at least 140 mmHg; diastolic blood pressure of at least 90 mmHg; previous measurement of fasting blood glucose of at least 99 mg/dL; vascular disease; Polycystic Ovarian Syndrome; or acanthosis nigricans.
• type II diabetes such as a family history of type II diabetes; one or more parents or siblings previously diagnosed with type II diabetes; dyslipidemia; total blood triglyceride levels of at least 200 mg/dL; blood high density lipoprotein level less than 35 mg
• the subject may have been diagnosed with type II diabetes.
• the subject may be refractory to treatment with at least one compound selected from the group consisting of: GLP-1 , exenatide-1, exendin, exendin analog, exendin agonist, liraglutide, exenatide LAR, a DPP-4 antagonist, a GLP-1 receptor agonist, and another GLP-1 agonist; or such compound may be contraindicated for administration to the subject.
• the methods may further comprise administering to said subject an antidiabetic agent or anti-obesity agent other than an anti-human CGRP antibody or antibody fragment.
• Said anti-diabetic agent or anti-obesity agent may comprise one or more of amylin, amylin agonist, sulfonylureas, calcitonin, glucagon, PPAR-gamma agonists, GPL-1 receptor agonists, dipeptidyl peptidase IV inhibitor, amylin analogs, biguanides, dopamine D2 receptor agonists, meglitinides, alpha-glucosidase inhibitor, antidyslipidemic bile acid sequestrant, exendin, exendin analog, exendin agonist, gastrin inhibitory peptide (GIP), incretin peptide, insulin, SGLT2 inhibitor, a glucose reabsorption inhibitor, fenofibrate, fibrate, an anti-ghrelin antibody or antibody fragment, an fibroblast growth factor receptor (FGFR)-l(IIIb), FGFR-l(IIIc), antibody or antibody fragment, and/or FGFR
• said anti-diabetic agent is metformin.
• the method may be effective to cause weight loss.
• the administered anti-human CGRP antibody or antibody fragment may not significantly increase insulin secretion in vivo, e.g., may not significantly increase insulin secretion above normal physiological levels in vivo, or may not significantly increase insulin secretion relative to the level of insulin secretion prior to administration of the anti-human CGRP antibody or antibody fragment.
• the administered anti-human CGRP antibody or antibody fragment may not result in an increased incidence in pancreatitis or the expression of markers or cytokines associated with pancreatic inflammation.
• composition may further comprise a pharmaceutically acceptable carrier.
• Said anti-human CGRP antibody or antibody fragment may be administered to said subject at a dosage between about 0.1 and 100.0 mg/kg of body weight of recipient subject.
• Said anti-human CGRP antibody or antibody fragment may be a human antibody.
• Said anti-human CGRP antibody or antibody fragment may be non-naturally occurring.
• Said anti-human CGRP antibody or antibody fragment may be a non-naturally occurring antibody fragment.
• Said anti-human CGRP antibody or antibody fragment may be a humanized antibody or fragment thereof.
• Said anti-human CGRP antibody or antibody fragment may be a chimeric antibody.
• Said anti-human CGRP antibody or antibody fragment may specifically bind to the same linear or conformational epitope(s) and/or may compete for binding to the same or overlapping linear or conformational epitope(s) on an intact CGRP polypeptide or fragment thereof as an anti-human CGRP antibody selected from the group consisting of: (a) Abl comprising the V L of SEQ ID NO:2 and the V H of SEQ ID NO:4; (b) Ab2 comprising the V L of SEQ ID NO: 12 and the V H of SEQ ID NO: 14; (c) Ab3 comprising the V L of SEQ ID NO:22 and the V H of SEQ ID NO:24; (d) Ab4 comprising the V L of SEQ ID NO:32 and the V H of SEQ ID NO:34; (e) Ab5 comprising the V L of SEQ ID NO:42 and the V H of SEQ ID NO:44; (f) Ab6 comprising the V L of SEQ ID NO:52 and the V H of SEQ
• Said anti-human CGRP antibody or antibody fragment may comprise at least one, at least two, at least three, at least four, at least five, or all six CDRs contained in an antibody selected from the group consisting of: (a) Abl comprising the VL of SEQ ID NO:2 and the V H of SEQ ID NO:4; (b) Ab2 comprising the V L of SEQ ID NO: 12 and the V H of SEQ ID NO: 14; (c) Ab3 comprising the V L of SEQ ID NO:22 and the V H of SEQ ID NO:24; (d) Ab4 comprising the V L of SEQ ID NO:32 and the V H of SEQ ID NO:34; (e) Ab5 comprising the V L of SEQ ID NO:42 and the V H of SEQ ID NO:44; (f) Ab6 comprising the V L of SEQ ID NO:52 and the V H of SEQ ID NO:54; (g) Ab7 comprising the V L of SEQ ID NO:62 and the V H of SEQ ID NO:
• Said anti-human CGRP antibody or antibody fragment may have a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to an antibody selected from the group consisting of: (a) Abl comprising the V L of SEQ ID NO:2 and the V H of SEQ ID NO:4; (b) Ab2 comprising the V L of SEQ ID NO:12 and the V H of SEQ ID NO: 14; (c) Ab3 comprising the V L of SEQ ID NO:22 and the V H of SEQ ID NO:24; (d) Ab4 comprising the V L of SEQ ID NO:32 and the V H of SEQ ID NO:34; (e) Ab5 comprising the V L of SEQ ID NO:42 and the V H of SEQ ID NO:44; (f) Ab6 comprising the V L of SEQ ID NO:52 and the V H of SEQ ID NO:54; (g) Ab7 comprising
• Said anti-human CGRP antibody or antibody fragment comprises an antibody selected from the group consisting of: (a) Abl comprising the VL of SEQ ID NO:2 and the V H of SEQ ID NO:4; (b) Ab2 comprising the V L of SEQ ID NO: 12 and the V H of SEQ ID NO: 14; (c) Ab3 comprising the V L of SEQ ID NO:22 and the V H of SEQ ID NO:24; (d) Ab4 comprising the V L of SEQ ID NO:32 and the V H of SEQ ID NO:34; (e) Ab5 comprising the V L of SEQ ID NO:42 and the V H of SEQ ID NO:44; (f) Ab6 comprising the V L of SEQ ID NO: 52 and the V H of SEQ ID NO: 54; (g) Ab7 comprising the V L of SEQ ID NO:62 and the V H of SEQ ID NO:64; (h) Ab8 comprising the V L of SEQ ID NO:52 and the V H of SEQ ID NO:
• the anti-human CGRP antibody or antibody fragment may comprise a human, chimeric or humanized antibody.
• the anti-human CGRP antibody or antibody fragment may comprise a Fab, F(ab') 2 , scFv, IgNar, or MetMab or another monovalent antibody fragment.
• composition suitable for use in a method as described herein which may comprise an effective amount of an anti-human CGRP antibody or antibody fragment and an anti-diabetic or anti-obesity agent other than an anti-human CGRP antibody or antibody fragment.
• the anti-human CGRP antibody or antibody fragment may one described herein, e.g., which may specifically bind to the same linear or conformational epitope(s), may compete for binding to the same or overlapping linear or conformational epitope(s) on an intact CGRP polypeptide or fragment thereof as, may have a polypeptide sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to, or may comprises, an anti-human CGRP antibody selected from the group consisting of (a) Abl comprising the VL of SEQ ID NO:2 and the V H of SEQ ID NO:4; (b) Ab2 comprising the V L of SEQ ID NO: 12 and the V H of SEQ ID NO: 14; (c) Ab3 comprising the V L of SEQ ID NO:22 and the V H of SEQ ID NO:24; (d) Ab4 comprising the V L of SEQ ID NO:32 and the V H
• Said anti-diabetic or anti-obesity agent comprises one or more of amylin, amylin agonist, sulfonylureas, calcitonin, glucagon, PPAR-gamma agonists, GPL-1 receptor agonists, dipeptidyl peptidase IV inhibitor, amylin analogs, biguanides, dopamine D2 receptor agonists, meglitinides, alpha-glucosidase inhibitor, antidyslipidemic bile acid sequestrant, exendin, exendin analog, exendin agonist, gastrin inhibitory peptide (GIP), incretin peptide, insulin, SGLT2 inhibitor, a glucose reabsorption inhibitor, fenofibrate, fibrate, metformin, an anti-ghrelin antibody or antibody fragment, an fibroblast growth factor receptor (FGFR)-l(IIIb), FGFR-l(IIIc), antibody or antibody fragment, and/or
• FIG. 1A-D Blood glucose and plasma insulin levels before and after treatment. Results are expressed as the mean ⁇ SEM. ## p ⁇ 0.01 vs vehicle with an ANOVA one way + Dunnett's post test.
• A Blood glucose was measured in fed condition before treatments, 18h after treatment with vehicle, Abl4 and metformin and 42h after treatment with vehicle and AM 4.
• B Plasma insulin was measured in fed condition before treatments, 18h after treatment with metformin and 42h after treatment with vehicle and Abl4.
• HOMA-IR insulin ( ⁇ /mL) / 22.5) was calculated before treatment, 18h after treatment with metformin and 42h after treatment with vehicle and Abl4 D: Blood glucose was measured in fasted condition just before the clamp (24h after treatment with metformin and 48h after treatment with vehicle and AM 4). Legend: Leftmost bar in each group, vehicle; middle bar in each group, A 4 treatment; rightmost bar in each group, metformin treatment.
• FIG. 2A-C Glucose infusion rate evolution during clamp procedure (A), blood glucose mean during steady state (B) and plasma insulin levels at the end of the clamp (C). Results are expressed as the mean ⁇ SEM.
• A *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 vs vehicle with an ANOVA two way with Bonferroni's post test.
• FIG. 2A upper line, metformin treatment; middle line, Abl4 treatmen t; lower line, vehicle treatment (at 180 min time point).
• FIG. 2B-2C Leftmost bar in each group, vehicle; middle bar in each group, Abl4 treatment; rightmost bar in each group, metformin treatment.
• FIG. 4A-C In vivo tissues specific glucose utilization. Results are expressed as the mean ⁇ SEM. # p ⁇ 0.05, ## p ⁇ 0.01 vs vehicle with an ANOVA one way with Dunnett's post test.
• A glucose utilization in epididymal white adipose tissue (EWAT), inguinal white adipose tissue (I WAT), and skin (as negative control).
• B glucose utilization in mixed vastus lateralis muscle (VL) and glycolytic extensor digitorum longus muscle (EDL).
• C glucose utilization in oxidative soleus muscle and heart apex. Legend: Leftmost bar in each group, vehicle; middle bar in each group, Abl4 treatment; rightmost bar in each group, metformin treatment.
• FIG. 5 Average body weight over time for animals fed a high-fat fructose diet or control animals fed normal chow. Legend: Upper line, high fat high fructose diet; lower line, control chow.
• FIG. 6 Body weight gain over time for the animal groups shown in FIG. 5.
• Upper line high fat high fructose diet
• lower line control chow.
• Upper line high fat high fructose diet
• lower line control chow.
• FIG. 7 Body weight gain over time for high-fat diet fed animals after treatment with Abl4 (10, 30, or 100 mg/kg) or metformin, as well as vehicle-treated animals and control animals fed normal chow. Treatment was administered on day 0. Lines on graph in order from lowest to highest at day 7 are: normal chow (NC) plus vehicle; high fat diet (HFD) plus metformin; HFD plus Abl4 30 mg/kg; HFD plus Abl4 10 mg/kg; HFD plus vehicle; HFD plus AM 4 100 mg/kg.
• FIG. 8A Food intake for the animals shown in FIG. 7.
• HFD high fat diet
• NC normal chow
• FIG 8B Cumulative food intake for the animals shown in FIG. 7.
• order of bars from left to right is: normal chow (NC) plus vehicle; high fat diet (HFD) plus vehicle; HFD plus Abl4 10 mg/kg; HFD plus AM 4 30 mg/kg; HFD plus AM 4 100 mg/kg; HFD plus metformin.
• FIG. 9 Fasting blood glucose for high-fat diet fed animals after treatment with AM 4 (10, 30, or 100 mg/kg) or metformin, as well as vehicle-treated animals and control animals fed normal chow. Treatment was administered on day 0. Legend: order of bars from left to right is as in FIG. 8B.
• FIG. 10 Fasting plasma insulin for high-fat diet fed animals after treatment with AM4 (10, 30, or 100 mg/kg) or metformin, as well as vehicle-treated animals and control animals fed normal chow. Treatment was administered on day 0. Legend: order of bars from left to right in each group is as in FIG. 8B.
• FIG. 11 Plasma insulin (upper panel) and C peptide (lower left and right panels) before and during glucose clamp performed after 15 days of treatment with AM 4 or metformin. Animals were fed a high fat diet for 6 weeks prior to treatment. Legend: order of bars from left to right is as in FIG. 8B.
• FIG. 12 HOMA-IR for high-fat diet fed animals after treatment with AM4 (10, 30, or 100 mg/kg) or metformin, as well as vehicle-treated animals and control animals fed normal chow. Treatment was administered on day 0. Legend: order of bars from left to right is as in FIG. 8B.
• FIG. 13 Glucose infusion rate for glucose clamp performed after 15 days of treatment with AM 4 or metformin, as well as vehicle-treated animals and control animals fed normal chow. Animals were fed a high fat diet for 6 weeks prior to treatment. Glucose clamp was performed at two different insulin infusion rates (5mU/kg/min, steady state achieved at approx. 70-100 min, and 15mU/kg/min, steady state achieved at approx.
• FIG. 14 Mean glucose infusion rate during steady state for the glucose clamp experiments shown in FIG. 13. Glucose infusion rates are shown for the low and high insulin infusion rates (5mU/kg/min, steady state achieved at approx. 70-100 min, and 15mU/kg/min, steady state achieved at approx. 170-210 min.) Order of the bars in each group is as in FIG. 8B.
• FIG. 15 Mean glucose fluxes during the glucose clamp experiments shown in FIG. 13. Results are shown for the lower (5mU/kg/min) insulin infusion rate, steady state achieved at approx. 70-100 min. Legend: order of bars from left to right in each group is: high fat diet (HFD) plus vehicle; HFD plus AM 4 10 mg/kg; HFD plus AM 4 30 mg/kg; HFD plus AM 4 100 mg/kg; HFD plus metformin.
• HFD high fat diet
• FIG. 16 Mean glucose fluxes during the glucose clamp experiments shown in FIG. 13. Results are shown for the higher (15mU/kg/min) insulin infusion rate, steady state achieved at approx. 170-210 min. Legend: order of bars in each group is as in FIG. 15.
• FIG. 17 Mean toxicokinetic profiles of an anti-CGRP antibody (specifically, Ab6) following i.v. bolus injection into male Sprague-Dawley rats. Plasma concentration over time is shown for 168 hours (7 days), supporting weekly dosing as performed in the Examples below. Legend: square markers, AM 4 10 mg/kg/week; upward-pointing triangle markers, AM 4 30 mg/kg/week; diamond markers, AM 4 100 mg/kg/week.
• FIG. 18A-D 6 hours fasting HOMA-IR (A), blood glucose (B), plasma insulin (C) and body weight (D) in 8-week old ZDF rats. Results are expressed as mean ⁇ SEM.
• FIG. 19A-B Body weight (A) and body weight gain (B) follow-up. Results are expressed as mean ⁇ SEM.
• FIG. 19A pioglitazone treated rats at day 8; Abl4 60mg/kg/wk + metformin treated rats at day 28; FIG. 19B: Abl4 60mg/kg/wk + metformin treated rats at days 22 and 25); $$ pO.01 (FIG. 19B: Abl4 60mg/kg/wk + metformin treated rats at day 28); $$$ pO.001 vs vehicle ZDF (FIG. 19A: pioglitazone treated rats at all time points between days 11-28; vehicle treated ZDF lean rats at all time points) (2-way ANOVA + Bonferroni's post test).
• FIG. 20A-B Food intake follow-up (A) and cumulative food consumption (B). Results are expressed as mean ⁇ SEM. The order of the bars in FIG. 20B is the same as in FIGS. 18A-D.
• FIG. 21A-D Blood glucose (A), plasma insulin (B), HOMA-IR (C) and C peptide (D) in 6-hours (day 0) or overnight (days 12, 19, 26) fasting conditions. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIGS. 21A-D is the same as in FIGS. 18A-D.
• FIG. 22 Fructosamine levels. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIG. 22 is the same as in FIGS. 18A-D.
• FIG. 23 HbAlc levels. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIG. 23 is the same as in FIGS. 18A-D.
• FIG. 24A-B Plasma triglycerides (A) and free fatty acids (B) levels in 6-hours (day 0) or overnight (days 12, 19, and 26) fasting conditions. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIGS. 24A-D is the same as in FIGS. 18A-D.
• FIG. 25A-C Plasma Total cholesterol (A), HDL-cholesterol (B) and non
• FIG. 26A-C Plasma Total cholesterol (A), HDL-cholesterol (B) and non HDL-cholesterol (C) levels relative to the day 0. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIGS. 26A-C is the same as in FIGS. 18A-D.
• FIG. 27A-C Oral glucose tolerance test on day 26 in overnight fasting conditions (A), area under the curve (AUC) calculated from the blood glucose measured on TO (B) and calculated from relative value vs TO (C). Results are expressed as meaniSEM. The order of the bars in FIGS. 27B-C is the same as in FIGS. 18A-D.
• FIG. 28A-B Plasma insulin (A) and C peptide (B) levels during oral glucose tolerance test on day 26. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIGS. 28A-B is the same as in FIGS. 18A-D.
• FIG. 29A-B Relative expression from T-60 of plasma insulin (A) and C peptide (B) levels during oral glucose tolerance test on day 26. Results are expressed as mean ⁇ SEM. The order of the bars in each group in FIGS. 29A-B is the same as in FIGS. 18A-D.
• FIG. 30A-C Pancreas content: proinsulin (A), insulin (B) and proinsulin/insulin ratio (C). Results are expressed as mean ⁇ SEM. The order of bars
• FIGS. 30A-C (from left to right) in FIGS. 30A-C is: vehicle 1 and vehicle 2 treated ZDF lean rats; vehicle 1 and vehicle 2 treated ZDF rats; Abl4 60 mg/kg/week and vehicle 2 treated ZDF rats; vehicle 1 and metformin 200 mg/kg/day treated ZDF rats; and Abl4 60 mg/kg/week and metformin 200 mg/kg/day treated ZDF rats.
• FIG. 31 Pancreas immunohistochemical analysis: insulin labelling quantification.
• the order of bars (from left to right) in FIG. 31 is: vehicle 1 and vehicle 2 treated ZDF lean rats; vehicle 1 and vehicle 2 treated ZDF rats; Abl4 60 mg/kg/week and vehicle 2 treated ZDF rats; vehicle 1 and metformin 200 mg/kg/day treated ZDF rats; and
• anti-CGRP antibodies produced significantly increased glucose utilization in peripheral muscle when compared to metformin, without any apparent increase in the glucose utilization rate in white adipose tissue. Moreover, the anti-CGRP antibodies described herein increased glucose utilization in heart, whereas metformin produced a decrease in the glucose utilization rate in the heart. Additionally, anti-CGRP antibodies described herein inhibited hepatic glucose production, similarly to the effect obtained from administration of metformin.
• ZDF Zucker diabetic fatty
• Example 1 a hyperinsulinemic-euglycemic clamp study was performed with AM 4 to determine its effects on whole body insulin sensitivity as well as on the insulin sensitivity of specific tissues in normal rats that are normoglycemic, normoinsulinemic, and have normal whole body and tissue-specific insulin sensitivities.
• AM 4 was given intravenously as a single 100 mg/kg administration to normal rats 48 hrs prior to a hyperinsulinemic-euglycemic clamp procedure.
• Results from the evaluation of plasma glucose and insulin levels measured just prior to the clamp procedure indicated that AM 4 reduced plasma insulin levels relative to vehicle-treated controls without altering plasma glucose levels.
• the resulting reductions in HOMA-IR indicated improvements in whole body insulin sensitivity.
• the hyperinsulinemic-euglycemic clamp procedure confirmed this improvement in whole body insulin sensitivity by CGRP antagonism, where at steady state, both the glucose infusion rate and whole body glucose turnover (utilization) rate were elevated relative to vehicle-treated controls. Increased glucose infusion rate and whole body glucose turnover with a constant insulin infusion was indicative of increased whole body insulin sensitivity.
• CGRP antagonism also increased glucose utilization in glycolytic as well as oxidative skeletal muscle (vastus lateralis, indicative of mixed glycolytic plus oxidative; extensor digitorum longus, indicative of glycolytic, and soleus, indicative of oxidative). The greatest increases in glucose utilization occurred in the mixed metabolic vastus lateralis. These observations are indicative of increased skeletal muscle insulin sensitivity caused by CGRP antagonism. CGRP antagonism also increased cardiac glucose utilization. In contrast, glucose utilization rates in visceral or subcutaneous fat depots were not affected, suggesting that CGRP antagonism did not substantially increase insulin sensitivity in white adipose tissue.
• Example 2 the effects of CGRP antagonism by chronic administration of Abl4 on hepatic and peripheral insulin sensitivity in insulin-resistant animals were evaluated in rats made hyperinsulinemic and insulin-resistant but not hyperglycemic by prolonged feeding of a high fat/high fructose diet. Rats were fed a diet containing 69% fat and 14% fructose for seven weeks prior to initiation of compound administration to induce the metabolic syndrome. At the end of the seven week diet treatment period, rats continued to receive the high fat high fructose diet and in addition were given Abl4 intravenously at doses of 0 mg/kg (vehicle), lOmg/kg, 30 mg/kg, or 100 mg/kg once a week for 2 weeks.
• Example 3 the effects of chronic administration of Abl4 on glucose control was evaluated in ZDF rats that were progressing from a prediabetic (hyperinsulinemic, normoglycemic) state to an overtly diabetic (hypoinsulinemic, hyperglycemic) state. These animals develop prediabetes, characterized by marked hyperinsulinemia to compensate for their developing insulin resistance, but with little to no hyperglycemia, by seven weeks of age. This rapidly progresses to overt diabetes, characterized by hypoinsulinemia, as a result of pancreatic beta cell failure, and marked hyperglycemia by 10-12 weeks of age.
• the combination of AM 4 and metformin produced a substantially greater prevention of the rise in fasting blood glucose, the reduction in plasma insulin and C-peptide levels, the reductions in pancreatic proinsulin and insulin levels, and the reduction in pancreatic islet fibrosis than either compound alone. This suggests that the effects of metformin may be enhanced by a CGRP antagonist such as AM 4.
• the ZDF rat used in Example 3 is a very severe model of diabetes progression that advances rapidly from an insulin-resistant prediabetic state to overt diabetes with complete beta-cell destruction occurring over a time-course of only a few weeks. This limits the opportunity to evaluate modulations of disease progression, rendering compound-related improvements in disease progression and beta-cell protection difficult to demonstrate in these animals. Therefore, any demonstration of a modest delay in disease progression by the CGRP antagonist AM 4 as outlined above suggests the potential to also affect disease progression in the clinic. In addition, the ability to improve the overall treatment efficacy through combination of CGRP antagonism with metformin also supports improved efficacy of combination therapy in the clinic.
• CGRP antagonism has the ability to improve whole body insulin sensitivity, hepatic insulin sensitivity, and skeletal muscle insulin sensitivity. These improvements can be observed acutely or chronically in normal animals that are normoinsulinemic and normoglycemic and have normal insulin sensitivity, as well as in insulin-resistant animals that are hyperinsulinemic but not yet hyperglycemic. These results indicate that CGRP antagonism should decrease the insulin resistance that presents in patients with the metabolic syndrome, prediabetes, or other prediabetic conditions and further that CGRP antagonism may be capable of slowing the progression of these diseases to overt diabetes.
• the ability of the CGRP antagonist Abl4 to reduce the hyperinsulinemia present in insulin-resistant animals by reducing pancreatic insulin secretion suggests that AM 4 may have a pancreatic beta-cell sparing effect by allowing the pancreas of an insulin-resistant animal to rest. This may further delay the progression of the metabolic syndrome, prediabetes, and other prediabetic conditions to overt diabetes in the clinic.
• CGRP antagonism may have the ability to affect disease progression not only in the prediabetic states outlined above but also in overt diabetes.
• CGRP antagonist such as Abl4 may favorably affect insulin resistance and abnormal glucose control in a clinical setting both in patients with prediabetic conditions and also in patients with developing or overt diabetes.
• CGRP Calcitonin Gene Related Peptide
• CGRP-alpha ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF- N3 ⁇ 4 (SEQ ID NO: 281), wherein the N-terminal phenylalanine is amidated. Except where indicated otherwise, in general references to "CGRP” typically refer to CGRP- alpha. CGRP-alpha is referred to interchangeably as aCGRP or a-CGRP.
• CGRP-beta ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF- NH 2 (SEQ ID NO: 282), wherein the N-terminal phenylalanine is amidated; but also any membrane-bound forms of these CGRP amino acid sequences, as well as mutants (muteins), splice variants, isoforms, orthologs, homologues and variants of this sequence.
• CGRP-beta is referred to interchangeably as pCGRP or ⁇ -CGRP.
• normoglycemia refers to the state of having a normal blood glucose concentration.
• An exemplary normal blood glucose concentration in humans is between 70 mg/dl and 99 mg/dl in fasting adults, and between 70 mg/dl and 140 mg/dl in postprandial adults.
• Sustained normoglycemia refers to maintenance of normoglycemia for an extensive period of time, e.g., at least one day, at least two days, at least one week, at least two weeks, at least one month, or longer.
• Mating competent yeast species In the present invention this is intended to broadly encompass any diploid or tetraploid yeast which can be grown in culture. Such species of yeast may exist in a haploid, diploid, or other polyploid form. The cells of a given ploidy may, under appropriate conditions, proliferate for an indefinite number of generations in that form. Diploid cells can also sporulate to form haploid cells. Sequential mating can result in tetraploid strains through further mating or fusion of diploid strains. The present invention contemplates the use of haploid yeast, as well as diploid or other polyploid yeast cells produced, for example, by mating or spheroplast fusion.
• the mating competent yeast is a member of the Saccharomycetaceae family, which includes the genera Arxiozyma; Ascobotryozyma; Citeromyces; Debaryomyces; Dekkera; Eremothecium; Issatchenkia; Kazachstania; Kluyveromyces; Kodamaea; Lodderomyces; Pachysolen; Pichia; Saccharomyces; Saturnispora; Tetrapisispora; Torulaspora; Williopsis; and Zygosaccharomyces.
• Other types of yeast potentially useful in the invention include Yarrowia; Rhodosporidium; Candida; Hansenula; Filobasidium; Sporidiobol s; Bullera; Le cosporidium and Filobasidiella.
• the mating competent yeast is a member of the genus Pichia.
• the mating competent yeast of the genus Pichia is one of the following species: Pichia pastoris, Pichia methanolica, and Hansenula polymorpha (Pichia angusta).
• the mating competent yeast of the genus Pichia is the species Pichia pastoris.
• Haploid Yeast Cell A cell having a single copy of each gene of its normal genomic (chromosomal) complement.
• Polyploid Yeast Cell A cell having more than one copy of its normal genomic (chromosomal) complement.
• Diploid Yeast Cell A cell having two copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two haploid cells.
• Tetraploid Yeast Cell A cell having four copies (alleles) of essentially every gene of its normal genomic complement, typically formed by the process of fusion (mating) of two haploid cells. Tetraploids may carry two, three, four or more different expression cassettes. Such tetraploids might be obtained in S. cerevisiae by selective mating homozygotic heterothallic a/a and alpha alpha diploids and in Pichia by sequential mating of haploids to obtain auxotrophic diploids.
• a [met his] haploid can be mated with [ade his] haploid to obtain diploid [his]; and a [met arg] haploid can be mated with [ade arg] haploid to obtain diploid [arg]; then the diploid [his] x diploid [arg] to obtain a tetraploid prototroph. It will be understood by those of skill in the art that reference to the benefits and uses of diploid cells may also apply to tetraploid cells.
• Yeast Mating The process by which two haploid yeast cells naturally fuse to form one diploid yeast cell.
• Meiosis The process by which a diploid yeast cell undergoes reductive division to form four haploid spore products. Each spore may then germinate and form a haploid vegetatively growing cell line.
• Selectable Marker is a gene or gene fragment that confers a growth phenotype (physical growth characteristic) on a cell receiving that gene as, for example through a transformation event.
• the selectable marker allows that cell to survive and grow in a selective growth medium under conditions in which cells that do not receive that selectable marker gene cannot grow.
• Selectable marker genes generally fall into several types, including positive selectable marker genes such as a gene that confers on a cell resistance to an antibiotic or other drug, temperature when two temperature sensitive ("ts") mutants are crossed or a ts mutant is transformed; negative selectable marker genes such as a biosynthetic gene that confers on a cell the ability to grow in a medium without a specific nutrient needed by all cells that do not have that biosynthetic gene, or a mutagenized biosynthetic gene that confers on a cell inability to grow by cells that do not have the wild type gene; and the like. Suitable markers include but are not limited to: ZEO; G418; LYS3; MET1 ; MET3a; ADE1 ; ADE3; URA3; and the like.
• Expression Vector These DNA vectors contain elements that facilitate manipulation for the expression of a foreign protein within the target host cell. Conveniently, manipulation of sequences and production of DNA for transformation is first performed in a bacterial host, e.g. E. coli, and usually vectors will include sequences to facilitate such manipulations, including a bacterial origin of replication and appropriate bacterial selection marker. Selection markers encode proteins necessary for the survival or growth of transformed host cells grown in a selective culture medium. Host cells not transformed with the vector containing the selection gene will not survive in the culture medium. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media.
• Expression vectors for use in the methods of the invention will further include yeast specific sequences, including a selectable auxotrophic or drug marker for identifying transformed yeast strains.
• a drug marker may further be used to amplify copy number of the vector in a yeast host cell.
• the polypeptide coding sequence of interest is operably linked to transcriptional and translational regulatory sequences that provide for expression of the polypeptide in yeast cells.
• These vector components may include, but are not limited to, one or more of the following: an enhancer element, a promoter, and a transcription termination sequence. Sequences for the secretion of the polypeptide may also be included, e.g. a signal sequence, and the like.
• a yeast origin of replication is optional, as expression vectors are often integrated into the yeast genome.
• the polypeptide of interest is operably linked, or fused, to sequences providing for optimized secretion of the polypeptide from yeast diploid cells.
• Nucleic acids are "operably linked" when placed into a functional relationship with another nucleic acid sequence.
• DNA for a signal sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
• "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers do not have to be contiguous.
• Linking is accomplished by ligation at convenient restriction sites or alternatively via a PCR/recombination method familiar to those skilled in the art (Gateway® Technology; Invitrogen, Carlsbad California). If such sites do not exist, the synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.
• Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription and translation of particular nucleic acid sequences to which they are operably linked. Such promoters fall into several classes: inducible, constitutive, and repressible promoters (that increase levels of transcription in response to absence of a repressor). Inducible promoters may initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, e.g. , the presence or absence of a nutrient or a change in temperature.
• the yeast promoter fragment may also serve as the site for homologous recombination and integration of the expression vector into the same site in the yeast genome; alternatively a selectable marker is used as the site for homologous recombination. Pichia transformation is described in Cregg et al. Mol. Cell. Biol. 5:3376- 3385, 1985.
• Suitable promoters from Pichia include the AOX1 and promoter (Cregg et al. Mol. Cell. Biol. 9:1316-1323, (1989)); ICL1 promoter (Menendez et al. Yeast 20(13): 1097-108, (2003)); glyceraldehyde-3 -phosphate dehydrogenase promoter (GAP) (Waterham et al. Gene 186(l):37-44 (1997)); and FLD1 promoter (Shen et al. Gene 216(1):93-102 (1998)).
• the GAP promoter is a strong constitutive promoter and the AOX and FLD1 promoters are inducible.
• yeast promoters include ADH1 , alcohol dehydrogenase II, GAL4, PH03, PH05, Pyk, and chimeric promoters derived therefrom.
• non-yeast promoters may be used in the invention such as mammalian, insect, plant, reptile, amphibian, viral, and avian promoters. Most typically the promoter will comprise a mammalian promoter (potentially endogenous to the expressed genes) or will comprise a yeast or viral promoter that provides for efficient transcription in yeast systems.
• the polypeptides of interest may be recombinantly produced not only directly, but also as a fusion polypeptide with a heterologous polypeptide, e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
• a heterologous polypeptide e.g. a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide.
• the signal sequence may be a component of the vector, or it may be a part of the polypeptide coding sequence that is inserted into the vector.
• the heterologous signal sequence selected preferably is one that is recognized and processed through one of the standard pathways available within the host cell.
• the S. cerevisiae alpha factor pre-pro signal has proven effective in the secretion of a variety of recombinant proteins from P. pastoris.
• yeast signal sequences include the alpha mating factor signal sequence, the invertase signal sequence, and signal sequences derived from other secreted yeast polypeptides. Additionally, these signal peptide sequences may be engineered to provide for enhanced secretion in diploid yeast expression systems. Other secretion signals of interest also include mammalian signal sequences, which may be heterologous to the protein being secreted, or may be a native sequence for the protein being secreted. Signal sequences include pre-peptide sequences, and in some instances may include propeptide sequences.
• signal sequences are known in the art, including the signal sequences found on immunoglobulin chains, e.g., 28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chain, human Ig light chain, and the like.
• signal sequences found on immunoglobulin chains e.g., 28 preprotoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequences, human Ig heavy chain, human Ig light chain, and the like.
• Transcription may be increased by inserting a transcriptional activator sequence into the vector.
• These activators are cis-acting elements of DNA, usually about from 10 to 300 bp, which act on a promoter to increase its transcription.
• Transcriptional enhancers are relatively orientation and position independent, having been found 5' and 3' to the transcription unit, within an intron, as well as within the coding sequence itself. The enhancer may be spliced into the expression vector at a position 5' or 3' to the coding sequence, but is preferably located at a site 5' from the promoter.
• Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from 3' to the translation termination codon, in untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA.
• Plasmids from the transformants are prepared, analyzed by restriction endonuclease digestion and/or sequenced.
• recombination methods based on att sites and recombination enzymes may be used to insert DNA sequences into a vector. Such methods are described, for example, by Landy Ann. Rev. Biochem. 55:913-949 (1989); and are known to those of skill in the art. Such methods utilize intermolecular DNA recombination that is mediated by a mixture of lambda and E. coli -encoded recombination proteins. Recombination occurs between specific attachment (att) sites on the interacting DNA molecules.
• Att sites may be introduced into a sequence of interest by ligating the sequence of interest into an appropriate vector; generating a PCR product containing att B sites through the use of specific primers; generating a cDNA library cloned into an appropriate vector containing att sites; and the like.
• Folding refers to the three-dimensional structure of polypeptides and proteins, where interactions between amino acid residues act to stabilize the structure. While non-covalent interactions are important in determining structure, usually the proteins of interest will have intra- and/or intermolecular covalent disulfide bonds formed by two cysteine residues. For naturally occurring proteins and polypeptides or derivatives and variants thereof, the proper folding is typically the arrangement that results in optimal biological activity, and can conveniently be monitored by assays for activity, e.g. ligand binding, enzymatic activity, etc.
• the expression host may be further modified by the introduction of sequences encoding one or more enzymes that enhance folding and disulfide bond formation, i.e. foldases, chaperonins, etc.
• sequences may be constitutively or inducibly expressed in the yeast host cell, using vectors, markers, etc. as known in the art.
• sequences, including transcriptional regulatory elements sufficient for the desired pattern of expression are stably integrated in the yeast genome through a targeted methodology.
• the eukaryotic PDI is not only an efficient catalyst of protein cysteine oxidation and disulfide bond isomerization, but also exhibits chaperone activity. Co-expression of PDI can facilitate the production of active proteins having multiple disulfide bonds. Also of interest is the expression of BIP (immunoglobulin heavy chain binding protein); cyclophilin; and the like.
• BIP immunoglobulin heavy chain binding protein
• cyclophilin cyclophilin
• each of the haploid parental strains expresses a distinct folding enzyme, e.g. one strain may express BIP, and the other strain may express PDI or combinations thereof.
• the terms “desired protein” or “desired antibody” are used interchangeably and refer generally to a parent antibody specific to a target, i.e., CGRP or a chimeric or humanized antibody or a binding portion thereof derived therefrom as described herein.
• the term “antibody” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
• the archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g.
• antibodies human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other avians, etc., are considered to be "antibodies.”
• a source for producing antibodies useful as starting material according to the invention is rabbits. Numerous antibody-coding sequences have been described; and others may be raised by methods well-known in the art. Examples thereof include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single chain antibodies (such as scFvs), camelbodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small-modular immunopharmaceuticals (SMIPs), and antibody fragments such as Fab, F(ab') 2 and the like.
• antibodies or antigen binding fragments may be produced by genetic engineering.
• antibody-producing cells are sensitized to the desired antigen or immunogen.
• the messenger RNA isolated from antibody producing cells is used as a template to make cDNA using PCR amplification.
• a library of vectors, each containing one heavy chain gene and one light chain gene retaining the initial antigen specificity, is produced by insertion of appropriate sections of the amplified immunoglobulin cDNA into the expression vectors.
• a combinatorial library is constructed by combining the heavy chain gene library with the light chain gene library. This results in a library of clones, which co-express a heavy and light chain (resembling the Fab fragment or antigen binding fragment of an antibody molecule).
• the vectors that carry these genes are co-transfected into a host cell. When antibody gene synthesis is induced in the transfected host, the heavy and light chain proteins self- assemble to produce active antibodies that can be detected by screening with the antigen or immunogen.
• Antibody coding sequences of interest include those encoded by native sequences, as well as nucleic acids that, by virtue of the degeneracy of the genetic code, are not identical in sequence to the disclosed nucleic acids, and variants thereof.
• Variant polypeptides can include amino acid (“aa”) substitutions, additions or deletions. The amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acids, such as to alter a glycosylation site, or to minimize misfolding by substitution or deletion of one or more cysteine residues that are not necessary for function.
• Variants can be designed so as to retain or have enhanced biological activity of a particular region of the protein (e.g. , a functional domain, catalytic amino acid residues, etc).
• Variants also include fragments of the polypeptides disclosed herein, particularly biologically active fragments and/or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. Also included in the subject invention are polypeptides that have been modified using ordinary molecular biological techniques so as to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent.
• Chimeric antibodies may be made by recombinant means by combining the variable light and heavy chain regions (VL and VH), obtained from antibody producing cells of one species with the constant light and heavy chain regions from another.
• VL and VH variable light and heavy chain regions
• chimeric antibodies utilize rodent or rabbit variable regions and human constant regions, in order to produce an antibody with predominantly human domains.
• the production of such chimeric antibodies is well known in the art, and may be achieved by standard means (as described, e.g., in U.S. Patent No. 5,624,659, incorporated herein by reference in its entirety).
• the human constant regions of chimeric antibodies of the invention may be selected from IgGl , IgG2, IgG3, or IgG4 constant regions.
• Humanized antibodies are engineered to contain even more human-like immunoglobulin domains, and incorporate only the complementarity-determining regions of the animal-derived antibody. This is accomplished by examination of the sequence of the hyper-variable loops of the variable regions of the monoclonal antibody to fit them to the structure of the human antibody chains. Although facially complex, the process is straightforward in practice. See, e.g., U.S. Patent No. 6,187,287, incorporated fully herein by reference.
• immunoglobulin fragments comprising the epitope binding site (e.g., Fab, F(ab') 2 , or other fragments) may be synthesized.
• "Fragment,” or minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
• Fv immunoglobulins for use in the present invention may be produced by synthesizing a fused variable light chain region and a variable heavy chain region. Combinations of antibodies are also of interest, e.g. diabodies, which comprise two distinct Fv specificities.
• SMIPs small molecule immunopharmaceuticals
• camelbodies camelbodies
• nanobodies and IgNAR are encompassed by immunoglobulin fragments.
• Immunoglobulins and fragments thereof may be modified post-translationally, e.g. to add effector moieties such as chemical linkers, detectable moieties, such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent materials, radioactive materials, chemiluminescent moieties and the like, or specific binding moieties, such as streptavidin, avidin, or biotin, and the like may be utilized in the methods and compositions of the present invention. Examples of additional effector molecules are provided infra.
• a "heterologous" region or domain of a DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
• the heterologous region encodes a mammalian gene
• the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
• Another example of a heterologous region is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene). Allelic variations or naturally occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
• a "coding sequence” is an in-frame sequence of codons that (in view of the genetic code) correspond to or encode a protein or peptide sequence. Two coding sequences correspond to each other if the sequences or their complementary sequences encode the same amino acid sequences. A coding sequence in association with appropriate regulatory sequences may be transcribed and translated into a polypeptide. A polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
• a “promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for binding of regulatory molecules (e.g., transcription factors) which affect the transcription of the coding sequence.
• a coding sequence is "under the control" of the promoter sequence or "operatively linked” to the promoter when RNA polymerase binds the promoter sequence in a cell and transcribes the coding sequence into mRNA, which is then in turn translated into the protein encoded by the coding sequence.
• Vectors are used to introduce a foreign substance, such as DNA, RNA or protein, into an organism or host cell.
• Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for polypeptides).
• a "DNA vector” is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment.
• An "expression vector” is a DNA vector which contains regulatory sequences which will direct polypeptide synthesis by an appropriate host cell.
• Amplification of polynucleotide sequences is the in vitro production of multiple copies of a particular nucleic acid sequence.
• the amplified sequence is usually in the form of DNA.
• a variety of techniques for carrying out such amplification are described in a review article by Van Brunt (Bio/Technol., 8(4):291-294 (1990)).
• Polymerase chain reaction or PCR is a prototype of nucleic acid amplification, and use of PCR herein should be considered exemplary of other suitable amplification techniques.
• Antibodies consist of two identical light polypeptide chains of molecular weight approximately 23,000 Daltons (the "light chain”), and two identical heavy chains of molecular weight 53,000-70,000 (the “heavy chain”).
• the four chains are joined by disulfide bonds in a "Y" configuration wherein the light chains bracket the heavy chains starting at the mouth of the "Y” configuration.
• the "branch” portion of the "Y” configuration is designated the Fab region; the stem portion of the "Y” configuration is designated the Fc region.
• the amino acid sequence orientation runs from the N-terminal end at the top of the "Y" configuration to the C-terminal end at the bottom of each chain.
• the N-terminal end possesses the variable region having specificity for the antigen that elicited it, and is approximately 100 amino acids in length, there being slight variations between light and heavy chain and from antibody to antibody.
• variable region is linked in each chain to a constant region that extends the remaining length of the chain and that within a particular class of antibody does not vary with the specificity of the antibody (i.e., the antigen eliciting it).
• constant regions There are five known major classes of constant regions that determine the class of the immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE corresponding to ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions).
• the constant region or class determines subsequent effector function of the antibody, including activation of complement (Kabat, E.
• Light chains are classified as either ⁇ (kappa) or ⁇ (lambda). Each heavy chain class can be prepared with either kappa or lambda light chain. The light and heavy chains are covalently bonded to each other, and the "tail" portions of the two heavy chains are bonded to each other by covalent disulfide linkages when the immunoglobulins are generated either by hybridomas or by B cells.
• variable region refers to the domains within each pair of light and heavy chains in an antibody that are involved directly in binding the antibody to the antigen.
• Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
• Each light chain has a variable domain (VL) at one end and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
• CDR complementarity determining region
• hypervariable region refers to one or more of the hyper-variable or complementarity determining regions (CDRs) found in the variable regions of light or heavy chains of an antibody (See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include the hypervariable regions as defined by Kabat et al. ("Sequences of Proteins of Immunological Interest,” Kabat E., et al., US Dept. of Health and Human Services, 1983) or the hypervariable loops in 3 -dimensional structures of antibodies (Chothia and Lesk, J Mol.
• the CDRs in each chain are held in close proximity by framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen binding site.
• the CDRs there are select amino acids that have been described as the selectivity determining regions (SDRs) which represent the critical contact residues used by the CDR in the antibody-antigen interaction (Kashmiri, S., Methods, 36:25-34 (2005)).
• framework region refers to one or more of the framework regions within the variable regions of the light and heavy chains of an antibody ⁇ See Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These expressions include those amino acid sequence regions interposed between the CDRs within the variable regions of the light and heavy chains of an antibody.
• Exemplary embodiments of the present methods comprise administering anti- CGRP antibodies and fragments thereof to subject.
• Exemplary anti-CGRP antibodies and fragments are described in U.S. patent publication no. 2012/0294797, which is hereby incorporated by reference in its entirety, and additional exemplary anti-CGRP antibodies as described in the paragraphs that follow.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCTVSGLDLSSYYMQWVRQAPGKGLEWIGVIGINDN TYYASWA GRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
• the invention also includes chimeric antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2, and/or one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 3 or SEQ ID NO: 4.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 or the light chain sequence of SEQ ID NO: 2.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 3 or the heavy chain sequence of SEQ ID NO: 4.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 1 ; the variable heavy chain region of SEQ ID NO: 3; the complementarity-determining regions (SEQ ID NO: 5; SEQ ID NO: 6; and SEQ ID NO: 7) of the variable light chain region of SEQ ID NO: 1 ; and the complementarity-determining regions (SEQ ID NO: 8; SEQ ID NO: 9; and SEQ ID NO: 10) of the variable heavy chain region of SEQ ID NO: 3.
• the chimeric anti- CGRP antibody is Abl, comprising, or alternatively consisting of, SEQ ID NO: 2 and SEQ ID NO: 4, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 1 and the variable heavy chain sequence of SEQ ID NO: 3.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 1 and/or SEQ ID NO: 3 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl .
• anti-CGRP antibodies such as Abl or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to CGRP and possessing a light chain sequence comprising the sequence set forth below: QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTST LASGVPSRFSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW VDNALQSGNSQ ESVTEQDSKDSTYSLSSTLTLS ADYE HKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 12).
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• APIEKTIS AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 1 or the light chain sequence of SEQ ID NO: 12, and/or one or more of the polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14, or combinations of these polypeptide sequences.
• CDRs complementarity-determining regions
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• the invention also contemplates fragments of the antibody having binding specificity to CGRP.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 1 1 or SEQ ID NO: 12.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 13 or SEQ ID NO: 14.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 1 or the light chain sequence of SEQ ID NO: 12.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 13 or the heavy chain sequence of SEQ ID NO: 14.
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 1 1 ; the variable heavy chain region of SEQ ID NO: 13; the complementarity-determining regions (SEQ ID NO: 15; SEQ ID NO: 16; and SEQ ID NO: 17) of the variable light chain region of SEQ ID NO: 1 1 ; and the complementarity-determining regions (SEQ ID NO: 18; SEQ ID NO: 19; and SEQ ID NO: 20) of the variable heavy chain region of SEQ ID NO: 13.
• the humanized anti- CGRP antibody is Ab2, comprising, or alternatively consisting of, SEQ ID NO: 12 and SEQ ID NO: 14, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 1 1 and the variable heavy chain sequence of SEQ ID NO: 13.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 1 1 and/or SEQ ID NO: 13 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab2.
• anti-CGRP antibodies such as Ab2 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HE 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGI NDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTL VTVSS (SEQ ID NO: 23).
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22, and/or one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• the invention also contemplates fragments of the antibody having binding specificity to CGRP.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 21 or SEQ ID NO: 22.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 23 or SEQ ID NO: 24.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 21 or the light chain sequence of SEQ ID NO: 22.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 23 or the heavy chain sequence of SEQ ID NO: 24.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 21 ; the variable heavy chain region of SEQ ID NO: 23; the complementarity-determining regions (SEQ ID NO: 25; SEQ ID NO: 26; and SEQ ID NO: 27) of the variable light chain region of SEQ ID NO: 21 ; and the complementarity-determining regions (SEQ ID NO: 28; SEQ ID NO: 29; and SEQ ID NO: 30) of the variable heavy chain region of SEQ ID NO: 23.
• the chimeric anti-CGRP antibody is Ab3, comprising, or alternatively consisting of, SEQ ID NO: 22 and SEQ ID NO: 24, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 21 and the variable heavy chain sequence of SEQ ID NO: 23.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 21 and/or SEQ ID NO: 23 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab3.
• anti-CGRP antibodies such as Ab3 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGA TYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS
• the invention also includes chimeric antibodies having binding specificity to CGRP and possessing a heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGA
• GK (SEQ ID NO: 34).
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32, and/or one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 31 or SEQ ID NO: 32. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 33 or SEQ ID NO: 34.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 31 or the light chain sequence of SEQ ID NO: 32.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 33 or the heavy chain sequence of SEQ ID NO: 34.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 31 ; the variable heavy chain region of SEQ ID NO: 33; the complementarity-determining regions (SEQ ID NO: 35; SEQ ID NO: 36; and SEQ ID NO: 37) of the variable light chain region of SEQ ID NO: 31 ; and the complementarity-determining regions (SEQ ID NO: 38; SEQ ID NO: 39; and SEQ ID NO: 40) of the variable heavy chain region of SEQ ID NO: 33.
• the humanized anti-CGRP antibody is Ab4, comprising, or alternatively consisting of, SEQ ID NO: 32 and SEQ ID NO: 34, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 31 and the variable heavy chain sequence of SEQ ID NO: 33.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 31 and/or SEQ ID NO: 33 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab4.
• anti-CGRP antibodies such as Ab4 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42, and/or one or more of the polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 41 or SEQ ID NO: 42. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 43 or SEQ ID NO: 44.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 41 or the light chain sequence of SEQ ID NO: 42.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 43 or the heavy chain sequence of SEQ ID NO: 44.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 41 ; the variable heavy chain region of SEQ ID NO: 43; the complementarity-determining regions (SEQ ID NO: 45; SEQ ID NO: 46; and SEQ ID NO: 47) of the variable light chain region of SEQ ID NO: 41 ; and the complementarity-determining regions (SEQ ID NO: 48; SEQ ID NO: 49; and SEQ ID NO: 50) of the variable heavy chain region of SEQ ID NO: 43.
• the chimeric anti-CGRP antibody is Ab5, comprising, or alternatively consisting of, SEQ ID NO: 42 and SEQ ID NO: 44, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 41 and the variable heavy chain sequence of SEQ ID NO: 43.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 41 and/or SEQ ID NO: 43 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab5.
• anti-CGRP antibodies such as Ab5 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52, and/or one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 51 or SEQ ID NO: 52. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 53 or SEQ ID NO: 54.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 51 or the light chain sequence of SEQ ID NO: 52.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 53 or the heavy chain sequence of SEQ ID NO: 54.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 51 ; the variable heavy chain region of SEQ ID NO: 53; the complementarity-determining regions (SEQ ID NO: 55; SEQ ID NO: 56; and SEQ ID NO: 57) of the variable light chain region of SEQ ID NO: 51 ; and the complementarity-determining regions (SEQ ID NO: 58; SEQ ID NO: 59; and SEQ ID NO: 60) of the variable heavy chain region of SEQ ID NO: 53.
• the humanized anti- CGRP antibody is Ab6, comprising, or alternatively consisting of, SEQ ID NO: 52 and SEQ ID NO: 54, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 51 and the variable heavy chain sequence of SEQ ID NO: 53.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 51 and/or SEQ ID NO: 53 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab6.
• anti-CGRP antibodies such as Ab6 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to CGRP and possessing a light chain sequence comprising the sequence set forth below: QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQPPKQLIYSTST LASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEV VVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW VDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QEQLKESGGRLVTPGTSLTLTCTVSGIDLSNHYMQWVRQAPGKGLEWIGVVGIN GRTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTV SS (SEQ ID NO: 63).
• the invention also includes chimeric antibodies having binding specificity to
• SLSPGK (SEQ ID NO: 64).
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62, and/or one or more of the polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 61 or SEQ ID NO: 62. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 63 or SEQ ID NO: 64.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 61 or the light chain sequence of SEQ ID NO: 62.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 63 or the heavy chain sequence of SEQ ID NO: 64.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 61 ; the variable heavy chain region of SEQ ID NO: 63; the complementarity-determining regions (SEQ ID NO: 65; SEQ ID NO: 66; and SEQ ID NO: 67) of the variable light chain region of SEQ ID NO: 61 ; and the complementarity-determining regions (SEQ ID NO: 68; SEQ ID NO: 69; and SEQ ID NO: 70) of the variable heavy chain region of SEQ ID NO: 63.
• the chimeric anti-CGRP antibody is Ab7, comprising, or alternatively consisting of, SEQ ID NO: 62 and SEQ ID NO: 64, and having at least one of the biological activities set
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 61 and the variable heavy chain sequence of SEQ ID NO: 63.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 61 and/or SEQ ID NO: 63 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab7.
• anti-CGRP antibodies such as Ab7 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72, and/or one or more of the polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 71 or SEQ ID NO: 72. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 73 or SEQ ID NO: 74.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 71 or the light chain sequence of SEQ ID NO: 72.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 73 or the heavy chain sequence of SEQ ID NO: 74.
• CDRs complementarity-determining regions
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 71 ; the variable heavy chain region of SEQ ID NO: 73; the complementarity-determining regions (SEQ ID NO: 75; SEQ ID NO: 76; and SEQ ID NO: 77) of the variable light chain region of SEQ ID NO: 71 ; and the complementarity-determining regions (SEQ ID NO: 78; SEQ ID NO: 79; and SEQ ID NO: 80) of the variable heavy chain region of SEQ ID NO: 73.
• the humanized anti-CGRP antibody is Ab8, comprising, or alternatively consisting of, SEQ ID NO: 72 and SEQ ID NO: 74, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 71 and the variable heavy chain sequence of SEQ ID NO: 73.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 71 and/or SEQ ID NO: 73 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab8.
• anti-CGRP antibodies such as Ab8 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeas strains.
• Suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGTPLTLTCTVSGIGLSSYYMQWVRQSPGRGLEWIGVIGSDGK TYYATWAKGRFTI SKTS STTVDLRMASLTTEDTATYFCTRGDI WGPGTLVTVS S
• the invention also includes chimeric antibodies having binding specificity to
• GK (SEQ ID NO: 84).
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82, and/or one or more of the polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 83 or SEQ ID NO: 84.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 81 or the light chain sequence of SEQ ID NO: 82.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 83 or the heavy chain sequence of SEQ ID NO: 84.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 81 ; the variable heavy chain region of SEQ ID NO: 83; the complementarity-determining regions (SEQ ID NO: 85; SEQ ID NO: 86; and SEQ ID NO: 87) of the variable light chain region of SEQ ID NO: 81 ; and the complementarity-determining regions (SEQ ID NO: 88; SEQ ID NO: 89; and SEQ ID NO: 90) of the variable heavy chain region of SEQ ID NO: 83.
• the chimeric anti-CGRP antibody is Ab9, comprising, or alternatively consisting of, SEQ ID NO: 82 and SEQ ID NO: 84, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 81 and the variable heavy chain sequence of SEQ ID NO: 83.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 81 and/or SEQ ID NO: 83 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Ab9.
• anti-CGRP antibodies such as Ab9 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92, and/or one or more of the polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 whic h correspond to the complementarity- determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 91 or SEQ ID NO: 92. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 93 or SEQ ID NO: 94.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 91 or the light chain sequence of SEQ ID NO: 92.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 93 or the heavy chain sequence of SEQ ID NO: 94.
• CDRs complementarity-determining regions
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 91 ; the variable heavy chain region of SEQ ID NO: 93; the complementarity-determining regions (SEQ ID NO: 95; SEQ ID NO: 96; and SEQ ID NO: 97) of the variable light chain region of SEQ ID NO: 91 ; and the complementarity-determining regions (SEQ ID NO: 98; SEQ ID NO: 99; and SEQ ID NO: 100) of the variable heavy chain region of SEQ ID NO: 93.
• the humanized anti-CGRP antibody is AblO, comprising, or alternatively consisting of, SEQ ID NO: 92 and SEQ ID NO: 94, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 91 and the variable heavy chain sequence of SEQ ID NO: 93.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 91 and/or SEQ ID NO: 93 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of AblO.
• anti-CGRP antibodies such as AblO or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QSLEESGGRLVTPGGSLTLTCTVSGIDVTNYYMQWVRQAPGKGLEWIGVIGVNG KRYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVS
• the invention also includes chimeric antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102, and/or one or more of the polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 110 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 101 or SEQ ID NO: 102. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 103 or SEQ ID NO: 104.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 101 or the light chain sequence of SEQ ID NO: 102.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 1 10 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 103 or the heavy chain sequence of SEQ ID NO: 104.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 101 ; the variable heavy chain region of SEQ ID NO: 103; the complementarity-determining regions (SEQ ID NO: 105; SEQ ID NO: 106; and SEQ ID NO: 107) of the variable light chain region of SEQ ID NO: 101 ; and the complementarity-determining regions (SEQ ID NO: 108; SEQ ID NO: 109; and SEQ ID NO: 1 10) of the variable heavy chain region of SEQ ID NO: 103.
• the chimeric anti-CGRP antibody is Abl 1, comprising, or alternatively consisting of, SEQ ID NO: 102 and SEQ ID NO: 104, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 101 and the variable heavy chain sequence of SEQ ID NO: 103.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 101 and/or SEQ ID NO: 103 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of AM I .
• anti-CGRP antibodies such as Abl 1 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 115; SEQ ID NO: 1 16; and SEQ ID NO: 117 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 111 or the light chain sequence of SEQ ID NO: 1 12, and/or one or more of the polypeptide sequences of SEQ ID NO: 1 18; SEQ ID NO: 1 19; and SEQ ID NO: 120 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 1 13 or the heavy chain sequence of SEQ ID NO: 1 14, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 1 11 or SEQ ID NO: 1 12. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 1 13 or SEQ ID NO: 114.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 1 15; SEQ ID NO: 1 16; and SEQ ID NO: 1 17 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 1 1 1 or the light chain sequence of SEQ ID NO: 112.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 113 or the heavy chain sequence of SEQ ID NO: 114.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 1 1 1 ; the variable heavy chain region of SEQ ID NO: 113; the complementarity-determining regions (SEQ ID NO: 1 15; SEQ ID NO: 1 16; and SEQ ID NO: 117) of the variable light chain region of SEQ ID NO: 11 1 ; and the complementarity-determining regions (SEQ ID NO: 118; SEQ ID NO: 119; and SEQ ID NO: 120) of the variable heavy chain region of SEQ ID NO: 113.
• the humanized anti-CGRP antibody is Abl2, comprising, or alternatively consisting of, SEQ ID NO: 1 12 and SEQ ID NO: 1 14, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 1 1 1 and the variable heavy chain sequence of SEQ ID NO: 1 13.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 111 and/or SEQ ID NO: 113 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl2.
• anti-CGRP antibodies such as Abl2 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes chimeric antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes chimeric antibodies having binding specificity to CGRP and possessing a light chain sequence comprising the sequence set forth below: AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQPPKRLIYDASK LASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEV VVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRGE
• the invention further includes chimeric antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below: QSVEESGGGLVQPEGSLTLTCTASGFDFSSNAMWWVRQAPG GLEWIGIIYNGD GSTYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVT VSS (SEQ ID NO: 123).
• the invention also includes chimeric antibodies having binding specificity to
• VDGVEVHNAKT PREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
• KSLSLSPGK (SEQ ID NO: 124).
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122, and/or one or more of the polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124, or combinations of these polypeptide sequences.
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 121 or SEQ ID NO: 122. In another embodiment of the invention, antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 123 or SEQ ID NO: 124.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 121 or the light chain sequence of SEQ ID NO: 122.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 123 or the heavy chain sequence of SEQ ID NO: 124.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 121 ; the variable heavy chain region of SEQ ID NO: 123; the complementarity-determining regions (SEQ ID NO: 125; SEQ ID NO: 126; and SEQ ID NO: 127) of the variable light chain region of SEQ ID NO: 121 ; and the complementarity-determining regions (SEQ ID NO: 128; SEQ ID NO: 129; and SEQ ID NO: 130) of the variable heavy chain region of SEQ ID NO: 123.
• the chimeric anti-CGRP antibody is Abl3, comprising, or alternatively consisting of, SEQ ID NO: 122 and SEQ ID NO: 124, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 121 and the variable heavy chain sequence of SEQ ID NO: 123.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 121 and/or SEQ ID NO: 123 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl3.
• anti-CGRP antibodies such as Abl 3 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichia) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• the invention includes humanized antibodies having binding specificity to CGRP and possessing a variable light chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• the invention further includes humanized antibodies having binding specificity to CGRP and possessing a variable heavy chain sequence comprising the sequence set forth below:
• the invention also includes humanized antibodies having binding specificity to
• APIEKTIS A GQPREPQVYTLPPSREEMT NQVSLTCLVKGFYPSDIAVEWESN
• the invention further contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132, and/or one or more of the polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134, or combinations of these polypeptide sequences.
• CDRs complementarity-determining regions
• the antibodies of the invention or fragments thereof comprise, or alternatively consist of, combinations of one or more of the CDRs, the variable heavy and variable light chain sequences, and the heavy and light chain sequences set forth above, including all of them.
• the invention also contemplates fragments of the antibody having binding specificity to CGRP.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 131 or SEQ ID NO: 132.
• antibody fragments of the invention comprise, or alternatively consist of, the polypeptide sequence of SEQ ID NO: 133 or SEQ ID NO: 134.
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable light chain sequence of SEQ ID NO: 131 or the light chain sequence of SEQ ID NO: 132.
• CDRs complementarity-determining regions
• fragments of the antibody having binding specificity to CGRP comprise, or alternatively consist of, one or more of the polypeptide sequences of SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140 which correspond to the complementarity-determining regions (CDRs, or hypervariable regions) of the variable heavy chain sequence of SEQ ID NO: 133 or the heavy chain sequence of SEQ ID NO: 134.
• CDRs complementarity-determining regions
• the invention also contemplates antibody fragments which include one or more of the antibody fragments described herein.
• fragments of the antibodies having binding specificity to CGRP comprise, or alternatively consist of, one, two, three or more, including all of the following antibody fragments: the variable light chain region of SEQ ID NO: 131 ; the variable heavy chain region of SEQ ID NO: 133; the complementarity-determining regions (SEQ ID NO: 135; SEQ ID NO: 136; and SEQ ID NO: 137) of the variable light chain region of SEQ ID NO: 131 ; and the complementarity-determining regions (SEQ ID NO: 138; SEQ ID NO: 139; and SEQ ID NO: 140) of the variable heavy chain region of SEQ ID NO: 133.
• the humanized anti-CGRP antibody is Abl4, comprising, or alternatively consisting of, SEQ ID NO: 132 and SEQ ID NO: 134, and having at least one of the biological activities set forth herein.
• antibody fragments comprise, or alternatively consist of, Fab (fragment antigen binding) fragments having binding specificity for CGRP.
• the Fab fragment includes the variable light chain sequence of SEQ ID NO: 131 and the variable heavy chain sequence of SEQ ID NO: 133.
• This embodiment of the invention further contemplates additions, deletions, and variants of SEQ ID NO: 131 and/or SEQ ID NO: 133 in said Fab while retaining binding specificity for CGRP.
• Fab fragments may be produced by enzymatic digestion (e.g., papain) of Abl4.
• anti-CGRP antibodies such as AM 4 or Fab fragments thereof may be produced via expression in mammalian cells such as CHO, NSO or HEK 293 cells, fungal, insect, or microbial systems such as yeast cells (for example diploid yeast such as diploid Pichi ) and other yeast strains.
• suitable Pichia species include, but are not limited to, Pichia pastoris.
• antibody fragments may be present in one or more of the following non-limiting forms: Fab, Fab', F(ab') 2 , Fv and single chain Fv antibody forms.
• the anti-CGRP antibodies described herein further comprises the kappa constant light chain sequence comprising the sequence set forth below:
• VAAPSVFIFPPSDEQL SGTASVVCLLN FYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKH VYACEVTHQGLSSPVTKSFNRG EC SEQ ID NO: 283.
• the anti-CGRP antibodies described herein further comprises the gamma- 1 constant heavy chain polypeptide sequence comprising the sequence set forth below:
• PAPIEKTIS AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ ID NO: 284).
• the invention contemplates an isolated anti-CGRP antibody comprising a VH polypeptide sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133, or a variant thereof; and further comprising a VL polypeptide sequence selected from: SEQ ID NO: 1, 11, 21, 31 , 41 , 51, 61, 71 , 81 , 91, 101 , 1 11, 121, or 131 , or a variant thereof, wherein one or more of the framework residues (FR residues) in said VH or L polypeptide has been substituted with another amino acid residue resulting in an anti-CGRP antibody that specifically binds CGRP.
• VH polypeptide sequence selected from: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, or 133, or a variant thereof
• VL polypeptide sequence selected from: SEQ ID
• the invention contemplates humanized and chimeric forms of these antibodies.
• the chimeric antibodies may include an Fc derived from IgGl, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgGlO, IgGl l, IgGl 2, IgG13, IgG14, IgG15, IgG16, IgGl 7, IgGl 8 or IgGl 9 constant regions.
• the antibodies or VH or VL polypeptides originate or are selected from one or more rabbit B cell populations prior to initiation of the humanization process referenced herein.
• the anti-CGRP antibodies and fragments thereof do not have binding specificity for CGRP-R. In a further embodiment of the invention, the anti-CGRP antibodies and fragments thereof inhibit the association of CGRP with CGRP-R. In another embodiment of the invention, the anti-CGRP antibodies and fragments thereof inhibit the association of CGRP with CGRP-R and/or additional proteins and/or multimers thereof, and/or antagonize the biological effects thereof.
• antibodies and fragments thereof may be modified post- translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
• effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
• Antibodies or fragments thereof may also be chemically modified to provide additional advantages such as increased solubility, stability and circulating time (in vivo half-life) of the polypeptide, or decreased immunogenicity (See U.S. Pat. No. 4,179,337).
• the chemical moieties for derivatization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the antibodies and fragments thereof may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 1 1,000, 1 1,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• Branched polyethylene glycols are described, for example, in U.S. Pat. No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 18:2745-2750 (1999); and Caliceti et al, Bioconjug. Chem. 10:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
• Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to polypeptides via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof).
• antibodies or fragments thereof may have increased in vivo half- lives via fusion with albumin (including but not limited to recombinant human serum albumin or fragments or variants thereof (See, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)) or other circulating blood proteins such as transferrin or ferritin.
• albumin including but not limited to recombinant human serum albumin or fragments or variants thereof (See, e.g., U.S. Pat. No. 5,876,969, issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883, issued Jun. 16, 1998, herein incorporated by reference in their entirety)
• other circulating blood proteins such as transferrin or ferritin.
• polypeptides and/or antibodies of the present invention are fused with the mature form of human serum albumin (i.e., amino acids 1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0 322 094), which is herein incorporated by reference in its entirety.
• Polynucleotides encoding fusion proteins of the invention are also encompassed by the invention.
• further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, beta- galactosidase and luciferase.
• Further exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dansyl chloride.
• Further chemiluminescent moieties include, but are not limited to, luminol.
• Further exemplary bioluminescent materials include, but are not limited to, luciferin and aequorin.
• Further exemplary radioactive materials include, but are not limited to, Iodine 125 ( I), Carbon 14 ( 14 C), Sulfur 35 ( 35 S), Tritium ( 3 H) and Phosphorus 32 ( 32 P).
• exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5- fluorouracil dacarbazine; alkylating agents such as mechlorethamine, thiotepa chlorambucil, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1- methylnitrosourea, cyclophosphamide, mechlorethamine, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiammineplatinum (II) (DDP), cisplatin, carboplatin (Paraplatin); anthracyclines include daunorubicin (formerly daunomycin), doxorubicin (Adriamycin), detorubicin, carminomycin, i
• cytotoxic agents include paclitaxel (Taxol), ricin, pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxy anthracin dione, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, mitotane (0,P'-(DDD)), interferons, and mixtures of these cytotoxic agents.
• paclitaxel Texol
• ricin pseudomonas exotoxin
• gemcitabine cytochalasin B
• gramicidin D ethidium bromide
• emetine etoposide
• teniposide colchicine
• cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin.
• chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine and bleomycin.
• Toxic enzymes from plants and bacteria such as ricin, diphtheria toxin and Pseudomonas tox in may be conjugated to the humanized or chimeric antibodies, or binding fragments thereof, to generate cell-type-specific-killing reagents (Youle, et al, Proc.
• cytotoxic agents include cytotoxic ribonucleases as described by Goldenberg in U.S. Pat. No. 6,653,104.
• Embodiments of the invention also relate to radioimmunoconjugates where a radionuclide that emits alpha or beta particles is stably coupled to the antibody, or binding fragments thereof, with or without the use of a complex-forming agent.
• Such radionuclides include beta-emitters such as Phosphorus-32 CP), Scandium-47 ( 4/ Sc), Copper-67 ( G / Cu), Gallium-67 f'Ga), Yttrium-88 ( 88 Y), Yttrium-90 ( 90 Y), Iodine-125 ( 125 I), Iodine-131 ( 131 I), Samarium-153 ( 153 Sm), Lutetium- 177 ( 177 Lu), Rhenium-186 ( 186 Re) or Rhenium-188 ( 188 Re), and alpha-emitters such as Astatine-21 1 ( 21 1 At), Lead-212 ( 212 Pb), Bismuth-212 ( 212 Bi) or -213 ( 213 Bi) or Actinium- 225 ( 225 Ac).
• beta-emitters such as Phosphorus-32 CP), Scandium-47 ( 4/ Sc), Copper-67 ( G / Cu), Gallium-67 f'Ga), Yttrium-
• Embodiments described herein further include variants and equivalents that are substantially homologous to the antibodies, antibody fragments, diabodies, SMIPs, camelbodies, nanobodies, IgNAR, polypeptides, variable regions and CDRs set forth herein.
• These may contain, e.g., conservative substitution mutations, (i.e., the substitution of one or more amino acids by similar amino acids).
• conservative substitution refers to the substitution of an amino acid with another within the same general class, e.g., one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art.
• the invention contemplates polypeptide sequences having at least 90% or greater sequence homology to any one or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein. More preferably, the invention contemplates polypeptide sequences having at least 95% or greater sequence homology, even more preferably at least 98% or greater sequence homology, and still more preferably at least 99% or greater sequence homology to any one or more of the polypeptide sequences of antibody fragments, variable regions and CDRs set forth herein. Methods for determining homology between nucleic acid and amino acid sequences are well known to those of ordinary skill in the art.
• the invention further contemplates the above-recited polypeptide homologs of the antibody fragments, variable regions and CDRs set forth herein further having anti-CGRP activity.
• anti-CGRP activity are set forth herein.
• the invention further contemplates the generation and use of anti-idiotypic antibodies that bind any of the foregoing sequences.
• an anti-idiotypic antibody could be administered to a subject who has received an anti-CGRP antibody to modulate, reduce, or neutralize, the effect of the anti-CGRP antibody.
• Such anti-idiotypic antibodies could also be useful for treatment of an autoimmune disease characterized by the presence of anti-CGRP antibodies.
• a further exemplary use of such anti-idiotypic antibodies is for detection of the anti-CGRP antibodies of the present invention, for example to monitor the levels of the anti-CGRP antibodies present in a subject's blood or other bodily fluids.
• the present invention also contemplates anti-CGRP antibodies comprising any of the polypeptide or polynucleotide sequences described herein substituted for any of the other polynucleotide sequences described herein.
• the present invention contemplates antibodies comprising the combination of any of the variable light chain and variable heavy chain sequences described herein, and further contemplates antibodies resulting from substitution of any of the CDR sequences described herein for any of the other CDR sequences described herein.
• the invention contemplates one or more anti-human CGRP antibodies or antibody fragments thereof which specifically bind to the same linear or conformational epitope(s) and/or competes for binding to the same linear or conformational epitope(s) on an intact human CGRP polypeptide or fragment thereof as an anti-human CGRP antibody selected from Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, AblO, Abl 1, Abl 2, Abl 3, or Abl 4.
• Said one or more anti-human CGRP antibodies or antibody fragments thereof may be non-naturally occurring, such as humanized or chimeric antibodies, non-naturally occurring antibody fragments, antibodies incorporating a tag or label, etc.
• the anti-human CGRP antibody or fragment thereof specifically binds to the same linear or conformational epitope(s) and/or competes for binding to the same linear or conformational epitope(s) on an intact human CGRP polypeptide or a fragment thereof as Ab3, Ab6, AM 3, or Abl4.
• a preferred embodiment of the invention is directed to chimeric or humanized antibodies and fragments thereof (including Fab fragments) having binding specificity for CGRP and inhibiting biological activities mediated by the binding of CGRP to the CGRP receptor.
• the chimeric or humanized anti-CGRP antibodies are selected from Ab3, Ab6, Abl3, or Abl4.
• the anti-human CGRP antibody is an antibody which specifically binds to the same linear or conformational epitopes on an intact CGRP polypeptide or fragment thereof that is (are) specifically bound by Ab3, Ab6, AM 3, or Abl4 as ascertained by epitopic mapping using overlapping linear peptide fragments which span the full length of the native human CGRP polypeptide.
• the invention is also directed to an anti-CGRP antibody that binds with the same CGRP epitope and/or competes with an anti-CGRP antibody for binding to CGRP as an antibody or antibody fragment disclosed herein, including but not limited to an anti- CGRP antibody selected from Abl, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, AblO, A I, AM2, Abl3, or AM4.
• the invention is also directed to an isolated anti-CGRP antibody or antibody fragment comprising one or more of the CDRs contained in the VH polypeptide sequences selected from: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 1 13, 123, or 133, or a variant thereof, and/or one or more of the CDRs contained in the VL polypeptide sequences selected from: 1 , 1 1 , 21, 31, 41, 51, 61, 71 , 81 , 91 , 101, 1 1 1, 121, or 131 , or a variant thereof.
• the one or more anti-human CGRP antibodies discussed above are aglycosylated or if glycosylated contain only mannose residues; that contain an Fc region that has been modified to alter effector function, half- life, proteolysis, and/or glycosylation; are human, humanized, single chain or chimeric; and are a humanized antibody derived from a rabbit (parent) anti-human CGRP antibody.
• the invention further contemplates one or more anti-human CGRP antibodies wherein the framework regions (FRs) in the variable light region and the variable heavy regions of said antibody respectively are human FRs which are unmodified or which have been modified by the substitution of one or more human FR residues in the variable light or heavy chain region with the corresponding FR residues of the parent rabbit antibody, and wherein said human FRs have been derived from human variable heavy and light chain antibody sequences which have been selected from a library of human germline antibody sequences based on their high level of homology to the corresponding rabbit variable heavy or light chain regions relative to other human germline antibody sequences contained in the library.
• the anti-human CGRP antibody or fragment specifically binds to CGRP expressing human cells and/or to circulating soluble CGRP molecules in vivo, including CGRP expressed on or by human cells in a patient with a disease associated with cells that express CGRP.
• the invention further contemplates anti-human CGRP antibodies or fragments directly or indirectly attached to a detectable label or therapeutic agent.
• the invention also contemplates one or more nucleic acid sequences which result in the expression of an anti-human CGRP antibody or antibody fragment as set forth above, including those comprising, or alternatively consisting of, yeast or human preferred codons.
• the invention also contemplates vectors (including plasmids or recombinant viral vectors) comprising said nucleic acid sequence(s).
• the invention also contemplates host cells or recombinant host cells expressing at least one of the antibodies set forth above, including a mammalian, yeast, bacterial, and insect cells.
• the host cell is a yeast cell.
• the yeast cell is a diploidal yeast cell.
• the yeast cell is a Pichia yeast.
• the invention also contemplates a method of treatment comprising administering to a patient with a disease or condition associated with CGRP expressing cells a therapeutically effective amount of at least one anti-human CGRP antibody or fragment described herein.
• the invention also contemplates that the treatment method may involve the administration of two or more anti-CGRP antibodies or fragments thereof and disclosed herein. If more than one antibody is administered to the patient, the multiple antibodies may be administered simultaneously or concurrently, or may be staggered in their administration.
• the anti-CGRP activity of the anti-CGRP antibodies of the present invention, and fragments thereof having binding specificity to CGRP may also be described by their strength of binding or their affinity for CGRP.
• the anti-CGRP antibodies of the present invention bind to CGRP with a dissociation constant (KD) of less than or equal to 5xl0 "7 M, 10 "7 M, 5xl0 "8 M, 10 "8 M, 5xl0 "9 M, 10 "9 M, 5xl0 "10 M, 10 "10 M, 5xl0 "n M, 10 "1 1 M, 5xl0 "12 M, 1 (T 12 M, 5xl(T 13 M, or 10 "13 M.
• KD dissociation constant
• the anti-CGRP antibodies and fragments thereof bind CGRP with a dissociation constant of less than or equal to 10 " 1 1 M, 5x10 "12 M, or 10 "12 M.
• the anti-CGRP antibodies of the present invention, and fragments thereof having binding specificity to CGRP bind to a linear or conformational CGRP epitope.
• the anti-CGRP activity of the anti- CGRP antibodies of the present invention, and fragments thereof having binding specificity to CGRP bind to CGRP with an off-rate of less than or equal to 10 ⁇ 4 5x10 " 5 S “1 , 10 "5 S “1 , 5xl0 “6 S “1 , 10 “6 S “1 , 5xl(T 7 S “1 , or 10 "7 S “1 .
• the anti-CGRP activity of the anti- CGRP antibodies of the present invention, and fragments thereof having binding specificity to CGRP exhibit anti-CGRP activity by preventing, ameliorating or reducing the symptoms of, or alternatively treating, diseases and disorders associated with CGRP.
• diseases and disorders associated with CGRP are set forth herein.
• the anti-CGRP antibodies may be encoded by polynucleotide sequences set forth in the biological sequence listing contained herein, or other encoding polynucleotides as may be readily identified by one of ordinary skill in the art.
• Examples thereof include the polynucleotide of SEQ ID NO: 141 (encoding the polypeptide of SEQ ID NO: 1), the polynucleotide of SEQ ID NO: 142 (encoding the polypeptide of SEQ ID NO: 2), the polynucleotide of SEQ ID NO: 143 (encoding the polypeptide of SEQ ID NO: 3), the polynucleotide of SEQ ID NO: 144 (encoding the polypeptide of SEQ ID NO: 4), the polynucleotide of SEQ ID NO: 151 (encoding the polypeptide of SEQ ID NO: 1 1), the polynucleotide of SEQ ID NO: 152 (encoding the polypeptide of SEQ ID NO: 12) the polynucleotide of SEQ ID NO: 153 (encoding the polypeptide of SEQ ID NO: 13) the polynucleotide of SEQ ID NO: 154 (encoding the polypeptide of SEQ ID NO: 14) the poly
• the present invention contemplates the preparation and isolation of a clonal population of antigen-specific B cells that may be used for isolating at least one CGRP antigen-specific cell, which can be used to produce a monoclonal antibody against CGRP, which is specific to a desired CGRP antigen, or a nucleic acid sequence corresponding to such an antibody.
• Methods of preparing and isolating said clonal population of antigen-specific B cells are taught, for example, in U.S. patent publication no. US 2007/0269868 to Carvalho-Jensen et al., the disclosure of which is herein incorporated by reference in its entirety.
• Methods of preparing and isolating said clonal population of antigen-specific B cells are also taught herein in the examples.
• Methods of "enriching" a cell population by size or density are known in the art. See, e.g., U.S. Patent 5,627,052. These steps can be used in addition to enriching the cell population by antigen-specificity.
• the present invention contemplates methods for humanizing antibody heavy and light chains.
• Methods for humanizing antibody heavy and light chains which may be applied to anti-CGRP antibodies are taught, for example, in U.S. patent application publication no. US 2009/0022659 to Olson et al, and in U.S. patent no. 7,935,340 to Garcia-Martinez et al, the disclosures of each of which are herein incorporated by reference in their entireties.
• the invention also includes screening assays designed to assist in the identification of diseases and disorders associated with CGRP in patients exhibiting symptoms of a CGRP associated disease or disorder.
• the present invention includes assays that detect insulin insensitivity (resistance) or glucose utilization in a subject. Said subject may optionally be in a fasted state or post-prandial state.
• the anti-CGRP antibodies of the invention are used to detect the presence of CGRP in a biological sample obtained from a patient exhibiting symptoms of a disease or disorder associated with CGRP.
• the presence of CGRP, or elevated levels thereof when compared to pre-disease levels of CGRP in a comparable biological sample, may be beneficial in diagnosing a disease or disorder associated with CGRP.
• Another embodiment of the invention provides a diagnostic or screening assay to assist in diagnosis of diseases or disorders associated with CGRP in patients exhibiting symptoms of a CGRP associated disease or disorder identified herein, comprising assaying the level of CGRP expression in a biological sample from said patient using a post-translationally modified anti-CGRP antibody or binding fragment thereof.
• the anti- CGRP antibody or binding fragment thereof may be post-translationally modified to include a detectable moiety such as set forth previously in the disclosure.
• the CGRP level in the biological sample may be determined using a modified anti-CGRP antibody or binding fragment thereof as set forth herein, and comparing the level of CGRP in the biological sample against a standard level of CGRP (e.g., the level in normal biological samples).
• a standard level of CGRP e.g., the level in normal biological samples.
• the anti- CGRP antibodies of the invention may be used to correlate CGRP expression levels with a particular stage of impaired glucose metabolism. For example, correlating levels of circulating CGRP with glucose and/or insulin levels will allow for establishing the level of insulin insensitivity, or hyperglycemia.
• Insulin sensitivity may additionally be measured in a subject using methods known in the art, for example as described in Muniyappa et al. (Am J Physiol Endocrinol Metab 294:E15-E26, 2008) which is hereby incorporated by reference in its entirety.
• insulin sensitivity may be measured using a variety of methods including hyperinsulinemic euglycemic glucose clamp, the insulin suppression test, QUICKI, HOMA, 1 /insulin, or the Matusda index.
• hyperinsulinemic euglycemic glucose clamp the insulin suppression test
• QUICKI the insulin suppression test
• HOMA the insulin suppression test
• 1 /insulin or the Matusda index.
• Matusda index Matusda index
• the above-recited assay may also be useful in monitoring a disease or disorder, where the level of CGRP obtained in a biological sample from a patient believed to have a CGRP associated disease or disorder is compared with the level of CGRP in prior biological samples from the same patient, in order to ascertain whether the CGRP level in said patient has changed with, for example, a treatment regimen.
• a treatment regimen One skilled in the art would understand that by measuring CGRP in the patient at different intervals, the progression of the impairment to an individual's ability metabolize glucose can be determined.
• the invention is also directed to a method of in vivo imaging which detects the presence of cells expressing CGRP comprising administering a diagnostically effective amount of a diagnostic composition. Said detection can be useful as part of a planning regimen for the design of an effective treatment protocol for diabetes or patients at risk for developing diabetes.
• the methods of the invention include one or more compositions used for treating impaired glucose metabolism, such as insulin resistance, impaired insulin secretion or hyperglycemia in combination with the anti-CGRP antibodies disclosed herein.
• compositions used for treating impaired glucose metabolism such as insulin resistance, impaired insulin secretion or hyperglycemia in combination with the anti-CGRP antibodies disclosed herein.
• sulfonylureas PPAR-gamma agonists
• GPL-1 receptor agonists GPL-1 receptor agonists
• dipeptidyl peptidase IV inhibitor dipeptidyl peptidase IV inhibitor
• amylin analogs biguanides
• dopamine D2 receptor agonists meglitinides, alpha-glucosidase inhibitor, antidyslipidemic bile acid sequestrant, insulin, cytokine therapy, gene therapy, and antibody therapy, as well as an anti-CGRP antibody or fragment thereof.
• biguanides examples include: Metformin such as Glucophage and Glucophage XR (Bristol Myers Squibb/Merck Serono), Fortamet (Watson), Glumetza (Biovail/Depomed/Santarus), and generics.
• Metformin such as Glucophage and Glucophage XR (Bristol Myers Squibb/Merck Serono), Fortamet (Watson), Glumetza (Biovail/Depomed/Santarus), and generics.
• sulfonylureas examples include Glimepiride such as Amar yl (Sanofi ) and generics; Glipizide such as Glucotrol and Glucotrol XL (Pfizer) and generics; Glyburide/glibenclamide such as Diabeta (Sanofi), Micronase/Glynase (Pfizer) and generics; Metformin + glyburide such as Glucovance (Bristol Myers Squibb), Suguan M (Sanofi-Aventis), GlicoRest, GlucoNorm (Abiogen), Bi-Euglucon (Roche) and generics; Metformin + glipizide such as Metaglip (Bristol Myers Squibb), and generics.
• Glimepiride such as Amar yl (Sanofi ) and generics
• Glipizide such as Glucotrol and Glucotrol XL (Pfizer) and generic
• Examples of PPAR-gamma agonists include: Rosiglitazone such as Avandia (GlaxoSmith line); Pioglitazone such as Actos (Takeda) and generics; Rosiglitazone + metformin such as Avandamet (GlaxoSmithKline); Pioglitazone + metformin such as Actoplus Met XR (Takeda); Pioglitazone + glimepiride such as Avandaryl/Avaglim (GlaxoSmithKline); Pioglitazone + glimepiride such as Duetact/Tandemact/Sonias (Takeda).
• Rosiglitazone such as Avandia (GlaxoSmith line); Pioglitazone such as Actos (Takeda) and generics
• Rosiglitazone + metformin such as Avandamet (GlaxoSmithKline)
• Pioglitazone + metformin such as Act
• GLP-1 receptor agonists examples include: Exenatide such as Byetta (Bristol Myers Squibb/AstraZeneca); Liraglutide such as Victoza (Novo Nordisk); Exenatide LAR such as Bydureon (Bristol Myers Squibb/AstraZeneca).
• Dipeptidyl peptidase IV (DPP-IV or DPP4) inhibitors include: Sitagliptin such as Januvia, Merck; Vildagliptin such as Galvus (Novartis); Saxagliptin such as Onglyza (Bristol Myers Squibb/AstraZeneca); Alogliptin such as Nesina (Takeda/Furiex); Linagliptin such as Trazenta (Boehringer Ingelheim/Eli Lilly); Teneligliptin such as Tenelia (Mitsubishi Tanabe/Daiichi Sankyo); Sitagliptin + metformin such as Janumet (Merck) and Janumet XR (Merck); Sitagliptin + simvastatin such as JuviSync (Merck); Vildagliptin + metformin such as Eurcreas (Novartis); Saxagliptin + metformin such as Kombiglyze/Kombigly
• Meglitinides include: Repaglinide such as GlucoNorm/Prandin/NovoNorm (Daiichi Sankyo/Fournier Pharma/Novo Nordisk); Nateglinide such as Starlix (Novartis), Fastic (Daiichi Sankyo), Starsis (Astellas) and generics; Mitiglinide such as Glufast (Kissei/Takeda).
• Repaglinide such as GlucoNorm/Prandin/NovoNorm (Daiichi Sankyo/Fournier Pharma/Novo Nordisk)
• Nateglinide such as Starlix (Novartis), Fastic (Daiichi Sankyo), Starsis (Astellas) and generics
• Mitiglinide such as Glufast (Kissei/Takeda).
• Alpha-glucosidase inhibitors include: Acarbose such as Precose/Glucobay (Bayer) and generics; Miglitol such as Glyset (Pfizer), Diastabol (Sanofi), Seibule (Sanwa Kagaku) and generics; voglibose such as Basen (Takeda) and generics.
• Example of a bile acid sequestrants include: Colesevelam such as Cholestagel (Sanofi), Welchol (Daiichi Sankyo).
• Example of a Dopamine D2 receptor agonist includes Bromocriptine such as Cycloset (Santarus).
• Example of an amylin analogue includes Pramlintide such as Symlin (Bristol Myers Squibb/AstraZeneca).
• Examples of fast-acting insulins include: insulin lispro such as Humalog (Eli Lilly); Insulin aspart such as NovoLog (Novo Nordisk), NovoRapid (Novo Nordisk), Insulin glulisine such as Apidra (Sanofi).
• Examples of regular human insulins include: Humulin/Umuline Rapide (Eli Lilly), Novolin R (Novo Nordisk), Actrapid (Sanofi).
• Examples of intermediate- acting insulins include: Humulin N (Eli Lilly), Novolin N (Novo Nordisk).
• Examples of long-lasting insulins include: Insulin glargine such as Lantus (Sanofi) and insulin detemir such as Levemir (Novo Nordisk).
• the present invention further provides for a kit for detecting binding of an anti- CGRP antibody of the invention to CGRP.
• the kit may be used to detect the presence of a CGRP specifically reactive with an anti-CGRP antibody of the invention or an immunoreactive fragment thereof.
• the kit may also include an antibody bound to a substrate, a secondary antibody reactive with the antigen and a reagent for detecting a reaction of the secondary antibody with the antigen.
• a kit may be an ELISA kit and can comprise the substrate, primary and secondary antibodies when appropriate, and any other necessary reagents such as detectable moieties, enzyme substrates, and color reagents, for example as described herein.
• the diagnostic kit may also be in the form of an immunoblot kit.
• the diagnostic kit may also be in the form of a chemiluminescent kit (Meso Scale Discovery, Gaithersburg, MD).
• the diagnostic kit may also be a lanthanide- based detection kit (PerkinElmer, San Jose, CA).
• a biological sample includes, but is not limited to, sera, plasma, urine, saliva, mucous, pleural fluid, synovial fluid and spinal fluid.
• anti-CGRP antibodies described herein, or fragments thereof are useful for ameliorating or reducing the symptoms of, or treating, or preventing, diseases and disorders associated with CGRP.
• Anti-CGRP antibodies described herein, or fragments thereof, as well as combinations, can also be administered in a therapeutically effective amount to patients in need of treatment of diseases and disorders associated with CGRP in the form of a pharmaceutical composition as described in greater detail below.
• anti-CGRP antibodies described herein, or fragments thereof are useful for ameliorating or reducing the symptoms of, or treating, or preventing, impaired glucose tolerance, insulin resistance (insensitivity), impaired insulin secretion, lipotoxicity, hyperglycemia, pancreatic beta cell failure as a result of diabetes, pre-diabetes, Type 1 diabetes, Type 2 diabetes, or gestational diabetes.
• the anti-CGRP antibodies described herein, or fragments thereof may be administered to an individual at risk of developing diabetes, e.g., an individual diagnosed with pre-diabetes. Without intent to be limited by theory, it is believed that by restoring insulin sensitivity, the subject anti-CGRP antibodies may be able to delay or prevent the progression to diabetes.
• the anti-CGRP antibodies described herein, or fragments thereof may be administered to a patient that does not achieve normoglycemia with administration of another treatment, e.g., treatment with metformin, pioglitazone, a sulfonylurea, a glinide, an oral thiazolidinedione (TZD) such as pioglitazone, a glucagon-like peptide 1 (GLP-1) agonist such as exenatide, a DPP4 inhibitor such as sitagliptin, vildagliptin, saxagliptin, alogliptin, linagliptin, or teneligliptin, or a combination therapy such as metformin and pioglitazone, metformin and a sulfonylurea, metformin and a glinide, metformin and a TZD, metformin and pioglita
• another treatment e.g., treatment
• anti-CGRP antibodies described herein, or fragments thereof are administered for prevention or treatment of obesity, e.g., to individuals having a body mass index of at least 25. Without intent to be limited by theory, it is believed that the subject anti-CGRP antibodies may increase peripheral and/or hepatic glucose utilization, thereby increasing metabolic rate and contributing to weight loss.
• Said anti-CGRP antibodies may be administered in combination with another anti-obesity agent such as orlistat, rimonabant, sibutramine, a peptide YY (PYY, a 36 amino acid peptide that reduces appetite), a PYY analog, a CB-1 antagonist, rimonabant, a leptin, a leptin analog, or a phentermine.
• another anti-obesity agent such as orlistat, rimonabant, sibutramine, a peptide YY (PYY, a 36 amino acid peptide that reduces appetite), a PYY analog, a CB-1 antagonist, rimonabant, a leptin, a leptin analog, or a phentermine.
• the anti-CGRP antibodies described herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or antibody fragments are administered to a subject at a concentration of between about 0.1 and 100.0 mg/kg of body weight of recipient subject. In an embodiment of the invention, the anti-CGRP antibodies described herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or antibody fragments, are administered to a subject at a concentration of about 0.4 mg/kg of body weight of recipient subject.
• the anti-CGRP antibodies described herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or antibody fragments are administered to a recipient subject with a frequency of once every twenty- six weeks or less, such as once every sixteen weeks or less, once every eight weeks or less, once every four weeks or less, once every two weeks or less, once every week or less, or once daily or less.
• Fab fragments may be administered every two weeks or less, every week or less, once daily or less, multiple times per day, and/or every few hours.
• a patient receives Fab fragments of 0.1 mg/kg to 40 mg/kg per day given in divided doses of 1 to 6 times a day, or in a sustained release form, effective to obtain desired results.
• concentration of the antibody or Fab administered to a given patient may be greater or lower than the exemplary administration concentrations set forth above in the two preceding paragraphs.
• the anti-CGRP antibodies described herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or antibody fragments are administered to a subject in a pharmaceutical formulation.
• a "pharmaceutical composition” refers to a chemical or biological composition suitable for administration to a mammal. Such compositions may be specifically formulated for administration via one or more of a number of routes, including but not limited to buccal, epicutaneous, epidural, inhalation, intraarterial, intracardial, intracerebroventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intrathecal, intravenous, oral, parenteral, rectally via an enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal. In addition, administration can occur by means of injection, powder, liquid, gel, drops, or other means of administration.
• the anti-CGRP antibodies described herein, or CGRP binding fragments thereof, as well as combinations of said antibodies or antibody fragments may be optionally administered in combination with one or more active agents.
• active agents include analgesic, anti-histamine, antipyretic, antiinflammatory, antibiotic, antiviral, and anti-cytokine agents.
• Active agents include agonists, antagonists, and modulators of TNF-a, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL- 18, IFN-a, IFN- ⁇ , BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, Hepatocyte Growth Factor (HGF), Hepcidin, including antibodies reactive against any of the foregoing, and antibodies reactive against any of their receptors.
• TNF-a TNF-a
• HGF Hepatocyte Growth Factor
• Active agents also include but are not limited to 2-Arylpropionic acids, Aceclofenac, Acemetacin, Acetylsalicylic acid (Aspirin), Alclofenac, Alminoprofen, Amoxiprin, Ampyrone, Arylalkanoic acids, Azapropazone, Benorylate/Benorilate, Benoxaprofen, Bromfenac, Carprofen, Celecoxib, Choline magnesium salicylate, Clofezone, COX-2 inhibitors, Dexibuprofen, Dexketoprofen, Diclofenac, Diflunisal, Droxicam, Ethenzamide, Etodolac, Etoricoxib, Faislamine, fenamic acids, Fenbufen, Fenoprofen, Flufenamic acid, Flunoxaprofen, Flurbiprofen, Ibuprofen, Ibuproxam, Indometacin, Indoprofen, Kebuzone, Ketoprofen
• An anti-histamine can be any compound that opposes the action of histamine or its release from cells (e.g., mast cells).
• Anti-histamines include but are not limited to acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, cetirizine analogues, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, HSR 609, hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.
• Antibiotics include but are not limited to Amikacin, Aminoglycosides, Amoxicillin, Ampicillin, Ansamycins, Arsphenamine, Azithromycin, Azlocillin, Aztreonam, Bacitracin, Carbacephem, Carbapenems, Carbenicillin, Cefaclor, Cefadroxil, Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime, Cefixime, Cefoperazone, Cefotaxime, Cefoxitin, Cefpodoxime, Cefprozil, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol, Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin,
• Active agents also include Aldosterone, Beclometasone, Betamethasone, Corticosteroids, Cortisol, Cortisone acetate, Deoxycorticosterone acetate, Dexamethasone, Fludrocortisone acetate, Glucocorticoids, Hydrocortisone, Methylprednisolone, Prednisolone, Prednisone, Steroids, and Triamcinolone. Any suitable combination of these active agents is also contemplated.
• a "pharmaceutical excipient” or a “pharmaceutically acceptable excipient” is a carrier, usually a liquid, in which an active therapeutic agent is formulated.
• the active therapeutic agent is a humanized antibody described herein, or one or more fragments thereof.
• the excipient generally does not provide any pharmacological activity to the formulation, though it may provide chemical and/or biological stability, and release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19 th Ed., Grennaro, A., Ed., 1995 which is incorporated by reference.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible.
• the carrier is suitable for parenteral administration.
• the carrier can be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration.
• Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.
• compositions typically must be sterile and stable under the conditions of manufacture and storage.
• the invention contemplates that the pharmaceutical composition is present in lyophilized form.
• the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.
• the invention further contemplates the inclusion of a stabilizer in the pharmaceutical composition.
• the proper fluidity can be maintained, for example, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
• an agent such as, monostearate salts and gelatin, the absorption of the injectable compositions can be prolonged.
• the alkaline polypeptide can be formulated in a time-release formulation, for example in a composition which includes a slow release polymer.
• the active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are known to those skilled in the art. [437] For each of the recited embodiments, the compounds can be administered by a variety of dosage forms. Any biologically-acceptable dosage form known to persons of ordinary skill in the art, and combinations thereof, are contemplated.
• dosage forms include, without limitation, reconstitutable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cachets, inhalants, aerosol inhalants, patches, particle inhalants, implants, depot implants, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), infusions, and combinations thereof.
• Blood glucose was measured in fed conditions before treatment, 18h after treatment with metformin and Abl4 and 42h after treatment with Abl4. The 2 compounds did not affect blood glucose in these conditions. Blood glucose was measured in fasted condition, just before clamp and only metformin had a significant decreasing effect (17%). Plasma insulin, measured in fed condition before treatment and 18h after treatment with metformin and 42h after treatment with Abl4, was slightly decreased by the 2 compounds, as well as the insulin resistance index HOMA-IR (not significant).
• Plasma samples were obtained from AM 4 treated animals just prior to the clamp procedure, 48-hours post treatment, Abl4 concentration was determined. The results of this analysis confirmed systemic exposures ranging from 642 to 797 ⁇ g/mL Abl4 in rats undergoing the clamp procedure.
• Metformin increased glucose utilization in the mixed vastus lateralis muscle (VL, 49% p ⁇ 0.05), in the glycolytic extensor digitorum longus muscle (EDL, 19% NS) and decreased glucose utilization in the heart (-39%, pO.01), presumably due to the stimulation of myocardial fatty acid oxidationl .
• Abl4 tended to increase GIR and GTO (NS) and had a stronger effect than metformin on glucose utilization in VL (70%, pO.01), in EDL (26%, NS), and in the oxidative soleus muscle (27%, NS). It also tended to increase glucose utilization in the heart (21%, NS).
• Abl4 did not affect glucose utilization rate in white adipose tissues (deep and subcutaneous). [452] In conclusion, Abl4 had a good trend to improve whole body glucose utilization in normal rats after acute treatment with a significant effect upon the glucose utilization rate in muscles (VL).
• rats were anesthetized (isoflurane) and a catheter was implemented in the femoral vein. A recovering period was followed for 5-6 days before the clamp procedure.
• BG Blood glucose
• Plasma samples were kept at -80°C until insulin measurement (using ELISA method).
• a sample of blood ( ⁇ 200 ⁇ xL) was obtained just prior to the clamp procedure for each Group 2 animal, processed to plasma (-60 ⁇ ), and maintained at -80°C for subsequent determination of AM 4 concentrations utilizing a Meso Scale Discovery (MSD) ELISA platform.
• MSD Meso Scale Discovery
• Metformin was administered by p.o. route 24h and 4h before the TO of clamp procedure (at 02:00 pm the day before the clamp and at 10:00 am the day of the clamp).
• the hyperinsulinemic-euglycemic clamp was performed using H-glucose as a tracer and 0.3U/kg/h insulin infusion from 02:00 pm (TO) to 05:00 pm (T+3h).
• a glucose solution was infused in parallel and the infusion rate was adjusted to reach the steady state (-100 +/-10 mg/dL).
• Blood glucose was measured from the tip of the tail using glucometers every 10 minutes. Blood was collected ( ⁇ ) from the tip of the tail during the last hour (steady state) and the following parameters were assessed: Glucose infusion rate; Whole body glucose utilization rate; Hepatic glucose production rate; Whole body glycogen and glycolytic rates.
• VL vastus lateralis
• EDL extensor digitorum longus
• soleus muscles epididymal and inguinal white adipose tissues
• heart apex heart apex
• skin skin
• a piece of each tissue was dissolved in 1M NaOH and then neutralized with 1M HC1.
• D-2- ,4 C deoxyglucose 6-phosphate.
• D-2- 14 C deoxyglucose was differentially precipitated by the use of a zinc hydroxide (0.3M) solution or a perchloric acid solution (6%). Both radioactivity contents were measured to evaluate the glucose uptake expressed as ng/mg/min.
• Plasma insulin level was measured at the end of the clamp.
• Histograms were analyzed using an AN OVA one way with a Dunnett's post test and curves were analyzed using an ANOVA two ways with a Bonferroni's post test. A difference was considered significant when p value was ⁇ 0.05. NS: not significant.
• Metformin significantly increased GIR evolution from 60 minutes after the start of the infusion compared to vehicle group.
• AM 4 had a trend to increase the GIR evolution mostly after 130 minutes infusion (FIG. 2A).
• Glucoses fluxes were then calculated from 140 minutes to 180 minutes of infusion (FIG. 3).
• AM4 and metformin increased the glucose infusion rate by 18% (NS) and 27% (p ⁇ 0.05) respectively, as well as the glucose turn over by 18% (NS) and 30% (p ⁇ 0.05) respectively.
• the hepatic glucose production was totally inhibited by this supraphysiological dose of insulin in the 3 groups.
• AM 4 did not affect glycolysis rate whereas metformin increased it by 43% (NS).
• AM4 increased glycogen synthesis rate by 23%o similarly to metformin (NS).
• metformin increased glucose utilization in the mixed vastus lateralis muscle (VL, 49% p ⁇ 0.05), in the glycolytic extensor digitorum longus muscle (EDL, 19% NS) and decreased glucose utilization in the heart (- 39%, p ⁇ 0.01), which is a known effect of metformin thought to be due to the stimulation of myocardial fatty acid oxidation.
• Abl4 tended to increase glucose infusion rate and whole body glucose turn over (NS) and had a stronger effect than metformin on glucose utilization in the VL (70%, pO.01), in EDL (26%, NS), and in the oxidative soleus muscle (27%, NS). It also tended to increase glucose utilization in the heart (21%, NS). Similar to metformin, AM 4 did not affect glucose utilization rate in white adipose tissues (deep and subcutaneous).
• Abl4 Plasma Concentration Analysis The Abl4 plasma concentrations for Group 2 animals undergoing the clamp procedure ranged from 642 to 797 ⁇ g/mL and supported up to 48-hours of systemic exposure.
• This example assesses the ability of AM 4 to improve insulin sensitivity in a rat model of insulin resistance.
• rats were fed with a high fat (69%) and high fructose (14%) diet (HFD) for 6 weeks to induce glucose intolerance, with the plasma insulin level becoming significantly increased and glycaemia becoming slightly increased compared to control animals fed normal chow.
• the antibody used in this example consisted of the light and heavy polypeptide chains of SEQ ID NOs 132 and 134.
• HFD rats After 6 weeks of HFD rats were randomized to treatment groups according to their glucose intolerance (AUC calculation during an oral glucose tolerance test (OGTT)) and their HOMA-IR (insulin resistance index). HFD rats were treated for 15 days (i.v., two administrations one week apart) with Abl4 at 10, 30 and 100 mg/kg/ week, or daily with metformin 200mg/kg/day in drinking water.
• AUC glucose intolerance
• HOMA-IR insulin resistance index
• HGP Hepatic glucose production
• the high fat diet composition was as follows (Kcal%): Protein: 17.3%; Carbohydrate (fructose): 14%; Fat (lard) ): 68.7%; cholesterol 1.65%, cholic acid 0.65%.
• Metformin (200mg/kg/day) was administered in drinking water for ⁇ 2 weeks until the clamp procedure. Rats treated with metformin were treated with vehicle on day 1 and day 8 (via the caudal vein).
• test groups were as follows:
• AUC Area under the curve
• Body weight was measured once a week during the first six week of HFD. Body weight was measured 3 times per week during the first 10 days of treatment. Food consumption was measured over 48h or 72h, just before treatment and 3 times per week during treatment until surgery procedure (day 1 1).
• the 2-step hyperinsulinemic-euglycemic clamp was performed using H- glucose as a tracer (except in the normal chow group), and 5mU/kg/min insulin infusion from 02:00 pm (TO) to 04:00 pm (T+2h), followed by 15mU/kg/min insulin infusion from 04:00 pm to 05:30 pm (t+3.5h).
• a glucose solution was infused in parallel and the infusion rate was adjusted to reach the steady state (100 +/-10 mg/dL). Blood glucose was measured from the tip of the tail using glucometers every 10 minutes. Blood was collected ( ⁇ ) regularly from the tip of the tail during the steady states of each step.
• Glucose infusion rate in all groups
• Whole body glucose utilization rate except in the normal chow group
• Hepatic glucose production rate except in the normal chow group
• Whole body glycogen and glycolytic rates except in the normal chow group.
• VL Vastus lateralis
• EDL Extensor digitorum longus
• Soleus muscle heart apex epididymal white adipose tissue inguinal white adipose tissue skin
• T3.5h Plasma insulin and C peptide levels were measured just before the infusion starts ( ⁇ T-30min.), at the end of the steady state of step 1 (T2h) and step 2 (T3.5h). For that, blood collection was performed from the tip of the tail (- ⁇ ,, on EDTA).
• the HOMA-IR insulin resistance index
• AUC was higher (-9%, not shown) in HFD groups compared to the control chow-fed group, as well as the HOMA-IR (34%, not shown) and the body weight (-17%, p ⁇ 0.001 , FIG. 7)
• the plasma C-peptide level profile on day 15 was similar to the plasma insulin level, but the effects were more marked and less variable.
• the C-peptide was significantly increased by 67% in HFD vehicle group as compared to the control chow-fed group.
• Abl4 treatment at 10, 30, or 100 mg/kg decreased C-peptide level by 30 % (p ⁇ 0.05), 23 % (ns), and 29% (p ⁇ 0.05), respectively, and metformin decreased C- peptide by 13% (ns) (FIG. 1 1, lower left panel).
• HOMA-IR an index of insulin resistance
• AM 4 had no effect after 10 days of treatment whereas metformin had a decreasing effect (ns) by 36%.
• Ab 14 at 10, 30 or 100 mg/kg decreased HOMA-IR by 33% (ns), 17%) (ns) and 38% (p ⁇ 0.05), respectively, and metformin tended to decrease HOMA-IR by 18% (ns, FIG. 12).
• FIG. 13 shows the glucose infusion rate (GIR) over time during the 2-step hyper-insulinemic clamp.
• GIR glucose infusion rate
• GIR for the control chow-fed group was higher than the HFD vehicle group during both of the clamp steps, and confirmed that HFD rats had an insulin-resistant phenotype after 8-9 weeks of diet.
• Metformin had no effect on GIR during the first clamp step, while the GIR plateau was slightly higher (ns) in AM 4 treated groups. All treated groups were observed with GIR plateaus higher than the HFD vehicle group during the second clamp step, with significant differences observed for metformin and Abl4 at 30 or 100 mg/kg (FIG. 13).
• Statistical significance was evaluated using a two- way ANOVA with Bonferroni's post test versus HFD.
• the GIR was significantly different only for the normal chow control, vehicle treated rats at the 50 and 60 minute time points (pO.01 and p ⁇ 0.05 respectively).
• the GIR was significantly different for the HFD rats treated with 30 mg/kg Abl4 at the 160-210 minute time points (p ⁇ 0.05 at 160 minutes and p ⁇ 0.01 for the 170- 210 minute time points), for the HFD rats treated with 100 mg/kg Abl4 at the 170-210 minute time points (pO.01 at 190 minutes and p ⁇ 0.05 for the 170-180 and 200-210 minute time points), and for the HFD rats treated with metformin at the 170-210 minute time points (pO.01 at 180 and 190 minutes, and p ⁇ 0.05 at the 170 and 200-210 minute time points).
• the GIR means were calculated for each plateau (FIG. 14). GIR was significantly decreased in HFD vehicle group compared to control chow-fed group by 32% (p ⁇ 0.05) and 17% (p ⁇ 0.01) during the first and the second steps, respectively. Abl4 at 10, 30, or 100 mg/kg increased GIR (ns) during the first step (by 26, 37, and 29% respectively), and metformin had also an increasing (ns) effect by 1 1%. During the second step, all treatments increased GIR as compared to the HFD vehicle group: Abl4 10, 30, or lOOmg/kg by 19% (ns), 36% (p ⁇ 0.01), and 28% (p ⁇ 0.05), respectively, and metformin by 27% (p ⁇ 0.05).
• Plasma insulin was measured during the clamp procedure. As expected the insulin level was similar between all groups at the end of the two clamp steps. During the first clamp step the insulin concentration was approximately 14( U/mL, and was a physiological level expected during fed conditions. The insulin concentration after the second clamp step was approximately 49( ⁇ U/mL, which was a pharmacological level (FIG. 1 1, upper panel).
• C-peptide was also measured during the clamp procedure (FIG. 1 1 , lower right panel). During euglycemic conditions the insulin secretion by beta cells was inhibited, and the plasma C-peptide levels were therefore low and not interpretable.
• H-glucose was infused with insulin during the clamp procedure in all HFD groups (not in the control chow-fed group). The whole body glucose fluxes were then calculated.
• the glucose turn over (GTO) was similar in all groups, excluding that Abl4 at 30 mg/kg tended to increase GTO as compared to the HFD vehicle group (17%, ns). Glycolysis and glycogen synthesis tended to increase, 15 and 16%, respectively (ns, FIG. 15), following treatment with AM 4 at 30 mg/kg.
• Example 3 assesses the effect of AM 4 on glucose metabolism and on glycemic control in a model a rat model of diabetes, the Zucker diabetic fatty (ZDF) rat.
• ZDF Zucker diabetic fatty
• the antibody used in this example consisted of the light and heavy polypeptide chains of SEQ ID NOs 132 and 134.
• the antibody was administered once weekly via the caudal vein (i.v., 5mL/kg) on days 1, 8, 15, and 22 at two different doses 20 mg/kg/week (groups 3 and 7) or 60 mg/kg/week (groups 4 and 8). All other groups were treated once weekly with vehicle 1 (i.v., 5mL/kg).
• vehicle 1 i.v., 5mL/kg.
• the intravenous treatments were performed in the morning on day 1 , 8, 15, and 22 while under isoflurane anaesthesia.
• the volume of administration was individually adapted according to the most recent body weight.
• Metformin (Met) and pioglitazone (PIO) were administered once daily for 28 days, via per os route (p.o., 5mL/kg) between 8:00 and 10:00 am, except that on the day of OGTT or after intravenous treatments, some per os treatments were completed after 10:00am.
• Group 5, 7, and 8 were treated with 200mg/kg/day metformin, and group 6 was treated with lOmg/kg/day pioglitazone. All other groups were treated daily with vehicle 2 (p.o., 5mL/kg) for 28 days. The most recent body weight was used to calculate the average volume of administration in each group.
• a fasting (6 hours on day 0 from 8:00 am to 02:00 pm, and overnight on day 12, 19 and 26 from -6:00 pm to -8:00 am) was performed before each blood collection. Blood was collected at - 2:00 pm, from the tip of the tail, on day 0 (before screening, 150 ⁇ , potassium EDTA), and at -8:00 am prior to dosing on days 12 (1 ⁇ ⁇ ,, potassium EDTA), 19 (150 ⁇ , potassium EDTA) and 26, (1 ⁇ , potassium EDTA).
• Fasting blood glucose was measured on days 0, 12, 19, and 26.
• Fasting plasma insulin, peptide-C (ELISA method), free fatty acids, triglycerides, total cholesterol (colorimetric method), and HDL-cholesterol (phosphotungstate precipitation, colorimetric method) were measured on day 0, and prior to dosing on days 12, 19, and day 26.
• Non HDL-cholesterol was calculated as total cholesterol - HDL-cholesterol.
• Fructosamine was measured on days 0, 19 and 28.
• HbAl c DCA 2000 was measured on days 0 and 28.
• the oral glucose tolerance test was performed as follows. On day 25, rats were fasted at -6:00 pm and an oral glucose tolerance test was performed the day after (on day 26). At -8:00 am (T-60) a blood collection (1 ⁇ ⁇ , EDTA) was performed for biochemical parameters measurements. One hour after (-09:00 am), an oral glucose bolus (1.5g/kg) was administered (TO). Blood glucose was measured (glucometer or colorimetric method in case of high glycaemia) on T-60, TO, T15, T30, T60, T90, T120, and T180 minutes. Area under the curve (AUC) was calculated based on the blood glucose values measured at TO. Plasma insulin and C-peptide were measured (ELISA method) on T-60, T15 ( ⁇ 40 ⁇ of blood, EDTA), and T30 minutes ( ⁇ 40 ⁇ of blood, EDTA).
• pancreas was divided into 2 parts (longitudinal cut). One piece was fixed in 10% formalin solution for histopathological processing. The other piece of pancreas was flash frozen and kept at -80°C for determination of insulin and proinsulin levels.
• pancreas sample was homogenized in an acid buffer, and insulin and proinsulin content were measured using ELISA kits in the following groups:
• Rats were excluded from analysis if they were an outlier in all or almost all parameters. This resulted in exclusion of four rats, each from a different group.
• Pioglitazone significantly increased body weight compared to the ZDF vehicle rats, from 8 days of treatment and body weight gain was 3-fold higher at the end of the treatment (FIG. 19A and B). All other drug treatments had no significant effect on body weight compared to vehicle ZDF rats. AB14 60mg/kg + metformin 200mg/kg combination significantly increased body weight gain from 22 days of treatment (FIG. 19B).
• Pioglitazone significantly decreased overnight fasting blood glucose levels to a normal level from 12 days of treatment (pO.001 , FIG. 21A).
• AB14 decreased by about 15% the blood glucose after 12, 19 or 26 days of treatment (n.s, FIG. 21 A).
• metformin 200mg/kg had similar effect on day 12, but this effect was not observed at day 19 and 26.
• AB14 20 mg/kg + metformin combination slightly reduced blood glucose on day 12 (12%), ns), and showed no effect on day 19 and day 26 (FIG. 21 A).
• Pioglitazone seemed to have no protective effect on insulin secretion as it showed no effect on plasma insulin and C-peptide levels on days 12, 19 and 26 compared to vehicle ZDF rats (FIGs. 21 B and D). Hence the reduction in blood glucose levels was related to the insulin sensitizing effect of pioglitazone, which reduced HOMA-IR by 67%), 62%) and 54%, on days 12, 19 and 26 respectively, as compared with vehicle ZDF rats (FIG. 21 C).
• AB14 20mg/kg did not change plasma insulin and C-peptide levels, as well as HOMA-IR on days 12, 19 and 26 (FIG. 21B-D). Meanwhile, AB14 60mg/kg increased plasma insulin levels on days 12, 19 and 26 by 74%, 21% and 19%, respectively (ns vs. vehicle ZDF rats, FIG. 2 IB).
• metformin increased plasma insulin levels on days 12, 19 and 26 by 79%, 55% and 48%, respectively (ns, FIG. 2 IB). Metformin increased plasma C-peptide levels on days 12, 19 and 26 by 23%, 21 %, and 9%, (NS vs ZDF rats treated with vehicle, FIG. 2 I D).
• AB14 20mg/kg + metformin combination increased plasma insulin levels on days 12, 19 and 26 by 2-fold (NS, FIG. 2 IB).
• AB14 20mg/kg + metformin combination increased plasma C-peptide levels on days 12, 19 and 26 by 21%, 23% and 25%, respectively (NS, FIG. 21D).
• AB14 60mg/kg + metformin combination significantly increased plasma insulin levels on days 12, 19 and 26 by a factor 2.5, 2.3 and 2.7 respectively.
• AB 14 60mg/kg + metformin combination significantly increased plasma C-peptide levels from day 12 by 45% (day 12), 48% (day 19), and 52% (day 26) (p ⁇ 0.05 vs vehicle-treated ZDF rats, FIG. 2 ID).
• fructosamine was significantly higher (66%) in 8- week old vehicle-treated ZDF rats (208 ⁇ 6 vs 144 ⁇ 2 ⁇ , pO.001). Fructosamine levels remained in a similar range in lean rats during the treatment period, but increased in vehicle ZDF rats after 19 (253 ⁇ 5 ⁇ , pO.001) and 28 (234 ⁇ 6 ⁇ , p ⁇ 0.001) days of treatment (FIG. 22). As expected, pioglitazone significantly reduced fructosamine levels from day 19 (30% on day 19 and 25% on day 28, pO.001). AB14 20 and 60mg/kg had no effect on fructosamine levels.
• metformin showed a trend towards lower fructosamine levels only on day 19 (6%, ns).
• AB 14 20mg/kg + metformin combination showed a non significant trend towards lower fructosamine levels on days 19 and 28 by 10% and 8%, respectively.
• the AB14 60mg/kg + metformin combination showed a non significant trend towards lower fructosamine levels (9%) on day 28 (FIG. 22).
• HbAlc was higher in 8-week old ZDF rats (4.3 ⁇ 0.1% vs3.1 ⁇ 0.04%), although these values were in a normal range.
• HbAlc reached a pathological value of 8.8 ⁇ 0.2% on day 28, (p ⁇ 0.001 ZDF vs lean rats, FIG. 23).
• AB14 20 and 60mg/kg had no effect on HbAlc levels after 28 days of treatment.
• pioglitazone and metformin significantly decreased HbAlc by 44 and 15%, respectively (FIG. 23).
• metformin with AB 14 20mg/kg and with 60mg/kg significantly reduced HbAlc by 1 1 and 19% respectively (FIG. 23).
• metformin + AB14 20 mg/kg combination showed a trend towards higher plasma triglycerides on days 19 and 26 (by 13% and 23%, respectively, ns) as compared with the vehicle ZDF group.
• Metformin + AB 14 60 mg/kg combination showed a trend towards higher plasma triglycerides on days 12, 19 and 26 (by 9%, 48% and 43% respectively, significant from day 19, FIG. 24A).
• AB14 20 mg/kg and metformin had no effect.
• AB14 60 mg/kg increased total cholesterol by 8%, 14%), and 15% on days 12, 19, and 26 respectively compared to the vehicle ZDF group.
• metformin AB14 20 mg/kg increased total cholesterol by 15% and 10% on days 12 and 26 respectively
• AB14 60 mg/kg increased total cholesterol by 24%, 21% and 13% on days 12, 19 and 26 respectively compared to the vehicle ZDF group.
• pioglitazone increased plasma HDL-cholesterol by 38%, 17% and 19% on days 12, 19 and 26 respectively.
• Metformin alone had no effect.
• AB 14 20 mg/kg and 60 mg/kg, alone or in combination with metformin increased plasma HDL-cholesterol levels by 11 to 22% after 12, 19 and 26 days of treatment.
• Plasma non HDL-cholesterol levels (FIG. 26C) were similar in all ZDF groups after 12 days of treatment.
• AB 14 20mg/kg, AB14 60 mg/kg, metformin, alone or in combination with AB14 had no effect on non HDL-cholesterol levels.
• AB14 20mg/kg, AB14 60 mg/kg and metformin alone or in combination with 20 mg/kg or 60 mg/kg AB14 showed a non significant reduction on AUC (7%, 11%, 6%, 7% and 17%, respectively).
• the AB14 60 mg/kg + metformin combination was slightly more effective in reducing AUC when compared with AB14 or metformin alone (FIG. 27B).
• Plasma insulin and C-peptide levels were measured at 15 and 30 minutes after the glucose load. The concentration versus time profiles were similar for both insulin and C-peptide. Insulin and C-peptide levels were similar between the vehicle, AB14 20 mg/kg and AB14 60 mg/kg treated groups, slightly increased in the metformin and pioglitazone treated groups, and more increased in a dose-dependent manner in groups treated with AB14 20 mg/kg and AB14 60 mg/kg combined with metformin (FIG. 28A- B). The capacity of insulin or C-peptide secretion in response to the glucose charge was evaluated by expressing the results in relative values calculated from T-60 minutes.
• AB14 60 mg/kg alone or combined with metformin showed a trend to decrease insulin secretion by 19% and 18%, respectively (FIG. 29 A).
• C-peptide secretion in response to glucose load (FIG. 29B) was significantly reduced in vehicle-treated ZDF rats by -40% at time T15 and T30.
• pioglitazone significantly increased C-peptide secretion at time T15 and T30 by 21% and 22% respectively.
• pancreas proinsulin (FIG. 30A) and insulin (FIG. 30B) levels were significantly lower in 12-week old ZDF rats when compared with lean rats.
• proinsulin/insulin ratio was significantly increased in ZDF rats, while no change was observed with drug treatments (FIG. 30C).
• pancreas insulin measured by immunohistochemistry showed a reduction in insulin labelling in ZDF rats.
• drug treatments slightly prevented this insulin labeling reduction with a better effect when AB14 and metformin were combined (FIG. 31).
• AM 4 also completely prevented the reduction in pancreatic proinsulin levels observed in the vehicle-treated controls when pancreas tissue was obtained at the end of the study (day 28) and also partially prevented the reduction in pancreatic insulin levels when measured either directly or through immunohistochemical analyses. Furthermore, AM 4 consistently decreased the islet vacuolation, islet fibrosis, and islet hyperplasia noted in the vehicle-treated animals upon histological evaluation at the end of the study, further indicating a favorable impact on diabetic pancreatic islet pathology.

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