Modulation of glucose metabolism using anti-cgrp antibodies

Abstract

The present disclosure provides methods of preventing or treating metabolic disorders. In exemplary embodiments, methods of administering an anti-CGRP antibody, optionally in combination with a second agent, are provided, wherein glucose utilization is increased in the periphery and / or the liver, thereby preventing or treating diseases and disorders associated with insulin resistance. Also provided are compositions comprising an anti-CGRP antibody, optionally in combination with a second agent, suitable for administration to increase glucose utilization in the periphery and / or the liver, and thereby prevent or treat diseases and disorders associated with insulin resistance.

Description

[0001] The relevant application is made public.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 61 / 982,611 (Attorney’s No. 43257.3002), filed April 22, 2014, and U.S. Provisional Patent Application No. 61 / 842,745 (Attorney’s No. 43257.3001), filed July 3, 2013, each of which is incorporated herein by reference in its entirety. Invention Field

[0003] This invention relates to the use of antibodies and fragments thereof (including Fab fragments) against human calcitonin gene-related peptide (“CGRP”), said antibodies specifically binding to CGRP and promoting glucose uptake and utilization in peripheral tissues and / or inhibiting hepatic glucose production. Exemplary embodiments of the proposed method can preserve functional pancreatic β-cells, thereby slowing the progression to significant diabetes. The invention also relates to methods for screening diseases and conditions associated with insulin resistance (including conditions of glucose, carbohydrate, and lipid metabolism), and methods for preventing or treating diseases and conditions associated with insulin resistance, achieved by administering said antibodies or fragments thereof.

[0004] This application includes a biological sequence listing, which has been submitted by EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on July 1, 2014, named "43257o3013.txt", and is 203,678 bytes in size. Background of the Invention

[0006] Calcitonin gene-related peptide (CGRP) is produced as a multifunctional neuropeptide of 37 amino acids in length. Two forms of CGRP exist in humans: CGRP-α and CGRP-β, both exhibiting similar activities. Human CGRP-α and CGRP-β differ by 3 amino acids and originate from different genes. The CGRP peptide family also includes amyloidin, adrenaline, and calcitonin, each with different receptors and biological activities. (Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68 (2001)). Within the CGRP protein family, the amino acid residues at the putative receptor binding sites are conserved, but overall homology varies. For example, human CGRP shares 46% amino acid sequence identity with amyloidin, while human calcitonin shares 15% amino acid sequence identity with CGRP. (Wimalawansa, SJ, Endocrine Rev. 17(5):533-585 (1996)).

[0007] The biological effects of CGRP are mediated by the CGRP receptor (CGRP-R), which is composed of seven transmembrane components along with receptor-associated membrane proteins (RAMPs). CGRP-R further requires the activity of receptor component proteins (RCPs), which are essential for the efficient coupling of G proteins with adenylate cyclase and for cAMP production. Doods, H., Curr. Op. Invest. Drugs, 2(9):1261-68 (2001).

[0008] CGRP is distributed throughout the peripheral and central nervous systems and affects the cardiovascular, nervous, and endocrine systems. When CGRP is released from tissues such as the trigeminal nerve, it can lead to continuous activation and release of neuropeptides into the meninges, mediating neurogenic inflammation characterized by vasodilation, vascular leakage, and mast cell degradation. Durham, PL, New Eng. J. Med., 350(11):1073-75 (2004). CGRP is considered to play a significant role in the development of migraines. Elevated levels of identified CGRP in plasma from jugular venous blood have been shown during the headache phase of migraines, while other neuropeptides have not. Arulmozhi, DK et al., Vas. Pharma., 43:176-187 (2005). Furthermore, CGRP antagonism has been shown to be effective in treating migraines (Olesen et al., N Engl J Med. 2004, 11 March; 350(11):1104-10).

[0009] Besides neural tissue, CGRP receptors have been found in cardiovascular tissue, adrenal glands, pituitary glands, kidneys, pancreas, and bone. Wimalawansa, SJ, Endocrine Rev. 17(5):533-585 (1996). In in vitro studies using isolated pancreatic tissue, both CGRP and amyloidin inhibited insulin secretion, counteracted insulin-stimulated glycogen synthesis in a dose-dependent manner, and impaired the effect of insulin on isolated hepatocytes (Gomez-Foix et al., Biochem. J. 276:607-610, 1991). In addition, Leighton and Cooper (Nature, 335(6191):632-5, 1988) reported that rat CGRP-1 inhibited basal and insulin-stimulated glycogen synthesis in dissected rat soleus muscle in vitro.

[0010] Glucose homeostasis is maintained by balancing the glycogen synthesis and breakdown induced by the hormone glucagon with the tissue glucose utilization and uptake induced by the hormone insulin. The presence of glucose normally stimulates insulin production, which increases the transport of glucose into skeletal muscle, muscle cells, the brain, and fat cells. Insulin also typically inhibits lipid degradation within fat cells. In the prediabetic or earliest stages of type 2 diabetes, tissues develop insulin resistance, but pancreatic beta cells compensate by secreting increased levels of insulin. Eventually, as muscle and liver insulin resistance increases, the compensatory capacity of pancreatic beta cells is exhausted, necessitating exogenous insulin.

[0011] Poor insulin synthesis and glucose utilization, resulting in an inability to strictly regulate glucose homeostasis, can have profound metabolic and health-damaging effects. The most common is the development of persistent hyperglycemia (hyperglycemia), leading to insulin resistance and a diagnosis of type 2 diabetes. In 2011, 25,600,000 people aged 20 or older in the United States were diagnosed with diabetes, of whom 95% had type 2 diabetes. (Centers for Disease Control and Prevention. National Diabetes Fact Sheet, 2011. Atlanta, GA: Centers for Disease Control and Prevention, USDapartment of Health and Human Services; 2011.) The healthcare costs for people with diabetes are up to twice the average for those without diabetes due to the increased risk of heart attack, stroke, kidney complications, and neuropathy. Imperatore et al., Am J Epidemiol. 160(6):531-539 (2004). Without significant changes, the CDC predicts that by 2050, one in three adults in the United States will have diabetes. Boyle et al. Popul. Health Metr. 8:29 (2010). The total number of people diagnosed with diabetes worldwide in 2011 was estimated at 366 million, and this number is projected to increase to 552 million by 2030. (International Diabetes Foundation, IDF Diabetes Atlas, 5th edition)

[0012] The role of CGRP in glucose metabolism is not clearly defined in the literature. Studies on isolated hepatocytes have confirmed that CGRP and amylase inhibit the rate of insulin-stimulated glycogen synthesis (Gomez-Foix et al., Biochem. J. 276:607-610, 1991). Beaumont et al., Br J Pharmacol. July; 115(5):713-5 (1995) found that CGRP receptors do not mediate the role of muscle glucose metabolism. Tanaka et al., Exp. Clin Endocrinol Diabetes 121:280-285 (2013) demonstrated that anti-CGRP antibodies slightly prolonged and slightly altered the first phase of insulin secretion in a rat model; however, this study did not report whether the antibody cross-reacted with other calcitonin family peptides that could confound the results. Finally, previous U.S. patents have claimed to demonstrate that CGRP is an amylase agonist and that administration of CGRP peptides (as opposed to CGRP antagonists) can treat diabetes (see, for example, U.S. Patents Nos. 5,641,744 and 5,175,145). Invention Overview

[0014] In one aspect, this disclosure provides a method for increasing glucose utilization in the peripheral and / or liver in an individual in need, comprising administering to the individual an effective amount of a composition comprising an anti-human CGRP antibody or an antibody fragment.

[0015] In one aspect, this disclosure provides a method for reducing insulin resistance in an individual in need, comprising administering to the individual an effective amount of a composition comprising an anti-human CGRP antibody or an antibody fragment.

[0016] In one aspect, this disclosure provides a method for treating, preventing, or controlling obesity in an individual in need, comprising administering to the individual an effective amount of a composition comprising an anti-human CGRP antibody or an antibody fragment.

[0017] In one aspect, this disclosure provides a method for achieving sustained normal blood glucose levels in an individual in need, comprising administering to the individual an effective amount of a composition comprising an anti-human CGRP antibody or an antibody fragment.

[0018] In one aspect, this disclosure provides a method for increasing the ratio of lean tissue to body fat in an individual in need, comprising administering to the individual an effective amount of a composition comprising an anti-human CGRP antibody or an antibody fragment.

[0019] The proposed method can effectively treat or delay the onset of type II diabetes and / or obesity. For example, it can delay the need for exogenous insulin. The method can effectively prevent or slow the loss of pancreatic β-cells. For example, without being theoretically limited, it is believed that the method can allow pancreatic β-cells in insulin-resistant humans or non-human animals to rest, thereby preventing the loss of functional pancreatic β-cells.

[0020] The individual may have been diagnosed with prediabetes or may exhibit one or more risk factors for developing type 2 diabetes.

[0021] The individuals can be premenopausal, perimenopausal, menopausal, or postmenopausal.

[0022] The individual may exhibit one or more symptoms of prediabetes, such as fasting blood glucose levels between 100 mg / dL and 125 mg / dL; blood glucose levels between 140 mg / dL and 199 mg / dL two hours after ingesting a 75 g glucose solution or a glucose solution containing 1.75 g of glucose per kilogram of body weight up to a maximum dose of 75 g; and / or glycated hemoglobin between 5.7% and 6.4%.

[0023] The individual may exhibit one or more symptoms of diabetes, such as a fasting blood glucose level greater than 125 mg / dL; a blood glucose level of at least 200 mg / dL two hours after ingesting a 75 g glucose solution or a glucose solution containing 1.75 g of glucose per kilogram of body weight up to a maximum dose of 75 g; and / or a glycated hemoglobin level of at least 6.5%.

[0024] The individual may exhibit one or more risk factors for developing type 2 diabetes, such as a family history of type 2 diabetes; one or more parents or siblings previously diagnosed with type 2 diabetes; dyslipidemia; total blood triglyceride levels of at least 200 mg / dL; high-density lipoprotein levels of less than 35 mg / dL; obesity; and a body mass index greater than 25 kg / m². 2 History of gestational diabetes; previous birth weight of infants greater than 9 lbs; hypertension; systolic blood pressure of at least 140 mmHg; diastolic blood pressure of at least 90 mmHg; previous fasting blood glucose level of at least 99 mg / dL; vascular disease; polycystic ovary syndrome; or acanthosis nigricans.

[0025] The individual may have been diagnosed with type II diabetes.

[0026] The individual may be unresponsive to treatment with at least one compound selected from the group consisting of: GLP-1, exenatide-1, exendin, exendin analogs, exendin agonists, liraglutide, exenatide LAR, DPP-4 antagonists, GLP-1 receptor agonists, and another GLP-1 agonist; or the compound may be contraindicated for the individual.

[0027] The method may further include administering to the individual an antidiabetic or antiobesity agent other than an anti-human CGRP antibody or antibody fragment. The antidiabetic or antiobesity agent may comprise one or more of the following: amyloids, amyloid agonists, sulfonylureas, calcitonin, glucagon, PPAR-γ agonists, GLP-1 receptor agonists, dipeptidyl peptidase IV inhibitors, amyloid analogs, biguanides, dopamine D2 receptor agonists, megatitinides, alpha-glucosidase inhibitors, bile acid sequestrants for dyslipidemia, insulin secretagogues, insulin secretagogue analogs, insulin secretagogue agonists, gastrostatin (GIP), secretin peptide, insulin, SGLT2 inhibitors, glucose reabsorption inhibitors, fenofibrate, fibrates, anti-ghrelin antibodies or antibody fragments, fibroblast growth factor receptor (FGFR)-1 (IIIb), FGFR-1 (IIIc), antibodies or antibody fragments, and / or FGFR-4 (IIIc), anti-CD38 antibodies or antibody fragments, anti-MIC-1 antibodies, or MIC-1 binding fragments, metformin, or a combination of any of the foregoing.

[0028] In one exemplary embodiment, the antidiabetic agent is metformin.

[0029] The method described can effectively reduce weight.

[0030] The administered anti-human CGRP antibody or antibody fragment does not significantly increase insulin secretion in the body, for example, it does not significantly increase insulin secretion above normal physiological levels in the body, or it does not significantly increase insulin secretion relative to insulin secretion levels prior to administration of the anti-human CGRP antibody or antibody fragment.

[0031] The administered anti-human CGRP antibody or antibody fragment does not lead to an increased incidence of pancreatitis or an increased expression of markers or cytokines associated with pancreatic inflammation.

[0032] The composition may also contain a pharmaceutically acceptable carrier.

[0033] The anti-human CGRP antibody or antibody fragment may be administered to the individual at a dose between about 0.1 and 100.0 mg / kg of the recipient's body weight.

[0034] The anti-human CGRP antibody or antibody fragment may be a human antibody. The anti-human CGRP antibody or antibody fragment may be non-naturally occurring. The anti-human CGRP antibody or antibody fragment may be a non-naturally occurring antibody fragment. The anti-human CGRP antibody or antibody fragment may be a humanized antibody or a fragment thereof. The anti-human CGRP antibody or antibody fragment may be a chimeric antibody.

[0035] The anti-human CGRP antibody or antibody fragment can specifically bind to the same linear or conformational epitopes on the intact CGRP polypeptide or fragment thereof, and / or competitively bind to the same or overlapping linear or conformational epitopes, such as anti-human CGRP antibodies selected from the group consisting of: (a) Ab1, which contains V of SEQ ID NO:2 L V with SEQ ID NO:4 H (b)Ab2, which contains V of SEQ ID NO:12 L V with SEQ ID NO:14 H (c)Ab3, which contains V of SEQ ID NO:22 L V of SEQ ID NO:24 H ;(d)Ab4, which contains V of SEQ ID NO:32 L V of SEQ ID NO:34 H ; (e)Ab5, which contains V of SEQ ID NO:42 L V of SEQ ID NO:44 H (f)Ab6, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H ;(g)Ab7, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(h)Ab8, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H (i)Ab9, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(j)Ab10, which contains V of SEQ ID NO:72 L V with SEQ ID NO:74 H ;(k)Ab11, which contains V of SEQ ID NO:82 L V of SEQ ID NO:84 H; (l)Ab12, which contains V of SEQ ID NO:92 L V with SEQ ID NO:94 H ;(m)Ab13, which contains V of SEQ ID NO:102 L V with SEQ ID NO:104 H ; and (n)Ab14, which contains V of SEQ ID NO:112 L V of SEQ ID NO:114 H .

[0036] The 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 antibodies selected from the group consisting of: (a) Ab1, which comprises V of SEQ ID NO:2 L V with SEQ ID NO:4 H (b)Ab2, which contains V of SEQ ID NO:12 L V with SEQ ID NO:14 H (c)Ab3, which contains V of SEQ ID NO:22 L V with SEQ ID NO:24 H ;(d)Ab4, which contains V of SEQ ID NO:32 L V of SEQ ID NO:34 H (e)Ab5, which contains V of SEQ ID NO:42 L V of SEQ ID NO:44 H (f)Ab6, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H ;(g)Ab7, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(h)Ab8, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H (i)Ab9, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(j)Ab10, which contains V of SEQ ID NO:72 L V with SEQ ID NO:74 H ;(k)Ab11, which contains V of SEQ ID NO:82 LV with SEQ ID NO:84 H ; (l)Ab12, which contains V of SEQ ID NO:92 L V with SEQ ID NO:94 H ;(m)Ab13, which contains V of SEQ ID NO:102 L V with SEQ ID NO:104 H ; and (n)Ab14, which contains V of SEQ ID NO:112 L V with SEQ ID NO:114 H .

[0037] The anti-human CGRP antibody or antibody fragment may contain a polypeptide sequence that is 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) Ab1, which contains V of SEQ ID NO:2 L V with SEQ ID NO:4 H (b)Ab2, which contains V of SEQ ID NO:12 L V with SEQ ID NO:14 H (c)Ab3, which contains V of SEQ ID NO:22 L V with SEQ ID NO:24 H ;(d)Ab4, which contains V of SEQ ID NO:32 L V of SEQ ID NO:34 H (e)Ab5, which contains V of SEQ ID NO:42 L V of SEQ ID NO:44 H (f)Ab6, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H ;(g)Ab7, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(h)Ab8, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H (i)Ab9, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(j)Ab10, which contains V of SEQ ID NO:72 L V with SEQ ID NO:74H ;(k)Ab11, which contains V of SEQ ID NO:82 L V with SEQ ID NO:84 H ; (l)Ab12, which contains V of SEQ ID NO:92 L V with SEQ ID NO:94 H ;(m)Ab13, which contains V of SEQ ID NO:102 L V with SEQ ID NO:104 H ; and (n)Ab14, which contains V of SEQ ID NO:112 L V with SEQ ID NO:114 H .

[0038] The anti-human CGRP antibody or antibody fragment comprises an antibody selected from the group consisting of: (a) Ab1, which contains V of SEQ ID NO:2. L V with SEQ ID NO:4 H (b)Ab2, which contains V of SEQ ID NO:12 L V with SEQ ID NO:14 H (c)Ab3, which contains V of SEQ ID NO:22 L V with SEQ ID NO:24 H ;(d)Ab4, which contains V of SEQ ID NO:32 L V of SEQ ID NO:34 H (e)Ab5, which contains V of SEQ ID NO:42 L V of SEQ ID NO:44 H (f)Ab6, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H ;(g)Ab7, which contains V of SEQ ID NO:62 L V of SEQ ID NO:64 H ;(h)Ab8, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H (i)Ab9, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(j)Ab10, which contains V of SEQ ID NO:72 L V with SEQ ID NO:74 H;(k)Ab11, which contains V of SEQ ID NO:82 L V with SEQ ID NO:84 H ; (l)Ab12, which contains V of SEQ ID NO:92 L V with SEQ ID NO:94 H ;(m)Ab13, which contains V of SEQ ID NO:102 L V with SEQ ID NO:104 H ; and (n)Ab14, which contains V of SEQ ID NO:112 L V with SEQ ID NO:114 H .

[0039] 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 Fab, F(ab')2, scFv, IgNar, or MetMab, or another monovalent antibody fragment.

[0040] In another aspect, this disclosure provides a composition suitable for use with, for example, the methods described herein as cited in the preceding paragraphs, the composition comprising an effective amount of an anti-human CGRP antibody or antibody fragment, and an antidiabetic or anti-obesity agent other than the anti-human CGRP antibody or antibody fragment. The anti-human CGRP antibody or antibody fragment may be one of those described herein, for example, specifically binding to the same linear or conformational epitope on the intact CGRP polypeptide or a fragment thereof, competitively binding to the same or overlapping linear or conformational epitopes on the intact CGRP polypeptide or a fragment thereof, and, like anti-human CGRP antibodies selected from the group consisting of, having 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% of the same polypeptide sequence as anti-human CGRP antibodies selected from the group consisting of, or comprising anti-human CGRP antibodies selected from the group consisting of: (a) Ab1, which comprises V of SEQ ID NO:2. L V with SEQ ID NO:4 H (b)Ab2, which contains V of SEQ ID NO:12 L V with SEQ ID NO:14 H (c)Ab3, which contains V of SEQ ID NO:22 L V with SEQ ID NO:24 H ;(d)Ab4, which contains V of SEQ ID NO:32 L V of SEQ ID NO:34H (e)Ab5, which contains V of SEQ ID NO:42 L V of SEQ ID NO:44 H (f)Ab6, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H ;(g)Ab7, which contains V of SEQ ID NO:62 L V of SEQ ID NO:64 H ;(h)Ab8, which contains V of SEQ ID NO:52 L V with SEQ ID NO:54 H (i)Ab9, which contains V of SEQ ID NO:62 L V with SEQ ID NO:64 H ;(j)Ab10, which contains V of SEQ ID NO:72 L V with SEQ ID NO:74 H ;(k)Ab11, which contains V of SEQ ID NO:82 L V with SEQ ID NO:84 H ; (l)Ab12, which contains V of SEQ ID NO:92 L V with SEQ ID NO:94 H ;(m)Ab13, which contains V of SEQ ID NO:102 L V with SEQ ID NO:104 H ; and (n)Ab14, which contains V of SEQ ID NO:112 L V with SEQ ID NO:114 H .

[0041] The antidiabetic or antiobesity agent comprises one or more of the following: amyloids, amyloid agonists, sulfonylureas, calcitonin, glucagon, PPAR-γ agonists, GLP-1 receptor agonists, dipeptidyl peptidase IV inhibitors, amyloid analogs, biguanides, dopamine D2 receptor agonists, megatitinides, alpha-glucosidase inhibitors, bile acid sequestrants for dyslipidemia, insulin secretagogues, insulin secretagogue analogs, insulin secretagogue agonists, gastrostatin (GIP), secretin peptide, insulin, SGLT2 inhibitors, glucose reabsorption inhibitors, fenofibrate, fibrates, metformin, anti-ghrelin antibodies or antibody fragments, fibroblast growth factor receptor (FGFR)-1 (IIIb), FGFR-1 (IIIc), antibodies or antibody fragments, and / or FGFR-4 (IIIc), anti-CD38 antibodies or antibody fragments, anti-MIC-1 antibodies or MIC-1 binding fragments, or combinations of any of the foregoing. For example, the antidiabetic or anti-obesity agent may contain metformin. Brief description of the attached diagram

[0043] Figure 1A-D. Blood glucose and plasma insulin levels before and after treatment. Results are expressed as mean ± SEM. In the case of ANOVA univariate plus Dunnett's post-hoc test, ##p<0.01, relative to the medium. A: Blood glucose measured before treatment, 18 h after treatment with the medium, Ab14, and metformin, and 42 h after treatment with the medium and Ab14, under fed conditions. B: Plasma insulin measured before treatment, 18 h after treatment with metformin, and 42 h after treatment with the medium and Ab14, under fed conditions. C: HOMA-IR (insulin resistance index = glucose (mM) × insulin (μU / mL) / 22.5) calculated before treatment, 18 h after treatment with metformin, and 42 h after treatment with the medium and Ab14. D: Blood glucose measured under fasting conditions just before clamping (24 h after treatment with metformin and 48 h after treatment with the medium and Ab14). Legend: The leftmost bar in each group represents the medium; the middle bar in each group represents Ab14 treatment; the rightmost bar in each group represents metformin treatment.

[0044] Figures 2A-C. Development of glucose infusion rate during clamping procedure (A), mean blood glucose during steady state (B), and plasma insulin level at the end of clamping procedure (C). Results are expressed as mean ± SEM. A: In the case of ANOVA bivariate plus Bonferroni's post-test, *p<0.05, **p<0.01, ***p<0.001, relative to the mediator. Legend for Figure 2A: Top line, metformin treatment; Middle line, Ab14 treatment; Bottom line, mediator treatment (at time 180 min). Legend for Figures 2B-2C: Leftmost bar in each group, mediator; Middle bar in each group, Ab14 treatment; Rightmost bar in each group, metformin treatment.

[0045] Figure 3. Measured glucose flux. Results are expressed as mean ± SEM. In the case of ANOVA with Dunnett post-hoc test, #p < 0.05, relative to the medium. The clamp procedure was performed under 6-hour fasting conditions. 0.3 U / kg / h insulin and 3 H-glucose perfusion for 180 minutes. Mean values ​​of glucose infusion rate, systemic turnover, hepatic glucose production (HGP), glycolysis, and glycogen synthesis were calculated corresponding to steady-state values ​​between 140 and 180 minutes. Legend: Leftmost bar in each group, mediator; middle bar in each group, Ab14 treatment; rightmost bar in each group, metformin treatment.

[0046] Figures 4A-C. Tissue-specific glucose utilization in vivo. Results are expressed as mean ± SEM. In a one-way ANOVA with post-hoc Dunnett test, #p<0.05, ##p<0.01, relative to the medium. A: Glucose utilization in epididymal white adipose tissue (EWAT), inguinal white adipose tissue (IWAT), and skin (as negative control). B: Glucose utilization in mixed lateral extensor digitorum longus (VL) and glycolytic extensor digitorum longus (EDL). C: Glucose utilization in oxidized soleus muscle and apical region of the heart. Legend: Leftmost bar in each group, medium; middle bar in each group, Ab14 treatment; rightmost bar in each group, metformin treatment.

[0047] Figure 5. Mean body weight over time in animals fed a high-fat, high-fructose diet or in control animals fed a normal diet. Legend: Top line, high-fat, high-fructose diet; bottom line, control diet.

[0048] Figure 6. Weight gain over time for the animal groups shown in Figure 5. Top line: high-fat, high-fructose diet; bottom line: control diet. Legend: Top line, high-fat, high-fructose diet; bottom line, control diet.

[0049] Figure 7. Weight gain over time in animals treated with Ab14 (10, 30, or 100 mg / kg) or metformin on a high-fat diet, in animals treated with a carrier, and in control animals on a control diet. Treatment was administered on day 0. The lines on the graph, from lowest to highest on day 7, represent: normal diet (NC) plus carrier; high-fat diet (HFD) plus metformin; HFD plus Ab14 30 mg / kg; HFD plus Ab14 10 mg / kg; HFD plus carrier; HFD plus Ab14 100 mg / kg.

[0050] Figure 8A. Food intake of the animal groups shown in Figure 7. The lines on the graph, from lowest to highest on day 7, are: High-fat diet (HFD) plus metformin; HFD plus Ab14 30 mg / kg; HFD plus Ab14 10 mg / kg; HFD plus Ab14 100 mg / kg; HFD plus a carrier; Normal food (NC) plus a carrier.

[0051] Figure 8B. Cumulative food intake of the animal groups shown in Figure 7. Legend: The bars from left to right are: Normal food (NC) plus a medium; High-fat diet (HFD) plus a medium; HFD plus Ab14 10 mg / kg; HFD plus Ab14 30 mg / kg; HFD plus Ab14 100 mg / kg; HFD plus metformin.

[0052] Figure 9. Fasting blood glucose levels in animals treated with Ab14 (10, 30, or 100 mg / kg) or metformin on a high-fat diet, as well as in animals treated with the drug and in control animals on a control diet. Treatment was administered on day 0. Legend: The order of the bars from left to right is the same as in Figure 8B.

[0053] Figure 10. Fasting plasma insulin levels in animals treated with Ab14 (10, 30, or 100 mg / kg) or metformin on a high-fat diet, as well as in animals treated with the drug and in control animals on a control diet. Treatment was administered on day 0. Legend: The order of the bars from left to right in each group is the same as in Figure 8B.

[0054] Figure 11. Plasma insulin (top) and C-peptide levels (bottom left and bottom right) before and during clamping, 15 days after treatment with Ab14 or metformin. Animals were fed a high-fat diet for 6 weeks prior to treatment. Legend: The order of the bars from left to right is the same as in Figure 8B.

[0055] Figure 12. HOMA-IR of animals treated with Ab14 (10, 30, or 100 mg / kg) or metformin on a high-fat diet, animals treated with the carrier, and control animals on a control diet. Treatment was administered on day 0. Legend: The order of the bars from left to right is the same as in Figure 8B.

[0056] Figure 13. Glucose infusion rates after glucose clamping performed on animals treated with Ab14 or metformin for 15 days, as well as on animals treated with a mediator and on a control diet. Animals were on a high-fat diet for 6 weeks prior to treatment. Glucose clamping was performed at two different insulin infusion rates (5 mU / kg / min, reaching steady state at approximately 70–100 min, and 15 mU / kg / min, reaching steady state at approximately 170–210 min). Legend: Circle, normal diet; Medium square, high-fat diet (HFD) plus mediator; Upward-pointing triangle, HFD plus Ab14 10 mg / kg; Downward-pointing triangle, HFD plus Ab14 30 mg / kg; Rhombus, HFD plus Ab14 100 mg / kg; Large square, HFD plus metformin. The error bars shown are the mean plus or minus the SEM.

[0057] Figure 14. Mean glucose infusion rate during steady state in the glucose clamping experiment shown in Figure 13. Glucose infusion rates show low and high insulin infusion rates (5 mU / kg / min, reaching steady state at approximately 70–100 min, and 15 mU / kg / min, reaching steady state at approximately 170–210 min). The order of the bars in each group is as shown in Figure 8B.

[0058] Figure 15. Mean glucose flux during the glucose clamping experiment shown in Figure 13. This indicates a low insulin infusion rate (5 mU / kg / min), reaching steady state at approximately 70–100 min. Legend: The order of the bars from left to right in each group is: High-fat diet (HFD) plus a mediator; HFD plus Ab14 10 mg / kg; HFD plus Ab14 30 mg / kg; HFD plus Ab14 100 mg / kg; HFD plus metformin.

[0059] Figure 16. Average glucose flux during the glucose clamping experiment shown in Figure 13. This indicates a high insulin infusion rate (15 mU / kg / min), reaching steady state at approximately 170–210 min. Legend: The order of the bars in each group is the same as in Figure 15.

[0060] Figure 17. Mean toxicokinetics of anti-CGRP antibody (specifically, Ab6) following intravenous bolus injection into male Sprague-Dawley rats. Plasma concentrations over 168 hours (7 days) are shown, supporting the weekly dosing regimen performed in the examples below. Legend: Square marker, Ab14 10 mg / kg / week; triangle pointing upwards, Ab14 30 mg / kg / week; rhombus marker, Ab14 100 mg / kg / week.

[0061] Figure 18 AD. 6-hour fasting HOMA-IR (A), blood glucose (B), plasma insulin (C), and body weight (D) of 8-week-old ZDF rats. Results are expressed as mean ± SEM.

[0062] #p<0.05; ###p<0.001, relative to the medium ZDF (Mann Whitney). Figure 18 The order of the bars in AD (from left to right) is as follows: (1) lean ZDF rats treated with mediator 1 and mediator 2; (2) ZDF rats treated with mediator 1 and mediator 2; (3) ZDF rats treated with Ab14 20 mg / kg / week and mediator 2; (4) ZDF rats treated with Ab14 60 mg / kg / week and mediator 2; (5) ZDF rats treated with mediator 1 and metformin (“met”) 200 mg / kg / day; (6) ZDF rats treated with mediator 1 and pioglitazone 10 mg / kg / day; (7) ZDF rats treated with Ab14 20 mg / kg / week and metformin 200 mg / kg / day; and (8) ZDF rats treated with Ab14 60 mg / kg / week and metformin 200 mg / kg / day.

[0063] Figure 19A -B. Tracking of body weight (A) and weight gain (B). Results are expressed as mean ± SEM.

[0064] $p<0.05( Figure 19A Rats treated with pioglitazone on day 8; rats treated with Ab1460 mg / kg / week + metformin on day 28; Figure 19B Rats treated with Ab1460 mg / kg / week + metformin on days 22 and 25); $$p<0.01( Figure 19B Rats treated with Ab1460 mg / kg / week + metformin on day 28; $$$p<0.001, relative to the mediator ZDF ( Figure 19ARats treated with pioglitazone at all time points between days 11 and 28; ZDF lean rats treated with the medium at all time points (two-factor ANOVA + post-hoc test of ponferroni).

[0065] Figure 20A -B. Tracking of food intake (A) and cumulative food intake (B). Results are expressed as mean ± SEM. Figure 20B The order of the central columns is as follows Figure 18 The same as in AD.

[0066] $p<0.05( Figure 20A Rats treated with pioglitazone on days 20 and 22; $$p<0.01( Figure 20A Rats treated with pioglitazone on day 15); $$$p<0.001, relative to the mediator ZDF (Figure 20a: mediator-treated lean ZDF rats at all time points) (two-factor ANOVA + post-hoc test of pamprolium).

[0067] ##p<0.01, relative to the medium ZDF (Mann-Whiteney)

[0068] Figures 21A-D. Fasting blood glucose (A), plasma insulin (B), HOMA-IR (C), and C-peptide (D) at 6 hours (day 0) or overnight (days 12, 19, and 26). Results are expressed as mean ± SEM. The order of the bars in each group within Figures 21A-D is as follows. Figure 18 The same as in AD.

[0069] #p<0.05; ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0070] *p<0.05; **p<0.01; ***p<0.001, relative to the medium ZDF (Kruskal-Wallis test + Dunn's post test))

[0071] ++p<0.01; compared to the metformin group and the AB14 20mg / kg + metformin group (one-way ANOVA + Newman-Keuls post-test)

[0072] $p<0.05; $$p<0.01, relative to the medium ZDF (two-factor ANOVA + Pomferroni post-hoc test)

[0073] Figure 22 Fructosamine levels. Results are expressed as mean ± SEM. Figure 22 The order of the columns in each group is as follows: Figure 18 The same as in AD.

[0074] ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0075] **p<0.01, relative to the medium ZDF (Kruskal-Wallis test + Dunn post-hoc test)

[0076] $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pompherelli post-hoc test)

[0077] Figure 23 HbA1c levels. Results are expressed as mean ± SEM. Figure 23 The order of the columns in each group is as follows: Figure 18 The same as in AD.

[0078] ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0079] ***p<0.001, relative to the medium ZDF (Kruskal-Wallis test + Dunn post-hoc test)

[0080] +p<0.05; relative to the AB14 60mg / kg group (one-way ANOVA + Newman-Coyles post-hoc test)

[0081] $p<0.05; $$p<0.01; $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pompheroni post-hoc test)

[0082] Figure 24 AB. Plasma triglyceride (A) and free fatty acid (B) levels under fasting conditions at 6 hours (day 0) or overnight (days 12, 19, and 26). Results are expressed as mean ± SEM. Figure 24 The order of the bars in each group within AB is as follows: Figure 18 The same as in AD.

[0083] ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0084] *p<0.05; **p<0.01; ***p<0.001, relative to the medium ZDF (Kruskal-Wallis test + Dunn post-hoc test)

[0085] +p<0.05; ++p<0.01; +++p<0.001, relative to the metformin group or the Ab1460mg / kg + metformin group (one-way ANOVA + Newman-Coyles post-hoc test).

[0086] $p<0.05; $$p<0.01; $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pompheroni post-hoc test)

[0087] Figure 25A -C. Plasma total cholesterol (A), HDL-cholesterol (B), and non-HDL-cholesterol (C) levels. Results are expressed as mean ± SEM. Figure 25A The order of the bars in each group within -C is as follows: Figure 18 The same as in AD.

[0088] #p<0.05; ##p<0.01; ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0089] *p<0.05; **p<0.01; ***p<0.001, relative to the medium ZDF (one-way ANOVA + Dunnett post-hoc test)

[0090] +p<0.05; ++p<0.01, relative to the metformin group or the Ab14+metformin group (one-way ANOVA + Newman-Coyle post-hoc test).

[0091] $$p<0.01, relative to the medium ZDF (two-factor ANOVA + Pomferroni post-hoc test)

[0092] Figure 26A -C. Plasma total cholesterol (A), HDL-cholesterol (B), and non-HDL-cholesterol (C) levels relative to day 0. Results are expressed as mean ± SEM. Figure 26A The order of the bars in each group within -C is as follows: Figure 18 The same as in AD.

[0093] #p<0.05; ##p<0.01; ###p<0.001, relative to the medium ZDF (unpaired t-test)

[0094] *p<0.05; **p<0.01, relative to the medium ZDF (one-way ANOVA + Dunnett post-hoc test)

[0095] +p<0.05; relative to the metformin group (one-way ANOVA + Neumann-Coyle post-hoc test)

[0096] $p<0.05; $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pomferroni post-hoc test)

[0097] Figure 27A-C. Oral glucose tolerance test on day 26 under overnight fasting conditions (A), area under the curve (AUC) calculated from blood glucose measured at T0 (B) and area under the curve (C) calculated from relative values ​​relative to T0. Results are expressed as mean ± SEM. Figure 27B The order of the bars in each group within -C is as follows: Figure 18 The same as in AD.

[0098] $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pompheroni post-hoc test) ( Figure 27A ZDF lean rats treated with the medium and rats treated with pioglitazone at all time points.

[0099] ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0100] ***p<0.001, relative to the medium ZDF (Kruskal-Wallis test + Dunn post-hoc test)

[0101] Figure 28 AB. Plasma insulin (A) and C-peptide (B) levels on day 26 during the oral glucose tolerance test. Results are expressed as mean ± SEM. Figure 28 The order of the bars in each group within AB is as follows: Figure 18 The same as in AD.

[0102] ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0103] *p<0.05, relative to the medium ZDF (Kruskal-Wallis test + Dunn post-hoc test)

[0104] $$p<0.01; $$$p<0.001, relative to the medium ZDF (two-factor ANOVA + Pompherelli post-hoc test)

[0105] Figure 29 A. Relative expression of T-60 plasma insulin (A) and C-peptide (B) levels on day 26 during the oral glucose tolerance test. Results are expressed as mean ± SEM. Figure 29 The order of the bars in each group within AB is as follows: Figure 18 The same as in AD.

[0106] #p<0.05; ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0107] *p<0.05, relative to the medium ZDF (one-way ANOVA + Dunnett post-hoc test)

[0108] +p<0.05; relative to the metformin group (one-way ANOVA + Neumann-Coyle post-hoc test)

[0109] $p<0.05; $$p<0.01, relative to the medium ZDF (two-factor ANOVA + Pomferroni post-hoc test)

[0110] Figures 30A-C. Pancreatic contents: proinsulin (A), insulin (B), and proinsulin / insulin ratio (C). Results are expressed as mean ± SEM. The order of the bars in Figures 30A-C (from left to right) is: lean ZDF rats treated with mediator 1 and mediator 2; ZDF rats treated with mediator 1 and mediator 2; ZDF rats treated with Ab14 60 mg / kg / week and mediator 2; ZDF rats treated with mediator 1 and metformin 200 mg / kg / day; and ZDF rats treated with Ab14 60 mg / kg / week and metformin 200 mg / kg / day.

[0111] #p<0.05; ###p<0.001, relative to the medium ZDF (Mann-Whiteney)

[0112] *p<0.05, relative to the medium ZDF (one-way ANOVA + Newman-Coyle post-hoc test)

[0113] Figure 31 Immunohistochemical analysis of the pancreas: insulin labeling and quantification. Figure 31 The order of the middle (from left to right) bars is as follows: lean ZDF rats treated with mediator 1 and mediator 2; ZDF rats treated with mediator 1 and mediator 2; ZDF rats treated with Ab14 60 mg / kg / week and mediator 2; ZDF rats treated with mediator 1 and metformin 200 mg / kg / day; and ZDF rats treated with Ab14 60 mg / kg / week and metformin 200 mg / kg / day. Invention Details

[0115] The inventors have discovered that anti-CGRP antibodies significantly increase glucose utilization in peripheral muscle compared to metformin, without significantly increasing glucose utilization in white adipose tissue. Furthermore, the anti-human CGRP antibodies described herein increase glucose utilization in the heart, while metformin decreases it. Additionally, the anti-human CGRP antibodies described herein inhibit hepatic glucose production, with effects similar to those obtained from metformin administration.

[0116] The anti-human CGRP antibody Ab14 (a potent functional antagonist) was evaluated in preclinical animal models of normal and altered glucose metabolism to determine its effects on insulin sensitivity and glycemic control in the following rats: normal rats (Example 1), obese (DIO) rats induced with a high-fat / high-fructose diet for 6 weeks to induce metabolic syndrome (Example 2), and Zucker diabetic obese (ZDF) rats that progressed from a pre-diabetic (hyperinsulinemia, normal glycemia) state to an overtly diabetic (hypoinsulinemia, hyperglycemia) state (Example 3).

[0117] In Example 1, Ab14 was used in a hyperinsulinemia-eukaryotic clamping study to determine its effects on systemic insulin sensitivity and tissue-specific insulin sensitivity in normal rats with normal blood glucose levels, normal insulinemia, and normal systemic and tissue-specific insulin sensitivity. Normal rats were administered Ab14 intravenously at a single dose of 100 mg / kg 48 hours prior to the hyperinsulinemia-eukaryotic clamping procedure. Assessment of plasma glucose and insulin levels measured just before the clamping procedure revealed that, relative to the mediator-treated control, Ab14 reduced plasma insulin levels without altering plasma glucose levels. The resulting decrease in HOMA-IR indicated improved systemic insulin sensitivity.

[0118] The hyperinsulinemia-euglucose clamp procedure confirmed this improvement in systemic insulin sensitivity induced by CGRP antagonism, with both glucose infusion rate and systemic glucose conversion (utilization) being increased at steady state compared to the mediator-treated control. The increased glucose infusion rate and systemic glucose conversion, combined with a constant insulin infusion, indicate increased systemic insulin sensitivity.

[0119] Consistent with increased glucose infusion rate and systemic glucose turnover, Ab14, relative to the mediator-treated control, increased hepatic glucose utilization related to glycolysis and glycogen synthesis, and decreased hepatic glucose production. These observations suggest that CGRP antagonism increases hepatic insulin sensitivity, leading to increased hepatic utilization, supplying more internalized glucose for energy production and storage, while simultaneously inhibiting re-hepatic glucose production.

[0120] CGRP antagonism also increases glucose utilization in both glycolytic and oxidative skeletal muscle (lateral extensor digitorum, indicating mixed glycolysis plus oxidation; extensor digitorum longus, indicating glycolysis; and soleus, indicating oxidation). The greatest increase in glucose utilization occurs in the mixed-metabolism lateral extensor digitorum. These observations suggest that increased skeletal muscle insulin sensitivity is caused by CGRP antagonism. CGRP antagonism also increases cardiac glucose utilization. In contrast, glucose utilization in visceral or subcutaneous fat deposits is unaffected, indicating that CGRP antagonism does not substantially increase insulin sensitivity in white adipose tissue.

[0121] As mentioned above, the animals used in this study were normoglycemic rats with normal systemic and hepatic tissue-specific insulin sensitivity, making it more difficult to demonstrate improvements in insulin sensitivity in these animals. Therefore, although improvements in some individual endpoints assessed in this study did not reach statistical significance, the observed tendency in the same direction does suggest, as mentioned, that increased research motivation can lead to statistical significance for additionally measured parameters. Furthermore, because results from hyperinsulinemia-eukaryotic clamp studies typically performed in laboratory animals are highly convertible to those performed in a clinical setting, these observations suggest the potential of CGRP antagonism to improve systemic and tissue-specific insulin sensitivity in humans.

[0122] In Example 2, the effect of chronic Ab14-induced CGRP antagonism on hepatic and peripheral insulin sensitivity in insulin-resistant animals was assessed in rats with hyperinsulinemia induced by a long-term high-fat / high-fructose diet and those that were insulin-resistant but not hyperglycemic. Metabolic syndrome was induced in rats by feeding them a diet containing 69% fat and 14% fructose for 7 weeks prior to compound administration. At the end of the 7-week dietary treatment period, rats continued to receive a high-fat / high-fructose diet and were given Ab14 intravenously once weekly at doses of 0 mg / kg (mediator), 10 mg / kg, 30 mg / kg, or 100 mg / kg for 2 weeks.

[0123] When compared with the mediator-treated control group on a high-fat diet, the CGRP antagonism had no effect on food intake or weight, indicating that the effect of the CGRP antagonism on the additional parameters assessed below was not due to calorie restriction or weight loss.

[0124] At the end of the treatment period, all doses of Ab14 reduced HOMA-IR relative to the cartel-treated control, indicating improved systemic insulin sensitivity. This decrease in HOMA-IR was primarily due to the reduction in plasma insulin levels observed at all doses of Ab14. This reduction in plasma insulin was a result of decreased insulin production, not increased insulin degradation, as both plasma C-peptide (a byproduct of pancreatic insulin synthesis) and plasma insulin were reduced. While the two lower doses of Ab14 only reduced plasma glucose levels, the 100 mg / kg dose of Ab14 resulted in a significant reduction in plasma glucose levels relative to the cartel-treated control.

[0125] Immediately following the last day of treatment, a two-step hyperinsulinemia-euglucose clamping procedure was performed, first with a physiological glucose infusion followed by a supraphysiological insulin infusion. Compared to the mediator-treated control, all three doses of Ab14 increased steady-state glucose infusion rate after both physiological and supraphysiological insulin infusion concentrations. This was consistent with improvements in systemic insulin sensitivity. Compared to the mediator-treated control, all three doses of Ab14 also increased systemic glucose conversion (utilization), increased hepatic glucose utilization from glycolysis and glycogen synthesis, and inhibited hepatic glucose production after physiological insulin infusion concentrations, consistent with improvements in both systemic and hepatic insulin sensitivity. Hepatic glucose production was also completely prevented after supraphysiological insulin infusion concentrations.

[0126] The similarity between the acute effects of CGRP antagonism in normal rats (Example 1) and the chronic effects in normoglycemic, hyperinsulinemic, and insulin-resistant rats (Example 2) suggests the potential of CGRP antagonism for long-term treatment of established insulin resistance. Furthermore, as mentioned above, because the results of hyperinsulinemic-eukaryotic clamp studies typically performed in laboratory animals are highly translatable into clinical settings, these observations suggest the potential of CGRP antagonism to improve systemic and tissue-specific insulin sensitivity in insulin-resistant humans with prediabetes or metabolic syndrome. Finally, the ability of CGRP antagonism to reduce plasma glucose levels in these normoglycemic animals (although only at the highest dose evaluated) suggests the potential of CGRP antagonism to also reduce plasma glucose levels in hyperglycemic patients.

[0127] In Example 3, the effect of chronic administration of Ab14 on glycemic control was evaluated in ZDF rats that progressed from a prediabetic (hyperinsulinemia, normal blood glucose) state to a fully diabetic (hypoinsulinemia, hyperglycemia) state. These animals developed prediabetes before 7 weeks of age, characterized by significant hyperinsulinemia to compensate for their developing insulin resistance, but with mild or no hyperglycemia. They then rapidly progressed to fully diabetic, characterized by hypoinsulinemia and significant hyperglycemia, before 10–12 weeks of age due to pancreatic β-cell depletion.

[0128] ZDF rats were selected at 8 weeks of age based on their HOMA-IR and treated weekly with 20 or 60 mg / kg Ab14 for 28 days. In addition to evaluating the effect of the CGRP antagonist Ab14 on glycemic control in this animal model, the effect of combining CGRP antagonism with the commercially available drug metformin (200 mg / kg / day) was also evaluated. Metformin alone partially prevented the rise in fasting blood glucose, partially prevented the decrease in plasma insulin and C-peptide levels, completely prevented the decrease in pancreatic proinsulin levels, partially prevented the decrease in pancreatic insulin levels, and reduced islet vacuolar formation, hyperplasia, and fibrosis, to a degree similar to that described above for high-dose Ab14. However, the combination of Ab14 and metformin produced substantially greater effects than either compound alone in preventing the rise in fasting blood glucose, the decrease in plasma insulin and C-peptide levels, the decrease in pancreatic proinsulin and insulin levels, and the reduction in islet fibrosis. This suggests that CGRP antagonists such as Ab14 can enhance the effects of metformin.

[0129] Furthermore, after 28 days of treatment, compared to the control group treated with the mediator, the combination of Ab14 and metformin resulted in a significant decrease in HbA1c (a marker of hemoglobin glycation) levels and a smaller decrease in fructosamine levels. This is consistent with the fact that the combination of Ab14 and metformin produces a greater reduction in plasma glucose levels than either agent alone. These results suggest that the combination of a CGRP antagonist and metformin will favorably influence hyperglycemia-mediated diabetic complications.

[0130] Similarly, during the oral glucose tolerance test (oGTT) performed on day 26 of the study, the combination of high-dose Ab14 and metformin showed improvements in glucose variability and glucose AUC compared to animals treated with the mediator. Furthermore, because β-cell destruction in these animals had progressed beyond their ability to cope with glucose stimulation and increase insulin secretion before day 26 of the study, it was expected that if the oGTT had been performed two weeks earlier or at another time point before complete β-cell destruction, a greater improvement in glucose variability and glucose AUC would have been observed with the Ab14 plus metformin combination.

[0131] The ZDF rats used in Example 3 are a very severe model of diabetes progression, which progresses rapidly from an insulin-resistant prediabetic state to overt diabetes with complete β-cell destruction in just a few weeks. This limits the opportunity to assess the regulation of disease progression, making it difficult to demonstrate compound-related improvements in disease progression and β-cell protection in these animals. Therefore, any evidence that the CGRP antagonist Ab14, as outlined above, moderately delays disease progression suggests that it may also affect disease progression clinically. Furthermore, the ability to improve overall therapeutic efficacy through combination therapy with CGRP antagonism and metformin also demonstrates the clinical efficacy of combination therapy.

[0132] The results of the embodiments in this application demonstrate that CGRP antagonism has the ability to improve systemic insulin sensitivity, hepatic insulin sensitivity, and skeletal muscle insulin sensitivity. These improvements can be observed acutely or persistently in normal animals with normal insulin blood levels, normal blood glucose levels, and normal insulin sensitivity, as well as in insulin-resistant animals with hyperinsulinemia but not yet hyperglycemia. These results suggest that CGRP antagonism should reduce insulin resistance present in patients with metabolic syndrome, prediabetes, or other prediabetic conditions, and that CGRP antagonism may be able to slow the progression of these conditions to full-blown diabetes.

[0133] Furthermore, the ability of the CGRP antagonist Ab14 to reduce hyperinsulinemia in insulin-resistant animals by decreasing insulin secretion suggests that Ab14 may have a protective effect on pancreatic β-cells by resting the pancreas in insulin-resistant animals. Clinically, this could further delay the progression of metabolic syndrome, prediabetes, and other prediabetic conditions to overt diabetes.

[0134] Furthermore, the ability of Ab14 to lower plasma glucose levels in insulin-resistant, hyperinsulinemic but normal-glucose rats, to slow the progression of prediabetes to marked diabetes in ZDF rats, and to maintain lowered plasma glucose levels in markedly diabetic animals with little or no residual capacity to increase insulin production, suggests that the antagonistic effects of CGRP may have the ability to influence not only the disease progression of prediabetes as outlined above, but also the progression of marked diabetes.

[0135] Therefore, the results of these studies together clearly demonstrate that CGRP antagonists such as Ab14 can advantageously influence insulin resistance and abnormal glycemic control in patients with prediabetes as well as those with developing or obvious diabetes in a clinical context.

[0136] Finally, the ability of the CGRP antagonist Ab14 to enhance the in vivo effects of metformin in ZDF rats suggests that the Ab14-metformin combination therapy has the potential to be clinically effective for treating patients with prediabetes, patients with developing diabetes, and patients with established diabetes, compared to Ab14 or metformin alone.

[0137] definition

[0138] It should be understood that the present invention is not limited to the specific methodologies, schemes, cell lines, animal species or genus, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is defined solely by the appended claims. Unless the context clearly specifies otherwise, the singular forms "a / a kind," "and," and "described" as used herein include the plural objects referred to. Thus, for example, reference to "a cell" includes a plurality of such cells, and reference to "described protein" includes reference to one or more proteins and their equivalents known to those skilled in the art, and so on. Unless explicitly stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0139] Calcitonin gene-related peptide (CGRP): As used herein, CGRP contains not only the following Homo sapiens CGRP-α and Homo sapiens CGRP-β amino acid sequences, which are available from American Peptides (Sunnyvale CA) and Bachem (Torrance, CA):

[0140] CGRP-α :ACDTATCVTHRLAGLLSRSGGVVK NNFVPTNVGSKAF-NH2 (SEQ ID NO:281), wherein the N-terminal phenylalanine is amidated. Unless otherwise specified, references to "CGRP" generally refer to CGRP-α. CGRP-α is interchangeably referred to as αCGRP or α-CGRP.

[0141] CGRP-β :ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-NH2 (SEQ ID NO:282), wherein the N-terminal phenylalanine is amidated; and also includes any membrane-bound form of these CGRP amino acid sequences, as well as mutants (mutant proteins), splice variants, isomers, orthogonal homologs, homologs, and variants of this sequence. CGRP-β is interchangeably referred to as βCGRP or β-CGRP.

[0142] Normal blood glucose levels: In this disclosure, the term normal blood glucose levels or normal blood glucose refers to a state with normal blood glucose concentrations. Indicative normal blood glucose concentrations in the human body are between 70 mg / dL and 99 mg / dL in fasting adults, and between 70 mg / dL and 140 mg / dL in postprandial adults. Sustained normal blood glucose levels refer to a prolonged period of maintenance of normal blood glucose levels, such as at least one day, at least two days, at least one week, at least two weeks, at least one month, or longer.

[0143] Pairing of competent yeast species: In this invention, this is intended to broadly include any diploid or tetraploid yeast capable of growing in a culture. These yeast species can exist in haploid, diploid, or other polyploid forms. Under suitable conditions, a given polyploid cell can proliferate in an unlimited number of generations in the aforementioned form. Diploid cells can also form spores to form haploid cells. Successive pairings can produce tetraploid strains via the pairing or fusion of further diploid strains. This invention envisions the use of haploid yeasts, and, for example, the use of diploid or other polyploid yeast cells produced by pairing or protoplast fusion.

[0144] In one embodiment of the invention, the pairing competent yeast is a member of the Saccharomycetaceae family, including: *Arxiozyma*, *Ascobotryozyma*, *Citeromyces*, *Debaryomyces*, *Dekkera*, *Eremothecium*, *Issatchenkia*, *Kazachstania*, and *Kluyveromyces*. The genera *Kluyveromyces*, *Kodamaea*, *Lodderomyces*, *Pachysolen*, *Pichia*, *Saccharomyces*, *Saturnispora*, *Tetrapisispora*, *Torulaspora*, *Williopsis*, and *Zygosaccharomyces* may be useful in this invention. Other yeasts that may be useful in this invention include: *Yarrowia*, *Rhodosporidium*, *Candida*, *Hansenula*, *Filobasidium*, *Sporidiobolus*, *Bullera*, *Leucosporidium*, and *Filobasidiella*.

[0145] In one embodiment of the invention, the pairing competent yeast is a member of the genus *Pichia*. In another embodiment of the invention, the pairing competent yeast of the genus *Pichia* is one of the following species: *Pichia pastoris*, *Pichia methanolica*, and *Hansenula polymorpha* (*Pichia angusta*). In an exemplary embodiment of the invention, the pairing competent yeast of the genus *Pichia* is the species *Pichia pastoris*.

[0146] Haploid yeast cells: Cells in which each gene in the normal genome (chromosome) set has a single copy.

[0147] Polyploid yeast cells: Cells whose normal genome (chromosome) set has more than one copy.

[0148] Diploid yeast cells: These are cells whose normal genome complements essentially have two copies of each gene (alleles) and are usually formed through the fusion (pairing) of two haploid cells.

[0149] Tetraploid yeast cells: These are cells whose normal genome complements essentially have four copies of each gene (alleles), and are typically formed through the fusion (pairing) of two haploid cells. Tetraploids can carry two, three, four, or more different expression cartridges. These tetraploids can be obtained by selectively pairing isomorphic heterothallic a / a and α / α diploids in *Saccharomyces cerevisiae*, and by successive haploid pairing in *Pichia pastoris* to obtain auxotrophic diploids. For example, a [met his] haploid can pair with an [ade his] haploid to obtain a diploid [his]; and a [met arg] haploid can pair with an [ade arg] haploid to obtain a diploid [arg]; then the diploid [his] x diploid [arg] yields a tetraploid prototrophic microorganism. Those skilled in the art will understand that the advantages and uses of the aforementioned diploid cells can also be applied to tetraploid cells.

[0150] Yeast pairing: The process by which two haploid yeast cells naturally fuse to form a diploid yeast cell.

[0151] Meiosis: The process by which a diploid yeast cell undergoes meiotic division to form four haploid spores. Each spore can then grow and form a viable haploid cell line.

[0152] Optional markers: Optional markers are genes or gene segments that, for example, through a transformation event, confer a growth phenotype (physiological growth characteristic) on cells that receive that gene. Optional markers allow cells to survive and grow in selective growth media where cells without the optional marker gene cannot grow. Optional marker genes typically fall into several categories, including: positive optional marker genes, such as genes that confer resistance to antibiotics or other drugs, or genes that confer temperature resistance when two temperature-sensitive (“ts”) mutants are hybridized or one ts mutant is transformed; negative optional marker genes, such as biosynthetic genes that confer the ability to grow in media lacking a specific nutrient required by all cells without the biosynthetic gene, or mutagenic biosynthetic genes that confer the ability to grow because cells without the wild-type gene cannot; and analogs. Suitable markers include, but are not limited to: ZEO; G418; LYS3; MET1; MET3a; ADE1; ADE3; URA3; and analogs.

[0153] Expression vectors: These DNA vectors contain elements that facilitate the manipulation of foreign protein expression within the target host cell. Conveniently, the manipulation of sequences and the production of DNA for transformation are performed first within a bacterial host, such as *E. coli*, and the vector typically includes sequences that facilitate these operations, including the bacterial source of replication and appropriate bacterial selection markers. Selection markers encode proteins essential for the survival or growth of transformed host cells in selective media. Host cells cannot survive in these media without transformation by a vector containing a selection gene. Typically, the proteins encoded by selection genes (a) confuse resistance to antibiotics or other toxins, (b) supplement auxotrophic deficiencies, or (c) supply key nutrients unavailable from complex media. Illustrative vectors and methods for transforming yeast are described, for example, in Burke, D., Dawson, D., & Stearns, T. (2000). “Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual.” Plainview, NY: Cold Spring Harbor Laboratory Press.

[0154] The expression vector provided for use in the methods of the present invention may also include yeast-specific sequences, including optional auxotrophic or pharmaceutical markers for identifying the transformed yeast strain. The pharmaceutical markers may also be used to select the number of vector copies amplified within the yeast host cell.

[0155] The polypeptide encoding the target sequence is operatively linked to transcriptional and translational regulatory sequences that provide for polypeptide expression within yeast cells. These vector components may include, but are not limited to, one or more of the following: enhancer elements, promoters, and transcription termination sequences. Sequences relating to the secretion of the polypeptide, such as signal sequences and analogs, may also be included. The origin of yeast replication is optional, as the expression vector is often incorporated into the yeast genome. In one embodiment of the invention, the target polypeptide is operatively linked to, or fused to, a sequence from diploid yeast cells to provide optimized polypeptide secretion.

[0156] When a nucleic acid is functionally associated with another nucleic acid sequence, the nucleic acid is "operationally ligated." For example, if the DNA of a signal sequence is expressed as a pre-secretion protein involved in the polypeptide, then the DNA of the signal sequence is operably ligated to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of the sequence, then the promoter or enhancer is operably ligated to the coding sequence. Generally, "operationally ligated" means that the ligated DNA sequences are adjacent, and in the case of a pre-secretion sequence, adjacent and located in the same reading frame. However, enhancers do not necessarily have to be adjacent. Ligation is accomplished by conjugation at a suitable restriction site, or optionally by PCR / recombinant methods familiar to those skilled in the art. Technology; Invitrogen, Carlsbad, California) to achieve this. If these sites are not present, synthetic oligonucleotide conjugates or linkers are used according to conventional practice.

[0157] A promoter is an untranslated sequence located 5' upstream of the start codon of a structural gene (typically in the range of about 100 to 1000 bp) that controls the transcription and translation of a specific nucleic acid sequence operatively linked to it. These promoters belong to several categories: inducible, constitutive, and repressible promoters (which increase the level of transcription in response to the absence of a repressor). Inducible promoters can respond to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature, and under their control initiate an increased level of transcription from DNA.

[0158] Yeast promoter fragments can also act as sites for homologous recombination, as well as the same sites where expression vectors are incorporated into the yeast genome; alternatively, optional markers can be used as sites for homologous recombination. Transformation of Pichia pastoris is described in Cregg et al., Mol. Cell. Biol. 5:3376-3385, 1985.

[0159] Examples of suitable promoters from Pichia pastoris include the AOX1 promoter (Cregg et al., Mol. Cell. Biol. 9:1316-1323, (1989)); the ICL1 promoter (Menendez et al., Yeast 20(13):1097-108, (2003)); the glyceraldehyde-3-phosphate dehydrogenase promoter (GAP) (Waterham et al., Gene 186(1):37-44, (1997)); and the FLD1 promoter (Shen et al., Gene 216(1):93-102, (1998)). The GAP promoter is a strongly constitutive promoter, while the AOX and FLD1 promoters are inducible promoters.

[0160] Other yeast promoters include ADH1, alcohol dehydrogenase II, GAL4, PHO3, PHO5, Pyk, and chimeric promoters derived therefrom. Furthermore, non-yeast promoters, such as those from mammals, insects, plants, reptiles, amphibians, viruses, and birds, can be used in this invention. Most typically, the promoters will contain mammalian promoters (which may be endogenous to the expressed gene), or yeast promoters or viral promoters that provide efficient transcription in the yeast system.

[0161] The target peptide can be generated directly through recombinant synthesis or as a fusion peptide carrying a heterologous peptide, such as a signal sequence or another peptide with a specific cleavage site at the N-terminus of a mature protein or peptide. Generally, the signal sequence can be a component of the vector or a portion of the peptide-coding sequence inserted into the vector. The selected heterologous signal sequence is preferably an identified and processed one via a standard intracellular pathway. The proto-signal of the brewer's yeast α-factor has been shown to be effective in secreting various recombinant proteins from Pichia pastoris. Other yeast signal sequences include α-pairing factor signal sequences, invertase signal sequences, and signal sequences derived from other secreted yeast peptides. Furthermore, these signal peptide sequences can be engineered to enhance secretion in diploid yeast expression systems. Other targeted secretion signals also include mammalian signal sequences, which can be heterologous to the protein to be secreted or can be the native sequence of the protein to be secreted. Signal sequences include propeptide sequences and, in some cases, may include propeptide sequences. Many of these signal sequences are known in the art, including signal sequences found on immunoglobulin chains, such as the K28 protoxin sequence, PHA-E, FACE, human MCP-1, human serum albumin signal sequence, human Ig heavy chain, human Ig light chain, and analogues. For example, see Hashimoto et al., Protein Eng 11(2)75(1998); and Kobayashi et al., Therapeutic Apheresis 2(4)257(1998).

[0162] Transcription can be enhanced by inserting transcription activator sequences into a vector. These activators are cis-DNA acting elements, typically about 10 to 300 bp, that act on the promoter to enhance its transcription. Transcription enhancers are relatively localized and position-independent; they have been found at the 5' and 3' of transcription units, within introns, and within the coding sequence itself. Enhancers can be spliced ​​into the expression vector at the 5' or 3' position of the coding sequence, but are preferably located at the 5' site of the promoter.

[0163] Expression vectors used in eukaryotic host cells can also contain sequences necessary for transcription termination and mRNA stabilization. These sequences are typically derived from the 3' of the translation stop codon in the untranslated region of eukaryotic or viral DNA or cDNA. The nucleotide segments contained in these regions are transcribed into untranslated, polyadenylated fragments of the mRNA.

[0164] Construct a suitable vector containing one or more of the components listed above using standard ligation techniques or PCR / recombinant methods. The isolated plasmid or DNA fragment is cut, modified, and religated in the manner required to produce the desired plasmid, or generated via recombination. To analyze and confirm the correct sequence within the constructed plasmid, host cells are transformed using the ligation mixture, and successful transformants are selected, as appropriate, by appropriate antibiotic resistance (e.g., ampicillin or zeocin). Plasmids from the transformants are prepared and analyzed by restriction endonuclease digestion and / or sequencing.

[0165] As an alternative to fragment restriction and ligation, DNA sequences can be inserted into vectors using att-site-based recombination methods and recombinases. Such methods are described, for example, in Landy Ann.'s Rev. Biochem. 58:913-949 (1989) and are known to those skilled in the art. These methods utilize intramolecular DNA recombination mediated by a mixture of λ and recombinant proteins encoded by *E. coli*. Recombination occurs between specific linking (att) sites of interacting DNA molecules. For a description of att sites, see Weisberg and Landy (1983), “Site-Specific Recombination in Phage Lambda, in Lambda II,” Weisberg (ed.) (Cold Spring Harbor, NY: Cold Spring Harbor Press), pp. 211-250. DNA segments located flanking the recombination site are exchanged to create a hybrid sequence at the att site composed of sequences donated by each parent vector after recombination. Recombination can occur between DNAs of any topology.

[0166] The att site can be introduced into the target sequence in the following ways: by ligating the target sequence into an appropriate vector; by generating a PCR product containing the att B site using specific primers; or by generating a cDNA library cloned into an appropriate vector containing the att site.

[0167] As used herein, folding refers to the three-dimensional structure of polypeptides and proteins, in which interactions between amino acid residues function to stabilize the structure. While non-covalent interactions are important in determining structure, target proteins typically possess intramolecular and / or intermolecular covalent disulfide bonds formed by two cysteine ​​residues. For naturally occurring proteins and polypeptides or their derivatives and variants, appropriate folding generally results in an arrangement that best suits biological activity and can be easily monitored through activity assays, such as ligand binding and enzyme activity.

[0168] In some cases, such as when the desired product is of synthetic origin, bioactivity-based assays may be less meaningful. The appropriate folding of such molecules can be determined based on physiological properties, active considerations, model studies, and other factors.

[0169] The expression host can be further modified by introducing sequences encoding one or more enzymes (i.e., folding enzymes, chaperone proteins, etc.) that enhance folding and disulfide bond formation. Such sequences can be constitutively or inducibly expressed in yeast host cells using vectors, markers, etc., as known in the art. These sequences (including transcriptional regulatory elements sufficient to facilitate the desired expression pattern) are preferably stably integrated into the yeast genome via a targeted approach.

[0170] For example, eukaryotic PDI is not only an effective catalyst for protein cysteine ​​oxidation and disulfide isomerization, but also exhibits molecular chaperone activity. Co-expression of PDI can facilitate the production of active proteins with multiple disulfide bonds. There is also interest in the expression of BIP (immunoglobulin heavy chain binding protein), cyclic proteins, and analogues. In one embodiment of the invention, different haploid parental strains express different folding enzymes; for example, one strain may express BIP, while another strain may express PDI or a combination thereof.

[0171] The terms "required protein" and "required antibody" are used interchangeably and generally refer to a parental antibody or a chimeric or humanized antibody derived from or bound to the target CGRP, as described herein. The term "antibody" is intended to include any molecular structure containing a polypeptide chain with a specific shape suitable for identifying epitopes, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. Prototype antibody molecules are immunoglobulins, and all types of immunoglobulins IgG, IgM, IgA, IgE, IgD, etc., from all sources (e.g., human, rodent, rabbit, cow, sheep, pig, dog, other mammals, chicken, other birds, etc.) are considered "antibodies." According to the invention, rabbits are a suitable source for generating antibodies from which starting materials can be used. Many antibody coding sequences have been described; and others can be cultured using methods well known in the art. Examples include chimeric antibodies, human antibodies and other non-human mammalian antibodies, humanized antibodies, single-chain antibodies (such as scFv), camel antibodies, nanobodies, IgNAR (single-chain antibodies derived from sharks), small molecule immunotherapies (SMIPs), and antibody fragments such as Fab, F(ab')2, etc.See Streltsov VA et al., Structure of a shark IgNAR antibody variable domain and modeling of an early-developmental isotype, Protein Sci. Nov; 14(11):2901-9(2005), electronic version 2005 September 30; Greenberg AS et al., A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks, Nature, Mar 9;374(6518):168-73(1995); Nuttall SD et al., Isolation of the new antigen receptor from wobbegong sharks, and use as a scaffold for the display of protein looplibraries, MolImmunol. Aug;38(4):313-26(2001); Hamers-Casterman C et al., Naturallyoccurring antibodies devoid of light Chains, Nature. June 3, 1993; 363(6428):446-8; Gill DS et al., Biopharmaceutical drug discovery using novel protein scaffolds, Curr Opin Biotechnol. Dec; 17(6):653-8 (2006), e-version October 19, 2006.

[0172] For example, antibodies or antigen-binding fragments can be generated through genetic engineering. In this technique, as with other methods, the antibody-producing cells are sensitive to the desired antigen or immunogen. Messenger RNA isolated from antibody-producing cells is used as a template to prepare cDNA using PCR amplification. Vector libraries are generated by inserting appropriate segments of amplified immunoglobulin cDNA into expression vectors, each containing a heavy chain gene and a light chain gene that retains the initial antigen specificity. Combinatorial libraries are constructed by combining the heavy chain gene library with the light chain gene library. This results in a clonal library co-expressing the heavy chain and light chain (similar to Fab fragments or antigen-binding fragments of antibody molecules). Vectors carrying these genes are co-transfected into host cells. In the transfected host, antibody-induced gene synthesis causes the heavy and light chain proteins to self-assemble to produce active antibodies, which can be detected by antigen or immunogen screening.

[0173] The target antibody encoding sequences include those encoded by natural sequences, as well as nucleic acids that are sequence-dissimilar to the published nucleic acids due to codon degeneracy, and their variants. Variant peptides can include amino acid (“aa”) substitutions, additions, or deletions. Amino acid substitutions can be conserved amino acid substitutions or substitutions that eliminate non-essential amino acids, such as altering glycosylation sites, or minimizing misfolding by substituting or deleting one or more non-functionally essential cysteine ​​residues. Variant strains can be engineered to retain or enhance the biological activity of specific domains of the protein (e.g., functional domains, catalytic amino acid residues, etc.). Variant strains also include fragments of the peptides disclosed herein, particularly biologically active fragments and / or fragments corresponding to functional domains. Techniques for in vitro mutagenesis of cloned genes are known. This invention also includes peptides that have been modified using common molecular biotechnologies to improve their resistance to proteolytic degradation, optimize their solubility properties, or make them more suitable as therapeutic agents.

[0174] The variable light chain region and heavy chain region (V) of antibodies derived from a species can be used to generate antibodies from cells. L and V H Chimeric antibodies are prepared by recombination methods combining a variable region from a rodent or rabbit with a human constant region. Chimeric antibodies typically utilize variable regions from rodents or rabbits with human constant regions to produce antibodies that are primarily human domains. The production of such chimeric antibodies is well known in the art and can be achieved by standard methods (such as those described, for example, in U.S. Patent No. 5,624,659, which is incorporated herein by reference in its entirety). It is also contemplated that the human constant region of the chimeric antibody of the present invention can be selected from the constant regions of IgG1, IgG2, IgG3, or IgG4.

[0175] Humanized antibodies are engineered to contain even more human-like immunoglobulin domains and incorporate only the complementarity-determining regions of animal-derived antibodies. This is achieved by examining the sequence of the hypervariable loop in the variable region of a monoclonal antibody to adapt it to the structure of a human antibody chain. Despite its apparent complexity, the method is simple in practice. See, for example, U.S. Patent No. 6,187,287, which is incorporated herein by reference in its entirety.

[0176] In addition to intact immunoglobulins (or their recombinant counterparts), immunoglobulin fragments (e.g., Fab, F(ab')2, or other fragments) containing epitope binding sites can be synthesized. These "fragments," or minimal immunoglobulins, can be designed using recombinant immunoglobulin technology. For example, "Fv" immunoglobulins for use in this invention can be generated by synthesizing fused variable light chain and variable heavy chain regions. Combinations of antibodies are also of interest, such as dimeric antibodies containing two distinct Fv specificities. In another embodiment of the invention, the immunoglobulin fragment comprises SMIPs (small molecule immunopharmaceuticals), camel antibodies, nanobodies, and IgNARs.

[0177] Immunoglobulins and their fragments can be post-translational modified, for example, by adding effector moieties that can be used in the methods and compositions of the present invention, such as chemical linkers, detectable moieties (such as fluorescent dyes, enzymes, toxins, substrates, bioluminescent substances, radioactive substances, chemiluminescent moieties, etc.), or specific binding moieties (such as streptavidin, avidin, or biotin, etc.). Examples of additional effector molecules are provided below.

[0178] If a polypeptide sequence is produced by translating a polynucleotide sequence according to a genetic codon, then the polynucleotide sequence "corresponds" to the polypeptide sequence (i.e., the polynucleotide sequence "encodes" the polypeptide sequence). If two sequences encode the same polypeptide sequence, then one polynucleotide sequence "corresponds" to the other polynucleotide sequence.

[0179] "Heterologous" regions or domains of DNA constructs are identifiable segments of DNA within a larger DNA molecule that have not been found to be associated with that larger molecule in nature. Therefore, when a heterologous region encodes a mammalian gene, the DNA flanking that gene is typically not adjacent to the mammalian genomic DNA in the genome of the source organism. Another example of a heterologous region is a construct in which the coding sequence itself does not exist in nature (e.g., cDNA containing introns or synthetic sequences with codons different from those of the natural gene). Allelic variations or naturally occurring mutations do not produce heterologous regions of DNA as defined herein.

[0180] A "coding sequence" is an in-frame codon sequence (considering the genetic codon) that corresponds to or encodes a protein or peptide sequence. Two coding sequences correspond to each other if the sequence or its complement encodes the same amino acid sequence. The coding sequence, along with appropriate regulatory sequences, can be transcribed and translated into a polypeptide. Polyadenylation signals and transcription termination sequences are typically located at the 3' end of the coding sequence. A "promoter sequence" is a DNA regulatory region that binds to intracellular RNA polymerase and initiates transcription of the downstream (3' direction) coding sequence. Promoter sequences typically contain additional sites for binding to regulatory molecules (e.g., transcription factors) that influence transcription of the coding sequence. When RNA polymerase binds to the intracellular promoter sequence and transcribes the coding sequence into mRNA, the coding sequence is "controlled" or "operationally linked" to the promoter, and the mRNA is then translated into the protein encoded by the coding sequence.

[0181] Vectors are used to introduce foreign substances (such as DNA, RNA, or proteins) into an organism or host cell. Typical vectors include recombinant viruses (for polynucleotides) and liposomes (for peptides). A "DNA vector" is a replicon, such as a plasmid, bacteriophage, or granule, which can be linked to another polynucleotide segment to cause replication of the linker segment. An "expression vector" is a DNA vector containing a regulatory sequence that guides peptide synthesis through a suitable host cell. This generally means that a promoter binds to RNA polymerase and initiates the transcription of mRNA, and that a ribosome binding site and initiation signal guide the translation of mRNA into a peptide. A polynucleotide sequence is incorporated into the expression vector at the appropriate site and within the correct reading frame, and then the vector is used to transform a suitable host cell, enabling the production of a peptide encoded by the polynucleotide sequence.

[0182] "Amplification" of polynucleotide sequences is the in vitro production of multiple copies of a specific nucleic acid sequence. The amplified sequence is usually in DNA form. Various techniques for performing such amplification are described in Van Brunt's review article (Bio / Technol., 8(4):291-294(1990)). Polymerase chain reaction, or PCR, is the prototype of nucleic acid amplification, and the use of PCR in this article should be regarded as an example of other suitable amplification techniques.

[0183] The general structure of vertebrate antibodies is now well understood (Edelman, GM, Ann. NY Acad. Sci., 190:5 (1971)). Antibodies consist of two identical light polypeptide chains (“light chains”) with a molecular weight of approximately 23,000 Daltons and two identical heavy chains (“heavy chains”) with a molecular weight of 53,000–70,000 Daltons. The four chains are linked by disulfide bonds to form a “Y” configuration, with the heavy chain supporting the light chain from the opening of the “Y” configuration. The “branching” portion of the “Y” configuration is called the Fab region; the stem portion of the “Y” configuration is called the F… C The amino acid sequence is located from the N-terminus at the top of the "Y" configuration to the C-terminus at the bottom of each chain. The N-terminus has a variable region, which is specific to the antigen that elicits it and is approximately 100 amino acids in length, with slight differences between the light and heavy chains and between antibodies.

[0184] The variable regions within each chain connect to the constant regions, which extend the remaining length of the chain and do not vary with antibody specificity (i.e., the antigen that elicits it) in a particular type of antibody. There are five known major types of constant regions that determine the type of immunoglobulin molecule (IgG, IgM, IgA, IgD, and IgE, corresponding to γ, μ, α, δ, and ε (gamma, mu, alpha, delta, or epsilon) heavy chain constant regions). The constant region or type determines the subsequent effector function of the antibody, including complement activation (Kabat, EA, Structural Concepts in Immunology and Immunochemistry, 2nd ed., pp. 413-436, Holt, Rinehart, Winston (1976)), and other cellular responses (Andrews, DW, et al., Clinical Immunobiology, pp. 1-18, WBSanders (1980); Kohl, S., et al., Immunology, 48:187 (1983)); while the variable region determines the antigen it reacts to. Light chains are classified as κ (kappa) or λ (lambda). Each heavy chain type can be prepared with κ or λ light chains. When immunoglobulins are produced by fusion tumors or B cells, 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 bonds.

[0185] The term "variable region" or "VR" refers to the regions within each pair of light and heavy chains of an antibody, which directly participate in the binding of the antibody to the antigen. Each heavy chain has a variable domain (VR) at one end. H ), followed by some constant regions. Each light chain has a variable region (V) at one end.L Furthermore, there is 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 variable domain of the light chain is aligned with the variable domain of the heavy chain.

[0186] The terms “complementarity-determining region,” “hypervariant region,” or “CDR” refer to one or more hypervariable or complementarity-determining regions (CDRs) present within the variable regions of the light or heavy chain of an antibody (see Kabat, E.A. et al., Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., (1987)). These terms include hypervariable regions as defined by Kabat et al. (“Sequences of Proteins of Immunological Interest,” Kabat E. et al., USDept. of Health and Human Services, 1983) or hypervariable loops in the three-dimensional structure of an antibody (Chothia and Lesk, J Mol. Biol. 196 901-917 (1987)). CDRs within each chain are kept close together through structural regions and, together with CDRs of another chain, contribute to the formation of antigen-binding sites. The CDR contains selected amino acids that have been described as selection-determining regions (SDRs), which represent key contact residues used by the CDR in antibody-antigen interactions (Kashmiri, S., Methods, 36:25-34 (2005)).

[0187] The terms “framework region” or “FR” refer to one or more 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 terms include amino acid sequence regions inserted between CDRs within the variable regions of the light and heavy chains of the antibody.

[0188] Anti-CGRP antibodies with CGRP-binding activity and their binding fragments

[0189] An exemplary embodiment of the method of the present invention includes administering an anti-CGRP antibody and a fragment thereof to an individual. Exemplary anti-CGRP antibodies and fragments are described in U.S. Patent Publication No. 2012 / 0294797, which is incorporated herein by reference in its entirety, while other exemplary anti-CGRP antibodies are described in the following paragraphs.

[0190] Antibody Ab1

[0191] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQTASPVSAAVGSTVTINCQASQSVYDNNYLAWYQQKPGQP PKQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLG SYDCSSGDCFVFGGGTEVVVKR (SEQ ID NO:1).

[0192] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQTASPVSAAVGSTVTINCQASQSV YDNNYLAWYQQKPGQPPKQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDLECADAATYYCLGSYDCSSGDCFVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:2).

[0193] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCTVSG LDLSSYYMQWVRQAPGKGLEWIGVIGINDNTYYASWAKGRFTIS RASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS (SEQ ID NO:3).

[0194] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCTVSGLDL SSYYMQWVRQAPGKGLEWIGVIGINDNTYYASWAKGRFTISRASSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:4).

[0195] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0196] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:1 or SEQ ID NO:2. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:3 or SEQ ID NO:4.

[0197] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:1 or the light chain sequence of SEQ ID NO:2.

[0198] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:8; SEQ ID NO:9; and SEQ ID NO:10, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:8; SEQ ID NO:9; and SEQ ID NO:10, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:3 or the heavy chain sequence of SEQ ID NO:4.

[0199] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:1 (SEQ ID NO:5; SEQ ID NO:6; and SEQ ID NO:7); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:3 (SEQ ID NO:8; SEQ ID NO:9; and SEQ ID NO:10).

[0200] In a particularly preferred embodiment of the invention, the chimeric anti-CGRP antibody is Ab1, which comprises, or optionally consists of, SEQ ID NO:2 and SEQ ID NO:4, and has at least one of the biological activities stated herein.

[0201] In another preferred embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab1, the Fab fragment comprises a variable light chain sequence of SEQ ID NO:1 and a variable heavy chain sequence of SEQ ID NO:3. This embodiment of the invention also contemplates the addition, deletion, and variants of SEQ ID NO:1 and / or SEQ ID NO:3 in the Fab fragment while preserving CGRP binding specificity.

[0202] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab1 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab1 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia diploid) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0203] Antibody Ab2

[0204] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSSGDCFVFGGGTKVEIKR (SEQ ID NO: 11).

[0205] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ SVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:12).

[0206] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO:13).

[0207] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG LDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:14).

[0208] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:15; SEQ ID NO:16; and SEQ ID NO:17, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:11 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0209] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:11 or SEQ ID NO:12. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:13 or SEQ ID NO:14.

[0210] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:15; SEQ ID NO:16; and SEQ ID NO:17, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:15; SEQ ID NO:16; and SEQ ID NO:17, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:11 or the light chain sequence of SEQ ID NO:12.

[0211] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:18; SEQ ID NO:19; and SEQ ID NO:20, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:18; SEQ ID NO:19; and SEQ ID NO:20, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:13 or the heavy chain sequence of SEQ ID NO:14.

[0212] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, one, two, three or more (including all) of the following antibody fragments: the variable light chain region of SEQ ID NO:11; the variable heavy chain region of SEQ ID NO:13; the complementarity-determining region of the variable light chain region of SEQ ID NO:11 (SEQ ID NO:15; SEQ ID NO:16; and SEQ ID NO:17); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:13 (SEQ ID NO:18; SEQ ID NO:19; and SEQ ID NO:20).

[0213] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab2, which comprises, or optionally consists of, SEQ ID NO:12 and SEQ ID NO:14, and has at least one of the biological activities stated herein.

[0214] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab2, the Fab fragment comprises the variable light chain sequence of SEQ ID NO:11 and the variable heavy chain sequence of SEQ ID NO:13. This embodiment of the invention also contemplates the addition, deletion, and variants of SEQ ID NO:11 and / or SEQ ID NO:13 in the Fab fragment while preserving CGRP binding specificity.

[0215] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab2 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab2 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0216] Antibody Ab3

[0217] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSSGDCFVFGGGTKVEIKR (SEQ ID NO: 21).

[0218] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ SVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:22).

[0219] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO:23).

[0220] This invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG LDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:24).

[0221] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:23 or the heavy chain sequence of SEQ ID NO:24, or a combination of these polypeptide sequences. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations thereof, or optionally all of the sequences described above.

[0222] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:21 or SEQ ID NO:22. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:23 or SEQ ID NO:24.

[0223] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:21 or the light chain sequence of SEQ ID NO:22.

[0224] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:28; SEQ ID NO:29; and SEQ ID NO:30, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:28; SEQ ID NO:29; and SEQ ID NO:30, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:23 or the heavy chain sequence of SEQ ID NO:24.

[0225] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:21 (SEQ ID NO:25; SEQ ID NO:26; and SEQ ID NO:27); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:23 (SEQ ID NO:28; SEQ ID NO:29; and SEQ ID NO:30).

[0226] In one embodiment of the invention, the chimeric anti-CGRP antibody is Ab3, which comprises, or optionally consists of, SEQ ID NO:22 and SEQ ID NO:24, and has at least one of the biological activities stated herein.

[0227] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab3, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:21 and / or SEQ ID NO:23 in the Fab fragment while preserving CGRP binding specificity.

[0228] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab3 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab3 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0229] Antibody Ab4

[0230] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPP KQLIYDASTLASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLG SYDCTNGDCFVFGGGTEVVVKR (SEQ ID NO:31).

[0231] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQTPSPVSAAVGSTVTINCQASQSV YHNTYLAWYQQKPGQPPKQLIYDASTLASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:32).

[0232] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGATYYASWAKGRFTISK TSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS (SEQ ID NO:33).

[0233] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCSVSGIDLS GYYMNWVRQAPGKGLEWIGVIGINGATYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:34).

[0234] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:35; SEQ ID NO:36; and SEQ ID NO:37, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0235] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:31 or SEQ ID NO:32. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:33 or SEQ ID NO:34.

[0236] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:35; SEQ ID NO:36; and SEQ ID NO:37, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:35; SEQ ID NO:36; and SEQ ID NO:37, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:31 or the light chain sequence of SEQ ID NO:32.

[0237] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:38; SEQ ID NO:39; and SEQ ID NO:40, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:38; SEQ ID NO:39; and SEQ ID NO:40, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:33 or the heavy chain sequence of SEQ ID NO:34.

[0238] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:31 (SEQ ID NO:35; SEQ ID NO:36; and SEQ ID NO:37); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:33 (SEQ ID NO:38; SEQ ID NO:39; and SEQ ID NO:40).

[0239] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab4, which comprises, or optionally consists of, SEQ ID NO:32 and SEQ ID NO:34, and has at least one of the biological activities stated herein.

[0240] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab4, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:31 and / or SEQ ID NO:33 in the Fab fragment while preserving CGRP binding specificity.

[0241] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab4 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab4 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0242] Antibody Ab5

[0243] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVP KQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCTNGDCFVFGGGTKVEIKR (SEQ ID NO: 41).

[0244] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ SVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:42).

[0245] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO:43).

[0246] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG IDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:44).

[0247] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:45; SEQ ID NO:46; and SEQ ID NO:47, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:43 or the heavy chain sequence of SEQ ID NO:44, or a combination of these polypeptide sequences. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations thereof, or optionally all of the sequences described above.

[0248] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:41 or SEQ ID NO:42. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:43 or SEQ ID NO:44.

[0249] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:45; SEQ ID NO:46; and SEQ ID NO:47, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:45; SEQ ID NO:46; and SEQ ID NO:47, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:41 or the light chain sequence of SEQ ID NO:42.

[0250] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:43 or the heavy chain sequence of SEQ ID NO:44.

[0251] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:41 (SEQ ID NO:45; SEQ ID NO:46; and SEQ ID NO:47); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:43 (SEQ ID NO:48; SEQ ID NO:49; and SEQ ID NO:50).

[0252] In one embodiment of the invention, the chimeric anti-CGRP antibody is Ab5, which comprises, or optionally consists of, SEQ ID NO:42 and SEQ ID NO:44, and has at least one of the biological activities stated herein.

[0253] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab5, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:41 and / or SEQ ID NO:43 in the Fab fragment while preserving CGRP binding specificity.

[0254] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab5 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab5 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0255] Antibody Ab6

[0256] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVP KQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCTNGDCFVFGGGTKVEIKR (SEQ ID NO: 51).

[0257] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ SVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:52).

[0258] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO:53).

[0259] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:54).

[0260] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:55; SEQ ID NO:56; and SEQ ID NO:57, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0261] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:51 or SEQ ID NO:52. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:53 or SEQ ID NO:54.

[0262] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:55; SEQ ID NO:56; and SEQ ID NO:57, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:55; SEQ ID NO:56; and SEQ ID NO:57, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:51 or the light chain sequence of SEQ ID NO:52.

[0263] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:58; SEQ ID NO:59; and SEQ ID NO:60, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:58; SEQ ID NO:59; and SEQ ID NO:60, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:53 or the heavy chain sequence of SEQ ID NO:54.

[0264] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:51 (SEQ ID NO:55; SEQ ID NO:56; and SEQ ID NO:57); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:53 (SEQ ID NO:58; SEQ ID NO:59; and SEQ ID NO:60).

[0265] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab6, which comprises, or optionally consists of, SEQ ID NO:52 and SEQ ID NO:54, and has at least one of the biological activities stated herein.

[0266] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab6, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:51 and / or SEQ ID NO:53 in said Fab, while preserving CGRP binding specificity.

[0267] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab6 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab6 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0268] Antibody Ab7

[0269] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQTASPVSAAVGSTVTINCQASQSVYNYNYLAWYQQKPGQP PKQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCL GSYDCSTGDCFVFGGGTEVVVKR (SEQ ID NO:61).

[0270] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQTASPVSAAVGSTVTINCQASQSV YNYNYLAWYQQKPGQPPKQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSTGDCFVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:62).

[0271] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QEQLKESGGRLVTPGTSLTLTCTVS GIDLSNHYMQWVRQAPGKGLEWIGVVGINGRTYYASWAKGRFTI SRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSS (SEQ ID NO:63).

[0272] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QEQLKESGGRLVTPGTSLTLTCTVSGID LSNHYMQWVRQAPGKGLEWIGVVGINGRTYYASWAKGRFTISRTSSTTVDLKMTRLTTEDTATYFCARGDIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:64).

[0273] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:65; SEQ ID NO:66; and SEQ ID NO:67, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0274] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:61 or SEQ ID NO:62. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:63 or SEQ ID NO:64.

[0275] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:65; SEQ ID NO:66; and SEQ ID NO:67, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:65; SEQ ID NO:66; and SEQ ID NO:67, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:61 or the light chain sequence of SEQ ID NO:62.

[0276] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:68; SEQ ID NO:69; and SEQ ID NO:70, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:68; SEQ ID NO:69; and SEQ ID NO:70, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:63 or the heavy chain sequence of SEQ ID NO:64.

[0277] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:61 (SEQ ID NO:65; SEQ ID NO:66; and SEQ ID NO:67); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:63 (SEQ ID NO:68; SEQ ID NO:69; and SEQ ID NO:70).

[0278] In a particularly preferred embodiment of the invention, the chimeric anti-CGRP antibody is Ab7, which comprises, or optionally consists of, SEQ ID NO:62 and SEQ ID NO:64, and has at least one of the biological activities stated herein.

[0279] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab7, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:61 and / or SEQ ID NO:63 in said Fab, while preserving CGRP binding specificity.

[0280] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab7 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab7 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0281] Antibody Ab8

[0282] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQSVYNYNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSTGDCFVFGGGTKVEIKR (SEQ ID NO: 71).

[0283] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ SVYNYNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSTGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:72).

[0284] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIDLSNHYMQWVRQAPGKGLEWVGVVGINGRTYYASWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS (SEQ ID NO:73).

[0285] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG IDLSNHYMQWVRQAPGKGLEWVGVVGINGRTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:74).

[0286] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:75; SEQ ID NO:76; and SEQ ID NO:77, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0287] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:71 or SEQ ID NO:72. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:73 or SEQ ID NO:74.

[0288] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:75; SEQ ID NO:76; and SEQ ID NO:77, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:75; SEQ ID NO:76; and SEQ ID NO:77, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:71 or the light chain sequence of SEQ ID NO:72.

[0289] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:78; SEQ ID NO:79; and SEQ ID NO:80, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:78; SEQ ID NO:79; and SEQ ID NO:80, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:73 or the heavy chain sequence of SEQ ID NO:74.

[0290] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:71 (SEQ ID NO:75; SEQ ID NO:76; and SEQ ID NO:77); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:73 (SEQ ID NO:78; SEQ ID NO:79; and SEQ ID NO:80).

[0291] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab8, which comprises, or optionally consists of, SEQ ID NO:72 and SEQ ID NO:74, and has at least one of the biological activities stated herein.

[0292] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab8, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:71 and / or SEQ ID NO:73 in the Fab fragment while preserving CGRP binding specificity.

[0293] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab8 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab8 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as CHO, NSO, or HEK 293 cells, yeast cells (e.g., diploid yeasts, such as Pichia pastoris), and other yeast strains. Suitable Pichia species include, but are not limited to, Pichia pastoris.

[0294] Antibody Ab9

[0295] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQTPSPVSA AVGSTVTINCQASQNVYNNNYLAWYQQKPGQPPKQLIYSTSTLA SGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVVVKR (SEQ ID NO:81).

[0296] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQTPSPVSAAVGSTVTINCQASQNV YNNNYLAWYQQKPGQPPKQLIYSTSTLASGVSSRFRGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSRGDCFVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:82).

[0297] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCTVSGI GLSSYYMQWVRQSPGRGLEWIGVIGSDGKTYYATWAKGRFTISK TSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSS (SEQ ID NO:83).

[0298] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGTPLTLTCTVSGIGLS SYYMQWVRQSPGRGLEWIGVIGSDGKTYYATWAKGRFTISKTSSTTVDLRMASLTTEDTATYFCTRGDIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:84).

[0299] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:85; SEQ ID NO:86; and SEQ ID NO:87, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:83 or the heavy chain sequence of SEQ ID NO:84, or a combination of these polypeptide sequences. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally consists of, all of the CDRs, variable heavy chain and variable light chain sequences, and one or more of the heavy chain and light chain sequences described above.

[0300] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:81 or SEQ ID NO:82. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:83 or SEQ ID NO:84.

[0301] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:85; SEQ ID NO:86; and SEQ ID NO:87, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:85; SEQ ID NO:86; and SEQ ID NO:87, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:81 or the light chain sequence of SEQ ID NO:82.

[0302] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:88; SEQ ID NO:89; and SEQ ID NO:90, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:88; SEQ ID NO:89; and SEQ ID NO:90, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:83 or the heavy chain sequence of SEQ ID NO:84.

[0303] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:81 (SEQ ID NO:85; SEQ ID NO:86; and SEQ ID NO:87); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:83 (SEQ ID NO:88; SEQ ID NO:89; and SEQ ID NO:90).

[0304] In one embodiment of the invention, the chimeric anti-CGRP antibody is Ab9, which comprises, or optionally consists of, SEQ ID NO:82 and SEQ ID NO:84, and has at least one of the biological activities stated herein.

[0305] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab9, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:81 and / or SEQ ID NO:83 in said Fab, while preserving CGRP binding specificity.

[0306] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab9 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab9 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0307] Antibody Ab10

[0308] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSRGDCFVFGGGTKVEIKR (SEQ ID NO: 91).

[0309] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ NVYNNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 92).

[0310] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIGLSSYYMQWVRQAPGKGLEWVGVIGSDGKTYYATWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVTVSS (SEQ ID NO:93).

[0311] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG IGLSSYYMQWVRQAPGKGLEWVGVIGSDGKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:94).

[0312] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:95; SEQ ID NO:96; and SEQ ID NO:97, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0313] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:91 or SEQ ID NO:92. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:93 or SEQ ID NO:94.

[0314] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:95; SEQ ID NO:96; and SEQ ID NO:97, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:95; SEQ ID NO:96; and SEQ ID NO:97, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:91 or the light chain sequence of SEQ ID NO:92.

[0315] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:98; SEQ ID NO:99; and SEQ ID NO:100, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:98; SEQ ID NO:99; and SEQ ID NO:100, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:93 or the heavy chain sequence of SEQ ID NO:94.

[0316] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:91 (SEQ ID NO:95; SEQ ID NO:96; and SEQ ID NO:97); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:93 (SEQ ID NO:98; SEQ ID NO:99; and SEQ ID NO:100).

[0317] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab10, which comprises, or optionally consists of, SEQ ID NO:92 and SEQ ID NO:94, and has at least one of the biological activities stated herein.

[0318] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab10, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:91 and / or SEQ ID NO:93 in said Fab, while preserving CGRP binding specificity.

[0319] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab10 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab10 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as CHO, NSO, or HEK 293 cells, yeast cells (e.g., diploid yeasts, such as Pichia pastoris), and other yeast strains. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0320] Antibody Ab11

[0321] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQTASPVSPAVGSTVTINCRASQSVYYNNYLAWYQQKPGQPP KQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLG SYDCSNGDCFVFGGGTEVVVKR (SEQ ID NO:101).

[0322] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQTASPVSPAVGSTVTINCRASQSV YYNNYLAWYQQKPGQPPKQLIYSTSTLASGVSSRFKGSGSGTQFTLTISDVQCDDAATYYCLGSYDCSNGDCFVFGGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:102).

[0323] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGGSLTLTCTVSG IDVTNYYMQWVRQAPGKGLEWIGVIGVNGKRYYASWAKGRFTIS KTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS (SEQ ID NO:103).

[0324] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QSLEESGGRLVTPGGSLTLTCTVSGIDV TNYYMQWVRQAPGKGLEWIGVIGVNGKRYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:104).

[0325] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:105; SEQ ID NO:106; and SEQ ID NO:107, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0326] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:101 or SEQ ID NO:102. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:103 or SEQ ID NO:104.

[0327] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:105; SEQ ID NO:106; and SEQ ID NO:107, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:105; SEQ ID NO:106; and SEQ ID NO:107, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:101 or the light chain sequence of SEQ ID NO:102.

[0328] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:108; SEQ ID NO:109; and SEQ ID NO:110, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:108; SEQ ID NO:109; and SEQ ID NO:110, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:103 or the heavy chain sequence of SEQ ID NO:104.

[0329] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:101 (SEQ ID NO:105; SEQ ID NO:106; and SEQ ID NO:107); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:103 (SEQ ID NO:108; SEQ ID NO:109; and SEQ ID NO:110).

[0330] In one embodiment of the invention, the chimeric anti-CGRP antibody is Ab11, which comprises, or optionally consists of, SEQ ID NO:102 and SEQ ID NO:104, and has at least one of the biological activities stated herein.

[0331] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab11, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:101 and / or SEQ ID NO:103 in said Fab, while preserving CGRP binding specificity.

[0332] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab11 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab11 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0333] Antibody Ab12

[0334] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCRASQSVYYNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSNGDCFVFGGGTKVEIKR (SEQ ID NO: 111).

[0335] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCRASQS VYYNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSNGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:112).

[0336] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIDVTNYYMQWVRQAPGKGLEWVGVIGVNGKRYYASWAKG RFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVS S (SEQ ID NO:113).

[0337] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG IDVTNYYMQWVRQAPGKGLEWVGVIGVNGKRYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:114).

[0338] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:115; SEQ ID NO:116; and SEQ ID NO:117, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:111 or the light chain sequence of SEQ ID NO:112, and / or one or more of the polypeptide sequences of SEQ ID NO:118; SEQ ID NO:119; and SEQ ID NO:120, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:113 or the heavy chain sequence of SEQ ID NO:114, or a combination of these polypeptide sequences. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally consists of, all of the CDRs, variable heavy chain and variable light chain sequences, and one or more of the heavy chain and light chain sequences described above.

[0339] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:111 or SEQ ID NO:112. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:113 or SEQ ID NO:114.

[0340] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:115; SEQ ID NO:116; and SEQ ID NO:117, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:115; SEQ ID NO:116; and SEQ ID NO:117, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:111 or the light chain sequence of SEQ ID NO:112.

[0341] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:118; SEQ ID NO:119; and SEQ ID NO:120, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:118; SEQ ID NO:119; and SEQ ID NO:120, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:113 or the heavy chain sequence of SEQ ID NO:114.

[0342] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, one, two, three or more (including all) of the following antibody fragments: the variable light chain region of SEQ ID NO:111; the variable heavy chain region of SEQ ID NO:113; the complementarity-determining region of the variable light chain region of SEQ ID NO:111 (SEQ ID NO:115; SEQ ID NO:116; and SEQ ID NO:117); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:113 (SEQ ID NO:118; SEQ ID NO:119; and SEQ ID NO:120).

[0343] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab12, which comprises, or optionally consists of, SEQ ID NO:112 and SEQ ID NO:114, and has at least one of the biological activities stated herein.

[0344] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab12, the Fab fragment comprises the variable light chain sequence of SEQ ID NO:111 and the variable heavy chain sequence of SEQ ID NO:113. This embodiment of the invention also contemplates the addition, deletion, and variants of SEQ ID NO:111 and / or SEQ ID NO:113 in said Fab, while preserving CGRP binding specificity.

[0345] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab12 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab12 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0346] Antibody Ab13

[0347] In one embodiment, the present invention includes a chimeric antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: AIVMTQTPSSKSVPVGDTVTINCQASESLYNNNALAWFQQKPGQP PKRLIYDASKLASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCG GYRSDSVDGVAFAGGTEVVVKR (SEQ ID NO:121).

[0348] The present invention also includes a chimeric antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: AIVMTQTPSSKSVPVGDTVTINCQASES LYNNNALAWFQQKPGQPPKRLIYDASKLASGVPSRFSGGGSGTQFTLTISGVQCDDAATYYCGGYRSDSVDGVAFAGGTEVVVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:122).

[0349] The present invention also includes a chimeric antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: QSVEESGGGLVQPEGSLTLTCTASG FDFSSNAMWWVRQAPGKGLEWIGIIYNGDGSTYYASWVNGRFSI SKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTVSS (SEQ ID NO:123).

[0350] The present invention also includes a chimeric antibody having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: QSVEESGGGLVQPEGSLTLTCTASGFDF SSNAMWWVRQAPGKGLEWIGCIYNGDGSTYYASWVNGRFSISKTSSTTVTLQLNSLTVADTATYYCARDLDLWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ IDNO:124).

[0351] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:125; SEQ ID NO:126; and SEQ ID NO:127, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) 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. In another embodiment of the invention, the antibody or fragment thereof of the present invention comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations of one or more of the heavy chain and light chain sequences described above.

[0352] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:121 or SEQ ID NO:122. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:123 or SEQ ID NO:124.

[0353] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:125; SEQ ID NO:126; and SEQ ID NO:127, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:125; SEQ ID NO:126; and SEQ ID NO:127, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:121 or the light chain sequence of SEQ ID NO:122.

[0354] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:128; SEQ ID NO:129; and SEQ ID NO:130, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:128; SEQ ID NO:129; and SEQ ID NO:130, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:123 or the heavy chain sequence of SEQ ID NO:124.

[0355] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:121 (SEQ ID NO:125; SEQ ID NO:126; and SEQ ID NO:127); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:123 (SEQ ID NO:128; SEQ ID NO:129; and SEQ ID NO:130).

[0356] In one embodiment of the invention, the chimeric anti-CGRP antibody is Ab13, which comprises, or optionally consists of, SEQ ID NO:122 and SEQ ID NO:124, and has at least one of the biological activities stated herein.

[0357] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab13, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:121 and / or SEQ ID NO:123 in the Fab fragment while preserving CGRP binding specificity.

[0358] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab13 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab13 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0359] Antibody Ab14

[0360] In one embodiment, the present invention includes a humanized antibody having CGRP binding specificity and having a variable light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQNVYNNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGS YDCSRGDCFVFGGGTKVEIKR (SEQ ID NO: 131).

[0361] The present invention also includes a humanized antibody having CGRP binding specificity and having a light chain sequence comprising the sequence stated below: QVLTQSPSSLSASVGDRVTINCQASQ NVYNNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSRGDCFVFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:132).

[0362] The present invention also includes a humanized antibody having CGRP binding specificity and having a variable heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCA VSGIGLSSYYMQWVRQAPGKGLEWVGVIGSDGKTYYATWAKGR FTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVTVSS (SEQ ID NO:133).

[0363] The present invention also includes humanized antibodies having CGRP binding specificity and having a heavy chain sequence comprising the sequence stated below: EVQLVESGGGLVQPGGSLRLSCAVSG IGLSSYYMQWVRQAPGKGLEWVGVIGSDGKTYYATWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCTRGDIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDARVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQID NO:134).

[0364] The present invention also contemplates antibodies comprising one or more of the polypeptide sequences of SEQ ID NO:135; SEQ ID NO:136; and SEQ ID NO:137, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) 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, wherein the polypeptide sequence corresponds to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:133 or the heavy chain sequence of SEQ ID NO:134, or a combination of these polypeptide sequences. In another embodiment of the invention, the antibody or fragment thereof comprises, or optionally comprises, all of the CDRs, variable heavy chain and variable light chain sequences, and combinations thereof, or optionally all of the sequences described above.

[0365] The present invention also contemplates fragments of antibodies having CGRP binding specificity. In one embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:131 or SEQ ID NO:132. In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, the polypeptide sequence of SEQ ID NO:133 or SEQ ID NO:134.

[0366] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:135; SEQ ID NO:136; and SEQ ID NO:137, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:135; SEQ ID NO:136; and SEQ ID NO:137, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable light chain sequence of SEQ ID NO:131 or the light chain sequence of SEQ ID NO:132.

[0367] In another embodiment of the invention, the antibody fragment having CGRP binding specificity comprises one or more of the polypeptide sequences of SEQ ID NO:138; SEQ ID NO:139; and SEQ ID NO:140, or optionally consists of one or more of the polypeptide sequences of SEQ ID NO:138; SEQ ID NO:139; and SEQ ID NO:140, said polypeptide sequences corresponding to the complementarity-determining region (CDR, or hypervariable region) of the variable heavy chain sequence of SEQ ID NO:133 or the heavy chain sequence of SEQ ID NO:134.

[0368] The present invention also contemplates antibody fragments comprising one or more of the antibody fragments described herein. In one embodiment of the invention, an antibody fragment having CGRP binding specificity comprises, or optionally comprises, 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 region of the variable light chain region of SEQ ID NO:131 (SEQ ID NO:135; SEQ ID NO:136; and SEQ ID NO:137); and the complementarity-determining region of the variable heavy chain region of SEQ ID NO:133 (SEQ ID NO:138; SEQ ID NO:139; and SEQ ID NO:140).

[0369] In one embodiment of the invention, the humanized anti-CGRP antibody is Ab14, which comprises, or optionally consists of, SEQ ID NO:132 and SEQ ID NO:134, and has at least one of the biological activities stated herein.

[0370] In another embodiment of the invention, the antibody fragment comprises, or optionally consists of, a Fab (antigen-binding fragment) fragment having CGRP binding specificity. Regarding antibody Ab14, the Fab fragment comprises 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 also contemplates the addition, deletion, and variants of SEQ ID NO:131 and / or SEQ ID NO:133 in the Fab fragment while preserving CGRP binding specificity.

[0371] In one embodiment of the invention described herein (hereinafter), the Fab fragment can be produced by enzymatic digestion of Ab14 (e.g., papain). In another embodiment of the invention, anti-CGRP antibodies, such as Ab14 or its Fab fragment, can be produced by expression in mammalian cells, fungi, insects, or microbial systems such as yeast cells (e.g., diploid yeasts, such as Pichia pastoris) and other yeast strains, such as CHO, NSO, or HEK 293 cells. Suitable Pichia pastoris species include, but are not limited to, Pichia pastoris.

[0372] In another embodiment, the antibody fragment may be presented in one or more of the following non-limiting forms: Fab, Fab', F(ab')2, Fv, and single-chain Fv antibody forms. In a preferred embodiment, the anti-CGRP antibody described herein further comprises a κ constant light chain sequence comprising the sequence described below:

[0373] VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVD NALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC (SEQ ID NO: 283).

[0374] In another preferred embodiment, the anti-CGRP antibody described herein further comprises a γ-1 constant heavy chain polypeptide sequence comprising the sequence set forth below:

[0375] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:284).

[0376] In another embodiment, the present invention contemplates an isolated anti-CGRP antibody comprising V selected from the following H Polypeptide sequence: SEQ ID NO: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 or 133, or a variant thereof; and also contains V selected from the following L Polypeptide sequence: SEQ ID NO: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 or 131, or a variant thereof, wherein the V H or V L One or more framework residues (FR residues) in a polypeptide are replaced by another amino acid residue to produce an anti-CGRP antibody that specifically binds to CGRP. The present invention anticipates humanized and chimeric forms of these antibodies. The chimeric antibodies may include Fc derived from the constant regions of IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG9, IgG10, IgG11, IgG12, IgG13, IgG14, IgG15, IgG16, IgG17, IgG18, or IgG19.

[0377] In one embodiment of the invention, prior to initiating the humanization process mentioned herein, the antibody or V H or V L The polypeptide is derived from or selected from one or more rabbit B cell populations.

[0378] In another embodiment of the invention, the anti-CGRP antibody and its fragment do not have CGRP-R binding specificity. In another embodiment of the invention, the anti-CGRP antibody and its fragment inhibit the association of CGRP with CGRP-R. In another embodiment of the invention, the anti-CGRP antibody and its fragment inhibit the association of CGRP with CGRP-R and / or with additional proteins and / or their multimers, and / or antagonize their biological effects.

[0379] As described above, antibodies and their fragments can be post-translational modified to incorporate effector parts, such as chemical linkers, detectable parts, such as, for example, fluorescent dyes, enzymes, substrates, bioluminescent substances, radioactive substances, and chemiluminescent or functional parts, such as, for example, streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, and radioactive substances.

[0380] Antibodies or fragments thereof may also be chemically modified to provide additional advantages, such as increased peptide solubility, stability, and cycling time (in vivo half-life), or reduced immunogenicity (see U.S. Patent No. 4,179,337). The chemical moiety used for derivatization may be selected from water-soluble polymers, such as polyethylene glycol, ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, etc. Antibodies and fragments thereof may be modified at random or predetermined positions within the molecule and may include one, two, three, or more linked chemical moieties.

[0381] The polymer can have any molecular weight and can be branched or unbranched. For polyethylene glycol, the molecular weight is preferably between about 1 kDa and about 100 kDa for ease of handling and preparation (the term "about" indicates that some molecules are heavier and some are lighter than the stated molecular weight in the preparation of polyethylene glycol). Other sizes may be used depending on the desired therapeutic profile (e.g., the required duration of sustained release, the effect if any biological activity is present, ease of handling, the degree or lack of antigenicity, and other known effects of polyethylene glycol on therapeutic proteins or analogs). For example, the average molecular weight of polyethylene glycol can be approximately 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 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 glycol is described in, for example, U.S. Patent 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 contents of which are incorporated herein by reference.

[0382] Several linking methods are available to those skilled in the art, see, for example, EP 0 401384, which is incorporated herein by reference (coupling PEG with G-CSF), and also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting the PEGylation of GM-CSF using trifluoroethanesulfonyl chloride). For example, polyethylene glycol can be covalently linked via amino acid residues through reactive groups, such as free amino or carboxyl groups. Reactive groups are those that can bind to activated polyethylene glycol molecules. Amino acid residues having free amino groups may include lysine residues and N-terminal amino acid residues; those having free carboxyl groups may include aspartic acid residues, glutamic acid residues, and C-terminal amino acid residues. Thiol groups can also serve as reactive groups for linking polyethylene glycol molecules. For therapeutic purposes, linkage to an amino group, such as to an N-terminal or lysine group, is preferred.

[0383] As shown above, polyethylene glycol (PEG) can be linked to proteins via bonds to any number of amino acid residues. For example, PEG can be linked to peptides via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine ​​residues. One or more reactive chemistry methods can be used to link PEG to specific amino acid residues (such as lysine, histidine, aspartic acid, glutamic acid, or cysteine) or to residues of more than one type (such as lysine, histidine, aspartic acid, glutamic acid, or cysteine, and combinations thereof).

[0384] Optionally, the antibody or a fragment thereof may increase the in vivo half-life by fusing with albumin (including, but not limited to, recombinant human serum albumin or fragments or variants thereof (see, for example, U.S. Patent No. 5,876,969, issued March 2, 1999; EP Patent 0 413622; and U.S. Patent No. 5,766,883, issued June 16, 1998, all of which are incorporated herein by reference in their entirety)) or other circulating blood proteins, such as transferrin or ferritin. In a preferred embodiment, the polypeptides and / or antibodies of the present invention (including fragments or variants thereof) are fused with a mature form of human serum albumin (i.e., amino acids 1 to 585 of human serum albumin as shown in Figures 1 and 2 of EP Patent 0 322094, which is incorporated herein by reference in its entirety). The present invention also covers polynucleotides encoding the fusion proteins of the present invention.

[0385] Regarding the detectable portion, other exemplary enzymes include, but are not limited to, wasabi peroxidase, acetylcholinesterase, alkaline phosphatase, β-galactosidase, and luciferase. Other exemplary fluorescent materials include, but are not limited to, rhodamine, fluorescein, fluorescent isothiocyanate, umbelliferone, dichlorotriazineamine, phycoerythrin, and dansyl chloride. Other exemplary chemiluminescent components include, but are not limited to, luminol. Other exemplary bioluminescent substances include, but are not limited to, luciferin and jellyfish luminescent proteins. Other exemplary radioactive substances include, but are not limited to, iodine-125 (…). 125 I), carbon 14( 14 C), Sulfur 35 ( 35 S), tritium ( 3 H) and phosphorus 32 ( 32 P).

[0386] In terms of functional components, exemplary cytotoxic agents include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, and dacarbazine; alkylating agents such as nitrogen mustard, thiotepiperazine, melphalan, carmustine (BSNU), mitomycin C, lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, streptozotocin, mitomycin C, cis-dichlorodiamineplatin(II) (DDP), and cisplatin combined with carboplatin (… Paraplatin; anthracyclines include daunorubicin (formerly known as daunomycin), doxorubicin, detoxin, erythromycin, idarubicin, epirubicin, mitoxantrone, and bifenthrin; antibiotics include bleomycin (actinomycin D), bleomycin, chalcogenin, scintillans, and atrazomycin (AMC); and antimitotic agents such as vinca alkaloids, vincristine, and vinblastine. Other cytotoxic agents include paclitaxel (Taxol), ricin, Pseudomonas exotoxin, gemcitabine, cytosine B, bacitracin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, anthraquinone, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparagine, corticosteroids, mitotane (O,P'-(DDD)), interferon, and mixtures of these cytotoxic agents.

[0387] Other cytotoxic agents include, but are not limited to, chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel, gemcitabine, cazithromycin, doxorubicin, 5-fluorouracil, mitomycin C, actinomycin D, cyclophosphamide, vincristine, and bleomycin. Toxic enzymes derived from plants and bacteria, such as ricin, diphtheria toxin, and pseudomonadoxins, can bind to humanized or chimeric antibodies or their binding fragments to produce cell type-specific killing agents (Youle et al., Proc. Nat'l Acad. Sci. USA 77:5483 (1980); Gilliland et al., Proc. Nat'l Acad. Sci. USA 77:4539 (1980); Krolick et al., Proc. Nat'l Acad. Sci. USA 77:5419 (1980)).

[0388] Other cytotoxic agents include cytotoxic ribonucleases such as those described by Goldenborg in U.S. Patent No. 6,653,104. Embodiments of the invention also relate to radioimmunobinding agents in which a radionuclide emitting α or β particles is stably coupled to an antibody or a binding fragment thereof, with or without the use of a complex-forming agent. Such radionuclides include β emitters, such as phosphorus-32 (…). 32 P), Scandium-47 ( 47 Sc), Copper-67 ( 67 Cu), Gallium-67 ( 67 Ga), Y-88( 88 Y), Yttrium-90 ( 90 Y), Iodine-125 ( 125 I), iodine-131( 131 I), Samarium-153 153 Sm), Luc-177 ( 177 Lu), Rhenium-186 186 Re) or Rhenium-188 188 Re), and alpha emitters, such as astatine-211 ( 211 At), Lead-212 ( 212 Pb), Bismuth-212 ( 212 Bi) or bismuth-213( 213 Bi) or Actinium-225 225 Ac).

[0389] Methods for associating antibodies or their binding fragments with detectable moieties are known in the art, such as those described by way of example: Hunter et al., Nature 144:945 (1962); David et al., Biochemistry 13:1014 (1974); Pain et al., J. Immunol. Meth. 40:219 (1981); and Nygren, J., Histochem. and Cytochem. 30:407 (1982).

[0390] The embodiments described herein also include variants and equivalents substantially homologous to the antibodies, antibody fragments, dimer antibodies, SMIPs, camel antibodies, nanobodies, IgNARs, peptides, variable regions, and CDRs stated herein. These may contain, for example, conserved substitution mutations (i.e., one or more amino acids are substituted by similar amino acids). For example, a conserved substitution refers to the substitution of an amino acid with another of the same general kind, such as replacing an acidic amino acid with another acidic amino acid, replacing a basic amino acid with another basic amino acid, or replacing a neutral amino acid with another neutral amino acid. The purpose of conserved amino acid substitution is well known in the art.

[0391] In another embodiment, the present invention anticipates a polypeptide sequence having at least 90% or greater sequence homology with any one or more of the polypeptide sequences of the antibody fragments, variable regions, and CDRs stated herein. More preferably, the present invention anticipates a polypeptide sequence having at least 95% or greater sequence homology with any one or more of the polypeptide sequences of the antibody fragments, variable regions, and CDRs stated herein, even more preferably at least 98% or greater sequence homology, and even more preferably at least 99% or greater sequence homology. Methods for determining homology between nucleic acid and amino acid sequences are well known to those skilled in the art.

[0392] In another embodiment, the invention also contemplates antibody fragments, variable regions, and peptide homologues of the CDR described above, which also possess anti-CGRP activity as set forth herein. Non-limiting examples of anti-CGRP activity are set forth herein.

[0393] In another embodiment, the invention also contemplates the generation and use of anti-idiotype antibodies that bind to any of the foregoing sequences. In one exemplary embodiment, such anti-idiotype antibodies can be administered to an individual who has already received anti-CGRP antibodies to modulate, reduce, or neutralize the effects of the anti-CGRP antibodies. Such anti-idiotype antibodies can also be used to treat autoimmune diseases characterized by the presence of anti-CGRP antibodies. Another exemplary use of such anti-idiotype antibodies is for detecting the anti-CGRP antibodies of the present invention, for example, monitoring the level of anti-CGRP antibodies present in an individual's blood or other bodily fluids.

[0394] The present invention also contemplates anti-CGRP antibodies comprising any of the polypeptide or polynucleotide sequences described herein in place of any of the other polynucleotide sequences described herein. For example, and not as a limitation, the present invention contemplates antibodies comprising combinations of any of the variable light chain and variable heavy chain sequences described herein, and also contemplates antibodies resulting from any of the CDR sequences described herein replacing any of the other CDR sequences described herein.

[0395] Other exemplary embodiments of the present invention

[0396] In another embodiment, the present invention contemplates one or more anti-human CGRP antibodies or antibody fragments thereof that, like anti-human CGRP antibodies selected from the following, specifically bind to the same linear or conformational epitopes on intact human CGRP peptides or fragments thereof, and / or competitively bind to the same linear or conformational epitopes: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13, or Ab14. The 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, incorporating labeled or labeled antibodies, etc. In a preferred embodiment, the anti-human CGRP antibody or antibody fragment thereof, like Ab3, Ab6, Ab13, or Ab14, specifically binds to the same linear or conformational epitopes on intact human CGRP peptides or fragments thereof, and / or competitively binds to the same linear or conformational epitopes.

[0397] A preferred embodiment of the present invention relates to chimeric or humanized antibodies and fragments thereof (including Fab fragments) that have CGRP binding specificity and inhibit biological activity mediated by the binding of CGRP to the CGRP receptor. In one embodiment of the present invention, the chimeric or humanized anti-CGRP antibody is selected from Ab3, Ab6, Ab13, or Ab14.

[0398] In another embodiment of the invention, the anti-human CGRP antibody is an antibody that, when determined by epitope mapping of overlapping linear peptide fragments spanning the full length of a natural human CGRP polypeptide, specifically binds to the same linear or conformational epitopes of Ab3, Ab6, Ab13, or Ab14 on the intact CGRP polypeptide or fragments thereof.

[0399] The present invention also relates to an anti-CGRP antibody or antibody fragment as disclosed herein, which binds to the same CGRP epitope and / or competes with anti-CGRP antibodies for binding to CGRP, including but not limited to anti-CGRP antibodies selected from the following: Ab1, Ab2, Ab3, Ab4, Ab5, Ab6, Ab7, Ab8, Ab9, Ab10, Ab11, Ab12, Ab13 or Ab14.

[0400] In another embodiment, the invention also relates to an isolated anti-CGRP antibody or antibody fragment comprising V selected from the following H The polypeptide sequence contains one or more CDRs: 3, 13, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123 or 133, or variants thereof, and / or selected from the following V... L The polypeptide sequence contains one or more CDRs: 1, 11, 21, 31, 41, 51, 61, 71, 81, 91, 101, 111, 121 or 131, or variants thereof.

[0401] The present invention also anticipates that one or more anti-human CGRP antibodies discussed above are glycosylated or, if glycosylated, contain only mannose residues; contain an Fc region that has been modified to alter effector function, half-life, proteolytic and / or glycosylation activity; are human, humanized, single-chain or chimeric; and are humanized antibodies derived from rabbit (parental) anti-human CGRP antibodies.

[0402] The present invention also contemplates one or more anti-human CGRP antibodies, wherein the structural regions (FRs) of the variable light chain region and the variable heavy chain region of the antibody are human FRs, which are either unmodified or modified by replacing 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 the human FRs are derived from human variable heavy and light chain antibody sequences selected from a library of human reproductive antibody sequences based on their high level of homology with the corresponding rabbit variable heavy or light chain regions relative to other human reproductive antibody sequences contained in the library.

[0403] In one embodiment of the invention, an anti-human CGRP antibody or fragment can specifically bind to CGRP-expressing human cells and / or circulating soluble CGRP molecules in vivo, including CGRP expressed on or through human cells of patients suffering from diseases associated with CGRP-expressing cells.

[0404] The present invention also anticipates the direct or indirect linking to anti-human CGRP antibodies or fragments that can detect markers or therapeutic agents.

[0405] The present invention also contemplates one or more nucleic acid sequences that result in the expression of anti-human CGRP antibodies or antibody fragments as described above, said nucleic acid sequences comprising or optionally consisting of yeast or human preferred codons. The present invention also contemplates vectors (including plasmids or recombinant viral vectors) comprising said nucleic acid sequences. The present invention also contemplates host cells or recombinant host cells expressing at least one of the antibodies described above, including mammalian cells, yeast cells, bacterial cells, and insect cells. In a preferred embodiment, the host cell is a yeast cell. In another preferred embodiment, the yeast cell is a diploid yeast cell. In an exemplary embodiment, the yeast cell is Pichia pastoris.

[0406] The present invention also contemplates a treatment method comprising administering to a patient suffering from a disease or symptom associated with CGRP-expressing cells at least one anti-human CGRP antibody or fragment described herein a therapeutically effective amount. The present invention also contemplates that the treatment method involves administering two or more anti-CGRP antibodies or fragments thereof disclosed herein. If more than one antibody is administered to a patient, the multiple antibodies may be administered simultaneously or in parallel, or they may be administered alternately.

[0407] The anti-CGRP activity of the CGRP-binding specific anti-CGRP antibody and its fragment of the present invention can also be described by its binding force to CGRP or the strength of its affinity for CGRP. In one embodiment of the present invention, the CGRP-binding specific anti-CGRP antibody and its fragment of the present invention have a dissociation constant (K) lower than or equal to the following: D Combined with CGRP: 5x10 -7 M, 10 -7 M, 5x10 -8 M, 10 -8 M, 5x10 -9 M, 10 -9 M, 5x10 -10 M, 10 -10 M, 5x10 -11 M, 10 - 11 M, 5x10 -12 M, 10-12 M, 5x10 -13 M or 10 -13 M. Preferably, the anti-CGRP antibody and its fragment are at a ratio of less than or equal to 10. -11 5x10 -12 M or 10 -12 The dissociation constant of M binds to CGRP. In another embodiment of the invention, the anti-CGRP antibody and fragment thereof of the invention, which have CGRP binding specificity, bind to linear or conformational CGRP epitopes.

[0408] In another embodiment of the invention, the anti-CGRP activity of the anti-CGRP antibody and its fragment having CGRP binding specificity is less than or equal to 10. -4 S -1 5x10 -5 S -1 10 -5 S -1 5x10 -6 S -1 10 -6 S -1 5x10 -7 S -1 Or 10 -7 S -1 The rate of detachment from CGRP binds to it.

[0409] In another embodiment of the invention, the anti-CGRP activity of the CGRP-binding specific anti-CGRP antibody and fragment thereof of the present invention is manifested by preventing, improving, or reducing symptoms of CGRP-related diseases or conditions, or optionally treating CGRP-related diseases or conditions. Non-limiting examples of CGRP-related diseases or conditions are set forth herein.

[0410] Polynucleotides encoding anti-CGRP antibody peptides

[0411] In an exemplary embodiment, the anti-CGRP antibody may be encoded by a polynucleotide sequence listed in the biological sequence listing herein, or by other coding polynucleotides readily identifiable by those skilled in the art. Examples 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:11), 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 polynucleotide of SEQ ID NO:161 (encoding the polypeptide of SEQ ID NO:21), the polynucleotide of SEQ ID NO:162 (encoding the polypeptide of SEQ ID NO:22), the polynucleotide of SEQ ID NO:163 (encoding the polypeptide of SEQ ID NO:23), and the polynucleotide of SEQ ID NO:164 (encoding the polypeptide of SEQ ID NO:24). The following polynucleotides are listed: SEQ ID NO:171 (encoding the polypeptide of SEQ ID NO:31), SEQ ID NO:172 (encoding the polypeptide of SEQ ID NO:32), SEQ ID NO:173 (encoding the polypeptide of SEQ ID NO:33), SEQ ID NO:174 (encoding the polypeptide of SEQ ID NO:34), SEQ ID NO:181 (encoding the polypeptide of SEQ ID NO:41), SEQ ID NO:182 (encoding the polypeptide of SEQ ID NO:42), SEQ ID NO:183 (encoding the polypeptide of SEQ ID NO:43), SEQ ID NO:184 (encoding the polypeptide of SEQ ID NO:44), SEQ ID NO:191 (encoding the polypeptide of SEQ ID NO:51), SEQ ID NO:192 (encoding the polypeptide of SEQ ID NO:52), SEQ ID NO:193 (encoding the polypeptide of SEQ ID NO:53), and SEQ ID NO:194 (encoding the polypeptide of SEQ ID NO:54).The polynucleotides SEQ ID NO:201 (encoding the polypeptide of SEQ ID NO:61), SEQ ID NO:202 (encoding the polypeptide of SEQ ID NO:62), SEQ ID NO:203 (encoding the polypeptide of SEQ ID NO:63), SEQ ID NO:204 (encoding the polypeptide of SEQ ID NO:64), SEQ ID NO:211 (encoding the polypeptide of SEQ ID NO:71), SEQ ID NO:212 (encoding the polypeptide of SEQ ID NO:72), SEQ ID NO:213 (encoding the polypeptide of SEQ ID NO:73), SEQ ID NO:214 (encoding the polypeptide of SEQ ID NO:74), SEQ ID NO:221 (encoding the polypeptide of SEQ ID NO:81), SEQ ID NO:222 (encoding the polypeptide of SEQ ID NO:82), SEQ ID NO:223 (encoding the polypeptide of SEQ ID NO:83), and SEQ ID NO:224 (encoding the polypeptide of SEQ ID NO:84) are listed. The polynucleotides SEQ ID NO:231 (encoding the polypeptide of SEQ ID NO:91), SEQ ID NO:232 (encoding the polypeptide of SEQ ID NO:92), SEQ ID NO:233 (encoding the polypeptide of SEQ ID NO:93), SEQ ID NO:234 ​​(encoding the polypeptide of SEQ ID NO:94), SEQ ID NO:241 (encoding the polypeptide of SEQ ID NO:101), SEQ ID NO:242 (encoding the polypeptide of SEQ ID NO:102), SEQ ID NO:243 (encoding the polypeptide of SEQ ID NO:103), SEQ ID NO:244 (encoding the polypeptide of SEQ ID NO:104), SEQ ID NO:251 (encoding the polypeptide of SEQ ID NO:111), SEQ ID NO:252 (encoding the polypeptide of SEQ ID NO:112), SEQ ID NO:253 (encoding the polypeptide of SEQ ID NO:113), and SEQ ID NO:254 (encoding the polypeptide of SEQ ID NO:113) are listed. The polypeptide of SEQ ID NO:114), the polynucleotide of SEQ ID NO:261 (encoding the polypeptide of SEQ ID NO:121), the polynucleotide of SEQ ID NO:262 (encoding the polypeptide of SEQ ID NO:122), SEQ IDThe polynucleotide NO:263 (a polypeptide encoding SEQ ID NO:123), the polynucleotide NO:264 (a polypeptide encoding SEQ ID NO:124), the polynucleotide NO:271 (a polypeptide encoding SEQ ID NO:131), the polynucleotide NO:272 (a polypeptide encoding SEQ ID NO:132), the polynucleotide NO:273 (a polypeptide encoding SEQ ID NO:133), or the polynucleotide NO:274 (a polypeptide encoding SEQ ID NO:134).

[0412] B-cell screening and isolation

[0413] In one embodiment, the present invention is contemplated for use in the preparation and isolation of an antigen-specific B cell clonal population of at least one CGRP antigen-specific cell, which can be used to generate monoclonal antibodies against CGRP, specific for a desired CGRP antigen or a nucleic acid sequence corresponding to such an antibody. For example, methods for the preparation and isolation of such antigen-specific B cell clonal populations are taught in U.S. Patent Publication No. US2007 / 0269868 to Carvalho-Jensen et al., the disclosure of which is incorporated herein by reference in its entirety. Methods for the preparation and isolation of such antigen-specific B cell clonal populations are also taught in the examples herein. Methods for “enriching” cell populations by size or density are known in the art. See, for example, U.S. Patent No. 5,627,052. These steps can be used in addition to enriching the cell population by antigen specificity.

[0414] Methods for humanized antibodies

[0415] In another embodiment, the present invention is contemplated for use in methods for humanizing the heavy and light chains of antibodies. For example, methods for humanizing the heavy and light chains of antibodies applicable to anti-CGRP antibodies are taught in U.S. Patent Application Publication No. US2009 / 0022659 to Olson et al. and U.S. Patent No. 7,935,340 to Garcia-Martinez et al., the disclosures of which are incorporated herein by reference in their entirety.

[0416] Screening Analysis

[0417] The invention also includes screening assays designed to assist in identifying patients exhibiting symptoms of CGRP-related diseases or conditions. For example, the invention includes assays to detect insulin insensitization (resistance) or glucose utilization in an individual. The individual may optionally be in a fasting or post-meal state.

[0418] In one embodiment of the invention, the anti-CGRP antibody or its CGRP-binding fragment is used to detect the presence of CGRP in a biological sample obtained from a patient exhibiting symptoms of a CGRP-related disease or condition. The presence of CGRP or an elevated level thereof, when compared to pre-disease levels of CGRP in comparable biological samples, can be helpful in diagnosing CGRP-related diseases or conditions.

[0419] Another embodiment of the invention provides a diagnostic or screening analysis to assist in the diagnosis of CGRP-related diseases or conditions in patients exhibiting symptoms of CGRP-related diseases or conditions identified herein, comprising analyzing CGRP expression levels in biological samples from said patients using a post-translationally modified anti-CGRP antibody or a binding fragment thereof. The anti-CGRP antibody or its binding fragment may be post-translationally modified to include a detectable portion as previously stated in this disclosure.

[0420] CGRP levels in biological samples can be determined using modified anti-CGRP antibodies or their binding fragments as described herein, and compared to standard CGRP levels (e.g., levels in normal biological samples). Skilled clinicians will understand that some variability can exist between normal biological samples and will take these into account when evaluating results. In one embodiment of the invention, the anti-CGRP antibody of the invention can be used to obtain an association between CGRP expression levels and a specific stage of impaired glucose metabolism. For example, establishing an association between circulating CGRP levels and glucose and / or insulin levels would allow for the establishment of levels of insulin insensitization or hyperglycemia. Additionally, methods known in the art can be used to measure an individual's insulin sensitivity, such as those described by Muniyappa et al. (Am J Physiol Endocrinol Metab 294:E15-E26, 2008), which is incorporated herein by reference in its entirety. In short, a variety of methods can be used to measure insulin sensitivity, including hyperinsulinemia-eukaryotic glucose clamping, insulin suppression test, QUICKI, HOMA, 1 / insulin, or Matsuda index. Those skilled in the art will be able to measure CGRP in many individuals to establish CGRP expression ranges corresponding to clinically defined stages of diabetes development or prediabetes.

[0421] The measurements listed above can also be used to monitor diseases or conditions, where CGRP levels obtained from biological samples from patients believed to have CGRP-related diseases or conditions are compared with CGRP levels from previous biological samples from the same patient to determine whether the patient's CGRP levels have changed with, for example, a treatment regimen. Those skilled in the art will understand that by measuring a patient's CGRP at different intervals, the progression of impairment in an individual's ability to metabolize glucose can be determined.

[0422] The present invention also relates to an in vivo imaging method for detecting the presence of cells expressing CGRP, the method comprising administering a diagnostically effective amount of a diagnostic composition. The detection can be used as part of the planning of an effective treatment regimen for patients with diabetes or at risk of developing diabetes.

[0423] In one embodiment, the method of the present invention includes one or more compositions of the anti-CGRP antibodies disclosed herein for treating glucose dysmegma, such as insulin resistance, poor insulin secretion, or hyperglycemia. Of particular interest are one or more of, for example, sulfonylureas, PPAR-γ agonists, GLP-1 receptor agonists, dipeptidyl peptidase IV inhibitors, amyloid analogs, biguanides, dopamine D2 receptor agonists, megatitinides, alpha-glucosidase inhibitors, bile acid sequestrants for dyslipidemia, insulin, cytokine therapy, gene therapy, and antibody therapy, as well as anti-CGRP antibodies or fragments thereof. Examples of biguanides include metformin, such as Glucophage and Glucophage XR (Bristol Myers Squibb / Merck Serono), Fortamet (Watson), Glumetza (Biovail / Depomed / Santarus), and generic drugs. Examples of sulfonylureas include glimepiride, such as Amaryl (Sanofi) and generic versions; glipizide, such as Glucotrol and Glucotrol XL (Pfizer) and generic versions; glibenclamide, such as Diabeta (Sanofi), Micronase / Glynase (Pfizer) and generic versions; metformin + glibenclamide, such as Glucovance (Bristol Myers Squibb), Suguan M (Sanofi-Aventis), GlicoRest, GlucoNorm (Abiogen), Bi-Euglucon (Roche) and generic versions; and metformin + glipizide, such as Metalip (Bristol Myers Squibb) and generic versions. Examples of PPAR-γ agonists include: rosiglitazone, such as Avandia (GlaxoSmithKline); pioglitazone, such as Actos (Takeda) and generic versions; rosiglitazone + metformin, such as Avandamet (GlaxoSmithKline); pioglitazone + metformin, such as Actoplus Met XR (Takeda); pioglitazone + glimepiride, such as Avandaryl / Avaglim (GlaxoSmithKline); and pioglitazone + glimepiride, such as Duetact / Tandemact / Sonias (Takeda).Examples of GLP-1 receptor agonists include: exenatide, such as Byetta (Bristol Myers Squibb / AstraZeneca); liraglutide, such as Victoza (Novo Nordisk); and exenatide LAR, such as Bydureon (Bristol Myers Squibb / AstraZeneca). Examples of 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); tenegliptin, such as Teneligliptin (Mitsubishi Tanabe / Daiichi Sankyo); sitagliptin plus metformin, such as Janumet (Merck) and Janumet. XR (Merck); Sitagliptin + Simvastatin, such as JuviSync (Merck); Vildagliptin + Metformin, such as Eurcreas (Novartis); Segliptin + Metformin, such as Kombiglyze / Kombiglyze XR (AstraZeneca / Bristol Myers Squibb); Alogliptin + Pioglitazone, such as Liovel (Takeda / Furiex); Ringerliptin + Metformin, such as Jentadueto (BoehringerIngelheim / Eli Lilly). Examples of meglinides include: repaglinide, such as GlucoNorm / Prandin / NovoNorm (Daiichi Sankyo / Fournier Pharma / Novo Nordisk); nateglinide, such as Starlix (Novartis), Fastic (Daiichi Sankyo), Starsis (Astellas), and generic drugs; and mitiglinide, such as Glufast (Kissei / Takeda).Examples of alpha-glucosidase inhibitors include: acarbose, such as Precose / Glucobay (Bayer) and generic versions; miglitol, such as Glyset (Pfizer), Diastabol (Sanofi), Seibule (Sanwa Kagaku) ​​and generic versions; and voglibose, such as Basen (Takeda) and generic versions. Examples of bile acid sequestrants include: colesevelam, such as Cholestagel (Sanofi) and Welchol (Daiichi Sankyo). Examples of dopamine D2 receptor agonists include: bromocriptine, such as Cycloset (Santarus). Examples of amyloid analogs include: Pramlintide, such as Symlin (Bristol Myers Squibb / AstraZeneca). Examples of rapid-acting insulins include: lispro insulin, such as Humalog (EliLilly); aspart insulin, such as NovoLog (Novo Nordisk) and NovoRapid (Novo Nordisk); and lisglutinin insulin, such as Apidra (Sanofi). Examples of conventional human insulins include: Humulin / Umuline Rapide (EliLilly), Novolin R (Novo Nordisk), and Actrapid (Sanofi). Examples of intermediate-acting insulins include: Humulin N (Eli Lilly) and Novolin N (Novo Nordisk). Examples of long-acting insulins include: glargine insulin, such as Lantus (Sanofi), and detemir insulin, such as Leavemir (Novo Nordisk).

[0424] This invention also provides a kit for detecting the binding of the anti-CGRP antibody of this invention to CGRP. Specifically, the kit can be used to detect the presence of CGRP that specifically reacts with the anti-CGRP antibody of this invention or its immunoreactive fragment. The kit may also include an antibody that binds to a substrate, a secondary antibody that is antigen-reactive, and reagents for detecting the reaction between the secondary antibody and the antigen. This kit may be an ELISA kit and may include, as appropriate, a substrate, primary and secondary antibodies, and any other desired reagents, such as a detectable fraction, enzyme substrate, and chromogenic reagent, as described herein. The diagnostic kit may also be in the form of an immunoblotting kit. The diagnostic kit may also be in the form of a chemiluminescence kit (Meso Scale Discovery, Gaithersburg, MD). The diagnostic kit may also be a lanthanide-based detection kit (PerkinElmer, San Jose, CA).

[0425] Clinicians with expertise in the field will understand biological samples, including but not limited to serum, plasma, urine, saliva, mucus, pleural fluid, synovial fluid, and cerebrospinal fluid.

[0426] Methods to improve or reduce symptoms of CGRP-related diseases or conditions, or to treat or prevent CGRP-related diseases or conditions.

[0427] In another embodiment of the invention, the anti-CGRP antibody or fragment thereof described herein is useful for improving or reducing symptoms of CGRP-related diseases or conditions, or for treating or preventing CGRP-related diseases or conditions. The anti-CGRP antibody or fragment thereof described herein, as well as combinations thereof, can also be administered to patients requiring treatment for CGRP-related diseases or conditions in the form of pharmaceutical compositions described in more detail below.

[0428] In another embodiment of the invention, the anti-CGRP antibody or fragment thereof described herein is suitable for improving or reducing, or treating or preventing, the following symptoms: poor glucose tolerance, insulin resistance (insensitization), poor insulin secretion, lipotoxicity, hyperglycemia, and pancreatic β-cell failure due to diabetes, prediabetes, type 1 diabetes, type 2 diabetes, or gestational diabetes.

[0429] In exemplary embodiments, the anti-CGRP antibody or fragment thereof described herein may be administered to individuals at risk of developing diabetes, such as those diagnosed with prediabetes. Without being bound by theory, it is believed that by restoring insulin sensitivity, the anti-CGRP antibody may delay or prevent the progression to diabetes.

[0430] In other exemplary embodiments, the anti-CGRP antibody or fragment thereof described herein may be administered to a patient whose blood glucose levels have not been normalized by another treatment, such as metformin, pioglitazone, sulfonylureas, meglitinides, oral thiazolidinediones (TZDs) such as pioglitazone, GLP-1 agonists such as exenatide, and DPP4 inhibitors such as sitagliptin, vildagliptin, selenoglitazone, alogliptin, and ringagliptin. Treatment with tergliptin, or combination therapy, such as metformin and pioglitazone, metformin and sulfonylureas, metformin and meglitinides, metformin and TZD, metformin and pioglitazone, metformin and GLP-1 agonists, metformin and exenatide, sitagliptin and metformin, sitagliptin and simvastatin, vildagliptin and metformin, segglitazone and metformin, alogliptin and pioglitazone, or lingagliptin and metformin.

[0431] In other exemplary embodiments, the anti-CGRP antibody or fragment thereof described herein may be administered to individuals, for example, with a body mass index of at least 25, for the prevention or treatment of obesity. Without being theoretically limited, it is believed that this anti-CGRP antibody may increase glucose utilization in the peripheral and / or liver, thereby increasing metabolic rate and resulting in weight loss. The anti-CGRP antibody may be administered in combination with another anti-obesity agent, such as orlistat, rimonaban, sibutramine, peptide YY (PYY, a 36-amino acid peptide that reduces appetite), PYY analogs, CB-1 antagonists, rimonaban, leptin, leptin analogs, or phentermine.

[0432] application

[0433] In one embodiment of the invention, the anti-CGRP antibody described herein or a CGRP-binding fragment thereof, and combinations of said antibody or antibody fragments, are administered to the individual at a concentration between about 0.1 and 100.0 mg / kg of the recipient's body weight. In one embodiment of the invention, the anti-CGRP antibody described herein or a CGRP-binding fragment thereof, and combinations of said antibody or antibody fragments, are administered to the individual at a concentration of about 0.4 mg / kg of the recipient's body weight. In another embodiment of the invention, the anti-CGRP antibody described herein or a CGRP-binding fragment thereof, and combinations of said antibody or antibody fragments, are administered to the recipient at 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 weekly or less, or once daily or less.

[0434] The Fab fragment can be administered every 2 weeks or less, weekly or less, once daily or less, multiple times daily, and / or every few hours. In one embodiment of the invention, a patient receives 0.1 mg / kg to 40 mg / kg of the Fab fragment daily to achieve the desired results, administered in divided doses of 1 to 6 times daily, or in a sustained-release form.

[0435] It should be understood that the concentration of antibody or Fab administered to a particular patient may be higher or lower than the illustrative concentrations listed in the two preceding paragraphs above.

[0436] Those skilled in the art can determine the effective dosage and frequency of administration through routine experiments, for example, by the guidance of the disclosures herein and the following teachings: Goodman, LS, Gilman, A., Brunton, LL, Lazo, JS, & Parker, KL (2006). Goodman & Gilman's the pharmacological basis of therapyeutics. New York: McGraw-Hill; Howland, RD, Mycek, MJ, Harvey, RA, Champe, PC, & Mycek, MJ (2006). Pharmacology. Lippincott's illustrated reviews. Philadelphia: Lippincott Williams & Wilkins; and Golan, DE (2008). Principles of pharmacology: the pathophysiologic basis of drug therapy. Philadelphia, Pa., [et al.]: Lippincott Williams & Wilkins.

[0437] In another embodiment of the invention, the anti-CGRP antibody described herein or a CGRP-binding fragment thereof, and a combination of said antibody or antibody fragment, are administered to an individual in the form of a pharmaceutical preparation.

[0438] "Pharmaceutical composition" means a chemical or biological composition suitable for administration to mammals. These compositions may be specifically formulated for administration via one or more routes, including but not limited to buccal, epidermal, intradural, inhalation, intra-arterial, intracardiac, intraventricular, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, intraspinal, intravenous, oral, parenteral, rectal via enema or suppository, subcutaneous, subdermal, sublingual, transdermal, and transmucosal routes. Furthermore, they may be administered by injection, powder, liquid, gel, drops, or other means.

[0439] In one embodiment of the invention, the anti-CGRP antibody described herein or a CGRP-binding fragment thereof, and combinations of said antibody or antibody fragment, may optionally be administered in combination with one or more active agents. Such active agents include analgesics, antihistamines, antipyretics, anti-inflammatory agents, antibiotics, antivirals, and anticytokine agents. Active agents include TNF-α, IL-2, IL-4, IL-6, IL-10, IL-12, IL-13, IL-18, IFN-α, IFN-γ, BAFF, CXCL13, IP-10, VEGF, EPO, EGF, HRG, hepatocyte growth factor (HGF), agonists, antagonists, and modulators of iron-regulating hormones, including antibodies that respond to any of the foregoing and antibodies that respond to any of their receptors. The active agents also include, but are not limited to, 2-arylpropionic acid, aceclofenac, acetylmethacin, acetylsalicylic acid (aspirin), alclofenac, alminoprofen, amoxiprin, ampyrone, aromatic alkyl acids, azapropazone, benorylate / benorilate, benoxaprofen, bromofenac, and carbofen. (Carprofen), Celecoxib, Magnesium Choline Salicylate, Clofezone, COX-2 Inhibitors, Dexibuprofen, Dexketoprofen, Diclofenac, Diflunisal, Droxicam, Ethenzamide, Etodolac, Etoricoxib, Faislamine, Fenamic Acid, Fenbufen, Fenoprofen, Flufenamic Acidacid), flunoprofen, flupiprofen, ibuprofen, isobutroxam, indomethacin, indoprofen, kebuzone, ketoprofen, ketorolac, lornoxicam, loxoprofen, lumiracoxib, magnesium salicylate, meclofenamic acid, mefenamic acid, meloxicam, metamizole, methyl salicylate, mofebutazone, nabumetone, naproxen, N-arylanthranilic acid acid), nerve growth factor (NGF), oxametacin, oxaprozin, oxicam, oxyphenbutazone, parecoxib, phenazone, phenylbutazone, piroxicam, pirprofen, profens, proglumetacin, pyrazolidine derivatives, rofecoxib, salicyl salicylate, salicylamide, salicylates, substance P, sulfadiazine, sulindac, suprofen, tenoxicam, tiaprofenic acid, tolfenamic acid The drugs include acetic acid, tolmetin, and valdecoxib.

[0440] Antihistamines are any compound that prevents the action of histamine or the release of histamine from cells (such as mast cells). Antihistamines include, but are not limited to, acrivastine, astemizole, azatadine, azelastine, betatastine, brompheniramine, buclizine, cetirizine, cetirizine analogs, chlorpheniramine, clemastine, CS 560, cyproheptadine, desloratadine, dexchlorpheniramine, ebastine, epinastine, fexofenadine, and HSR. 609. Hydroxyzine, levocabastine, loratidine, methscopolamine, mizolastine, norastemizole, phenindamine, promethazine, pyrilamine, terfenadine, and tranilast.

[0441] Antibiotics include, but are not limited to, amikacin, aminoglycosides, amoxicillin, ampicillin, ansamycins, arsphenamine, azithromycin, azlocillin, aztreonam, bacitracin, carbacephem, carbapenems, carbenicillin, cefaclor, and cephalosporins. Cefadroxil, Cefalexin, Cefalothin, Cefalotin, Cefamandole, Cefazolin, Cefdinir, Cefditoren, Cefepime, Cefixime, Cefotaxime, Cefoxitin, Cefoxitin, Cefopodoxime, Cefprozil, and other similar drugs are listed. Ceftazidime, Ceftibuten, Ceftizoxime, Ceftobiprole, Ceftriaxone, Cefuroxime, Cephalosporins, Chloramphenicol, Cilastatin, Ciprofloxacin, Clarithromycin, Clindamycin, Cloxacillin, Colistin Colistin, Co-trimoxazole, Dalfopristin, Demeclocycline, Dicloxacillin, Diirithromycin, Doripenem, Doxycycline, Enoxacin, Ertapenem, Erythromycin, Ethambutol, Flucloxacillin, FosfomycinFurazolidone, Fusidic acid, Gatifloxacin, Geldanamycin, Gentamicin, Glycopeptides, Herbimycin, Imipenem, Isoniazid, Kanamycin, Levofloxacin, Lincomycin, Linezolid, Lomefloxacin, Loracarbef, Macrolides, Mafenide, Meropenem, Meticillin-resistant, Metronidazole, Meropenem (Mezlocillin), Minocycline, Monobactams, Moxifloxacin, Mupirocin, Nafcillin, Neomycin, Netilmicin, Nitrofurantoin, Norfloxacin, Ofloxacin, Oxacillin, Oxytetracycline, Paromomycin, Penicillin, Penicillins, Piperacillin, Platensimycin, Polymyxin B B) Peptides, Prontosil, Pyrazinamide, Quinolones, Quinupristin, Rifampicin, Roxithromycin, Spectinomycin, Streptomycin, Sulfacetamide, Sulfamethizole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Sulfonamides, Teicoplanin, Telithromycin, TetracyclineTetracyclines, Ticarcillin, Tinidazole, Tobramycin, Trimethoprim, Trimethoprim-Sulfamethoxazole, Troleandomycin, Trovafloxacin, and Vancomycin.

[0442] The active agents also include aldosterone, beclometasone, betamethasone, corticosteroids, cortisol, cortisone acetate, deoxycorticosterone acetate, dexamethasone acetate, fludrocortisone acetate, glucocorticoids, hydrocortisone, methylprednisolone, prednisolone, prednisone, steroids, and triamcinolone. Any suitable combination of these active agents is also anticipated.

[0443] "Pharmaceutical excipient" or "pharmaceuticalally acceptable excipient" is a carrier in which an active therapeutic agent is formulated, typically a liquid. In one embodiment of the invention, the active therapeutic agent is a humanized antibody, or one or more fragments thereof, as described herein. Excipients typically do not provide any pharmaceutical activity to the formulation, but they may provide chemical and / or biological stability, as well as release characteristics. Exemplary formulations can be found, for example, in Remington's Pharmaceutical Sciences, 19th edition, ed. Grennaro, A., 1995, which is incorporated herein by reference.

[0444] As used herein, "pharmaceutically acceptable carrier" or "excipient" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, or sublingual administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions, as well as sterile powders for the ad-hoc preparation of sterile injectable solutions or dispersions. The use of such media and reagents for pharmaceutically active substances is well known in the art. Their use in the pharmaceutical compositions of the present invention is contemplated, except for any conventional media and reagents incompatible with the active compound. Complementary active compounds may also be incorporated into the compositions.

[0445] Pharmaceutical compositions typically must be sterile and stable under the conditions of preparation and storage. This invention contemplates pharmaceutical compositions in lyophilized form. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. This invention also contemplates the inclusion of stabilizers in the pharmaceutical compositions. For example, in the case of dispersions, this can be achieved by maintaining the desired particle size and by using surfactants to maintain appropriate flowability.

[0446] In many cases, it is preferable to include an isotonic agent in the composition, such as sugars, polyols such as mannitol, sorbitol, or sodium chloride. The absorption of injectable compositions can be prolonged by including agents such as monostearates and gelatin. Furthermore, basic peptides can be formulated into time-release formulations, for example, compositions comprising sustained-release polymers. Active compounds can be prepared using carriers that prevent the compound from being rapidly released, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic acid-polyglycolic acid copolymers (PLG) can be used. Numerous methods for preparing such formulations are known to those skilled in the art.

[0447] For each of the listed embodiments, the compound can be administered via various dosage forms. Any biologically acceptable dosage form known to those skilled in the art is available, and combinations thereof are contemplated. Examples of such dosage forms include, but are not limited to, reconstituteable powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible particles, capsules, inhalers, aerosol inhalers, patches, particle inhalers, implants, reservoir implants, injectables (including subcutaneous, intramuscular, intravenous, and intradermal), injectables, and combinations thereof.

[0448] The above description of the various embodiments of the present invention is not intended to be exhaustive or to limit the precise forms disclosed herein. Although specific embodiments and examples of the invention have been described herein for illustrative purposes, various equivalent modifications that will be recognized by those skilled in the art will fall within the scope of the invention. The teachings provided herein regarding the invention can be applied to purposes other than those described above.

[0449] Based on the detailed description above, these and other variations can be made to the present invention. Generally, the terminology used in the following claims should not be construed as limiting the invention to the specific embodiments disclosed in the specification and claims. Therefore, the invention is not limited to the disclosure, and its scope is entirely determined by the following claims.

[0450] The invention may be practiced in ways other than those specifically described in the foregoing description and embodiments. Given the foregoing teachings, many modifications and variations can be made to the invention, and therefore fall within the scope of the appended claims.

[0451] Certain teachings relating to obtaining a clonal population of antigen-specific B cells are disclosed in U.S. Provisional Patent Application No. 60 / 801,412, filed May 19, 2006, the disclosure of which is incorporated herein by reference in its entirety.

[0452] Teachings relating to the humanization of rabbit-derived monoclonal antibodies and preferred sequence modifications to maintain antigen-binding affinity are disclosed in International Application No. PCT / US2008 / 064421, filed May 21, 2008, which corresponds to International Application Publication No. WO / 2008 / 144757 entitled “Novel Rabbit Antibody Humanization Methods and Humanized Rabbit Antibodies”, the disclosure of which is incorporated herein by reference in its entirety.

[0453] Certain teachings on the production of antibodies or fragments thereof using paired competent yeast and the corresponding methods are disclosed in U.S. Patent Application No. 11 / 429,053 (U.S. Patent Application Publication No. US2006 / 0270045), filed May 8, 2006, the disclosure of which is incorporated herein by reference in its entirety.

[0454] Certain CGRP antibody polynucleotides and peptides are disclosed in the sequence listing filed with this patent application, and the disclosure of the sequence listing is incorporated herein by reference in its entirety.

[0455] The entire disclosure of all documents (including patents, patent applications, journal articles, abstracts, manuals, books or other disclosures) cited in the background, detailed description and embodiments of this invention is incorporated herein by reference in its entirety.

[0456] The following examples are set forth to provide a complete disclosure and description of how and how one skilled in the art can do and use the invention, and are not intended to limit the scope of the invention. Efforts have been made to ensure the accuracy of the numbers involved (e.g., quantity, temperature, concentration, etc.), but some experimental errors and inaccuracies should be allowed. Unless otherwise specified, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric pressure or close to atmospheric pressure. Example

[0457] Example 1

[0458] Normal rats were treated with Ab14 100 mg / kg (administered intravenously as a single dose 48 h prior to the clamping procedure) and the effect was compared with that of metformin 500 mg / kg (administered orally twice, 24 h and 4 h prior to the clamping procedure). The antibodies used in this example consist of the light and heavy polypeptide chains of SEQ ID NO 132 and 134.

[0459] Blood glucose levels were measured before treatment, 18 hours after treatment with metformin and Ab14, and 42 hours after treatment with Ab14 under fed conditions. Neither compound affected blood glucose under these conditions. Blood glucose was measured under fasting conditions, just before clamping, and only metformin showed a significant reducing effect (17%). Both compounds slightly reduced plasma insulin levels measured before treatment, 18 hours after metformin treatment, and 42 hours after Ab14 treatment under fed conditions, as did the insulin resistance index HOMA-IR (not significant).

[0460] The concentration of Ab14 was determined from plasma samples taken from animals treated with Ab14 exactly 48 hours prior to the clamping procedure. The results of this analysis confirmed that the systemic exposure to Ab14 in rats undergoing the clamping procedure ranged from 642 to 797 μg / mL.

[0461] To assess the effect on systemic insulin sensitivity, 0.3 U / kg / h insulin and 3H-glucose was used in the clamping procedure. Rats were fasted for 6 hours prior to perfusion (180 minutes). Steady state was reached 140 minutes after perfusion, and glucose infusion rate (GIR), systemic glucose turnover (GTO), hepatic glucose production (HGP), glycolysis, and glycogen synthesis were calculated from 140 to 180 minutes. Tissue-specific glucose utilization was measured by administering a bolus of 14-C-2-deoxyglucose 1 hour before the end of clamping. As expected, metformin significantly increased GIR (27%) and GTO (30%) by increasing glycolysis and glycogen synthesis. Metformin increased glucose utilization in mixed lateral extensor digitorum (VL, 49% p < 0.05), glycolytic extensor digitorum longus (EDL, 19% NS), and decreased glucose utilization in the heart (-39%, p < 0.01), presumably due to stimulation of myocardial fatty acid oxidation. Ab14 tends to increase GIR and GTO (NS), and has a stronger effect than metformin on glucose utilization in VL (70%, p<0.01), EDL (26%, NS), and oxidized soleus muscle (27%, NS). Ab14 also tends to increase intracardiac glucose utilization (21%, NS). Similar to metformin, Ab14 does not affect glucose utilization in white adipose tissue (deep and subcutaneous).

[0462] In summary, Ab14 tends to improve systemic glucose utilization in normal rats after acute treatment and has a significant effect on glucose utilization in muscle (VL).

[0463] method

[0464] Male Stowe rats were kept in cages (1500cm) throughout the experimental period. 2 The cages are 21cm x 100cm in size. The bedding is changed weekly. Animals are housed in groups of 3-4 during the acclimatization period, and then individually after surgery until the restraint procedure. A reversed 12-hour light cycle (lights off at 8:00 AM), 22±2°C, and 55±10% relative humidity are maintained. Standard diet (RM1(E)801492, SDS) and tap water are provided for free access.

[0465] After a 2-week acclimatization period, the rats were anesthetized (with isoflurane) and a catheter was inserted into the femoral vein. A 5-6 day recovery period followed before the clamping procedure.

[0466] Blood glucose (BG) was measured using a glucometer from the tail tip between 7:30 AM and 8:00 AM (just before lights out), and blood was collected (on EDTA) immediately after plasma insulin measurement. The following table describes the procedure:

[0467]

[0468] Plasma samples were kept at -80°C until insulin was measured (using the ELISA method).

[0469] Blood samples (~200 μL) were obtained from each Group 2 animal just before the clamping procedure, processed into plasma (~60 μL), and maintained at -80°C for subsequent determination of Ab14 concentration using the Meso Scale Discovery (MSD) ELISA platform.

[0470] The mediator and Ab14 were administered intravenously 48 hours before the T0 procedure (2 days prior to the clamping procedure at 02:00 PM).

[0471] Metformin was administered orally 24 hours and 4 hours before the T0 procedure (at 2:00 p.m. the day before the procedure and at 10:00 a.m. on the day of the procedure).

[0472] Rats were fasted for 6 hours before the start of the restraint procedure (approximately 8 hours, just after blood collection).

[0473] use 3 H-glucose was used as a tracer, and a 0.3 U / kg / h insulin infusion was administered from 2:00 PM (T0) to 5:00 PM (T+3h) using a hyperinsulinemia-eukaryotic clamp. Glucose solution was infused concurrently, with the infusion rate adjusted to achieve steady state (~100 + / - 10 mg / dL). Blood glucose was measured every 10 minutes from the tail tip using a glucometer. During the last hour (steady state), 10 μL of blood was collected from the tail tip, and the following parameters were assessed: glucose infusion rate; systemic glucose utilization; hepatic glucogenesis; and systemic glycogen and glycolysis rates.

[0474] To determine the glucose utilization rate of individual tissues, in D-[3- 3 Sixty minutes before the end of the H-glucose infusion, each rat was injected with 100 μCi of deoxy-D-glucose 2- via femoral vein. 14 C( 14 C-2-DOG). A 10 μL sample of blood was taken from the tip of the tail vein at 0, 5, 10, 15, 20, 25, 30, 45, and 60 minutes after injection to determine plasma levels. 14 The disappearance of C-2-DOG and glucose concentration. At the end of the experiment, the lateral femoral leg (VL), glycolytic extensor digitorum longus (EDL) and soleus muscles, epididymis and inguinal white adipose tissue, apex of the heart, and skin (as a negative control) were peeled off, rapidly frozen, and maintained at -80°C. One slice of each tissue was dissolved in 1M NaOH and then neutralized with 1M HCl. D-2-14 C-deoxyglucose 6-phosphate. D-2- is obtained by using zinc hydroxide (0.3M) solution or perchloric acid solution (6%). 14 C-deoxyglucose differential precipitation. Two radioactive concentrations were measured to assess glucose uptake, expressed as ng / mg / min.

[0475] Plasma insulin levels were measured at the end of the clamping procedure.

[0476] Statistical analyses were performed using GraphPad Prism software. Histograms were analyzed using one-way ANOVA with the Dunnett post-hoc test, and curves were analyzed using two-way ANOVA with the Pomferroni post-hoc test. A p-value < 0.05 was considered significant. NS: Not significant.

[0477] Results and discussion

[0478] Blood glucose and insulin measurements. Under fed conditions, blood glucose levels were unaffected 42 h after treatment with Ab14 100 mg / kg and 18 h after treatment with metformin 500 mg / kg compared to the mediator group (Figure 1A). However, Ab14 and metformin reduced plasma insulin levels by 11% and 18%, respectively (not significant, Figure 1B). The insulin resistance index HOMA-IR then decreased in a similar manner compared to the mediator group (Figure 1C). On the other hand, under 6-hour fasting conditions, metformin significantly reduced blood glucose by 17% after 30 h of treatment (Figure 1D).

[0479] Systemic glucose flux. Hyperinsulinemia-eukaryotic clamping was performed 48 hours after a single administration of Ab14 100 mg / kg and 4 hours after a final administration of metformin 500 mg / kg, under 6-hour fasting conditions.

[0480] Compared to the mediator group, metformin significantly increased GIR development from 60 minutes after infusion initiation. Ab14 tended to increase GIR development, primarily after 130 minutes of infusion (Figure 2A).

[0481] The glucose infusion rate reached a plateau in all groups at 140 min. During this steady-state period, blood glucose levels were similar across all groups (Figure 2B). Plasma insulin levels were similar across all groups at the end of the clamp procedure (Figure 2C). The plasma insulin levels at the end of the clamp procedure were almost identical to those measured under feeding conditions, indicating that the insulin dose used to achieve hyperinsulinemia was a physiological dose.

[0482] Next, the glucose flux infused from 140 to 180 minutes was calculated (Figure 3). Ab14 and metformin increased the glucose infusion rate by 18% (NS) and 27% (p<0.05), respectively, and the glucose conversion by 18% (NS) and 30% (p<0.05), respectively. This supraphysiological dose of insulin completely inhibited hepatic glucose production in all three groups. Ab14 did not affect the rate of glycolysis, while metformin increased the rate of glycolysis by 43% (NS). Ab14 increased the rate of glycogen synthesis by 23%, similar to metformin (NS).

[0483] It also determined the glucose utilization rate of individual tissues, in D-[3- 3 Sixty minutes before the end of the H-glucose infusion, each rat was injected with 100 μCi of deoxy-D-glucose 2- via femoral vein bolus. 14 C( 14 (C-2-DOG). Two radioactive concentrations were measured to assess glucose uptake, expressed as ng / mg / min. As shown in Figures 4A-C, metformin increased glucose utilization in mixed lateral femoral muscle (VL, 49% p<0.05) and glycolytic extensor digitorum longus muscle (EDL, 19% NS), and decreased glucose utilization in the heart (-39%, p<0.01), presumably due to stimulation of myocardial fatty acid oxidation, a known effect of metformin. Ab14 tended to increase glucose infusion rate and systemic glucose turnover (NS), and had a stronger effect than metformin on glucose utilization in VL (70%, p<0.01), EDL (26%, NS), and oxidized soleus muscle (27%, NS). Ab14 also tended to increase glucose utilization in the heart (21%, NS). Similar to metformin, Ab14 did not affect glucose utilization in white adipose tissue (deep and subcutaneous).

[0484] Ab14 plasma concentration analysis. Ab14 plasma concentrations in Group 2 animals undergoing the clamping procedure ranged from 642 to 797 μg / mL, supporting systemic exposure up to 48 hours.

[0485] Conclusion: Acute treatment with Ab14 slightly decreases plasma insulin and tends to increase systemic glucose utilization by increasing glycogen synthesis and muscle glucose utilization.

[0486] Example 2

[0487] This example evaluates the ability of Ab14 to improve insulin sensitivity in an insulin-resistant rat model. In this model, glucose intolerance was induced by feeding rats a high-fat (69%) and high-fructose (14%) diet (HFD) for 6 weeks, and plasma insulin levels were significantly increased and glycemia was slightly increased compared to control animals fed a normal diet. The antibody used in this example consists of the light and heavy polypeptide chains of SEQ ID NOs 132 and 134.

[0488] Rats fed with HFD for 6 weeks were treated with Ab14 for 2 weeks, and a two-step hyperinsulinemia-eukaryotic clamp was performed to assess insulin sensitivity. Physiological doses of insulin were used during the first step, and pharmaceutical doses were used during the second step to assess the effects on peripheral and hepatic insulin sensitivity under both conditions.

[0489] Summarize

[0490] After 6 weeks of HFD, rats were randomized to treatment groups based on glucose intolerance (calculated from AUC during the oral glucose tolerance test (OGTT)) and their HOMA-IR (insulin resistance index). HFD rats were treated for 15 days with Ab14 (intravenously, twice weekly at intervals) at doses of 10, 30, and 100 mg / kg / week, or with metformin 200 mg / kg / day in drinking water.

[0491] Weight and food intake were measured three times a week until day 10 of treatment. HOMA-IR was measured on days 10 and 15. A two-step clamping technique (5 mU / kg / min followed by 15 mU / kg / min insulin) was performed on day 15 or 16. 3 H-glucose tracer infusion was used to assess glucose turnover (GTO) during clamping procedures.

[0492] At the end of the study, compared with control rats fed a normal diet, HFD rats showed significant increases in body weight, fasting blood glucose, plasma insulin and C-peptide, and a significant decrease in glucose infusion rate (GIR) during the 2-step hyperinsulinemic normoglycemic clamp procedure.

[0493] When compared with the HFD plus mediator control group, Ab14 treatment had no effect on weight or food intake, while the metformin group significantly reduced both parameters.

[0494] After 15 days of treatment, Ab14 at 100 mg / kg significantly reduced HOMA-IR by 38% (by lowering fasting blood glucose and plasma insulin). Metformin had a non-significant (ns) reduction in HOMA-IR on day 15. Ab14 treatment also significantly reduced C-peptide (30% at 10 mg / kg and 29% at 100 mg / kg).

[0495] When compared with the HFD mediator control group, an increased GIR (ns) was observed in the Ab14 treatment group during the first step of the clamping procedure, while metformin had a slightly smaller but significant increase in GIR. Treatment with 30 or 100 mg / kg of Ab14 and metformin significantly increased GIR during the second step of the clamping procedure (36%, 28%, and 27%, respectively).

[0496] Compared with the HFD mediator control group, 30 mg / kg Ab14 tended to increase GTO (17%, ns) during the first step of the clamping procedure. 30 mg / kg Ab14 had a slight increasing effect (ns) on glycolysis and glycogen synthesis during both clamping steps.

[0497] During the first step, Ab14 or metformin treatment resulted in only a slight but significant reduction in hepatic glucose production (HGP) (between 11-20%). During the second step, compared with the HFD mediator control group, 10 mg / kg of Ab14 did not significantly reduce HGP (78%), while 30 or 100 mg / kg of Ab14 and metformin completely inhibited HGP.

[0498] In summary, improved insulin resistance (mainly in the liver) was observed after intravenous administration of Ab14 in a rat model of HFD.

[0499] method

[0500] Eighty-two Stowe rats (starting at 8 weeks of age, with an average weight of approximately 250 grams) were kept in cages (904 cm) throughout the experimental period. 2 The cages were kept in enclosures measuring 23 cm x 23 cm. Bedding was changed three times a week. Animals were housed in groups of 2-3 during the acclimatization, HFD, and treatment periods. Rats were then individually housed post-surgery until the restraint procedure was performed. Rats were housed under a reversed 12-hour light cycle (lights off at 8:00 AM) and at 22±2°C and 55±10% relative humidity. An acclimatization period of at least 5 days was provided before initiating HFD feeding. During the acclimatization period, a standard diet (RM1(E)801492, SDS) and free access to tap water were provided.

[0501] After the adaptation period, 10 rats were fed normal food (NC), while 72 rats were fed HFD (RD1, SAFE) throughout the experiment.

[0502] The composition of a high-fat diet is as follows (kcal%): Protein: 17.3%; Carbohydrates (fructose): 14%; Fat (lard): 68.7%; Cholesterol: 1.65%; Bile acids: 0.65%.

[0503] Six weeks after HFD (High-Frequency Diet), rats fasted for 6 hours and underwent an oral glucose tolerance test. Rats exhibiting the lowest AUC (~17%) were then excluded from the study. The remaining rats were then randomly assigned to different groups based on their AUC (glucose tolerance index) and HOMA-IR (insulin resistance index).

[0504] Ab14 (10, 30, 100 mg / kg) and the carrier were administered weekly via intravenous route (via the tail vein under isoflurane anesthesia) on the mornings of the 1st and 8th days of treatment.

[0505] Metformin (200 mg / kg / day) was administered in drinking water for 2 weeks until the clamping procedure was completed. Rats treated with metformin were administered via a vehicle (through the tail vein) on days 1 and 8.

[0506] The test group is as follows:

[0507] Group 1: NC + intravenous administration of a medium (n=10)

[0508] Group 2: HFD + intravenous administration of a medium (n=10)

[0509] Group 3: HFD + Ab14 10mg / kg intravenously (n=10)

[0510] Group 4: HFD + Ab14 30mg / kg intravenously (n=10)

[0511] Group 5: HFD + Ab14 100mg / kg intravenously (n=10)

[0512] Group 6: HFD + metformin 200 mg / kg in drinking water + intravenous administration of a carrier (n = 10)

[0513] During the final week of HFD feeding, prior to screening, water intake was measured three times a week to assess metformin dilution in tap water.

[0514] After 6 weeks of normal diet and HFD, 82 rats were fasted from 08:00 AM to 02:00 PM (6 hours). At 02:00 PM (t0), a glucose bolus (2.5 g / kg) was administered. Blood glucose was measured at t-30, 0, 15, 30, 60, 90, 120, and 150 min (using a glucometer, collecting blood drops from the tip of the tail). At t-30, blood (40 μL, on EDTA) was collected from the tip of the tail to measure plasma insulin (ELISA method).

[0515] The area under the curve (AUC) was calculated. The 12 HFD rats exhibiting the highest AUC were considered to have less glucose intolerance and were excluded from the study. The remaining 60 rats were randomly assigned to 6 groups based on their homogeneous AUC, HOMA-IR, and body weight.

[0516] During the first 6 weeks of HFD, body weight was measured weekly. During the first 10 days of treatment, body weight was measured 3 times a week. Food intake was measured over 48 hours or 72 hours, exactly before treatment, and 3 times a week during treatment until the surgical procedure (day 11).

[0517] All rats were fasting from 08:00 AM before treatment began (on the day of the OGTT) and on day 10 of treatment. At 01:30 PM, blood (40 μL, on EDTA) was collected from the tip of the tail. Plasma insulin was measured (using a blood glucose meter) and (ELISA method).

[0518] On day 11, the rats were anesthetized (with isoflurane) and a catheter was inserted into the femoral vein. A 4-day recovery period followed before the clamping procedure.

[0519] On the morning of the clamping procedure, rats were fasting for 6 hours (from 8:00 AM to 2:00 PM). Just before the clamping procedure (around 1:00 PM), blood samples (~160 μL, on EDTA) were collected from the tail tip of each rat in groups 3, 4, 5, and 6, processed into plasma (~60 μL), and maintained at -80°C until Ab14 concentration was assessed. Although antibody concentrations in the control and metformin groups were not analyzed, similar amounts of blood were collected from rats in groups 1, 2, and 7 (and discarded).

[0520] On day 15 or 16, a two-step hyperinsulinemia-eukopenic purpura clamp procedure is performed, which uses... 3H-glucose was used as a tracer (in addition to the normal food group), and insulin was infused at 5 mU / kg / min from 02:00 PM (T0) to 04:00 PM (T+2h), followed by insulin infusion at 15 mU / kg / min from 04:00 PM to 05:30 PM (t+3.5h). Glucose solution was infused concurrently, and the infusion rate was adjusted to reach steady state (100 ± 10 mg / dL). Blood glucose was measured every 10 minutes from the tail tip using a glucometer. During the steady-state period of each step, blood (10 μL) was regularly collected from the tail tip.

[0521] Assess the following parameters: glucose infusion rate (in all groups); systemic glucose utilization (except for the normal diet group); hepatic glucose production rate (except for the normal diet group); and systemic glycogen and glycolysis rates (except for the normal diet group).

[0522] In addition to the normal food group, a bolus injection was administered 1 hour before the end of the restraint experiment. 14 C-2DOG, and samples of the following tissues collected at the end of the clamping procedure and retained for further evaluation:

[0523] Vas lateralis (VL); extensor digitorum longus (EDL); soleus muscle apex; epididymal white adipose tissue; inguinal white adipose tissue skin (negative control).

[0524] Plasma insulin and C-peptide levels were measured just before the start of infusion (~T-30 min), at the end of steady state in steps 1 (T2 h) and 2 (T3.5 h). For this purpose, blood was collected from the tail tip (~100 μL, on EDTA).

[0525] Statistical analyses were performed using GraphPad Prism software. ANOVA (two-way) with the Pomferroni post-hoc test was used to analyze the curves. Histograms were analyzed using the t-test to compare the HFD plus vector control group and the normal food plus vector control group. Univariate ANOVA with the Dunnett post-hoc test was used to analyze the histograms to compare the Ab14 and metformin groups against the HFD plus vector control group. A p-value < 0.05 was considered statistically significant. NS: Not significant.

[0526] result

[0527] Animal Model and Screening. Eight-week-old rats were fed a high-fat, fructose-rich diet (HFD, 69% fat and 16% fructose) for 7 weeks before treatment. The HFD group weighed 522 ± 5 g, compared to 448 ± 13 g in the control group fed a normal diet. Therefore, the HFD group showed a 16% increase in body weight on day 42 (t-test p < 0.001 (Figure 5)). After 7 weeks, the HFD group showed a body weight increase of 187 ± 3 g, compared to 158 ± 9 g in the control group fed a normal diet (t-test p < 0.001 on day 42, Figure 6).

[0528] During week 7 of HFD, an oral glucose tolerance test was performed to assess glucose intolerance in the HFD population. Blood glucose levels in the HFD population remained elevated up to 150 min after glucose administration (not shown). The calculated AUC relative to T0 was significantly higher (9%) in the HFD rats compared to rats fed a control diet (not shown).

[0529] HOMA-IR (insulin resistance index) was calculated using the t-30 OGTT. Rats exhibiting higher AUC and higher HOMA-IR were randomly assigned to 6 groups. Compared to the control diet group, the HFD group had a higher AUC (~9%, not shown), and higher HOMA-IR (34%, not shown) and body weight (~17%, p<0.001, Figure 7).

[0530] Body weight and food intake were tracked. Body weight was tracked 10 days after treatment. Ab14 treatment had no effect on body weight. The body weight of rats fed the control diet was still significantly lower than that of the HFD-mediated group (Figure 7). Ab14 (30 mg / kg) slightly reduced body weight gain (ns), while metformin 200 mg / kg significantly reduced body weight gain from the second day of treatment.

[0531] As expected, food intake was lower in the HFD-mediated group than in the control food group. Ab14 treatment had no effect on tracked food intake (Figure 8A) or cumulative food intake (not shown in Figure 8B). Metformin significantly reduced cumulative food intake by 25%. Food intake measurements were interrupted by fasting in animals between days 9 and 10 prior to the surgical procedure.

[0532] Biochemical parameters. Compared with the control group consuming the control food, fasting blood glucose in the HFD-mediated group increased by 5% (ns), 11% (ns), and 20% on days 0, 10, and 15, respectively (p<0.001). Ab14 or metformin treatment had no effect on day 10. Compared with the HFD-mediated group, Ab14 100 mg / kg treatment had a significant effect on reducing fasting blood glucose on day 15 (Figure 9).

[0533] Compared to the control food group, fasting plasma insulin in the HFD mediator group increased by 33% (ns), 49% (ns), and 67% on days 0, 10, and 15, respectively (p<0.01). Ab14 treatment had no effect on day 10, while metformin reduced plasma insulin by 37% (ns). After 15 days of treatment, Ab14 treatment at 10, 30, or 100 mg / kg reduced plasma insulin by 26, 16, or 18% (ns), respectively, while metformin reduced plasma insulin by 11% (ns, Figure 10).

[0534] As expected, plasma C-peptide levels on day 15 were similar to those of plasma insulin levels, but with a more pronounced effect and less variation. Compared to the control food group, the HFD mediator group showed a significant 67% increase in C-peptide. Ab14 treatment at 10, 30, or 100 mg / kg reduced C-peptide levels by 30% (p<0.05), 23% (ns), and 29% (p<0.05), respectively, while metformin reduced C-peptide levels by 13% (ns) (Figure 11, lower left).

[0535] Compared to the control food group, the insulin resistance index HOMA-IR in the HFD mediator group increased by 36% (ns) on day 0, 42% (ns) on day 10, and 98% on day 15 (p<0.01). Compared to the HFD mediator, Ab14 had no effect after 10 days of treatment, while metformin showed a 36% reduction (ns). After 15 days of treatment, Ab14 treatment at 10, 30, or 100 mg / kg reduced HOMA-IR by 33% (ns), 17% (ns), and 38% (p<0.05), respectively, while metformin tended to reduce HOMA-IR by 18% (ns, Figure 12).

[0536] Hyperinsulinemia suppression. Figure 13 shows the glucose infusion rate (GIR) over time during the two-step hyperinsulinemia period. During the first step (5 mU / kg / min insulin), hepatic glucose production (HGP) was incompletely suppressed, and the glucose infusion rate (GIR) was lower when hepatic glucose production was suppressed than during the second step (15 mU / kg / min insulin).

[0537] During both clamping steps, the GIR in the control food group was higher than that in the HFD mediator group, confirming the insulin resistance phenotype in HFD rats after 8–9 weeks of dieting. During the first clamping step, metformin had no effect on GIR, while the GIR flat area in the Ab14 treatment group was slightly higher (ns). During the second clamping step, the GIR flat area was observed to be higher in all treatment groups than in the HFD mediator group, and significant differences were observed between metformin and Ab14 at 30 or 100 mg / kg (Figure 13). Statistical significance relative to HFD was assessed using a two-factor ANOVA plus a post-hoc test of pamprolium. During the first clamping step, only at time points 50 and 60 minutes were the GIRs of the control and mediator-treated rats on the normal food diet significantly different (p<0.01 and p<0.05, respectively). During the second clamping procedure, the GIRs of HFD rats treated with 30 mg / kg Ab14 were significantly different at time points 160–210 minutes (p<0.05 for time point 160 minutes, p<0.01 for time points 170–210 minutes), the GIRs of HFD rats treated with 100 mg / kg Ab14 were significantly different at time points 170–210 minutes (p<0.01 for time point 190 minutes, p<0.05 for time points 170–180 minutes and 200–210 minutes), and the GIRs of HFD rats treated with metformin were significantly different at time points 170–210 minutes (p<0.01 for time points 180 minutes and 190 minutes, p<0.05 for time points 170 minutes and 200–210 minutes).

[0538] The mean GIR for each flat zone was calculated (Figure 14). Compared with the food control group, the GIR in the HFD mediator group was significantly reduced by 32% (p<0.05) and 17% (p<0.01) during the first and second steps, respectively. Ab14 at 10, 30, or 100 mg / kg increased the GIR during the first step (ns) (26, 37, and 29%, respectively), while metformin also had an 11% increase (ns) effect. During the second step, all treatments increased the GIR when compared with the HFD mediator group: Ab14 at 10, 30, or 100 mg / kg by 19% (ns), 36% (p<0.01), and 28% (p<0.05), respectively, while metformin increased it by 27% (p<0.05).

[0539] As expected, the mean blood glucose levels during the two clamping steps corresponded to normal blood glucose levels. Although there were significant differences between the control food group and the HFD mediator group during the first clamping step, glucose levels remained within the normal range, and the biological status was the same in both groups (Figure 14).

[0540] Plasma insulin was measured during the clamping procedure. As expected, insulin levels were similar across all groups at the end of the two clamping steps. During the first clamping step, insulin concentrations were approximately 140 μU / mL, which is the expected physiological level under feeding conditions. After the second clamping step, insulin concentrations were approximately 490 μU / mL, which is the pharmaceutical level (Figure 11, top).

[0541] C-peptide levels were also measured during the clamping procedure (Figure 11, bottom right). During normal glycemic conditions, β-cells suppress insulin secretion, and therefore the low plasma C-peptide levels are not explainable.

[0542] All HFD groups during the clamping procedure 3 H-glucose was infused along with insulin (not in the control food group). Systemic glucose flux was then calculated. During the first clamping procedure, glucose turnover (GTO) was similar in all groups except for Ab14 at 30 mg / kg, which tended to increase GTO (17%, ns) compared to the HFD mediator group. Following treatment with Ab14 at 30 mg / kg, glycolysis and glycogen synthesis propensity increased by 15% and 16%, respectively (ns, Figure 15).

[0543] During the second clamping step, GTO, glycolysis, and glycogen synthesis were similar in all treatment groups, with a slight increase in glycogen synthesis (10%, ns) observed in the Ab14 30 mg / kg treatment group compared to the HFD mediator group. Ab14 at 30 mg / kg (p<0.05) and 100 mg / kg (ns) completely inhibited HGP, similar to metformin (ns), and Ab14 treatment at 10 mg / kg reduced HGP by 78% (ns, Figure 16).

[0544] Conclusion: In the HFD rat model, Ab14 treatment reduced HOMA-IR by decreasing fasting blood glucose and plasma insulin levels. Furthermore, Ab14 significantly improved hepatic insulin sensitivity; however, no clear effect of Ab14 on systemic peripheral insulin sensitivity was observed. As expected, metformin treatment also improved hepatic insulin sensitivity.

[0545] Example 3

[0546] This embodiment evaluates the effects of Ab14 on glucose metabolism and glycemic control in Zucker's diabetic obese (ZDF) rats, a rat model of diabetes. The effect of chronic administration of Ab14 on glycemic control was assessed in ZDF rats progressing from a prediabetic (hyperinsulinemia, normigable) state to a fully diabetic (hypoinsulinemia, hyperglycemia) state. These animals develop prediabetes a few weeks before age, characterized by significant hyperinsulinemia to compensate for their progressive insulin resistance, but with mild or no hyperglycemia. They rapidly progress to fully diabetic state before 10-12 weeks of age due to pancreatic β-cell depletion, characterized by hypoinsulinemia and significant hyperglycemia. The antibody used in this embodiment consists of the light and heavy polypeptide chains of SEQ ID NOs 132 and 134.

[0547] method

[0548] Eighty-one ZDF fa / fa rats (Charles River Laboratories, France) and ten lean ZDF® / + rats (controls) were housed in 1-2 animal groups in well-ventilated and well-fed cages under a normal 12-hour light cycle (lights off at 8 p.m.), at 22 ± 2°C and 50 ± 10% relative humidity. The rats were 7 weeks old at arrival and acclimatized for one week prior to the start of the study. They were fed the standard ZDF rat diet (Purina 5008, Charles River) and provided with free access to tap water. All animals were monitored for any signs of ill health, adverse reactions to treatment, or morbidity at least daily throughout the study.

[0549] Eight-week-old male ZDF fa / fa rats were induced with hyperinsulinemia and mild diabetes. Due to the variability in blood glucose and insulin levels in this state, ZDF rats were selected based on their HOMA-IR.

[0550] For the AB14 treatment groups, two different doses of 20 mg / kg / week (groups 3 and 7) or 60 mg / kg / week (groups 4 and 8) were administered weekly via the tail vein (intravenous, 5 mL / kg) on ​​days 1, 8, 15, and 22. All other groups were treated weekly with vehicle 1 (intravenous, 5 mL / kg). Intravenous treatment was administered on the mornings of days 1, 8, 15, and 22 under isoflurane anesthesia. The volume administered was adjusted according to recent body weight.

[0551] Metformin (Met) and pioglitazone (PIO) were administered orally (5 mL / kg) once daily for 28 days, between 8:00 and 10:00 AM. Some oral administrations were completed after 10:00 AM, except on the day of the OGTT or after intravenous administration. Groups 5, 7, and 8 were treated with 200 mg / kg / day of metformin, while group 6 was treated with 10 mg / kg / day of pioglitazone. All other groups were treated daily with mediator 2 (orally, 5 mL / kg) for 28 days. The mean volume administered to each group was calculated using the most recent body weight.

[0552] The test group is summarized in the table below. Met: metformin.

[0553]

[0554]

[0555] After a one-week acclimatization period, all rats were weighed and fasted for 6 hours (from 8:00 AM to 2:00 PM). Blood (~150 μL, EDTA) was collected from the tip of the tail between 8:00 PM and 2:00 PM. Blood glucose (using a glucometer), plasma, and insulin (ELISA) were measured, and HOMA-IR (insulin resistance index) was calculated. Eleven ZDFfa / fa rats exhibiting extreme HOMA-IR values ​​were excluded from the study but continued to be held in captivity for 28 days for plasma collection at the end of the study. The remaining 70 rats were then randomly assigned to seven groups based on their HOMA-IR and body weight. Lean rats remained in Group 1 and were treated with only the agonist.

[0556] During the 4-week treatment period, weight was measured twice a week.

[0557] Food intake was measured just before the screening procedure, and then twice a week during the first three weeks of treatment. During the OGTT (week 4), food intake was measured once a week.

[0558] Fasting was performed before each blood collection (6 hours from 8:00 AM to 2:00 PM on day 0, and overnight from 6:00 PM to 8:00 AM on days 12, 19, and 26). Blood was collected from the tail tip from 2:00 PM on day 0 (before screening, 150 μL, EDTA potassium), and from 8:00 AM on day 12 (110 μL, EDTA potassium), day 19 (150 μL, EDTA potassium), and day 26 (110 μL, EDTA potassium) before administration.

[0559] Fasting blood glucose (using a glucometer) was measured on days 0, 12, 19, and 26. Fasting plasma insulin, peptide-C levels (ELISA), free fatty acids, triglycerides, total cholesterol (colorimetric method), and HDL-cholesterol (phosphotungstic acid precipitation, colorimetric method) were measured on days 0, 12, 19, and 26 prior to drug administration. Total cholesterol-HDL-cholesterol was calculated as non-HDL-cholesterol. Fructosamine was measured on days 0, 19, and 28. HbA1c (DCA 2000) was measured on days 0 and 28.

[0560] The oral glucose tolerance test (OGTT) was performed as follows. On day 25, rats were fasted from afternoon to 6:00 PM, and the OGTT was performed the following day (day 26). Blood samples (110 μL, EDTA) were collected from morning to 8:00 AM (T-60) for measuring biochemical parameters. One hour later (from morning to 9:00 AM), an oral glucose bolus (1.5 g / kg) was administered (T0). Blood glucose was measured at T-60, T0, T15, T30, T60, T90, T120, and T180 minutes (using a glucometer or colorimetric method in the case of hyperglycemia). The area under the curve (AUC) was calculated based on the blood glucose value measured at T0. Plasma insulin and C-peptide were measured (ELISA) at T-60, T15 (~40 μL blood, EDTA), and T30 minutes (~40 μL blood, EDTA).

[0561] Eighty rats were anesthetized with isoflurane after a two-hour fast on day 28 (from 8:00 AM to 10:00 AM). Blood (~3000 μL from the abdominal vein, treated with K2-EDTA) was collected to determine the plasma concentration of AB14. Plasma (three aliquots of ~200 μL) was maintained at -80°C until testing. The pancreas was then removed. The rats were euthanized by removing the abdominal vein and aorta.

[0562] Each pancreas was divided into two parts (longitudinal cut). One part was fixed in 10% formalin solution for histopathological processing. The other part of the pancreas was rapidly frozen and held at -80°C to determine insulin and proinsulin levels.

[0563] After 28 days in captivity, 11 ZDF fa / fa rats excluded from the study were sacrificed. The rats were anesthetized with isoflurane. Blood was collected from the abdominal vein (maximum volume of EDTA potassium). Frozen plasma samples (two aliquots, 1 mL each) were used for further testing. The rats were euthanized by excision of the abdominal vein and aorta.

[0564] Each pancreatic sample was homogenized in acidic buffer and the insulin and proinsulin levels in the following groups were measured using an ELISA kit:

[0565] Group 1: Lean rats + vector (n=10)

[0566] Group 2: ZDF rats + vector (n=10)

[0567] Group 4: ZDF rats + AB14 60mg / kg / week (n=10)

[0568] Group 5: ZDF rats + metformin 200 mg / kg / day (n = 10)

[0569] Group 8: ZDF rats + AB14 60mg / kg / week + metformin 200mg / kg / day (n=10)

[0570] Pancreatic samples were fixed in 4% formalin solution over a period of 24-48 hours; the volume of formalin was 5-10 times higher than the sample volume to ensure proper fixation. After 48 hours, the samples were placed in 70% ethanol. The samples were then encased in paraffin for use in the following tissue processing groups:

[0571] Group 1: Lean rats + vector (n=10)

[0572] Group 2: ZDF rats + vector (n=10)

[0573] Group 4: ZDF rats + AB14 60mg / kg / week (n=10)

[0574] Group 5: ZDF rats + metformin 200 mg / kg / day (n = 10)

[0575] Group 8: ZDF rats + AB14 60mg / kg / week + metformin 200mg / kg / day (n=10)

[0576] After the islets of Langerhans are delineated, the surface area and intensity of insulin are quantified by analyzing the labeled (brown) and unlabeled (blue) areas.

[0577] When the Fisher test does not show a significant difference in variance, the Student test is used to compare the means of the ZDF-mediated rats and lean rats. If not, the nonparametric Mann-Whitney test is used.

[0578] The mean values ​​of treated ZDF rats were compared with those of vector-treated ZDF rats using a one-way ANOVA plus Dunnett's post-hoc test. If Bartlett's test showed a significant difference in variance, a nonparametric Kruskal-Wallis test plus Dunn's post-hoc test was used.

[0579] The mean of AB14 20 mg / kg alone was compared with metformin alone or in combination with metformin 200 mg / kg and AB14 20 mg / kg using a one-way ANOVA with a post-hoc Newman-Coyle test.

[0580] The mean of AB14 60 mg / kg alone was compared with metformin 200 mg / kg alone or in combination with AB14 60 mg / kg using a one-way ANOVA with a post-hoc Newman-Coyle test.

[0581] The curves were analyzed using a two-factor ANOVA with the Pomferroni post-hoc test.

[0582] If a rat is an outlier in all or almost all parameters, it will be excluded from the analysis. This resulted in the exclusion of four rats from different groups.

[0583] result

[0584] As expected, HOMA-IR was significantly increased in 8-week-old ZDF rats compared to lean rats (~111 vs. 3.5). Figure 18 A). ZDF rats exhibit mild hyperglycemia (~180 mg / dL relative to 113 mg / dL). Figure 18 B) and hyperinsulinemia (~250 relative to 12.6 μU / mL, Figure 18 C). The ZDF rats gained a slight increase in body weight ( Figure 18 D).

[0585] Compared to lean rats, the body weight of ZDF rats remained high throughout the treatment period. Figure 19A The weight gain was similar between lean rats and ZDF rats. Figure 19B ).

[0586] Compared with ZDF-mediated rats, pioglitazone significantly increased body weight from 8 days of treatment, and by the end of the treatment period, the weight gain was up to 3 times higher. Figure 19A And B). Compared with the ZDF-mediated rats, all other drug treatments had no significant effect on body weight. The combination of AB14 60 mg / kg + metformin 200 mg / kg significantly increased body weight gain after 22 days of treatment ( Figure 19B ).

[0587] Compared to lean rats, ZDF-mediated rats showed a 2-fold increase in food intake (significant on day 13). Rats treated with pioglitazone showed a tendency for higher food intake compared to ZDF-mediated rats (significant on days 15, 20, and 22). Figure 20A )。 When compared to the lean group, the cumulative food intake of the vehicle ZDF group increased by 94% (p < 0.01), and when compared to the vehicle ZDF group, the pioglitazone group increased by 14% (NS, Figure 20B )。 When compared to vehicle-treated ZDF rats, the other treatments had no effect on food intake.

[0588] During the 26-day treatment period, whether fasted for 6 hours or after an overnight fast, the fasting blood glucose of lean rats remained within the normal range (Figure 21A). This was associated with normal insulin levels (Figure 21B). In vehicle ZDF rats, the overnight fasting blood glucose reached 362 ± 32 mg / dL on day 12 (at approximately 10 weeks of age), and remained significantly higher than that of lean rats until the end of the treatment period (Figure 21A). This was associated with decreased plasma insulin levels measured on days 12, 19, and 26 (49.2 ± 6.7, 41.2 ± 4.8, and 36.6 ± 2.7 μU / mL – p < 0.001, relative to lean rats, Figure 21B), and with decreased plasma C-peptide levels (2813 ± 249, 2472 ± 195, 2156 ± 165 pM - < p < 0.001, relative to lean rats (Figure 21D). The development of HOMA-IR in lean rats and vehicle ZDF rats reflected the changes in blood glucose and plasma insulin levels (Figure 21C).

[0589] Pioglitazone significantly reduced the level of overnight blood glucose to normal levels from day 12 of treatment (p < 0.001, Figure 21A). Both doses of AB14 reduced blood glucose by approximately 15% after 12, 19, or 26 days of treatment (n.s., Figure 21A). Compared to AB14, metformin 200 mg / kg had a similar effect on day 12, but this effect was not observed on days 19 and 26. Compared to vehicle-treated ZDF rats, the combination of AB14 20 mg / kg + metformin slightly reduced blood glucose on day 12 (12%, ns), and showed no effect on days 19 and 26 (Figure 21A). In contrast, the combination of AB14 60 mg / kg with metformin significantly reduced the level of blood glucose on day 12 (38%, p < 0.01, relative to vehicle-treated ZDF rats). Although not statistically significant, a decrease in blood glucose was still observed on days 19 and 26 (22% and 27%, respectively, Figure 21A).

[0590] Pioglitazone appeared 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 ZDF-mediated rats (Figures 21B and D). Therefore, the reduction in blood glucose levels was associated with the insulin-sensitizing effect of pioglitazone, which reduced HOMA-IR by 67%, 62%, and 54% on days 12, 19, and 26, respectively, compared to ZDF-mediated rats (Figure 21C).

[0591] Compared with ZDF rats treated with the vector, AB14 20 mg / kg did not alter plasma insulin and C-peptide levels, or HOMA-IR, on days 12, 19, and 26 (Fig. 21B-D). Meanwhile, AB14 60 mg / kg increased plasma insulin levels by 74%, 21%, and 19% (ns, relative to ZDF rats treated with the vector, Fig. 21B) on days 12, 19, and 26, respectively.

[0592] AB14 60 mg / kg increased plasma C-peptide levels by 10% (ns) on day 12, and had no effect on days 19 and 26 (Figure 21D).

[0593] Compared with ZDF rats treated with the vehicle, metformin increased plasma insulin levels by 79%, 55%, and 48% on days 12, 19, and 26, respectively (ns, Fig. 21B). Metformin increased plasma C-peptide levels by 23%, 21%, and 9% on days 12, 19, and 26, respectively (ns, relative to ZDF rats treated with the vehicle, Fig. 21D).

[0594] Compared with ZDF rats treated with the same mediator, the combination of AB14 20 mg / kg + metformin increased plasma insulin levels by 2-fold on days 12, 19, and 26 (NS, Figure 21B). The combination of AB14 20 mg / kg + metformin increased plasma C-peptide levels by 21%, 23%, and 25% on days 12, 19, and 26, respectively (NS, Figure 21D).

[0595] Compared with vector-treated ZDF rats, the combination of AB14 60 mg / kg + metformin significantly increased plasma insulin levels by 2.5, 2.3, and 2.7-fold on days 12, 19, and 26, respectively. The combination of AB14 60 mg / kg + metformin significantly increased plasma C-peptide levels by 45% (day 12), 48% (day 19), and 52% (day 26) from day 12 (p < 0.05, relative to vector-treated ZDF rats, Figure 21D).

[0596] In this model where insulin secretion decreases over time, the increase in HOMA-IR reflects the improvement in insulin secretion. Therefore, compared with the ZDF mediator group, the group treated with metformin alone or in combination with AB14 showed an increase in HOMA-IR, which was maintained on days 12, 19 and 26 (Figure 21C).

[0597] Compared with lean rats, 8-week-old ZDF rats treated with the vector had significantly higher fructosamine levels (208±6 μM vs. 144±2 μM, p<0.001) (66%). Fructosamine levels in lean rats remained within a similar range during treatment, but increased significantly after 19 days (253±5 μM, p<0.001) and 28 days (234±6 μM, p<0.001) in the vector-treated ZDF rats. Figure 22 As expected, pioglitazone significantly reduced fructosamine levels from day 19 (30% on day 19 and 25% on day 28, p < 0.001). AB14 20 and 60 mg / kg had no effect on fructosamine levels. Compared with vector-treated rats, metformin showed a tendency for lower fructosamine levels only on day 19 (6%, ns). Compared with vector-treated ZDF rats, the combination of AB14 20 mg / kg + metformin showed a non-significant tendency for lower fructosamine levels by 10% and 8% on days 19 and 28, respectively. The combination of AB14 60 mg / kg + metformin similarly showed a non-significant tendency for lower fructosamine levels (9%) on day 28. Figure 22 ).

[0598] Compared with lean rats, 8-week-old ZDF rats had higher HbA1c (4.3±0.1% vs. 3.1±0.04%), but these values ​​fell within the normal range.

[0599] In 12-week-old ZDF rats, HbA1c reached a pathological value of 8.8 ± 0.2% on day 28 (p < 0.001, ZDF compared to lean rats). Figure 23 Compared with ZDF rats treated with carcasses, AB14 at 20 and 60 mg / kg had no effect on HbA1c levels after 28 days of treatment. After 28 days of treatment, pioglitazone and metformin significantly reduced HbA1c by 44% and 15%, respectively. Figure 23 The combination of metformin and AB14 20 mg / kg and the combination of metformin and AB14 60 mg / kg significantly reduced HbA1c by 11% and 19%, respectively. Figure 23 ).

[0600] Compared with lean rats, 8-week-old ZDF rats had significantly increased plasma triglyceride levels (~8 mM vs. ~0.7 mM). Figure 24 A).

[0601] Compared with the mediator, pioglitazone significantly reduced plasma triglyceride levels from day 12 of treatment. AB14 20 mg / kg slightly reduced plasma triglyceride levels on days 12 and 19 (15% and 7%, ns, respectively), and had no effect on day 26. AB14 60 mg / kg slightly reduced plasma triglyceride levels by 14%, 9%, and 12% (ns) on days 12, 19, and 26, respectively. Metformin increased plasma triglyceride levels by 26%, 40%, and 49% (significant from day 19) on days 12, 19, and 26, respectively. When compared with the mediator ZDF group, the combination of metformin + AB14 20 mg / kg showed a tendency to higher plasma triglyceride levels on days 19 and 26 (13% and 23%, ns, respectively). The combination of metformin and AB14 60 mg / kg showed a tendency for higher plasma triglycerides on days 12, 19, and 26 (9%, 48%, and 43%, respectively, with a significant increase from day 19). Figure 24 A).

[0602] After a 6-hour fast, plasma free fatty acid levels in 8-week-old ZDF rats were higher than in lean rats (~0.85 mM vs. ~0.59 mM). After overnight fasting (maximum lipolysis state), free fatty acid levels at weeks 10 and 11 were similar (~1.3 mM). After 12 weeks of treatment, compared to lean rats, the lower free fatty acid levels indicated decreased lipolysis capacity in ZDF rats (1.05 ± 0.06 mM vs. 1.38 ± 0.03 mM). Figure 24 B). Compared with ZDF-mediated rats, rats treated with pioglitazone showed lower plasma free fatty acid levels, reaching 35% (p<0.001) on day 12, 17% (ns) on day 19, and 30% (p<0.05) on day 26. AB14 20 and 60 mg / kg had no effect. When compared with ZDF-mediated rats, metformin increased free fatty acid levels by 14%, 25%, and 8% on days 12, 19, and 26, respectively, but this was not significant. Metformin alone or in combination with AB14 20 mg / kg had similar effects. On the other hand, when compared with the ZDF-mediated group, metformin in combination with AB14 60 mg / kg showed an increased effect only on day 19 (20%, NS, ...). Figure 24 B).

[0603] Compared with lean rats, 8-week-old ZDF rats had higher plasma total cholesterol and HDL-cholesterol levels, which gradually increased over the following 4 weeks. Figure 25A -B, Figure 26A -B). At 8 weeks of age, plasma non-HDL-cholesterol levels were similar in lean rats and ZDF rats, but from 10 weeks onwards, plasma non-HDL-cholesterol levels in both ZDF rats and lean rats increased over time. Figure 25C and Figure 26C Because there were significant differences in total cholesterol and HDL-cholesterol on day 0 among the ZDF groups (Figure 25), the results are expressed as relative values ​​from day 0 (Figure 26). Figure 26A As shown, pioglitazone tends to prevent an increase in plasma total cholesterol levels over time. AB14 20 mg / kg had no effect when combined with metformin. Compared with the ZDF mediator group, AB14 60 mg / kg increased total cholesterol by 8%, 14%, and 15% on days 12, 19, and 26, respectively. Compared with the ZDF mediator group, when combined with metformin, AB14 20 mg / kg increased total cholesterol by 15% and 10% on days 12 and 26, respectively, while AB14 60 mg / kg increased total cholesterol by 24%, 21%, and 13% on days 12, 19, and 26, respectively. Compared with the mediator, pioglitazone increased plasma HDL-cholesterol by 38%, 17%, and 19% on days 12, 19, and 26, respectively. Metformin alone had no effect. Treatment with AB14 at 20 mg / kg and 60 mg / kg, alone or in combination with metformin, for 12, 19, and 26 days resulted in an increase in plasma HDL-cholesterol levels of 11% to 22%. At day 12 of treatment, plasma non-HDL-cholesterol levels in all ZDF groups ( Figure 26C The results were similar. Compared with the mediators, AB14 20 mg / kg, AB14 60 mg / kg, metformin alone, or metformin in combination with AB14 had no effect on non-HDL-cholesterol levels. Only pioglitazone significantly reduced plasma non-HDL-cholesterol levels by 49% and 47% on days 19 and 26, respectively.

[0604] An oral glucose tolerance test was performed 26 days after treatment. Compared with carboxylated lean ZDF rats, carboxylated ZDF rats were expected to have higher blood glucose levels before and after glucose loading. Figure 27A Compared with ZDF rats treated with the vehicle, only ZDF rats treated with pioglitazone showed significantly reduced blood glucose levels at all time points. Compared with the vehicle, the combination of AB14 60 mg / kg and metformin tended to reduce blood glucose levels at t-60 minutes. Figure 27AOther drug treatments did not show significant effects. Compared with lean rats, the area under the curve (AUC) of blood glucose in ZDF rats treated with the vehicle was significantly increased by 3.7 times. Compared with ZDF rats treated with the vehicle, rats treated with pioglitazone showed a significant 54% reduction in blood glucose AUC. AB14 20 mg / kg, AB14 60 mg / kg, and metformin alone or in combination with AB14 20 mg / kg or 60 mg / kg showed no significant reduction in AUC (7%, 11%, 6%, 7%, and 17%, respectively). When compared with AB14 or metformin alone, the combination of AB14 60 mg / kg + metformin was slightly effective in reducing AUC. Figure 27B ).

[0605] Plasma insulin and C-peptide levels were measured at 15 and 30 minutes after glucose loading. The concentration-time curves for both insulin and C-peptide were similar. Insulin and C-peptide levels were similar in the mediator, AB14 20 mg / kg, and AB14 60 mg / kg treatment groups. Metformin and pioglitazone treatment groups showed a slight increase in insulin and C-peptide levels, and the groups treated with AB14 20 mg / kg and AB14 60 mg / kg in combination with metformin showed a greater dose-dependent increase in insulin and C-peptide levels. Figure 28 (AB). The ability to secrete insulin or C-peptide in response to glucose challenge was assessed, expressed as a relative value calculated at T-60 minutes. As expected, ZDF-mediated rats significantly lost their ability to secrete insulin and C-peptide in response to glucose challenge compared to lean rats. Figure 29 AB). Compared with ZDF rats treated with other mediators, rats treated with pioglitazone increased insulin secretion by 20% (p<0.05) and 5% at times T15 and T30, respectively. All other treatments had no effect on insulin secretion at time T15. Metformin decreased insulin secretion by 26% at time T30 (p<0.01). AB14 20 mg / kg alone had no effect at time T30 and showed a tendency to decrease insulin secretion (14%, ns) when combined with metformin. AB14 60 mg / kg alone or in combination with metformin showed a tendency to decrease insulin secretion by 19% and 18%, respectively. Figure 29 A). Compared with lean rats, the C-peptide secretion in response to glucose loading in ZDF rats treated with the medium at times T15 and T30 was significantly lower. Figure 29 B) Significantly reduced by up to 40%. When compared with ZDF rats treated with the mediator, only pioglitazone significantly increased C-peptide secretion by 21% and 22% at times T15 and T30, respectively.

[0606] As expected, pancreatic proinsulin (Fig. 30A) and insulin (Fig. 30B) levels were significantly reduced in 12-week-old ZDF rats compared to lean rats. AB14 60 mg / kg and metformin completely prevented the decrease in proinsulin, and AB14 60 mg / kg in combination with metformin significantly increased proinsulin levels (p < 0.05, relative to the mediator) (Fig. 30A). AB14 60 mg / kg slightly increased pancreatic insulin levels, and metformin or AB14 60 mg / kg in combination with metformin significantly increased insulin levels (p < 0.05, relative to the mediator) (Fig. 30B). The proinsulin / insulin ratio was significantly increased in ZDF rats compared to lean rats; however, no change was observed with drug treatment. Figure 30C ).

[0607] This study reported no signs of toxicity and focused on microscopic changes in the pancreas of lean rats or ZDF rats treated with the mediator, AB14, metformin, or a combination of AB14 and metformin.

[0608] A higher incidence and severity of focal to multifocal large / giant islets were observed in ZDF rats that provided a mediator control, corresponding to islet hyperplasia and islet fibrosis (Tables 1 and 2).

[0609] Table 1. Observation of histopathological incidence. Unless otherwise specified, all treatment groups were ZDF rats. Met.: metformin 200 mg / kg / day.

[0610]

[0611]

[0612] Table 2. Histopathological analysis: mean scores of basic findings in each group. Unless otherwise specified, all treatment groups were ZDF rats. Met.: Metformin 200 mg / kg / day.

[0613]

[0614]

[0615] Compared to lean rats, ZDF rats treated with the drug showed milder islet cell vacuolation and increased incidence and severity of islet fibrosis. All drug treatments, AB14 60 mg / kg, metformin, or a combination of metformin and AB14, consistently tended to reduce vacuolation and the severity of islet fibrosis. These effects were more pronounced in the combination of AB14 and metformin.

[0616] As expected, immunohistochemical analysis of pancreatic insulin levels showed a reduction in insulin labeling in ZDF rats. As observed from insulin content measurements (Figure 30B), drug treatment slightly prevented this reduction in insulin labeling, with the effect being more pronounced when AB14 was combined with metformin. Figure 31 ).

[0617] discuss

[0618] Eight-week-old ZDF rats exhibited marked insulin resistance and severe hyperinsulinemia, but only mild hyperglycemia, after intravenous administration of Ab14 at doses of 0 mg / kg (carrier), 20 mg / kg, or 60 mg / kg weekly for four weeks. During this period, the carrier-treated controls progressed to marked diabetes and, by day 12 of the study, developed severe hypoinsulinemia and definitive hyperglycemia, consistent with complete pancreatic β-cell failure prior to age 10 weeks. This was confirmed at study end by direct measurement of pancreatic insulin and proinsulin levels, both of which were significantly reduced, and by immunohistochemical assessment of pancreatic insulin markers, which were also remarkably reduced. Furthermore, histological analysis performed at study end confirmed an increased incidence and severity of islet cell vacuolation, islet hyperplasia (large / giant islets), and islet fibrosis in these animals, consistent with diabetic islet lesions.

[0619] In contrast, both doses of AB14 partially prevented the rise in fasting blood glucose in the mediator-treated control group on day 12 of the study, and this partial preventive effect was also observed on days 19 and 26 of the study. Furthermore, the high dose of Ab14 also partially prevented the observed decrease in plasma insulin and C-peptide levels in the mediator-treated control group on day 12 of the study. This partial preventive effect was also observed on days 19 and 26 of the study, but to a lesser extent, indicating a moderate delay in disease progression (pancreatic β-cell failure) and demonstrating the compound's partial protective effect against pancreatic β-cells. In fact, when pancreatic tissue was obtained at the end of the study (day 28), the high dose of Ab14 also completely prevented the decrease in pancreatic proinsulin levels in the mediator-treated control group, and a partial preventive effect on the decrease in pancreatic insulin levels was also observed when measured directly or by immunohistochemical analysis. Furthermore, at the end of the study's histological evaluation, animals treated with the vector showed a consistent reduction in islet vacuolation, islet fibrosis, and islet hyperplasia by Ab14, further indicating a beneficial effect on diabetic islet lesions.

[0620] As demonstrated in Example 2, Ab14 had no effect on food intake or weight when compared with the control group treated with the mediator, indicating that the effect of Ab14 on the parameters assessed above was not due to calorie restriction or weight loss.

Claims

1. Use of a humanized anti-human calcitonin gene-related peptide (CGRP) antibody or antibody fragment in the manufacture of a medicament for treating, preventing, or managing hyperglycemia in an individual in need thereof, wherein the humanized anti-human CGRP antibody or antibody fragment comprises a variable light chain polypeptide of SEQ ID NO: 51 and a variable heavy chain polypeptide of SEQ ID NO:

53.

2. The use of claim 1, wherein the use is to achieve sustained normoglycemia in the individual.

3. The use of claim 1, wherein the individual is premenopausal, perimenopausal, menopausal, or postmenopausal.

4. The use of claim 1, wherein the humanized anti-human CGRP antibody or antibody fragment is used with an anti-diabetic agent or an anti-obesity agent.

5. The use of claim 4, wherein the anti-diabetic agent or anti-obesity agent comprises one or more of amylin, an amylin agonist, a sulfonylurea, a calcitonin, a glucagon, a PPAR-gamma agonist, a GLP-1 receptor agonist, a dipeptidyl peptidase IV inhibitor, an amylin analog, a biguanide, a dopamine D2 receptor agonist, a meglitinide, an alpha glucosidase inhibitor, an anti-dyslipidemic bile acid sequestrant, an exendin, an exendin analog, an exendin agonist, a gastric inhibitory polypeptide (GIP), a secretin peptide, insulin, an SGLT2 inhibitor, a glucose reabsorption inhibitor, fenofibrate, a fibrate, an anti-ghrelin antibody or antibody fragment, a fibroblast growth factor receptor (FGFR)-1 (IIIb), FGFR-1 (IIIc), an antibody or antibody fragment, and / or FGFR-4 (IIIc), an anti-CD38 antibody or antibody fragment, an anti-MIC-1 antibody, or MIC-1 binding fragment, metformin, or a combination of any of the foregoing.

6. The use of any one of claims 1-5, wherein the medicament does not cause an increased incidence of pancreatitis or an increased expression of markers or cytokines associated with pancreatic inflammation.

7. The use of any one of claims 1-5, wherein the medicament further comprises a pharmaceutically acceptable carrier.

8. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment is administered to the individual at a dose of between 0.1 and 100.0 mg / kg of body weight of the recipient individual.

9. The use of claim 1, wherein the humanized anti-human CGRP antibody or antibody fragment comprises a light chain polypeptide of SEQ ID NO: 52 and a heavy chain polypeptide of SEQ ID NO:

54.

10. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment comprises a Fab, F(ab')2, or scFv.

11. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody comprises an Fc region that has been modified to alter effector function, half-life, proteolysis, and / or glycosylation.

12. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment is non-glycosylated.

13. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment is glycosylated and comprises only mannose residues.

14. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment is expressed in Pichia pastoris (G 1 1 1 ). Pichia pastoris ) 15. The use of any one of claims 1-5, wherein the humanized anti-human CGRP antibody or antibody fragment is expressed in CHO cells.

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