BR122020017648B1 - COMPOSITION FOR THE TREATMENT OR REDUCTION OF THE INCIDENCE OF MIGRAINE COMPRISING A MONOCLONAL ANTIBODY THAT INHIBITS THE CALCITONIN GENE-RELATED PEPTIDE PATHWAY - Google Patents

Abstract

The invention features methods for preventing or treating CGRP-associated disorders, such as vasomotor symptoms and/or headaches (e.g., migraine, cluster headache, and tension headache) by administering an anti-CGRP antagonist antibody. Compositions for use in the disclosed methods are also provided. G1 antagonist antibody and G1-derived antibodies directed to CGRP are also described.

Description

[001] Divided from BR1120160214234, filed on 03/20/2015.

CROSS-REFERENCE TO RELATED ORDERS

[002] This application claims priority to U.S. Provisional Patent Application No. 61/968,897, filed March 21, 2014, Provisional Patent Application No. 62/083,809, filed November 24, 2014, and U.S. Provisional Patent Application No. 62/119,778, filed February 23, 2015, which applications are incorporated herein by reference in their entireties for all purposes.

Background

[003] CGRP (calcitonin gene-related peptide) is a 37-amino acid neuropeptide that belongs to the peptide family that includes calcitonin, adrenomedullin, and amylin. In humans, there are two forms of CGRP (α-CGRP and β-CGRP) and they have similar activities. They vary in three amino acids and have differential distribution. At least two subtypes of CGRP receptors may also be responsible for differential activities. CGRP is a neurotransmitter in the central nervous system and has been shown to be a potent vasodilator in the periphery, where CGRP-containing neurons are closely associated with blood vessels. CGRP-mediated vasodilation is also associated with neurogenic inflammation, as part of a cascade of events that results in plasma leakage and vasodilation of the microvasculature, and is present in migraine.

[004] CGRP has been highlighted for its possible association with vasomotor symptoms (Wyon et al. Scand. J. Urol. Nephrol. 35: 92-96 (2001); Wyon et al. Menopause 7(1):25-30 (2000)). Vasomotor symptoms (VMS), such as hot flashes and night sweats, are the most common symptoms associated with menopause, occurring in 60% to 80% of all women after natural or surgically induced menopause. Hot flashes may be an adaptive response of the central nervous system (CNS) to decreasing sex steroids (Freedman Am. J. Human Biol. 13:453464 (2001)). To date, the most effective therapies for hot flashes are hormone-based treatments, including estrogens and/or some progestins. Hormonal treatments may be effective in relieving hot flashes, but they are not suitable for all women. The psychological and emotional symptoms observed, such as nervousness, fatigue, irritability, insomnia, depression, memory loss, headaches, anxiety, jitteriness or inability to concentrate are thought to be caused by sleep deprivation following hot flashes and night sweats (Kramer et al., In: Murphy et al., 3rd Supp. Int'l Symposium on Recent Advances in Urological Cancer Diagnosis and Treatment-Proceedings, Paris, France: SCI: 3-7 (1992)).

[005] Men also experience hot flashes after withdrawal of steroid (androgen) hormones. This is true in cases of age-related androgen depletion (Katovich, et al., Proceedings of the Society for Experimental Biology & Medicine, 1990, 193(2): 129-35), as well as in extreme cases of hormone deprivation associated with prostate cancer treatments (Berendsen, et al., European Journal of Pharmacology, 2001, 419(1): 47-54). One third of these patients will experience persistent and frequent symptoms severe enough to cause significant discomfort and distress.

[006] CGRP is a potent vasodilator that has been implicated in the pathology of other vasomotor symptoms, such as forms of vascular headache, including migraine (with or without aura) and cluster headache. Durham, N. Engl. J. Med. 350:10731075, 2004. Serum levels of CGRP in the external jugular vein are elevated in patients during migraine. Goadsby et al., Ann. Neurol. 28:183-7, 1990. Intravenous administration of human α-CGRP induced headache and migraine in patients suffering from migraine without aura, suggesting that CGRP has a causative role in migraine. Lassen et al., Cephalalgia 22:54-61, 2002.

[007] The possible involvement of CGRP in migraine has been the basis for the development and testing of a series of compounds that inhibit CGRP release (e.g. sumatriptan), antagonize the CGRP receptor (e.g. dipeptide derivative BIBN4096BS (Boerhringer Ingelheim); CGRP(8-37)) or interact with one or more of the receptor-associated proteins such as receptor activity membrane protein (RAMP) or receptor component protein (RCP), both of which affect the binding of CGRP to its receptors. Brain, S. et al., Trends in Pharmacological Sciences 23:51-53, 2002. Alpha 2-adrenoceptor subtypes and adenosine A1 receptors also control (inhibit) CGRP release and trigeminal activation (Goadsby et al., Brain 125:1392-401, 2002). The adenosine A1 receptor agonist GR79236 (metrafadil), which has been shown to inhibit neurogenic vasodilation and trigeminal nociception in humans, may also have antimigraine activity (Arulmani et al., Cephalalgia 25:1082-1090, 2005; Giffin et al., Cephalalgia 23:287-292, 2003.)

[008] Confounding this theory is the observation that treatment with compounds that exclusively inhibit neurogenic inflammation (e.g., NK1 tachykinin antagonists) or trigeminal activation (e.g., 5HT1D receptor agonists) has been shown to be relatively ineffective as acute migraine treatments, leading some investigators to question whether inhibiting CGRP release is the primary mechanism of action of effective migraine treatments. Arulmani et al., Eur. J. Pharmacol. 500:315-330, 2004.

[009] Migraine is a common complex neurological condition that is characterized by severe episodic attacks of headache and associated features, which may include nausea, vomiting, and sensitivity to light, sound, or movement. In some patients, the headache is preceded or accompanied by an aura. The headache may be severe and may also be unilateral in certain patients.

[0010] Migraine attacks are a daily nuisance. In Western Europe and the United States, the overall prevalence of migraine sufferers is 11% of the general population (6% males; 15-18% females). Furthermore, the average frequency of attacks in an individual is 1.5/month. Although there are a number of treatments available to alleviate or reduce symptoms, preventive therapy is recommended for those patients with more than 3-4 monthly migraine attacks. Goadsby et al. New Engl. J. Med. 346(4): 257-275, 2002.

[0011] The variety of pharmacologic interventions that have been used to treat migraine and the variability in responses among patients are a testament to the diverse nature of this disorder. Thus, relatively nonselective drugs such as ergot alkaloids (e.g., ergotamine, dihydroergotamine, methysergide), which exhibit serotonergic, adrenergic, as well as noradrenergic and dopaminergic activity, have been used for over 80 years to treat migraine. Other treatments include opiates (e.g., oxycodone) and beta-adrenergic antagonists (e.g., propranolol). Some patients, usually those with milder symptoms, are able to control their symptoms with over-the-counter medications such as one or more nonsteroidal anti-inflammatory agents (NSAIDs), such as a combination of aspirin, acetaminophen, and caffeine (e.g., Excedrin® Migraine).

[0012] More recently, some patients with migraine have been treated with topiramate, an anticonvulsant that blocks voltage-gated sodium channels and certain glutamate (AMPA-kainate) receptors, potentiates GABA-A receptor activity, and blocks carbonic anhydrase. The relatively recent success of serotonin 5HT-1B/1D and/or 5HT-1A receptor agonists such as sumatriptan in some patients has led investigators to propose a serotonergic etiology of the disorder. Unfortunately, although some patients have responded well to this treatment, others have been relatively resistant to its effects.

[0013] Dysfunction of an ion channel in the brainstem aminergic nuclei has been postulated to underlie the disorder. However, the precise pathophysiology of migraine is not yet fully understood. One form of migraine, familial hemiplegic migraine, has been shown to be associated with missense mutations in the α1 subunit of the voltage-gated P/Q-type calcium channel, and it is anticipated that other ion channel mutations will also be found in other patient populations. Although blood vessel dilation is associated with and exacerbates migraine pain symptoms, these neurovascular events are believed to be a result of the condition rather than its cause. In general, dysfunction of brainstem pathways that modulate sensory inputs is considered a unifying feature of migraine. Goadsby, P.J. et al., New Engl. J. Med. 346(4): 257-275, 2002.

BRIEF SUMMARY OF THE INVENTION

[0014] In some aspects, the invention disclosed herein relates to anti-CGRP antibodies and methods of anti-CGRP antagonist antibodies for treating or preventing vasomotor symptoms. Examples of vasomotor symptoms are provided herein, such as hot flushes. In some cases, the anti-CGRP antagonist antibodies are used to treat or prevent a headache, such as a migraine with or without aura, migraine hemiplegia, cluster headaches, migrainous neuralgia, chronic headaches, tension headaches, and headaches resulting from other medical conditions (such as from infection or increased pressure in the skull due to a tumor).

[0015] In one aspect, the present invention provides a method for treating or preventing symptoms of at least one vasomotor symptom in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody.

[0016] In one aspect, the present invention provides a method of treating or preventing headache (e.g., migraine and cluster headache) in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody.

[0017] In another aspect, the invention provides a method for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of headache (e.g., migraine and cluster headache) in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody.

[0018] In one aspect, the invention provides a method for treating or reducing the incidence of at least one vasomotor symptom and/or headache in a subject. In one embodiment, the method comprises administering to the subject for multiple days an amount of a monoclonal antibody (e.g., the monoclonal anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the amount administered on each of those multiple days is less than 1000 mg. In one embodiment, the method comprises administering to the subject for multiple days an amount of a monoclonal antibody (e.g., the monoclonal anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the amount administered on each of those multiple days is between 100 and 2000 mg. In some embodiments, the headache is migraine (e.g., chronic migraine or episodic migraine). In some embodiments, two of the multiple days are more than seven days apart. In some embodiments, the incidence of headache is reduced for at least seven days after a single administration. In some embodiments, the amount of the monoclonal antibody administered on a first day is different from (e.g., more than) the amount of the monoclonal antibody administered on a second day. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous. In some embodiments, administration is intravenous. In some embodiments, administration comprises use of a pre-filled syringe comprising the amount of the monoclonal antibody. In some embodiments, the monoclonal antibody is formulated at a concentration of 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the monthly headache hours experienced by the subject following said administration are reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80 or more) from a pre-administration level in the subject. The monthly headache hours can be reduced by more than 60 hours. In some embodiments, the monthly headache hours experienced by the subject following said administration are reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50% or more) from a pre-administration level in the subject. The monthly headache hours can be reduced by 40% or more. In some embodiments, the monthly headache days experienced by the subject following said administration are reduced by 3 or more days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days) from a level pre-administration to the subject. In some embodiments, the method further comprises administering to the subject a second agent simultaneously or sequentially with the monoclonal antibody. The second agent can be any of 5-HT1 agonists, triptans, ergot alkaloids, and nonsteroidal anti-inflammatory drugs. In some embodiments, the second agent is an agent taken by the subject prophylactically. In some embodiments, the subject's monthly use of the second agent is reduced by at least 15% following administration of the monoclonal antibody. In some embodiments, the second agent is a triptan. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is a human or humanized monoclonal antibody. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set forth in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0019] In one aspect, the invention provides a method for reducing the number of monthly headache hours suffered by a subject. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is present in an amount effective to decrease the number of monthly headache hours by at least 20 (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or more headache hours) after a single dose. In some embodiments, the number of monthly headache hours is reduced by at least 50 hours. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to reduce the number of monthly headache hours by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more) after a single dose. In some embodiments, the number of monthly headache hours is reduced by at least about 30%. In some embodiments, the monoclonal antibody is an anti-CGRP antagonist antibody. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set forth in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0020] In one aspect, the invention provides a method for reducing the number of monthly headache days suffered by a subject. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is an amount effective to reduce the number of monthly headache days by at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more headache days) after a single dose. In some embodiments, the number of monthly headache days is reduced by at least about 6 headache days. In some embodiments, the monoclonal antibody is an anti-CGRP antagonist antibody. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set out in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0021] In one aspect, the invention provides a method of decreasing the use of a headache medication in a subject, comprising administering to the subject a monoclonal antibody (e.g., an anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease the subject's monthly headache medication use by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more). In some embodiments, the headache medication is selected from the group consisting of 5-HT1 agonists, triptans, opiates, α-adrenergic antagonists, ergot alkaloids, and nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the headache medication is a triptan. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set out in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0022] In one aspect, the invention provides a method for treating or reducing the incidence of headache (e.g., migraines) in a subject, comprising administering to the subject a single dose of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) in an amount that modulates the CGRP pathway, wherein the amount of the monoclonal antibody is between 100-2000 mg.

[0023] In another embodiment, the invention provides methods for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of headache (e.g., migraine and cluster headache) in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody in combination with at least one additional agent useful for treating headache. Such additional agents include 5-HT1 agonists (and agonists acting at other 5-HT1 sites) and nonsteroidal anti-inflammatory drugs (NSAIDs).

[0024] Examples of 5-HT1 agonists that may be used in combination with an anti-CGRP antibody include a class of compounds known as triptans, such as sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, and frovatriptan. Ergot alkaloids and related compounds are also known to have 5-HT agonist activity and have been used to treat headaches such as migraine. Such compounds include ergotamine tartrate, ergonovine maleate, and ergoloid mesylates (e.g., dihydroergocornine, dihydroergocristine, dihydroergocryptine, and dihydroergotamine mesylate (DHE 45)).

[0025] Examples of NSDAIDs that may be used in combination with an anti-CGRP antibody include aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib; rofecoxib; meloxicam; JTE-522; L-745337; NS398; or a pharmaceutically acceptable salt thereof.

[0026] In another aspect, the invention provides a method for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of hot flashes in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody.

[0027] In another aspect, the invention provides methods for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of hot flashes in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody in combination with at least one additional agent useful for treating hot flashes. Such additional agents include, but are not limited to, hormone-based treatments, including estrogens and/or progestins.

[0028] In one embodiment, the anti-CGRP antagonist antibody used in any of the methods described above is any of the antibodies described herein.

[0029] In some embodiments, the anti-CGRP antagonist antibody recognizes a human CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to α-CGRP and β-CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to human and rat CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to the C-terminal fragment at amino acids 25-37 of CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to a C-terminal epitope at amino acids 25-37 of CGRP.

[0030] In some embodiments, the anti-CGRP antibody is a monoclonal antibody. In some embodiments, the anti-CGRP antagonist antibody is humanized. In some embodiments, the antibody is human. In some embodiments, the anti-CGRP antagonist antibody is a G1 antibody (as described herein). In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDR(s) (such as one, two, three, four, five, or in some embodiments, all six CDRs) of the G1 antibody or the G1 variants shown in Table 6. In still other embodiments, the anti-CGRP antagonist antibody comprises the heavy chain variable region amino acid sequence shown in Figure 5 (SEQ ID NO: 1) and the light chain variable region amino acid sequence shown in Figure 5 (SEQ ID NO: 2).

[0031] In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert (including partially immunologically inert), e.g., does not trigger complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), does not activate microglia, or reduces one or more of these activities. In some embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In other embodiments, the antibody comprises a human IgG2 heavy chain constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence). Eur. J. Immunol. (1999) 29:2613-2624. In some embodiments, the heavy chain constant region of the antibody is a human IgG1 heavy chain having any of the following mutations: 1) A327A330P331 to G327S330S331; 2) E233L234L235G236 (SEQ ID NO: 48) to P233V234A235 with G236 deleted; 3) E233L234L235 to P233V234A235; 4) E233L234L235G236A327A330P331 (SEQ ID NO: 49) toP233V234A235G327S330S331 (SEQ ID NO: 50) with G236 deleted; 5) E233L234L235A327A330P331 (SEQ ID NO: 51) to P233V234A235G327S330S331 (SEQ ID NO: 50); and 6) N297 to A297 or any other amino acid except N. In some embodiments, the antibody heavy chain constant region is a human IgG4 heavy chain with any of the following mutations: E233F234L235G236 (SEQ ID NO: 52) to P233V234A235 with G236 deleted; E233F234L235 to P233V234A235; and S228L235 to P228E235.

[0032] In still other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the oligosaccharide-binding residue (such as Asn297) and/or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically or by expression in a glycosylation-deficient host cell.

[0033] The binding affinity (K) of an anti-CGRP antagonist antibody to CGRP (such as human α-CGRP, as measured by plasmon resonance at a suitable temperature, such as 25 or 37 °C) can be from about 0.02 to about 200 nM. In some embodiments, the binding affinity is between about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, about 60 pM, about 50 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some embodiments, the binding affinity is less than about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM. In some embodiments, the binding affinity is less than about 50 nM.

[0034] The anti-CGRP antagonist antibody can be administered prior to, during, and/or after the headache. In some embodiments, the anti-CGRP antagonist antibody is administered prior to the headache attack (e.g., migraine and cluster headache). Administration of an anti-CGRP antagonist antibody can be by any means known in the art, including: orally, intravenously, subcutaneously, intraarterially, intramuscularly, intranasally (e.g., with or without inhalation), intracardially, intraspinally, intrathoracicly, intraperitoneally, intraventricularly, sublingually, transdermally, and/or via inhalation. Administration can be systemic, e.g., intravenously, or localized.

[0035] In some embodiments, the anti-CGRP antagonist antibody can be administered together with another agent, such as another agent to treat headaches.

[0036] In another aspect, the invention provides the use of an anti-CGRP antagonist antibody for the production of a medicament for use in any of the methods described herein, for example: for treating or preventing headaches.

[0037] In another aspect, the invention provides a pharmaceutical composition for preventing or treating headaches (e.g., migraine and cluster headaches) comprising an effective amount of an anti-CGRP antagonist antibody in combination with one or more pharmaceutically acceptable excipients.

[0038] In another aspect, the invention provides a kit for use in any of the methods described herein. In some embodiments, the kit comprises a container, a composition comprising an anti-CGRP antagonist antibody described herein, in combination with a pharmaceutically acceptable carrier, and instructions for use of the composition in any of the methods described herein.

[0039] The present invention also provides anti-CGRP antagonist antibodies and polypeptides derived from the G1 antibody or its variants shown in Table 6. Accordingly, in one aspect, the invention provides a G1 antibody (interchangeably referred to as "G1") that is produced by expression vectors having ATCC Accession Nos. PTA-6866 and PTA-6867. For example, in one embodiment, it is an antibody comprising a heavy chain produced by the expression vector having ATCC Accession No. PTA-6867. In another embodiment, it is an antibody comprising a light chain produced by the expression vector having ATCC Accession No. PTA-6866. The amino acid sequences of the G1 light chain and heavy chain variable regions are shown in Figure 5. Portions of the complementarity determining region (CDR) of the G1 antibody (including Chothia and Kabat CDRs) are also shown in Figure 5. Reference to any part or all of the G1 region is understood to encompass sequences produced by expression vectors having ATCC Accession No. PTA-6866 and PTA-6867 and/or the sequences illustrated in Figure 5. In some embodiments, the invention also provides G1 antibody variants having the amino acid sequences set forth in Table 6.

[0040] In one aspect, the invention provides an antibody comprising a VH domain that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to SEQ ID NO: 1.

[0041] In another aspect, the invention provides an antibody comprising a VL domain that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to SEQ ID NO: 2.

[0042] In another aspect, the invention provides an antibody comprising a fragment or a region of the G1 antibody or variants thereof shown in Table 6. In another embodiment, the fragment is a fragment of the light chain of the G1 antibody. In another embodiment, the fragment is a heavy chain of the G1 antibody. In yet another embodiment, the fragment contains one or more variable regions of a light chain and/or heavy chain of the G1 antibody. In yet another embodiment, the fragment contains one or more variable regions of a light chain and/or heavy chain of the G1 antibody shown in Figure 5. In yet another embodiment, the fragment contains one or more CDRs of a light chain and/or heavy chain of the G1 antibody.

[0043] In another aspect, the invention provides polypeptides (which may or may not be an antibody) comprising a VH CDR3 as set forth in SEQ ID NO: 5, or a sequence that differs from SEQ ID NO: 5 by 1, 2, 3, 4, or 5 amino acid substitutions. In a particular embodiment, these amino acid substitutions are conservative substitutions.

[0044] In another aspect, the invention provides polypeptides (which may or may not be an antibody) comprising a VL CDR3 as set forth in SEQ ID NO: 8, or a sequence that differs from SEQ ID NO: 8 by 1, 2, 3, 4, or 5 amino acid substitutions. In a particular embodiment, these amino acid substitutions are conservative substitutions.

[0045] In another aspect, the invention provides polypeptides (which may or may not be an antibody) comprising one or more of the following: a) one or more CDR(s) of antibody G1 or variants thereof shown in Table 6; b) CDR H3 of antibody G1 heavy chain or variants thereof shown in Table 6; c) CDR L3 of antibody G1 light chain or variants thereof shown in Table 6; d) three CDRs of antibody G1 light chain or variants thereof shown in Table 6; e) three CDRs of antibody G1 heavy chain or variants thereof shown in Table 6; f) three light chain CDRs and three heavy chain CDRs of the G1 antibody or its variants shown in Table 6. In some embodiments, the invention further provides polypeptides (which may or may not be an antibody) comprising one or more of the following: a) one or more (one, two, three, four, five, or six) CDR(s) derived from the G1 antibody or its variants shown in Table 6; b) a CDR derived from the G1 antibody heavy chain CDR H3; and/or c) a CDR derived from the G1 antibody light chain CDR L3. In some embodiments, the CDR is a CDR shown in Figure 5. In some embodiments, one or more CDRs derived from the G1 antibody or its variants shown in Table 6 are at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to at least one, at least two, at least three, at least four, at least five, or at least six CDRs of G1 or its variants.

[0046] In some embodiments, the CDR is a Kabat CDR. In other embodiments, the CDR is a Chothia CDR. In other embodiments, the CDR is a combination of a Kabat and a Chothia CDR (also referred to as a "combined CDR" or "extended CDR"). In other words, for any embodiment containing more than one CDR, the CDRs may be Kabat, Chothia, and/or combined.

[0047] In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence of KASKXaaVXaaTYVS (SEQ ID NO: 53), wherein Xaa at position 5 is R, W, L, L, or N; and wherein Xaa at position 7 is T, A, D, G, R, S, W, or V. In some embodiments, the amino acid sequence of KASKXaaVXa-aTYVS (SEQ ID NO: 53) is an antibody light chain CDR1.

[0048] In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence of XaaXaaSNRYXaa (SEQ ID NO: 54), wherein Xaa at position 1 is L or A; wherein Xaa at position 2 is A or H; and wherein Xaa at position 7 is L, T, I, or S. In some embodiments, the amino acid sequence of XaaXaaSNRYXaa (SEQ ID NO: 54) is a CDR2 of the antibody light chain.

[0049] In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence of EIRSXaaSDXaaXaaATXaaYAXaaAVKG (SEQ ID NO: 55), wherein Xaa at position 5 is E, R, K, Q, or N; wherein Xaa at position 8 is A, L, N, E, H, S, L, R, C, F, Y, V, D, or P; wherein Xaa at position 9 is S, G, T, Y, C, E, G, A, P, I, N, R, V, D, or H; wherein Xaa at position 12 is H or F; wherein Xaa at position 15 is E or D. In some embodiments, the amino acid sequence of EIRSXaaSDXaaXaaATXaaYAXaaAVKG (SEQ ID NO: 55) is an antibody heavy chain CDR2.

[0050] In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence of SEQ ID NO: 1, wherein the amino acid residue at position 99 of SEQ ID NO: 1 is L or is substituted with A, N, S, T, V, or R; and wherein the amino acid residue at position 100 of SEQ ID NO: 1 is A or is substituted with L, R, S, V, Y, C, G, T, K, or P.

[0051] In some embodiments, the antibody is a human antibody. In other embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is monoclonal. In some embodiments, the antibody (or polypeptide) is isolated. In some embodiments, the antibody (or polypeptide) is substantially pure.

[0052] The constant region of the heavy chain of antibodies can be of any type of constant region, such as IgG, IgM, IgD, IgA and IgE; and any isotypes, such as IgG1, IgG2, IgG3 and IgG4.

[0053] In some embodiments, the antibody comprises a modified constant region as described herein.

[0054] In another aspect, the invention provides a polynucleotide (which may be isolated) comprising a polynucleotide encoding a fragment or a region of the G1 antibody or its variants shown in Table 6. In another embodiment, the fragment is a fragment of the light chain of the G1 antibody. In another embodiment, the fragment is a heavy chain of the G1 antibody. In yet another embodiment, the fragment contains one or more variable regions of a light chain and/or heavy chain of the G1 antibody. In yet another embodiment, the fragment contains one or more (i.e., one, two, three, four, five, or six) complementarity determining regions (CDRs) of a light chain and/or heavy chain of the G1 antibody.

[0055] In another aspect, the invention provides a polynucleotide (which may be isolated) comprising a polynucleotide encoding the G1 antibody or its variants shown in Table 6. In some embodiments, the polynucleotide comprises one or both of the polynucleotides set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

[0056] In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) or polypeptides described herein.

[0057] In another aspect, the invention provides vectors (including cloning and expression vectors) and host cells comprising any of the polynucleotides disclosed herein. In some embodiments, the vector is pDb.CGRP.hFcGI, having ATCC No. PTA-6867. In other embodiments, the vector is pEb.CGRP.hKGI, having ATCC No. PTA-6866.

[0058] In another aspect, the invention provides a host cell comprising a polynucleotide encoding any of the antibodies described herein.

[0059] In another aspect, the invention provides a complex of CGRP linked to any of the antibodies or polypeptides described herein. In some embodiments, the antibody is a G1 antibody or variants thereof shown in Table 6.

[0060] In another aspect, the invention provides a pharmaceutical composition comprising an effective amount of any of the polypeptides (including antibodies, such as an antibody comprising one or more CDRs of the G1 antibody) or polynucleotides described herein and a pharmaceutically acceptable excipient.

[0061] In another aspect, the invention provides a method of generating a G1 antibody, comprising culturing a host cell or descendant thereof under conditions that allow for the production of the G1 antibody, wherein the host cell comprises an expression vector encoding the G1 antibody; and, in some embodiments, promoting purification of the G1 antibody. In some embodiments, the expression vector comprises one or both of the polynucleotide sequences set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

[0062] In another aspect, the invention provides methods of producing any of the antibodies or polypeptides described herein by expressing one or more polynucleotides encoding the antibody (which may be expressed separately as a single light or heavy chain or the light and heavy chain are expressed from a vector) or the polypeptide in a suitable cell, generally followed by recovery and/or isolation of the antibody or polypeptides of interest.

[0063] The anti-CGRP antagonist antibody and polypeptides, and the polynucleotides encoding the antibodies and polypeptides of the present invention, can be used to treat, prevent, ameliorate, control or reduce the incidence of diseases associated with abnormal functioning of CGRP, such as headache (e.g., migraine, cluster headache, chronic headache and tension headache) and other conditions that can be treated or prevented by antagonizing CGRP activity.

[0064] In another aspect, the invention provides kits and compositions comprising one or more of the compositions described herein. Such kits, generally in suitable packaging and provided with suitable instructions, are useful for any of the methods described herein.

[0065] In one aspect, the invention provides a composition for use in accordance with any of the methods described herein.

[0066] In one aspect, the invention provides a composition for use in treating or reducing the incidence of at least one vasomotor symptom and/or headache in a subject. In one embodiment, the use comprises administering to the subject for multiple days an amount of a monoclonal antibody (e.g., the monoclonal anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the amount administered on each of those multiple days is less than 1000 mg. In one embodiment, the use comprises administering to the subject for multiple days an amount of a monoclonal antibody (e.g., the monoclonal anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the amount administered on each of those multiple days is between 100 and 2000 mg. In some embodiments, the headache is migraine (e.g., chronic migraine or episodic migraine). In some embodiments, two of the multiple days are more than seven days apart. In some embodiments, the incidence of headache is reduced for at least seven days after a single administration. In some embodiments, the amount of the monoclonal antibody administered on a first day is different from (e.g., more than) the amount of the monoclonal antibody administered on a second day. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous. In some embodiments, administration is intravenous. In some embodiments, administration comprises use of a pre-filled syringe comprising the amount of the monoclonal antibody. In some embodiments, the monoclonal antibody is formulated at a concentration of 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the monthly headache hours experienced by the subject following said administration are reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80 or more) from a pre-administration level in the subject. The monthly headache hours can be reduced by more than 60 hours. In some embodiments, the monthly headache hours experienced by the subject following said administration are reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50% or more) from a pre-administration level in the subject. The monthly headache hours can be reduced by 40% or more. In some embodiments, the monthly headache days experienced by the subject following said administration are reduced by 3 or more days (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days) from a pre-administration level in the subject. In some embodiments, the use further comprises administering to the subject a second agent simultaneously or sequentially with the monoclonal antibody. The second agent can be any of 5-HT1 agonists, triptans, ergot alkaloids, and nonsteroidal anti-inflammatory drugs. In some embodiments, the second agent is an agent taken by the subject prophylactically. In some embodiments, the subject's monthly use of the second agent is reduced by at least 15% following administration of the monoclonal antibody. In some embodiments, the second agent is a triptan. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is a human or humanized monoclonal antibody. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set forth in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0067] In one aspect, the invention provides a composition for use in decreasing the number of monthly headache hours suffered by a subject. In one embodiment, the use comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is present in an amount effective to decrease the number of monthly headache hours by at least 20 (e.g., 25, 30, 35, 40, 45, 50, 55, 60, 65, 70 or more headache hours) after a single dose. In some embodiments, the number of monthly headache hours is reduced by at least 50 hours. In one embodiment, the use comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to reduce the number of monthly headache hours by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more) after a single dose. In some embodiments, the number of monthly headache hours is reduced by at least about 30%. In some embodiments, the monoclonal antibody is an anti-CGRP antagonist antibody. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set forth in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0068] In one aspect, the invention provides a composition for use in reducing a number of monthly headache days experienced by a subject. In one embodiment, the use comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the monoclonal antibody is an amount effective to reduce the number of monthly headache days by at least 3 (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more headache days) after a single dose. In some embodiments, the number of monthly headache days is reduced by at least about 6 headache days. In some embodiments, the monoclonal antibody is an anti-CGRP antagonist antibody. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set out in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0069] In one aspect, the invention provides a composition used to decrease the use of a headache medication in a subject, comprising administering to the subject a monoclonal antibody (e.g., an anti-CGRP antagonist antibody) that modulates the CGRP pathway, wherein the monoclonal antibody is in an amount effective to decrease the subject's monthly headache medication use by at least 15% (e.g., 20%, 25%, 30%, 35%, 40%, or more). In some embodiments, the headache medication is selected from the group consisting of 5-HT1 agonists, triptans, opiates, α-adrenergic antagonists, ergot alkaloids, and nonsteroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the headache medication is a triptan. In some embodiments, the amount of the monoclonal antibody is less than 1000 mg. In some embodiments, the subject receives less than 3 doses per month. In some embodiments, administration is subcutaneous or intravenous. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, the monoclonal antibody is administered at a volume of less than 2 mL. In some embodiments, the subject is a human. In some embodiments, the monoclonal antibody is human or humanized. In some embodiments, the monoclonal antibody comprises (a) an antibody having a CDR H1 as set forth in SEQ ID NO: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set out in SEQ ID NO: 8; or (b) a variant of an antibody according to (a), as shown in Table 6.

[0070] In one aspect, the invention provides a composition used for treating or reducing the incidence of headache (e.g., migraines) in a subject, comprising administering to the subject a single dose of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) in an amount that modulates the CGRP pathway, wherein the amount of the monoclonal antibody is between 100-2000 mg.

Brief Description of Figures

[0071] Figure 1 is a table showing the binding affinities of 12 murine antibodies for different alanine-substituted human α-CGRP fragments. Binding affinities were measured at 25 °C using Biacore by flowing the Fabs past the CGRPs on the chip. Boxed values represent the loss of affinity of alanine mutants relative to the parental fragment, 25-37 (italicized), except K35A, which was derived from a parental 19-37. An "a" indicates the affinities for 19-37 and fragments 25-37 are the mean ± standard deviation of two independent measurements on different sensor chips. A "b" indicates those interactions deviated from a simple biomolecular interaction model due to a biphasic dissociation rate, so their affinities were determined using a conformational change model. Grayscale legend: white (1.0) indicates parental affinity; light gray (less than 0.5) indicates an affinity greater than parental; dark gray (more than 2) indicates an affinity less than parental; and black indicates that no binding was detected.

[0072] Figures 2A and 2B show the effect of administration of CGRP 8-37 (400 nmol/kg), antibody 4901 (25 mg/kg), and antibody 7D11 (25 mg/kg) on skin blood flow measured as blood cell flux following a 30 second electrical pulse stimulation. CGRP 8-37 was administered intravenously (iv) 3-5 min prior to electrical pulse stimulation. Antibodies were administered intraperitoneally (ip) 72 hours prior to electrical pulse stimulation. Each point on the graphs represents an AUC for a rat treated under the indicated conditions. Each line on the graphs represents the mean AUC for rats treated under the indicated condition. AUC (area under the curve) is equal to Δflux x Δtime. "Δflux" represents the change in flux units following electrical pulse stimulation; and "Δtime" represents the period of time required for the level of blood cell flow to return to the level prior to stimulation by electrical pulses.

[0073] Figure 3 shows the effect of different dosage administration of antibody 4901 (25 mg/kg, 5 mg/kg, 2.5 mg/kg or 1 mg/kg) on skin blood flow measured as blood cell flux following electrical pulse stimulation for 30 seconds. The antibodies were administered intravenously (IV) 24 hours prior to electrical pulse stimulation. Each point on the graphs represents an AUC of rat treated under the indicated conditions. The line on the graphs represents the mean AUC of rats treated with the indicated condition.

[0074] Figures 4A and 4B show the effect of administration of antibody 4901 (1 mg/kg or 10 mg/kg, i.v.), antibody 7E9 (10 mg/kg, i.v.), and antibody 8B6 (10 mg/kg, i.v.) on skin blood flow measured as blood cell flux following 30 second electrical pulse stimulation. Antibodies were administered intravenously (IV) followed by electrical pulse stimulation 30 min, 60 min, 90 min, and 120 min after antibody administration. The Y-axis represents the percentage of AUC compared to the AUC level when no antibody was administered (time 0). The X-axis represents the time period (minutes) between antibody administration and electrical pulse stimulation. "*" indicates P < 0.05 and "**" indicates P < 0.01 compared with time 0. Data were analyzed using one-way ANOVA with a Dunnett's multiple comparison test.

[0075] Figure 5 shows the amino acid sequence of the heavy chain variable region (SEQ ID NO: 1) and light chain variable region (SEQ ID NO: 2) of the G1 antibody. The Kabat CDRs are in bold and the Chothia CDRs are underlined. The amino acid residues for the heavy chain and light chain variable region are numbered sequentially.

[0076] Figure 6 shows epitope mapping of the G1 antibody by peptide competition using Biacore. Human n-biotinylated α-CGRP was captured on a SA sensor chip. G1 Fab (50 nM) in the absence of a competitor peptide or preincubated for 1 h with 10 uM of a competitor peptide was dropped onto the chip. The binding of G1 Fab to human α-CGRP on the chip was measured. The Y-axis represents the percentage of binding blocked by the presence of the competitor peptide compared to binding in the absence of the competitor peptide.

[0077] Figure 7 shows the effect of administration of G1 antibody (1 mg/kg or 10 mg/kg, i.v.) or vehicle (PBS, 0.01% Tween 20) on skin blood flow measured as blood cell flux after electrical pulse stimulation for 30 seconds. G1 antibody or vehicle was administered intravenously (IV) followed by electrical nerve pulse stimulation 30 min, 60 min, 90 min, and 120 min after antibody administration. The Y-axis represents the percentage of AUC compared to the AUC level when no antibody or vehicle (defined as 100%) was administered (time 0). The X-axis represents the time period (minutes) between antibody administration and electrical pulse stimulation. "*" indicates P < 0.05 and "**" indicates P < 0.01 compared to vehicle. Data were analyzed using two-way ANOVA and Bonferroni post hoc tests.

[0078] Figure 8A shows the effect of administration of G1 antibody (1 mg/kg, 3 mg/kg, or 10 mg/kg, i.v.) or vehicle (PBS, 0.01% Tween 20) on skin blood flow measured as blood cell flux following electrical nerve stimulation for 30 seconds 24 hours after dosing. G1 antibody or vehicle was administered intravenously (i.v.) 24 hours prior to electrical nerve stimulation. The Y-axis represents the total area under the curve (change in blood cell flux multiplied by the change in time from stimulation to flow return to baseline, AUC). The X-axis represents varying doses of G1 antibody. "*" indicates P < 0.05, and "**" indicates P < 0.01, compared to vehicle. Data were analyzed using ANOVA and Dunnett's multiple comparison test.

[0079] Figure 8B shows the effect of administration of G1 antibody (0.3 mg/kg, 1 mg/kg, 3 mg/kg, or 10 mg/kg, i.v.) or vehicle (PBS, 0.01% Tween 20) on skin blood flow measured as blood cell flux following electrical nerve stimulation for 30 seconds 7 days after dosing. G1 antibody or vehicle was administered intravenously (i.v.) 7 days prior to electrical nerve stimulation. The Y-axis represents total AUC. The X-axis represents varying doses of G1 antibody. "**" indicates P < 0.01, and "***" indicates P < 0.001, compared to vehicle. Data were analyzed using ANOVA and Dunnett's multiple comparison test.

[0080] Figure 8C is a curve-fit analysis of the data from Figures 8A and 8B. G1 antibody or vehicle was administered intravenously (i.v.) 24 hours or 7 days prior to electrical nerve pulse stimulation. The Y-axis represents total AUC. The X-axis represents varying doses of G1 antibody in "mg/kg" on a logarithmic scale to determine EC50.

[0081] Figure 9 shows the effect of mu7E9 antibody (10 mg/kg), BIBN4096BS, or vehicle (PBS, 0.01% Tween 20) on the change in diameter of the middle meningeal artery following an electric field stimulus. The mu7E9 antibody, BIBN4096BS, or vehicle were administered intravenously (i.v.) at the 0 minute time point, after a baseline response to electrical stimulation had been established. The Y-axis represents the change in diameter of the middle meningeal artery following an electric field stimulus. The settled diameter corresponds to 0%. The X-axis represents the time (minutes) of electrical pulse stimulation. "*" indicates P < 0.05 and "**" indicates P < 0.01 compared to vehicle. Data were analyzed using ANOVA and a Dunnett's multiple comparison test.

[0082] Figure 10 shows the effect of various doses of G1 antibody (1 mg/kg, 3 mg/kg, or 10 mg/kg, i.v.) or vehicle (PBS, 0.01% Tween 20) on the change in middle meningeal artery diameter after electric field stimulation. G1 antibody or vehicle was administered intravenously (i.v.) 7 days prior to electric field stimulation. The Y-axis represents the change in middle meningeal artery diameter. The settled diameter corresponds to 0%. The X-axis represents the stimulus voltage. "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001, compared with vehicle. Data were analyzed using two-way ANOVA and Bon-Ferroni post hoc tests.

[0083] Figure 11A shows the effect of mu4901 antibody (10 mg/kg) or vehicle (PBS, 0.01% Tween 20), administered intravenously (i.v.) 24 hours earlier, on the reduction in core temperature induced by subcutaneous injection of naloxone (1 mg/kg) in morphine-addicted rats. The Y-axis represents the temperature difference from baseline. The X-axis represents the time measured since the point of naloxone injection.

[0084] Figure 11B shows the effect of mu4901 antibody (10 mg/kg) or vehicle (PBS, 0.01% Tween 20), administered intravenously (i.v.) 24 hours earlier, on the increase in back surface temperature induced by subcutaneous injection of naloxone (1 mg/kg) in morphine-addicted rats. The Y-axis represents the temperature difference from baseline. The X-axis represents the time measured since the point of naloxone injection.

[0085] Figure 12 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0086] Figure 13 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0087] Figure 14 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0088] Figure 15 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0089] Figure 16 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0090] Figure 17 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

[0091] Figure 18 is a graph illustrating the results of a clinical study comparing the effects of different doses of G1 antibody with placebo.

Detailed Description

[0092] In some aspects, the invention disclosed herein provides methods for treating and/or preventing vasomotor symptoms (e.g., hot flashes) in a subject by administering to the subject a therapeutically effective amount of an anti-CGRP antagonist antibody.

[0093] In some aspects, the invention disclosed herein provides methods for treating and/or preventing headaches (e.g., migraine, cluster headache, chronic headache, and tension headaches) in a subject by administering to the subject a therapeutically effective amount of an anti-CGRP antagonist antibody. In some cases, the headache is a migraine.

[0094] In some aspects, the invention disclosed herein also provides anti-CGRP antagonist antibodies and polypeptides derived from G1 and its variants shown in Table 6. In some embodiments, the invention also provides methods of making and using such antibodies and polypeptides.

[0095] Throughout this application, various publications (including patents and patent applications) are referenced. The disclosures of these publications in their entireties are incorporated herein by reference.

General Techniques

[0096] The practice of the various aspects of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of one of ordinary skill in the art. Such techniques are fully explained in the literature, such as in Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather & P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths & D.G. Newell, eds., 1993-1998) J. Wiley &Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D.M. Weir and C.C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F.M. Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Definitions

[0097] An "antibody" is an immunoglobulin molecule capable of binding to a specific target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies, but also their fragments (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv), mutants thereof, fusion proteins comprising a part of the antibody (such as domain antibodies), and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site. An antibody includes an antibody of any class, such as IgG, IgA, or IgM (or subclass thereof), and the antibody need not be of any specific class. Depending on the antibody amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are termed alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0098] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies comprising the population are identical except for possible naturally occurring mutations, which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any specific method. For example, monoclonal antibodies to be used in accordance with the present invention can be made by the hybridoma method first described by Kohler and Milstein, 1975, Nature, 256:495, or can be produced by recombinant DNA methods such as those described in U.S. Pat. No. 4,816,567. Monoclonal antibodies can also be isolated from phage libraries generated using the techniques described in McCafferty et al., 1990, Nature, 348:552-554, for example.

[0099] As used herein, "humanized" antibodies refer to non-human (e.g., murine) antibody forms of antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of the antibodies) that contain minimal sequence derived from non-human immunoglobulins. Most humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced with residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit with the desired specificity, affinity, and biological activity. In some cases, residues from the Fv framework region (FR) of the human immunoglobulin are replaced with corresponding non-human residues. In addition, the humanized antibody may comprise residues that are not found in either the recipient antibody or in the CDR or framework sequences, but are included to refine and optimize the performance of the antibody. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody will ideally also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Antibodies may have modified Fc regions as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that are altered with respect to the parent antibody, which are also referred to as one or more CDRs "derived from" one or more CDRs of the parent antibody.

[00100] As used herein, "human antibody" denotes an antibody that has an amino acid sequence that corresponds to that of an antibody produced by a human and/or that was produced using any of the techniques for producing human antibodies known in the art or disclosed herein. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. An example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely knocked out. This approach is described in U.S. Patent Nos. 5,545,807 ; 5,545,806 ; 5,569,825 ; 5,625,126; 5,633,425; and 5,661,016. Alternatively, the human antibody can be prepared by immortalizing human B lymphocytes that produce an antibody directed against a target antigen (such B lymphocytes can be recovered from an individual or can have been immunized in vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Patent No. 5,750,373.

[00101] As used herein, the terms "calcitonin gene-related peptide" and "CGRP" refer to any form of calcitonin gene-related peptide and variants thereof that retain at least some of the activity of CGRP. For example, CGRP may be α-CGRP or β-CGRP. As used herein, CGRP includes all mammalian species of native sequence CGRP, e.g., humans, canines, felines, equines, and bovines.

[00102] As used herein, an "anti-CGRP antagonist antibody" (interchangeably referred to as "anti-CGRP antibody") refers to an antibody that is capable of binding to CGRP and inhibiting the biological activity of CGRP and/or the downstream pathway(s) mediated by CGRP signaling. An anti-CGRP antagonist antibody encompasses antibodies that modulate, block, antagonize, suppress, or reduce (including significantly) the biological activity of CGRP or otherwise antagonize the CGRP pathway, including downstream pathways mediated by CGRP signaling, such as receptor binding and/or triggering of a cellular response to CGRP. For purposes of the present invention, it will be explicitly understood that the term “anti-CGRP antagonist antibody” encompasses all of the above-identified terms, titers, and functional states and characteristics in which CGRP itself, the biological activity of CGRP (including but not limited to its ability to mediate any aspect of headache), or the consequences of the biological activity are substantially abrogated, diminished, or neutralized to any significant degree. In some embodiments, an anti-CGRP antagonist antibody binds to CGRP and prevents the binding of CGRP to the CGRP receptor. In other embodiments, an anti-CGRP antibody binds to CGRP and prevents the activation of a CGRP receptor. Examples of anti-CGRP antagonist antibodies are provided herein.

[00103] As used herein, the terms "G1" and "G1 antibody" are used interchangeably to refer to an antibody produced by expression vectors having ATCC deposit numbers PTA-6867 and ATCC PTA-6866. The amino acid sequences of the light chain and heavy chain variable regions are shown in Figure 5. The CDR portions of the G1 antibody (including Chothia and Kabat CDRs) are schematically represented in Figure 5. The polynucleotides encoding the heavy and light chain variable regions are shown in SEQ ID NO: 9 and SEQ ID NO: 10. Characterization of G1 is described in the Examples.

[00104] The terms "polypeptide", "oligopolypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that, since the polypeptides of this invention are based on an antibody, the polypeptides may occur in the form of single chains or associated chains.

[00105] "Polynucleotide" or "nucleic acid", as used interchangeably herein, refers to polymers of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and their analogs. If present, modification of the nucleotide backbone may be imparted before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "capping", replacement of one or more of the naturally occurring nucleotides with an analogue, internucleotide modifications such as, for example, those with uncharged bonds (e.g., methyl phosphonates, phosphotriesters, phosphoamidates, cabamates, etc.) and with charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those containing pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified bonds (e.g., alpha-anomeric nucleic acids, etc.), as well as forms unmodified polynucleotide(s). In addition, any of the hydroxyl groups commonly present on sugars may be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages for additional nucleotides, or may be conjugated to solid or semi-solid supports. The 5' and 3' OH ends may be phosphorylated or replaced by amines or organic capping group moieties of 1 to 20 carbon atoms. Other hydroxyls may also be derived from standard protecting groups. The polynucleotides may also contain analogous forms of ribose or deoxyribose sugars that are generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro or 2'-azido-ribose, carbocyclic sugar analogs, α-anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranose sugars, furanose sugars, heptuloses, acyclic analogs and basic nucleoside analogs such as methyl-riboside. One or more phosphodiester linkages may be replaced by alternative linking groups. Such alternative linking groups include, but are not limited to, embodiments in which the phosphate is replaced by P(O)S ("thioate"), P(S)S ("dithioate"), (O)NR2 ("amidate"), P(O)R, P(O)OR', CO, or CH2 ("formacetal"), in which each R or R' is independently H or substituted or unsubstituted alkyl (1-20C), optionally containing an ether, aryl, alkenyl, cycloalkyl, cycloalkenyl, or araldyl (-O-) linkage. Not all linkages in a polynucleotide need to be identical. The foregoing description applies to all polynucleotides referred to herein, including RNA and DNA.

[00106] A "variable region" of an antibody refers to either the antibody light chain variable region or the antibody heavy chain variable region, either alone or in combination. The heavy and light chain variable regions consist of four framework regions (FR) connected by three complementarity determining regions (CDRs), also known as the hypervariable regions. The CDRs in each chain are held together in close proximity by the FRs and, with the CDRs of the other chain, contribute to the formation of the antigen-binding site of antibodies. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al. Sequences of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of Health, Bethesda MD)); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al-lazikani et al (1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR may refer to a CDR defined by either approach or by a combination of both approaches.

[00107] A "constant region" of an antibody refers to either the constant region of the antibody light chain or the constant region of the antibody heavy chain, either alone or in combination.

[00108] An epitope that "preferentially binds" or "specifically binds" (used interchangeably herein) to an antibody or a polypeptide is a term well understood in the art, and methods for determining such preferential or specific binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, more rapidly, for a longer duration, and/or with greater affinity with a specific cell or substance than with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an epitope of CGRP is an antibody that binds to that epitope with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other CGRP epitopes or non-CGRP epitopes. It is also understood from reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding means preferential binding.

[00109] As used herein, "substantially pure" refers to material that is at least 50% pure (i.e., free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, more preferably at least 98% pure, most preferably at least 99% pure.

[00110] A "host cell" includes a cultured cell or an individual cell that may be or have been a recipient for vector(s) for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transfected in vivo with polynucleotide(s) of this invention.

[00111] The term "Fc region" is used to define a C-terminal region of an immunoglobulin heavy chain. The "Fc region" may be a native sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is usually defined to extend from an amino acid residue at position Cys226, or from Pro230, to the carboxyl terminus thereof. The numbering of residues in the Fc region is that of the Kabat EU index. Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3.

[00112] As used herein, "Fc receptor" and "FcR" describe a receptor that binds to the Fc region of an antibody. The preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one that binds an IgG antibody (a gamma receptor) and includes FcyRI, FcyRII, and FcyRIII receptors, including allelic variant subclasses and alternative spliced forms of these receptors. FCYRII receptors include FCYRIIA (an "activating receptor") and FcyRIIB (an "inhibitory receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcRs are reviewed in Ravetch & Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J. Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and Kim et al., 1994, J. Immunol., 24:249).

[00113] "Complement-dependent cytotoxicity" and "CDC" refer to the lysis of a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), can be performed.

[00114] A "functional Fc region" possesses at least one "effector function" of a native sequence Fc region. Examples of "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptor; BCR), etc. Such effector functions generally require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be assessed using various assays known in the art for assessing such antibody effector functions.

[00115] A "native Fc region sequence" comprises an amino acid sequence identical to the amino acid sequence of an Fc region found in nature. A "variant Fc region" comprises an amino acid sequence that differs from that of a native sequence Fc region by virtue of at least one amino acid modification, and yet retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to a native sequence Fc region or an Fc region of a source polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in a native sequence Fc region or in the Fc region of the source polypeptide. The variant Fc region herein will preferably have at least about 80% sequence identity to a native Fc region sequence and/or to an Fc region of a parent polypeptide, and more preferably at least about 90% identity to such a sequence, most preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% identity to such a sequence.

[00116] As used herein, "cell-mediated antibody-dependent cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcRs) (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize antibody bound to a target cell and subsequently cause lysis of the target cell. The ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay as described in U.S. Patent Nos. 5,500,362 or 5,821,337. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or additionally, the ADCC activity of the molecule of interest can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., 1998, PNAS (USA), 95:652-656.

[00117] As used herein, "treatment" is an approach to achieving beneficial or desired clinical outcomes. For purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: improvement in any aspect of the headache, including decreasing severity, alleviating pain intensity and other associated symptoms, reducing the frequency of recurrence, increasing the quality of life of those suffering from the headache, and decreasing the dose of other medications needed to treat the headache. For migraine, other associated symptoms include, but are not limited to, nausea, vomiting, and sensitivity to light, sound, and/or movement. For cluster headache, other associated symptoms include, but are not limited to, swelling under or around the eyes, excessive tearing, red eyes, runny or stuffy nose, and flushing of the face.

[00118] "Reducing the incidence" of headache denotes reducing the severity (which may include reducing the need for and/or amount of (e.g., exposure to) other drugs and/or therapies commonly used for such a condition, including, for example, ergotamine, dihydroergotamine, or triptans for migraine), duration, and/or frequency (including, for example, delaying or increasing the time until the next episodic attack in an individual). As understood by those skilled in the art, individuals may vary in their response to treatment, and thus, for example, a "method for reducing the incidence of headaches in an individual" reflects the administration of the anti-CGRP antagonist antibody based on the reasonable expectation that such administration is likely to cause such a reduction in incidence in that particular individual.

[00119] "Improving" headache or one or more headache symptoms means a reduction or improvement of one or more headache symptoms compared to no administration of an anti-CGRP antagonist antibody. "Improving" also includes the shortening or reduction in duration of a symptom.

[00120] As used herein, "controlling headache" refers to maintaining or reducing the severity or duration of one or more headache symptoms or the frequency of headache attacks in an individual (compared to the pre-treatment level). For example, the duration or severity of headache or the frequency of attacks is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, or 70% in the individual compared to the pre-treatment level.

[00121] As used herein, a "headache hour" refers to one hour during which an individual suffers from a headache. Headache hours may be expressed in terms of whole hours (e.g., one hour headache, two hours headache, three hours headache, etc.) or in terms of whole and partial hours (e.g., 0.5 hour headache, 1.2 hour headache, 2.67 hour headache, etc.). One or more headache hours may be described in relation to a specific time interval. For example, "daily headache hours" may refer to the number of headache hours an individual suffers in the span of a day (e.g., in a 24-hour period). In another example, "weekly headache hours" may refer to the number of headache hours an individual suffers in the span of a week (e.g., in a 7-day period). As can be appreciated, a one-week interval may or may not correspond to a calendar week. In another example, "monthly headache hours" may refer to the number of headache hours an individual suffers in a one-month interval. As can be appreciated, a one-month interval (e.g., a period of 28-31 days) may vary in terms of the number of days to months, depending on the particular month, and may or may not correspond to a calendar month. In another example, "yearly headache hours" may refer to the number of headache hours an individual suffers in a one-year interval. As can be appreciated, a one-year interval (e.g., a period of 365 or 366 days) may vary in terms of the number of days, depending on the particular year, and may or may not correspond to a calendar year. In some embodiments, a headache hour may be a reference to a specific type of headache (e.g., migraine, cluster headache, chronic headache, and tension headache). For example, a "migraine hour" may refer to an hour during which an individual suffers from a migraine.

[00122] As used herein, a "headache day" refers to a day during which an individual suffers from a headache. Headache days may be expressed in terms of full days (e.g., one headache day, two headache days, three headache days, etc.) or in terms of full and partial days (e.g., 0.5 day headache, 1.2 day headache, 2.67 day headache, etc.). One or more headache days may be described in relation to a specific time interval. For example, "weekly headache days" may refer to the number of headache days an individual suffers in the span of a week (e.g., in a 7-day period). As can be appreciated, a span of a week may or may not correspond to a calendar week. In another example, "monthly headache days" may refer to the number of headache days an individual suffers in a one-month interval. As can be appreciated, a one-month interval (e.g., a 28-31 day period) may vary in terms of the number of days to months, depending on the particular month, and may or may not correspond to a calendar month. In another example, "yearly headache days" may refer to the number of headache days an individual suffers in a one-year interval. As can be appreciated, a one-year interval (e.g., a 365- or 366-day period) may vary in terms of the number of days, depending on the particular year, and may or may not correspond to a calendar year. In some embodiments, a headache day may be a reference to a specific type of headache (e.g., migraine, cluster headache, chronic headache, and tension headache). For example, a "migraine day" may refer to a day during which an individual suffers from a migraine.

[00123] As used herein, "delaying" the development of headache means to defer, prevent, delay, postpone, stabilize, and/or postpone the progression of the disease. Such delay may be for varying lengths of time, depending on the history of the disease and/or individual being treated. As is apparent to those skilled in the art, a sufficient or significant delay may, in effect, include prevention, whereby the individual does not develop headache (e.g., migraine). A method of "delaying" the development of a symptom is a method that reduces the likelihood of developing the symptom within a given time interval and/or reduces the extent of symptoms over a given period of time, when compared to not using the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWV AVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDRLNYYD SSGYYHYKYYGMAVWGQGTTVTVSS

[00124] "Development" or "progression" of headache means initial manifestations and/or progression of the disease. Development of headache can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that may be undetectable. For purposes of the present disclosure, development or progression refers to the biological field of symptoms. "Development" includes occurrence, recurrence, and onset. As used herein "onset" or "occurrence" of headache includes initial onset and/or recurrence.

[00125] As used herein, an "effective dose" or "effective amount" of drug, compound, or pharmaceutical composition is an amount sufficient to effect beneficial or desired results. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, decreasing the severity, or delaying the onset of disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that present during the development of the disease. For therapeutic use, beneficial or desired results include clinical results such as reducing pain intensity, duration or frequency of headache attack, and decreasing one or more symptoms resulting from headache (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes presented during the development of the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications needed to treat the disease, increasing the effect of another medication, and/or slowing the progression of the disease in patients. An effective dosage may be administered in one or more administrations. For the purposes of this invention, an effective dosage of a drug, compound, or pharmaceutical composition is a dosage sufficient to effect prophylactic or therapeutic treatment directly or indirectly. As understood in the clinical context, an effective dosage of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective dosage" may be considered in the context of the administration of one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result can be or is achieved.

[00126] An "individual" or an "individual" is a mammal, most preferably a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

[00127] As used herein, the term "vector" refers to a construct, which is capable of delivering and preferentially expressing one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[00128] As used herein, "expression control sequence" means a nucleic acid sequence that directs the transcription of a nucleic acid. An expression control sequence may be a promoter, such as a constitutive or an inducible promoter, or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

[00129] As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and is non-reactive with the individual's immune system. Examples include, but are not limited to, any of the conventional pharmaceutical carriers such as phosphate buffered saline, water, emulsions such as oil/water emulsion, and various types of wetting agents. The preferred diluents for aerosol or parenteral administration are phosphate buffered saline, or (0.9%) normal saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th ed., A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing, 2000).

[00130] The term "kon" as used herein is intended to refer to the rate constant for the association of an antibody with an antigen.

[00131] The term "koff", as used herein, is intended to refer to the rate constant for the dissociation of an antibody from the antibody/antigen complex.

[00132] The term "KD" as used herein is intended to refer to the equilibrium dissociation constant of an antibody-antigen interaction.

[00133] As used herein, the term "vasomotor symptom," is intended to refer to conditions related to vasodilation. This vasodilation may be associated or potentially associated with headache (such as migraine with or without aura; hemiplegic migraine; chronic migraine; episodic migraine; high frequency episodic migraine; cluster headache; hemicranial neuralgia; chronic headaches, tension headaches, headaches resulting from other medical conditions (such as infection or increased pressure in the skull due to a tumor); chronic paroxysmal hemicrania, miscellaneous headaches not associated with a structural lesion; headache associated with a nonvascular intracranial disorder; headache associated with substance administration or withdrawal; headache associated with noncephalic infection; headache associated with a metabolic disorder; headache associated with a disorder of the skull, neck, eyes, ears, nose, sinuses, teeth, mouth, or other facial or cranial structure; cranial neuralgias and nerve trunk pain and headache). deafferentation), hot flushes (or hot flashes), cold flashes, insomnia, sleep disturbances, mood disorders, irritability, excessive sweating, night sweats, daytime sweats, fatigue and the like, caused by, inter alia, thermoregulatory dysfunction.

[00134] As used herein, the terms "flush", "hot flush" and "heat" are art recognized terms that refer to an episodic disturbance of body temperature, typically consisting of a sudden flushing of the skin, usually accompanied by sweating in an individual.

A. Methods for the prevention or treatment of vasomotor symptoms and/or headache

[00135] In one aspect, the invention provides a method for treating or reducing the incidence of at least one vasomotor symptom in a subject. In another aspect, the invention provides a method of treating or reducing the incidence of headache (e.g., migraine) in a subject. In some embodiments, the method comprises administering to the subject an effective amount of an antibody or antibody-derived polypeptides that modulate the CGRP pathway (e.g., an anti-CGRP antagonist monoclonal antibody). In some embodiments, the at least one vasomotor symptom can be associated with the headache (e.g., migraine) and/or hot flashes.

[00136] In another aspect, the invention provides a method for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of flushing in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody. In some embodiments, the at least one vasomotor symptom can be associated with the headache (e.g., migraine) and/or hot flushes.

[00137] In another aspect, the invention provides methods for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of headache (e.g., migraine) in a subject or symptoms associated with headache (e.g., diarrhea or sensitivity to light), comprising administering to the subject an effective amount of an antibody that modulates the CGRP pathway or an anti-CGRP antagonist antibody in combination with at least one additional agent useful for treating headache.

[00138] Such additional agents include, but are not limited to, 5-HT agonists and NSAIDs. For example, the antibody and at least one additional agent may be administered concomitantly, that is, they may be given in close enough temporal proximity to allow their individual therapeutic effects to overlap. For example, the amount of 5-HT agonist or NSAID administered in combination with an anti-CGRP antibody should be sufficient to reduce the frequency of headache relapses in patients or produce more lasting efficacy compared to administration of either agent in the absence of the other. This procedure may be used to treat headaches that fall into any of a wide variety of classes, including: migraine with or without aura; hemiplegic migraine; chronic migraine; episodic migraine; high frequency episodic migraine; cluster headache; migraine neuralgia; chronic headaches; tension headaches; headaches resulting from other medical conditions (e.g., infection or increased pressure in the skull due to a tumor); chronic paroxysmal hemicrania; miscellaneous headaches not associated with a structural lesion; headache associated with a nonvascular intracranial disorder; headache associated with the administration of a substance or its withdrawal; headache associated with a noncephalic infection; headache associated with a metabolic disorder; headache associated with a disorder of the skull, neck, eyes, ears, nose, sinuses, teeth, mouth, or other cranial or facial structure; cranial neuralgias; and nerve trunk pain and deafferentation pain.

[00139] Additional non-limiting examples of additional agents that may be administered in combination with an anti-CGRP antagonist antibody include one or more of the following:(i) an opioid analgesic, e.g., morphine, heroin, hydromorphone, oxymorphone, levorphanol, levallorphan, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, or pentazocine;(ii) a nonsteroidal anti-inflammatory drug (NSAID), e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, namic, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib; rofecoxib; meloxicam; JTE-522; L-745337; NS398; or a pharmaceutically acceptable salt thereof;(iii) a barbiturate sedative, for example, amobarbital, aprobarbital, butabarbital, butabital, mephobarbital, metharbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutal, teamilal or thiopental or a pharmaceutically acceptable salt thereof;(iv) a barbiturate analgesic, for example, butalbital or a pharmaceutically acceptable salt thereof or a composition comprising butalbital.(v) a benzodiazepine having a sedative action, for example, chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam or a pharmaceutically acceptable salt thereof;(vi) an H1 antagonist having a sedative action, for example, diphenhydramine, pyrilamine, promethazine, chlorpheniramine or chlorcyclizine or a pharmaceutically acceptable salt thereof;(vii) a sedative such as glutethimide, meprobamate, methaqualone, dichloralphenazone or a pharmaceutically acceptable salt thereof;(viii) a skeletal muscle relaxant, for example, baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or orphrenadine or a pharmaceutically acceptable salt thereof;(ix) an NMDA receptor antagonist, for example, dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine, pyrroloquinoline quinone or cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid or a salt thereof pharmaceutically acceptable;(x) an alpha-adrenergic e.g., doxazosin, tamsulosin, clonidine or 4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline;(xi) tricyclic antidepressants e.g., desipramine, imipramine, amitriptyline or nortriptyline;(xii) an anticonvulsant e.g., carbamazepine or valproate;(xiii) a tachykinin (NK) antagonist, particularly an NK-3, NK-2 or NK-1 antagonist e.g., (αR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11- tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphtridine-6-13-di-one(TAK-637), 5 - [[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophe- nyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one(MK-869), lanepitant, dapitant or 3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylamino]-2-phenyl-piperidine(2S,3S);(xiv) a muscarinic antagonist, for example, oxybutin, tolterodine, propiverine, trospium chloride or darifenacin;(xv) a COX-2 inhibitor, e.g. celecoxib, rofecoxib or valdecoxib;(xvi) a non-selective COX inhibitor (preferably with GI protection), e.g. nitroflurbiprofen (HCT-1026);(xvii) a coal tar analgesic, in particular paracetamol;(xviii) a neuroleptic such as droperidol;(xix) a vanilloid receptor agonist (e.g. resiniferatoxin) or antagonist (e.g. capsazepine); (xx) ) a beta-adrenergic such as propranolol;(xxi) a local anesthetic such as mexiletine;(xxii) a corticosteroid such as dexamethasone;(xxiii) a serotonin receptor agonist or antagonist;(xxiv) ) a cholinergic (nicotinic) analgesic;(xxv) ) Tramadol (registered trademark);(xxvi) a PDEV inhibitor such as sildenafil, vardenafil, or taladafil;(xxvii) an alpha-2-delta ligand such as gabapentin or pregabalin;(xxviii) a cannabinoid; and(xxix) i) an antidepressant such as amitriptyline (Elavil), trazodone (Desyrel), and imipramine (Tofranil) or anticonvulsants such as phenytoin (Dilantin) or carbamazepine (Tegretol).

[00140] Those skilled in the art will be able to determine appropriate dosage amounts for particular agents to be used in combination with an anti-CGRP antibody. For example, sumatriptan can be administered in a dosage from about 0.01 to about 300 mg. In some cases, sumatriptan can be administered in a dosage of 2 mg to 300 mg. When administered non-parenterally, the typical dosage of sumatriptan is about 25 to about 100 mg, with about 50 mg generally being preferred, and when administered parenterally, the preferred dosage is about 6 mg. However, these dosages can be varied according to standard methods in the art so that they are optimized for a particular patient or for a specific combination therapy. Also, for example, celecoxib can be administered in an amount of between 50 and 500 mg.

[00141] In another aspect, the invention provides methods for ameliorating, controlling, reducing the incidence of, or delaying the development or progression of hot flashes in a subject, comprising administering to the subject an effective amount of an anti-CGRP antagonist antibody in combination with at least one additional agent useful for treating hot flashes. Such additional agents include, but are not limited to, hormone-based treatments, including estrogens and/or some progestins.

[00142] In another aspect, the disclosure provides a method of treating or reducing the incidence of headache (e.g., migraine) in a subject comprising administering to the subject on a plurality of days an amount of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) that modulates the CGRP pathway. In some embodiments, the amount of the monoclonal antibody administered on each of the plurality of days can range from 0.1 mg - 5,000 mg, 1 mg - 5,000 mg, 10 mg - 5,000 mg, 100 mg - 5,000 mg, 1,000 mg - 5,000 mg, 0.1 mg - 4,000 mg, 1 mg - 4,000 mg, 10 mg - 4,000 mg, 100 mg - 4,000 mg, 0.1 mg - 3,000 mg, 1 mg - 3,000 mg, 10 mg - 3,000 mg, 100 mg - 3,000 mg, 1,000 mg - 3,000 mg, 0.1 mg - 2,000 mg, 1 mg - 2000 mg, 10 mg - 2000 mg, 100 mg - 2000 mg, 1,000 mg - 2,000 mg, 0.1 mg - 1,000 mg, 1 mg - 1,000 mg, 10 mg - 1000 mg or 100 mg - 1000 mg. In some embodiments, the amount is between 100 - 2000 mg.

[00143] In another aspect, the disclosure provides a method of treating or reducing the incidence of headache (e.g., migraine) in a subject comprising administering to the subject a single dose of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) in an amount that modulates the CGRP pathway. In some embodiments, the single dose can be an amount of antibody between 0.1 mg - 5,000 mg, 1 mg - 5,000 mg, 10 mg - 5,000 mg, 100 mg - 5,000 mg, 1,000 mg - 5,000 mg, 0.1 mg - 4,000 mg, 1 mg - 4,000 mg, 10 mg - 4,000 mg, 100 mg - 4,000 mg, 0.1 mg - 3,000 mg, 1 mg - 3,000 mg, 10 mg - 3,000 mg, 10 mg - 3,000 mg, 100 mg - 3,000 mg, 1000 mg - 3,000 mg, 1000 mg - 3,000 mg, 0.1 mg - 2,000 mg, 1 mg - 2,000 mg, 10 mg - 2000 mg, 100 mg-2000 mg, 1,000 mg-2,000 mg, 0.1 mg-1,000 mg, 1 mg-1000 mg, 10 mg-1000 mg, or 100 mg-1000 mg. In some embodiments, the single dose can be an amount of antibody between 100-2000 mg.

[00144] In another aspect, the disclosure provides a method of treating or reducing the incidence of at least one vasomotor symptom in a subject comprising administering to the subject on a plurality of days an amount of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) that modulates the CGRP pathway. In some embodiments, the amount of the monoclonal antibody administered on each of the plurality of days can range from 0.1 mg - 5,000 mg, 1 mg - 5,000 mg, 10 mg - 5,000 mg, 100 mg - 5,000 mg, 1,000 mg - 5,000 mg, 0.1 mg - 4,000 mg, 1 mg - 4,000 mg, 10 mg - 4,000 mg, 100 mg - 4,000 mg, 0.1 mg - 3,000 mg, 1 mg - 3,000 mg, 10 mg - 3,000 mg, 100 mg - 3,000 mg, 1,000 mg - 3,000 mg, 0.1 mg - 2,000 mg, 1 mg - 2000 mg, 10 mg - 2000 mg, 100 mg - 2000 mg, 1,000 mg - 2,000 mg, 0.1 mg - 1,000 mg, 1 mg - 1,000 mg, 10 mg - 1000 mg or 100 mg - 1000 mg. In some embodiments, the amount is between 100 - 2000 mg.

[00145] In another aspect, the disclosure provides a method of decreasing the number of monthly headache hours experienced by a subject, comprising administering to the subject an amount of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache hours by at least 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more headache hours after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache hours by at least 20 headache hours, following a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache hours by at least 40 headache hours. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache hours by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache hours by at least 15% after a single dose.

[00146] In another aspect, the disclosure provides a method of decreasing the number of monthly headache days experienced by a subject, comprising administering to the subject an amount of a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache days by at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more headache days after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache days by at least 3 headache days after a single dose. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the number of monthly headache days by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more after a single dose.

[00147] In another aspect, the disclosure provides a method for decreasing the use of an anti-headache medication in a subject, comprising administering to the subject a monoclonal antibody (e.g., an anti-CGRP antagonist monoclonal antibody) that modulates the CGRP pathway. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the subject's monthly anti-headache medication usage by at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more. In some embodiments, the monoclonal antibody can be in an amount effective to decrease the subject's monthly anti-headache medication usage by at least 15%. The anti-headache medication can be any type of anti-headache medication described elsewhere herein. Non-limiting examples of anti-headache medications include 5-HT1 agonists (and agonists acting at other 5-HT1 sites), triptans (e.g., sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, afrovatriptan), ergot alkaloids (e.g., ergotamine tartrate, ergonovine maleate, and ergoloid mesylates (e.g., dihydroergocornine, dihydroergocristine, dihydroergocryptine, and dihydroergotamine mesylate (DHE 45)), and nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, piroxicam, su- ndac, tolmetin, or zomepirac, cyclooxygenase-2 (COX-2) inhibitors (celecoxib; rofecoxib; meloxicam; JTE-522, L-745337; NS398; or a pharmaceutically acceptable salt thereof), opiates (e.g., oxycodone), and α-adrenergic antagonists (e.g., propranolol).

[00148] With respect to all methods described herein, references to antibodies (e.g., monoclonal antibodies that modulate the CGRP pathway, anti-CGRP antagonist antibodies, anti-CGRP monoclonal antagonist antibodies) also include compositions comprising one or more of these agents. Accordingly, such a composition may be used in accordance with a method referring to an antibody described herein. Such compositions may further comprise suitable excipients, such as pharmaceutically acceptable excipients as described elsewhere herein. The present invention may be used alone or in combination with other conventional methods of treatment.

[00149] An antibody described herein (e.g., a monoclonal antibody, an anti-CGRP antagonist antibody, an anti-CGRP antagonist monoclonal antibody) may be administered to a subject or individual at any therapeutic dose, by any suitable route, and in any suitable formulation. It should be apparent to one of skill in the art that the examples described herein are not intended to be limiting, but to be illustrative of available techniques. Accordingly, in some embodiments, an antibody described herein can be administered to a subject according to known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, sublingual, intraarterial, intrasynovial, via insufflation, intrathecal, oral, inhalation, intranasal (e.g., with or without inhalation), buccal, rectal, transdermal, intracardiac, intraosseous, intradermal, transmucosal, vaginal, intravitreal, periarticular, local, epicutaneous, or topical routes. Administration can be systemic, e.g., intravenous administration, or localized. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers are useful for administration. Liquid formulations may be nebulized directly and the lyophilized powder may be nebulized after reconstitution. Alternatively, an antibody described herein may be aerosolized using a fluorocarbon formulation and a metered dose inhaler, or inhaled as a lyophilized and milled powder.

[00150] In some embodiments, an antibody described herein can be administered via site-specific or targeted local delivery techniques. Examples of local or targeted site-specific delivery techniques include various implantable antibody depot sources or local delivery catheters, such as infusion catheters, a catheter, or a needle catheter, synthetic grafts, adventitial sheaths, shunts and stents or other implantable devices, site-specific carriers, direct injection, or direct application. See, for example, PCT Publication No. WO 00/53211 and U.S. Patent No. 5,981,568.

[00151] Various formulations of an antibody described herein can be used for administration. In some embodiments, an antibody can be administered neat. In some embodiments, the antibody and a pharmaceutically acceptable excipient can be in various formulations. Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient can impart shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizing agents, wetting and emulsifying agents, salts to vary osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients as well as formulations for parenteral and nonparenteral drug delivery are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[00152] In some embodiments, these agents, including the antibodies described herein, can be formulated for administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Accordingly, these agents can be combined with pharmaceutically acceptable carriers, such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dose, timing, and repetition, will depend on the particular individual and the individual's medical history.

[00153] In some embodiments, these agents, including the antibodies described herein, can be formulated for peripheral administration. Such formulations can be administered via any suitable peripherally route, including intravenously and subcutaneously. An agent prepared for peripheral administration can include a substance, drug, and/or the antibody that is not administered centrally, spinally, intrathecally, or directly to the CNS. Non-limiting examples of peripheral routes of administration include a route that is oral, sublingual, buccal, topical, rectal, via inhalation, transdermal, subcutaneous, intravenous, intra-arterial, intramuscular, intracardiac, intraosseous, intradermal, intraperitoneal, transmucosal, vaginal, intravitreal, intra-articular, peri-articular, local, or epi-cutaneous.

[00154] Therapeutic formulations of antibodies used in accordance with the present invention may be prepared for storage and/or use by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000)), and may in some cases be in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed. A therapeutic formulation of an antibody may comprise one or more pharmaceutically acceptable carriers, excipients or stabilizers examples of such species include buffers such as phosphate, citrate, and other non-limiting organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues); polypeptide proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids (e.g., at concentrations of 0.1 mM to 100 mM, 0.1 mM to 1 mM, 0.01 mM to 50 mM, 1 mM to 50 mM, 1 mM to 30 mM, 1 mM to 20 mM, 10 mM to 25 mM) such as glycine, glutamine, methionine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents (e.g., in concentrations of 0.001 mg/ml to 1 mg/mL, 0.001 mg/ml to 1 mg/mL, 0.001 mg/ml to 0.1 mg/mL, 0.001 mg/ml to 0.01 mg/ml, 0.01 mg/mL to 0.1 mg/ml) such as EDTA (e.g., disodium EDTA dihydrate); sugars (e.g., in concentrations of 1 mg/mL to 500 mg/mL, 10 mg/mL to 200 mg/mL, 10 mg/mL to 100 mg/mL, 50 mg/mL to 150 mg/ml) such as sucrose, mannitol, trehalose, or sorbitol; counterion-forming salt such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants (e.g., at concentrations of 0.01 mg/mL to 10 mg/mL, 0.01 mg/mL to 1 mg/mL, 0.1 mg/mL to 1 mg/mL, 0.01 mg/mL to 0.5 mg/mL) such as TweenTM (e.g., polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80)), PLURONICSTM, or polyethylene glycol (PEG).

[00155] An antibody formulation can be characterized in terms of any of a variety of physical properties. For example, a liquid antibody formulation can have any pH suitable for therapeutic effect, safety, and storage. For example, the pH of a liquid antibody formulation can be from pH 4 to about pH 9, from pH 5 to pH 8, from pH 5 to pH 7, or from pH 6 to pH 8. In some embodiments, a liquid antibody formulation can have a pH of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10, or higher, or lower.

[00156] In another example, a liquid antibody formulation may have any viscosity suitable for therapeutic efficacy, safety, and storage. For example, the viscosity of a liquid antibody formulation can be 0.5 centipoise (cP) to 100 cP, 1 cP to 50 cP, 1 cP to 20 cP, 1 cP to 15 cP, or 5 cP to 15 cP at 25° C. In some embodiments, a liquid antibody formulation can have a viscosity of 0.5 cP, 1 cP, 1.2 cP, 1.4 cP, 1.6 cP, 1.8 cP, 2.0 cP, 2.2 cP, 2.4 cP, 2.6 cP, 2.8 cP, 3.0 cP, 3.2 cP, 3.4 cP, 3.6 cP, 3.8 cP, 4.0 cP, 4.2 cP, 4.4 cP, 4.6 cP, 4.8 cP, 5.0 cP, 5.2 cP, 5.4 cP, 5.6 cP, 5.8 cP, 6.0 cP, 6.2 cP, 6.4cP, 6.6 cP, 6.8 cP, 7.0 cP, 7.2 cP, 7.4 cP, 7.6 cP, 7.8 cP, 8.0 cP, 8.2 cP, 8.4 cP, 8.6cP, 8.8 cP, 9.0 cP, 9.2 cP, 9.4 cP, 9.6 cP, 9.8 cP, 10.0 cP, 10.2 cP, 10.4 cP, 10.6 cP, 10.8 cP, 11.0 cP, 11.2 cP, 11.4 cP, 11.6 cP, 11.8 cP, 12.0 cP, 12.2 cP, 12.4 cP, 12.6cP, 12.8 cP, 13.0 cP, 13.2 cP, 13.4 cP, 13.6 cP, 13.8 cP, 14.0 cP, 14.2 cP, 14.4 cP,14.6 cP, 14.8 cP, or 15.0 cP at 25°C or the viscosity may be higher or lower.

[00157] In another example, a liquid antibody formulation may have any conductivity suitable for therapeutic efficacy, safety, and storage. For example, the conductivity of a liquid antibody formulation may be 0.1 millisiemens per centimeter (mS/cm) to 15 mS/cm, 0.1 mS/cm to 10 mS/cm, 0.1 mS/cm to 5 mS/cm, 0.1 mS/cm to 2 mS/cm, or 0.1 mS/cm to 1.5 mS/cm. In some embodiments, a liquid antibody formulation can have a conductivity of 0.19 mS/cm, 0.59 mS/cm, 1.09 mS/cm, 1.19 mS/cm, 1.29 mS/cm, 1.39 mS/cm, 1.49 mS/cm, 1.59 mS/cm, 1.69 mS/cm, 1.79 mS/cm, 1.89 mS/cm, 1.99 mS/cm, 2.09 mS/cm, 2.19 mS/cm, 2.29 mS/cm, 2.39 mS/cm, 2.49 mS/cm, 2.59 mS/cm, 2.69 mS/cm, 2.79 mS/cm, 2.89 mS/cm, 2.99 mS/cm, 3.09 mS/cm, 3.19 mS/cm, 3.29 mS/cm, 3.39 mS/cm, 3.49 mS/cm, 3.59 mS/cm, 3.69 mS/cm, 3.79 mS/cm, 3.89 mS/cm, 3.99 mS/cm, 4.09 mS/cm, 4.19 mS/cm, 4.29 mS/cm, 4.39 mS/cm, 4.49 mS/cm, 4.59 mS/cm, 4.69 mS/cm, 4.79 mS/cm, 4.89 mS/cm, 4.99 mS/cm, 5.09 mS/cm, 6.09 mS/cm, 6.59 mS/cm, 7.09 mS/cm, 7.59 mS/cm, 8.09 mS/cm, 8.59 mS/cm, 9.09 mS/cm, 9.59 mS/cm, 10.09 mS/cm, 10.59 mS/cm, 11.09 mS/cm, 11.59 mS/cm, 12.09 mS/cm, 12.59 mS/cm, 13.09 mS/cm, 13.59 mS/cm, 14.09 mS/cm, 14.59 mS/cm or 15.09 mS/cm or the conductivity may be higher or lower.

[00158] In another example, a liquid antibody formulation may have any osmolality suitable for therapeutic efficacy, safety, and storage. For example, the osmolality of a liquid antibody formulation may be from 50 milliosmoles per kilogram (mOsm/kg) to 5,000 mOsm/kg, 50 mOsm/kg to 2,000 mOsm/kg, 50 mOsm/kg to 1,000 mOsm/kg, 50 mOsm/kg to 750 mOsm/kg, or 50 mOsm/kg to 500 mOsm/kg. In some embodiments, a liquid antibody formulation can have an osmolality of 50 mOsm/kg, 60 mOsm/kg, 70 mOsm/kg, 80 mOsm/kg, 90 mOsm/kg, 100 mOsm/kg 120 mOsm/kg, 140 mOsm/kg, 160 mOsm/kg, 180 mOsm/kg, 200 mOsm/kg, 220 mOsm/kg, 240 mOsm/kg, 260 mOsm/kg, 280 mOsm/kg, 300 mOsm/kg, 320 mOsm/kg, 340 mOsm/kg, 360 mOsm/kg, 380 mOsm/kg, 400 mOsm/kg, 420 mOsm/kg, 440 mOsm/kg, 460 mOsm/kg, 480 mOsm/kg, 500 mOsm/kg, 520 mOsm/kg, 540 mOsm/kg, 560 mOsm/kg, 580 mOsm/kg, 600 mOsm/kg, 620 mOsm/kg, 640 mOsm/kg, 660 mOsm/kg, 680 mOsm/kg, 700 mOsm/kg, 720 mOsm/kg, 740 mOsm/kg, 760 mOsm/kg, 780 mOsm/kg, 800 mOsm/kg, 820 mOsm/kg, 840 mOsm/kg, 860 mOsm/kg, 880 mOsm/kg, 900 mOsm/kg, 920 mOsm/kg, 940 mOsm/kg, 960 mOsm/kg, 980 mOsm/kg, 1000 mOsm/kg, 1050 mOsm/kg, 1100 mOsm/kg, 1150 mOsm/kg, 1200 mOsm/kg, 1250 mOsm/kg, 1300 mOsm/kg, 1350 mOsm/kg, 1400 mOsm/kg, 1450 mOsm/kg, 1500 mOsm/kg, or the osmolality may be higher or lower.

[00159] Liposomes containing the antibody can be prepared by methods known in the art, such as described in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang, et al., Proc. Natl Acad. Sci. USA 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with increased circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with the desired diameter.

[00160] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[00161] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in which the matrices are in the form of shaped articles, e.g., films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol), polylactides (U.S. Patent 3,773,919), L-glutamic acid-7-ethyl-L-glutamate copolymers, nondegradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(-)-3-hydroxybutyric acid.

[00162] Formulations to be used for in vivo administration should generally be sterile. This is readily achieved, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle.

[00163] Compositions according to the present invention may be in unit dosage forms, such as tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories, for oral, parenteral or rectal administration, or administration by inhalation or insufflation. In some cases, a unit dosage form may be provided in a pre-filled container (e.g., a pre-filled syringe) useful in administering the dosage unit to a subject.

[00164] To prepare solid compositions, such as tablets, the main active ingredient may be mixed with a pharmaceutical carrier, for example, conventional tabletting ingredients such as cornstarch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, for example, water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When referring to such preformulation compositions as homogeneous, it is understood that the active ingredient is uniformly dispersed throughout the composition, so that the composition can be readily subdivided equally into effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from about 0.1 to about 500 mg of the active ingredient of the present invention. The tablets or pills of the novel composition may be coated or otherwise compounded to provide a dosage form providing the advantage of prolonged action. For example, the tablet or pill may comprise an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former. The two components may be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

[00165] Suitable surfactants include, in particular, non-ionic agents such as Tween (e.g. Tween™ 20, 40, 60, 80 or 85) and other sorbitans (e.g. Span™ 20, 40, 60, 80 or 85). Compositions with a surfactant will conveniently comprise between 0.05 and 5% surfactant and may be between 0.1 and 2.5%. It will be appreciated that other ingredients may be added, for example mannitol or other pharmaceutically acceptable carriers, if required.

[00166] Suitable emulsions may be prepared using commercially available fatty emulsions such as IntralipidTM, LiposynTM, InfonutrolTM, LipofundinTM and LipiphysanTM. The active ingredient may be either dissolved in a pre-mixed emulsion composition or, alternatively, may be dissolved in an oil (e.g. soya oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed by mixing with a phospholipid (e.g. egg phospholipids, soya phospholipids or soya lecithin) and water. It will be appreciated that other ingredients may be added, for example glycerol or glucose, to adjust the tonicity of the emulsion. Suitable emulsions will typically contain oil up to 20%, for example between 5 and 20%. The fat emulsion may comprise fat droplets between 0.1 and 1.0 lm, particularly 0.1 and 0.5 lm, and has a pH in the range of 5.5 to 8.0.

[00167] Emulsion compositions may be those prepared by mixing an antibody with IntralipidTM or its components (soybean oil, egg phospholipids, glycerol and water).

[00168] Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents, or mixtures thereof, and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as set forth above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents, preferably sterile, may be nebulized using gases. Nebulized solutions may be breathed directly from the nebulization device or the nebulization device may be connected to a face mask, tent or intermittent positive pressure breathing machine. Solution, suspension or powder compositions may also be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner.

[00169] In some embodiments, a formulation comprising an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be prepared by any suitable route of administration, with an amount of antibody ranging from 0.1 mg to 3000 mg, 1 mg to 1000 mg, 100 to 1000 mg, or 100 to 500 mg. In some cases, a formulation comprising an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can comprise an amount of antibody of no more than or at least 0.1 mg, 1 mg, 100 mg, 1 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg and 3000 mg.

[00170] In some embodiments, a liquid formulation comprising an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be prepared by any suitable route of administration, with an antibody concentration ranging from 0.1-500 mg/mL, 0.1-375 mg/mL, 0.1-250 mg/mL, 0.1-175 mg/mL, 0.1-100 mg/mL, 1 mg/mL-500 mg/mL, 1 mg/mL-375 mg/mL, 1 mg/mL-300 mg/mL, 1 mg/mL-250 mg/mL, 1 mg/mL-200 mg/mL, 1 mg/mL-150 mg/mL, 1 mg/mL-250 mg/mL, 1 mg/mL-375 mg/mL, 1 mg/mL-400 mg/mL, 1 mg/mL-5 ... mg/mL to 100 mg/mL, 10 mg/mL to 500 mg/mL, 10 mg/mL to 375 mg/mL, 10 mg/mL to 250 mg/mL, 10 mg/mL to 150 mg/mL, 10 mg/mL to 100 mg/mL, 100 mg/mL to 500 mg/mL, 100 mg/mL to 450 mg/mL, 100 mg/mL to 400 mg/mL, 100 mg/mL to 350 mg/mL, 100 mg/mL to 300 mg/mL, 100 mg/mL to 250 mg/mL, 100 mg/mL to 200 mg/mL or 100 mg/mL to 150 mg/mL. In some embodiments, a liquid formulation can comprise an antibody described herein, at a concentration of at most at least or less than 0.1, 0.5, 1, 5, 10, 15 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 mg/mL.

[00171] An antibody formulation may comprise one or more components, including the antibody and other species described elsewhere herein. The antibody and other components may be in any suitable amount and/or any suitable concentration for therapeutic efficacy of the antibody, safety, and storage. In one example, an antibody formulation may be a solution comprising 51.4 mg/mL antibody (e.g., G1 antibody, another antagonistic anti-CGRP antibody, a monoclonal antibody that modulates the CGRP pathway), 20 mM histidine, 0.1 mg/mL methionine, 84 mg/mL trehalose dihydrate, 0.05 mg/mL disodium EDTA dihydrate, and 0.2 mg/mL polysorbate 80.

[00172] In another example, an antibody formulation may comprise 200 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 15 mM arginine, 78 mg/mL sucrose, 0.3 mg/mL EDTA, and 0.1 mg/mL polysorbate 80.

[00173] In another example, an antibody formulation may comprise 175 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 20 mM glycine, 88 mg/mL trehalose dihydrate, 0.015 mg/mL EDTA, and 0.25 mg/mL polysorbate 80.

[00174] In another example, an antibody formulation may comprise 225 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 23 mM asparagine, 84 mg/mL sorbitol, 0.1 mg/mL EDTA, and 0.15 mg/mL polysorbate 60.

[00175] In another example, an antibody formulation may comprise 150 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 17 mM asparagine, 74 mg/mL mannitol, 0.025 mg/mL EDTA, and 0.2 mg/mL polysorbate 80.

[00176] In another example, an antibody formulation may comprise 100 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 16 mM arginine, 87 mg/mL mannitol, 0.025 mg/mL EDTA, and 0.15 mg/mL polysorbate 20.

[00177] In another example, an antibody formulation may comprise 250 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 25 mM histidine, 74 mg/mL mannitol, 0.025 mg/mL EDTA, and 0.25 mg/mL polysorbate 20.

[00178] In another example, an antibody formulation may comprise 50 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 19 mM arginine, 84 mg/mL sucrose, 0.05 mg/mL EDTA, and 0.3 mg/mL polysorbate 80.

[00179] In another example, an antibody formulation may comprise 125 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 22 mM glycine, 79 mg/mL trehalose dihydrate, 0.15 mg/mL EDTA, and 0.15 mg/mL polysorbate 80.

[00180] In another example, an antibody formulation can be a solution comprising 175 mg/mL antibody (e.g., G1 antibody, another antagonistic anti-CGRP antibody, a monoclonal antibody that modulates the CGRP pathway), 20 mM histidine, 0.1 mg/mL methionine, 84 mg/mL trehalose dihydrate, 0.05 mg/mL disodium EDTA dihydrate, and 0.2 mg/mL polysorbate 80.

[00181] In another example, an antibody formulation may comprise 200 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 30 mM arginine, 78 mg/mL sucrose, 0.3 mg/mL EDTA, and 0.1 mg/mL polysorbate 80.

[00182] In another example, an antibody formulation may comprise 175 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 20 mM glycine, 88 mg/mL trehalose dihydrate, 0.015 mg/mL EDTA, and 0.15 mg/mL polysorbate 80.

[00183] In another example, an antibody formulation may comprise 150 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 20 mM histidine, 84 mg/mL sucrose, 0.05 mg/mL EDTA, and 0.2 mg/mL polysorbate 80.

[00184] In another example, an antibody formulation may comprise 225 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 23 mM histidine, 84 mg/mL sorbitol, 0.1 mg/mL EDTA, and 0.15 mg/mL polysorbate 60.

[00185] In another example, an antibody formulation may comprise 150 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 17 mM asparagine, 74 mg/mL mannitol, 0.3 mg/mL EDTA, and 0.2 mg/mL polysorbate 80.

[00186] In another example, an antibody formulation may comprise 100 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 16 mM arginine, 87 mg/mL mannitol, 0.025 mg/mL EDTA, and 0.25 mg/mL polysorbate 20.

[00187] In another example, an antibody formulation may comprise 250 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 25 mM histidine, 89 mg/mL sorbitol, 0.025 mg/mL EDTA, and 0.25 mg/mL polysorbate 20.

[00188] In another example, an antibody formulation may comprise 125 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 29 mM arginine, 84 mg/mL sucrose, 0.05 mg/mL EDTA, and 0.3 mg/mL polysorbate 80.

[00189] In another example, an antibody formulation may comprise 150 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 25 mM asparagine, 84 mg/mL mannitol, 0.05 mg/mL EDTA, and 0.2 mg/mL polysorbate 80.

[00190] In another example, an antibody formulation may comprise 145 mg/mL antibody (e.g., G1 antibody, another anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway), 22 mM histidine, 72 mg/mL trehalose dihydrate, 0.05 mg/mL EDTA, and 0.1 mg/mL polysorbate 80.

[00191] An antibody described herein may be administered using any suitable method, including by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.). Antibodies may also be administered by inhalation, as described herein. In some cases, an antibody may be administered nasally with or without inhalation. Generally, for administration of an antibody described herein, an initial candidate dosage may be about 2 mg/kg. For purposes of the present invention, a typical daily dosage may range from about any one of 3μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to 100 mg/kg or more, depending on the factors mentioned above. For example, dosages of about 1 mg/kg, about 2.5 mg/kg, about 5 mg/kg, about 10 mg/kg, and about 25 mg/kg may be used. For repeated administrations over several days or longer, depending on the condition, treatment is continued until a desired suppression of symptoms occurs, or until sufficient therapeutic levels are achieved, for example, to reduce pain. An exemplary dosage regimen comprises administration of an initial dose of about 8.5 mg/kg, followed by a weekly maintenance dose of about 2.8 mg/kg of an antibody, or followed by a maintenance dose of about 2.8 mg/kg every other week. Another exemplary dosage regimen comprises administering a dose of 100 mg, 125 mg, 150 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 675 mg, or 900 mg to an individual once monthly subcutaneously. Another exemplary dosage regimen comprises administering an initial dose of 675 mg subcutaneously, followed by a monthly dose of 225 mg of the antibody subcutaneously. However, other dosage regimens may be useful, depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, in some embodiments, once-weekly and four-times-weekly doses are contemplated. The progress of this therapy is readily monitored by conventional techniques and assays. The dosage regimen (including the CGRP antagonist used) may vary over time.

[00192] In some embodiments, the dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein and administered to a subject can range from 0.1 Dg to 3000 mg, 1 mg to 1000 mg, 100 to 1000 mg, 100 to 500 mg, 0.1 mg to 5000 mg, 1 mg to 4000 mg, 250 mg to 1000 mg, 500 mg to 1000 mg, 100 mg to 900 mg, 400 mg to 900 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, or 200 mg to 3000 mg. mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg. In some embodiments, the dose or amount of an antibody described herein and administered to an individual can be, can be at most, can be less than, or can be at least 0.1DDg, 1Dg, 100Dg, 1 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg and 3000 mg. In some embodiments, the amount is between 100 - 2000 mg.

[00193] In some embodiments, the dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein and administered to a subject can range from 0.1 to 500, 0.1 to 100, 0.1 to 50, 0.1 to 20, 0.1 to 10, 1 to 10, 1 to 7, 1 to 5, or 0.1 to 3 mg/kg of body weight. In some embodiments, the dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein and administered to a subject can be, can be at most, can be less than, or can be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500 mg/kg body weight.

[00194] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein is administered to a subject may vary. In some embodiments, a single dose of antibody may be administered to a subject through therapy. In some embodiments, the frequency at which a dose or amount of an antibody is administered to a subject is constant (e.g., administered once per month). In some embodiments, the frequency at which a dose or amount of an antibody described herein is administered to a subject is variable (e.g., an initial dose followed by a dose at one month, followed by additional doses at three months and seven months). In some embodiments, the frequency at which an antibody is administered to a subject is, is at least, is less than, or is at most, one, two, three, four, five, or six times per day. In some embodiments, the frequency with which an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) is administered to a subject is, is at least, is less than, or is at most one, two, three, four, five, or six doses per day.

[00195] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein is administered to a subject is, is at least, is less than, or is at most, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty time(s) for each one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty- one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-five, sixty, sixty-five, seventy, seventy-five, eighty, eighty-five, ninety, ninety-five, one hundred, one hundred twenty-five, one hundred fifty, one hundred eighty, or two hundred days.

[00196] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein is administered to a subject is, is at least, is less than, or is at most, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty time(s) for every one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty- one, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-nine, forty, forty-one, forty-two, forty-three, forty-four, forty-five, forty-six, forty-seven, forty-eight, forty-nine, fifty, fifty-five, sixty, sixty-five, seventy, seventy-five, eighty, eighty-five, ninety, ninety-five, or one hundred weeks. In some embodiments, the frequency with which an antibody (e.g., antibody, monoclonal that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP monoclonal antagonist antibody) described herein is administered to a subject is less than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or eighty doses per week.

[00197] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, monoclonal anti-CGRP antagonist antibody) is administered to a subject is, is at least, is less than, or is at most, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty times per each month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, every twelve months, every thirteen months, every fourteen months, every fifteen months, every sixteen months, every seventeen months, or every 18 months. In some embodiments, the frequency with which an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP monoclonal antagonist antibody) described herein is administered to a subject is less than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, or more doses per month. In some embodiments, a dose or amount of an antibody can be administered (e.g., subcutaneously or intravenously) to a subject once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, or more per month.

[00198] In some embodiments, an antibody in a dose or amount of 50 mg, 100 mg to 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2050 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg, 2950 mg, 3000 mg, or more may be administered (e.g., subcutaneously or intravenously) to an individual once a month. In some embodiments, an antibody in a dose or amount of between 0.1 mg to 5000 mg, 1 mg to 4000 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg can be administered (e.g., subcutaneously or intravenously) to an individual once monthly. In some embodiments, between 100 - 2000 mg of antibody is administered once monthly.

[00199] In some embodiments, an antibody in a dose or amount of 50 mg, 100 mg to 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2050 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg, 2950 mg, 3000 mg, or more may be administered (e.g., subcutaneously or intravenously) to an individual once every three months. In some embodiments, an antibody in a dose or amount of between 0.1 mg to 5000 mg, 1 mg to 4000 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg may be administered (e.g., subcutaneously or intravenously) to an individual once every three months. In some embodiments, between 450 mg to 2000 mg is administered once every three months, or less.

[00200] In some embodiments, an antibody in a dose or amount of 50 mg, 100 mg to 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2050 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg, 2950 mg, 3000 mg, or more may be administered (e.g., subcutaneously or intravenously) to an individual once every six months. In some embodiments, an antibody in a dose or amount of between 0.1 mg to 5000 mg, 1 mg to 4000 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg may be administered (e.g., subcutaneously or intravenously) to an individual once every six months. In some embodiments, between 450 mg to 2000 mg is administered once every six months, or less.

[00201] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) is administered to an individual (e.g., subcutaneously or intravenously) is, is at least, is less than, or is at most one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty times per quarter. As can be appreciated, a "quarter" can refer to a period of one quarter or can also refer to a calendar quarter, such as a time period from January 1 - March 31, April 1 - June 30, July 1 - September 30, or October 1 - December 31. In some cases, a "quarter" may refer to a period of time of about three months.

[00202] In some embodiments, an antibody in a dose or amount of 50 mg, 100 mg to 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350 mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2050 mg, 2100 mg, 2150 mg, 2200 mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg, 2950 mg, 3000 mg, or more may be administered (e.g., subcutaneously or intravenously) to an individual once every quarter. In some embodiments, an antibody in a dose or amount of between 0.1 mg to 5000 mg, 1 mg to 4000 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg may be administered (e.g., subcutaneously or intravenously) to an individual once every quarter.

[00203] In some embodiments, the frequency at which a dose or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) is administered is, is at least, is less than, or is at most, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty times per year, every two years, every three years, every four years, or every five years. In some embodiments, the frequency with which an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) is administered to an individual is less than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five doses per year.

[00204] In some embodiments, an antibody in a dose or amount of 50 mg, 100 mg to 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 950 mg, 1000 mg, 1050 mg, 1100 mg, 1150 mg, 1200 mg, 1250 mg, 1300 mg, 1350mg, 1400 mg, 1450 mg, 1500 mg, 1550 mg, 1600 mg, 1650 mg, 1700 mg, 1750 mg, 1800 mg, 1850 mg, 1900 mg, 1950 mg, 2000 mg, 2050 mg, 2100 mg, 2150 mg, 2200mg, 2250 mg, 2300 mg, 2350 mg, 2400 mg, 2450 mg, 2500 mg, 2550 mg, 2600 mg, 2650 mg, 2700 mg, 2750 mg, 2800 mg, 2850 mg, 2900 mg, 2950 mg, 3000 mg, or more may be given to an individual once every year. In some embodiments, an antibody in a dose or amount of between 0.1 mg to 5000 mg, 1 mg to 4000 mg, 10 mg to 3000 mg, 10 mg to 2000 mg, 100 mg to 2000 mg, 150 mg to 2000 mg, 200 mg to 2000 mg, 250 mg to 2000 mg, 300 mg to 2000 mg, 350 mg to 2000 mg, 400 mg to 2000 mg, 450 mg to 2000 mg, 500 mg to 2000 mg, 550 mg to 2000 mg, 600 mg to 2000 mg, 650 mg to 2000 mg, 700 mg to 2000 mg, 750 mg to 2000 mg, 800 mg to 2000 mg, 850 mg to 2000 mg, 900 mg to 2000 mg, 950 mg to 2000 mg, or 1000 mg to 2000 mg may be administered (e.g., subcutaneously or intravenously) to an individual once every year. In some embodiments, between 450 mg to 2000 mg is administered once every year, or less.

[00205] In some embodiments, a method may comprise administering an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein to a subject on a plurality of days. Two, three, four, five, six, seven, eight or more days of the plurality of days may be more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more days apart. In some embodiments, two of the plurality of days are more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, or more days apart. Furthermore, in some embodiments, the amount of antibody administered on the first day of a plurality of days can be different (e.g., greater or less) than the amount of antibody administered on the second day.

[00206] In some embodiments, an initial dose (e.g., a loading dose) of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP monoclonal antagonist antibody) described herein can be administered to a subject, followed by administration of one or more additional doses, at desired intervals. In some embodiments, the initial dose and one or more of the additional doses are the same dose. In some embodiments, the one or more additional doses are a different dose than the initial dose. In some embodiments, the frequency at which the one or more additional doses are administered is constant (e.g., every month). In some embodiments, the frequency at which the one or more additional doses are administered is variable (e.g., an additional dose administered one month after the initial dose, followed by one more additional dose three months after the initial dose). Any desirable and/or therapeutic regimen of initial loading dose, additional doses, and frequency (e.g., including those described herein) of additional doses may be used. An exemplary regimen includes an initial loading dose of 675 mg of anti-CGRP antagonist antibody administered subcutaneously, followed by subsequent 225 mg maintenance doses of the antibody administered subcutaneously at one-month intervals.

[00207] In some embodiments, an initial dose of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP monoclonal antagonist antibody) of 0.1μg, 1μg, 100 μg, 1 mg, 10 mg, 25 mg, 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1500 mg, 2000 mg and 3000 mg can be administered to an individual, followed by one or more additional doses of the antibody 0.1μg, 1μg, 100μg, 1mg, 10mg, 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg , 300 mg, 325 mg, 350 mg, 375 mg, 400mg, 450 mg, 475 mg, 500 mg,525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1000 mg, 1500 mg, 2000 mg and 3000 mg.

[00208] In some embodiments, a dose or amount of antibody (e.g., antibody, monoclonal that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be divided into sub-doses and administered as multiple sub-doses, depending on, for example, the route of administration and/or the particular formulation administered. For example, in cases where the dose is administered subcutaneously, the subcutaneous dose can be divided into multiple sub-doses and each sub-dose administered at a different site in order to avoid, for example, a larger single subcutaneous injection at a single site. For example, a 900 mg subcutaneous dose can be divided into four sub-doses of 225 mg each and each 225 mg dose administered at a different site, which can help minimize the volume injected at each site. The division of sub-doses may be equal (e.g. 4 equal sub-doses) or it may be different (e.g. 4 sub-doses, two of the sub-doses twice as large as the other sub-doses).

[00209] In some embodiments, the number of doses of antibody administered to a subject over the course of treatment may vary depending on, for example, achieving a reduction in the incidence of a vasomotor symptom and/or headache in the subject. In some embodiments, the vasomotor symptom is associated with a form of headache (e.g., migraine, chronic migraine, episodic migraine, other type of headache, etc.). For example, the number of doses administered over the course of treatment may be, may be at least, or may be at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. In some cases (for example, where an individual has chronic migraine), Treatment may be given indefinitely. In some cases, treatment may be acute such that a maximum of 1, 2, 3, 4, 5, or 6 doses are administered to an individual for treatment.

[00210] In some embodiments, a dose (or sub-dose) or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be formulated into a liquid formulation and administered (e.g., by subcutaneous injection, via intravenous injection) to a subject. In such cases, the volume of antibody comprising the liquid formulation can vary depending on, for example, the concentration of antibody in the liquid formulation, the desired dose of the antibody, and/or the route of administration used. For example, the volume of liquid formulation comprising an antibody described herein and administered (e.g., via an injection, such as, for example, a subcutaneous injection or intravenous injection) to an individual can be from 0.001 mL to 10.0 mL, 0.01 mL to 5.0 mL, 0.1 mL to 5 mL, 0.1 mL to 3 mL, 0.5 mL to 2.5 mL, or 1 mL to 2.5 mL. For example, the volume of the liquid formulation comprising an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein and administered (e.g., via an injection, such as, for example, a subcutaneous injection, an intravenous injection) to a subject may be, may be at least, may be less than, or may be at most, 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mL.

[00211] In some embodiments, a dose (or sub-dose) or amount of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be provided in pre-filled containers useful in administering the antibody to a subject. Such pre-filled containers can be designed for self-administration or for administration by another. For example, a dose (or sub-dose) or amount of antibody described herein can be provided as a liquid formulation in pre-filled syringes. In such examples, the pre-filled syringes can be designed for self-administration or for administration by another. In some cases, the pre-filled syringes can be designed for subcutaneous and/or intravenous administration.

[00212] For purposes of the present invention, the appropriate dosage of an antibody may depend on the antibody (or compositions thereof) used, the type and severity of the vasomotor symptom, the type and severity of the headache (e.g., migraine) or other condition being treated, whether the agent is administered for preventive or therapeutic purposes, prior therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. Typically, the clinician will administer the antibody until a dosage that achieves the desired result is reached. The dose and/or frequency may vary throughout the course of treatment.

[00213] Empirical considerations, such as half-life, will generally contribute to determination of dosage. For example, antibodies that are compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to prolong the half-life of the antibody and to prevent the antibody from being attacked by the host immune system. The frequency of administration can be determined and adjusted during the course of therapy, and is generally, but not necessarily, based on the treatment and/or suppression and/or amelioration and/or delay of the headache (e.g., migraine) or other condition. Alternatively, sustained continuous release formulations of antibodies may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

[00214] In one embodiment, dosages for an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein can be determined empirically, in subjects administered one or more administrations of the antibody. Subjects are given incremental doses of an antibody. To assess the efficacy of an antibody, an indicator of disease can be followed.

[00215] Administration of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) in accordance with the methods of the present invention may be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those skilled in the art. Administration of an antibody may be substantially continuous over a preselected period of time or may be a series of spaced doses, for example, either before, during, or after the development of headache (e.g., migraine); before; during; before and after; during and after; before and during; or before, during, and after the development of headache. Administration may be before, during, and/or after any event that may give rise to headache.

[00216] In some embodiments, more than one antibody may be present. At least one, at least two, at least three, at least four, at least five different, or more antibodies may be present. Generally, antibodies may have complementary activities that do not adversely affect each other. An antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein may also be used in conjunction with other CGRP antagonists or CGRP receptor antagonists. For example, one or more of the following CGRP antagonists may be used: an antisense molecule directed to a CGRP (including an antisense molecule directed to a nucleic acid encoding CGRP), a CGRP inhibitory compound, a structural analog of CGRP, a dominant negative mutation of a CGRP receptor that binds to a CGRP, and an anti-CGRP receptor antibody. An antibody may also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

[00217] Diagnosis or assessment of headache is well established in the art. Assessment can be performed based on subjective measures, such as patient characterization of symptoms. For example, migraine can be diagnosed based on the following criteria: 1) episodic headache attacks lasting 4 to 72 hours; 2) having two of the following symptoms: unilateral, throbbing pain, worsening on movement, and pain of moderate or severe intensity; and 3) one of the following symptoms: nausea or vomiting, and photophobia or phonophobia. Goadsby et al., N. Engl. J. Med. 346:257-270, 2002. In some embodiments, assessment of headache (e.g., migraine) can be via headache time, as described elsewhere herein. For example, assessment of headache (e.g., migraine) may be in terms of daily headache hours, weekly headache hours, monthly headache hours, and/or annual headache hours. In some cases, headache hours may be as reported by the individual.

[00218] The efficacy of the treatment can be assessed by methods well known in the art. For example, pain relief can be assessed. Accordingly, in some embodiments, pain relief is subjectively observed after 1, 2, or a few hours after administration of an anti-CGRP antibody. In some embodiments, the frequency of headache attacks is subjectively observed after administration of an anti-CGRP antibody.

[00219] In some embodiments, a method for treating or reducing the incidence of headache in a subject as described herein can reduce the incidence of headache following a single administration of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein for an extended period of time. For example, the incidence of headache can be reduced for at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more days after a single administration.

[00220] In some embodiments, a method for treating or reducing the incidence of headache in a subject as described herein can reduce the number of hours of headache experienced by a subject from a pre-administration level following administration of one or more doses of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein to the subject. For example, hours of daily headache experienced by the individual following administration of one or more doses of an antibody to the individual may be reduced by 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours of headache from a pre-administration level in the individual. In some cases, daily headache hours experienced by the individual following administration of one or more doses of an antibody to the individual may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, relative to a pre-administration level in the individual. In another example, weekly headache hours in the subject following administration of one or more doses of an antibody to the subject may be reduced by 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more headache hours from a pre-administration level in the subject. In some cases, weekly headache hours experienced by the individual following administration of one or more doses of an antibody to the individual may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, relative to a pre-administration level in the individual. In another example, monthly headache hours in the subject following administration of one or more doses of an antibody to the subject may be reduced by 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125 or more headache hours from a pre-administration level. In some cases, weekly headache hours experienced by the individual following administration of one or more doses of an antibody to the individual may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, relative to a pre-administration level in the individual.

[00221] In some embodiments, a method for treating or reducing the incidence of headache in a subject as described herein can reduce the number of headache days experienced by a subject from a pre-administration level following administration of one or more doses of an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody) described herein to the subject. For example, weekly headache days in the subject following administration of one or more doses of an antibody to the subject can be reduced by 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7 headache days from a pre-administration level in the subject. In some cases, weekly headache days experienced by the individual following administration of one or more doses of an antibody to the individual may be reduced by 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, relative to a pre-administration level in the individual. In another example, monthly headache days in the subject following administration of one or more doses of an antibody to the subject may be reduced by 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more headache days from a pre-administration level.

[00222] In some embodiments, a method may comprise administering to a subject one or more additional agents simultaneously or sequentially with an antibody (e.g., monoclonal antibody that modulates the CGRP pathway, anti-CGRP antagonist antibody, anti-CGRP antagonist monoclonal antibody). In some embodiments, an additional agent may be an anti-headache medication, such as an example of anti-headache medications (e.g., 5-HT1 agonists, triptans, ergot alkaloids, opiates, β-adrenergic antagonists, NSAIDs) described elsewhere herein. In some embodiments, a therapeutic effect may be greater compared to the use of an antibody or one or more additional agents alone. Thus, a synergistic effect between an antibody and one or more additional agents may be achieved. In some embodiments, one or more agents may be taken by a subject prophylactically.

B. anti-CGRP antagonist antibodies

[00223] In some embodiments, the methods of the invention utilize an antibody, which may be an anti-CGRP antagonist antibody. An anti-CGRP antagonist antibody may refer to any antibody molecule that blocks, suppresses, or reduces (including significantly) the biological activity of CGRP, including downstream pathways mediated by CGRP signaling, such as binding to and/or triggering a cellular response to the CGRP receptor.

[00224] An anti-CGRP antagonist antibody can exhibit any one or more of the following characteristics: (a) bind to CGRP; (b) block CGRP from binding to its receptor(s); (c) block or decrease CGRP receptor activation (including activation of cAMP); (d) inhibit the biological activity of CGRP or downstream pathways mediated by CGRP signaling function; (e) prevent, ameliorate, or treat any aspect of headache (e.g., migraine); (f) increase CGRP clearance; and (g) inhibit (reduce), produce, or release CGRP synthesis. Anti-CGRP antagonist antibodies are known in the art. See, for example, Tan et al., Clin. Sci. (Lond). 89:565-73, 1995; Sigma (Missouri, US), product number C7113 (clone #4901); Plourde et al., Peptides 14:1225–1229, 1993.

[00225] In some embodiments, the antibody reacts with CGRP in a manner that inhibits CGRP, and/or the CGRP pathway, including downstream pathways mediated by CGRP signaling function. In some embodiments, the anti-CGRP antagonist antibody recognizes human CGRP. In some embodiments, the human anti-CGRP antagonist antibody binds to both α-CGRP and β-CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to both human and rat CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to the C-terminal fragment at amino acids 25-37 of CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to a C-terminal epitope at amino acids 25-37 of CGRP.

[00226] Antibodies useful in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (scFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., a domain antibody), humanized antibodies, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies may be murine, rat, human, or of any other origin (including chimeric or humanized antibodies).

[00227] In some embodiments, the anti-CGRP antibody is a monoclonal antibody. In some embodiments, the anti-CGRP antagonist antibody is humanized. In some embodiments, the antibody is human. In some embodiments, the anti-CGRP antagonist antibody is a G1 antibody (as described herein). In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDR(s) (such as one, two, three, four, five, or in some embodiments, all six CDRs) of the G1 antibody or the G1 variants shown in Table 6. In still other embodiments, the anti-CGRP antagonist antibody comprises the heavy chain variable region amino acid sequence shown in Figure 5 (SEQ ID NO: 1) and the light chain variable region amino acid sequence shown in Figure 5 (SEQ ID NO: 2).

[00228] In some embodiments, the antibody comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR) selected from the groups consisting of: (a) LCVR17 (SEQ ID NO: 58) and HCVR22 (SEQ ID NO: 59); (b) LCVR18 (SEQ ID NO: 60) and HCVR23 (SEQ ID NO: 61); (c) LCVR19 (SEQ ID NO: 62) and HCVR24 (SEQ ID NO: 63); (d) LCVR20 (SEQ ID NO: 64) and HCVR25 (SEQ ID NO: 65); (e) LCVR21 (SEQ ID NO: 66) and HCVR26 (SEQ ID NO: 67); (f) LCVR27 (SEQ ID NO: 68) and HCVR28 (SEQ ID NO: 69); (g) LCVR29 (SEQ ID NO: 70) and HCVR30 (SEQ ID NO: 71); (h) LCVR31 (SEQ ID NO: 72) and HCVR32 (SEQ ID NO: 73); (i) LCVR33 (SEQ ID NO: 74) and HCVR34 (SEQ ID NO: 75); (j) LCVR35 (SEQ ID NO: 76) and HCVR36 (SEQ ID NO: 77); and (k) LCVR37 (SEQ ID NO: 78) and HCVR38 (SEQ ID NO: 79). The sequences of these regions are provided herein. Other examples of antibodies are described in US20110305711, US20120294802, US20120294797 and US20100172895, which are incorporated herein by reference.

[00229] In some embodiments, the antibody comprises a modified constant region, such as a constant region that is immunologically inert described herein. In some embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In other embodiments, the antibody comprises a human IgG2 heavy chain constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence). Eur. J. Immunol. (1999) 29:2613-2624. In some embodiments, the antibody comprises an IgG4 constant region comprising the following mutations: E233F234L235 to P233V234A235. In still other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the oligosaccharide-binding residue (such as Asn297) and/or flanking residues that are part of the N-glycosylation recognition sequence in the constant region. In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation, enzymatically or by expression in a glycosylation-deficient host cell.

[00230] The binding affinity (K) of an anti-CGRP antibody for CGRP (such as human α-CGRP) can be from about 0.02 to about 200 nM. In some embodiments, the binding affinity is between approximately 200 nM, approximately 100 nM, about 50 nM, about 10 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 60 pM, approximately 50 pM, approximately 20 pM, about 15 pM, about 10 pM, about 5 pM, or about 2 pM. In some embodiments, the binding affinity is less than about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, or approximately 50 pM.

[00231] One way to determine the binding affinity of antibodies for CGRP is by measuring the binding affinity of monofunctional Fab fragments of the antibody. To obtain monofunctional Fab fragments, an antibody (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of an anti-CGRP Fab fragment of an antibody can be determined by surface plasmon resonance (Biacore3000™ surface plasmon resonance (SPR), BIAcore system, INC, Piscataway NJ), equipped with pre-immobilized streptavidin (SA) sensor chips using HBS-EP running buffer (0.01 M HEPES, pH 7.4, 0.15 NaCl, 3 mM EDTA, 0.005% v/v Surfactant P20). Biotinylated human CGRP (or any other CGRP) can be diluted in HBS-EP buffer to a concentration of less than 0.5 μg/mL and injected into individual chip channels using varying contact times to achieve two antigen density ranges, either 50–200 response units (RU) for detailed kinetic studies or 800–1,000 RU for screening assays. Regeneration studies have shown that 25 mM NaOH in 25% v/v ethanol effectively removes bound Fab, maintaining CGRP activity on the chip for over 200 injections. Typically, serial dilutions (spanning estimated K concentrations of 0.1–10x) of purified Fab samples were injected over 1 min at 100 μL/hr and dissociation times of up to two hours were allowed. Fab protein concentrations are determined by ELISA and/or SDS-PAGE electrophoresis using a Fab fragment of known concentration (as determined by amino acid analysis) as a standard. Kinetic association rates (kon) and dissociation rates (koff) were obtained simultaneously by fitting the overall data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using the BIAevaluation program. Equilibrium dissociation constant (KD) values are calculated as koff/kon. This protocol is suitable for use in determining the binding affinity of an antibody to any CGRP, including human CGRP, CGRP from other mammals (such as mouse CGRP, rat CGRP, primate CGRP), as well as different forms of CGRP (such as the α and β forms). The binding affinity of an antibody is usually measured at 25 °C, but can also be measured at 37 °C.

[00232] Antibodies, including anti-CGRP antagonist antibodies, may be made by any method known in the art. The route and schedule of immunization of the host animal are generally in accordance with established and conventional techniques for stimulation and production of antibodies, as further described herein. General techniques for production of human and mouse antibodies are known in the art and are described herein.

[00233] It is contemplated that any mammalian subject, including humans, or antibody-producing cells thereof may be engineered to serve as the basis for the production of mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and/or intradermally with an amount of immunogen, including as described herein.

[00234] Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al, In. In Vitro, 18:377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from the Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used in the hybridization. Generally, the technique involves the fusion of myeloma cells and lymphoid cells using a fusogen such as polyethylene glycol, or by electrical means well known to those skilled in the art. Following fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT), to eliminate unhybridized parental cells. Any of the media described herein, supplemented with or without serum, can be used to culture hybridomas secreting monoclonal antibodies. As another alternative to the cell fusion technique, EBV-immortalized B cells can be used to produce monoclonal antibodies (e.g., anti-CGRP monoclonal antibodies) of the present invention. The hybridomas are expanded and subcloned, if desired, and the supernatants are tested for anti-immunogenic activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[00235] Hybridomas that can be used as a source of antibodies encompass all derivatives, progeny cells of the parental hybridomas that produce monoclonal antibodies specific for CGRP, or a portion thereof.

[00236] Hybridomas producing such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from body fluids or culture media by conventional immunoglobulin purification procedures such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired. Undesirable activity, if present, can be removed, for example, by running the preparation over adsorbents made of the immunogen bound to a solid phase and eluting or releasing the desired antibodies away from the immunogen. Immunization of a host animal with a human CGRP, or a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoylsulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups, can produce a population of antibodies (e.g., monoclonal antibodies).

[00237] If desired, an antibody (e.g., anti-CGRP antagonist monoclonal or polyclonal antibody) of interest can be sequenced and the polynucleotide sequence can then be cloned into an expression or propagation vector. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to "humanize" the antibody or to improve the affinity, or other characteristics of the antibody. For example, the constant region can be engineered to more closely resemble human constant regions to avoid immune response if the antibody is used in clinical trials and treatments in humans. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity for CGRP and greater efficacy in inhibiting CGRP. It will be apparent to one skilled in the art that one or more polynucleotide alterations can be made to the anti-CGRP antagonist antibody and still maintain its CGRP binding ability.

[00238] Humanizing a monoclonal antibody can comprise four general steps. These are: (1) determining the nucleotide sequences and predicted amino acid sequence of the starting antibody's light and heavy chain variable domains; (2) designing the humanized antibody, i.e., deciding which antibody framework region to use during the humanization process; (3) actual humanization methodologies/techniques; and (4) transfection and expression of the humanized antibody. See, for example, U.S. Patent Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

[00239] A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having modified rodent and rodent V regions and their associated complementarity determining regions (CDRs) fused to human constant domains. See, e.g., Winter et al. Nature 349:293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA 86:4220-4224 (1989), Shaw et al. J Immunol. 138:4534-4538 (1987), and Brown et al. Cancer Res. 47:3577-3583 (1987). Other references describe rodent CDRs grafted onto a human framework (FR) region prior to fusion with an appropriate human antibody constant domain. See, e.g., Riechmann et al. Nature 332:323-327 (1988), Verhoeyen et al. Science 239:1534-1536 (1988), and Jones et al. Nature 321:522-525 (1986). Another reference describes rodent CDRs supported by recombinant rodent foliar framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize the undesirable immune response to rodent anti-human antibody molecules, which limits the duration and efficacy of therapeutic applications of such moieties in human recipients. For example, the antibody constant region can be engineered so that it is immunologically inert (e.g., does not cause complement lysis). See, e.g., PCT Publication No. PCT/GB99/01441; UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that can also be used are described by Daugherty et al., Nucl. Acids Res. 19:2471-2476 (1991) and in U.S. Patent Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01/27160.

[00240] In yet another alternative, fully human antibodies can be obtained using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals that are engineered to produce a more desirable (e.g., fully human antibodies), or more robust immune response, can also be used for the generation of humanized or human antibodies. Examples of such technology are Xenomouse™ from Abgenix, Inc. (Fremont, CA) and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ).

[00241] In one alternative, antibodies may be made recombinantly and expressed using any method known in the art. In another alternative, antibodies may be made recombinantly by phage display technology. See, e.g., U.S. Patent Nos. 5,565, 332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., Annu. Rev. Immunol. 12:433-455 (1994). Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) may be used to produce human antibodies and antibody fragments in vitro from variable immunoglobulin (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V-domain genes are cloned in-frame into a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody that exhibits those properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of formats; for reviews see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed, and antibodies to a diverse array of antigens (including self antigens) can be isolated essentially following the techniques described by Mark et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). In a natural immune response, antibody genes accumulate mutations at a high rate (somatic hypermutation). Some of the introduced changes will confer higher affinity, and B cells displaying high-affinity surface immunoglobulin are preferentially replicated and differentiated during subsequent antigen challenge. This natural process can be mimicked by employing a technique known as "chain shuffling." Marks, et al., Bio/Technol. 10:779-783 (1992). In this method, the affinity of "primary" human antibodies obtained by phage display can be improved by sequentially replacing the heavy and light chain V region genes with naturally occurring variant repertoires (repertoires) of V domain genes obtained from unimmunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for preparing very large phage antibody repertoires (also known as "the master of all libraries") has been described by Waterhouse et al., Nucl. Acids Res. 21:2265-2266 (1993). Genetic shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibody has similar affinities and specificities to the starting rodent antibody. According to this method, which is also referred to as "epitope imprinting", the heavy or light chain V domain gene of rodent antibodies obtained by phage display is replaced with a repertoire of human V domain genes, creating rodent-human chimeras. Antigen selection results in the isolation of human variable regions capable of restoring a functional antigen-binding site, i.e., the epitope governs (imprints) the choice of partner. When the process is repeated to replace the remaining rodent V domain, a human antibody is obtained (see PCT Publication No. WO 93/06213, published April 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that have no rodent-derived framework or CDR residues.

[00242] It is apparent that although the above discussion refers to humanized antibodies, the general principles discussed are applicable to the customization of antibodies for use in, for example, dogs, cats, primates, cattle, and horses. It is further apparent that one or more aspects of humanization of an antibody described herein may be combined, for example, CDR grafting, framework mutation, and CDR mutation.

[00243] Antibodies can be made recombinantly by first isolating antibodies and antibody-producing cells from host animals, obtaining the gene sequence, and using the gene sequence to express the antibody recombinantly in host cells (e.g., CHO cells). Another method that can be employed is to express the antibody sequence in plants (e.g., tobacco) or transgenic milk. Methods for expressing antibodies recombinantly in plants or milk have been disclosed. See, for example, Peeters, et al. Vaccine 19:2756 (2001); Lonberg, N. and D. Huszar Int.Rev.Immunol 13:65 (1995); and Pollock, et al., J Immunol Methods 231:147(1999). Methods for preparing antibody derivatives, e.g., humanized, single-chain, etc., are known in the art.

[00244] Immunoassays and flow cytometric sorting techniques, such as fluorescence activated cell sorting (FACS), can also be used to isolate antibodies that are specific for CGRP.

[00245] Antibodies can be bound to many different carriers. Carriers can be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylases, glass, natural and modified celluloses, polyacrylamides, agaroses, and magnetite. The nature of the carrier can be soluble or insoluble. Those skilled in the art will be aware of other suitable carriers for binding antibodies, or will be able to determine them using routine experimentation. In some embodiments, the carrier comprises a myocardial-targeting moiety.

[00246] DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of monoclonal antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into expression vectors (such as the expression vectors described in PCT Publication No. WO 87/04462), which are then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that otherwise do not produce immunoglobulin protein, to achieve synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT Publication No. WO 87/04462. The DNA may also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al., Proc. Nat. Acad. Sci. 81:6851 (1984), or by covalently joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of an anti-CGRP monoclonal antibody herein.

[00247] Antibodies (antagonist antibodies, e.g., anti-CGRP) and antibody-derived polypeptides can be identified or characterized using methods known in the art, wherein the reduction, enhancement, or neutralization of a biological activity of CGRP is detected and/or measured. For example, the antagonist anti-CGRP antibody can also be identified by incubating a candidate agent with CGRP and monitoring for any one or more of the following characteristics: (a) bind to CGRP; (b) block CGRP from binding to its receptor(s); (c) block or decrease CGRP receptor activation (including activation of cAMP); (d) inhibit the biological activity of CGRP or downstream pathways mediated by CGRP signaling function; (e) prevent, ameliorate, or treat any aspect of headache (e.g., migraine); (f) increase CGRP clearance; and (g) inhibit (reduce), produce, or release the synthesis of CGRP. In some embodiments, an anti-CGRP antagonist antibody or polypeptide is identified by incubating a candidate agent with CGRP and monitoring binding and/or the reduction and/or neutralization of a biological activity of CGRP. The binding assay can be performed with purified CGRP polypeptide(s), or with cells naturally expressing, or transfected to express, CGRP polypeptide(s). In one embodiment, the binding assay is a competitive binding assay, wherein the ability of a candidate antibody to compete with a known anti-CGRP antagonist antibody for CGRP binding is assessed. The assay can be performed in various formats, including ELISA format. In other embodiments, an anti-CGRP antagonist antibody is identified by incubating a candidate agent with CGRP and monitoring inhibition of binding and activation of the CGRP helper receptor expressed on the surface of a cell.

[00248] After initial identification, the activity of a candidate antibody (e.g., anti-CGRP antagonist antibody) can be further confirmed and refined by known bioassays to test for targeted biological activities. Alternatively, bioassays can be used to screen candidates directly. For example, CGRP promotes a number of measurable changes in responding cells. These include, but are not limited to, stimulation of cAMP in the cell (e.g., SK-N-MC cells). Antagonist activity can also be measured using animal models, such as measuring cutaneous vasodilation induced by mouse saphenous nerve stimulation. Escott et al., Br. J. Pharmacol. 110:772-776, 1993. Animal models of headache (such as migraine) can further be used to test the efficacy of antagonist antibodies or polypeptides. Reuter, et al., Functional Neurology (15) Suppl.3, 2000. Some of the methods for identification and characterization of anti-CGRP antagonist antibody or polypeptide are described in detail in the Examples.

[00249] Antibodies, including anti-CGRP antagonist antibodies, can be characterized using methods well known in the art. For example, one method is to identify the epitope to which it binds, or "epitope mapping." There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving the crystal structure of an antibody-antigen complex, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In a further example, epitope mapping can be used to determine the sequence to which an anti-CGRP antibody binds the antagonist antibody. Epitope mapping is commercially available from several sources, e.g., PEPSCAN systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands). The epitope may be a linear epitope, i.e., contained in a single stretch of amino acids, or a conformational epitope formed by a three-dimensional interaction of amino acids that cannot necessarily be contained in a single stretch. Peptides of varying lengths (e.g., at least 4–6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used for binding assays with an anti-CGRP antagonist antibody. In another example, the epitope to which the anti-CGRP antagonist antibody binds can be determined in a systematic screen using overlapping peptides derived from the CGRP sequence and determination of binding by the anti-CGRP antagonist antibody. In gene fragment expression assays, the open reading frame encoding CGRP is fragmented randomly or by specific genetic constructs and the reactivity of the expressed CGRP fragments with the antibody to be tested is determined. Gene fragments can, for example, be produced by PCR and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. Antibody binding to the radiolabeled CGRP fragments is then determined by immunoprecipitation and gel electrophoresis. Certain epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined library of overlapping peptide fragments can be tested for binding to the test antibody in single-binding assays. In a further example, domain mutagenesis of an antigen, domain alanine permutation experiments and binding scanning mutagenesis can be performed to identify residues necessary, sufficient, and/or required for epitope binding. For example, domain permutation experiments can be performed using a mutant CGRP in which various fragments of the CGRP polypeptide have been replaced (swapped) with sequences from a closely related but antigenically distinct protein (e.g., another member of the neurotrophin family of proteins). By assessing antibody binding to the mutant CGRP, the importance of the particular CGRP fragment for antibody binding can be assessed.

[00250] Yet another method that can be used to characterize an antibody, including an anti-CGRP antagonist antibody, is to use competition assays with other antibodies known to bind to the same antigen, i.e., various fragments of CGRP, to determine whether the anti-CGRP antagonist antibody binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.

[00251] An expression vector can be used to direct expression of an antibody, including an anti-CGRP antagonist antibody. One of skill in the art is familiar with the administration of expression vectors to achieve expression of an exogenous protein in vivo. See, for example, U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes local or systemic administration, including by injection, oral administration, particle gun or characterized administration, and topical administration. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglion, or into a coronary artery, atrium, ventricle, or pericardium.

[00252] Targeted delivery of therapeutic compositions containing an expression vector, or subgenomic polynucleotides may also be used. Receptor-mediated DNA delivery techniques are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol. Concentration ranges of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or nonviral origin (see generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous or heterologous mammalian promoters. Expression of the coding sequence may be constitutive or regulated.

[00253] Virus-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2200651; and EP Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-1246) (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA linked to killed adenoviruses as described in Curiel, Hum. Gene Ther. (1992) 3:147 may also be employed.

[00254] Non-viral delivery vehicles and methods may also be used, including but not limited to polycationic condensed DNA linked or unlinked to killed adenoviruses alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); linker-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicles (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO 97/42338), and charge-neutralizing or nucleic acid fusion with cell membranes. Naked DNA may also be employed. Exemplary naked DNA introduction methods are described in PCT Publication No. WO 90/11092 and US Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in US Patent No. 5,422,120; PCT Publication Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0,524,968. Additional approaches are described in Philip, Mol. Biol Cell. (1994) 14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

C. G1 antibody and related antibodies, polypeptides, polynucleotides, vectors and host cells

[00255] This invention encompasses compositions, including pharmaceutical compositions, comprising the G1 antibody and its variants shown in Table 6 or polypeptide derived from the G1 antibody and its variants shown in Table 6; and polynucleotides comprising sequences encoding G1 and its variants or the polypeptide. In some embodiments, the compositions comprise one or more antibodies or polypeptides (which may or may not be an antibody) that bind to CGRP, and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to CGRP. These compositions can further comprise suitable excipients, such as pharmaceutically acceptable excipients, including buffers, which are well known in the art.

[00256] In some embodiments, the anti-CGRP antagonist antibody, and polypeptides of the invention are characterized by any (one or more) of the following characteristics: (a) bind to CGRP; (b) block CGRP from binding to its receptor(s); (c) block or decrease CGRP receptor activation (including activation of cAMP); (d) inhibit the biological activity of CGRP or downstream pathways mediated by CGRP signaling function; (e) prevent, ameliorate, or treat any aspect of headache (e.g., migraine); (f) increase CGRP clearance; and (g) inhibit (reduce), produce, or release CGRP synthesis.

[00257] In some embodiments, the invention provides any of the following, or compositions (including pharmaceutical compositions) comprising any of the following: (a) the G1 antibody or variants thereof shown in Table 6; (b) a fragment or a region of the G1 antibody or variants thereof shown in Table 6; (c) a light chain of the G1 antibody or variants thereof shown in Table 6; (d) a heavy chain of the G1 antibody or variants thereof shown in Table 6; (e) one or more of the variable region(s) from a light chain and/or a heavy chain of the G1 antibody or variants thereof shown in Table 6; (f) one or more CDR(s) (one, two, three, four, five, or six CDRs) of the G1 antibody or variants thereof shown in Table 6; (g) CDR H3 of the heavy chain of the G1 antibody; (h) CDR L3 of the light chain of the G1 antibody or its variants shown in Table 6; (i) three CDRs of the light chain of the G1 antibody or its variants shown in Table 6; (j) three CDRs of the heavy chain of the G1 antibody or its variants shown in Table 6; (k) three CDRs of the light chain and three CDRs of the heavy chain of the G1 antibody or its variants shown in Table 6; and (l) an antibody comprising any one of (b) through (k). In some embodiments, the invention also provides polypeptides comprising any one or more of the above.

[00258] The CDR portions of the G1 antibody (including Chothia and Kabat CDRs) are depicted schematically in Figure 5. Determination of the CDR regions is well within the skill of the art. It is understood that in some embodiments, the CDRs may be a combination of Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In some embodiments, the CDRs are the Kabat CDRs. In other embodiments, the CDRs are the Chothia CDRs. In other words, in embodiments with more than one CDR, the CDRs may be any of the Kabat, Chothia, combination CDRs, or combinations thereof.

[00259] In some embodiments, the invention provides a polypeptide (which may or may not be an antibody) that comprises at least one CDR, at least two, at least three, or at least four, at least five, or all six CDRs that are substantially identical to at least one CDR, at least two, at least three, at least four, at least five, or all six CDRs of G1 or variants thereof set forth in Table 6. Other embodiments include antibodies that have at least two, three, four, five, or six CDR(s) that are substantially identical to at least two, three, four, five, or six CDRs of G1 or G1 derivatives. In some embodiments, at least one, two, three, four, five, or six CDR(s) are at least about 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, or 99% identical to at least one, two, three, four, five, or six CDRs of G1 or the variants thereof shown in Table 6. It is understood that for purposes of this invention, binding specificity and/or overall activity is generally maintained, although the degree of activity may vary relative to G1 or the variants thereof shown in Table 6 (may be greater or lesser).

[00260] In some embodiments, the invention also provides a polypeptide (which may or may not be an antibody) comprising an amino acid sequence of G1 or its variants set forth in Table 6 that has any of the following: at least 5 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of a sequence of G1 or its variants set forth in Table 6, wherein at least 3 of the amino acids are from a variable region of G1 (Figure 5) or its variants set forth in Table 6. In one embodiment, the variable region is from a light chain of G1. In another embodiment, the variable region is from a heavy chain of G1. An exemplary polypeptide has contiguous amino acids (lengths described above) from both the heavy and light chain variable regions of G1. In another embodiment, the 5 (or more) contiguous amino acids are from a complementarity determining region (CDR) of G1 shown in Figure 5. In some embodiments, the contiguous amino acids are from a variable region of G1.

[00261] The binding affinity (K) of an anti-CGRP antibody and CGRP antagonist for polypeptide (such as human α-CGRP) can be from about 0.06 to about 200 nM. In some embodiments, the binding affinity is between approximately 200 nM, 100 nM, approximately 50 nM, approximately 10 nM, approximately 1 nM, approximately 500 pM, approximately 100 pM, approximately 60 pM, approximately 50 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, or approximately 2 pM. In some embodiments, the binding affinity is less than about 250 nM, about 200 nM, about 100 nM, about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, or about 50 pM.

[00262] In some embodiments, the invention also provides methods of producing any of these antibodies and polypeptides. The antibodies of the present invention can be made by processes known in the art. The polypeptides can be produced by proteolytic or other degradation of the antibodies, by recombinant methods (i.e., individual or fusion polypeptides) as described above, or by chemical synthesis. Antibody polypeptides, especially shorter polypeptides of up to about 50 amino acids, are conveniently made by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, an antibody can be produced by an automated polypeptide synthesizer using the solid phase method. See also, U.S. Patent Nos. 5,807,715; 4,816,567; and 6,331,415.

[00263] In another alternative, the antibodies can be made recombinantly, using procedures that are well known in the art. In one embodiment, a polynucleotide comprising a sequence encoding the heavy chain and/or light chain of G1 antibody variable regions set forth in SEQ ID NO: 9 and SEQ ID NO: 10. In another embodiment, the polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 9 and SEQ ID NO: 10 are cloned into one or more vectors for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector in a host cell and the host cell can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

[00264] In some embodiments, the invention also encompasses single chain variable region fragments ("scFv") of antibodies of this invention, such as G1. Single chain variable region fragments are made by linking light and heavy chain variable regions and/or through the use of a short linker peptide. Bird et al. (1988) Science 242: 423-426. An example of a linker peptide is (GGGGS)3 (SEQ ID NO: 57) which links approximately 3.5 nm between the carboxyl terminus of one variable region and the amino terminus of the other variable region. Linkers of other sequences have been designed and used. Bird et al. (1988). The linkers may in turn be modified for additional functions, such as drug binding or binding to solid supports. Single chain variants may be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a plasmid containing polynucleotide encoding the appropriate scFv can be introduced into a suitable host cell, whether eukaryotic, such as yeast, plant, insect or mammalian cells, or prokaryotic, such as E. coli. The polynucleotides encoding the scFv of interest can be made by routine manipulations, such as polynucleotide ligation. The resulting scFv can be isolated using standard protein purification techniques known in the art.

[00265] Other forms of single chain antibodies, such as diabodies, are also encompassed. Diabodies are a form of bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with complementary domains from another chain and creating two antigen-binding sites (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2:1121-1123).

[00266] For example, bispecific antibodies, monoclonal antibodies that have binding specificities for at least two different antigens, can be prepared using the antibodies described herein. Methods for preparing bispecific antibodies are known in the art (see, e.g., Suresh et al, 1986, Methods in Enzymology. 121: 210). Traditionally, recombinant production of bispecific antibodies has been based on the coexpression of two pairs of immunoglobulin heavy and light chains, with the two heavy chains having different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

[00267] According to one approach to making bispecific antibodies, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2 and CH3 regions. It is preferred to have the first heavy chain constant region (CH1) that contains the site necessary for light chain binding, present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimal yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains into an expression vector when expression of at least two polypeptide chains in equal proportions results in high yields or when the proportions have no particular significance.

[00268] In one approach, bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. This asymmetric structure, with an immunoglobulin light chain on only one half of the bispecific molecule, facilitates separation of the desired bispecific compound from the undesired immunoglobulin chain combinations. This approach is described in PCT Publication No. WO 94/04690, published March 3, 1994.

[00269] Heteroconjugate antibodies, which comprise two covalently linked antibodies, are also within the scope of the invention. Such antibodies have been used to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for treatment of HIV infection (PCT Application Publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies may be produced using any convenient cross-linking methods. Suitable cross-linking agents and techniques are well known in the art, and are described in U.S. Patent No. 4,676,980.

[00270] Chimeric or hybrid antibodies can also be prepared in vitro using known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by the formation of a thioether bond. Iminothiolate and methyl-4-mercaptobutyrimidate are examples of suitable reagents for this purpose.

[00271] The humanized antibody comprising one or more CDRs from the G1 antibody or its variants shown in Table 6, or one or more CDRs derived from the G1 antibody or its variants shown in Table 6 can be prepared using any methods known in the art. For example, four general steps can be used to humanize a monoclonal antibody.

[00272] In some embodiments, the invention encompasses modifications to the G1 antibody or its variants shown in Table 6, including functionally equivalent antibodies that do not significantly affect their properties and variants that have improved or reduced activity and/or affinity. For example, the amino acid sequence of the G1 antibody or its variants shown in Table 6, can be mutated to obtain an antibody with the desired binding affinity for CGRP. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Modification of polypeptides is exemplified in the Examples. Examples of modified polypeptides include polypeptides with conservative substitutions of amino acid residues, one or more deletions or additions of amino acids that do not significantly detrimentally alter functional activity, or the use of chemical analogs.

[00273] Amino acid sequence insertions include amino- and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or the antibody fused to an epitope tag. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody of an enzyme or a polypeptide that increases the serum half-life of the antibody.

[00274] Substitutional variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted in its place. Sites of greatest interest for substitutional mutagenesis include the hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in Table 1 under the heading "conservative substitutions." If these substitutions result in a change in biological activity, then more substantial changes, termed "exemplary substitutions" in Table 1, or as further described above in reference to amino acid classes, can be introduced and the products screened.

[00275] Table 1: Amino Acid Substitutions

[00276] Substantial modifications in the biological properties of the antibody are achieved by selective substitutions that differ significantly in their effects on maintaining (a) the structure of the polypeptide backbone at the site of substitution, e.g., as a helical or sheet conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the total side chain. Naturally occurring residues are divided into groups based on common side chain properties:

[00277] (1) Nonpolar: Norleucine, Met, Ala, Val, Leu, Ile;

[00278] (2) Uncharged polar: Cys, Ser, Thr, Asn, Gln;

[00279] (3) Acids (negatively charged): Asp, Glu;

[00280] (4) Basic (positively charged): Lys, Arg;

[00281] (5) Residues influencing chain orientation: Gly, Pro; and

[00282] (6) Aromatics: Trp, Tyr, Phe, His.

[00283] Non-conservative substitutions are made to exchange a member of one of these classes for another class.

[00284] Any cysteine residue not involved in maintaining the proper conformation of the antibody may also be replaced, usually with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) may be added to the antibody to improve its stability particularly when the antibody is an antibody fragment such as an Fv fragment.

[00285] Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as the variable region. Changes to the variable region can alter binding affinity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made within a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made within a CDR domain. In still other embodiments, the CDR domain is CDR H3 and/or CDR L3.

[00286] Modifications include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as, for example, glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at conservative positions in their constant regions (Jefferis and Lund, 1997, Chem. Immunol. 65:111-128; Wright and Morrison, 1997, TibTECH 15:26-32). The oligosaccharide side chains of immunoglobulins affect protein function (Boyd et al., 1996, Mol. Immunol. 32:1311-1318; Wittwe and Howard, 1990, Biochem. 29:4175-4180) and the intramolecular interaction between glycoprotein moieties, which can affect the conformation and three-dimensional surface presentation of the glycoprotein (Hefferis and Lund, supra; Wyss and Wagner, 1996, Current Opin. Biotech. 7:409-416). Oligosaccharides may also serve to target a given glycoprotein to certain molecules based on specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β(1,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing the formation of bisecting GlcNAc, were reported to have enhanced ADCC activity (Umana et al., 1999, Mature Biotech. 17:176-180).

[00287] Antibody glycosylation is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[00288] Addition of glycosylation sites to the antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration may also be by the addition of, or substitution of, one or more serine or threonine residues to the original antibody sequence (for O-linked glycosylation sites).

[00289] The glycosylation pattern of antibodies can also be altered without altering the underlying nucleotide sequence. Glycosylation depends to a large extent on the host cell used to express the antibody. Since the cell type used for expression of recombinant glycoproteins, e.g. antibodies as potential therapeutic agents, is rarely the native cell, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al., 1997, J. Biol. Chem. 272: 9062-9070).

[00290] In addition to the choice of host cells, factors that affect glycosylation during recombinant antibody production include growth mode, media formulation, culture density, oxygenation, pH, purification schemes, and the like. Several methods have been proposed to alter the glycosylation pattern achieved in a given host organism, including introducing or overexpressing certain enzymes involved in oligosaccharide production (U.S. Patent Nos. 5,047,335; 5,510,261; and 5,278,299). Glycosylation, or certain types of glycosylation, can be enzymatically removed from the glycoprotein, for example using endoglycosidase H (Endo H), N-glycosidase F, endoglycosidase F1, endoglycosidase F2, endoglycosidase F3. In addition, the recombinant host cell can be genetically modified to be deficient in the processing of certain types of polysaccharides. These and other techniques are well known in the art.

[00291] Other methods of modification include the use of coupling techniques known in the art, including, but not limited to, enzymatic means, oxidative substitution, and chelation. The modifications can be used, for example, for attachment of labels for immunoassay. Modified G1 polypeptides can be made using procedures established in the art and can be screened using standard assays known in the art, some of which are described below and in the Examples.

[00292] In some embodiments of the invention, the antibody comprises a modified constant region, such as a constant region that is immunologically inert or partially inert, e.g., does not trigger complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia; or has reduced activities (compared to the unmodified antibody) in any one or more of the following: triggering complement-mediated lysis, stimulating antibody-dependent cell-mediated cytotoxicity (ADCC), or activating microglia. Different modifications of the constant region can be used to achieve the optimal level and/or combination of effector functions. See, e.g., Morgan et al., Immunology 86:319-324 (1995); Lund et al., J. Immunology 157:4963-9 157:4963-4969 (1996); Idusogie et al., J. Immunology 164:4178-4184 (2000); Tao et al., J. Immunology 143:2595-2601 (1989); and Jefferis et al., Immunological Reviews 163:59-76 (1998). In some embodiments, the constant region is modified as described in Eur. J. Immunol. (1999) 29:2613-2624; PCT Application No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In other embodiments, the antibody comprises a human IgG heavy chain constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence). Eur. J. Immunol. (1999) 29:2613-2624. In still other embodiments, the constant region is aglycosylated for N-linked glycosylation. In some embodiments, the constant region is aglycosylated for N-linked glycosylation by mutating the glycosylated amino acid residue or flanking residues that are part of the N-glycosylation recognition sequence of the constant region. For example, the N-glycosylation site of N297 can be mutated to A, Q, K, or H. See, Tao et al., J. Immunology 143:2595-2601 (1989); and Jefferis et al., Immunological Reviews 163:59-76 (1998). In some embodiments, the constant region is aglycosylated for N-linked glycosylation. The constant region can be aglycosylated for N-linked glycosylation enzymatically (such as carbohydrate removal by the enzyme PNGase), or by expression in a glycosylation-deficient host cell.

[00293] Other antibody modifications include antibodies that have been modified as described in PCT Publication No. WO 99/58572, published November 18, 1999. These antibodies comprise, in addition to a binding domain directed toward the target molecule, an effector domain having an amino acid sequence homologous to substantially all or part of a constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding to the target molecule without triggering significant complement-dependent lysis or cell-mediated killing of the target. In some embodiments, the effector domain is capable of specifically binding to FcRn and/or FcYRIIb. These are typically based on chimeric domains derived from two or more human immunoglobulin heavy chain CH2 domains. Antibodies modified in this manner are particularly suitable for use in chronic antibody therapy in order to avoid inflammatory and other adverse reactions to conventional antibody therapy.

[00294] In some embodiments, the invention includes affinity-matured embodiments. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio/Technology, 10:779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al., 1995, Gene, 169:147-155; Yelton et al., 1995, J. Immunol., 155:1994-2004; Jackson et al., 1995, J. Immunol., 154(7):3310-9; Hawkins et al, 1992, J. Mol. Biol., 226:889-896; and WO2004/058184).

[00295] The following methods can be used to adjust the affinity of an antibody and to characterize a CDR. One way to characterize a CDR of an antibody and/or alter (such as improve) the binding affinity of a polypeptide, such as an antibody, is called "library scanning mutagenesis." Generally, library scanning mutagenesis works as follows. One or more amino acid positions of the CDR are replaced with two or more (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids, using art recognized methods. This generates small libraries of clones (in some embodiments, one for each amino acid position that is analyzed), each with a complexity of two or more members (if two or more amino acids are substituted at each position). Generally, the library also includes a clone comprising the native (unsubstituted) amino acid. A small number of clones, e.g., about 20-80 clones (depending on the complexity of the library), from each library are screened for binding affinity for the target polypeptide (or other binding target), and candidates with increased, the same, decreased, or no binding are identified. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore surface plasmon resonance analysis, which detects differences in binding affinity of about two-fold or greater. Biacore is particularly useful when the starting antibody already binds with relatively high affinity, e.g., a K of about 10 nM or lower. Tracking using Biacore surface plasmon resonance is described in the Examples, here.

[00296] Binding affinity can be determined using Kinexa Biocensor, scintillation proximity assays, ELISA, ORIGEN (IGEN) immunoassays, fluorescence quenching, fluorescence transfer, and/or yeast display. Binding affinity can also be screened using a suitable bioassay.

[00297] In some embodiments, each amino acid position in a CDR is replaced (in some embodiments, one at a time) with all 20 naturally occurring amino acids using art-recognized mutagenesis methods (some of which are described herein). This generates small clone libraries (in some embodiments, one for each amino acid position that is analyzed), each with a complexity of 20 members (if 20 amino acids are replaced at each position).

[00298] In some embodiments, the library to be screened comprises substitutions at two or more positions, which may be located in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions at two or more positions of a CDR. The library may comprise substitution at two or more positions in two or more CDRs. The library may comprise substitution at 3, 4, 5, or more positions, said positions found in two, three, four, five, or six CDRs. The substitution may be prepared using low-redundancy codons. See, for example, Table 2 of Balint et al., (1993) Gene 137(1): 109-18.

[00299] The CDR may be CDRH3 and/or CDRL3. The CDR may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH3. The CDR may be a Kabat CDR, a Chothia CDR, or an extended CDR.

[00300] Candidates with improved binding can be sequenced, thereby identifying a CDR substitution mutant that results in improved affinity (also termed an "improved" substitution). Candidates that bind can also be sequenced, thereby identifying a CDR substitution that maintains binding.

[00301] Multiple rounds of screening may be performed. For example, candidates (each comprising an amino acid substitution at one or more of the positions of one or more CDRs) with improved binding are also useful for designing a second library containing at least the original amino acid and substituted at each improved CDR position (i.e., amino acid position in the CDR at which a substitution mutant showed improved binding). Preparation and screening or selection of this library is discussed further below.

[00302] Library scanning mutagenesis also provides a means for characterizing a CDR, in that the frequency of clones with improved binding, the same binding, decreased binding, or no binding also provides information about the importance of each amino acid position to the stability of the antibody-antigen complex. For example, if a CDR position retains binding when altered for all 20 amino acids, that position is identified as a position that is unlikely to be required for antigen binding. Conversely, if a CDR position retains binding in only a small percentage of substitutions, that position is identified as a position that is important for CDR function. Thus, library scanning mutagenesis methods generate information about positions in the CDR that can be altered for many different amino acids (including all 20 amino acids), and positions in the CDR that cannot be altered or that can only be altered for a few amino acids.

[00303] The affinity-enhanced candidates may be combined into a second library, which includes the improved amino acid, the starting amino acid at that position, and may further include additional substitutions at that position, depending on the complexity of the library that is desired or permitted using the desired screening or selection method. Furthermore, if desired, the adjacent amino acid position may be randomized to at least two or more amino acids. Randomization of adjacent amino acids may allow additional conformational flexibility in the mutant CDR, which may in turn permit or facilitate the introduction of a greater number of improving mutations. The library may also comprise substitution at positions that do not show improved affinity in the first round of screening.

[00304] The second library is screened or selected for library members with improved and/or altered binding affinity using any method known in the art, including screening using BIAcore surface plasmon resonance analysis, and selection using any method known in the art for selection, including phage display, yeast display, and ribosome display.

[00305] In some embodiments, the invention also encompasses fusion proteins comprising one or more fragments or regions of the antibodies (such as G1) or polypeptides of the present invention. In one embodiment, a fusion polypeptide is provided that it comprises at least 10 contiguous amino acids of the light chain variable region set forth in SEQ ID NO: 2 (Figure 5) and/or at least 10 amino acids of the heavy chain variable region set forth in SEQ ID NO: 1 (Figure 5). In other embodiments, a fusion polypeptide is provided that comprises at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the light chain variable region set forth in SEQ ID NO:2 (Figure 5) and/or at least about 10, at least about 15, at least about 20, at least about 25, or at least about 30 contiguous amino acids of the heavy chain variable region set forth in SEQ ID NO:1 (Figure 5). In another embodiment, the fusion polypeptide comprises a light chain variable region and/or a heavy chain variable region of G1 as shown in SEQ ID NO:2 and SEQ ID NO:1 of Figure 5. In another embodiment, the fusion polypeptide comprises one or more G1 CDR(s). In still other embodiments, the fusion polypeptide comprises CDR H3 and/or CDR L3 of antibody G1. For purposes of this invention, a G1 fusion protein contains one or more G1 antibodies and another amino acid sequence to which it is not linked in the native molecule, e.g., a heterologous sequence or a homologous sequence from another region. Examples of heterologous sequences include, but are not limited to, a "tag" such as a FLAG tag or a 6His tag (SEQ ID NO: 56). Tags are well known in the art.

[00306] A G1 fusion polypeptide can be created by methods known in the art, for example, recombinantly or synthetically. Typically, the G1 fusion proteins of this invention are made by preparing an expression of a polynucleotide encoding them using recombinant methods described herein, although they can also be prepared by other means known in the art, including, for example, chemical synthesis.

[00307] In some aspects, the present invention also provides compositions comprising antibodies or polypeptides derived from G1 conjugated (e.g., linked) to an agent that facilitates coupling to a solid support (such as biotin or avidin). For simplicity, reference will generally be made to G1 or antibodies with the understanding that these methods are applicable to any of the CGRP binding embodiments described herein. Conjugation generally refers to the linking of these components as described herein. Linking (which is generally the attachment of these components in close association, at least for administration) may be accomplished in any number of ways. For example, a direct reaction between an agent and an antibody is possible when each possesses a substituent capable of reacting with the other. For example, a nucleophilic group, such as an amino or sulfhydryl group, may be capable of reacting with a carbonyl-containing group, such as an anhydride or an acid halide, or with an alkyl group containing a good leaving group (e.g., a halide) on the other.

[00308] An antibody or polypeptide may be linked to a labeling agent (alternatively referred to as a "label") such as a fluorescent molecule, a radioactive molecule, or any other markers known in the art. Labels are known in the art that generally provide (directly or indirectly) a signal.

[00309] In some embodiments, the invention also provides compositions (including pharmaceutical compositions) and kits comprising antibody G1, and/or any or all of the antibodies or polypeptides described herein.

[00310] In some embodiments, the invention also provides isolated polynucleotides encoding the antibodies and polypeptides of the present invention (including an antibody comprising the heavy chain and light chain variable region polypeptide sequences shown in Figure 5), and vectors and host cells comprising the polynucleotide.

[00311] In some embodiments, the invention provides polynucleotides (or compositions, including pharmaceutical compositions) comprising polynucleotides encoding any of the following: (a) the G1 antibody or variants thereof shown in Table 6; (b) a fragment or a region of the G1 antibody or variants thereof shown in Table 6; (c) a light chain of the G1 antibody or variants thereof shown in Table 6; (d) a heavy chain of the G1 antibody or variants thereof shown in Table 6; (e) one or more of the variable region(s) from a light chain and/or a heavy chain of the G1 antibody or variants thereof shown in Table 6; (f) one or more CDR(s) (one, two, three, four, five, or six CDRs) of the G1 antibody or variants thereof shown in Table 6; (g) CDR H3 of the heavy chain of the G1 antibody; (h) CDR L3 of the light chain of the G1 antibody or variants thereof shown in Table 6; (i) three CDRs of the light chain of the G1 antibody or variants thereof shown in Table 6; (j) three CDRs of the heavy chain of the G1 antibody or variants thereof shown in Table 6; (k) three CDRs of the light chain and three CDRs of the heavy chain of the G1 antibody or variants thereof shown in Table 6; and (l) an antibody comprising any one of (b) through (k). In some embodiments, the polynucleotide comprises one or both of the polynucleotide(s) set forth in SEQ ID NO: 9 and SEQ ID NO: 10.

[00312] In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides that have impaired effector function. The polynucleotides can be produced by procedures known in the art.

[00313] In another aspect, the invention provides compositions (such as a pharmaceutical composition), comprising any of the polynucleotides of the invention. In some embodiments, the composition comprises an expression vector comprising a polynucleotide encoding the G1 antibody as described herein. In another embodiment, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In still other embodiments, the composition comprises one or both of the polynucleotides set forth in SEQ ID NO: 9 and SEQ ID NO: 10. Expression vectors, and administration of polynucleotide compositions are further described herein.

[00314] In another aspect, the invention provides a method of making any of the polynucleotides described herein.

[00315] Polynucleotides complementary to such sequences are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded and may be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to a DNA molecule in a one-to-one manner, and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide of the present invention, and a polynucleotide may, but need not, be linked to other molecules and/or support materials.

[00316] The polynucleotides may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or a portion thereof) or may comprise a variant of such a sequence. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions such that the immunoreactivity of the encoded polypeptide is not diminished, relative to a native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. The variants preferably exhibit at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to a polynucleotide sequence encoding a native antibody or a portion thereof.

[00317] Two polypeptide or polynucleotide sequences are said to be "identical" if the nucleotide or amino acid sequence in the two sequences is the same when aligned for maximum correspondence, as described below. Comparison between two sequences is typically accomplished by comparing the sequences across a comparison window to identify and compare local regions of sequence similarity. A "comparison window," as used herein, refers to a segment of at least about 20 contiguous positions, generally from 30 to about 75, from 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.

[00318] Optimal alignment of sequences for comparison can be conducted using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, WI) using the default parameters. This program incorporates several alignment schemes described in the following references: Dayhoff, M.O. (1978) A model of evolutionary change in proteins - Matrices for the detection of distant relationships. In Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. 183, Academic Press, Inc., San Diego, CA; Higgins, D.G. and Sharp, P.M., 1989, CABIOS 5:151-153; Myers, E.W. and Muller W., 1988, CABIOS 4:11-17; Robinson, E.D., 1971, Comb. Theor. 11:105; Santou, N., Nes, M., 1987, Mol. Biol. Evolution. 4:406-425; Sneath, P.H.A. and Sokal, R.R., 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, W.J. and Lipman, D.J., 1983, Proc. Natl. Academic. Sci. USA 80: 726-730.

[00319] Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less, typically 5 to 15 percent, or 10 to 12 percent, compared to the reference sequences (which comprise no additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which identical nucleic acid bases or amino acid residues occur in both sequences to yield the number of matching positions, dividing the number of matching positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the results by 100 to yield the percent sequence identity.

[00320] Variants may also, or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under moderately stringent conditions to a naturally occurring DNA sequence encoding a native antibody (or a complementary sequence).

[00321] Suitable "moderately stringent conditions" include prewashing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization at 50°C-65°C, 5 X SSC, overnight; followed by two washes at 65°C for 20 minutes, with each of 2X, 0.5X, and 0.2X SSC containing 0.1% SDS.

[00322] As used herein, "highly stringent conditions" or "very stringent conditions" are those that: (1) employ low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent such as formamide, e.g., 50% (w/w) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) employ 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55°C. One of skill in the art will recognize how to adjust temperature, ionic strength, etc., as necessary to accommodate factors such as probe length and the like.

[00323] It will be appreciated by those skilled in the art that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides bear minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. Furthermore, alleles of genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations, such as nucleotide deletions, additions, and/or substitutions. The resulting mRNA and protein may, but need not, have an altered structure or function. Alleles can be identified using conventional techniques (such as hybridization, amplification, and/or sequence database comparison).

[00324] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or by PCR. Methods of chemical synthesis of polynucleotides are well known in the art and need not be described in detail herein. One skilled in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.

[00325] For the preparation of polynucleotides using recombinant methods, a polynucleotide comprising a desired sequence can be inserted into a suitable vector and the vector can in turn be introduced into a suitable host cell for replication and amplification, as discussed herein. Polynucleotides can be inserted into host cells by any means known in the art. Cells are transformed by introduction of an exogenous polynucleotide by direct uptake, endocytosis, transfection, F mating, or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrating vector (such as a plasmid) or integrated into the genome of the host cell. The thus amplified polynucleotide can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al. (1989).

[00326] Alternatively, PCR allows the reproduction of DNA sequences. PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as by PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston (1994).

[00327] RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, as described in Sambrook et al., (1989), for example.

[00328] Suitable cloning vectors can be constructed according to standard techniques or can be selected from a large number of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell intended to be used, useful cloning vectors will generally be capable of self-replication, may possess a unique target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector. Suitable examples include bacterial plasmids and viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as PSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

[00329] Expression vectors are generally replicable polynucleotide constructs that contain a polynucleotide according to any of the various aspects of the invention. It is implied that an expression vector is to be replicated in host cells, either as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) described in PCT Publication No. WO 87/04462. Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.

[00330] Vectors containing the polynucleotides of interest may be introduced into the host cell by any of a number of suitable means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus). The choice of whether to introduce vectors or polynucleotides will often depend on the characteristics of the host cell.

[00331] In some aspects, the invention also provides host cells comprising any of the polynucleotides described herein. Any host cells capable of overexpressing heterologous DNAs can be used for the purpose of isolating the genes encoding the antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS cells, HeLa cells, and CHO cells. See also PCT Publication No. WO 87/04462. Suitable non-mammalian host cells include prokaryotic (such as E. coli or B. subtilis) and yeast (such as S. cerevisiae, S. pombe; or K. lactis) cells. Preferably, the host cells express the cDNA at a level about 5-fold higher, more preferably 10-fold higher, even more preferably 20-fold higher than that of the corresponding endogenous antibody or protein of interest, if present, in the host cells. Screening of the host cells for specific binding to AD1-40 is performed by an immunoassay or FACS. A cell overexpressing the antibody or protein of interest can be identified.

D. Compositions

[00332] In some embodiments, the compositions used in the method of the invention comprise an effective amount of an antibody (e.g., anti-CGRP antagonist antibody, monoclonal antibody that modulates the CGRP pathway), or an antibody-derived polypeptide described herein. Examples of such compositions, as well as how to formulate them, are also described in a previous and lower section. In one embodiment, the composition further comprises a CGRP antagonist. In some embodiments, the composition comprises one or more monoclonal antibodies that modulate the CGRP pathway. In some embodiments, the composition comprises one or more anti-CGRP antagonist antibodies. In some embodiments, the anti-CGRP antagonist antibody recognizes human CGRP. In some embodiments, the anti-CGRP antagonist antibody is humanized. In some embodiments, the anti-CGRP antagonist antibody comprises a constant region that does not trigger an unwanted or undesirable immune response, such as antibody-mediated lysis or ADCC. In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDR(s) of G1 antibody (such as one, two, three, four, five, or in some embodiments, all six CDRs of G1). In some embodiments, the anti-CGRP antagonist antibody is human.

[00333] It is understood that the compositions may comprise more than one antibody (e.g., more than one anti-CGRP antagonist antibody--a mixture of anti-CGRP antagonist antibodies that recognize different epitopes of CGRP). Other exemplary compositions comprise more than one anti-CGRP antagonist antibodies that recognize the same epitope(s), or different species of anti-CGRP antagonist antibodies that bind to different epitopes of CGRP.

[00334] A composition may further comprise carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the dosages and concentrations employed. A therapeutic formulation of an antibody may comprise one or more pharmaceutically acceptable carriers, excipients or stabilizers, with non-limiting examples of such species including buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids (e.g., at concentrations of 0.1 mM to 100 mM, 0.1 mM to 1 mM, 0.01 mM to 50 mM, 1 mM to 50 mM, 1 mM to 30 mM, 1 mM to 20 mM, 10 mM to 25 mM) such as glycine, glutamine, methionine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents (e.g., at concentrations of 0.001 mg/mL to 1 mg/mL, 0.001 mg/mL to 1 mg/mL, 0.001 mg/mL to 0.1 mg/mL, 0.001 mg/mL to 0.01 mg/mL) such as EDTA (e.g., disodium EDTA dihydrate); sugars (e.g., at concentrations of 1 mg/mL to 500 mg/mL, 10 mg/mL to 200 mg/mL, 10 mg/mL to 100 mg/mL, 50 mg/mL to 150 mg/mL) such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants (e.g., at concentrations of 0.01 mg/mL to 10 mg/mL, 0.01 mg/mL to 1 mg/mL, 0.1 mg/mL to 1 mg/mL, 0.01 mg/mL to 0.5 mg/mL) such as TWEEN™ (e.g., polysorbate (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80)), PLURONICS™ or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[00335] An antibody (e.g., an anti-CGRP antagonist antibody) and compositions thereof may also be used in conjunction with other agents that serve to enhance and/or complement the effectiveness of the agents.

E. Kits

[00336] In one aspect, the invention also provides kits for use in the present methods. The kits can include one or more containers comprising an antibody described herein (e.g., an anti-CGRP antagonist antibody (such as a humanized antibody)) or polypeptide described herein and instructions for use in accordance with any of the methods described herein. Generally, these instructions include a description of administering the antibody to treat, ameliorate, or prevent headache (e.g., migraine) in accordance with any of the methods described herein. The kit can further comprise a description of selecting a suitable subject for treatment based on identifying whether such subject has headache, or whether the subject is at risk for headache. In still other embodiments, the instructions comprise a description of administering an antibody (e.g., the anti-CGRP antagonist antibody) to a subject at risk for headache (e.g., migraine).

[00337] In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is human. In other embodiments, the antibody is a monoclonal antibody. In other embodiments. In some embodiments, the antibody comprises one or more CDR(s) of antibody G1 (such as one, two, three, four, five, or in some embodiments, all six CDRs of G1).

[00338] Instructions for use of an antibody (e.g., anti-CGRP antagonist antibody) typically include information such as the dosage, dosing schedule, and route of administration for the intended treatment. Containers may be unit doses, batch packages (e.g., multidose packages), or subunit doses. Instructions provided in kits are instructions on a label or insert (e.g., a sheet of paper included with the kit) typically written, but machine-readable instructions (e.g., instructions held on a magnetic or optical storage disk) are also acceptable.

[00339] The label or package insert indicates that the composition is used for the treatment, amelioration, and/or prevention of headache (e.g., migraine). Instructions may be provided for the practice of any of the methods described herein.

[00340] The kits of the present invention are in suitable packaging. Suitable packaging includes, but is not limited to, ampoules, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also contemplated are packaging for use in combination with a specific device, such as an inhaler, nasal delivery device (e.g., an atomizer), or an infusion device such as a minipump. A kit may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CGRP antagonist antibody and/or a monoclonal antibody that modulates the CGRP pathway. The container may further comprise a second pharmaceutically active agent.

[00341] Kits may optionally provide additional components such as buffers and interpretive information. Typically, the kit comprises a container and a label or informational insert(s) on or associated with the container.

[00342] The following Examples are provided to illustrate, but not limit, the invention.

Examples Example 1: Generation and characterization of monoclonal antibodies directed against CGRP

[00343] Generation of anti-CGRP antibodies. To generate anti-CGRP antibodies that have cross-species reactivity between mouse and human CGRP, mice were immunized with 25-100 μg of human α-CGRP or β-CGRP conjugated to KLH in adjuvant (50 μl per 100 μl, paw per mouse total) at various intervals. Immunization was generally performed as described in Geerligs HJ et al., 1989, J. Immunol. Methods 124:95-102; Kenney JS et al., 1989, J. Immunol. Methods 121:157-166; and Wicher K et al., 1989, Int. Arch. Allergy Appl. Immunol. 89:128-135. Mice were first immunized with 50 μg of human α-CGRP or β-CGRP conjugated to KLH in CFA (complete Freund's adjuvant). After 21 days, mice were secondarily immunized with 25 μg of human β-CGRP (for mice immunized with the first human α-CGRP) or α-CGRP (for mice immunized with the first human β-CGRP) conjugated to KLH in IFA (incomplete Freund's adjuvant). Twenty-three days later, after the second immunization, a third immunization was performed with 25 μg of mouse α-CGRP conjugated to KLH in IFA. Ten days later, antibody titers were tested by ELISA. Further immunization was performed with 25 μg of peptide (mouse α-CGRP-KLH) in IFA 34 days after the third immunization. Final booster was performed with 100 μg soluble peptide (mouse α-CGRP) 32 days after further immunization.

[00344] Splenocytes were obtained from the immunized mouse and fused with NSO myeloma cells at a ratio of 10:1 with polyethylene glycol 1500. The hybrids were plated in 96-well plates in DMEM containing 20% horse serum and 2-oxaloacetate/pyruvate/insulin (Sigma), and hypoxanthine/aminopterin/thymidine selection was initiated. On day 8, 100 μl of DMEM containing 20% horse serum was added to all wells. The supernatants of the hybrids were screened using antibody capture immunoassay. Determination of the antibody class was done with antibodies specific for the second class.

[00345] A panel of monoclonal antibody-producing cell lines were selected based on their binding to human and mouse CGRP for further characterization. These antibodies and characteristics are presented below in Tables 2 and 3.

[00346] Purification and preparation of Fab fragment. Monoclonal antibodies selected for further characterization were purified from hybridoma culture supernatants using protein A affinity chromatography. The supernatants were equilibrated to pH 8. The supernatants were then loaded onto a MabSelect protein A column (Amersham Biosciences #17-5199-02) equilibrated with PBS at pH 8. The column was washed with 5 column volumes of PBS, pH 8. Antibodies were eluted with 50 mM citrate-phosphate buffer, pH 3. The eluted antibodies were neutralized with 1 M phosphate buffer, pH 8. The purified antibodies were dialyzed against PBS, pH 7.4. Antibody concentrations were determined by SDS-PAGE using a murine monoclonal antibody standard curve.

[00347] Fabs were prepared by papain proteolysis of full length antibodies using the Immunopure Fab kit (Pierce #44885) and purified by flow through protein A chromatography following the manufacturer's instructions. Concentrations were determined by ELISA and/or SDS-PAGE electrophoresis using a Fab standard of known concentration (determined by amino acid analysis), and by A280 using 1OD = 0.6 mg/ml (or theoretical equivalent based on amino acid sequence).

[00348] Determination of Fab Affinity. Affinities of anti-CGRP monoclonal antibodies were determined at either 25°C or 37°C using the Biacore3000™ surface plasmon resonance (SPR) system (BIAcore, Inc., Piscataway NJ) with the manufacturer's own running buffer, HBS-EP (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/w polysorbate 20). Affinity was determined by capturing N-terminally biotinylated CGRP peptides (custom ordered from GenScript Corporation, New Jersey or Global Peptide Services, Colorado) via streptavidin pre-immobilized on SA chip and measuring binding kinetics of the Fab antibody titrated across the CGRP surface. Biotinylated CGRP was diluted in HBS-EP and injected onto the chip at a concentration of less than 0.001 mg/ml. Using variable flow times between individual chip channels, two ranges of antigen density were achieved: <50 response units (RU) for detailed kinetic studies and approximately 800 RU for concentration and screening studies. Two- or three-fold serial dilutions at concentrations typically spanning 1 μM–0.1 nM (aimed at 0.1–10x estimated KD) of purified Fab fragments were allowed for 1 min at 100 μL/min and dissociation times of 10 min were injected. After each binding cycle, the surfaces were regenerated with 25 mM NaOH in 25% w/w ethanol, which was tolerated over hundreds of cycles. Kinetics of association rate (Kon) and dissociation rate (koff) were obtained simultaneously by fitting the data to a 1:1 Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods Enzymology 6. 99-110) using the BIAe-valuation program. The global equilibrium dissociation constants (KD) or "affinities" were calculated from the ratio KD = koff/kon. The affinities of the murine Fab fragments are presented in Tables 2 and 3.

[00349] Epitope mapping of murine anti-CGRP antibodies. To determine the epitope that human anti-CGRP antibodies bind on α-CGRP, the affinities of Fab fragments for various CGRP binding fragments were measured as described above by capturing N-terminally biotinylated CGRP fragments of amino acids 19-37 and amino acids 25-37 onto an SA sensor chip. Figure 1 shows their binding affinities measured at 25°C. As shown in Figure 1, all antibodies except antibody 4901 bind to human α-CGRP fragments 19-37 and 25-37 with similar affinity to their binding affinity to full-length human α-CGRP (1-37). Antibody 4901 binds to human α-CGRP fragment 25-37 with six-fold lower affinity than binding to full-length human α-CGRP fragment, primarily due to a loss of off-rate. The data indicate that these anti-CGRP antibodies generally bind to the C-terminal end of CGRP.

[00350] Alanine scanning was performed to further characterize the amino acids in human α-CGRP involved in anti-CGRP antibody binding. Different variants of human α-CGRP with single alanine substitutions were generated by peptide synthesis. Their amino acid sequences are presented in Table 4, along with all other peptides used in the Biacore analysis. The affinities of the Fab fragments of the anti-CGRP antibodies to these variants were determined using Biacore, as described above. As shown in Figure 1, all 12 antibodies target a C-terminal epitope, with amino acid F37 being the most important residue. Mutation of F37 to alanine significantly reduced the affinity or even completely eliminated binding of the anti-CGRP antibodies to the peptide. The next most important amino acid residue is G33, however, only the high affinity antibodies (7E9, 8B6, 10A8 and 7D11) were influenced by the alanine substitution at this position. The amino acid residue S34 also plays a significant, but minor, role in the binding of these four high affinity antibodies.

[00351] Table 2. Characteristics of human anti-CGRP monoclonal antibodies that bind to α-CGRP and their antagonist activity Note: Antibody 4901 is commercially available (Sigma, Product No. C7113).nd = not determined

[00352] Table 3. Characteristics of mouse anti-CGRP monoclonal antibodies that bind to α-CGRP and have antagonistic activity "nd" indicates that no testing was performed for the antibody.

[00353] Table 4 Amino acid sequences of human α-CGRP fragments (SEQ ID NOS: 15-40) and related peptides (SEQ ID NOS: 41-47). All peptides are C-terminally amidated except SEQ ID NOS: 36-40. Bold residues indicate point mutations.

Example 2: Screening for anti-CGRP antagonist antibodies using in vitro assays.

[00354] Mouse anti-CGRP antibodies were further screened for in vitro antagonist activity using a cell-based cAMP activation binding assay.

[00355] Antagonist activity measured by cAMP assay. Five microliters of human or mouse α-CGRP (50 nM final concentration) in the presence or absence of an anti-CGRP antibody (1-3000 nM final concentration), or of mouse α-CGRP or human α-CGRP (0.1 nM-10 μM final concentration; as a positive control for c-AMP activation) was dispensed into a 384-well plate (Nunc, Cat. No. 264657). Ten microliters of cells (human SK-N-MC if human α-CGRP is used, or mouse L6 from ATCC if mouse α-CGRP is used) in stimulation buffer (20 mM HEPES, pH 7.4, 146 mM NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 500 μM 3-isobutyl-1-methylxanthine (IBMX)) were added to the wells of the plate. The plate was incubated at room temperature for 30 min.

[00356] Following incubation, cAMP activation was performed using the HitHunterTM Enzyme Fragment Complementation Assay (Applied Biosystems) following the manufacturer's instructions. The assay is based on a genetically engineered β-galactosidase enzyme consisting of two fragments termed Acceptor Enzyme (EA) and Donor Enzyme (ED). When the two fragments are separated, the enzyme is inactive. When the fragments are brought together they can spontaneously recombine to form an active enzyme by a process called complementation. The CEF assay platform utilizes an ED-cAMP peptide conjugate in which cAMP is recognized by anti-cAMP antibodies. This ED fragment is capable of reassociation with EA to form an active enzyme. In the assay, the anti-cAMP antibody is optimally titrated to bind the ED-cAMP conjugate and inhibit enzyme formation. cAMP levels in cell lysate samples compete with the cAMP-ED conjugate for binding to the anti-cAMP antibody. The amount of free ED conjugate in the assay is proportional to the cAMP concentration. Therefore, cAMP is measured by the formation of an active enzyme that is quantified by the turnover of luminescent β-galactosidase substrate. The cAMP activation assay was performed by adding 10 μl of lysis buffer and the anti-cAMP antibody (1:1 ratio) followed by incubation at room temperature for 60 min. Then, 10 μl of the ED-cAMP reagent was added to each well and incubated for 60 min at room temperature. After incubation, 20 μl of the EA reagent and CL mixture (containing the substrate) (1:1 ratio) was added to each well and incubated for 1–3 h or overnight at room temperature. The plate was read at 1 second/well on the PMT instrument or 30 seconds/spot on the imager. Antibodies that inhibit cAMP activation by α-CGRP were identified (referred to as "yes") in Tables 2 and 3 above. The data in Tables 2 and 3 indicate that antibodies that demonstrated antagonistic activity in the assay generally have high affinity. For example, antibodies having a K (determined at 25°C) of about 80 nM or less to human α-CGRP or having a K (determined at 37°C) of about 47 nM or less to mouse α-CGRP showed antagonistic activity in this assay.

[00357] Radioligand Binding Assay. Binding assay was performed to measure the IC50 of anti-CGRP antibody in blocking CGRP from receptor binding as described previously. Zimmermann et al., Peptides 16:421-4, 1995; Mallee et al., J. Biol. Chem. 277:14294-8, 2002. Membranes (25 μg) from SK-N-MC cells were incubated for 90 min at room temperature in incubation buffer (50 mM Tris-HCL, pH 7.4, 5 mM MgCl2, 0.1% BSA) containing 10 pM 125I-human α-CGRP in a total volume of 1 mL. To determine the inhibition concentrations (IC50), antibodies or unlabeled CGRP (as a control) from a stock solution greater than approximately 100-fold were dissolved at various concentrations in the incubation buffer and incubated simultaneously with the membranes and 10 pM 125I-human α-CGRP. The incubation was terminated by filtration through a glass microfiber filter (GF/B, 1μm) that had been blocked with 0.5% polyethylenimine. The dose-response curves were plotted and Ki values were determined using the equation: Ki = IC50/(1+(([ligand]/KD); where the equilibrium dissociation constant KD = 8 pM for human α-CGRP to the CGRP1 receptor as present on SK-N-MC cells, and Bmax = 0.025 pmol/mg protein. The reported IC50 value (in terms of IgG molecules) was converted to binding sites (by multiplying by 2) so that it can be compared with the affinities (KD) determined by Biacore (see Table 2).

[00358] Table 2 shows the IC50 of murine antibodies 7E9, 8B6, 6H2, and 4901. The data indicate that antibody affinity generally correlates with IC50: antibodies with higher affinity (lower KD values) have lower IC50 in the radioligand binding assay.

Example 3: Effect of anti-CGRP antagonist antibodies on cutaneous vasodilation induced by mouse saphenous nerve stimulation

[00359] To test the antagonistic activity of anti-CGRP antibodies, the effect of the antibodies on cutaneous vasodilation by stimulation of the rat saphenous nerve was tested using a previously described rat model. Escott et al., Br. J. Pharmacol. 110:772-776, 1993. In this mouse model, electrical stimulation of the saphenous nerve induces the release of CGRP from nerve endings, resulting in an increase in skin blood flow. Blood flow in the skin of the foot of Sprague Dawley mice (170-300 g, from Charles River Hollister) was measured after saphenous nerve stimulation. The mice were maintained under 2% isoflurane anesthesia. Bretylium tosylate (30 mg/kg, administered IV) was given at the beginning of the experiment to minimize vasoconstriction due to concomitant stimulation of the sympathetic fibers of the saphenous nerve. Body temperature was maintained at 37°C by use of a rectal probe thermostatically attached to a temperature-pad heating blanket. Compounds, including antibodies, positive control (CGRP 8-37), and vehicle (PBS, 0.01% Tween 20) were administered intravenously via the right femoral vein, except for the experiment shown in Figure 3, the test compound and control were injected via the tail vein, and for the experiments shown in Figures 2A and 2B, antibodies 4901 and 7D11 were injected intraperitoneally (IP). Positive control compound CGRP 8-37 (vasodilation antagonist), because of its short half-life, was given 3–5 min before nerve stimulation at 400 nmol/kg (200 μl). Tan et al., Clin. Sci. 89:656-73, 1995. Antibodies were administered at different doses (1 mg/kg, 2.5 mg/kg, 5 mg/kg, 10 mg/kg, and 25 mg/kg).

[00360] For the experiments presented in Figures 2A and 2B, antibody 4901 (25 mg/kg), antibody 7D11 (25 mg/kg), or vehicle control (PBS with 0.01% Tween 20) was administered intraperitoneally (IP) 72 hours prior to electrical pulse stimulation. For the experiment presented in Figure 3, antibody 4901 (1 mg/kg, 2.5 mg/kg, 5 mg/kg, or 25 mg/kg) or vehicle control (PBS with 0.01% Tween 20) was administered intravenously 24 hours prior to electrical pulse stimulation. Following administration of antibody or vehicle control, the saphenous nerve of the right hindlimb was surgically exposed, cut proximally, and covered with plastic wrap to prevent drying. A laser Doppler probe was placed over the middorsal side of the skin of the hind paw, which is the region innervated by the saphenous nerve. Skin blood flow, measured as blood cell flow, was monitored with a laser Doppler flow meter. When a stable baseline flow (less than 5% variation) had been established for at least 5 min, the nerve was placed over bipolar platinum electrodes and electrically stimulated with 60 pulses (2 Hz, 10 V, 1 ms, for 30 s) and then again 20 min later. Cumulative change in skin blood flow was calculated by the area under the flow-time curve (AUC, which is equal to the change in flow multiplied by the change in time) for each flow response to electrical pulse stimulation. The blood flow response to the two stimulations was averaged. The animals were maintained under anesthesia for a period of 1 to 3 h.

[00361] As shown in Figure 2A and Figure 2B, increased blood flow stimulated by electronic pulse application in the saphenous nerve was inhibited by the presence of CGRP 8-37 (400 nmol/kg, administered i.v.), antibody 4901 (25 mg/kg, administered i.p.), or antibody 7D11 (25 mg/kg, administered i.p.), compared to control. CGRP 8-37 was administered 3-5 min prior to saphenous nerve stimulation; and antibodies were administered 72 h prior to saphenous nerve stimulation. As shown in Figure 3, increased blood flow stimulated by electronic pulse application in the saphenous nerve was inhibited by the presence of antibody 4901 at different doses (1 mg/kg, 2.5 mg/kg, 5 mg/kg, and 25 mg/kg) administered intravenously 24 h prior to saphenous nerve stimulation.

[00362] For the experiments presented in Figures 4A and 4B, the saphenous nerve was surgically exposed prior to antibody administration. The saphenous nerve of the right hind limb was surgically exposed, cut proximally, and covered with plastic wrap to prevent drying. A laser Doppler probe was placed over the middorsal side of the skin of the hind paw, which is the region innervated by the saphenous nerve. Skin blood flow, measured as blood cell flux, was monitored with a laser Doppler flow meter. Thirty to forty-five minutes after bretylium tosylate injection, when a stable baseline flow (less than 5% variation) had been established for at least 5 minutes, the nerve was placed over bipolar platinum electrodes and electrically stimulated (2 Hz, 10 V, 1 ms, for 30 sec) and again 20 minutes later. The mean blood flow response to these two stimulations was used to establish the baseline (time 0) response to electrical stimulation. The 4901 antibody (1 mg/kg or 10 mg/kg), 7E9 antibody (10 mg/kg), 8B6 antibody (10 mg/kg), or vehicle (PBS with 0.01% Tween 20) were then administered intravenously (i.v.). The nerve was subsequently stimulated (2 Hz, 10 V, 1 ms, for 30 seconds) at 30 min, 60 min, 90 min, and 120 min after antibody or vehicle administration. Animals were maintained under anesthesia for a period of approximately three hours. Cumulative change in skin blood flow was calculated by the area under the flow-time curve (AUC, which is equal to the change in flow multiplied by the change in time) for each flow response to electrical impulse stimulation.

[00363] As shown in Figure 4A, increased blood flow stimulated by application of electronic pulses in the saphenous nerve was significantly inhibited by the presence of 1 mg/kg i.v. administered antibody 4901 when electronic pulse stimulation was applied at 60 min, 90 min, and 120 min after antibody administration, and increased blood flow stimulated by application of electronic pulses in the saphenous nerve was significantly inhibited by the presence of 10 mg/kg i.v. administered antibody 4901 when electronic pulse stimulation was applied at 30 min, 60 min, 90 min, and 120 min after antibody administration. Figure 4B shows that the increase in blood flow stimulated by electronic pulse application in the saphenous nerve was significantly inhibited by the presence of 7E9 antibody (10 mg/kg, i.v. administered) when electronic pulse stimulation was applied at 30 min, 60 min, 90 min, and 120 min after antibody administration, and by the presence of 8B6 antibody (10 mg/kg, i.v. administered) when electronic pulse stimulation was applied 30 min after antibody administration.

[00364] These data indicate that antibodies 4901, 7E9, 7D11, and 8B6 are effective in blocking CGRP activity as measured by cutaneous vasodilation induced by mouse saphenous nerve stimulation.

Example 4 Characterization of the G1 anti-CGRP antibody and its variants

[00365] The amino acid sequences for the light chain region and the variable heavy chain region of the anti-CGRP antibody G1 are shown in Figure 5. The following methods were used for expression and characterization of antibody G1 and its variants.

[00366] Expression Vectors Used. Expression of the Fab fragment of the antibodies was under the control of an IPTG-inducible LacZ promoter similar to that described in Barbas (2001) Phage display: a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press pg. 2.10. Vector pComb3X), however, modifications included the addition and expression of the following additional domains: the human kappa light chain constant domain and the CH1 constant domain of human immunoglobulin IgG2, the C region of the Ig gamma-2 chain, protein accession number P01859; Immunoglobulin kappa light chain (Homo sapiens), protein accession number CAA09181.

[00367] Small-scale Fab preparation. From E. coli transformed (either using electroporated competent TG1 cells or chemically competent Top 10 cells) with a Fab library, single colonies were used to inoculate both a master plate (LB agar + carbenicillin (50 μg/mL) + 2% glucose) and a working plate (2 ml/well, 96-well/plate) in which each well contained 1.5 ml LB + carbenicillin (50 μg/mL) + 2% glucose. A gas permeable sealing adhesive (ABgene, Surrey, UK) was applied to the plate. Both plates were incubated at 30 °C for 12-16 h; the working plate was shaken vigorously. The master plate was stored at 4 °C until needed, while cells from the working plate were pelleted (4000 rpm, 4 °C, 20 min) and resuspended in 1.0 ml LB carbenicillin (50 μg/ml) + 0.5 mM IPTG to induce Fabs expression by vigorous shaking for 5 h at 30 °C. The induced cells were centrifuged at 4000 rpm, 4 °C for 20 min and resuspended in 0.6 ml Biacore HB-SEP buffer (10 mM Hepes pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/w P20). Lysis of HB-SEP resuspended cells was performed by freezing (−80 °C) and then thawing at 37 °C. Cell lysates were centrifuged at 4000 rpm, 4 °C for 1 hour to separate debris from the Fab-containing supernatants, which were subsequently filtered 0.2 µm using a Millipore MultiScreen 96-Well Filter Plate Assay System and vacuum manifold. BIAcore was used to analyze the filtered supernatants by injection through CGRPs on the sensor chip. Affinity-selected clones expressing Fabs were rescued from the master plate, which provided template DNA for PCR, sequencing, and plasmid preparation.

[00368] Large scale Fab preparation. To obtain kinetic parameters, Fabs were expressed on a larger scale as follows. Erlenmeyer flasks containing 150 ml of LB + carbenicillin (50 ug/mL) + 2% glucose were inoculated with 1 ml of an overnight "starter" culture from an affinity-selected E. coli Fab-expressing clone. The remainder of the starter culture (~3 ml) was used to prepare plasmid DNA (QIAprep mini-prep kit, Qiagen) for sequencing and further manipulation. The large culture was incubated at 30 °C with vigorous shaking until an OD600 nm of 1.0 was reached (typically 12-16 h). Cells were pelleted by centrifugation at 4000 rpm, 4 °C for 20 min, and resuspended in 150 ml LB + IPTG (50 μg/ml) + 0.5 mM carbenicillin. After 5 h expression at 30 °C, cells were pelleted by centrifugation at 4000 rpm, 4 °C for 20 min, resuspended in 10 ml Biacore HBS-EP buffer, and lysed using a single freeze (-80 °C)/thaw (37 °C) cycle. Cell lysates were pelleted by centrifugation at 4000 rpm, 4 °C for 1 h, and the supernatant was collected and filtered (0.2 μm). Filtered supernatants were loaded onto Ni-NTA Superflow Sepharose (Qiagen, Valencia, CA) columns equilibrated with PBS, pH 8, then washed with 5 column volumes of PBS, pH 8. Individual Fabs in different fractions eluted with PBS (pH 8) 300 mM imidazole. Fractions containing Fabs were pooled and dialyzed into PBS, then quantified by ELISA prior to affinity characterization.

[00369] Total Antibody Preparation. For expression of full-length antibodies, variable regions of the heavy and light chains were cloned into mammalian expression vectors and transfected using Lipofectamine into HEK 293 cells for transient expression. Antibodies were purified using protein A using standard methods.

[00370] Vector pDb.CGRP.hFcGI is an expression vector comprising the G1 antibody heavy chain, and is suitable for transient or stable expression of the heavy chain. Vector pDb.CGRP.hFcGI has nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 7-612); a synthetic intron (nucleotides 613-1679); DHFR coding region (nucleotides 688-1253); human growth hormone signal peptide (nucleotides 1899-1976); G1 heavy chain variable region (nucleotides 1977-2621); human IgG2 constant region heavy chain containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence; see Eur. J. Immunol. (1999) 29:26132624). Vector pDb.CGRP.hFcGI was deposited with the ATCC on July 15, 2005, and has been assigned ATCC Accession No. PTA-6867.

[00371] Vector pEb.CGRP.hKGI is an expression vector comprising the G1 antibody light chain, and is suitable for transient expression of the light chain. Vector pEb.CGRP.hKGI has nucleotide sequences corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 2-613); human EF-1 intron (nucleotides 614-1149); human growth hormone signal peptide (nucleotides 1160-1237); antibody G1 light chain variable region (nucleotides 1238-1558); human kappa chain constant region (nucleotides 1559-1882). Vector pEb.CGRP.hKGI was deposited with the ATCC on July 15, 2005, and has been assigned ATCC Accession No. PTA-6866.

[00372] Biacore Assay for Affinity Determination. Affinities of the G1 monoclonal antibody and its variants were determined at either 25 °C or 37 °C using the Biacore3000™ surface plasmon resonance (SPR) system (BIAcore, Inc., Piscataway NJ). Affinity was determined by capturing N-terminally biotinylated CGRP or fragments via pre-immobilized streptavidin (SA sensor chip) and measuring the binding kinetics of the antibody to the Fab fragments or G1 variants titrated between the CGRP or fragment on the chip. All assays were conducted in Biacore HBS-EP running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% w/w polysorbate 20). CGRP surfaces were prepared by diluting N-biotinylated CGRP to a concentration of less than 0.001 mg/mL in HBS-EP buffer and injecting it through the SA sensor chip using varying contact times. Low-capacity surfaces corresponding to capture levels <50 response units (RU) were used for high-resolution kinetic studies, while high-capacity surfaces (approximately 800 RU of captured CGRP) were used for concentration studies, screening, and affinity solution determinations. Kinetic data were obtained by serially diluting G1 Fab antibody in two- or three-fold increments to concentrations spanning 1uM–0.1 nM (aiming for 0.1–10x the estimated KD). Samples were typically injected for one minute at 100 μL/min and allowed to dissociate for at least 10 minutes. After each binding cycle, the surfaces were regenerated with 25 mM NaOH in 25% w/w ethanol, which was tolerated over hundreds of cycles. An entire titration series (usually generated in duplicate) was globally fitted to a 1:1 Langmuir binding model using the BIAevaluation program. This yielded a unique pair of association and dissociation kinetic constants (kon and koff, respectively) for each binding interaction, the ratio of which gave the equilibrium dissociation constant (KD = koff/kon). Affinities (KD values) thus determined are presented in Tables 6 and 7.

[00373] High-resolution analysis of binding interactions with extremely slow off rates. For interactions with extremely slow off rates (in particular, the Fab-binding G1 antibody to human □-CGRP on-chip at 25 °C), affinities were obtained in a two-part experiment. The protocol described above was used with the following modifications. The rate constant of association (kon) was determined by injecting a 2-fold titration series (in duplicate) measuring 550 nm-1 nM for 30 seconds at 100 uL/min and allowing only a 30-second dissociation phase. The rate constant of dissociation (koff) was determined by injecting three concentrations (high, medium, and low) of the same titration series in duplicate for 30 seconds and allowing a 2-hour dissociation phase. The affinity (KD) of each interaction was obtained by combining the kon and koff values obtained in the two types of experiments, as shown in Table 5.

[00374] Solution affinity determination by Biacore. The affinity of G1 antibody solution for mouse DD-CGRP and human F37A(19-37)D-CGRP was measured by Biacore at 37 °C. High capacity CGRP chip surface was used (high affinity human D-CGRP was chosen for detection purposes) and HBS-EP running buffer was flowed at 5 uL/min. G1 antibody Fab fragment at a constant concentration of 5 nM (intended to be equal to or less than the expected KD from the solution-based interaction) was preincubated with competing peptide, either mouse DD-CGRP or human F37A(19-37)D-CGRP, at final concentrations ranging from 1 nM to 1 uM in 3 serial dilutions. Fab solutions of G1 antibodies in the absence or presence of the solution-based competitor peptide were injected via CGRP onto the chip and the depletion of the binding responses detected on the chip surface as a result of the competition solution was monitored.

[00375] These binding responses were converted to "free Fab concentrations" using a calibration curve, which was constructed by titrating Fab G1 antibody alone (5, 2.5, 1.25, 0.625, 0.325, and 0 nM) across the CGRP on the chip. "Free Fab concentrations" were plotted against the solution-based competitor peptide concentration used to generate each data point and fitted to a solution affinity model using the BIAe-valuation program. The solution affinities determined (indirectly) in this manner are shown in Tables 5 and 7 and were used to validate the affinities obtained when Fabs are injected directly through N-biotinylated CGRPs on an SA chip. The close agreement between the affinities determined by these two methods confirms that titrating an N-biotinylated version of CGRP to the chip does not alter its native solution binding activity.

[00376] Table 5 below shows the binding affinities of antibody G1 to human α-CGRP, human β-CGRP, rat α-CGRP, and rat β-CGRP determined by BIAcore by flowing Fab fragments through N-biotinylated CGRPs on an SA chip. To better resolve the binding affinities of interactions with extremely slow offrates, the affinities were also determined in a two-part experiment; to complement this assay orientation, the affinity of the rat α-CGRP solution interaction was also determined (as described above). The close agreement between the affinities measured in both assay orientations confirms that the binding affinity of native rat α-CGRP in solution is not altered when it is N-biotinylated and titrated to an SA chip.

[00377] Table 5. Binding affinities of G1 antibody Fabs titrated via on-chip CGRPs

[00378] *Affinities for α-CGRPs (rat and human) were determined in a two-part high-resolution experiment in which the dissociation phase was monitored for 2 h (values for kon, koff, and KD represent the mean of n replicate experiments with the mean deviation expressed as a percent variance). Affinities for β-CGRPs (rat and human) were determined by bulk analysis using only a 20 min dissociation phase, which was not precise enough to quantify their extremely slow offrates (their offrates are probably slower than indicated here and therefore their affinities are probably even higher). G1 Fab antibody dissociated extremely slowly from all CGRPs (except rat α-CGRP) with offrates that approached the resolution limit of the Biacore assay (especially at 25°C).

[00379] ** Affinity solution determined by measuring the decrease in binding responses detected by on-chip CGRP to G1 Fab antibody pre-incubated with mice based on competing α-CGRP solutions.

[00380] Table 6 below shows antibodies having the amino acid sequence variation compared to the G1 antibody and their affinities for both rat α-CGRP and human α-CGRP. All amino acid substitutions of the variants shown in Table 6 are described relative to the G1 sequence. The binding affinities of the Fab fragments were determined by Biacore by flowing them through CGRPs on an SA chip.

[00381] Table 6. Amino acid sequences and binding affinity data for . G1 antibody variants determined at 37°C by Biacore.

[00382] All CDRs including both Kabat and Chothia CDRs. Amino acid residues are numbered sequentially (see Figure 5). All clones have L3+H1+H3 sequences identical to G1.

[00383] KD = koff/kon. All koff values were determined in a screening mode, except those that are underlined, which were obtained by bulk analysis of a Fab concentration series (G1 was analyzed in a high resolution mode). The underlined KD values were therefore determined experimentally by measuring kon. Other kon values were estimated to be the same as M25.

[00384] nd = not determined

[00385] To determine the epitope on human α-CGRP that is recognized by the G1 antibody, Biacore assays described above were used. Human α-CGRP was purchased as an N-biotinylated version to allow its high affinity capture through SA sensor chips. The binding of the G1 Fab fragment to human α-CGRP on the chip in the absence or presence of a CGRP peptide was determined. Typically, a 2000:1 mol peptide/Fab solution (e.g., 10 μM peptide in 50 nM G1 Fab) was injected across human α-CGRP on the chip. Figure 6 shows the percentage of binding blocked by competitor peptide. The data presented in Figure 6 indicate that the peptides that block 100% binding of Fab G1 to human α-CGRP are 1-37 (WT), 8-37, 26-37, P29A (19-37), K35A (19-37), K35E (19-37), and K35M (19-37) of human α-CGRP; 1-37 of β-CGRP (WT); 1-37 of rat α-CGRP (WT); and 1-37 of rat β-CGRP (WT). All of these peptides are amidated at the C-terminus. Peptides F37a (19-37) and 19-37 (the latter not C-terminally amidated) from human α-CGRP also blocked approximately 80% to 90% of the binding of G1 Fab to human α-CGRP. Peptide 1-36 (not C-terminally amidated) from human α-CGRP blocked approximately 40% of the binding of G1 Fab to human α-CGRP. Peptide fragment 19-36 (C-terminally amidated) from human α-CGRP; peptide fragments 1-13 and 1-19 from human α-CGRP (neither of which are C-terminally amidated); and human amylin, calcitonin, and adrenomedullin (all C-terminally amidated) did not compete with binding of G1 Fab to human α-CGRP on the ChIP. These data demonstrate that G1 targets a C-terminal epitope of CGRP and that both the identity of the most terminal residue (F37) and its amidation are important for binding.

[00386] The binding affinities of G1 Fab for human α-CGRP variants (at 37°C) were also determined. Table 7 below shows the affinities as measured directly by titration of G1 Fab between N-biotinylated human α-CGRP and variants on the chip. The data in Table 7 indicate that antibody G1 binds to a C-terminal epitope with F37 and G33 being the most important residues. G1 does not bind to CGRP when an additional amino acid residue (alanine) is added at the C-terminus (which is amidated).

[00387] Table 7. Binding affinities of Fab G1 to human α-CGRP and variants measured at 37°C (see Table 4 for their amino acid sequences)

[00388] The above data indicate that the epitope that the G1 antibody binds is at the C-terminal end of human α-CGRP, and amino acids 33 and 37 in human α-CGRP are important for G1 antibody binding. In addition, amidation of residue F37 is important for binding.

Example 5: Effect of anti-CGRP antagonist G1 antibody on cutaneous vasodilation induced by rat saphenous nerve stimulation

[00389] To test the antagonist activity of anti-CGRP G1 antibody, the effect of the antibody on cutaneous vasodilation by stimulation of the rat saphenous nerve was tested using a rat model described in Example 3. Briefly, rats were maintained under 2% isoflurane anesthesia. Bretylium tosylate (30 mg/kg, administered IV) was given at the beginning of the experiment to minimize vasoconstriction due to concomitant stimulation of the sympathetic fibers of the saphenous nerve. Body temperature was maintained at 37°C by use of a rectal probe thermostatically attached to a temperature-controlled heating blanket. The saphenous nerve of the right hind limb was surgically exposed, cut proximally, and covered with plastic wrap to prevent drying. A laser Doppler probe was placed over the mid-dorsal side of the skin of the hind paw, which is the region innervated by the saphenous nerve. Skin blood flow, measured as blood cell flux, was monitored with a laser Doppler flow meter. In experiments to determine the effects of antibody within two hours after injection Thirty to forty-five minutes after injection of bretylium tosylate, when a stable baseline flow (less than 5% variation) had been established for at least 5 minutes, the nerve was placed over bipolar platinum electrodes and stimulated electrically (2 Hz, 10 V, 1 ms, for 30 sec) and again 20 minutes later. The mean of the blood flow response to these two stimulations was used to establish the baseline (time 0) response to electrical stimulation. G1 antibody (1 mg/kg or 10 mg/kg) or vehicle (PBS with 0.01% Tween 20 equal to 10 mg/kg G1 volume) were then administered intravenously (IV). The nerve was subsequently stimulated (2 Hz, 10 V, 1 ms, for 30 seconds) at 30 min, 60 min, 90 min and 120 min after antibody administration. The animals were kept under anesthesia for a period of about three hours. Cumulative change in skin blood flow was calculated by the area under the flow-time curve (AUC, which is equal to the change in flow multiplied by the change in time) for each flow response to electrical impulse stimulation.

[00390] As shown in Figure 7, the increase in blood flow stimulated by application of electronic impulses in the saphenous nerve was significantly inhibited by the presence of G1 antibody at 1 mg/kg (administered IV), compared to vehicle, when the saphenous nerve was electrically stimulated at 90 min after administration of the antibody. Increase in blood flow stimulated by application of electronic impulses in the saphenous nerve was significantly inhibited by the presence of G1 antibody at 10 mg/kg (administered IV), compared to vehicle, when the saphenous nerve was electrically stimulated at 90 minutes and 120 minutes after administration of the antibody.

[00391] In experiments to determine the effects of antibodies at longer time points in the saphenous nerve assay, rats were injected IV with the indicated doses of antibody 24 hours or 7 days prior to preparing the animal for saphenous nerve stimulation as described above. In these experiments it was possible to establish a baseline response in individual rats to electrical pulse stimulation prior to dosing, and the groups so treated were compared with animals treated with vehicle (PBS, 0.01% Tween 20) at 24 hours or 7 days.

[00392] As shown in Figures 8A and 8B, increases in blood flow in the skin of the dorsomedial hind paw evoked by saphenous nerve stimulation were significantly inhibited in the groups of animals dosed with 10 mg/kg or 3 mg/kg G1 at either 24 hours or 7 days prior to stimulation, compared to the groups dosed with vehicle at the same time points.

[00393] Figure 8C depicts a curve-fitting analysis applied to the dose-response data represented in Figures 8A and 8B to determine the dose required for 50% maximal effect (EC50). The 24-hour EC50 is 1.3 mg/kg and the 7-day EC50 is slightly lower (0.8 mg/kg).

Example 6: Acute effect of anti-CGRP antagonist antibody mu7E9 assay in a dural artery (closed cranial window)

[00394] Closed Cranial Window Model: The purpose of this experiment was to determine the acute effect of anti-CGRP antagonist antibodies and compare it with the acute effect of the CGRP receptor antagonist BIBN4096BS. Experiments were performed as previously described (Williamson et al., Cephalalgia 17(4):518-24 (1997)) with the following modifications. Sprague Dawley rats (300-400 g) were anesthetized with 70 mg/kg pentobarbital IP. Anesthesia was maintained with pentobarbital 20 mg/kg/hour IV. Rats were cannulated in the jugular vein for administration of all drugs. Blood pressure was monitored with a probe (mikro-tip catheter, Millar Instruments) threaded through the femoral artery into the abdominal aorta. The rats were tracheostomized and the respiratory rate was maintained at 75 breaths per minute in a volume of 3.5 ml. After fixation of the head with a stereotaxic instrument and removal of the scalp, a 2x6 mm window in the left parietal zone just lateral to the sagittal suture was made by thinning the bone with a dental drill. Using a micromanipulator, a bipolar platinum electrode was lowered onto the surface and covered with heavy mineral oil. Lateral to the electrode window another 5x6 mm window was created and filled with heavy mineral oil, through which the diameter of a branch of the middle meningeal artery (MMA) was continuously monitored with a CCD camera and a video dimension analyzer (Living Systems). The rats were rested for not less than 45 min after preparation. A baseline response to electrical stimulation was established (15 V, 10 Hz, 0.5 ms pulses, 30 s), and then rats were dosed IV with the experimental compound (10 mg/kg mu7E9, 300αg/kg BIBN4096BS, or PBS 0.01% Tween 20). Additional electrical stimulations were performed at 5 (BIBN4096BS), 30, 60, 90, and 120 min after dosing. All data were recorded using a graphing program (ADInstruments).

[00395] As shown in Figure 9 mu7E9 at 10 mg/kg significantly blocks MMA dilation evoked by electric field stimulation within 60 minutes after dosing and maintains the effect throughout the duration of the assay (120 minutes). For comparison BIBN4096BS blocks MMA dilation within 5 minutes after dosing but the effect completely disappeared by 90 minutes. The magnitude of blockade is comparable between BIBN4096BS and mu7E9.

Example 7: Chronic effect of anti-CGRP G1 antagonist antibody assay on a dural artery (closed cranial window)

[00396] The objective of this experiment was to determine whether anti-CGRP antibody could still block electrically stimulated MMA dilation 7 days after dosing. Preparation of the rats was identical to that in the acute experiment described above (Example 6) with the following exceptions. Rats were injected IV (10mg/kg, 3mg/kg or 1mg/kg G1) 7 days prior to creation of the preparation and stimulation of the closed cranial window. It was impossible to establish a baseline dilation response to electrical stimulation prior to dosing as in the acute experiment so the antibody groups were compared to a control group dosed with MMA dilation in a vehicle (PBS, 0.01% Tween 20). After the rats were allowed to rest for not less than 45 minutes, the dura mater was electrically stimulated at 30 minute intervals. The stimulations were in pulses of 2.5 V, 5 V, 10 V, 15 V and 20 V, all at 10 Hz, 0.5 ms for 30 seconds.

[00397] As shown in Figure 10 G1 at 10 mg/kg and 3 mg/kg significantly blocked MMA dilation evoked by electrical stimulation in the 10 to 20 volt range. These data demonstrate that G1 can block electrically stimulated MMA dilation for up to 7 days after dosing.

Example 8: Morphine withdrawal model of hot flashes

[00398] The rat morphine withdrawal model is an established rodent model for mechanisms of menopausal hot flashes (Sipe et al, Brain Res 1028(2):191-202 (2004); Merchenthaler et al, Maturitas 30:307-316 (1998); Katovich et al, Brain Res. 494:85-94 (1989); Simpkins et al, Life Sciences 32:1957-1966 (1983)). Basically, rats are addicted to morphine by implanting morphine pellets under the skin. After addiction, the animals are injected with naloxone (an opiate antagonist), which sends them into immediate withdrawal. This re-withdrawal is accompanied by an increase in skin temperature, a decrease in core body temperature, an increase in heart rate, and an increase in serum luteinizing hormone. These are all similar in magnitude and speed to what occurs in human hot flashes (Simpkins et al, Life Sciences 32: 1957-1966 (1983)). Furthermore, when rats are treated with estradiol prior to withdrawal induction, hot flash symptoms are reduced (Merchenthaler et al, Maturitas 30: 307-316 (1998)). This is why the morphine withdrawal model is believed to mimic clinical hot flashes.

[00399] Ovariectomized rats were ordered from Charles River Laboratories. Not less than 7 days after ovariectomy morphine dependence was created by implanting a morphine pellet (75 mg morphine base) subcutaneously. Two days later 2 more pellets were implanted. The next day rats were injected intravenously with 10 mg/kg 4901 [**] or vehicle (PBS, 0.01% Tween). Two days after the second pellet rats were anesthetized with ketamine (90 mg/kg) and lightly restrained. A surface temperature thermocouple was taped to the base of the tail and a rectal thermocouple was used to measure core temperature. Data were recorded using graphing software (ADInstruments). After recording 15 minutes of stable baseline temperature, naloxone (1 mg/kg) was injected subcutaneously. Temperature was recorded continuously for the next 60 minutes. The results are shown in Figures 11A and 11B.

Example 9: Treatment of Chronic Migraine

[00400] A 45-year-old male human subject is identified as having had chronic migraine for at least three months. Identification of having had chronic migraine is achieved by noting a history of frequent headaches suggestive of chronic migraine (e.g., 15 days per month) for at least three months prior to screening. Verification of headache frequency is achieved through prospectively collected baseline headache information on at least 15 days, with at least 8 days per month meeting any of the following: i. qualifying as a migraine attack; and/or ii. preceded or accompanied by migraine aura.

[00401] In order to reduce the incidence of migraine in the subject, the subject is administered a 225 mg dose of antagonistic anti-CGRP antibody (e.g., G1 antibody). The antagonistic anti-CGRP antibody is provided as a liquid formulation at a concentration of 150 mg/mL. The 225 mg dose is administered as a 1.5 mL subcutaneous injection into the back of an upper arm of the subject's body. Alternatively, the dose may be delivered to the subject via intravenous infusion. In such cases, 5.85 mL of 150 mg/mL CGRP antibody may be combined with 0.9% Sodium Chloride Solution (normal saline) in an IV bag for a total bag volume of 130 mL. 100 mL of the IV bag volume is infused intravenously to the subject over one hour for a total dose of 225 mg. The dosage is repeated every twenty-eight days until a reduction in the incidence of migraine is observed. A reduction in the incidence of chronic migraine is examined using a variety of criteria that include the number of headache days, the number of hours during which headaches occur (i.e., headache hours), the severity of the headaches, and the number of migraine days observed in the individual.

Example 10: Treatment of Chronic Migraine

[00402] A 37-year-old female human subject is identified as having had chronic migraine for at least three months. Identification of having had chronic migraine is achieved by noting a history of frequent headaches suggestive of chronic migraine (e.g., 15 days per month) for at least three months prior to screening. Verification of headache frequency is achieved through prospectively collected baseline headache information on at least 15 days, with at least 8 days per month meeting any of the following: i. qualifying as a migraine attack; and/or ii. preceded or accompanied by migraine aura.

[00403] In order to reduce the incidence of migraine in the subject, the subject is administered an initial loading dose of 675 mg of antagonistic anti-CGRP antibody (e.g., G1 antibody). The antagonistic anti-CGRP antibody is provided as a liquid formulation at a concentration of 150 mg/mL. The 675 mg loading dose is administered as three 225 mg 1.5 mL subcutaneous injections to various regions (e.g., back of upper arms, lower abdomen/belly/waist, front of thighs, etc.) of the subject's body. Dosing is repeated every twenty-eight days at 225 mg (e.g., via a 1.5 mL subcutaneous injection in the subject's upper arm) until a reduction in the incidence of migraine is observed. A reduction in the incidence of chronic migraine is examined using a variety of criteria that include the number of headache days, the number of hours during which headaches occur (i.e., headache hours), the severity of the headaches, and the number of migraine days observed in the individual.

Example 11: Treatment of Chronic Migraine

[00404] A 23-year-old male human subject is identified as having had chronic migraine for at least three months. Identification of having had chronic migraine is achieved by observing a history of frequent headaches suggestive of chronic migraine (e.g., 15 days per month) for at least three months prior to screening. Verification of headache frequency is achieved through prospectively collected baseline headache information on at least 15 days, with at least 8 days per month meeting any ONE of the following: i. qualifying as a migraine attack; and/or ii. preceded or accompanied by migraine aura.

[00405] In order to reduce the incidence of migraine in the subject, the subject is administered a 900 mg dose of an anti-CGRP antagonist antibody (e.g., G1 antibody). The anti-CGRP antagonist antibody is provided as a liquid formulation at a concentration of 150 mg/mL. The 900 mg dose is administered as four 225 mg x 1.5 mL subcutaneous injections to various regions (e.g., back of upper arm, lower abdomen/belly/waist, front of thighs, etc.) of the subject's body. Dosing is repeated every twenty-eight days until a reduction in the incidence of migraine is observed. A reduction in the incidence of chronic migraine is examined using a variety of criteria that include the number of headache days, the number of hours during which headaches occur (e.g., headache hours), the severity of the headaches, and the number of migraine days observed in the subject.

Example 12: Treatment of Episodic Migraine

[00406] A 28-year-old male human subject is identified as having high-frequency episodic migraine. Subjects are identified as having high-frequency episodic migraine using criteria that include: having a history of headaches on more than 8 days per month for at least 3 months prior to screening; and verification of headache frequency through prospectively collected baseline information demonstrating headaches (of any type) of 8 to 14 days, with at least 8 days meeting criteria for at least one of the following: i. migraine; ii. probable migraine; and/or iii. use of triptan or ergotamine compounds.

[00407] In order to reduce the incidence of migraine in the subject, the subject is administered a 675 mg dose of an anti-CGRP antagonist antibody (e.g., G1 antibody). The anti-CGRP antagonist antibody is provided as a liquid formulation at a concentration of 150 mg/mL. The 675 mg dose is administered as three 225 mg x 1.5 mL subcutaneous injections to various regions (e.g., back of upper arm, lower abdomen/belly/waist, front of thighs, etc.) of the subject's body. Dosing is repeated every twenty-eight days until a reduction in the incidence of migraine is observed. A reduction in the incidence of episodic migraine is examined using a variety of criteria that include the number of headache days, the number of hours during which headaches occur (i.e., headache hours), the severity of the headaches, and the number of migraine days observed in the individual.

Example 13: Treatment of Episodic Migraine

[00408] A 52-year-old female human subject is identified as having high-frequency episodic migraine. Subjects are identified as having high-frequency episodic migraine using criteria that include: having a history of headaches on more than 8 days per month for at least 3 months prior to screening; and verification of headache frequency through prospectively collected baseline information demonstrating headaches (of any type) of 8 to 14 days, with at least 8 days meeting criteria for at least one of the following: i. migraine; ii. probable migraine; and/or iii. use of triptan or ergotamine compounds.

[00409] In order to reduce the incidence of migraine in the subject, the subject is administered a 225 mg dose of an anti-CGRP antagonist antibody (e.g., G1 antibody). The anti-CGRP antagonist antibody is provided as a liquid formulation at a concentration of 150 mg/mL. The 225 mg dose is administered as a 1.5 mL subcutaneous injection into the back of one upper arm of the subject's body. Dosing is repeated every twenty-eight days until a reduction in the incidence of migraine is observed. A reduction in the incidence of episodic migraine is examined using a variety of observations that include the number of headache days, the number of hours during which headaches occur, the severity of the headaches, and the number of migraine days in the subject.

Example 14: Nonclinical Toxicology and Pharmacokinetics

[00410] Anti-CGRP G1 antagonist antibody was well tolerated in 1-month IV repeat-dose toxicity studies in Sprague-Dawley (SD) rats and cynomolgus monkeys and no target organ toxicity was determined in either of these studies. No observed adverse event level (NOAEL) of 100 mg/kg/week was established for both rat and monkey studies. This dose level corresponded to systemic exposure, with a maximum concentration (Cmax) of 2570 and 3440 μg/mL and areas under the curve (AUC(0-168h)) of 194000 μgΦ/mL and 299000 μgΦ/mL (Day 22) in rats and monkeys, respectively.

[00411] In a 3-month IV/SC rat study, no target organ toxicities were identified and G1 was well tolerated up to the highest dose tested, 300 mg/kg. In a 3-month monkey study, perivascular inflammation of the ciliary artery as a result of immune complex deposition was observed at ^100 mg/kg. This finding was attributed to the monkey's immunogenic response to a humanized antibody and was not considered to be clinically relevant. The highest dose tested of 300 mg/kg in the present monkey study is at least 10-fold higher than the highest anticipated clinical dose of 2000 mg or 29 mg/kg on a mg/kg basis (assuming an average individual weight of 70 kg).

Example 15: Clinical Pharmacokinetics

[00412] The PK of G1 antibody after single IV exposure was examined in four randomized, double-blind, placebo-controlled studies examining doses ranging from 10 to 2,000 mg. Maximum plasma concentrations (Cmax) were achieved shortly after the end of the 1-hour intravenous infusion. The median time to Cmax (Tmax) ranged from 1.0 to 3.0 hours, followed by a multiphasic decline. Cmax and total exposure increased in a nearly linear fashion with increasing doses of G1. Terminal half-life (t%) ranged from 36.4 to 48.3 days. There is no evidence of G1 metabolism in the liver; the primary mode of metabolism is by proteasomal degradation.

[00413] A study defined the pharmacokinetics of 30 mg and 300 mg doses administered twice, two weeks apart. Maximum concentrations and the area under the concentration-time profile increased with increasing dose. The apparent terminal half-life (t%) after the second dose was 41.2 days (30 mg) and 50.0 days (300 mg) (arithmetic mean). The plasma accumulation ratios of G1 after two doses administered IV 15 days apart were 1.5 (30 mg) and 1.4 (300 mg).

Example 16: Clinical Safety and Pharmacokinetics

[00414] In six studies, G1 antibody was administered to 118 healthy men and women, while 57 male and female subjects received placebo. The study included single IV doses ranging from 0.2 mg up to 2,000 mg, two IV doses of up to 300 mg administered once every 14 days, and SC administration of 225 and 900 mg. The six studies included: two single IV escalating dose PK and pharmacodynamic (PD) studies in healthy men (studies B0141001 and B0141002); a double-cohort placebo-controlled crossover study to examine the acute effects of IV administration of G1 antibody on the capsaicin dilation response in healthy volunteers (B0141006); a parallel-group repeated dose study of G1 antibody in healthy male and female volunteers (B0141007); a single-dose study evaluating the safety and tolerability of doses up to 2,000 mg administered IV to healthy female volunteers (B0141008), and a study comparing the relative safety and bioavailability between IV and SC administration (G1-SC-IV).

[00415] The six studies are summarized below in Table 11. Of the five IV studies (B0141001, B0141002, B0141006, B0141007, and B0141008), three had virtually identical designs and assessments. Study B014100 tested doses of 0.2 mg, 1 mg, and 3 mg given as a single one-hour IV infusion. The study had a parallel design. Participants were confined to the clinic for seven days following infusion, with multiple assessments on each of those days. Following discharge, patients were reassessed one week after discharge (day 14), and then one, two, and three months post-infusion. Study B0141002 tested doses ranging from 10 mg to 1000 mg as a single administration. Finally, Study B0141008 tested doses of 300 mg, 1000 mg, 1500 mg, or 2000 mg. Study B0141006 was different from the others as it also aimed to integrate pharmacodynamic readouts by measuring capsaicin inhibition of dilation up to one week after IV infusion of the G1 antibody.

[00416] For IV studies, adverse event (AE) profiles were reported only for the first dosing period. Study B0141007 tested various doses of G1 antibody at 30 or 300 mg IV given two weeks apart using a parallel design. Each eligible subject was assigned a randomization sequence through an interactive web-based system that contained treatment assignment. The randomization scheme was developed by the chief statistician. Participants in all studies were generally healthy men and women (18 to 65 years of age); all participants gave written informed consent. All studies were approved by the appropriate research review boards (IRBs). AEs were defined as any untoward medical occurrence in clinical trial participants with or without a causal relationship to study drug. AEs observed after administration of study drug or placebo were termed “treatment-emergent” AEs (TEAEs) regardless of the potential for causality with study drug. All subjects experiencing TEAEs were followed at appropriate time intervals until the event had resolved or until the event had stabilized and/or reached a new baseline. All TEAEs were classified as mild, moderate, or severe. Serious AEs (SAEs) were defined a priori as any untoward medical event that at any dose resulted in death, was life-threatening (i.e., the subject was at imminent risk of death at the time of the event), required hospitalization or prolongation of existing hospitalization, resulted in persistent disability/incapacity (i.e., a substantial disruption of the subject's ability to perform normal life functions), resulted in a birth anomaly/defect, or any other clinically important event. Treatment-related AEs (TRAEs) were considered when one of the following was present: 1) a temporal relationship between the first symptoms of the AE and administration of the investigational drug could be identified; 2) the AE could not be readily explained by the patient's clinical status, intercurrent illness, or concomitant therapies; 3) AE decreased with discontinuation of investigational product or dose reduction.

[00417] Blood pressure, heart rate, and oral temperature were measured at screening, pre-dose, immediately after the end of the infusion, and multiple times during patient confinement in the clinics, as well as at all clinic visits. Laboratory tests included serum chemistries, hematology, and urinalysis. Hematology, chemistry, coagulation, and urine safety laboratory tests were performed at multiple study times. ECGs were recorded at screening, pre-dose on Day 1, immediately after the end of the infusion, and five additional times during the first day, as well as at all clinic visits. QTcF values were derived using the Fridericia heart rate correction (QTcF) formula. Absolute values and changes from baseline for the ECG parameters QT interval, heart rate, QTcF interval, PR interval, and QRS interval were assessed by cohort, treatment, and time post-dose. In addition to the safety assessments described above, Protocol B014008 included comprehensive ophthalmic assessments at baseline and at three time points after dosing (Day 28, Day 84, and Day 168).

[00418] Clinical data and vital signs were summarized using descriptive tables and summary statistics. Laboratory and other safety data were summarized as a function of any abnormalities (values outside the reference range), as well as any clinically relevant abnormalities, which were defined a priori. Summary tables were stratified by dose, and data were pooled across studies. In addition, comparisons of consolidated data for all G1 antibody exposures were contrasted with placebo. Placebo was also contrasted with G1 antibody doses of 100 mg and higher (100 mg, 300 mg, 1000 mg, 1500 mg, and 2000 mg), and with G1 antibody doses of 1000 mg and higher (1000 mg, 1500 mg, and 2000 mg).

[00419] In the IV/SC (G1-SC-IV) study, thirty-six subjects were randomized to receive a single administration of the G1 antibody (225 or 900 mg) or placebo, delivered either as a subcutaneous (SC) bolus injection or as a 1-hour IV infusion. Subjects were confined to the clinical research unit for seven days following dosing, and returned to the clinic periodically for additional outpatient visits through study day 90. ECGs were performed extensively on Day 1 (pre-dose, Hours 1, 6, 12), Day 3, Day 7 while subjects were confined, and once at the end of the study (Day 90). Vital signs, including temperature, blood pressure, and heart rate, were collected pre-dose, Days 1, 3, 7, and 90. Table 11

[00420] Across the wide range of dosages evaluated in the five IV studies (0.2 to 2,000 mg), IV G1 antibody was acceptably tolerated. Table 8 summarizes the overall adverse event (AE) rate per dose for the IV studies. Based on these tolerability results, no overt safety concerns emerged. Across all trials in the IV studies, participants receiving placebo reported an average of 1.3 treatment-emergent adverse events (TEAEs). These are all events reported regardless of the investigator's opinion of relationship to study drug. Across all IV G1 doses, the rate was 1.4 TEAEs/subject. Subjects receiving G1 doses of 100 mg or higher had an average of 1.5 TEAEs; those receiving doses of 1,000 mg or higher had an average of 1.6 TEAEs. Table 8

[00421] AE = adverse events; n = any event, treatment related or not; (N) = treatment related by the investigator. Note: For protocol B0141006 (placebo and 300 mg), only data for the first active treatment period were included, due to its cross-over nature.

[00422] In IV studies, treatment-related adverse events (TRAEs or AEs that may be related to therapy according to the principal investigator) were reported in 21.2% of subjects receiving IV G1, compared with 17.7% in those receiving placebo. At doses of 100 mg G1 or higher, TRAEs occurred in 22.4% of subjects. At doses of 1,000 mg or higher, TRAEs occurred in 21.7% of subjects. The G1 antibody did not appear to be associated with any clinically relevant patterns of changes in vital signs (systolic and diastolic blood pressure [BP], temperature, and heart rate [HR]), electrocardiogram (ECG) abnormalities (including QTcB and QTcF), infusion site reactions, or clinical laboratory findings. There were limited effects on liver function tests (aspartate aminotransferase [AST], alanine aminotransferase [ALT], total bilirubin, and alkaline phosphatase), with a Grade 1 increase in total bilirubin in one subject receiving placebo (Study B0141001), and a Grade 1 increase in ALT in one subject receiving placebo (Study B0141002). Clinically significant liver function changes were not observed among subjects receiving any of the doses of G1 studied. There was no evidence of differences between G1 and placebo in hematologic tests assessing renal function, electrolytes, or in urine tests.

[00423] In the IV/SC (G1-SC-IV) study, safety and tolerability were comparable between SC and IV routes of delivery. Mean heart rate and blood pressure (diastolic and systolic) were not affected by G1 antibody treatment, nor were there any significant changes in any of the cardiovascular parameters following SC G1 antibody treatment. A summary of the TRAEs observed during the SC study is shown below in Table 12. Table 12

[00424] In single-dose studies (B0141001, B0141002, B0141006 and B0141008), pharmacokinetic (PK) parameters were calculated for doses ranging from 30 mg to 2,000 mg. Mean group terminal half-life (t1/2) ranged from approximately 40 to 48 days. Cmax and total exposure (assessed by AUCinf) increased with increasing dose. The increase in AUCinf appeared to be approximately dose proportional between 30 and 1,000 mg and appeared to be greater than dose proportional between 1,000 and 2,000 mg. The volume of distribution was small, between 6-10 L.

[00425] In the two-dose study (B0141007), the apparent terminal half-life after a second dose ranged from 41 to 50 days. Plasma concentrations accumulated after the second dose, with an accumulation ratio of approximately 1.5. Furthermore, in the IV/SC (G1-SC-IV) study, pharmacokinetic evaluations indicated that G1 had a similar terminal half-life when given SC as IV.

Example 17: Prevention of chronic migraine in a G1 antibody clinical trial

[00426] A multicenter, randomized, double-blind, double-dummy, parallel-group, placebo-controlled, multiple-dose study comparing anti-CGRP G1 antagonist antibody to placebo was conducted in subjects with chronic migraine. Eligible subjects entered a baseline, 28-day pre-treatment period. Subjects had their headache and health information captured daily throughout the study using an electronic headache diary system. No changes in migraine medications were permitted during the study or pre-treatment period. Inclusion criteria were as follows: (1) males or females aged 18 to 65 years; (2) a signed and dated informed consent document indicating that the subject had been informed of all pertinent aspects of the study, including any known and potential risks and available alternative treatments; (3) chronic migraine meeting the diagnostic criteria listed in the International Classification of Headache Disorders (ICHD-III beta version, 2013); (4) subjects may use up to two different daily migraine preventive medications for migraine (e.g., topiramate, propranolol, amitriptyline) or for other medical conditions (e.g., propranolol being used for hypertension) if the dose and regimen have been stable for at least 2 months prior to the start of the 28-day run-in period; (5) Body Mass Index (BMI) of 17.5 to 34.5 kg/m2, and a total body weight between 50 kg and 120 kg, inclusive; (6) subject is either not of reproductive potential as defined in the Methods or if subject is of reproductive potential, they agree either to remain abstinent or to use (or have their partner use) an acceptable method of birth control for the anticipated duration of the study; (7) demonstrate compliance with the electronic headache diary during the pre-treatment headache data entry period for a minimum of 24/28 days (85% compliance). Chronic migraine diagnostic criteria in (3) above were as follows: (a) history of frequent headaches suggesting chronic migraine (15 days per month) for at least three months prior to screening; (b) verification of headache frequency through prospectively collected baseline information during the 28-day pre-treatment phase demonstrating headaches on at least 15 days, with at least 8 days per month that meet either of (i) qualifying as a migraine attack, (ii) preceded or accompanied by migraine aura, or (iii) relieved by ergotamine derivatives or triptans.

[00427] Exclusion criteria require that subjects do not meet any of the following: (1) Onset of chronic migraine after the age of 50 years; (2) Subject has received onabotulinumtoxinA for migraine or for any medical or cosmetic reasons requiring injections in the head, face, or neck during the six months prior to screening; (3) Subject uses medications containing opioids (including codeine) or barbiturates (including Fiorinal®, Fioracet®, or any other combination containing butalbital) on more than 4 days per month for the treatment of migraine or for any other reason; (4) Failed >2 medication categories or >3 preventive medications (within two medication categories) due to lack of efficacy for prophylactic treatment of episodic or chronic migraine after an adequate therapeutic trial; (5) clinically significant hematologic, renal, endocrine, pulmonary, gastrointestinal, genitourinary, neurologic, or ocular disease at the discretion of the investigator; (6) individuals with evidence or clinical history of clinically significant psychiatric problems, including major depression, panic disorder, or generalized anxiety disorder (according to criteria from the Diagnostic and Statistical Manual, Fifth Edition [DSM-5]); (7) screening systolic blood pressure greater than 160 mm Hg or less than 90 mm Hg; (8) screening diastolic blood pressure greater than 110 mm Hg or less than 50 mm Hg; (9) history of clinically significant cardiovascular disease or vascular ischemia (such as myocardial, neurologic [e.g., cerebral ischemia], extremity ischemia, or other ischemic event); (10) history of prior or current cancer, with the exception of individuals with basal cell carcinoma that has been excised; (11) pregnant or lactating women; (12) History of hypersensitivity reactions to injected proteins, including monoclonal antibodies; (13) Treatment with an investigational drug within 30 days of study entry; (14) Clinically significant abnormality on the baseline 12-core ECG, including sinus pauses >2 seconds, second or third heart block, or other abnormalities judged clinically significant by the investigator; (15) A baseline 12-core ECG demonstrating QTcF >450 msec for men and 470 msec for women at screening (if QTcF exceeded these values, the ECG was repeated and the average of the three QTcF values used to determine the subject's potential eligibility; QTc obtained using Fridericia calculation); (16) any finding that in the judgment of the investigator is clinically significantly abnormal, including hematology values, blood chemistries, coagulation tests, or urinalysis (abnormal tests were allowed to be repeated for confirmation); (17) liver enzymes (alanine aminotransferase [ALT], aspartate aminotransferase [AST], alkaline phosphatase) >1.3 times the upper limit of normal (ULN) after confirmation on a repeat test; (18) serum creatinine >1.5 times the ULN, clinically significant proteinuria (urine dipstick +4), or evidence of renal disease.

[00428] Subjects confirmed to have chronic migraine and high compliance with the daily pain diary during the pretreatment period were randomized at visit 2 (day 1) to one of three treatment arms. Randomization was performed using an electronic, interactive web-based response system. Subjects were stratified based on gender and baseline migraine medication use. Treatment was administered once monthly (every 28 days) for a total of three treatments over a 3-month period. Treatment administration occurred at visit 2 (day 1; first dose), visit 3 (day 29; second dose), and visit 4 (day 57; the third and final dose). Final study exit assessments were performed at visit 5 (day 85), approximately 28 days after the third and final dose. Injections were administered subcutaneously over a 3-month period (every 28 days) to subjects in each of the following groups: (1) those randomized to the 900 mg arm received four active injections every 28 days; (2) those randomized to the 675/225 mg arm received three active and one placebo injection during the first treatment, and one active and three placebo injections for the second and third treatments; (3) those randomized to placebo received four placebo injections every 28 days. Active injections contained 225 mg of G1 antibody. Endpoints were obtained from an electronic daily pain diary, an interactive web-based system that recorded data for the previous 24-hour period. Overall headache duration was recorded numerically in hours, as well as the number of hours with headache at each level of severity. Headache severity was subjectively assessed by the subject at predefined time points as follows: no pain, mild pain, moderate pain, and severe pain. Subjects were also asked to record whether the following associated symptoms were present or absent at predefined time points: photophobia, phonophobia, nausea, and vomiting. An additional endpoint was derived from monitoring the use of triptans (e.g., sumatriptan) as an acute anti-headache medication by study subjects throughout the course of the study. A summary of the disposition and demographics of study subjects is presented in Table 9. Table 9

[00429] The mean decrease in number of headache hours relative to baseline at each of Weeks 1, 2, and 3 (W1, W2, and W3, respectively) for each of the groups is graphically represented in FIGURE 15. The results show a significant decrease in both treatment groups relative to the placebo group at each of W1, W2, and W3, including the first week.

[00430] The mean decrease in number of headache hours relative to baseline at each of Months 1, 2, and 3 (M1, M2, and M3, respectively) for each of the groups is graphically represented in FIGURE 12. The results show a significant decrease in both treatment groups relative to the placebo group at all three time points, including after the first dose. Statistical significance relative to the placebo group for the data in Figure 12 is provided by the indicated p-values.

[00431] Mean number of headache hours in each group at baseline and at visits 2, 3, and 4 (V2, V3, and V4, respectively) is graphically represented in FIGURE 13. The results show a significant decrease in both treatment groups relative to the placebo group at all three time points, including after the first dose.

[00432] The mean decrease in number of hours of moderate or severe headache from baseline at each of Months 1, 2, and 3 (M1, M2, and M3, respectively) for each of the groups is depicted in FIGURE 14. The results show a statistically significant decrease in both treatment groups relative to the placebo group at all three time points, including after the first dose. Statistical significance relative to the placebo group is provided by the indicated p-values.

[00433] The mean decrease in number of uses of triptans as an acute rescue medication relative to baseline at each of Months 1, 2, and 3 (M1, M2, and M3, respectively) for each of the groups is graphically represented in FIGURE 16. The results show a significant decrease in triptan use in both treatment groups relative to the placebo group at all three time points, including after the first dose. Statistical significance relative to the placebo group is provided by the p-values shown in FIGURE 16.

[00434] A significant decrease in the number of headache hours was also observed in subjects using preventive medications (e.g., topiramate and amitriptyline or propranolol) relative to a placebo group.

[00435] Both doses were well tolerated, and no safety concerns emerged. A summary of treatment-emergent adverse events (TEAE) by group is provided in Table 10. The difference in related TEAE is almost entirely explained by mild injection-related events (erythema, some discomfort). Serious TEAE were not drug-related (no drug-related adverse events). Table 10

Example 18: Prevention of high-frequency episodic migraine in a G1 antibody clinical trial

[00436] A multicenter, randomized, double-blind, placebo-controlled, parallel-group study comparing anti-CGRP antibody G1 with placebo was conducted in subjects with high-frequency episodic migraine (HFEM). The study design followed that of Example 17, with two differences. First, the inclusion criteria (3) was for subjects meeting the criteria for episodic migraine according to the second edition of The International Headache Society (Olesen and Steiner 2004), who experience high-frequency migraine as follows: (a) History of headaches on more than 8 days per month for at least 3 months prior to screening; (B) ascertainment of headache frequency through prospectively collected baseline information during the 28-day pretreatment phase demonstrating headache (of any type) for 8 to 14 days, with at least 8 days meeting criteria for at least one of (i) migraine, (ii) probable migraine, or (iii) use of triptan or ergotamine compounds. Second, dosing schedules were altered for the groups receiving G1. Specifically, injections were administered subcutaneously over a 3-month period (every 28 days) to subjects in each of the following groups: (1) those randomized to the 675 mg arm received 675 mg of G1 every 28 days; (2) those randomized to the 225 mg arm received 225 mg of G1 every 28 days; and (3) those randomized to placebo received a placebo injection every 28 days. A summary of the disposition and demographics of study subjects is presented in Table 13. Study endpoints included reduction in the number of migraine days and reduction in the number of headache days of any severity.Table 13

[00437] The mean decrease in number of migraine days relative to baseline at each of Months 1, 2, and 3 (M1, M2, and M3, respectively) for each of the groups is depicted in FIGURE 17. The results show a statistically significant decrease in both treatment groups relative to the placebo group at all three time points, including after the first dose. Statistical significance relative to the placebo group is provided by the indicated p-values.

[00438] The mean decrease in number of headache days of any severity relative to baseline at each of Months 1, 2, and 3 (M1, M2, and M3, respectively) for each of the groups is depicted in FIGURE 18. The results show a statistically significant decrease in both treatment groups relative to the placebo group at all three time points, including after the first dose. Statistical significance relative to the placebo group is provided by the indicated p-values.

[00439] Both doses were well tolerated, and no safety concerns emerged. A summary of treatment-emergent adverse events (TEAE) by group is provided in Table 14. The four serious TEAEs were due to one case of fibular fracture, one case of tremor due to medication discontinuation, and two cases of migraine requiring emergency room (ER) treatment. Table 14

Example 19: Non-clinical safety

[00440] Two studies evaluating the safety of G1 antibody were conducted in cynomolgus monkeys. In the first study, the safety of a single dose of G1 antibody was evaluated. In the second study, the safety of repeat doses of G1 antibody was evaluated. Each of the studies and their results are further described in detail below. For both the single and repeat dose studies, G1 antibody was formulated as a 51.4 mg/mL solution in 20 mM histidine, 84 mg/mL trehalose dihydrate, 0.2 mg/mL polysorbate 80, 0.05 mg/mL disodium EDTA dihydrate, and 0.1 mg/mL L-methionine, at pH ~5.5. Vehicle was formulated identically without G1 antibody. Furthermore, in both studies, blood samples were collected periodically for analysis of plasma G1 antibody concentration using a validated ELISA method.

[00441] Data were first aggregated into summary tables and figures using GraphPad Prism (version 6.0) and Excel 2010 (Microsoft). For single exposure study, telemetry data were analyzed using ANOVA. Analysis was performed using SAS Release 8.2. In order to normalize the QT interval over an R-R interval

[00442] intervals, Individual Animal Correction Factors (IACFs) were generated for each animal by relating each RR-interval to its associated QT interval. A linear regression of the QT/RR-interval relationship was determined for the data set. The slope of this linear regression was used as the IACF for the associated animal across all treatments. This IACF was used to calculate the corrected QT interval (QTc) using the following equation:

[00443] QT-I(c) = QT interval corrected for heart rate = QT-I - [(RR - 300)*(IACF)].

[00444] For the multiple dose study, 1-way ANOVA was also used to analyze the data. If the analysis of variance was significant (P < 0.05), Dunnett's post-hoc test was used for comparisons between groups. For each sex, the treated group was compared with the control (vehicle) group at the 5% two-tailed probability level.

Single dose telemetry study

[00445] Eight adult male cynomolgus monkeys (Charles River Primates) were surgically instrumented with rangefinders and allowed to recover for at least two weeks. Implants (DSI TL11M2-D70-PCT) and receivers (RMC-1) were manufactured by Data Sciences International.

[00446] Animals were acclimated to telemetry data acquisition cages at least overnight prior to dosing. During acclimation, pre-study recording of hemodynamic parameters was performed to verify that transducers and equipment were functioning properly. During telemetry data acquisition, animals were housed individually in cages equipped with telemetry receivers. On non-collection days, animals were housed in cages without telemetry receivers. Animals were maintained on a daily 12-hour light/12-hour dark cycle, with water ad libitum, and fed a certified primate diet.

[00447] For the first phase of the study, animals (8 males) were administered vehicle only, and telemetry data were collected beginning ~1 hour pre-dose through 22 hours post-dose. Six days after vehicle administration, the same animals received a single IV administration of G1 antibody (100 mg/kg, a dose ~10-fold higher than the pharmacological EC50 in cynomolgus monkeys). Telemetered electrocardiographic and hemodynamic data were again continuously recorded from all animals. Additionally, these animals were monitored for ~24 hours on days 3, 7, 10, and 14 after receiving the single dose of G1 antibody. ECG telemetry and blood pressure signals were transmitted via the implanted radio-telemetry devices to receivers mounted in each cage. The acquired signals were passed through a data exchange matrix (DSI DEM model) and into a PC-based data acquisition system (DSI Ponemah P3 software version 3.4); the data analysis software was Emka Technologies version 2.4.0.20

[00448] (Emka Technologies). The analog/digital sampling rate was 1,000 Hz for the telemetered ECG data and 500 Hz for the blood pressure data. Data were recorded as 1-min averages.

[00449] Group mean systolic blood pressure (SBP) was similar before and after G1 antibody treatment throughout the first day after administration and on subsequent days (animals telemetered on days 3, 7, 10, and 14 at identical time intervals as day 1). At hours 1-4 after administration when G1 antibody blood concentrations were at peak levels (mean concentration of 3,500 mg/mL at 4 hours), mean SBPs were 111 mmHg, compared to 113 mmHg at the same time interval after vehicle administration. Additionally, SBP was 110 mmHg on days 3 and 7, 109 mmHg on day 10, and 110 mmHg on day 14 following G1 antibody administration. Similar SBP data were recorded for other time intervals. Since this was a crossover study design, the treated animals served as their own controls. When the data were analyzed as differences in blood pressure after administration of the G1 antibody compared with vehicle treatment, there are small statistically significant reductions in SBP at the last time point on days 7, 10, and 14.

[00450] Following G1 antibody treatment, diastolic blood pressure (DBP) was noted to be approximately 3 mmHg lower than mean values obtained following vehicle administration. From hours 5-22, the group means for the vehicle and G1 antibody groups were similar. The same trend was observed on other days, when a slight decrease in DBP (ranging from 2.62-3.5 mmHg) occurred in the first measured interval, with some changes of similar magnitude seen sporadically on days 7-10 in the interval of hours 7-22. Similar to what was seen for DBP, small decreases in heart rate were seen during the first assessment (hours 1-4) relative to vehicle treatment. Differences were undetectable during the interim assessments and were once again seen between hours 18-22 on all days.

[00451] Furthermore, with respect to ECG results, there were no statistically significant changes in QTc interval at any time point, relative to vehicle treatment. Although statistically significant changes in RR, PR, RS and QT were seen over the 14 day period, when compared to vehicle, they were all smaller in absolute value.

Repeated dose safety study

[00452] The repeat-dose safety study included 48 adult, sex-matched (6 per sex per group) G1 antibody-naïve cynomolgus monkeys (Charles River Primates). Animals received vehicle or G1 antibody as an intravenous injection once weekly for 14 weeks at doses of 10 mg/kg, 100 mg/kg, or 300 mg/kg. In each group, two animals of each sex were allowed to recover for an additional 4 months after the end of dosing. ECG and blood pressure measurements were recorded once during the pre-study phase, twice after steady state was achieved (prior to dosing and 4 hours post-dose on day 85), and once ~1 week after the end of dosing (day 103 of the recovery phase). Animals were anesthetized with ketamine and ECGs were recorded using eight leads. ECG measurements (including heart rate) were performed using data captured using the Life Science Suite Ponemah Physiology Platform system via DSI, using leads I, II, aVF, CG4RL and CV4LL as standard. Heart rate correction for QT interval (QTc) was calculated using the Bazett formula.

[00453] Blood pressure was recorded prior to the first dose, after 12 weeks of dosing (13 doses), and approximately 1 week after the end of dosing. No significant changes were noted in SBP or DBP in any of the treated animal groups relative to vehicle-treated animals. Group mean heart rates were relatively consistent across dose groups and time points measured, with no statistical differences measured. Plasma G1 antibody concentrations were measured during the first week of dosing and at the time of blood pressure and ECG assessments, demonstrating accumulation with repeated weekly dosing.

[00454] Furthermore, with respect to ECG, there were no significant differences in QT interval across all doses and time points. Furthermore, no significant or relevant changes in ECG were observed for any of the ECG parameters assessed throughout the study.

[00455] In summary, the G1 antibody was very well tolerated in both studies, with no clinically significant changes observed in any hemodynamic parameter, nor any relevant changes observed in any ECG parameter. In cynomolgus monkeys, cardiovascular and hemodynamic parameters do not appear to be affected by long-term inhibition of CGRP with G1 antibody.

[00456] It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in the light will be suggested to persons skilled in the art and are intended to be included within the meaning and scope of this application. All publications, patents and patent applications herein are incorporated by reference in their entirety, for all purposes, to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

Biological Material Deposit

[00457] The following materials have been deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia 20110-2209, USA (ATCC):

[00458] Vector pEb.CGRP.hKGI is a polynucleotide encoding the G1 light chain variable region and the kappa light chain constant region; and vector pDb.CGRP.hFcGI is a polynucleotide encoding the heavy chain variable region and the G1 constant region of the IgG2 heavy chain containing the following mutations: A330P331 to S330S331 (amino acid numbering with reference to the wild-type IgG2 sequence; see Eur. J. Immunol. (1999) 29:2613-2624).

[00459] These deposits were made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Micro-Organisms for the Purposes of Patent Procedure and its Regulations (Budapest Treaty). This ensures the maintenance of a viable deposit culture for 30 years from the date of deposit. The deposit will be made available by the ATCC under the terms of the Budapest Treaty, and subject to an agreement between Rinat Neuroscience Corp. and the ATCC, which ensures the permanent and unrestricted availability of the progeny of the deposit culture to the public after grant of the relevant U.S. patent or after opening to the public of any U.S. or foreign patent application, whichever occurs first, and ensures the availability of the progeny to anyone determined by the U.S. Commissioner of Patents and Trademarks to be entitled thereto in accordance with 35 U.S.C. Section 122 and the Commissioner's rules thereunder (including 37 C.F.R. Section 1.14 with particular reference to 886 O.G. 638).

[00460] The assignee of this application has agreed that if a culture of the deposited materials should die or be lost or destroyed when cultivated under suitable conditions, the materials will be promptly replaced upon notice with another of the same. The availability of the deposited material shall not be construed as a license to practice the invention in contravention of the rights granted under the authority of any government in accordance with its patent laws.

[00461] Antibody Sequences

[00462] Heavy chain amino acid sequence variable region G1 (SEQ ID NO:1)

[00463] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKG LEWVAEIRSESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAY FDYGLAIQNYWGQGTLVTVSS

[00464] Light chain amino acid sequence variable region G1 (SEQ ID NO: 2)

[00465] EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPR LLIYGASNRYLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGT KLEIK

[00466] G1 CDR H1 (Extended CDR) (SEQ ID NO:3)

[00467] GFTFSNYWIS

[00468] G1 CDR H2 (extended CDR) (SEQ ID NO:4)

[00469] EIRSESDASATHYAEAVKG

[00470] G1 CDR H3 (SEQ ID NO:5)

[00471] YFDYGLAIQNY

[00472] G1 CDR L1 (SEQ ID NO:6)

[00473] KASKRVTTYVS

[00474] G1 CDR L2 (SEQ ID NO:7)

[00475] GASNRYL

[00476] G1 CDR L3 (SEQ ID NO:8)

[00477] SQSYNYPYT

[00478] G1 heavy chain nucleotide sequence variable region (SEQ ID NO:9)

[00479] gaagttcagctggttgaatccggtggtggtctggttcagccaggtggttccctgcgtctg- tcctgcgctgcttccggtttcaccttctccaactactggatctcctgggttcgtcaggctcctggtaaaggtctggaatgggt tgctgaaatccgttccgaatccgacgcgtccgctacccattacgctgaagctgttaaagg-tcgtttcaccatctcccgtgacaacgctaagaactccctgtacctgcagatgaactccctgcgtgctgaagacaccgct gtttactactgcctggcttactttgactacggtctggctatccagaactactggggtcaggg- taccctggttaccgtttcctcc

[00480] G1 light chain nucleotide sequence variable region (SEQ ID NO:10)

[00481] gaaatcgttctgacccagtccccggctaccctgtccctgtccccaggtgaacgtgctac- cctgtcctgcaaagcttccaaacgggttaccacctacgtttcctggtaccagcagaaacccggtcaggctcctcgtctg ctgatctacggtgcttccaaccgttacctcggtatcccagctcgtttctccggttccggtt-ccggtaccgacttcaccctgaccatctcctccctggaacccgaagacttcgctgtttactactgcagtcagtcctacaact acccctacaccttcggtcagggtaccaaactggaaatcaaa

[00482] Amino acid sequence of complete G1 heavy chain antibody (including modified IgG2 as described herein) (SEQ ID NO: 11)

[00483] EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKG LEWVAEIRSESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAY FDYGLAIQNYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSN TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCK VSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPMLDSDGSFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK

[00484] Complete amino acid sequence of antibody light chain G1 (SEQ ID NO:12)

[00485] EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPR LLIYGASNRYLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGT KLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[00486] Nucleotide sequence of complete heavy chain antibody G1 (including modified IgG2 as described herein) (SEQ ID NO: 13)

[00487] gaagttcagctggttgaatccggtggtggtctggttcagccaggtggttccctgcgtctg- tcctgcgctgcttccggtttcaccttctccaactactggatctcctgggttcgtcaggctcctggtaaaggtctggaatgggt tgctgaaatccgttccgaatccgacgcgtccgctacccattacgctgaagctgttaaagg-tcgtttcaccatctcccgtgacaacgctaagaactccctgtacctgcagatgaactccctgcgtgctgaagacaccgct gtttactactgcctggcttactttgactacggtctggctatccagaactactggggtcaggg-taccctggttaccgtttcctccgcctccaccaagggcccatctgtcttcccactggccccatgctcccgcagcacctccg agagcacagccgccctgggctgcctggtcaaggactacttcccagaacctgtgaccgtg-tcctggaactctggcgctctgaccagcggcgtgcacaccttcccagctgtcctgcagtcctcaggtctctactccctcag cagcgtggtgaccgtgccatccagcaacttcggcacccagacctacacctgcaacgtagat-cacaagccaagcaacaccaaggtcgacaagaccgtggagagaaagtgttgtgtggagtgtccaccttgtccagccc ctccagtggccggaccatccgtgttcctgttccctccaaagccaaaggacac-cctgatgatctccagaaccccagaggtgacctgtgtggtggtggacgtgtcccacgaggacccagaggtgcagttca actggtatgtggacggagtggaggtgcacaacgccaagaccaagccaagagaggagcagtt-caactccaccttcagagtggtgagcgtgctgaccgtggtgcaccaggactggctgaacggaaaggagtataagtgta aggtgtccaacaagggactgccatccagcatcgagaagaccatctccaagaccaagggacag-ccaagagagccacaggtgtataccctgcccccatccagagaggagatgaccaagaaccaggtgtccctgacctgtc tggtgaagggattctatccatccgacatcgccgtggagtgggagtccaacggacagcca-gagaacaactataagaccacccctccaatgctggactccgacggatccttcttcctgtattccaagctgaccgtggaca agtccagatggcagcagggaaacgtgttctcttgttccgtgatgcacgaggccctgcacaac-cactatacccagaagagcctgtccctgtctccaggaaagtaa

[00488] Complete antibody nucleotide sequence of G1 light chain (SEQ ID NO: 14)

[00489] gaaatcgttctgacccagtccccggctaccctgtccctgtccccaggtgaacgtgctac- cctgtcctgcaaagcttccaaacgggttaccacctacgtttcctggtaccagcagaaacccggtcaggctcctcgtctg ctgatctacggtgcttccaaccgttacctcggtatcccagctcgtttctccggttccggtt-ccggtaccgacttcaccctgaccatctcctccctggaacccgaagacttcgctgtttactactgcagtcagtcctacaact acccctacaccttcggtcagggtaccaaactggaaatcaaacgcactgtggctgcac-catctgtcttcatcttccctccatctgatgagcagttgaaatccggaactgcctctgttgtgtgcctgctgaataacttctatcc gcgcgaggccaaagtacagtggaaggtggataacgccctccaatccggtaactcccagga-gagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgaccctgagcaaagcagac tacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagttctccagtca-caaagagcttcaaccgcggtgagtgctaa

[00490] Amino acid sequence comparison of human and rat CGRP (human α-CGRP (SEQ ID NO:15); human β-CGRP (SEQ ID NO:43); rat α-CGRP (SEQ ID NO:41); and rat β-CGRP (SEQ ID NO:44)):

[00491] NH2-ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-CONH2 (human α-CGRP)

[00492] NH2-ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-CONH2 (human β-CGRP)

[00493] NH2-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-CONH2 (α-CGRP mouse)

[00494] NH2-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF-CONH2 (mouse β-CGRP)

[00495] Light chain variable region LCVR17 amino acid sequence (SEQ ID NO:58)

[00496] DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPK LLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGT KLEIK

[00497] HCVR22 heavy chain variable region amino acid sequence (SEQ ID NO:59)

[00498] QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR LSDYVSGFSYWGQGTLVTVSS

[00499] LCVR18 light chain variable region amino acid sequence (SEQ ID NO:60)

[00500] DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPK LLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGT KLEIK

[00501] HCVR23 heavy chain variable region amino acid sequence (SEQ ID NO:61)

[00502] QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR LSDYVSGFSYWGQGTLVTVSS

[00503] LCVR19 light chain variable region amino acid sequence (SEQ ID NO:62)

[00504] DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPK LLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGT KVEIK

[00505] HCVR24 heavy chain variable region amino acid sequence (SEQ ID NO:63)

[00506] QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDTAVYYCARL SDYVSGFGYWGQGTTVTVSS

[00507] LCVR20 light chain variable region amino acid sequence (SEQ ID NO:64)

[00508] DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKAPK LLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK

[00509] HCVR25 heavy chain variable region amino acid sequence (SEQ ID NO:65)

[00510] QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPG QGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTLVTVSS

[00511] LCVR21 light chain variable region amino acid sequence (SEQ ID NO:66)

[00512] DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKAPK LLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGT KVEIK

[00513] HCVR26 heavy chain variable region amino acid sequence (SEQ ID NO:67)

[00514] QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPG QGLEWMGAIYEGTGKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS

[00515] Light chain variable region LCVR27 amino acid sequence (SEQ ID NO:68)

[00516] QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKV PKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR

[00517] HCVR28 heavy chain variable region amino acid sequence (SEQ ID NO:69)

[00518] EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGK GLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGD IWGQGTLVTVSS

[00519] Light chain variable region LCVR29 amino acid sequence (SEQ ID NO:70)

[00520] QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCF VFGGGTKVEIKR

[00521] Heavy chain HCVR30 variable region amino acid sequence (SEQ ID NO:71)

[00522] EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGK GLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGD IWGQGTLVTVSS

[00523] Light chain variable region LCVR31 amino acid sequence (SEQ ID NO:72)

[00524] QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKV PKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCF VFGGGTKVEIKR

[00525] Heavy chain variable region HCVR32 amino acid sequence (SEQ ID NO:73)

[00526] EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGK GLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGD IWGQGTLVTVSS

[00527] Light chain variable region LCVR33 amino acid sequence (SEQ ID NO:74)

[00528] QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQP PKQLIYDASTLASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDC FVFGGGTEVVVKR

[00529] Heavy chain variable region HCVR34 amino acid sequence (SEQ ID NO:75)

[00530] QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGL EWIGVIGINGATYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGP GTLVTVSS

[00531] Light chain variable region LCVR35 amino acid sequence (SEQ ID NO:76)

[00532] QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKV PKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCF VFGGGTKVEIKR

[00533] Heavy chain variable region HCVR36 amino acid sequence (SEQ ID NO:77)

[00534] EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGK GLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGD IWGQGTLVTVSS

[00535] Light chain variable region LCVR37 amino acid sequence (SEQ ID NO:78)

[00536] QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAP KLLIYDNNKRPSGIPDRFSGSKSGTSTTLGITGLQTGDEADYYCGTWDSRLSAVVFG GGTKLTVL

[00537] Heavy chain variable region HCVR38 amino acid sequence (SEQ ID NO:79)

[00538] QVQLVESGGGVVQPGRSLRLSCAASGFTFSFGMHWVRQAPGK GLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDR LNYYDSSGYYHYKYYGMAVWGQGTTVTVSS

Claims (8)

1. Composition for the treatment or reduction of the incidence of migraine in an individual CHARACTERIZED by the fact that it comprises a monoclonal antibody that inhibits the calcitonin gene-related peptide (CGRP) pathway at a concentration of 150 mg/mL, in which the monoclonal antibody comprises a CDR H1 as set forth in SEQ ID: 3; a CDR H2 as set forth in SEQ ID NO: 4; a CDR H3 as set forth in SEQ ID NO: 5; a CDR L1 as set forth in SEQ ID NO: 6; a CDR L2 as set forth in SEQ ID NO: 7; and a CDR L3 as set forth in SEQ ID NO: 8. 2. Composition according to claim 1, CHARACTERIZED by the fact that the composition is a subcutaneous or intravenous formulation. 3. Composition according to claim 1, CHARACTERIZED by the fact that said composition is contained in a pre-filled syringe in a volume of less than 2 mL. 4. Composition according to claim 3, CHARACTERIZED by the fact that said composition is contained in a pre-filled syringe in a volume of 1.5 mL. 5. Composition according to claim 1, CHARACTERIZED by the fact that it comprises the monoclonal antibody at a concentration of 225 mg/1.5 mL. 6. Composition according to claim 1, CHARACTERIZED by the fact that the migraine is chronic migraine. 7. Composition according to claim 1, CHARACTERIZED by the fact that the migraine is episodic migraine. 8. Composition according to claim 1, CHARACTERIZED by the fact that the monoclonal antibody is human or humanized.