HK1233955A1 - Antagonist antibodies directed against calcitonin gene-related peptide and methods using same - Google Patents

Description

Antagonist antibodies to calcitonin gene-related peptide and methods of use thereof

Cross Reference to Related Applications

This application claims priority to U.S. provisional patent application No.61/968,897 filed 3-21 2014, U.S. provisional patent application No.62/083,809 filed 11-24 2014, and U.S. provisional patent application No.62/119,778 filed 2-23 2015, which are incorporated herein by reference in their entirety for all purposes.

Background

CGRP (calcitonin gene related peptide) is a 37 amino acid neuropeptide belonging to a family of peptides including calcitonin, adrenomedullin and dextrin. In humans, two forms of CGRP (α -CGRP and β -CGRP) exist and have similar activities. They differ by three amino acids and show different distributions. At least two CGRP receptor subtypes may also result in differential activity. 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 tightly bound to blood vessels. CGRP-mediated vasodilation is also associated with neurogenic inflammation and is present in migraine as part of a cascade of events leading to plasma extravasation and vasodilation of the microvasculature.

CGRP has received attention 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 who naturally or surgically induce menopause. Hot flashes may be an adaptive response of the Central Nervous System (CNS) to sex steroid decline (Freedman am. J. human biol.13:453-464 (2001)). To date, the most effective therapy for flushing is hormone-based therapy, including estrogens and/or some progestins. Hormone therapy is effective in reducing flushing, but is not suitable for all women. The psychological and emotional symptoms observed, such as nervousness, fatigue, irritability, insomnia, depression, memory loss, headaches, anxiety, nervousness or lack of concentration, are believed to be caused by sleep deprivation following hot flashes and night sweats (Kramer et al, in: Murphy et al, 3. supplement Int' l Symposium on Recent Advances in urologic Cancer Diagnosis and Treatment-Proceedings, Paris, France: SCI:3-7 (1992)).

Men also experience hot flashes after steroid hormone (androgen) depletion. This is also true in the case of age-related androgen decline (Katovich et al, Proceedings of the Society for Experimental Biology & Medicine,1990,193(2):129-35) and in the extreme case of prostate cancer treatment-related hormone deprivation (Berendsen et al, European Journal of Pharmacology,2001,419(1): 47-54). One third of these patients will experience long-term and frequent symptoms that are severe enough to cause significant discomfort and inconvenience.

CGRP is a potent vasodilator involved in the pathology of other vasomotor symptoms, such as all forms of vascular headache, including migraine (with or without precursor) and cluster headache. Durham, N.Engl.J.Med.350:1073-1075, 2004. Serum levels of CGRP in the external jugular vein were elevated during migraine headache in the patient. Goadsby et al, Ann. neuron.28: 183-7, 1990. Intravenous administration of human α -CGRP induced headache and migraine in patients with promonteless migraine, suggesting that CGRP has an initiating role in migraine. Lassen et al, Cephalalgia 22:54-61,2002.

The possible association of CGRP with migraine has been the basis for the development and testing of a variety of compounds that inhibit the release of CGRP (e.g., sumatriptan), antagonize the CGRP receptor (e.g., dipeptide derivative BIBN4096BS (Boerhringer Ingelheim), CGRP (8-37)) or interact with one or more receptor-associated proteins such as receptor-active membrane protein (RAMP) or Receptor Component Protein (RCP), both of which affect the binding of CGRP to its receptor. Brann, S.et al, Trends in pharmaceutical Sciences 23:51-53,2002. The alpha-2 adrenoreceptor subtype and adenosine A1 receptor also control (inhibit) CGRP release and trigeminal activation (Goadsby et al Brain 125: 1392-cells 401, 2002). Adenosine A1 receptor agonist GR79236(metrafadil), which has been shown to inhibit neurogenic vasodilation and trigeminal nociception in humans, may also have anti-migraine activity (Arulmani et al, Cephalalgia 25:1082-1090, 2005; Giffin et al, Cephalalgia 23:287-292, 2003.)

The following observations refute this theory: with specific inhibition of neurogenic inflammation (e.g., tachykinin NK1 receptor antagonists) or trigeminal activation (e.g., 5 HT)_1DReceptor agonists) have been shown to be relatively ineffective as acute treatment for migraine, leading some researchers to question whether inhibition of CGRP release is the primary mechanism of action for effective anti-migraine therapy. Arulmani et al, Eur.J.Pharmacol.500:315-330, 2004.

Migraine is a complex, common neurological condition characterized by severe, episodic headache attacks and associated symptoms, which can include nausea, vomiting, sensitivity to light, sound or movement. In some patients, the headache is preceded or accompanied by a precursor. The headache can be severe and in some patients also unilateral.

Migraine attacks are destructive to daily life. The overall prevalence of migraine sufferers in the united states and western europe is 11% of the general population (6% male; 15-18% female). Furthermore, the median frequency of episodes in an individual is 1.5 times per month. While there are a number of treatments available to alleviate or reduce symptoms, prophylactic treatment is recommended for those patients with migraine attacks more than 3-4 times per month. Goadsby et al, New Engl. J. Med.346(4): 257; 275; 2002).

The diversity of pharmacological interventions used to treat migraine and the variability of responses among patients are evidence of the different nature of the disease. Thus, 5-hydroxytryptamine as well as adrenergic, noradrenergic, and dopaminergic are exhibitedActive, relatively non-selective drugs such as ergot alkaloids (e.g., ergotamine, dihydroergotamine, mexican) have been used for more than eighty years for the treatment of migraine other treatments include opiates (e.g., oxycodone) and β -adrenergic antagonists (e.g., propranolol) some patients (usually those with minor symptoms) are able to control their symptoms with over-the-counter drugs such as one or more non-steroidal anti-inflammatory agents (NSAIDs) such as aspirin, combinations of acetaminophen and caffeine (e.g.,Migraine)。

recently, some migraine sufferers have been treated with topiramate, an anticonvulsant that blocks voltage-dependent sodium channels and certain glutamate receptors (AMPA-kainic acid), potentiates GABA-a receptor activity, and blocks carbonic anhydrase. Relatively more recently, the success of 5-hydroxytryptamine 5HT-1B/1D and/or 5HT-1a receptor agonists (such as sumatriptan) has led researchers to come up with the 5-hydroxytryptamine etiology of the disease in some patients. Unfortunately, while some patients respond well to this treatment, others are relatively resistant to their effects.

It is postulated that ion channel dysfunction in the nucleus of amine-competent brainstem cells leads to the disease, however the precise pathophysiology of migraine is still not well understood. One form of migraine (familial hemiplegic migraine) has been shown to be associated with missense mutations in the α 1 subunit of voltage-gated P/Q-type calcium channels, and it is believed that it is likely that other ion channel mutations are also present in other patient populations. Although vasodilation is associated with migraine and exacerbates its pain symptoms, such neurovascular events are currently considered to be the result, rather than the cause, of the condition. In summary, dysfunction of the brainstem pathway that regulates sensory input is considered a unifying feature of migraine. Goadsby, P.J., et al, New Engl.J.Med.346(4): 257-.

Disclosure of Invention

In some aspects, the invention disclosed herein relates to anti-CGRP antagonist antibodies and methods of using anti-CGRP antagonist antibodies to treat or prevent vasomotor symptoms. Examples of vasomotor symptoms, such as hot flashes, are provided herein. In some cases, anti-CGRP antagonist antibodies are used to treat or prevent headache, such as migraine with or without precursor, hemiplegic migraine, cluster headache, migrainous neuralgia, chronic headache, tension headache, and headache resulting from other medical conditions (such as infections caused by tumors or increased intracranial pressure).

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

In one aspect, the invention provides methods for treating or preventing headache (e.g., migraine and cluster headache) in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody.

In another aspect, the invention provides methods for ameliorating, controlling, reducing the onset of, or delaying the development or progression of headache (e.g., migraine and cluster headache) in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody.

In one aspect, the present invention provides a method of treating or reducing the onset of at least one vasomotor symptom and/or headache in a subject. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over a plurality of days, wherein the amount administered per day over said plurality of days is less than 1000 mg. In one embodiment, the method comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over multiple days, wherein the amount administered per day over said multiple days is between 100-2000 mg. In some embodiments, the headache is a migraine (e.g., chronic migraine or episodic migraine). In some embodiments, two of the multiple days are separated by more than seven days. In some embodiments, the onset of headache is reduced for at least seven days after a single administration. In some embodiments, the amount of monoclonal antibody administered on the first day is different (e.g., greater) than the amount of monoclonal antibody administered on the second day. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous administration. In some embodiments, the administration is intravenous administration. In some embodiments, the administering comprises utilizing a pre-filled syringe comprising an 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 in a volume of less than 2 mL. In some embodiments, the amount of monoclonal antibody is less than 1000 mg. In some embodiments, the number of monthly headache episodes experienced by the subject following said administration is reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80, or more) from the subject's pre-administration level. The number of hours per month of headache can be reduced by more than 60 hours. In some embodiments, the number of hours per month headache experienced by the subject after said administration is reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50% or more) relative to the subject's pre-administration level. The number of headache hours per month can be reduced by 40% or more. In some embodiments, the number of monthly headache days experienced by the subject following said administration is reduced from the pre-administration level of the subject 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). In some embodiments, the method further comprises administering a second agent (agent) to the subject simultaneously or sequentially with the monoclonal antibody. The second agent can be any one of a 5-HT1 agonist, triptan (triptan), ergot alkaloid, and a non-steroidal anti-inflammatory drug. In some embodiments, the second agent is an agent that is prophylactically ingested by the subject. In some embodiments, the monthly use of the second agent by the subject is reduced by at least 15% following administration of the monoclonal antibody. In some embodiments, the second agent is 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the invention provides a method of reducing the number of monthly headache hours experienced 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 amount of the monoclonal antibody is effective to reduce the number of headache hours per month by at least 20 hours (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 headache hours per month is reduced by at least about 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 amount of the monoclonal antibody is 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 headache hours per month 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 monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the invention provides a method of reducing the number of monthly headache days experienced 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 amount of the monoclonal antibody is effective to reduce the number of headache days per month by at least 3 days (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 headache days per month 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 monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the invention provides a method of reducing the use of an anti-headache agent in a subject, comprising administering to the subject a monoclonal antibody that modulates a CGRP pathway (e.g., an anti-CGRP antagonist antibody), wherein the amount of the monoclonal antibody is effective to reduce the monthly use of the anti-headache agent by the subject by at least 15% (e.g., 20%, 25%, 30%, 35%, 40% or more). In some embodiments, the anti-headache agent is selected from the group consisting of 5-HT1 agonists, triptans, opiates, beta-adrenergic antagonists, ergot alkaloids, and non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the anti-headache agent is triptan. In some embodiments, the amount of monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 the CDR H1 shown in SEQ ID NO:3, the CDR H2 shown in SEQ ID NO:4, the CDR H3 shown in SEQ ID NO:5, the CDR L1 shown in SEQ ID NO:6, the CDR L2 shown in SEQ ID NO:7, and the CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

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

In another embodiment, the present invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the development or progression of headache (e.g., migraine and cluster headache) in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody in combination with at least one additional agent for treating headache. Such additional agents include 5-HT 1-like agonists (as well as agonists acting at other 5-HT1 sites) and non-steroidal anti-inflammatory drugs (NSAIDs).

Examples of 5-HT1 agonists that can be used in combination with anti-CGRP antibodies 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). These compounds include ergotamine tartrate, ergonovine maleate, and dihydroergotamine mesylate (e.g., dihydroergocornine mesylate, dihydroergotamine mesylate, and dihydroergotamine mesylate (DHE 45)).

Examples of NSAIDs that may be used in combination with the anti-CGRP antibody include aspirin, diclofenac, diflunisal, 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-745,337; NS 398; or a pharmaceutically acceptable salt thereof.

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

In one embodiment, the anti-CGRP antagonist antibody for use in any of the methods described above is any antibody described herein.

In some embodiments, the anti-CGRP antagonist antibody recognizes human CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to human 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 a C-terminal fragment having amino acids 25-37 of CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to a C-terminal epitope within amino acids 25-37 of CGRP.

In some embodiments, the anti-CGRP antagonist 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 antibody G1 (as described herein). In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDRs (such as one, two, three, four, five, or in some embodiments, all six CDRs) of a variant of antibody G1 or G1 shown in table 6. In other embodiments, the anti-CGRP antagonist antibody comprises the amino acid sequence of the heavy chain variable region shown in FIG. 5(SEQ ID NO:1) and the amino acid sequence of the light chain variable region shown in FIG. 5(SEQ ID NO: 2).

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 has one or more of these activities reduced. In some embodiments, the constant region is as defined in Eur.J.Immunol. (1999)29: 2613-2624; PCT patent application No. PCT/GB 99/01441; and/or modified as described in UK patent application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering refers to wild type IgG2 sequence). Eur.J.Immunol. (1999)29: 2613-2624. In some embodiments, the heavy chain constant region of the antibody is human heavy chain IgG1 with any of the following mutations: 1) a327a330P331 to G327S330S 331; 2) E233L234L235G236(SEQ ID NO:48) to P233V234A235, wherein G236 is deleted; 3) E233L234L235 to P233V234a 235; 4) E233L234L235G236A327A330P331(SEQ ID NO:49) to P233V234A235G327S330S331(SEQ ID NO:50), wherein G236 is deleted; 5) E233L234L235A327A330P331(SEQ ID NO:51) to P233V234A235G327S330S331(SEQ ID NO: 50); and 6) N297 to A297 or any other amino acid other than N. In some embodiments, the heavy chain constant region of the antibody is human heavy chain IgG4 with any of the following mutations: E233F234L235G236(SEQ ID NO:52) to P233V234A235, wherein G236 is deleted; E233F234L235 to P233V234a 235; and S228L235 to P228E 235.

In other embodiments, the constant region is not N-linked glycosylated. In some embodiments, the constant region is not N-linked glycosylated due to mutation of an oligosaccharide linking 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 not N-linked glycosylated. The constant region may be N-linked glycosylated due to enzymatic cleavage or expression in a glycosylation deficient host cell.

Binding affinity (K) of an anti-CGRP antagonist antibody to CGRP, such as human α -CGRP, as determined by surface plasmon resonance at an appropriate temperature, such as 25 or 37 ℃_D) Can be about 0.02 to about 200 nM. In some embodiments, the binding affinity is any of about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 60pM, about 50pM, about 20pM, about 15pM, about 10pM, about 5pM, or about 2 pM. In some embodiments, the binding affinity is less than any of about 250nM, about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50 pM. In some embodiments, the binding affinity is less than about 50 nM.

The anti-CGRP antagonist antibody may be administered before, during and/or after headache. In some embodiments, the anti-CGRP antagonist antibody is administered prior to the onset of headache (e.g., migraine and cluster headache). Administration of the anti-CGRP antagonist antibody can be by any means known in the art, including: oral, intravenous, subcutaneous, intraarterial, intramuscular, intranasal (e.g., inhaled or non-inhaled), intracardiac, intraspinal, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal and/or by inhalation. Administration may be systemic, e.g., intravenous, or local.

In some embodiments, the anti-CGRP antagonist antibody may be administered in combination with another agent, such as another agent used to treat headache.

In another aspect, the invention provides the use of an anti-CGRP antagonist antibody for the manufacture of a medicament for any of the methods described herein, e.g., for treating or preventing headache.

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

In another aspect, the invention provides a kit for 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 using the composition in any of the methods described herein.

The present invention also provides anti-CGRP antagonist antibodies and polypeptides derived from antibody G1 or variants thereof shown in table 6. Thus, in one aspect, the invention provides antibody G1 (interchangeably referred to as "G1") made by expression vectors having ATCC accession numbers PTA-6866 and PTA-6867. For example, in one embodiment is an antibody comprising a heavy chain made by the expression vector with ATCC accession No. PTA-6867. In another embodiment is an antibody comprising a light chain made by the expression vector having ATCC accession No. PTA-6866. The amino acid sequences of the heavy and light chain variable regions of G1 are shown in fig. 5. The Complementarity Determining Region (CDR) portions of antibody G1, including Chothia and Kabat CDRs, are also shown in figure 5. It should be understood that reference to any portion or the entire region of G1 encompasses the sequence made by the expression vectors with ATCC accession nos. PTA-6866 and PTA-6867, and/or the sequence depicted in fig. 5. In some embodiments, the invention also provides antibody variants of G1 having the amino acid sequence depicted in table 6.

In one aspect, the invention provides a polypeptide comprising at least 85%, at least 86%, at least 87% amino acid sequence as set forth in SEQ ID NO. 1,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% amino acid sequence identity to the V_HAntibodies to the domains.

In another aspect, the invention provides a polypeptide comprising a V having 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% amino acid sequence identity to SEQ ID NO 2_LAntibodies to the domains.

In another aspect, the invention provides an antibody comprising a fragment or region of antibody G1 or a variant thereof shown in table 6. In one embodiment, the fragment is the light chain of antibody G1. In another embodiment, the fragment is the heavy chain of antibody G1. In yet another embodiment, the fragment comprises one or more variable regions of the light chain and/or heavy chain of antibody G1. In yet another embodiment, the fragment comprises one or more variable regions of a light chain and/or a heavy chain as set forth in figure 5. In yet another embodiment, the fragment comprises one or more CDRs of the light chain and/or heavy chain of antibody G1.

In another aspect, the invention provides a vector comprising V as set forth in SEQ ID NO.5_HCDR3, or a polypeptide (which may or may not be an antibody) that differs from SEQ ID NO.5 by a sequence of 1,2,3,4, or 5 amino acid substitutions. In particular embodiments, such amino acid substitutions are conservative substitutions.

In another aspect, the invention provides a vector comprising V as set forth in SEQ ID NO 8_LCDR3, or a polypeptide (which may or may not be an antibody) that differs from SEQ ID NO. 8 by a sequence of 1,2,3,4, or 5 amino acid substitutions. In particular embodiments, such amino acid substitutions are conservative substitutions.

In another aspect, the invention provides a polypeptide (which may or may not be an antibody) comprising any one or more of: a) one or more CDRs of antibody G1 or a variant thereof shown in table 6; b) CDR H3 of the heavy chain of antibody G1 or a variant thereof shown in table 6; c) the CDR L3 of the light chain of antibody G1 or a variant thereof shown in table 6; d) three CDRs of the light chain of antibody G1 or variants thereof shown in table 6; e) three CDRs of the heavy chain of antibody G1 or variants thereof shown in table 6; f) table 6 shows the three CDRs of the light chain and the three CDRs of the heavy chain of antibody G1 or variants thereof. In some embodiments, the invention also provides a polypeptide (which may or may not be an antibody) comprising any one or more of: a) one or more (one, two, three, four, five or six) CDRs derived from antibody G1 or a variant thereof shown in table 6; b) CDRs derived from CDR H3 of the heavy chain of antibody G1; and/or c) a CDR derived from CDR L3 of the light chain of antibody G1. In some embodiments, the CDR is the CDR shown in fig. 5. In some embodiments, the one or more CDRs derived from antibody G1 or variants thereof set forth 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 variants thereof.

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 Kabat and Chothia CDRs (also referred to as "combined CDRs" or "extended CDRs"). In other words, for any given embodiment that includes more than one CDR, the CDR can be any one of Kabat, Chothia, and/or combinations thereof.

In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence KASKXaaVxaaaTYVS (SEQ ID NO:53) wherein Xaa at position 5 is R, W, G, 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 KASKXaaVXaaTYVS (SEQ ID NO:53) is the CDR1 of the light chain of the antibody.

In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence XaaXaaSNRYXaa (SEQ id no:54), wherein Xaa at position 1 is G 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 XaaXaaSNRYXaa (SEQ ID NO:54) is the CDR2 of an antibody light chain.

In some embodiments, the polypeptide (such as an antibody) comprises the amino acid sequence EIRSXaaSDXaaATXaaYAXaaAVKG (SEQ ID NO:55), wherein Xaa at position 5 is E, R, K, Q or N; wherein Xaa at position 8 is A, G, 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, L, A, P, I, N, R, V, D or M; 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 EIRSXaaSDXaaATXaaYAXaAVKG (SEQ ID NO:55) is the CDR2 of an antibody heavy chain.

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.

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.

The heavy chain constant region of the antibody can be from any type of constant region, such as IgG, IgM, IgD, IgA, and IgE; and any isotype such as IgG1, IgG2, IgG3, and IgG 4.

In some embodiments, the antibody comprises a modified constant region described herein.

In another aspect, the invention provides a polynucleotide (which may be isolated): which comprises a polynucleotide encoding a fragment or region of antibody G1 or a variant thereof shown in table 6. In one embodiment, the fragment is the light chain of antibody G1. In another embodiment, the fragment is the heavy chain of antibody G1. In yet another embodiment, the fragment comprises one or more variable regions of the light chain and/or heavy chain of antibody G1. In yet another embodiment, the fragment comprises one or more (i.e., one, two, three, four, five, or six) Complementarity Determining Regions (CDRs) of the light chain and/or heavy chain of antibody G1.

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

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

In another aspect, the invention provides vectors (including expression and cloning vectors) and host cells comprising any of the polynucleotides disclosed herein. In some embodiments, the vector is pdb.cgrp.hfcgi with ATCC No. pta-6867. In other embodiments, the vector is peb.cgrp.hkgi with ATCC No. pta-6866.

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

In another aspect, the invention provides a complex of any of the antibodies or polypeptides described herein bound to CGRP. In some embodiments, the antibody is antibody G1 shown in table 6 or a variant thereof.

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 antibody G1) or polynucleotides described herein and a pharmaceutically acceptable excipient.

In another aspect, the invention provides a method of producing antibody G1, comprising culturing a host cell or progeny thereof under conditions that allow for production of antibody G1, wherein the host cell comprises an expression vector encoding antibody G1; and in some embodiments, purifying the antibody G1. 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.

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

anti-CGRP antagonist antibodies and polypeptides, and polynucleotides encoding the antibodies and polypeptides of the invention are useful for treating, preventing, ameliorating, controlling or reducing the onset of diseases associated with CGRP dysfunction, 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.

In another aspect, the invention provides kits and compositions comprising any one or more of the compositions described herein. These kits are generally in suitable packaging and with appropriate instructions, and can be used in any of the methods described herein.

In one aspect, the invention provides a composition for use according to any of the methods described herein.

In one aspect, the present invention provides a composition for treating or reducing the onset of at least one vasomotor symptom and/or headache in a subject. In one embodiment, the use comprises administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over a plurality of days, wherein the amount administered per day over said plurality of days is less than 1000 mg. In one embodiment, the use comprises administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over a plurality of days, wherein the amount administered per day over said plurality of days is between 100-2000 mg. In some embodiments, the headache is a migraine (e.g., chronic migraine or episodic migraine). In some embodiments, two of the multiple days are separated by more than seven days. In some embodiments, the onset of headache is reduced for at least seven days after a single administration. In some embodiments, the amount of monoclonal antibody administered on the first day is different (e.g., greater) than the amount of monoclonal antibody administered on the second day. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous administration. In some embodiments, the administration is intravenous administration. In some embodiments, the administering comprises utilizing a pre-filled syringe comprising an 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 in a volume of less than 2 mL. In some embodiments, the amount of monoclonal antibody is less than 1000 mg. In some embodiments, the number of monthly headache episodes experienced by the subject following said administration is reduced by 40 or more hours (e.g., 45, 50, 55, 60, 65, 70, 75, 80, or more) from the subject's pre-administration level. The number of hours per month of headache can be reduced by more than 60 hours. In some embodiments, the number of hours per month headache experienced by the subject after said administration is reduced by 25% or more (e.g., 30%, 35%, 40%, 45%, 50% or more) relative to the pre-administration level in the subject. The number of headache hours per month can be reduced by 40% or more. In some embodiments, the number of monthly headache days experienced by the subject following said administration is reduced from the pre-administration level of the subject 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). 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 one of a 5-HT1 agonist, triptan (triptan), ergot alkaloid, and a non-steroidal anti-inflammatory drug. In some embodiments, the second agent is an agent that is prophylactically ingested by the subject. In some embodiments, the monthly use of the second agent by the subject is reduced by at least 15% following administration of the monoclonal antibody. In some embodiments, the second agent is 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the present invention provides a composition for reducing the number of monthly headache hours 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 amount of the monoclonal antibody is effective to reduce the number of headache hours per month by at least 20 hours (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 headache hours per month is reduced by at least about 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 amount of the monoclonal antibody is 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 headache hours per month 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 monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the invention provides a composition for reducing the 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 amount of the monoclonal antibody is effective to reduce the number of headache days per month by at least 3 days (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 headache days per month 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 monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 CDR H1 shown in SEQ ID NO:3, CDR H2 shown in SEQ ID NO:4, CDR H3 shown in SEQ ID NO:5, CDR L1 shown in SEQ ID NO:6, CDR L2 shown in SEQ ID NO:7, and CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

In one aspect, the invention provides a composition for reducing the use of an anti-headache agent in a subject, comprising administering to the subject a monoclonal antibody that modulates a CGRP pathway (e.g., an anti-CGRP antagonist antibody), wherein the amount of the monoclonal antibody is effective to reduce the monthly use of the anti-headache agent by the subject by at least 15% (e.g., 20%, 25%, 30%, 35%, 40% or more). In some embodiments, the anti-headache agent is selected from the group consisting of 5-HT1 agonists, triptans, opiates, beta-adrenergic antagonists, ergot alkaloids, and non-steroidal anti-inflammatory drugs (NSAIDs). In some embodiments, the anti-headache agent is triptan. In some embodiments, the amount of monoclonal antibody is less than 1000 mg. In some embodiments, the subject is administered less than 3 doses per month. In some embodiments, the administration is subcutaneous or intravenous administration. In some embodiments, the monoclonal antibody is formulated at a concentration of at least 150 mg/mL. In some embodiments, wherein the monoclonal antibody is administered in 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 the CDR H1 shown in SEQ ID NO:3, the CDR H2 shown in SEQ ID NO:4, the CDR H3 shown in SEQ ID NO:5, the CDR L1 shown in SEQ ID NO:6, the CDR L2 shown in SEQ ID NO:7, and the CDR L3 shown in SEQ ID NO: 8; or (b) variants of the antibody according to (a) as shown in table 6.

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

Brief Description of Drawings

FIG. 1 is a table showing the binding affinities of 12 murine antibodies to different alanine-substituted human α -CGRP fragments binding affinities were measured at 25 ℃ using Biacore by flowing Fab through the CGRP on the chip in-box values indicate the loss of affinity of the alanine mutant relative to the parent fragment 25-37 (italics), with the exception of K35A, which was derived from the 19-37 parent.^a"means for 19-37 and 25-37 sheetsThe affinity of the segments is the mean ± standard deviation of two independent measurements on different sensor chips. "^b"indicates that these interactions deviate from the simple bimolecular interaction model due to the biphasic off-rates, and therefore their affinities were determined using the conformational change model. Grayscale legend: white (1.0) represents parental affinity; light gray (less than 0.5) indicates higher affinity than the parent; dark grey (greater than 2) indicates lower affinity than the parent; and black indicates no binding detected.

FIGS. 2A and 2B show the effect of administration of CGRP8-37 (400nmol/kg), antibody 4901(25mg/kg) and antibody 7D11(25mg/kg) on skin blood flow, measured as blood cell flux 30 seconds after electrical pulse stimulation. (iv) CGRP8-37 administered intravenously 3-5 minutes prior to electrical pulse stimulation. Antibodies (IP) were administered intraperitoneally 72 hours prior to electrical pulse stimulation. Each point in the graph represents the AUC of one rat treated under the specified conditions. Each line in the graph represents the mean AUC of rats treated under the specified conditions. AUC (area under the curve) is equal to Δ flux × Δ time. "delta flux" means the change in flux units following electrical pulse stimulation; and "delta time" represents the time period required for the blood cell flux level to return to the level before stimulation by the electrical pulse.

Figure 3 shows the effect of administration of different doses of antibody 4901(25mg/kg, 5mg/kg, 2.5mg/kg, or 1mg/kg) on skin blood flow, measured as blood cell flux 30 seconds after electrical pulse stimulation. (IV) the antibody was administered intravenously 24 hours prior to electrical pulse stimulation. Each point in the graph represents the AUC of one rat treated under the specified conditions. The line in the graph represents the mean AUC of rats treated under the specified conditions.

Fig. 4A and 4B show the effect of administration of antibody 4901(1mg/kg or 10mg/kg, intravenous), antibody 7E9(10mg/kg, intravenous), and antibody 8B6(10mg/kg, intravenous) on skin blood flow, measured as blood cell flux 30 seconds after electrical pulse stimulation. The antibody was administered intravenously (i.v.) and then electrical pulse stimulation was performed 30 min, 60 min, 90 min and 120 min after antibody administration. The Y-axis represents the AUC percentage compared to the AUC level when no antibody was administered (time 0). The X-axis represents the time (minutes) between antibody administration and electrical pulse stimulation. Compared to time 0, "# indicates P <0.05," # indicates P < 0.01. Data were analyzed using one-way analysis of variance and Dunnett's multiple comparison test.

FIG. 5 shows the amino acid sequences of the heavy chain variable region (SEQ ID NO:1) and the light chain variable region (SEQ ID NO:2) of antibody G1. The Kabat CDR is bold text, Chothia CDR underlined. The amino acid residues of the heavy and light chain variable regions are numbered sequentially.

Figure 6 shows an epitope map of antibody G1 plotted by peptide competition using Biacore. N-biotinylated human α -CGRP was captured on an SA sensor chip. The G1Fab (50nM) was flowed onto the chip in the absence of competitor peptide or preincubated with 10uM competitor peptide for 1 h. The binding of G1Fab to human α -CGRP on the chip was determined. The Y-axis represents the percentage of blocked binding in the presence of competing peptide compared to binding in the absence of competing peptide.

FIG. 7 shows the effect of administration of antibody G1(1mg/kg or 10mg/kg, intravenously) or vehicle (PBS, 0.01% Tween20) on skin blood flow, measured as blood cell flux 30 seconds after electrical pulse stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) followed by neuro-electrical pulse stimulation 30, 60, 90 and 120 minutes after antibody administration. The Y-axis represents the AUC percentage compared to the AUC level when no antibody or vehicle (defined as 100%) was administered (time 0). The X-axis represents the time (minutes) between antibody administration and electrical pulse stimulation. In comparison to vehicle, "×" indicates P <0.05 and "×" indicates P < 0.01. Data were analyzed using two-way analysis of variance and Bonferroni post-hoc tests.

FIG. 8A shows the effect of administration of antibody G1(1mg/kg, 3mg/kg, or 10mg/kg, intravenously) or vehicle (PBS, 0.01% Tween20) on skin blood flow, measured as blood cell flux 30 seconds after electrical pulse stimulation 24 hours after dosing. Antibody G1 or vehicle was administered intravenously (i.v.) 24 hours prior to neural electric pulse stimulation. The Y-axis represents the area under the total curve (change in blood cell flux multiplied by the change in time from stimulation until flux returns to baseline, AUC). The X-axis represents different doses of antibody G1. In comparison to vehicle, "×" indicates P <0.05 and "×" indicates P < 0.01. Data were analyzed using one-way analysis of variance and Dunn multiple comparison test.

FIG. 8B shows the effect of administration of antibody G1(0.3mg/kg, 1mg/kg, 3mg/kg, or 10mg/kg, intravenously) or vehicle (PBS, 0.01% Tween20) on skin blood flow, measured as blood cell flux 30 seconds after 7 days post-dose electrical pulse stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) 7 days prior to neural electric pulse stimulation. The Y-axis represents the total AUC. The X-axis represents different doses of antibody G1. Compared to vehicle, "×" indicates P <0.01 and "×" indicates P < 0.001. Data were analyzed using one-way analysis of variance and Dunn multiple comparison test.

Fig. 8C is a curve fit analysis of the data of fig. 8A and 8B. Antibody G1 or vehicle was administered intravenously (i.v.) 24 hours or 7 days prior to neuro-electrical pulse stimulation. The Y-axis represents the total AUC. The X-axis represents different doses of antibody G1, expressed in "mg/kg" on the logarithmic scale, to determine EC₅₀。

FIG. 9 shows the effect of antibody mu7E9(10mg/kg), BIBN4096BS or vehicle (PBS, 0.01% Tween20) on the change in meningeal artery diameter following electric field stimulation. After baseline establishment in response to electrical stimulation, antibody mu7E9, BIBN4096BS or vehicle was administered intravenously (i.v.) at time point 0 minutes. The Y-axis represents the change in diameter of the middle cerebral artery following electric field stimulation. The resting diameter corresponds to 0%. The X-axis represents the time (minutes) of electrical pulse stimulation. In comparison to vehicle, "×" indicates P <0.05 and "×" indicates P < 0.01. Data were analyzed using one-way analysis of variance and the Dunett multiple comparison test.

FIG. 10 shows the effect of different doses of antibody G1(1mg/kg, 3mg/kg or 10mg/kg, intravenous) or vehicle (PBS, 0.01% Tween20) on the change in diameter of the meningeal artery after electric field stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) 7 days before electric field stimulation. The Y-axis represents the change in diameter of the middle cerebral artery. The resting diameter corresponds to 0%. The X-axis represents the stimulation voltage. In comparison to vehicle, "+" indicates P <0.05, "+" indicates P <0.01, and "+" indicates P < 0.001. Data were analyzed using two-way analysis of variance and Bonferroni post-hoc tests.

Figure 11A shows the effect of intravenous administration (i.v.) of antibody mu4901(10mg/kg) or vehicle (PBS, 0.01% Tween20) 24 hours prior to subcutaneous injection of naloxone (1mg/kg) to morphine-addicted rats on the reduction of body core temperature induced by said subcutaneous injection. The Y-axis represents the difference in temperature from the baseline. The X-axis represents the time measured from the point of naloxone injection.

Figure 11B shows the effect of intravenous administration (i.v.) of antibody mu4901(10mg/kg) or vehicle (PBS, 0.01% Tween20) 24 hours prior to subcutaneous injection of naloxone (1mg/kg) to morphine-addicted rats on tail surface temperature elevation induced by the subcutaneous injection. The Y-axis represents the difference in temperature from the baseline. The X-axis represents the time measured from the point of naloxone injection.

Figure 12 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 13 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 14 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 15 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 16 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 17 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Figure 18 is a graph showing the results of a clinical study comparing the effect of different doses of antibody G1 with placebo.

Detailed Description

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.

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

In some aspects, the invention disclosed herein also provides anti-CGRP antagonist antibodies and polypeptides derived from G1 or variants thereof shown in table 6. In some embodiments, the invention also provides methods of making and using these antibodies and polypeptides.

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

General techniques

The practice of 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 the art. Such techniques are well explained in the literature, such as Molecular Cloning A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; oligonucleotide Synthesis (m.j. gait eds., 1984); methods in Molecular Biology, human Press; cell Biology, ALaborory Notebook (J.E. Cellis eds., 1998) Academic Press; animal Cell Culture (r.i. freshney eds, 1987); introduction to Cell and Tissue Culture (J.P.Mather and P.E.Roberts, 1998) Plenum Press; cell and Tissue Culture Laboratory Procedures (A.Doyle, J.B.Griffiths and D.G.Newell eds., 1993-1998) J.Wiley and Sons; methods in enzymology (Academic Press, Inc.); handbook of Experimental Immunology (eds. d.m.weir and c.c.blackwell); gene Transfer Vectors for Mammalian Cells (eds. J.M.Miller and M.P.Calos, 1987); current Protocols in Molecular Biology (ed. F.M. Ausubel et al, 1987); PCR The Polymerase Chain Reaction (Mullis et al eds., 1994); current Protocols in immunology (J.E.Coligan et al, 1991); short Protocols in Molecular Biology (Wileyand Sons, 1999); immunobiology (c.a. janeway and p.travers, 1997); antibodies (p.finch, 1997); antibodies a practical prophach (D.Catty. eds., IRL Press, 1988-1989); monoclonal antigens a practical proproach (edited by P.shepherd and C.dean, Oxford university Press, 2000); using Antibodies a Laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M.Zantetti and J.D.Capra, eds., Harwood Academic Publishers, 1995).

Definition of

An "antibody" is an immunoglobulin molecule capable of specifically binding a 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 fragments thereof (such as Fab, Fab ', F (ab')₂Fv), single chain (ScFv), mutants, fusion proteins comprising an antibody portion (such as a domain antibody), and any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site. Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subclasses thereof), and antibodies are not necessarily of any particular class. Immunoglobulins can be classified into different classes based on the antibody amino acid sequence of the constant domain of the antibody heavy chain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these categories can be further divided into subcategories (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. Corresponds to notThe heavy chain constant domains of immunoglobulins of the same class are designated α, gamma, and mu, respectively.

As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that 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 that the property of the antibody is achieved in a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be prepared by the hybridoma method first described by Kohler and Milstein,1975, Nature,256:495, or may be prepared by recombinant DNA methods such as those described in U.S. Pat. No.4,816,567. The monoclonal antibodies can also be isolated from phage libraries generated using techniques such as those described in McCafferty et al, 1990, Nature,348: 552-554.

As used herein, a "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that: which are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof comprising minimal sequences derived from non-human immunoglobulins (such as Fv, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). In most cases, the humanized antibody is a human immunoglobulin (recipient) antibody: wherein residues of the recipient's Complementarity Determining Regions (CDRs) are replaced with residues of CDRs (donor antibodies) of a non-human species, such as mouse, rat or rabbit, having the desired specificity, affinity and biological activity. In some instances, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that: this residue is present neither in the recipient antibody nor in the imported CDR or framework sequences, but is includedThese residues are included to further refine and optimize antibody performance. Generally, a humanized antibody will comprise substantially all (at least one, and typically two) variable domains, in which 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 also optimally comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified as described in WO 99/58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, six) that have been altered relative to the original antibody, also referred to as one or more CDRs "derived from" the one or more CDRs of the original antibody.

As used herein, "human antibody" means an antibody having an amino acid sequence corresponding to an antibody produced by a human and/or made using any of the techniques for making human antibodies known in the art or disclosed herein. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising murine light chain and human heavy chain polypeptides. Human antibodies can be made using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses a human antibody (Vaughan et al, 1996, Nature Biotechnology, 14: 309-. Human antibodies can also be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which endogenous immunoglobulin genes have been partially or completely inactivated). This method is described in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425 and 5,661,016. Alternatively, the human antibody may be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes may be recovered from the individual, or may be 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. Pat. No.5,750,373.

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 a portion of the activity of CGRP. For example, the CGRP may be an α -CGRP or a β -CGRP. As used herein, CGRP includes the native sequence CGRP of all mammalian species (e.g., human, canine, feline, equine, and bovine).

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 CGRP biological activity and/or CGRP signaling mediated downstream pathways. anti-CGRP antagonist antibodies encompass antibodies that modulate, block, antagonize, inhibit or reduce (including significantly) CGRP biological activity, or antagonize CGRP pathways, including CGRP signaling-mediated downstream pathways such as receptor binding and/or the initiation of a cellular response to CGRP. For the purposes of the present invention, it is to be expressly understood that: the term "anti-CGRP antagonist antibody" encompasses all terms, topics and functional states and characteristics as defined herein before, whereby CGRP itself, CGRP bioactivity (including but not limited to its ability to mediate headache in any aspect) or the consequences of bioactivity are substantially eliminated, reduced or neutralized to any meaningful degree. In some embodiments, the anti-CGRP antagonist antibody binds to CGRP and prevents CGRP binding to the CGRP receptor. In other embodiments, the anti-CGRP antibody binds to CGRP and prevents activation of the CGRP receptor. Examples of anti-CGRP antagonist antibodies are provided herein.

As used herein, the terms "G1" and "antibody G1" are used interchangeably to refer to antibodies prepared by the expression vectors with accession nos. ATCC PTA-6867 and ATCC PTA-6866. The amino acid sequences of the heavy and light chain variable regions are shown in FIG. 5. The CDR portions of antibody G1 (including Chothia and Kabat CDRs) are illustrated in fig. 5. The polynucleotides encoding the heavy and light chain variable regions are shown in SEQ ID NO 9 and SEQ ID NO 10. The characterization of G1 is as described in the examples.

The terms "polypeptide", "oligopeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and the amino acids may be interrupted by non-amino acids. The term also encompasses amino acid polymers modified either naturally or by intervention; the modification is, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation to a labeling component. Also included in this definition are, for example, polypeptides that comprise one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It will be appreciated that because the polypeptides of the invention are antibody-based, the polypeptides may exist as single chains or related chains.

"Polynucleotide" or "nucleic acid" are used interchangeably herein to refer to a polymer 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 a DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. Modification of the nucleotide structure, if present, may be performed before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. Other types of modifications include, for example, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those having uncharged bonds (e.g., methylphosphonates, phosphotriesters, phosphoamides, carbamates, etc.) and charged bonds (e.g., phosphorothioates, phosphorodithioates, etc.), those comprising pendant moieties such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those having intercalators (e.g., acridine, psoralen, etc.), polynucleotides comprising chelation, as well as unmodified forms thereofIn addition, any hydroxyl group typically present in a sugar may be substituted, for example, with a phosphonate group, a phosphate group, or activated to prepare an additional linkage to an additional nucleotide, or may be conjugated to a solid support.5 'and 3' terminal OH may be phosphorylated, or substituted with an amine or an organic capping group having 1 to 20 carbon atoms other hydroxyl groups may also be derivatized to standard protecting groups₂("amide"), P (O) R, P (O) OR', CO OR CH₂("methylal") alternative embodiment, wherein each R or R' is independently H or a substituted or unsubstituted alkyl (1-20 carbons) optionally containing a (-O-) bond, aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl (araldyl). All linkages in a polynucleotide need not be identical. The above description applies to all polynucleotides referred to herein, including RNA and DNA.

The "variable region" of an antibody refers to the variable region of an antibody light chain or the variable region of an antibody heavy chain, alone or in combination. The variable regions of the heavy and light chains are each composed of four Framework Regions (FRs) connected by three Complementarity Determining Regions (CDRs), also known as hypervariable regions. The CDRs of each chain are held together tightly by the FRs, with the CDRs of the other chain contributing to the formation of the antigen-binding site of the antibody. There are at least two techniques for determining CDRs: (1) methods based on sequence variability across species (i.e., Kabat et al, Sequences of Proteins of Immunological Interest (5 th edition, 1991, national institutes of Health, Bethesda MD)); and (2) methods 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 method or a combination of both methods.

The "constant region" of an antibody refers to the constant region of an antibody light chain or the constant region of an antibody heavy chain, alone or in combination.

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

As used herein, "substantially pure" refers to a 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, more preferably at least 99% pure.

"host cell" includes a single cell or cell culture that may be or has been the recipient of a vector for incorporation of a polynucleotide insertion sequence. Host cells include progeny of a single host cell that, due to natural, accidental, or deliberate mutation, is not necessarily identical (in morphology or in genomic DNA complement) to the original parent cell. Host cells include cells transfected in vivo with a polynucleotide of the invention.

The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can 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 human IgG heavy chain Fc region is generally defined as the stretch from amino acid residue position Cys226 or from Pro230 to its carboxy terminus. The numbering of residues in the Fc region is that of the EU index in Kabat. Kabat et al, Sequences of Proteins of immunological Interest, 5 th edition, public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically comprises two constant domains, CH2 and CH 3.

As used herein, "Fc receptor" and "FcR" describe receptors that bind the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, a preferred FcR is one that binds an IgG antibody (gamma receptor) and includes receptors of the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants or alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. FcR in ravatch and 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).

"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) that complexes with a cognate antigen. To assess complement activation, CDC assays such as those described by Gazzano-Santoro et al, J.Immunol.methods, 202:163(1996) can be performed.

A "functional Fc region" has at least one effector function of a native sequence Fc region. Exemplary "effector functions" include C1q binding, Complement Dependent Cytotoxicity (CDC), Fc receptor binding, antibody dependent cell mediated cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptor, BCR), and the like. Such effector functions typically require an Fc region in combination with a binding domain (e.g., an antibody variable domain), and can be assessed using various assays known in the art for assessing effector function of such antibodies.

A "native sequence Fc region" comprises an amino acid sequence that is identical to the amino acid sequence of a naturally occurring Fc region. A "variant Fc region" comprises an amino acid sequence that differs from a native sequence Fc region by at least one amino acid modification, but retains at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution as compared to the native sequence Fc region or to the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, and preferably from about one to about five amino acid substitutions in the native sequence Fc region or in the Fc region of the parent polypeptide. Variant Fc regions herein preferably have at least about 80% sequence identity to the native sequence Fc region and/or to the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity thereto, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity thereto.

As used herein, "antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells that express Fc receptors (fcrs) (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC activity of a molecule of interest can be assessed using an in vitro ADCC assay such as that described in U.S. patent No.5,500,362 or 5,821,337. Effector cells useful in such assays include Peripheral Blood Mononuclear Cells (PBMC) and NK cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be assessed in vivo, for example in an animal model such as that disclosed in Clynes et al, 1998, PNAS (USA), 95: 652-.

As used herein, "treatment" is a method for obtaining a beneficial or desired clinical result. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: improvements in any aspect of headache, including lessening the severity, alleviating the intensity of pain and other associated symptoms, reducing the frequency of relapse, increasing the quality of life of patients suffering from headache, and reducing the dosage of other medications required to treat headache. For migraine, other related symptoms include, but are not limited to, nausea, vomiting, and sensitivity to light, sound, and/or motion. For cluster headache, other related symptoms include, but are not limited to, swelling under or around the eyes, tears, redness, rhinorrhea or nasal congestion, and flushing.

By "reducing the" onset "of a" headache "is meant any of lessening the severity (which may include reducing the need and/or amount (e.g., exposure) of other medications and/or treatments commonly used for this condition, including, for example, ergotamine, dihydroergotamine, or triptan for migraine), duration, and/or frequency (including, for example, delaying or increasing the time of the next contingent episode in an individual). As will be appreciated by those skilled in the art, individuals may differ in their response to treatment, as such, for example, a "method of reducing the onset of headache in an individual" reflects administration of an anti-CGRP antagonist antibody based on a reasonable expectation that such administration is likely to cause a reduction in such onset in that particular individual.

By "ameliorating" headache or one or more symptoms of headache is meant reducing or ameliorating one or more symptoms of headache as compared to not administering an anti-CGRP antagonist antibody. "improving" also includes shortening or reducing the duration of symptoms.

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

As used herein, "headache hour" refers to the number of hours during which a subject experiences headache. Headache hours can be expressed in whole hours (e.g., one headache hour, two headache hours, three headache hours, etc.) or in whole and partial hours (e.g., 0.5 headache hours, 1.2 headache hours, 2.67 headache hours, etc.). One or more headache hours can be described in terms of a particular time interval. For example, "headache hours per day" may refer to the number of headache hours experienced by a subject within a one-day interval (e.g., a 24-hour period). In another example, "headache hours per week" can refer to the number of headache hours experienced by a subject over a one-week interval (e.g., a 7-day period). It will be appreciated that the week interval may or may not correspond to a calendar week. In another example, "number of headache hours per month" can refer to the number of headache hours experienced by a subject over a one month interval. It will be appreciated that a monthly interval (e.g., a 28-31 day period) may vary by day for a particular month, and may or may not correspond to a calendar month. In yet another example, "annual headache hours" can refer to the number of headache hours experienced by a subject within a one-year interval. It is to be understood that the one-year intervals (e.g., 365-day or 366-day periods) may vary by day for a particular year, and may or may not correspond to calendar years. In some embodiments, the headache hours can be combined with a particular type of headache (e.g., migraine, cluster headache, chronic headache, and tension headache). For example, "migraine hour" may refer to the number of hours during which a subject experiences migraine.

As used herein, "headache day" refers to the number of days during which a subject experiences headache. Headache days can be expressed as whole days (e.g., one headache day, two headache days, three headache days, etc.) or as whole and partial days (e.g., 0.5 headache day, 1.2 headache days, 2.67 headache days, etc.). One or more headache days can be described in terms of a particular time interval. For example, "headache day per week" can refer to the number of headache days experienced by a subject within a one week interval (e.g., a 7 day period). It will be appreciated that the week interval may or may not correspond to a calendar week. In another example, "monthly headache days" can refer to the number of headache days experienced by a subject within a monthly interval. It will be appreciated that a monthly interval (e.g., a 28-31 day period) may vary by day for a particular month, and may or may not correspond to a calendar month. In yet another example, "annual headache days" can refer to the number of headache days experienced by a subject within a one-year interval. It is to be understood that the one-year intervals (e.g., 365-day or 366-day periods) may vary by day for a particular year, and may or may not correspond to calendar years. In some embodiments, the headache day can be associated with a particular type of headache (e.g., migraine, cluster headache, chronic headache, and tension headache). For example, "migraine day" may refer to the number of days during which a subject experiences migraine.

As used herein, "delaying" the progression of headache means delaying, impeding, slowing, stopping, stabilizing and/or delaying the progression of the disease. The delay may be of varying lengths of time, depending on the medical history and/or the individual to be treated. As will be apparent to those skilled in the art, a sufficient or significant delay may actually encompass prevention, as the individual will not suffer from a headache (e.g., migraine). A method of "delaying" the development of a symptom is a method of reducing the probability of the development of the symptom over a given time frame and/or reducing the extent of the symptom over a given time frame, as compared to not using the method. Such comparisons are typically based on clinical studies conducted using a statistically significant number of subjects.

"development" or "progression" of headache refers to the initial manifestation and/or subsequent progression of the condition. The development of headache can be detected and assessed using standard clinical techniques well known in the art. However, development also refers to progression that is undetectable. For the purposes of this disclosure, development or progression refers to the biological process of a symptom. "development" includes occurrence, recurrence and pathogenesis. As used herein, "onset" or "occurrence" of headache includes incipient and/or recurrent.

As used herein, an "effective dose" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to achieve a beneficial or desired result. For prophylactic use, beneficial or desired results include results such as elimination or reduction of risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes present during disease development. For therapeutic use, beneficial or desired results include the following clinical results: such as reducing the intensity, duration, or frequency of headache episodes, as well as reducing one or more symptoms (biochemical, histological, and/or behavioral) caused by headache, including its complications and intermediate pathological phenotypes present during the development of the disease, improving the quality of life of patients with the disease, reducing the dosage of other drugs required to treat the disease, enhancing the effect of another drug, and/or delaying the progression of the disease in the patient. An effective dose may be administered in one or more administrations. For the purposes of this disclosure, an effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to effect, directly or indirectly, prophylactic or therapeutic treatment. As understood in the clinical setting, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, an "effective dose" may be considered in the context of administration of one or more therapeutic agents, and a single agent is considered to be administered in an effective amount if the desired result is achieved or has been achieved in combination with one or more other agents.

An "individual" or "subject" is a mammal, more preferably a human. Mammals also include, but are not limited to, domestic animals, sport animals, pets, primates, horses, dogs, cats, mice, and rats.

As used herein, "vector" means a construct capable of delivery, preferably expression, of 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.

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

As used herein, a "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 be non-reactive to the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, water, emulsions such as oil/water emulsions, and various types of wetting agents. Preferred diluents for aerosol or parenteral administration are phosphate buffered saline or physiological (0.9%) saline. Compositions comprising such carriers are formulated by well-known conventional methods (see, e.g., Remington's pharmaceutical Sciences, 18 th edition, A.Gennaro eds., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy, 20 th edition, Mack Publishing, 2000).

As used herein, the term "k_on"is intended to mean the rate constant for binding of an antibody to an antigen.

As used herein, the term "k_off"is intended to mean the rate constant at which an antibody dissociates from an antibody/antigen complex.

As used hereinBy the term "K_D"is intended to mean the equilibrium dissociation constant of an antibody-antigen interaction.

As used herein, the term "vasomotor symptoms" is intended to refer to conditions associated with vasodilation. Such vasodilation may be associated or may be associated with the following symptoms: headache (such as migraine with or without precursor; hemiplegic migraine; chronic migraine; episodic migraine; cluster headache; migrainous neuralgia; chronic headache; tension headache; headache caused by infections or increased intracranial pressure caused by tumors; chronic episodic migraine; other headache not associated with structural damage; headache associated with non-vascular intracranial diseases; headache associated with substance administration or withdrawal; headache associated with non-head infections; headache associated with metabolic disorders; headache associated with cranial, cervical, ocular, otic, nasal, sinus, dental, oral or other facial or cranial structural diseases; cranial neuralgia; and nerve trunk pain and afferent nerve block pain), in particular hot flush (or hot flush) caused by thermoregulatory dysfunction, Cold, insomnia, sleep disorders, mood disorders, irritability, perspiration, night sweats, daytime perspiration, fatigue, and the like.

As used herein, the terms "hot flashes", and "hot flashes" are art-recognized terms that refer to occasional fluctuations in body temperature, typically consisting of sudden skin flashes, typically accompanied by perspiration of the subject.

A. Method for preventing or treating vasomotor symptoms and/or headache

In one aspect, the present invention provides a method of treating or reducing the onset of at least one vasomotor symptom in a subject. In another aspect, the invention provides methods of treating or reducing the onset of headache (e.g., migraine) in a subject. In some embodiments, the method comprises administering to the individual an effective amount of an antibody or polypeptide derived from an antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody). In some embodiments, the at least one vasomotor symptom may be associated with headache (e.g., migraine) and/or hot flashes.

In another aspect, the invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the development or progression of at least one vasomotor symptom 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 may be associated with headache (e.g., migraine) and/or hot flashes.

In another aspect, the present invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the development or progression of headache (e.g., migraine) or a symptom associated with headache (e.g., dysentery or photosensitivity) in an individual comprising administering to the individual 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 for treating headache.

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 in combination, i.e., their administrations are close enough in time to allow their separate therapeutic effects to overlap. For example, the amount of 5-HT agonist or NSAID administered in combination with the anti-CGRP antibody should be sufficient to reduce the frequency of headache recurrence or produce a more sustained efficacy in the patient compared to administration of either of these agents in the absence of the other. This procedure can be used to treat headaches, which fall into a broad category that includes: migraine with or without aura; hemiplegic migraine; chronic migraine headache; episodic migraine headache; high frequency episodic migraine; cluster headache; migraine-associated neuralgia; chronic headache; tension headache; headache due to other medical conditions (such as infections caused by tumors or increased intracranial pressure); chronic paroxysmal migraine; other headaches not associated with structural damage; headache associated with non-vascular intracranial disorders; headache associated with substance administration or withdrawal thereof; headache associated with non-head infections; headache associated with metabolic disorders; headaches associated with disorders of the head, neck, eyes, ears, nose, sinuses, teeth, mouth or other facial or cranial structures; cranial nerve pain; as well as nerve trunk pain and afferent blocking pain.

Additional non-limiting examples of additional agents that may be administered in combination with the anti-CGRP antagonist antibody include one or more of the following agents:

(i) opioid analgesics, for example, morphine, heroin, hydromorphone, oxymorphone, levorphanol, methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone, propoxyphene, nalmefene, nalprofen, naloxone, naltrexone, buprenorphine, butorphanol, nalbuphine, or pentazocine;

(ii) non-steroidal anti-inflammatory drugs (NSAIDs), for example, aspirin, diclofenac, diflunisal, 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-745,337; NS 398; or a pharmaceutically acceptable salt thereof;

(iii) barbiturates, for example, amobarbital, alprenol, sec-butyl barbital, butabarbital, tolbarbital, methabarbital, methohexital, pentobarbital, phenobarbital, secobarbital, talbutane, semetol (theomylal), or pentothiobarbital or a pharmaceutically acceptable salt thereof;

(iv) barbiturate analgesics, for example, butabital or a pharmaceutically acceptable salt thereof or a composition comprising butabital.

(v) Benzodiazepines having a sedative effect, for example, methotrexate, chlordiazepoxide, diazepam, flurazepam, lorazepam, oxazepam, temazepam or triazolam or a pharmaceutically acceptable salt thereof;

(vi) h with sedative effect₁Antagonists, for example diphenhydramine, pyrilamine, promethamine, chlorpheniramine or cloxazine or pharmaceutically acceptable salts thereof;

(vii) sedatives such as glutethimide, tranquilizer (meprobamate), methaqualone or dichlofenoxaprop or their pharmaceutically acceptable salts;

(viii) skeletal muscle relaxants, for example, baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol, or nordiphenhydramine, or pharmaceutically acceptable salts thereof;

(ix) NMDA receptor antagonists, for example, dextromethorphan ((+) -3-hydroxy-N-methyl morphinan) or its metabolite dextrorphan ((+) -3-hydroxy-N-methyl morphinan), ketamine, memantine, pyrroloquinoline quinone or cis-4- (phosphonomethyl) -2-piperidinecarboxylic acid or a pharmaceutically acceptable salt thereof;

(x) α -adrenergic agents, for example, 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, for example, desipramine, imipramine, amitriptyline or nortriptyline;

(xii) Anticonvulsants, for example, carbamazepine or valproic acid;

(xiii) Tachykinin (NK) antagonists, in particular NK-3, NK-2 or NK-1 antagonists, such as (alpha R,9R) -7- [3, 5-bis (trifluoromethyl) benzyl ] -8,9,10, 11-tetrahydro-9-methyl-5- (4-methylphenyl) -7H- [1,4] diazepine [2,1-g ] [1,7] naphthyridine-6-13-dione (TAK-637), 5- [ [ (2R,3S) -2- [ (1R) -1- [3, 5-bis (trifluoromethyl) phenyl ] ethoxy-3- (4-fluorophenyl) -4-morpholinyl ] methyl ] -1, 2-dihydro-3H-1, 2, 4-triazol-3-one (MK-869), lanopiptan, dapitaptan or 3- [ [ 2-methoxy-5- (trifluoromethoxy) phenyl ] methylamino ] -2-phenyl-piperidine (2S, 3S);

(xiv) Muscarinic antagonists, such as oxybutynin, tolterodine, propiverine, trospium chloride or darifenacin;

(xv) COX-2 inhibitors, e.g., celecoxib, rofecoxib, or valdecoxib;

(xvi) Non-selective COX inhibitors (preferably with GI protection), for example, nitroflurbiprofen (HCT-1026);

(xvii) Coal tar analgesics, particularly acetaminophen;

(xviii) Neuroleptics, such as dapiprol;

(xix) A vanilloid receptor agonist (e.g., resiniferatoxin) or antagonist (e.g., capsazepine);

(xix) Beta-adrenergic drugs, such as propranolol;

(xx) Local anesthetics, such as mexiletine;

(xxi) Corticosteroids, such as dexamethasone;

(xxii) A 5-hydroxytryptamine receptor agonist or antagonist;

(xxiii) Cholinergic (nicotinic) analgesics;

(xxiv) Tramadol (trade mark);

(xxv) PDEV inhibitors such as sildenafil, vardenafil or tadalafil;

(xxvi) Alpha-2-ligands such as gabapentin or pregabalin;

(xxvii) Cannabinoids (canabinoids); and

(xxviii) Antidepressants such as amitriptyline (Elavil), trazodone (Desyrel) and imipramine (tofranel) or anticonvulsants such as phenytoin (Dilantin) or carbamazepine (Tegretol).

One skilled in the art will be able to determine the appropriate dosage of a particular agent to be used in combination with the anti-CGRP antibody. For example, sumatriptan can be administered in a dose of about 0.01 to about 300 mg. In some cases, sumatriptan can be administered at a dose of 2mg to 300 mg. A typical dose of sumatriptan is about 25 to about 100mg when administered parenterally, about 50mg being generally preferred, and a preferred dose is about 6mg when administered parenterally. However, these dosages can be varied according to standard methods in the art so that they can be optimized for a particular patient or for a particular combination therapy. Additionally, for example, celecoxib can be administered in an amount between 50 and 500 mg.

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

In another aspect, the present disclosure provides a method of treating or reducing the onset of a headache (e.g., migraine) in a subject, comprising administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over multiple days. In some embodiments, the amount of monoclonal antibody administered daily over the multiple days may be between 0.1mg-5000mg, 1mg-5000mg, 10mg-5000mg, 100mg-5000mg, 1000 mg-5000mg, 0.1mg-4000mg, 1mg-4000mg, 10mg-4000mg, 100mg-4000mg, 1000 mg-4000mg, 0.1mg-3000mg, 1mg-3000mg, 10mg-3000mg, 100mg-3000mg, 1000 mg-3000mg, 0.1mg-2000mg, 1mg-2000mg, 10mg-2000mg, 100mg-2000mg, 1000 mg-2000mg, 0.1mg-1000mg, 1mg-1000mg, 10mg-1000mg, or 100mg-1000 mg. In some embodiments, the amount is between 100-2000 mg.

In another aspect, the present disclosure provides a method of treating or reducing the onset of headache (e.g., migraine) in a subject comprising administering a single dose of a monoclonal antibody (e.g., a monoclonal anti-CGRP antagonist antibody) to the subject in an amount that modulates the CGRP pathway. In some embodiments, the single dose may be an amount of antibody between 0.1mg-5000mg, 1mg-5000mg, 10mg-5000mg, 100mg-5000mg, 1000 mg-5000mg, 0.1mg-4000mg, 1mg-4000mg, 10mg-4000mg, 100mg-4000mg, 1000 mg-4000mg, 0.1mg-3000mg, 1mg-3000mg, 10mg-3000mg, 100mg-3000mg, 1000 mg-3000mg, 0.1mg-2000mg, 1mg-2000mg, 10mg-2000mg, 100mg-2000mg, 1000 mg-2000mg, 0.1mg-1000mg, 1mg-1000mg, 10mg-1000mg, or 100mg-1000 mg. In some embodiments, the single dose may be an amount of antibody between 100-2000 mg.

In another aspect, the present disclosure provides a method of treating or reducing the onset of at least one vasomotor symptom in a subject, comprising administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody) over multiple days. In some embodiments, the amount of monoclonal antibody administered daily over the multiple days may be between 0.1mg-5000mg, 1mg-5000mg, 10mg-5000mg, 100mg-5000mg, 1000 mg-5000mg, 0.1mg-4000mg, 1mg-4000mg, 10mg-4000mg, 100mg-4000mg, 1000 mg-4000mg, 0.1mg-3000mg, 1mg-3000mg, 10mg-3000mg, 100mg-3000mg, 1000 mg-3000mg, 0.1mg-2000mg, 1mg-2000mg, 10mg-2000mg, 100mg-2000mg, 1000 mg-2000mg, 0.1mg-1000mg, 1mg-1000mg, 10mg-1000mg, or 100mg-1000 mg. In some embodiments, the amount is between 100-2000 mg.

In another aspect, the present disclosure provides a method of reducing the number of monthly headache hours experienced by a subject comprising administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody). In some embodiments, the amount of the monoclonal antibody is effective to reduce the number of headache hours per month 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 amount of the single clone is effective to reduce the number of headache hours per month by at least 20 headache hours after a single dose. In some embodiments, the amount of the monoclonal antibody is effective to reduce the number of headache hours per month by at least 40 headache hours. In some embodiments, the amount of the monoclonal antibody is effective to reduce 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 amount of the single clone is effective to reduce the number of monthly headache hours by at least 15% after a single dose.

In another aspect, the present disclosure provides a method of reducing the number of monthly headache days experienced by a subject comprising administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody). In some embodiments, the amount of the monoclonal antibody is effective to reduce the number of headache days per month 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 amount of the monoclonal antibody is effective to reduce the number of headache days per month by at least 3 headache days after a single dose. In some embodiments, the amount of the monoclonal antibody is effective to reduce the number of days to headache per month 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 another aspect, the present disclosure provides a method of reducing the use of an anti-headache agent in a subject, comprising administering to the subject a monoclonal antibody that modulates the CGRP pathway (e.g., a monoclonal anti-CGRP antagonist antibody). In some embodiments, the amount of the monoclonal antibody is effective to reduce monthly use of the anti-headache agent 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 by the subject. In some embodiments, the amount of the monoclonal antibody is effective to reduce monthly use of the anti-headache agent by the subject by at least 15%. The anti-headache agent may be any type of anti-headache agent described elsewhere herein. Non-limiting examples of anti-headache agents include 5-HT1 agonists (as well as agonists acting at other 5-HT1 sites), triptans (e.g., sumatriptan, zolmitriptan, naratriptan, rizatriptan, eletriptan, almotriptan, frovatriptan), ergot alkaloids (e.g., ergotamine tartrate, ergotamine maleate, and dihydroergotamine mesylate (e.g., dihydroergotamine mesylate, and dihydroergotamine mesylate (DHE 45)), and non-steroidal anti-inflammatory agents (NSAIDs) (e.g., aspirin, diclofenac, diflunisal, etodolac, fenbufen, fenoprofen, flurbiprofen, ibuprofen, indomethacin, ketoprofen, etc.) 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-745,337; NS 398; or a pharmaceutically acceptable salt thereof), opiates (e.g., oxycodone), and beta-adrenergic antagonists (e.g., propranolol).

With respect to all methods described herein, reference to an antibody (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) also includes compositions comprising one or more of these agents. Thus, such compositions may be used according to methods involving the antibodies described herein. These compositions may also comprise suitable excipients, such as the pharmaceutically acceptable excipients described elsewhere herein. The present invention may be used alone or in combination with other conventional therapeutic methods.

The antibodies described herein (e.g., monoclonal antibodies, anti-CGRP antagonist antibodies, monoclonal anti-CGRP antagonist antibodies) can be administered to an individual or subject at any therapeutic dose, by any suitable route, and in any suitable formulation. It will be apparent to those skilled in the art that the examples described herein are not intended to be limiting, but rather illustrative of available technology. Thus, in some embodiments, the antibodies described herein can be administered to an individual according to known methods, such as intravenous administration, e.g., in a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, sublingual, intraarterial, intrasynovial, by insufflation, intrathecal, oral, inhalation, intranasal (e.g., inhalation or non-inhalation), buccal, rectal, transdermal, intracardiac, intraosseous, intradermal, transmucosal, vaginal, intravitreal, periarticular, topical, epidermal or topical routes. Administration may be systemic, e.g., intravenous, or local. Commercially available nebulizers of liquid formulations, including jet nebulizers and ultrasonic nebulizers, can be used for administration. Liquid formulations can be directly nebulized, while lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, or inhaled as lyophilized and ground powders.

In some embodiments, the antibodies described herein can be administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable reservoirs or local delivery catheters for antibodies, such as infusion catheters, indwelling catheters or needle catheters, synthetic grafts, adventitial wraps, shunts and stents or other implantable devices, site-specific vectors, direct injection or direct administration. See, for example, PCT publication No. wo 00/53211 and U.S. patent No.5,981,568.

Various formulations of the antibodies described herein can be used for administration. In some embodiments, the antibody is administered in pure form. In some embodiments, the antibody and pharmaceutically acceptable excipient may be in the form of various formulations. Pharmaceutically acceptable excipients are known in the art and are relatively inert substances that facilitate administration of a pharmacologically effective substance. For example, an excipient may impart a form or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts of varying osmolality, encapsulating agents, buffering agents, and skin penetration enhancers. Excipients and formulations for parenteral and parenteral drug delivery are disclosed in Remington, The Science and Practice of pharmacy, 20 th edition, Mack Publishing (2000).

In some embodiments, these agents (including the antibodies described herein) can be formulated for administration by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). Thus, these agents may be combined with a pharmaceutically acceptable vehicle, such as saline, Ringer's solution, dextrose solution, and the like. The particular dosage regimen, i.e., dosage, schedule and repeat administration, will depend upon the particular individual and the individual's medical history.

In some embodiments, these agents (including the antibodies described herein) can be formulated for peripheral administration. Such formulations may be administered peripherally by any suitable peripheral route, including intravenously and subcutaneously. Agents prepared for peripheral administration may include substances, drugs and/or antibodies delivered non-centrally, spinally, intrathecally or directly introduced into the CNS. Non-limiting examples of peripheral routes of administration include oral, sublingual, buccal, topical, rectal, by inhalation, transdermal, subcutaneous, intravenous, intraarterial, intramuscular, intracardiac, intraosseous, intradermal, intraperitoneal, transmucosal, vaginal, intravitreal, intraarticular, periarticular, topical, or epidermal routes.

Therapeutic formulations of antibodies for use according to The present disclosure may be prepared for storage and/or use by mixing The antibody of The desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington, The science and Practice of Pharmacy, 20 th edition, Mack Publishing (2000)), and in some cases may be in The form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Therapeutic formulations of the antibodies may comprise one or more pharmaceutically acceptable carriers, excipients, or stabilizers, non-limiting examples of which includeBuffers such as phosphoric acid, citric acid and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine di-ammonium; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; 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; an amino acid (e.g., at a concentration of 0.1mM to 100mM, 0.1mM to 1mM, 0.01mM to 50mM, 1mM to 30mM, 1mM to 20mM, 10mM to 25mM), such as glycine, glutamine, methionine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents (e.g., at a concentration of 0.001mg/mL to 1mg/mL, 0.001mg/mL to 0.1mg/mL, 0.001mg/mL to 0.01mg/mL, 0.01mg/mL to 0.1mg/mL), such as EDTA (e.g., disodium ethylenediaminetetraacetate dihydrate); a sugar (e.g., at a concentration of 1mg/mL to 500mg/mL, 10mg/mL to 200mg/mL, 10mg/mL to 100mg/mL, 50mg/mL to 150mg/mL), such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a nonionic surfactant (e.g., at a concentration of 0.01mg/mL to 10mg/mL, 0.01mg/mL to 1mg/mL, 0.1mg/mL to 1mg/mL, 0.01mg/mL to 0.5mg/mL) such as, for example, TWEEN^TM(e.g., polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80)), PLURONICS^TMOr polyethylene glycol (PEG).

Antibody formulations can be characterized with respect to any of a variety of physical properties. For example, the liquid antibody formulation can have any suitable pH for therapeutic efficacy, safety, and storage. For example, the pH of the liquid antibody formulation may be from pH4 to about pH9, pH5 to pH8, pH5 to pH7, or pH6 to pH 8. In some embodiments, the 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.

In another example, the liquid antibody formulation can have any suitable viscosity for therapeutic efficacy, safety, and storage. For example, the viscosity of the liquid antibody formulation can be 0.5 to 100, 1 to 50, 1 to 20, 1 to 15, or 5 to 15 centipoise (cP) at 25 ℃. In some embodiments, the liquid antibody formulation may have a viscosity of 0.5cP, 1cP, 1.2cP, 1.4cP, 1.6cP, 1.8cP, 2.0cP, 2.2cP, 2.4cP, 2.6cP, 2.8cP, 3.0cP, 3.2cP, 3.4cP, 3.6cP, 3.8cP, 4.0cP, 4.2cP, 4.4cP, 4.6cP, 4.8cP, 5.0cP, 5.2cP, 5.4cP, 5.6cP, 5.8cP, 6.0cP, 6.2cP, 6.4cP, 6.6cP, 6.8cP, 7.0cP,7.2cP, 7.4cP, 7.6cP, 7.8, 8.0cP, 8.2cP, 8.4cP, 8.6cP, 8.8cP, 8.0cP, 8.2cP, 8.4cP, 8.6cP, 8.9.0 cP,7.2cP, 7.4cP, 7.6cP, 7.8cP, 13 cP, 13.12 cP, 13 cP, 13.6cP, 13 cP, or more.

In another example, the liquid antibody formulation can have any suitable conductivity for therapeutic efficacy, safety, and storage. For example, the conductivity of the liquid antibody formulation can be from 0.1 milliSiemens/centimeter (mS/cm) to 15mS/cm, from 0.1mS/cm to 10mS/cm, from 0.1mS/cm to 5mS/cm, from 0.1mS/cm to 2mS/cm, or from 0.1mS/cm to 1.5 mS/cm. In some embodiments, the liquid antibody formulation may have a concentration of 0.19mS/cm, 0.59mS/cm, 1.09mS/cm, 1.19mS/cm, 1.29mS/cm, 1.39mS/cm, 1.49mS/cm, 1.59mS/cm, 1.69mS/cm, 1.79mS/cm, 1.89mS/cm, 1.99mS/cm, 2.09mS/cm, 2.19mS/cm, 2.29mS/cm, 2.39mS/cm, 2.49mS/cm, 2.59mS/cm, 2.69mS/cm, 2.79mS/cm, 2.89mS/cm, 2.99mS/cm, 3.09mS/cm, 3.19mS/cm, 3.29mS/cm, 3.39 cm, 3.89mS/cm, 3.19mS/cm, 3.3.29 mS/cm, 3.1 mS/cm, 3., 4.39mS/cm, 4.49mS/cm, 4.59mS/cm, 4.69mS/cm, 4.79mS/cm, 4.89mS/cm, 4.99mS/cm, 5.09mS/cm, 6.09mS/cm, 6.59mS/cm, 7.09mS/cm, 7.59mS/cm, 8.09mS/cm, 8.59mS/cm, 9.09mS/cm, 9.59mS/cm, 10.09mS/cm, 10.59mS/cm, 11.09mS/cm, 11.59mS/cm, 12.09mS/cm, 12.59mS/cm, 13.09mS/cm, 13.59mS/cm, 14.09mS/cm, 14.59mS/cm, or 15.09mS/cm, or higher or lower conductivity, or higher.

In another example, the liquid antibody formulation can have any suitable osmolality for therapeutic efficacy, safety, and storage. For example, the osmolality (osmolality) of the liquid antibody formulation can be 50 milliosmoles per kilogram (mOsm/kg) to 5000mOsm/kg, 50mOsm/kg to 2000mOsm/kg, 50mOsm/kg to 1000mOsm/kg, 50mOsm/kg to 750mOsm/kg, or 50mOsm/kg to 500 mOsm/kg. In some embodiments, the liquid antibody preparation may have a concentration of 50mOsm/kg, 60mOsm/kg, 70mOsm/kg, 80mOsm/kg, 90mOsm/kg, 100mOsm/kg, 120mOsm/kg, 140mOsm/kg, 160mOsm/kg, 180mOsm/kg, 200mOsm/kg, 220mOsm/kg, 240mOsm/kg, 260mOsm/kg, 280mOsm/kg, 300mOsm/kg, 320mOsm/kg, 340mOsm/kg, 360mOsm/kg, 380mOsm/kg, 400mOsm/kg, 420mOsm/kg, 440mOsm/kg, 460mOsm/kg, 480mOsm/kg, 500mOsm/kg, 520mOsm/kg, 540mOsm/kg, 560mOsm/kg, 580mOsm/kg, 620mOsm/kg, 660mOsm/kg, 180mOsm/kg, or similar to the antibody preparation, or similar to that, 700mOsm/kg, 720mOsm/kg, 740mOsm/kg, 760mOsm/kg, 780mOsm/kg, 800mOsm/kg, 820mOsm/kg, 840mOsm/kg, 860mOsm/kg, 880mOsm/kg, 900mOsm/kg, 920mOsm/kg, 940mOsm/kg, 960mOsm/kg, 980mOsm/kg, 1000mOsm/kg, 1050mOsm/kg, 1100mOsm/kg, 1150mOsm/kg, 1200mOsm/kg, 1250mOsm/kg, 1300mOsm/kg, 1350mOsm/kg, 1400mOsm/kg, 1450mOsm/kg, 1500mOsm/kg, or the osmolality may be higher or lower.

Liposomes containing antibodies can be prepared by methods known in the art, such as 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. patent nos. 4,485,045 and 4,544,545. Liposomes having increased circulation time are disclosed, for example, in U.S. Pat. No.5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to give liposomes with the desired diameter.

The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacylate) 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, 20 th edition, Mack Publishing (2000).

Can be prepared into sustained release preparation. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (hydroxyethyl 2-methacrylate) or' poly (vinyl alcohol)), polylactides (U.S. Pat. No.3,773,919), copolymers of L-glutamic acid and 7-ethyl-L-glutamic acid, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate and poly-D- (-) -3-hydroxybutyric acid.

Formulations for in vivo administration should generally be sterile. This is readily accomplished by, for example, filtration through sterile filtration membranes. The therapeutic antibody composition is typically placed in a container having a sterile access port, such as an intravenous bag or vial having a stopper that can be pierced by a hypodermic needle.

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

For preparing solid compositions such as tablets, the principal active ingredient may be mixed with a pharmaceutically acceptable carrier, e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, e.g., water, to form a solid preformulation composition comprising a homogeneous mixture of a compound of the present invention or a non-toxic pharmaceutically acceptable salt thereof. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. The solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500mg of the active ingredient of the present invention. The tablets or pills of the novel compositions may be coated or compounded so as to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill may contain an inner dosage and an outer dosage component, the latter being in the form of a coating over the former. The two components may be separated by an enteric layer which acts to resist disintegration in the stomach and allows the inner component to pass intact through the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include, in particular, nonionic agents, such as polyoxyethylene sorbitan (e.g., Tween @)^TM20. 40, 60, 80 or 85) and other sorbitan (e.g. Span)^TM20. 40, 60, 80, or 85). Compositions with surfactants 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, such as mannitol or other pharmaceutically acceptable vehicles, may be added if desired.

Suitable emulsions are commercially available fat emulsions such as Intralipid^TM、Liposyn^TM、Infonutrol^TM、Lipofundin^TMAnd Lipiphysan^TMAnd (4) preparation. The active ingredient may be dissolved in a pre-mixed emulsion composition, or may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) and in an emulsion formed when mixed with a phospholipid (e.g., lecithin, soybean lecithin, or soybean lecithin) and water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable emulsions typically comprise up to 20% oil, for example between 5 and 20%. The fat emulsion may comprise fat droplets of between 0.1 and 1.01 m, in particular between 0.1 and 0.51 m, and have a pH in the range of 5.5 to 8.0.

The emulsion composition may be prepared by combining the antibody with an Intralipid^TM.Or their components (soybean oil, lecithin, glycerin and water).

Compositions for inhalation or insufflation include solutions and suspensions or mixtures thereof in pharmaceutically acceptable aqueous or organic solvents, as well as powders. The liquid or solid composition may comprise suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by oral or nasal inhalation routes to produce local or systemic effects. Compositions in preferably sterile pharmaceutically acceptable solvents may be nebulized using a gas. The nebulized solution can be inhaled directly from the nebulizing device, or the nebulizing device can be connected to a face mask, tent, or intermittent positive pressure ventilator. The solution, suspension or powder composition may preferably be administered orally or nasally from a device which delivers the formulation in a suitable manner.

In some embodiments, a formulation comprising an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) can be prepared for any suitable route of administration with an antibody amount in the range of 0.1mg to 3000mg, 1mg to 1000mg, 100 to 1000mg, or 100 to 500 mg. In some cases, a formulation comprising an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) can comprise an antibody amount of up to or at least 0.1mg, 1mg, 100mg, 1mg, 10mg, 25mg, 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, 675mg, 700mg, 725mg, 750mg, 775mg, 800mg, 825mg, 850mg, 875mg, 900mg, 925mg, 950mg, 975mg, 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, or 3000 mg.

In some embodiments, a liquid formulation comprising an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) can be prepared for any suitable route of administration, wherein the antibody concentration is between 0.1 to 500mg/mL, 0.1 to 375mg/mL, 0.1 to 250mg/mL, 0.1 to 175mg/mL, 0.1 to 100mg/mL, 1mg/mL to 500mg/mL, 1mg/mL to 375mg/mL, 1mg/mL to 300mg/mL, 1mg/mL to 250mg/mL, 1mg/mL to 200mg/mL, 1mg/mL to 150mg/mL, 1mg/mL to 100mg/mL, 10mg/mL to 500mg/mL, 10mg/mL to 375mg/mL, 10mg/mL to 250mg/mL, a, 10mg/mL to 150mg/mL, 10mg/mL to 100mg/mL, 100mg/mL to 500mg/mL, 100mg/mL to 450mg/mL, 100mg/mL to 400mg/mL, 100mg/mL to 350mg/mL, 100mg/mL to 300mg/mL, 100mg/mL to 250mg/mL, 100mg/mL to 200mg/mL, or 100mg/mL to 150 mg/mL. In some embodiments, the liquid formulation can comprise an antibody described herein at a concentration of up to, at least, or less than 0.1, 0.5, 1,5, 10, 1520, 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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

The antibodies described herein can be administered using any suitable method, including by injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). The antibodies can also be administered by inhalation, as described herein. In some cases, the antibody may be administered nasally (inhaled or non-inhaled). Generally, for administration of the antibodies described herein, the initial candidate dose may be about 2 mg/kg. For the purposes of the present invention, typical daily dosages may range from about any of 3 μ g/kg to 30 μ g/kg to 300 μ g/kg to 3mg/kg to 30mg/kg to 100mg/kg or more, depending on the factors described above. For example, dosages of about 1mg/kg, about 2.5mg/kg, about 5mg/kg, about 10mg/kg and about 25mg/kg may be used. For repeated administration for several days or longer, depending on the condition, the treatment is maintained until the desired suppression of symptoms occurs, or until a sufficient level of treatment is achieved, e.g., to reduce pain. An exemplary dosing regimen includes administering an initial dose of about 8.5mg/kg, followed by a maintenance dose of about 2.8mg/kg antibody every week, or then a maintenance dose of about 2.8mg/kg every other week. Another exemplary dosing regimen includes subcutaneously administering a dose of 100mg, 125mg, 150mg, 200mg, 225mg, 250mg, 275mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 675mg, or 900mg once a month to the subject. Another exemplary dosing regimen includes an initial dose of 675mg administered subcutaneously followed by a dose of 225mg of antibody administered subcutaneously per month. However, other dosage regimens may be used depending on the pharmacokinetic decay pattern that the practitioner wishes to achieve. For example, in some embodiments, one to four times per week administration is contemplated. The course of treatment is readily monitored by conventional techniques and assays. The dosing regimen, including the CGRP antagonist used, may vary over time.

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

In some embodiments, the dose or amount of an antibody described herein and administered to a subject (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) may be in the range of 0.1 to 500, 0.1 to 100, 0.1 to 50, 0.1 to 20, 0.1 to 10,1 to 7,1 to 5, or 0.1 to 3mg/kg body weight. In some embodiments, the dose or amount of an antibody described herein and administered to a subject (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) may be, may be at most, may be less than, or may 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, 11.5, 12.0, 12.5, 12.0, 12.5, 13.0, 19, 35, 40, 19, 40, 35, 19, 40, 19, 40, 19, 40, 35, 40, 19, 40, 45, 40, 19, 40, 120. 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500mg/kg body weight.

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

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

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

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

In some embodiments, a dose or amount of 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 1750mg, 2500mg, 2550mg, 2600mg, 2650mg, 2700mg, 2750mg, 2800mg, 28050 mg, 2900mg, 2950mg, 3000mg, or more of the antibody can be administered (e.g., subcutaneously or intravenously) once per month to a subject. In some embodiments, a dose or amount of between 0.1mg to 5000mg, 1mg to 4000mg, 10mg to 3000mg, 10mg to 2000mg, 100mg to 2000mg, 150mg to 2000mg, 200mg to 2000mg, 250mg to 2000mg, 300mg to 2000mg, 350mg to 2000mg, 400mg to 2000mg, 450mg to 2000mg, 500mg to 2000mg, 550mg to 2000mg, 600mg to 2000mg, 650mg to 2000mg, 700mg to 2000mg, 750mg to 2000mg, 800mg to 2000mg, 850mg to 2000mg, 900mg to 2000mg, 950mg to 2000mg, or 1000mg to 2000mg of the antibody may be administered (e.g., subcutaneously or intravenously) once/month to the subject. In some embodiments, between 100 and 2000mg of antibody is administered once/month.

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

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

In some embodiments, a dose or amount of an antibody (e.g., a monoclonal antibody that modulates a CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) is administered to a subject (e.g., subcutaneously or intravenously) at a frequency of at least, less than, or at most one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty/quarterly. It is understood that "quarterly" may refer to a quarter year period of time, or may also refer to a calendar quarterly, such as a 1-3-31, 4-1-6-30, 7-1-9-30, or 10-1-12-31 month period of time. In some cases, "quarterly" may refer to a period of time of about three months.

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

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

In some embodiments, a dose or amount of 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg, 400mg, 450mg, 500mg, 550mg, 600mg, 650mg, 700mg, 750mg, 800mg, 850mg, 900mg, 950mg, 1000mg, 1050mg, 1100mg, 1150mg, 1200mg, 1250mg, 1300mg, 1350mg, 1400mg, 1450mg, 1500mg, 1550mg, 1600mg, 1650mg, 1700mg, 1750mg, 1800mg, 1850mg, 1900mg, 1950mg, 2000mg, 2050mg, 2100mg, 2150mg, 2200mg, 2250mg, 2300mg, 2350mg, 2450mg, 2500mg, 2550mg, 2600mg, 2650mg, 2700mg, 2750mg, 2800mg, 28050 mg, 2900mg, 2950mg, 3000mg, or more of the antibody may be administered to a subject once per year. In some embodiments, an antibody in a dose or amount between 0.1mg to 5000mg, 1mg to 4000mg, 10mg to 3000mg, 10mg to 2000mg, 100mg to 2000mg, 150mg to 2000mg, 200mg to 2000mg, 250mg to 2000mg, 300mg to 2000mg, 350mg to 2000mg, 400mg to 2000mg, 450mg to 2000mg, 500mg to 2000mg, 550mg to 2000mg, 600mg to 2000mg, 650mg to 2000mg, 700mg to 2000mg, 750mg to 2000mg, 800mg to 2000mg, 850mg to 2000mg, 900mg to 2000mg, 950mg to 2000mg, or 1000mg to 2000mg may be administered to a subject once a year. In some embodiments, between 450mg and 2000mg is administered once a year or less frequently.

In some embodiments, the methods may comprise administering an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) to the subject over multiple days. Two, three, four, five, six, seven, eight or more of the plurality of days can be separated by 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. In some embodiments, two of the multiple days are separated by more than one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty or more days. Further, in some embodiments, the amount of antibody administered on a first day of the multiple days can be different (e.g., higher or lower) than the amount of antibody administered on a second day.

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

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

In some embodiments, a dose or amount of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) can be divided into sub-doses and administered in multiple sub-doses depending, for example, on the route of administration and/or the particular formulation being administered. For example, in the case where the dose is administered subcutaneously, the subcutaneous dose may be divided into multiple sub-doses, and each sub-dose administered at a different site, to avoid, for example, a larger single subcutaneous injection at a single site. For example, a subcutaneous dose of 900mg may be divided into four sub-doses of 225mg each, and each 225mg dose administered to a different site, which may help to minimize the volume injected at each site. The sub-dose distribution may be equal (e.g., 4 equal sub-doses) or may be unequal (e.g., 4 sub-doses, with 2 sub-doses being as large as the other sub-doses).

In some embodiments, the number of doses of antibody administered to a subject during a course of treatment may vary depending on, for example, the vasomotor symptoms and/or the reduced onset of headache achieved in the subject. In some embodiments, the vasomotor symptoms are associated with forms of headache (e.g., migraine, chronic migraine, episodic migraine, other types of headache, etc.). For example, the number of doses administered during a course of treatment can be, can be at least, or can 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 (e.g., in cases where the subject has chronic migraine), treatment may be given indefinitely. In some cases, the treatment may be acute such that no more than 1,2,3,4, 5, or 6 doses are administered to the subject for treatment.

In some embodiments, a dose (or sub-dose) or amount of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) can be formulated in a liquid formulation and administered (e.g., by subcutaneous injection, by intravenous injection) to a subject. In such cases, the volume of the liquid formulation comprising the antibody may vary depending on, for example, the concentration of the antibody in the liquid formulation, the desired dose of the antibody, and/or the route of administration used. For example, the volume of a liquid formulation comprising an antibody described herein and administered (e.g., by injection, such as, for example, subcutaneous injection or intravenous injection) to a subject can be 0.001mL to 10.0mL, 0.01mL to 5.0mL, 0.1mL to 5mL, 0.1mL to 3mL, 0.5mL to 2.5mL, or 1mL to 2.5 mL. For example, the amount of a liquid formulation comprising an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) and administered (e.g., by injection, such as, e.g., subcutaneous injection, intravenous injection) to a subject can be, can be at least, can be less than, or can 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, 2.5, 3.5, 3.4, 3.5, 4, 3.5, 4, 3.8, 3.0, 3.5, 4, 4.8, 3.0, 3.5, 4, 3.5, or 4 mL.

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

For the purposes of the present invention, the appropriate dosage of the antibody will depend on the antibody (or composition thereof) used, the type and severity of vasomotor symptoms, the type and severity of headache (e.g., migraine) or other condition to be treated (whether the agent is administered for prophylactic or therapeutic purposes), previous treatment, the patient's medical history and response to the agent, and the judgment of the attending physician. The antibody is typically administered by the clinician until a dosage is reached that achieves the desired result. The dosage and/or frequency may vary during the course of treatment.

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

In one embodiment, the dosage of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) in an individual administered one or more times the antibody may be determined empirically. Increasing doses of the antibody are administered to the individual. To assess the efficacy of the antibody, indicators of disease can be followed.

Administration of antibodies (e.g., monoclonal antibodies that modulate the CGRP pathway, anti-CGRP antagonist antibodies, monoclonal anti-CGRP antagonist antibodies) according to the methods of the invention may be continuous or intermittent, depending on, for example, the physiological condition of the recipient (whether the purpose of administration is therapeutic or prophylactic), and other factors known to those of skill in the art. Administration of the antibody may be substantially continuous over a preselected period of time, or may be a series of spaced doses, for example, before, during or after the development of a headache (e.g., migraine); before; during the period; before and after; during and after; before and during; or before, during and after the development of headache. Administration can be before, during, and/or after any event that may result in headache.

In some embodiments, more than one antibody may be provided. At least one, at least two, at least three, at least four, at least five different, or more antibodies can be provided. Generally, those antibodies may have complementary activities that do not adversely affect each other. The antibodies described herein (e.g., monoclonal antibodies that modulate the CGRP pathway, anti-CGRP antagonist antibodies, monoclonal anti-CGRP antagonist antibodies) can also be used in combination with other CGRP antagonists or CGRP receptor antagonists. For example, one or more of the following CGRP antagonists may be used: antisense molecules directed to CGRP (including antisense molecules directed to nucleic acids encoding CGRP), CGRP inhibitory compounds, CGRP structural analogs, dominant negative mutations in CGRP receptors that bind CGRP, and anti-CGRP receptor antibodies. The antibody may also be used in combination with other agents that act to enhance and/or complement the effectiveness of the agent.

Diagnosis and assessment of headache is well established in the art. The assessment may be made based on subjective measures such as characterization of patient symptoms. For example, migraine can be diagnosed according to the following criteria: 1) episodic headache attacks last 4 to 72 hours; 2) has two of the following symptoms: unilateral pain, palpitations, increased distension and contraction, and moderate or heavy intensity pain; 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, the assessment of headache (e.g., migraine) can be performed by headache hours as described elsewhere herein. For example, the assessment of headache (e.g., migraine) can be in terms of hours of headache daily, hours of headache weekly, hours of headache monthly, and/or hours of headache annually. In some cases, the number of hours of headache can be reported by the subject.

Therapeutic efficacy can be assessed by methods well known in the art. For example, pain reduction can be assessed. Thus, in some embodiments, pain reduction is subjectively observed 1,2, or several hours after administration of the anti-CGRP antibody. In some embodiments, the frequency of headache episodes is subjectively observed after administration of the anti-CGRP antibody.

In some embodiments, the methods described herein for treating or reducing the onset of headache in a subject can reduce the onset of headache following a single administration of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) over an extended period of time. For example, the onset of headache pain can be reduced by 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.

In some embodiments, the methods described herein for treating or reducing the onset of headache in a subject may reduce the number of hours of headache experienced by the subject from pre-administration levels following administration of one or more doses of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) to the subject. For example, after administering one or more doses of the antibody to the subject, the hours of headache experienced by the subject per day can 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 the pre-administration level of the subject. In some cases, following administration of one or more doses of the antibody to a subject, the hours of headache experienced by the subject per day 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 the pre-administration level of the subject. In another example, the headache hours experienced by the subject per week after administration of one or more doses of the antibody to the subject can 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 the pre-administration level of the subject. In some cases, following administration of one or more doses of the antibody to a subject, the headache hours per week experienced by 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%, 99% or more relative to the pre-administration level of the subject. In another example, the number of hours of headache per month experienced by the subject after administration of one or more doses of the antibody to the subject can be reduced from pre-administration levels 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 hours of headache. In some cases, following administration of one or more doses of the antibody to a subject, the headache hours per week experienced by 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%, 99% or more relative to the pre-administration level of the subject.

In some embodiments, the methods of treating or reducing the onset of headache in a subject described herein, following administration of one or more doses of an antibody described herein (e.g., a monoclonal antibody that modulates the CGRP pathway, an anti-CGRP antagonist antibody, a monoclonal anti-CGRP antagonist antibody) to the subject, may reduce the number of headache days experienced by the subject from pre-administration levels. For example, after administering one or more doses of the antibody to the subject, the number of headache days per week experienced by the subject may be reduced from the subject's pre-administration levels 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. In some cases, the number of days per week of headache experienced by a subject following administration of one or more doses of the 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%, 99% or more relative to the pre-administration level of the subject. In another example, the number of headache days per month experienced by the subject after administration of one or more doses of the antibody to the subject can be reduced from pre-administration levels by 0.5, 1,2,3,4, 5,6, 7,8, 9,10, 15, 20 or more headache days.

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

B. anti-CGRP antagonist antibodies

In some embodiments, the antibodies used in the methods of the invention may be anti-CGRP antagonist antibodies. An anti-CGRP antagonist antibody may refer to any antibody molecule that blocks, inhibits or reduces (including significantly) CGRP biological activity, including CGRP signaling mediated downstream pathways such as receptor binding and/or initiation of a cellular response to CGRP.

The anti-CGRP antagonist antibody may exhibit any one or more of the following characteristics: (a) binding to CGRP; (b) blocking CGRP binding to its receptor; (c) block or reduce CGRP receptor activation (including cAMP activation); (d) inhibiting CGRP biological activity or CGRP signal transduction function-mediated downstream pathways; (e) preventing, ameliorating or treating headache in any respect (e.g., migraine); (f) increasing CGRP clearance; and (g) inhibiting (reducing) CGRP synthesis, production or release. anti-CGRP antagonist antibodies are known in the art. See, e.g., Tan et al, Clin.Sci. (Lond).89: 565-; sigma (Missouri, US), product number C7113 (clone # 4901); plourde et al, Peptides 14: 1225-.

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 anti-CGRP antagonist antibody binds to human 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 a C-terminal fragment having amino acids 25-37 of CGRP. In some embodiments, the anti-CGRP antagonist antibody binds to a C-terminal epitope within amino acids 25-37 of CGRP.

Antibodies used in the invention can encompass monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., 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 an immunoglobulin molecule comprising an antigen recognition site of the desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be murine, rat, human, or any other origin (including chimeric or humanized antibodies).

In some embodiments, the anti-CGRP antagonist 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 antibody G1 (as described herein). In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDRs (such as one, two, three, four, five, or in some embodiments, all six CDRs) of a variant of antibody G1 or G1 shown in table 6. In other embodiments, the anti-CGRP antagonist antibody comprises the amino acid sequence of the heavy chain variable region shown in FIG. 5(SEQ ID NO:1) and the amino acid sequence of the light chain variable region shown in FIG. 5(SEQ ID NO: 2).

In some embodiments, the antibody comprises a Light Chain Variable Region (LCVR) and a Heavy Chain Variable Region (HCVR) selected from the group 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). 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.

In some embodiments, the antibody comprises a modified constant region, such as an immunologically inert constant region described herein. In some embodiments, the constant region is as defined in Eur.J.Immunol. (1999)29: 2613-2624; PCT patent application No. PCT/GB 99/01441; and/or modified as described in UK patent application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering refers to wild type IgG2 sequence). Eur.J.Immunol. (1999)29: 2613-2624. In some embodiments, the antibody comprises a constant region of IgG4 comprising the following mutations: E233F234L235 to P233V234a 235. In other embodiments, the constant region is not N-linked glycosylated. In some embodiments, the constant region is not N-linked glycosylated due to mutation of an oligosaccharide linking 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 not N-linked glycosylated. The constant region may be N-linked glycosylated due to enzymatic cleavage or expression in a glycosylation deficient host cell.

Binding affinity (K) of anti-CGRP antagonist antibodies to CGRP, such as human α -CGRP_D) Can be about 0.02 to about 200 nM. In some embodiments, the binding affinity is any of about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 60pM, about 50pM, about 20pM, about 15pM, about 10pM, about 5pM, or about 2 pM. In some embodiments, the binding affinity is less than any of about 250nM, about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50 pM.

One way to determine the binding affinity of an antibody to CGRP is to measure the binding affinity of a monofunctional Fab fragment of the antibody. To obtain monofunctional Fab fragments, antibodies (e.g., IgG) can be cleaved with papain or expressed recombinantly. The affinity of anti-CGRP Fab fragments of antibodies can be determined by surface plasmon resonance (Biacore 3000) equipped with a pre-immobilized streptavidin sensor chip (SA)^TMSurface Plasmon Resonance (SPR) system, Biacore, INC, Piscataway NJ) was determined using HBS-EP running buffer (0.01M HEPES, pH 7.4, 0.15NaCl, 3mM 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 0.5ug/mL and injected into a single chip channel using variable contact times to achieve two antigen density ranges, 50-200 Response Units (RU) for detailed kinetic studies, or 800-. Regeneration studies showed that 25mM NaOH dissolved in 25% v/v ethanol effectively removed bound Fab while maintaining the activity of the on-chip CGRP over 200 injections. Typically, serial dilutions (concentration range 0.1-10X expected)K_D) The purified Fab sample of (a) was injected at 100 μ L/min for 1min and then subjected to dissociation for a period of up to 2 hours. The concentration of Fab protein is determined by ELISA and/or SDS-PAGE electrophoresis using known concentrations of Fab (determined by amino acid analysis) as standards. Kinetic association Rate (k)_on) And dissociation rate (k)_off) The BIAevaluation program was used to obtain simultaneously by fitting the data globally to a 1:1Langmuir binding model (Karlsson, r.roos, h.fagerstat, l.petersson, B. (1994). Methods Enzymology 6.99-110). Equilibrium dissociation constant (K)_D) Value of k_off/k_onThe protocol is applicable to determining the binding affinity of an antibody to any CGRP, including human CGRP, other mammalian CGRPs (such as mouse CGRP, rat CGRP, primate CGRP) and different forms of CGRP (such as types α and β).

Antibodies, including anti-CGRP antagonist antibodies, may be prepared by any method known in the art. As further described herein, host animal immunization pathways and programs are generally consistent with established and conventional techniques for antibody stimulation and production. General techniques for generating human and mouse antibodies are known in the art and are described herein.

It is contemplated that any mammalian subject, including humans or antibody producing cells thereof, can be manipulated as a basis for the generation of mammalian, including human, hybridoma cell lines. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar and/or intradermally with an amount of an immunogen, including the immunogens described herein.

Hybridomas can be prepared from lymphocytes and immortal myeloma cells using general somatic hybridization techniques described by Kohler, B, and Milstein, C. (1975) Nature 256:495-497 or modified by Buck, D.W. et al, In Vitro, 18:377-381 (1982). Useful myeloma Cell lines (including but not limited to X63-Ag8.653 and those from SalkInstitate, Cell Distribution Center, San Diego, Calif., USA) can be used for hybridization. Generally, the technique involves fusing myeloma cells and lymphocytes using a fusing agent such as polyethylene glycol, or by electrical methods well known to those skilled in the art. After fusion, the cells are isolated from the fusion medium and grown on a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate non-hybridized mother cells. Any of the media described herein, with or without serum supplementation, can be used to culture monoclonal antibody-secreting hybridomas. As another alternative to cell fusion techniques, EBV immortalized B cells can be used to produce monoclonal antibodies (e.g., monoclonal anti-CGRP antibodies) of the invention. The hybridomas are expanded and subcloned, if necessary, and supernatants are assayed for anti-immunogen activity by conventional immunoassay procedures (e.g., radioimmunoassay, enzyme immunoassay, or fluoroimmunoassay).

Hybridomas useful as antibody sources encompass all derivatives, progeny cells of the parental hybridoma that produce monoclonal antibodies specific for CGRP or portions thereof.

Hybridomas that produce such antibodies can be grown in vitro or in vivo using known procedures. Monoclonal antibodies can be isolated from the culture medium or body fluids by conventional immunoglobulin purification procedures, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and, if desired, ultrafiltration. When present, the preparation can be performed, for example, by running on an adsorbent made of the immunogen attached to the solid phase, and the undesired activity is removed from the desired antibody elution or release of the immunogen. Immunization of host animals with human CGRP or fragments comprising the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using bifunctional or derivatizing agents such as maleimidobenzoyl sulphosuccinimide ester (conjugated through a cysteine residue), N-hydroxysuccinimide (conjugated through a lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R1N ═ C ═ NR where R and R1 are different alkyl groups, can produce populations of antibodies (e.g., monoclonal antibodies).

If desired, the antibody of interest (e.g., monoclonal or polyclonal anti-CGRP antagonist antibody) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequences encoding the antibody of interest can be maintained in a vector for the host cell, and the host cell can then be expanded and frozen for future use. In one alternative, the polynucleotide sequences may be used for genetic manipulation to "humanize" or enhance the affinity of the antibody, or other characteristics of the antibody. For example, constant regions can be engineered to more closely resemble human constant regions, thereby avoiding immune responses when the antibodies are used in clinical trials and treatments in humans. It may be advantageous to genetically manipulate antibody sequences to achieve greater affinity for CGRP and greater efficacy in CGRP inhibition. It will be apparent to those skilled in the art that one or more polynucleotide changes may be made to an anti-CGRP antagonist antibody while still maintaining the ability to bind CGRP.

Humanized monoclonal antibodies may comprise four general steps. They are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains; (2) designing a humanized antibody, i.e., determining which antibody framework regions are used during the humanization process; (3) actual humanization methods/techniques and (4) transfection and expression of humanized antibodies. See, for example, U.S. Pat. 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.

Described are "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin, including chimeric antibodies having rodent or modified rodent V regions and their associated Complementarity Determining Regions (CDRs) fused to human constant domains. See, e.g., Winter et al, Nature 349: 293-. Other references describe rodent CDRs grafted onto human supporting Framework Regions (FRs) prior to fusion with appropriate human antibody constant domains. See, for example, Riechmann et al, Nature 332: 323-. Another reference describes rodent CDRs supported by recombinant surface modified rodent framework regions. See, for example, 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 effectiveness of therapeutic administration of those moieties in human recipients. For example, the antibody constant region can be engineered to be immunologically inert (e.g., not triggering complement lysis). See, e.g., PCT publication No. PCT/GB99/01441, British patent application No. 9809951.8. Other methods of humanizing antibodies that may also be used are disclosed in Daugherty et al, Nucl. acids Res.19:2471-2476(1991) and U.S. Pat. Nos. 6,180,377, 6,054,297, 5,997,867, 5,866,692, 6,210,671 and 6,350,861, and PCT publication No. WO 01/27160.

In yet another alternative, fully human antibodies can be obtained using commercially available mice engineered to express specific human immunoglobulins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or stronger immune response may also be used to generate humanized or human antibodies. An example of such a technique is Xenomouse from Abgenix, Inc. (Fremont, CA)^TMAnd from Metarex, Inc. (Princeton, NJ)And TC Mouse^TM。

In one alternative, the antibody may be recombinantly produced and expressed using any method known in the art. In another alternative, the antibody may be recombinantly produced by phage display technology. See, e.g., U.S. Pat. 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)) can be used to generate human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene profiles of unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame to the major or minor coat protein genes of filamentous phage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Since the filamentous particle comprises a single-stranded DNA copy of the phage genome, the result of the selection being made in addition to the functional properties of the antibody is the selection of the gene encoding the antibody exhibiting these properties. Thus, the phage mimics some of the properties of the B cell. Phage display can be performed in a variety of ways; for review see, e.g., Johnson, KevinS. and Chiswell, David J., Current Opinion in Structural Biology 3:564, 571 (1993). Multiple sources of V gene segments are available for phage display. Clackson et al, Nature 352:624-628(1991) isolated a series of various anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. V gene profiles from non-immunized human donors can be constructed and antibodies to a range of various antigens, including self-antigens, can be substantially isolated according to 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 the innate immune response, antibody genes accumulate mutations at high rates (somatic high-frequency mutations). Some of the introduced changes will confer high affinity and B cells that show high affinity surface immunoglobulins preferentially replicate and differentiate during subsequent antigen challenge. This natural process can be simulated using a technique called "chain shuffling". Marks et al, Bio/Technol.10:779-783 (1992)). In this method, the affinity of "original" human antibodies obtained by phage display can be improved by subsequent replacement of the heavy and light chain V region genes with a naturally occurring variant repertoire of V domain genes obtained from an unimmunized donor. This technique allows the generation of antibodies and antibody fragments with affinities in the pM-nM range. A strategy for generating very large phage antibody profiles (also referred to as "all source libraries") is described in Waterhouse et al, Nucl. acids Res.21:2265-2266 (1993). Gene shuffling can also be used to derive human antibodies from rodent antibodies, where the human antibodies have similar affinity and specificity as the starting rodent antibody. According to this method (also known as "epitope blotting"), the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced by a human V domain gene bank, resulting in a rodent-human chimera. The result of the selection on the antigen is the isolation of human variable regions capable of restoring a functional antigen binding site, i.e. epitopes determining the choice of (imprinted) partners. When this process is repeated to replace the remaining rodent V domains, human antibodies are obtained (see PCT publication No. WO 93/06213, published on month 4 and 1, 1993). Unlike traditional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that are free of framework or CDR residues of rodent origin.

It will be apparent that although the above discussion relates to humanized antibodies, the general principles discussed apply to the tailoring of antibodies for use in, for example, dogs, cats, primates, horses and cattle. It will also be apparent that one or more aspects of the humanized antibodies described herein, such as CDR grafting, framework mutations, and CDR mutations, may be combined.

Antibodies can be recombinantly produced by: antibodies and antibody-producing cells are first isolated from a host animal, a gene sequence is obtained, and the gene sequence is used to recombinantly express the antibody in a host cell (e.g., a CHO cell). Another method that can be used is to express the antibody sequence in a plant (e.g., tobacco) or transgenic milk. Methods for recombinant expression of antibodies in plants or milk have been disclosed. See, e.g., Peeters et al, Vaccine 19:2756 (2001); lonberg, n. and d.huskzarint.rev.immunol 13:65 (1995); and Pollock et al, J Immunol Methods 231:147 (1999). Methods for making antibody derivatives, e.g., humanized, single chain, etc., are known in the art.

Immunoassays and flow cytometric sorting techniques, such as Fluorescence Activated Cell Sorting (FACS), can also be used to isolate antibodies specific for CGRP.

The antibody may be bound to a variety of different carriers. The support may be activated and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified cellulose, polyacrylamide, agarose, and magnetite. The carrier may be soluble or insoluble in nature. Those skilled in the art will know of other suitable carriers for binding antibodies, or will be able to determine such carriers using routine experimentation. In some embodiments, the vector includes a moiety that targets the myocardium.

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 the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, such as that disclosed in PCT publication No. wo 87/04462, and then transfected into a host cell, such as an e.coli (e.coli) cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce immunoglobulin, to synthesize a monoclonal antibody in the recombinant host cell. 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 for the homologous murine sequences (Morrison et al, Proc. Nat. Acad. Sci.81:6851(1984)) or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Thus, "chimeric" or "hybrid" antibodies having the binding specificity of the anti-CGRP monoclonal antibodies herein are prepared.

Antibodies (e.g., anti-CGRP antagonist antibodies) and polypeptides derived from the antibodies can be identified or characterized using methods known in the art, whereby a reduction, improvement, or neutralization of CGRP biological activity is detected and/or measured. For example, anti-CGRP antagonist antibodies can also be identified by: incubating the candidate agent with CGRP and monitoring any one or more of the following characteristics: (a) binding to CGRP; (b) blocking CGRP binding to its receptor; (c) block or reduce CGRP receptor activation (including cAMP activation); (d) inhibiting CGRP biological activity or CGRP signal transduction function-mediated downstream pathways; (e) preventing, ameliorating or treating headache in any respect (e.g., migraine); (f) increasing CGRP clearance; and (g) inhibiting (reducing) CGRP synthesis, production or release. In some embodiments, an anti-CGRP antagonist antibody or polypeptide is identified by the following method: candidate agents are incubated with CGRP and binding of CGRP and/or concomitant reduction or neutralization of its biological activity is monitored. Binding assays may be performed using purified CGRP polypeptide, or using cells naturally expressed or transfected to express CGRP polypeptide. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete for CGRP binding with known anti-CGRP antagonists is assessed. The assay can be performed in a variety of ways, including ELISA. In other embodiments, the anti-CGRP antagonist antibody is identified by the following method: candidate agents are incubated with CGRP and binding and concomitant inhibition of CGRP receptor activation expressed on the cell surface is monitored.

After initial identification, the activity of a candidate antibody (e.g., an anti-CGRP antagonist antibody) can be further determined and refined by bioassays known to test the biological activity of the target. Alternatively, the bioassay may be used directly to screen candidates. For example, CGRP promotes multiple measurable changes in responsive cells. These changes include, but are not limited to, stimulation of cAMP in cells (e.g., SK-N-MC cells). Antagonist activity can also be measured using animal models, such as measuring cutaneous vasodilation induced by stimulation of the rat saphenous nerve. Escott et al, Br.J.Pharmacol.110:772-776, 1993. Animal models of headache, such as migraine, can also be used to test the efficacy of antagonist antibodies or polypeptides. Reuter et al, Functional Neurology (15) supplement 3, 2000. Some methods for identifying and characterizing anti-CGRP antagonist antibodies or polypeptides are described in detail in the examples.

Antibodies, including anti-CGRP antagonist antibodies, can be characterized using methods well known in the art. For example, one approach is to identify the epitope or "epitope mapping" to which the antibody binds. There are a variety of methods known in the art for mapping and characterizing the location of epitopes on proteins, including the resolution of the crystal structure of antibody-antigen complexes, 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 further examples, epitope mapping can be used to determine the sequence to which an anti-CGRP antagonist antibody binds. Epitope mapping is commercially available from a variety of sources, such as Pepscan Systems (Edelhertweg 15,8219PH Lelystad, the Netherlands). The epitope may be a linear epitope, i.e. a conformational epitope comprised in a single chain of amino acids, or formed by three-dimensional interactions of amino acids not necessarily comprised in a single chain. Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., by recombinant means) and used in binding assays with anti-CGRP antagonist antibodies. In another example, the epitope to which an anti-CGRP antagonist antibody binds can be determined in a systematic screen by using overlapping peptides derived from CGRP sequences and determining the binding of the anti-CGRP antagonist antibody. The open reading frame encoding the CGRP is fragmented randomly or by specific genetic constructs, as determined by gene fragment expression, and the reactivity of the expressed fragment of CGRP with the antibody to be tested is determined. The gene fragment can be prepared, for example, by the following method: PCR, then in vitro transcription and translation into protein in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled CGRP fragment was 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 a test antibody in a simple binding assay. In further examples, mutagenesis of the antigen binding domain, domain exchange experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain swapping experiments can be performed using mutant CGRP in which various fragments of the CGRP polypeptide are replaced (swapped) with sequences of closely related, but antigenically distinct proteins, such as another member of the neurotrophin family. By assessing the binding of an antibody to a mutant CGRP, the importance of binding of a particular CGRP fragment to the antibody can be assessed.

Another method that can be used to characterize antibodies, including anti-CGRP antagonist antibodies, is to use a competition assay with other antibodies known to bind to the same antigen (i.e., various fragments on 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.

Expression vectors may be used to direct the expression of antibodies, including anti-CGRP antagonist antibodies. The person skilled in the art is familiar with the administration of expression vectors to obtain in vivo expression of exogenous proteins. See, for example, U.S. patent nos. 6,436,908, 6,413,942, and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter administration, and external use. In another embodiment, the expression vector is administered directly to the sympathetic trunk or ganglia, or to the coronary arteries, atria, ventricles, or pericardium.

Targeted delivery of therapeutic compositions comprising expression vectors or subgenomic polynucleotides may also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al, Trends Biotechnol. (1993)11: 202; chiou et al, general therapeutics: Methods And Applications Of Direct Gene Transfer (eds. J.A.Wolff) (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. In a gene therapy regimen, a therapeutic composition comprising a polynucleotide is administered topically in the range of about 100ng to about 200mg of DNA. Concentration ranges of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500g, and about 20 μ g to about 100 μ g of DNA may also be used during a gene therapy regimen. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral 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 mammalian promoters or heterologous promoters. Expression of a coding sequence may be constitutive or regulated.

Viral-based vectors for delivering a desired polynucleotide and expressing in a desired cell are well known in the art. Exemplary virus-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication No. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British patent No.2,200,651; and European patent No. 0345242), alphavirus-based vectors (e.g., sindbis virus vectors, semliki forest virus (ATCC VR-67, ATCCVR-1247), Ross river virus (ATCC VR-373, ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)) and adeno-associated virus (AAV) vectors (see, e.g., PCT publication No. WO 94/12649; WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Administration of DNA that kills adenovirus-associated can also be used, as described by Curiel, hum.

Non-viral delivery vehicles and methods can also be utilized, including but not limited to killing adenovirus-linked or unlinked polycationic aggregated DNA alone (see, e.g., Curiel, hum. Gene Ther. (1992)3: 147); ligand-linked DNA (see, e.g., Wu, j.biol.chem. (1989)264: 16985); eukaryotic cells deliver vehicle cells (see, e.g., U.S. Pat. No.5,814,482; PCT publication No. WO 95/07994; WO 96/17072; WO 95/30763 and WO 97/42338) and nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary naked DNA introduction methods are described in PCT publication No. WO90/11092 and U.S. Pat. No.5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Pat. Nos. 5,422,120; PCT publication nos. wo 95/13796; WO 94/23697; WO 91/14445 and EP 0524968. Additional methods are described in Philip, mol.cell Biol. (1994)14:2411 and Wffindin, Proc.Natl.Acad.Sci. (1994)91: 1581.

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

The present invention encompasses compositions, including pharmaceutical compositions comprising antibody G1 and variants thereof shown in table 6 or polypeptides derived from antibody G1 and variants thereof shown in table 6; and polynucleotides comprising sequences encoding G1 and variants or polypeptides thereof. In some embodiments, the compositions comprise one or more antibodies or polypeptides (which may or may not be antibodies) that bind CGRP, and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind CGRP. These compositions may also contain suitable excipients, such as pharmaceutically acceptable excipients (including buffers), which are well known in the art.

In some embodiments, the anti-CGRP antagonist antibodies and polypeptides of the invention are characterized by any (one or more) of the following features: (a) binding to CGRP; (b) blocking CGRP binding to its receptor; (c) block or reduce CGRP receptor activation (including cAMP activation); (d) inhibiting CGRP biological activity or a downstream pathway mediated by CGRP signaling function; (e) preventing, ameliorating or treating headache in any respect (e.g., migraine); (f) increasing CGRP clearance; and (g) inhibiting (reducing) CGRP synthesis, production or release.

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

The CDR portions of antibody G1 (including Chothia and Kabat CDRs) are illustrated in fig. 5. Determination of CDR regions is 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 Kabat CDRs. In other embodiments, the CDR is a Chothia CDR. In other words, in embodiments with more than one CDR, the CDR can be any one of Kabat, Chothia, a combination CDR, or a combination thereof.

In some embodiments, the invention provides a polypeptide (which may or may not be an antibody) comprising at least one CDR, at least two, at least three, at least four, at least five, or all six CDRs substantially identical to at least one CDR, at least two, at least three, or at least four, at least five, or all six CDRs of G1 or variants thereof shown in table 6. Other embodiments include antibodies having at least two, three, four, five, or six CDRs that are substantially identical to G1 or at least two, three, four, five, or six CDRs derived from G1. In some embodiments, the at least one, two, three, four, five, or six CDRs 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 variants thereof shown in table 6. It is to be understood that for purposes of the present invention, the binding specificity and/or overall activity is generally maintained, although the degree of activity may vary (may be greater or lesser) as compared to G1 shown in table 6 or variants thereof.

In some embodiments, the invention also provides a polypeptide (which may or may not be an antibody) comprising the amino acid sequence of G1 shown in table 6 or a variant thereof having any of: at least 5 consecutive amino acids, at least 8 consecutive amino acids, at least about 10 consecutive amino acids, at least about 15 consecutive amino acids, at least about 20 consecutive amino acids, at least about 25 consecutive amino acids, at least about 30 consecutive amino acids of the sequence of G1 or a variant thereof shown in table 6, wherein at least 3 amino acids are from the variable region of G1 (fig. 5) or a variant thereof shown in table 6. In one embodiment, the variable region is from the light chain of G1. In another embodiment, the variable region is from the heavy chain of G1. Exemplary polypeptides have contiguous amino acids (length as described above) from both the heavy and light chain variable regions of G1. In another embodiment, 5 (or more) consecutive amino acids are from the Complementarity Determining Regions (CDRs) of G1 shown in fig. 5. In some embodiments, the contiguous amino acids are from the variable region of G1.

Binding affinity (K) of anti-CGRP antagonist antibodies and polypeptides to CGRP, such as human α -CGRP_D) Can be about 0.06 to about 200 nM. In some embodiments, the binding affinity is any of about 200nM, 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, about 60pM, about 50pM, about 20pM, about 15pM, about 10pM, about 5pM, or about 2 pM. In some embodiments, the binding affinity is less than any of about 250nM, about 200nM, about 100nM, about 50nM, about 10nM, about 1nM, about 500pM, about 100pM, or about 50 pM.

In some embodiments, the invention also provides methods of making any of these antibodies or polypeptides. The antibodies of the invention can be prepared by procedures known in the art. The polypeptides may be prepared by proteolytic or other degradation of the antibody, by recombinant methods described above (i.e., single chain or fusion polypeptides), or by chemical synthesis. Polypeptides of antibodies, particularly shorter polypeptides of up to about 50 amino acids, are conveniently prepared by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be prepared by an automated polypeptide synthesizer using a solid phase method. See also, U.S. patent nos. 5,807,715; 4,816,567 and 6,331,415.

In another alternative, the antibody may be recombinantly produced using procedures well known in the art. In one embodiment, the polynucleotide comprises a sequence encoding the variable region of the heavy and/or light chain of antibody G1 shown in SEQ ID NO 9 and SEQ ID NO 10. In another embodiment, a polynucleotide comprising the nucleotide sequences set forth in SEQ ID NO 9 and SEQ ID NO 10 is cloned into one or more vectors for expression or propagation. The sequences encoding the antibody of interest can be maintained in a vector for the host cell, and the host cell can then be expanded and frozen for future use. Vectors (including expression vectors) and host cells are also described herein.

In some embodiments, the invention also encompasses single chain variable fragments ("scFv") of antibodies of the invention, such as G1. Single chain variable fragments are prepared by linking the light and/or heavy chain variable regions using short linking peptides. Bird et al (1988) Science 242: 423-. An example of a linker peptide is (GGGGS)3(SEQ ID NO:57) which is bridged between the carboxy terminus of one variable region and the amino terminus of the other variable region by about 3.5 nm. Other sequences of linkers were designed and used. Bird et al (1988). The linker can then be modified to perform additional functions, such as linking to a drug or to a solid support. Single-stranded variants can be prepared recombinantly or synthetically. For synthetic preparation of scFv, an automated synthesizer can be used. For recombinant production of an scFv, a suitable plasmid comprising a polynucleotide encoding an scFv can be introduced into a suitable host cell, a eukaryotic cell such as a yeast, plant, insect, or mammalian cell, or a prokaryotic cell such as e. Polynucleotides encoding the scFv of interest can be prepared by conventional procedures, such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

Other forms of single chain antibodies, such as diabodies, are also contemplated. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but a shorter linker is used to allow pairing between the two domains on the same chain, thereby pairing the domains with the complementary domains of the other chain and forming two antigen binding sites (see, e.g., Holliger, P. et al, (1993) Proc. Natl. Acad. Sci.USA 90: 6444-.

For example, bispecific antibodies, monoclonal antibodies having binding specificities for at least two different antigens can be prepared using the antibodies disclosed herein. Methods for making 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 co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, 1983, Nature 305, 537-539).

According to one method of making bispecific antibodies, antibody variable domains having the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant domain sequences. Preferably with an immunoglobulin heavy chain constant domain (comprising at least a portion of the hinge, CH2, and CH3 regions). Preferably, a first heavy chain constant region (CH1) (comprising the site necessary for light chain binding) is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used for construction provide optimal yields, this provides greater flexibility in adjusting the mutual ratios of the three polypeptide fragments. However, when at least two polypeptide chains are expressed in equal proportions resulting in high yields, or when the ratio is of no particular significance, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

In one approach, a bispecific antibody is composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has immunoglobulin light chains in only half of the bispecific molecules, facilitating the isolation of the desired bispecific compound from the undesired immunoglobulin chain combinations. This method is described in PCT publication No. wo 94/04690, published 3.3.1994.

Heteroconjugate antibodies comprising 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. Pat. No.4,676,980), as well as in the treatment of HIV infection (PCT patent application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). The heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. Pat. No.4,676,980.

Chimeric or hybrid antibodies can also be prepared in vitro using known synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may use a disulfide exchange reaction or by forming a thioether linkage configuration. Examples of suitable reagents for this purpose include iminothiolate and methyl 4-mercaptobutyrimidate.

Humanized antibodies comprising one or more CDRs of antibody G1, or variants thereof, shown in table 6, or derived from one or more CDRs of antibody G1, or variants thereof, shown in table 6, can be made using any method known in the art. For example, a monoclonal antibody can be humanized using four general steps.

In some embodiments, the invention encompasses modifications of antibody G1 or variants thereof shown in table 6, including functionally equivalent antibodies and variants with increased or decreased activity and/or affinity that have no significant effect on their properties. For example, the amino acid sequence of antibody G1 or a variant thereof shown in table 6 may be mutated to obtain an antibody having the desired binding affinity for CGRP. Modification of polypeptides is routine practice in the art and need not be described in detail herein. Modifications of the polypeptides are exemplified in the examples. Examples of modified polypeptides include polypeptides having conservative substitutions of amino acid residues, deletions or additions of one or more amino acids that do not significantly adversely alter functional activity, or the use of chemical analogs.

Amino acid sequence insertions include amino and/or carboxyl 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 antibodies with an N-terminal methionyl residue or antibodies fused to an epitope tag. Other insertional variants of the antibody molecule include the N or C terminus of the antibody fused to an enzyme or polypeptide, which increases the serum half-life of the antibody.

Substitution variants have at least one amino acid residue in the antibody molecule removed and a different residue inserted at that position. The most interesting sites for substitution mutagenesis include the hypervariable regions, but FR alterations are also envisaged. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". If such substitutions result in an alteration in biological activity, more substantial changes, designated "exemplary substitutions" in Table 1, or as described further below with reference to amino acid species, can be introduced and the products screened.

Table 1: amino acid substitutions

Substantial modification of the biological properties of antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure, e.g., the folded or helical conformation, of the main backbone of the polypeptide in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) steric hindrance of the side chains. Naturally occurring residues are divided into several groups according to the nature of the common side chains:

(1) non-polar: norleucine, Met, Ala, Val, Leu, Ile;

(2) the polarity is uncharged: cys, Ser, Thr, Asn, Gln;

(3) acidic (negatively charged): asp and Glu;

(4) basic (positively charged): lys, Arg;

(5) residues that influence chain orientation: gly, Pro; and

(6) aromatic: trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging members of one of these classes for another.

Any cysteine residue not involved in maintaining the correct conformation of the antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine bonds may be added to the antibody to improve its stability, particularly when the antibody is an antibody fragment such as an Fv fragment.

Amino acid modifications can range from changing or modifying one or more amino acids to complete redesign of a region, such as a variable region. Changes in 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 other embodiments, the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and non-glycosylated polypeptides, as well as polypeptides with other post-translational modifications such as, for example, glycosylation, acetylation, and phosphorylation by different sugars. Antibodies are glycosylated at conserved 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 influence the function of the protein (Boyd et al, 1996, mol. Immunol.32: 1311-1318; Wittwe and Howard, 1990, biochem.29:4175-4180) and the intramolecular interaction between parts of the glycoprotein, which can influence the conformation and the three-dimensional surface of the glycoprotein presented (Hefferis and Lund, supra; Wys and Wagner, 1996, Current Opin. Biotech.7: 409-416). Oligosaccharides can also be used to target a given glycoprotein to certain molecules based on a particular recognition structure. Glycosylation of antibodies has also been reported to affect antibody-dependent cell-mediated cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated expression of β (1,4) -N-acetylglucosaminyltransferase III (GnTIII), glycosyltransferase catalyzed bisecting GlcNAc formation, are reported to have improved ADCC activity (Umana et al, 1999, MatureBiotech.17: 176-180).

Glycosylation of antibodies is usually N-linked or O-linked. N-linked refers to the attachment of a sugar 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 recognition sequences for enzymatic attachment of the sugar moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide forms 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, but 5-hydroxyproline or 5-hydroxylysine may also be used.

Glycosylation site addition to the antibody is conveniently achieved by altering the amino acid sequence so that it comprises one or more of the tripeptide sequences described above (for N-linked glycosylation sites). Alterations may also be made by adding one or more serine or threonine residues to the sequence or substitution of the original antibody (for O-linked glycosylation sites).

The glycosylation pattern of the antibody can also be altered without altering the basic nucleotide sequence. Glycosylation is largely dependent on the host cell used to express the antibody. Since the cell type used as a potential therapeutic for the expression of recombinant glycoproteins (e.g., antibodies) is rarely a native cell, variations in the glycosylation pattern of antibodies can be expected (see, e.g., Hse et al, 1997, J.biol.chem.272: 9062-.

Factors that influence glycosylation during recombinant production of antibodies include, in addition to host cell selection, growth pattern, medium formulation, culture density, oxygenation, pH, purification protocols, and the like. Various methods have been proposed to achieve altered glycosylation patterns in specific host organisms, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. Pat. 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, recombinant host cells can be genetically engineered to be deficient in certain types of polysaccharide processing. These and similar techniques are well known in the art.

Other methods of modification include the use of ligation techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. Modifications can be used, for example, for attachment of immunoassay labels. The modified G1 polypeptide can be prepared using procedures established in the art, and can be screened using standard assays known in the art, some assays being described below and in the examples.

In some embodiments of the invention, the antibody comprises a modified constant region such as an immunologically or partially inert constant region, e.g., does not trigger complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia; or a reduction in any one or more of the following activities (as compared to an unmodified antibody): 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 optimal levels and/or combinations of effector functions. See, e.g., Morgan et al, Immunology 86:319-324 (1995); lund et al, J.immunology 157: 4963-; idusogene et al, J.immunology 164: 4178-; 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 as defined in Eur.J.Immunol. (1999)29: 2613-2624; PCT patent application No. PCT/GB 99/01441; and/or modifications as described in UK patent application No. 9809951.8. In other embodiments, the antibody comprises a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering refers to wild type IgG2 sequence). Eur.J.Immunol. (1999)29: 2613-2624. In other embodiments, the constant region is not N-linked glycosylated. In some embodiments, the constant region is not N-linked glycosylated due to mutation of glycosylated amino acid residues or flanking residues that are part of an N-glycosylation recognition sequence in the constant region. For example, the N-glycosylation site N297 can be mutated to A, Q, K or H. See Tao et al, J.immunology 143:2595-2601 (1989); and Jefferis et al, ImmunologicalReviews 163:59-76 (1998). In some embodiments, the constant region is not N-linked glycosylated. The constant region may be due to enzymatic cleavage (such as removal of the sugar by the enzyme PNGase), or expressed in a glycosylation deficient host cell without N-linked glycosylation.

Other antibody modifications include antibodies modified as described in published PCT publication No. WO 99/58572, 11/18/1999. In addition to the binding domain directed to the target molecule, these antibodies also comprise an effector domain having an amino acid sequence that is substantially homologous to all or a portion of the constant domain of a human immunoglobulin heavy chain. These antibodies are capable of binding to a target molecule without triggering significant complement-dependent lysis, or cell-mediated target destruction. In some embodiments, the effector domain is capable of specifically binding FcRn and/or fcyriib. These are generally based on heavy chain C derived from two or more human immunoglobulins_H2 domain. Antibodies modified in this manner are particularly useful in chronic antibody therapy to avoid inflammatory and other adverse reactions to conventional antibody therapy.

In some embodiments, the invention includes embodiments of affinity maturation. For example, affinity matured antibodies can be prepared 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- & WO 2004/058184).

The following methods can be used to adjust the affinity of the antibody and to characterize the CDRs. One method of characterizing the CDRs of an antibody and/or altering (such as increasing) the binding affinity of a polypeptide, such as an antibody, is referred to as "library scanning mutagenesis". Generally, library scanning mutagenesis works as follows. One or more amino acid positions in a CDR are substituted 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 methods known in the art. This generates libraries of small clones (in some embodiments, one clone per amino acid position analyzed), each library having a complexity of two or more members (if there are two or more amino acid substitutions at each position). Generally, the library further comprisesCloning of natural (unsubstituted) amino acids. 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 to the target polypeptide (or other binding target) and candidates are identified that bind more, the same, less or no. Methods for determining binding affinity are well known in the art. Binding affinity can be determined using Biacore surface plasmon resonance analysis, which detects binding affinity differences of about 2-fold or greater. When the starting antibody has been raised to a relatively high affinity, e.g., a K of about 10nM or less_DIn combination, Biacore is particularly useful. The use of Biacore surface plasmon resonance screening is described in the examples herein.

Binding affinity can be determined using Kinexa biocensors, scintillation proximity assays, ELISA, ORIGEN Immunoassays (IGEN), fluorescence quenching, fluorescence transfer, and/or yeast display assays. Binding affinities may also be screened using a suitable bioassay.

In some embodiments, each amino acid position in the CDR is substituted (in some embodiments, one at a time) for all 20 natural amino acids using art-recognized mutagenesis methods (some of which are described herein). This generates small clone libraries (in some embodiments, one clone per amino acid position analyzed), each library having a complexity of 20 members (if there are all 20 amino acid substitutions per position).

In some embodiments, the library to be screened comprises substitutions at two or more positions, which may be in the same CDR or in two or more CDRs. Thus, the library may comprise substitutions at two or more positions in one CDR. The library may comprise substitutions at two or more positions in two or more CDRs. The library may comprise substitutions at 3,4, 5 or more positions present in two, three, four, five or six CDRs. Such substitutions can be made using low redundancy codons. See, e.g., Table 2 of Balint et al, (1993) Gene 137 (1: 109-18).

The CDRs may be CDRH3 and/or CDRL 3. The CDRs may be one or more of CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and/or CDRH 3. The CDRs may be Kabat CDRs, Chothia CDRs, or extended CDRs.

Candidates with improved binding can be sequenced to identify CDR substitution mutants that produce improved affinity (also referred to as "improved" substitutions). The bound candidates can also be sequenced, thereby identifying CDR substitutions that remain bound.

Multiple rounds of screening can be performed. For example, candidates with improved binding (each candidate comprising an amino acid substitution at one or more positions of one or more CDRs) are also used to design a second library comprising at least the initial and substituted amino acids at each improved CDR position (i.e., the amino acid position of the CDR at which the substitution mutant exhibits improved binding). The preparation of the library and screening or selection is discussed further below.

Library scanning mutagenesis also provides a method of characterization of CDRs, binding being the same, binding being reduced or not binding also provides information on the importance of each amino acid position for antibody-antigen complex stability in terms of cloning frequency with improved binding. For example, if a position of a CDR remains bound when changed to all 20 amino acids, that position is identified as an unnecessary position for antigen binding. Conversely, if a position of a CDR remains bound for only a small percentage of substitutions, that position is identified as a position that is important to the function of the CDR. Thus, library scanning mutagenesis generates information about positions in the CDRs that can be changed to a number of different amino acids (including all 20 amino acids), as well as positions in the CDRs that cannot be changed or can be changed to only a few amino acids.

Candidates with improved affinity may be combined into a second library comprising the improved amino acid, the original amino acid at that position, and additional substitutions at that position, as desired or allowed by the desired screening or selection method. In addition, adjacent amino acid positions can be randomly divided into at least two or more amino acids, if desired. Randomization of adjacent amino acids may allow additional conformational flexibility of the mutated CDRs, which in turn may allow or facilitate the introduction of a number of improved mutations. The library may also comprise substitutions at positions that do not show improved affinity in the first round of screening.

Library members having improved and/or altered binding affinities are screened or selected in the second library using any method known in the art, including screening using Biacore surface plasmon resonance analysis, and selection using any selection method known in the art, including phage display, yeast display, and ribosome display.

In some embodiments, the invention also encompasses fusion proteins comprising one or more fragments or regions from an antibody (such as G1) or polypeptide of the invention. In one embodiment, fusion polypeptides are provided comprising at least 10 contiguous amino acids of the variable light chain region set forth in SEQ ID NO:2 (FIG. 5) and/or at least 10 amino acids of the variable heavy chain region set forth in SEQ ID NO:1 (FIG. 5). In other embodiments, fusion polypeptides are provided that comprise 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 variable light chain region set forth in SEQ ID NO:2 (FIG. 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 variable heavy chain region set forth in SEQ ID NO:1 (FIG. 5). In another embodiment, the fusion polypeptide comprises the light chain variable region and/or the heavy chain variable region of G1 shown in SEQ ID NO:2 and SEQ ID NO:1 of FIG. 5. In another embodiment, the fusion polypeptide comprises one or more CDRs of G1. In other embodiments, the fusion polypeptide comprises the CDR H3 and/or CDR L3 of antibody G1. For the purposes of the present invention, a G1 fusion protein comprises one or more G1 antibodies and another amino acid sequence to which the antibody is not linked in the native molecule, e.g., a heterologous or homologous sequence from another region. Exemplary 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.

The G1 fusion polypeptide can be produced by methods known in the art, such as synthesis or recombination. Typically, the G1 fusion proteins of the invention are prepared by expressing the polynucleotides encoding them using recombinant methods described herein, but can also be prepared by other methods known in the art, including, for example, chemical synthesis.

In some aspects, the invention also provides compositions comprising an antibody or polypeptide derived from G1 conjugated (e.g., linked) to an agent that facilitates attachment to a solid support, such as biotin or avidin. For the sake of brevity, the following understanding is generally mentioned with respect to G1 or antibodies: these methods are applicable to any of the CGRP binding embodiments described herein. Conjugation generally refers to linking these components as described herein. Linking (typically tightly fixing the components for at least administration) can be achieved in a variety of ways. For example, when the reagent and the antibody each have a surrogate capable of reacting with the other, they may react directly with each other. For example, a nucleophilic group such as an amino or mercapto group on one can react with a carbonyl-containing group such as an anhydride or acyl halide on the other, or with an alkyl group containing a good leaving group (e.g., halide).

The 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 label known in the art. Labels are known in the art and typically provide a signal (directly or indirectly).

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.

In some embodiments, the invention also provides isolated polynucleotides encoding the antibodies and polypeptides of the invention (including antibodies comprising polypeptide sequences of the light and heavy chain variable regions shown in fig. 5), as well as vectors and host cells comprising the polynucleotides.

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

In another aspect, the invention provides polynucleotides encoding any of the antibodies (including antibody fragments) and polypeptides described herein, such as antibodies and polypeptides having impaired effector function. Polynucleotides can be prepared by procedures known in the art.

In another aspect, the invention provides a composition (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 a G1 antibody described herein. In other embodiments, the composition comprises an expression vector comprising a polynucleotide encoding any of the antibodies or polypeptides described herein. In other embodiments, the composition comprises any one or both of the polynucleotides set forth in SEQ ID NO 9 and SEQ ID NO 10. Also described herein are expression vectors, and administration of polynucleotide compositions.

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

The invention also encompasses polynucleotides complementary to any such sequence. 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 DNA molecules in a one-to-one manner), as well as mRNA molecules (which do not contain introns). Additional coding or non-coding sequences may, but need not, be present within the polynucleotides of the invention, and the polynucleotides may, but need not, be linked to other molecules and/or vector materials.

The polynucleotide may comprise a native sequence (i.e., an endogenous sequence encoding an antibody or portion thereof) or may comprise a variant of such a sequence. A polynucleotide variant comprises one or more substitutions, additions, deletions and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the naturally-occurring immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide is typically assessed as described herein. 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 portion thereof.

Two polynucleotide or polypeptide sequences are considered "identical" if the nucleotide or amino acid sequences in the two sequences are identical when aligned for maximum correspondence, as described below. Comparison between two sequences is typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity. As used herein, a "comparison window" refers to a segment of at least about 20 consecutive positions, typically 30 to about 75, 40 to about 50 consecutive positions, wherein after optimal alignment of two sequences, a sequence can be compared to a reference sequence of the same number of consecutive positions.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software package (DNASTAR, Inc., Madison, Wis.) using default parameters. The program includes a number of alignment schemes, described in the following references: dayhoff, M.O. (1978) A model of evolution change in proteins-substrates for detecting displacement relationships, described in Dayhoff, M.O. (eds.) Atlas of protein Sequence and Structure, National biological Research Foundation, Washington DC, Vol.5, supplement 3, pp.345 and 358; hein J, 1990, Unified apparatus to alignment and olefins, page 626-; higgins, D.G. and Sharp, P.M., 1989, CABIOS 5: 151-; 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.Evol.4: 406-425; sneath, p.h.a. and Sokal, r.r., 1973, Numerical taxomones and Practice of Numerical taxomones, FreemanPress, San Francisco, CA; wilbur, W.J., and Lipman, D.J., 1983, Proc. Natl. Acad. Sci. USA80: 726-.

Preferably, the "percentage of sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein for optimal alignment of the two sequences, a portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5% to 15%, or 10% to 12%, as compared to the reference sequence (which does not comprise additions or deletions). The percentage is calculated by the following method: determining the number of positions of identical nucleic acid bases or amino acid residues present in both sequences to yield the number of matched positions; dividing the number of matched positions by the total number of positions in the reference sequence (i.e., the window size); and multiplying the result by 100 to yield the percentage of sequence identity.

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

Suitable "moderately stringent conditions" include a pre-wash in a solution of 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); 5 XSSC hybridization overnight at 50 ℃ to 65 ℃; then washed twice at 65 ℃ for 20 minutes using 2 ×, 0.5 × and 0.2 × SSC containing 0.1% SDS.

As used herein, "highly stringent conditions" or "high stringency conditions" are: (1) washing with low ionic strength and high temperature, e.g. 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium dodecyl sulphate at 50 ℃; (2) during hybridization with a denaturing agent, such as formamide, for example 50% (v/v) formamide containing 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5 and 750mM sodium chloride, 75mM sodium citrate at 42 ℃; or (3) washing with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS, and 10% dextran sulfate at 42 ℃ in 0.2 XSSC (sodium chloride/sodium citrate) and 55 ℃ in 50% formamide, followed by high stringency washing in 0.1 XSSC containing EDTA at 55 ℃. The skilled person will know how to adjust the temperature, ionic strength, etc. as required to accommodate factors such as probe length.

One of ordinary skill in the art will recognize that, due to the degeneracy of the genetic code, there are a number of nucleotide sequences that encode the polypeptides described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotide changes due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles comprising the polynucleotide sequences provided herein are within the scope of the invention. An allele is an endogenous gene that is altered by one or more mutations, such as deletions, additions and/or substitutions, of nucleotides. The resulting mRNA and protein may, but need not, have altered structure or function. Alleles can be identified using standard techniques, such as hybridization, amplification, and/or database sequence comparison.

The polynucleotides of the invention may be obtained using chemical synthesis, recombinant methods or PCR. Chemical polynucleotide synthesis is 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 commercial DNA synthesizers to prepare the desired DNA sequence.

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

Alternatively, PCR allows DNA sequences to replicate. PCR techniques are well known in The art and are described in U.S. Pat. Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, and PCR, The Polymerase Chain Reaction, eds, Mullis et al, Birkauswer Press, Boston (1994).

RNA can be obtained by the following method: isolated DNA in a suitable vector is used and inserted 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 shown, for example, in Sambrook et al, (1989).

Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a wide variety of cloning vectors available in the art. Although the cloning vector selected may vary depending on the host cell intended for use, useful cloning vectors are typically self-replicating, may have a single target for a particular restriction endonuclease, and/or may carry a marker gene that can be used to select clones containing the vector. Suitable examples include plasmids and bacterial viruses such as pUC18, pUC19, Bluescript (e.g., pBSSK +) and its derivatives mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors such as pSA3 and pAT 28. These and various other cloning vectors are available from commercial suppliers such as BioRad, Strategene and Invitrogen.

The expression vector is typically a replicable polynucleotide construct comprising a polynucleotide according to any of the various aspects of the present invention. This means that the expression vector must be capable of replication in the host cell, either as episomes or as an integral part of the chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the expression vectors disclosed in PCT publication No. WO 87/04462. The carrier component may generally include, but is 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 control elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also typically required.

The vector comprising the polynucleotide of interest can be produced by any of a variety of suitable methods, including electroporation; transfection with calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran or other substances; bombardment of particles; carrying out liposome transfection; and infection (e.g., where the vector is an infectious agent, such as vaccinia virus) into the host cell. The choice of vector or polynucleotide to introduce will generally depend on the characteristics of the host cell.

In some aspects, the invention also provides a host cell comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate the gene encoding the antibody, polypeptide or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, COS, HeLa, and CHO cells. See also PCT publication No. WO87/04462. Suitable non-mammalian host cells include prokaryotes such as E.coli or Bacillus subtilis and yeasts such as Saccharomyces cerevisiae (S.cerevisae), Schizosaccharomyces pombe (S.pombe), or Kluyveromyces lactis (K.lactis). Preferably, the host cell has a cDNA expression level that is about 5-fold, more preferably 10-fold, even more preferably 20-fold that of the corresponding endogenous antibody or protein of interest (if present) in the host cell. Screening for host cells that specifically bind to A1-40 was accomplished by immunoassay or FACS. Cells that overexpress the antibody or protein of interest can be identified.

D. Composition comprising a metal oxide and a metal oxide

In some embodiments, the compositions for use in the methods of the invention comprise an effective amount of an antibody (e.g., an anti-CGRP antagonist antibody, a monoclonal antibody that modulates the CGRP pathway) or antibody-derived polypeptide described herein. Examples of such compositions and how to formulate are also described in the previous section and below. 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 undesired or unwanted immune response such as antibody-mediated lysis or ADCC. In some embodiments, the anti-CGRP antagonist antibody comprises one or more CDRs of antibody G1 (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.

It is understood that the composition 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 antibody recognizing the same epitope, or different substances of anti-CGRP antagonist antibodies binding to different epitopes of CGRP.

The compositions may also contain pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The science and practice of Pharmacy, 20 th edition, (2000) Lippincott Williams and Wilkins eds., K.E. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed. Therapeutic formulations of the antibodies may comprise one or more pharmaceutically acceptable carriers, excipients, or stabilizers, non-limiting examples of which include buffers such as phosphateCitric acid and other organic acids; salts, such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; chlorhexidine di-ammonium; benzalkonium chloride, benzethonium chloride; phenol, butanol or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; 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; an amino acid (e.g., at a concentration of 0.1mM to 100mM, 0.1mM to 1mM, 0.01mM to 50mM, 1mM to 30mM, 1mM to 20mM, 10mM to 25mM), such as glycine, glutamine, methionine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars including glucose, mannose, or dextrins; chelating agents (e.g., at a concentration of 0.001mg/mL to 1mg/mL, 0.001mg/mL to 0.1mg/mL, 0.001mg/mL to 0.01mg/mL), such as EDTA (e.g., disodium ethylenediaminetetraacetate dihydrate); a sugar (e.g., at a concentration of 1mg/mL to 500mg/mL, 10mg/mL to 200mg/mL, 10mg/mL to 100mg/mL, 50mg/mL to 150mg/mL), such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and/or a nonionic surfactant (e.g., at a concentration of 0.01mg/mL to 10mg/mL, 0.01mg/mL to 1mg/mL, 0.1mg/mL to 1mg/mL, 0.01mg/mL to 0.5mg/mL) such as, for example, TWEEN^TM(e.g., polysorbates (e.g., polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80)), PLURONICS^TMOr polyethylene glycol (PEG). Pharmaceutically acceptable excipients are also described herein.

Antibodies (e.g., anti-CGRP antagonist antibodies) and compositions thereof can also be used in combination with other agents that act to enhance and/or supplement the effectiveness of the agent.

E. Reagent kit

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

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 CDRs of antibody G1 (such as one, two, three, four, five, or in some embodiments, all six CDRs of G1).

Instructions for use of the antibodies (e.g., anti-CGRP antagonist antibodies) generally include information about the dosage, dosing schedule, and route of administration of the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. The instructions provided in the kit are typically a label or written instructions on a manual (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions on a magnetic or optical disk) are also acceptable.

The label or instructions indicate that the composition can be used to treat, ameliorate and/or prevent headache (such as migraine). Instructions may be provided for practicing any of the methods described herein.

The kit of the invention has suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar (r) or plastic bags), and the like. Packages for use in conjunction with a particular device, such as an inhaler, nasal administration device (e.g., nebulizer), or infusion device, such as a micropump, are also contemplated. The kit may have a sterile access port (e.g., the container may be an intravenous bag or vial having a stopper that is pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous bag or vial having a stopper that is 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 also contain a second pharmaceutically active agent.

The kit may optionally provide additional components, such as buffers and explanatory information. Typically, the kit includes a container and a label or instructions on or associated with the container.

The following examples are provided to illustrate, but not to limit, the invention.

Examples

Example 1: generation and characterization of monoclonal antibodies against CGRP

Production of anti-CGRP antibodies. To generate anti-CGRP antibodies with cross-species reactivity to rat and human CGRP, mice were immunized with 25-100 μ g of human α -CGRP or β -CGRP conjugated to KLH in adjuvant at different intervals (50 μ l per footpad, 100 μ l total per mouse). Immunization is generally performed as described by Geerligs HJ et al, 1989, J.Immunol.methods 124: 95-102; kenney JS et al, 1989, J.Immunol.methods 121: 157-; and Wicher K et al, 1989, int. Arch. allergy appl. Immunol.89: 128-135. Mice were immunized for the first time with 50 μ g of human α -CGRP or β -CGRP conjugated to KLH in CFA (complete Freund's adjuvant). After 21 days, mice were immunized a second time with 25 μ g of human β -CGRP conjugated to KLH in IFA (incomplete Freund's adjuvant) (mice using human α -CGRP for the first immunization) or α -CGRP (mice using human β -CGRP for the first immunization). Twenty three days after the second immunization, a third immunization was performed using 25 μ g of rat α -CGRP conjugated to KLH in IFA. After ten days, antibody titers were tested using ELISA. A fourth immunization was performed 34 days after the third immunization with 25. mu.g of peptide in IFA (rat. alpha. -CGRP-KLH). At 32 days after the fourth immunization, a final boost was performed with 100. mu.g of soluble peptide (rat. alpha. -CGRP).

Splenocytes were obtained from immunized mice and fused with NSO myeloma cells at a 10:1 ratio using polyethylene glycol 1500. The hybrids were seeded in 96-well plates in DMEM containing 20% horse serum and 2-oxaloacetate/pyruvate/insulin (Sigma) and hypoxanthine/aminopterin/thymidine screening was initiated. On day 8, 100 μ l of DMEM containing 20% horse serum was added to all wells. Supernatants from hybrids were screened using antibody capture immunoassay. Class-specific secondary antibodies are used to determine antibody classes.

A panel of monoclonal antibody-producing cell lines were selected for further characterization based on binding to human and rat CGRP. These antibodies and characteristics are shown in tables 2 and 3 below.

Purification and Fab fragment preparation. Monoclonal antibodies were purified from hybridoma culture supernatants using protein a affinity chromatography for further characterization of selection. The supernatant was equilibrated to pH8. The supernatant was then loaded onto a protein A column MabSelect (Amersham Biosciences #17-5199-02) equilibrated to pH8 with PBS. The column was washed with 5 column volumes of PBS (pH 8). The antibody was eluted with 50mM citrate-phosphate buffer (pH 3). The eluted antibody was neutralized with 1M phosphate buffer (pH 8). The purified antibody was dialyzed against PBS (pH7.4). Antibody concentrations were determined by SDS-PAGE using a murine monoclonal antibody standard curve.

Fab was prepared by papain digestion of whole antibodies using the Immunopure Fab kit (Pierce #44885) and purified by flow-through protein a chromatography according to the manufacturer's instructions. Concentrations were determined by ELISA and/or SDS-PAGE electrophoresis using standard Fab of known concentration (determined by amino acid analysis) and determined by a280 using 1OD ═ 0.6mg/ml (or determining the theoretical equivalent based on amino acid sequence).

Affinity assay for Fab. Using a Biacore3000^TMThe affinity of the anti-CGRP monoclonal antibody was determined at 25 ℃ or 37 ℃ using a Surface Plasmon Resonance (SPR) system (Biacore, INC, Piscataway NJ) and the manufacturer's own running buffer HBS-EP (10mM HEPES pH7.4,150mM NaCl,3mM EDTA, 0.005% v/v polysorbate P20). Affinity was determined by: the N-terminally biotinylated CGRP Peptide (custom made from GenScript Corporation, New Jersey or Global Peptide Services, Colorado) was captured by pre-immobilized streptavidin on an SA chip and the binding kinetics of the titrated antibody Fab on the entire CGRP surface was determined. Biotinylated CGRP was diluted to HBS-EP and injected onto the chip at a concentration of less than 0.001 mg/ml. Using variable flow times on a single chip channel, two antigen density ranges were achieved:<50 Response Units (RU) for detailed kinetic studies, about 800RU for concentration studies and screening two-fold or three-fold serial dilutions (typical concentration range 1. mu.M-0.1 nM (target 0.1-10 × estimated K)_D) Purified Fab fragment of (g) was injected at 100. mu.L/min for 1 minute and then subjected to dissociation for a period of 10 minutes. After each binding cycle, a surface was regenerated using 25mM NaOH in 25% v/v ethanol, which surface was tolerant to more than a few hundred cycles. Kinetic association Rate (k)_on) And dissociation rate (k)_off) The BIAevaluation program was used to obtain simultaneously by fitting the data to a 1:1Langmuir binding model (Karlsson, r.roos, h.fagerstam, l.petersson, B. (1994). Methods Enzymology 6.99-110). From the ratio K_D＝k_off/k_onCalculating the global equilibrium dissociation constant (K)_D) Or "affinity". The affinities of murine Fab fragments are shown in tables 2 and 3.

Epitope mapping of murine anti-CGRP antibodies. To determine the epitope to which the anti-CGRP antibody binds human α -CGRP, the binding affinity of the Fab fragment to the various CGRP fragments was determined by capturing the N-terminally biotinylated CGRP fragment at amino acids 19-37 and amino acids 25-37 on the SA sensor chip as described above. FIG. 1 shows the binding affinity determined at 25 ℃. As shown in FIG. 1, all antibodies, except antibody 4901, bound to human α -CGRP fragments 19-37 and 25-37 with an affinity similar 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 to full-length human α -CGRP fragment, primarily due to a decrease in off-rate. The data indicate that these anti-CGRP antibodies typically bind to the C-terminus of CGRP.

Alanine scans were performed to further characterize the amino acids in human α -CGRP involved in anti-CGRP antibody binding. Different human α -CGRP variants with a single alanine substitution were generated by peptide synthesis. The amino acid sequences of these and all other peptides used for Biacore analysis are shown in table 4. The affinity of Fab fragments of anti-CGRP antibodies to these variants was determined using Biacore as described above. As shown in fig. 1, all 12 antibodies target the C-terminal epitope, with amino acid F37 being the most critical residue. The mutation of F37 to alanine significantly reduced the affinity, even completely abrogating the binding of anti-CGRP antibodies to the peptide. The next important amino acid residue is G33, however, only the high affinity antibodies (7E9, 8B6, 10a8 and 7D11) were affected by the alanine substitution at this position. Amino acid residue S34 also played a significant, but minor, role in the binding of these four high affinity antibodies.

TABLE 2 characterization of the binding of anti-CGRP monoclonal antibodies to human alpha-CGRP and their antagonist activity

Note: antibody 4901 is commercially available (Sigma, product No. C7113).

n.d. ═ not determined

TABLE 3 binding characteristics of anti-CGRP monoclonal antibodies to rat alpha-CGRP and antagonist Activity

"n.d." means that the antibody was not tested.

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 except SEQ ID NO 36-40 were C-terminally amidated. Bold residues represent point mutations.

Example 2: anti-CGRP antagonist antibodies were screened using an in vitro assay.

Antagonist activity of murine anti-CGRP antibodies was further screened in vitro using cell-based cAMP activation and binding assays.

Five microliters of human or rat α -CGRP (final concentration 50nM), or rat α -CGRP or human α -CGRP (final concentration 0.1 nM-10. mu.M; as a positive control for c-AMP activation) were dispensed into 384 well plates (Nunc, catalog No.264657) in the presence or absence of anti-CGRP antibody (final concentration 1-3000 nM.) Ten microliters of stimulation buffer (20mM HEPES pH7.4, 146mM NaCl, 5mM KCl, 1mM CaCl)₂、1mMMgCl₂And 500uM 3-isobutyl-1-methylxanthine (IBMX)) were added to the wells of the plate (human SK-N-MC if human α -CGRP was used, or rat L6 from ATCC if rat α -CGRP was used).

After incubation, HitHunter was used^TMEnzyme Fragment completion Assay (applied biosystems) was followed for cAMP activation according to the manufacturer's instructions. The test is carried outThe EFC assay platform utilizes an ED-cAMP peptide conjugate in which cAMP is recognized by anti-cAMP the ED fragment is capable of reassociating with EA to form an active enzyme in which anti-cAMP antibodies are optimally titrated to bind to the ED-cAMP conjugate and inhibit enzyme formation in the assay, the level of cAMP in a cell lysate sample competes with the ED-cAMP conjugate for binding to the anti-cAMP antibody the amount of ED conjugate free in the assay is proportional to the concentration of cAMP, thus, cAMP is measured by the formation of active enzyme, the formation is quantified by the conversion of the-galactosidase luminescent substrate, the activation assay is performed by adding 10 μ l lysis buffer and anti-cAMP antibody (1:1 ratio), then incubating for 60min, then 10 μ l ED-cAMP reagent is added to each well, and after incubation for 60min, the assay is performed with a high rate of substrate for each well of an enzyme receptor (EA) and Enzyme Donor (ED) to indicate that the assay has a high rate of inhibition of activity in a CGRP 2-cAMP by adding a high rate of an antibody in a PMT assay, typically indicated by adding a high rate of an assay in a PMT-cAMP 2-cAMP assay at room temperature, an assay, where the assay is indicated by adding a high rate of an assay for each well, an assay, where the assay is indicated by adding an assay, where the addition of an antibody, where the assay is indicated by adding an antibody, where the assay is indicated by an assay of an antibody, where the assay is indicated by an assay, where the following incubation of an assay, where the assay is indicated_D(measured at 25 ℃) or has a K of about 47nM or less to rat α -CGRP_DThe antibody (measured at 37 ℃) showed antagonist activity.

Radioligand binding assay. Binding assays were performed to measure the IC of anti-CGRP antibodies blocking CGRP binding to the receptor as described above₅₀. Zimmermann et al, Peptides 16: 421-; mallee et al, J.biol.chem.277:14294-8, 2002. Membranes of SK-N-MC cells (25. mu.g) were packed in a cell containing 10pM 1²⁵I-human α -CGRP incubation buffer (50mM Tris-HCl pH7.4,5mM MgCL)₂0.1% BSA) (total volume 1mL) was incubated at room temperature for 90 min. To determine the Inhibitory Concentration (IC)₅₀) From stock solutions about 100 times higherAntibody or unlabeled CGRP (as control) was dissolved in incubation buffer at various concentrations and incubated with membrane and 10pM¹²⁵I-human α -CGRP incubation at the same time the incubation was stopped by filtration through a glass microfiber filter paper (GF/B,1 μm), which had been blocked with 0.5% polyethyleneimine, dose response curves were plotted and the formula K was used_i＝IC₅₀V. (1+ (ligand/K)_D) Determination of K_iValues wherein the equilibrium dissociation constant K of human α -CGRP and CGRP1 receptors present in SK-N-MC cells_D8pM, and B_max0.025pmol/mg protein. IC to be reported₅₀The value (in the case of IgG molecules) is converted to a binding site (multiplying it by 2) so that it can bind to a Biacore-determined affinity (K)_D) (see Table 2) comparison.

Table 2 shows the IC's of murine antibodies 7E9, 8B6, 6H2 and 4901₅₀. Data show that antibody affinity is generally associated with IC₅₀And (3) correlation: higher affinity (smaller K) in radioligand binding assays_DValue) has a smaller IC₅₀。

Example 3: effect of anti-CGRP antagonist antibodies on stimulation of cutaneous vasodilation in rat saphenous nerves

To test antagonist activity of anti-CGRP antibodies, the effect of the antibodies on stimulation of cutaneous vasodilation by the rat saphenous nerve was tested using the rat model described previously. Escott et al, Br.J.Pharmacol.110:772-776, 1993. In this rat model, electrical stimulation of the saphenous nerve induces release of CGRP from the nerve endings, resulting in increased skin blood flow. Blood flow was measured in the plantar skin of male Sprague Dawley rats (170-300g from Charles River Hollester) after cryptoneurostimulation. Rats were maintained under anesthesia with 2% isoflurane. Benzalkonium tosylate (30mg/kg, administered intravenously) was given at the beginning of the experiment to minimize vasoconstriction due to concomitant stimulation of the sympathetic nerve fibers of the saphenous nerve. Body temperature was maintained at 37 ℃ using a rectal probe thermostatically connected to a temperature controlled heating plate. Compounds including antibodies, positive controls (CGRP 8-37) and vehicle (PBS, 0.01% Tween 20) were administered intravenously via the right femoral vein, and in addition to the experiments shown in figure 3, compounds and controls were tested by tail vein injection, and for the experiments shown in figures 2A and 2B, antibodies 4901 and 7D11 were injected Intraperitoneally (IP). The positive control compound CGRP 8-37 (vasodilation antagonist), due to its short half-life, was administered at 400nmol/kg (200. mu.l) 3-5min prior to neural stimulation. Tan et al, Clin.Sci.89:656-73, 1995. The antibody was administered at different doses (1mg/kg, 2.5mg/kg, 5mg/kg, 10mg/kg and 25 mg/kg).

For the experiments shown in fig. 2A and 2B, antibody 4901(25mg/kg), antibody 7D11(25mg/kg), or vehicle control (PBS with 0.01% Tween 20) was administered intraperitoneally 72 hours prior to electrical pulse stimulation. For the experiments shown in FIG. 3, antibody 4901(1mg/kg, 2.5mg/kg, 5mg/kg, or 25mg/kg) or vehicle control (PBS with 0.01% Tween 20) was administered intravenously 24 hours prior to electrical pulse stimulation. Following antibody or vehicle control administration, the saphenous nerve of the right hindlimb was surgically exposed, cut proximally and covered with a plastic wrap to prevent desiccation. The laser doppler probe is placed on the medial dorsal aspect of the hindpaw skin, which is the area of the saphenous innervation. Skin blood flow is measured as a blood cell flux, which is monitored using a laser doppler flow meter. When a stable baseline flux (less than 5% change) was established for at least 5min, the nerves were placed on a platinum bipolar electrode, electrically stimulated with 60 pulses (2Hz, 10V, 1ms, for 30 seconds), and then the stimulation was repeated after 20 minutes. The cumulative change in skin blood flow is estimated by the area under the flux-time curve (AUC, equal to the flux change times the time change) for each flux response of the electrical pulse stimulation. The blood flow response of the two stimuli was averaged. The animals were maintained under anesthesia for a period of one to three hours.

As shown in fig. 2A and 2B, the increase in blood flow applied to saphenous nerve stimulation by electrical pulses was inhibited by the presence of CGRP 8-37(400nmol/kg, administered intravenously), antibody 4901(25mg/kg, administered intraperitoneally), or antibody 7D11(25mg/kg, administered intraperitoneally), as compared to controls. CGRP 8-37 is administered 3-5min prior to saphenous nerve stimulation; and the antibody was administered 72 hours prior to saphenous nerve stimulation. As shown in fig. 3, the increase in blood flow applied to saphenous nerve stimulation by the electrical pulse was inhibited by the presence of different doses (1mg/kg, 2.5mg/kg, 5mg/kg, and 25mg/kg) of antibody 4901 administered intravenously 24h prior to saphenous nerve stimulation.

For the experiments shown in fig. 4A and 4B, the saphenous nerve was surgically exposed prior to antibody administration. The saphenous nerve of the right hindlimb was surgically exposed, cut proximally and covered with a plastic wrap to prevent desiccation. The laser doppler probe is placed on the medial dorsal aspect of the hindpaw skin, which is the area of the saphenous innervation. Skin blood flow is measured as a blood cell flux, which is monitored using a laser doppler flow meter. Thirty to forty-five minutes after the injection of benzalkonium tosylate, when a stable baseline flux (less than 5% change) was established for at least 5min, the nerves were placed on a platinum bipolar electrode and subjected to electrical stimulation (2Hz, 10V, 1ms, 30 seconds duration), which was repeated after 20 minutes. The mean of the blood flow response of these two stimulations was used to establish the baseline response of the electrical stimulation (time 0). Antibody 4901(1mg/kg or 10mg/kg), antibody 7E9(10mg/kg), antibody 8B6(10mg/kg), or vehicle (PBS with 0.01% Tween 20) was then administered intravenously (i.v.). Nerves were then stimulated (2Hz, 10V, 1ms, up to 30 seconds) 30min, 60min, 90min and 120min after antibody or vehicle administration. The animals were maintained under anesthesia for a period of approximately three hours. The cumulative change in skin blood flow is estimated by the area under the flux-time curve (AUC, equal to the flux change times the time change) for each flux response of the electrical pulse stimulation.

As shown in fig. 4A, the increase in blood flow applied to saphenous nerve stimulation by the electrical pulse was significantly inhibited by the presence of intravenously administered 1mg/kg of antibody 4901 when electrical pulse stimulation was applied at 60min, 90min, and 120min after antibody administration, and by the presence of intravenously administered 10mg/kg of antibody 4901 when electrical pulse stimulation was applied at 30min, 60min, 90min, and 120min after antibody administration. Fig. 4B shows that the increase in blood flow with electrical pulses applied to the saphenous nerve stimulation was significantly inhibited by the presence of antibody 7E9(10mg/kg, administered intravenously) when electrical pulse stimulation was applied 30min, 60min, 90min, and 120min after antibody administration, and by the presence of antibody 8B6(10mg/kg, administered intravenously) when electrical pulse stimulation was applied 30min after antibody administration.

These data indicate that antibodies 4901, 7E9, 7D11, and 8B6 are effective in blocking CGRP activity as measured by the vasodilation of skin caused by stimulation of the rat saphenous nerve.

Example 4 characterization of anti-CGRP antibody G1 and variants thereof

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

The expression vector used. Expression of the Fab fragment of the antibody under the control of the IPTG inducible lacZ promoter is analogous to Barbas (2001) phase display: a laboratory manual, Cold Spring Harbor, NY, Cold Spring Harbor laboratory Press pg.2.10. Vector pComb3X), however, modifications include the addition and expression of the following additional domains: human kappa light chain constant domain and CH1 constant domain of IgG2 human immunoglobulin, Ig γ -2 chain C region, protein accession number P01859; immunoglobulin kappa light chain (homo sapiens), protein accession number CAA 09181.

Small scale Fab preparation. Coli transformed from Fab libraries (using electroporation competent TG1 cells or chemically competent Top 10 cells) were inoculated with a single colony simultaneously with master plates (agar LB + carbenicillin (50ug/mL) + 2% glucose) and working plates (2 mL/well, 96 wells/plate) where each well contained 1.5mL LB + carbenicillin (50ug/mL) + 2% glucose. A breathable adhesive sealing film (ABgene, Surrey, UK) was applied to the flat panel. Incubating the two plates at 30 ℃ for 12-16 h; the work plate was shaken vigorously. The master plate was stored at 4 ℃ until use, and the cells from the working plate were pelleted by centrifugation (4000rpm,4 ℃,20min) and resuspended in 1.0mL LB + carbenicillin (50ug/mL) +0.5mM IPTG, and Fab expression was induced by vigorous shaking for 5h at 30 ℃. The induced cells were centrifuged at 4000rpm for 20min at 4 ℃ and resuspended in 0.6 mM of LBiacore HB-SEP buffer (10mM Hepes pH7.4,150mM NaCl,3mM EDTA, 0.005% v/v P20). Lysis of the HB-SEP resuspended cells was achieved by freezing (-80 ℃) followed by thawing at 37 ℃. Cell lysates were centrifuged at 4000rpm for 1 hour at 4 ℃ to separate debris from the Fab-containing supernatants, followed by Filtration using a Millipore MultiScreen Assay System96-Well Filtration Plate (0.2um) and vacuum manifold. The filtered supernatants were injected over all CGRPs on the sensor chip to analyze them using Biacore. Clones were selected for affinity recovery of Fab expression from master plates, which provided template DNA for PCR, sequencing and plasmid preparation.

Large scale Fab preparation. To obtain kinetic parameters, the Fab was expressed on a large scale as follows. 1mL of the "initial" overnight culture from the affinity selection Fab expressing E.coli clones was used to inoculate Erlenmeyer flasks containing 150mL LB + carbenicillin (50ug/mL) + 2% glucose. The remainder of the initial culture (approximately 3mL) was used to prepare plasmid DNA (QIAprep mini-prep, Qiagen kit) for sequencing and further manipulation. Incubate the bulk culture with vigorous shaking at 30 ℃ until OD_600nmUp to 1.0 (typically 12-16 h). The cells were pelleted by centrifugation at 4000rpm for 20min at 4 ℃ and resuspended in 150mL LB + carbenicillin (50ug/mL) +0.5mM IPTG. After 5h at 30 ℃ the cells were pelleted by centrifugation at 4000rpm for 20min at 4 ℃ and resuspended in 10mL Biacore HBS-EP buffer and lysed using a single freeze (-80 ℃) thaw (37 ℃) cycle. The cell lysate was pelleted by centrifugation at 4000rpm for 1 hour at 4 ℃ and the supernatant was collected and filtered (0.2 um). The filtered supernatant was loaded onto a Ni-ntacuperflow sepharose (Qiagen, valencia. ca) column equilibrated with PBS pH8, and then washed with 5 column volumes of PBS (pH 8). Individual fabs were eluted in different fractions using PBS (pH8) +300mM imidazole. Fab-containing fractions were collected and dialyzed in PBS, then quantified by ELISA, and then affinity characterized.

And (4) preparing a whole antibody. For expression of whole antibodies, the heavy and light chain variable regions were cloned into mammalian expression vectors and transfected into HEK 293 cells using lipofectamine for transient expression. Antibodies were purified using protein a using standard methods.

Vector pdb.cgrp.hfcgi is an expression vector comprising the heavy chain of the G1 antibody and is suitable for transient or stable expression of the heavy chain. Vector pdb.cgrp.hfcgi has a nucleotide sequence corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 7-612); synthesis of an intron (nucleotide 613-; the DHFR coding region (nucleotide 688-1253); human growth hormone signal peptide (nucleotides 1899-1976); the heavy chain variable region of G1 (nucleotides 1977-2621); a human heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering refers to the wild-type IgG2 sequence; see Eur. J. Immunol. (1999)29: 2613-2624). Vector pdb.cgrp.hfcgi was deposited at ATCC on 2005, 7/15 and assigned ATCC accession No. PTA-6867.

The vector peb. cgrp. hkgi is an expression vector comprising the light chain of the G1 antibody and is suitable for transient expression of the light chain. The vector peb.cgrp.hkgi has a nucleotide sequence corresponding to the following regions: the murine cytomegalovirus promoter region (nucleotides 2-613); the human EF-1 intron (nucleotide 614-1149); human growth hormone signal peptide (nucleotide 1160) -1237); the variable region of the light chain of antibody G1 (nucleotides 1238-1558); the human kappa chain constant region (nucleotides 1559-1882). Vector peb.cgrp.hkgi was deposited at ATCC on 2005, 7/15 and assigned ATCC accession No. PTA-6866.

Biacore analysis for affinity determination. Using a Biacore3000^TMThe affinity of the G1 monoclonal antibody and its variants was determined by a Surface Plasmon Resonance (SPR) system (Biacore, INC, Piscataway NJ) at 25 ℃ or 37 ℃. Affinity was determined by: the N-terminal biotinylated CGRP or fragment was captured by pre-immobilized streptavidin (SA sensor chip) and the binding kinetics of antibody G1Fab fragment or variant titrated across all CGRP or fragments on the chip was measured. All Biacore assays were performed in HBS-EP running buffer (10mM HEPES pH7.4,150mM NaCl,3mM EDTA, 0.005% v/v polysorbate P20). CGRP surfaces were prepared as follows: the N-biotinylated CGRP was diluted to a concentration of less than 0.001mg/mL in HBS-EP buffer and injected throughout the SA sensor chip using variable contact times.Correspond to<A low capacity surface at a capture level of 50 Response Units (RU) was used for high resolution kinetics studies, while a high capacity surface (about 800RU of captured CGRP) was used for concentration studies, screening and solution affinity assays. Kinetic data were obtained by serially diluting the antibody G1Fab in two-or three-fold increments to 1uM-0.1nM (target 0.1-10 Xpredicted K)_D) The concentration range of (c). The sample is typically injected at 100. mu.L/min for 1 minute, followed by dissociation for a period of at least 10 minutes. After each binding cycle, the surface was regenerated using 25mM NaOH in 25% v/v ethanol, which was tolerant to more than several hundred cycles. The entire titration series (usually generated in duplicate) was fit globally to a 1:1Langmuir binding model using the BIAevaluation program. This returns unique association and dissociation kinetic rate constant pairs (k respectively) for each binding interaction_onAnd k_off) Their ratio yields the equilibrium dissociation constant (K)_D＝k_off/k_on). Affinity (K) thus determined_DValues) are listed in tables 6 and 7.

High resolution analysis of binding interactions with very low off-rates. For interactions with very low off-rates (in particular, binding of antibody G1Fab to-CGRP on chip at 25 ℃), affinity was obtained in two part experiments. The above scheme was used with the following modifications. Binding Rate constant (k)_on) Is determined by: a2-fold titration series (in duplicate) in the range of 550nM to 1nM was injected at 100uL/min for 30 seconds and only a 30 second off-phase was allowed. Dissociation rate constant (k)_off) Is determined by: the same titration series of three concentrations (high, medium and low) was injected in duplicate for 30 seconds and allowed a2 hour off-phase. Affinity (K) of each interaction_D) Obtained by the following method: combining k obtained in two types of experiments_onAnd k_offValues, as shown in table 5.

Solution affinity was determined by Biacore solution affinity of rat α -CGRP and F37A (19-37) antibody G1 to human α -CGRP solution affinity was determined by Biacore at 37 deg.C using a high capacity CGRP chip surface (high affinity human α -CGRP was selected for detection) andand HBS-EP running buffer was flowed at 5 uL/min. A constant concentration of 5nM (target equal to or less than the predicted K based on solution-based interaction)_DThe antibody G1Fab fragment of (1 nM) was pre-incubated with the competitor peptide rat α -CGRP or F37A (19-37) human α -CGRP with a final concentration ranging from 3-fold serial dilutions of 1nM to 1 uM. in the absence or presence of the solution-based competitor peptide, the antibody G1Fab solution was injected over all CGRPs on the chip and the lack of binding response detected at the chip surface as a result of monitoring the solution competition.

Table 5 below shows the binding affinity of antibody G1 to human α -CGRP, human β -CGRP, rat α -CGRP and rat β -CGRP as determined by Biacore by flowing Fab fragments through N-biotinylated CGRP on SA chips. To better resolve the affinity of binding interactions with very low off-rates, affinity was also determined by a two-part experiment to complement this analytical localization, and the solution affinity of the rat α -CGRP interaction was also determined (as described above). Close agreement of the measured affinities in the two assay locations confirmed that the binding affinity of native rat α -CGRP in solution was unchanged when N-biotinylated and confined to the SA chip.

TABLE 5 binding affinity of the fully CGRP titrated antibody G1Fab on the chip

Affinity of α -CGRP (rat and human) was determined in a high resolution two-part experiment in which the off-phase was monitored for 2 hours (k)_on、k_offAnd K_DValues of (b) represent the mean of n replicates, with standard deviation expressed as percentage of variance.) β -CGRP (rat and human) whose affinity was determined by global analysis using only a 20min dissociation phase, was not accurate enough to quantify its extreme dissociation rate (their dissociation rate may be slower as described herein, so their affinity may be even higher.) antibody G1Fab dissociates at very low rates from all CGRPs (except α -rat CGRP) with an dissociation rate that reached the resolution limit of the Biacore assay (especially at 25 ℃).

Solution affinity was determined by measuring the lack of binding response by detecting antibody G1Fab by CGRP on a chip, which was preincubated with solution-based rat α -CGRP competitor.

Table 6 below shows antibodies with amino acid sequence variations compared to antibody G1 and its affinity for both rat and human α -CGRP. All amino acid substitutions of the variants shown in table 6 are described with respect to the sequence of G1. The binding affinity of the Fab fragments was determined by Biacore by flowing the Fab fragments through all CGRPs on the SA chip.

TABLE 6 amino acid sequence and binding affinity data for antibody G1 variants determined by Biacore at 37 ℃.

All CDRs include Kabat and Chothia CDRs. Amino acid residues are numbered sequentially (see figure 5). All clones had the same sequence of L3+ H1+ H3 as G1.

K_D＝k_off/k_onExcept thatUnderlinedExcept that all k are_offValues were all determined in the screening mode, which were obtained by global analysis of the Fab concentration series (G1 analyzed in the high resolution mode). Therefore, the temperature of the molten metal is controlled,underlinedK of_DValue by determining k_onTo be determined experimentally. Other k are contemplated_onThe value is the same as M25.

n.d. ═ not determined

To determine the epitope on human α -CGRP recognized by antibody G1, the Biacore assay described above was used. Human α -CGRP was purchased as N-biotinylated type to enable high affinity capture by SA sensor chips. The binding of the G1Fab fragment to human α -CGRP on the chip was determined in the absence or presence of CGRP peptide. Typically, a 2000:1 molar peptide/Fab solution (e.g., 10uM peptide in 50nM G1 Fab) is injected into the human α -CGRP on the chip. Figure 6 shows the percentage of binding blocked by the competing peptide. The data presented in FIG. 6 indicate that the peptides that block 100% of the binding of G1Fab 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 beta-CGRP (WT). All these peptides are C-terminally amidated. Peptides F37A (19-37) and 19-37 of human α -CGRP (the latter C-terminal being unamidated) also blocked the binding of about 80% to 90% of G1Fab to human α -CGRP. Peptides 1-36 of human α -CGRP (which were not amidated at the C-terminus) blocked the binding of about 40% of the G1Fab to human α -CGRP. Peptide fragment 19-36 of human α -CGRP (C-terminally amidated); peptide fragments 1-13 and 1-19 of human α -CGRP (both not amidated at the C-terminus); and human dextrin, calcitonin and adrenomedullin (all C-terminally amidated) do not compete for the binding of G1Fab to human α -CGRP on the chip. These data show that G1 targets the C-terminal epitope of CGRP, and that identification of the most terminal residue (F37) and its amidation are both important for binding.

The binding affinity of the G1Fab to variants of human α -CGRP was also determined (at 37 ℃). Table 7 below shows that the affinity can be determined directly by titrating all N-biotinylation human α -CGRP and variants on the chip with G1 Fab. The data in table 7 indicate that antibody G1 binds to the C-terminal epitope, with F37 and G33 being the most important residues. When an additional amino acid residue (alanine) is added to the C-terminus (amidation), G1 does not bind CGRP.

TABLE 7 binding affinities of the G1Fab to human α -CGRP and variants measured at 37 ℃ (see Table 4 for amino acid sequence)

The above data indicate that the epitope bound by antibody G1 is on the C-terminus of human α -CGRP and that amino acids 33 and 37 on human α -CGRP are important for binding of antibody G1. In addition, amidation of the residue F37 is also important for binding.

Example 5: effect of anti-CGRP antagonist antibody G1 on stimulation of cutaneous vasodilation caused by the saphenous nerve of rats

To test the antagonist activity of anti-CGRP antibody G1, the effect of the antibody on stimulation of cutaneous vasodilation by the rat saphenous nerve was tested using the rat model described in example 3. Briefly, rats were maintained under anesthesia with 2% isoflurane. Benzalkonium tosylate (30mg/kg, administered intravenously) was given at the beginning of the experiment to minimize vasoconstriction due to concomitant stimulation of the sympathetic nerve fibers of the saphenous nerve. Body temperature was maintained at 37 ℃ using a rectal probe thermostatically connected to a temperature controlled heating mantle. The saphenous nerve of the right hindlimb was surgically exposed, cut proximally and covered with a plastic wrap to prevent desiccation. The laser doppler probe is placed on the medial dorsal aspect of the hindpaw skin, which is the area of the saphenous innervation. Skin blood flow is measured as a blood cell flux, which is monitored using a laser doppler flow meter. Thirty to forty-five minutes after the injection of benzalkonium tosylate, in experiments to determine the effect of antibodies within two hours of injection, when a stable baseline flux (change less than 5%) was established for at least 5min, the nerves were placed on a platinum bipolar electrode and electrically stimulated (2Hz, 10V, 1ms, for 30 seconds), with stimulation repeated after 20 minutes. The mean of the blood flow response of these two stimulations was used to establish the baseline response of the electrical stimulation (time 0). Antibody G1(1mg/kg or 10mg/kg) or vehicle (PBS volume containing 0.01% Tween 20 equals 10mg/kgG1) was then administered intravenously (i.v.). Nerves were then stimulated (2Hz, 10V, 1ms, up to 30 seconds) 30min, 60min, 90min and 120min after antibody administration. The animals were maintained under anesthesia for a period of approximately three hours. The cumulative change in skin blood flow is estimated by the area under the flux-time curve (AUC, equal to the flux change times the time change) for each flux response of the electrical pulse stimulation.

As shown in fig. 7, when the saphenous nerve was electrically stimulated 90min after antibody administration, the increase in blood flow applied to saphenous nerve stimulation by the electrical pulse was significantly inhibited by the presence of 1mg/kg (intravenously administered) of antibody G1, as compared to vehicle. When the saphenous nerve was electrically stimulated 90 and 120 minutes after antibody administration, the increase in blood flow stimulated by the electrical pulse applied to the saphenous nerve was significantly inhibited by the presence of 10mg/kg (intravenously administered) of antibody G1, as compared to vehicle.

As described above, in experiments to determine the effect of antibodies at longer time points in the saphenous nerve assay, rats were injected intravenously with the indicated dose of antibody 24 hours or 7 days before preparing the animals for saphenous nerve stimulation. In these experiments, it was not possible to establish a baseline response to electrical pulse stimulation in a single rat prior to dosing, so the treatment group corresponded to animals given vehicle (PBS, 0.01% Tween 20) at 24 hours or 7 days.

As shown in fig. 8A and 8B, the increase in blood flow to the skin of the middle dorsal hind paw caused by the saphenous nerve stimulation was significantly suppressed in the animal groups administered with 10mg/kg or 3mg/kg G1 24 hours or 7 days before stimulation, compared to the vehicle group administered at the same time point.

FIG. 8C shows a curve fitting analysis applied to the dose response data shown in FIGS. 8A and 8B to determine 50% maximal Effect (EC)₅₀) The required dosage. EC of 24 hours₅₀1.3mg/kg, 7 days EC₅₀Slightly smaller (0.8 mg/kg).

Example 6: acute Effect of anti-CGRP antagonist antibody mu7E9 in the dural artery (closed cranial Window) assay

Closed type skull window model: the aim 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 BIBN4096 BS. The experiments were performed as described above (Williamson et al, Cephalalgia 17(4):518-24(1997)) with the following modifications. Sprague Dawley rats (300-400g) were anesthetized by intraperitoneal administration of 70mg/kg pentobarbital. Intravenous administration of 20mg/kg/h pentobarbital maintained the anesthetic state. The cannula was inserted into the jugular vein of the rat to deliver all the drugs. Blood pressure was monitored using a probe (mikro-tip catheter, Millar Instruments) passed through the femoral artery into the abdominal aorta. The trachea of the rat was cut and the respiratory rate was maintained at a volume of 3.5mL of 75 breaths/min. After the head was fixed in the stereotactic instrument and the scalp was removed, a 2X 6mm window was made in the left apical area lateral to the sagittal suture by thinning the bone with a dental drill. The platinum bipolar electrode was moved down to the surface using a micromanipulator and covered with heavy mineral oil. On the side of the electrode window, a further 5X 6mm window was made and filled with heavy mineral oil, through which the diameter of a bundle of mesencephalic arteries (MMA) was continuously monitored with a CCD camera and a videometric analyzer (Living Systems). After the preparation work, the rats were placed in the supine position for not less than 45 minutes. Baseline responses to electrical stimulation were established (15V, 10hz, 0.5ms pulse, 30 seconds) and rats were then given test compounds (10mg/kg mu7E9, 300g/kg BIBN4096BS or PBS 0.01% Tween 20 intravenously, additional electrical stimulation was performed 5(BIBN4096BS), 30, 60, 90 and 120 minutes post-dose all data were recorded using graphical software (instruments).

As shown in FIG. 9, mu7E9 at 10mg/kg significantly blocked the electric field-stimulated dilation of MMA within 60 minutes after administration, and the effect was maintained throughout the measurement period (120 minutes). By comparison, BIBN4096BS blocked MMA dilation within 5 minutes of administration, but the effect completely disappeared at 90 minutes. The magnitude of the blockade is equivalent to between BIBN4096BS and mu7E 9.

Example 7: chronic Effect of anti-CGRP antagonist antibody G1 in the dural artery (closed cranial Window) assay

The purpose of this experiment was to determine whether anti-CGRP antibodies still blocked electrically stimulated MMA dilation 7 days after dosing. The preparation of rats was the same as the acute experiment described above (example 6) with the following exceptions. Rats were injected intravenously (10mg/kg, 3mg/kg or 1mg/kg G1) 7 days before closed cranial window preparation and stimulation. As with the acute experiment, it was not possible to establish a baseline expansion response to electrical stimulation prior to dosing, so the antibody group was compared to the MMA expansion in the vehicle (PBS, 0.01% Tween 20) administered to the control group. After the rat was placed in the supine position for not less than 45 minutes, the dura mater was electrically stimulated at 30-minute intervals. Stimulation was performed at 2.5V, 5V, 10V, 15V and 20V, all at 10Hz, with 0.5ms pulses for 30 seconds.

As shown in fig. 10, 10mg/kg and 3mg/kg G1 significantly blocked the electrical stimulation-induced dilation of MMA in the range of 10 to 20 volts. The data show that G1 blocks electrically stimulated MMA dilation up to 7 days after dosing.

Example 8: does oneCoffee hot flash model for withdrawal

The morphine withdrawal rat model is an established rodent model of the climacteric hot flush mechanism (Sipe et al, BrainRes.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 particles under the skin. At the time of addiction, the animals are immediately put into withdrawal by injecting the animals with naloxone (opioid antagonist). The 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 similar in magnitude and arrangement to the occurrence of hot flashes in humans (Simpkins et al, Life sciences 32:1957-1966 (1983)). Furthermore, hot flashes are reduced if rats are treated with estradiol prior to induction of withdrawal (Merchenthaler et al, Maturitas 30: 307-. This is why the morphine withdrawal model is believed to mimic clinical hot flashes.

Ovariectomized rats were ordered from Charles River Laboratories. Morphine dependence was established by subcutaneous implantation of morphine particles (75mg of morphine base) no less than 7 days after ovariectomy. Two days later, 2 granules were implanted. On the following days, rats were injected intravenously with 10mg/kg 4901 [. prime ] or vehicle (PBS, 0.01% tween). Two days after the second implantation of the pellets, rats were anesthetized with ketamine (90mg/kg) and slightly restrained. The base of the tail was measured using a surface temperature thermocouple and the core body temperature was measured using a rectal thermocouple. Data were recorded using chart software (ADInstructions). Naloxone (1mg/kg) was injected subcutaneously after recording a stable baseline temperature for 15 minutes. The temperature was recorded continuously for a further 60 minutes. The results are shown in FIGS. 11A and 11B.

Example 9: treatment of chronic migraine

A human male subject aged 45 years is identified as having chronic migraine for at least three months. Identification of patients with chronic migraine is achieved by observing a history of frequent headache prior to screening, which suggests that chronic migraine (e.g., 15 days/month) persists for at least three months. The examination of headache frequency is achieved by baseline information collected in the future, which is shown in headache for at least 15 days, and at least 8 days/month, which meet any of the following criteria: i. identified as a migraine attack; with or accompanied by migraine precursors.

To reduce the onset of migraine in a subject, the subject is administered a 225mg dose of an anti-CGRP antagonist antibody (e.g., antibody G1). The anti-CGRP antagonist antibody is provided in a liquid formulation having a concentration of 150 mg/mL. The 225mg dose was administered by subcutaneous injection of 1.5mL in the upper arm back of the subject's body. Alternatively, the dose may be provided to the subject by intravenous infusion. In this case, 5.85mL of 150mg/mL anti-CGRP antibody can be combined in an intravenous infusion bag with 0.9% sodium chloride solution (saline) to a total volume of 130mL in the infusion bag. Subjects were infused intravenously with a volume of 100mL iv bag over the course of one hour for a total dose of 225 mg. Dosing was repeated every twenty-eight days until a reduction in migraine attacks was observed. The reduction in the onset of chronic migraine is examined using a number of criteria including the number of headache days observed in the subject, the number of hours that the headache occurred (e.g., headache hours), the severity of the headache, and the number of migraine days.

Example 10: treatment of chronic migraine

A human female subject aged 37 years is identified as having chronic migraine for at least three months. Identification of patients with chronic migraine is achieved by observing a history of frequent headache prior to screening, which suggests that chronic migraine (e.g., 15 days/month) persists for at least three months. The examination of headache frequency is achieved by baseline information collected in the future, which is shown in headache for at least 15 days, and at least 8 days/month, which meet any of the following criteria: i. identified as a migraine attack; with or accompanied by migraine precursors.

To reduce the onset of migraine in a subject, the subject is administered an initial loading dose of 675mg of an anti-CGRP antagonist antibody (e.g., antibody G1). The anti-CGRP antagonist antibody is provided in a liquid formulation having a concentration of 150 mg/mL. The 675mg loading dose was administered by three subcutaneous injections of 1.5mL of 225mg into various regions of the subject's body (e.g., upper arm back, lower abdominal cavity/abdomen/waist, front thigh, etc.). 225mg (e.g., 1.5mL by one subcutaneous injection through the subject's arm) is repeated every twenty-eight days until a reduction in migraine attack is observed. The reduction in the onset of chronic migraine is examined using a number of criteria including the number of headache days observed in the subject, the number of hours that the headache occurred (e.g., headache hours), the severity of the headache, and the number of migraine days.

Example 11: treatment of chronic migraine

A human male subject aged 23 years is identified as having chronic migraine for at least three months. Identification of patients with chronic migraine is achieved by observing a history of frequent headache prior to screening, which suggests that chronic migraine (e.g., 15 days/month) persists for at least three months. The examination of headache frequency is achieved by baseline information collected in the future, which is shown in headache for at least 15 days, and at least 8 days/month, which meet any of the following criteria: i. identified as a migraine attack; with or accompanied by migraine precursors.

To reduce the onset of migraine in a subject, the subject is administered a 900mg dose of an anti-CGRP antagonist antibody (e.g., antibody G1). The anti-CGRP antagonist antibody is provided in a liquid formulation having a concentration of 150 mg/mL. The 900mg dose is administered by four 225mg subcutaneous injections of 1.5mL into various regions of the subject's body (e.g., upper arm back, lower abdominal cavity/abdomen/waist, front thigh, etc.). Dosing was repeated every twenty-eight days until a reduction in migraine attacks was observed. The reduction in the onset of chronic migraine is examined using a number of criteria including the number of headache days observed in the subject, the number of hours that the headache occurred (e.g., headache hours), the severity of the headache, and the number of migraine days.

Example 12: treatment of episodic migraine

Human male subjects aged 28 years were identified as having high frequency of sporadic migraine. The subject is identified as having a high frequency of sporadic migraine using criteria comprising: having a history of headache for at least 3 months over 8 days/month prior to screening; and examining the headache frequency by baseline information collected in the future, which information shows that among 8 to 14 days of headache (of any type), at least 8 days meet at least one of the following criteria: i. migraine headache; possible migraine; use of triptan or ergot compounds.

To reduce the onset of migraine in a subject, the subject is administered a 675mg dose of an anti-CGRP antagonist antibody (e.g., antibody G1). The anti-CGRP antagonist antibody is provided in a liquid formulation having a concentration of 150 mg/mL. The 675mg dose was administered by three subcutaneous injections of 1.5mL of 225mg into various regions of the subject's body (e.g., upper arm back, lower abdominal cavity/abdomen/waist, front thigh, etc.). Dosing was repeated every twenty-eight days until a reduction in migraine attacks was observed. Reduction of sporadic migraine attacks is examined using a number of criteria including the number of headache days observed in the subject, the number of hours that the headache occurred (e.g., headache hours), the severity of the headache, and the number of migraine days.

Example 13: treatment of episodic migraine

Human female subjects aged 52 years were identified as having high frequency episodic migraine headaches. The subject is identified as having a high frequency of sporadic migraine using criteria comprising: having a history of headache for at least 3 months over 8 days/month prior to screening; and examining the headache frequency by baseline information collected in the future, which information shows that among 8 to 14 days of headache (of any type), at least 8 days meet at least one of the following criteria: i. migraine headache; possible migraine; use of triptan or ergot compounds.

To reduce the onset of migraine in a subject, the subject is administered a 225mg dose of an anti-CGRP antagonist antibody (e.g., antibody G1). The anti-CGRP antagonist antibody is provided in a liquid formulation having a concentration of 150 mg/mL. The 225mg dose was administered by subcutaneous injection of 1.5mL in the upper arm back of the subject's body. Dosing was repeated every twenty-eight days until a reduction in migraine attacks was observed. A reduction in sporadic migraine attacks is examined using a plurality of observations, including the number of days of headache, the number of hours headache occurred, the severity of headache, and the number of days of migraine observed in the subject.

Example 14: non-clinical toxicology and pharmacokinetics

anti-CGRP antagonist antibody G1 was well tolerated in 1 month intravenous repeat dose toxicity studies in Sprague-dawley (sd) rats and cynomolgus monkeys, and no target organ toxicity was determined in any of these studies. Levels of no adverse events (NOAEL) of 100 mg/kg/week were established for rat and monkey studies. This dose level corresponds to the maximum concentration in rats and monkeys exposed systemically to 2,570 and 3,440 μ g/mL (Cmax) and the area under the curve (AUC (0-168h)) of 194,000 μ g-h/mL and 299,000 μ g-h/mL (day 22), respectively.

In the 3 month intravenous/subcutaneous rat study, no target organ toxicity was identified and G1 was well tolerated up to the highest tested dose of 300 mg/kg. Perivascular inflammation of the ciliary arteries was observed at ≥ 100mg/kg in a 3-month monkey study as a result of immunocomplex deposition. These findings are due to the immunogenic response of monkeys to humanized antibodies and are not considered to be of clinical relevance. In this monkey study, the highest tested dose of 300mg/kg was at least 10-fold (in mg/kg) greater than the highest expected clinical dose of 2,000mg or 29mg/kg (assuming the subjects averaged 70kg body weight).

Example 15: clinical pharmacokinetics

After a single intravenous exposure, the pharmacokinetics of antibody G1 were tested by four randomized placebo-controlled double-blind studies, testing doses between 10 and 2,000 mg. The maximum plasma concentration (Cmax) was reached immediately after the 1 hour intravenous infusion was completed. The median time to Cmax (Tmax) ranged from 1.0 to 3.0 hours, followed by a biphasic decline. Cmax and total exposure increased approximately linearly with increasing dose of G1. The terminal half-life (t1/2) is in the range of 36.4 to 48.3 days. There is no evidence that G1 metabolism occurs in the liver, the primary mode of metabolism being proteasomal degradation.

One study defined the pharmacokinetics of 30mg and 300mg doses administered twice, two weeks apart. The area under the maximum concentration and concentration-time curves increases with increasing dose. After the second dose, the apparent terminal half-life (t1/2) was 41.2 days (30mg) and 50.0 days (300mg) (arithmetic mean). The plasma accumulation ratio of G1 was 1.5(30mg) and 1.4(300mg) after two intravenous doses administered 15 days apart.

Example 16: clinical safety and pharmacokinetics

In six studies, antibody G1 was administered to 118 healthy males and females, while 57 male and female subjects received placebo. The study included a single intravenous dose ranging from 0.2mg up to 2,000mg, two intravenous doses up to 300mg, administered once every 14 days, and 225 and 900mg administered subcutaneously. Six studies included: two intravenous single dose escalation PK and Pharmacodynamic (PD) studies in healthy men (studies B0141001 and B0141002); two placebo controlled crossover studies to examine the acute effect of intravenous administration of antibody G1 on capsaicin flush response in healthy volunteers (B0141006); a parallel group repeat dose study of antibody G1 in healthy male and female volunteers (B0141007); single dose studies to evaluate safety and resistance to intravenous administration of up to 2,000mg doses to healthy female volunteers (B0141008) and studies comparing relative safety and bioavailability between intravenous and subcutaneous administration (G1-SC-IV).

The six studies are summarized in table 11 below. In five intravenous studies (B0141001, B0141002, B0141006, B0141007 and B0141008), three times had virtually the same design and evaluation. Study B014100 tested 0.2mg, 1mg and 3mg doses given by a single one hour intravenous infusion. The study has a parallel design. Participants were confined to the clinic seven days after infusion, with multiple assessments per day over the several days. Patients were again evaluated one week after discharge (day 14) and then one, two and three months after infusion. Study B0141002 tested doses ranging from 10mg to 1000mg as a single administration. Finally, study B0141008 tested doses of 300mg, 1000mg, 1500mg or 2000 mg. Study B0141006 differed from other studies in that it also aimed to integrate pharmacodynamic readings by measuring capsaicin flush inhibition up to one week after intravenous infusion of antibody G1.

For the intravenous study, Adverse Event (AE) curves only report the first dose phase. Study B0141007 tested multiple doses of antibody G1 using a parallel design, which antibody G1 was administered 30 or 300mg intravenously two weeks apart. Randomized sequences were assigned to each eligible subject by an interactive network system comprising a treatment schedule. The randomization architecture was developed by a competent statistician. Participants for all studies were typically healthy males and females (ages 18 to 65); all participants signed informed consent. All studies were approved by the ethical review board (IRB). An AE is defined as any unfortunate medical event in a clinical study participant that is causally related to the presence or absence of the study drug. AEs observed after study drug or placebo administration were referred to as "AEs occurred in treatment" (teae), regardless of whether there was a potential causal relationship with the study drug. All subjects undergoing TEAE were followed at appropriate time intervals until the event was resolved or until the event stabilized and/or a new baseline was reached. All TEAEs were classified as mild, moderate or severe. By inference, severe ae (sae) is defined as any unfortunate medical event that results from any dose that causes death, is life-threatening (i.e., the subject is at risk of immediate death at the time of the event), requires hospitalization or prolonged current hospitalization, results in persistent or significant disability/disability (e.g., substantially destroys the subject's ability to function normally in life), results in congenital abnormalities/birth defects or any other medically significant event. A treatment-related ae (trae) when one of the following occurs: 1) a plausible temporal relationship between the onset of the AE and the administration of the study product can be identified; 2) AEs are not readily explained by the clinical status, complication, or concomitant treatment of the patient; 3) AE abrogated termination or dose reduction of study products.

Blood pressure, pulse rate and oral temperature were measured at screening, before dosing, after the end of infusion and during the period when the patient was confined to the clinic, as well as at all visits. Laboratory tests include serum chemistry, hematology, and urinalysis. Hematology, chemistry, coagulation and urine safety laboratory tests were performed at various study times. ECG was recorded at screening, before dosing on day 1, after the end of infusion and at five other times during day 1, and at all visits. QTcF values were obtained using the fridericia (QTcF) heart rate correction formula. The absolute values of the ECG parameters QT interval, heart rate, QTcF interval, PR interval and QRS interval as well as the changes from baseline were assessed by group, treatment and time post-dose. In addition to the safety assessments described above, protocol B014008 also included a full ophthalmologic assessment at baseline and at three time points post-dose (day 28, day 84 and day 168).

Clinical data and vital signs were summarized using descriptive tables and summary statistics. Laboratory and other safety data are summarized as a function of any changes (values outside the reference range), as well as any clinically relevant changes (defined by reasoning). Summary tables are stratified by dose and all study data is collected. In addition, comparison of the consolidated data for all antibody G1 exposures was compared to placebo. Placebo was also compared to antibody G1 doses at 100mg and higher (100mg, 300mg, 1000mg, 1500mg and 2000mg) and antibody G1 doses at 1000mg and higher (1000mg, 1500mg and 2000 mg).

In the intravenous/subcutaneous study (G1-SC-IV), thirty-six subjects randomly received a single administration of antibody G1(225 or 900mg) or placebo delivered by Subcutaneous (SC) bolus injection or 1 hour intravenous infusion. Subjects were confined to the clinical study unit until seven days post-dose and returned periodically to the clinic for additional outpatient examinations until study day 90. ECG was performed on a large scale on day 1 (pre-dose, 1, 6, 12 hours), day 3, day 7, while subjects were confined once at study completion (day 90). Vital signs including temperature, blood pressure and heart rate were collected prior to dosing, on days 1, 3, 7 and 90.

TABLE 11

The intravenous administration of antibody G1 gave acceptable tolerability over a broad dose range assessed in five intravenous studies (0.2 to 2,000 mg). Table 8 summarizes the overall Adverse Event (AE) rates by dose for the intravenous study. According to these tolerability results, no significant safety issues arise. In all trials of the intravenous study, participants who received placebo reported an average occurrence of adverse events (TEAE) occurring in 1.3 treatments. These are all reported events, regardless of the investigator's view of the study drug relationship. This ratio was 1.4 TEAEs/subject in all intravenous G1 doses. Subjects receiving a G1 dose of 100mg or greater had an average of 1.5 TEAEs; those subjects receiving doses of 1,000mg or higher had an average of 1.6 TEAEs.

TABLE 8

AE is an adverse event; n-any event that is treatment-related or not; (N) is a treatment contemplated by researchers. Note: for regimen B0141006 (placebo and 300mg), only data for the first active treatment phase are included due to its crossover nature

In the intravenous study, 21.2% of subjects receiving intravenous G1 reported a treatment-related adverse event (TRAE or AE that may be relevant to treatment according to the study leader), compared to only 17.7% of subjects receiving placebo. At doses of 100mg G1 or higher, TRAE appeared in 22.4% of participants. At doses of 1,000mg or higher, TRAE occurs in 21.7% of participants. Antibody G1 appears to be unrelated to changes in vital signs (systolic and diastolic blood pressure [ BP ], temperature and heart rate [ HR ]), Electrocardiogram (ECG) abnormalities (including QTcB and QTcF), any clinically relevant pattern of infusion site reactions, or clinical laboratory findings. An increase in total bilirubin of grade 1 in one subject receiving placebo (study B0141001), and an increase in ALT of grade 1 in one subject receiving placebo (study B0141002) had limited efficacy on liver function tests (aspartate aminotransferase [ AST ], alanine aminotransferase [ ALT ], total bilirubin and alkaline phosphatase). No clinically significant liver function abnormalities were found in subjects receiving any of the study doses G1. In hematological tests to assess kidney function, electrolytes, or in urine tests, there was no evidence of a difference between G1 and placebo.

In the intravenous/subcutaneous study (G1-SC-IV), safety and tolerability were similar between the subcutaneous and intravenous delivery routes. Mean heart rate and blood pressure (diastolic and systolic) were not affected by treatment with antibody G1, nor did it produce any meaningful changes in any cardiovascular parameters after subcutaneous administration of antibody G1. A summary of the TRAEs observed during the subcutaneous study is shown in table 12 below.

TABLE 12

	900mg(N＝6)	225mg(N＝6)	Placebo (N ═ 6)
				Gastrointestinal diseases	2(33.3％)	0	1(16.7％)
CNS	0	1(16.7％)	0
				Infection and infestation	0	0	0
Skeletal muscle and connective tissue	0	0	0
				Respiratory property	0	0	0
Reproductive and mammary disorders	0	0	0
				Injury by injury	0	0	0
Pregnancy	0	0	0
				Kidney (A)	1(16.7％)	0	0
Blood vessel	0	0	0

In a single dose study (B0141001, B0141002, B0141006 and B0141008), Pharmacokinetic (PK) parameters were calculated for doses ranging from 30mg to 2,000 mg. Group mean terminal half-life (t)_1/2) In the range of about 40 to 48 days. C_maxAnd total exposure (by AUC)_inf) Evaluation) increased with increasing dose. AUC_infThe increase in (b) appears to be approximately equal to the dose ratio between 30 and 1,000mg, and appears to be greater than the dose ratio between 1,000 and 2,000 mg. The volume of distribution is very low, between 6-10L.

In the two dose study (B0141007), the apparent terminal half-life was between 41 and 50 days after the second dose. Plasma concentrations accumulated after the second dose at a rate of about 1.5. Furthermore, in the intravenous/subcutaneous study (G1-SC-IV), pharmacokinetic assessments indicate that G1 has a similar terminal half-life when delivered subcutaneously compared to intravenous delivery.

Example 17: prevention of chronic migraine in clinical studies with antibody G1

A multicenter, randomized, double-blind, double-simulated, placebo-controlled, parallel-group, multi-dose study comparing anti-CGRP antagonist antibody G1 to placebo was conducted in subjects with chronic migraine, eligible subjects entered a baseline 28-day break-in period, throughout the study, subjects may collect their Headache and health information using an electronic Headache diary system daily, migraine medications are unchanged during the break-in period or study period, inclusion criteria are as follows, (1) male or female aged 18 to 65 years, (2) an informed consent document is signed and dated indicating that subjects are aware of all relevant aspects of the study, including any known and potential risks and available alternative therapies, (3) chronic migraine is up to the International Headache disease Classification (International Classification of Header, ICHD-III β edition, 2013) listed diagnostic criteria, (4) if the dose and regimen are stable for at least 2 months before the start of the 28-day break-in period, the most two different medications may be used daily for migraine treatment, such as migraine treatment, high-quality (5 kg) of migraine, 5-year migraine, 5-day, or 5-year-day napolol (BMm) treatment of migraine²A total body weight between 50kg and 120kg, inclusive; (6) subjects were infertile as defined by the method, or if subjects were fertile, and agreed to maintain abstinence or use (or their companion use) an acceptable method of birth control for the planned duration of the study; (7) headache data was entered for a minimum of 24/28 days during the break-in period, in compliance with the electronic headache diary specifications (85% compliance). The criteria for diagnosing chronic migraine in the above (3) are as follows: (a) screening for pre-frequent headacheThe medical history suggests chronic migraine (15 days/month) persists for at least three months; (b) the examination of headache frequency was achieved by baseline information collected in the future during a28 day break-in period, which was shown to meet any of the following criteria for at least 8 days/month in at least 15 days of headache: (i) identified as a migraine attack, (ii) with or accompanied by a migraine precursor, or (iii) alleviated by an ergot or triptan derivative.

The exclusion criteria required that the subject not satisfy any of the following conditions: (1) chronic migraine attacks after more than 50 years of age; (2) subjects received botulinum neurotoxin a (onabotulinum) for migraine treatment during the first six months of screening, or required injections in the head, face or neck for any medical or cosmetic reason; (3) subjects use compositions containing opioids (including codeine) or barbiturates (includingOr any other combination comprising butabital) for migraine treatment or for any other reason more than 4 days per month; (4) after appropriate treatment trials, due to the lack of efficacy in the prophylactic treatment of occasional or chronic migraine,>2 kinds of drugs or>3 prophylactic drugs (within two drug classes) were not effective; (5) according to the judgment of a researcher, clinically significant blood, kidney, endocrine, lung, gastrointestinal, urogenital, neurological or ocular diseases are suffered; (6) the subject has evidence or history of clinically significant psychiatric problems including major depression, panic disorder, or generalized anxiety disorder (according to Diagnostic and Statistical Manual, 5 th edition [ DSM-5)]Standard of (d); (7) systolic blood pressure at screening is greater than 160mmHg or less than 90 mmHg; (8) at screening, the diastolic pressure is greater than 110mmHg or less than 50 mmHg; (9) has clinically significant cardiovascular disease or vascular ischemia (such as myocardial, neural [ e.g., cerebral ischemia)]Peripheral limb ischemia or other ischemic event); (10) those subjects who had a previous or current history of cancer, except for resection of basal cell carcinoma; (11) pregnant or lactating women; (12) has a history of hypersensitivity to injected proteins (including monoclonal antibodies); (13) study enrollment period at 30 daysInternal use study drug treatment; (14) clinically significant baseline 12 lead surface (12-lead surface) ECG abnormalities, including sinus asystole>2 seconds, second or third degree heart block or other abnormalities that the investigator judges to be clinically significant; (15) at screening, baseline 12 lead ECG shows, male QTcF>450ms, female 470ms (if QTcF exceeds these values, the ECG is repeated and the mean of three QTcF's is used to determine the eligibility of the potential subject; QTc is obtained using the Fridericia algorithm); (16) any finding that a researcher judges a clinically significant abnormality, including hematology values, blood chemistry, coagulation tests, or urinalysis (abnormal tests allow for repeat confirmation); (17) liver enzymes (alanine aminotransferase [ ALT ] after confirmation in duplicate tests]Aspartate aminotransferase [ AST ]]Alkaline phosphatase)>1.3 times Upper Limit of Normal (ULN); (18) serum creatinine>1.5 times ULN, clinically significant proteinuria (urine test paper +4), or evidence of kidney disease.

Subjects identified as having chronic migraine headaches during the break-in period and highly matched the headache diary were randomly assigned to one of three treatment groups at visit 2 (day 1). The random assignment is made using an electronic interactive network response system. Subjects were graded according to gender and baseline migraine drug use. Treatments were administered once a month (every 28 days), for a total of three treatments over a3 month period. Therapeutic administration was given at visit 2 (day 1; first dose), visit 3 (day 29; second dose) and visit 4 (day 57; third and final dose). Final study withdrawal assessments were performed at the 5 th visit (day 85) approximately 28 days after the third and final dose. Injections were administered subcutaneously to subjects in each of the following groups over a3 month period (every 28 days): (1) those subjects randomized to the 900mg group received four positive injections every 28 days; (2) those subjects randomized to the 675/225mg group received three positive and one placebo injections for the first treatment and one positive and three placebo injections for the second and third treatments; (3) those subjects randomized to the placebo group received four placebo injections every 28 days. The active injection contained 225mg of antibody G1. The endpoint was derived from an electronic headache diary, which is a network interactive system that recorded the above-mentioned 24-hour period data. The total headache duration is recorded numerically in hours and hours of headache for each severity level. Headache severity was assessed subjectively by the subjects at predetermined time points: no pain, mild pain, moderate pain and severe pain. The subject was also asked to record whether the following associated symptoms were present at the predetermined time point: photophobia, phonophobia, nausea and vomiting. Additional endpoints derive from monitoring study subjects throughout the study's course of use of triptan (e.g., sumatriptan) as an acute anti-headache agent. A summary of the subjects' treatment and demographics in the study are provided in table 9.

TABLE 9

	Placebo	675/225mg	900mg	Total of
					Random	89	88	87	264
Complete the process	77(86.5％)	72(81.8％)	76(87.4％)	225
					Analysis by ITT	89(100％)	87(98.9％)	85(97.7％)	261(98.9％)
Security analysis	89(100％)	88(100％)	86(98.9％)	263(99.6％)
					Unfinished	12(13.5％)	16(18.2％)	11(12.6％)	39(14.8％)
Age (average year)	40.7	40	41.5	40.75
					% of female	85.4％	86.3％	86.2％	85.9％
Number of years migraine headache occurs	20.4	15.8	18.7	18.3

The average reduction in headache hours per group relative to baseline per week on weeks 1, 2 and 3 (W1, W2 and W3, respectively) is graphically represented in fig. 15. The results show that at each week of W1, W2, and W3 (including every first week), both treatment groups were significantly reduced relative to the placebo group.

The average reduction in headache hours per group relative to baseline per month for months 1, 2 and 3 (M1, M2 and M3, respectively) is graphically represented in fig. 12. The results show that at all three time points (including after the first dose), both treatment groups were significantly reduced relative to the placebo group. For the data of fig. 12, statistical significance relative to placebo is provided as the indicated p-value.

The average number of headache hours per group at baseline and at visits 2, 3 and 4 (V2, V3 and V4, respectively) is graphically represented as in fig. 13. The results show that at all three time points (including after the first dose), both treatment groups were significantly reduced relative to the placebo group.

The average reduction in moderate or severe headache days per group from baseline per month at months 1, 2 and 3 (M1, M2 and M3, respectively) is shown in figure 14. The results show that at all three time points (including after the first dose), both treatment groups had a statistically significant reduction relative to the placebo group. Statistical significance relative to placebo is provided as the indicated p-value.

The average reduction in the number of times of use of triptan as a rescue medication per group versus baseline per month for months 1, 2 and 3 (M1, M2 and M3, respectively) is graphically represented in fig. 16. The results show that at all three time points (including after the first dose), triptan usage was significantly reduced in both treatment groups relative to the placebo group. Statistical significance relative to placebo is provided by the p-value shown in figure 16.

A significant reduction in the number of hours of headache was also observed in subjects using prophylactic drugs (e.g., topiramate and amitriptyline or propranolol) relative to the placebo group.

Both doses were well tolerated and no safety issues occurred. A summary of adverse events occurring during Treatment (TEAE) by group is provided in table 10. Differences in associated TEAEs can be almost completely explained by injection-related mild events (erythema, some discomfort). Severe TEAE did not involve drugs (no drug related adverse events).

Watch 10

Example 18: prevention of high frequency episodic migraine in clinical studies with antibody G1

A multicenter, randomized, double-blind, placebo-controlled, parallel-group study comparing anti-CGRP antibody G1 to placebo was performed in subjects with High Frequency Episodic Migraine (HFEM). The study design was as in example 17, but with two differences. First, according to The International Headache Society second edition (Olesen and Steiner2004), The inclusion criteria for subjects experiencing high frequency migraine headaches, meeting The incidental migraine criteria (3) are: (a) the history of headache prior to screening exceeds 8 days/month for at least 3 months; (b) the examination of headache frequency was achieved by collecting baseline information during a28 day break-in period in the future, which was shown in 8 to 14 days of headache (of any type), at least 8 days meeting any of the following criteria: (i) migraine, (ii) possibly migraine, or (iii) use of triptan or ergot compounds. Second, the dosing schedule was changed to the group receiving G1. In particular, the injections were administered subcutaneously to subjects in each of the following groups over a period of 3 months (every 28 days): (1) those subjects randomized to the 675mg group received 675mg G1 every 28 days; (2) those subjects randomized to the 225mg group received 225mg G1 every 28 days; and (3) those subjects randomized to the placebo group received placebo injections every 28 days. A summary of the subjects' treatments and demographics in the study are provided in table 13. The endpoints of the study included a reduction in the number of migraine days and a reduction in the number of days of any severe headache.

Watch 13

	Placebo	225mg	675mg	Total of
					Random	104	96	97	297
Analysis by ITT	104(100％)	95(99％)	96(99％)	295(99％)
					Security analysis	104(100％)	96(100％)	97(100％)	297(100％)
Age (average year)	42.0	40.8	40.7	41.2
					% of female	88％	91％	85％	88％
% of caucasian people	82％	77％	76％	78％

The average reduction in migraine days per group from baseline per month at months 1, 2, and 3 (M1, M2, and M3, respectively) is shown in fig. 17. The results show that at all three time points (including after the first dose), both treatment groups had a statistically significant reduction relative to the placebo group. Statistical significance relative to placebo is provided as the indicated p-value.

The mean reduction in the number of headache days of any severity for each group from baseline per month at months 1, 2 and 3 (M1, M2 and M3, respectively) is represented in figure 18. The results show that at all three time points (including after the first dose), both treatment groups had a statistically significant reduction relative to the placebo group. Statistical significance relative to placebo is provided as the indicated p-value.

Both doses were well tolerated and no safety issues occurred. A summary by group of adverse events (TEAEs) occurring during treatment is provided in table 14. Four severe TEAEs were due to one condition of fibular fracture, one condition of tremor due to withdrawal, and two migraine cases requiring Emergency Room (ER) treatment.

TABLE 14

Example 19: non-clinical safety

Two studies were performed in cynomolgus monkeys to evaluate the safety of antibody G1. In a first study, the safety of a single dose of antibody G1 was evaluated. In a second study, the safety of repeated doses of antibody G1 was assessed. Each study and its results are described in further detail below. For both single and repeated dose studies, antibody G1 was formulated as a 51.4mg/mL solution: 20mM histidine, 84mg/mL trehalose dihydrate, 0.2mg/mL polysorbate 80, 0.05mg/mL disodium EDTA dihydrate, and 0.1mg/mL L-methionine (pH about 5.5). The vehicle without antibody G1 was formulated in the same way. In addition, in both studies, blood samples were periodically collected for analysis of antibody G1 plasma concentrations using a certified ELISA method.

Data were first compiled into summary tables and graphs using GraphPad Prism (version 6.0) and Excel 2010 (Microsoft). For single exposure studies, telemetry data was analyzed using ANOVA. Analysis was performed using SAS Release 8.2. To normalize QT intervals within the range of R-R intervals, a single animal correction factor (IACF) was generated for each animal by correlating each RR interval with its associated QT interval. Linear regression of the QT/RR interval relationship of the data set was determined. The slope of this linear regression was used as the IACF for all animals of interest in treatment. The corrected QT interval (QTc) was calculated using the IACF by the following formula:

QT-I (c) heart rate corrected QT interval QT-I- [ (RR-300) × (IACF) ].

For multiple dose studies, data was also analyzed using one-way analysis of variance. If ANOVA is significant (P.ltoreq.0.05), comparisons between groups are made using Dunnett's post-hoc test. For each gender, the treatment group was compared to the control (vehicle) group at a 5% two-tailed probability level.

Single dose telemetry study

Eight adult male cynomolgus monkeys (Charles River Primates) were surgically instrumented with a telemeter and allowed to recover for at least two weeks. The implant (DSI TL11M2-D70-PCT) and the receptor (RMC-1) were manufactured by Data sciences International.

Animals were acclimated to the telemetry data acquisition cage at least overnight prior to dosing. During the acclimation period, pre-study recordings of hemodynamic parameters were performed to verify that the sensors and devices were functioning properly. During telemetry data acquisition, animals were individually housed in cages equipped with telemetry receivers. On the non-collection day, animals were housed in cages without telemetry receivers. Animals were maintained in a 12 hour light, 12 hour dark diurnal cycle with free intake of water and a validated primate diet.

For the first phase of the study, animals (8 males) were dosed with vehicle only and telemetry data was collected from about 1 hour before dosing to 22 hours after dosing. Six days after vehicle administration, the same animals received a single intravenous administration of antibody G1(100mg/kg, at a dose approximately 10-fold higher than pharmacological EC50 for cynomolgus monkeys). Telemetric electrocardiogram and hemodynamic data were again continuously recorded for all animals. In addition, these animals were monitored for approximately 24 hours on days 3, 7, 10, and 14 after receiving a single dose of antibody G1. The telemetered ECG and blood pressure signals are transmitted by implanted radio telemetry to a receiver mounted in each cage. The acquired signals enter a PC-based data acquisition system (DSI software Ponemah P3 version 3.4) through a data conversion matrix (DSI model DEM); the data analysis software was Emka Technologies version 2.4.0.20(Emka Technologies). The analog/digital sampling rates were 1,000Hz (telemetered ECG data) and 500Hz (blood pressure data). Data are reported as 1min average.

The group mean Systolic Blood Pressure (SBP) before and after antibody G1 treatment was similar on the first and subsequent days post-dose (animals were telemetered on days 3, 7, 10 and 14 with the same time interval as day 1). At 1-4 hours post-dose, when antibody G1 blood concentration was at the maximum level (mean concentration at 4 hours was 3,500 μ G/mL), the mean SBP was 111mmHg and the same time interval after vehicle administration was 113 mmHg. In addition, SBP was 110mmHg at days 3 and 7, 109mmHg at day 10, and 110mmHg at day 14 after antibody G1 administration. Similar SBP data for other time intervals is recorded. Since this is a cross-design study, the treated animals were used as controls for themselves. The reduction in SBP at the latter time intervals was less statistically significant at days 7, 10 and 14 when data were analyzed for differences in blood pressure after administration with antibody G1, compared to vehicle treatment.

Following treatment with antibody G1, the recorded diastolic pressure (DBP) was about 3mmHg less than the mean obtained after vehicle administration. From 5-22 hours, the group mean values for the vehicle group and the antibody G1 group were similar. The same trend was also observed for the other days when a slight decrease in the DBP (in the range of 2.62-3.5 mmHg) occurred in the first interval of the assay, with small changes of similar magnitude occasionally observed at 7-22 hour intervals from day 7 to 10. Similar to the observed DBP, a small drop in heart rate was observed during the first assessment period (1-4 hours) relative to vehicle treatment. During the intermediate evaluation, the difference was undetectable and was again observable during 18-22 hours of the day.

Furthermore, with respect to ECG findings, QTc interval changes at any time point were not statistically significant relative to vehicle treatment. Although statistically significant changes in RR, PR, RS and QT were observed over the 14 day period compared to vehicle, their absolute values were small.

Repeat dose safety study

Repeated dose safety studies included 48 adult, gender-matched (6 per gender per group), cynomolgus monkeys (Charles River Primates) not exposed to antibody G1. Animals received weekly intravenous injections of vehicle or antibody G1 for 14 weeks at doses of 10mg/kg, 100mg/kg, or 300 mg/kg. In each group, two animals of each sex were allowed to recover for an additional 4 months after dosing was complete.

ECG and blood pressure measurements were recorded once during the pre-study period, two measurements were performed after steady state (day 85 before dosing and 4 hours after dosing), and once about 1 week after dosing was complete (day 103 of recovery). Animals were anesthetized with ketamine and ECGs were recorded using eight leads. ECG (including heart rate) measurements were performed using the captured data using the Life Science Suite Ponemah physics Platform software system with DSI using leads I, II, aVF, CG4RL and CV4LL as standards. Heart rate correction for the QT interval (QTc) was calculated using Bazett's formula.

Blood pressure was recorded before the first dose, 12 weeks after administration (13 doses) and approximately 1 week after the end of administration. No significant changes in SBP or DBP were recorded in any of the treatment groups relative to vehicle-treated animals. The group mean heart rates were relatively consistent between the dose group and the time points of the determinations, and no statistical differences were determined. Plasma concentrations of antibody G1 were determined during the first week of dosing and when blood pressure and ECG were assessed, showing that repeated dosing weekly has an accumulating effect.

Furthermore, there was no significant difference in QTc interval between all doses and time points with respect to ECG findings. In addition, no significant or relevant ECG changes were observed for any ECG parameters evaluated during the study.

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

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

Preservation of biological materials

The following materials are deposited at the American Type culture Collection (American Type culture Collection), University Board 10801, Mass., 20110-:

vector peb. cgrp. hkgi is a polynucleotide encoding the light chain variable and light chain kappa constant regions of G1; vector pdb.cgrp.hfcgi is a polynucleotide encoding the heavy chain variable region of G1 and the heavy chain IgG2 constant region comprising the following mutations: A330P331 to S330S331 (amino acid numbering refers to the wild-type IgG2 sequence; see Eur. J. Immunol. (1999)29: 2613-2624).

These deposits were prepared according to the provisions of the Budapest Treaty on the International Recognition of the department of microorganisms for the Purpose of the Patent Procedure and the rules for its implementation (Budapest Treaty) and the Budapest Treaty for the Purpose of the Patent Procedure (Budapest Treaty). This ensures that the deposit remains in viable culture for 30 years from the date of deposit. The ATCC ensures availability of the deposit under the terms of the budapest treaty and subject to the agreement between Rinat Neuroscience corp. and ATCC, which ensures that upon issuance of the relevant U.S. patent or publication of any U.S. or foreign patent application, first arrived at the priority, the public can permanently and indefinitely obtain the progeny of the deposit, and that the progeny can be obtained by personnel designated by the U.S. patent and trademark specialist who claim the deposit under 35USC Section 122 and the specialist rules therefor (including 37CFR Section 1.14, specifically relating to 886 OG 638).

The assignee of the present patent application has agreed that if a culture of the deposited material is to die or be lost or damaged when cultured under appropriate conditions, the material is quickly replaced with the same additional material upon notification. The availability of the deposited material is not to be construed as a license to practice the invention in contravention of the rights granted under the authority of any government agency in accordance with its patent laws.

Antibody sequences

G1 heavy chain variable region amino acid sequence (SEQ ID NO: 1)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQGTLVTVSS

G1 light chain variable region amino acid sequence (SEQ ID NO: 2)

EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLLIYGASNRYLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIK

G1 CDR H1 (extended CDR) (SEQ ID NO: 3)

GFTFSNYWIS

G1 CDR H2 (extended CDR) (SEQ ID NO: 4)

EIRSESDASATHYAEAVKG

G1 CDR H3(SEQ ID NO：5)

YFDYGLAIQNY

G1 CDR L1(SEQ ID NO：6)

KASKRVTTYVS

G1 CDR L2(SEQ ID NO：7)

GASNRYL

G1 CDR L3(SEQ ID NO：8)

SQSYNYPYT

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

GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCTGCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCTCCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTCCGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACCATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTCTGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCC

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

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAA

G1 heavy chain full antibody amino acid sequence (including modified IgG2 as described herein) (SEQ ID NO: 11)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISWVRQAPGKGLEWVAEIRSESDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPSSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

G1 light chain full antibody amino acid sequence (SEQ ID NO: 12)

EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSWYQQKPGQAPRLLIYGASNRYLGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

G1 heavy chain Total antibody nucleotide sequence (including modified IgG2 as described herein) (SEQ ID NO: 13)

GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCTGCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCTCCTGGGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTCCGAATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACCATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCGTGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTCTGGCTATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCCGCCTCCACCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTGACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCATCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAGCAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCACCTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAGCCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGTGGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGAGTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCACCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGGAAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGAAGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTGCCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAGCCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTTCTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACGTGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAGAGCCTGTCCCTGTCTCCAGGAAAGTAA

G1 light chain full antibody nucleotide sequence (SEQ ID NO: 14)

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCGCGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA

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)):

NH₂-ACDTATCVTHRLAGLLSRSGGVVKNNFVPTNVGSKAF-CONH₂(human α -CGRP)

NH₂-ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-CONH₂(human β -CGRP)

NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-CONH₂(rat α -CGRP)

NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF-CONH₂(rat β -CGRP)

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

DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK

Heavy chain variable region HCVR22 amino acid sequence (SEQ ID NO: 59)

QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGDTRYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWGQGTLVTVSS

Light chain variable region LCVR18 amino acid sequence (SEQ ID NO: 60)

DIQMTQSPSSLSASVGDRVTITCRASQDIDNYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK

Heavy chain variable region HCVR23 amino acid sequence(SEQ ID NO：61)

QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFSYWGQGTLVTVSS

Light chain variable region LCVR19 amino acid sequence (SEQ ID NO: 62)

DIQMTQSPSSLSASVGDRVTITCRASKDISKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIK

Heavy chain variable region HCVR24 amino acid sequence (SEQ ID NO: 63)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFADRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS

Light chain variable region LCVR20 amino acid sequence (SEQ ID NO: 64)

DIQMTQSPSSLSASVGDRVTITCRASRPIDKYLNWYQQKPGKAPKLLIYYTSEYHSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGDALPPTFGQGTKLEIK

Heavy chain variable region HCVR25 amino acid sequence (SEQ ID NO: 65)

QVQLVQSGAEVKKPGASVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTLVTVSS

Light chain variable region LCVR21 amino acid sequence (SEQ ID NO: 66)

DIQMTQSPSSLSASVGDRVTITCRASQDIDKYLNWYQQKPGKAPKLLIYYTSGYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGDALPPTFGGGTKVEIK

Heavy chain variable region HCVR26 amino acid sequence (SEQ ID NO: 67)

QVQLVQSGAEVKKPGSSVKVSCKASGYTFGNYWMQWVRQAPGQGLEWMGAIYEGTGKTVYIQKFAGRVTITADKSTSTAYMELSSLRSEDTAVYYCARLSDYVSGFGYWGQGTTVTVSS

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

QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR

Heavy chain variable region HCVR28 amino acid sequence (SEQ ID NO: 69)

EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS

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

QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKR

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

EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS

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

QVLTQSPSSLSASVGDRVTINCQASQSVYDNNYLAWYQQKPGKVPKQLIYSTSTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCSSGDCFVFGGGTKVEIKR

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

EVQLVESGGGLVQPGGSLRLSCAVSGLDLSSYYMQWVRQAPGKGLEWVGVIGINDNTYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS

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

QVLTQTPSPVSAAVGSTVTINCQASQSVYHNTYLAWYQQKPGQPPKQLIYDASTLASGVPSRFSGSGSGTQFTLTISGVQCNDAAAYYCLGSYDCTNGDCFVFGGGTEVVVKR

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

QSLEESGGRLVTPGTPLTLTCSVSGIDLSGYYMNWVRQAPGKGLEWIGVIGINGATYYASWAKGRFTISKTSSTTVDLKMTSLTTEDTATYFCARGDIWGPGTLVTVSS

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

QVLTQSPSSLSASVGDRVTINCQASQSVYHNTYLAWYQQKPGKVPKQLIYDASTLASGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCLGSYDCTNGDCFVFGGGTKVEIKR

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

EVQLVESGGGLVQPGGSLRLSCAVSGIDLSGYYMNWVRQAPGKGLEWVGVIGINGATYYASWAKGRFTISRDNSKTTVYLQMNSLRAEDTAVYFCARGDIWGQGTLVTVSS

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

QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSTTLGITGLQTGDEADYYCGTWDSRLSAVVFGGGTKLTVL

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

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSFGMHWVRQAPGKGLEWVAVISFDGSIKYSVDSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCARDRLNYYDSSGYYHYKYYGMAVWGQGTTVTVSS

Claims (43)

1. A method of treating or reducing the onset of headache pain in a subject comprising administering to said subject an amount of a monoclonal antibody that modulates the CGRP pathway over a plurality of days, wherein the amount administered per day over said plurality of days is between 100-2000 mg.

2. A method of treating or reducing the onset of at least one vasomotor symptom in a subject, comprising administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway over a plurality of days, wherein the amount administered per day over the plurality of days is between 100-2000 mg.

3. The method of claim 1, wherein the headache is migraine.

4. The method of claim 3, wherein the migraine is a chronic migraine.

5. The method of claim 3, wherein the migraine is episodic migraine.

6. The method of claim 1, wherein the onset of headache is reduced by at least seven days after a single administration.

7. The method of claim 1, wherein the number of monthly headache hours experienced by the subject after the administration is reduced by 40 or more hours from the subject's pre-administration level.

8. The method of claim 1, wherein the number of monthly headache days experienced by the subject following the administration is reduced from the pre-administration level of the subject by 3 or more days.

9. The method of claim 1, wherein the number of hours per month headache experienced by the subject following said administration is reduced by 25% or more relative to the subject's pre-administration level.

10. The method of any one of claims 1 or 2, wherein the monoclonal antibody is an anti-CGRP antagonist antibody.

11. The method of any one of claims 1 or 2, wherein the amount of monoclonal antibody is less than 1000 mg.

12. The method of any one of claims 1 or 2, wherein two of the multiple days are separated by more than seven days.

13. The method of any one of claims 1 or 2, wherein the amount of the monoclonal antibody administered on a first day is different from the amount of the monoclonal antibody administered on a second day.

14. The method of claim 13, wherein the amount of the monoclonal antibody administered on the first day is higher than the amount of the monoclonal antibody administered on the second day.

15. The method of any one of claims 1 or 2, wherein less than 3 doses per month are administered to the subject.

16. The method of any one of claims 1 or 2, wherein the administration is subcutaneous or intravenous administration.

17. The method of any one of claims 1 or 2, wherein the administering comprises utilizing a pre-filled syringe comprising the amount of the monoclonal antibody.

18. The method of any one of claims 1 or 2, wherein the monoclonal antibody is formulated at a concentration of at least 150 mg/mL.

19. The method of any one of claims 1 or 2, wherein the monoclonal antibody is administered in a volume of less than 2 mL.

20. The method of any one of claims 1 or 2, comprising administering a second agent to the subject simultaneously or sequentially with the monoclonal antibody.

21. The method of claim 20, wherein the second agent is selected from the group consisting of: 5-HT1 agonists, triptans, ergot alkaloids, and non-steroidal anti-inflammatory drugs.

22. The method of claim 20, wherein the subject's monthly use of the second agent is reduced by at least 15% following administration of the monoclonal antibody.

23. The method of claim 21, wherein the second agent is triptan.

24. The method of any one of claims 1 or 2, wherein the subject is a human.

25. The method of any one of claims 1 or 2, wherein the monoclonal antibody is human or humanized.

26. The method according to any one of claims 1 or 2, wherein the monoclonal antibody comprises (a) an antibody having CDR H1 shown in SEQ ID No.3, CDR H2 shown in SEQ ID No.4, CDR H3 shown in SEQ ID No.5, CDR L1 shown in SEQ ID No.6, CDR L2 shown in SEQ ID No. 7 and CDR L3 shown in SEQ ID No. 8; or (b) variants of the antibodies according to (a) shown in table 6.

27. A method of reducing the number of monthly headache hours experienced by a subject comprising administering to the subject an amount of a monoclonal antibody that modulates a CGRP pathway, wherein the amount of the monoclonal antibody is effective to reduce the number of monthly headache hours by at least 20 hours after a single dose.

28. A method of reducing the number of monthly headache hours experienced by a subject comprising administering to the subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the amount of the monoclonal antibody is effective to reduce the number of monthly headache hours by at least 15% after a single dose.

29. A method of reducing the number of monthly headache days experienced by a subject comprising administering to said subject an amount of a monoclonal antibody that modulates the CGRP pathway, wherein the amount of said monoclonal antibody is effective to reduce the number of monthly headache days by at least 3 days after a single dose.

30. A method of reducing the use of an anti-headache agent in a subject comprising administering to said subject a monoclonal antibody that modulates the CGRP pathway, wherein the amount of said monoclonal antibody is effective to reduce the monthly use of said anti-headache agent by said subject by at least 15%.

31. The method of claim 30, wherein the anti-headache agent is selected from the group consisting of 5-HT1 agonists, triptans, opiates, β -adrenergic antagonists, ergot alkaloids, and non-steroidal anti-inflammatory drugs.

32. The method of claim 31, wherein the anti-headache agent is triptan.

33. The method of any one of claims 27-30, wherein the monoclonal antibody is an anti-CGRP antagonist antibody.

34. The method of any one of claims 27-30, wherein the amount of monoclonal antibody is less than 1000 mg.

35. The method of any one of claims 27-30, wherein the subject is administered less than 3 doses per month.

36. The method of any one of claims 27-30, wherein the administration is subcutaneous or intravenous administration.

37. The method of any one of claims 27-30, wherein the monoclonal antibody is formulated to a concentration of at least 150 mg/mL.

38. The method of any one of claims 27-30, wherein the monoclonal antibody is administered in a volume of less than 2 mL.

39. The method of any one of claims 27-30, wherein the subject is a human.

40. The method of any one of claims 27-30, wherein the monoclonal antibody is human or humanized.

41. The method of any one of claims 27-30, wherein the monoclonal antibody comprises (a) an antibody having CDR H1 shown in SEQ ID No.3, CDR H2 shown in SEQ ID No.4, CDR H3 shown in SEQ ID No.5, CDR L1 shown in SEQ ID No.6, CDR L2 shown in SEQ ID No. 7, and CDR L3 shown in SEQ ID No. 8; or (b) variants of the antibodies according to (a) shown in table 6.

42. A method of treating or reducing the onset of headache pain in a subject comprising administering a single dose of a monoclonal antibody to the subject in an amount that modulates the CGRP pathway, wherein the amount of the monoclonal antibody is between 100-2000 mg.

43. A composition for use according to any one of the preceding claims.