HK1190411A - Antagonist antibodies directed against calcitonin gene-related peptide and methods using same - Google Patents

Abstract

The application relates to antagonist antibodies directed against calcitonin gene-related peptide and methods using same. The invention features methods for preventing or treating CGRP associated disorders such as vasomotor symptoms, including headaches (e.g., migraine, cluster headache, and tension headache) and hot flushes, by administering an anti-CGRP antagonist antibody. Antagonist antibody G1 and antibodies derived from G1 directed to CGRP are also described.

Description

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

The application is a divisional application of Chinese invention patent application 200680042443.0(PCT/IB2006/003181), with the application date of the parent application being 2006, 11, and 2, and is entitled "antagonist antibody against calcitonin gene-related peptide and method of use thereof". This application claims priority from U.S. patent application No.60/736, 623, filed on 14/11/2005, which is incorporated herein by reference.

Technical Field

The present invention relates to the use of anti-CGRP antagonist antibodies for preventing, ameliorating or treating vasomotor symptoms such as CGRP related headaches (e.g., migraine) and hot flashes.

Background

CGRP (calcitonin gene related peptide) is a 37 amino acid neuropeptide belonging to a family of peptides including calcitonin, adrenomedullin and amylin. In humans, there are two forms of CGRP (. alpha. -CGRP and. beta. -CGRP) which have similar activities. They differ in three amino acids and show different distributions. At least two CGRP receptor subtypes may also account for 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 as part of the cascade of events leading to protoplasmic extravasation and microvascular vasodilation, which is present in migraine.

CGRP has been noted 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 undergo natural or surgically-induced menopause. Hot flashes are likely an adaptive response of the Central Nervous System (CNS) to declining sex steroids (Freedman am. J. Humanbiol. 13: 453-464 (2001)). To date, the most effective therapy for flushing is hormone-based treatment, including estrogens and/or some progestins. Hormone therapy is effective in alleviating flushing, but is not suitable for all women. The psychological and emotional symptoms observed (e.g., nervousness, fatigue, irritability, insomnia, depression, memory loss, headache, anxiety, nervousness, or inability to concentrate) are believed to be caused by hot flashes and sleep deficiencies after sweating (Kramer et al, In: Murphy et al, 3. rd Int' l Symposium on Recent Advances In Urological Cancer diagnosis Treatment-Proceedings, Paris, France: SCI: 3-7 (1992)).

Male steroid hormones (androgens) also experience hot flashes after reduction. This is 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 hormone loss associated with prostate cancer treatment (Berendsen, et al., European Journal of Pharmacology, 2001, 419 (1): 47-54). One third of these patients experience long-term and frequent symptoms that are severe enough to cause significant discomfort and inconvenience.

CGRP is a potent vasodilator associated with the pathology of other vasomotor symptoms, such as all forms of vascular headache, including migraine (with or without aura) and cluster headache. Durham, n.engl.j.med.350: 1073-1075, 2004. CGRP levels in the external jugular vein increased during migraine headache in the patient. Goadsby et al, Ann. neuron.28: 183-7, 1990. Intravenous administration of human α -CGRP induced headaches and migraines in patients with migraine without aura, suggesting that CGRP has a causal role in migraine. Lassen et al, Cephalalgia 22: 54-61, 2002.

The possible association of CGRP with migraine is the basis for the development and testing of a large number of compounds that inhibit the release of CGRP (e.g., termatatriptan), antagonize CGRP receptors (e.g., the 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 module protein (RCP), all of which affect the binding of CGRP to their receptors. Brain, s.et al, Trends in pharmaceutical Sciences 23: 51-53, 2002. The A-2 adrenoreceptor subtype and adenosine A1 receptor also regulate (inhibit) CGRP release and trigeminal activation (Goadsbyet al, Brain 125: 1392-. Receptors directed to GR79236 (metafacial) adenosine A1, which have 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).

What confounds this theory is the following observation: by inhibiting neurogenic inflammation only (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, raising 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 neurogenic condition characterized by severe, episodic headaches and associated features that can include nausea, vomiting, sensitivity to light, sound or motion. 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). In addition, the median frequency of episodes in an individual is 1.5 times per month. Although a large number of treatments are available to alleviate or reduce symptoms, prophylactic treatment is recommended for patients with more than 3-4 migraine attacks per month. Goadsby et al.New Engl.J.Med.346 (4): 257-275, 2002.

Variations in pharmacological intervention and the diversity of responses among patients for the treatment of migraine are evidence of the different characteristics of the condition. Thus, it was shown that 5-hydroxytryptamine can be obtainedAnd adrenergic, noradrenergic, and dopaminergic activities, such as ergot alkaloids (e.g., ergotamine, dihydroergotamine, mexican), have been used for over eighty years for the treatment of migraine. Other treatments include opiates (e.g. oxycodone) and beta-adrenergic antagonists (e.g. propranolol). Some patients (usually those with milder symptoms) are able to control their symptoms with over-the-counter medications, such as one or more non-steroidal anti-inflammatory agents (NSAIDs), such as aspirin, paracetamol, and caffeine (e.g., such asMigraine).

More recently, some migraine sufferers have been treated with topiramate, an anticonvulsant that blocks voltage-dependent sodium channels and certain glutamate receptors (AMPA kainate), 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 (e.g., termatatriptan) in some patients has led researchers to suggest a 5-hydroxytryptamine-competent etiology for this disorder. Unfortunately, while some patients respond well to this treatment, others are relatively resistant to its effects.

The disease is explained by the postulated malfunction of ion channels in the brainstem cell nucleus by amino acids, 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 other ion channel mutations are likely to be found in other patient populations as well. Although vasodilation is associated with migraine and exacerbates the 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): 257-275, 2002.

Various publications (including patents and patent applications) are cited in this application. The disclosures of these publications in their entireties are hereby incorporated by reference.

Disclosure of Invention

The invention disclosed herein relates to anti-CGRP antagonist antibodies and methods of administering anti-CGRP antagonist antibodies for treating or preventing vasomotor symptoms, such as headaches, e.g., migraine with or without aura, hemiplegic migraine, cluster headache, migraine neuralgia, prolonged headache, tension headache, and headache caused by other medical conditions, such as infections caused by tumors or increased intracranial pressure. Other vasomotor symptoms include hot flashes.

In one aspect, the invention provides a method for treating or preventing at least one vasomotor symptom in an individual, comprising administering to the individual 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 onset 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 another embodiment, the invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the onset 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 useful in the treatment of headache. Such other agents include 5-HT 1-like agonists (agonists acting at other 5-HT1 sites) and non-steroidal anti-inflammatory drugs (NSAIDs).

Examples of 5-HT1 agonists that may be used in combination with anti-CGRP antibodies include a class of compounds known as triptans (triptans), such as tertramadol, zolmitriptan, naratriptan, rizatriptan, iritriptan, almotriptan and frovatriptan. Ergot alkaloids and related compounds have also been shown to have 5-HT agonist activity and are used to treat headaches such as migraine. These compounds include ergotamine, ergotamine maleate and mesylline (e.g., dihydroergocornine, dihydroergocristine, dihydroergocryptine and dihydroergotamine mesylate (DHE 45)).

Examples of NSAIDs that may be used in combination with the anti-CGRP antibody include naproxen, flurbiprofen, ketoprofen, oxaprozin, etodolac, indomethacin, ketorolac, nabumetone, mefanamic acid (mefanamic acid), and piroxicam. Other NSAIDs include cyclooxygenase 2(COX-2) inhibitors. The group members include: celecoxib; rofecoxib; meloxicam; JTE-522; l-745, 337; NS398 and pharmaceutically acceptable salts thereof.

In another aspect, the invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the onset or development of hot flashes in an individual comprising administering to the individual 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 onset or development 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 agent suitable for treating hot flashes. Such other agents include, but are not limited to, hormone-based therapies including estrogens and/or progestins.

In one embodiment, the anti-CGRP antagonist antibody used 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 both 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, an anti-CGRP antagonist antibody comprises one or more CDRs (e.g., one, two, three, four, five, or in some embodiments all six CDRs) of an antibody G1 or G1 variant shown in table 6. In still 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 that is, for example, immunologically inert (including being partially immunologically inert, e.g., does not elicit complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), activates microglia), or has reduced one or more of these activities. In some embodiments, the constant region is modified, as described in eur.j.immunol. (1999) 29: 2613-2624; PCT application No. PCT/GB 99/01441; and/or 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 (with reference to the amino acid sequence of 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 to P233V234a235, G236 is deleted; 3) E233L234L235 to P233V234a 235; 4) E233L234L235G236a327a330P331 to P233V234a235G327S330S331, G236 are deleted; 5) E233L234L235a327a330P331 to P233V234a235G327S330S331 and 6) N297 to a297 or any other amino acid 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 to P233V234a235, G236 is deleted; E233F234L235 to P233V234a 235; and S228L235 to P228E 235.

In still other embodiments, the N-linked glycosylation of the constant region is not glycosylated (aglycosylated). In some embodiments, the constant region is not glycosylated for N-linked glycosylation by mutating the oligosaccharide attachment residue (e.g., Asn297) and/or the flanking residues that are part of the N-glycosylation recognition sequence in the constant region. In some embodiments, the constant region is not glycosylated for N-linked glycosylation. The constant region may be left unglycosylated for N-linked glycosylation either enzymatically or by expression in a glycosylation deficient host cell.

Affinity (K) of anti-CGRP antagonist antibodies to CGRP (e.g., human α -CGRP measured by surface plasmon resonance at a suitable temperature such as 25 or 37 ℃)_D) And may be about 0.02 to about 200 nM. In some embodiments, the 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 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.

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 anti-CGRP antagonist antibodies can be by any means known in the art: oral, intravenous, subcutaneous, intraarterial, intramuscular, cardial, intravertebral, intrathoracic, intraperitoneal, intraventricular, sublingual, transdermal and/or via inhalation. Administration can be systemic (e.g., intravenous) or local.

In some embodiments, the anti-CGRP antagonist antibody may be administered in combination with another agent (e.g., 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 use in any of the methods described herein (e.g., for treating or preventing headache).

In another aspect, the invention provides a pharmaceutical composition for preventing or treating headache, such as 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 use in any of the methods described herein. In some embodiments, the kit comprises a container, a composition comprising an anti-CGRP antagonist antibody described herein (in combination with a pharmaceutically acceptable vehicle), and instructions for using the composition in any of the methods described herein.

The invention also provides anti-CGRP antagonist antibodies and polypeptides from antibody G1 shown in table 6 or variants thereof. Thus, in one aspect, the invention is antibody G1 (interchangeably referred to as "G1") produced by the expression vectors of ATCC accession nos. PTA-6866 and PTA-6867. For example, in one embodiment is an antibody comprising a heavy chain produced by the expression vector with ATCC accession No. pta-6867. In yet another embodiment is an antibody comprising a light chain, said antibody produced by the expression vector with 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 fig. 5. It should be understood that: reference to any local or global region of G1 includes the sequences produced by the expression vectors with ATCC accession nos. PTA-6866 and PTA-6867 and/or the sequences shown in fig. 5. The invention also provides G1 antibody variants having the amino acid sequence shown in table 6.

In one aspect, the invention comprises V_HAntibodies to the domains V_HAnd SEQ ID NO: 1 at least 85%, at least 86%, at least 87%, at least 88%, at least 89% >, and,At least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

In another aspect, the invention comprises V_LThe antibody of (1), the V_LAnd SEQ ID NO: 2 are at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical.

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

In another aspect, the invention provides a polypeptide (which may or may not be an antibody) comprising the amino acid sequence of SEQ ID NO: v disclosed in 5_HCDR3, or a variant of SEQ ID NO: 5 by 1, 2, 3,4 or 5 amino acid substitutions. In particular embodiments, such amino acid substitutions are conservative amino acid substitutions.

In another aspect, the invention provides a polypeptide (which may or may not be an antibody) comprising the amino acid sequence of SEQ ID NO: v disclosed in 8_LCDR3, or a variant of SEQ ID NO: 8 by 1, 2, 3,4 or 5 amino acid substitutions. In particular embodiments, such amino acid substitutions are conservative amino acid 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 (one, two, three, four, five or six) CDRs from antibody G1 or a variant thereof shown in table 6; b) the CDRs from CDR H3 of the heavy chain of antibody G1 shown in table 6; and/or c) the CDRs 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, one or more CDRs from antibody G1 or a variant 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 a variant thereof.

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

In some embodiments, the polypeptide (e.g., antibody) comprises the amino acid sequence kaskxaaxaatyvs, 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 kaskxaavxaaatyvs is the CDR 1of the antibody light chain.

In some embodiments, the polypeptide (e.g., antibody) comprises the amino acid sequence XaaXaaSNRYXaa, 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 is the CDR2 of an antibody light chain.

In some embodiments, a polypeptide (e.g., an antibody) comprises the amino acid sequence eirsxasdxaaxaaatxaayxaaxaaavkg, 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 eirsxaassdxaaaatxaayxaaaavkg is the CDR2 of an antibody heavy chain.

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

In some embodiments, the antibodies of the invention are human antibodies. In other embodiments, the antibodies of the invention are humanized antibodies. 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 an antibody can be from any type of constant region (e.g., IgG, IgM, IgD, IgA, and IgE) and any isotype (e.g., 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) comprising a polynucleotide encoding a fragment or region of antibody G1, or a variant thereof, as set forth in table 6. In one embodiment, the fragment is the light chain of antibody G1. In other embodiments, the fragment is the heavy chain of antibody G1. In yet another embodiment, the fragment contains one or more variable regions from the light and/or heavy chain of antibody G1. In yet another embodiment, the fragment contains one or more (i.e., one, two, three, four, five, or six) Complementarity Determining Regions (CDRs) from the light chain and/or heavy chain of antibody G1.

In another aspect, the invention is a polynucleotide (which may be isolated) comprising a polynucleotide encoding antibody G1 or a variant thereof as set forth in table 6. In some embodiments, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10 or both of the polynucleotides set forth in figure 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 vectors and cloning vectors) and host cells comprising any of the polynucleotides disclosed herein. In some embodiments, the vector is pdb.cgrp.hfcgi of ATCC No. pta-6867. In other embodiments, the vector is peb.cgrp.hkgi of ATCC No. pta-6866.

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

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

In another aspect, the invention is 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 is 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 antibody G1. In some embodiments, the expression vector comprises SEQ ID NO: 9 and SEQ ID NO: 10 or both.

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

anti-CGRP antagonist antibodies and polypeptides and polynucleotides encoding the antibodies and polypeptides of the invention may be used to treat, prevent, ameliorate, control or reduce the onset of diseases associated with abnormal function of CGRP, such as headache (e.g., migraine, cluster headache, chronic headache and tension headache) and other conditions which 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, which are typically suitably packaged and provided with appropriate instructions, are suitable for use in any of the methods described herein.

Brief description of the drawings

FIG. 1 is a table showing the affinity of 12 murine antibodies for different alanine substituted human α -CGRP fragments. Avidity was measured by flowing Fab over CGRP on a chip using Biacore at 25 ℃. In-frame values represent loss of affinity of the alanine mutant relative to the parent fragment 25-37 (italics), with the exception of K35A from the 19-37 parent.^″a″The affinities for the 19-37 and 25-37 fragments are shown as the mean. + -. standard deviation of two independent measurements on different sensor chips.^″b″Indicating that these interactions deviate from the single bimolecular interaction model due to biphasic off-rates (offrate), and thus their affinities were determined using the conformational change model. Grey scale key: white (1.0) indicates parental affinity; light grey (less than 0.5) indicates higher affinity than the parent; dark grey (more than 2) indicates lower affinity than the parent; black indicates no binding detected.

FIGS. 2A and 2B show the effect of administration of CGRP 8-37(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. (iii) intravenous (iv) administration of CGRP 8-37 3-5 minutes prior to electrical pulse stimulation. Antibodies were administered Intraperitoneally (IP) 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 x Δ time. "delta flux" refers to the change in flux units following electrical pulse stimulation; "delta time" refers to the time required for the blood cell flux level to return to the level prior to stimulation by the electrical pulse.

FIG. 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 the blood cell flux 30 seconds after electrical pulse stimulation. Antibodies were administered Intravenously (IV) 24 hours prior to electrical pulse stimulation. Each dot 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 indicated conditions.

Fig. 4A and 4B show the effect of administration of antibody 4901(1mg/kg or 10mg/kg, Lv.), antibody 7E9(10mg/kg, i.v.), and antibody 8B6(10mg/kg, i.v.) 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 minutes, 60 minutes, 90 minutes, and 120 minutes 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, and" "+" indicates P < 0.01. Data were analyzed using a one-way ANOVA with 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. Kabat CDR is bold, Chothia CDR underlined. The amino acid residues of the heavy and light chain variable regions are numbered consecutively.

Figure 6 shows epitope mapping of antibody G1 by peptide competition using Biacore. N biotinylated human α -CGRP was captured on an SA sensing chip. Without competitor peptide or after 1 hour incubation with 10 μ M competitor peptide, G1Fab (50nM) was poured onto the chip. The binding of G1Fab to human α -CGRP on the chip was measured. The Y-axis represents the percent binding blocked by the presence of competitor peptide compared to the absence of competitor peptide.

FIG. 7 shows the effect on skin blood flow using antibody G1(1mg/kg or 10mg/kg, intravenous) or vehicle (PBS, 0.01% Tween 20), measured as the blood cell flux 30 seconds after electrical pulse stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) followed by neuro-electrical pulse stimulation at 30, 60, 90 and 120 minutes after antibody administration. The Y-axis represents the AUC percentage compared to the AUC level (defined as 100%) when no antibody or vehicle was administered (time 0). The X-axis represents the time (minutes) between antibody administration and electrical pulse stimulation. Compared to the vehicle, "+" indicates P < 0.05, and "+" indicates P < 0.01. Data were analyzed by two-way ANOVA and Bonferroni post-test.

FIG. 8A shows the effect of administration of antibody G1(1mg/kg, 3mg/kg or 10mg/kg, intravenously) or vehicle (PBS, 0.01% Tween 20) on skin blood flow, measured as blood cell flux 30 seconds after 24 hours post-dose electrical pulse stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) 24 hours prior to neuro-electrical pulse stimulation. The Y-axis represents the total area under the curve (change in blood cell flux multiplied by the time difference from stimulation to baseline for flux recovery, AUC). The X-axis represents different doses of antibody G1. Compared to the vehicle, "+" indicates P < 0.05, and "+" indicates P < 0.01. Data were analyzed using one-way ANOVA and Dunn's multiple comparative tests.

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% Tween 20) 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 neuro-electrical pulse stimulation. The Y-axis represents the total AUC. The X-axis represents different doses of antibody G1. Compared to the vehicle, "+" indicates P < 0.01, and "+" indicates P < 0.001. Data were analyzed using one-way ANOVA and Dunn's multiple comparative tests.

Fig. 8C is a curve fit analysis of the data from fig. 8A and 8B. Antibody G1 or vehicle 24 hours or 7 days prior to neuroelectric pulse stimulationIntravenous (i.v.) administration. The Y-axis represents the total AUC. The X-axis represents the different G1 doses in "mg/kg" using a logarithmic scale to determine EC_5O。

FIG. 9 shows the effect of antibody mu7E9(10mg/kg), BIBN4096BS or vehicle (PBS, 0.01% Tween 20) on arterial diameter changes in meninges after electric field stimulation. Antibody mu7E9, BIBN4096BS or vehicle was administered intravenously (i.v.) at a time point of 0 minutes after establishment of a baseline response to electrical stimulation. The Y-axis represents the change in diameter of the artery in the meninges after electric field stimulation. The resting diameter corresponds to 0%. The X-axis represents the time (minutes) of electrical pulse stimulation. Compared to the vehicle, "+" indicates P < 0.05 and "+" indicates P < 0.01. Data were analyzed using one-way ANOVA and Dunett's multiple comparative tests.

FIG. 10 shows the effect of different doses of antibody G1(1mg/kg, 3mg/kg or 10mg/kg, venous) or vehicle (PBS, 0.01% Tween 20) on the change in diameter of arteries in meninges after electric field stimulation. Antibody G1 or vehicle was administered intravenously (i.v.) 7 days prior to electric field stimulation. The Y-axis represents the change in diameter of the artery in the meninges. The resting diameter corresponds to 0%. The X-axis represents the stimulation voltage. Compared to the vehicle, "+" indicates P < 0.05, "+" indicates P < 0.01, and "+" indicates P < 0.001. Data were analyzed using two-way ANOVA and Bonferroni posttests.

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

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

Detailed Description

The invention disclosed herein provides methods for treating and/or preventing vasomotor symptoms such as headache (e.g., migraine, cluster headache, chronic headache, and tension headache) or hot flashes in an individual by administering to the individual a therapeutically effective amount of an anti-CGRP antagonist antibody.

The invention disclosed herein also provides anti-CGRP antagonist antibodies and polypeptides from G1 shown in table 6 or variants thereof. The invention also provides methods of making and using these antibodies and polypeptides.

General techniques

The practice 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, for example, molecular cloning: a Laboratory Manual, second edition (Sambrook et a1., 1989) CoIdspring Harbor Press; oligonucleotide Synthesis (mj. gate, ed., 1984); method in Molecular Biology, Humana Press; cell Biology: a Laboratory Notebook (J.E. Cellis, ed., 1998) Academic Press; animal Cell Culture (r.i. freshney, ed., 1987); introduction to Cell and Tissue Culture (J.P.Mather 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; methodsin enzymology (Academic Press, Inc.); handbook of Experimental Immunology (d.m.well and cc.blackwell, eds.); gene Transfer Vectors for Mammaliancells (J.M.Miller and M.P.Calos, eds., 1987); current Protocols in molecular biology (f.m. ausubel et al, eds., 1987); and (3) PCR: the Polymerase Chain Reaction, (Mullis et al, eds., 1994); current Protocols in Immunology (j.e. coligan et al, eds., 1991); short Protocols in Molecular Biology (Wiley and Sons, 1999); lmmicrobiology (ca. janeway and p. travers, 1997); antibodies (p.finch, 1997); antibodies: a practical proproach (D.Catty., ed., IRL Press, 1988-; monoclone antigens: a practical approach (P.shepherd and C Dean, eds., Oxford University Press, 2000); using antibodies: a Laboratory manual (E.Harlow and D.Lane (Cold Spring Harbor Laboratory Press, 1999); the reagents (M.Zantetti and J.D.Capra, eds., Harwood Academic Publishers, 1995).

Definition of

An "antibody" is an immunoglobulin molecule that is capable of specifically binding a target (e.g., a carbohydrate, polynucleotide, lipid, polypeptide, etc.) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. The term as used herein includes not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab ', F (ab')₂Fv), single chain (ScFv), mutants, fusion proteins comprising an antibody portion (e.g., domain antibodies), and any other modified configuration of an immunoglobulin molecule that comprises an antigen recognition site. Antibodies include any class of antibody, such as IgG, IgA, or IgM (or subfamilies thereof), and antibodies need not be of any particular class. Immunoglobulins can be assigned to different species depending on the amino acid sequence of the constant domain of the antibody heavy chain. There are five major immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different immunoglobulin families are referred to as α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different families of immunoglobulins are well known.

As used herein, "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous 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. In addition, 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 obtained by substantially homogeneous population of the antibody, and is not to be construed as requiring production of the antibody by any particular method. For example, the composition can be prepared by first providing a solution prepared by Kohler and Milstein, 1975, Nature, 256: 495 or the monoclonal antibody to be used according to the invention can be made by recombinant DNA methods as described in u.s.pat. No.4,816,567. Monoclonal antibodies can also be isolated from phage libraries using, for example, McCafferty et al, 1990, Nature, 348: 552, 554.

"humanized" antibodies, as used herein, refers to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or fragments thereof containing minimal sequence from non-human immunoglobulins (e.g., Fv, Fab, Fab ', F (ab')₂Or other antigen binding subsequences of antibodies). For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which the Complementarity Determining Regions (CDRs) from the recipient are replaced with residues from a CDR from a non-human species (donor antibody), such as mouse, rat or rabbit, having the desired specificity, affinity and biological activity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are replaced with corresponding non-human residues. In addition, humanized antibodies may comprise residues that are not present in the recipient antibody nor in the introduced CDRs or framework regions, but 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 most suitably also comprises at least a portion of an immunoglobulin constant region or domain (Fc), typically of a human immunoglobulinA constant region or domain. 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 are altered from the original antibody, also referred to as one or more CDRs "from" one or more original antibody CDRs.

As used herein, "human antibody" refers to 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 produced using a variety of techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses human antibodies (Vaughan et al, 1996, Nature Biotechnology, 14: 309-. 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 approach is described in U.S. patent nos.5,545,807; 5,545,806; 5,569,825; 5,625,126, respectively; 5,633,425 and 5,661, 016. Alternatively, human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies to the target antigen (such B lymphocytes can be recovered from an individual or can be immunized in vitro). See, e.g., Cole et al, Monoclonal Antibodies and cancer therapy, Alan R.Liss, p.77 (1985); boemer et al, 1991, j.immunol, 147 (1): 86-95 and U.S. patent No.5,750,373.

The terms "calcitonin gene-related peptide" and "CGRP" as used herein refer to any form of calcitonin gene-related peptide and variants thereof that maintain at least a portion of the activity of CGRP. For example, the CGRP may be an α -CGRP or a β -CGRP. CGRP as used herein includes the native sequence CGRP of all mammalian species, such as human, canine, feline, equine, and bovine.

As used herein, an "anti-CGRP antagonist antibody" (used interchangeably with "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 include antibodies that are capable of (including significantly) blocking, antagonizing, inhibiting, or reducing CGRP biological activity, including downstream pathways of CGRP signaling such as receptor binding and/or stimulation of CGRP cellular responses. For the purposes of the present invention, it is to be expressly understood that: the term "anti-CGRP antagonist antibody" includes all previously defined terms, topics and functional states and characteristics whereby CGRP itself, CGRP bioactivity (including but not limited to its ability to mediate any aspect of headache) or the consequences of bioactivity are substantially nullified, reduced or neutralized to any meaningful degree. In some embodiments, the anti-CGRP antagonist antibody binds to CGRP and prevents CGRP from binding to 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.

The terms "G1" and "antibody G1" are used interchangeably herein and refer to the antibodies produced by the expression vectors with accession numbers ATCC PTA-6867 and ATCC PTA-6866. FIG. 5 shows the amino acid sequences of the heavy and light chain variable regions. The CDR portions (including Chothia and Kabat CDRs) of antibody G1 are depicted in fig. 5. The polynucleotides encoding the heavy and light chain variable regions are shown in SEQ ID NOs: 9 and SEQ ID NO: 10 in (b). Characterization of G1 is 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 multimer may be linear or branched, and may contain modified amino acids, which may be blocked by non-amino acids. The term also includes amino acid polymers that have been modified either naturally or by interference; for example disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other processing or modification, such as conjugation to a tag component. Also included in the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. It is understood that because the polypeptides of the invention are antibody-based, the polypeptides can exist as single chains or conjugated chains.

"Polynucleotide" or "nucleic acid" as used interchangeably herein refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and their analogs. If there is a modification to the nucleotide structure, the modification can be given before or after assembly of the multimer. The sequence of nucleotides may be blocked by non-nucleotide components. The polynucleotide may be further modified after multimerization, for example by conjugation to a tag 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 modification with uncharged linkages (e.g., methyl phosphates, phosphotriesters, phosphoamides, carbamates, etc.) and with charged linkages (phosphorothioates, phosphorodithioates, etc.), modifications containing pendant moieties such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, ply-L-lysine, etc.), modifications with intercalators (e.g., acridine, psoralen, etc.), modifications containing chelators (e.g., metals, radioactive metals, boron, metal oxides, etc.), modifications containing alkylylators, modifications with modified linkages (e.g., alpha anomeric nucleic acids), and unmodified forms of the polynucleotide. In addition, any hydroxyl groups typically present in sugars may be replaced by phosphate groups, protected by standard protecting groups, or activated to make additional bonds to additional nucleotides, or may be conjugated to a solid support. The 5 'and 3' terminal OH groups may be phosphorylated or partially substituted with amines or organic capping groups of 1 to 20 carbon atoms. OthersHydroxyl groups can also be evolved as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, D-anomeric sugars, epimeric sugars such as arabinose, xylose, or lyxose, pyranose, furanose, sedoheptulose, acyclic analogs, and non-basic nucleoside analogs such as methylribonucleosides. One or more phosphodiester linkages may be replaced with alternative linking groups. Such alternative linking groups include, but are not limited to, those in which the phosphate is replaced with P (O) S ("thioate"), P (S) S ("dithioate"), (O) NR₂("amidate"), P (O) R, P (O) OR', CO OR CH₂An embodiment of ("" formaltai ""), wherein each R or R' is independently H or substituted or unsubstituted with an alkyl (1-20C) group optionally containing an (-O-) linkage, an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group, or an araldyl group. It is not necessary that all linkages in a polynucleotide be identical. The foregoing 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 each consist of four Framework Regions (FR) connected by three Complementarity Determining Regions (CDRs), also referred to as hypervariable regions. The CDRs in each chain are held together tightly by the FRs and, together with the CDRs from the other chain, contribute to the formation of the antigen binding site of the antibody. There are at least two techniques for determining CDRs: (1) a pathway based on sequence variability across species (Kabat et al. sequence of Proteins of Immunological Interest, (5th ed., 1991, National Institutes of health, Bethesda MD)); and (2) a pathway 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 pathway or a combination of both pathways.

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.

Epitopes that "preferentially bind" or "specifically bind" (used interchangeably herein) to an antibody or polypeptide are terms well known in the art, as are methods for determining such specific or preferential binding. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or binds with a particular cell or substance more frequently, more rapidly, for a longer duration, and/or with greater affinity than it does with other cells or substances. An antibody "specifically binds" or "preferentially binds" to a target if it binds to the target with greater affinity, avidity, more readily, and/or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to CGRP is an antibody that binds to this epitope with greater affinity, avidity, more readily, and/or with greater duration than it binds 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 to a first target may specifically or preferentially bind to a second target, or may not specifically or preferentially bind. Likewise, "specific binding" or "preferential binding" does not necessarily require (although it may include) specific binding. Typically, but not necessarily, reference to binding indicates 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 individual cells or cell cultures that may be or have been the recipient of a vector for incorporation into a polynucleotide insert. Host cells include progeny of a single host cell, and because of natural, accidental, or deliberate mutation, the progeny are not necessarily identical to the original parent cell (either morphologically or over genomic DNA complement). 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 Fc region boundaries of immunoglobulin heavy chains may vary, the human IgG heavy chain Fc region is generally defined as the stretch from the amino acid residue at position Cys226 or Pro230 to its carboxy terminus. The ordering of residues in the Fc region is the EU index ordering as in Kabat. Kabat et al, Sequences of Proteins of National Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, Md., 1991. The Fc region of an immunoglobulin typically contains two constant domains, CH2 and CH 3.

"Fc receptor" and "FcR" are used herein to describe receptors that bind the Fc region of an antibody. A preferred FcR is a native sequence human FcR. In addition, a preferred FcR is one which 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 indirect 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 regions. Ravech and Kinet, 1991, ann.rev.immunol., 9: 457-92; capel et al, 1994, immunoassays, 4: 25-34; and de Haas et al, 1995, j.lab.clin.med., 126: FcRs are reviewed in 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) complexed with an alloantigen. To evaluate complement activation, for example, Gazzano-Santoro et al, j.immunol.methods, 202: 163 (1996).

A "functional Fc region" possesses 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; downregulation of cell surface receptors (e.g., B cell receptors; BCR), and the like. Such effector functions typically require the Fc region to be combined with a binding domain (e.g., an antibody variable domain) and can be evaluated using a variety of assays known in the art for assessing effector functions 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 maintains 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 as compared to the Fc region of the parent polypeptide, e.g., from about one to about ten amino acid substitutions, 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. The variant Fc region herein preferably has at least about 80% sequence identity with the native sequence Fc region and/or with the Fc region of the parent polypeptide, most preferably at least about 90% sequence identity therewith, more preferably at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity therewith.

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, followed by causing lysis of the target cell. ADCC activity of a molecule of interest can be assessed using in vitro ADCC assays, such as the assays described in U.S. patent No.5,500,362 or 5,821,337. Suitable effector cells for these experiments include Peripheral Blood Mononuclear Cells (PBMC) and NK cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example, as described in methods such as Clynes et al, 1998, pnas (usa), 95: 652-656.

As used herein, "treatment" is a means 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 include 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, hyperdacryosis, redness of the eyes, nasal leakage or nasal congestion, and a flushing face.

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

"ameliorating" headache or one or more symptoms of headache means a reduction or improvement in one or more symptoms of headache compared to not administering the 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 of headache or the frequency of episodes in an individual is reduced by at least any of about 10%, 20%, 30%, 40%, 50%, 60% or 70% as compared to pre-treatment levels.

As used herein, "delaying the onset of" headache refers to delaying, impeding, slowing, delaying, stabilizing, and/or delaying the progression of the disease. The delay can be of varying lengths of time depending on the disease history and/or the individual being treated. As will be appreciated by those skilled in the art, a sufficient or significant delay may effectively include prophylaxis, as the individual will not develop a headache (e.g., migraine). A method of "delaying" the onset of symptoms is a method that reduces the likelihood of onset of symptoms and/or reduces the extent of symptoms in a given time frame when compared to the absence of the method. Such comparisons are typically based on clinical studies using a statistically significant number of subjects.

The "onset" or "progression" of headache is meant to indicate the initial manifestation and/or subsequent progression of the condition. The occurrence of headache is detectable and evaluated using standard clinical techniques well known in the art. However, occurrence also indicates undetectable progression. For the purposes of the present invention, biological processes indicative of symptoms occur or develop. "onset" includes onset, recurrence and onset. As used herein, "onset" or "onset" of headache includes initial onset and/or recurrence.

An "effective dose" or "effective amount" of a drug, compound or pharmaceutical composition as used herein is an amount sufficient to produce a beneficial or desired result. For prophylactic use, beneficial or desired results include, for example, 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, complications thereof, and intermediate pathological phenotypes present at the time of disease onset. For therapeutic use, beneficial or desired results include results such as a reduction in pain intensity, duration, or frequency of headache episodes and a reduction in one or more (biochemical, histological, and/or behavioral) symptoms caused by headache (including its complications and intermediate pathological phenotypes present at the time of disease onset), an improvement in the quality of life of patients with the disease, a reduction in the dosage of other medications required to treat the disease, an enhancement in the effect of another medication, and/or a delay in the development of the disease in patients. An effective dose may be administered in one or more administrations. For the purposes of the present invention, 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 context, an effective dose of a drug, compound or pharmaceutical composition may be achieved in combination or non-combination with other drugs, compounds or pharmaceutical compositions. Thus, an "effective dose" may be considered in the context of administering one or more therapeutic agents if the desired result is achieved or attained; a single dose, when combined with one or more other agents, is considered to be provided in an effective amount if the desired result is achieved or attained.

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

As used herein, "vector" means a construct capable of delivering and preferably expressing one or more genes or sequences of interest into 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 bound to 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" refers to a nucleic acid sequence that directs the transcription of a nucleic acid. The expression control sequence may be a promoter (e.g., 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, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to maintain 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 (e.g., 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, 18th edition, A.Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of pharmacy 20th Ed. Mack Publishing, 2000).

The term "k" is used herein_on"is intended to mean the binding rate constant of an antibody to an antigen.

The term "k" is used herein_off"is intended to mean the rate constant at which an antibody dissociates from an antibody/antigen complex.

The term "K" is used herein_D"is intended to mean the equilibrium dissociation constant of an antibody-antigen interaction.

The term "vasomotor symptoms" as used herein is intended to mean conditions associated with vasodilation, including, but not limited to, headaches (e.g., migraine, … … others), hot flashes, cold flashes (or hot flashes), insomnia, sleep disturbances, mood disorders, irritability, profuse perspiration, night sweats, diurnal perspiration, fatigue, and the like, particularly as a result of thermoregulatory dysfunction.

The terms "flushing", "hot flashes", and "transient hot flashes" are used herein as art-recognized terms, and refer to occasional disorders of body temperature, typically consisting of sudden skin flashes, usually accompanied by perspiration of the subject.

A. Method for preventing or treating vasomotor symptoms

In one aspect, the invention provides methods for treating or preventing at least one vasomotor symptom, such as headache (e.g., migraine) or hot flashes, in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody or a polypeptide derived therefrom.

In another aspect, the invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the onset or development of at least one vasomotor symptom, such as headache (e.g., migraine) or hot flashes 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 onset or development of a headache (e.g., migraine) in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody in combination with at least one other agent useful in the treatment of headache.

Such other agents include, but are not limited to: 5-HT agonists and NSAIDs. For example, the antibody and at least one other agent may be administered concomitantly, i.e., they may be administered in close enough temporal proximity to allow the therapeutic effects of their individuals 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 recurrence of headache in the patient, or produce a longer lasting effect than when one of these agents is administered in the absence of the other. This step can be used to treat headaches falling in any of a variety of categories, including: migraine with or without aura; cluster headache; migraine-associated neuralgia; chronic headache; tension headache; headache due to other medical conditions (e.g., increased intracranial pressure caused by infection or tumor); chronic episodic migraine; various headaches not associated with structural damage; headache associated with non-vascular intracranial disorders; headache associated with substance administration and its withdrawal; headache associated with non-head infections; headache associated with metabolic disorders; headache associated with cranial, cervical, ocular, ear, nose, sinoatrial node, tooth, mouth or other facial or cranial structural disorders; cerebral neuralgia and nerve trunk pain and afferent denervation (afferent) pain.

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 at a dose of from about 0.01 to about 300 mg. When administered parenterally, a typical dose of sumatriptan is from about 25 to about 100 mg. Generally about 50mg is preferred, and when administered parenterally, a preferred dose is about 6 mg. However, these dosages can be varied according to standard methods in the art to optimize them for a particular patient or for a particular combination therapy. Additionally, celecoxib, for example, may be administered in amounts between 50 and 500 mg.

In another aspect, the invention provides a method for ameliorating, controlling, reducing the onset of, or delaying the onset or development 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 useful for treating hot flashes. Such other agents include, but are not limited to, hormone-based therapies including estrogens and/or some progestins.

With respect to all of the methods described herein, reference to an anti-CGRP antagonist antibody also includes compositions comprising one or more of these agents. These compositions may further comprise suitable excipients well known in the art, such as pharmaceutically acceptable excipients including buffers. The present invention may be used alone or in combination with other conventional therapeutic methods.

The anti-CGRP antagonist antibody may be administered to the subject by any suitable route. Those skilled in the art will understand that: the embodiments described herein are not intended to be limiting, but rather illustrative of available technologies. Thus, in some embodiments, the anti-CGRP antagonist antibody is administered to the individual according to known methods, e.g., intravenously (e.g., as a bolus injection or by continuous infusion over a period of time), by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarticular, sublingual, intrasynovial, by insufflation, intraspinal, intraoral, inhalation, or topical routes. Administration can be systemic (e.g., intravenous) or local. Commercially available nebulizers for liquid formulations (including jet nebulizers and ultrasonic nebulizers) are suitable for administration. The liquid formulation can be sprayed directly, and the lyophilized powder can be sprayed after reconstitution. Alternatively, the anti-CGRP antagonist antibody may be aerosolized using fluorocarbon formulations and pressurized metered dose aerosols, or inhaled as a lyophilized and ground powder. In one embodiment, the anti-CGRP antagonist antibody is administered by site-specific or targeted local delivery techniques. Examples of site-specific or targeted local delivery techniques include various implantable sources of anti-CGRP antagonist antibodies (depot sources) or local delivery catheters, such as infusion catheters, indwelling catheters or trocars, synthetic grafts, adventitial wraps (adventitial wraps), shunts and stents or other implantable devices, site-specific vehicles, direct injection or direct application. See, for example, PCT publication No. wo 00/53211 and u.s patent No.5,981,568.

Various formulations of anti-CGRP antagonist antibodies are available for administration. In some embodiments, the anti-CGRP antagonist antibody may be administered alone. In some embodiments, the anti-CGRP antagonist antibody and pharmaceutically acceptable excipient may be present in a variety of formulations. Pharmaceutically acceptable excipients are substances known in the art and are relatively inert substances which facilitate the administration of a pharmacologically effective substance. For example, the excipient may have a shape or consistency, or act as a diluent. Suitable excipients include, but are not limited to, stabilizers, wetting and emulsifying agents, salts of varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. Excipients and formulations for parenteral and non-parenteral drug delivery are disclosed in Remington, The Science and Practice of Pharmacy 20th ed. mack Publishing (2000).

In some embodiments, these agents are 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 dosing regimen (i.e., dose, time and repetition) will depend on the particular individual and the medical history of that individual.

The anti-CGRP antibody may be administered using any suitable method, including injection (e.g., intraperitoneal, intravenous, subcutaneous, intramuscular, etc.). The anti-CGRP antibody may also be administered by inhalation as described herein. Typically, for administration of anti-CGRP antibodies, the initial candidate dose may be about 2 mg/kg. For the purposes of the present invention, typical daily dosages may range from about 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 mentioned 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 over several days or longer, treatment is continued depending on the condition until the desired suppression of symptoms occurs or until a sufficient therapeutic level is reached, e.g., to reduce pain. An exemplary dosing regimen includes administering an initial dose of about 2mg/kg, followed by a weekly maintenance dose of about 1mg/kg of anti-CGRP antibody, or followed by a maintenance dose of about 1mg/kg every other week. However, other methods of administration may be useful depending on the pharmacokinetic decay pattern that the physician wishes to achieve. For example, in some embodiments, one to four times a week dosing is contemplated. The course of the therapy is readily detected by routine techniques and experimentation. The dosing regimen, including the CGRP antagonist used, may vary over time.

For the purposes of the present invention, the appropriate dosage of anti-CGRP antagonist antibody will depend on the anti-CGRP antagonist antibody (or composition thereof) used, the type and severity of the headache (e.g., migraine) to be treated, whether the agent is being used for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the agent, and the judgment of the attending physician. Typically, the clinician will administer the anti-CGRP antagonist antibody until a dose is reached that achieves the desired result. The dose and/or frequency may vary with the progress of the treatment.

Empirical considerations (e.g., half-life) often aid in the determination of dosage. For example, antibodies compatible with the human immune system (e.g., 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 as the treatment progresses and is typically, but not necessarily, based on the treatment and/or inhibition and/or amelioration and/or delay of headache (e.g., migraine). Alternatively, a sustained continuous release formulation of the anti-CGRP antagonist antibody may be appropriate. Various formulations and devices for achieving sustained release are known in the art.

In one embodiment, the dosage of anti-CGRP antagonist antibody in an individual who has been administered anti-CGRP antagonist antibody one or more times may be determined empirically. The individual is administered an increasing dose of an anti-CGRP antagonist antibody. To assess the efficacy of anti-CGRP antagonist antibodies, one can follow the indications for the disease.

Administration of an anti-CGRP antagonist antibody 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 the skilled practitioner. Administration of the anti-CGRP antagonist antibody may be substantially continuous over a preselected period of time, or may be a series of spaced doses, such as any one of before, during or after the onset 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 occurrence of a headache. Administration can be before, during, and/or after any event that may cause headache.

In some embodiments, more than one anti-CGRP antagonist antibody may be present. At least one, at least two, at least three, at least four, at least species different or more anti-CGRP antagonist antibodies may be present. Typically, these anti-CGRP antagonist antibodies may have complementary activities that do not adversely affect each other. Antagonist anti-CGRP antibodies may also be used to bind to other CGRP antagonists or CGRP receptor antagonists. For example, one or more of the following CGRP antagonists may be used: anti-antisense molecules against CGRP (including anti-antisense molecules against nucleic acids encoding CGRP), CGRP inhibitory compounds, CGRP structural analogs, dominant negative mutations of CGRP receptors that bind CGRP, anti-CGRP receptor antibodies. anti-CGRP antagonist antibodies may also be used in combination with other agents that act to enhance and/or complement the effectiveness of the agent.

The therapeutic formulations of anti-CGRP antagonist antibodies for use according to the invention are prepared as lyophilized formulations or as aqueous solutions for storage as described below: will haveThe antibody of The desired purity is mixed with an optional pharmaceutically acceptable carrier, excipient or stabilizer (Remington, The Science and practice of Pharmacy 20th ed. mack Publishing (2000)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages employed, and may include buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, phenethylammonium 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; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG).

Liposomes containing anti-CGRP antagonist antibodies are prepared by methods known in the art, for example as described in Epstein, et al, proc.natl.acad.sci.usa 82: 3688 (1985); hwang, et al, proc.natl acad.sci.usa 77: 4030 (1980); and U.S. Pat. nos.4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. patent No.5,013,556. Particularly useful liposomes can be produced by reverse phase evaporation methods with a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes having the desired diameter are obtained by filtering the liposomes through a filter of defined pore size.

In colloidal delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, the active ingredient may also be entrapped in microcapsules, for example hydroxymethylcellulose or gelatin microcapsules and poly (methylmethanolate) microcapsules, respectively, prepared for example by coacervation techniques or by interfacial polymerization. Such techniques are disclosed in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

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

Formulations for in vivo administration must be sterile. This is easily achieved by, for example, filtration through sterile filtration membranes. Therapeutic anti-CGRP antagonist antibody compositions are typically placed in a container (e.g., an intravenous solution bag or bottle with a stopper penetrable by a hypodermic needle) having a sterile access port.

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.

To prepare solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical 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 containing 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 uniformly throughout the composition so that the composition may be readily subdivided into equivalent 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. Tablets or pills of the novel compositions may be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, a tablet or pill may comprise an inner dosage and an outer dosage component, the latter being in the form of an outer membrane on 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 into the duodenum or to be delayed in release. A variety of materials may be used for such enteric layers or coatings, including amounts of polyacids and mixtures of polyacids with materials such as shellac, cetyl alcohol and cellulose acetate.

Suitable surfactants include, inter alia: non-ionic agents, e.g. polyoxyethylene sorbitan (e.g. Tween)^TM20. 40, 60, 80 or 85) and other sorbitans (e.g. Span)^TM20. 40, 60, 80, or 85). The surfactant-containing composition should conveniently contain between 0.05% and 5% surfactant and may be between 0.1% and 2.5%. It will be appreciated that other ingredients may be added if necessary, for example mannitol or other pharmaceutically acceptable vehicle.

Suitable emulsions may be prepared using commercially available fat emulsions, such as Intralipid^TM、Liposyn^TM、Infonutrol^TM、Lipofundin^TMAnd Lipiphysan^TM. The active ingredient may be dissolved in a pre-mixed emulsion composition, or it may be dissolved in an oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil, or almond oil) or an emulsion formed by mixing a phospholipid (e.g., lecithin, soybean phospholipid, or soybean lecithin) with water. It will be appreciated that other ingredients, such as glycerol or glucose, may be added to adjust the tonicity of the emulsion. Suitable milksThe agent typically contains up to 20% oil, for example between 5% and 20%. The fat emulsion comprises fat droplets of between 0.1 and 1.0Im, especially between 0.1 and 0.5Im, and has a pH in the range of 5.5 to 8.0.

The emulsion composition may be prepared by combining an anti-CGRP antagonist antibody with an Intralipid^TMOr a composition prepared by mixing the components thereof (soybean oil, lecithin, glycerin and water).

Compositions for inhalation or insufflation include solutions and suspensions and powders in pharmaceutically acceptable, aqueous or organic solvents or mixtures thereof. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as disclosed above. In some embodiments, the composition is administered via the oral or nasal respiratory route for local or systemic effect. The composition, preferably in a sterile pharmaceutically acceptable solvent, can be sprayed by using a gas. The sprayed solution may be breathed directly from the nebulizing device, or the nebulizing device may be combined with a face mask, tent, or intermittent positive pressure ventilator. The solution, suspension or powder composition may be administered from a device that delivers the formulation in a suitable manner, preferably orally or nasally.

Diagnosis or assessment of headache is well established in the art. The assessment may be determined from subject measurements (e.g., patient characteristics of symptoms). For example, migraine can be diagnosed based on the following criteria: 1) episodic episodes of headache last 4 to 72 hours; 2) two of the following symptoms are present: unilateral pain, restlessness, increased intensity with exercise, and moderate or severe intensity pain; and 3) one of the following symptoms: nausea or vomiting, and photophobia or phonophobia (phonophobia). Godsby et al, n.engl.j.med.346: 257-270, 2002.

The effectiveness of the treatment can be assessed by methods well known in the art. For example, pain relief can be assessed. Thus, in some embodiments, pain relief is subjectively observed after 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.

B.anti-CGRP antagonist antibodies

The methods of the invention employ anti-CGRP antagonist antibodies, by which is meant any antibody molecule that blocks, inhibits or reduces (including significantly) the biological activity of CGRP, including downstream pathways mediated by CGRP signaling, such as receptor binding and/or the initiation of a cellular response to CGRP.

The anti-CGRP antagonist antibody should exhibit any one or more of the following characteristics: (a) combining CGRP; (b) blocking CGRP binding to its receptor; (c) blocking or reducing CGRP receptor activation (including cAMP activation); (d) inhibiting CGRP biological activity or down-regulating downstream pathways mediated by CGRP signaling function; (e) preventing, ameliorating or treating any aspect of headache (e.g., migraine); (f) the clearance rate of the CGRP is improved; 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: 565-73, 1995; sigma (Missouri, US), product number C7113(clone # 4901); ploudde et al, Peptides 14: 1225-1229, 1993.

For the purposes of the present invention, antibodies react with CGRP in a manner that inhibits CGRP and/or 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 both 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 suitable for use in the present invention may include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab ', F (ab')₂Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chain (ScFv), mutants thereof, fusion proteins comprising an antibody portion (e.g., domain antibodies), humanized antibodies,and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site of a desired specificity, including glycosylation variants of an antibody, amino acid sequence variants of an antibody, and covalently modified antibodies. The antibody may be canine, rat, human, or of 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, an anti-CGRP antagonist antibody comprises one or more CDRs (e.g., one, two, three, four, five, or in some embodiments all six CDRs) of an antibody G1 or G1 variant shown in table 6. In still 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, e.g., a constant region that is immunologically inert as described herein. In some embodiments, the constant region is as defined in eur.j.immunol. (1999) 29: 2613-2624; PCT 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 (with reference to the amino acid sequence of wild-type IgG2 sequence). Eur.j.immunol. (1999) 29: 2613-2624. In some embodiments, the antibody comprises an IgG4 constant region with the following mutations: E233F234L235 to P233V234a 235. In still other embodiments, the constant region is not glycosylated for N-linked glycosylation (aglycosylated). In some embodiments, the constant region is not glycosylated for N-linked glycosylation by mutating the oligosaccharide attachment residue (e.g., Asn297) and/or the flanking residues that are part of the N-glycosylation recognition sequence in the constant region. In some embodiments, the constant region is not glycosylated for N-linked glycosylation. The constant region may be left unglycosylated for N-linked glycosylation either enzymatically or by expression in a glycosylation deficient host cell.

Affinity (K) of anti-CGRP antagonist antibodies to CGRP (e.g., human α -CGRP)_D) And may be about 0.02 to about 200 nM. In some embodiments, the 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 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 affinity of an antibody for CGRP is by measuring the 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. Can be detected by surface plasmon resonance (Biacore 3000) equipped with an immobilized streptavidin sensing chip (SA)^TMSurface Plasmon Resonance (SPR) system, Biacore, INC, Piscataway NJ) the affinity of anti-CGRP Fab fragments of antibodies was determined using HBS-EP electrophoresis buffer (0.01M HEPES, pH7.4, 0.15NaCl, 3mM EDTA, 0.005% v/v surfactant P20). Biotinylated human CGRP (or any other CGRP) can be diluted to a concentration of less than 0.5ug/mL in HBS-EP buffer and injected across individual chip channels using different contact times to achieve two ranges of antigen density: 50-200 Response Units (RU) for detailed kinetic studies or 800-1,000RU for screening experiments. Regeneration studies showed that 25mM NaOH in 25% v/v ethanol effectively removed bound Fab while maintaining CGRP activity for more than 200 injections on the chip. Typically, serial dilutions of purified Fab samples (K assessed against 0.1-10 ×)_DAt a concentration of 100 μ L/min) for 1 minute and allowing a dissociation time of up to 2 hours. The concentration of the Fab protein was determined by ELISA and/or SDS-PAGE electrophoresis using known concentrations of Fab (determined by amino acid analysis) as a standard. Using the BlAevaluation program, the data was integrated by combining the data with a 1: 1 Langmuir binding model (Karlsson,roos, H.Fagerstam, L.Petersson, B. (1994). Methods Enzymology 6.99-110) were fitted, while obtaining the kinetic binding rate (k)_on) And dissociation rate (k)_off). Equilibrium dissociation constant (K)_D) Value as k_off/k_onAnd (4) calculating. This protocol is applicable to determining the affinity of an antibody for any CGRP, including human CGRP, another mammalian CGRP (e.g., mouse CGRP, rat CGRP, primate CGRP), and different forms of CGRP (e.g., alpha and beta forms). The affinity of an antibody is usually measured at 25 ℃, but can also be measured at 37 ℃.

anti-CGRP antagonist antibodies can be made by any method known in the art. The route and schedule of immunization of the host animal is generally consistent with established and conventional techniques for antibody stimulation and production as further described herein. General techniques for producing human and mouse antibodies are known in the art and described herein.

The invention comprises the following steps: any mammalian subject (including humans) or antibody-producing cells derived therefrom can be processed for use as a basis for the production of mammalian (including human) hybridoma cell lines. Typically, the host cell is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantar, and/or intradermally with an amount of an immunogen (including immunogens described herein).

Kohler, b.and Milstein, c. (1975) Nature 256: 495-497 or the equivalent of Buck, d.w., et al, In Vitro, 18: 377-381(1982) modified general somatic hybridization techniques for the preparation of hybridomas from lymphocytes and immortal myeloma cells. Available myeloma lines (including but not limited to X63-Ag8.653 and myeloma lines from Saik Institute, Cell Distribution Center, San Diego, Calif., USA) may be used in the hybridization. Generally, this technique involves fusing myeloma cells and lymphocytes using a fusion promoting agent known to those skilled in the art (e.g., polyethylene glycol) or by electrical means. After fusion, the cells are isolated from the fusion medium and cultured on a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to eliminate unhybridized parental cells. Hybridomas secreting monoclonal antibodies can be cultured using any of the media lai described herein, supplemented with serum or unsupplemented with serum. As an alternative to cell fusion techniques, EBV immortalized B cells can be used to produce anti-CGRP monoclonal antibodies of the invention. If desired, the hybridomas are expanded and subcloned, and the supernatants are tested for anti-immunogenic activity by conventional immunoassay procedures, such as radioimmunoassay, enzyme immunoassay, or fluorescent immunoassay.

Hybridomas useful as antibody sources include all derived progeny cells of the parent hybridoma that produce a monoclonal antibody or portion thereof specific for CGRP.

Hybridomas producing such antibodies can be cultured in vitro or in vivo using known procedures. If desired, monoclonal antibodies can be isolated from the culture medium or body fluids by conventional immunoglobulin purification steps such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. If there is undesired activity, this can be removed, for example, by flowing the formulation through an adsorbent consisting of the immunogen attached to a solid phase and eluting or releasing the desired antibody from the immunogen. Immunization of host animals with human CGRP or fragments containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin (keyholelet hemocyanin), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide (maleimidobenzoyl sulfosuccinimide) ester (conjugated through a cysteine residue), N-hydroxysuccinimide ester (conjugated through a lysine residue), acrolein, succinic anhydride, SOCl2, or R1N ═ C ═ NR, where R or R1 are different alkyl groups, enables a population of antibodies (e.g., monoclonal antibodies).

If desired, the anti-CGRP antagonist antibody of interest (monoclonal or polyclonal) 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 may be maintained in a vector in the host cell, and the host cell may then be expanded or frozen for further use. Alternatively, the polynucleotide sequences may be used in genetic manipulation to "humanize" the antibody or to improve the affinity or other properties of the antibody. For example, the constant region may be engineered to more resemble a human constant region, thereby avoiding an immune response if the antibody is used in clinical trials and treatment in humans. It may be desirable to genetically engineer antibody sequences to obtain greater affinity for CGRP and greater potency in inhibiting CGRP. It will be appreciated by those skilled in the art that one or more polynucleotide changes may be made to an anti-CGRP antagonist antibody and still maintain its ability to bind CGRP.

There are four general steps for humanizing monoclonal antibodies. The steps are as follows: (1) determination of the nucleotide and predicted amino acid sequences of the light and heavy variable domains of the original antibody (2) design of the humanized antibody, i.e., determination of which antibody framework regions to use in the humanization process (3) the actual humanization method/technique and (4) transfection and expression of the humanized antibody. See, for example, U.S. patent nos.4,816,567, 5,807,715, 5,866,692, 6,331,415, 5,530,101, 5,693,761, 5,693,762, 5,585,089, and 6,180,370.

A number of "humanized" antibody molecules comprising an antigen-binding site derived from a non-human immunoglobulin have been described, including chimeric antibodies having rodent or modified rodent V regions fused to human constant regions and Complementarity Determining Regions (CDRs) to which they bind. See, e.g., Winter et al. Nature 349: 293-: 4220-4224(1989), Shaw et al.J. Immunol.138: 4534-: 3577-3583(1987). Other references describe rodent CDRs grafted into human supporting Framework Regions (FRs) prior to fusion with appropriate human antibody constant regions. See, e.g., Riechmann et al Nature 332: 323-327(1988), Verhoeyen et al science 239: 1534-1536(1988) and Jones et al. Nature321: 522-525(1986). Another reference describes rodent CDRs supported by recombinantly modified rodent framework regions. See, for example, european patent application No. 0519596. These "humanized" molecules are designed to minimize undesirable immune responses to rodent anti-human antibody molecules that limit the duration and effectiveness of therapeutic applications of these moieties in human recipients. For example, the antibody constant region can be engineered such that it is immunologically inert (e.g., does not trigger complement lysis). See, e.g., PCT publication No. PCT/GB 99/01441; UK patent application No. 9809951.8. Other methods of humanizing antibodies that may also be used are described by Daugherty et al, nucleic acids res.19: 2471-2476(1991) and U.S. Pat. Nos.6,180,377; 6,054,297; 5,997,867, respectively; 5,866,692, respectively; 6,210,671 and 6,350,861 and PCT publication No. WO 01/27160.

Still alternatively, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or more viable immune response may also be used to produce humanized or human antibodies. An example of such a technique is Xenomouse from Abgenix, Inc. (Fremont, CA)^TMAnd TCMouse from Medarex, Inc (Princeton, NJ)^TM。

Alternatively, the antibody may be recombinantly produced and expressed using any method known in the art. Alternatively, antibodies can be recombinantly produced by phage display technology. See, for example, U.S. patent nos.5,565,332; 5,580,717; 5,733,743, respectively; and 6,265,150; and Winter et al, annu.rev.immunol.12: 433-455(1994). Alternatively, human antibodies and antibody fragments can be produced in vitro from a gene repertoire of immunoglobulin variable (V) domains from an unimmunized donor using phage display technology (McCafferty et al, Nature 348: 552-553 (1990)). According to this technique, antibody V domains are cloned in-frame into the major or minor coat proteins of filamentous phages (such as M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on functional characteristics of the antibody also results in selection of genes encoding antibodies exhibiting these characteristics. Thus, the phage mimics some of the characteristics of B cells. Phage display can be performed in a variety of formats; for reviews see, e.g., Johnson, Kevin s.andchisweli, David j., Current Opinion in Structural Biology 3: 564-571(1993). Several sources of V gene segments can be used for phage display. Clackson et al, Nature 352: 624-628(1991) different anti-oxazolone antibody arrays isolated from small random combinatorial libraries of V genes from the spleens of immunized mice. Substantially in accordance with Mark et al, j.moi.biol.222: 581-597(1991) or Griffith et al, EMBO J.12: 725-734(1993) can construct V gene pools from non-immunized human donors and isolate antibodies against arrays of different antigens, including self-antigens. In the innate immune response, antibody genes are mutated cumulatively at a high rate (somatic hypermutation). Some of the introduced changes confer higher affinity and preferentially replicate and differentiate B cells displaying high affinity surface immunoglobulins in subsequent antigen priming. This natural process can be simulated by using a technique known as "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 as follows: the V region genes of the heavy and light chains were replaced consecutively with a pool (repertoire) of naturally occurring variants of the V domain genes from non-immunized donors. This technique allows the production of antibodies and antibody fragments with affinities in the pM-nM range. The strategy to make a very large phage antibody repertoire (also known as "all of the mother libraries") has been described by Waterhouse et al, nucleic 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 original rodent antibody. According to this method (which is also referred to as "epitope imprinting"), the heavy or light chain V domain genes of rodent antibodies obtained by phage display technology are replaced with a human V domain gene repository, creating a rodent-human chimera. Selection on the antigen results in the isolation of human variable regions that are capable of reconstituting a functional antigen-binding site, i.e. the epitope governs (imprinting) the selection of the partner. When this procedure is repeated to replace the remaining rodent V domains, a human antibody is obtained (see PCT publication No. WO 93/06213, April 1, 1993). Unlike conventional humanization of rodent antibodies by CDR grafting, this technique provides fully human antibodies that do not have rodent-derived framework or CDR residues.

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

Antibodies can be recombinantly produced as follows: cells producing multiple antibodies or one antibody are first isolated from a host animal, the gene sequence is obtained, and the antibody is recombinantly expressed in a host cell (e.g., a CHO cell) using the gene sequence. 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.huskzar int.rev.immunol 13: 65 (1995); and Pollock, et al, J ImmunolMethods 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.

Antibodies can be conjugated to a number of different carriers. The carrier may be active and/or inert. Examples of well-known carriers include polypropylene, polystyrene, polyethylene, dextran, nylon, amylase, glass, natural and modified celluloses, polyacrylamides, agarose, and magnetite. For the purposes of the present invention, the nature of the carrier may be soluble or insoluble. Those skilled in the art will recognize other suitable carriers for binding the antibody or will be able to ascertain the same using routine experimentation. In some embodiments, the carrier comprises a portion that targets the myocardium.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specifically binding to the genes of the genus encoding the heavy and light chains of the monoclonal antibody). The hybridoma cells act as a preferred source of such DNA. Once isolated, the DNA may 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 cell, simian COS cell, Chinese Hamster Ovary (CHO) cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. See, for example, PCT publication No. wo 87/04462. The DNA may also be modified, for example, by substituting homologous murine sequence positions for the coding sequences for human heavy and light chain constant domains (morrison et al, proc. nat. acad. sci.81: 6851(1984)), or by covalently joining all or part of the sequence encoding the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the anti-CGRP monoclonal antibodies herein.

anti-CGRP antagonist antibodies and polypeptides from the antibodies can be identified or characterized using methods known in the art to detect and/or measure a decrease, slowing, or neutralization of the biological activity of CGRP. For example, anti-CGRP antagonist antibodies may also be identified by incubating a candidate agent with CGRP and monitoring any one or more of the following characteristics: (a) combining CGRP; (b) blocking CGRP binding to its receptor; (c) blocking or reducing 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 any aspect of headache (e.g., migraine); (f) the clearance rate of the CGRP is improved; and (g) inhibiting (reducing) CGRP synthesis, production or release. In some embodiments, an anti-CGRP antagonist antibody or polypeptide is identified by incubating a candidate agent with CGRP and monitoring binding and/or concomitant reduction or neutralization of biological activity of the CGRP. Binding assays may be performed with purified CGRP polypeptides, or with cells that naturally express CGRP polypeptides or are transfected to express CGRP polypeptides. In one embodiment, the binding assay is a competitive binding assay in which the ability of a candidate antibody to compete with known anti-CGRP antagonists for binding to CGRP is evaluated. The test can be performed in a variety of formats, including ELISA formats. In other embodiments, anti-CGRP antagonist antibodies are identified by incubating the candidate agent with CGRP and monitoring binding and concomitant inhibition of CGRP receptor activation expressed on the cell surface.

After initial identification, the activity of the candidate anti-CGRP antagonist antibodies can be further confirmed and refined by bioassays known to examine targeted biological activity. Alternatively, the candidates may be screened directly using a bioassay. For example, CGRP promotes a large number of 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. The efficacy of the antagonist antibody or polypeptide can be further tested using an animal model of headache, e.g., migraine. Reuter, et al, Functional Neurology (15) supply.3, 2000. Some of the methods for identifying and characterizing anti-CGRP antagonist antibodies are detailed in the examples.

anti-CGRP antagonist antibodies can be characterized using methods well known in the art. For example, one approach is to identify the epitope to which it binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing epitope positions on proteins, including solving the crystal structure of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11of Harbor and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1999. In additional 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., contained in a single-chain amino acid) or a conformational epitope (which is not necessarily contained in a single chain) formed by three-dimensional interaction of amino acids. Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or (e.g., recombinantly) synthesized 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 from the CGRP sequence and determining the binding of the anti-CGRP antagonist antibody. Based on the gene fragment expression assay, the open reading frame encoding CGRP is fragmented, either randomly or by specific genetic construction, and the reactivity of the CGRP fragment to be expressed with the antibody to be tested is determined. Gene fragments can be produced, for example, by PCR, and subsequently transcribed and translated into protein in vitro 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 library of defined overlapping peptide fragments may be tested for binding to a test antibody in a simple binding assay. In an additional example, antigen binding domain mutagenesis, domain switching experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and/or necessary for epitope binding. For example, domain switching experiments can be performed using mutant CGRP in which multiple fragments of the CGRP polypeptide have been replaced (switched) into sequences from closely related, but antigenically distinct proteins (e.g., another member of the neurotrophin protein family). The importance of a particular CGRP fragment for antibody binding can be assessed by assessing antibody binding to mutant CGRP.

Yet another method that can be used to characterize an anti-CGRP antagonist antibody is to use a competition assay that uses other antibodies known to bind the same antigen, i.e., multiple fragments on CGRP, to determine whether the anti-CGRP antagonist antibody binds the same epitope as the other antibodies. Competitive assays are well known to those skilled in the art.

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

Targeted delivery of therapeutic compositions containing 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, Gene Therapeutics: methods AndApplications Of Direct Gene Transfer (J.A. Wolff, ed.) (1994); wu et al, j.biol.chem. (1988) 263: 621 of the first and second substrates; 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 gene therapy protocols, for topical administration, therapeutic compositions containing polynucleotides are administered in the range of about 100mg to about 200mg of DNA. Concentrations in the range of about 500ng to about 50mg, about 1 μ g to about 2mg, about 5 μ g to about 500 μ g, and about 20 μ g to about 100 μ g of DNA may also be used in gene therapy protocols. 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 may be induced using endogenous mammalian or heterologous promoters. Expression of a coding sequence may be constitutive or regulated.

Viral-based vectors for delivering and expressing a desired polynucleotide in a desired host cell are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT publication Nos. WO 90/07936; WO 94/03622; WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S. Pat. Nos.5,219,740 and 4,777,127; GB patent No.2,200,651; and EP patent No. 0345242), alphavirus-based vectors (e.g., Sindbis viral vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross river virus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)) and adeno-associated virus (AAV) vectors (see, e.g., PCT publication Nos. WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655). Also useful are compositions such as Curiel, hum. Gene Ther (1992) 3: 147 with killed adenovirus.

Non-viral delivery vehicles and methods may also be used, including but not limited to polycationic condensed DNA alone linked or not linked to killed adenovirus (see, e.g., Curiel, hum. genether. (1992) 3: 147); ligand-linked DNA (see, e.g., Wu, j.biol.chem. (1989) 264: 16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. patent No.5,814,482; PCT publication nos. WO 95/07994; WO 96/17072; WO 95/30763; and WO97/42338) 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 patent Nos. WO 90/11092 and U.S. Pat. No.5,580,859. Liposomes that can function as gene delivery vehicles are described in U.S. patent nos.5,422,120; PCT publication nos. wo 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Other pathways are described in Philip, moi. cell Biol. (1994) 14: 2411, and Woffendin, proc.natl.acad.sd. (1994) 91: 1581.

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

The present invention includes compositions (including pharmaceutical compositions) comprising antibody G1 and variants thereof shown in table 6, or a polypeptide derived from antibody G1 and variants thereof shown in table 6; and polynucleotides comprising sequences encoding antibody G1 and variants or polypeptides thereof. Compositions for use herein include one or more antibodies or polypeptides (which may be or are inappropriate antibodies) that bind to CGRP and/or one or more polynucleotides comprising sequences encoding one or more antibodies or polypeptides that bind to CGRP. These compositions may further comprise suitable excipients well known in the art, such as pharmaceutically acceptable excipients (including buffers).

The anti-CGRP antagonist antibodies and polypeptides of the invention are characterized by any (one or more) of the following characteristics: (a) combining CGRP; (b) blocking CGRP binding to its receptor; (c) blocking or reducing CGRP receptor activation (including cAMP activation); (d) inhibiting CGRP biological activity or down-regulating downstream pathways mediated by CGRP signaling function; (e) preventing, ameliorating or treating any aspect of headache (e.g., migraine); (f) the clearance rate of the CGRP is improved; and (g) inhibiting (reducing) CGRP synthesis, production or release.

Accordingly, 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 from the light and/or heavy chain of antibody G1 shown in table 6, or a variant thereof; (f) one or more CDRs (one, two, three, four, five or six CDRs) from antibody G1 or a variant thereof shown in table 6; (g) CDR H3 from the heavy chain of antibody G1; (h) CDR L3 from the light chain of antibody G1 or a variant thereof shown in table 6; (i) three CDRs from the light chain of antibody G1 or variants thereof shown in table 6; (j) three CDRs from the heavy chain of antibody G1 or variants thereof shown in table 6; (k) three CDRs from the light chain and three CDRs from the heavy chain of antibody G1 shown in table 6 or variants thereof; and (I) an antibody comprising any one of (b) to (k). The invention also provides polypeptides comprising one or more of the above.

The CDR portions of antibody G1 (including the CDRs of Chothia and Kabat) 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 where there is more than one CDR, the CDR can be any 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, or at least four, at least five, or at least all six CDRs, which are substantially identical to at least one CDR, at least two, at least three, at least four, at least five, or all six CDRs of G1 or variants thereof shown in table 6. Other embodiments include antibodies having at least two, three, four, five, or six CDRs that are substantially identical to at least two, three, four, five, or six CDRs of G1 or 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 should be understood that: for the purposes of the present invention, the binding specificity and/or overall activity is generally constant, although the degree of activity may vary (may be greater or less) compared to G1 or variants thereof shown in table 6.

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 contiguous amino acids, at least 8 contiguous amino acids, at least about 10 contiguous amino acids, at least about 15 contiguous amino acids, at least about 20 contiguous amino acids, at least about 25 contiguous amino acids, at least about 30 contiguous amino acids of the sequence of G1 or a variant thereof shown in table 6, wherein at least three amino acids are from the variable region of G1 or a variant thereof shown in table 6 (figure 5). 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 G1 heavy and light chain variable regions. In another embodiment, 5 (or more) consecutive amino acids are from the Complementarity Determining Region (CDR) of G1 shown in fig. 5. In some embodiments, the contiguous amino acids are from the variable region of G1.

Affinity (K) of anti-CGRP antagonist antibodies and polypeptides for CGRP (e.g., human α -CGRP)_D) And may be about 0.06 to about 200 nM. In some embodiments, the 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 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.

The invention also provides methods of making any of these antibodies or polypeptides. The antibodies of the invention can be made by procedures known in the art. The polypeptides may be produced by proteolytic or other degradation of the antibody, by recombinant methods as described above (i.e., single or fused polypeptides), or by chemical synthesis. Polypeptides of antibodies, particularly shorter polypeptides of up to about 50 amino acids, are conveniently made by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available. For example, antibodies can be produced 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.

Alternatively, antibodies can be recombinantly produced using procedures well known in the art. In one embodiment, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10 encoding the heavy and/or light chain variable region of antibody G1. In another embodiment, the polypeptide comprising 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 may be maintained in a vector in the host cell, which may then be expanded or frozen for future use. Vectors (including expression vectors) and host cells are further described herein.

The invention also includes single chain variable fragments ("scFv") of the antibodies of the invention (e.g., G1). The G1 single chain variable region fragment was made by joining the variable regions of the light and/or heavy chains using short linking peptides. Birdet al (1988) Science 242: 423-426. An example of a linker peptide is (GGGGS)3, which is linked approximately 3.5nm between the carboxy terminus of one variable region and the amino terminus of the other variable region. Other sequence structures have been designed and used. Bird et al (1988). The linker may then be modified to have additional functionality, for example binding to a drug or binding to a solid support. Single-stranded variants can be produced recombinantly or synthetically. For synthetic production of scFv, an automated synthesizer can be used. For recombinant production of scFv, a suitable plasmid containing a polynucleotide encoding an scFv can be introduced into a suitable host cell, either eukaryotic (such as yeast, plant, insect or mammalian cells) or prokaryotic (such as E.coli). Polynucleotides encoding the scFv of interest can be made by conventional procedures such as ligation of polynucleotides. The resulting scFv can be isolated using standard protein purification techniques known in the art.

The invention also includes other forms of single chain antibodies such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thereby forcing the domain to pair with the complementary domain of the other chain and generating two antigen binding sites (see for example 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 Caluelo, 1983, Nature 305, 537-539).

According to one approach to making bispecific antibodies, antibody variable region domains with the desired binding specificity (antibody-antigen binding site) are fused to immunoglobulin constant region sequences. The fusion preferably carries an immunoglobulin heavy chain constant domain comprising at least a portion of the hinge, CH2 and CH3 regions. Preferably, the first heavy chain constant region (CH1) is present in at least one fusion, said constant region comprising the site necessary for light chain binding. The DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and transfected into a suitable host organism. This provides great flexibility in adjusting the ratio of the three polypeptide fragments to each other in embodiments that construct an optimal yield using moderate ratios of the three polypeptide chains. However, when equal ratios of at least two polypeptide chains result in high yields, or when the ratios are not of particular importance, it is possible to insert two or three polypeptide chains into one expression vector.

In one approach, bispecific antibodies consist of a hybrid immunoglobulin heavy chain with a first binding specificity on one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) on the other arm. This asymmetric structure (immunoglobulin light chain in only one half of the bispecific molecule) facilitates the separation of the desired bispecific compound from the undesired immunoglobulin chain combination. This pathway 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) and to treat HIV infection (PCT application publication Nos. WO 91/00360 and WO 92/200373; EP 03089). Heteroconjugate antibodies can be made using any convenient crosslinking method. Suitable crosslinking agents and techniques are well known in the art and are described in U.S. patent No.4,676,980.

Chimeric or hybrid antibodies can also be prepared in vitro using known synthetic protein chemistry, including chemistry involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Suitable reagents for this purpose include iminothiolate (iminothiolate) and methyl-4-mercaptobutylimidazole (mercaptobutyliminate).

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

The present invention includes modifications to antibody G1 shown in table 6, or variants thereof, including functionally equivalent antibodies that do not significantly affect its properties and variants with enhanced or reduced activity and/or avidity. 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 a desired 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, one or more amino acid deletions or additions which do not significantly adversely alter functional activity, or the use of chemical homologs.

Amino acid sequence insertions include amino and/or carboxy-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with N-terminal methionyl residues or antibodies fused to epitope tags. Other insertional variants of the antibody molecule include fusions of enzymes or polypeptides to the N-or C-terminus of the antibody, which increase the serum half-life of the antibody.

A substitution variant is one in which at least one amino acid residue in the antibody molecule is removed and a different residue is inserted in its place. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR alterations are also contemplated. Conservative substitutions are shown in table 1 under the heading "conservative substitutions". If such substitutions result in a change in biological activity, more significant changes (referred to as "exemplary substitutions" in Table 1) or changes as described further below with respect to the amino acid family can be introduced.

Table 1: amino acid substitutions

Substitution modifications in antibody biological characteristics are accomplished by selecting substitutions that maintain a significant difference in the effect of (a) the polypeptide backbone structure, e.g., sheet or helical conformation, in the region of the substitution, (b) the charge or hydrophobicity at the target site of the molecule, or (c) the volume of the side chain. Naturally occurring residues are grouped according to common side chain features:

(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 GIu;

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

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

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

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

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

Amino acid modifications range from changing or modifying one or more amino acids to completely redesigning a region, such as the variable region. Changes in the variable region may alter avidity and/or specificity. In some embodiments, no more than one to five conservative amino acid substitutions are made in a CDR domain. In other embodiments, no more than one to three conservative amino acid substitutions are made in a CDR domain. In still other embodiments, the CDR domain is CDR H3 and/or CDR L3.

Modifications also include glycosylated and unglycosylated polypeptides, as well as polypeptides with other post-translational modifications, such as glycosylation with different sugars, acetylation, and phosphorylation. Antibodies are glycosylated at positions conserved in their constant regions (Jefferis and Lund, 1997, chem. Immunol.65: 111-128; Wright and Morrison, 1997, TibTECH 15: 26-32). The oligosaccharide side chains of immunoglobulins affect the function of the protein (Boyd et al, 1996, MoI. Immunol.32: 1311-1318; Wittweand Howard, 1990, biochem.29: 4175-4180) and the intramolecular interactions between the glycoprotein moieties, which can affect the conformation of the glycoprotein and the three-dimensional surface represented (Hefferis and Lund, supra; Wys and Wagner, 1996, Current Opin. Biotech.7: 409-416).

Oligosaccharides may also act to target a given glycoprotein to certain molecules based on specific recognition structures. Glycosylation of antibodies has also been reported to affect antibody-dependent cellular cytotoxicity (ADCC). In particular, CHO cells with tetracycline-regulated β (1, 4) -N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase that catalyzes the formation of bisecting GlcNac, have been reported to have improved ADCC activity (Umana et al, 1999, Mature Biotech.17: 176-.

Typically, glycosylation of an antibody is N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences of asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine are recognition sequences for the binding of a carbohydrate moiety to an asparagine side chain enzyme, where X is any amino acid except proline. Thus, the presence of these tripeptide sequences in a polypeptide creates potential glycosylation sites. O-linked glycosylation refers to the binding of one of N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most often serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

The addition of glycosylation sites to the antibody is conveniently accomplished as described below: the amino acid sequence is altered so that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations (for O-linked glycosylation sites) can also be made by adding or substituting one or more serine or threonine residues to the original antibody sequence.

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

In addition to the choice of host cell, factors that influence glycosylation during recombinant production of antibodies include: growth pattern, medium formulation, culture density, oxygenation, pH, purification scheme, etc. Various methods have been proposed for altering the glycosylation pattern achieved in a particular host organism, including the introduction or overexpression of certain enzymes involved in oligosaccharide production (U.S. patent nos.5,047,335; 5,510,261 and 5.278,299). Enzymatic removal of glycosylation or certain types of glycosylation from glycoproteins can be performed, 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 processing certain types of polysaccharides. These and similar techniques are well known in the art.

Other methods of modification include the use of coupling techniques known in the art, including but not limited to enzymatic means, oxidative substitution, and chelation. Modifications such as binding tags for immunoassays can be used. The modified G1 polypeptide was made using procedures established in the art and can be screened using standard assays known in the art, some of which are described below and in the examples.

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 elicit complement-mediated lysis, does not stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or does not activate microglia; or reduced activity (as compared to an unmodified antibody) in any one or more of: initiate complement-mediated lysis, stimulate antibody-dependent cell-mediated cytotoxicity (ADCC), or activate 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.immunology157: 4963-9157: 4963 + 4969 (1996); idulogie et al, j.immunology 164: 4178-4184 (2000); tao et al, j.immunology 143: 2595-2601 (1989); and Jefferis et al, Immunological Reviews 163: 59-76(1998). In some embodiments, the constant region is as defined in eur.j.immunol. (1999) 29: 2613-2624; PCT 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 sequence referred to wild type IgG2 sequence). Eur.j.immunol. (1999) 29: 2613-2624. In still other embodiments, the constant region is not glycosylated for N-linked glycosylation. In some embodiments, the constant region is not glycosylated for N-linked glycosylation by mutating glycosylated amino acid residues or flanking residues that are part of the 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, Immunological Reviews 163: 59-76(1998). In some embodiments, the constant region is not glycosylated for N-linked glycosylation. The expression of the polypeptide(s) can be achieved by an enzyme (e.g. by removal of carbohydrates by the enzyme PNGase) or by expression in a host cell deficient in glycosylation such that: for N-linked glycosylation, the constant region is not glycosylated.

Other antibody modifications include antibodies modified as described in PCT publication No. WO99/58572, published 11/18/1999. These antibodies include, in addition to a binding domain for a target molecule, 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 Fc γ RIIb. These are typically based on heavy chain C from two or more human immunoglobulins_H2 domain. Antibodies modified in this manner are particularly useful in chronic antibody therapy to avoid inflammation and other adverse effects to conventional antibody therapy.

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

The following methods can be used to modulate the affinity of an antibody and characterize the CDRs. Characterization of antibodiesOne method of somatic CDR and/or altering (e.g., promoting) the avidity of polypeptides, such as antibodies, is referred to as "library scanning mutagenesis". In general, library scanning mutagenesis functions as follows. One or more amino acid positions in a CDR are substituted with two or more (e.g., 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) amino acids using art-recognized methods. This results in a small library of clones (in some embodiments, one for each amino acid position analyzed), each clone having a complexity of two or more members (if two or more amino acids are substituted at each position). Typically libraries also include clones containing natural (unsubstituted) amino acids. Small clones, e.g., about 20-80 clones (depending on the complexity of the library) are screened for affinity for the target polypeptide (or other binding target) and candidates with increased, identical, reduced or no binding are identified. Methods for determining affinity are well known in the art. Avidity can be determined using Biacore surface plasmon resonance analysis, with approximately 2-fold or greater differences in avidity being monitored by the high method. When the initial antibody has been raised to a relatively high affinity (e.g., a K of about 10mM or less)_D) In combination, Biacore is particularly suitable. Screening for suitable Biacore surface plasmon resonances is described in the examples herein.

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

In some embodiments, each amino acid position in the CDRs is substituted (in some embodiments, one at a time) with 20 natural amino acids using approved mutagenesis methods (some of which are described herein). This results in a small library of clones (in some embodiments, one for each amino acid position analyzed), each having a complexity of 20 members (if all 20 amino acids are substituted at each position).

In some embodiments, the library to be screened comprises substitutions in two or more positions, which may be in the same CDR or two or more CDRs. Thus, the library may comprise substitutions in two or more positions in one CDR. The library may comprise substitutions in two or more positions of two or more CDRs. The library may comprise substitutions in 3,4, 5 or more positions present in two, three, four, five or six CDRs. Substitutions can be made using low redundancy codons. See, e.g., 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, extended CDRs.

Candidates with improved binding can be sequenced, thereby identifying CDR substitution mutants (also referred to as "improved" substitutions) that result in improved affinity. Candidates that bind may also be sequenced to identify CDR substitutions that retain binding.

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

Library scanning mutagenesis also provides a means for characterizing CDRs, as well as the importance of involvement of each amino acid position in the stability of the antibody-antigen complex, as regards the frequency of clones with improved binding, identical binding, reduced binding or no binding. For example, if the position of the CDR maintains binding when changed to all 20 amino acids, that position is identified as unlikely to be a position required for antigen binding. Conversely, if a position of a CDR maintains binding only for a small fraction of substitutions, that position is identified as a position important to the function of the CDR. Thus, library scanning mutagenesis methods yield information concerning positions in the CDR that can be changed to many different amino acids (including all 20 amino acids) and positions in the CDR that cannot be changed or can only be changed to a small number of amino acids.

Candidates with improved affinity may be combined in a second library that includes the improved amino acid, the original amino acid at that position, and may further include other substitutions at that position, depending on the complexity of the library that is desired or allowed using the desired screening or selection method. In addition, adjacent amino acid positions can be randomized to at least two or more amino acids, if desired. Randomization of adjacent amino acids may allow for additional conformational flexibility in the mutant CDRs, which may subsequently allow or facilitate the introduction of a greater number of improved mutations. The library also includes substitutions at positions in the first round of screening that do not show improved affinity.

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

The invention also includes fusion peptides comprising one or more fragments or regions from an antibody (e.g., G1) or polypeptide of the invention. In one embodiment, there is provided a polypeptide comprising SEQ ID NO: 2 (fig. 5) and/or at least 10 consecutive amino acids of the variable light chain region shown in SEQ ID NO: 1 (fig. 5) to a variable heavy chain region of at least 10 amino acids. In other embodiments, there is provided a polypeptide comprising SEQ ID NO: 2 (fig. 5), 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 and/or the amino acid sequence of SEQ id no: 1 (fig. 5), 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 fig. 1. In another embodiment, the nucleic acid sequence as set forth in SEQ ID NO: 2 and SEQ ID NO: 1, the fusion polypeptide comprises the variable region of the light chain and/or the variable region of the heavy chain of G1. In another embodiment, the fusion polypeptide comprises one or more CDRs of G1. In still 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 contains one or more G1 antibodies and another amino acid sequence (e.g., a heterologous sequence or a homologous sequence from another region) that does not bind the G1 antibody in the native molecule. Exemplary heterologous sequences include, but are not limited to, a "tag," such as a FLAG tag or a 6His tag. Labels are well known in the art.

The G1 fusion polypeptide can be created by methods known in the art (e.g., synthetically or recombinantly). Typically, the G1 fusion proteins of the invention are made by preparing polynucleotides encoding them using recombinant methods described herein, although they may also be made by other means known in the art, including, for example, chemical synthesis.

The invention also provides compositions comprising an antibody or polypeptide from G1 conjugated to (e.g., linked to) a reagent that facilitates coupling of a copper solid support (e.g., biotin or avidin). Briefly, these methods are generally applied to any of the CGRP binding embodiments described herein with reference to G1 or antibodies. Conjugation generally refers to linking these components as described herein. The attachment may be accomplished in any of a number of ways (which generally immobilize the components in intimate association for at least administration). For example, when one substance has subunits capable of reacting with other substances, a direct reaction between the reagent and the antibody is possible. For example, a nucleophilic group (e.g., amino or thiol) on one species may be capable of reacting with a carbonyl-containing group (e.g., anhydride or acid halide) or an alkyl group containing a good leaving group (e.g., halide) on another species.

The antibody or polypeptide of the invention 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 that typically provide a signal (directly or indirectly) are known in the art.

The invention also provides compositions (including pharmaceutical compositions) and kits comprising antibody G1 and (as defined by the disclosure) any or all of the antibodies and/or polypeptides disclosed herein.

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 figure 5), and vectors and host cells comprising the polynucleotides.

Accordingly, 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 from the light and/or heavy chain of antibody G1 shown in table 6, or a variant thereof; (f) one or more CDRs (one, two, three, four, five or six CDRs) from antibody G1 or a variant thereof shown in table 6; (g) CDR H3 from the heavy chain of antibody G1; (h) CDR L3 from the light chain of antibody G1 or a variant thereof shown in table 6; (i) three CDRs from the light chain of antibody G1 or variants thereof shown in table 6; (j) three CDRs from the heavy chain of antibody G1 or variants thereof shown in table 6; (k) three CDRs from the light chain and three CDRs from the heavy chain of antibody G1 shown in table 6 or variants thereof; and (I) an antibody comprising any one of (b) to (k). In some embodiments, the polynucleotide comprises SEQ ID NO: 9 and SEQ ID NO: 10, or a polynucleotide shown in seq id no.

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

In another aspect, the invention provides a composition (e.g., a pharmaceutical composition) comprising any of the polynucleotides of the invention. In some embodiments, the compositions comprise 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 still other embodiments, the composition comprises SEQ ID NO: 9 and SEQ ID NO: 10, or a combination thereof. The administration of the expression vector and polynucleotide composition is further described herein.

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

Polynucleotides complementary to any such sequence are also encompassed by the present invention. Polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include HnRNA molecules (which contain introns and correspond in a one-to-one manner to DNA molecules) and 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, which may (but need not) be linked to other molecules and/or support 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. Polynucleotide variants contain one or more substitutions, additions, deletions, and/or insertions such that the immunoreactivity of the encoded polypeptide is not reduced relative to the native immunoreactive molecule. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. Variants typically exhibit at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 99% identity to the polynucleotide sequence encoding the native antibody or fragment thereof.

Two polynucleotide or polypeptide sequences are said to be "identical" if the sequences of nucleotides or amino acids in the two sequences are identical when aligned for maximum correspondence as described below. Typically, the comparison between two sequences is 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 contiguous positions (typically 30 to about 75, 40 to about 50), where a sequence can be compared to the same number of contiguous positions of a reference sequence after optimal alignment of the two sequences.

Optimal alignment of sequences for comparison can be performed using the Megalign program in the Lasergene bioinformatics software suite (DNASTAR, inc., Madison, W1) software, using default parameters. This program incorporates several alignment schemes described in the following references: dayhoff, M.O. (1978) A model of evolution change in proteins-substrates for detecting differences in relationships, in Dayhoff, M.O. (ed.) Atlas of Protein Sequence and Structure, National biological Research Foundation, Washington DC Vol.5, Suppl.3, pp.345.358; hein J., 1990, Unified Approach to Alignment and phenols pp.626-645Methods in Enzymology vol.183, Academic Press, Inc., San Diego, Calif.; higgins, d.g.and Sharp, p.m., 1989, cabaos 5: 151-153; myers, e.w. and Muller w., 1988, cabaos 4: 11-17; robinson, e.d., 1971, comb. 105; santou, n., Nes, m., 1987, moi.biol.evol.4: 406-425; sneath, p.h.a.and Sokal, r.r., 1973, Numerical taxomones and practice of Numerical taxomones, Freeman Press, San Francisco, CA; wilbur, w.j.and Lipman, d.j., 1983, proc.natl.acad.sci.usa 80: 726-730.

Preferably, the "percent sequence identity" is determined by comparing two optimally aligned sequences over a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20% or less, typically 5% to 15% or 10% to 12%, as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentages are calculated as follows: the number of positions at which the same nucleic acid base or amino acid residue is present in both sequences is determined to give the number of matched positions, the number of matched positions is divided by the total number of positions in the reference sequence (i.e., the window size) and the result is multiplied by 100 to give the percentage of sequence identity.

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

Suitable "moderately stringent conditions" include a prewash in 5 XSSC, 0.5% SDS, 1.0mM EDTA (pH 8.0); overnight hybridization at 50 ℃ to 65 ℃ in 5 XSSC; then washed twice with 2X, 0.5X and 0.2X SSC containing 0.1% SDS at 65 ℃ for 20 minutes each.

As used herein, "high stringency conditions" or "high stringency conditions" means: (1) low ionic strength and high temperature are used for washing, for example at 50 ℃ with 0.015M sodium chloride/0.0015M sodium citrate/0.1% sodium lauryl sulfate; (2) at 42 ℃ hybridization using denaturants such as formamide, for example containing 0.1% bovine serum albumin/0.1% FicolI/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer pH6.5, and 750mM sodium chloride, 75mM sodium citrate; or (3) washing at 42 ℃ with 50% formamide, 5 XSSC (0.75M NaCl, 0.075M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 XDenhardt's solution, sonicated salmon sperm DNA (50. mu.g/ml), 0.1% SDS and 10% dextran sulfate in 0.2 XSSC (sodium chloride/sodium citrate) at 42 ℃ and in 50% formamide at 55 ℃ followed by a high stringency wash consisting of EDTA-containing 0.1 XSSC at 55 ℃. The skilled person will understand how to adjust the temperature, ionic strength, etc., as necessary to accommodate factors such as probe length, etc.

It will be appreciated by those of ordinary skill in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences that encode a polypeptide as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are of particular interest to the present invention. In addition, alleles of genes 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 (e.g., 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 (e.g., hybridization, amplification, and/or database sequence comparison).

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

To prepare a polynucleotide using recombinant methods, as discussed further herein, a polynucleotide comprising a desired sequence can be inserted into a suitable vector, which can then be introduced into a suitable host cell for replication and amplification. The polynucleotide may be inserted into the host cell by any means known in the art. Cells are transformed by introduction (by direct uptake, endocytosis, transfection, F mating or electroporation) of the exogenous polynucleotide. The exogenous polynucleotide may be introduced and maintained in the cell as an unincorporated vector (e.g., 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 for the reproduction of DNA sequences. PCR techniques are well known in the art and are described in U.S. patent nos.4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: the Polymerase Chain Reaction, Mullis et al, eds., Birkauswer Press, Boston (1994).

RNA can be obtained using isolated DNA in a suitable vector and inserted into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example as disclosed in Sambrook et al, (1989).

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

Expression vectors are generally replicable polynucleotide constructs comprising a polynucleotide of the invention. This means that the expression vector must be replicable 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 (including adenovirus, adeno-associated virus, retrovirus) vectors, cosmids, and expression vectors disclosed in PCT publication No. WO 87/04462. The carrier component typically includes, but is not limited to, one or more of the following: a single sequence; an origin of replication; one or more marker genes; suitable transcriptional regulatory elements (e.g., promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational regulatory elements, such as a ribosome binding site, a translation initiation site, and a stop codon, are also typically required.

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

The invention also provides a host cell comprising any of the polynucleotides disclosed herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating 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 (e.g.E.coli or B.subtillis) and yeasts (e.g.S.cerevisae, S.pombe; or K.lactis). Preferably, the host cell expresses the cDNA at a level about 5-fold higher, more preferably about 10-fold higher, even more preferably 20-fold higher than the corresponding endogenous antibody or protein of interest if present in the host cell. Screening for host cells that specifically bind to AD 1-40 was performed 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

The compositions used in the methods of the invention comprise an effective amount of an anti-CGRP antagonist antibody or anti-CGRP antagonist antibody-derived polypeptide described herein. Examples of such compositions and methods of how to formulate are also described in the previous sections and below. In one embodiment, the composition further comprises a CGRP antagonist. In another embodiment, the composition comprises one or more anti-CGRP antagonist antibodies. In other embodiments, the anti-CGRP antagonist antibody recognizes human CGRP. In still other embodiments, the anti-CGRP antagonist antibody is humanized. In still other embodiments, the anti-CGRP antagonist antibody comprises a constant region that does not elicit an unwanted or undesirable immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the anti-CGRP antagonist antibody comprises one or more CDRs of antibody G1 (e.g., one, two, three, four, five, or in some embodiments all six CDRs from G1). In some embodiments, the anti-CGRP antagonist antibody is human.

It should be understood that: the composition may comprise more than one anti-CGRP antagonist antibody (e.g., a mixture of anti-CGRP antagonist antibodies recognizing different epitopes of CGRP). Other exemplary compositions comprise more than one anti-CGRP antagonist antibody recognizing the same epitope, or different classes of anti-CGRP antagonist antibodies binding to different epitopes of CGRP.

The compositions used in The present invention may further comprise a pharmaceutically acceptable carrier, excipient or stabilizer (Remington: The Science and practice of pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed.K.E.Hoover) in The form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at dosages and concentrations, and may contain buffers such as phosphate, citrate, or other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (for example octadecyl dimethyl benzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, phenethylammonium 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; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants, e.g. TWEEN^TM、PLURONICS^TMOr polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

anti-CGRP antagonist antibodies and compositions thereof may also be used in combination with other agents that act to enhance and/or supplement the effectiveness of the agent.

E. Reagent kit

The invention also provides a kit for use in the rapid method. The kits of the invention comprise one or more containers comprising an anti-CGRP antagonist antibody (e.g., a humanized antibody) or polypeptide as described herein, and instructions for use according to any of the methods of the invention described herein. In general, these instructions include a description of administering an anti-CGRP antagonist antibody to treat, ameliorate or prevent headache (e.g., migraine) according to any of the methods described herein. The kit further comprises a description of selecting an individual suitable for treatment, the selection based on identifying whether the individual has a headache or whether the individual is at risk for a headache. In still other embodiments, the instructions include a description of administering an anti-CGRP antagonist antibody to an individual at risk of headache (e.g., 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 still other embodiments, the antibody comprises one or more CDRs (e.g., one, two, three, four, five, or in some embodiments all six CDRs from G1) of antibody G1.

Instructions for use of the anti-CGRP antagonist antibodies will generally include information such as dosage, dosing schedule and route of administration for the planned 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 kits of the invention are typically written instructions on a label or on an inner page of a package (e.g., a paper sheet included in the kit), but may also be machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disc).

The label or package insert indicates that the composition is for use in treating, ameliorating and/or preventing headache (e.g., migraine). Instructions for practicing any of the methods described herein can be provided.

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to: tubes, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Also of note are packages for administration in combination with a specific instrument, e.g., an inhaler, a nasal administration instrument (e.g., a nebulizer) or a fusion instrument such as a mini-pump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or bottle with a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or bottle with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CGRP antagonist antibody. The container may further comprise a second pharmaceutically active agent.

The kit may optionally provide other components such as buffers and instructional information. Typically, the kit contains a container and a label or package insert on or associated with the container.

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

Examples

Example 1: generation and characterization of monoclonal anti-bodies to CGRP

anti-CGRP antibodies were generated. To generate anti-CGRP antibodies with cross-species reactivity to rat and human CGRP, mice were immunized at different intervals with 25-100 μ g of human α -CGRP or β -CGRP conjugated to KLH in adjuvant (50 μ l per footpad, 100 μ l per mouse). Immunization is generally as described by georlis HJ et al, 1989, j.immunol.methods 124: 95-102; kenney JS et al, 1989, j. immunological. methods 121: 157-166; and Wicher K et al, 1989, int. 128-135. Mice were first immunized with 50 μ g of human α -CGRP or β -CGRP conjugated to KLH in CFA (complete Freund's adjuvant). Mice were immunized a second time 21 days later with 25 μ g of human β -CGRP conjugated to KLH in IFA (incomplete Freund's adjuvant) (for mice first immunized with human α -CGRP) or α -CGRP (for mice first immunized with human β -CGRP). Twenty three days after the second immunization a third immunization was performed with 25 μ g rat α -CGRP conjugated to KLH in IFA. Ten days later, antibody titers were checked 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). Final boosting was performed 32 days after the fourth immunization with 100. mu.g of lytic peptide (rat. alpha. -CGRP).

Splenocytes were obtained from immunized mice and fused with NSO myeloma cells at a ratio of 10: 1 with polyethylene glycol 1500. Hybrid 96-well plates were placed in DMEM containing 20% horse serum and 2-oxaloacetate/pyruvate/insulin (Sigma) and hypoxanthine/aminopterin/thymidine selection was initiated. 100 μ l DMEM containing 20% horse serum was added to all wells on day 8. The supernatants of the hybrids were screened by using an antibody capture immunoassay. The determination of the antibody species is performed using a second antibody that is species-specific.

Based on their binding to human and rat CGRP, a panel of monoclonal antibody-producing cell lines was selected for further analysis. These antibodies and characteristics are shown in tables 2 and 3 below.

Purification and Fab fragment preparation. Monoclonal antibodies selected for further analysis were purified from the supernatants of hybridoma cultures using protein a affinity chromatography. The supernatant was equilibrated to pH 8. The supernatant was then applied to a protein A column MabSelect (Amersham biosciences #17-5199-02) equilibrated to pH8 with PBS. The column was washed with 5 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, pH 7.4. Antibody concentrations were determined by SDS-PAGE using a standard curve of murine monoclonal antibodies.

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

Affinity assay for Fab. The affinity of the anti-CGRP monoclonal antibody is 25 DEG COr Biacore3000 at 37 deg.C^TMSurface Plasmon Resonance (SPR) system (Biacore, INC, Piscataway NJ) was assayed with 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 capturing the N-terminal biotinylated CGRP Peptide (a custom-made product from genscript corporation, New Jersey or Global Peptide Services, Colorado) from streptavidin pre-immobilized on SA chips and measuring the binding kinetics of antibody Fab titrated across the CGRP surface. Biotinylated CGRP was diluted into HBS-EP and injected onto the chip at a concentration of less than 0.001 mg/ml. Two antibody density ranges were achieved using different flow times across the channels of the individual chips: < 50 Response Units (RU) for detailed kinetic studies and about 800 RU for concentration studies and screening. Will typically span 1 μ M-0.1nM (estimated K for 0.1-10 ×)_D) Two-or three-fold serial dilutions of purified Fab fragment at concentrations were injected at 100 μ L/min and allowed for a dissociation time of 10 min. After each binding cycle, the surface was regenerated with 25mM NaOH in 25% v/v ethanol, which was able to withstand hundreds of cycles. Kinetic binding rates (k. times. enzyme 6.99-110) were obtained by fitting the data to a Langmuir binding model (Karlsson, R. Roos, H. Fagerstam, L. Petersson, B. (1994). Methods enzyme 6.99-110) using the BIAevaluation program_on) And dissociation rate (k)_off). From the ratio KD ═ k_off/k_onCalculation of the Overall equilibrium dissociation constant (K)_D). 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 to human α -CGRP, the affinity of the Fab fragment for various CGRP fragments was measured by capturing the N-terminally biotinylated CGRP fragment amino acids 19-37 and amino acids 25-37 on an SA-sensing chip as described above. FIG. 1 shows their affinity measured at 25 ℃. As shown in FIG. 1, any antibodies other than antibody 4901 bind to human α -CGRP fragments 19-37 and 25-37 with similar affinity for full-length human α -CGRP (1-37). Antibody 4901 binds human α -CGRP fragment 25-37 with six-fold lower affinity than to full-length human α -CGRP fragment, primarily due to the loss of detachment rate. The data indicate that anti-CGRP antibodies typically bind to the C-terminus of CGRP.

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

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

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

n.d. ═ not determined

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

The body 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). Except for SEQ ID NOS: all peptides except 36-40 were C-terminally amidated. Residues in bold represent point mutations.

Example 2: screening for anti-CGRP antagonist antibodies Using in vitro assays

Murine anti-CGRP antibodies were further screened in vitro for antagonist activity using cell-based cAMP activation and binding assays.

Antagonist activity was measured by cAMP assay. Five microliters of human or rat α -CGRP (final concentration 50nM) were dispersed in 384-well plates (Nunc, Cat. No.264657) in the presence or absence of anti-CGRP antibody (final concentration 1-3000nM) or rat α -CGRP or human α -CGRP (final concentration 0.1nM-10 μ M; as a positive control for c-AMP activation). Ten microliters of cells (20mM HEPES, pH7.4, 146mM NaCl, 5mM KCl, 1mM C.alpha.Cl) in stimulation buffer (human SK-N-MC if human. alpha. -CGRP, rat L6 from ATCC if rat. alpha. -CGRP) were added to the wells of the plates₂、1mM MgCl₂And 500. mu.M 3-isopropyl-1-methylxanthine (IBMX)). The plates were incubated at room temperature for 30 minutes.

Following incubation, cAMP activation was performed using a HitHunter enzyme fragment complementation assay (Applied Biosystems) according to the manufacturer's instructions. The experiment is based on a genetically engineered beta-galactosidase enzyme consisting of two fragments called Enzyme Acceptor (EA) and Enzyme Donor (ED). When the two fragments are separated, the enzyme is inactivated. When the fragments are brought together, they can spontaneously recombine to form the active enzyme by a process called complementation. The EFC test platform uses an ED-cAMP peptide conjugate in which cAMP is recognized by anti-cAMP. The ED fragment is capable of recombining with EA to form an active enzyme. In this assay, anti-cAMP antibodies are optimally titrated to bind the ED-cAMP conjugate and the inhibitory enzyme form. Cell lysatecAMP levels in the sample compete with the ED-cAMP conjugate for binding to anti-cAMP antibodies. The amount of free ED in the assay is proportional to the concentration of cAMP. Thus, cAMP is measured by the formation of an active enzyme, which is quantified by the inversion of the β -galactosidase luminescence substrate. cAMP activation experiments were performed by adding 10. mu.l lysis buffer and anti-cAMP antibody (1: 1 ratio) followed by incubation at room temperature for 60 min. Then 10. mu.l ED-cAMP reagent was added to each well and incubated for 60 minutes at room temperature. After incubation, 20. mu.l of EA reagent and CL mix (containing substrate) (1: 1 ratio) were added to each well and incubated at room temperature for 1-3 hours or overnight. Plate readings were taken at 1 second/well on PMT instrument or 30 seconds/plate on imager. Antibodies that inhibit cAMP activation by α -CGRP are identified in tables 2 and 3 above (referred to as "yes"). The data in tables 2 and 3 indicate that antibodies demonstrated antagonist activity in the assay generally have high affinity. For example, human α -CGRP has a K of about 80nM or less_D(measured at 25 ℃) or about 47nM or less for rat alpha-CGRP_DAntibodies (measured at 37 ℃) showed antagonist activity in this assay.

Radioligand binding assay. Binding assays to measure IC of anti-CGRP antibodies in blocking CGRP binding receptors were performed as described previously₅₀. Zimmermann et al, Peptides 16: 421-4, 1995; mallee et al, j.biol.chem.277: 14294-8, 2002. Membranes (25. mu.g) from SK-N-MC cells were combined in a total volume of 1mL with 10pM¹²⁵I-human alpha-CGRP incubation buffer (50mM Tris-HCL, pH7.4, 5mM MgCL)₂0.1% BSA) at room temperature for 90 minutes. To determine the Inhibitory Concentration (IC)₅₀) Antibodies or unlabeled CGRP from approximately 100-fold higher concentration storage solutions (as controls) were solubilized to different concentrations in incubation buffer and compared to membranes and 10pM¹²⁵I-human α -CGRP were incubated for the same time. The incubation was stopped by filtration through glass microfibers (GF/B, 1 μm) blocked with 0.5% polyethylemimine. Dose response curves were plotted and obtained by using the equation KI ═ IC₅₀V. (1+ ([ ligand ]]/K_D) Determination of K_IA value; wherein for human alpha-CGRP for CGRP1 receptor present in SK-N-MC cellsEquilibrium dissociation constant K_D＝8pM，B_max0.025pmol/mg protein. Reported IC₅₀The value (in terms of IgG molecules) is converted to binding sites (multiplied by 2) so that it can bind to the affinity (K) determined by Biacore_D) Comparison (see table 2).

Table 2 shows the IC's of murine antibodies 7E9, 8B6, 6H2 and 4901₅₀The value is obtained. Data indicate that antibody affinity generally corresponds to IC₅₀: with higher affinity (lower K)_DValue) has a lower IC in radioligand binding assays₅₀。

Example 3: effect of anti-CGRP antagonist antibodies on cutaneous vasodilation induced by rat saphenous nerve stimulation By using

To test antagonist activity of anti-CGRP antibodies, the effect of the antibodies on cutaneous vasodilation by rat saphenous nerve stimulation was tested using the previously described rat model. Escott et al, br.j.pharmaco 1.110: 772-776, 1993. In this rat model, the electrical stimulation of the saphenous nerve induces release of CGRP from the nerve endings, resulting in increased skin blood flow. Blood flow in the skin of the male SpragueDwaley rats (170-300g from Charles River Hollester) was measured after saphenous nerve stimulation. Rats were maintained under anesthesia with 2% isoflurane. Benzalkonium bromide (30mg/kg, administered intravenously) was given at the beginning of the experiment to minimize vasoconstriction of the sympathetic nerve fibers of the saphenous nerve with stimulation. Body temperature was maintained at 37 ℃ by using a rectal probe connected to a temperature controlled heating blanket. In addition to the experiment shown in fig. 3, where test compounds and controls were injected via the tail vein, compounds including antibodies, positive controls (CGRP 8-37) and vehicle (PBS, 0.01% tween 20) were administered intravenously via the right femoral vein, and for the experiments shown in fig. 2A and 2B, antibodies 4901 and 7D11 were injected Intraperitoneally (IP). Due to the half-life of the positive control compound CGRP 8-37 (vasodilator antagonist), it was administered at 400nmol/kg (200. mu.l) 3-5 minutes 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 FIGS. 2A and 2B, antibody 4901(25mg/kg), antibody 7D11(25mg/k), or vehicle control (PBS with 0.01% Tween 20) was administered Intraperitoneally (IP) 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 containing 0.01% Tween 20) was administered intravenously 24 hours prior to electrical pulse stimulation. Following administration of the antibody or vehicle control, the saphenous nerve of the right hind limb was surgically exposed, cut proximally and covered with plastic film to prevent desiccation. The laser doppler probe was placed on the dorsal-medial side of the hind paw skin, which is the region of saphenous innervation. Skin blood flow (measured as blood cell flux) was monitored with a laser doppler flow meter. When a stable baseline flux (less than 5% change) was established for at least 5 minutes, the nerve was placed on a platinum bipolar electrode and stimulated with electrical stimulation (2Hz, 10V, 1ms, 30 seconds) for 60 pulses, and after 20 minutes, stimulated again. For each flux in response to electrical pulse stimulation, the cumulative change in skin blood flow is assessed by the area under the flux-time curve (AUC, which is equal to the change in flux multiplied by the change in time). The average of the blood flow in response to these two stimuli was calculated. Animals were kept under anesthesia for a period of one to three hours.

As shown in fig. 2A and 2B, the increase in blood flow stimulated by the application of the electrical pulse on the saphenous nerve was inhibited by the presence of CGRP 8-37(400nmol/kg, intravenous administration), antibody 4901(25mg/kg, intraperitoneal administration), or antibody 7D11(25mg/kg, intraperitoneal administration), as compared to the control. CGRP 8-37 is administered 3-5 minutes prior to saphenous nerve stimulation; the antibody was administered 72 hours prior to saphenous nerve stimulation. As shown in fig. 3, the increase in blood flow stimulated by application of electrical pulses to the saphenous nerve was inhibited by the presence of antibody 4901, which was administered intravenously at different doses (1mg/kg, 2.5mg/kg, 5mg/kg, and 25mg/kg) 24 hours 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 hind limb was surgically exposed, cut proximally and covered with plastic film to prevent desiccation. The laser doppler probe was placed on the dorsocentric portion of the hind paw skin, which is the region of the saphenous innervation. Skin blood flow (measured as blood cell flux) was monitored with a laser doppler flow meter. Thirty to forty-five minutes after the injection of benzalkonium bromide, a stable baseline flux (less than 5% change) was established for at least 5 minutes, the nerve was placed on a platinum bipolar electrode and stimulated again with electrical stimulation (2Hz, 10V, 1ms, for 30 seconds) after 20 minutes. The mean of the blood flow flux in response to these two stimuli was used to establish a baseline response to 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.). Subsequently, nerves were stimulated (2Hz, 10V, 1ms, for 30 seconds) 30 min, 60 min, 90 min and 120 min after antibody or vehicle administration. Animals were kept under anesthesia for a period of approximately three hours. For each flux in response to electrical pulse stimulation, the cumulative change in skin blood flow is assessed by the area under the flux-time curve (AUC, which is equal to the change in flux multiplied by the change in time).

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

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

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. Antibody G1 and its antibodies were expressed and characterized using the following method.

The expression vector used. Expression of antibody Fab fragments was localized to the same sites as barkas (2001) Phagedisplay: under the control of a similar IPTG inducible promoter lacZ as described in 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 the CH1 constant domain of IgG2 human immunoglobulin, Ig γ -2 chain C region, protein number P01859; immunoglobulin kappa light chain (human), protein No. CAA 09181.

Small scale Fab preparation. From e.coli (e.coli) transformed with Fab libraries (using electroporation competent TG1 cells or chemically competent Top 10 cells), single clones were used to inoculate both the master plate (agar LB + carbenicillin (50ug/mL) + 2% glucose) and the working plate (2 mL/well, 96 wells/plate), with each well containing 1.5mL _ LB + carbenicillin (50ug/mL) + 2% glucose. Breathable adhesive seals (ABgene, Surrey, UK) were used for the plates. Incubating each plate at 30 ℃ for 12-16 hours; the work plate was shaken vigorously. The master plate was stored at 4 ℃ until needed, and the cells from the working plate were centrifuged (400rpm, 4 ℃,20 min) and resuspended in 1.0mL LB + carbenicillin (50ug/mL) +0.5mM IPTG, and Fab expression was induced by vigorous shaking at 30 ℃ for 5 hours. The induced cells were centrifuged at 4000rpm at 4 ℃ for 20 minutes and resuspended in 0.6mL BiacoreHB-SEP buffer (10mM Hepes pH7.4, 150mM NaCl, 3mM EDTA, 0.005% v/v P20). Lysis of HB-SEP suspended cells was accomplished by freezing (-80 ℃) and then thawing at 37 ℃. The cell lysate was centrifuged at 4000rpm for 1 hour at 4 ℃, the debris was separated from the Fab-containing supernatant, and the supernatant was subsequently filtered (0.2 μm) using a Millipore Multiscreen Assay System 96-well filter plate and a vacuum manifold (vacuum manifold). The filtered supernatant was analyzed using Biacore by injecting it over CGRP on a sensor chip. Affinity-selective clones expressing Fab were recovered from the master plate, providing 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. Erlenmeyer flasks containing 150mL LB + carbenicillin (50ug/mL) + 2% glucose were inoculated with 1mL of "initial" overnight culture from affinity-selected Fab-expressing E.coli clones. Plasmid DNA (QIAprep mini-prep, Qiagen kit) was prepared for sequencing and further processing using the remainder of the initial culture (. about.3 mL). Large cultures were incubated at 30 ℃ with vigorous shaking until an OD of 1.0 was reached_600nm(typically for 12-16 hours). Cells were pelleted by centrifugation at 4000rpm for 20 minutes at 4 ℃ and resuspended in 150mL LB + carbenicillin (50ug/mL) +0.5mM IPTG. After 5 hours of expression at 30 ℃, the cells were pelleted by centrifugation at 4000rpm, 4 ℃ for 20 minutes, suspended in 10mL of 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-NTA upflow sepharose (Qiagen, valencia. ca) column equilibrated with PBS, pH8, followed by washing with 5 volumes of PBS, pH 8. Individual fabs were eluted in different fractions with PBS (pH 8) +300mM imidazole. Fab-containing fractions were pooled and dialyzed against PBS and then quantified by ELISA prior to affinity characterization.

And (4) preparing a complete antibody. To express the intact antibody, the heavy and light chain variable regions were cloned into mammalian expression vectors and transfected into HEK 293 cells using lipofectamine for transient expression. The antibody was 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 7-613); a synthetic 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); human heavy chain IgG2 constant region containing the following mutations: A330P331 to S330S331 (amino acids encoding the reference wild type IgG2 sequence; see Eur. J. Immunol. (1999) 29: 2613-2624). Vector pdb.cgrp.hfcgi was deposited at ATCC on july 15 of 2005 and was designated ATCC No. pta-6867.

The vector peb. cgrp. hkgi is a 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. hgki was deposited and designated ATCC No. pta-6866 at july 15 of 2005.

Biacore assay for affinity assay. Affinity of the G1 monoclonal antibody and its variants Using a Biacore3000 at 25 ℃ or 37 ℃^TMSurface Plasmon Resonance (SPR) system (Biacore, INC, Piscataway NJ). Affinity was determined by capturing the N-terminal biotinylated CGRP peptide or fragment by pre-immobilized streptavidin (SA-sensing chip) and measuring the binding kinetics of the antibody G1Fab fragment or variant titrated across the CGRP or fragment on the chip. 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 by diluting N-biotinylated CGRP to a concentration of less than 0.001mg/ml in HBS-EP buffer and injecting across the SA-sensing chip using various contact times. Low capacity surfaces corresponding to < 50 Response Unit (RU) capture levels were used for high resolution kinetics studies, while high capacity surfaces (about 800 RU of captured CGRP) were used for concentration studies, screening and solution affinity assays. Increase by serially diluting the antibody G1Fab two-fold or three-fold to span 1uM-0.1nM (estimated K against 0.1-10X)_D) Kinetic data were obtained for the concentrations of (c). Typically, the sample is injected at 100 μ Ι/min for 1 minute and allowed for a dissociation time of at least 10 minutes. After each binding cycle, use25mM NaOH in 25% v/v ethanol regenerates the surface, which can withstand hundreds of cycles. The entire titration series (typically generated in duplicate) was fit to the Langmuir binding model as a whole by using the BIAevaluation program. This results in a unique pair of binding and dissociation kinetic rate constants (k, respectively) for each binding interaction_onAnd k_off) The ratio gives the equilibrium dissociation constant (K)_D＝k_off/k_on). Affinity (K) determined in this manner_DValues) are listed in tables 6 and 7.

High resolution analysis of binding interactions with extremely low off rates (offrates). For interactions with extremely low off-rates (especially the binding of antibody G1Fab to human α -CGRP on-chip at 25 ℃), affinity was obtained in two part experiments. The above protocol with the following modifications was used. The association rate constant (k) was determined by injecting a 2-fold titer series (in duplicate) spanning 550nM-1nM at 100 uL/min for 30 seconds and allowing only 30 seconds of dissociation phase_on). The dissociation rate constant (k) was determined by injecting the same titer series of three concentrations (high, medium and low) in duplicate for 30 seconds and allowing a2 hour dissociation phase_off). The affinity (K) of each interaction was obtained by combining the sums obtained in the two types of experiments_D) As shown in table 5.

The solution affinity was determined by Biacore. The solution affinity of antibody G1 for rat α -CGRP and F37A (19-37) human D-CGRP was measured by Biacore at 37 ℃. High capacity CGRP chip surfaces (high affinity human α -CGRP was selected for detection purposes) were used and HBS-EP running buffer was flowed at 5 uL/min. A constant concentration of 5nM of the antibody G1Fab fragment (to be at or below the expected K for solution-based interactions)_D) Preincubated with competing peptides spanning 1nM to 1. mu.M at final concentrations in 3-fold serial dilutions, either rat α -CGRP or F37A (19-37) human α -CGRP. In the absence or presence of solution-based competitor peptide, antibody G1Fab solution was injected across the CGRP on the chip and monitored for the absence of binding response detected on the chip surface due to solution competition. Using a calibration curve to correct for thisThese binding responses were converted to "free Fab concentrations" and curves were constructed by titrating the antibody G1Fab alone (5, 2.5, 1.25, 0.625, 0.325 and 0nM) across the CGRP on the chip. The "free Fab concentration" was plotted against the concentration of the solution-based competing peptide used to generate each data point and fitted to the solution affinity model using BIAevaluation software. The solution affinities determined by this approach (directly) are shown in tables 5 and 7 and used to confirm the affinities obtained when Fab was injected directly onto the SA chip through the N-biotinylated CGRR. The close agreement of the affinities determined by these two methods confirms that the binding of the N biotinylated form of CGRP to the chip does not alter its native solution binding activity.

Table 5 below shows the 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 over N biotinylated CGRP on SA chips. To better analyze the affinity of binding interactions with very low off-rates, the affinity was also determined in two-part experiments to supplement the direction of the experiment, and the solution affinity of the rat α -CGRP interaction was also determined (as described above). The close agreement of the affinities measured in the two experimental directions confirms that the affinity of native rat α -CGRP is unchanged when it is N-biotinylated in solution and bound to the SA chip.

TABLE 5 affinity of antibody G1Fab titrated through CGRP on a chip

Affinity of α -CGRP (rat and human) was determined in a high resolution two-part experiment in which the dissociation phase was monitored for 2 hours (k)_on、k_offAnd K_DValues of (d) represent the mean of n replicates, and standard deviations are expressed as percent variance). Affinity for β -CGRP (rat and human) was determined by bulk analysis using only 20 min dissociation phase, which was not accurate enough to quantify their extreme off-rates (their off-rates)The rates may be slower than set forth herein and thus their affinities may be even higher). The antibody G1Fab dissociates extremely slowly from all CGRPs (except the a-rat CGRP) at an off-rate that reaches the resolution limit of Biacore experiments (especially at 25 ℃).

Solution affinity determined by measuring the absence of binding response detected by CGRP on a chip for antibody G1Fab preincubated with solution-based rat α -CGRP competitor.

Table 6 below shows antibodies with amino acid sequence changes compared to antibody G1 and their affinity for rat α -CGRP 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 affinity of Fab fragments was determined by Biacore by flowing them through CGRP on an SA chip.

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

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

KD＝k_off/k_onAll k are obtained by global analysis of the Fab concentration series (analysis of G1 in high resolution mode), except that underlined_offValues were determined in the screening mode. Thus, underlined K_DValue is measured by_onAnd (4) carrying out experimental determination. Other k_onThe value was evaluated as the same as M25.

n.d. ═ not determined

To determine the epitope on human α -CGRP recognized by antibody G1, the Biacore experiment described above was used. The N-biotinylated version of human α -CGRP was purchased, enabling high affinity capture by SA-sensing chip. The binding of the G1Fab fragment to human α -CGRP on the chip in the absence or presence of VGRP peptide was determined. Typically, a 2000: 1mol peptide/Fab solution (e.g., 10uM peptide in 50nM G1 Fab) is injected over the human α -CGRP onto the chip. Figure 6 shows the percent binding blocked by the competitor peptide. The data shown in FIG. 6 indicate that peptides blocking 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 amidated at the C-terminus. Peptides F37A (19-37) and 19-37 of human α -CGRP, which were not amidated at the C-terminus, also blocked the binding of G1Fab to human α -CGRP by about 80% to 90%. Peptides 1-36 of human α -CGRP (which were not amidated at the C-terminus) blocked the binding of G1Fab to human α -CGRP by about 40%. Peptide fragment 19-36 of human α -CGRP (amidated at the C-terminus); peptide fragments 1-13 and 1-19 of human α -CGRP (neither of which is amidated at the C-terminus); and human amylin, calcitonin and adrenomedullin (all amidated at the C-terminus) do not compete with G1Fab for binding to human α -CGRP on the chip. These data demonstrate that G1 targets the C-terminal epitope of CGRP, and that recognition of the most terminal residue (F37) and its amidation are both important for binding.

The binding of the G1Fab to the human α -CGRP variant was also determined (at 37 ℃). Table 7 shows the affinities measured directly by crossing the G1Fab over the N-biotinylated human α -CGRP on the chip and the variant titration. The data in table 7 show that: antibody G1 binds to a C-terminal epitope, with F37 and G33 being the most important residues. When an additional amino acid residue (alanine) is added at the C-terminus (which is amidated), G1 does not bind CGRP.

TABLE 7.37 ℃ affinity of the G1Fab for human α -CGRP and variants (see Table 4 for their amino acid sequences)

On-chip CGRP	kon(1/Ms)	koff(1/s)	KD(nM)
				1-37(WT)	4.68×105	7.63×10-5	0.16 (high resolution K)D＝0.06)
19-37	4.60×105	7.30×10-5	0.16
				25-37	3.10×105	8.80×10-5	0.28
F27A(25-37)	3.25×105	1.24×10-4	0.38
				V28A(25-37)	3.32×105	9.38×10-5	0.28
P29A(25-37)	2.26×105	1.78×10-4	0.79
				T30A(25-37)	1.79×105	8.41×10-5	0.47
N31A(25-37)	2.17×105	1.14×10-4	0.53
				V32A(25-37)	2.02×105	3.46×10-4	1.71
G33A (25-37)	2.07×105	0.0291	141

On-chip CGRP	kon(1/Ms)	koff(1/s)	KD(nM)
				S34A(25-37)	2.51×105	7.64×10-4	3.04
K35A (19-37)	2.23×105	2.97×10-4	1.33
				K35E(19-37)	5.95×104	5.79×10-4	9.73
K35M(19-37)	2.63×105	1.34×10-4	0.51
				K35Q(19-37)	1.95×105	2.70×10-4	1.38
F37A(25-37)	8.90×104	8.48×1-3	95 (solution K)D＝172nM)
				38A(25-38A)	-	-	No binding detected

The data above indicate that the epitope bound by antibody G1 is located 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. Amidation of the residue F37 is also important for binding.

Example 5: anti-CGRP antagonist antibody G1 was applied to rat cryptoneurostimulation-induced skin Effect of tube dilation

To test the antagonist activity of anti-CGRP antibody G1, the effect of the antibody on cutaneous vasodilation due to rat saphenous nerve stimulation was tested using the rat model described in example 3. Briefly, rats were maintained under anesthesia with 2% isoflurane. Benzalkonium bromide (30mg/kg, administered intravenously) was given at the beginning of the experiment to minimize vasoconstriction of the sympathetic nerve fibers of the saphenous nerve with stimulation. Body temperature was maintained at 37 ℃ by using a rectal probe connected to a temperature controlled heating blanket. The saphenous nerve of the right hind limb was surgically exposed, cut proximally and covered with plastic film to prevent desiccation. The laser doppler probe was placed on the dorsocentric portion of the hind paw skin, which is the region of the saphenous innervation. Skin blood flow (measured as blood cell flux) was monitored with a laser doppler flow meter. In experiments in which the effect of the antibody within two hours of injection was measured thirty to forty-five minutes after the injection of benzalkonium bromide, when a stable baseline flux (less than 5% change) was established for at least 5 minutes, the nerve was placed on a platinum bipolar electrode and stimulated again with electrical stimulation (2Hz, 10V, 1ms, for 30 seconds) after 20 minutes. The mean of the blood flow flux in response to these two stimuli was used to establish a baseline response to electrical stimulation (time 0). Antibody G1(1mg/kg or 10mg/kg) or vehicle (PBS containing 0.01% Tween 20 in an equal volume to 10mg/kg G1) was then administered intravenously (i.v.). Subsequently, nerves were stimulated (2Hz, 10V, 1ms, for 30 seconds) 30 min, 60 min, 90 min and 120 min after antibody administration. Animals were kept under anesthesia for a period of approximately three hours. For each flux in response to electrical pulse stimulation, the cumulative change in skin blood flow is assessed by the area under the flux-time curve (AUC, which is equal to the change in flux multiplied by the change in time).

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

In experiments to determine the effect of antibodies at longer time points in saphenous vein experiments, rats were injected intravenously with the prescribed dose of antibody 24 hours or 7 days prior to preparation of animals for the above saphenous nerve stimulation. In these experiments, it was possible to establish a baseline response to electrical pulse stimulation in individual rats prior to dosing, comparing the treated groups with animals dosed with vehicle (PBS, 0.01% tween 20) at 24 hours or 7 days.

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

Figure 8C shows a curve fitting analysis of the dose response data shown in figures 8A and 8B to determine the dose required for 50% maximal effect (EC 50). The 24 hour EC50 was 1.3mg/kg, while the 7 day EC50 was slightly lower (0.8 mg/kg).

Example 6: urgency of anti-CGRP antagonist antibody G1 in dural artery (closed cranial window) experiments Sexual function

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. Experiments were performed as described previously (Williamson et al, Cephalalgia 17 (4): 518-24(1997)) with the following modifications. Sprague Dawley rats (300-400g) were anesthetized with 70mg/kg intraperitoneal pentobarbital. Anesthesia was maintained with 20mg/kg/hr intravenous pentobarbital. Rats were cannulated through the jugular vein for delivery of all drugs. Blood pressure was monitored with a probe (mikro-tip catheter, Millar instruments) threaded through the femoral artery into the abdominal aorta. The rat was tracheotomized and the respiration rate was maintained at 75 breaths per minute with a volume of 3.5 mL. The head was fixed to the orienting instrument and the scalp was removed, and a 2x6mm window was made in the lateral left wall region of the sagittal suture by thinning the bone with a dental drill. The platinum bipolar electrode was lowered onto the surface using a micromanipulator and covered with heavy mineral oil. A further window of 5x6mm was created on the side of the electrode window and filled with heavy mineral oil, through which the diameter of the midbrain artery (MMA) branch was continuously monitored with a CCD camera and video size analyzer (Living Systems). After preparation, rats were allowed to rest for no less than 45 minutes. A baseline response to electrical stimulation (15V, 10hz, 0.5ms pulse, duration 30 seconds) was established, and rats were then dosed intravenously with test compound (10mg/kg mu7E9, 300. mu.g/kg BIBN4096BS or PBS 0.01% Tween 20). Additional electrical stimulation was performed at 5(BIBN4096BS), 30, 60, 90 and 120 minutes post-dose. All data were recorded using graphics software (ADInstructions). As shown in fig. 9, mu7E9 at 10mg/kg significantly blocked electric field stimulation-stimulated MMA expansion within 60 minutes post-dose and maintained this effect for the duration of the experiment (120 minutes). In contrast, BIBN4096BS blocked MMA dilation within 5 minutes of administration, but the effect completely disappeared at 90 minutes. The strength of the closure was comparable between BIBN4096BS and mu7E 9.

Example 7: anti-CGRP antagonist antibody G1 in meningeal artery (closed cranial window) experiments Chronic effects in (1)

The purpose of this experiment was to determine whether anti-CGRP antibody was still able to block electrically stimulated MMA dilation 7 days after administration. Rats were prepared identically to 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 creation of the closed cranial window formulation and stimulation. It was not possible to establish a baseline expansion response to electrical stimulation prior to dosing as in the acute experiment, so the antibody group was compared to the MMA expansion in the vehicle (PBS, 0.01% tween 20) dosed control group. After the rats were allowed to rest for not less than 45 minutes, the dura mater was electrically stimulated at 30-minute intervals. The stimulation was at 2.5V, 5V, 10V, 15V and 20V, all at 10hz, 0.5ms pulse for 30 seconds.

As shown in fig. 10, 10mg/kg and 3mg/kg of G1 significantly blocked the distension of MMA stimulated by electrical stimulation in the 10 to 20 volt range. This data demonstrates that G1 is able to block electrically stimulated MMA dilation up to 7 days post-administration.

Example 8: morphine withdrawal hot flash model

The morphine withdrawal rat model is a well-established rodent model for the menopausal hot flash mechanism (Sipe et al, Brain Res.1028 (2): 191-202 (2004); Merchenthaler et al, Maturitas 30: 307-316 (1998); Katovich et al, Brain Res.494: 85-94 (1989); Simpkins et al, Life Sciences 32: 1957-1966 (1983)). Rats were essentially addicted to morphine by subcutaneous implantation of morphine pellets. Animals were injected with naloxone (an opioid antagonist) after addiction, which allowed them to be taken off immediately. This 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 intensity and time to those occurring in human hot flashes (Simpkins et al, Life Sciences 32: 1957-. In addition, if rats are treated with estradiol prior to induction of withdrawal, symptoms of hot flashes are reduced (Merchenthaler et al, Maturitas 30: 307-316 (1998)). This is why the morphine withdrawal model is believed to mimic clinical hot flashes.

Ovariectomized rats were custom made from Charles River Laboratories. Morphine dependence was created by subcutaneous implantation of morphine pellets (75mg of morphine base) not less than 7 days after ovariectomy. Two more pellets were implanted two days later. Rats were injected intravenously with 10mg/kg 4901 [. multidot. ] or vehicle (PBS, 0.01% tween) the following day. Two days after the second implantation of the pellet, rats were anesthetized with ketamine (90mg/kg) and lightly inhibited. A surface temperature thermocouple was glued to the tail base and a rectal thermocouple was used to measure the core temperature. Data was recorded using Chart software (instruments). After recording the stable baseline temperature for 15 minutes, naloxone (1mg/kg) was injected subcutaneously. The temperature was continuously recorded during the next 60 minutes. The results are shown in FIGS. 11A and 11B.

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 mentioned herein are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Preservation of biological materials

The following materials have been deposited under the American Type Culture Collection, 10801University Boulevard, Manassas, Virginia 20110-:

material	Antibody No.	ATCC No.	Date of storage
				pDb.CGRP.hFcGI	G1 heavy chain	PTA-6867	7/15/2005
pEb.CGRP.hKGI	G1 light chain	PTA-6866	7/15/2005

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, containing the following mutations: A330P331 to S330S331 (amino acids encoding the reference wild type IgG2 sequence; see Eur. J. Immunol. (1999) 29: 2613-2624).

These deposits were made in compliance with the provisions of the Budapest Treaty (Budapest treat) and its regulations, which are internationally recognized for the preservation of microorganisms for patent procedures. This ensures that the deposited viable cultures are maintained for 30 years from the date of deposit. According to the provisions of the budapest treaty, deposits are available from ATCC and follow the an1 protocol between Rinat Neuroscience corp. and ATCC which ensures: progeny of the deposited culture are permanently and without limitation publicly available to the public after relevant U.S. patent publications or any U.S. or foreign patent application publication (grant first), and are assured of being available to persons authorized by U.S. Commission of Patents and Trademarks according to 35USC Section 122 and its Commission's rules, including 37 CFR Section 1.14, especially 886OG 638.

The assignee of the present patent application has agreed that if a culture of the preserved material is to die or be lost or damaged when cultured under appropriate conditions, it will be quickly replaced with another identical material upon notification. The availability of 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 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 NQ: 10)

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAA

G1 heavy chain full antibody amino acid sequence (including the description herein)Modified IgG2) (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 whole antibody nucleotide sequence (SEQ ID NO: 14):

GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACGTGCTACCCTGTCCTGCAAAGCTTCCAMCGGGTTACCACCTACGTTTCCTGGTACCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACCGTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTCACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGTCAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAACGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTGAAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCGCGCGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACCCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGAGTGCTAA

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 alpha-CGRP)

NH₂-ACNTATCVTHRLAGLLSRSGGMVKSNFVPTNVGSKAF-CONH₂(human beta-CGRP)

NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSEAF-CONH₂(rat alpha-CGRP)

NH₂-SCNTATCVTHRLAGLLSRSGGVVKDNFVPTNVGSKAF-CONH₂(rat beta-CGRP)

Claims (16)

1. Has 50nM or less affinity (K) for human alpha-CGRP_D) The antibody of (1), the affinity being measured by surface plasmon resonance at 37 ℃.

2. The antibody according to claim 1, comprising a heavy chain variable region amino acid sequence that differs from SEQ ID NO: 1 at least 90% identical V_HA domain.

3. The antibody of claim 2, wherein the amino acid sequence of SEQ ID NO: 1 the amino acid residue at position 99 is L or is replaced with A, N, S, T, V or R, and wherein SEQ ID NO: 1, amino acid residue 100 is a or is replaced with L, R, S, V, Y, C, G, T, K or P.

4. The antibody according to claim 1, comprising a heavy chain variable region amino acid sequence that differs from SEQ ID NO: 2 at least 90% identical V_LA domain.

5. The antibody according to claim 1, comprising at least one CDR selected from:

seq ID NO: 3 CDR H1;

seq ID NO: 4 CDR H2;

seq ID NO: CDR H3 shown in FIG. 5;

d.SEQ ID NO: CDR L1 shown in fig. 6;

e.SEQ ID NO: CDR L2 shown in FIG. 7;

seq ID NO: CDR L3 shown in fig. 8;

g. variants of L1, L2 and H2 shown in table 6.

6. The antibody according to claim 1, comprising:

seq ID NO: v shown in 5_HCDR3, or a CDR that differs from SEQ ID NO: 5 different sequences; and

seq ID NO: v shown in 8_LCDR3, or a CDR that differs from SEQ ID NO: 8 different sequences.

7. An antibody comprising an amino acid sequence that differs from SEQ ID NO: 1 at least 90% identical V_HA domain and a polypeptide that differs in amino acid sequence from SEQ ID NO: 2 at least 90% identical V_LA domain.

8. The antibody of claim 7, wherein the antibody is an IgG, IgM, IgE, IgA or IgD molecule, or is derived therefrom.

9. The antibody according to claim 7, comprising a heavy chain produced by the expression vector of ATCC accession No. pta-6867.

10. The antibody according to claim 7, comprising a light chain produced by the expression vector of ATCC accession No. pta-6866.

11. A pharmaceutical composition comprising an antibody according to claim 1 and a pharmaceutically acceptable excipient.

12. A method of preventing or treating at least one vasomotor symptom in an individual comprising administering to the individual an effective amount of an anti-CGRP antagonist antibody.

13. The method of claim 12, wherein the vasomotor symptom is migraine with or without aura, hemiplegic migraine, cluster headache, migrainous neuralgia, prolonged headache, or tension headache.

14. The method according to claim 12, wherein the vasomotor symptom is hot flashes.

15. The method of claim 12, wherein said anti-CGRP antagonist antibody is any antibody of any one of claims 1 to 10.

16. The method of claim 12, wherein the anti-CGRP antagonist antibody is an antibody produced by the expression vectors of ATCC accession nos. PTA-6867 and PTA-6866.