U.S. patent application number 15/135378 was filed with the patent office on 2017-03-16 for methods for treating visceral pain by administering antagonist antibodies directed against calcitonin gene-related peptide.
The applicant listed for this patent is Labrys Biologics, Inc.. Invention is credited to Ariel Ates Pios, Kristian Todd Poulsen, David Louis Shelton, Joerg Zeller.
Application Number | 20170073412 15/135378 |
Document ID | / |
Family ID | 42983393 |
Filed Date | 2017-03-16 |
United States Patent
Application |
20170073412 |
Kind Code |
A1 |
Pios; Ariel Ates ; et
al. |
March 16, 2017 |
METHODS FOR TREATING VISCERAL PAIN BY ADMINISTERING ANTAGONIST
ANTIBODIES DIRECTED AGAINST CALCITONIN GENE-RELATED PEPTIDE
Abstract
The invention features methods for preventing or treating
visceral pain, including pain associated with functional bowel
disorder, inflammatory bowel disease and interstitial cystitis, by
administering an anti-CGRP antagonist antibody.
Inventors: |
Pios; Ariel Ates; (Pacifica,
CA) ; Poulsen; Kristian Todd; (San Francisco, CA)
; Shelton; David Louis; (Oakland, CA) ; Zeller;
Joerg; (Ann Arbor, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Labrys Biologics, Inc. |
Redwood City |
CA |
US |
|
|
Family ID: |
42983393 |
Appl. No.: |
15/135378 |
Filed: |
April 21, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14855959 |
Sep 16, 2015 |
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15135378 |
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14295583 |
Jun 4, 2014 |
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14855959 |
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14057747 |
Oct 18, 2013 |
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14295583 |
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13392860 |
Feb 27, 2012 |
8623366 |
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PCT/IB2010/053787 |
Aug 23, 2010 |
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14057747 |
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61237901 |
Aug 28, 2009 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/26 20130101;
A61P 13/12 20180101; C07K 2317/76 20130101; C07K 16/18 20130101;
A61P 1/00 20180101; A61P 13/08 20180101; A61P 1/18 20180101; C07K
2317/56 20130101; C07K 2317/55 20130101; A61P 25/04 20180101; A61P
1/14 20180101; A61P 13/10 20180101; A61P 9/08 20180101; A61P 29/00
20180101; A61P 15/00 20180101; C07K 2317/92 20130101; A61P 1/04
20180101; A61P 13/00 20180101; A61K 2039/505 20130101; A61P 15/04
20180101; C07K 2317/94 20130101; A61P 25/00 20180101; C07K 2317/565
20130101; A61P 15/12 20180101 |
International
Class: |
C07K 16/26 20060101
C07K016/26 |
Claims
1. A method of treating visceral pain and/or one or more symptoms
of visceral pain in an individual, comprising administration of a
therapeutically effective amount of an anti-CGRP antagonist
antibody to an individual suffering from or at risk for visceral
pain.
2. The method of claim 1, wherein the visceral pain is associated
with a functional bowel disorder (FBD).
3. The method of claim 2, wherein the FBD is selected from the
group consisting of gastro-esophageal reflux, dyspepsia, irritable
bowel syndrome (IBS) and functional abdominal pain syndrome
(FAPS).
4. The method of claim 1, wherein the visceral pain is associated
with inflammatory bowel disease (IBD).
5. The method of claim 4, wherein the IBD is selected from the
group consisting of Crohn's disease, ileitis and ulcerative
colitis.
6. The method of claim 1, wherein the visceral pain is associated
with renal colic, dysmenorrhea, cystitis, menstrual period, labor,
menopause, prostatitis or pancreatitis.
7. The method of claim 6, wherein the visceral pain is associated
with interstitial cystitis (IC).
8. The method of claim 1, wherein the anti-CGRP antagonist antibody
binds CGRP with a K.sub.D of 50 nM or less (as measured by surface
plasmon resonance at 37.degree. C.); and/or has a half life in-vivo
of at least 7 days.
9. The method of claim 1, wherein the anti-CGRP antagonist antibody
specifically binds to the C-terminal region of CGRP.
10. The method of claim 9, wherein the anti-CGRP antagonist
antibody specifically recognizes the epitope defined by the
sequence GSKAF (SEQ ID NO: 39).
11. The method of claim 1, wherein the anti-CGRP antibody comprises
a VH domain having the amino acid sequence shown in SEQ ID NO: 1 or
19.
12. The method of claim 1, wherein the anti-CGRP antibody comprises
a VL domain having the amino acid sequence shown in SEQ ID NO: 2 or
20.
13. The method of claim 1, wherein the anti-CGRP antibody comprises
at least one CDR selected from the group consisting of: (a) CDR H1
as set forth in SEQ ID NO: 3, 21, 33, 34, 36 or 37; (b) CDR H2 as
set forth in SEQ ID NO: 4, 22, 35 or 38; (c) CDR H3 as set forth in
SEQ ID NO: 5 or 23; (d) CDR L1 as set forth in SEQ ID NO: 6 or 24;
(e) CDR L2 as set forth in SEQ ID NO: 7 or 25; and (f) CDR L3 as
set forth in SEQ ID NO: 8 or 26.
14. The method of claim 1, wherein the anti-CGRP antibody comprises
a VH domain having the amino acid sequence shown in SEQ ID NO: 1
and a VL domain having the amino acid sequence shown in SEQ ID NO:
2.
15. The method of claim 1, wherein the anti-CGRP antibody is
produced by the expression vectors with ATCC Accession Nos.
PTA-6867 and/or PTA-6866.
16. The method of claim 1, wherein the anti-CGRP antibody
comprises: the antibody G1 heavy chain full antibody amino acid
sequence shown in SEQ ID NO: 11, with or without the C-terminal
lysine; and the antibody G1 light chain full antibody amino acid
sequence shown in SEQ ID NO: 12.
17. The method of claim 1, wherein the anti-CGRP antibody
comprises: the antibody G2 heavy chain full antibody amino acid
sequence shown in SEQ ID NO: 29; and the antibody G2 light chain
full antibody amino acid sequence shown in SEQ ID NO: 30.
18. A pharmaceutical composition for treatment and/or prevention of
visceral pain and/or symptoms of visceral pain in an individual,
comprising an anti-CGRP antagonist antibody and a pharmaceutically
acceptable carrier wherein the composition is prepared to be
peripherally administered.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/855,959, filed Sep. 16, 2015, which is a
continuation of U.S. patent application Ser. No. 14/295,583, filed
Jun. 4, 2014, which is a continuation of U.S. patent application
Ser. No. 14/057,747, filed Oct. 18, 2013, which is a continuation
of U.S. patent application Ser. No. 13/392,860, filed Feb. 27, 2012
(now U.S. Pat. No. 8,623,366), which is a national stage of PCT
International Application No. PCT/IB2010/053787, filed Aug. 23,
2010, which claim the priority benefit of provisional patent
application U.S. Ser. No. 61/237,901 filed on Aug. 28, 2009, said
applications are incorporated herein by reference in their
entireties.
REFERENCE TO SEQUENCE LISTING
[0002] This application is being filed electronically and includes
an electronically submitted sequence listing in .txt format. The
.txt file contains a sequence listing entitled
"44306704304SeqList.txt" created on Apr. 21, 2016 and having a size
of 28 KB. The sequence listing contained in this .txt file is part
of the specification and is herein incorporated by reference in its
entirety.
FIELD
[0003] The invention relates to a method of treating and/or
preventing visceral pain and/or symptoms of visceral pain using an
anti-CGRP antibody, and to an anti-CGRP antibody for use in the
prevention and/or treatment of visceral pain and/or symptoms of
visceral pain.
BACKGROUND
[0004] Visceral pain is a leading cause of patient visits to the
clinic, yet effective treatments for visceral pain are limited.
Visceral pain is difficult to manage clinically and often requires
the use of opiates. Although widely used, the severe dose-limiting
adverse effects of opiates often result in diminished efficacy.
Additionally, opiates carry the risk of abuse and physical
dependence and induce constipation and other unwanted adverse
effects, which are contraindicated in many cases and diminish
quality of life.
[0005] Visceral pain is pain associated with the viscera, which
encompass the internal organs of the body. These organs include,
e.g., the heart, lungs, reproductive organs, bladder, ureters, the
digestive organs, liver, pancreas, spleen, and kidneys. There are a
variety of conditions in which visceral pain may exist, such as,
for example, pancreatitis, labor, abdominal surgery associated with
ileus, cystitis, menstrual period, or dysmenorrhea. Likewise,
kidney pain, epigastric pain, pleural pain, and painful biliary
colic, appendicitis pain may all be considered to be visceral pain.
Substernal pain or pressure from early myocardial infarction is
also visceral. Diseases of the stomach, dudenum or colon can cause
visceral pain. Commonly encountered gastrointestinal (GI) disorders
that cause visceral pain include functional bowel disorder (FBD)
and inflammatory bowel disease (IBD). These GI disorders include a
wide range of disease states that are currently only moderately
controlled, including, with respect to FBD, gastro-esophageal
reflux, dyspepsia, irritable bowel syndrome (IBS) and functional
abdominal pain syndrome (FAPS), and, with respect to IBD, Crohn's
disease, ileitis and ulcerative colitis, all of which regularly
produce visceral pain.
[0006] IBS affects 10-20% of adults and adolescents worldwide
(Longstreth et al., 2006, Gastroenterology 130(5):1480-91). The
primary reason these patients seek medical attention is chronic
visceral pain believed to be due to enhanced visceral sensitivity
(Aziz, 2006, Gastroenterology 131(2):661-4). Patients with IBS have
been shown to have a lower visceral sensory threshold to colorectal
distension and that this is highly correlated to the visceral pain
symptoms (Delafoy et al, 2006, Gut 55(7):940-5). Colorectal
distension after trinitrobenzene sulfonic acid (TNBS) induced
colitis in rats is an animal model that has been used by many
researchers to explore the mechanisms of visceral hypersensitivity
(Gay et al, 2006, Neuroimmunomodulation 23; 13(2):114-121; Delafoy
et al, 2006; Adam et al., 2006, Pain 123(1-2):179-86).
[0007] Interstitial cystitis (IC) is a painful bladder syndrome
characterized in the clinic by urinary urgency, frequency and
chronic pelvic pain. Clinical studies indicate that this involves
visceral sensory afferent nerve hypersensitivity where the
sensation of bladder fullness occurs at lower than patients
indicates an increase in nerve density in the submucosa of the
bladder and evidence of neurogenic inflammation further normal
volumes and bladder fullness is perceived as painful.
Histopathology of IC suggests the involvement of visceral
afferents.
[0008] Visceral pain can be produced in response to, for example,
inflammation, distention, or increased pressure. It is not always
elicited by visceral injury. In addition, visceral pain is diffuse,
may be referred to other locations; and may be associated with
other autonomic and motor reflexes (e.g., nausea, lower-back muscle
tension from renal colic) (Lancet 1999, 353, 2145-48).
[0009] CGRP (calcitonin gene-related peptide) is a 37 amino acid
neuropeptide, which belongs to a family of peptides that includes
calcitonin, adrenomedullin and amylin. In humans, two forms of CGRP
(.alpha.-CGRP and .beta.-CGRP) exist and have similar activities.
They vary by three amino acids and exhibit differential
distribution. At least two CGRP receptor subtypes may also account
for differential activities. CGRP is a neurotransmitter in the
central nervous system, and has been shown to be a potent
vasodilator in the periphery, where CGRP-containing neuronal
processes are closely associated with blood vessels. CGRP-mediated
vasodilatation is also associated with neurogenic inflammation, as
part of a cascade of events that results in extravasation of plasma
and vasodilation of the microvasculature and is present in
migraine.
[0010] Spinally administered small molecule selective CGRP
antagonists have been shown to be useful in the treatment of
neuropathic and nociceptive pain conditions (Adwanikar et al, Pain,
2007, 132(1-2):53-66) suggesting that removal of endogenous CGRP
signalling in the spinal cord has an antinociceptive effect.
Reports have established that blocking CGRP signalling is effective
in reversing visceral hypersensitivity (VH) by systemically
injecting CGRP 8-37, a CGRP receptor antagonist (Delafoy et al.,
2006; Plourde et al., 1997, Am J Physiol. 273(1 Pt 1):G191-6; Julia
and Bueno, 1997, Am J Physiol. 272(1 Pt 1):G141-6). However, CGRP
8-37 has a very short half-life in-vivo and would therefore not be
a useful therapeutic. Thus, there is a critical medical need to
identify new therapeutics for the treatment and prevention of
visceral pain.
[0011] Throughout this application various publications (including
patents and patent applications) are referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference.
SUMMARY OF THE INVENTION
[0012] The present invention provides a method for preventing
and/or treating visceral pain and/or symptoms of visceral pain in
an individual, the method comprising administering a
therapeutically effective amount of an anti-CGRP antagonist
antibody to an individual suffering from or at risk for visceral
pain.
[0013] In some embodiments, the anti-CGRP antagonist antibody
administered peripherally. In other embodiments, the anti-CGRP
antagonist antibody is administered orally, sublingually, via
inhalation, transdermally, subcutaneously, intravenously,
intra-arterially, intra-articularly, peri-articularly, locally
and/or intramuscularly.
[0014] In some embodiments, the visceral pain is associated with a
functional bowel disorder (FBD). The FBD may be gastro-esophageal
reflux, dyspepsia, irritable bowel syndrome (IBS) and functional
abdominal pain syndrome (FAPS). In some embodiments, the visceral
pain is associated with inflammatory bowel disease (IBD). The IBD
may be Crohn's disease, ileitis or ulcerative colitis. In some
embodiments, the visceral pain is associated with renal colic,
dysmenorrhea, cystitis, menstrual period, labor, menopause,
prostatitis or pancreatitis. In some embodiments, the visceral pain
is associated interstitial cystitis (IC).
[0015] In some embodiments, the anti-CGRP antagonist antibody binds
to CGRP; blocks CGRP from binding to its receptor; blocks or
decreases CGRP receptor activation; inhibits blocks, suppresses or
reduces CGRP biological activity; increases clearance of CGRP;
and/or inhibits CGRP synthesis, production or release.
[0016] In some embodiments, the anti-CGRP antagonist antibody is a
human antibody or a humanized antibody. In some embodiments, the
anti-CGRP antagonist antibody is a monoclonal antibody. In some
embodiments, the anti-CGRP antagonist antibody can bind CGRP with a
K.sub.D of 50 nM or less (as measured by surface plasmon resonance
at 37.degree. C. and/or has a half life in-vivo of at least 7
days).
[0017] In some embodiments, the anti-CGRP antagonist antibody
specifically binds to the C-terminal region of CGRP. In some
embodiments, the anti-CGRP antagonist antibody specifically
recognizes the epitope defined by the sequence GSKAF (SEQ ID NO:
39). In some embodiments, the anti-CGRP antagonist antibody
comprises a VH domain that is at least 90% identical in amino acid
sequence to SEQ ID NO: 1 or 19.
[0018] In some embodiments, the anti-CGRP antagonist antibody
comprises a VL domain that is at least 90% identical in amino acid
sequence to SEQ ID NO: 2 or 20. In some embodiments, the anti-CGRP
antagonist antibody further comprises a VH domain that is at least
90% identical in amino acid sequence to SEQ ID NO: 1 or 19. In
other embodiments, the anti-CGRP antibody comprises at least one
CDR selected from the group consisting of: (a) CDR H1 as set forth
in SEQ ID NO: 3, 21, 33, 34, 36 or 37; (b) CDR H2 as set forth in
SEQ ID NO: 4, 22, 35 or 38; (c) CDR H3 as set forth in SEQ ID NO: 5
or 23; (d) CDR L1 as set forth in SEQ ID NO: 6 or 24; (e) CDR L2 as
set forth in SEQ ID NO: 7 or 25; (f) CDR L3 as set forth in SEQ ID
NO: 8 or 26; and (g) variants of L1, L2 and H2.
[0019] In some embodiments, the anti-CGRP antibody comprises the
antibody G1 heavy chain full antibody amino acid sequence shown in
SEQ ID NO: 11, with or without the C-terminal lysine. In some
embodiments, the anti-CGRP antibody comprises the antibody G1 light
chain full antibody amino acid sequence shown in SEQ ID NO: 12. In
some embodiments, the anti-CGRP antibody comprises the antibody G1
heavy chain full antibody amino acid sequence shown in SEQ ID NO:
11, with or without the C-terminal lysine; and the antibody G1
light chain full antibody amino acid sequence shown in SEQ ID NO:
12.
[0020] In some embodiments, the anti-CGRP antibody comprises the
antibody G2 heavy chain full antibody amino acid sequence shown in
SEQ ID NO: 29, with or without the C-terminal lysine. In some
embodiments, the anti-CGRP antibody comprises the antibody G2 light
chain full antibody amino acid sequence shown in SEQ ID NO: 30. In
some embodiments, the anti-CGRP antibody comprises the antibody G2
heavy chain full antibody amino acid sequence shown in SEQ ID NO:
29; and the antibody G2 light chain full antibody amino acid
sequence shown in SEQ ID NO: 30.
[0021] In some embodiments, the anti-CGRP antagonist antibody
comprises a VH domain that is at least 90% identical in amino acid
sequence to SEQ ID NO: 1 and a VL domain that is at least 90%
identical in amino acid sequence to SEQ ID NO: 2. In some
embodiments, the anti-CGRP antagonist antibody comprises a heavy
chain produced by the expression vector with ATCC Accession No.
PTA-6867. In some embodiments, the anti-CGRP antagonist antibody
comprises a light chain produced by the expression vector with ATCC
Accession No. PTA-6866. In some embodiments, the anti-CGRP
antagonist antibody is produced by the expression vectors with ATCC
Accession Nos. PTA-6867 and PTA-6866.
[0022] In some embodiments, the anti-CGRP is administered by
subcutaneous or intravenous injection between once, twice, three or
four times per month. In some embodiments, the anti-CGRP antagonist
antibody is administered at a concentration of between 5 to 100
mg/ml. In some embodiments, the anti-CGRP antagonist antibody is
administered at a concentration of between 1 to 100 mg/kg of body
weight.
[0023] In some embodiments, the anti-CGRP antagonist antibody does
not produce CNS impairment of motor coordination or attention. In
some embodiments, the anti-CGRP antagonist antibody is not
administered centrally, spinally or intrathecally. In some
embodiments, the anti-CGRP antagonist antibody is not a centrally,
spinally or intrathecal penetrating molecule.
[0024] In some embodiments, the anti-CGRP antagonist antibody is
administered separately, sequentially or simultaneously in
combination with one or more further pharmacologically active
compounds. In some embodiments, the one or more further
pharmacologically active compounds is/are selected from: an opioid
analgesic, e.g. morphine, heroin, hydromorphone, oxymorphone,
levorphanol, levallorphan, methadone, meperidine, fentanyl,
cocaine, codeine, dihydrocodeine, oxycodone, hydrocodone,
propoxyphene, nalmefene, nalorphine, naloxone, naltrexone,
buprenorphine, butorphanol, nalbuphine or pentazocine; a
nonsteroidal antiinflammatory drug (NSAID), e.g. aspirin,
diclofenac, diflusinal, etodolac, fenbufen, fenoprofen, flufenisal,
flurbiprofen, ibuprofen, indomethacin, ketoprofen, ketorolac,
meclofenamic acid, mefenamic acid, nabumetone, naproxen, oxaprozin,
phenylbutazone, piroxicam, sulindac, tolmetin or zomepirac, or a
pharmaceutically acceptable salt thereof; a barbiturate sedative,
e.g. amobarbital, aprobarbital, butabarbital, butabital,
mephobarbital, metharbital, methohexital, pentobarbital,
phenobartital, secobarbital, talbutal, theamylal or thiopental or a
pharmaceutically acceptable salt thereof; a benzodiazepine having a
sedative action, e.g. chlordiazepoxide, clorazepate, diazepam,
flurazepam, lorazepam, oxazepam, temazepam or triazolam or a
pharmaceutically acceptable salt thereof; an H1 antagonist having a
sedative action, e.g. diphenhydramine, pyrilamine, promethazine,
chlorpheniramine or chlorcyclizine or a pharmaceutically acceptable
salt thereof; a sedative such as glutethimide, meprobamate,
methaqualone or dichloralphenazone or a pharmaceutically acceptable
salt thereof; a skeletal muscle relaxant, e.g. baclofen,
carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or
orphrenadine or a pharmaceutically acceptable salt thereof; an NMDA
receptor antagonist, e.g. dextromethorphan
((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan
((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,
pyrroloquinoline quinone or
cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid or a
pharmaceutically acceptable salt thereof; an alpha-adrenergic, e.g.
doxazosin, tamsulosin, clonidine or
4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-
-2-yl)-5-(2-pyridyl) quinazoline; a tricyclic antidepressant, e.g.
desipramine, imipramine, amytriptiline or nortriptiline; an
anticonvulsant, e.g. carbamazepine or valproate; a tachykinin (NK)
antagonist, particularly an NK-3, NK-2 or NK-1 antagonist, e.g.
(.alpha.R,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-m-
ethyl-5-(4-methylphenyl)-7H-[[1,4]diazocino[2,1-g][1,7]naphthridine-6-13-d-
ione (TAK-637),
5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorop-
henyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one
(MK-869), lanepitant, dapitant or
3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylamino]-2-phenyl-piperidine
(2S,3S); a muscarinic antagonist, e.g oxybutin, tolterodine,
propiverine, tropsium chloride or darifenacin; a COX-2 inhibitor,
e.g. celecoxib, rofecoxib or valdecoxib; a non-selective COX
inhibitor (preferably with GI protection), e.g. nitroflurbiprofen
(HCT-1026); a coal-tar analgesic, in particular paracetamol;
neuroleptic such as droperidol; a vanilloid receptor agonist (e.g.
resinferatoxin) or antagonist (e.g. capsazepine); a beta-adrenergic
such as propranolol; a local anaesthetic, such as mexiletine; a
corticosteriod, such as dexamethasone; a serotonin receptor agonist
or antagonist; a cholinergic (nicotinic) analgesic; Tramadol.RTM.;
a PDEV inhibitor, such as sildenafil, vardenafil or taladafil; an
alpha-2-delta ligand such as gabapentin or pregabalin; and a
canabinoid.
[0025] The present invention further provides the use of an
anti-CGRP antagonist antibody for the manufacture of a medicament
for the prevention and/or treatment of visceral pain and/or
symptoms of visceral pain. In some embodiments, the medicament is
prepared to be peripherally administered. In some embodiments, the
anti-CGRP antagonist antibody acts peripherally on
administration.
[0026] The present invention further provides a pharmaceutical
composition for treatment and/or prevention of visceral pain and/or
symptoms of visceral pain in an individual, comprising an anti-CGRP
antagonist antibody and a pharmaceutically acceptable carrier. In
some embodiments the composition is prepared to be peripherally
administered.
[0027] The present invention further provides a kit comprising: a
pharmaceutical composition for treatment and/or prevention of
visceral pain and/or symptoms of visceral pain in an individual,
and instructions for the peripheral administration of a
therapeutically effective amount of said pharmaceutical composition
to an individual for treatment and/or prevention of visceral pain
and/or symptoms of visceral pain.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIGS. 1A and 1B depict a visceral pain model. (A) Antibody
4901 ("4901") or PBS control ("Vehicle") was administered
intravenously into animals injected with trinitrobenzene sulfonic
acid (TNBS) after abdominal laparotomy. Visceral pain threshold in
the animals was tested using balloon distension. Pain threshold is
indicated in mm Hg (y-axis). Sham represents animals injected with
a control (30% ethanol) solution instead of TNBS after laparotomy.
(B) CGRP receptor antagonist CGRP 8-37 or PBS control ("Vehicle")
was administered intravenously into TNBS-treated animals after
abdominal laparotomy. As in (A), visceral pain threshold in the
animals was tested using balloon distension, and pain threshold is
indicated in mm Hg (y-axis).
[0029] FIG. 2 depicts a visceral pain model. Antibody 4901 ("4901")
or PBS control ("Vehicle") was administered intravenously into
animals. Bladder motility, measured as number of contractions
(y-axis), was tested at 1 h, 3 h and 5 h after turpentine-induced
bladder inflammation.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The invention disclosed herein provides methods for treating
and/or preventing visceral pain in an individual by administering
to the individual a therapeutically effective amount of an
anti-CGRP antagonist antibody.
[0031] The invention disclosed herein also provides anti-CGRP
antagonist antibodies and polypeptides derived from G1 or its
variants shown in Table 6 of WO2007/054809, which is hereby
incorporated by reference in its entirety. The invention also
provides methods of making and using these antibodies and
polypeptides.
General Techniques
[0032] 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 explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M.
J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press;
Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998)
Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds.,
1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic
Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and
C. C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells
(J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in
Molecular Biology (F. M. Ausubel et al., eds., 1987); PCR: The
Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Wiley and Sons, 1999);
Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P.
Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL
Press, 1988-1989); Monoclonal antibodies: a practical approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995).
DEFINITIONS
[0033] An "antibody" is an immunoglobulin molecule capable of
specific binding to a target, such as a carbohydrate,
polynucleotide, lipid, polypeptide, etc., through at least one
antigen recognition site, located in the variable region of the
immunoglobulin molecule. As used herein, the term encompasses not
only intact polyclonal or monoclonal antibodies, but also fragments
thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv),
mutants thereof, fusion proteins comprising an antibody portion
(such as domain antibodies), and any other modified configuration
of the immunoglobulin molecule that comprises an antigen
recognition site. An antibody includes an antibody of any class,
such as IgG, IgA, or IgM (or sub-class thereof), and the antibody
need not be of any particular class. Depending on the antibody
amino acid sequence of the constant domain of its heavy chains,
immunoglobulins can be assigned to different classes. There are
five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM,
and several of these may be further divided into subclasses
(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The
heavy-chain constant domains that correspond to the different
classes of immunoglobulins are called alpha, delta, epsilon, gamma,
and mu, respectively. The subunit structures and three-dimensional
configurations of different classes of immunoglobulins are well
known.
[0034] 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.
Furthermore, in contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler and Milstein, 1975,
Nature, 256:495, or may be made by recombinant DNA methods such as
described in U.S. Pat. No. 4,816,567. The monoclonal antibodies may
also be isolated from phage libraries generated using the
techniques described in McCafferty et al., 1990, Nature,
348:552-554, for example.
[0035] As used herein, "humanized" antibodies refer to forms of
non-human (e.g. murine) antibodies that are specific chimeric
immunoglobulins, immunoglobulin chains, or fragments thereof (such
as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of
antibodies) that contain minimal sequence derived from non-human
immunoglobulin. For the most part, humanized antibodies are human
immunoglobulins (recipient antibody) in which residues from a
complementarity determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat, or rabbit having the desired
specificity, affinity, and biological activity. In some instances,
Fv framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore, the
humanized antibody may comprise residues that are found neither in
the recipient antibody nor in the imported CDR or framework
sequences, but are included to further refine and optimize antibody
performance. In general, the humanized antibody will comprise
substantially all of at least one, and typically two, variable
domains (e.g., a heavy chain variable domain and a light chain
variable domain), 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 optimally
also will comprise at least a portion of an immunoglobulin constant
region or domain (Fc), typically that of a human immunoglobulin.
Antibodies may have Fc regions modified as described in WO
99/58572. Other forms of humanized antibodies have one or more CDRs
(one, two, three, four, five, six) which are altered with respect
to the original antibody, which are also termed one or more CDRs
"derived from" one or more CDRs from the original antibody.
[0036] As used herein, "human antibody" means an antibody having an
amino acid sequence corresponding to that of an antibody produced
by a human and/or has been made using any of the techniques for
making human antibodies known in the art or disclosed herein. This
definition of a human antibody includes antibodies comprising at
least one human heavy chain polypeptide or at least one human light
chain polypeptide. One such example is an antibody comprising
murine light chain and human heavy chain polypeptides. Human
antibodies can be produced using various techniques known in the
art. In one embodiment, the human antibody is selected from a phage
library, where that phage library expresses human antibodies
(Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et
al., 1998, PNAS, (USA) 95:6157-6162; Hoogenboom and Winter, 1991,
J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol.,
222:581). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous immunoglobulin genes have been partially or
completely inactivated. This approach is described in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and
5,661,016. Alternatively, the human antibody may be prepared by
immortalizing human B lymphocytes that produce an antibody directed
against a target antigen (such B lymphocytes may be recovered from
an individual or may have been immunized in vitro). See, e.g., Cole
et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.
77, 1985; Boerner et al., 1991, J. Immunol., 147 (1):86-95; and
U.S. Pat. No. 5,750,373.
[0037] As used herein, the term "calcitonin gene-related peptide"
and "CGRP" refers to any form of calcitonin gene-related peptide
and variants thereof that retain at least part of the activity of
CGRP. For example, CGRP may be .alpha.-CGRP or .beta.-CGRP. As used
herein, CGRP includes all mammalian species of native sequence
CGRP, e.g., human, canine, feline, equine, and bovine.
[0038] As used herein, an "anti-CGRP antagonist antibody"
(interchangeably termed "anti-CGRP antibody") refers to an antibody
that is able to bind to CGRP and inhibit CGRP biological activity
and/or downstream pathway(s) mediated by CGRP signaling. An
anti-CGRP antagonist antibody encompasses antibodies that block,
antagonize, suppress or reduce (including significantly) CGRP
biological activity, including downstream pathways mediated by CGRP
signaling, such as receptor binding and/or elicitation of a
cellular response to CGRP. For purpose of the present invention, it
will be explicitly understood that the term "anti-CGRP antagonist
antibody" encompasses all the previously identified terms, titles,
and functional states and characteristics whereby the CGRP itself,
a CGRP biological activity (including but not limited to its
ability to mediate any aspect of visceral pain), or the
consequences of the biological activity, are substantially
nullified, decreased, or neutralized in any meaningful degree. In
some embodiment, an anti-CGRP antagonist antibody binds CGRP and
prevents CGRP binding to a CGRP receptor. In other embodiments, an
anti-CGRP antibody binds CGRP and prevents activation of a CGRP
receptor. Examples of anti-CGRP antagonist antibodies are provided
herein.
[0039] As used herein, the terms "G1" and "antibody G1" are used
interchangeably to refer to an antibody produced by the expression
vectors having deposit numbers ATCC-PTA-6867 and ATCC-PTA-6866. The
amino acid sequence of the heavy chain and light chain variable
regions are shown in SEQ ID NOs: 1 and 2. The CDR portions of
antibody G1 (including Chothia and Kabat CDRs) are diagrammatically
depicted in FIG. 5 of WO2007/054809, the content of which is herein
incorporated by reference in its entirety. The polynucleotides
encoding the heavy and light chain variable regions are shown in
SEQ ID NOs: 9 and 10. The characterization of antibody G1 is
described in the Examples of WO2007/054809.
[0040] As used herein, the terms "G2" and "antibody G2" are used
interchangeably to refer to an anti-rat CGRP mouse monoclonal
antibody as described in Wong H C et al. Hybridoma 12:93-106, 1993.
The amino acid sequence of the heavy chain and light chain variable
regions are shown in SEQ ID NOs: 19 and 20. The polynucleotides
encoding the heavy and light chain variable regions are shown in
SEQ ID NOs: 27 and 28. The CDR portions of antibody G2 are provided
in SEQ ID NOs: 21 to 26.
[0041] The terms "polypeptide", "oligopeptide", "peptide" and
"protein" are used interchangeably herein to refer to chains of
amino acids of any length, preferably, relatively short (e.g.,
10-100 amino acids). The chain may be linear or branched, it may
comprise modified amino acids, and/or may be interrupted by
non-amino acids. The terms also encompass an amino acid chain that
has been modified naturally or by intervention; for example,
disulfide bond formation, glycosylation, lipidation, acetylation,
phosphorylation, or any other manipulation or modification, such as
conjugation with a labeling component. Also included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), as well as other modifications known in the art. It
is understood that the polypeptides can occur as single chains or
associated chains.
[0042] As known in the art, "polynucleotide," or "nucleic acid," as
used interchangeably herein, refer to chains of nucleotides of any
length, and include 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 a chain by DNA or RNA polymerase. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and their analogs. If present, modification
to the nucleotide structure may be imparted before or after
assembly of the chain. The sequence of nucleotides may be
interrupted by non-nucleotide components. A polynucleotide may be
further modified after polymerization, such as by conjugation with
a labeling component. Other types of modifications include, for
example, "caps", substitution of one or more of the naturally
occurring nucleotides with an analog, internucleotide modifications
such as, for example, those with uncharged linkages (e.g., methyl
phosphonates, phosphotriesters, phosphoamidates, carbamates, etc.)
and with charged linkages (e.g., phosphorothioates,
phosphorodithioates, etc.), those containing pendant moieties, such
as, for example, proteins (e.g., nucleases, toxins, antibodies,
signal peptides, poly-L-lysine, etc.), those with intercalators
(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,
metals, radioactive metals, boron, oxidative metals, etc.), those
containing alkylators, those with modified linkages (e.g., alpha
anomeric nucleic acids, etc.), as well as unmodified forms of the
polynucleotide(s). Further, any of the hydroxyl groups ordinarily
present in the sugars may be replaced, for example, by phosphonate
groups, phosphate groups, protected by standard protecting groups,
or activated to prepare additional linkages to additional
nucleotides, or may be conjugated to solid supports. The 5' and 3'
terminal OH can be phosphorylated or substituted with amines or
organic capping group moieties of from 1 to 20 carbon atoms. Other
hydroxyls may also be derivatized to standard protecting groups.
Polynucleotides can also contain analogous forms of ribose or
deoxyribose sugars that are generally known in the art, including,
for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or
2'-azido-ribose, carbocyclic sugar analogs, alpha- or beta-anomeric
sugars, epimeric sugars such as arabinose, xyloses or lyxoses,
pyranose sugars, furanose sugars, sedoheptuloses, acyclic analogs
and abasic nucleoside analogs such as methyl riboside. One or more
phosphodiester linkages may be replaced by alternative linking
groups. These alternative linking groups include, but are not
limited to, embodiments wherein phosphate is replaced by
P(O)S("thioate"), P(S)S ("dithioate"), (O)NR.sub.2 ("amidate"),
P(O)R, P(O)OR', CO or CH.sub.2 ("formacetal"), in which each R or
R' is independently H or substituted or unsubstituted alkyl (1-20
C) optionally containing an ether (--O--) linkage, aryl, alkenyl,
cycloalkyl, cycloalkenyl or araldyl. Not all linkages in a
polynucleotide need be identical. The preceding description applies
to all polynucleotides referred to herein, including RNA and
DNA.
[0043] A "variable region" of an antibody refers to the variable
region of the antibody light chain or the variable region of the
antibody heavy chain, either alone or in combination. The variable
regions of the heavy and light chain each consist of four framework
regions (FR) connected by three complementarity determining regions
(CDRs) also known as hypervariable regions. The CDRs in each chain
are held together in close proximity by the FRs and, with the CDRs
from the other chain, contribute to the formation of the
antigen-binding site of antibodies. There are at least two
techniques for determining CDRs: (1) an approach based on
cross-species sequence variability (i.e., Kabat et al. Sequences of
Proteins of Immunological Interest, (5th ed., 1991, National
Institutes of Health, Bethesda Md.)); and (2) an approach based on
crystallographic studies of antigen-antibody complexes (Al-lazikani
et al (1997) J. Molec. Biol. 273:927-948)). As used herein, a CDR
may refer to CDRs defined by either approach or by a combination of
both approaches.
[0044] A "constant region" of an antibody refers to the constant
region of the antibody light chain or the constant region of the
antibody heavy chain, either alone or in combination.
[0045] As used herein, "immunospecific" binding of antibodies
refers to the antigen specific binding interaction that occurs
between the antigen-combining site of an antibody and the specific
antigen recognized by that antibody (i.e., the antibody reacts with
the protein in an ELISA or other immunoassay, and does not react
detectably with unrelated proteins).
[0046] An epitope that "preferentially binds" or "specifically
binds" (used interchangeably herein) to an antibody or a
polypeptide is a term well understood in the art, and methods to
determine such specific or preferential binding are also well known
in the art. A molecule is said to exhibit "specific binding" or
"preferential binding" if it reacts or associates more frequently,
more rapidly, with greater duration and/or with greater affinity
with a particular cell or substance than it does with alternative
cells or substances. An antibody "specifically binds" or
"preferentially binds" to a target if it binds with greater
affinity, avidity, more readily, and/or with greater duration than
it binds to other substances. For example, an antibody that
specifically or preferentially binds to a CGRP epitope is an
antibody that binds 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 moiety
or epitope) that specifically or preferentially binds to a first
target may or may not specifically or preferentially bind to a
second target. As such, "specific binding" or "preferential
binding" does not necessarily require (although it can include)
exclusive binding. Generally, but not necessarily, reference to
binding means preferential binding.
[0047] As used herein, "substantially pure" refers to material
which is at least 50% pure (i.e., free from 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.
[0048] A "host cell" includes an individual cell or cell culture
that can be or has been a recipient for vector(s) for incorporation
of polynucleotide inserts. Host cells include progeny of a single
host cell, and the progeny may not necessarily be completely
identical (in morphology or in genomic DNA complement) to the
original parent cell due to natural, accidental, or deliberate
mutation. A host cell includes cells transfected with a
polynucleotide(s) of this invention.
[0049] The term "Fc region" is used to define a C-terminal region
of an immunoglobulin heavy chain. The "Fc region" may be a native
sequence Fc region or a variant Fc region. Although the boundaries
of the Fc region of an immunoglobulin heavy chain might vary, the
human IgG heavy chain Fc region is usually defined to stretch from
an amino acid residue at position Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. The numbering of the residues
in the Fc region is that of the EU index as in Kabat. Kabat et al.,
Sequences of Proteins of Imunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md., 1991.
The Fc region of an immunoglobulin generally comprises two constant
domains, CH2 and CH3.
[0050] As used herein, "Fc receptor" and "FcR" describe a receptor
that binds to the Fc region of an antibody. The preferred FcR is a
native sequence human FcR. Moreover, a preferred FcR is one which
binds an IgG antibody (a gamma receptor) and includes receptors of
the Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses,
including allelic variants and alternatively spliced forms of these
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. FcRs are reviewed in
Ravetch and Kinet, 1991, Ann. Rev. Immunol., 9:457-92; Capel et
al., 1994, Immunomethods, 4:25-34; and de Haas et al., 1995, J.
Lab. Clin. Med., 126:330-41. "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., 1976, J. Immunol., 117:587; and
Kim et al., 1994, J. Immunol., 24:249).
[0051] "Complement dependent cytotoxicity" and "CDC" refer to the
lysing of a target in the presence of complement. The complement
activation pathway is initiated by the binding of the first
component of the complement system (C1q) to a molecule (e.g. an
antibody) complexed with a cognate antigen. To assess complement
activation, a CDC assay, e.g. as described in Gazzano-Santoro et
al., J. Immunol. Methods, 202:163 (1996), may be performed.
[0052] 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; down-regulation of cell surface
receptors (e.g. B cell receptor; BCR), etc. Such effector functions
generally require the Fc region to be combined with a binding
domain (e.g. an antibody variable domain) and can be assessed using
various assays known in the art for evaluating such antibody
effector functions.
[0053] A "native sequence Fc region" comprises an amino acid
sequence identical to the amino acid sequence of an Fc region found
in nature. A "variant Fc region" comprises an amino acid sequence
which differs from that of a native sequence Fc region by virtue of
at least one amino acid modification, yet retains at least one
effector function of the native sequence Fc region. Preferably, the
variant Fc region has at least one amino acid substitution compared
to a native sequence Fc region or to the Fc region of a parent
polypeptide, e.g. from about one to about ten amino acid
substitutions, and preferably from about one to about five amino
acid substitutions in a native sequence Fc region or in the Fc
region of the parent polypeptide. The variant Fc region herein will
preferably possess at least about 80% sequence identity with a
native sequence Fc region and/or with an Fc region of a parent
polypeptide, and 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.
[0054] As used herein "antibody-dependent cell-mediated
cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which
nonspecific cytotoxic cells that express Fc receptors (FcRs) (e.g.
natural killer (NK) cells, neutrophils, and macrophages) recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC activity of a molecule of interest can be
assessed using an in vitro ADCC assay, such as that described in
U.S. Pat. No. 5,500,362 or 5,821,337. Useful effector cells for
such assays include peripheral blood mononuclear cells (PBMC) and
NK cells. Alternatively, or additionally, ADCC activity of the
molecule of interest may be assessed in vivo, e.g., in an animal
model such as that disclosed in Clynes et al., 1998, PNAS (USA),
95:652-656.
[0055] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, one or more of the following: improvement in any
aspect of visceral pain including lessening severity, alleviation
of pain intensity, and other associated symptoms, reducing
frequency of recurrence, increasing the quality of life of those
suffering from the visceral pain, and decreasing dose of other
medications required to treat the visceral pain. Other associated
symptoms include, but are not limited to, cramps, aches, diffuse
pain, pressure, fullness, squeezing, nausea, vomiting, and
sensitivity to light, sound, and/or movement.
[0056] "Reducing incidence" of visceral pain means any of reducing
severity (which can include reducing need for and/or amount of
(e.g., exposure to) other drugs and/or therapies generally used for
this condition, including, for example, opiates (e.g., oxycodone,
morphine, butorphanol, nalbuphine, etc.), duration, and/or
frequency. As is understood by those skilled in the art,
individuals may vary in terms of their response to treatment, and,
as such, for example, a "method of reducing incidence of visceral
pain in an individual" reflects administering the anti-CGRP
antagonist antibody based on a reasonable expectation that such
administration may likely cause such a reduction in incidence in
that particular individual.
[0057] "Ameliorating" visceral pain and/or a symptom associated
with visceral pain means a lessening or improvement of one or more
symptoms of visceral pain and/or symptoms associated with visceral
pain as compared to not administering an anti-CGRP antagonist
antibody. "Ameliorating" also includes shortening or reduction in
duration of a symptom.
[0058] "Palliating" visceral pain and/or a symptom associated with
visceral pain means lessening the extent of one or more undesirable
clinical manifestations of visceral pain in an individual or
population of individuals treated with an anti-CGRP antagonist
antibody in accordance with the invention.
[0059] As used herein, "controlling visceral pain" refers to
maintaining or reducing severity or duration of one or more
symptoms of visceral pain or frequency of visceral pain as compared
to the level before treatment. For example, the duration or
severity of visceral pain, or frequency of visceral pain, can be
reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, or
70% in the individual as compared to the level before
treatment.
[0060] As used therein, "delaying" the development of visceral pain
means to defer, hinder, slow, retard, stabilize, and/or postpone
progression of the visceral pain. This delay can be of varying
lengths of time, depending on the history of the disease and/or
individuals being treated. As is evident to one skilled in the art,
a sufficient or significant delay can, in effect, encompass
prevention, in that the individual does not develop visceral pain.
A method that "delays" development of the symptom is a method that
reduces probability of developing the symptom in a given time frame
and/or reduces extent of the symptoms in a given time frame, when
compared to not using the method. Such comparisons are typically
based on clinical studies, using a number of subjects sufficient to
show a statistically significant difference between treated and
untreated subjects.
[0061] "Development" or "progression" of visceral pain means
initial manifestations and/or ensuing progression of the disorder.
Development of visceral pain can be detectable and assessed using
standard clinical techniques as well known in the art. However,
development also refers to progression that may be undetectable.
For purpose of this invention, development or progression refers to
the biological course of the symptoms. "Development" includes
occurrence, recurrence, and onset. As used herein "onset" or
"occurrence" of visceral pain includes initial onset and/or
recurrence.
[0062] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides, or embedding in a
semi-solid or solid matrix for sectioning purposes. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0063] As used herein, an "effective dosage" or "effective amount"
of drug, compound, or pharmaceutical composition is an amount
sufficient to effect beneficial or desired results. For
prophylactic use, beneficial or desired results include results
such as eliminating or reducing the risk, lessening the severity,
or delaying the outset of the disease, including biochemical,
histological and/or behavioral symptoms of the disease, its
complications and intermediate pathological phenotypes presenting
during development of the disease. For therapeutic use, beneficial
or desired results include clinical results such as reducing pain
intensity, duration, or frequency of visceral pain attack, and
decreasing one or more symptoms resulting from visceral pain
(biochemical, histological and/or behavioral), including its
complications and intermediate pathological phenotypes presenting
during development of the pain, increasing the quality of life of
those suffering from the pain, decreasing the dose of other
medications required to treat the pain, enhancing effect of another
medication, and/or delaying the progression of the pain of
patients. An effective dosage can be administered in one or more
administrations. For purposes of this invention, an effective
dosage of drug, compound, or pharmaceutical composition is an
amount sufficient to accomplish prophylactic or therapeutic
treatment either directly or indirectly. As is understood in the
clinical context, an effective dosage of a drug, compound, or
pharmaceutical composition may or may not be achieved in
conjunction with another drug, compound, or pharmaceutical
composition. Thus, an "effective dosage" may be considered in the
context of administering one or more therapeutic agents, and a
single agent may be considered to be given in an effective amount
if, in conjunction with one or more other agents, a desirable
result may be or is achieved.
[0064] An "individual" or a "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.
[0065] As used herein, "vector" means a construct, which is capable
of delivering, and preferably expressing, one or more gene(s) or
sequence(s) of interest in a host cell. Examples of vectors
include, but are not limited to, viral vectors, naked DNA or RNA
expression vectors, plasmid, cosmid or phage vectors, DNA or RNA
expression vectors associated with cationic condensing agents, DNA
or RNA expression vectors encapsulated in liposomes, and certain
eukaryotic cells, such as producer cells.
[0066] As used herein, "expression control sequence" means a
nucleic acid sequence that directs transcription of a nucleic acid.
An expression control sequence can be a promoter, such as a
constitutive or an inducible promoter, or an enhancer. The
expression control sequence is operably linked to the nucleic acid
sequence to be transcribed.
[0067] As used herein, "pharmaceutically acceptable carrier" or
"pharmaceutical acceptable excipient" includes any material which,
when combined with an active ingredient, allows the ingredient to
retain biological activity and is non-reactive with the subject's
immune system. Examples include, but are not limited to, any of the
standard pharmaceutical carriers such as a phosphate buffered
saline solution, water, emulsions such as oil/water emulsion, and
various types of wetting agents. Preferred diluents for aerosol or
parenteral administration are phosphate buffered saline or normal
(0.9%) saline. Compositions comprising such carriers are formulated
by well known conventional methods (see, for example, 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).
[0068] The term "k.sub.on", as used herein, is intended to refer to
the rate constant for association of an antibody to an antigen.
[0069] The term "k.sub.off", as used herein, is intended to refer
to the rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0070] The term "K.sub.D", as used herein, is intended to refer to
the equilibrium dissociation constant of an antibody-antigen
interaction.
Methods for Preventing or Treating Visceral Pain
[0071] Disclosed herein are methods for the prevention and/or
treatment of visceral pain and/or symptoms of visceral pain and a
medicament for prevention and/or treatment of visceral pain and/or
one or more symptoms of visceral pain in an individual.
[0072] In some embodiments, the invention provides a method of
preventing and/or treating visceral pain and/or one or more
symptoms of visceral pain in an individual, comprising peripheral
administration to the individual of an effective amount of an
anti-CGRP antagonist antibody.
[0073] In other embodiments, the invention provides a method of
ameliorating, controlling, reducing incidence of, or delaying the
development or progression of visceral pain and/or one or more
symptoms of visceral pain in an individual, comprising peripheral
administration to the individual of an effective amount of an
anti-CGRP antagonist antibody.
[0074] In some embodiments, the invention provides the use of an
anti-CGRP antagonist antibody for the manufacture of a medicament
for the prevention and/or treatment of visceral pain and/or one or
more symptoms of visceral pain, wherein the medicament is prepared
for peripheral administration or wherein the medicament is
administered peripherally.
[0075] In other embodiments, the invention provides an anti-CGRP
antagonist antibody for use in the prevention and/or treatment of
visceral pain and/or symptoms of visceral pain wherein the antibody
is prepared for peripheral administration or wherein the antibody
is administered peripherally.
[0076] In other embodiments, the invention provides the use of an
anti-CGRP antagonist antibody for the manufacture of a medicament
for ameliorating, controlling, reducing incidence of, or delaying
the development or progression of visceral pain and/or symptoms of
visceral pain, wherein the medicament is prepared for peripheral
administration or wherein the medicament is administered
peripherally.
[0077] In some embodiments, the individual is preferably a mammal,
for example a companion animal such as a horse, cat or dog or a
farm animal such as a sheep, cow or pig. Most preferably the mammal
is a human.
[0078] In some embodiments, the medicament and/or anti-CGRP
antagonist antibody is prepared for oral, sublingual, buccal,
topical, rectal, inhalation, transdermal, subcutaneous,
intravenous, intra-arterial, intramuscular, intracardiac,
intraosseous, intradermal, intraperitoneal, transmucosal, vaginal,
intravitreal, intra-articular, peri-articular, central, local or
epicutaneous administration.
[0079] In some embodiments, the medicament is prepared for
peripheral administration prior to and/or during and/or after the
development of visceral pain.
[0080] In some embodiments, the anti-CGRP antagonist antibody acts
peripherally on administration. In one embodiment, the anti-CGRP
antagonist antibody is not administered centrally, spinally or
intrathecally.
[0081] In some embodiments, the visceral pain is associated with
and/or caused by a disease such as, for example, a functional bowel
disorder (FBD) or inflammatory bowel disease (IBD). In embodiments
where the visceral pain is associated with FBD, the FBD may be, for
example without limitation, gastro-esophageal reflux, dyspepsia,
irritable bowel syndrome (IBS) or functional abdominal pain
syndrome (FAPS). Most preferably, the disease is IBS. In
embodiments where the visceral pain is associated with IBD, the IBD
may be, for example without limitation, Crohn's disease, ileitis or
ulcerative colitis. Other types of visceral pain include the pain
associated with, for example, cancer, renal colic, dysmenorrhea,
cystitis, including interstitial cystitis (IC), surgery associated
with the ileus, menstrual period, labor, menopause, bone fracture,
diverticulitis, peritonitis, pericarditis, hepatitis, appendicitis,
colitis, cholecystitis, endometriosis, chronic and/or acute
pancreatitis, myocardial infarction, kidney pain, pleural pain,
prostatitis, pelvic pain, and trauma to an organ.
[0082] In some embodiments, the methods and uses of the invention
may be for amerliorating visceral pain and/or one or more symptoms
associated with visceral pain in an individual having FBD, IBD or
IC.
[0083] Diagnosis or assessment of visceral pain is well-established
in the art. Assessment may be performed based on measures known in
the art, such as patient characterization of pain using various
pain scales. See, e.g., Katz et al, Surg Clin North Am., 1999, 79
(2):231-52; Caraceni et al. J Pain Symptom Manage, 2002,
23(3):239-55. For example, the verbal descriptor scale (VDS), the
visual analog scale (VAS), the Prince Henry Hospital Pain Scale
(PHHPS), the numeric rating scale (NRS), and the Faces Pain Scale,
and variations thereof, may be employed to assess pain and evaluate
response to the treatment. There are also commonly used scales to
measure disease state such as the Functional Bowel Disorder
Severity Index (FBDSI) (Drossman et al., 1995, Digestive Diseases
and Sciences 40(5):986-995) and the IBS Severity Scoring System
(Francis et al., 1997, Aliment Pharmacol Ther., 11(2):395-402).
Such scales may be employed to evaluate response to the
treatment.
[0084] In some embodiments, ameliorating, controlling, reducing
incidence of, or delaying the development or progression of FBD
pain and/or symptoms of FBD pain is measured by one or more of the
FBDSI, VDS, VAS, PHHPS, NRS and Faces Pain Scale. In another
embodiment, ameliorating, controlling, reducing incidence of, or
delaying the development or progression of IBS pain and/or symptoms
of IBS pain is measured by one or more of the IBS Severity Scoring
System, VDS, VAS, PHHPS, NRS and Faces Pain Scale.
[0085] In some embodiments, ameliorating, controlling, reducing
incidence of, or delaying the development or progression of IC pain
and/or symptoms of IC pain is measured by one or more of the VDS,
VAS, PHHPS, NRS and Faces Pain Scale.
Anti-CGRP Antagonist Antibodies
[0086] In some embodiments, the anti-CGRP antagonist antibody binds
to CGRP. Preferably, the anti-CGRP antagonist antibody binds to
CGRP and inhibits the ability of CGRP to bind to the CGRP receptor.
In some embodiments, the anti-CGRP antagonist antibody binds to
both human and rodent CGRP, preferably human and rat CGRP. More
preferably, the antibody binds to human CGRP. In preferred
embodiments, the anti-CGRP antagonist antibody binds to human
.alpha.-CGRP or to human .alpha.-CGRP and/or .beta.-CGRP. Most
preferably, the anti-CGRP antagonist antibody is an antibody that
exhibits any one or more of the following functional
characteristics: (a) binds to CGRP; (b) blocks CGRP from binding to
its receptor(s); (c) blocks or decreases CGRP receptor activation,
including cAMP activation; (d) inhibits, blocks, suppresses or
reduces CGRP biological activity, including downstream pathways
mediated by CGRP signalling, such as receptor binding and/or
elicitation of a cellular response to CGRP; (e) prevents,
ameliorates, or treats any aspect of visceral pain; (f) increases
clearance of CGRP; and (g) inhibits (reduces) CGRP synthesis,
production or release.
[0087] In some embodiments, the anti-CGRP antagonist antibody binds
to a fragment of CGRP, more preferably to a fragment of CGRP as
well as to the full length CGRP. Preferably, the anti-CGRP
antagonist antibody binds to the C-terminal region or fragment of
CGRP. The C-terminal region or fragment of CGRP preferably
comprises amino acids 19-37 or 25-37 or 29-37, or, alternatively,
amino acids 30-37, or, further alternatively, amino acids 31-37 of
CGRP. In a further embodiment, the C-terminal region or fragment of
CGRP preferably comprises amino acids 32-37, most preferably amino
acids 33-37 of CGRP. Preferably, the CGRP is either .alpha.-CGRP or
.beta.-CGRP, further preferably human or rodent, further preferably
human or rat, more preferably human, further preferably human
.alpha.-CGRP or .beta.-CGRP, most preferably human
.alpha.-CGRP.
[0088] In some embodiments, the anti-CGRP antagonist antibody
specifically binds to the amino acid sequence GSKAF (SEQ ID NO:
39). Preferably the sequence GSKAF (SEQ ID NO: 39) of CGRP is the
epitope to which the anti-CGRP antagonist antibody binds.
[0089] In some embodiments, an anti-CGRP antagonist antibody is
provided which specifically binds to an epitope defined by amino
acids G33 to F37 of CGRP. The anti-CGRP antagonist antibody may
specifically bind to the epitope defined by the amino acid sequence
GSKAF (SEQ ID NO: 39). In some embodiments, the present invention
provides the use of such an antibody in the uses and methods
defined in the various aspects of the present invention.
[0090] In some embodiments, the anti-CGRP antagonist antibody
inhibits or prevents activation of the CGRP receptor. Preferably
the anti-CGRP antibody has an IC50 of between about 0.0001 (0.1 nM)
to about 500 .mu.M. In some preferred embodiments, the IC50 is
between about 0.0001 .mu.M.quadrature. and any of about 250 .mu.M,
100 .mu.M, 50 .mu.M, 10 .mu.M, 1 .mu.M, 500 nM, 250 nM, 100 nM, 50
nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, or 0.5 nM as measured in an in
vitro binding assay. In some further preferred embodiments, IC50 is
less than any of about 500 .mu.M, or about 100 .mu.M, or about 50
.mu.M, as measured in an in vitro binding assay. In a further more
preferred embodiment IC50 is about 1.2 nM or 31 nM.
[0091] In some embodiments, the anti-CGRP antagonist antibody used
is capable of competing with an antibody herein above described for
the binding of CGRP or to a fragment of CGRP, or to a fragment of
CGRP as well as the full length CGRP, preferably to the C-terminal
region or fragment of CGRP. In preferred embodiments, the
C-terminal region or fragment of CGRP comprises amino acids 19-37,
25-37, 29-37, 30-37, or 31-37 of CGRP. In a further embodiment, the
C-terminal region or fragment of CGRP preferably comprises amino
acids 32-37, most preferably 33-37, of CGRP.
[0092] In some embodiments, the anti-CGRP antagonist antibody binds
to CGRP, a region of CGRP, or a fragment of CGRP with a binding
affinity (K.sub.D) of between about 0.00001 .mu.M (0.01 nM) to
about 500 .mu.M. In some embodiments, the binding affinity
(K.sub.D) is between about 0.00001 .mu.M.quadrature. and any of
about 250 .mu.M, 100 .mu.M, 50 .mu.M, 10 .mu.M, 1 .mu.M, 500 nM,
250 nM, 100 nM, 50 nM, 20 nM, 15 nM, 10 nM, 5 nM, 1 nM, 0.5 nM, 1
nM, 0.05 nM, or 0.01 nM as measured in an in vitro binding assay.
In some embodiments, the binding affinity (K.sub.D) is less than
any of about 500 .mu.M, or 100 .mu.M, 50 .mu.M, or 10 .mu.M, as
measured in an in vitro binding assay. In further more preferred
embodiments, binding affinity (K.sub.D) is about 0.04 nM or 16
nM.
[0093] The anti-CGRP antagonist antibody as used in the present
invention may be selected from the group of: monoclonal antibodies,
polyclonal antibodies, antibody fragments (e.g., Fab, Fab',
F(ab')2, Fv, Fc, ScFv etc.), chimeric antibodies, bispecific
antibodies, heteroconjugate antibodies, single chain (ScFv)
antibodies, mutants thereof, fusion proteins comprising an antibody
portion (e.g., a domain antibody), humanized antibodies, and any
other modified configuration of the immunoglobulin molecule that
comprises an antigen recognition site of the required specificity,
including glycosylation variants of antibodies, amino acid sequence
variants of antibodies, and covalently modified antibodies. The
anti-CGRP antagonist antibody may be murine, rat, human, or any
other origin (including chimeric or humanized antibodies). In some
embodiments, the anti-CGRP antagonist antibody may be humanized but
is more preferably human. In some embodiments, the anti-CGRP
antagonist antibody is isolated. In some embodiments, the anti-CGRP
antagonist antibody is substantially pure. Where the anti-CGRP
antagonist antibody is an antibody fragment, the fragment
preferably retains the functional characteristics of the original
antibody, i.e., the CGRP binding and/or antagonist activity as
described in the functional characteristics above.
[0094] Examples of anti-CGRP antagonist antibodies are known in the
art. Hence, according to a preferred embodiment of the present
invention the anti-CGRP antagonist antibody as used in the present
invention is preferably an anti-CGRP antibody as generally or
specifically disclosed in any of (i) WO2007/054809, (ii)
WO2007/076336, (iii) Tan et al., Clin. Sci. (Lond). 89:565-73,
1995, (iv) Sigma (Missouri, US), product number C7113 (clone
#4901), (v) Plourde et al., Peptides 14:1225-1229, 1993, or which
comprises or consists of:
[0095] (a) a fragment of said antibody (e.g., Fab, Fab', F(ab')2,
Fv, Fc, ScFv etc.),
[0096] (b) a light chain of said antibody,
[0097] (c) a heavy chain of said antibody,
[0098] (d) one or more variable region(s) from a light chain and/or
a heavy chain of said antibody,
[0099] (e) one or more CDR(s) (one, two, three, four, five or six
CDRs) of said antibody,
[0100] (f) CDR H3 from the heavy chain of said antibody,
[0101] (g) CDR L3 from the light chain of said antibody,
[0102] (h) three CDRs from the light chain of said antibody,
[0103] (i) three CDRs from the heavy chain of said antibody,
[0104] (j) three CDRs from the light chain and three CDRs from the
heavy chain, of said antibody,
[0105] (k) any one or more of (a) through (j).
[0106] In some embodiments, the anti-CGRP antagonist antibody is
antibody G2 or antibody G1. According to a most preferred
embodiment of the present the anti-CGRP antagonist antibody used is
the anti-CGRP antibody G1 as specifically disclosed in PCT Patent
Application Pub. No. WO2007/054809, or comprising its variants
shown in Table 6 of WO2007/054809, also including functionally
equivalent antibodies to G1, i.e., comprising conservative
substitutions of amino acid residues or one or more deletions or
additions of amino acids which do not significantly affect their
functional characteristics e.g. CGRP binding or antagonist activity
and variants which have enhanced or decreased activity and/or
binding. As used herein, the terms "G1" and "antibody G1" are used
interchangeably to refer to an antibody produced by expression
vectors having deposit numbers of ATCC PTA-6867 and ATCC PTA-6866
as disclosed in application WO2007/054809. Functional
characteristics of antibody G1 are described in PCT Patent
Application Nos. PCT/IB2009/050849 and PCT/IB2009/050852, both
filed Mar. 3, 2009, and incorporated herein by reference in their
entireties.
[0107] According to a further embodiment of the present invention,
the anti-CGRP antagonist antibody comprises or consists of a
polypeptide selected from: (a) antibody G1 or its variants shown in
Table 6 of WO2007/054809; (b) a fragment or a region of antibody G1
or its variants shown in Table 6 of WO2007/054809; (c) a light
chain of antibody G1 or its variants shown in Table 6 of
WO2007/054809; (d) a heavy chain of antibody G1 or its variants
shown in Table 6 of WO2007/054809 (e) one or more variable
region(s) from a light chain and/or a heavy chain of antibody G1 or
its variants shown in Table 6 of WO2007/054809; (f) one or more
CDR(s) (one, two, three, four, five or six CDRs) of antibody G1 or
its variants shown in Table 6 of WO2007/054809; (g) CDR H3 from the
heavy chain of antibody G1 or its variants shown in Table 6 of
WO2007/054809; (h) CDR L3 from the light chain of antibody G1 or
its variants shown in Table 6 of WO2007/054809; (i) three CDRs from
the light chain of antibody G1 or its variants shown in Table 6 of
WO2007/054809; (j) three CDRs from the heavy chain of antibody G1
or its variants shown in Table 6 of WO2007/054809; (k) three CDRs
from the light chain and/or three CDRs from the heavy chain, of
antibody G1 or its variants shown in Table 6 of WO2007/054809; and
(i) an antibody comprising any one of (b) through (k). The
invention also provides polypeptides comprising any one or more of
the above. In some embodiments, the at least one, two, three, four,
five, or six CDR(s) are at least about 85%, 86%, 87%, 88%, 89%,
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to at
least one, two, three, four, five or six CDRs of G1 or its variants
shown in Table 6 of WO2007/054809. Determination of CDR regions is
well within the ability of the skilled person. It is understood
that in some embodiments, CDRs can be a combination of the Kabat
and Chothia CDR. In some embodiments, the CDRs are the Kabat CDRs.
In other embodiments, the CDRs are the Chothia CDRs.
[0108] The anti-CGRP antagonist antibody preferably comprises or
consists of a fragment or a region of the antibody G1 (e.g., Fab,
Fab', F(ab')2, Fv, Fc, ScFv etc.) or its variants shown in Table 6
of WO2007/054809. Preferably, said fragment or region has the
functional characteristics of an anti-CGRP antagonist antibody such
as, for example, CGRP binding activity and/or antagonist activity,
and comprises or consists of one or more of: (i) a light chain,
(ii) a heavy chain, (iii) a fragment containing one or more
variable regions from a light chain and/or a heavy chain, and (iv)
one or more CDRs from a light chain and/or a heavy chain of the
antibody G1.
[0109] In some embodiments, the anti-CGRP antagonist antibody
comprises a light chain variable region (LCVR) comprising a peptide
with a sequence selected from the group consisting of SEQ ID NOs:
28-32 and/or a heavy chain variable region (HCVR) comprising a
peptide with a sequence selected from the group consisting of SEQ
ID NOs: 34-38 of patent application WO2007/076336.
[0110] Further preferably, the anti-CGRP antagonist antibody
comprises an LCVR polypeptide of a SEQ ID NO as shown in Table 1 of
patent application WO2007/076336 and further comprises a HCVR
polypeptide of a SED ID NO as shown in Table 1 of patent
application WO2007/076336.
[0111] According to a further embodiment of the invention, the
anti-CGRP antagonist antibody used comprises a light chain CDR
(CDRL) selected from the group consisting of SEQ ID NOs: 8-13
and/or a heavy chain CDR (CDRH) selected from the group consisting
of SEQ ID NOs: 14-22 of patent application WO2007/076336.
[0112] Methods of making and isolating the anti-CGRP antagonist
antibodies of application WO2007/076336 and data demonstrating the
CGRP binding and antagonist characterisation of the same are
described in application WO2007/076336.
[0113] In some embodiments, the anti-CGRP antagonist antibody for
use in the present invention comprises a VH domain that is 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%, at least about 99% or 100% identical in amino acid sequence to
SEQ ID NO: 1 or SEQ ID NO: 19 presented herein.
[0114] In some embodiments, the anti-CGRP antagonist antibody
comprises a VL domain that is 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%, at least about 99% or
100% identical in amino acid sequence to SEQ ID NO: 2 or SEQ ID NO:
20 presented herein.
[0115] In some embodiments, the anti-CGRP antagonist antibody
comprises a VH domain and a VL domain that 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%, at
least about 99% or 100% identical in amino acid sequence to SEQ ID
NO: 1 and 2 respectively or SEQ ID NO: 19 and 20 presented herein,
respectively.
[0116] In some embodiments, the anti-CGRP antagonist antibody
comprises a VH domain that is at least 90% identical in amino acid
sequence to SEQ ID NO: 1 and a VL domain that is at least 90%
identical in amino acid sequence to SEQ ID NO: 2 presented
herein.
[0117] Alternatively, the anti-CGRP antagonist antibody can
comprise a VH domain that is at least 90% identical in amino acid
sequence to SEQ ID NO: 19 and a VL domain that is at least 90%
identical in amino acid sequence to SEQ ID NO: 20 presented
herein.
[0118] In some embodiments, the anti-CGRP antagonist antibody
comprises at least one CDR selected from the group consisting of:
(a). CDR H1 as set forth in SEQ ID NO: 3, 21, 33, 34, 36 or 37;
(b). CDR H2 as set forth in SEQ ID NO: 4, 22, 35 or 38; (c). CDR H3
as set forth in SEQ ID NO: 5 or 23; (d). CDR L1 as set forth in SEQ
ID NO: 6 or 24; (e) CDR L2 as set forth in SEQ ID NO: 7 or 25; (f).
CDR L3 as set forth in SEQ ID NO: 8 or 26; and (g). variants of CDR
L1, CDR L2 and CDR H2 as shown in Table 6 of WO2007/054809.
[0119] In some embodiments, the anti-CGRP antagonist antibody heavy
chain constant region may be from any types of constant region,
such as IgG, IgM, IgD, IgA, and IgE; and any isotypes, such as
IgGI, IgG2, IgG3, and IgG4.
[0120] In some embodiments, the anti-CGRP antagonist antibody
comprises a heavy chain produced by the expression vector with ATCC
Accession No. PTA-6867. Further preferably the anti-CGRP antagonist
antibody comprises a light chain produced by the expression vector
with ATCC Accession No. PTA-6866. In some embodiments, the
anti-CGRP antagonist antibody comprises the antibody G1 heavy chain
full antibody amino acid sequence (including modified IgG2 as
described herein) shown in SEQ ID NO: 11, with or without the
C-terminal lysine. The anti-CGRP antagonist antibody also includes
an antibody lacking a terminal lysine on the heavy chain, as this
is normally lost in a proportion of antibodies during manufacture.
In some embodiments, the anti-CGRP antagonist antibody comprises
the antibody G1 light chain full antibody amino acid sequence shown
in SEQ ID NO: 12. In some embodiments, the anti-CGRP antagonist
antibody comprises the antibody G2 heavy chain full antibody amino
acid sequence shown in SEQ ID NO: 29. In some embodiments, the
anti-CGRP antagonist antibody comprises the antibody G2 light chain
full antibody amino acid sequence shown in SEQ ID NO: 30. In some
embodiments, the anti-CGRP antagonist antibody is produced by the
expression vectors with ATCC Accession Nos. PTA-6867 and
PTA-6866.
[0121] In some embodiments, the anti-CGRP antagonist antibody for
use in the present invention is antibody G1 or antibody G2 defined
herein. In preferred embodiments, the anti-CGRP antagonist antibody
for use in the present invention is antibody G1, or an antigen
binding fragment thereof.
[0122] According to further embodiments of the invention, the
anti-CGRP antagonist antibody comprises a modified constant region
as for example described in WO2007/054809. Preferably, the modified
constant region is immunologically inert, including partially
immunologically inert, such that it does not trigger complement
mediated lysis, does not stimulate antibody-dependent cell mediated
cytotoxicity (ADCC), does not activate microglia. Preferably, the
modified constant region is reduced in one or more of these
activities. Most preferably, the constant region is modified as
described in Eur. J. Immunol., 1999, 29:2613-2624; PCT Application
No. PCT/GB99/01441; and/or UK Patent Application No. 9809951.8. In
some embodiments, the anti-CGRP antagonist antibody comprises a
human heavy chain IgG2 constant region comprising the following
mutations: A330, P331 to S330, S331 (amino acid numbering with
reference to the wildtype IgG2 sequence). Eur. J. Immunol., 1999,
29:2613-2624.
[0123] Methods of making and isolating the anti-CGRP antagonist
antibodies of application WO2007/054809 and data demonstrating the
CGRP binding and antagonist characterisation of the same are
described in application WO2007/054809. Sequences of SEQ ID NO: 1
to 14 of said application are provided herein as SEQ ID NO: 1 to
14, respectively.
Dosage and Administration
[0124] In some embodiments, the anti-CGRP antagonist antibody is
peripherally administered between, for example, about once to about
7 times per week, further preferably between about once to about
four times per month, further preferably between about once to
about six times per 6 month period, further preferably about once
to about twelve times per year. Preferably, the anti-CGRP
antagonist antibody is peripherally administered in a period
selected from: about once daily, once every two, three, four, five
or six days, weekly, once every two weeks, once every three weeks,
monthly, once every two months, once every three months, once every
four months, once every five months, once every six months, once
every seven months, once every eight months, once every nine
months, once every ten months, once every eleven months or yearly.
According to preferred embodiments, the anti-CGRP antagonist
antibody is administered via a route selected from one or more of:
orally, sublingually, buccally, topically, rectally, via
inhalation, transdermally, subcutaneously, intravenously,
intra-arterially or intramuscularly, via intracardiac
administration, intraosseously, intradermally, intraperitoneally,
transmucosally, vaginally, intravitreally, epicutaneously,
intra-articularly, peri-articularly or locally.
[0125] According to a further embodiment of the present invention,
the medicament is prepared for peripheral administration between
about once to about 7 times per week, further preferably between
about once to about four times per month, further preferably
between about once to about six times per 6 month period, further
preferably about once to about twelve times per year. Preferably,
the medicament is prepared to be peripherally administered in a
period selected from: about once daily, once every two, three,
four, five or six days, weekly, once every two weeks, once every
three weeks, monthly, once every two months, once every three
months, once every four months, once every five months, once every
six months, once every seven months, once every eight months, once
every nine months, once every ten months, once every eleven months
or yearly. According to preferred embodiments, the medicament is
prepared to be peripherally administered via a route selected from
one or more of: orally, sublingually, buccally, topically,
rectally, via inhalation, transdermally, subcutaneously,
intravenously, intra-arterially or intramuscularly, via
intracardiac administration, intraosseously, intradermally,
intraperitoneally, transmucosally, vaginally, intravitreally,
epicutaneously, intra-articularly, peri-articularly or locally.
[0126] According to a further embodiment of the present invention,
an antibody concentration of between about 0.1 to about 200 mg/ml;
preferably at any one of about 0.5, 1, 5, 10, 15 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190 or 200 mg/ml+/-10% error, most
preferably at about 50 mg/ml.
[0127] According to a further embodiment of the present invention
the medicament is prepared for peripheral administration with an
antibody concentration of between 0.1 to 200 mg/kg of body weight;
preferably at any one of about 0.5, 1, 5, 10, 15 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140, 150, 160, 170, 180, 190 or 200 mg/kg of body weight+/-10%
error, most preferably at about 10 mg/kg.
[0128] According to a preferred embodiment of the present invention
the anti-CGRP antagonist antibody has a half life in-vivo of more
than any one of about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,
26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120,
122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146,
148, 150, 152, 154, 156, 158, 160, 62, 164, 166, 168, 170, 172,
174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198,
200, 202, 204, 206, 208 or 210 days+/-1 day, further preferably
more than any one of about 7, 8, 9, 10, 11, or 12 months.
[0129] Preferably, the anti-CGRP antagonist antibody has a half
life in-vivo of more than 6 days.
[0130] According to a further preferred embodiment of the present
invention, the medicament and/or the anti-CGRP antagonist antibody
does not produce effects of central nervous system and/or cognitive
impairment. Preferably the medicament and/or the anti-CGRP
antagonist antibody does not induce any one or more of the
following: amnesia, confusion, depersonalization, hypesthesia,
abnormal thinking, trismus, vertigo, akathisia, apathy, ataxia,
circumoral paresthesia, CNS stimulation, emotional lability,
euphoria, hallucinations, hostility, hyperesthesia, hyperkinesia,
hypotonia, incoordination, libido increase, manic reaction,
myoclonus, neuralgia, neuropathy, psychosis, seizure, abnormal
speech, stupor, suicidal ideation; dizziness, somnolence, Insomnia,
anxiety, tremor, depression or paresthesia. Most preferably the
medicament and/or the anti-CGRP antagonist antibody does not induce
impairment of motor coordination or attention.
[0131] According to a further embodiment of the present invention,
the medicament and/or the anti-CGRP antagonist antibody does not
produce respiratory, liver renal or gastrointestinal
impairment.
[0132] According to a further embodiment of the present invention,
the medicament and/or the anti-CGRP antagonist antibody does not
produce effects of physical and/or psychological dependence.
Preferably the medicament and/or the anti-CGRP antagonist antibody
does not demonstrate affinity for opiate, benzodiazepine,
phencyclidine (PCP), or N-methyl-D-aspartic acid (NMDA) receptors,
or CNS stimulant, or produce any sedating or euphoric effect.
[0133] In some embodiments, the anti-CGRP antagonist antibody, on
administration, ameliorates, controls, reduces incidence of, or
delays the development or progression of central pain
sensation.
[0134] In other embodiments, the effect of the anti-CGRP antagonist
antibody is equal and/or superior to the effects of NSAIDS and/or
opiates in the same models of visceral pain. In one embodiment, the
anti-CGRP antagonist antibody is effective in treating refractory
pain populations.
[0135] According to further embodiments of the present invention,
there is provided the use or method according to any other aspect
of the invention wherein the anti-CGRP antagonist antibody is
administered separately, sequentially or simultaneously in
combination with one or more further pharmacologically active
compounds or agents, preferably compounds or agents useful for
treating visceral pain. Preferably, the additional agent(s) is/are
selected from one or more of:
[0136] (i) an opioid analgesic, e.g. morphine, heroin,
hydromorphone, oxymorphone, levorphanol, levallorphan, methadone,
meperidine, fentanyl, cocaine, codeine, dihydrocodeine, oxycodone,
hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone,
naltrexone, buprenorphine, butorphanol, nalbuphine or
pentazocine;
[0137] (ii) a nonsteroidal antiinflammatory drug (NSAID), e.g.
aspirin, diclofenac, diflusinal, etodolac, fenbufen, fenoprofen,
flufenisal, flurbiprofen, ibuprofen, indomethacin, ketoprofen,
ketorolac, meclofenamic acid, mefenamic acid, nabumetone, naproxen,
oxaprozin, phenylbutazone, piroxicam, sulindac, tolmetin or
zomepirac, cyclooxygenase-2 (COX-2) inhibitors, celecoxib;
rofecoxib; meloxicam; JTE-522; L-745,337; NS398; or a
pharmaceutically acceptable salt thereof;
[0138] (iii) a barbiturate sedative, e.g. amobarbital,
aprobarbital, butabarbital, butabital, mephobarbital, metharbital,
methohexital, pentobarbital, phenobartital, secobarbital, talbutal,
theamylal or thiopental or a pharmaceutically acceptable salt
thereof;
[0139] (iv) a benzodiazepine having a sedative action, e.g.
chlordiazepoxide, clorazepate, diazepam, flurazepam, lorazepam,
oxazepam, temazepam or triazolam or a pharmaceutically acceptable
salt thereof;
[0140] (v) an H.sub.1 antagonist having a sedative action, e.g.
diphenhydramine, pyrilamine, promethazine, chlorpheniramine or
chlorcyclizine or a pharmaceutically acceptable salt thereof;
[0141] (vi) a sedative such as glutethimide, meprobamate,
methaqualone or dichloralphenazone or a pharmaceutically acceptable
salt thereof;
[0142] (vii) a skeletal muscle relaxant, e.g. baclofen,
carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol or
orphrenadine or a pharmaceutically acceptable salt thereof;
[0143] (viii) an NMDA receptor antagonist, e.g. dextromethorphan
((+)-3-hydroxy-N-methylmorphinan) or its metabolite dextrorphan
((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,
pyrroloquinoline quinone or
cis-4-(phosphonomethyl)-2-piperidinecarboxylic acid or a
pharmaceutically acceptable salt thereof;
[0144] (ix) an alpha-adrenergic, e.g. doxazosin, tamsulosin,
clonidine or
4-amino-6,7-dimethoxy-2-(5-methanesulfonamido-1,2,3,4-tetrahydroisoquinol-
-2-yl)-5-(2-pyridyl) quinazoline;
[0145] (x) a tricyclic antidepressant, e.g. desipramine,
imipramine, amytriptiline or nortriptiline;
[0146] (xi) an anticonvulsant, e.g. carbamazepine or valproate;
[0147] (xii) a tachykinin (NK) antagonist, particularly an NK-3,
NK-2 or NK-1 antagonist, e.g.
(.alpha.R,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-m-
ethyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]naphthridine-6-13-di-
one (TAK-637),
5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorop-
henyl)-4-morpholinyl]methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one
(MK-869), lanepitant, dapitant or
3-[[2-methoxy-5-(trifluoromethoxy)phenyl]methylamino]-2-phenyl-piperidine
(2S,3S);
[0148] (xiii) a muscarinic antagonist, e.g oxybutin, tolterodine,
propiverine, tropsium chloride or darifenacin;
[0149] (xiv) a COX-2 inhibitor, e.g. celecoxib, rofecoxib or
valdecoxib;
[0150] (xv) a non-selective COX inhibitor (preferably with GI
protection), e.g. nitroflurbiprofen (HCT-1026);
[0151] (xvi) a coal-tar analgesic, in particular paracetamol;
[0152] (xvii) a neuroleptic such as droperidol;
[0153] (xviii) a vanilloid receptor agonist (e.g. resinferatoxin)
or antagonist (e.g. capsazepine);
[0154] (xix) a beta-adrenergic such as propranolol;
[0155] (xx) a local anaesthetic, such as mexiletine;
[0156] (xxi) a corticosteriod, such as dexamethasone;
[0157] (xxii) a serotonin receptor agonist or antagonist;
[0158] (xxiii) a cholinergic (nicotinic) analgesic;
[0159] (xxiv) tramadol;
[0160] (xxv) a PDEV inhibitor, such as sildenafil, vardenafil or
taladafil;
[0161] (xxvi) an alpha-2-delta ligand such as gabapentin or
pregabalin;
[0162] (xxvii) a canabinoid; and
[0163] (xxviii) an antidepressant, such as amitriptyline
(Elavil.RTM.), trazodone (Desyrel.RTM.), and imipramine
(Tofranil.RTM.) or anticonvulsants such as phenytoin
(Dilantin.RTM.) or carbamazepine (Tegretol.RTM.)).
[0164] According to a further aspect of the present invention there
is provided a pharmaceutical composition for the prevention and/or
treatment of visceral pain and/or symptoms of visceral pain or for
ameliorating, controlling, reducing incidence of, or delaying the
development or progression of visceral pain and/or symptoms of
visceral pain in an individual, comprising an anti-CGRP antagonist
antibody and a pharmaceutically acceptable carrier and/or an
excipient, wherein the composition is prepared to be peripherally
administered.
Kits
[0165] According to a further aspect of the present invention there
is provided a kit comprising a pharmaceutical composition as
defined above, and instructions for the peripheral administration
of an effective amount of said pharmaceutical composition to an
individual for the prevention and/or treatment of visceral pain
and/or symptoms of visceral pain or for ameliorating, controlling,
reducing incidence of, or delaying the development or progression
of visceral pain and/or symptoms of visceral pain.
[0166] The kit may include one or more containers containing an
anti-CGRP antagonist antibody or polypeptide described herein and
instructions for use in accordance with any of the methods and uses
of the invention. The kit may further comprise a description of
selecting an individual suitable for treatment based on identifying
whether that individual has visceral pain or is at risk of having
visceral pain. The instructions for the peripheral administration
of the pharmaceutical composition may include information as to
dosage, dosing schedule and routes of administration for the
intended treatment.
[0167] Preferred features of each aspect of the invention apply
equally to each other aspect mutatis mutandis.
EXAMPLES
[0168] The following examples are offered for illustrative purposes
only, and are not intended to limit the scope of the present
invention in any way. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
Example 1
Visceral Pain Model
[0169] This Example illustrates the effect of anti-CGRP antagonist
antibody treatment in a visceral pain model.
[0170] Patients with IBS have been shown to have a lower visceral
sensory threshold to colorectal distension and that this is highly
correlated to the visceral pain symptoms (Delafoy et al, 2006).
Colorectal distension after TNBS-induced colitis in rats is an
animal model that has been used by many researchers to explore the
mechanisms of visceral hypersensitivity (Gay et al, 2006, Delafoy
et al, 2006, Adam et al., 2006). In this Example, the rat TNBS
colitis model was used to test the effect of a function-blocking
antibody for CGRP. In the model, as in the human IBS studies, the
visceral pain threshold is measured by response to balloon
distension of the colon.
[0171] After overnight fasting, rats were anesthetized with
ketamine (80 mg/ml)/xylazine (12 mg/ml) at a dosage of 1 ml/kg. An
abdominal laparotomy was performed and a TNBS solution (50 mg at
1.5 ml/kg in 30% ethanol, "TNBS treatment group") or 30% ethanol
solution ("sham group") was injected into the proximal colon 1 cm
distal from ceacum. The sham group was used as a non-colitis
control. On the fifth day following surgery, the TNBS treatment
group was subdivided into two groups. One group received anti-CGRP
antagonist monoclonal antibody 4901 (commercially available at
Sigma (Missouri, US), product number C7113, clone #4901) at 10
mg/kg intravenously. The other group received vehicle (PBS, 0.01%
tween 20) as a negative control.
[0172] The seventh day following surgery, after a second overnight
fast, TNBS treated rats sustaining a weight loss of no greater than
11% were tested for visceral pain threshold with balloon
distension. A 5 cm latex balloon attached to a catheter was
inserted into the distal colon with the base of the balloon 5 cm
from the anus. The catheter was fixed to the tail with tape to
prevent balloon movement. After a 30-minute acclimation period the
balloon was inflated sequentially from 5 mmHg to 80 mmHg in
30-second intervals. Balloon distension was halted at the threshold
pressure required to elicit a stereotypical rodent visceral pain
posture known as the alpha position (repeated waves of contraction
of oblique musculature with inward turning of the hindpaw) and this
was recorded as the visceral pain threshold.
[0173] Rats undergoing sham procedure had a threshold of 36.8+/-2.6
(N=5, mean+/-se) on day 7 post surgery (FIG. 1A). TNBS treated rats
that received antibody 4901 (10 mg/kg) on day 5 had a threshold of
32.3+/-4.1 (N=9) on day 7 and were statistically significantly
different (one-way ANOVA plus Dunnet's multiple comparison
post-test) from the day 7 threshold of TNBS treated rats that
received vehicle on day 5 (21.0+/-3.0, N=10) (FIG. 1A). The effect
of 4901 was comparable to CGRP receptor antagonist CGRP 8-37 (FIG.
1B). This result demonstrated that an anti-CGRP antagonist antibody
was effective in significantly shifting the visceral pain threshold
towards the sham threshold, i.e., reversing pain, in a visceral
pain model.
Example 2
Interaction Analysis and Binding Assay
[0174] Interaction analysis was conducted at 25.degree. C. and at
37.degree. C. on a Biacore 3000.TM. system equipped with
streptavidin-coated (SA) sensor chips (Biacore AB, Uppsala, Sweden)
using standard Biacore running buffers (HBS-P or HBS-EP).
N-LC-biotinylated human and rat .alpha.- and .beta.-CGRPs were
captured on individual flow cells at low levels (typically 100
response units) to provide the reaction surfaces, while an
unmodified flow cell served as a reference channel. Purified Fab
fragments of antibodies G1 and G2 were generated. Typically, Fabs
were prepared as a two-fold serial dilution using 0.5 .mu.M as the
top concentration and injected for 1-min at 100 allowing up to two
hours for the dissociation time. Surfaces were regenerated with a
mixture of 50% v/v ethanol+25 mM NaOH for G1 Fab and 2:1 v/v Pierce
Gentle Elution Buffer/4M NaCl for G2 Fab. Fab injections were
duplicated to demonstrate that the assay was reproducible. The
binding responses were double-referenced and fit globally to a
simple model using BiaEvaluation v. 4.0 software. Affinities were
deduced from the quotient of the kinetic rate constants
(K.sub.D=k.sub.off/k.sub.on).
[0175] The results for antibody G1 are expressed in Table 1 below.
Antibody G1 binds human .alpha.- and .beta.-CGRP with similar and
tight affinities (K.sub.D=163 and 155 .mu.M, respectively when
analyzed side-by-side on the same chip at 37.degree. C., allowing a
20-min dissociation time). Human and cynomolgus monkey have
identical sequences; therefore, the human data also apply to
cynomolgus. G1 also binds rat CGRPs but discriminates between
.alpha.- and .beta.-isoforms (K.sub.D=2.57 nM and <150 .mu.M,
respectively, at 37.degree. C.).
TABLE-US-00001 TABLE 1 K.sub.D of G1 antibody measured at
25.degree. C. and 37.degree. C. against human and rat CGRPs.
N-biotin-CGRP Temp T.sub.1/2 K.sub.D on chip (.degree. C.) k.sub.on
(1/Ms) k.sub.off (1/s) (h) (nM) .alpha.-human/cyno 25 1.86 .times.
10.sup.5 7.80 .times. 10.sup.-6 24.68 0.042 .alpha.-human/cyno 37
5.87 .times. 10.sup.5 3.63 .times. 10.sup.-5 5.30 0.063
.beta.-human/cyno 37 4.51 .times. 10.sup.5 <6.98 .times.
10.sup.-5 2.76 <0.155 .alpha.-rat 25 5.08 .times. 10.sup.4 6.18
.times. 10.sup.-5 3.12 1.22 .alpha.-rat 37 1.55 .times. 10.sup.5
3.99 .times. 10.sup.-4 0.48 2.57 .beta.-rat 37 5.16 .times.
10.sup.5 <7.85 .times. 10.sup.-5 2.45 <0.152
[0176] The dissociation of G1 Fab from .alpha.-human, .beta.-human,
and .beta.-human-CGRPs occurs very slowly. As such, the offrate
(k.sub.off) cannot be measured accurately unless the dissociation
phase is monitored for a very long time. As a general
rule-of-thumb, the binding response decays at least 5% over the
allowed dissociation time for the report of an accurate offrate.
However, monitoring long dissociation times on the Biacore is
hindered by baseline drift, which is particularly challenging when
working at the low surface capacities required for kinetic
analyses. In this study, the dissociation phase was followed for
two hours over the .alpha.-CGRPs but only 20 mins over the
.beta.-CGRPs. As a result, the offrates for .beta.-CGRPs cannot be
resolved as accurately as those for .alpha.-CGRPs. However, when
assayed side-by-side on the same chip under identical conditions
and using a 20-min dissociation time, G1 had virtually the same
binding kinetics for .alpha.-human/cyno, .beta.-human/cyno, and
.beta.-rat CGRPs (K.sub.D=150 .mu.M at 37.degree. C.).
[0177] The results for antibody G2 are expressed in Table 2.
Antibody G2 binds .alpha.-rat CGRP with tighter affinity
(K.sub.D=0.9 nM at 25.degree. C.) than the .alpha.- and
.beta.-human CGRPs (K.sub.D=19 nM and 20 nM respectively at
25.degree. C.). G2 binding .beta.-rat CGRP was not examined in this
assay format, but showed comparable binding characteristics to
.alpha.- and .beta.-human CGRPs in a reverse orientation assay
format (data not shown).
TABLE-US-00002 TABLE 2 K.sub.D of G2 antibody measured at
25.degree. C. and 37.degree. C. against human and rat CGRPs.
N-biotin CGRP Temp T.sub.1/2 K.sub.D on chip (.degree. C.) k.sub.on
(1/MS) k.sub.off (1/s) (min) (nM) .alpha.-rat 25 2.31 .times.
10.sup.5 2.14 .times. 10.sup.-4 53.98 0.9 .alpha.-rat 37 5.0
.times. 10.sup.5 1.7 .times. 10.sup.-3 6.80 3.4 .alpha.-human 25
6.03 .times. 10.sup.4 1.15 .times. 10.sup.-3 10.05 19.1
.alpha.-human 37 9.3 .times. 10.sup.4 3.9 .times. 10.sup.-3 2.96
41.9 .beta.-human 25 8.14 .times. 10.sup.4 1.62 .times. 10.sup.-3
7.13 19.9
[0178] A binding assay was performed to measure the IC50 of
anti-CGRP G1 antibody in blocking human .alpha.-CGRP from binding
to the CGRP1-receptor. Membranes (25 from SK-N-MC cells were
incubated for 90 minutes at 25.degree. C. in incubation buffer (50
mM Tris-HCl, pH 7.4, 5 mM MgCl2, 0.1% BSA) containing 10 .mu.M
.sup.125I-human .alpha.-CGRP and varying concentrations of
anti-CGRP antibody (0.015 nM-33 nM) in a total volume of 1 ml.
Incubation was terminated by filtration through a glass microfiber
filter (GF/B, 1 .mu.m) which had been blocked with 0.5%
polyethylenimine. The protein-bound radioactivity was determined in
a gamma counter. Dose response curves were plotted and K values
were determined using the equation: K=IC50/(1+([ligand]/K.sub.D);
where the equilibrium dissociation constant K.sub.D=8 .mu.M for
human .alpha.-CGRP to CGRP1-receptor as present in SK-N-MC cells.
The reported IC50 value (in terms of IgG molecules) was converted
to binding sites (by multiplying it by 2 to allow for the fact that
the Biacore was analysis of Fab fragments) so that it could be
compared with the affinities (K.sub.D) determined by Biacore. The
IC50 observed (1.8 nM) was 23-fold higher than the K.sub.D observed
by Biacore (42 .mu.M) at equivalent temperature. This mismatch
reflects a possible lack of sensitivity of the binding assay.
Example 3
Visceral Pain Model
[0179] This Example illustrates the effect of anti-CGRP antagonist
antibody treatment in a visceral pain model.
[0180] In this Example, the rat interstitial cystitis model was
used to test the effect of a function-blocking antibody for CGRP.
In the model, visceral hypersensitivity was measured by bladder
motility in response to turpentine irritation of the bladder.
[0181] Female rats were maintained under urethane anesthesia during
cystometry and were not allowed to recover. Body temperature was
maintained at 37.degree. C. by the use of a rectal probe,
thermostatically connected to a temperature controlled heating pad.
One group of rats (n=7) received anti-CGRP antagonist monoclonal
antibody 4901 (commercially available at Sigma (Missouri, US),
product number C7113, clone #4901) at 10 mg/kg intravenously.
Another group (n=7) received vehicle (PBS, 0.01% tween 20) as a
negative control.
[0182] Twenty-four hours after dosing with 4901 or vehicle, rats
were anesthetized, and the bladder was catheterized transurethrally
with PE50 tubing (1 mm OD) to allow 0.06 ml/min filling (using a
syringe pump) of the bladder with normal saline. The tubing has a T
joint proximal to the bladder to allow monitoring of the bladder
pressure with a pressure transducer. Pressure and contractions were
monitored during a 14-minute interval (0.84 ml total volume) to
determine bladder motility. After establishing a baseline
cystometrogram, bladder irritation was created by infusing 0.5 ml
of 50% turpentine oil for 1 hour. The bladder was then drained and
subsequent tests of bladder motility were performed immediately
post (1 h), 3 h and 5 h post-turpentine.
[0183] Rats that received antibody 4901 (10 mg/kg) 24 hours prior
to the cystometrogram procedure had fewer bladder contractions at
all time points measured compared to rats that received vehicle
(FIG. 2). This result demonstrated that an anti-CGRP antagonist
antibody was effective in reducing bladder motility in response to
turpentine irritation, i.e., reversing pain, in a visceral pain
model.
[0184] Deposit of Biological Material
[0185] The following materials have been deposited with the
American Type Culture
[0186] Collection, 10801 University Boulevard, Manassas, Va.
20110-2209, USA (ATCC):
TABLE-US-00003 ATCC Material Antibody No. Accession No. Date of
Deposit pDb.CGRP.hFcGI G1 heavy chain PTA-6867 Jul. 15, 2005
pEb.CGRP.hKGI G1 light chain PTA-6866 Jul. 15, 2005
[0187] Vector pEb.CGRP.hKGI is a polynucleotide encoding the G1
light chain variable region and the light chain kappa constant
region; and vector pDb.CGRP.hFcGI is a polynucleotide encoding the
G1 heavy chain variable region and the heavy chain IgG2 constant
region containing the following mutations: A330P331 to S330S331
(amino acid numbering with reference to the wildtype IgG2 sequence;
see Eur. J. Immunol., 1999, 29:2613-2624).
[0188] These deposits were made under the provisions of the
Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for the Purpose of Patent Procedure and the
Regulations thereunder (Budapest Treaty). This assures maintenance
of a viable culture of the deposit for 30 years from the date of
deposit. The deposit will be made available by ATCC under the terms
of the Budapest Treaty, and subject to an agreement between Rinat
Neuroscience Corp. and ATCC, which assures permanent and
unrestricted availability of the progeny of the culture of the
deposit to the public upon issuance of the pertinent U.S. patent or
upon laying open to the public of any U.S. or foreign patent
application, whichever comes first, and assures availability of the
progeny to one determined by the U.S. Commissioner of Patents and
Trademarks to be entitled thereto according to 35 USC Section 122
and the Commissioner's rules pursuant thereto (including 37 CFR
Section 1.14 with particular reference to 886 OG 638).
[0189] The assignee of the present application has agreed that if a
culture of the materials on deposit should die or be lost or
destroyed when cultivated under suitable conditions, the materials
will be promptly replaced on notification with another of the same.
Availability of the deposited material is not to be construed as a
license to practice the invention in contravention of the rights
granted under the authority of any government in accordance with
its patent laws.
[0190] Below are given antibody sequences useful for various
embodiments disclosed herein.
Antibody Sequences
TABLE-US-00004 [0191] Antibody G1 heavy chain variable region amino
acid sequence (SEQ ID NO: 1)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISVVVRQAPGKGLEVVVAEIRSESDASA
THYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQNYWGQGTLVT VSS
Antibody G1 light chain variable region amino acid sequence (SEQ ID
NO: 2)
EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSVVYQQKPGQAPRLLIYGASNRYLGIPAR
FSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIK Antibody G1 CDR H1
(extended CDR) (SEQ ID NO: 3) GFTFSNYWIS Antibody G1 CDR H2
(extended CDR, same as Kabat CDR) (SEQ ID NO: 4)
EIRSESDASATHYAEAVKG Antibody G1 CDR H3 (SEQ ID NO: 5) YFDYGLAIQNY
Antibody G1 CDR L1 (SEQ ID NO: 6) KASKRVTTYVS Antibody G1 CDR L2
(SEQ ID NO: 7) GASNRYL Antibody G1 CDR L3 (SEQ ID NO: 8) SQSYNYPYT
Antibody G1 heavy chain variable region nucleotide sequence (SEQ ID
NO: 9) GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCT
GCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCTCCTG
GGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTCCG
AATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACC
ATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCG
TGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTCTGGC
TATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCC Antibody G1 light
chain variable region nucleotide sequence (SEQ ID NO: 10)
GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACG
TGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCTGGTA
CCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACC
GTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTC
ACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGT
CAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAA Antibody G1
heavy chain full antibody amino acid sequence (including modified
IgG2 as described herein) (SEQ ID NO: 11)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWISVVVRQAPGKGLEVVVAEIRSE
SDASATHYAEAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCLAYFDYGLAIQN
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNS
GALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTV
ERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQF
NVVYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP
SSIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK
Antibody G1 light chain full antibody amino acid sequence (SEQ ID
NO: 12) EIVLTQSPATLSLSPGERATLSCKASKRVTTYVSVVYQQKPGQAPRLLIYGASNRYL
GIPARFSGSGSGTDFTLTISSLEPEDFAVYYCSQSYNYPYTFGQGTKLEIKRTVAAP
SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDS
KDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Antibody G1 heavy
chain full antibody nucleotide sequence (including modified IgG2 as
described herein) (SEQ ID NO: 13)
GAAGTTCAGCTGGTTGAATCCGGTGGTGGTCTGGTTCAGCCAGGTGGTTCCCT
GCGTCTGTCCTGCGCTGCTTCCGGTTTCACCTTCTCCAACTACTGGATCTCCTG
GGTTCGTCAGGCTCCTGGTAAAGGTCTGGAATGGGTTGCTGAAATCCGTTCCG
AATCCGACGCGTCCGCTACCCATTACGCTGAAGCTGTTAAAGGTCGTTTCACC
ATCTCCCGTGACAACGCTAAGAACTCCCTGTACCTGCAGATGAACTCCCTGCG
TGCTGAAGACACCGCTGTTTACTACTGCCTGGCTTACTTTGACTACGGTCTGGC
TATCCAGAACTACTGGGGTCAGGGTACCCTGGTTACCGTTTCCTCCGCCTCCA
CCAAGGGCCCATCTGTCTTCCCACTGGCCCCATGCTCCCGCAGCACCTCCGA
GAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCAGAACCTGTG
ACCGTGTCCTGGAACTCTGGCGCTCTGACCAGCGGCGTGCACACCTTCCCAG
CTGTCCTGCAGTCCTCAGGTCTCTACTCCCTCAGCAGCGTGGTGACCGTGCCA
TCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCAAG
CAACACCAAGGTCGACAAGACCGTGGAGAGAAAGTGTTGTGTGGAGTGTCCAC
CTTGTCCAGCCCCTCCAGTGGCCGGACCATCCGTGTTCCTGTTCCCTCCAAAG
CCAAAGGACACCCTGATGATCTCCAGAACCCCAGAGGTGACCTGTGTGGTGGT
GGACGTGTCCCACGAGGACCCAGAGGTGCAGTTCAACTGGTATGTGGACGGA
GTGGAGGTGCACAACGCCAAGACCAAGCCAAGAGAGGAGCAGTTCAACTCCA
CCTTCAGAGTGGTGAGCGTGCTGACCGTGGTGCACCAGGACTGGCTGAACGG
AAAGGAGTATAAGTGTAAGGTGTCCAACAAGGGACTGCCATCCAGCATCGAGA
AGACCATCTCCAAGACCAAGGGACAGCCAAGAGAGCCACAGGTGTATACCCTG
CCCCCATCCAGAGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGT
GAAGGGATTCTATCCATCCGACATCGCCGTGGAGTGGGAGTCCAACGGACAG
CCAGAGAACAACTATAAGACCACCCCTCCAATGCTGGACTCCGACGGATCCTT
CTTCCTGTATTCCAAGCTGACCGTGGACAAGTCCAGATGGCAGCAGGGAAACG
TGTTCTCTTGTTCCGTGATGCACGAGGCCCTGCACAACCACTATACCCAGAAG
AGCCTGTCCCTGTCTCCAGGAAAGTAA Antibody G1 light chain full antibody
nucleotide sequence (SEQ ID NO: 14)
GAAATCGTTCTGACCCAGTCCCCGGCTACCCTGTCCCTGTCCCCAGGTGAACG
TGCTACCCTGTCCTGCAAAGCTTCCAAACGGGTTACCACCTACGTTTCCTGGTA
CCAGCAGAAACCCGGTCAGGCTCCTCGTCTGCTGATCTACGGTGCTTCCAACC
GTTACCTCGGTATCCCAGCTCGTTTCTCCGGTTCCGGTTCCGGTACCGACTTC
ACCCTGACCATCTCCTCCCTGGAACCCGAAGACTTCGCTGTTTACTACTGCAGT
CAGTCCTACAACTACCCCTACACCTTCGGTCAGGGTACCAAACTGGAAATCAAA
CGCACTGTGGCTGCACCATCTGTCTTCATCTTCCCTCCATCTGATGAGCAGTTG
AAATCCGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCGCGCGA
GGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCCGGTAACTCCCAG
GAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCA
CCCTGACCCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAA
GTCACCCATCAGGGCCTGAGTTCTCCAGTCACAAAGAGCTTCAACCGCGGTGA GTGCTAA Amino
acid sequence comparison of human and rat CGRP (human .alpha.-CGRP
(SEQ ID NO: 15); human .beta.-CGRP (SEQ ID NO: 16); rat
.alpha.-CGRP (SEQ ID NO: 17); and rat .beta.-CGRP (SEQ ID NO: 18)):
(SEQ ID NO: 15) ##STR00001## (SEQ ID NO: 16) ##STR00002## (SEQ ID
NO: 17) ##STR00003## (SEQ ID NO: 18) ##STR00004## Antibody G2 heavy
chain variable region amino acid sequence (SEQ ID NO: 19)
EVQLQQSGPELVKPGASVKMSCKASGYTFTSSVMHVVVKQKPGQGLEWIGYINPY
NDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAKGGNDGYWGQG TTLTVSS
Antibody G2 light chain variable region amino acid sequence (SEQ ID
NO: 20) EIVLTQSPTTMAASPGEKITITCSASSSISSIYLHVVYQQKPGFSPKVLIYRASNLASG
VPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSTIPFTFGSGTKLEIK Antibody G2 CDR
H1 (Kabat CDR) (SEQ ID NO: 21) SSVMH Antibody G2 CDR H2 (extended
CDR) (SEQ ID NO: 22) YINPYNDGTKYNEKFKG Antibody G2 CDR H3 (SEQ ID
NO: 23) GGNDGY Antibody G2 CDR L1 (SEQ ID NO: 24) SASSSISSIYLH
Antibody G2 CDR L2 (SEQ ID NO: 25) RASNLAS Antibody G2 CDR L3 (SEQ
ID NO: 26) QQGSTIPFT Antibody G2 heavy chain variable region
nucleotide sequence (SEQ ID NO: 27)
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAG
TGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTGTTATGCACT
GGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCT
TACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACT
TCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCCTGACCTCTGA
GGACTCTGCGGTCTATTACTGTGCAAAAGGGGGTAACGATGGCTACTGGGGCC
AAGGCACTACTCTCACAGTCTCCTCA
Antibody G2 light chain variable region nucleotide sequence (SEQ ID
NO: 28) GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAA
GATCACTATCACCTGTAGTGCCAGCTCAAGTATAAGTTCCATTTACTTGCATTG
GTATCAGCAGAAGCCAGGATTCTCCCCTAAAGTCTTGATTTATAGGGCATCCAA
TCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT
ACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCC
AGCAGGGTAGTACTATACCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATA AAA Antibody
G2 heavy chain full antibody amino acid sequence (not including Fc
domain) (SEQ ID NO: 29)
EVQLQQSGPELVKPGASVKMSCKASGYTFTSSVMHVVVKQKPGQGLEWIGYINPY
NDGTKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCAKGGNDGYWGQG
TTLTVSSAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSS
GVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRD Antibody G2
light chain full antibody amino acid sequence (SEQ ID NO: 30)
EIVLTQSPTTMAASPGEKITITCSASSSISSIYLHVVYQQKPGFSPKVLIYRASNLASG
VPARFSGSGSGTSYSLTIGTMEAEDVATYYCQQGSTIPFTFGSGTKLEIKRADAAP
TVSIFPPSSEQLTSGGASVVCFLNNFYPRDINVKWKIDGSERQNGVLNSVVTDQDS
KDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Antibody G2 heavy
chain full antibody nucleotide sequence (not including Fc domain)
(SEQ ID NO: 31)
GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGGTAAAGCCTGGGGCTTCAG
TGAAGATGTCCTGCAAGGCTTCTGGATACACATTCACTAGCTCTGTTATGCACT
GGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATATTAATCCT
TACAATGATGGTACTAAGTACAATGAGAAGTTCAAAGGCAAGGCCACACTGACT
TCAGACAAATCCTCCAGCACAGCCTACATGGAACTCAGCAGCCTGACCTCTGA
GGACTCTGCGGTCTATTACTGTGCAAAAGGGGGTAACGATGGCTACTGGGGC
CAAGGCACTACTCTCACAGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTAT
CCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATG
CCTGGTCAAGGGCTATTTCCCTGAGCCAGTGACAGTGACCTGGAACTCTGGAT
CCCTGTCCAGCGGTGTGCACACCTTCCCAGCTGTCCTGCAGTCTGACCTCTAC
ACTCTGAGCAGCTCAGTGACTGTCCCCTCCAGCACCTGGCCCAGCGAGACCG
TCACCTGCAACGTTGCCCACCCGGCCAGCAGCACCAAGGTGGACAAGAAAATT GTGCCCAGGGAT
Antibody G2 light chain full antibody nucleotide sequence (SEQ ID
NO: 32) GAAATTGTGCTCACCCAGTCTCCAACCACCATGGCTGCATCTCCCGGGGAGAA
GATCACTATCACCTGTAGTGCCAGCTCAAGTATAAGTTCCATTTACTTGCATTG
GTATCAGCAGAAGCCAGGATTCTCCCCTAAAGTCTTGATTTATAGGGCATCCAA
TCTGGCTTCTGGAGTCCCAGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTT
ACTCTCTCACAATTGGCACCATGGAGGCTGAAGATGTTGCCACTTACTACTGCC
AGCAGGGTAGTACTATACCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATA
AAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCA
GTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAG
AGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGTGTCC
TGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGC
ACCCTCACATTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGA
GGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTCAACAGGAATG AGTGTTAA
Antibody G1 CDR H1 (Chothia CDR) (SEQ ID NO: 33) GFTFSNY Antibody
G1 CDR H1 (Kabat CDR) (SEQ ID NO: 34) NYWIS Antibody G1 CDR H2
(Chothia CDR) (SEQ ID NO: 35) RSESDASA Antibody G2 CDR H1 (extended
CDR) (SEQ ID NO: 36) GYTFTSSVMH Antibody G2 CDR H1 (Chothia CDR)
(SEQ ID NO: 37) GYTFTSS Antibody G2 CDR H2 (Chothia CDR) (SEQ ID
NO: 38) NPYNDG
[0192] Although the disclosed teachings have been described with
reference to various applications, methods, kits, and compositions,
it will be appreciated that various changes and modifications can
be made without departing from the teachings herein and the claimed
invention below. The foregoing examples are provided to better
illustrate the disclosed teachings and are not intended to limit
the scope of the teachings presented herein. While the present
teachings have been described in terms of these exemplary
embodiments, the skilled artisan will readily understand that
numerous variations and modifications of these exemplary
embodiments are possible without undue experimentation. All such
variations and modifications are within the scope of the current
teachings.
[0193] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the described
subject matter in any way. It will be appreciated that there is an
implied "about" prior to the temperatures, concentrations, times,
etc discussed in the present teachings, such that slight and
insubstantial deviations are within the scope of the present
teachings herein. In this application, the use of the singular
includes the plural unless specifically stated otherwise. Also, the
use of "comprise", "comprises", "comprising", "contain",
"contains", "containing", "include", "includes", and "including"
are not intended to be limiting. It is to be understood that both
the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention.
[0194] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated by reference in their entirety. In the event that one
or more of the incorporated literature and similar materials
differs from or contradicts this application, including but not
limited to defined terms, term usage, described techniques, or the
like, this application controls.
[0195] The foregoing description and Examples detail certain
specific embodiments of the invention and describes the best mode
contemplated by the inventors. It will be appreciated, however,
that no matter how detailed the foregoing may appear in text, the
invention may be practiced in many ways and the invention should be
construed in accordance with the appended claims and any
equivalents thereof.
Sequence CWU 1
1
391122PRTArtificial sequenceSynthetic polypeptide, Antibody G1
heavy chain variable region 1Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile
Arg Ser Glu Ser Asp Ala Ser Ala Thr His Tyr Ala Glu 50 55 60 Ala
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70
75 80 Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr 85 90 95 Tyr Cys Leu Ala Tyr Phe Asp Tyr Gly Leu Ala Ile Gln
Asn Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 2107PRTArtificial sequenceSynthetic polypeptide, Antibody G1
light chain variable region 2Glu Ile Val Leu Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Lys Ala Ser Lys Arg Val Thr Thr Tyr 20 25 30 Val Ser Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala
Ser Asn Arg Tyr Leu Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70
75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Ser Gln Ser Tyr Asn Tyr Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
310PRTArtificial sequenceSynthetic polypeptide, Antibody G1 CDR H1
3Gly Phe Thr Phe Ser Asn Tyr Trp Ile Ser 1 5 10 419PRTArtificial
sequenceSynthetic polypeptide, Antibody G1 CDR H2 4Glu Ile Arg Ser
Glu Ser Asp Ala Ser Ala Thr His Tyr Ala Glu Ala 1 5 10 15 Val Lys
Gly 511PRTArtificial sequenceSynthetic polypeptide, Antibody G1 CDR
H3 5Tyr Phe Asp Tyr Gly Leu Ala Ile Gln Asn Tyr 1 5 10
611PRTArtificial sequenceSynthetic polypeptide, Antibody G1 CDR L1
6Lys Ala Ser Lys Arg Val Thr Thr Tyr Val Ser 1 5 10 77PRTArtificial
sequenceSynthetic polypeptide, Antibody G1 CDR L2 7Gly Ala Ser Asn
Arg Tyr Leu 1 5 89PRTArtificial sequenceSynthetic polypeptide,
Antibody G1 CDR L3 8Ser Gln Ser Tyr Asn Tyr Pro Tyr Thr 1 5
9366DNAArtificial sequenceSynthetic oligonucleotide, Antibody G1
heavy chain variable region 9gaagttcagc tggttgaatc cggtggtggt
ctggttcagc caggtggttc cctgcgtctg 60tcctgcgctg cttccggttt caccttctcc
aactactgga tctcctgggt tcgtcaggct 120cctggtaaag gtctggaatg
ggttgctgaa atccgttccg aatccgacgc gtccgctacc 180cattacgctg
aagctgttaa aggtcgtttc accatctccc gtgacaacgc taagaactcc
240ctgtacctgc agatgaactc cctgcgtgct gaagacaccg ctgtttacta
ctgcctggct 300tactttgact acggtctggc tatccagaac tactggggtc
agggtaccct ggttaccgtt 360tcctcc 36610321DNAArtificial
sequenceSynthetic oligonucleotide, Antibody G1 light chain variable
region 10gaaatcgttc tgacccagtc cccggctacc ctgtccctgt ccccaggtga
acgtgctacc 60ctgtcctgca aagcttccaa acgggttacc acctacgttt cctggtacca
gcagaaaccc 120ggtcaggctc ctcgtctgct gatctacggt gcttccaacc
gttacctcgg tatcccagct 180cgtttctccg gttccggttc cggtaccgac
ttcaccctga ccatctcctc cctggaaccc 240gaagacttcg ctgtttacta
ctgcagtcag tcctacaact acccctacac cttcggtcag 300ggtaccaaac
tggaaatcaa a 32111448PRTArtificial sequenceSynthetic polypeptide,
Antibody G1 heavy chain 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Ile Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Arg
Ser Glu Ser Asp Ala Ser Ala Thr His Tyr Ala Glu 50 55 60 Ala Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Leu Ala Tyr Phe Asp Tyr Gly Leu Ala Ile Gln Asn Tyr
Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr 130 135 140 Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190 Val Pro
Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp 195 200 205
His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys 210
215 220 Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro
Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro 260 265 270 Glu Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Phe Arg Val Val 290 295 300 Ser Val Leu Thr
Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys
Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 325 330
335 Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Met Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445
12214PRTArtificial sequenceSynthetic polypeptide, Antibody G1 light
chain 12Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Lys Ala Ser Lys Arg Val
Thr Thr Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Tyr Leu Gly
Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val
Tyr Tyr Cys Ser Gln Ser Tyr Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly
Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120
125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu
Cys 210 131347DNAArtificial sequenceSynthetic oligonucleotide,
Antibody G1 heavy chain 13gaagttcagc tggttgaatc cggtggtggt
ctggttcagc caggtggttc cctgcgtctg 60tcctgcgctg cttccggttt caccttctcc
aactactgga tctcctgggt tcgtcaggct 120cctggtaaag gtctggaatg
ggttgctgaa atccgttccg aatccgacgc gtccgctacc 180cattacgctg
aagctgttaa aggtcgtttc accatctccc gtgacaacgc taagaactcc
240ctgtacctgc agatgaactc cctgcgtgct gaagacaccg ctgtttacta
ctgcctggct 300tactttgact acggtctggc tatccagaac tactggggtc
agggtaccct ggttaccgtt 360tcctccgcct ccaccaaggg cccatctgtc
ttcccactgg ccccatgctc ccgcagcacc 420tccgagagca cagccgccct
gggctgcctg gtcaaggact acttcccaga acctgtgacc 480gtgtcctgga
actctggcgc tctgaccagc ggcgtgcaca ccttcccagc tgtcctgcag
540tcctcaggtc tctactccct cagcagcgtg gtgaccgtgc catccagcaa
cttcggcacc 600cagacctaca cctgcaacgt agatcacaag ccaagcaaca
ccaaggtcga caagaccgtg 660gagagaaagt gttgtgtgga gtgtccacct
tgtccagccc ctccagtggc cggaccatcc 720gtgttcctgt tccctccaaa
gccaaaggac accctgatga tctccagaac cccagaggtg 780acctgtgtgg
tggtggacgt gtcccacgag gacccagagg tgcagttcaa ctggtatgtg
840gacggagtgg aggtgcacaa cgccaagacc aagccaagag aggagcagtt
caactccacc 900ttcagagtgg tgagcgtgct gaccgtggtg caccaggact
ggctgaacgg aaaggagtat 960aagtgtaagg tgtccaacaa gggactgcca
tccagcatcg agaagaccat ctccaagacc 1020aagggacagc caagagagcc
acaggtgtat accctgcccc catccagaga ggagatgacc 1080aagaaccagg
tgtccctgac ctgtctggtg aagggattct atccatccga catcgccgtg
1140gagtgggagt ccaacggaca gccagagaac aactataaga ccacccctcc
aatgctggac 1200tccgacggat ccttcttcct gtattccaag ctgaccgtgg
acaagtccag atggcagcag 1260ggaaacgtgt tctcttgttc cgtgatgcac
gaggccctgc acaaccacta tacccagaag 1320agcctgtccc tgtctccagg aaagtaa
134714645DNAArtificial sequenceSynthetic oligonucleotide, Antibody
G1 light chain 14gaaatcgttc tgacccagtc cccggctacc ctgtccctgt
ccccaggtga acgtgctacc 60ctgtcctgca aagcttccaa acgggttacc acctacgttt
cctggtacca gcagaaaccc 120ggtcaggctc ctcgtctgct gatctacggt
gcttccaacc gttacctcgg tatcccagct 180cgtttctccg gttccggttc
cggtaccgac ttcaccctga ccatctcctc cctggaaccc 240gaagacttcg
ctgtttacta ctgcagtcag tcctacaact acccctacac cttcggtcag
300ggtaccaaac tggaaatcaa acgcactgtg gctgcaccat ctgtcttcat
cttccctcca 360tctgatgagc agttgaaatc cggaactgcc tctgttgtgt
gcctgctgaa taacttctat 420ccgcgcgagg ccaaagtaca gtggaaggtg
gataacgccc tccaatccgg taactcccag 480gagagtgtca cagagcagga
cagcaaggac agcacctaca gcctcagcag caccctgacc 540ctgagcaaag
cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc
600ctgagttctc cagtcacaaa gagcttcaac cgcggtgagt gctaa 6451537PRTHomo
sapiensPolypeptide, Alpha-CGRP 15Ala Cys Asp Thr Ala Thr Cys Val
Thr His Arg Leu Ala Gly Leu Leu 1 5 10 15 Ser Arg Ser Gly Gly Val
Val Lys Asn Asn Phe Val Pro Thr Asn Val 20 25 30 Gly Ser Lys Ala
Phe 35 1637PRTHomo sapiensPolypeptide, Beta-CGRP 16Ala Cys Asn Thr
Ala Thr Cys Val Thr His Arg Leu Ala Gly Leu Leu 1 5 10 15 Ser Arg
Ser Gly Gly Met Val Lys Ser Asn Phe Val Pro Thr Asn Val 20 25 30
Gly Ser Lys Ala Phe 35 1737PRTRattus norvegicusPolypeptide,
Alpha-CGRP 17Ser Cys Asn Thr Ala Thr Cys Val Thr His Arg Leu Ala
Gly Leu Leu 1 5 10 15 Ser Arg Ser Gly Gly Val Val Lys Asp Asn Phe
Val Pro Thr Asn Val 20 25 30 Gly Ser Glu Ala Phe 35 1837PRTRattus
norvegicusPolypeptide, Beta-CGRP 18Ser Cys Asn Thr Ala Thr Cys Val
Thr His Arg Leu Ala Gly Leu Leu 1 5 10 15 Ser Arg Ser Gly Gly Val
Val Lys Asp Asn Phe Val Pro Thr Asn Val 20 25 30 Gly Ser Lys Ala
Phe 35 19115PRTArtificial sequenceSynthetic polypeptide, Antibody
G2 heavy chain variable region 19Glu Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 20 25 30 Val Met His Trp
Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr
Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Gly Gly Asn Asp Gly Tyr Trp Gly Gln Gly
Thr Thr Leu Thr 100 105 110 Val Ser Ser 115 20108PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 light chain variable
region 20Glu Ile Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser
Pro Gly 1 5 10 15 Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser
Ile Ser Ser Ile 20 25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly
Phe Ser Pro Lys Val Leu 35 40 45 Ile Tyr Arg Ala Ser Asn Leu Ala
Ser Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val
Ala Thr Tyr Tyr Cys Gln Gln Gly Ser Thr Ile Pro 85 90 95 Phe Thr
Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 215PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 Kabat CDR H1 21Ser Ser
Val Met His 1 5 2217PRTArtificial sequenceSynthetic polypeptide,
Antibody G2 CDR H2 22Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr
Asn Glu Lys Phe Lys 1 5 10 15 Gly 236PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 CDR H3 23Gly Gly Asn Asp
Gly Tyr 1 5 2412PRTArtificial sequenceSynthetic polypeptide,
Antibody G2 CDR L1 24Ser Ala Ser Ser Ser Ile Ser Ser Ile Tyr Leu
His 1 5 10 257PRTArtificial sequenceSynthetic polypeptide, Antibody
G2 CDR L2 25Arg Ala Ser Asn Leu Ala Ser 1 5 269PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 CDR L3 26Gln Gln Gly Ser
Thr Ile Pro Phe Thr 1 5 27345DNAArtificial sequenceSynthetic
oligonucleotide, Antibody G2 heavy chain variable region
27gaggtccagc tgcagcagtc tggacctgag ctggtaaagc ctggggcttc agtgaagatg
60tcctgcaagg cttctggata cacattcact agctctgtta tgcactgggt gaagcagaag
120cctgggcagg gccttgagtg gattggatat attaatcctt acaatgatgg
tactaagtac 180aatgagaagt tcaaaggcaa ggccacactg acttcagaca
aatcctccag cacagcctac 240atggaactca gcagcctgac ctctgaggac
tctgcggtct attactgtgc aaaagggggt 300aacgatggct actggggcca
aggcactact ctcacagtct cctca 34528324DNAArtificial sequenceSynthetic
oligonucleotide, Antibody G2 light chain variable region
28gaaattgtgc tcacccagtc tccaaccacc atggctgcat ctcccgggga gaagatcact
60atcacctgta gtgccagctc aagtataagt tccatttact tgcattggta tcagcagaag
120ccaggattct cccctaaagt cttgatttat agggcatcca atctggcttc
tggagtccca 180gctcgcttca gtggcagtgg gtctgggacc tcttactctc
tcacaattgg caccatggag 240gctgaagatg ttgccactta ctactgccag
cagggtagta ctataccatt cacgttcggc 300tcggggacaa agttggaaat aaaa
32429216PRTArtificial sequenceSynthetic polypeptide, Antibody G2
heavy chain 29Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys
Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Ser Ser 20 25 30 Val Met His Trp Val Lys Gln Lys Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn
Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr
Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75
80 Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Gly Gly Asn Asp Gly Tyr Trp Gly Gln Gly Thr Thr
Leu Thr 100 105 110 Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr
Pro Leu Ala Pro 115 120 125 Gly Ser Ala Ala Gln Thr Asn Ser Met Val
Thr Leu Gly Cys Leu Val 130 135 140 Lys Gly Tyr Phe Pro Glu Pro Val
Thr Val Thr Trp Asn Ser Gly Ser 145 150 155 160 Leu Ser Ser Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu 165 170 175 Tyr Thr Leu
Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro Ser 180 185 190 Glu
Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val 195 200
205 Asp Lys Lys Ile Val Pro Arg Asp 210 215 30215PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 light chain 30Glu Ile
Val Leu Thr Gln Ser Pro Thr Thr Met Ala Ala Ser Pro Gly 1 5 10 15
Glu Lys Ile Thr Ile Thr Cys Ser Ala Ser Ser Ser Ile Ser Ser Ile 20
25 30 Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Phe Ser Pro Lys Val
Leu 35 40 45 Ile Tyr Arg Ala Ser Asn Leu Ala Ser Gly Val Pro Ala
Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Gly Thr Met Glu 65 70 75 80 Ala Glu Asp Val Ala Thr Tyr Tyr Cys
Gln Gln Gly Ser Thr Ile Pro 85 90 95 Phe Thr Phe Gly Ser Gly Thr
Lys Leu Glu Ile Lys Arg Ala Asp Ala 100 105 110 Ala Pro Thr Val Ser
Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser 115 120 125 Gly Gly Ala
Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Arg Asp 130 135 140 Ile
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val 145 150
155 160 Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Met 165 170 175 Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
His Asn Ser 180 185 190 Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
Ser Pro Ile Val Lys 195 200 205 Ser Phe Asn Arg Asn Glu Cys 210 215
31648DNAArtificial sequenceSynthetic oligonucleotide, Antibody G2
heavy chain 31gaggtccagc tgcagcagtc tggacctgag ctggtaaagc
ctggggcttc agtgaagatg 60tcctgcaagg cttctggata cacattcact agctctgtta
tgcactgggt gaagcagaag 120cctgggcagg gccttgagtg gattggatat
attaatcctt acaatgatgg tactaagtac 180aatgagaagt tcaaaggcaa
ggccacactg acttcagaca aatcctccag cacagcctac 240atggaactca
gcagcctgac ctctgaggac tctgcggtct attactgtgc aaaagggggt
300aacgatggct actggggcca aggcactact ctcacagtct cctcagccaa
aacgacaccc 360ccatctgtct atccactggc ccctggatct gctgcccaaa
ctaactccat ggtgaccctg 420ggatgcctgg tcaagggcta tttccctgag
ccagtgacag tgacctggaa ctctggatcc 480ctgtccagcg gtgtgcacac
cttcccagct gtcctgcagt ctgacctcta cactctgagc 540agctcagtga
ctgtcccctc cagcacctgg cccagcgaga ccgtcacctg caacgttgcc
600cacccggcca gcagcaccaa ggtggacaag aaaattgtgc ccagggat
64832648DNAArtificial sequenceSynthetic oligonucleotide, Antibody
G2 light chain 32gaaattgtgc tcacccagtc tccaaccacc atggctgcat
ctcccgggga gaagatcact 60atcacctgta gtgccagctc aagtataagt tccatttact
tgcattggta tcagcagaag 120ccaggattct cccctaaagt cttgatttat
agggcatcca atctggcttc tggagtccca 180gctcgcttca gtggcagtgg
gtctgggacc tcttactctc tcacaattgg caccatggag 240gctgaagatg
ttgccactta ctactgccag cagggtagta ctataccatt cacgttcggc
300tcggggacaa agttggaaat aaaacgggct gatgctgcac caactgtatc
catcttccca 360ccatccagtg agcagttaac atctggaggt gcctcagtcg
tgtgcttctt gaacaacttc 420taccccagag acatcaatgt caagtggaag
attgatggca gtgaacgaca aaatggtgtc 480ctgaacagtt ggactgatca
ggacagcaaa gacagcacct acagcatgag cagcaccctc 540acattgacca
aggacgagta tgaacgacat aacagctata cctgtgaggc cactcacaag
600acatcaactt cacccatcgt caagagcttc aacaggaatg agtgttaa
648337PRTArtificial sequenceSynthetic polypeptide, Antibody G1
Chothia CDR H1 33Gly Phe Thr Phe Ser Asn Tyr 1 5 345PRTArtificial
sequenceSynthetic polypeptide, Antibody G1 Kabat CDR H1 34Asn Tyr
Trp Ile Ser 1 5 358PRTArtificial sequenceSynthetic polypeptide,
Antibody G1 Chothia CDR H2 35Arg Ser Glu Ser Asp Ala Ser Ala 1 5
3610PRTArtificial sequenceSynthetic polypeptide, Antibody G2
extended CDR H1 36Gly Tyr Thr Phe Thr Ser Ser Val Met His 1 5 10
377PRTArtificial sequenceSynthetic polypeptide, Antibody G2 Chothia
CDR H1 37Gly Tyr Thr Phe Thr Ser Ser1 5 386PRTArtificial
sequenceSynthetic polypeptide, Antibody G2 Chothia CDR H2 38Asn Pro
Tyr Asn Asp Gly 1 5 395PRTArtificial sequenceSynthetic polypeptide
39Gly Ser Lys Ala Phe1 5
* * * * *