U.S. patent application number 14/090664 was filed with the patent office on 2014-07-03 for prostaglandin e2 binding proteins and uses thereof.
This patent application is currently assigned to AbbVie, Inc.. The applicant listed for this patent is AbbVie, Inc.. Invention is credited to Eileen Belanger, Tariq Ghayur, Jijie Gu, Maria C. Harris, Paul R. Hinton, Charles W. Hutchins, Veronica M. Juan, Junjian Liu, John Mankovich, Jianwei Shen, Rong-Rong Zhu.
Application Number | 20140186377 14/090664 |
Document ID | / |
Family ID | 41507421 |
Filed Date | 2014-07-03 |
United States Patent
Application |
20140186377 |
Kind Code |
A1 |
Gu; Jijie ; et al. |
July 3, 2014 |
PROSTAGLANDIN E2 BINDING PROTEINS AND USES THEREOF
Abstract
The present invention encompasses prostaglandin E.sub.2
(PGE.sub.2) binding proteins. The invention relates to antibodies
that are wild-type, chimeric, CDR grafted and humanized. Preferred
antibodies have high affinity for prostaglandin E.sub.2 and
neutralize prostaglandin E.sub.2 activity in vitro and in vivo. An
antibody of the invention can be a full-length antibody, or an
antigen-binding portion thereof. Methods of making and methods of
using the antibodies of the invention are also provided. The
antibodies, or antigen-binding portions, of the invention are
useful for detecting prostaglandin E.sub.2 and for inhibiting
prostaglandin E.sub.2 activity, e.g., in a human subject suffering
from a disorder in which prostaglandin E.sub.2 activity is
detrimental.
Inventors: |
Gu; Jijie; (Shrewsbury,
MA) ; Hutchins; Charles W.; (Green Oaks, IL) ;
Zhu; Rong-Rong; (Southborough, MA) ; Shen;
Jianwei; (Lake Bluff, IL) ; Harris; Maria C.;
(Shrewsbury, MA) ; Belanger; Eileen; (Northbridge,
MA) ; Ghayur; Tariq; (Holliston, MA) ; Hinton;
Paul R.; (Sunnyvale, CA) ; Juan; Veronica M.;
(Menlo, CA) ; Mankovich; John; (Andover, MA)
; Liu; Junjian; (Shrewsbury, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie, Inc. |
North Chicago |
IL |
US |
|
|
Assignee: |
AbbVie, Inc.
North Chicago
IL
|
Family ID: |
41507421 |
Appl. No.: |
14/090664 |
Filed: |
November 26, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12499646 |
Jul 8, 2009 |
8624002 |
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14090664 |
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61134264 |
Jul 8, 2008 |
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61197258 |
Oct 23, 2008 |
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Current U.S.
Class: |
424/175.1 ;
435/188; 435/252.33; 435/254.11; 435/254.2; 435/254.21; 435/320.1;
435/345; 435/419; 435/69.6; 435/7.92; 530/387.3; 530/388.9;
530/389.8; 530/391.3; 530/391.7; 536/23.53 |
Current CPC
Class: |
A61P 25/32 20180101;
A61P 15/08 20180101; A61P 25/16 20180101; Y02A 50/412 20180101;
A61P 1/18 20180101; A61P 3/12 20180101; A61P 7/02 20180101; A61P
9/10 20180101; A61P 25/14 20180101; C07K 2317/56 20130101; Y02A
50/478 20180101; A61P 17/08 20180101; A61P 31/22 20180101; A61K
2039/505 20130101; A61P 27/14 20180101; A61P 9/00 20180101; A61P
9/06 20180101; A61P 7/04 20180101; A61P 17/14 20180101; A61P 33/00
20180101; A61K 31/405 20130101; A61P 27/16 20180101; A61P 7/00
20180101; A61P 9/14 20180101; A61P 13/08 20180101; A61P 29/00
20180101; A61P 5/14 20180101; A61P 25/04 20180101; A61P 37/02
20180101; A61P 19/00 20180101; A61P 19/02 20180101; A61P 31/18
20180101; A61P 11/06 20180101; A61P 17/02 20180101; A61P 37/06
20180101; A61P 27/02 20180101; A61P 35/02 20180101; C07K 2317/76
20130101; Y02A 50/30 20180101; A61P 31/12 20180101; A61P 35/00
20180101; A61P 37/08 20180101; C07K 2317/21 20130101; C07K 2317/622
20130101; A61P 21/00 20180101; A61P 21/04 20180101; Y02A 50/41
20180101; Y02A 50/386 20180101; A61P 31/10 20180101; C07K 16/18
20130101; A61K 31/573 20130101; A61P 31/00 20180101; Y02A 50/481
20180101; A61P 13/00 20180101; A61P 11/00 20180101; A61P 3/10
20180101; A61P 5/18 20180101; A61P 13/12 20180101; A61K 39/39533
20130101; A61P 13/10 20180101; A61P 15/06 20180101; A61P 17/04
20180101; A61P 9/12 20180101; A61P 25/00 20180101; A61P 31/04
20180101; A61P 5/00 20180101; A61P 15/00 20180101; A61P 19/10
20180101; C07K 2317/565 20130101; A61P 1/04 20180101; C07K 2317/24
20130101; Y02A 50/401 20180101; A61P 1/16 20180101; A61P 17/00
20180101; C07K 16/26 20130101; C07K 2317/55 20130101; A61P 7/06
20180101; A61P 9/04 20180101; A61P 11/04 20180101; A61P 17/06
20180101; A61P 43/00 20180101; A61P 11/02 20180101; A61P 25/28
20180101; A61P 1/00 20180101 |
Class at
Publication: |
424/175.1 ;
530/389.8; 530/387.3; 530/388.9; 530/391.3; 530/391.7; 435/188;
536/23.53; 435/320.1; 435/252.33; 435/345; 435/419; 435/254.11;
435/254.2; 435/254.21; 435/69.6; 435/7.92 |
International
Class: |
C07K 16/18 20060101
C07K016/18; A61K 31/573 20060101 A61K031/573; A61K 31/405 20060101
A61K031/405; A61K 39/395 20060101 A61K039/395 |
Claims
1-92. (canceled)
93. An antibody or antigen-binding fragment thereof, wherein the
antibody or antigen-binding fragment is capable of binding
prostaglandin E2 (PGE2), and the antibody or antigen-binding
fragment comprises complementarity determining region (CDR)
sequences of SEQ ID NO: 44 (GYTFTKYWLG, DIYPYGDYTHYNEKFKD, and
SDGSSTY) and SEQ ID NO: 45 (TSSQNIVHSNGNTYLE, KVSNRFSG, and
FQVSHVPYTF).
94. An antibody or antigen-binding fragment thereof, wherein the
antibody or antigen-binding fragment is capable of binding
prostaglandin E2, and the antibody or antigen-binding fragment
comprises variable domain sequences selected from the group
consisting of (a) SEQ ID NO: 44 and SEQ ID NO: 45; and (b) SEQ ID
NO: 40 and SEQ ID NO: 41.
95. The antibody or antigen-binding fragment according to claim 93
or claim 94, wherein the antibody or antigen-binding fragment (a)
is capable of modulating a biological function of prostaglandin
E.sub.2; (b) is capable of neutralizing prostaglandin E.sub.2; (c)
is capable of preventing the binding of prostaglandin E.sub.2 to
one or more prostaglandin E.sub.2 receptors selected from the group
consisting of EP1, EP2, EP3, and EP4; (d) is capable of preventing
the binding of prostaglandin E.sub.2 to prostaglandin E.sub.2
receptors EP1, EP2, EP3, and EP4; (e) is capable of binding
prostaglandin E.sub.2 with an EC.sub.50 selected from the group
consisting of about 1.times.10.sup.-6 to about 1.times.10.sup.-7 M,
about 1.times.10.sup.-7 to about 1.times.10.sup.-8 M, about
1.times.10.sup.-8 to about 1.times.10.sup.-9 M, about
1.times.10.sup.-9 to about 1.times.10.sup.-10 M, about
1.times.10.sup.-10 to about 1.times.10.sup.-11 M and about
1.times.10.sup.-11 to about 1.times.10.sup.-12 M using an ELISA
assay; and/or (f) is capable of inhibiting calcium influx induced
by prostaglandin E.sub.2 to the prostaglandin E.sub.2 receptor EP4
in an EP4 receptor mediated FLIPR assay with an IC.sub.50 selected
from the group consisting of about 1.times.10.sup.-6 to about
1.times.10.sup.-7 M, about 1.times.10.sup.-7 to about
1.times.10.sup.-8 M, about 1.times.10.sup.-8 to about
1.times.10.sup.-9 M, about 1.times.10.sup.-9 to about
1.times.10.sup.-10 M, about 1.times.10.sup.-10 to about
1.times.10.sup.-11 M, and about 1.times.10.sup.-11 to about
1.times.10.sup.-12 M.
96. The antibody or antigen-binding fragment according to claim 93
or claim 94, wherein the antibody or antigen-binding fragment has
been affinity matured.
97. The antibody or antigen-binding fragment according to claim 93
or claim 94, further comprising a linker polypeptide.
98. The binding protein according to claim 93, wherein the binding
protein further comprises human acceptor framework sequences.
99. The antibody or antigen-binding fragment according to claim 93
or claim 94, wherein the antibody or antigen-binding fragment is
selected from the group consisting of an immunoglobulin molecule, a
disulfide linked Fv, a monoclonal antibody, an scFv, a chimeric
antibody, a single domain antibody, a CDR-grafted antibody, a
diabody, a humanized antibody, a multispecific antibody, a Fab, a
dual specific antibody, a Fab', a bispecific antibody, a F(ab')2,
and an Fv.
100. The antibody or antigen-binding fragment according to claim 93
or claim 94, further comprising a heavy chain immunoglobulin
constant domain selected from the group consisting of a human IgM
constant domain, a human IgG4 constant domain, a human IgG1
constant domain, a human IgE constant domain, a human IgG2 constant
domain, a human IgG3 constant domain, and a human IgA constant
domain
101. The antibody or antigen-binding fragment according to claim
100, wherein the immunoglobulin constant domain has an amino acid
sequence selected from the group consisting of SEQ ID NOs: 1-4.
102. An antibody conjugate comprising the antibody or
antigen-binding fragment according to claim 93 or claim 94, the
antibody conjugate further comprising an immunoadhesion molecule,
an imaging agent, a therapeutic agent, or a cytotoxic agent.
103. The antibody conjugate of claim 102, wherein the imaging agent
is a radiolabel, an enzyme, a fluorescent label, a luminescent
label, a bioluminescent label, a magnetic label, or biotin.
104. The antibody conjugate of claim 103, wherein the radiolabel is
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111 In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153SM.
105. The antibody conjugate of claim 102, wherein the therapeutic
or cytotoxic agent is an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, a toxin, or an apoptotic
agent.
106. The antibody or antigen-binding fragment according to claim 93
or claim 94, wherein: (a) the antibody or antigen-binding fragment
possesses a human glycosylation pattern; (b) the antibody or
antigen-binding fragment is crystallized; (c) the antibody or
antigen-binding fragment is a carrier-free pharmaceutical
controlled release crystal; (d) the antibody or antigen-binding
fragment of (a)-(c) has a greater half-life in vivo than a soluble
counterpart; and/or (e) the antibody or antigen-binding fragment of
(a)-(d) retains biological activity after crystallization.
107. An isolated nucleic acid encoding the antibody or
antigen-binding fragment of claim 93 or claim 94.
108. A vector comprising the isolated nucleic acid according to
claim 107, wherein the vector optionally comprises pcDNA, pTT,
pTT3, pEFBOS, pBV, pJV, pBJ, or pA2.
109. A host cell comprising the vector according to claim 108,
wherein the host cell is optionally selected from the group
consisting of a prokaryotic cell, an Escherichia coli cell, a
eukaryotic cell, a protist cell, an animal cell, a plant cell, a
fungal cell, a mammalian cell, an avian cell, an insect cell, a CHO
cell, a COS cell, a HEK293 cell, a yeast cell, a Saccharomyces
cerevisiae cell, and an insect Sf9 cell.
110. A method of producing an antibody or antigen-binding fragment
capable of binding prostaglandin E.sub.2, the method comprising the
step of culturing the host cell of claim 109 under conditions
sufficient to produce the antibody or antigen-binding fragment.
111. A composition for the release of an antibody or
antigen-binding fragment, the composition comprising: (a) a
crystallized antibody or antigen-binding fragment according to
claim 106; (b) an ingredient selected from the group consisting of
albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol, and
polyethylene glycol; and/or (c) at least one polymeric carrier
optionally selected from the group consisting of poly (acrylic
acid), poly (cyanoacrylates), poly (amino acids), poly
(anhydrides), poly (depsipeptide), poly (esters), poly (lactic
acid), poly (lactic-co-glycolic acid) or PLGA, poly
(b-hydroxybutryate), poly (caprolactone), poly (dioxanone); poly
(ethylene glycol), poly ((hydroxypropyl) methacrylamide,
poly[(organo) phosphazene], poly (ortho esters), poly (vinyl
alcohol), poly (vinylpyrrolidone), maleic anhydride-alkyl vinyl
ether copolymers, pluronic polyols, albumin, alginate, cellulose
and cellulose derivatives, collagen, fibrin, gelatin, hyaluronic
acid, oligosaccharides, glycaminoglycans, sulfated polyeaccharides,
and a blend and/or a copolymer thereof.
112. A pharmaceutical composition comprising the antibody or
antigen-binding fragment of claim 93 or claim 94 and a
pharmaceutically acceptable carrier.
113. The pharmaceutical composition of claim 112, wherein the
pharmaceutically acceptable carrier functions as an adjuvant useful
to increase the absorption or dispersion of the antibody or
antigen-binding fragment thereof, wherein the adjuvant is
optionally hyaluronidase.
114. The pharmaceutical composition of claim 113, optionally
further comprising an additional therapeutic agent selected from
the group consisting of a therapeutic agent, an imaging agent, a
cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor, a
co-stimulation molecule blocker, an adhesion molecule blocker, an
anti-cytokine antibody or functional fragment thereof,
methotrexate, cyclosporine, rapamycin, FK506, a detectable label or
reporter, a TNF antagonist, an anti-rheumatic, a muscle relaxant, a
narcotic, a non-steroid anti-inflammatory drug (NSAID), an
analgesic, an anesthetic, a sedative, a local anesthetic, a
neuromuscular blocker, an antimicrobial, an antipsoriatic, a
corticosteroid, an anabolic steroid, an erythropoietin, an
immunization, an immunoglobulin, an immunosuppressive, a growth
hormone, a hormone replacement drug, a radiopharmaceutical, an
antidepressant, an antipsychotic, a stimulant, an asthma
medication, a beta agonist, an inhaled steroid, an oral steroid, an
epinephrine or analog thereof, a cytokine, and a cytokine
antagonist.
115. Use of the antibody or antigen-binding fragment according to
claim 93 or claim 94 to reduce prostaglandin E.sub.2 activity in a
human subject.
116. The use of claim 115, wherein the human subject is suffering
from a disorder selected from the group consisting of an autoimmune
disease, an inflammatory disease, a tumor, rheumatoid arthritis,
allergic arthritis, Guillain Barre syndrome, infectious
mononucleosis, a viral lymphadenopathy, a herpes virus infection,
multiple sclerosis, a demyelinating disease, a hematological
disorder, hemolytic anemia, thrombocytopenia, an endocrinologic
disorder, diabetes mellitus, Addison's disease, idiopathic
hypoparathyroidism, chronic lymphocytic thyroiditis, a collagen
disorder, systemic lupus erythematosus, a reproductive disorder,
amenorrhoea, infertility, recurrent abortion, eclampsia, a headneck
tumor, lung cancer, gastric cancer, prostate cancer, pancreatic
cancer, a gastrointestinal organ disease, inflammatory bowel
disease, ulcerative colitis, Crohn's disease, pain, pain related
with osteoarthritis, an ocular disorder, or age-related mascular
degeneration, rheumatoid arthritis, osteoarthritis, juvenile
chronic arthritis, septic arthritis, Lyme arthritis, psoriatic
arthritis, reactive arthritis, spondyloarthropathy, systemic lupus
erythematosus, Crohn's disease, ulcerative colitis, inflammatory
bowel disease, insulin dependent diabetes mellitus, thyroiditis,
asthma, allergic diseases, psoriasis, dermatitis scleroderma, graft
versus host disease, organ transplant rejection, acute or chronic
immune disease associated with organ transplantation, sarcoidosis,
atherosclerosis, disseminated intravascular coagulation, Kawasaki's
disease, Grave's disease, nephrotic syndrome, chronic fatigue
syndrome, Wegener's granulomatosis, Henoch-Schoenlein purpurea,
microscopic vasculitis of the kidneys, chronic active hepatitis,
uveitis, septic shock, toxic shock syndrome, sepsis syndrome,
cachexia, infectious diseases, parasitic diseases, acquired
immunodeficiency syndrome, acute transverse myelitis, Huntington's
chorea, Parkinson's disease, Alzheimer's disease, stroke, primary
biliary cirrhosis, hemolytic anemia, malignancies, heart failure,
myocardial infarction, Addison's disease, sporadic, polyglandular
deficiency type I and polyglandular deficiency type II, Schmidt's
syndrome, adult (acute) respiratory distress syndrome, alopecia,
alopecia areata, seronegative arthropathy, arthropathy, Reiter's
disease, psoriatic arthropathy, ulcerative colitic arthropathy,
enteropathic synovitis, chlamydia, yersinia and salmonella
associated arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, acquired immunodeficiency
related diseases, hepatitis B, hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, Lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, drug-induced hepatitis, non-alcoholic steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental
disorders, depression, schizophrenia, Th2 Type and Th1 Type
mediated diseases, acute pain, chronic pain, cancer, lung cancer,
breast cancer, stomach cancer, bladder cancer, colon cancer,
pancreatic cancer, ovarian cancer, prostate cancer, rectal cancer,
hematopoietic malignancies, leukemia, lymphoma, abetalipoprotemia,
acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti cd3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aortic and peripheral
aneuryisms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chronic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic ateriosclerotic disease, diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, epstein-barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant
rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular
nephritis, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallerrorden-Spatz disease,
Hashimoto's thyroiditis, hay fever, heart transplant rejection,
hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis A, His bundle arrythmias, HIV infection/HIV neuropathy,
Hodgkin's disease, hyperkinetic movement disorders, hypersensitity
reactions, hypersensitivity pneumonitis, hypertension, hypokinetic
movement disorders, hypothalamic-pituitary-adrenal axis evaluation,
idiopathic Addison's disease, idiopathic pulmonary fibrosis,
antibody mediated cytotoxicity, asthenia, infantile spinal muscular
atrophy, inflammation of the aorta, influenza A, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma,
kidney transplant rejection, legionella, leishmaniasis, leprosy,
lesions of the corticospinal system, lipedema, liver transplant
rejection, lymphederma, malaria, malignant lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia,
metabolic/idiopathic, migraine headache, mitochondrial multi.system
disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Mencel
Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,
mycobacterium avium intracellulare, mycobacterium tuberculosis,
myelodyplastic syndrome, myocardial infarction, myocardial ischemic
disorders, nasopharyngeal carcinoma, neonatal chronic lung disease,
nephritis, nephrosis, neurodegenerative diseases, neurogenic I
muscular atrophies, neutropenic fever, non-Hodgkins lymphoma,
occlusion of the abdominal aorta and its branches, occulsive
arterial disorders, okt3 therapy, orchitis/epidydimitis,
orchitis/vasectomy reversal procedures, organomegaly, osteoporosis,
pancreas transplant rejection, pancreatic carcinoma, paraneoplastic
syndrome/hypercalcemia of malignancy, parathyroid transplant
rejection, pelvic inflammatory disease, perennial rhinitis,
pericardial disease, peripheral atherlosclerotic disease,
peripheral vascular disorders, peritonitis, pernicious anemia,
pneumocystis carinii pneumonia, pneumonia, POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), post perfusion syndrome,
post pump syndrome, post-MI cardiotomy syndrome, preeclampsia,
progressive supranucleo palsy, primary pulmonary hypertension,
radiation therapy, Raynaud's phenomenon and disease, Raynoud's
disease, Refsum's disease, regular narrow QRS tachycardia,
renovascular hypertension, reperfusion injury, restrictive
cardiomyopathy, sarcomas, scleroderma, senile chorea, senile
dementia of Lewy body type, seronegative arthropathies, shock,
sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
subacute sclerosing panencephalitis, syncope, syphilis of the
cardiovascular system, systemic anaphalaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, vital
encephalitis/aseptic meningitis, vital-associated hemaphagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, acute coronary syndromes, acute
idiopathic polyneuritis, acute inflammatory demyelinating
polyradiculoneuropathy, acute ischemia, adult Still's disease,
alopecia areata, anaphylaxis, anti-phospholipid antibody syndrome,
aplastic anemia, arteriosclerosis, atopic eczema, atopic
dermatitis, autoimmune dermatitis, autoimmune disorder associated
with Streptococcus infection, autoimmune enteropathy, autoimmune
hearing loss, Autoimmune Lymphoproliferative Syndrome (ALPS),
autoimmune myocarditis, autoimmune premature ovarian failure,
blepharitis, bronchiectasis, bullous pemphigoid, cardiovascular
disease, catastrophic antiphospholipid syndrome, celiac disease,
cervical spondylosis, chronic ischemia, cicatricial pemphigoid,
clinically isolated Syndrome (CIS) with risk for multiple
sclerosis, conjunctivitis, childhood onset psychiatric disorder,
chronic obstructive pulmonary disease (COPD), dacryocystitis,
dermatomyositis, diabetic retinopathy, diabetes mellitus, disk
herniation, disk prolapse, drug induced immune hemolytic anemia,
endocarditis, endometriosis, endophthalmitis, episcleritis,
erythema multiforme, erythema multiforme major, gestational
pemphigoid, Guillain-Barre Syndrome (GBS), hay fever, Hughes
syndrome, idiopathic Parkinson's disease, idiopathic interstitial
pneumonia, IgE-mediated allergy, immune hemolytic anemia, inclusion
body myositis, infectious ocular inflammatory disease, inflammatory
demyelinating disease, inflammatory heart disease, inflammatory
kidney disease, IPF/UIP, iritis, keratitis, keratojuntivitis sicca,
Kussmaul disease or Kussmaul-Meier Disease, Landry's paralysis,
Langerhan's cell histiocytosis, livedo reticularis, macular
degeneration, microscopic polyangiitis, morbus bechterev, motor
neuron disorders, mucous membrane pemphigoid, multiple organ
failure, myasthenia gravis, myelodysplastic syndrome, myocarditis,
nerve root disorders, neuropathy, non-A non-B hepatitis, optic
neuritis, osteolysis, pauciarticular JRA, peripheral artery
occlusive disease (PAOD), peripheral vascular disease (PVD),
peripheral artery disease (PAD), phlebitis, polyarteritis nodosa
(or periarteritis nodosa), polychondritis, polymyalgia rheumatica,
poliosis, polyarticular JRA, polyendocrine deficiency syndrome,
polymyositis, polymyalgia rheumatica (PMR), post-pump syndrome,
primary parkinsonism, prostatitis, pure red cell aplasia, primary
adrenal insufficiency, recurrent neuromyelitis optica, restenosis,
rheumatic heart disease, SAPHO (synovitis, acne, pustulosis,
hyperostosis, and osteitis), scleroderma, secondary amyloidosis,
shock lung, scleritis, sciatica, secondary adrenal insufficiency,
silicone associated connective tissue disease, Sneddon-Wilkinson
dermatosis, spondilitis ankylosans, Stevens-Johnson Syndrome (SJS),
systemic inflammatory response syndrome, temporal arteritis,
toxoplasmic retinitis, toxic epidermal necrolysis, transverse
myelitis, TRAPS (Tumor Necrosis Factor Receptor Associated Periodic
Syndrome), Type 1 allergic reaction, Type II diabetes, urticaria,
usual interstitial pneumonia (UIP), vasculitis, vernal
conjunctivitis, viral retinitis, Vogt-Koyanagi-Harada syndrome (VKH
syndrome), wet macular degeneration, and wound healing.
117. The use of claim 115, wherein the human subject is suffering
from: (a) rheumatoid arthritis, allergic arthritis, juvenile
arthritis, ankylosing spondylitis, or osteoarthritis; (b) certain
illnesses induced by viruses, optionally selected from Guillain
Barre syndrome, infectious mononucleosis, other viral
lymphadenopathies, and infections by herpes virus; (c) multiple
sclerosis or other demyelinating diseases; (d) hematological
disorders, optionally selected from hemolytic anemias and
thrombocytopenias; (e) endocrinologic disorders, optionally
selected from diabetes mellitus, Addison's disease, idiopathic
hypoparathyroidism, and chronic lymphocytic thyroiditis; (f)
collagen disorders and/or systemic lupus erythematosus; (g)
disorders of reproduction, optionally selected from amenorrhoea,
infertility, recurrent abortions, and eclampsia; (h) tumors,
optionally selected from head and neck tumors, lung cancers,
gastric cancers, prostate cancers, and pancreatic cancers; (i)
inflammatory bowel diseases, optionally selected from Crohn's
disease and ulcerative colitis; (j) pain associated with
osteoarthritis and other disorders; or (k) ocular disorders and/or
age-related macular degeneration (AMD).
118. The use of claim 115, wherein the antibody or antigen-binding
fragment is optionally administered before, concurrently, or after
the administration of a second agent, wherein the second agent is
optionally selected from the group consisting of methotrexate;
leflunomide; low doses of corticosteroids; prednisone; cortisone;
an anti-malarial medication; hydroxychloroquine; gold;
sulfasalazine; penicillamine; cyclophosphamide; cyclosporine;
minocycline; acetaminophen; aspirin; ibuprofen; naproxen;
celecoxib; Infliximab; etanercept; adalimumab; abatacept;
rituximab; anakinra; a biologic agent or oral delivery agent
targeting IL-6, IL-6R, IL-17, IL-18, IL-23, or B7.1/B7.2;
acetaminophen; aspirin; ibuprofen; naproxen; celecoxib; steroids;
artificial joint fluid; Synvisc.TM.; Hyalgan.TM.; adalimumab;
Azasan.TM.; Asacol.TM.; azathioprine; Azulfidine.TM.; budesonide;
Entocort.TM.; Flagyl.TM.; Imuran.TM.; infliximab; mercaptopurine;
metronidazole; protostat; Purinethol.TM.; Remicade.TM.;
sulfasalazine; acetocot; acetylsalicylic acid; acuprin 81;
adalimumab; Aleve.TM.; amcort; Anaprox.TM.; Aristocort.TM.;
aspirin; aspirtab; Azmacort.TM.; Bufferin.TM.; buffex;
Cataflam.TM.; Celebrex.TM.; Clinoril.TM.; cortisone; diclofenac;
Dipentum.TM.; Easprin.TM.; etanercept; Indocin.TM.; indomethacin;
infliximab; naproxen; triamcinolone; Voltaren.TM.; Avonex.TM.,
Azasan.TM., Azathioprine, Betaseron.TM., Bubbli-Pred.TM.,
Copaxone.TM., Cotolone, Glatiramer, Imuran.TM., Interferon Beta-1a,
Interferon Beta-1b Solution, Key-Pred, Key-Pred SP, Mitoxantrone,
Natalizumab, Novantrone.TM., Orapred.TM., Orapred ODT.TM.,
Pediapred.TM., Pred-Ject-50, Predacort 50, Predalone 50,
Predate-50, Prednisolone, Prelone.TM., Rebif.TM.; Tysabri.TM.;
Abraxane.TM., Adriamycin, Adrucil.TM., Aldara.TM., Alemtuzumab,
Alimta.TM., Aminolevulinic Acid, Anastrozole, Aprepitant,
Arimidex.TM., Aromasin.TM., Arranon.TM., Arsenic Trioxide,
Avastin.TM. (Bevacizumab), Azacitidine, Bevacizumab, Bexarotene,
Bortezomib, Campath.TM. (Alemtuzumab), Camptosar.TM. (Irinotecan
Hydrochloride), Capecitabine, Carboplatin, Cetuximab, Cisplatin,
Clafen (Cyclophosphamide), Clofarabine, Clofarex (Clofarabine),
Clolar.TM. (Clofarabine), Cyclophosphamide, Cytarabine, Cytosar-U
(Cytarabine), Cytoxan (Cyclophosphamide), Dacogen.TM. (Decitabine),
Dasatinib, Decitabine, DepoCyt.TM. (Liposomal Cytarabine),
DepoFoam.TM. (Liposomal Cytarabine), Dexrazoxane Hydrochloride,
Docetaxel, Doxil.TM. (Doxorubicin Hydrochloride Liposome),
Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome,
Dox-SL (Doxorubicin Hydrochloride Liposome), Efudex.TM.
(Fluorouracil), Ellence.TM. (Epirubicin Hydrochloride),
Eloxatin.TM. (Oxaliplatin), Emend.TM. (Aprepitant), Epirubicin
Hydrochloride, Erbitux.TM. (Cetuximab), Erlotinib Hydrochloride,
Evacet (Doxorubicin Hydrochloride Liposome), Evista.TM. (Raloxifene
Hydrochloride), Exemestane, Faslodex.TM. (Fulvestrant), Femara.TM.
(Letrozole), Fluoroplex.TM. (Fluorouracil), Fluorouracil,
Fulvestrant, Gefitinib, Gemcitabine Hydrochloride, Gemtuzumab
Ozogamicin, Gemzar.TM. (Gemcitabine Hydrochloride), Gleevec.TM.
(Imatinib Mesylate), Herceptin.TM. (Trastuzumab), Hycamtin.TM.
(Topotecan Hydrochloride), Imatinib Mesylate, Imiquimod, Iressa.TM.
(Gefitinib), Irinotecan Hydrochloride, Ixabepilone, Ixempra.TM.
(Ixabepilone), Keoxifene (Raloxifene Hydrochloride), Kepivance.TM.
(Palifermin), Lapatinib Ditosylate, Lenalidomide, Letrozole,
Levulan.TM. (Aminolevulinic Acid), LipoDox.TM.(Doxorubicin
Hydrochloride Liposome), Liposomal Cytarabine, Methazolastone
(Temozolomide), Mylosar (Azacitidine), Mylotarg.TM. (Gemtuzumab
Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized
Nanoparticle Formulation), Nelarabine, Neosar.TM.
(Cyclophosphamide), Nexavar.TM. (Sorafenib Tosylate), Nilotinib,
Nolvadex (Tamoxifen Citrate), Oncaspar.TM. (Pegaspargase),
Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle
Formulation, Palifermin, Panitumumab, Paraplat (Carboplatin),
Paraplatin.TM. (Carboplatin), Pegaspargase, Pemetrexed Disodium,
Platinol-AQ (Cisplatin), Platinol.TM. (Cisplatin), Raloxifene
Hydrochloride, Revlimid (Lenalidomide), Rituxan.TM. (Rituximab),
Rituximab, Sclerosol Intrapleural Aerosol (Talc), Sorafenib
Tosylate, Sprycel.TM. (Dasatinib), Sterile Talc Powder (Talc),
Steritalc.TM. (Talc), Sunitinib Malate, Sutent.TM. (Sunitinib
Malate), Synovir (Thalidomide), Talc, Tamoxifen Citrate, Tarabine
PFS (Cytarabine), Tarceva.TM. (Erlotinib Hydrochloride),
Targretin.TM. (Bexarotene), Tasigna.TM. (Nilotinib), Taxol.TM.
(Paclitaxel), Taxotere.TM. (Docetaxel), Temodar.TM. (Temozolomide),
Temozolomide, Temsirolimus, Thalomid.TM. (Thalidomide),
Thalidomide, Totect.TM. (Dexrazoxane Hydrochloride), Topotecan
Hydrochloride, Torisel.TM. (Temsirolimus), Trastuzumab,
Trisenox.TM. (Arsenic Trioxide), Tykerb.TM. (Lapatinib Ditosylate),
Vectibix.TM. (Panitumumab), Velcade.TM. (Bortezomib), Vidaza.TM.
(Azacitidine), Vorinostat, Xeloda.TM. (Capecitabine), Zinecard.TM.
(Dexrazoxane Hydrochloride), Zoledronic Acid, Zolinza.TM.
(Vorinostat), and Zometa.TM. (Zoledronic Acid); and/or wherein the
antibody or antigen-binding fragment is optionally adapted for
parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal
administration.
119. A method of determining the presence of PGE2 or fragment
thereof in a test sample by an immunoassay, and/or wherein the
immunoassay comprises contacting the test sample with at least one
antibody or antigen-binding fragment of claim 93 or claim 94, and
at least one detectable label, and/or wherein the method optionally
comprises: (a) a direct assay comprising: (i) contacting the test
sample with at least one antibody or antigen-binding fragment,
wherein the antibody or antigen-binding fragment binds to an
epitope on the PGE2 or fragment thereof so as to form a first
complex; (ii) contacting the complex with the at least one
detectable label, wherein the detectable label binds to the
antibody or antigen-binding fragment or an epitope on the PGE2 or
fragment thereof that is not bound by the antibody or
antigen-binding fragment to form a second complex; and (iii)
detecting the presence of the PGE2 or fragment thereof in the test
sample based on the signal generated by the detectable label in the
second complex, wherein the presence of the PGE2 or fragment
thereof is directly correlated with the signal generated by the
detectable label, or (b) an indirect assay comprising: (i)
contacting the test sample with at least one antibody or
antigen-binding fragment, wherein the antibody or antigen-binding
fragment binds to an epitope on the PGE2 or fragment thereof so as
to form a first complex; (ii) contacting the complex with the at
least one detectable label, wherein the detectable label competes
with the PGE2 or fragment thereof for binding to the antibody or
antigen-binding fragment so as to form a second complex; and (iii)
detecting the presence of the PGE2 or fragment thereof in the test
sample based on the signal generated by the detectable label in the
second complex, wherein the presence of the PGE2 or fragment
thereof is indirectly correlated with the signal generated by the
detectable label, wherein if the test sample is from a patient, the
method optionally further comprises diagnosing, prognosticating, or
assessing the efficiency of therapeutic/prophylactic treatment of
the patient.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 12/499,646, filed Jul. 8, 2009, which claims the benefit of
priority of U.S. provisional application No. 61/197,258, filed Oct.
23, 2008, and U.S. provisional application No. 61/134,264, filed
Jul. 8, 2008, all of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to binding proteins and
compositions thereof, for example antibodies and antigen binding
portions specific to lipid metabolites such as prostaglandin E2
(PGE.sub.2), and methods of making, characterizing, and using them
in the prevention, diagnosis, and treatment of diseases.
BACKGROUND OF THE INVENTION
[0003] Bioactive lipids such as prostaglandin (PG), thromboxane
(TX), leukotriene (LT), and sphingosine-1-phosphate play a critical
physiological role in the etiology of various disorders. (Wymann, M
P and Schneiter R, Nat. Rev. Mol. Cell. Biol. 9(2):162-76(2008)).
During inflammation, cellular phospholipases, especially
phospholipases A2 and C, are activated and degrade cell membrane
phospholipids to arachidonic acid (AA). AA is metabolized by two
major routes, the cyclooxygenase (COX) and lipooxygenase (LO)
pathways. The COX pathway produces prostaglandins (PGD.sub.2,
PGE.sub.2, PGF.sub.2.alpha., prostacyclin or PGI.sub.2, and
thromboxane A2 or TXA.sub.2). The LO pathway has two branches; the
5-LO pathway produces leukotrienes (e.g., LTA4, LTB4, LTC4, LTD4,
LTE4, and LTF4) and the 15-LO pathway produces lipoxins (e.g.,
LXA4, LXB4). Prostanoids such as prostaglandin (PG), thromboxane
(TX) and leukotriene (LT) have various physiological activities for
maintaining local homeostasis in the body (The Pharmacological
Basis of Therapeutics, Gilman, et al., eds., 7th Ed., p. 660,
Macmillan Publishing Co., New York (1985)). The products of COX, PG
G2/PG H2, are converted to specific PGs by the actions of tissue
specific isomerases to yield PGI.sub.2, TXA.sub.2, PGD.sub.2,
PGE.sub.2, and PGF.sub.2.alpha.. The biological functions of PGs
are mediated by tissue-specific cell surface rhodopsin-like seven
transmembrane spanning G protein-coupled receptors (GPCRs). The
precise physiological/pathological role of each PG is determined by
the cellular context, receptor expression profile, ligand affinity,
and differential coupling to signal transduction pathways (Haluska
et al., Annu Rev. Pharm. Tox. 10:213 (1989); Prostanoids and their
Receptors. In Comprehensive Medicinal Chemistry, p. 643, Pergamon
Press, Oxford (1990)). PGs play a wide variety of physiological
roles in the regulation of modulation of vasomotricity, the
sleep/wake cycle, intestinal secretion, lipolysis, glomerular
filtration, mast cell degranulation, neurotransmission, platelet
aggregation, leuteolysis, myometrial contraction and labor,
inflammation and arthritis, patent ductus arteriosus, cell growth
and differentiation, and immune responses generally.
Patho-physiologically, PGs have been implicated in a variety of
diseases including pain and inflammation, cancer, neurological
diseases, cardiovascular diseases, and hypertension.
[0004] Prostaglandin E.sub.2 (PGE.sub.2) is a member of the
prostanoid family. PGE.sub.2 participates widely in the contraction
and relaxation of the gastrointestinal tract, secretion of gastric
acid, relaxation of smooth muscle, and release of
neurotransmitters. Four subtype receptors for PGE.sub.2 have been
identified, including EP1, EP2, EP3, and EP4 (Negishi, M. et al.,
J. Lipid Mediators Cell Signalling, 12:379-391 (1995)), each of
which is involved in a different signal transduction pathway.
[0005] PGE.sub.2 is the main product of the COX pathway of AA
metabolism. It is the major PG synthesized in the joints and plays
an important role in inflammation and the pathogenesis of
arthritis. Five PGE.sub.2 synthases have been identified. (Smith W
L, Am. J. Physiol. 263(2 Pt 2):F181-91 (1992)). Of these five,
membrane PGE synthase (mPGES)-1 appears to be the key PGE.sub.2
convertase enzyme responsible for PGE.sub.2 production. MPGES-1
displays the highest catalytic activity relative to other PGE
synthases and functions in conjunction with COX-1 and/or COX-2, to
convert PGH.sub.2 to PGE.sub.2. Studies using mPGES-1 KO mice
(Kamei, D., et al., J. Biol. Chem., 279(32):33684-95 (2004);
Trebino, C. E., et al., Proc. Natl. Acad. Sci. USA 100(15):9044-9
(2003), specific PGE.sub.2 receptor isoform KO mice (McCoy, J. M.,
et al. J. Clin. Invest., 110(5):651-8 (2002); Majima, M., et al.
Trends Pharmacol. Sci., 24(10):524-9 (2003); and Amano, H., et al.,
J. Exp. Med., 197(2):221-32 (2003); and anti-PGE.sub.2 specific
antibodies (Portanova, J. P., et al., J. Exp. Med., 184(3):883-91
(1996); Zhang, Y., et al., J. Pharmacol. Exp. Ther.,
283(3):1069-75(1997) suggest that PGE.sub.2 plays a major role in
animal models of rheumatoid arthritis (RA), pain and inflammation
and cancer development. In the absence of mPGES-1, levels of COX-1,
COX-2, and other PGE.sub.2 synthases remain relatively unaltered.
The mPGES-1 KO mice are viable, fertile, and develop normally
compared to wild type mice. However, they display a drastic
reduction in both basal levels of PGE.sub.2 production as well as
in PGE.sub.2 production from macrophages following challenge with
various inflammatory stimuli. In addition, production of TXA2 is
increased. The mPGES-1 KO mice show reduced incidence and severity
of arthritis development and show resistance to pain and
inflammation in various models. Several laboratories have
independently generated various EP receptor isoform KO mice. These
mice are viable, fertile and develop normally. Studies using
specific EP isoform KO mice demonstrate that the various functions
of PGE.sub.2 are mediated via specific EP isoforms. For example,
the lack of EP4 isoform clearly affects the severity of arthritis
development in mice, whereas the lack of EP3 influences tumor
development and progression by modulating VEGF production by
stromal cells and angiogenesis.
[0006] Defects in the biosynthesis and metabolism of prostaglandins
are now believed to play an important part in the etiology of
autoimmune and inflammatory disorders. For example, the synovial
tissues from patients suffering from rheumatoid arthritis produce
larger amounts of PGE.sub.2 and prostaglandin F.sub.2.alpha.
(PGF.sub.2.alpha.) compared to the synovial tissues from unaffected
subjects (Blotman, F., et al., Rev. Rhum. Mal. Osteoartic,
46(4):243-7 (1979)). Similarly, an increased synthesis of PGE.sub.2
and PGF.sub.2.alpha. occurs in patients exhibiting systemic and
gastrointestinal symptoms secondary to food intolerance. Thus,
migraine headaches secondary to the ingestion of certain foods
could be the result of an increased synthesis of 2-series
prostaglandins. Multiple sclerosis is also associated with an
imbalance in the normal levels of the prostaglandins, PGE.sub.1 and
PGE.sub.2. Many aspects of reproduction, for example, fertility,
pregnancy and labor, may be regulated by prostaglandins.
Prostaglandins also play a major role in reproductive physiology.
Excessive prostaglandin synthesis causes dysmenorrhea and
parturition, which may be induced by administering prostaglandins
intravenously or by insertion of a prostaglandin pessary. (Wang L.
et al., Occup. Environ. Med. 61(12):1021-1026 (2004)). Excessive
synthesis of PGE.sub.2 also plays a major role in disorders of
reproduction, such as infertility, repeated miscarriage,
preeclampsia, and eclampsia. A need therefore exists for antibodies
specific to PGE.sub.2 that block or modulate its biological
functions, which may be used to prevent and treat the diseases
associated with excess production of PGE.sub.2 as well as
diagnostic purposes.
[0007] The generation of a highly specific, high affinity (K.sub.D
is about 300 pM) anti-PGE.sub.2 mAb, 2B5, has been reported. (Mnich
S J, et al. J. Immunol. 155(9):4437-44 (1995)). The efficacy of 2B5
relative to indomethacin, a COX-1,2 inhibitor, was determined in
animal models of pain and inflammation in mice and adjuvant-induced
arthritis in rats. (Portanova J P et al., J. Exp. Med.
184(3):883-91 (1996)). These studies clearly showed that 2B5 was as
effective as indomethacin in reducing pain and inflammation as well
as the severity of arthritis, suggesting that PGE.sub.2 is a key
participant in the COX-1, 2 pathway of AA metabolism in these
animal models.
[0008] Inhibition of pan-PG production by COX-inhibitors has been a
well-established therapeutic strategy for decades. Two isoforms of
COX, COX-1 and COX-2, are known, each of which are encoded by a
distinct gene. The two isoforms carry out essentially the same
catalytic reaction and have similar tertiary structures (Garavito R
M, et al., Annu Rev. Biophys. Biomol. Struct. 32:183-206 (2003)).
COX-1 is constitutively expressed in nearly all tissues and is
believed to be largely responsible for the normal "house keeping"
functions, such as gastric cytoprotection and homeostasis. COX-2,
by contrast, is constitutively expressed in particular tissues, and
is highly inducible at sites of inflammation and cancer. Thus,
COX-2-mediated PG production is thought to play an important role
at the site of inflammation and cancer. The traditional
non-steroidal anti-inflammatory drugs (NSAIDs), e.g., aspirin,
indomethacin, ibuprofen) inhibit both COX isoforms. These compounds
are the most widely used drugs for pain, rheumatoid arthritis (RA),
osteoarthritis (OA), and cardiovascular diseases and now are under
consideration for the prevention of colon cancer and AD. The main
liabilities of traditional NSAIDs are gastric and renal adverse
events, in high-risk populations, which are believed to be due to
inhibition of COX-1. Therefore, the second generation of NSAIDs,
the COX-2 selective inhibitors (e.g., celecoxib, Celebrex.TM.;
rofecoxib, Vioxx.TM.; valdecoxib, Bextra.TM.), are believed to have
a better therapeutic profile. This assumption has resulted in their
widespread use for pain, RA, and OA. Since the approval of the
first COX-2 inhibitor in 1999 the combined sales of COX-2
inhibitors in 2004 was approximately US $5 billion. However,
recently some COX-2 selective inhibitors were taken off the market,
and are under FDA review, due to cardiovascular side-effects in
high risk patients for certain COX-2 inhibitors. The liabilities
associated with COX inhibitors probably arose due to their ability
to inhibit all PGs, and in particular due to their ability to
differentially interfere with PGI.sub.2 and TXA.sub.2 production,
both of which play an important role in maintaining cardiovascular
homeostasis (Martinez-Gonzalez J. et al., Curr. Pharm. Des.
13(22):2215-2227 (2007)). The inhibition of COX may make more AA
available to the LO pathways, thus increasing the production of
leukotrienes and lipoxins, which may contribute to COX
inhibition-associated adverse effects. Recent studies using COX-1
and/or COX-2 knockout mice and COX-1 and COX-2 specific inhibitors
also suggest that assumptions concerning the physiological roles of
the two COX-isoforms may not be correct. (Loftin, C. D., et al.
Prostaglandins Other Lipid Mediat. 68-69: 177-85 (2002)). These
studies suggest that both COX-1 and COX-2 play an important role in
supplying PGs to maintain tissue homeostasis and both isoforms may
contribute to disease development, such as pain, inflammation and
cancer. Therefore, blocking detrimental PGE.sub.2 downstream of
COX-1 and COX-2 pathway with a specific antibody appears to be an
attractive approach for the treatment of certain human
diseases.
[0009] Another example of an important bioactive prostaglandin is
PGD.sub.2. PGD.sub.2 is the major cyclooxygenase product of
arachidonic acid produced from mast cells on immunological
challenge (Lewis, et al., J. Immunol. 129:1627-1631 (1982)).
Activated mast cells, a major source of PGD.sub.2, are one of the
key players in driving the allergic response in conditions such as
asthma, allergic rhinitis, allergic conjunctivitis, allergic
dermatitis and other diseases (Brightling, et al., Clin. Exp.
Allergy 33:550-556 (2003)). Recent studies have shown that
PGD.sub.2 exerts its effects through two different
G-protein-coupled receptors (GPCRs), the D-prostanoid receptor (DP)
and the chemoattractant receptor-homologous molecule expressed on T
helper type-2 cells (CRTH2), expressed in various human tissues.
The PGD.sub.2/CRTH2 system mediates the chemotaxis of eosinophils,
basophils, and Th2 cells, which are involved in the induction of
allergic inflammation (Ulven T et al., Curr. Top. Med. Chem.
6(13):1427-1444 (2006)). Many of the actions of PGD.sub.2 are
mediated through its action on the D-type prostaglandin ("DP")
receptor, a G protein-coupled receptor expressed on epithelium and
smooth muscle. In asthma, the respiratory epithelium has long been
recognized as a key source of inflammatory cytokines and chemokines
that drive the progression of the disease (Holgate, et al., Am. J.
Respir. Crit. Care Med. 162:113-117 (2000)). In an experimental
murine model of asthma, the DP receptor is dramatically
up-regulated on airway epithelium on antigen challenge (Matsuoka,
et al., Science 287:2013-2017 (2000)). The DP receptor is involved
in human allergic rhinitis, a frequent allergic disease that is
characterized by the symptoms of sneezing, itching, rhinorea and
nasal congestion. DP antagonists have been shown to be effective at
alleviating the symptoms of allergic rhinitis in multiple species,
and more specifically have been shown to inhibit the
antigen-induced nasal congestion, the most manifest symptom of
allergic rhinitis (Jones, et al., Am. J. Resp. Crit. Care Med.
167:A218 (2003); Arimura, et al., S-5751. J. Pharmacol. Exp. Ther.
298(2):411-9 (2001)). DP antagonists are also effective in
experimental models of allergic conjunctivitis and allergic
dermatitis (Arimura et al., S-5751. J. Pharmacol. Exp. Ther.
298(2):411-9 (2001); Torisu, et al., Bioorg. & Med. Chem.
12:5361-5378 (2004)). A need therefore also exists for antibodies
specific to PGD.sub.2 and blocking or modulating its biological
functions therefore may be used to prevent and treat the diseases
associated with excess production of PGD.sub.2.
[0010] Sphingosine-1-phosphate (S1P) is another example of a
bioactive lipid that induces many cellular effects, including those
that result in platelet aggregation, cell proliferation, cell
morphology, tumor cell invasion, endothelial cell chemotaxis, and
endothelial cell in vitro angiogenesis. S1P receptors are therefore
good targets for therapeutic applications such as wound healing and
tumor growth inhibition. S1P signals cells in part via a set of G
protein-coupled receptors named S1P1, S1P2, S1P3, S1P4, and S1P5
(formerly called EDG-1, EDG-5, EDG-3, EDG-6, and EDG-8,
respectively). These receptors share 50-55% amino acid and cluster
identity with three other receptors (LPA1, LPA2, and LPA3 (formerly
EDG-2, EDG-4 and EDG-7)) for the structurally-related
lysophosphatidic acid (LPA). (Ishii, I. et al., Mol. Pharmacol.
58(5):895-902 (2000)). A conformational shift is induced in the
G-Protein Coupled Receptor (GPCR) when the ligand binds to that
receptor, causing GDP to be replaced by GTP on the .alpha.-subunit
of the associated G-proteins and subsequent release of the
G-proteins into the cytoplasm. The .alpha.-subunit then dissociates
from the .beta..gamma.-subunit, and each subunit can then associate
with effector proteins, which activate second messengers leading to
a cellular response. Eventually the GTP on the G-proteins is
hydrolyzed to GDP, and the subunits of the G-proteins re-associate
with each other and then with the receptor. Amplification plays a
major role in the general GPCR pathway. The binding of one ligand
to one receptor leads to the activation of many G-proteins, each
capable of associating with many effector proteins, leading to an
amplified cellular response. S1P receptors make good drug targets,
because individual receptors are both tissue- and
response-specific. Tissue specificity of the S1P receptors is
important, because development of an agonist or antagonist
selective for one receptor localizes the cellular response to
tissues containing that receptor, limiting unwanted side effects.
Response specificity of the S1P receptors is also important because
it allows for development of agonists or antagonists that initiate
or suppress certain cellular responses without affecting other
responses. For example, the response specificity of the S1P
receptors could allow for an S1P mimetic that initiates platelet
aggregation without affecting cell morphology.
[0011] S1P is formed as a metabolite of sphingosine in its reaction
with sphingosine kinase, and is abundantly stored in platelet
aggregates where high levels of sphingosine kinase exist and
sphingosine lyase is absent. S1P is released during platelet
aggregation, accumulates in serum and is also found in malignant
ascites. S1P biodegradation most likely proceeds via hydrolysis by
ectophosphohydrolases, specifically the sphingosine 1-phosphate
phosphohydrolases. A need therefore exists for antibodies specific
to S1P for modulating its biological functions either by blocking
its interaction with receptors or stabilizing S1P and enhancing its
biological effects, for use in preventing or treating autimmune
diseases, inflammatory diseases, and cancers.
[0012] Due to the role of PGE2 in a variety of human disorders,
therapeutic strategies have been designed to inhibit or counteract
PGE2 activity. In particular, therapeutic antibodies suitable for
delivery to humans that bind to, and neutralize, PGE2 have not been
reported. There exists a need in the art for improved antibodies
capable of binding and neutralizing PGE2.
SUMMARY OF THE INVENTION
[0013] The present invention relates to binding proteins specific
to lipid metabolites, such as prostaglandin E2 (PGE.sub.2). The
PGE.sub.2 binding proteins of the invention include, but are not
limited to, antibodies, antigen binding fragments, and antigen
binding fragments with various scaffolds, that are capable of
binding PGE.sub.2.
[0014] One aspect of the invention pertains to binding proteins
capable of binding PGE.sub.2. In an embodiment, the binding
proteins of the invention have neutralizing, stabilizing,
antagonist, and/or agonist activities. In another embodiment, the
binding proteins are capable of modulating a biological function of
PGE.sub.2. For example, the binding proteins are capable of at
least partially neutralizing PGE.sub.2.
[0015] In one aspect of the invention, the binding proteins are
capable of binding PGE.sub.2 and preventing the binding of
PGE.sub.2 to one or more PGE.sub.2 receptors (e.g., EP1, EP2, EP3,
and EP4). In an embodiment of the invention, the binding proteins
are capable of binding PGE.sub.2 and preventing the binding of
PGE.sub.2 to the EP1, EP2, EP3, and EP4 receptors.
[0016] The invention provides methods of making, characterizing and
using the PGE.sub.2 binding proteins as a monotherapy or as a
combination therapy with other therapeutic agents; and methods in
the prevention and/or treatment of diseases mediated by PGE.sub.2,
for example, autoimmune and inflammatory diseases such as, for
example, rheumatoid arthritis, Crohn's disease, osteoarthritis,
AMD, lymphadenopathies, hemolytic anemias, purpura, ankylosing
spondylitis, multiple sclerosis, diabetes mellitus, cancer, pain,
bone loss/restoration, atherosclelotic diseases, disorders of
reproduction, and other diseases. The binding proteins of the
invention can also be used in the diagnosis of such diseases.
[0017] In an embodiment of the invention, a binding protein is an
isolated antibody, or antigen binding fragment thereof, that binds
PGE.sub.2. Such binding may be demonstrated in a biotinylated
PGE.sub.2 ELISA based assay with an EC.sub.50 selected from the
group consisting of about 1.times.10.sup.-6 to about
1.times.10.sup.-7 M, about 1.times.10.sup.-7 to about
1.times.10.sup.-8 M, about 1.times.10.sup.-8 to about
1.times.10.sup.-9 M, about 10.sup.-9 to about 10.sup.-10 M, about
1.times.10.sup.-10 to about 1.times.10.sup.-11 M and about
10.sup.-11 to about 10.sup.-12 M. In another embodiment, binding of
the binding proteins to PGE.sub.2 is demonstrated in a
.sup.3H-labelled PGE.sub.2 based radioimmunoassay with a K.sub.D
selected from the group consisting of about 1.times.10.sup.-6 to
about 1.times.10.sup.-7 M, about 1.times.10.sup.-7 to about
1.times.10.sup.-8 M, about 1.times.10.sup.-8 to about
1.times.10.sup.-9 M, about 10.sup.-9 to about 10.sup.-10 M, about
1.times.10.sup.-10 to about 1.times.10.sup.-11 M, and about
10.sup.-11 to about 10.sup.-12. In another embodiment, binding of
the binding proteins to PGE.sub.2 is demonstrated in a FLIPR
wherein PGE.sub.2-induced calcium influx mediated through its
receptor EP4 is inhibited by binding of the binding proteins to
PGE.sub.2, with an IC.sub.50 selected from the group consisting of
about 1.times.10.sup.-6 to about 1.times.10.sup.-7 M, about
1.times.10.sup.-7 to about 1.times.10.sup.-8 M, about
1.times.10.sup.-8 to about 1.times.10.sup.-9 M, about 10.sup.-9 to
about 10.sup.-10 M, about 1.times.10.sup.-10 to about
1.times.10.sup.-11 M, and about 10.sup.-11 to about 10.sup.-12.
[0018] In an embodiment, the antibodyinhibits the binding of
biotinylated PGE.sub.2 to the EP1, EP2, EP3, and/or EP4 receptor on
the cell surface in FACS-based receptor binding assay, or the
antibody inhibits the binding of biotinylated PGE.sub.2 to the EP1,
EP2, EP3, and/or EP4 receptor on the membrane preparation made
using receptor expressing cells in ELISA-based receptor binding
assay with an IC.sub.50 of about 1.times.10.sup.-6 to about
1.times.10.sup.-7 M, about 1.times.10.sup.-7 to about
1.times.10.sup.-8 M, about 1.times.10.sup.-8 to about
1.times.10.sup.-9 M, about 10.sup.-9 to about 10.sup.-10 M, about
1.times.10.sup.-10 to about 1.times.10.sup.-11 M, and about
10.sup.-11 to about 10.sup.-12 or the antibody inhibits the binding
of .sup.3H-PGE.sub.2 to the EP1, EP2, EP3, and/or EP4 receptor on
the cell surface or on the membrane preparation in an .sup.3H
PGE.sub.2 based radioimmunoassay with a IC.sub.50 of about
1.times.10.sup.-6 to about 1.times.10.sup.-7 M, about
1.times.10.sup.-7 to about 1.times.10.sup.-8 M, about
1.times.10.sup.-8 to about 1.times.10.sup.-9 M, about 10.sup.-9 to
about 10.sup.-10 M, about 1.times.10.sup.-10 to about
1.times.10.sup.-11 M, and about 10.sup.-11 to about 10.sup.-12
and/or the antibody inhibits PGE.sub.2 induced calcium flux in a
EP4 mediated FLIPR assay with an IC.sub.50 of about
1.times.10.sup.-6 to about 1.times.10.sup.-7 M, about
1.times.10.sup.-7 to about 1.times.10.sup.-8 M, about
1.times.10.sup.-8 to about 1.times.10.sup.-9 M, about 10.sup.-9 to
about 10.sup.-10 M, about 1.times.10.sup.-10 to about
1.times.10.sup.-11 M, and about 10.sup.-11 to about 10.sup.-12. In
an embodiment, the antibody, or antigen binding fragment thereof
binds PGE.sub.2 and inhibits the binding of PGE.sub.2 to at least
one of its receptors in a cell surface-based receptor binding assay
or in a radioimmunoassay-based receptor binding assay by about
70-100% at a concentration of 100 nM.
[0019] In an embodiment, the antibody is 19C9, 4F10, 15F10, K1B,
K7H, K3A, L11, L21, 2B5-7.0, 2B5-8.0 or 2B5-9.0, or a variant
thereof. In an embodiment, the variant is a humanized variant, such
as Hu2B5.P1 or Hu2B5.P2.
[0020] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that binds PGE.sub.2
and inhibits paw edema by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or 100% in a carrageenan-induced rodent paw edema model.
In a particular embodiment, the antibody inhibits paw edema by
greater than about 10% in a carrageenan induced rodent paw edema
model
[0021] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that binds PGE.sub.2
and inhibits paw swelling or mean arthritis score by about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in a rodent collagen
induced arthritis model. In a particular embodiment, the antibody
inhibits paw swelling or mean arthritis score by greater than 10%
in a rodent collagen induced arthritis model.
[0022] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that binds PGE.sub.2
and inhibits paw edema by about 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90% or 100% in a carrageenan-induced rodent paw edema model.
In a particular embodiment, the antibody inhibits paw edema by
greater than about 10% in a carrageenan induced rodent paw edema
model
[0023] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that binds PGE.sub.2
and inhibits paw swelling or mean arthritis score by about 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in a rodent collagen
induced arthritis model. In a particular embodiment, the antibody
inhibits paw swelling or mean arthritis score by greater than 10%
in a rodent collagen induced arthritis model.
[0024] In another embodiment, the binding protein of the invention
has an off rate constant (k.sub.off) to PGE.sub.2 of at most about
10.sup.-3 s.sup.-1; at most about 10.sup.-4 s.sup.-1; at most about
10.sup.-5 s.sup.-1; or at most about 10.sup.-6 s.sup.-1, as
measured by radioimmunoassay. Preferably, the binding protein of
the invention has an off rate constant (k.sub.off) to PGE.sub.2 of
about 10.sup.-3 s.sup.-1 to about 10.sup.-4 s.sup.-1; of about
10.sup.-4 s.sup.-1 to about 10.sup.-5 s.sup.-1; or of about
10.sup.-5 s.sup.-1 to about 10.sup.-6 s.sup.-1, as measured by
radioimmunoassay.
[0025] In another embodiment, the binding protein of the invention
has a dissociation constant (K.sub.D) to PGE.sub.2 determined by a
radioimmunoassay of at most about 10.sup.-6 M; at most about
10.sup.-7 M; at most about 10.sup.-8 M; at most about 10.sup.-9 M;
at most about 10.sup.-10 M; at most about 10.sup.-11 M; at most
about 10.sup.-12 M; or at most 10.sup.-13M. Preferably, the binding
protein of the invention has a dissociation constant (K.sub.D) to
PGE.sub.2 of about 10.sup.-7 M to about 10.sup.-8 M; of about
10.sup.-8 M to about 10.sup.-9 M; of about 10.sup.-9 M to about
10.sup.-10 M; of about 10.sup.-10 to about 10.sup.-11 M; of about
10.sup.-11 M to about 10.sup.-12 M; or of about 10.sup.-12 to M
about 10.sup.-13M. One aspect of the invention provides at least
one PGE2 anti-idiotype antibody to at least one PGE2 binding
protein of the present invention. The anti-idiotype antibody
includes any protein or peptide containing molecule that comprises
at least a portion of an immunoglobulin molecule such as, but not
limited to, at least one complementarily determining region (CDR)
of a heavy or light chain or a ligand binding portion thereof, a
heavy chain or light chain variable region, a heavy chain or light
chain constant region, a framework region, or; any portion thereof,
that can be incorporated into a binding protein of the present
invention.
[0026] In another aspect, the invention provides an isolated
antibody, or antigen binding fragment thereof, that binds
prostaglandin E.sub.2 and inhibits the binding of prostaglandin
E.sub.2 to at least one of E1, E2, E3, and E4 receptor in a cell
surface-based receptor binding assay with an IC.sub.50 selected
from the group consisting of about 1.times.10.sup.-6 to
1.times.10.sup.-7 M, 1.times.10.sup.-7 to 1.times.10.sup.-8M,
1.times.10.sup.-8 to 1.times.10.sup.-9 M, 10.sup.-9 to 10.sup.-10
M, 1.times.10.sup.-10 to 1.times.10.sup.-11 M and 10.sup.-11 to
10.sup.-12 M, or in an ELISA-based receptor binding assay with an
IC.sub.50 selected from the group consisting of about
1.times.10.sup.-6 to 1.times.10.sup.-7 M, 1.times.10.sup.-7 to
1.times.10.sup.-8 M, 1.times.10.sup.-8 to 1.times.10.sup.-9 M,
10.sup.-9 to 10.sup.-10 M, 1.times.10.sup.-10 to 1.times.10.sup.-11
M and 10.sup.-11 to 10.sup.-12M.
[0027] In another embodiment, the antibody, or antigen binding
fragment thereof binds prostaglandin E.sub.2 and inhibits the
binding of prostaglandin E.sub.2 to at least one of the E1, E2, E3,
and E4 receptors in a cell surface-based receptor binding assay or
in a .sup.3H-PGE.sub.2 based radioimmunoassay using cells
expressing or cell membrane preparation the expressing at least one
of the E1, E2, E3, and E4 receptors by about 70-100% at a
concentration of about 100 nM. In an embodiment, the antibody is
selected from the group consisting of 19C9, 4F10, 15F10, K1B, K7H,
K3A, L11, L21, 2B5-7.0, 2B5-8.0 and 2B5-9.0. In another embodiment,
the antibody, or antigen binding fragment thereof, is capable of
modulating a biological function of prostaglandin E.sub.2, such as
neutralizing prostaglandin E.sub.2. The antibody, or antigen
binding fragment thereof, is selected from the group consisting of
an immunoglobulin molecule, a monoclonal antibody, a chimeric
antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a
Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single
domain antibody, a diabody, a multispecific antibody, a dual
specific antibody, and a bispecific antibody. In an embodiment, the
antibody, or antigen binding fragment thereof, is a humanized
antibody. In another embodiment, the antibody is selected from the
group consisting of Hu2B5.P1 and Hu2B5.P2. The invention also
provides a pharmaceutical composition comprising the antibody, or
antigen binding fragment thereof, and a pharmaceutically acceptable
carrier. In an embodiment, the pharmaceutical composition further
comprising at least one additional therapeutic agent for treating a
disorder in which prostaglandin E.sub.2 activity is
detrimental.
[0028] In another aspect, the invention provides a method of
generating an antibody, or fragment thereof, that binds to
prostaglandin E.sub.2 comprising the steps of immunizing a
non-human animal with prostaglandin E.sub.2-thyroglobulin,
collecting a body fluid or organ comprising an anti-prostaglandin
E.sub.2 antibody, and isolating the anti-prostaglandin E.sub.2
antibody.
[0029] In another aspect, the invention provides a humanized
antibody comprising an antigen binding domain, capable of binding
prostaglandin E.sub.2, comprising at least one CDR region
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 54-59. In another embodiment, the
invention provides a humanized antibody comprising an antigen
binding domain comprising at least one CDR region comprising an
amino acid sequence at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least
about 85%, at least about 90%, at least about 95%, or at least
about 98% homologous to an amino acid sequence selected from the
group consisting of SEQ ID NOs: 54-59. In an embodiment, the
humanized antibody comprises an amino acid sequence selected from
the group consisting of SEQ ID NOs: 78, 79, 80, and 81.
[0030] In one aspect the invention binding protein capable of
binding PGE2, said antigen binding domain comprising at least one
CDR comprising an amino acid sequence selected from the group
consisting of: CDR-H1: GYTFTKYWLG (SEQ ID NO: 54), CDR-H2:
DIYPGYDYTHYNEKFKD (SEQ ID NO: 55), CDR-H3: SDGSSTY (SEQ ID NO: 56),
CDR-L1: TSSQNIVHSNGNTYLE (SEQ ID NO: 57), CDR-L2: KVSNRFSG (SEQ ID
NO: 58), CDR-L3: FQVSHVPYT (SEQ ID NO: 59).
[0031] In another embodiment, the invention provides a binding
protein, or fragment thereof, comprising an antigen binding domain
comprising at least one CDR comprising an amino acid sequence
selected from the group consisting of SEQ ID NOs: 6, 7, 8, 10, 11,
12, 14, 15, 16, 18, 19, 20, 22, 23, 26, 27, 28, 30, 31, 32, 34, 35,
37, 38, and 39. In another embodiment, the binding protein, or
fragment thereof, comprises an antigen binding domain comprising at
least one VH region comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 5, 13, 21, 25, 33, 40, 42, and
44. In yet another embodiment, the binding protein, or fragment
thereof, comprises an antigen binding domain comprising at least
one VL region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 9, 17, 24, 29, 36, 41, 43, and 45.
In still another embodiment, the binding protein, or fragment
thereof, comprises an antigen binding domain comprising at least
one CDR region comprising an amino acid sequence selected from the
group consisting of SEQ ID NOs: 54-59.
[0032] In an embodiment, the binding protein comprises at least 3
CDRs, for example, selected from a VH CDR set selected from the
group consisting of SEQ ID NOs: 6,7, and 8; SEQ ID NOs: 14, 15, and
16; SEQ ID NOs: 14, 22, and 23, SEQ ID NOs: 26, 27, and 28, and 32;
SEQ ID NOs: 26, 34, and 35; and SEQ ID NOs: 54, 55, and 56. In
another embodiment, the at least 3 CDRs are selected from a VL CDR
set selected from the group consisting of SEQ ID NOs: 10, 11, and
12; SEQ ID NOs: 17, 18, and 19; SEQ ID NOs: 30, 31, and 32; SEQ ID
NOs: 37, 38, and 39; SEQ ID NOs: 42, 43, and 44; and SEQ ID NOs:
57, 58, and 59. In still another embodiment, the at least 3 CDRs
comprise a VH CDR set of amino acid sequences of SEQ ID NOs: 54,
55, and 56 and/or a VL CDR set of amino acid sequences of SEQ ID
NOs: 57, 58, and 59.
[0033] In another embodiment, the binding protein comprises at
least two variable domain CDR sets, for example, selected from a
group consisting of SEQ ID NOs: 6, 7, 8 and SEQ ID NOs: 10, 11, 12;
SEQ ID NOs: 14, 15, 16 and SEQ ID NOs: 18, 19, 20; SEQ ID NOs: 14,
22, 23 and SEQ ID NOs: 10, 11, 12; SEQ ID NOs: 26, 27, 28 and SEQ
ID NOs: 30, 31, 32; and SEQ ID NOs: 26, 34, 35 and SEQ ID NOs: 37,
38, 39.
[0034] In another embodiment, the binding protein of the invention
comprises two variable domains that have amino acid sequences
selected from the group consisting of SEQ ID NO:5 and SEQ ID NO:9;
SEQ ID NO:13 and SEQ ID NO:17; SEQ ID NO:21 and SEQ ID NO:24; SEQ
ID NO:25 and SEQ ID NO:29; SEQ ID NO:33 and SEQ ID NO:36; SEQ ID
NO:40 and SEQ ID NO:41; SEQ ID NO:42 and SEQ ID NO:43; and SEQ ID
NO:44 and SEQ ID NO:45. In another embodiment, the two variable
domains have amino acid sequences selected from the group
consisting of SEQ ID NO:60 and SEQ ID NO:61; SEQ ID NO:62 and SEQ
ID NO:63; SEQ ID NO:64 and SEQ ID NO:65; SEQ ID NO:66 and SEQ ID
NO:67; SEQ ID NO:68 and SEQ ID NO:69; SEQ ID NO:70 and SEQ ID
NO:71; SEQ ID NO:72 and SEQ ID NO:73; SEQ ID NO:74 and SEQ ID
NO:75; and SEQ ID NO:76 and SEQ ID NO:77. In another embodiment,
the two variable domains have amino acid sequences selected from
the group consisting of SEQ ID NO:78 and SEQ ID NO:79; and SEQ ID
NO:80 and SEQ ID NO:81.
[0035] In another aspect, the invention provides a humanized
antibody, or fragment thereof, that binds to prostaglandin E.sub.2,
the humanized antibody comprising at least one VH region comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, and 80. In another
embodiment, the humanized antibody, or fragment thereof, comprises
at least one VL region comprising an amino acid sequence selected
from the group consisting of SEQ ID NOs: 61 63, 65, 67, 69, 71, 73,
75, 77, 79, and 81. In yet another embodiment, the at least one VH
region or at least one VL region comprises human acceptor framework
sequences that comprise at least one amino acid substitution,
wherein the amino acid sequence of the framework sequence is at
least 65% identical to the sequence of the human acceptor framework
sequence. For example, the human acceptor framework may comprise at
least one framework amino acid substitution at a key residue, the
key residue selected from the group consisting of a residue
adjacent to a CDR, a glycosylation site residue, a rare residue, a
residue capable of interacting with prostaglandin E.sub.2, a
residue capable of interacting with a CDR, a canonical residue, a
contact residue between heavy chain variable region and light chain
variable region, a residue within a Vernier zone, and a residue in
a region that overlaps between a Chothia-defined variable heavy
chain CDR1 and a Kabat-defined first heavy chain framework.
[0036] In another embodiment the binding protein further comprises
a human acceptor framework.
[0037] In one embodiment of the invention the human heavy chain and
light chain acceptor sequences are selected from the sequences
described in Table 7 and Table 8 (Example 4.2.1). Other human heavy
chain and light chain acceptor sequences are well known in the art
and are suitable for use with the invention. In an embodiment the
binding protein is a CDR grafted antibody or antigen binding
portion thereof capable of binding PGE2. In another embodiment, the
binding protein is a humanized antibody or antigen binding portion
thereof capable of binding PGE2. In an embodiment, the CDR grafted
antibody or humanized antibody, or antigen binding portion thereof,
comprise one or more CDRs disclosed herein, for example, three or
more, four or more, five or more, or six or more CDRs. In another
embodiment, the CDR grafted antibody or humanized antibody, or
antigen binding portion thereof, comprises a human acceptor
framework. The said human acceptor framework can be any acceptor
framework of a human immunoglobulin. In a particular embodiment,
the human acceptor framework is any one of the human acceptor
frameworks disclosed herein. In an embodiment, the CDRs are
incorporated into a human antibody variable domain of a human
acceptor framework. In an embodiment, the human antibody variable
domain is a consensus human variable domain. In another embodiment,
the human acceptor framework comprises at least one Framework
Region amino acid substitution at a key residue, wherein the key
residue is selected from the group consisting of a residue adjacent
to a CDR; a glycosylation site residue; a rare residue; a residue
capable of interacting with PGE2; a residue capable of interacting
with a CDR; a canonical residue; a contact residue between heavy
chain variable region and light chain variable region; a residue
within a Vernier zone; and a residue in a region that overlaps
between a Chothia-defined variable heavy chain CDR1 and a
Kabat-defined first heavy chain framework. In an embodiment, the
human acceptor framework human acceptor framework comprises at
least one Framework Region amino acid substitution, wherein the
amino acid sequence of the framework is at least 65% identical to
the sequence of said human acceptor framework and comprises at
least 70 amino acid residues identical to said human acceptor
framework. In an embodiment, the framework region amino acid
substitution at a key residue is selected from the group consisting
of M (human) at position 48 to I (mouse), V (human) at position 68
to A (mouse), M (human) at position 70 to L (mouse), and T (human)
at position 72 to V (mouse) in the heavy chain variable region; and
I (human) at position 2 to V (mouse) and V (human) at position 3 to
L (mouse) in the light chain variable region.
[0038] In another aspect, the invention provides an antibody
construct comprising any one of the binding protein and a linker
polypeptide and/or an immunoglobulin constant domain. In an
embodiment, the antibody construct is selected from the group
consisting of an immunoglobulin molecule, a monoclonal antibody, a
chimeric antibody, a CDR-grafted antibody, a humanized antibody, a
Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a
single domain antibody, a diabody, a multispecific antibody, a dual
specific antibody, and a bispecific antibody. In an embodiment, the
antibody construct comprises a heavy chain immunoglobulin constant
domain selected from the group consisting of a human IgM constant
domain, a human IgG1 constant domain, a human IgG2 constant domain,
a human IgG3 constant domain, a human IgG4 constant domain, a human
IgE constant domain, and a human IgA constant domain. In an
embodiment, the antibody construct comprises an immunoglobulin
constant domain having an amino acid sequence selected from the
group consisting of SEQ ID NO.:1; SEQ ID NO.:2; SEQ ID NO.:3; SEQ
ID NO.:4; and SEQ ID NO.:5.
[0039] In another embodiment, the invention provides an anti-PGE2
antibody conjugate comprising an anti-PGE2 antibody construct and
an agent selected from the group consisting of an immunoadhesion
molecule, an imaging agent, a therapeutic agent, and a cytotoxic
agent. In an embodiment, the agent is an imaging agent selected
from the group consisting of a radiolabel, an enzyme, a fluorescent
label, a luminescent label, a bioluminescent label, a magnetic
label, and biotin. In another embodiment, the imaging agent is a
radiolabel selected from the group consisting of 3H, .sup.14C,
.sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I,
.sup.177Lu, .sup.166Ho, and .sup.153Sm, for example. In another
embodiment, the agent is a therapeutic or cytotoxic agent selected
from the group consisting of an anti-metabolite, an alkylating
agent, an antibiotic, a growth factor, a cytokine, an
anti-angiogenic agent, an anti-mitotic agent, an anthracycline, a
toxin, and an apoptotic agent.
[0040] In another embodiment, the binding protein is glycosylated.
In a particular embodiment, the PGE2 binding protein has a human
glycosylation pattern.
[0041] In another embodiment, the PGE2 binding protein, antibody
construct or antibody conjugate is crystallized (e.g., exists as a
crystal). In an embodiment, the crystal is a carrier-free
pharmaceutical controlled release crystal. In another embodiment
the crystallized binding protein, crystallized antibody construct
or crystallized antibody conjugate has a greater half life in vivo
than its soluble counterpart. In another embodiment the
crystallized binding protein, crystallized antibody construct or
crystallized antibody conjugate retains biological activity after
crystallization.
[0042] One aspect of the invention pertains to a DVD binding
protein comprising binding proteins capable of PGE2. Preferably,
the DVD binding protein is capable of binding two PGE2 binding
sites or binding PGE2 and a second target. The second target is
selected from the group consisting of CSF1 (MCSF), CSF2 (GM-CSF),
CSF3 (GCSF), FGF2, IFN.alpha.1, IFN.beta.1, IFN.gamma., histamine
and histamine receptors, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12.alpha.,
IL-12.beta., IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, KITLG,
PDGFB, IL-2R.beta., IL-4R, IL-5R.alpha., IL-8R.alpha., IL-8R.beta.,
IL-12R.beta.1, IL-12R.beta.2, IL-13R.alpha.1, IL-13R.alpha.2,
IL-18R1, TSLP, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13,
CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2,
CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1,
GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STAT6, TBX21, TGFB1, TNFSF6,
YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase. In
an embodiment, the DVD binding protein is capable of recognizing
PGE2 and IL-1.beta., PGE2 and IL-9; PGE2 and IL-4; PGE2 and IL-5;
PGE2 and IL-25; PGE2 and TARC; PGE2 and MDC; PGE2 and MIF; PGE2 and
TGF-.beta.; PGE2 and LHR agonist; PGE2 and CL25; PGE2 and SPRR2a;
PGE2 and SPRR2b; or PGE2 and ADAM8. In an embodiment, the DVD
binding protein is capable of binding PGE2 and TNF.alpha..
[0043] In another aspect, the invention provides isolated nucleic
acid, encoding a PGE2 binding protein, antibody construct or
antibody conjugate. A further embodiment provides vectors
comprising the isolated nucleic acids of the invention, wherein the
vector is selected from the group consisting of pcDNA; pTT
(Durocher et al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3
(pTT with additional multiple cloning site); pEFBOS (Mizushima, S.
and Nagata, S., (1990) Nucleic Acids Research Vol 18, No. 17); pBV;
pJV; pA2; and pBJ.
[0044] In another aspect, the invention provides host cells
transformed with the vectors of the invention. In an embodiment,
the host cell is a prokaryotic cell (e.g., E. coli). In another
embodiment, the host cell is a eukaryotic cell, e.g., a protist
cell, an animal cell, an avian cell, a plant cell, a fungal cell
(e.g., a yeast cell such as, for example, Saccharomyces
cerevisiae), a mammalian cell (e.g., a CHO, COS, and HEK293), an
insect cell (e.g., Sf9).
[0045] In another aspect, the invention provides a method of
producing a protein that binds PGE2, comprising culturing any one
of the host cells of the invention in a culture medium under
conditions sufficient to produce a binding protein that binds PGE2.
Another embodiment provides a binding protein produced according to
the methods of the invention.
[0046] In another aspect, the invention provides a formulation that
comprises a crystallized PGE2 binding protein, crystallized
antibody construct or crystallized antibody conjugate, an
ingredient, and/or at least one polymeric carrier. In an
embodiment, the polymeric carrier is a polymer selected from the
group consisting of: poly (acrylic acid), poly (cyanoacrylates),
poly (amino acids), poly (anhydrides), poly (depsipeptide), poly
(esters), poly (lactic acid), poly (lactic-co-glycolic acid) or
PLGA, poly (b-hydroxybutryate), poly (caprolactone), poly
(dioxanone); poly (ethylene glycol), poly ((hydroxypropyl)
methacrylamide, poly[(organo)phosphazene], poly (ortho esters),
poly (vinyl alcohol), poly (vinylpyrrolidone), maleic
anhydride-alkyl vinyl ether copolymers, pluronic polyols, albumin,
alginate, cellulose and cellulose derivatives, collagen, fibrin,
gelatin, hyaluronic acid, oligosaccharides, glycaminoglycans,
sulfated polyeaccharides, and a blend and/or copolymer thereof. In
another embodiment, the ingredient is selected from the group
consisting of albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol.
[0047] In another aspect, the invention provides a method for
treating a subject comprising the step of administering to the
subject an effective amount of a PGE2 binding protein, antibody
construct, conjugate, or composition. In an embodiment, the subject
is a mammal, such as a human suffering from an inflammatory disease
or other disorder described herein. In an embodiment, the invention
provides a method of reducing, ameliorating, or preventing one or
more symptoms of such a disease or disorder, such as a symptom of
(a) rheumatoid arthritis, allergic arthritis, juvenile arthritis,
ankylosing spondylitis and osteoarthritis; (b) certain illnesses
induced by viruses, such as Guillain Bane syndrome, infectious
mononucleosis, other viral lymphadenopathies and infections with
herpes virus; (c) multiple sclerosis and other demyelinating
diseases; (d) hematological disorders, such as hemolytic anemias
and thrombocytopenias; (e) endocrinologic disorders, such as
diabetes mellitus, Addison's disease, idiopathic hypoparathyroidism
and chronic lymphocytic thyroiditis; (f) collagen disorders, such
as systemic lupus erythematosus; and (g) disorders of reproduction
such as amenorrhoea, infertility, recurrent abortions and
eclampsia; and (h) tumors such as headneck tumor, lung cancer,
gastric cancer, prostate cancer, pancreatic cancer etc; and (i)
inflammatory bowel diseases than include Crohn's disease and
ulcerative colitis; and (j) pain associated with osteoarthritis and
other disorders; and (k) ocular disorders such as age-related
macular degeneration (AMD).
[0048] In another aspect, the invention provides a pharmaceutical
composition comprising a PGE2 binding protein, antibody, construct,
conjugate, or composition and a pharmaceutically acceptable
carrier. In an embodiment, the pharmaceutically acceptable carrier
functions as an adjuvant useful to increase the absorption or
dispersion of the binding protein. In an embodiment, the adjuvant
is hyaluronidase. The pharmaceutical composition may further
comprises at least one additional agent for diagnosing or treating
a disorder in which PGE2 activity is detrimental, for example, an
agent selected from the group consisting of a therapeutic agent, an
imaging agent; a cytotoxic agent; an angiogenesis inhibitor (e.g.,
an anti-VEGF antibody or VEGF-trap); a kinase inhibitor (e.g., a
KDR or TIE-2 inhibitor); a co-stimulation molecule blocker (e.g.,
an anti-B7.1, anti-B7.2, CTLA4-Ig, or anti-CD20); an adhesion
molecule blocker (e.g., an anti-LFA-1, anti-E/L selectin, or small
molecule inhibitor); an anti-cytokine antibody or functional
fragment thereof (e.g., an anti-IL-18, anti-TNF, or
anti-IL-6/cytokine receptor antibody); methotrexate; cyclosporine;
rapamycin; FK506; a detectable label or reporter; a TNF antagonist;
an anti-rheumatic; a muscle relaxant; a narcotic; a non-steroid
anti-inflammatory drug (NSAID); an analgesic; an anesthetic; a
sedative; a local anesthetic; a neuromuscular blocker; an
antimicrobial; an antipsoriatic; a corticosteroid; an anabolic
steroid; an erythropoietin; an immunization; an immunoglobulin; an
immunosuppressive; a growth hormone; a hormone replacement drug; a
radiopharmaceutical; an antidepressant; an antipsychotic; a
stimulant; an asthma medication; a beta agonist; an inhaled
steroid; an oral steroid; an epinephrine or analog thereof; a
cytokine; and a cytokine antagonist.
[0049] In another aspect, the invention provides a method for
inhibiting and/or reducing PGE2 activity comprising contacting PGE2
with a PGE2 binding protein such that PGE2 activity is inhibited
and/or reduced. In an embodiment, the invention provides a method
for inhibiting and/or reducing PGE2 activity in a subject suffering
from a disorder in which PGE2 activity is detrimental, comprising
administering to the subject a binding protein such that PGE2
activity in the subject is inhibited and/or reduced. In another
embodiment, the method comprises administering to a subject a PGE2
binding protein of the invention such that treatment is
achieved.
[0050] In another aspect, the invention provides a method of
treating (e.g., curing, suppressing, ameliorating, delaying or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a PGE2-associated disorder in a subject. The method
includes: administering to the subject a PGE2 binding protein
(particularly an antagonist), e.g., an anti-PGE2 antibody or
fragment thereof, in an amount sufficient to treat or prevent the
PGE2-associated disorder. The PGE2 antagonist, e.g., the anti-PGE2
antibody or fragment thereof, can be administered to the subject,
alone or in combination with other therapeutic modalities.
[0051] In one embodiment, the subject is a mammal, e.g., a human
suffering from one or more PGE2-associated disorders (e.g.,
characterized by excessive PGE2 levels or biosynthesis). In an
embodiment, The invention provides methods for the treatment of
inflammatory disorders and disorders of immunity in a subject,
which disorders may be characterized by excessive PGE.sub.2
biosynthesis, which methods comprise administering to the subject
an effective amount of an antibody specific to PGE.sub.2. Disorders
that may be treated by the method according to the invention
include autoimmune and inflammatory diseases and tumors in which
excessive PGE.sub.2 synthesis has been implicated. Such disorders
include: (a) rheumatoid arthritis, allergic arthritis, juvenile
arthritis, ankylosing spondylitis and osteoarthritis; (b) certain
illnesses induced by viruses, such as Guillain Barre syndrome,
infectious mononucleosis, other viral lymphadenopathies and
infections with herpes virus; (c) multiple sclerosis and other
demyelinating diseases; (d) hematological disorders, such as
hemolytic anemias and thrombocytopenias; (e) endocrinologic
disorders, such as diabetes mellitus, Addison's disease, idiopathic
hypoparathyroidism and chronic lymphocytic thyroiditis; (f)
collagen disorders, such as systemic lupus erythematosus; and (g)
disorders of reproduction such as amenorrhoea, infertility,
recurrent abortions and eclampsia; and (h) tumors such as headneck
tumor, lung cancer, gastric cancer, prostate cancer, pancreatic
cancer etc; and (i) inflammatory bowel diseases than include
Crohn's disease and ulcerative colitis; and (j) pain associated
with osteoarthritis and other disorders; and (k) ocular disorders
such as age-related macular degeneration (AMD). In another aspect,
this application provides a method for detecting the presence of
PGE2 in a sample in vitro (e.g., a biological sample, such as
serum, plasma, tissue, biopsy). The subject method can be used to
diagnose a disorder, e.g., an immune cell-associated disorder. The
method includes: (i) contacting the sample or a control sample with
the anti-PGE2 antibody or fragment thereof as described herein; and
(ii) detecting formation of a complex between the anti-PGE2
antibody or fragment thereof, and the sample or the control sample,
wherein a statistically significant change in the formation of the
complex in the sample relative to the control sample is indicative
of the presence of the PGE2 in the sample.
[0052] In yet another aspect, this application provides a method
for detecting the presence of PGE2 in vivo (e.g., in vivo imaging
in a subject). The subject method can be used to diagnose a
disorder, e.g., a PGE2-associated disorder. The method includes:
(i) administering the anti-PGE2 antibody or fragment thereof as
described herein to a subject or a control subject under conditions
that allow binding of the antibody or fragment to PGE2; and (ii)
detecting formation of a complex between the antibody or fragment
and PGE2, wherein a statistically significant change in the
formation of the complex in the subject relative to the control
subject is indicative of the presence of PGE2.
[0053] In another aspect, the binding proteins of the invention are
useful for treating a disorder selected from the group consisting
of arthritis, osteoarthritis, juvenile chronic arthritis, septic
arthritis, Lyme arthritis, psoriatic arthritis, reactive arthritis,
spondyloarthropathy, systemic lupus erythematosus, Crohn's disease,
ulcerative colitis, inflammatory bowel disease, insulin dependent
diabetes mellitus, thyroiditis, asthma, allergic diseases,
psoriasis, dermatitis scleroderma, graft versus host disease, organ
transplant rejection, acute or chronic immune disease associated
with organ transplantation, sarcoidosis, atherosclerosis,
disseminated intravascular coagulation, Kawasaki's disease, Grave's
disease, nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis
of the kidneys, chronic active hepatitis, uveitis, septic shock,
toxic shock syndrome, sepsis syndrome, cachexia, infectious
diseases, parasitic diseases, acquired immunodeficiency syndrome,
acute transverse myelitis, Huntington's chorea, Parkinson's
disease, Alzheimer's disease, stroke, primary biliary cirrhosis,
hemolytic anemia, malignancies, heart failure, myocardial
infarction, Addison's disease, sporadic, polyglandular deficiency
type I and polyglandular deficiency type II, Schmidt's syndrome,
adult (acute) respiratory distress syndrome, alopecia, alopecia
areata, seronegative arthopathy, arthropathy, Reiter's disease,
psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic
synovitis, chlamydia, yersinia and salmonella associated
arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis B, Hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Th1
Type mediated diseases, acute and chronic pain (different forms of
pain), and cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer and hematopoietic
malignancies (leukemia and lymphoma), Abetalipoprotemia,
Acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti cd3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aordic and peripheral
aneuryisms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chromic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, Dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic ateriosclerotic disease, Diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's Syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, Epstein-Barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant
rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular
nephritis, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallerrorden-Spatz disease,
Hashimoto's thyroiditis, hay fever, heart transplant rejection,
hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy,
Hodgkin's disease, hyperkinetic movement disorders, hypersensitity
reactions, hypersensitivity pneumonitis, hypertension, hypokinetic
movement disorders, hypothalamic-pituitary-adrenal axis evaluation,
idiopathic Addison's disease, idiopathic pulmonary fibrosis,
antibody mediated cytotoxicity, Asthenia, infantile spinal muscular
atrophy, inflammation of the aorta, influenza a, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma,
kidney transplant rejection, legionella, leishmaniasis, leprosy,
lesions of the corticospinal system, lipedema, liver transplant
rejection, lymphederma, malaria, malignamt Lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia,
metabolic/idiopathic, migraine headache, mitochondrial multi.system
disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Mencel
Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,
mycobacterium avium intracellulare, mycobacterium tuberculosis,
myelodyplastic syndrome, myocardial infarction, myocardial ischemic
disorders, nasopharyngeal carcinoma, neonatal chronic lung disease,
nephritis, nephrosis, neurodegenerative diseases, neurogenic I
muscular atrophies, neutropenic fever, non-Hodgkins lymphoma,
occlusion of the abdominal aorta and its branches, occulsive
arterial disorders, okt3 therapy, orchitis/epidydimitis,
orchitis/vasectomy reversal procedures, organomegaly, osteoporosis,
pancreas transplant rejection, pancreatic carcinoma, paraneoplastic
syndrome/hypercalcemia of malignancy, parathyroid transplant
rejection, pelvic inflammatory disease, perennial rhinitis,
pericardial disease, peripheral atherlosclerotic disease,
peripheral vascular disorders, peritonitis, pernicious anemia,
pneumocystis carinii pneumonia, pneumonia, POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), post perfusion syndrome,
post pump syndrome, post-MI cardiotomy syndrome, preeclampsia,
Progressive supranucleo Palsy, primary pulmonary hypertension,
radiation therapy, Raynaud's phenomenon and disease, Raynoud's
disease, Refsum's disease, regular narrow QRS tachycardia,
renovascular hypertension, reperfusion injury, restrictive
cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile
Dementia of Lewy body type, seronegative arthropathies, shock,
sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the
cardiovascular system, systemic anaphalaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, vital
encephalitis/aseptic meningitis, vital-associated hemaphagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, Acute coronary syndromes, Acute
Idiopathic Polyneuritis, Acute Inflammatory Demyelinating
Polyradiculoneuropathy, Acute ischemia, Adult Still's Disease,
Alopecia areata, Anaphylaxis, Anti-Phospholipid Antibody Syndrome,
Aplastic anemia, Arteriosclerosis, Atopic eczema, Atopic
dermatitis, Autoimmune dermatitis, Autoimmune disorder associated
with Streptococcus infection, Autoimmune Enteropathy, Autoimmune
hearingloss, Autoimmune Lymphoproliferative Syndrome (ALPS),
Autoimmune myocarditis, Autoimmune premature ovarian failure,
Blepharitis, Bronchiectasis, Bullous pemphigoid, Cardiovascular
Disease, Catastrophic Antiphospholipid Syndrome, Celiac Disease,
Cervical Spondylosis, Chronic ischemia, Cicatricial pemphigoid,
Clinically isolated Syndrome (CIS) with Risk for Multiple
Sclerosis, Conjunctivitis, Childhood Onset Psychiatric Disorder,
Chronic obstructive pulmonary disease (COPD), Dacryocystitis,
dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Disk
herniation, Disk prolaps, Drug induced immune hemolytic anemia,
Endocarditis, Endometriosis, endophthalmitis, Episcleritis,
Erythema multiforme, erythema multiforme major, Gestational
pemphigoid, Guillain-Barre Syndrome (GBS), hay fever, Hughes
Syndrome, Idiopathic Parkinson's Disease, idiopathic interstitial
pneumonia, IgE-mediated Allergy, Immune hemolytic anemia, Inclusion
Body Myositis, Infectious ocular inflammatory disease, Inflammatory
demyelinating disease, Inflammatory heart disease, Inflammatory
kidney disease, IPF/UIP, Iritis, Keratitis, Keratojuntivitis sicca,
Kussmaul disease or Kussmaul-Meier Disease, Landry's Paralysis,
Langerhan's Cell Histiocytosis, Livedo reticularis, Macular
Degeneration, Microscopic Polyangiitis, Morbus Bechterev, Motor
Neuron Disorders, Mucous membrane pemphigoid, Multiple Organ
failure, Myasthenia Gravis, Myelodysplastic Syndrome, Myocarditis,
Nerve Root Disorders, Neuropathy, Non-A Non-B Hepatitis, Optic
Neuritis, Osteolysis, Pauciarticular JRA, peripheral artery
occlusive disease (PAOD), peripheral vascular disease (PVD),
peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa
(or periarteritis nodosa), Polychondritis, Polymyalgia Rheumatica,
Poliosis, Polyarticular JRA, Polyendocrine Deficiency Syndrome,
Polymyositis, polymyalgia rheumatica (PMR), Post-Pump Syndrome,
primary parkinsonism, Prostatitis, Pure red cell aplasia, Primary
Adrenal Insufficiency, Recurrent Neuromyelitis Optica, Restenosis,
Rheumatic heart disease, SAPHO (synovitis, acne, pustulosis,
hyperostosis, and osteitis), Scleroderma, Secondary Amyloidosis,
Shock lung, Scleritis, Sciatica, Secondary Adrenal Insufficiency,
Silicone associated connective tissue disease, Sneddon-Wilkinson
Dermatosis, spondilitis ankylosans, Stevens-Johnson Syndrome (SJS),
Systemic inflammatory response syndrome, Temporal arteritis,
toxoplasmic retinitis, toxic epidermal necrolysis, Transverse
myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1 allergic
reaction, Type II Diabetes, Urticaria, Usual interstitial pneumonia
(UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,
Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular
degeneration, and Wound healing.
[0054] In an embodiment, diseases that can be treated or diagnosed
with the compositions and methods of the invention include, but are
not limited to, primary and metastatic cancers, including
carcinomas of breast, colon, rectum, lung, oropharynx, hypopharynx,
esophagus, stomach, pancreas, liver, gallbladder and bile ducts,
small intestine, urinary tract (including kidney, bladder and
urothelium), female genital tract (including cervix, uterus, and
ovaries as well as choriocarcinoma and gestational trophoblastic
disease), male genital tract (including prostate, seminal vesicles,
testes and germ cell tumors), endocrine glands (including the
thyroid, adrenal, and pituitary glands), and skin, as well as
hemangiomas, melanomas, sarcomas (including those arising from bone
and soft tissues as well as Kaposi's sarcoma), tumors of the brain,
nerves, eyes, and meninges (including astrocytomas, gliomas,
glioblastomas, retinoblastomas, neuromas, neuroblastomas,
Schwannomas, and meningiomas), solid tumors arising from
hematopoietic malignancies such as leukemias, and lymphomas (both
Hodgkin's and non-Hodgkin's lymphomas).
[0055] The method comprises administering to the subject a PGE2
antagonist, e.g., a PGE2 antibody or a fragment thereof, in an
amount sufficient to treat (e.g., reduce, ameliorate) or prevent
one or more symptoms. The PGE2 antibody can be administered
therapeutically or prophylactically, or both. The PGE2 antagonist,
e.g., the anti-PGE2 antibody, or fragment thereof, can be
administered to the subject, alone or in combination with other
therapeutic modalities as described herein. Preferably, the subject
is a mammal, e.g., a human suffering from a PGE2-associated
disorder as described herein.
[0056] In another aspect, the binding proteins of the invention are
useful for treating a disorder selected from the group consisting
of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,
Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Cerebellar
Astrocytoma, Cerebral Astrocytoma, Basal Cell Carcinoma, Bile Duct
Cancer, Extrahepatic, Bladder Cancer, Bone Cancer,
Osteosarcoma/Malignant Fibrous Histiocytoma Brain Stem Glioma,
Brain Tumor, Brain Stem Glioma, Cerebral strocytoma/Malignant
Glioma, Ependymoma, Medulloblastoma, Supratentorial Primitive
Neuroectodermal Tumors, Visual Pathway and Hypothalamic Glioma,
Breast Cancer, Bronchial Adenomas/Carcinoids, Carcinoid Tumor,
Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown Primary,
Central Nervous System Lymphoma, Primary Cerebellar Astrocytoma,
Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous
Leukemia Chronic Myeloproliferative Disorders, Colon Cancer,
Colorectal Cancer, Cutaneous T-Cell Lymphoma, Endometrial Cancer,
Ependymoma, Esophageal Cancer, Ewing Family of Tumors, Extracranial
Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile
Duct Cancer, Eye Cancer, Intraocular Melanoma Retinoblastoma,
Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal
Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),
Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Ovarian
Germ Cell Tumor, Gestational Trophoblastic Tumor, Glioma, Brain
Stem Glioma, Cerebral Astrocytoma Glioma, Childhood Visual Pathway
and Hypothalamic Glioma, Hairy Cell Leukemia, Head and Neck Cancer,
Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal
Cancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine
Pancreas), Kaposi Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal
Cancer, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,
Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy
Cell Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-Small
Cell Lung Cancer, Small Cell Lung Cancer, AIDS-Related Lymphoma,
Burkitt Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin Lymphoma,
Non-Hodgkin Lymphoma, Primary Central Nervous System Lymphoma,
Waldenstrom Macroglobulinemia, Malignant Fibrous Histiocytoma of
Bone/Osteosarcoma, Medulloblastoma, Melanoma, Intraocular (Eye)
Melanoma, Merkel Cell Carcinoma, Malignant Mesothelioma, Metastatic
Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple
Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell
Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,
Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia,
Chronic Myeloid Leukemia, Multiple Myeloma, Myeloproliferative
Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal
Cancer, Neuroblastoma, Oral Cancer, Oral Cavity Cancer, Lip and
Oropharyngeal Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma
of Bone, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ
Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer, Islet Cell Pancreatic Cancer, Paranasal Sinus and Nasal
Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal
Cancer, Pheochromocytoma, Pineoblastoma and Supratentorial
Primitive Neuroectodermal Tumors, Pituitary Tumor, Plasma Cell
Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Prostate
Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and
Ureter, Transitional Cell Cancer, Retinoblastoma, Salivary Gland
Cancer, Sarcoma, Ewing Family of Tumors, Kaposi Sarcoma, Soft
Tissue Sarcoma, Uterine Sarcoma, Sezary Syndrome, Skin Cancer
(Nonmelanoma), Skin Cancer (Melanoma), Merkel Cell Skin Carcinoma,
Small Intestine Cancer, Squamous Cell Carcinoma, Metastatic
Squamous Neck Cancer with Occult Primary, Stomach (Gastric) Cancer,
Supratentorial Primitive Neuroectodermal Tumors, Cutaneous T-Cell
Lymphoma, Testicular Cancer, Throat Cancer, Thymoma, Thymoma and
Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the
Renal Pelvis and Ureter, Gestational Trophoblastic Tumor, Ureter
and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer,
Uterine Cancer, Endometrial Uterine Sarcoma, Vaginal Cancer, Visual
Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom
Macroglobulinemia, Wilms Tumor.
[0057] In another aspect the invention provides a method of
treating a patient suffering from a disorder in which PGE2 is
detrimental comprising the step of administering any one of the
binding proteins disclosed above before, concurrently, or after the
administration of a second agent, as discussed above. In an
embodiment the second therapeutic agent that can be coadministered
and/or coformulated with one or more PGE.sub.2 antagonists, (e.g.,
anti-PGE2 antibodies or fragments thereof) include, but are not
limited to, one or more of: inhaled steroids; oral steroids;
beta-agonists, e.g., short-acting or long-acting beta-agonists;
antagonists of leukotrienes or leukotriene receptors; combination
drugs such as ADVAIR.TM.; IgE inhibitors, e.g., anti-IgE antibodies
(e.g., XOLAIR.TM.); phosphodiesterase inhibitors (e.g., PDE4
inhibitors); xanthines; anticholinergic drugs; mast
cell-stabilizing agents such as cromolyn; IL-4 inhibitors; IL-5
inhibitors; eotaxin/CCR3 inhibitors; antagonists of histamine or
its receptors including H1, H2, H3, and H4, and antagonists of
prostaglandin D or its receptors (DPI and CRTH2). Such combinations
can be used to treat asthma and other respiratory disorders.
Additional examples of therapeutic agents that can be
coadministered and/or coformulated with one or more anti-PGE2
antibodies or fragments thereof include one or more of: TNF
antagonists (e.g., a soluble fragment of a TNF receptor, e.g., p55
or p75 human TNF receptor or derivatives thereof, e.g., 75 kD
TNFR-IgG (75 kD TNF receptor-IgG fusion protein, ENBREL.TM.)); TNF
enzyme antagonists, e.g., TNF converting enzyme (TACE) inhibitors;
muscarinic receptor antagonists; TGF-beta antagonists; interferon
gamma; perfenidone; chemotherapeutic agents, e.g., methotrexate,
leflunomide, or a sirolimus (rapamycin) or an analog thereof, e.g.,
CCI-779; COX2 and cPLA2 inhibitors; NSAIDs; immunomodulators; p38
inhibitors, TPL-2, MK-2 and NFkB inhibitors, among others.
Additional second agent is selected from the group consisting of
budenoside, epidermal growth factor, corticosteroids, cyclosporin,
sulfasalazine, aminosalicylates, 6-mercaptopurine, azathioprine,
metronidazole, lipoxygenase inhibitors, mesalamine, olsalazine,
balsalazide, antioxidants, thromboxane inhibitors, IL-1 receptor
antagonists, anti-IL-1.beta., monoclonal antibodies, anti-IL-6
monoclonal antibodies, growth factors, elastase inhibitors,
pyridinyl-imidazole compounds, antibodies or agonists of TNF, LT,
IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF,
FGF, and PDGF, antibodies of CD2, CD3, CD4, CD8, CD25, CD28, CD30,
CD40, CD45, CD69, CD90 or their ligands, methotrexate, cyclosporin,
FK506, rapamycin, mycophenolate mofetil, leflunomide, NSAIDs,
ibuprofen, corticosteroids, prednisolone, phosphodiesterase
inhibitors, adenosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, IRAK, NIK, IKK, p38, MAP kinase
inhibitors, IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme inhibitors, T-cell signaling inhibitors,
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors, soluble p55 TNF receptor, soluble p75
TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, anti-inflammatory
cytokines, IL-4, IL-10, IL-11, and TGF.beta..
[0058] In a particular embodiment, the invention provides a method
of treating a patient suffering from a disorder in which
prostaglandin E.sub.2 is detrimental, the method comprising the
step of administering a binding protein of the invention before,
concurrently, or after the administration of a second agent,
wherein the second agent is selected from the group consisting of
the drugs that currently are used for the treatment of various
human diseases and disorders. A list of such drugs is available
from the internet. This list is updated frequently to reflect the
state-of-art for the treatment of various human diseases. A list of
such drugs is also available from the most updated drug guide
(Complete Guide to Prescription & Nonprescription Drugs 2008.
by H. Winter Griffith, Stephen Moore, ISBN-13: 978-0399533723). The
anti-PGE.sub.2 binding protein can be combined with any of the
therapies in the above list for a particular disease conditions.
For example, the anti-PGE.sub.2 binding protein can be combined
with one or more agents for the treatment of rheumatoid arthritis
and juvenile rheumatoid arthritis. Examples of these agents
include, but are not limited to, methotrexate, leflunomide, low
doses of corticosteroids such as prednisone or cortisone,
anti-malarial medications such as hydroxychloroquine, gold,
sulfasalazine, penicillamine, cyclophosphamide, cyclosporine,
minocycline, Acetaminophen, aspirin, ibuprofen, naproxen,
celecoxib, Infliximab, etanercept, adalimumab, abatacept,
rituximab, anakinra and other new biologic agents and oral delivery
agents targeting IL-6, IL-6R, IL-17, IL-18, IL-23, B7.1/B7.2. The
antiPGE.sub.2 binding protein can be combined with one or more
agents for the treatment of osteoarthritis. Examples of these
agents include but are not limited to acetaminophen, aspirin,
ibuprofen, naproxen, celecoxib, steroids, artificial joint fluid
such as Synvisc.TM., and Hyalgan.TM.. The anti-PGE.sub.2 binding
protein can be combined with one or more agents for the treatment
of Crohn's disease. Examples of these agents include but are not
limited to adalimumab, Azasan.TM., Asacol.TM., azathioprine,
Azulfidine.TM., budesonide, Entocort.TM., Flagyl.TM., Imuran.TM.,
infliximab, mercaptopurine, metronidazole, protostat,
Purinethol.TM., Remicade.TM., and sulfasalazine. The anti-PGE.sub.2
binding protein can be combined with one or more agents for the
treatment of ankylosing spondylitis. Examples of these agents
include but are not limited to acetocot, acetylsalicylic acid,
acuprin 81, adalimumab, Aleve.TM., amcort, Anaprox.TM.,
Aristocort.TM., aspirin, aspirtab, Azmacort.TM., Bufferin.TM.,
buffex, Cataflam.TM., Celebrex.TM., Clinoril.TM., cortisone,
diclofenac, Dipentum.TM., Easprin.TM., etanercept, Indocin.TM.,
indomethacin, infliximab, naproxen, Remicade.TM., triamcinolone,
and Voltaren.TM.. The anti-PGE.sub.2 binding protein can be
combined with one or more agents for the treatment of multiple
sclerosis. Examples of these agents include but are not limited to
Avonex.TM., Azasan.TM., Azathioprine, Betaseron.TM.,
Bubbli-Pred.TM., Copaxone.TM., Cotolone, Glatiramer, Imuran.TM.,
Interferon Beta-1a, Interferon Beta-1b Solution, Key-Pred, Key-Pred
SP, Mitoxantrone, Natalizumab, Novantrone.TM., Orapred.TM., Orapred
ODT.TM., Pediapred.TM., Pred-Ject-50, Predacort 50, Predalone 50,
Predate-50, Prednisolone, Prelone.TM., Rebif.TM., and Tysabri.TM..
The anti-PGE.sub.2 binding protein can be combined with one or more
agents or treatment procedures for the treatment of various human
cancers and malignancies. Besides a list available from the
internet and from the most updated drug guide (Complete Guide to
Prescription & Nonprescription Drugs 2008. by H. Winter
Griffith, Stephen Moore, ISBN-13: 978-0399533723), NCI also
maintains drug information about certain drugs that are approved by
the U.S. Food and Drug Administration (FDA) for the treatment of
cancer or conditions related to cancer. Examples of these agents
include but are not limited to Abraxane.TM., Adriamycin,
Adrucil.TM., Aldara.TM., Alemtuzumab, Alimta.TM., Aminolevulinic
Acid, Anastrozole, Aprepitant, Arimidex.TM., Aromasin.TM.,
Arranon.TM., Arsenic Trioxide, Avastin.TM. (Bevacizumab),
Azacitidine, Bevacizumab, Bexarotene, Bortezomib, Campath.TM.
(Alemtuzumab), Camptosar.TM. (Irinotecan Hydrochloride),
Capecitabine, Carboplatin, Cetuximab, Cisplatin, Clafen
(Cyclophosphamide), Clofarabine, Clofarex (Clofarabine), Clolar.TM.
(Clofarabine), Cyclophosphamide, Cytarabine, Cytosar-U
(Cytarabine), Cytoxan (Cyclophosphamide), Dacogen.TM. (Decitabine),
Dasatinib, Decitabine, DepoCyt.TM. (Liposomal Cytarabine),
DepoFoam.TM. (Liposomal Cytarabine), Dexrazoxane Hydrochloride,
Docetaxel, Doxil.TM. (Doxorubicin Hydrochloride Liposome),
Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome,
Dox-SL (Doxorubicin Hydrochloride Liposome), Efudex.TM.
(Fluorouracil), Ellence.TM. (Epirubicin Hydrochloride),
Eloxatin.TM. (Oxaliplatin), Emend.TM. (Aprepitant), Epirubicin
Hydrochloride, Erbitux.TM. (Cetuximab), Erlotinib Hydrochloride,
Evacet (Doxorubicin Hydrochloride Liposome), Evista.TM. (Raloxifene
Hydrochloride), Exemestane, Faslodex.TM. (Fulvestrant), Femara.TM.
(Letrozole), Fluoroplex.TM. (Fluorouracil), Fluorouracil,
Fulvestrant, Gefitinib, Gemcitabine Hydrochloride, Gemtuzumab
Ozogamicin, Gemzar.TM. (Gemcitabine Hydrochloride), Gleevec.TM.
(Imatinib Mesylate), Herceptin.TM. (Trastuzumab), Hycamtin.TM.
(Topotecan Hydrochloride), Imatinib Mesylate, Imiquimod, Iressa.TM.
(Gefitinib), Irinotecan Hydrochloride, Ixabepilone, Ixempra.TM.
(Ixabepilone), Keoxifene (Raloxifene Hydrochloride), Kepivance.TM.
(Palifermin), Lapatinib Ditosylate, Lenalidomide, Letrozole,
Levulan.TM. (Aminolevulinic Acid), LipoDox.TM.(Doxorubicin
Hydrochloride Liposome), Liposomal Cytarabine, Methazolastone
(Temozolomide), Mylosar (Azacitidine), Mylotarg.TM. (Gemtuzumab
Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-stabilized
Nanoparticle Formulation), Nelarabine, Neosar.TM.
(Cyclophosphamide), Nexavar.TM. (Sorafenib Tosylate), Nilotinib,
Nolvadex (Tamoxifen Citrate), Oncaspar.TM. (Pegaspargase),
Oxaliplatin, Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle
Formulation, Palifermin, Panitumumab, Paraplat (Carboplatin),
Paraplatin.TM. (Carboplatin), Pegaspargase, Pemetrexed Disodium,
Platinol-AQ (Cisplatin), Platinol.TM. (Cisplatin), Raloxifene
Hydrochloride, Revlimid (Lenalidomide), Rituxan.TM. (Rituximab),
Rituximab, Sclerosol Intrapleural Aerosol (Talc), Sorafenib
Tosylate, Sprycel.TM. (Dasatinib), Sterile Talc Powder (Talc),
Steritalc.TM. (Talc), Sunitinib Malate, Sutent.TM. (Sunitinib
Malate), Synovir (Thalidomide), Talc, Tamoxifen Citrate, Tarabine
PFS (Cytarabine), Tarceva.TM. (Erlotinib Hydrochloride),
Targretin.TM. (Bexarotene), Tasigna.TM. (Nilotinib), Taxol.TM.
(Paclitaxel), Taxotere.TM. (Docetaxel), Temodar.TM. (Temozolomide),
Temozolomide, Temsirolimus, Thalomid.TM. (Thalidomide),
Thalidomide, Totect.TM. (Dexrazoxane Hydrochloride), Topotecan
Hydrochloride, Torisel.TM. (Temsirolimus), Trastuzumab,
Trisenox.TM. (Arsenic Trioxide), Tykerb.TM. (Lapatinib Ditosylate),
Vectibix.TM. (Panitumumab), Velcade.TM. (Bortezomib), Vidaza.TM.
(Azacitidine), Vorinostat, Xeloda.TM. (Capecitabine), Zinecard.TM.
(Dexrazoxane Hydrochloride), Zoledronic Acid, Zolinza.TM.
(Vorinostat), and Zometa.TM. (Zoledronic Acid).
[0059] In a preferred embodiment the PGE2 binding protein
pharmaceutical compositions disclosed above are administered to the
subject by at least one mode selected from parenteral,
subcutaneous, intramuscular, intravenous, intrarticular,
intrabronchial, intraabdominal, intracapsular, intracartilaginous,
intracavitary, intracelial, intracerebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, and
transdermal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0060] The foregoing and other objects, features and advantages of
the present invention, as well as the invention itself, will be
more fully understood from the following description of preferred
embodiments when read together with the accompanying drawings, in
which:
[0061] FIG. 1 provides measurement of the binding of two hybridoma
derived mAbs 15F10.3C9 and 1F7.1D5 to Biotin-PGE.sub.2 in an ELISA
described in Example 1.1.A.
[0062] FIG. 2 provides measurement of the binding of two hybridoma
derived mAbs 19C9.4B10 and 4F10.3B9 to Biotin-PGE.sub.2 in an ELISA
described in Example 1.1.A.
[0063] FIG. 3 provides measurement of the binding of PROfusion.TM.
derived mAbs K1B, K7H, K3A, L11 and L21 to Biotin-PGE.sub.2 in an
ELISA described in Example 1.1.A.
[0064] FIG. 4 provides the binding of recombinant anti-PGE.sub.2
mAbs 2B5-7.0, 2B5-8.0 and 2B5-9.0 to Biotin-PGE.sub.2 in an ELISA
described in Example 1.1.A. The hybridoma derived antibody 2B5 is
the positive control in this assay.
[0065] FIG. 5 provides anti-PGE.sub.2 mAb 2B5-8.0 to neutralize
PGE.sub.2 induced Ca.sup.++ influx in EP4 transfected HEK293
G.alpha.16 stable cell line measured by FLIPR as described in
Example 1.1.C 1. The hybridoma derived antibody 2B5 is the positive
control in this assay.
[0066] FIG. 6 provides humanized anti-PGE.sub.2 mAb 2B5.5, 2B5.6,
2B5.7 and 2B5.8 to neutralize PGE.sub.2 induced Ca.sup.++ influx in
EP4 transfected HEK293 G.alpha.16 stable cell line measured by
FLIPR as described in Example 1.1.C 1.
[0067] FIG. 7 provides humanized anti-PGE.sub.2 mAb 2B5.1, 2B5.2,
2B5.3 and 2B5.4 to neutralize PGE.sub.2 induced Ca.sup.++ influx in
EP4 transfected HEK293 G.alpha.16 stable cell line measured by
FLIPR as described in Example 1.1.C 1.
[0068] FIG. 8 provides alignment of VH regions (SEQ ID NOS 40, 42,
44, and 82, respectively, in order of appearance) and VL regions
(SEQ ID NOS 41, 43, 45, and 83, respectively, in order of
appearance) of anti-PGE2 antibodies 2B5.7, 2B5.8 and 2B5.9 as
described in Example 3.
[0069] FIG. 9 provides efficacy of anti-PGE.sub.2 antibody,
anti-murine TNF antibody and their combination in a
collagen-induced arthritis model measured by MAS (mean arthritis
score).
DETAILED DESCRIPTION OF THE INVENTION
[0070] This invention pertains to Prostaglandin E2 (PGE.sub.2)
binding proteins, particularly anti-PGE.sub.2 antibodies, or
antigen-binding fragments thereof, that bind PGE.sub.2. Various
aspects of the invention relate to antibodies and antibody
fragments, and pharmaceutical compositions thereof, as well as
nucleic acids, recombinant expression vectors and host cells for
making such antibodies and fragments. Methods of using the
antibodies of the invention to detect PGE.sub.2, to inhibit one or
more PGE.sub.2 activities, either in vitro or in vivo; and to
regulate gene expression are also encompassed by the invention.
[0071] Unless otherwise defined herein, scientific and technical
terms used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. The meaning and scope of the terms should be clear,
however, in the event of any latent ambiguity, definitions provided
herein take precedent over any dictionary or extrinsic definition.
Further, unless otherwise required by context, singular terms shall
include pluralities and plural terms shall include the singular. In
this application, the use of "or" means "and/or" unless stated
otherwise. Furthermore, the use of the term "including", as well as
other forms, such as "includes" and "included", is not limiting.
Also, terms such as "element" or "component" encompass both
elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0072] Generally, nomenclatures used in connection with, and
techniques of, cell and tissue culture, molecular biology,
immunology, microbiology, genetics and protein and nucleic acid
chemistry and hybridization described herein are those well known
and commonly used in the art. The methods and techniques of the
present invention are generally performed according to conventional
methods well known in the art and as described in various general
and more specific references that are cited and discussed
throughout the present specification unless otherwise indicated.
Enzymatic reactions and purification techniques are performed
according to manufacturer's specifications, as commonly
accomplished in the art or as described herein. The nomenclatures
used in connection with, and the laboratory procedures and
techniques of, analytical chemistry, synthetic organic chemistry,
and medicinal and pharmaceutical chemistry described herein are
those well known and commonly used in the art. Standard techniques
are used for chemical syntheses, chemical analyses, pharmaceutical
preparation, formulation, and delivery, and treatment of
patients.
[0073] That the present invention may be more readily understood,
select terms are defined below.
[0074] The term "polypeptide" as used herein, refers to any
polymeric chain of amino acids. The terms "peptide" and "protein"
are used interchangeably with the term polypeptide and also refer
to a polymeric chain of amino acids. The term "polypeptide"
encompasses native or artificial proteins, protein fragments and
polypeptide analogs of a protein sequence. A polypeptide may be
monomeric or polymeric.
[0075] The term "isolated protein" or "isolated polypeptide" is a
protein or polypeptide that by virtue of its origin or source of
derivation is not associated with naturally associated components
that accompany it in its native state; is substantially free of
other proteins from the same species; is expressed by a cell from a
different species; or does not occur in nature. Thus, a polypeptide
that is chemically synthesized or synthesized in a cellular system
different from the cell from which it naturally originates will be
"isolated" from its naturally associated components. A protein may
also be rendered substantially free of naturally associated
components by isolation, using protein purification techniques well
known in the art.
[0076] The term "recovering" as used herein, refers to the process
of rendering a chemical species such as a polypeptide substantially
free of naturally associated components by isolation, e.g., using
protein purification techniques well known in the art.
[0077] The terms "Prostaglandin E2" (abbreviated herein as
PGE.sub.2), as used herein, refers to the prostaglandin having the
following structure or a variant thereof that retains some or all
PGE.sub.2 activities:
##STR00001##
[0078] "Biological activity" as used herein, refers to inherent
biological properties of the cytokine Biological properties of
PGE.sub.2 include but are not limited to binding to a PGE.sub.2
receptor.
[0079] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody, a
protein, or a peptide with a second chemical species, mean that the
interaction is dependent upon the presence of a particular
structure (e.g., an antigenic determinant or epitope) on the
chemical species; for example, an antibody recognizes and binds to
a specific protein structure rather than to proteins generally. If
an antibody is specific for epitope "A", the presence of a molecule
containing epitope A (or free, unlabeled A), in a reaction
containing labeled "A" and the antibody, will reduce the amount of
labeled A bound to the antibody.
[0080] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative anitbody formats are known in the art.
Nonlimiting embodiments of which are discussed below.
[0081] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or VH)
and a heavy chain constant region. The heavy chain constant region
is comprised of three domains, CH1, CH2 and CH3. Each light chain
is comprised of a light chain variable region (abbreviated herein
as LCVR or VL) and a light chain constant region. The light chain
constant region is comprised of one domain, CL. The VH and VL
regions can be further subdivided into regions of hypervariability,
termed complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can
be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class
(e.g., IgG 1, IgG2, IgG 3, IgG4, IgA1 and IgA2) or subclass.
[0082] The term "antigen-binding portion" or "antigen binding
fragment" of an antibody (or simply "antibody portion"), as used
herein, refers to one or more fragments of an antibody that retain
the ability to specifically bind to an antigen (e.g., PGE.sub.2).
The antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Such antibody embodiments may
also have bispecific, dual specific, or multi-specific formats,
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the VL, VH, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the VH and CH1 domains; (iv) a Fv
fragment consisting of the VL and VH domains of a single arm of an
antibody, (v) a dAb fragment (Ward et al., (1989) Nature
341:544-546, Winter et al., PCT publication WO 90/05144 A1), which
comprises a single variable domain; and (vi) an isolated
complementarity determining region (CDR). Furthermore, although the
two domains of the Fv fragment, VL and VH, are coded for by
separate genes, they can be joined, using recombinant methods, by a
synthetic linker that enables them to be made as a single protein
chain in which the VL and VH regions pair to form monovalent
molecules (known as single chain Fv (scFv); see e.g., Bird et al.
(1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl.
Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also
encompassed within the term "antigen-binding portion" of an
antibody. Other forms of single chain antibodies, such as diabodies
are also encompassed. Diabodies are bivalent, bispecific antibodies
in which VH and VL domains are expressed on a single polypeptide
chain, but using a linker that is too short to allow for pairing
between the two domains on the same chain, thereby forcing the
domains to pair with complementary domains of another chain and
creating two antigen binding sites (see e.g., Holliger, P., et al.
(1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et
al. (1994) Structure 2:1121-1123). Such antibody binding portions
are known in the art (Kontermann and Dubel eds., Antibody
Engineering (2001) Springer-Verlag, New York, pp. 790 (ISBN
3-540-41354-5).
[0083] The term "antibody construct" as used herein refers to a
polypeptide comprising one or more antigen binding portions of the
invention linked to a linker polypeptide or an immunoglobulin
constant domain. Linker polypeptides comprise two or more amino
acid residues joined by peptide bonds and are used to link one or
more antigen binding portions. Such linker polypeptides are well
known in the art (see e.g., Holliger, P., et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure
2:1121-1123). An immunoglobulin constant domain refers to a heavy
or light chain constant domain. Human IgG heavy chain and light
chain constant domain amino acid sequences are known in the art and
are represented in Table 1.
TABLE-US-00001 TABLE 1 Sequence Of Human IgG Heavy Chain Constant
Domain And Light Chain Constant Domain Sequence Sequence Protein
Identifier 12345678901234567890123456789012 Ig gamma-1 SEQ ID NO.:
1 ASTKGPSVFFLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK
KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK Ig gamma-1 SEQ ID NO.:
2 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDY constant region
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS mutant
LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFY
PSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK Ig Kappa constant SEQ ID NO.: 3
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY region
PREAKVQWKVDNALQSGNSQESVTEQDSKDST YSLSSTLTLSKADYEKHKVYACEVTHQGLSSP
VTKSFNRGEC Ig Lambda SEQ ID NO.: 4 QPKAAPSVTLFPPSSEELQANKATLVCLISDF
constant region YPGAVTVAWKADSSPVKAGVETTTPSKQSNNK
YAASSYLSLTPEQWKSHRSYSCQVTHEGSTVE KTVAPTECS
[0084] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecule, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. Examples of
such immunoadhesion molecules include use of the streptavidin core
region to make a tetrameric scFv molecule (Kipriyanov, S. M., et
al. (1995) Human Antibodies and Hybridomas 6:93-101) and use of a
cysteine residue, a marker peptide and a C-terminal polyhistidine
tag to make bivalent and biotinylated scFv molecules (Kipriyanov,
S. M., et al. (1994) Mol. Immunol. 31:1047-1058). Antibody
portions, such as Fab and F(ab').sub.2 fragments, can be prepared
from whole antibodies using conventional techniques, such as papain
or pepsin digestion, respectively, of whole antibodies. Moreover,
antibodies, antibody portions and immunoadhesion molecules can be
obtained using standard recombinant DNA techniques, as described
herein.
[0085] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds PGE.sub.2 is substantially free of
antibodies that specifically bind antigens other than PGE.sub.2).
An isolated antibody that specifically binds PGE.sub.2 may,
however, have cross-reactivity to other antigens, such as
Prostaglandin E1 (PGE.sub.1) molecules. Moreover, an isolated
antibody may be substantially free of other cellular material
and/or chemicals.
[0086] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0087] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell (described further in Section II C,
below), antibodies isolated from a recombinant, combinatorial human
antibody library (Hoogenboom H. R., (1997) TIB Tech. 15:62-70;
Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445;
Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145;
Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378),
antibodies isolated from an animal (e.g., a mouse) that is
transgenic for human immunoglobulin genes (see e.g., Taylor, L. D.,
et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and
Green L. L. (2002) Current Opinion in Biotechnology 13:593-597;
Little M. et al. (2000) Immunology Today 21:364-370) or antibodies
prepared, expressed, created or isolated by any other means that
involves splicing of human immunoglobulin gene sequences to other
DNA sequences. Such recombinant human antibodies have variable and
constant regions derived from human germline immunoglobulin
sequences. In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. One embodiment provides fully human antibodies capable of
binding PGE.sub.2 which can be generated using techniques well
known in the art, such as, but not limited to, using human Ig phage
libraries such as those disclosed in Jermutus et al., PCT
publication No. WO 2005/007699 A2.
[0088] The term "chimeric antibody" refers to antibodies which
comprise heavy and/or light chain variable region sequences from
one species and constant region sequences from another species,
such as antibodies having murine heavy and light chain variable
regions linked to human constant regions.
[0089] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and/or light chain variable region sequences from
one species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0090] The term "humanized antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences. In one embodiment, humanized anti-PGE.sub.2
antibodies and antigen binding portions are provided. Such
antibodies were generated by obtaining murine anti-PGE.sub.2
monoclonal antibodies using traditional hybridoma technology
followed by humanization using in vitro genetic engineering, such
as those disclosed in Kasaian et al PCT publication No. WO
2005/123126 A2.
[0091] The terms "Kabat numbering", "Kabat definitions" and "Kabat
labeling" are used interchangeably herein. These terms, which are
recognized in the art, refer to a system of numbering amino acid
residues which are more variable (i.e., hypervariable) than other
amino acid residues in the heavy and light chain variable regions
of an antibody, or an antigen binding portion thereof (Kabat et al.
(1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al.
(1991) Sequences of Proteins of Immunological Interest, Fifth
Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242). For the heavy chain variable region, the
hypervariable region ranges from amino acid positions 31 to 35 for
CDR1, amino acid positions 50 to 65 for CDR2, and amino acid
positions 95 to 102 for CDR3. For the light chain variable region,
the hypervariable region ranges from amino acid positions 24 to 34
for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid
positions 89 to 97 for CDR3.
[0092] As used herein, the terms "acceptor" and "acceptor antibody"
refer to the antibody or nucleic acid sequence providing or
encoding at least 80%, at least 85%, at least 90%, at least 95%, at
least 98% or 100% of the amino acid sequences of one or more of the
framework regions. In some embodiments, the term "acceptor" refers
to the antibody amino acid or nucleic acid sequence providing or
encoding the constant region(s). In yet another embodiment, the
term "acceptor" refers to the antibody amino acid or nucleic acid
sequence providing or encoding one or more of the framework regions
and the constant region(s). In a specific embodiment, the term
"acceptor" refers to a human antibody amino acid or nucleic acid
sequence that provides or encodes at least 80%, preferably, at
least 85%, at least 90%, at least 95%, at least 98%, or 100% of the
amino acid sequences of one or more of the framework regions. In
accordance with this embodiment, an acceptor may contain at least
1, at least 2, at least 3, at least 4, at least 5, or at least 10
amino acid residues that does (do) not occur at one or more
specific positions of a human antibody. An acceptor framework
region and/or acceptor constant region(s) may be, e.g., derived or
obtained from a germline antibody gene, a mature antibody gene, a
functional antibody (e.g., antibodies well-known in the art,
antibodies in development, or antibodies commercially
available).
[0093] As used herein, the term "CDR" refers to the complementarity
determining region within antibody variable sequences. There are
three CDRs in each of the variable regions of the heavy chain and
the light chain, which are designated CDR1, CDR2 and CDR3, for each
of the variable regions. The term "CDR set" as used herein refers
to a group of three CDRs that occur in a single variable region
capable of binding the antigen. The exact boundaries of these CDRs
have been defined differently according to different systems. The
system described by Kabat (Kabat et al., Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md. (1987) and (1991)) not only provides an unambiguous residue
numbering system applicable to any variable region of an antibody,
but also provides precise residue boundaries defining the three
CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and
coworkers (Chothia &Lesk, J. Mol. Biol. 196:901-917 (1987) and
Chothia et al., Nature 342:877-883 (1989)) found that certain
sub-portions within Kabat CDRs adopt nearly identical peptide
backbone conformations, despite having great diversity at the level
of amino acid sequence. These sub-portions were designated as L1,
L2 and L3 or H1, H2 and H3 where the "L" and the "H" designates the
light chain and the heavy chains regions, respectively. These
regions may be referred to as Chothia CDRs, which have boundaries
that overlap with Kabat CDRs. Other boundaries defining CDRs
overlapping with the Kabat CDRs have been described by Padlan
(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45
(1996)). Still other CDR boundary definitions may not strictly
follow one of the above systems, but will nonetheless overlap with
the Kabat CDRs, although they may be shortened or lengthened in
light of prediction or experimental findings that particular
residues or groups of residues or even entire CDRs do not
significantly impact antigen binding. The methods used herein may
utilize CDRs defined according to any of these systems, although
preferred embodiments use Kabat or Chothia defined CDRs.
[0094] As used herein, the term "canonical" residue refers to a
residue in a CDR or framework that defines a particular canonical
CDR structure as defined by Chothia et al., J. Mol. Biol.
196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992).
According to Chothia et al., critical portions of the CDRs of many
antibodies have nearly identical peptide backbone confirmations
despite great diversity at the level of amino acid sequence. Each
canonical structure specifies primarily a set of peptide backbone
torsion angles for a contiguous segment of amino acid residues
forming a loop.
[0095] As used herein, the terms "donor" and "donor antibody" refer
to an antibody providing one or more CDRs. In a preferred
embodiment, the donor antibody is an antibody from a species
different from the antibody from which the framework regions are
obtained or derived. In the context of a humanized antibody, the
term "donor antibody" refers to a non-human antibody providing one
or more CDRs.
[0096] As used herein, the term "framework" or "framework sequence"
refers to the remaining sequences of a variable region minus the
CDRs. Because the exact definition of a CDR sequence can be
determined by different systems, the meaning of a framework
sequence is subject to correspondingly different interpretations.
The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of light chain and CDR-H1,
CDR-H2, and CDR-H3 of heavy chain) also divide the framework
regions on the light chain and the heavy chain into four
sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 is
positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3
between FR3 and FR4. Without specifying the particular sub-regions
as FR1, FR2, FR3 or FR4, a framework region, as referred by others,
represents the combined FRs within the variable region of a single,
naturally occurring immunoglobulin chain. As used herein, a FR
represents one of the four sub-regions, and FRs represent two or
more of the four sub-regions constituting a framework region. See
Tables 5 and 6 for exemplary FR sequences.
[0097] As used herein, the term "germline antibody gene" or
"germline antibody gene fragment" refers to an immunoglobulin
sequence encoded by non-lymphoid cells that have not undergone the
maturation process that leads to genetic rearrangement and mutation
for expression of a particular immunoglobulin. (See, e.g., Shapiro
et al., Crit. Rev. Immunol. 22(3): 183-200 (2002); Marchalonis et
al., Adv Exp Med Biol. 484:13-30 (2001)). One of the advantages
provided by various embodiments of the present invention stems from
the recognition that germline antibody genes are more likely than
mature antibody genes to conserve essential amino acid sequence
structures characteristic of individuals in the species, hence less
likely to be recognized as from a foreign source when used
therapeutically in that species.
[0098] As used herein, the term "key" residues refer to certain
residues within the variable region that have more impact on the
binding specificity and/or affinity of an antibody, in particular a
humanized antibody. A key residue includes, but is not limited to,
one or more of the following: a residue that is adjacent to a CDR,
a potential glycosylation site (can be either N- or O-glycosylation
site), a rare residue, a residue capable of interacting with an
antigen, a residue capable of interacting with a CDR, a canonical
residue, a contact residue between heavy chain variable region and
light chain variable region, a residue within the Vernier zone, and
a residue in the region that overlaps between the Chothia
definition of a variable heavy chain CDR1 and the Kabat definition
of the first heavy chain framework.
[0099] As used herein, the term "humanized antibody" is an antibody
or a variant, derivative, analog or fragment thereof which
immunospecifically binds to an antigen of interest and which
comprises a framework (FR) region having substantially the amino
acid sequence of a human antibody and a complementary determining
region (CDR) having substantially the amino acid sequence of a
non-human antibody. As used herein, the term "substantially" in the
context of a CDR refers to a CDR having an amino acid sequence at
least 80%, preferably at least 85%, at least 90%, at least 95%, at
least 98% or at least 99% identical to the amino acid sequence of a
non-human antibody CDR. A humanized antibody comprises
substantially all of at least one, and typically two, variable
domains (e.g., Fab, Fab', F(ab') 2, FabC, Fv) in which all or
substantially all of the CDR regions correspond to those of a
non-human immunoglobulin (i.e., donor antibody) and all or
substantially all of the framework regions are those of a human
immunoglobulin consensus sequence. Preferably, a humanized antibody
also comprises at least a portion of an immunoglobulin constant
region (Fc), typically that of a human immunoglobulin. In some
embodiments, a humanized antibody contains both the light chain as
well as at least the variable domain of a heavy chain. The antibody
also may include the CH1, hinge, CH2, CH3, and CH4 regions of the
heavy chain. In some embodiments, a humanized antibody only
contains a humanized light chain. In some embodiments, a humanized
antibody only contains a humanized heavy chain. In specific
embodiments, a humanized antibody only contains a humanized
variable domain of a light chain and/or humanized heavy chain.
[0100] The humanized antibody can be selected from any class of
immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any
isotype, including without limitation IgG 1, IgG2, IgG3 and IgG4.
The humanized antibody may comprise sequences from more than one
class or isotype, and particular constant domains may be selected
to optimize desired effector functions using techniques well-known
in the art.
[0101] The framework and CDR regions of a humanized antibody need
not correspond precisely to the parental sequences, e.g., the donor
antibody CDR or the consensus framework may be mutagenized by
substitution, insertion and/or deletion of at least one amino acid
residue so that the CDR or framework residue at that site does not
correspond to either the donor antibody or the consensus framework.
In a preferred embodiment, such mutations, however, will not be
extensive. Usually, at least 80%, preferably at least 85%, more
preferably at least 90%, and most preferably at least 95% of the
humanized antibody residues will correspond to those of the
parental FR and CDR sequences. As used herein, the term "consensus
framework" refers to the framework region in the consensus
immunoglobulin sequence. As used herein, the term "consensus
immunoglobulin sequence" refers to the sequence formed from the
most frequently occurring amino acids (or nucleotides) in a family
of related immunoglobulin sequences (See, e.g., Winnaker, From
Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987). In a
family of immunoglobulins, each position in the consensus sequence
is occupied by the amino acid occurring most frequently at that
position in the family. If two amino acids occur equally
frequently, either can be included in the consensus sequence.
[0102] As used herein, "Vernier" zone refers to a subset of
framework residues that may adjust CDR structure and fine-tune the
fit to antigen as described by Foote and Winter (1992) J. Mol.
Biol. 224:487-499). Vernier zone residues form a layer underlying
the CDRs and may impact on the structure of CDRs and the affinity
of the antibody.
[0103] The term "multivalent binding protein" is used in this
specification to denote a binding protein comprising two or more
antigen binding sites. The multivalent binding protein is
preferably engineered to have the three or more antigen binding
sites, and is generally not a naturally occurring antibody. The
term "multispecific binding protein" refers to a binding protein
capable of binding two or more related or unrelated targets. Dual
variable domain (DVD) binding proteins as used herein, are binding
proteins that comprise two or more antigen binding sites and are
tetravalent or multivalent binding proteins. Such DVD binding
proteins may be monospecific, i.e., capable of binding one antigen
or multispecific, i.e., capable of binding two or more antigens.
DVD binding proteins comprising two heavy chain DVD polypeptides
and two light chain DVD polypeptides are refered to a DVD-Ig.TM..
Each half of a DVD-Ig comprises a heavy chain DVD polypeptide, and
a light chain DVD polypeptide, and two antigen binding sites. Each
binding site comprises a heavy chain variable domain and a light
chain variable domain with a total of 6 CDRs involved in antigen
binding per antigen binding site.
[0104] As used herein, the term "neutralizing" refers to
neutralization of biological activity of a cytokine or a lipid
metabolite when a binding protein specifically binds the cytokine
or a lipid metabolite. Preferably a neutralizing binding protein is
a neutralizing antibody whose binding to PGE.sub.2 results in
inhibition of a biological activity of PGE.sub.2. Preferably the
neutralizing binding protein binds PGE.sub.2 and reduces a
biological activity of PGE.sub.2 by at least about 10%, 20%, 40%,
60%, 80%, 85% or more Inhibition of a biological activity of
PGE.sub.2 by a neutralizing binding protein can be assessed by
measuring one or more indicators of PGE.sub.2 biological activity
well known in the art. For example inhibition of PGE.sub.2 induced
calcium influx by EP4 assay using HEK293 cells over-expressing EP4
receptor (see Example 1.1.C 1).
[0105] The term "activity" includes activities such as the binding
specificity/affinity of an antibody for an antigen, for example, an
anti-PGE.sub.2 antibody that binds to an PGE.sub.2 antigen and/or
the neutralizing potency of an antibody, for example, an
anti-PGE.sub.2 antibody whose binding to PGE.sub.2 inhibits the
biological activity of PGE.sub.2, e.g., for example inhibition of
PGE.sub.2 induced calcium influx by EP4 assay using HEK293 cells
over-expressing EP4 receptor (see Example 1.1.C 1).
[0106] The term "epitope" includes any polypeptide determinant
capable of specific binding to an immunoglobulin or T-cell
receptor. In certain embodiments, epitope determinants include
chemically active surface groupings of molecules such as amino
acids, sugar side chains, phosphoryl, or sulfonyl, and, in certain
embodiments, may have specific three-dimensional structural
characteristics, and/or specific charge characteristics. An epitope
is a region of an antigen that is bound by an antibody. In certain
embodiments, an antibody specifically binds an antigen when it
preferentially recognizes its target antigen in a complex mixture
of proteins and/or macromolecules.
[0107] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example, using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Jonsson, U., et
al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991)
Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol.
Recognit. 8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem.
198:268-277.
[0108] The term "k.sub.on", as used herein, is intended to refer to
the on rate constant for association of an antibody to an antigen
to form an antibody/antigen complex as is known in the art.
[0109] The term "k.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from an
antibody/antigen complex as is known in the art.
[0110] The term "K.sub.D", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction as is known in the art.
[0111] The term "labeled binding protein" as used herein, refers to
a protein with a label incorporated that provides for the
identification of the binding protein. Preferably, the label is a
detectable marker, e.g., incorporation of a radiolabeled amino acid
or attachment to a polypeptide of biotinyl moieties that can be
detected by marked avidin (e.g., streptavidin containing a
fluorescent marker or enzymatic activity that can be detected by
optical or colorimetric methods). Examples of labels for
polypeptides include, but are not limited to, the following:
radioisotopes or radionuclides (e.g., .sup.3H, .sup.14C, 35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, or .sup.153Sm); fluorescent labels (e.g., FITC,
rhodamine, lanthanide phosphors); enzymatic labels (e.g.,
horseradish peroxidase, luciferase, alkaline phosphatase);
chemiluminescent markers; biotinyl groups; predetermined
polypeptide epitopes recognized by a secondary reporter (e.g.,
leucine zipper pair sequences, binding sites for secondary
antibodies, metal binding domains, epitope tags); and magnetic
agents, such as gadolinium chelates.
[0112] The term "antibody conjugate" refers to a binding protein,
such as an antibody, chemically linked to a second chemical moiety,
such as a therapeutic or cytotoxic agent. The term "agent" is used
herein to denote a chemical compound, a mixture of chemical
compounds, a biological macromolecule, or an extract made from
biological materials. Preferably the therapeutic or cytotoxic
agents include, but are not limited to, pertussis toxin, taxol,
cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,
etoposide, tenoposide, vincristine, vinblastine, colchicin,
doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin and
analogs or homologs thereof.
[0113] The terms "crystal", and "crystallized" as used herein,
refer to an antibody, or antigen binding portion thereof, that
exists in the form of a crystal. Crystals are one form of the solid
state of matter, which is distinct from other forms such as the
amorphous solid state or the liquid crystalline state. Crystals are
composed of regular, repeating, three-dimensional arrays of atoms,
ions, molecules (e.g., proteins such as antibodies), or molecular
assemblies (e.g., antigen/antibody complexes). These
three-dimensional arrays are arranged according to specific
mathematical relationships. The fundamental unit, or building
block, that is repeated in a crystal is called the asymmetric unit.
Repetition of the asymmetric unit in an arrangement that conforms
to a given, well-defined crystallographic symmetry provides the
"unit cell" of the crystal. Repetition of the unit cell by regular
translations in all three dimensions provides the crystal. See
Giege, R. and Ducruix, A. Barrett, Crystallization of Nucleic Acids
and Proteins, a Practical Approach, 2nd ed., pp. 20 1-16, Oxford
University Press, New York, N.Y., (1999).
[0114] One embodiment provides a composition for the release of a
binding protein wherein the composition comprises a formulation
which in turn comprises a crystallized binding protein,
crystallized antibody construct or crystallized antibody conjugate
as disclosed above and an ingredient; and at least one polymeric
carrier. Preferably the polymeric carrier is a polymer selected
from one or more of the group consisting of: poly (acrylic acid),
poly (cyanoacrylates), poly (amino acids), poly (anhydrides), poly
(depsipeptide), poly (esters), poly (lactic acid), poly
(lactic-co-glycolic acid) or PLGA, poly (b-hydroxybutryate), poly
(caprolactone), poly (dioxanone); poly (ethylene glycol), poly
((hydroxypropyl) methacrylamide, poly[(organo)phosphazene], poly
(ortho esters), poly (vinyl alcohol), poly (vinylpyrrolidone),
maleic anhydride-alkyl vinyl ether copolymers, pluronic polyols,
albumin, alginate, cellulose and cellulose derivatives, collagen,
fibrin, gelatin, hyaluronic acid, oligosaccharides,
glycaminoglycans, sulfated polyeaccharides, blends and copolymers
thereof. Preferably the ingredient is selected from the group
consisting of albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-.beta.-cyclodextrin, methoxypolyethylene glycol and
polyethylene glycol. Another embodiment provides a method for
treating a mammal comprising the step of administering to the
mammal an effective amount of the composition disclosed above.
[0115] The term "polynucleotide" as referred to herein means a
polymeric form of two or more nucleotides, either ribonucleotides
or deoxynucleotides or a modified form of either type of
nucleotide. The term includes single and double stranded forms of
DNA but preferably is double-stranded DNA.
[0116] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide (e.g., of genomic, cDNA, or synthetic origin, or
some combination thereof) that, by virtue of its origin, the
"isolated polynucleotide" is not associated with all or a portion
of a polynucleotide with which the "isolated polynucleotide" is
found in nature; is operably linked to a polynucleotide that it is
not linked to in nature, or does not occur in nature as part of a
larger sequence.
[0117] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0118] The term "operably linked" refers to a juxtaposition wherein
the components described are in a relationship permitting them to
function in their intended manner. A control sequence "operably
linked" to a coding sequence is ligated in such a way that
expression of the coding sequence is achieved under conditions
compatible with the control sequences. "Operably linked" sequences
include both expression control sequences that are contiguous with
the gene of interest and expression control sequences that act in
trans or at a distance to control the gene of interest. The term
"expression control sequence" as used herein refers to
polynucleotide sequences that are necessary to effect the
expression and processing of coding sequences to which they are
ligated. Expression control sequences include appropriate
transcription initiation, termination, promoter and enhancer
sequences; efficient RNA processing signals such as splicing and
polyadenylation signals; sequences that stabilize cytoplasmic mRNA;
sequences that enhance translation efficiency (i.e., Kozak
consensus sequence); sequences that enhance protein stability; and
when desired, sequences that enhance protein secretion. The nature
of such control sequences differs depending upon the host organism;
in prokaryotes, such control sequences generally include promoter,
ribosomal binding site, and transcription termination sequence; in
eukaryotes, generally, such control sequences include promoters and
transcription termination sequence. The term "control sequences" is
intended to include components whose presence is essential for
expression and processing, and can also include additional
components whose presence is advantageous, for example, leader
sequences and fusion partner sequences. Protein constructs of the
present invention may be expressed, and purified using expression
vectors and host cells known in the art, including expression
cassettes, vectors, recombinant host cells, and methods for the
recombinant expression and proteolytic processing of recombinant
polyproteins and pre-proteins from a single open reading frame
(e.g., WO 2007/014162).
[0119] "Transformation", as defined herein, refers to any process
by which exogenous DNA enters a host cell. Transformation may occur
under natural or artificial conditions using various methods well
known in the art. Transformation may rely on any known method for
the insertion of foreign nucleic acid sequences into a prokaryotic
or eukaryotic host cell. The method is selected based on the host
cell being transformed and may include, but is not limited to,
viral infection, electroporation, lipofection, and particle
bombardment. Such "transformed" cells include stably transformed
cells in which the inserted DNA is capable of replication either as
an autonomously replicating plasmid or as part of the host
chromosome. They also include cells that transiently express the
inserted DNA or RNA for limited periods of time.
[0120] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which exogenous
DNA has been introduced. It should be understood that such terms
are intended to refer not only to the particular subject cell, but,
to the progeny of such a cell. Because certain modifications may
occur in succeeding generations due to either mutation or
environmental influences, such progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term "host cell" as used herein. Preferably host cells
include prokaryotic cells, eukaryotic cells, insect cells, or cells
selected from any of the Kingdoms of life. Preferred eukaryotic
cells include protist, fungal, plant and animal cells. Most
preferably host cells include but are not limited to the
prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293
and COS; the insect cell line Sf9; and the fungal cell S.
cerevisiae.
[0121] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The foregoing techniques and procedures
may be generally performed according to conventional methods well
known in the art and as described in various general and more
specific references that are cited and discussed throughout the
present specification. See e.g., Sambrook et al. Molecular Cloning:
A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (1989)).
[0122] "Transgenic organism", as known in the art and as used
herein, refers to an organism having cells that contain a
transgene, wherein the transgene introduced into the organism (or
an ancestor of the organism) expresses a polypeptide not naturally
expressed in the organism. A "transgene" is a DNA construct, which
is stably and operably integrated into the genome of a cell from
which a transgenic organism develops, directing the expression of
an encoded gene product in one or more cell types or tissues of the
transgenic organism.
[0123] The term "regulate" and "modulate" are used interchangeably,
and, as used herein, refers to a change or an alteration in the
activity of a molecule of interest (e.g., the biological activity
of PGE.sub.2). Modulation may be an increase or a decrease in the
magnitude of a certain activity or function of the molecule of
interest. Exemplary activities and functions of a molecule include,
but are not limited to, binding characteristics, enzymatic
activity, cell receptor activation, and signal transduction.
[0124] Correspondingly, the term "modulator," as used herein, is a
compound capable of changing or altering an activity or function of
a molecule of interest (e.g., the biological activity PGE.sub.2).
For example, a modulator may cause an increase or decrease in the
magnitude of a certain activity or function of a molecule compared
to the magnitude of the activity or function observed in the
absence of the modulator. In certain embodiments, a modulator is an
inhibitor, which decreases the magnitude of at least one activity
or function of a molecule. Exemplary inhibitors include, but are
not limited to, proteins, peptides, antibodies, peptibodies,
carbohydrates or small organic molecules. Peptibodies are
described, e.g., in WO01/83525.
[0125] The term "agonist", as used herein, refers to a modulator
that, when contacted with a molecule of interest, causes an
increase in the magnitude of a certain activity or function of the
molecule compared to the magnitude of the activity or function
observed in the absence of the agonist. Particular agonists of
interest may include, but are not limited to, PGE.sub.2 or
polypeptides, nucleic acids, carbohydrates, or any other molecules
that bind to PGE.sub.2.
[0126] The term "antagonist" or "inhibitor", as used herein, refer
to a modulator that, when contacted with a molecule of interest
causes a decrease in the magnitude of a certain activity or
function of the molecule compared to the magnitude of the activity
or function observed in the absence of the antagonist. Particular
antagonists of interest include those that block or modulate the
biological or immunological activity of PGE.sub.2. Antagonists and
inhibitors of PGE.sub.2 may include, but are not limited to,
proteins, nucleic acids, carbohydrates, or any other molecules,
which bind to PGE.sub.2.
[0127] The term "inhibit binding to the receptor" refers to the
ability of the binding protein to prevent the binding of PGE.sub.2
to one or more of its receptors. Such inhibition of binding to the
receptor would result in diminishing or abolishing the biological
activity mediated by binding of PGE.sub.2 to its receptor or
receptors.
[0128] As used herein, the term "effective amount" refers to the
amount of a therapy which is sufficient to reduce or ameliorate the
severity and/or duration of a disorder or one or more symptoms
thereof, prevent the advancement of a disorder, cause regression of
a disorder, prevent the recurrence, development, onset or
progression of one or more symptoms associated with a disorder,
detect a disorder, or enhance or improve the prophylactic or
therapeutic effect(s) of another therapy (e.g., prophylactic or
therapeutic agent).
[0129] The term "sample", as used herein, is used in its broadest
sense. A "biological sample", as used herein, includes, but is not
limited to, any quantity of a substance from a living thing or
formerly living thing. Such living things include, but are not
limited to, humans, mice, rats, monkeys, dogs, rabbits and other
animals. Such substances include, but are not limited to, blood,
serum, urine, synovial fluid, cells, organs, tissues, bone marrow,
lymph nodes, and spleen.
[0130] In a preferred embodiment the binding protein is a CDR
grafted antibody or antigen binding portion thereof capable of
binding PGE.sub.2. Preferably the CDR grafted antibody or antigen
binding portion thereof comprise one or more CDRs from 2B5-7.0, or
2B5-8.0 or 2B5-9.0 disclosed above. Preferably the CDR grafted
antibody or antigen binding portion thereof comprises a human
acceptor framework. More preferably the human acceptor framework is
any one of the human acceptor frameworks having >60% homology to
mouse antibody frameworks of 2B5-7.0, or 2B5-8.0 or 2B5-9.0.
[0131] In a preferred embodiment the binding protein is a humanized
antibody or antigen binding portion thereof capable of binding
PGE.sub.2. Preferably the humanized antibody or antigen binding
portion thereof comprises one or more CDRs disclosed above
incorporated into a human antibody variable domain of a human
acceptor framework. Preferably the human antibody variable domain
is a consensus human variable domain. More preferably the human
acceptor framework comprises at least one Framework Region amino
acid substitution at a key residue, wherein the key residue is
selected from the group consisting of a residue adjacent to a CDR;
a glycosylation site residue; a rare residue; a residue capable of
interacting with PGE.sub.2; a residue capable of interacting with a
CDR; a canonical residue; a contact residue between heavy chain
variable region and light chain variable region; a residue within a
Vernier zone; and a residue in a region that overlaps between a
Chothia-defined variable heavy chain CDR1 and a Kabat-defined first
heavy chain framework. Preferably the human acceptor framework
human acceptor framework comprises at least one Framework Region
amino acid substitution, wherein the amino acid sequence of the
framework is at least 65% identical to the sequence of the human
acceptor framework and comprises at least 70 amino acid residues
identical to the human acceptor framework.
[0132] In an embodiment, the humanized antibody, or antigen binding
portion thereof, comprises three or more CDRs disclosed above. In a
certain embodiment, the humanized antibody, or antigen binding
portion, thereof comprises six CDRs disclosed above.
[0133] One embodiment of the invention provides an antibody
construct comprising any one of the binding proteins disclosed
above and a linker polypeptide or an immunoglobulin. In a preferred
embodiment the antibody construct is selected from the group
consisting of an immunoglobulin molecule, a monoclonal antibody, a
chimeric antibody, a CDR-grafted antibody, a humanized antibody, a
Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a
single domain antibody, a diabody, a multispecific antibody, a dual
specific antibody, a DVD-Ig.TM. and a bispecific antibody. In a
preferred embodiment the antibody construct comprises a heavy chain
immunoglobulin constant domain selected from the group consisting
of a human IgM constant domain, a human IgG1 constant domain, a
human IgG2 constant domain, a human IgG3 constant domain, a human
IgG4 constant domain, a human IgE constant domain, and a human IgA
constant domain. In another embodiment the invention provides an
antibody conjugate comprising an the antibody construct disclosed
above and an agent an agent selected from the group consisting of;
an immunoadhension molecule, an imaging agent, a therapeutic agent,
and a cytotoxic agent. In a preferred embodiment the imaging agent
selected from the group consisting of a radiolabel, an enzyme, a
fluorescent label, a luminescent label, a bioluminescent label, a
magnetic label, and biotin. More preferably the imaging agent is a
radiolabel selected from the group consisting of: .sup.3H,
.sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In, .sup.125I,
.sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm. In a preferred
embodiment the therapeutic or cytotoxic agent is selected from the
group consisting of an anti-metabolite, an alkylating agent, an
antibiotic, a growth factor, a cytokine, an anti-angiogenic agent,
an anti-mitotic agent, an anthracycline, toxin, and an apoptotic
agent.
[0134] In another embodiment the antibody construct is
glycosylated. Preferably the glycosylation is a human glycosylation
pattern.
[0135] In another embodiment, the binding protein, antibody
construct or antibody conjugate disclosed above is a crystal.
Preferably, the crystal is a carrier-free pharmaceutical controlled
release crystal. In a preferred embodiment the crystallized binding
protein, crystallized antibody construct or crystallized antibody
conjugate has a greater half life in vivo than its soluble
counterpart. In another preferred embodiment the crystallized
binding protein, crystallized antibody construct or crystallized
antibody conjugate retains biological activity after
crystallization.
[0136] One aspect of the invention pertains to an isolated nucleic
acid encoding any one of the binding proteins, antibody constructs
or antibody conjugates disclosed above. A further embodiment
provides a vector comprising the isolated nucleic acid disclosed
above wherein the vector is selected from the group consisting of
pcDNA; pTT (Durocher et al., Nucleic Acids Research 2002, Vol 30,
No. 2); pTT3 (pTT with additional multiple cloning site; pEFBOS
(Mizushima, S. and Nagata, S. (1990) Nucleic acids Research Vol 18,
No. 17); pBV; pJV; pA2; and pBJ.
[0137] In another aspect a host cell is transformed with the vector
disclosed above. Preferably the host cell is a prokaryotic cell.
More preferably the host cell is E. coli. In another embodiment the
host cell is a eukaryotic cell. Preferably the eukaryotic cell is
selected from the group consisting of protist cell, animal cell,
plant cell and fungal cell. More preferably the host cell is a
mammalian cell including, but not limited to, CHO, HEK293, and COS;
or a fungal cell such as Saccharomyces cerevisiae; or an insect
cell such as Sf9.
[0138] Another aspect of the invention provides a method of
producing a binding protein that binds PGE.sub.2, comprising
culturing any one of the host cells disclosed above in a culture
medium under conditions sufficient to produce a binding protein
that binds PGE.sub.2. Another embodiment provides a binding protein
produced according to the method disclosed above.
[0139] The invention also provides a pharmaceutical composition
comprising a binding protein, antibody construct or antibody
conjugate as disclosed above and a pharmaceutically acceptable
carrier. In a further embodiment the pharmaceutical composition
comprises at least one additional therapeutic agent for treating a
disorder in which PGE.sub.2 activity is detrimental. Preferably the
additional agent is selected from the group consisting of a
therapeutic agent, an imaging agent, a cytotoxic agent, an
angiogenesis inhibitor (including but not limited to anti-VEGF
antibodies or VEGF-trap); a kinase inhibitor (including but not
limited to KDR and TIE-2 inhibitors); a co-stimulation molecule
blocker (including but not limited to anti-B7.1, anti-B7.2,
CTLA4-Ig, anti-CD20); an adhesion molecule blocker (including but
not limited to anti-LFA-1 Abs, anti-E/L selectin Abs, small
molecule inhibitors); an anti-cytokine antibody or functional
fragment thereof (including but not limited to anti-IL-18,
anti-TNF, anti-IL-6/cytokine receptor antibody); methotrexate;
cyclosporin; rapamycin; FK506; a detectable label or reporter; a
TNF antagonist; an antirheumatic; a muscle relaxant, a narcotic, a
non-steroid anti-inflammatory drug (NSAID), an analgesic, an
anesthetic, a sedative, a local anesthetic, a neuromuscular
blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an
anabolic steroid, an erythropoietin, an immunization, an
immunoglobulin, an immunosuppressive, a growth hormone, a hormone
replacement drug, a radiopharmaceutical, an antidepressant, an
antipsychotic, a stimulant, an asthma medication, a beta agonist,
an inhaled steroid, an epinephrine or analog, a cytokine, and a
cytokine antagonist.
[0140] In another aspect, the invention provides a method for
inhibiting PGE.sub.2 .quadrature.activity comprising contacting
PGE.sub.2 with a binding protein disclosed above such that
PGE.sub.2 activity is inhibited. In a related aspect the invention
provides a method for inhibiting PGE.sub.2 activity in a human
subject suffering from a disorder in which PGE.sub.2 activity is
detrimental, comprising administering to the human subject a
binding protein disclosed above such that PGE.sub.2 activity in the
human subject is inhibited and treatment is achieved.
[0141] In another aspect, the invention provides a method of
treating (e.g., curing, suppressing, ameliorating, delaying or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a PGE.sub.2-associated disorder, in a subject. The
method includes: administering to the subject a PGE.sub.2 binding
agent (particularly an antagonist), e.g., an anti-PGE.sub.2
antibody or fragment thereof as described herein, in an amount
sufficient to treat or prevent the PGE.sub.2-associated disorder.
The PGE.sub.2 antagonist, e.g., the anti-PGE.sub.2 antibody or
fragment thereof, can be administered to the subject, alone or in
combination with other therapeutic modalities as described
herein.
[0142] In one embodiment, the invention providing methods and
compositions for treating (e.g., reducing, ameliorating) or
preventing one or more symptoms in a mammalian subject, e.g., a
human suffering from one or more PGE.sub.2-associated disorders,
including, e.g., autoimmune and inflammatory diseases and tumors in
which excessive PGE.sub.2 synthesis has been implicated. Such
disorders include: (a) rheumatoid and allergic arthritis; (b)
certain illnesses induced by viruses, such as Guillain Bane
syndrome, infectious mononucleosis, other viral lymphadenopathies
and infections with herpes virus; (c) multiple sclerosis and other
demyelinating diseases; (d) hematological disorders, such as
hemolytic anemias and thrombocytopenias; (e) endocrinologic
disorders, such as diabetes mellitus, Addison's disease, idiopathic
hypoparathyroidism and chronic lymphocytic thyroiditis; (f)
collagen disorders, such as systemic lupus erythematosus; and (g)
disorders of reproduction such as amenorrhoea, infertility,
recurrent abortions and eclampsia; and (h) tumors such as headneck
tumor, lung cancer, gastric cancer, prostate cancer, pancreatic
cancer etc., and (i) gastrointestinal organ disorders (e.g.,
inflammatory bowel diseases (IBD), such as ulcerative colitis
and/or Crohn's disease); and (j) pain disorders such as pain
related with osteoarthritis and other disorders; and (k) ocular
disorders such as age-related mascular degeneration (AMD).
Accordingly, the disclosure includes the use of a PGE.sub.2 binding
agent (such as an anti-PGE.sub.2 antibody or fragment thereof) for
a treatment and the use of a PGE.sub.2 binding agent (such as an
anti-PGE.sub.2 antibody or fragment thereof) for preparing a
medicament for a treatment.
[0143] The method comprises administering to the subject a
PGE.sub.2 antagonist, e.g., a PGE.sub.2 antibody or a fragment
thereof, in an amount sufficient to treat (e.g., reduce,
ameliorate) or prevent one or more symptoms. The PGE.sub.2 antibody
can be administered therapeutically or prophylactically, or both.
The PGE.sub.2 antagonist, e.g., the anti-PGE.sub.2 antibody, or
fragment thereof, can be administered to the subject, alone or in
combination with other therapeutic modalities as described herein.
Preferably, the subject is a mammal, e.g., a human suffering from a
PGE.sub.2-associated disorder as described herein.
[0144] In another aspect, this application provides a method for
detecting the presence of PGE.sub.2 in a sample in vitro (e.g., a
biological sample, such as serum, plasma, tissue, biopsy). The
subject method can be used to diagnose a disorder, e.g., an immune
cell-associated disorder. The method includes: (i) contacting the
sample or a control sample with the anti-PGE.sub.2 antibody or
fragment thereof as described herein; and (ii) detecting formation
of a complex between the anti-PGE.sub.2 antibody or fragment
thereof, and the sample or the control sample, wherein a
statistically significant change in the formation of the complex in
the sample relative to the control sample is indicative of the
presence of the PGE.sub.2 in the sample.
[0145] In another aspect the invention provides a method of
treating a patient suffering from a disorder in which PGE.sub.2 is
detrimental comprising the step of administering any one of the
binding proteins disclosed above before, concurrent, or after the
administration of a second agent, as discussed above. In a
preferred embodiment the additional therapeutic agent that can be
coadministered and/or coformulated with one or more PGE.sub.2
antagonists, (e.g., anti-PGE.sub.2 antibodies or fragments thereof)
include, but are not limited to, one or more of MTX; oral steroids;
metalloproteinase inhibitors, sulfasalazine, azathioprine,
6-mercaptopurines, angiotensin converting enzyme inhibitors,
soluble cytokine receptors, soluble p55 TNF receptor, soluble p75
TNF receptor, sIL-1RI, sIL-1RII, sIL-6R, antiinflammatory
cytokines, IL-4, IL-10, IL-11, and TGF .beta..
[0146] In a preferred embodiment the pharmaceutical compositions
disclosed above are administered to the subject by at least one
mode selected from parenteral, subcutaneous, intramuscular,
intravenous, intrarticular, intrabronchial, intraabdominal,
intracapsular, intracartilaginous, intracavitary, intracelial,
intracerebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, and transdermal.
[0147] One aspect of the invention provides at least one PGE.sub.2
anti-idiotype antibody to at least one PGE.sub.2 binding protein of
the present invention. The anti-idiotype antibody includes any
protein or peptide containing molecule that comprises at least a
portion of an immunoglobulin molecule such as, but not limited to,
at least one complementarily determining region (CDR) of a heavy or
light chain or a ligand binding portion thereof, a heavy chain or
light chain variable region, a heavy chain or light chain constant
region, a framework region, or; any portion thereof, that can be
incorporated into a binding protein of the present invention.
I. Antibodies that Bind Prostaglandin E2
[0148] In invention provides murine monoclonal antibodies, or
antigen-binding portions thereof, that bind to PGE.sub.2 with high
affinity, a slow off rate and high neutralizing capacity. The
invention also provides chimeric antibodies that bind PGE.sub.2.
The invention also provides humanized antibodies, or
antigen-binding portions thereof, that bind PGE.sub.2. Preferably,
the antibodies, or portions thereof, are isolated antibodies.
Preferably, the antibodies of the invention are neutralizing human
anti-PGE.sub.2 and/or human anti-PGE.sub.2 antibodies.
A. Method Of Making Anti Prostaglandin E.sub.2 Antibodies
[0149] The antibodies and antibody fragments of the invention may
be generated by any art-known method, such as, for example,
hybridoma, microbe (e.g., phage, bacteria, yeast), recombinant,
ribosome, mRNA, and DNA displays, or a combination thereof. Many
methods can be used to manipulate antibodies to modify antibody
properties including humanization, affinity maturation, antibody
isotype switching, physiochemical property and pharmacokinetic
profile improvement, etc., which are well known in the art.
1. Anti-Prostaglandin E.sub.2 Monoclonal Antibodies Using Hybridoma
Technology
[0150] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, phage, and yeast display technologies, or a
combination thereof. Monoclonal antibodies can be produced, for
example, using hybridoma techniques including those known in the
art and taught, for example, in Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell
Hybridomas 563-681 (Elsevier, N.Y., 1981). The term "monoclonal
antibody" as used herein is not limited to antibodies produced
through hybridoma technology. The term "monoclonal antibody" refers
to an antibody that is derived from a single clone, including any
eukaryotic, prokaryotic, or phage clone, and not the method by
which it is produced.
[0151] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In one embodiment, the present invention provides methods of
generating monoclonal antibodies as well as antibodies produced by
the method comprising culturing a hybridoma cell secreting an
antibody of the invention wherein, preferably, the hybridoma is
generated by fusing splenocytes isolated from a mouse immunized
with an antigen of the invention with myeloma cells and then
screening the hybridomas resulting from the fusion for hybridoma
clones that secrete an antibody able to bind a polypeptide of the
invention (See Example 1.2). Briefly, mice can be immunized with a
carrier protein conjugated PGE.sub.2 known as hapten-carrier
protein conjugates. Here, the hapten is PGE.sub.2 and carrier
protein can be any of bovine thyroglobulins, keyhole limpet
hemocyanin, bovine serum albumin, ovalbumin etc. In a preferred
embodiment, the PGE.sub.2-thyroglobulin conjugate is administered
with an adjuvant to stimulate the immune response. Such adjuvants
include complete or incomplete Freund's adjuvant, RIBI (muramyl
dipeptides) or ISCOM (immunostimulating complexes). Such adjuvants
may protect the polypeptide from rapid dispersal by sequestering it
in a local deposit, or they may contain substances that stimulate
the host to secrete factors that are chemotactic for macrophages
and other components of the immune system. Preferably, if a
polypeptide is being administered, the immunization schedule will
involve two or more administrations of the polypeptide, spread out
over several weeks.
[0152] After immunization of an animal with a
PGE.sub.2-thyroglobulin conjugate, antibodies and/or
antibody-producing cells may be obtained from the animal. An
anti-PGE.sub.2 antibody-containing serum is obtained from the
animal by bleeding or sacrificing the animal. The serum may be used
as it is obtained from the animal, an immunoglobulin fraction may
be obtained from the serum, or the anti-PGE.sub.2 antibodies may be
purified from the serum. Serum or immunoglobulins obtained in this
manner are polyclonal, thus having a heterogeneous array of
properties.
[0153] Once an immune response is detected, e.g., antibodies
specific for the antigen PGE.sub.2 are detected in the mouse serum,
the mouse spleen is harvested and splenocytes isolated. The
splenocytes are then fused by well-known techniques to any suitable
myeloma cells, for example cells from cell line SP20 available from
the ATCC. Hybridomas are selected and cloned by limited dilution.
The hybridoma clones are then assayed by methods known in the art
for cells that secrete antibodies capable of binding PGE.sub.2.
Ascites fluid, which generally contains high levels of antibodies,
can be generated by immunizing mice with positive hybridoma
clones.
[0154] In another embodiment, antibody-producing immortalized
hybridomas may be prepared from the immunized animal. After
immunization, the animal is sacrificed and the splenic B cells are
fused to immortalized myeloma cells as is well known in the art.
See, e.g., Harlow and Lane, supra. In a preferred embodiment, the
myeloma cells do not secrete immunoglobulin polypeptides (a
non-secretory cell line). After fusion and antibiotic selection,
the hybridomas are screened using PGE.sub.2, or a portion thereof,
or a cell expressing PGE.sub.2. In a preferred embodiment, the
initial screening is performed using an enzyme-linked immunoassay
(ELISA) or a radioimmunoassay (RIA), preferably an ELISA. An
example of ELISA screening is provided in WO 00/37504.
[0155] Anti-PGE.sub.2 antibody-producing hybridomas are selected,
cloned and further screened for desirable characteristics,
including robust hybridoma growth, high antibody production and
desirable antibody characteristics, as discussed further below.
Hybridomas may be cultured and expanded in vivo in syngeneic
animals, in animals that lack an immune system, e.g., nude mice, or
in cell culture in vitro. Methods of selecting, cloning and
expanding hybridomas are well known to those of ordinary skill in
the art.
[0156] In a preferred embodiment, the hybridomas are mouse
hybridomas, as described above. In another preferred embodiment,
the hybridomas are produced in a non-human, non-mouse species such
as rats, sheep, pigs, goats, cattle or horses. In another
embodiment, the hybridomas are human hybridomas, in which a human
non-secretory myeloma is fused with a human cell expressing an
anti-PGE.sub.2 antibody.
[0157] Antibody fragments that recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab')2
fragments of the invention may be produced by proteolytic cleavage
of immunoglobulin molecules, using enzymes such as papain (to
produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
F(ab')2 fragments contain the variable region, the light chain
constant region and the CH1 domain of the heavy chain.
2. Anti-Prostaglandin E.sub.2 Monoclonal Antibodies Using SLAM
[0158] In another aspect of the invention, recombinant antibodies
are generated from single, isolated lymphocytes using a procedure
referred to in the art as the selected lymphocyte antibody method
(SLAM), as described in U.S. Pat. No. 5,627,052, PCT Publication WO
92/02551 and Babcock, J. S. et al. (1996) Proc. Natl. Acad. Sci.
USA 93:7843-7848. In this method, single cells secreting antibodies
of interest, e.g., lymphocytes derived from any one of the
immunized animals described in Section 1, are screened using an
antigen-specific hemolytic plaque assay, wherein the antigen
PGE.sub.2, a subunit of PGE.sub.2, or a fragment thereof, is
coupled to sheep red blood cells using a linker, such as biotin,
and used to identify single cells that secrete antibodies with
specificity for PGE.sub.2. Following identification of
antibody-secreting cells of interest, heavy- and light-chain
variable region cDNAs are rescued from the cells by reverse
transcriptase-PCR and these variable regions can then be expressed,
in the context of appropriate immunoglobulin constant regions
(e.g., human constant regions), in mammalian host cells, such as
COS, HEK293, or CHO cells. The host cells transfected with the
amplified immunoglobulin sequences, derived from in vivo selected
lymphocytes, can then undergo further analysis and selection in
vitro, for example by panning the transfected cells to isolate
cells expressing antibodies to PGE.sub.2. The amplified
immunoglobulin sequences further can be manipulated in vitro, such
as by in vitro affinity maturation methods such as those described
in PCT Publication WO 97/29131 and PCT Publication WO 00/56772.
3. Anti-Prostaglandin E.sub.2 Monoclonal Antibodies Using
Transgenic Animals
[0159] In another embodiment of the instant invention, antibodies
are produced by immunizing a non-human animal comprising some, or
all, of the human immunoglobulin locus with a PGE.sub.2-carrier
protein conjugate. In a preferred embodiment, the non-human animal
is a XENOMOUSE.TM. transgenic mouse, an engineered mouse strain
that comprises large fragments of the human immunoglobulin loci and
is deficient in mouse antibody production. See, e.g., Green et al.
Nature Genetics 7:13-21 (1994) and U.S. Pat. Nos. 5,916,771,
5,939,598, 5,985,615, 5,998,209, 6,075,181, 6,091,001, 6,114,598
and 6,130,364. See also WO 91/10741, published Jul. 25, 1991, WO
94/02602, published Feb. 3, 1994, WO 96/34096 and WO 96/33735, both
published Oct. 31, 1996, WO 98/16654, published Apr. 23, 1998, WO
98/24893, published Jun. 11, 1998, WO 98/50433, published Nov. 12,
1998, WO 99/45031, published Sep. 10, 1999, WO 99/53049, published
Oct. 21, 1999, WO 00 09560, published Feb. 24, 2000 and WO
00/037504, published Jun. 29, 2000. The XENOMOUSE.TM. transgenic
mouse produces an adult-like human repertoire of fully human
antibodies, and generates antigen-specific human Mabs. The
XENOMOUSE.TM. transgenic mouse contains approximately 80% of the
human antibody repertoire through introduction of megabase sized,
germline configuration YAC fragments of the human heavy chain loci
and x light chain loci. See Mendez et al., Nature Genetics
15:146-156 (1997), Green and Jakobovits J. Exp. Med. 188:483-495
(1998).
4. Anti-Prostaglandin E.sub.2 Monoclonal Antibodies Using
Recombinant Antibody Libraries
[0160] In vitro methods also can be used to make the antibodies of
the invention, wherein an antibody library is screened to identify
an antibody having the desired binding specificity. Methods for
such screening of recombinant antibody libraries are well known in
the art and include methods described in, for example, Ladner et
al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO
92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et
al. PCT Publication No. WO 92/20791; Markland et al. PCT
Publication No. WO 92/15679; Breitling et al. PCT Publication No.
WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047;
Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991)
Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod
Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;
McCafferty et al., Nature (1990) 348:552-554; Griffiths et al.
(1993) EMBO J 12:725-734; Hawkins et al. (1992) J Mol Biol
226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.
(1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio/Technology
9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137;
and Barbas et al. (1991) PNAS 88:7978-7982, US patent application
publication 20030186374, and PCT Publication No. WO 97/29131.
[0161] The recombinant antibody library may be from a subject
immunized with PGE.sub.2-carrier protein conjugate. Alternatively,
the recombinant antibody library may be from a naive subject, i.e.,
one who has not been immunized with PGE.sub.2-carrier protein
conjugate, such as a human antibody library from a human subject
who has not been immunized with PGE.sub.2-carrier protein
conjugate. Antibodies of the invention are selected by screening
the recombinant antibody library with an agent comprising PGE.sub.2
to thereby select those antibodies that recognize PGE.sub.2.
Methods for conducting such screening and selection are well known
in the art, such as described in the references in the preceding
paragraph. To select antibodies of the invention having particular
binding affinities for PGE.sub.2, such as those that dissociate
from PGE.sub.2 with a particular k.sub.off rate constant, the
art-known method of surface plasmon resonance can be used to select
antibodies having the desired k.sub.off rate constant. To select
antibodies of the invention having a particular neutralizing
activity for PGE.sub.2, such as those with a particular an
IC.sub.50, standard methods known in the art for assessing the
inhibition of PGE.sub.2 activity may be used.
[0162] For example, the antibodies of the present invention can
also be generated using various phage display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles that carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.
[0163] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988). Examples
of techniques that can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258, 498; Huston et al., Methods in Enzymology 203:46-88 (1991);
Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science
240:1038-1040 (1988).
[0164] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0165] In another approach the antibodies of the present invention
can also be generated using yeast display methods known in the art.
In yeast display methods, genetic methods are used to tether
antibody domains to the yeast cell wall and display them on the
surface of yeast. In particular, such yeast can be utilized to
display antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed in Wittrup, et al.
U.S. Pat. No. 6,699,658.
5. Anti-Prostaglandin E.sub.2 Monoclonal Antibodies Using PROfusion
mRNA Display
[0166] PROfusion.TM. technology is one of the mRNA display
technologies decribed above. PROfusion.TM. technology can be used
to display human antibody fragments (VH or VL or scFv) coupled to
their encoding DNA sequences for selecting against various
antigens. Examples of mRNA display methods that can be used to make
the antibodies of the present invention include those disclosed in
Szostak, et al. U.S. Pat. Nos. 6,207,446; 6,214,553; and Gold, et
al. U.S. Pat. No. 6,194,550 and in Roberts, R. W. and Szostak, J.
W. (1997) Proc. Natl. Acad. Sci. USA 94:12297-12302. In this
system, a covalent fusion is created between an mRNA and the
peptide or protein that it encodes by in vitro translation of
synthetic mRNAs that carry puromycin, a peptidyl acceptor
antibiotic, at their 3' end. Thus, a specific mRNA can be enriched
from a complex mixture of mRNAs (e.g., a combinatorial library)
based on the properties of the encoded peptide or protein, e.g.,
antibody, or portion thereof, such as binding of the antibody, or
portion thereof, to the dual specificity antigen. Nucleic acid
sequences encoding antibodies, or portions thereof, recovered from
screening of such libraries can be expressed by recombinant means
as described above (e.g., in mammalian host cells) and, moreover,
can be subjected to further affinity maturation by either
additional rounds of screening of mRNA-peptide fusions in which
mutations have been introduced into the originally selected
sequence(s), or by other methods for affinity maturation in vitro
of recombinant antibodies, as described above.
6. Affinity Maturation of Anti-Prostaglandin E.sub.2 Monoclonal
Antibodies
[0167] In vitro methods also can be used for the affinity
maturation of the antibodies of the invention, wherein a
mutagenesis antibody library is generated by introducing point
mutation(s) in CDRs and/or frameworks of one initial antibody using
error-prone PCR, or synthetic combinatorial oligo nucleotides, or
oligo nucleotides directed mutagenesis. The mutagenesis library
then can be screened to identify an antibody having the improved
binding specificity. Methods for such screening of recombinant
antibody libraries are well known in the art and include methods
described in, for example, Ladner et al. U.S. Pat. No. 5,223,409;
Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT
Publication No. WO 91/17271; Winter et al. PCT Publication No. WO
92/20791; Markland et al. PCT Publication No. WO 92/15679;
Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al.
PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No.
WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et
al. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989)
Science 246:1275-1281; McCafferty et al., Nature (1990)
348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et
al. (1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature
352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al.
(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc
Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982,
US patent application publication 20030186374, and PCT Publication
No. WO 97/29131.
[0168] Methods for conducting such screening and selection are well
known in the art, such as described in the references in the
preceding paragraph. To select antibodies of the invention having
particular binding affinities for PGE.sub.2, such as those that
dissociate from PGE.sub.2 with a particular k.sub.off rate
constant, the art-known method of surface plasmon resonance can be
used to select antibodies having the desired k.sub.off rate
constant. To select antibodies of the invention having a particular
neutralizing activity for PGE.sub.2, such as those with a
particular IC.sub.50, standard, methods known in the art for
assessing the inhibition of PGE.sub.2 activity may be used.
[0169] For example, the antibodies of the present invention can
also be affinity matured using various display methods known in the
art. In phage display methods, functional antibody domains are
displayed on the surface of phage particles that carry the
polynucleotide sequences encoding them. In a particular, such phage
can be utilized to display antigen-binding domains expressed from a
repertoire or combinatorial antibody library (e.g., human or
murine). Phage expressing an antigen binding domain that binds the
antigen of interest can be selected or identified with antigen,
e.g., using labeled antigen or antigen bound or captured to a solid
surface or bead. Phage used in these methods are typically
filamentous phage including fd and M13 binding domains expressed
from phage with Fab, Fv or disulfide stabilized Fv antibody domains
recombinantly fused to either the phage gene III or gene VIII
protein. Examples of phage display methods that can be used to make
the antibodies of the present invention include those disclosed in
Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al.,
J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur.
J. Immunol. 24:952-958 (1994); Persic et al., Gene 187 9-18 (1997);
Burton et al., Advances in Immunology 57:191-280 (1994); PCT
application No. PCT/GB91/01134; PCT publications WO 90/02809; WO
91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO
95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;
5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;
5,516,637; 5,780, 225; 5,658,727; 5,733,743 and 5,969,108.
[0170] As described in the above references, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies including human antibodies or any
other desired antigen binding fragment, and expressed in any
desired host, including mammalian cells, insect cells, plant cells,
yeast, and bacteria, e.g., as described in detail below. For
example, techniques to recombinantly produce Fab, Fab' and F(ab')2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
BioTechniques 12(6):864-869 (1992); and Sawai et al., AJRI 34:26-34
(1995); and Better et al., Science 240:1041-1043 (1988). Examples
of techniques that can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258, 498; Huston et al., Methods in Enzymology 203:46-88 (1991);
Shu et al., PNAS 90:7995-7999 (1993); and Skerra et al., Science
240:1038-1040 (1988).
[0171] Alternative to screening of recombinant antibody libraries
by phage display, other methodologies known in the art for
screening large combinatorial libraries can be applied to the
identification of dual specificity antibodies of the invention. One
type of alternative expression system is one in which the
recombinant antibody library is expressed as RNA-protein fusions,
as described in PCT Publication No. WO 98/31700 by Szostak and
Roberts, and in Roberts, R. W. and Szostak, J. W. (1997) Proc.
Natl. Acad. Sci. USA 94:12297-12302. In this system, a covalent
fusion is created between an mRNA and the peptide or protein that
it encodes by in vitro translation of synthetic mRNAs that carry
puromycin, a peptidyl acceptor antibiotic, at their 3' end. Thus, a
specific mRNA can be enriched from a complex mixture of mRNAs
(e.g., a combinatorial library) based on the properties of the
encoded peptide or protein, e.g., antibody, or portion thereof,
such as binding of the antibody, or portion thereof, to the dual
specificity antigen. Nucleic acid sequences encoding antibodies, or
portions thereof, recovered from screening of such libraries can be
expressed by recombinant means as described above (e.g., in
mammalian host cells) and, moreover, can be subjected to further
affinity maturation by either additional rounds of screening of
mRNA-peptide fusions in which mutations have been introduced into
the originally selected sequence(s), or by other methods for
affinity maturation in vitro of recombinant antibodies, as
described above.
[0172] In another approach, the antibodies of the present invention
can be generated using yeast display methods known in the art. In
yeast display methods, genetic methods are used to tether antibody
domains to the yeast cell wall and display them on the surface of
yeast. In particular, such yeast can be utilized to display
antigen-binding domains expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Examples of
yeast display methods that can be used to make the antibodies of
the present invention include those disclosed in Wittrup, et al.
U.S. Pat. No. 6,699,658.
B. Production Of Recombinant Prostaglandin E.sub.2 Antibodies
[0173] Antibodies of the present invention may be produced by any
of a number of techniques known in the art. For example, expression
from host cells, wherein expression vector(s) encoding the heavy
and light chains are transfected into a host cell by standard
techniques. The various forms of the term "transfection" are
intended to encompass a wide variety of techniques commonly used
for the introduction of exogenous DNA into a prokaryotic or
eukaryotic host cell, e.g., electroporation, calcium-phosphate
precipitation, DEAE-dextran transfection and the like. Although it
is possible to express the antibodies of the invention in either
prokaryotic or eukaryotic host cells, expression of antibodies in
eukaryotic cells is preferable, and most preferable in mammalian
host cells, because such eukaryotic cells (and in particular
mammalian cells) are more likely than prokaryotic cells to assemble
and secrete a properly folded and immunologically active
antibody.
[0174] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, used
with a DHFR selectable marker, e.g., as described in R. J. Kaufman
and P. A. Sharp (1982) Mol. Biol. 159:601-621), NS0 myeloma cells,
COS cells and SP2 cells. When recombinant expression vectors
encoding antibody genes are introduced into mammalian host cells,
the antibodies are produced by culturing the host cells for a
period of time sufficient to allow for expression of the antibody
in the host cells or, more preferably, secretion of the antibody
into the culture medium in which the host cells are grown.
Antibodies can be recovered from the culture medium using standard
protein purification methods.
[0175] Host cells can also be used to produce functional antibody
fragments, such as Fab fragments or scFv molecules. It will be
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding functional fragments of
either the light chain and/or the heavy chain of an antibody of
this invention. Recombinant DNA technology may also be used to
remove some, or all, of the DNA encoding either or both of the
light and heavy chains that is not necessary for binding to the
antigens of interest. The molecules expressed from such truncated
DNA molecules are also encompassed by the antibodies of the
invention. In addition, bifunctional antibodies may be produced in
which one heavy and one light chain are an antibody of the
invention and the other heavy and light chain are specific for an
antigen other than the antigens of interest by crosslinking an
antibody of the invention to a second antibody by standard chemical
crosslinking methods.
[0176] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are cultured to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture medium.
Still further the invention provides a method of synthesizing a
recombinant antibody of the invention by culturing a host cell of
the invention in a suitable culture medium until a recombinant
antibody of the invention is synthesized. The method can further
comprise isolating the recombinant antibody from the culture
medium.
Production of Anti-Prostaglandin E.sub.2 Chimeric Antibodies
[0177] A chimeric antibody is a molecule in which different
portions of the antibody are derived from different animal species,
such as antibodies having a variable region derived from a murine
monoclonal antibody and a human immunoglobulin constant region.
Methods for producing chimeric antibodies are known in the art and
discussed in detail in Example 2.1. See e.g., Morrison, Science
229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et
al., (1989) J. Immunol. Methods 125:191-202; U.S. Pat. Nos.
5,807,715; 4,816,567; and 4,816,397. In addition, techniques
developed for the production of "chimeric antibodies" (Morrison et
al., 1984, Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al.,
1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454)
by splicing genes from a mouse antibody molecule of appropriate
antigen specificity together with genes from a human antibody
molecule of appropriate biological activity can be used.
[0178] In one embodiment, the chimeric antibodies of the invention
are produced by replacing the heavy chain constant region of the
murine monoclonal anti PGE.sub.2 antibodies described in section 1
with a human IgG1 constant region. In a specific embodiment the
chimeric antibody of the invention comprises a heavy chain variable
region (V.sub.H) comprising the amino acid sequence of SEQ ID 60,
62, 64, 66, 68, 70, 72, 74, 76, 78, and 80 and light chain variable
region (V.sub.L) SEQ ID 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, and
81.
Anti-Prostaglandin E.sub.2 Humanized Antibodies
[0179] Humanized antibodies are antibody molecules derived from a
non-human species antibody that binds the desired antigen having
one or more complementarity determining regions (CDRs) from the
non-human species and framework regions from a human immunoglobulin
molecule. Known human Ig sequences are disclosed, e.g., Kabat et
al., Sequences of Proteins of Immunological Interest, U.S. Dept.
Health (1983). Such imported sequences can be used to reduce
immunogenicity or reduce, enhance or modify binding, affinity,
on-rate, off-rate, avidity, specificity, half-life, or any other
suitable characteristic, as known in the art.
[0180] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, preferably improve, antigen binding. These
framework substitutions are identified by methods well known in the
art, e.g., by modeling of the interactions of the CDR and framework
residues to identify framework residues important for antigen
binding and sequence comparison to identify unusual framework
residues at particular positions. (See, e.g., Queen et al., U.S.
Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988)).
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available that illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of the residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. I n this way, FR residues can be selected and combined
from the consensus and import sequences so that the desired
antibody characteristic, such as increased affinity for the target
antigen(s), is achieved. In general, the CDR residues are directly
and most substantially involved in influencing antigen binding.
Antibodies can be humanized using a variety of techniques known in
the art, such as, but not limited to, those described in Jones et
al., Nature 321:522 (1986); Verhoeyen et al., Science 239:1534
(1988)), Sims et al., J. Immunol. 151: 2296 (1993); Chothia and
Lesk, J. Mol. Biol. 196:901 (1987), Carter et al., Proc. Natl.
Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
151:2623 (1993), Padlan, Molecular Immunology 28(4/5):489-498
(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);
Roguska. et al., PNAS 91:969-973 (1994); PCT publication WO
91/09967, PCT/US98/16280, US96/18978, US91/09630, US91/05939,
US94/01234, GB89/01334, GB91/01134, GB92/01755; WO90/14443,
WO90/14424, WO90/14430, EP 229246, EP 592,106; EP 519,596, EP
239,400, U.S. Pat. Nos. 5,565,332, 5,723,323, 5,976,862, 5,824,514,
5,817,483, 5,814,476, 5,763,192, 5,723,323, 5,766886, 5,714,352,
6,204,023, 6,180,370, 5,693,762, 5,530,101, 5,585,089, 5,225,539;
and 4,816,567.
C. Production of Antibodies and Antibody-Producing Cell Lines
[0181] Preferably, anti-PGE.sub.2 antibodies of the present
invention exhibit a high capacity to reduce or to neutralize
PGE.sub.2 activity, e.g., as assessed by any one of several in
vitro and in vivo assays known in the art (e.g., see Example
1.1.C). For example, these antibodies neutralize PGE.sub.2-induced
calcium influx in EP4 assay with IC.sub.50 values in the range of
at least about 10.sup.-6 M, about 10.sup.-7 M, about 10.sup.-8 M,
about 10.sup.-9 M, about 10.sup.-10 M, about 10.sup.-11 M or about
10.sup.-12 M.
[0182] In preferred embodiments, the isolated antibody, or
antigen-binding portion thereof, binds PGE.sub.2, wherein the
antibody, or antigen-binding portion thereof, dissociates from
PGE.sub.2 with a k.sub.off rate constant of about 0.1 s.sup.-1 or
less, as determined by radioimmoassay or which inhibits PGE.sub.2
activity with an IC.sub.50 of about 1.times.10.sup.-6 M or less.
Alternatively, the antibody, or an antigen-binding portion thereof,
may dissociate from PGE.sub.2 with a k.sub.off rate constant of
about 1.times.10.sup.-2 s.sup.-1 or less, as determined by
radioimmunoassay, or may inhibit PGE.sub.2 activity with an
IC.sub.50 of about 1.times.10.sup.-7 M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from PGE.sub.2 with a k.sub.off rate constant of about
1.times.10.sup.-3 s.sup.-1 or less, as determined by
radioimmunoassay, or may inhibit PGE.sub.2 with an IC.sub.50 of
about 1.times.10.sup.-8 M or less. Alternatively, the antibody, or
an antigen-binding portion thereof, may dissociate from PGE.sub.2
with a k.sub.off rate constant of about 1.times.10.sup.-4 s.sup.-1
or less, as determined by radioimmunoassay, or may inhibit
PGE.sub.2 activity with an IC.sub.50 of about 1.times.10.sup.-9 M
or less. Alternatively, the antibody, or an antigen-binding portion
thereof, may dissociate from PGE.sub.2 with a k.sub.off rate
constant of about 1.times.10.sup.-5 s.sup.-1 or less, as determined
by radioimmunoassay, or may inhibit PGE.sub.2 activity with an
IC.sub.50 of about 1.times.10.sup.-10 M or less. Alternatively, the
antibody, or an antigen-binding portion thereof, may dissociate
from PGE.sub.2 with a k.sub.off rate constant of about
1.times.10.sup.-6 s.sup.-1 or less, as determined by
radioimmunoassay, or may inhibit PGE.sub.2 activity with an
IC.sub.50 of about 1.times.10.sup.-11 M or less.
[0183] The monoclonal antibodies of the invention block PGE.sub.2
binding to at least one of EP1, EP2, EP3, and EP4 receptors. Both
FACS-based receptor binding assay and .sup.3H-labeled PGE.sub.2
binding assay on cell surface demonstrate that both murine version
and humanized version anti-PGE2, are able to effectively block
PGE.sub.2 binding to its receptors. The crystal structure of
PGE.sub.2 complexed with the Fab portion of humanized
anti-PGE.sub.2 antibody Hu2B5.7 is envisioned by the invention
[0184] In certain embodiments, the antibody comprises a heavy chain
constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM
or IgD constant region. Preferably, the heavy chain constant region
is an IgG1 heavy chain constant region or an IgG4 heavy chain
constant region. Furthermore, the antibody can comprise a light
chain constant region, either a kappa light chain constant region
or a lambda light chain constant region. Preferably, the antibody
comprises a kappa light chain constant region. Alternatively, the
antibody portion can be, for example, a Fab fragment or a single
chain Fv fragment.
[0185] Replacements of amino acid residues in the Fc portion to
alter antibody effector function are known in the art (Winter, et
al. U.S. Pat. Nos. 5,648,260; 5,624,821). The Fc portion of an
antibody mediates several important effector functions, e.g.,
cytokine induction, ADCC, phagocytosis, complement dependent
cytotoxicity (CDC) and half-life/clearance rate of antibody and
antigen-antibody complexes. In some cases these effector functions
are desirable for therapeutic antibody but in other cases might be
unnecessary or even deleterious, depending on the therapeutic
objectives. Certain human IgG isotypes, particularly IgG1 and IgG3,
mediate ADCC and CDC via binding to Fc.gamma.Rs and complement C1q,
respectively. Neonatal Fc receptors (FcRn) are the critical
components determining the circulating half-life of antibodies. In
still another embodiment at least one amino acid residue is
replaced in the constant region of the antibody, for example, the
Fc region of the antibody, such that effector functions of the
antibody are altered. In another embodiment, the activity of the Fc
fragment can be greatly enhanced by sialylation of the N-linked
glycan of the Fc portion (e.g., with 2,6-linkage to the penultimate
galactose on the complex, biantennary glycan found at Asn 297 in
immunoglobulin G (IgG)) (Anthony, R M (2008) Science
320:373-376).
[0186] One embodiment provides a labeled binding protein wherein an
antibody or antibody portion of the invention is derivatized or
linked to another functional molecule (e.g., another peptide or
protein). For example, a labeled binding protein of the invention
can be derived by functionally linking an antibody or antibody
portion of the invention (e.g., by chemical coupling, genetic
fusion, noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
association of the antibody or antibody portion with another
molecule (e.g., such as a streptavidin core region or a
polyhistidine tag).
[0187] Useful detectable agents with which an antibody or antibody
portion of the invention may be derivatized include fluorescent
compounds. Exemplary fluorescent detectable agents include
fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
[0188] Another embodiment of the invention provides a crystallized
binding protein. Preferably the invention relates to crystals of
whole anti-PGE.sub.2 antibodies and fragments thereof as disclosed
herein, and formulations and compositions comprising such crystals.
In one embodiment the crystallized binding protein has a greater
half-life in vivo than the soluble counterpart of the binding
protein. In another embodiment the binding protein retains
biological activity after crystallization.
[0189] Crystallized binding protein of the invention may be
produced according methods known in the art and as disclosed in WO
02072636.
[0190] Another embodiment of the invention provides a glycosylated
binding protein wherein the antibody or antigen-binding portion
thereof comprises one or more carbohydrate residues. Nascent in
vivo protein production may undergo further processing, known as
post-translational modification. In particular, sugar (glycosyl)
residues may be added enzymatically, a process known as
glycosylation. The resulting proteins bearing covalently linked
oligosaccharide side chains are known as glycosylated proteins or
glycoproteins. Antibodies are glycoproteins with one or more
carbohydrate residues in the Fc domain, as well as the variable
domain. Carbohydrate residues in the Fc domain have important
effect on the effector function of the Fc domain, with minimal
effect on antigen binding or half-life of the antibody (R.
Jefferis, Biotechnol. Prog. 21 (2005), pp. 11-16). In contrast,
glycosylation of the variable domain may have an effect on the
antigen binding activity of the antibody. Glycosylation in the
variable domain may have a negative effect on antibody binding
affinity, likely due to steric hindrance (Co, M. S., et al., Mol.
Immunol. (1993) 30:1361-1367), or result in increased affinity for
the antigen (Wallick, S. C., et al., Exp. Med. (1988)
168:1099-1109; Wright, A., et al., EMBO J. (1991) 10:2717
2723).
[0191] One aspect of the present invention is directed to
generating glycosylation site mutants in which the O- or N-linked
glycosylation site of the binding protein has been mutated. One
skilled in the art can generate such mutants using standard
well-known technologies. Glycosylation site mutants that retain the
biological activity, but have increased or decreased binding
activity, are other objects of the present invention.
[0192] In still another embodiment, the glycosylation of the
antibody or antigen-binding portion of the invention is modified.
For example, an aglycoslated antibody can be made (i.e., the
antibody lacks glycosylation). Glycosylation can be altered to, for
example, increase the affinity of the antibody for antigen. Such
carbohydrate modifications can be accomplished by, for example,
altering one or more sites of glycosylation within the antibody
sequence. For example, one or more amino acid substitutions can be
made that result in elimination of one or more variable region
glycosylation sites to thereby eliminate glycosylation at that
site. Such a glycosylation may increase the affinity of the
antibody for antigen. Such an approach is described in further
detail in PCT Publication WO2003016466A2, and U.S. Pat. Nos.
5,714,350 and 6,350,861.
[0193] Additionally or alternatively, a modified antibody of the
invention can be made that has an altered type of glycosylation,
such as a hypofucosylated antibody having reduced amounts of
fucosyl residues or an antibody having increased bisecting GlcNAc
structures. Such altered glycosylation patterns have been
demonstrated to increase the ADCC ability of antibodies. Such
carbohydrate modifications can be accomplished by, for example,
expressing the antibody in a host cell with altered glycosylation
machinery. Cells with altered glycosylation machinery have been
described in the art and can be used as host cells in which to
express recombinant antibodies of the invention to thereby produce
an antibody with altered glycosylation. See, for example, Shields,
R. L. et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al.
(1999) Nat. Biotech. 17:176-1, as well as, European Patent No: EP
1,176,195; PCT Publications WO 03/035835; WO 99/54342 80.
[0194] Protein glycosylation depends on the amino acid sequence of
the protein of interest, as well as the host cell in which the
protein is expressed. Different organisms may produce different
glycosylation enzymes (e.g., glycosyltransferases and
glycosidases), and have different substrates (nucleotide sugars)
available. Due to such factors, protein glycosylation pattern, and
composition of glycosyl residues, may differ depending on the host
system in which the particular protein is expressed. Glycosyl
residues useful in the invention may include, but are not limited
to, glucose, galactose, mannose, fucose, n-acetylglucosamine and
sialic acid. Preferably the glycosylated binding protein comprises
glycosyl residues such that the glycosylation pattern is human.
[0195] It is known to those skilled in the art that differing
protein glycosylation may result in differing protein
characteristics. For instance, the efficacy of a therapeutic
protein produced in a microorganism host, such as yeast, and
glycosylated utilizing the yeast endogenous pathway may be reduced
compared to that of the same protein expressed in a mammalian cell,
such as a CHO cell line. Such glycoproteins may also be immunogenic
in humans and show reduced half-life in vivo after administration.
Specific receptors in humans and other animals may recognize
specific glycosyl residues and promote the rapid clearance of the
protein from the bloodstream. Other adverse effects may include
changes in protein folding, solubility, susceptibility to
proteases, trafficking, transport, compartmentalization, secretion,
recognition by other proteins or factors, antigenicity, or
allergenicity. Accordingly, a practitioner may prefer a therapeutic
protein with a specific composition and pattern of glycosylation,
for example, glycosylation composition and pattern identical, or at
least similar, to that produced in human cells or in the
species-specific cells of the intended subject animal.
[0196] Expressing glycosylated proteins different from that of a
host cell may be achieved by genetically modifying the host cell to
express heterologous glycosylation enzymes. Using techniques known
in the art a practitioner may generate antibodies or
antigen-binding portions thereof exhibiting human protein
glycosylation. For example, yeast strains have been genetically
modified to express non-naturally occurring glycosylation enzymes
such that glycosylated proteins (i.e., glycoproteins) produced in
these yeast strains exhibit protein glycosylation identical to that
of animal cells, especially human cells (U.S patent applications
20040018590 and 20020137134 and PCT publication WO2005100584
A2).
[0197] In addition to the binding proteins, the present invention
is also directed to an anti-idiotypic (anti-Id) antibody specific
for such binding proteins of the invention. An anti-Id antibody is
an antibody that recognizes unique determinants generally
associated with the antigen-binding region of another antibody. The
anti-Id can be prepared by immunizing an animal with the binding
protein or a CDR containing region thereof. The immunized animal
will recognize, and respond to the idiotypic determinants of the
immunizing antibody and produce an anti-Id antibody. The anti-Id
antibody may also be used as an "immunogen" to induce an immune
response in yet another animal, producing a so-called anti-anti-Id
antibody.
[0198] Further, it will be appreciated by one skilled in the art
that a protein of interest may be expressed using a library of host
cells genetically engineered to express various glycosylation
enzymes, such that member host cells of the library produce the
protein of interest with variant glycosylation patterns. A
practitioner may then select and isolate the protein of interest
with particular novel glycosylation patterns. Preferably, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
D. Uses of Anti-Prostaglandin E.sub.2 Antibodies
[0199] Given their ability to bind to PGE.sub.2, the anti-PGE.sub.2
antibodies, or portions thereof, of the invention can be used to
detect PGE.sub.2 (e.g., in a biological sample, such as serum or
plasma), using a conventional immunoassay, such as an enzyme linked
immunosorbent assays (ELISA), an radioimmunoassay (RIA) or tissue
immunohistochemistry. The invention provides a method for detecting
PGE.sub.2 in a biological sample comprising contacting a biological
sample with an antibody, or antibody portion, of the invention and
detecting either the antibody (or antibody portion) bound to
PGE.sub.2 or unbound antibody (or antibody portion), to thereby
detect PGE.sub.2 in the biological sample. The antibody is directly
or indirectly labeled with a detectable substance to facilitate
detection of the bound or unbound antibody. Suitable detectable
substances include various enzymes, prosthetic groups, fluorescent
materials, luminescent materials and radioactive materials.
Examples of suitable enzymes include horseradish peroxidase,
alkaline phosphatase, .beta.-galactosidase, or
acetylcholinesterase; examples of suitable prosthetic group
complexes include streptavidin/biotin and avidin/biotin; examples
of suitable fluorescent materials include umbelliferone,
fluorescein, fluorescein isothiocyanate, rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include
.sup.3H, .sup.14C, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, or .sup.153Sm.
[0200] Alternative to labeling the antibody, PGE.sub.2 can be
assayed in biological fluids by a competition immunoassay utilizing
PGE.sub.2 standards labeled with a detectable substance and an
unlabeled anti-PGE.sub.2 antibody. In this assay, the biological
sample, the labeled PGE.sub.2 standards and the anti-PGE.sub.2
antibody are combined and the amount of labeled PGE.sub.2 standard
bound to the unlabeled antibody is determined. The amount of
PGE.sub.2 in the biological sample is inversely proportional to the
amount of labeled PGE.sub.2 standard bound to the anti-PGE.sub.2
antibody. Similarly, PGE.sub.2 can also be assayed in biological
fluids by a competition immunoassay utilizing PGE.sub.2 standards
labeled with a detectable substance and an unlabeled anti-PGE.sub.2
antibody.
[0201] The antibodies and antibody portions of the invention
preferably are capable of neutralizing PGE.sub.2 activity both in
vitro and in vivo. Accordingly, such antibodies and antibody
portions of the invention can be used to inhibit PGE.sub.2
activity, e.g., in a cell culture containing PGE.sub.2, in human
subjects or in other mammalian subjects having PGE.sub.2 with which
an antibody of the invention cross-reacts. In one embodiment, the
invention provides a method for inhibiting PGE.sub.2 activity
comprising contacting PGE.sub.2 with an antibody or antibody
portion of the invention such that PGE.sub.2 activity is inhibited.
For example, in a cell culture containing, or suspected of
containing PGE.sub.2, an antibody or antibody portion of the
invention can be added to the culture medium to inhibit PGE.sub.2
activity in the culture.
[0202] In another embodiment, the invention provides a method for
reducing PGE.sub.2 activity in a subject, advantageously from a
subject suffering from a disease or disorder in which PGE.sub.2
activity is detrimental. The invention provides methods for
reducing PGE.sub.2 activity in a subject suffering from such a
disease or disorder, which method comprises administering to the
subject an antibody or antibody portion of the invention such that
PGE.sub.2 activity in the subject is reduced. Preferably, the
subject is a human subject. Alternatively, the subject can be a
mammal expressing PGE.sub.2 to which an antibody of the invention
is capable of binding. Still further the subject can be a mammal
into which PGE.sub.2 has been introduced (e.g., by administration
of PGE.sub.2 or by expression of a PGE.sub.2 synthetase transgene).
An antibody of the invention can be administered to a human subject
for therapeutic purposes. Moreover, an antibody of the invention
can be administered to a non-human mammal expressing PGE.sub.2 with
which the antibody is capable of binding for veterinary purposes or
as an animal model of human disease. Regarding the latter, such
animal models may be useful for evaluating the therapeutic efficacy
of antibodies of the invention (e.g., testing of dosages and time
courses of administration).
[0203] As used herein, the term "a disorder in which PGE.sub.2
activity is detrimental" is intended to include diseases and other
disorders in which the presence of PGE.sub.2 in a subject suffering
from the disorder has been shown to be or is suspected of being
either responsible for the pathophysiology of the disorder or a
factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which PGE.sub.2 activity is detrimental
is a disorder in which reduction of PGE.sub.2 activity is expected
to alleviate the symptoms and/or progression of the disorder. Such
disorders may be evidenced, for example, by an increase in the
concentration of PGE.sub.2 in a biological fluid of a subject
suffering from the disorder (e.g., an increase in the concentration
of PGE.sub.2 in serum, plasma, synovial fluid, etc. of the
subject), which can be detected, for example, using an
anti-PGE.sub.2 antibody as described above. Non-limiting examples
of disorders that can be treated with the antibodies of the
invention include those disorders discussed in the section below
pertaining to pharmaceutical compositions of the antibodies of the
invention.
[0204] PGE.sub.2 has been implicated as having a pivotal role in
causing pathological responses associated with rheumatoid
arthritis. However other mediators of differential immunological
pathways are also involved in arthritis, and blocking these
mediators, in addition to PGE.sub.2, may offer additional
therapeutic benefit. Thus, binding proteins of the invention may be
incorporated into DVD-Ig.TM. proteins where in the DVD-Ig.TM. is
capable of binding target pairs including, but not limited to,
PGE.sub.2 and a pro-inflammatory cytokine, such as tumor necrosis
factor-.alpha. (TNF-.alpha.). Blocking both PGE.sub.2 and
TNF-.alpha. may have beneficial effects that combining DMARD effect
of TNF-.alpha. and pain relief from blocking PGE.sub.2. In a
preferred embodiment, the DVD-Ig.TM. of the invention binds the
targets PGE.sub.2 and TNF.alpha. and is used for treating
rheumatoid arthritis.
[0205] One aspect of the invention pertains to a DVD-Ig.TM. binding
protein comprising binding proteins capable of binding PGE.sub.2.
Preferably the DVD-Ig.TM. binding protein is capable of binding
PGE.sub.2 and a second target. The second target is selected from
the group consisting of TNF, EGF, EGFR, IGF1, IGF2, IGF1/2, IGFR
Erb2, Erb3, VEGF, VEGFR, Muc-1, CSF1 (MCSF), CSF2 (GM-CSF), CSF3
(GCSF), FGF2, IFN.gamma.1, IFN.gamma.1, IFN.gamma., histamine and
histamine receptors, IL-1.alpha., IL-1.beta., IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12 .alpha., IL-12
.beta., IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, KITLG, PDGFB,
IL-2R .alpha., IL-4R, IL-5R .alpha., IL-8R .alpha., IL-8R .beta.,
IL-12R .beta.,1, IL-12R .beta.,2, IL-18R1, TSLP, CCL1, CCL2, CCL3,
CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22,
CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCL1, CCR2, CCR3, CCR4, CCR5,
CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3,
STATE, TBX21, TGFB1, TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R,
TB4R2, LTBR, and Chitinase. More preferably, the DVD binding
protein is capable of recognizing PGE.sub.2 and TNF .alpha.,
PGE.sub.2 and IL-6, PGE.sub.2 and IL-1.beta., PGE.sub.2 and IL-6R,
PGE.sub.2 and CTLA-4, PGE.sub.2 and EGF; PGE.sub.2 and IGF-1/2,
PGE.sub.2 and Erb2, PGE.sub.2 and Erb3, PGE.sub.2 and VEGF. Most
preferably, the DVD binding protein is capable of binding PGE.sub.2
and TNF .alpha.. Preferred DVD-Igs for treating autoimmune diseases
include but are not limited to a DVD-Ig containing an anti
PGE.sub.2 antibody and a target selected from the group consisting
of TNF .alpha., IL-1 .alpha., IL-1.beta., IL-6 or IL-6R, CTLA-4Ig,
BAFF, TALI, RANKL, or DKK1 or SOCT, MMP13, or MMP1 or MMP4. Target
pairs preferably for cancer therapy include PGE.sub.2+EGF or EGFR,
PGE.sub.2+IGF1 or IGF1R, PGE.sub.2+IGF2 or IGF2R, PGE.sub.2+IGF1/2,
PGE.sub.2+VEGF or VEGFR, PGE.sub.2+Erb2, PGE.sub.2+Erb3, and
PGE.sub.2+SIP.
[0206] PGE.sub.2 has been implicated as having a pivotal role in
causing pathological responses associated with rheumatoid arthritis
or cancer. However other mediators of differential immunological
pathways are also involved in arthritis or cancer, and blocking
these mediators, in addition to PGE.sub.2, may offer additional
therapeutic benefit. A list of the drugs that currently are used
for the treatment of various human diseases and disorders is
available on the internet. This list is updated frequently to
reflect state-of-art for the treatment of various human diseases.
Anti-PGE.sub.2 can be combined with any of the therapies in that
list for a particular disease conditions. A few examples are
provided below. Anti-PGE.sub.2 can be combined with one or more
agents for the treatment of rheumatoid arthritis and juvenile
rheumatoid arthritis. Examples of these agents include but are not
limited to the agents listed below, for example, drugs that
decrease pain and inflammation while decreasing the growth of
abnormal synovial tissue (the tissue that lines the inside of the
joint). These drugs include methotrexate and low doses of
corticosteroids (such as prednisone or cortisone). In some people,
these drugs also decrease joint destruction. Other medications used
to treat rheumatoid arthritis include: anti-malarial medications
(such as hydroxychloroquine), gold, sulfasalazine, penicillamine,
cyclophosphamide, cyclosporin and minocycline. In addition, more
than one drug may be prescribed. Newer biologic agents that block
the effects of specific inflammatory factors (cytokines) are now
available. Infliximab, etanercept and adalimumab block the cytokine
TNF.alpha., abatacept blocks T cell costimulation, rituximab
depletes B cells, anakinra blocks the cytokine interleukin-1, and
other new biologic agents target IL-6, IL-6R, IL-17, IL-18, IL-23,
B7.1/B7.2. Anti-PGE.sub.2 can combine with one or more agents for
the treatment of ankylosing spondylitis. Examples of these agents
include but are not limited to corticosteroids, cytotoxic drugs and
most recently anti-TNF.alpha. agents. Anti-PGE.sub.2 can be
combined with one or more agents for the treatment of multiple
sclerosis. Examples of these agents include but are not limited to
Avonex.TM., Azasan.TM., Azathioprine, Betaseron.TM.,
Bubbli-Pred.TM., Copaxone.TM., Cotolone, Glatiramer, Imuran.TM.,
Interferon Beta-1a, Interferon Beta-1b Solution, Key-Pred, Key-Pred
SP, Mitoxantrone, Natalizumab, Novantrone.TM., Orapred.TM., Orapred
ODT.TM., Pediapred.TM., Pred-Ject-50, Predacort 50, Predalone 50,
Predate-50, Prednisolone, Prelone.TM., Rebif.TM., and Tysabri.TM..
Anti-PGE.sub.2 can be combined with one or more agents or treatment
procedures for the treatment of pain. Anti-PGE.sub.2 can be
combined with one or more agents or treatment procedures for the
treatment of Crohn's disease. Anti-PGE.sub.2 can combine with one
or more agents or treatment procedures for the treatment of various
human cancers and malignancies.
E. Pharmaceutical Composition
[0207] The invention also provides pharmaceutical compositions
comprising an antibody, or antigen-binding portion thereof, of the
invention and a pharmaceutically acceptable carrier. The
pharmaceutical compositions comprising antibodies of the invention
are for use in, but not limited to, diagnosing, detecting, or
monitoring a disorder, in preventing, treating, managing, or
ameliorating a disorder or one or more symptoms thereof, and/or in
research. In a specific embodiment, the pharmaceutical composition
comprises one or more antibodies of the invention. In another
embodiment, the pharmaceutical composition comprises one or more
antibodies of the invention and one or more prophylactic or
therapeutic agents other than antibodies of the invention for
treating a disorder in which PGE.sub.2 activity is detrimental.
Preferably, the prophylactic or therapeutic agents known to be
useful for or that have been or are currently being used in the
prevention, treatment, management, or amelioration of a disorder or
one or more symptoms thereof. In accordance with these embodiments,
the composition may further comprise of a carrier, diluent or
excipient.
[0208] The antibodies and antibody portions of the invention can be
incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody or antibody portion of the
invention and a pharmaceutically acceptable carrier. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it will be preferable to
include isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of the antibody or antibody portion.
[0209] Various delivery systems are known and can be used to
administer one or more antibodies of the invention or the
combination of one or more antibodies of the invention and a
prophylactic agent or therapeutic agent useful for preventing,
managing, treating, or ameliorating a disorder or one or more
symptoms thereof, e.g., encapsulation in liposomes, microparticles,
microcapsules, recombinant cells capable of expressing the antibody
or antibody fragment, receptor-mediated endocytosis (see, e. g., Wu
and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a
nucleic acid as part of a retroviral or other vector, etc. Methods
of administering a prophylactic or therapeutic agent of the
invention include, but are not limited to, parenteral
administration (e.g., intradermal, intramuscular, intraperitoneal,
intravenous and subcutaneous), epidural administration,
intratumoral administration, and mucosal administration (e.g.,
intranasal and oral routes). In addition, pulmonary administration
can be employed, e.g., by use of an inhaler or nebulizer, and
formulation with an aerosolizing agent. See, e.g., U.S. Pat. Nos.
6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064; 5,855,913;
5,290,540; and 4,880,078; and PCT Publication Nos. WO 92/19244; WO
97/32572; WO 97/44013; WO 98/31346; and WO 99/66903. In one
embodiment, an antibody of the invention, combination therapy, or a
composition of the invention is administered using Alkermes
AIR.RTM. pulmonary drug delivery technology (Alkermes, Inc.,
Cambridge, Mass.). In a specific embodiment, prophylactic or
therapeutic agents of the invention are administered
intramuscularly, intravenously, intratumorally, orally,
intranasally, pulmonary, or subcutaneously. The prophylactic or
therapeutic agents may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local.
[0210] In a specific embodiment, it may be desirable to administer
the prophylactic or therapeutic agents of the invention locally to
the area in need of treatment. This may be achieved by, for
example, local infusion, by injection, or by means of an implant,
such an implant being of a porous or non-porous material, including
membranes and matrices, such as sialastic membranes, polymers,
fibrous matrices (e.g., Tissuel.RTM.), or collagen matrices. In one
embodiment, an effective amount of one or more antibodies of the
invention is administered locally to the affected area to a subject
to prevent, treat, manage, and/or ameliorate a disorder or a
symptom thereof. In another embodiment, an effective amount of one
or more antibodies of the invention is administered locally to the
affected area in combination with an effective amount of one or
more therapies (e.g., one or more prophylactic or therapeutic
agents) other than an antibody of the invention of a subject to
prevent, treat, manage, and/or ameliorate a disorder or one or more
symptoms thereof.
[0211] In another embodiment, the prophylactic or therapeutic agent
of the invention can be delivered in a controlled release or
sustained release system. In one embodiment, a pump may be used to
achieve controlled or sustained release (see Langer, supra; Sefton,
1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980,
Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In
another embodiment, polymeric materials can be used to achieve
controlled or sustained release of the therapies of the invention
(see e.g., Medical Applications of Controlled Release, Langer and
Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J.,
Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351;
Howard et al., 1989, J. Neurosurg. 7 1:105); U.S. Pat. No.
5,679,377; U.S. Pat. No. 5,916,597; U.S. Pat. No. 5,912,015; U.S.
Pat. No. 5,989,463; U.S. Pat. No. 5,128,326; PCT Publication No. WO
99/15154; and PCT Publication No. WO 99/20253. Examples of polymers
used in sustained release formulations include, but are not limited
to, poly(2-hydroxy ethyl methacrylate), poly(methyl methacrylate),
poly(acrylic acid), poly(ethylene-co-vinyl acetate),
poly(methacrylic acid), polyglycolides (PLG), polyanhydrides,
poly(N-vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide,
poly(ethylene glycol), polylactides (PLA),
poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In a
preferred embodiment, the polymer used in a sustained release
formulation is inert, free of leachable impurities, stable on
storage, sterile, and biodegradable. In yet another embodiment, a
controlled or sustained release system can be placed in proximity
of the prophylactic or therapeutic target, thus requiring only a
fraction of the systemic dose (see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138
(1984)).
[0212] Controlled release systems are discussed in the review by
Langer (1990, Science 249:1527-1533). Any technique known to one of
skill in the art can be used to produce sustained release
formulations comprising one or more therapeutic agents of the
invention. See, e.g., U.S. Pat. No. 4,526,938, PCT publication WO
91/05548, PCT publication WO 96/20698, Ning et al., 1996,
"Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft
Using a Sustained-Release Gel," Radiotherapy &Oncology
39:179-189, Song et al., 1995, "Antibody Mediated Lung Targeting of
Long-Circulating Emulsions," PDA Journal of Pharmaceutical Science
&Technology 50:372-397, Cleek et al., 1997, "Biodegradable
Polymeric Carriers for a bFGF Antibody for Cardiovascular
Application," Pro. Int'l. Symp. Control. Rel. Bioact. Mater.
24:853-854, and Lam et al., 1997, "Microencapsulation of
Recombinant Humanized Monoclonal Antibody for Local Delivery,"
Proc. Intl. Symp. Control Rel. Bioact. Mater. 24:759-760.
[0213] In a specific embodiment, where the composition of the
invention is a nucleic acid encoding a prophylactic or therapeutic
agent, the nucleic acid can be administered in vivo to promote
expression of its encoded prophylactic or therapeutic agent, by
constructing it as part of an appropriate nucleic acid expression
vector and administering it so that it becomes intracellular, e.g.,
by use of a retroviral vector (see U.S. Pat. No. 4,980,286), or by
direct injection, or by use of microparticle bombardment (e.g., a
gene gun; Biolistic, Dupont), or coating with lipids or
cell-surface receptors or transfecting agents, or by administering
it in linkage to a homeobox-like peptide which is known to enter
the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci.
USA 88:1864-1868). Alternatively, a nucleic acid can be introduced
intracellularly and incorporated within host cell DNA for
expression by homologous recombination.
[0214] A pharmaceutical composition of the invention is formulated
to be compatible with its intended route of administration.
Examples of routes of administration include, but are not limited
to, parenteral, e.g., intravenous, intradermal, subcutaneous, oral,
intranasal (e.g., inhalation), transdermal (e.g., topical),
transmucosal, and rectal administration. In a specific embodiment,
the composition is formulated in accordance with routine procedures
as a pharmaceutical composition adapted for intravenous,
subcutaneous, intramuscular, oral, intranasal, or topical
administration to human beings. Typically, compositions for
intravenous administration are solutions in sterile isotonic
aqueous buffer. Where necessary, the composition may also include a
solubilizing agent and a local anesthetic such as lignocaine to
ease pain at the site of the injection.
[0215] If the compositions of the invention are to be administered
topically, the compositions can be formulated in the form of an
ointment, cream, transdermal patch, lotion, gel, shampoo, spray,
aerosol, solution, emulsion, or other form known to one of skill in
the art. See, e.g., Remington's Pharmaceutical Sciences and
Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub.
Co., Easton, Pa. (1995). For non-sprayable topical dosage forms,
viscous to semi-solid or solid forms comprising a carrier or one or
more excipients compatible with topical application and having a
dynamic viscosity preferably greater than water are typically
employed. Suitable formulations include, without limitation,
solutions, suspensions, emulsions, creams, ointments, powders,
liniments, salves, and the like, which are, if desired, sterilized
or mixed with auxiliary agents (e.g., preservatives, stabilizers,
wetting agents, buffers, or salts) for influencing various
properties, such as, for example, osmotic pressure. Other suitable
topical dosage forms include sprayable aerosol preparations wherein
the active ingredient, preferably in combination with a solid or
liquid inert carrier, is packaged in a mixture with a pressurized
volatile (e.g., a gaseous propellant, such as freon) or in a
squeeze bottle. Moisturizers or humectants can also be added to
pharmaceutical compositions and dosage forms if desired. Examples
of such additional ingredients are well known in the art.
[0216] If the method of the invention comprises intranasal
administration of a composition, the composition can be formulated
in an aerosol form, spray, mist or in the form of drops. In
particular, prophylactic or therapeutic agents for use according to
the present invention can be conveniently delivered in the form of
an aerosol spray presentation from pressurized packs or a
nebuliser, with the use of a suitable propellant (e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas).
In the case of a pressurized aerosol the dosage unit may be
determined by providing a valve to deliver a metered amount.
Capsules and cartridges (composed of, e.g., gelatin) for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0217] If the method of the invention comprises oral
administration, compositions can be formulated in the form of
tablets, capsules, cachets, gelcaps, solutions, suspensions, and
the like. Tablets or capsules can be prepared by conventional means
with pharmaceutically acceptable excipients such as binding agents
(e.g., pregelatinised maize starch, polyvinylpyrrolidone, or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose, or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc, or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods well-known in the art. Liquid preparations for
oral administration may take the form of, but not limited to,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives,
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol, or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-p-hydroxybenzoates or sorbic acid). The
preparations may also contain buffer salts, flavoring, coloring,
and sweetening agents as appropriate. Preparations for oral
administration may be suitably formulated for slow release,
controlled release, or sustained release of a prophylactic or
therapeutic agent(s).
[0218] The method of the invention may comprise pulmonary
administration, e.g., by use of an inhaler or nebulizer, of a
composition formulated with an aerosolizing agent. See, e.g., U.S.
Pat. Nos. 6,019,968; 5,985,320; 5,985,309; 5,934,272; 5,874,064;
5,855,913; 5,290,540; and 4,880,078; and PCT Publication Nos. WO
92/19244; WO 97/32572; WO 97/44013; WO 98/31346; and WO 99/66903.
In a specific embodiment, an antibody of the invention, combination
therapy, and/or composition of the invention is administered using
Alkermes AIR.RTM. pulmonary drug delivery technology (Alkermes,
Inc., Cambridge, Mass.).
[0219] The method of the invention may comprise administration of a
composition formulated for parenteral administration by injection
(e.g., by bolus injection or continuous infusion). Formulations for
injection may be presented in unit dosage form (e.g., in ampoules
or in multi-dose containers) with an added preservative. The
compositions may take such forms as suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing agents.
Alternatively, the active ingredient may be in powder form for
constitution with a suitable vehicle (e.g., sterile pyrogen-free
water) before use.
[0220] The methods of the invention may additionally comprise
administration of compositions formulated as depot preparations.
Such long acting formulations may be administered by implantation
(e.g., subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compositions may be formulated
with suitable polymeric or hydrophobic materials (e.g., as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives (e.g., as a sparingly soluble
salt).
[0221] The methods of the invention encompass administration of
compositions formulated as neutral or salt forms. Pharmaceutically
acceptable salts include those formed with anions such as those
derived from hydrochloric, phosphoric, acetic, oxalic, tartaric
acids, etc., and those formed with cations such as those derived
from sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0222] Generally, the ingredients of compositions are supplied
either separately or mixed together in unit dosage form, for
example, as a dry lyophilized powder or water free concentrate in a
hermetically sealed container such as an ampoule or sachette
indicating the quantity of active agent. Where the mode of
administration is infusion, composition can be dispensed with an
infusion bottle containing sterile pharmaceutical grade water or
saline. Where the mode of administration is by injection, an
ampoule of sterile water for injection or saline can be provided so
that the ingredients may be mixed prior to administration.
[0223] In particular, the invention also provides that one or more
of the prophylactic or therapeutic agents, or pharmaceutical
compositions of the invention is packaged in a hermetically sealed
container such as an ampoule or sachette indicating the quantity of
the agent. In one embodiment, one or more of the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
is supplied as a dry sterilized lyophilized powder or water free
concentrate in a hermetically sealed container and can be
reconstituted (e.g., with water or saline) to the appropriate
concentration for administration to a subject. Preferably, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied as a dry sterile
lyophilized powder in a hermetically sealed container at a unit
dosage of at least 5 mg, more preferably at least 10 mg, at least
15 mg, at least 25 mg, at least 35 mg, at least 45 mg, at least 50
mg, at least 75 mg, or at least 100 mg. The lyophilized
prophylactic or therapeutic agents or pharmaceutical compositions
of the invention should be stored at between 2.degree. C. and
8.degree. C. in its original container and the prophylactic or
therapeutic agents, or pharmaceutical compositions of the invention
should be administered within 1 week, preferably within 5 days,
within 72 hours, within 48 hours, within 24 hours, within 12 hours,
within 6 hours, within 5 hours, within 3 hours, or within 1 hour
after being reconstituted. In an alternative embodiment, one or
more of the prophylactic or therapeutic agents or pharmaceutical
compositions of the invention is supplied in liquid form in a
hermetically sealed container indicating the quantity and
concentration of the agent. Preferably, the liquid form of the
administered composition is supplied in a hermetically sealed
container at least 0.25 mg/ml, more preferably at least 0.5 mg/ml,
at least 1 mg/ml, at least 2.5 mg/ml, at least 5 mg/ml, at least 8
mg/ml, at least 10 mg/ml, at least 15 mg/kg, at least 25 mg/ml, at
least 50 mg/ml, at least 75 mg/ml or at least 100 mg/ml. The liquid
form should be stored at between 2.degree. C. and 8.degree. C. in
its original container.
[0224] The antibodies and antibody-portions of the invention can be
incorporated into a pharmaceutical composition suitable for
parenteral administration. Preferably, the antibody or
antibody-portions will be prepared as an injectable solution
containing 0.1-250 mg/ml antibody. The injectable solution can be
composed of either a liquid or lyophilized dosage form in a flint
or amber vial, ampule or pre-filled syringe. The buffer can be
L-histidine (1-50 mM), optimally 5-10 mM, at pH 5.0 to 7.0
(optimally pH 6.0). Other suitable buffers include but are not
limited to, sodium succinate, sodium citrate, sodium phosphate or
potassium phosphate. Sodium chloride can be used to modify the
toxicity of the solution at a concentration of 0-300 mM (optimally
150 mM for a liquid dosage form). Cryoprotectants can be included
for a lyophilized dosage form, principally 0-10% sucrose (optimally
0.5-1.0%). Other suitable cryoprotectants include trehalose and
lactose. Bulking agents can be included for a lyophilized dosage
form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can
be used in both liquid and lyophilized dosage forms, principally
1-50 mM L-Methionine (optimally 5-10 mM). Other suitable bulking
agents include glycine, arginine, can be included as 0-0.05%
polysorbate-80 (optimally 0.005-0.01%). Additional surfactants
include but are not limited to polysorbate 20 and BRIJ surfactants.
The pharmaceutical composition comprising the antibodies and
antibody-portions of the invention prepared as an injectable
solution for parenteral administration, can further comprise an
agent useful as an adjuvant, such as those used to increase the
absorption, or dispersion of a therapeutic protein (e.g.,
antibody). A particularly useful adjuvant is hyaluronidase, such as
Hylenex.RTM. (recombinant human hyaluronidase). Addition of
hyaluronidase in the injectable solution improves human
bioavailability following parenteral administration, particularly
subcutaneous administration. It also allows for greater injection
site volumes (i.e., greater than 1 ml) with less pain and
discomfort, and minimum incidence of injection site reactions (see
WO2004078140 and US2006104968).
[0225] The compositions of this invention may be in a variety of
forms. These include, for example, liquid, semi-solid and solid
dosage forms, such as liquid solutions (e.g., injectable and
infusible solutions), dispersions or suspensions, tablets, pills,
powders, liposomes and suppositories. The preferred form depends on
the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or
infusible solutions, such as compositions similar to those used for
passive immunization of humans with other antibodies. The preferred
mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0226] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody or antibody
portion) in the required amount in an appropriate solvent with one
or a combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions are
prepared by incorporating the active compound into a sterile
vehicle that contains a basic dispersion medium and the required
other ingredients from those enumerated above. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and spray-drying that yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof. The proper fluidity of a
solution can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion and by the use of surfactants. Prolonged
absorption of injectable compositions can be brought about by
including in the composition an agent that delays absorption, for
example, monostearate salts and gelatin.
[0227] The antibodies and antibody-portions of the present
invention can be administered by a variety of methods known in the
art, although for many therapeutic applications, the preferred
route/mode of administration is subcutaneous injection, intravenous
injection or infusion. As will be appreciated by the skilled
artisan, the route and/or mode of administration will vary
depending upon the desired results. In certain embodiments, the
active compound may be prepared with a carrier that will protect
the compound against rapid release, such as a controlled release
formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0228] In certain embodiments, an antibody or antibody portion of
the invention may be orally administered, for example, with an
inert diluent or an assimilable edible carrier. The compound (and
other ingredients, if desired) may also be enclosed in a hard or
soft shell gelatin capsule, compressed into tablets, or
incorporated directly into the subject's diet. For oral therapeutic
administration, the compounds may be incorporated with excipients
and used in the form of ingestible tablets, buccal tablets,
troches, capsules, elixirs, suspensions, syrups, wafers, and the
like. To administer a compound of the invention by other than
parenteral administration, it may be necessary to coat the compound
with, or co-administer the compound with, a material to prevent its
inactivation.
[0229] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating disorders in which PGE.sub.2 activity is detrimental. For
example, an anti-PGE.sub.2 antibody or antibody portion of the
invention may be coformulated and/or coadministered with one or
more additional antibodies that bind other targets (e.g.,
antibodies that bind other cytokines or that bind cell surface
molecules). Furthermore, one or more antibodies of the invention
may be used in combination with two or more of the foregoing
therapeutic agents. Such combination therapies may advantageously
utilize lower dosages of the administered therapeutic agents, thus
avoiding possible toxicities or complications associated with the
various monotherapies.
[0230] In certain embodiments, an antibody to PGE.sub.2 or fragment
thereof is linked to a half-life extending vehicle known in the
art. Such vehicles include, but are not limited to, the Fc domain,
polyethylene glycol, and dextran. Such vehicles are described,
e.g., in U.S. application Ser. No. 09/428,082 and published PCT
Application No. WO 99/25044.
[0231] In a specific embodiment, nucleic acid sequences comprising
nucleotide sequences encoding an antibody of the invention or
another prophylactic or therapeutic agent of the invention are
administered to treat, prevent, manage, or ameliorate a disorder or
one or more symptoms thereof by way of gene therapy. Gene therapy
refers to therapy performed by the administration to a subject of
an expressed or expressible nucleic acid. In this embodiment of the
invention, the nucleic acids produce their encoded antibody or
prophylactic or therapeutic agent of the invention that mediates a
prophylactic or therapeutic effect.
[0232] Any of the methods for gene therapy available in the art can
be used according to the present invention. For general reviews of
the methods of gene therapy, see Goldspiel et al., 1993, Clinical
Pharmacy 12:488-505; Wu and Wu, 1991, Biotherapy 3:87-95;
Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol. 32:573-596;
Mulligan, Science 260:926-932 (1993); and Morgan and Anderson,
1993, Ann. Rev. Biochem. 62:191-217; May, 1993, TIBTECH
11(5):155-215. Methods commonly known in the art of recombinant DNA
technology which can be used are described in Ausubel et al.
(eds.), Current Protocols in Molecular Biology, John Wiley
&Sons, NY (1993); and Kriegler, Gene Transfer and Expression, A
Laboratory Manual, Stockton Press, NY (1990). Detailed descriptions
of various methods of gene therapy are disclosed in US20050042664
A1.
[0233] In another aspect, this application features a method of
treating (e.g., curing, suppressing, ameliorating, delaying or
preventing the onset of, or preventing recurrence or relapse of) or
preventing a PGE.sub.2-associated disorder, in a subject. The
method includes: administering to the subject a PGE.sub.2 binding
agent (particularly an antagonist), e.g., an anti-PGE.sub.2
antibody or fragment thereof as described herein, in an amount
sufficient to treat or prevent the PGE.sub.2-associated disorder.
The PGE.sub.2 antagonist, e.g., the anti-PGE.sub.2 antibody or
fragment thereof, can be administered to the subject, alone or in
combination with other therapeutic modalities as described
herein.
[0234] The invention provides methods for the treatment of
inflammatory disorders and disorders of immunity in a subject,
which disorders are characterised by excessive
PGE.sub.2biosynthesis, which methods comprise administering to the
subject an effective amount of a antibody specific to PGE.sub.2.
Disorders that may be treated by the method according to the
invention include autoimmune and inflammatory diseases and tumors
in which excessive PGE.sub.2 synthesis has been implicated. Such
disorders include: (a) rheumatoid and allergic arthritis; (b)
certain illnesses induced by viruses, such as Guillain Barre
syndrome, infectious mononucleosis, other viral lymphadenopathies
and infections with herpes virus; (c) multiple sclerosis and other
demyelinating diseases; (d) hematological disorders, such as
hemolytic anemias and thrombocytopenias; (e) endocrinologic
disorders, such as diabetes mellitus, Addison's disease, idiopathic
hypoparathyroidism and chronic lymphocytic thyroiditis; (f)
collagen disorders, such as systemic lupus erythematosus; and (g)
disorders of reproduction such as amenorrhoea, infertility,
recurrent abortions and eclampsia; and (h) tumors such as headneck
tumor, lung cancer, gastric cancer, prostate cancer, pancreatic
cancer, etc., and (i) inflammatory and/or autoimmune conditions of
the skin, gastrointestinal organs (e.g., inflammatory bowel
diseases (IBD), such as ulcerative colitis and/or Crohn's disease);
and (j) pain related with osteoarthritis and other disorders; and
(k) ocular disorders such as age-related mascular degeneration
(AMD). Accordingly, the disclosure includes the use of a PGE.sub.2
binding agent (such as an anti-PGE.sub.2 antibody or fragment
thereof described herein) for a treatment described herein and the
use of a PGE.sub.2 binding agent (such as an anti-PGE.sub.2
antibody or fragment thereof described herein) for preparing a
medicament for a treatment described herein.
[0235] Examples of PGE.sub.2-associated disorders include, but are
not limited to, a disorder selected from the group consisting of
arthritis, rheumatoid arthritis, osteoarthritis, juvenile chronic
arthritis, septic arthritis, Lyme arthritis, psoriatic arthritis,
reactive arthritis, spondyloarthropathy, systemic lupus
erythematosus, Crohn's disease, ulcerative colitis, inflammatory
bowel disease, insulin dependent diabetes mellitus, thyroiditis,
asthma, allergic diseases, psoriasis, dermatitis scleroderma,
atopic dermatitis, graft versus host disease, organ transplant
rejection, acute or chronic immune disease associated with organ
transplantation, sarcoidosis, atherosclerosis, disseminated
intravascular coagulation, Kawasaki's disease, Grave's disease,
nephrotic syndrome, chronic fatigue syndrome, Wegener's
granulomatosis, Henoch-Schoenlein purpurea, microscopic vasculitis
of the kidneys, chronic active hepatitis, uveitis, septic shock,
toxic shock syndrome, sepsis syndrome, cachexia, infectious
diseases, parasitic diseases, acquired immunodeficiency syndrome,
acute transverse myelitis, Huntington's chorea, Parkinson's
disease, Alzheimer's disease, stroke, primary biliary cirrhosis,
hemolytic anemia, malignancies, heart failure, myocardial
infarction, Addison's disease, sporadic, polyglandular deficiency
type I and polyglandular deficiency type II, Schmidt's syndrome,
adult (acute) respiratory distress syndrome, alopecia, alopecia
areata, seronegative arthopathy, arthropathy, Reiter's disease,
psoriatic arthropathy, ulcerative colitic arthropathy, enteropathic
synovitis, chlamydia, yersinia and salmonella associated
arthropathy, spondyloarthopathy, atheromatous
disease/arteriosclerosis, atopic allergy, autoimmune bullous
disease, pemphigus vulgaris, pemphigus foliaceus, pemphigoid,
linear IgA disease, autoimmune haemolytic anaemia, Coombs positive
haemolytic anaemia, acquired pernicious anaemia, juvenile
pernicious anaemia, myalgic encephalitis/Royal Free Disease,
chronic mucocutaneous candidiasis, giant cell arteritis, primary
sclerosing hepatitis, cryptogenic autoimmune hepatitis, Acquired
Immunodeficiency Disease Syndrome, Acquired Immunodeficiency
Related Diseases, Hepatitis B, Hepatitis C, common varied
immunodeficiency (common variable hypogammaglobulinaemia), dilated
cardiomyopathy, female infertility, ovarian failure, premature
ovarian failure, fibrotic lung disease, cryptogenic fibrosing
alveolitis, post-inflammatory interstitial lung disease,
interstitial pneumonitis, connective tissue disease associated
interstitial lung disease, mixed connective tissue disease
associated lung disease, systemic sclerosis associated interstitial
lung disease, rheumatoid arthritis associated interstitial lung
disease, systemic lupus erythematosus associated lung disease,
dermatomyositis/polymyositis associated lung disease, Sjogren's
disease associated lung disease, ankylosing spondylitis associated
lung disease, vasculitic diffuse lung disease, haemosiderosis
associated lung disease, drug-induced interstitial lung disease,
fibrosis, radiation fibrosis, bronchiolitis obliterans, chronic
eosinophilic pneumonia, lymphocytic infiltrative lung disease,
postinfectious interstitial lung disease, gouty arthritis,
autoimmune hepatitis, type-1 autoimmune hepatitis (classical
autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis
(anti-LKM antibody hepatitis), autoimmune mediated hypoglycaemia,
type B insulin resistance with acanthosis nigricans,
hypoparathyroidism, acute immune disease associated with organ
transplantation, chronic immune disease associated with organ
transplantation, osteoarthrosis, primary sclerosing cholangitis,
psoriasis type 1, psoriasis type 2, idiopathic leucopaenia,
autoimmune neutropaenia, renal disease NOS, glomerulonephritides,
microscopic vasulitis of the kidneys, lyme disease, discoid lupus
erythematosus, male infertility idiopathic or NOS, sperm
autoimmunity, multiple sclerosis (all subtypes), sympathetic
ophthalmia, pulmonary hypertension secondary to connective tissue
disease, Goodpasture's syndrome, pulmonary manifestation of
polyarteritis nodosa, acute rheumatic fever, rheumatoid
spondylitis, Still's disease, systemic sclerosis, Sjorgren's
syndrome, Takayasu's disease/arteritis, autoimmune
thrombocytopaenia, idiopathic thrombocytopaenia, autoimmune thyroid
disease, hyperthyroidism, goitrous autoimmune hypothyroidism
(Hashimoto's disease), atrophic autoimmune hypothyroidism, primary
myxoedema, phacogenic uveitis, primary vasculitis, vitiligo acute
liver disease, chronic liver diseases, alcoholic cirrhosis,
alcohol-induced liver injury, choleosatatis, idiosyncratic liver
disease, Drug-Induced hepatitis, Non-alcoholic Steatohepatitis,
allergy and asthma, group B streptococci (GBS) infection, mental
disorders (e.g., depression and schizophrenia), Th2 Type and Th1
Type mediated diseases, acute and chronic pain (different forms of
pain), and cancers such as lung, breast, stomach, bladder, colon,
pancreas, ovarian, prostate and rectal cancer and hematopoietic
malignancies (leukemia and lymphoma), Abetalipoprotemia,
Acrocyanosis, acute and chronic parasitic or infectious processes,
acute leukemia, acute lymphoblastic leukemia (ALL), acute myeloid
leukemia (AML), acute or chronic bacterial infection, acute
pancreatitis, acute renal failure, adenocarcinomas, aerial ectopic
beats, AIDS dementia complex, alcohol-induced hepatitis, allergic
conjunctivitis, allergic contact dermatitis, allergic rhinitis,
allograft rejection, alpha-1-antitrypsin deficiency, amyotrophic
lateral sclerosis, anemia, angina pectoris, anterior horn cell
degeneration, anti cd3 therapy, antiphospholipid syndrome,
anti-receptor hypersensitivity reactions, aordic and peripheral
aneuryisms, aortic dissection, arterial hypertension,
arteriosclerosis, arteriovenous fistula, ataxia, atrial
fibrillation (sustained or paroxysmal), atrial flutter,
atrioventricular block, B cell lymphoma, bone graft rejection, bone
marrow transplant (BMT) rejection, bundle branch block, Burkitt's
lymphoma, Burns, cardiac arrhythmias, cardiac stun syndrome,
cardiac tumors, cardiomyopathy, cardiopulmonary bypass inflammation
response, cartilage transplant rejection, cerebellar cortical
degenerations, cerebellar disorders, chaotic or multifocal atrial
tachycardia, chemotherapy associated disorders, chromic myelocytic
leukemia (CML), chronic alcoholism, chronic inflammatory
pathologies, chronic lymphocytic leukemia (CLL), chronic
obstructive pulmonary disease (COPD), chronic salicylate
intoxication, colorectal carcinoma, congestive heart failure,
conjunctivitis, contact dermatitis, cor pulmonale, coronary artery
disease, Creutzfeldt-Jakob disease, culture negative sepsis, cystic
fibrosis, cytokine therapy associated disorders, Dementia
pugilistica, demyelinating diseases, dengue hemorrhagic fever,
dermatitis, dermatologic conditions, diabetes, diabetes mellitus,
diabetic ateriosclerotic disease, Diffuse Lewy body disease,
dilated congestive cardiomyopathy, disorders of the basal ganglia,
Down's Syndrome in middle age, drug-induced movement disorders
induced by drugs which block CNS dopamine receptors, drug
sensitivity, eczema, encephalomyelitis, endocarditis,
endocrinopathy, epiglottitis, epstein-barr virus infection,
erythromelalgia, extrapyramidal and cerebellar disorders, familial
hematophagocytic lymphohistiocytosis, fetal thymus implant
rejection, Friedreich's ataxia, functional peripheral arterial
disorders, fungal sepsis, gas gangrene, gastric ulcer, glomerular
nephritis, graft rejection of any organ or tissue, gram negative
sepsis, gram positive sepsis, granulomas due to intracellular
organisms, hairy cell leukemia, Hallerrorden-Spatz disease,
hashimoto's thyroiditis, hay fever, heart transplant rejection,
hemachromatosis, hemodialysis, hemolytic uremic
syndrome/thrombolytic thrombocytopenic purpura, hemorrhage,
hepatitis (A), His bundle arrythmias, HIV infection/HIV neuropathy,
Hodgkin's disease, hyperkinetic movement disorders, hypersensitity
reactions, hypersensitivity pneumonitis, hypertension, hypokinetic
movement disorders, hypothalamic-pituitary-adrenal axis evaluation,
idiopathic Addison's disease, idiopathic pulmonary fibrosis,
antibody mediated cytotoxicity, Asthenia, infantile spinal muscular
atrophy, inflammation of the aorta, influenza a, ionizing radiation
exposure, iridocyclitis/uveitis/optic neuritis,
ischemia-reperfusion injury, ischemic stroke, juvenile rheumatoid
arthritis, juvenile spinal muscular atrophy, Kaposi's sarcoma,
kidney transplant rejection, legionella, leishmaniasis, leprosy,
lesions of the corticospinal system, lipedema, liver transplant
rejection, lymphederma, malaria, malignamt Lymphoma, malignant
histiocytosis, malignant melanoma, meningitis, meningococcemia,
metabolic/idiopathic, migraine headache, mitochondrial multi.system
disorder, mixed connective tissue disease, monoclonal gammopathy,
multiple myeloma, multiple systems degenerations (Mencel
Dejerine-Thomas Shi-Drager and Machado-Joseph), myasthenia gravis,
mycobacterium avium intracellulare, mycobacterium tuberculosis,
myelodyplastic syndrome, myocardial infarction, myocardial ischemic
disorders, nasopharyngeal carcinoma, neonatal chronic lung disease,
nephritis, nephrosis, neurodegenerative diseases, neurogenic I
muscular atrophies, neutropenic fever, non-hodgkins lymphoma,
occlusion of the abdominal aorta and its branches, occulsive
arterial disorders, okt3 therapy, orchitis/epidydimitis,
orchitis/vasectomy reversal procedures, organomegaly, osteoporosis,
pancreas transplant rejection, pancreatic carcinoma, paraneoplastic
syndrome/hypercalcemia of malignancy, parathyroid transplant
rejection, pelvic inflammatory disease, perennial rhinitis,
pericardial disease, peripheral atherlosclerotic disease,
peripheral vascular disorders, peritonitis, pernicious anemia,
pneumocystis carinii pneumonia, pneumonia, POEMS syndrome
(polyneuropathy, organomegaly, endocrinopathy, monoclonal
gammopathy, and skin changes syndrome), post perfusion syndrome,
post pump syndrome, post-MI cardiotomy syndrome, preeclampsia,
Progressive supranucleo Palsy, primary pulmonary hypertension,
radiation therapy, Raynaud's phenomenon and disease, Raynoud's
disease, Refsum's disease, regular narrow QRS tachycardia,
renovascular hypertension, reperfusion injury, restrictive
cardiomyopathy, sarcomas, scleroderma, senile chorea, Senile
Dementia of Lewy body type, seronegative arthropathies, shock,
sickle cell anemia, skin allograft rejection, skin changes
syndrome, small bowel transplant rejection, solid tumors, specific
arrythmias, spinal ataxia, spinocerebellar degenerations,
streptococcal myositis, structural lesions of the cerebellum,
Subacute sclerosing panencephalitis, Syncope, syphilis of the
cardiovascular system, systemic anaphalaxis, systemic inflammatory
response syndrome, systemic onset juvenile rheumatoid arthritis,
T-cell or FAB ALL, Telangiectasia, thromboangitis obliterans,
thrombocytopenia, toxicity, transplants, trauma/hemorrhage, type
III hypersensitivity reactions, type IV hypersensitivity, unstable
angina, uremia, urosepsis, urticaria, valvular heart diseases,
varicose veins, vasculitis, venous diseases, venous thrombosis,
ventricular fibrillation, viral and fungal infections, vital
encephalitis/aseptic meningitis, vital-associated hemaphagocytic
syndrome, Wernicke-Korsakoff syndrome, Wilson's disease, xenograft
rejection of any organ or tissue, Acute coronary syndromes, Acute
Idiopathic Polyneuritis, Acute Inflammatory Demyelinating
Polyradiculoneuropathy, Acute ischemia, Adult Still's Disease,
Alopecia areata, Anaphylaxis, Anti-Phospholipid Antibody Syndrome,
Aplastic anemia, Arteriosclerosis, Atopic eczema, Atopic
dermatitis, Autoimmune dermatitis, Autoimmune disorder associated
with Streptococcus infection, Autoimmune Enteropathy, Autoimmune
hearingloss, Autoimmune Lymphoproliferative Syndrome (ALPS),
Autoimmune myocarditis, Autoimmune premature ovarian failure,
Blepharitis, Bronchiectasis, Bullous pemphigoid, Cardiovascular
Disease, Catastrophic Antiphospholipid Syndrome, Celiac Disease,
Cervical Spondylosis, Chronic ischemia, Cicatricial pemphigoid,
Clinically isolated Syndrome (CIS) with Risk for Multiple
Sclerosis, Conjunctivitis, Childhood Onset Psychiatric Disorder,
Chronic obstructive pulmonary disease (COPD), Dacryocystitis,
dermatomyositis, Diabetic retinopathy, Diabetes mellitus, Disk
herniation, Disk prolaps, Drug induced immune hemolytic anemia,
Endocarditis, Endometriosis, endophthalmitis, Episcleritis,
Erythema multiforme, erythema multiforme major, Gestational
pemphigoid, Guillain-Barre Syndrome (GBS), Hay Fever, Hughes
Syndrome, Idiopathic Parkinson's Disease, idiopathic interstitial
pneumonia, IgE-mediated Allergy, Immune hemolytic anemia, Inclusion
Body Myositis, Infectious ocular inflammatory disease, Inflammatory
demyelinating disease, Inflammatory heart disease, Inflammatory
kidney disease, IPF/UIP, Iritis, Keratitis, Keratojuntivitis sicca,
Kussmaul disease or Kussmaul-Meier Disease, Landry's Paralysis,
Langerhan's Cell Histiocytosis, Livedo reticularis, Macular
Degeneration, Microscopic Polyangiitis, Morbus Bechterev, Motor
Neuron Disorders, Mucous membrane pemphigoid, Multiple Organ
failure, Myasthenia Gravis, Myelodysplastic Syndrome, Myocarditis,
Nerve Root Disorders, Neuropathy, Non-A Non-B Hepatitis, Optic
Neuritis, Osteolysis, Pauciarticular JRA, peripheral artery
occlusive disease (PAOD), peripheral vascular disease (PVD),
peripheral artery disease (PAD), Phlebitis, Polyarteritis nodosa
(or periarteritis nodosa), Polychondritis, Polymyalgia Rheumatica,
Poliosis, Polyarticular JRA, Polyendocrine Deficiency Syndrome,
Polymyositis, polymyalgia rheumatica (PMR), Post-Pump Syndrome,
primary parkinsonism, Prostatitis, Pure red cell aplasia, Primary
Adrenal Insufficiency, Recurrent Neuromyelitis Optica, Restenosis,
Rheumatic heart disease, SAPHO (synovitis, acne, pustulosis,
hyperostosis, and osteitis), Scleroderma, Secondary Amyloidosis,
Shock lung, Scleritis, Sciatica, Secondary Adrenal Insufficiency,
Silicone associated connective tissue disease, Sneddon-Wilkinson
Dermatosis, spondilitis ankylosans, Stevens-Johnson Syndrome (SJS),
Systemic inflammatory response syndrome, Temporal arteritis,
toxoplasmic retinitis, toxic epidermal necrolysis, Transverse
myelitis, TRAPS (Tumor Necrosis Factor Receptor, Type 1 allergic
reaction, Type II Diabetes, Urticaria, Usual interstitial pneumonia
(UIP), Vasculitis, Vernal conjunctivitis, viral retinitis,
Vogt-Koyanagi-Harada syndrome (VKH syndrome), Wet macular
degeneration, and Wound healing.
[0236] In another aspect, the binding proteins of the invention are
useful for treating a disorder selected from the group consisting
of Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,
Adrenocortical Carcinoma, Anal Cancer, Appendix Cancer, Cerebellar
Astrocytoma, Cerebral Astrocytoma, Basal Cell Carcinoma, Bile Duct
Cancer, Extrahepatic, Bladder Cancer, Bone Cancer,
Osteosarcoma/Malignant Fibrous Histiocytoma Brain Stem Glioma,
Brain Tumor, Brain Stem Glioma, Cerebral strocytoma/Malignant
Glioma, Ependymoma, Medulloblastoma, Supratentorial Primitive
Neuroectodermal Tumors, Visual Pathway and Hypothalamic Glioma,
Breast Cancer, Bronchial Adenomas/Carcinoids, Carcinoid Tumor,
Carcinoid Tumor, Gastrointestinal Carcinoma of Unknown Primary,
Central Nervous System Lymphoma, Primary Cerebellar Astrocytoma,
Cervical Cancer, Chronic Lymphocytic Leukemia, Chronic Myelogenous
Leukemia Chronic Myeloproliferative Disorders, Colon Cancer,
Colorectal Cancer, Cutaneous T-Cell Lymphoma, Endometrial Cancer,
Ependymoma, Esophageal Cancer, Ewing Family of Tumors, Extracranial
Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile
Duct Cancer, Eye Cancer, Intraocular Melanoma Retinoblastoma,
Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal
Carcinoid Tumor, Gastrointestinal Stromal Tumor (GIST),
Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Ovarian
Germ Cell Tumor, Gestational Trophoblastic Tumor, Glioma, Brain
Stem Glioma, Cerebral Astrocytoma Glioma, Childhood Visual Pathway
and Hypothalamic Glioma, Hairy Cell Leukemia, Head and Neck Cancer,
Hepatocellular (Liver) Cancer, Hodgkin Lymphoma, Hypopharyngeal
Cancer, Intraocular Melanoma, Islet Cell Carcinoma (Endocrine
Pancreas), Kaposi Sarcoma, Kidney (Renal Cell) Cancer, Laryngeal
Cancer, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia,
Chronic Lymphocytic Leukemia, Chronic Myelogenous Leukemia, Hairy
Cell Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Non-Small
Cell Lung Cancer, Small Cell Lung Cancer, AIDS-Related Lymphoma,
Burkitt Lymphoma, Cutaneous T-Cell Lymphoma, Hodgkin Lymphoma,
Non-Hodgkin Lymphoma, Primary Central Nervous System Lymphoma,
Waldenstrom Macroglobulinemia, Malignant Fibrous Histiocytoma of
Bone/Osteosarcoma, Medulloblastoma, Melanoma, Intraocular (Eye)
Melanoma, Merkel Cell Carcinoma, Malignant Mesothelioma, Metastatic
Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple
Endocrine Neoplasia Syndrome, Multiple Myeloma/Plasma Cell
Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes,
Myelodysplastic/Myeloproliferative Diseases, Myelogenous Leukemia,
Chronic Myeloid Leukemia, Multiple Myeloma, Myeloproliferative
Disorders, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal
Cancer, Neuroblastoma, Oral Cancer, Oral Cavity Cancer, Lip and
Oropharyngeal Cancer, Osteosarcoma/Malignant Fibrous Histiocytoma
of Bone, Ovarian Cancer, Ovarian Epithelial Cancer, Ovarian Germ
Cell Tumor, Ovarian Low Malignant Potential Tumor, Pancreatic
Cancer, Islet Cell Pancreatic Cancer, Paranasal Sinus and Nasal
Cavity Cancer, Parathyroid Cancer, Penile Cancer, Pharyngeal
Cancer, Pheochromocytoma, Pineoblastoma and Supratentorial
Primitive Neuroectodermal Tumors, Pituitary Tumor, Plasma Cell
Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Prostate
Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and
Ureter, Transitional Cell Cancer, Retinoblastoma, Salivary Gland
Cancer, Sarcoma, Ewing Family of Tumors, Kaposi Sarcoma, Soft
Tissue Sarcoma, Uterine Sarcoma, Sezary Syndrome, Skin Cancer
(Nonmelanoma), Skin Cancer (Melanoma), Merkel Cell Skin Carcinoma,
Small Intestine Cancer, Squamous Cell Carcinoma, Metastatic
Squamous Neck Cancer with Occult Primary, Stomach (Gastric) Cancer,
Supratentorial Primitive Neuroectodermal Tumors, Cutaneous T-Cell
Lymphoma, Testicular Cancer, Throat Cancer, Thymoma, Thymoma and
Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the
Renal Pelvis and Ureter, Gestational Trophoblastic Tumor, Ureter
and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer,
Uterine Cancer, Endometrial Uterine Sarcoma, Vaginal Cancer, Visual
Pathway and Hypothalamic Glioma, Vulvar Cancer, Waldenstrom
Macroglobulinemia, and Wilms Tumor.
[0237] In another aspect, the invention provides a method for
detecting the presence of PGE.sub.2 in a sample in vitro (e.g., a
biological sample, such as serum, plasma, tissue, biopsy). The
subject method can be used to diagnose a disorder, e.g., an immune
cell-associated disorder. The method includes: (i) contacting the
sample or a control sample with the anti-PGE.sub.2 antibody or
fragment thereof as described herein; and (ii) detecting formation
of a complex between the anti-PGE.sub.2 antibody or fragment
thereof, and the sample or the control sample, wherein a
statistically significant change in the formation of the complex in
the sample relative to the control sample is indicative of the
presence of the PGE.sub.2 in the sample.
[0238] In yet another aspect, this application provides a method
for detecting the presence of PGE.sub.2 in vivo (e.g., in vivo
imaging in a subject). The subject method can be used to diagnose a
disorder, e.g., a PGE.sub.2-associated disorder. The method
includes: (i) administering the anti-PGE.sub.2 antibody or fragment
thereof as described herein to a subject or a control subject under
conditions that allow binding of the antibody or fragment to
PGE.sub.2; and (ii) detecting formation of a complex between the
antibody or fragment and PGE.sub.2, wherein a statistically
significant change in the formation of the complex in the subject
relative to the control subject is indicative of the presence of
PGE.sub.2.
[0239] Antibodies of the invention, or antigen binding portions
thereof, can be used alone or in combination to treat such
diseases. It should be understood that the antibodies of the
invention or antigen binding portion thereof can be used alone or
in combination with an additional agent, e.g., a therapeutic agent,
the additional agent being selected by the skilled artisan for its
intended purpose. For example, the additional agent can be a
therapeutic agent art-recognized as being useful to treat the
disease or condition being treated by the antibody of the present
invention. The additional agent also can be an agent that imparts a
beneficial attribute to the therapeutic composition, e.g., an agent
that affects the viscosity of the composition.
[0240] It should further be understood that the combinations that
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative for purposes and not intended to be limited. The
combinations, which are part of this invention, can be the
antibodies of the present invention and at least one additional
agent selected from the lists below. The combination can also
include more than one additional agent, e.g., two or three
additional agents if the combination is such that the formed
composition can perform its intended function.
[0241] The combination therapy can include one or more PGE.sub.2
antagonists, e.g., anti-PGE.sub.2 antibodies or fragments thereof,
coformulated with, and/or coadministered with, one or more
additional therapeutic agents, e.g., one or more cytokine and
growth factor inhibitors, immunosuppressants, anti-inflammatory
agents (e.g., systemic anti-inflammatory agents), anti-fibrotic
agents, metabolic inhibitors, enzyme inhibitors, and/or cytotoxic
or cytostatic agents, as described in more herein. Examples of
preferred additional therapeutic agents that can be coadministered
and/or coformulated with one or more PGE.sub.2 antagonists, e.g.,
anti-PGE.sub.2 antibodies or fragments thereof, include, but are
not limited to, one or more of: inhaled steroids; beta-agonists,
e.g., short-acting or long-acting beta-agonists; antagonists of
leukotrienes or leukotriene receptors; combination drugs such as
ADVAIR.TM.; IgE inhibitors, e.g., anti-IgE antibodies (e.g.,
XOLAIR.TM.); phosphodiesterase inhibitors (e.g., PDE4 inhibitors);
xanthines; anticholinergic drugs; mast cell-stabilizing agents such
as cromolyn; IL-4 inhibitors; IL-5 inhibitors; eotaxin/CCR3
inhibitors; antagonists of histamine or its receptors including HI,
H2, H3, and H4, and antagonists of prostaglandin D or its receptors
(DPI and CRTH2). Such combinations can be used to treat asthma and
other respiratory disorders. Additional examples of therapeutic
agents that can be coadministered and/or coformulated with one or
more anti-PGE2 antibodies or fragments thereof include one or more
of: TNF antagonists (e.g., a soluble fragment of a TNF receptor,
e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g.,
75 kD TNFR-IgG (75 kD TNF receptor-IgG fusion protein,
ENBREL.TM.)); TNF enzyme antagonists, e.g., TNF converting enzyme
(TACE) inhibitors; muscarinic receptor antagonists; TGF-beta
antagonists; interferon gamma; perfenidone; chemotherapeutic
agents, e.g., methotrexate, leflunomide, or a sirolimus
(raparnycin) or an analog thereof, e.g., CCI-779; COX2 and cPLA2
inhibitors; NSAIDs; immunomodulators; p38 inhibitors, TPL-2, MK-2
and NFkB inhibitors, among others. Other combinations are cytokine
suppressive anti-inflammatory drug(s) (CSAIDs); antibodies to or
antagonists of other human cytokines or growth factors, for
example, IL1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-15,
IL-16, IL-18, IL-21, IL-31, interferons, EMAP-II, GM-CSF, FGF, EGF,
PDGF, and edothelin-1, as well as the receptors of these cytokines
and growth factors. Antibodies of the invention, or antigen binding
portions thereof, can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30, CD40, CD45,
CD69, CD80 (B7.1), CD86 (B7.2), CD90, CTLA or their ligands
including CD154 (gp39 or CD40L).
[0242] Preferred combinations of therapeutic agents may interfere
at different points in the inflammatory cascade. Preferred examples
include TNF antagonists like chimeric, humanized or human TNF
antibodies, D2E7, (PCT Publication No. WO 97/29131), CA2
(Remicade.TM.), CDP 571, and soluble p55 or p75 TNF receptors,
derivatives, thereof, (p75TNFR1gG (Enbrel.TM.) or p55TNFR1gG
(Lenercept), and also TNF converting enzyme (TACE) inhibitors;
similarly IL-1 inhibitors (Interleukin-1-converting enzyme
inhibitors, IL-1RA etc.) may be effective for the same reason.
[0243] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or antibody portion of the
invention. A "therapeutically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired therapeutic result. A therapeutically effective amount
of the antibody or antibody portion may be determined by a person
skilled in the art and may vary according to factors such as the
disease state, age, sex, and weight of the individual, and the
ability of the antibody or antibody portion to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody,
or antibody portion, are outweighed by the therapeutically
beneficial effects. A "prophylactically effective amount" refers to
an amount effective, at dosages and for periods of time necessary,
to achieve the desired prophylactic result. Typically, since a
prophylactic dose is used in subjects prior to or at an earlier
stage of disease, the prophylactically effective amount will be
less than the therapeutically effective amount.
[0244] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
containing a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0245] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody or antibody
portion of the invention is 0.1-20 mg/kg, more preferably 1-10
mg/kg. It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0246] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods of the
invention described herein are obvious and may be made using
suitable equivalents without departing from the scope of the
invention or the embodiments disclosed herein. Having now described
the present invention in detail, the same will be more clearly
understood by reference to the following examples, which are
included for purposes of illustration only and are not intended to
be limiting of the invention.
EXEMPLIFICATION
Example 1
Generation and Isolation of Anti Prostaglandin E.sub.2 Monoclonal
Antibodies
Example 1.1
Assays to Identify Anti Human Prostaglandin E.sub.2 Antibodies
[0247] The following assays were used to identify and characterize
anti-prostaglandin E.sub.2 antibodies unless otherwise stated.
Example 1.1.A
ELISA
[0248] Enzyme linked immunosorbent assays to screen for antibodies
that bind prostaglandin E.sub.2 were performed according to at
least one of the two following methods.
Method 1
[0249] ELISA plates (Costar 3369, Corning, N.Y.) were coated with
50 .mu.l of anti-host Fc IgG (Sigma, St. Louis, Mo.) at 2 .mu.g/ml
in PBS (Invitrogen Carlsbad, Calif.). Following an overnight
incubation at 4.degree. C., washed with PBS and the plate was
blocked with 200 .mu.l Superblock (Pierce #37535, Rockford, Ill.).
The IgG containing samples were diluted to 1 .mu.g/ml in Assay
Buffer (10% Superblock in PBS containing 0.05% Surfactamps (Pierce
#37535, Rockford, Ill.) and 50 .mu.l/well added to each well and
incubated for 1 hour at room temperature. Plates were washed four
times with Tween-Tris Buffered Solution (TTBS). PGE2-biotinamide
(Cayman Chemicals, Ann Arbor, Mich.) was diluted to 30 nM and
serially diluted 1:3 in Assay Buffer. The titration curve samples
were added to each IgG sample at 50 .mu.l/well and incubated for 1
hour at room temperature. The plates were washed as previously
described and 50 .mu.l of 1:5000 dilution of streptavidin polyhrp40
(Fitzgerald Industries, Concord, Mass.) in Assay Buffer was added
to each well and incubated for 45 minutes at room temperature. A
final wash step was performed and the plates were developed using a
single step TMB system (Sigma #T8665, St. Louis, Mo.) and 100
.mu.l/well 2N H.sub.2SO.sub.4. Plates were read at 450 nm on a
Molecular Devices Spectramax plate reader (Sunnyvale, Calif.).
EC.sub.50 was determined using GraphPad Prism 5 (GraphPad Software,
La Jolla, Calif.).
Method 2
[0250] Alternatively, prostaglandin binding was determined using a
.sup.3H-PGE.sub.2 ELISA. Plates were coated at 50 .mu.L/well with 5
.mu.g/ml of goat anti-human IgG (Fc) (Thermo Scientific #31170,
Hudson, N.H.) or goat anti-mouse IgG (Fc) (Thermo Scientific
#31125, Hudson, N.H.) in PBS and incubated overnight at 4.degree.
C. The following day, plates were flicked and blotted dry. Plates
were blocked with 200 .mu.L/well of Superblock (Thermo Scientific
#37515, Hudson, N.H.) for 1 hour at room temperature. Plates were
flicked and blotted dry. Monoclonal antibodies were diluted to 0.04
.mu.g/ml in phosphate buffer solution with Tween 20 (PBST) (Abbott
Bioresearch Center, Worcester, Mass.) and 10% Superblock and 50
.mu.L of each antibody was added to each well of the pre-blocked
ELISA plate at 2 ng/well and incubated for 1 hour at room
temperature. Wells were washed 3 times with PBS+0.1% Tween-20. A
serial 3-fold titration of .sup.3H-PGE.sub.2 (Perkin Elmer #
NET-428, Waltham, Mass.) was prepared in PBST/10% Superblock. Fifty
microliters of the .sup.3H-PGE.sub.2 solution was added to each
well of the plate and incubated for 1 hour at room temperature.
Wells were washed manually 6 times with PBST/10% Superblock and 50
.mu.L of scintillation fluid (Perkin Elmer #6013621, Waltham,
Mass.) was added to each well. Plates were read using a TopCount
reader (Perkin Elmer, Waltham, Mass.) with a 5 minute count delay.
An EC.sub.50 number was determined using GraphPad Prism 5 (GraphPad
Software, La Jolla, Calif.).
Example 1.1.B
PGE.sub.2 Competition ELISA
[0251] Competition enzyme linked immunosorbent assays were
performed to determine prostaglandin binding specificity for
antibodies that bind prostaglandin E.sub.2, according to at least
one of the two following methods.
Method 1
[0252] ELISA plates (Costar 3369, Corning, N.Y.) were coated with
50 .mu.l/well of anti-host Fc IgG (Sigma, St. Louis, Mo.) at 2
.mu.g/ml in PBS (Invitrogen, Carlsbad, Calif.). Following an
overnight incubation at 4.degree. C., the plate was blocked with
200 .mu.l Superblock (Pierce #37535, Rockford, Ill.). The IgG
samples were diluted to 6 .mu.g/ml in Assay Buffer (10% Superblock
in PBS containing 0.05% Surfactamps (Pierce #37535, Rockford,
Ill.). The PGE.sub.2-biotinamide was diluted to 3 nM in Assay
Buffer. A titration curve in Assay Buffer was prepared for the
prostaglandins PGA.sub.2 (Cayman Chemicals, Ann Arbor, Mich.),
PGD.sub.2 (Cayman Chemicals, Ann Arbor, Mich.) and PGE.sub.2
(Cayman Chemicals, Ann Arbor, Mich.) starting at 300 nM by a 1:10
serial dilution. The reagents were added to tubes at a volume of 50
.mu.l each/tube and preincubated for 1 hour at room temperature.
Following the preincubation, the mix was transferred to the blocked
plates and allowed to incubate for 1 hour at room temperature.
Next, the plates were washed four times with Tween 20-Tris buffered
solution (TTBS). Streptavidin polyhrp40 in Assay Buffer (Fitzgerald
Industries, Concord, Mass.) at a 1:5000 dilution was then added to
the wells and incubated for 45 minutes at room temperature. A final
wash step was performed and the plates were developed using a
single step TMB system (Sigma #T8665, Sigma, St. Louis, Mo.) and
100 .mu.l/well 2N H.sub.2SO.sub.4. Plates were read at 450 nm on a
Molecular Devices Spectramax plate reader (Sunnyvale, Calif.).
Wells in which unlabeled prostaglandins competed with the
PGE.sub.2-biotinamide for binding resulted in a decrease of signal.
IC.sub.50 number was determined using GraphPad Prism 5 (GraphPad
Software, La Jolla, Calif.). The cross reactivity index was then
calculated by IC.sub.50 of PGE.sub.2/IC.sub.50 of other
prostaglandin(s).
Method 2
[0253] Alternatively, prostaglandin selectivity was determined
using a .sup.3H-PGE.sub.2 competitition ELISA. Plates were coated
with 50 .mu.L/well of 5 .mu.g/ml of goat anti-human IgG (Fc)
(Thermo Scientific #31170, Hudson, N.H.) or goat anti-mouse IgG
(Fc) (Thermo Scientific #31125, Hudson, N.H.) in PBS and incubated
overnight at 4.degree. C. The following day plates were flicked and
blotted dry. Plates were blocked with 200 .mu.L/well of Superblock
(Thermo Scientific #37515, Hudson, N.H.), 1 hour at room
temperature. Plates were flicked and blotted dry. Monoclonal
antibodies were diluted to 0.04 .mu.g/ml in PBST (Abbott
Bioresearch Center, Worcester, Mass.)/10% Superblock and 50 .mu.L
of each was added to each well (2 ng/well) of the pre-blocked ELISA
plate and incubated for 1 hour at room temperature. Wells were
washed 3 times with PBS/0.1% Tween-20. .sup.3H-PGE.sub.2 (Perkin
Elmer # NET-428, Waltham, Mass.) was diluted in PBST/10% Superblock
to 6nM (2.times. stock). Each prostaglandin (Cayman Chemicals, Ann
Arbor, Mich.) was prepared in PBST+10% Superblock at various
concentrations ranging from 2000 .mu.M (2.times. stock) to 0.00004
04 (2.times.). Equal volumes of the .sup.3H-PGE.sub.2 solution and
of each prostaglandin dilution were mixed. Fifty microliters of
this mixture was then added to each well of the plate and incubated
for 1 hour at room temperature. Wells were washed manually 6 times
with PBST/10% Superblock and 50 .mu.L of scintillation fluid
(Perkin Elmer #6013621, Waltham, Mass.) added to each well. Plates
were read using a TopCount reader (Perkin Elmer, Waltham, Mass.)
with a 5 minute count delay. IC.sub.50 number was determined using
GraphPad Prism 5 (GraphPad Software, La Jolla, Calif.). The cross
reactivity index was then calculated by IC.sub.50 of
PGE.sub.2/IC.sub.50 of other prostaglandin(s)
Example 1.1.C
Measurement of Functional Activity of Anti Prostaglandin E.sub.2
Antibodies
[0254] To examine the functional activity of the anti-PGE.sub.2
antibodies of the invention, the antibodies were used in the
following in vitro and in vivo assays that measure the ability of
an antibody to inhibit PGE.sub.2 activity.
Example 1.1.C.1
EP4 Bioassay
[0255] The ability of anti-PGE.sub.2 antibody to inhibit cellular
response of PGE.sub.2 in vitro was determined in a Ca.sup.++ flux
assay in HEK293 G.alpha.16 cells (Abbott Bioresearch Center,
Worcester, Mass.) stably transfected with human EP4 receptor. In
brief, an expression plasmid encoding one of the four human
PGE.sub.2 receptors, EP4, and an expression plasmid encoding
G.alpha.16, were co-transfected into human embryonic kidney cell
line 293 cells (ATCC# CRL1573, Manassas, Va.). The stable clones
co-expressing both human EP4 and G.alpha.16 protein were selected
using standard methods (Joseph Sambrook and David W. Russell.
Molecular Cloning: A Laboratory Manual Publisher. Published by Cold
Spring Harbor Laboratory Press, 2001) and used for the EP4
bioassay.
[0256] The HEK293 G.alpha.16 cells were plated in black/clear
poly-D-lysine plates (Corning #3667, Corning, N.Y.) and incubated
with a Ca.sup.++-sensitive dye (Molecular Devices, Sunnyvale,
Calif.) for 90 minutes. Stock PGE.sub.2 (in 200 proof ethanol) was
diluted with FLIPR buffer [containing 1.times.HBSS (Invitrogen
Carlsbad, Calif.), 20 mM HEPES (Invitrogen Carlsbad, Calif.), 0.1%
BSA (Sigma, St. Louis, Mo.) and 2.5 mM Probenecid (Sigma, St.
Louis, Mo.)]. Anti-PGE.sub.2 antibodies or isotype matched control
antibodies were also pre-diluted in FLIPR buffer. 25 .mu.l of
PGE.sub.2 or pre-incubated PGE.sub.2/antibody mixture was added to
the wells pre-plated with cells. The dose response of PGE.sub.2 was
determined on a serial titration of PGE.sub.2 and using FLIPR1 or
Tetra (Molecular Devices, Sunnyvale, Calif.) and EC.sub.50 was
determined using GraphPad Prism 5 (GraphPad Software, La Jolla,
Calif.). For testing antibodies, PGE.sub.2 at EC.sub.50
concentration was incubated with varying concentrations of test
antibodies or isotype matched antibody (negative control) (ABC) for
20 minutes, and added to dye-loaded human EP4 in HEK293 G.alpha.16
cells. Ca.sup.++ flux was monitored using FLIPR1 and data was
analyzed using GraphPad Prism 5 (GraphPad Software, La Jolla,
Calif.).
Example 1.1.C.2
Competitive Inhibition of PGE.sub.2 Binding to PGE.sub.2 Receptors
by Anti Prostaglandin E.sub.2 Antibodies Using
.sup.3H-PGE.sub.2
[0257] Competitive inhibition of PGE.sub.2 binding to PGE.sub.2
receptors, for example EP4 or EP3, by an anti-PGE.sub.2 antibody
are determined using a cell-based or membrane based receptor
binding assay using .sup.3H-PGE.sub.2 (ProstaglandinE2,
[5,6,8,11,12,14,15-3H(N)], Perkin Elmer, Waltham, Mass. Cat#
NET428250UC).
[0258] Cells endogenously expressing or stably overexpressing EP4
receptor (i.e., HEK293-EP4 cells or HEK293-EP4-G.alpha.16 cells
used for EP4 bioassay) (10.sup.5 cells/mL) are grown overnight in a
24-well plate in DMEM medium (Invitrogen, Carlsbad, Calif.)/10% FCS
(Sigma #T8665, Sigma, St. Louis, Mo.). The medium is removed and
100 .mu.l binding buffer (medium without FCS) is added. The plate
is placed on ice for 10 minutes. Non-radioactive PGE.sub.2 (0-1
.mu.M) is added together with tracer (40 pM of .sup.3H-PGE.sub.2)
in 100 .mu.l volume. Equilibrium receptor binding is performed for
90 minutes at 4.degree. C. The medium is removed and the cells are
washed four times with 200 .mu.l cold medium. The cells are
harvested by adding 20 .mu.l 0.5 M NaOH. The lysate is transferred
to a liquid scintillation plate. 100 .mu.l Aquasafe 500 (Zinsser
Analytic, Frankfurt, Germany) plus LSC cocktail (Lumac LSC,
Groningen, The Netherlands) is added to each well and mixed. The
cell-bound radioactivity is determined by liquid scintillation
counting. For most agonist-receptor interactions, it is assumed
that receptor binding inhibition by agonist (PGE.sub.2) follows a
one-site model. The EC.sub.50, K.sub.i and K.sub.d values are
calculated using the GraphPad Prism 5 (GraphPad Software, La Jolla,
Calif.).
[0259] Inhibition of an anti-PGE.sub.2 antibody on the binding of
.sup.3H-PGE.sub.2 (ProstaglandinE2, [5,6,8,11,12,14,15-3H(N)],
Perkin Elmer, Waltham, Mass. Cat# NET428250UC) to the EP3 receptor
was performed using membrane preparations from cells that
over-express the EP3 receptor (Millipore, Billerica, Mass.). Before
the binding assay, 50 .mu.l/well of 0.3% Polyethyleneimine (PEI)
(Sigma, St. Louis, Mo.) was added to a Unifilter-96 GF/B filter
plate (Perkin Elmer, Waltham, Mass.) and placed at 4.degree. C. for
one hour until ready for use. A 1:3 dilution of antibody was
prepared at 2.times. concentration in binding buffer (50 mM HEPES
pH 7.0, 10 mM MgCl.sub.2, 1 mM EDTA, 0.2% BSA). .sup.3H-PGE.sub.2
was prepared at 2.times. concentration in binding buffer. 50 .mu.l
of a serial 3 fold dilution .sub.[gl]of antibody was then added to
each well containing 50 .mu.l of 200 pM .sup.3H-PGE.sub.2, mixed
well and allowed to sit at room temperature for 10 minutes. Frozen
membranes were thawed and resuspended in binding buffer. 5 .mu.g of
membrane was added to each well. Mixtures were incubated at room
temperature for 60 minutes before filtering onto pretreated GF/B
filtration plates using a Packard 96 well harvester. Plates were
then dried for one hour before adding Microscint.TM.20 (Perkin
Elmer, Waltham, Mass.). Plates were then sealed and counted on the
TopCount reader (Perkin Elmer, Waltham, Mass.). Non-specific
binding was determined in the presence of 100 .mu.M cold PGE.sub.2.
The measured radioactivity (cpm) was used to determine IC.sub.50
values using Graphpad Prism (GraphPad Software, La Jolla,
Calif.).
Example 1.1.C.3
Competitive Inhibition of PGE.sub.2 Binding to PGE.sub.2 Receptors
by Anti Prostaglandin E, Antibodies Using a Cell Based FACS
Assay
[0260] Competitive inhibition of PGE.sub.2 binding to PGE.sub.2
receptors, for example EP4, by an anti-PGE.sub.2 antibody can be
determined using a cell-based FACS assay using
PGE.sub.2-biotinimide (Cayman Chemical, Ann Arbor, Mich.
Cat#10006987) and Streptavidin-R-Phycoerythrin (SA-RPE; Invitrogen,
Carlsbad, Calif., Cat#15-4301). Cells (1.times.10.sup.6)
endogenously expressing or stably overexpressing EP4 (i.e.,
HEK293-EP4 cells or HEK293-EP4-G.alpha.16 cells used for EP4
bioassay) are cultivated in DMEM medium (Invitrogen, Carlsbad,
Calif.)/10% FCS (Sigma #T8665, Sigma, St. Louis, Mo.). Cells are
harvested and washed several times with 500 .mu.l washing buffer
(PBS/1% BSA). Cells are resuspended in 500 .mu.l FACS binding
buffer (medium without FCS). 20 .mu.l PGE.sub.2-biotinimide is
added to the cell suspension and incubated at 4.degree. C. for 1
hour. Cells are washed with washing buffer three times. The cells
are resuspended in 500 .mu.l FACS binding buffer and 20 .mu.l
SA-RPE is added to the cells and incubated at 4.degree. C. for 30
minutes. Cells are then resuspended in 500 .mu.l FACS binding
buffer and the binding of PGE.sub.2 on the cell surface is analyzed
by flow cytometry. The inhibition of an anti-PGE.sub.2 antibody can
be determined by pre-incubating the cells with a titration of
anti-PGE.sub.2 antibody before incubation with
PGE.sub.2-biotinimide and SA-RPE.
Example 1.2
Generation of Anti-Prosta Landing E.sub.2 Monoclonal Antibodies by
Hybridoma Approach
[0261] Anti-prostaglandin E.sub.2 mouse monoclonal antibodies were
obtained as follows:
Example 1.2.A
Immunization of Mice with a Prostaglandin E.sub.2-Thyroglobulin
Conjugate
[0262] Twenty micrograms of PGE.sub.2/thyroglobulin conjugate mixed
with complete Freund's adjuvant (Pierce, Rockford, Ill.) or
Immunoeasy adjuvant (Qiagen, Valencia, Calif.) was injected
subcutaneously into five 6-8 week-old Balb/C mice, five C57B/6
mice, and five AJ mice on Day 1. On days 24, 38, and 49, 25 .mu.g
of PGE.sub.2/thyroglobulin conjugate mixed with incomplete Freund's
adjuvant or Immunoeasy adjuvant was injected subcutaneously into
the same mice. On days 84, 112, or 144, mice were injected
intravenously with 1 .mu.g PGE.sub.2/thyroglobulin conjugate.
Example 1.2.B
Generation of Hybridomas
[0263] Splenocytes obtained from the immunized mice described in
Example 1.2.A were fused with SP2/O--Ag-14 cells at a ratio of 5:1
according to the established method described in Kohler, G. and
Milstein, Nature, 256:495 (1975) to generate hybridomas. Fusion
products were plated in selection media containing azaserine
(Pierce, Rockford, Ill.) and hypoxanthine (Pierce, Rockford, Ill.)
in 96-well plates at a density of 2.5.times.10.sup.6 spleen cells
per well. Seven to ten days post fusion, macroscopic hybridoma
colonies were observed. Supernatant from each well containing
hybridoma colonies was tested by ELISA (as described in Example
1.1.A) for the presence of antibody to PGE.sub.2.
[0264] PGE.sub.2 was conjugated to several different carrier
proteins, including bovine thyroglobulins, keyhole limpet
hemocyanin, bovine serum albumin, and ovalbumin. (Amiram, et al.
Eur. J. Biochem. 53:145-150 (1975)). Mice were immunized with one
of these conjugated PGE.sub.2-protein complexes as described in
Example 1.2.A. Spleen cells from immunized mice were then fused to
generate hybridomas as described in Example 1.2.B. The hybridomas
producing antibodies specific for PGE.sub.2 were isolated and the
antibodies characterized using a biotinylated PGE.sub.2 ELISA as
described in Example 1.1.A.
Example 1.2.C
Identification and Characterization of Anti-Prostaglandin E.sub.2
Monoclonal Antibodies
[0265] Hybridomas producing antibodies that bind PGE.sub.2,
generated according to Examples 1.2.B, were scaled up and cloned by
limiting dilution.
[0266] Several antibodies, named 19C9, 4F10 and 15F10 specific for
PGE.sub.2 were isolated. The affinities of these antibodies were
determined by ELISA using biotinylated PGE.sub.2 as described in
Example 1.1.A (FIGS. 1 and 2). The specificity of these antibodies
for PGE.sub.2 was further determined by a competition ELISA with
various prostaglandins as described in Example 1.1.B (Table 2).
TABLE-US-00002 TABLE 2 Affinity and Cross Reactivity of 19C9, 4F10,
15F10, and 2B5 Antibodies 19C9 4F10 15F10 2B5 EC50 (nM) 16 6 2
0.048 23 14 5 0.033 Cross PGE1: <5.6% PGE1: <3.2% PGE1:
<2.9% PGE1: ~20% Reactivity PGA2: ~0.1% PGA2: ~0.1% PGA2:
<0.1% PGA2: ~0.6% Index PGD2: <0.01% PGD2: <0.01% PGD2:
<0.01% PGD2: <0.01%
Example 2
Human Anti Prostaglandin E.sub.2 Antibodies by In Vitro Display
Technology
Example 2.1
Human Anti Prostaglandin E.sub.2 Antibodies Selected from
Non-Immune Human Antibody Libraries by In Vitro Display
Technology
[0267] Using PROfusion.TM. mRNA display, human anti-PGE.sub.2
antibodies were selected from non-immune human antibody libraries
by in vitro display technology in the single chain Fv (scFv)
format. Antibody amino acid sequences that encoded biotinylated
PGE.sub.2-binding scFv proteins were collected using streptavidin
or neutravidin magnetic beads and further enriched from the
libraries by multiple rounds of selection. Bulk output scFv nucleic
acid sequences were subcloned into plasmid DNA suitable for
bacterial propagation and individual bacterial colonies were picked
for scFv sequence analysis and confirmation of their PGE.sub.2
binding by the same antigen binding assay used in the library
selection. VH and VL DNA of PGE.sub.2-binding scFv clones was then
separately subcloned into respective human IgG-expressing heavy
chain and light chain vectors, and transfected into COS7 cells for
IgG expression. The human IgG-containing COS7 media were then used
to confirm PGE.sub.2 binding by ELISA as described in Example 1.1.A
(Table 3).
TABLE-US-00003 TABLE 3 Binding of PROfusion Library Derived
PGE.sub.2 Antibodies To PGE.sub.2- Biotinamide (OD.sub.450) Biotin-
Antibody PGE.sub.2 (.mu.M) K1B K7H K3A L11 L12A L21 L20 Control 45
0.88 0.24 0.58 0.07 0.26 0.46 0.12 0.07 15 0.82 0.29 0.78 0.07 0.22
0.41 0.09 0.05 5 0.67 0.13 0.67 0.07 0.18 0.31 0.07 0.05 2 0.27
0.10 0.61 0.07 0.13 0.32 0.06 0.08 1 0.29 0.06 0.51 0.05 0.09 0.25
0.06 0.05 0.3 0.21 0.04 0.34 0.04 0.09 0.16 0.05 0.03 0.1 0.13 0.03
0.22 0.04 0.07 0.10 0.06 0.04 0 0.07 0.04 0.11 0.04 0.08 0.08 0.07
0.03
[0268] Table 4 provides a list of amino acid sequences of VH and VL
regions of human anti-PGE.sub.2 antibodies derived from the
PROfusion.TM. mRNA display library.
TABLE-US-00004 TABLE 4 List of Amino Acid Sequences of VH and VL
regions SEQ ID Sequence No. Protein region
123456789012345678901234567890 5 VH PGE2LNK1B
EVQLVQSGAEVKRPGASVKVSCKTSGYTFT NYDINWVRLAPGQGLEWMGCMNPTTGKTGY
AQKFQGRVTMTRDTTIATAYMELSRLTSED TAVYYCARGRGYSPGYGVAYADYWGQGTLV TVSS
6 VH PGE2LNK1B Residues 31-35 NYDIN CDR-H1 of SEQ ID NO.: 5 7 VH
PGE2LNK1B Residues 50-66 CMNPTTGKTGYAQKFQG CDR-H2 of SEQ ID NO.: 5
8 VH PGE2LNK1B Residues 99- GRGYHPGYGVAYADY CDR-H3 113 of SEQ ID
NO.: 5 9 VL PGE2LNK1B DIQLTQSPSSLPASVGDRVTITCRASQSIS
TYLNWYQQTPGKAPSLLIYAASSLQSGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQQ
SYSPPPTFGGGTKVEIKR 10 VL PGE2LNK1B Residues 24-34 RASQSISTYLN CDR
-L1 of SEQ ID NO.: 9 11 VL PGE2LNK1B Residues 50-56 AASSLQS CDR-L2
of SEQ ID NO.: 9 12 VL PGE2LNK1B Residues 89-97 QQSYSPPPT CDR-L3 of
SEQ ID NO.: 9 13 VH PGE2LNK3A EVQLVQSGAETKKPGASVEVSCKASGYSFT
EYGISWVRQAPGQGPEWMGCISPYNGKLHY AQEFQGRVTMTTGTSTNTAYMELGSLRSDD
TAVYYCARGGFSFYDSSGYYYVTDHWGQGT LVTVSS 14 VH PGE2LNK3A Residues
31-35 EYGIS CDR-H1 of SEQ ID NO.: 13 15 VH PGE2LNK3A Residues 50-66
CISPYNGKLHYAQEFQG CDR-H2 of SEQ ID NO.: 13 16 VH PGE2LNK3A Residues
99- GGFSFYDSSGYYYVTDH CDR-H3 115 of SEQ ID NO.: 13 17 VL PGE2LNK3A
DIRLTQSPSSLSASVGDRVTITCRASQSIG SYLNWYQQKSGKAPKLLIYAASKLQSGVPS
RFSGSGFGTDFTLTISSLQPEDSATYYCQQ SDTTPFTFGQGTKLEIKR 18 VL PGE2LNK3A
Residues 24-34 RASQSIGSYLN CDR-L1 of SEQ ID NO.: 17 19 VL PGE2LNK3A
Residues 50-56 AASKLQS CDR-L2 of SEQ ID NO.: 17 20 VL PGE2LNK3A
Residues 89-97 QQSDTTPFT CDR-L3 of SEQ ID NO.: 17 21 VH PGE2LNK7H
EVQLVQSGSELKKPGASVKVSCKASGYSFT EYGISWVRQAPGQGPEWMGCISPYNGKLHY
AQKFLGRVTMTTDTSTNTAYMELRSLKSDD TAVYYCARGGFSSYDSSGYYYVTDHWGQGT
LVTVSS 14 VH PGE2LNK7H Residues 31-35 EYGIS CDR-H1 of SEQ ID NO.:
21 22 VH PGE2LNK7H Residues 50-66 CISPYNGKLHYAQKFLG CDR-H2 of SEQ
ID NO.: 21 23 VH PGE2LNK7H Residues 99- GGFSSYDSSGYYYVTDH CDR-H3
115 of SEQ ID NO.: 21 24 VL PGE2LNK7H
DIRLTQSPSSLPASVGDRVTITCRASQSIS TYLNWYQQTPGKAPSILIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQ SYSPPPTFGGGTKVEIKR 10 VL PGE2LNK7H
Residues 24-34 RASQSISTYLN CDR-L1 of SEQ ID NO.: 24 11 VL PGE2LNK7H
Residues 50-56 AASSLQS CDR-L2 of SEQ ID NO.: 24 12 VL PGE2LNK7H
Residues 89-97 QQSYSPPPT CDR-L3 of SEQ ID NO.: 24 25 VH PGE2LNL11
EVQLVQSGPELKKPGTSVKVSCKASGYTLT TYAMNWVRQAPGQGLEWMGWIDTSTGNPTY
APGFLGRFVFSLDTSLSTTYLQISSLKPDD TAVYYCARSSHTRPGDFWGQGTLVTVSS 26 VH
PGE2LNL11 Residues 31-35 TYAMN CDR-H1 of SEQ ID NO.: 25 27 VH
PGE2LNL11 Residues 50-66 WIDTSTGNPTYAPGFLG CDR-H2 of SEQ ID NO.: 25
28 VH PGE2LNL11 Residues 99- SSHTRPGDF CDR-H3 113 of SEQ ID NO.: 25
29 VL PGE2LNL11 QSGLTQPPSVSGTPGQRVTISCSGSESNVG
TNSVNWYQQLPGAAPRLLIRGNSDRPSGVP DRFSASKSGTSASLAISRLQSEDEADYFCG
ACDGRLSGLYVFGTGTKVTVL 30 VL PGE2LNL11 Residues 23-35 SGSESNVGTNSVN
CDR-L1 of SEQ ID NO.: 29 31 VL PGE2LNL11 Residues 51-56 GNSDPP
CDR-L2 of SEQ ID NO.: 29 32 VL PGE2LNL11 Residues 90- GACDGRLSGLYV
CDR-L3 101 of SEQ ID NO.: 29 33 VH PGE2LNL21
EVQLVQSGSELKKPGTSVKVSCKASGYTLT TYAMNWVRQAPGQGLEWMGWIGTSTGNPTY
AQGFTGRFVFSLDTSVNTAHLQIYSLKAED TALYYCARSSLTRPADYWGQGTLVTVSS 26 VH
PGE2LNL21 Residues 31-35 TYAMN CDR Hl of SEQ ID NO.: 33 34 VH
PGE2LNL21 Residues 50-66 WIGTSTGNPTYAQGFTG CDR-H2 of SEQ ID NO.: 33
35 VH PGE2LNL21 Residues 99- SSLTRPADY CDR-H3 107 of SEQ ID NO.: 33
36 VL PGE2LNL21 QSGLTQPPSVSGAPGQRVTISCFGSSSNIG
AGYDVHWYQQLPGAAPKLLIFGNNNRPSGV PDRFSGSKSGTSASLAITGLQAEDEADYYC
QSCDSSLSGAVFGTGTRVTVL 37 VL PGE2LNL21 Residues 23-36 FGSSSNIGAGYDVH
CDR-L1 of SEQ ID NO.: 36 38 VL PGE2LNL21 Residues 52-57 GNNNRP
CDR-L2 of SEQ ID NO.: 36 39 VL PGE2LNL21 Residues 91- QSCDSSLSGAV
CDR-L3 101 of SEQ ID NO.: 36
Example 3
Generation and Characterization of Recombinant Anti Prostaglandin
E.sub.2 Antibodies According to Solved Protein Sequence by a
Combination of Edman Sequencing, Mass Spectrum Analysis and
BLAST
[0269] The protein sequence of hybridoma-derived mouse antibodies
specific for PGE.sub.2 was generated by analyzing amino acid
sequences using a combination of Edman degradation, mass spectrum
analysis and BLAST (Basic Local Alignment Search Tool, NCBI, NIH,
Bethesda, Md.) as described previously (Pham, V. et al. Analyt.
Biochem. 352:77-86 (2006)). 0.45 mg of anti-PGE.sub.2 antibody was
reduced with 100 mM DTT (Invitrogen, Carlsbad, Calif.) to light
chain and heavy chain. The light chains and heavy chains of the
anti-PGE.sub.2 antibodies were separated by reverse phase HPLC on
Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia,
Md.) with a Vydac C-18 reverse phase column (H-P Separations Group,
Hesperia, Calif.). The molecular weights of the light and heavy
chains were measured on the Applied Biosystems API QSTAR Pulsar i
mass spectrometer (Applied Biosystems, Foster City, Calif.) and
Agilent Q-TOF mass spectrometer (Agilent, Palo Alto, Calif.). N
terminal sequencing of the light chains was performed in solution
on PE Applied Biosystems 494/785A/140C/610A Protein-Peptide
Sequencer (Applied Biosystems, Foster City, Calif.). 45 uL of light
chain of the anti-PGE2 2B5 antibody was loaded on the center of the
filter and 42 cycles were performed. The N-terminal of heavy chain
of the anti-PGE.sub.2 antibody was blocked with pyroglutamic acid
and it could not be sequenced directly by Edman degradation. Prior
to N-terminal Edman sequencing the heavy chain, the heavy chain N
terminal was de-blocked using pyroglutamate aminopeptidase (Sigma,
St. Louis, Mo.). 80 .mu.g of anti-PGE.sub.2 antibody was reduced
with 50 mM DTT at 37.degree. C. for 30 minutes. 0.42 .mu.l of 0.5 M
EDTA, pH 7.5 (Invitrogen, Carlsbad, Calif.) was added to the
reduced sample to a final EDTA concentration of 1 mM. 50 .mu.l
reconstituted recombinant pyrococcus furiosus pyroglutamate
aminopeptidase (Sigma, St. Louis, Mo.) was added to the sample.
After incubating the sample solution at 40.degree. C. for 15 hours,
the temperature was increased to 60.degree. C. for an additional
two hours. An additional 10 .mu.l of the reconstituted pyrococcus
furiosus pyroglutamate aminopeptidase was added and the sample was
incubated at 60.degree. C. for an additional hour. 4 .mu.l of the
sample was used for LC/MS analysis at 15 hour, 17 hour, and 18 hour
time points to monitor the extent of the de-blocking process. When
the de-blocking reaction was complete, the solution was
concentrated by speed-vacuum (Eppendorf, Westbury, N.Y.) to about
100 .mu.l. The de-blocked heavy chain was separated from light
chain by SDS-PAGE and then transferred to PVDF membrane
(Invitrogen, Carlsbad, Calif.) for Edman Sequencing (Niall, H D,
Methods Enzymol. 27:942-1010 (1973)).
[0270] To obtain the internal peptide sequence of an anti-PGE.sub.2
antibody, the antibody was digested with multiple proteases with or
without alkylation treatment. The sample was first reduced with
DTT. The reduced sample was either digested directly with a
protease or alkylated with iodoacetamide (Sigma, St. Louis, Mo.)
before digestion. Proteases used in this study include trypsin,
glu-C, asp-N and chymotrypsin (Sigma, St. Louis, Mo.). Fractions of
protease digested peptides were separated by HPLC and each fraction
was collected in a separate eppendorf tube for either MS or Edman
sequencing. For LC/MS/MS analysis, MALDI-MS (Applied Biosystems,
Foster City, Calif.), nano-LC/ESI-MS/MS (Applied Biosystems, Foster
City, Calif.) with either LCQ-deca, API QStar Pulsar (Applied
Biosystems, Foster City, Calif.) and Agilent Q-TOF (Agilent, Palo
Alto, Calif.) were used. HPLC conditions were mobile Phase A=0.1%
Formic acid; mobile Phase B=80% ACN/20% 0.1% Formic acid. A 1-3
hour gradient (5-50% B) was applied. For Edman sequencing, the
fractions containing peptides resulting from protease digestion
were transferred to PVDF membrane by a ProSorb cartridge (Applied
Biosystems, Foster City, Calif.). Each fraction was diluted to 100
.mu.l of 0.1% TFA solution (Sigma, St. Louis, Mo.). After wetting
the PVDF membrane (Invitrogen, Carlsbad, Calif.) in the reservoir
with 10 .mu.l methanol (Sigma, St. Louis, Mo.), the sample was
added to the reservoir. The sample was removed from the reservoir
and the PVDF membrane was air dried. The PVDF membrane was punched
out, 5 .mu.l of 10% diluted Biobrene solution (Sigma, St. Louis,
Mo.) was added and the membrane dried completely. After washing the
PVDF membrane with 15 .mu.l 0.1% TFA for 15 seconds, the surface
was wiped with filter paper. 4 .mu.l of methanol was added to the
PVDF membrane and allowed to dry thoroughly. The dried PVDF
membrane was used for Edman Sequencing.
[0271] Germline sequences of the VH and VL of an anti-PGE.sub.2
antibody were determined by alignment of the solved variable
regions of heavy chain and light chain according to the above
methods with the VH and VL database of mouse germline sequences
(Ig-BLAST, NCBI, NIH, Bethesda, Md.). For the regions unsolved by
MS and Edman sequencing, the closest germline sequence was
assigned. The possible hot spot mutations were identified manually
to match the experimental molecular weight of the heavy chain and
light chain of an anti-PGE.sub.2 antibody determined by MS
respectively. The protein sequence of an anti-PGE.sub.2 antibody
was solved using the above method.
[0272] Several versions of the recombinant anti-PGE.sub.2
antibodies (2B5-7.0, 2B5-8.0 and 2B5-9.0) were constructed based on
this solved protein sequence, each having different residues in a
few unsolved positions (Table 5). The testing of these recombinant
antibodies is described in Example 4. Although the amino acid
sequence of antibody CDRs are critical for binding specificity,
potency and affinity of the antibody, subsititutions, alterations,
deletions or additions of a few residues in the frameworks and even
CDRs may still largely maintain the binding specificity, potency,
and affinity of an antibody. A version of an antibody with at least
one or a few such substitution(s), alterations, deletions or
additions is still within the scope of the invention. An alignment
of VH and VL regions of anti-PGE.sub.2 antibodies (2B5-7.0, 2B5-8.0
and 2B5-9.0) is shown in Figure. 8.
TABLE-US-00005 TABLE 5 Several Versions Of The Solved Protein
Sequences For A Mouse Anti-PGE2 Antibody SEQ ID Protein Sequence
No. region 123456789012345678901234567890 40 VH 2B5-7.0
QVQLQQSGPELVRPGSSVKISCKASGYTFTK YWLGWVKQRPGHGLEWIGDIYPGYDYTHYNE
KFKDKATLTVDTSSSTAYMQLSSLTSEDSAV YFCARSDGSSTYWGQGTLVTVSA 41 VL
2B5-7.0 DVLMTQTPLSLPVSLGDQASISCTSSQNIVH
SNGNTYLEWYLQRPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTVFTLKISRVEAEDLGVYYC
FQVSHVPYTFGGGTKLEIKR 42 VH 2B5-8.0 QVQLQQSGPELVRPGSSVKISCKASGYTFTK
YWLGWVKQRPGHGLEWIGDIYPGYDYTHYNE KFKDKATLTVDTSSSTAYMQLSSLTSEDSAI
YYCARSDGSSTYWGQGTLVTVSA 43 VL 2B5-8.0
DVLMTQTPLSLPVSLGDQASISCTSSQNIVH SNGNTYLEWYLQRPGQSPKLLIYKVSNRFSG
VPDRFSGSGSGTVFTLKISRVEAEDLGVYYC FQVSHVPYTFGGGTKLEIKR 44 VH 2B5-9.0
QVQLQQSGPELVRPGSSVKISCKASGYTFTK YWLGWVKQRPGHGLEWIGDIYPYGDYTHYNE
KFKDKATLTVDTSSSTAYMQLSSLTSEDSAV YFCARSDGSSTYWGQGTLVTVSA 45 VL
2B5-9.0 DVLMTQTPLSLPVSLGDQASISCTSSQNIVH
SNGNTYLEWYLQRPGQSPKLLIYKVSNRFSG VPDRFSGSGSGTVFTLKISRVEAEDLGVYYC
FQVSHVPYTFGGGTKLEIKR
Example 4
Recombinant Anti-Prostaglandin E.sub.2 Antibodies
Example 4.1
Construction and Expression of Recombinant Anti-Prostaglandin
E.sub.2 Antibodies
[0273] DNA encoding the heavy chain variable regions of mouse
anti-PGE2 antibodies 2B5-7.0, 2B5-8.0 or 2B5-9.0 was fused to a
cDNA fragment encoding either a human IgG1 constant region, a mouse
IgG1 constant, or a mouse IgG2a constant region by homologous
recombination in bacteria. (Zhang, Y et al. Nature Biotechnol.
18(12):1314-7 (2000)). DNA encoding the light chain variable
regions of 2B5-7.0, 2B5-8.0 or 2B5-9.0 was fused to a human kappa
constant region or mouse kappa constant region. Id. Full-length
antibodies were transiently expressed in 293 cells by
co-transfection of heavy and light chain cDNAs ligated into the
pTT3 expression plasmids. (Durocher, Y et al. Nucleic Acids Res.
30(2):E9 (2002)). Cell supernatants containing recombinant chimeric
antibody were purified by Protein A Sepharose chromatography and
bound antibody was eluted by addition of acid buffer. Antibodies
were neutralized and dialyzed into PBS. (Making and Using
Antibodies: A Practical Handbook. Edited by Gary C. Howard and
Matthew R. Kaser. Published by CRC (2006)).
[0274] The purified chimeric anti-PGE.sub.2 monoclonal antibodies
2B5-7.0, 2B5-8.0 and 2B5-9.0 were then tested for their ability to
bind to PGE.sub.2 in an ELISA assay as described in Example 1.1.A
(Table 6) and for their selectivity in a competition ELISA as
described in Example 1.1.B (Table 6). All three recombinant
anti-PGE.sub.2 monoclonal antibodies 2B5-7.0, 2B5-8.0 and 2B5-9.0
potently bound to PGE.sub.2 with a similar specificity for
PGE.sub.2. 2B5-8.0 demonstrated the highest binding ability to
PGE.sub.2 and was selected for further characterization in an EP4
bioassay to characterize its ability to neutralize PGE.sub.2
bioactivity and in a .sup.3H-PGE.sub.2 competition ELISA to
characterize its prostaglandin selectivity using a full panel of
prostaglandins. 2B5-8.0 potently inhibited PGE.sub.2 induced
calcium influx in the EP4 bioassay as described in Examples 1.1.0
(Table 6).
TABLE-US-00006 TABLE 6 Characterization Of PGE.sub.2 Binding,
Prostaglandin Binding Selectivity And PGE.sub.2 Neutralization
Potency Of Engineered Anti-PGE.sub.2 Mabs Anti-PGE.sub.2 mAb 2B5
2B5-7.0 2B5-8.0 2B5-9.0 PGE.sub.2 binding in Biotin-PGE.sub.2 ELISA
2.99 2.46 1.03 3.04 (EC50, nM) PG Selectivity in PGE.sub.1 41 27 28
23 Biotin-PGE.sub.2 PGA.sub.2 0.29 0.11 0.21 0.24 Competition ELISA
PGD.sub.2 <0.01 <0.01 <0.01 <0.01 (CRI %) PGE.sub.2
binding in .sup.3H-PGE.sub.2 ELISA 315 253 (EC50, pM) PG
Selectivity PGE2 100 100 in .sup.3H-PGE.sub.2 PGE1 12 3.6
competition PGA2 0.17 0.04 ELISA PGD2 0.04 <1 .times. 10.sup.-4
(CRI %) PGF2.alpha. 0.25 0.05 PGI2 NA 0.16 Iloprost NA 0.01
Carbaprostacyclin NA 0.18 Pinane TXA2 NA 0.00033 15R-Pinane TXA2 NA
0.00043 Carbocyclic TXA2 NA 0.0005 TXB2 <0.01 <0.01 6-keto
PGF1.alpha. 0.4 0.64 PGB2 0.03 0.00038 8-iso PGF2.alpha. 0.02 0.092
13,14-dihydroxyl-15- <0.01 0.012 keto PGE2 2,3-dinor-6-keto- NA
0.019 PGF1.alpha. 15-keto PGE2 <0.01 0.013 19R-hydroxy PGE2
<0.01 0.09 LTE4 <0.01 0.012 5(S)-HETE <0.01 0.01
Arachidonic acid <0.01 <0.01 PGE.sub.2 Neutralization Potency
in 38 44 Cellular EP4 assay (IC50, pM)
Example 4.2
Construction and Expression of Humanized Anti-Prostaglandin E.sub.2
Antibodies
Example 4.2.1
Selection of Human Antibody Frameworks
[0275] Humanization was based on amino acid sequence homology, CDR
cluster analysis, frequency of use among expressed human
antibodies, and available information on the crystal structures of
human antibodies. Taking into account possible effects on antibody
binding, VH-VL pairing, and other factors, murine residues were
mutated to human residues where murine and human framework residues
were different, with a few exceptions. Additional humanization
strategies were designed based on an analysis of human germline
antibody sequences, or a subgroup thereof, that possessed a high
degree of homology, i.e., sequence similarity, to the actual amino
acid sequence of the murine antibody variable regions.
[0276] Homology modeling was used to identify residues unique to
the murine antibody sequences that are predicted to be critical to
the structure of the antibody CDRs. A reference protein sharing
sequence similarity to the target protein of interest and for which
three dimensional coordinates are known was used to obtain initial
coordinates and guidance for their further refinement. The primary
sequences of the reference and target proteins are aligned such
that coordinates of identical portions of the two proteins are
aligned. Coordinates for mismatched portions of the two proteins,
e.g., from residue mutations, insertions, or deletions, are
constructed from generic structural templates and energy refined to
insure consistency with the already aligned model coordinates. This
computational protein structure may be further refined or employed
directly in modeling studies.
[0277] The murine variable heavy and variable light chain gene
sequence of 2B5-8.0 was separately aligned against 44 human
immunoglobulin germline variable heavy chain or 46 germline
variable light chain sequences (derived from NCBI Ig BlAST website
at http://www.ncbi.nlm.nih.gov/igblast/retrieveig.html.) using
Vector NTI software. A combination of BLAST searching and visual
inspection was used to identify suitable reference structures.
Sequence identity of 25% between the reference and target amino
acid sequences is considered the minimum necessary to attempt a
homology modeling exercise. Sequence alignments were constructed
manually and model coordinates were generated with the program
Jackal (Petrey, D., et al. Proteins 53 (Suppl. 6):430-435 (2003)).
For 2B5-8.0 humanization, based on a homology search against human
V and J segment sequences, the VH segment VH1-18 and the J segment
JH4 were selected to provide the frameworks for the humanized heavy
chain variable region for 2B5-8.0. For the 2B5-8.0 light chain
variable region, the VL segment 01 and the J segment JK4 were used
(see Tables 7 and 8). The identity of the framework amino acids
between 2B5-8.0 VH and the acceptor human VH1-18 and JH4 segments
was 80.2%, while the identity between 2B5-8.0 VL and the acceptor
human 01 and JK4 segments was 90.3%. Although a specific pair of
preferred human framework acceptors VH/JH and VL/JK was selected as
acceptors for humanization of 2B5-8.0, it is known in the art that
other human framework acceptors with sequence identity of minimum
25% to mouse framework can also be used for humanization of 2B5-8.0
and are therefor within the scope of this invention.
TABLE-US-00007 TABLE 7 Heavy Chain Acceptor Sequences For 2B5-8.0
Humanization SEQ ID Protein Sequence No. region
12345678901234567890123456789012 46 VH1-18 &
QVQLQQSGPELVRPGSSVKISCKAS JH4 FR1 47 VH1-18 & WVKQRPGHGLEWIG
JH4 FR2 48 VH1-18 & KATLTVDTSSSTAYMQLSSLTSEDSAIYYCAR JH4 FR3 49
VH1-18 & WGQGTLVTVSA JH4 FR4
TABLE-US-00008 TABLE 8 Light Chain Acceptor Sequences For 2B5-8.0
Humanization SEQ ID Protein Sequence No. region
12345678901234567890123456789012 50 01 & JK4 FR1
DVLMTQTPLSLPVSLGDQASISC 51 01 & JK4 FR2 WYLQRPGQSPKLLIY 52 01
& JK4 FR3 VPDRFSGSGSGTVFTLKISRVEAEDLGVYYC 53 01 & JK4 FR4
FGGGTKLEIKR
[0278] The primary sequences of the murine and human framework
regions of the selected antibodies share significant identity.
Residue positions that differed were candidates for inclusion of
the murine residue in the humanized sequence in order to retain the
observed binding potency of the murine antibody. Such framework
region amino acid substitutions (human residues that are back
mutated to mouse residues) at a key residue are called framework
back mutations, wherein the key residue is selected from the group
consisting of a residue adjacent to a CDR; a glycosylation site
residue; a rare residue; a residue capable of interacting with
PGE2; a residue capable of interacting with a CDR; a canonical
residue; a contact residue between heavy chain variable region and
light chain variable region; a residue within a Vernier zone; and a
residue in a region that overlaps between a Chothia-defined
variable heavy chain CDR1 and a Kabat-defined first heavy chain
framework. In an embodiment, the human acceptor framework comprises
at least one Framework Region amino acid substitution, wherein the
amino acid sequence of the framework is at least 65% identical to
the sequence of the human acceptor framework and comprises at least
70 amino acid residues identical to the human acceptor framework.
For humanization of 2B5-8.0, the framework region amino acid
substitution at a key residue is selected from the group consisting
of M (human) to I (mouse) at position 48, V (human) to A (mouse) at
position 68, M (human) to L (mouse) at position 70, and T (human)
to V (mouse) at position 72 in the heavy chain variable region; and
I (human) to V (mouse) at position 2 and V (human) to L (mouse) at
position 3 in the light chain variable region.
[0279] The likelihood that a given framework residue would impact
the binding properties of the antibody depends on its proximity to
the CDR residues. Therefore, using the model structures, the
residues that differ between the murine and human sequences were
ranked according to their distance from any atom in the CDRs that
likely contact PGE.sub.2. Those residues that fell within 4.5 .ANG.
of any CDR atom were identified as most important and were deemed
candidates for retention of the murine residue in the humanized
antibody (i.e., frame work back mutation).
[0280] For humanization of the 2B5-8.0 antibody variable regions,
the general approach provided in the present invention was as
follows. First, a molecular model of the 2B5-8.0 antibody variable
regions was constructed with the aid of the computer programs ABMOD
and ENCAD (Levitt, M., J. Mol. Biol. 168: 595-620 (1983)). Next,
based on a homology search against human V and J segment sequences,
the VH segment VH1-18 (The Immunoglobulin Facts Book. 2001,
authored by Marie-Paule Lefranc and Gerald Lefranc, published by
Academic Press) and the J segment JH4 (Id.) were selected to
provide the frameworks for the humanized heavy chain variable
region for 2B5-8.0. For the 2B5-8.0 light chain variable region,
the VL segment 01 (Id.) and the J segment JK4 (Id.) were used. The
identity of the framework amino acids between 2B5-8.0 VH and the
acceptor human VH1-18 and JH4 segments was 80.2%, while the
identity between 2B5-8.0 VL and the acceptor human 01 and JK4
segments was 90.3%. The computer model did not identify any
significant contact residues with the CDRs that need to be back
mutated. No further replacements were done.
[0281] Nine different versions of humanized 2B5-8.0, named as
HU2B5.1, HU2B5.2, HU2B5.3, HU2B5.4, HU2B5.5, HU2B5.6, HU2B5.7,
HU2B5.8 and HU2B5.9, were designed. The nine antibodies were
different in the framework back mutations at positions 48, 68, 70,
and 72 in the heavy chain variable region and positions 2 and 3 in
the light chain variable region as described above.
TABLE-US-00009 TABLE 9 CDRs of Mouse Anti-PGE.sub.2 Antibody
2B5-8.0 SEQ ID Protein Sequence No. region
123456789012345678901234567890 54 CDR H1 GYTFTKYWLG 55 CDR-H2
DIYPGYDYTHYNEKFKD 56 CDR-H3 SDGSSTY 57 CDR-L1 TSSQNIVHSNGNTYLE 58
CDR-L2 KVSNRFSG 59 CDR-L3 FQVSHVPYT
TABLE-US-00010 TABLE 10 Nine Humanized Anti-PGE.sub.2 Antibodies
With CDRs of 2B5-8.0 SEQ ID Protein Sequence No. region
123456789012345678901234567890 60 VH Hu2B5.1
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWIGDIYPGYDYTHY
NEKFKDRATLTVDTSTSTAYMELRSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 61 VL
HU2B5.1 DVVMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 62 VH HU2B5.2
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWIGDIYPGYDYTHY
NEKFKDRATLTVDTSTSTAYMELSSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 63 VL
HU2B5.2 DVVMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 64 VH HU2B5.3
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWIGDIYPGYDYTHY
NEKFKDRATLTVDTSTSTAYMELRSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 65 VL
HU2B5.3 DVLMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 66 VH HU2B5.4
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWIGDIYPGYDYTHY
NEKFKDRATLTVDTSTSTAYMELSSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 67 VL
HU2B5.4 DVLMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 68 VH HU2B5.5
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWMGDIYPGYDYTHY
NEKFKDRVTLTTDTSTSTAYMELRSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 69 VL
HU2B5.5 DVVMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 70 VH HU2B5.6
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWMGDIYPGYDYTHY
NEKFKDRVTLTTDTSTSTAYMELSSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 71 VL
HU2B5.6 DVVMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 72 VH HU2B5.7
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWMGDIYPGYDYTHY
NEKFKDRVTLTTDTSTSTAYMELRSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 73 VL
HU2B5.7 DVLMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 74 VH HU2B5.8
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWMGDIYPGYDYTHY
NEKFKDRVTLTTDTSTSTAYMELSSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 75 VL
HU2B5.8 DVLMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR 76 VH HU2B5.9
EVQLVQSGAEVKKPGASVKVSCKASGYTFT KYWLGWVRQAPGQGLEWMGDIYPGYDYTHY
NEKFKDRVTMTTDTSTSTAYMELRSLRSDD TAVYYCARSDGSSTYWGQGTLVTVSS 77 VL
HU2B5.9 DVVMTQTPLSLPVTPGEPASISCTSSQNIV
HSNGNTYLEWYLQKPGQSPQLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDVGV
YYCFQVSHVPYTFGGGTKVEIKR
Example 4.2.2
Construction of Humanized Antibodies
[0282] The in silico designed humanized antibodies described in
Example 4.2.1 were constructed de novo using oligonucleotides. For
each variable region cDNA, 6 oligonucleotides of 60-80 nucleotides
each were designed to overlap each other by 20 nucleotides at the
5' and/or 3' end of each oligonucleotide. In an annealing reaction,
all 6 oligos were combined, boiled, and annealed in the presence of
dNTPs. DNA polymerase I, Large (Klenow) fragment (New England
Biolabs #M0210, Beverley, Mass.) was added to fill-in the
approximately 40 bp gaps between the overlapping oligonucleotides.
PCR was then performed to amplify the entire variable region gene
using two outermost primers containing overhanging sequences
complementary to the multiple cloning site in a modified pTT3
vectors. The PCR products derived from each cDNA assembly were
separated on an agarose gel and the band corresponding to the
predicted variable region cDNA size was excised and purified. The
variable heavy region was inserted in-frame into a cDNA fragment
encoding a wild-type human IgG1 constant region, or a human IgG1
constant region containing 2 hinge-region amino acid mutations by
homologous recombination in bacteria. (Zhang, Y et al. Nature
Biotechnol. 18(12):1314-7 (2000)). The mutations were a leucine to
alanine change at position 234 (EU numbering) and a leucine to
alanine change at position 235 (Lund et al. J. Immunol., 147:2657
(1991)). The variable light chain region was inserted in-frame into
a human kappa constant region by homologous recombination.
Bacterial colonies were isolated, plasmid DNA extracted, and cDNA
inserts were sequenced in their entirety. pTT3 vectors containing
correct humanized heavy and light chains corresponding to each
antibody were co-transfected into HEK293 cells to transiently
produce full-length humanized anti-PGE.sub.2 antibodies. Cell
supernatants containing recombinant chimeric antibody were purified
by Protein A Sepharose chromatography and bound antibody was eluted
by the addition of 0.1N acetic acid/0.15 M NaCl (pH3.0). Antibodies
were neutralized and dialyzed in PBS.
Example 4.2.3
Alternative Construction of Humanized Anti-PGE2 Antibodies
[0283] This example describes the humanization of an anti-PGE.sub.2
antibody. Humanization of the murine monoclonal antibody 2B5-8.0
was carried out essentially according to the procedure of Queen,
C., et al., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989).
First, human V segments with high homology to the 2B5-8.0 VH or VL
amino acid sequences were identified. Next, the
complementarity-determining region (CDR) sequences together with
framework amino acids important for maintaining the structures of
the CDRs were grafted into the selected human framework sequences.
In addition, human framework amino acids that were found to be rare
in the corresponding V region subgroup were substituted with
consensus amino acids to reduce potential immunogenicity.
[0284] For humanization of the 2B5-8.0 variable regions, the
general approach provided in the present invention was followed.
First, a molecular model of the 2B5-8.0 variable regions was
constructed with the aid of the computer programs ABMOD and ENCAD
(Levitt, M., J. Mol. Biol. 168: 595-620 (1983)). Next, based on a
homology search against human V and J segment sequences, the VH
segment MUC1-1'CL (Griffiths, A. D., et al., EMBO J. 12: 725-734
(1993)) and the J segment JH4 (Ravetch, J. V., et al., Cell 27:
583-591 (1981)) were selected to provide the frameworks for the
Hu2B5-8.0 heavy chain variable region. For the Hu2B5-8.0 light
chain variable region, the VL segment TR1.37'CL (Portolano, S., et
al., J. Immunol. 151: 2839-2851 (1993)) and the J segment JK2
(Hieter, P.A., et al., J. Biol. Chem. 257: 1516-1522 (1982)) were
used. The identity of the framework amino acids between 2B5-8.0 VH
and the acceptor human MUC1-1'CL and JH4 segments was 76%, while
the identity between 2B5-8.0 VL and the acceptor human TR1.37'CL
and JK2 segments was 84%.
[0285] At framework positions in which the computer model suggested
significant contact with the CDRs, the amino acids from the mouse V
regions were substituted for the original human framework amino
acids. For humanization of 2B5-8.0, the framework region amino acid
substitution at a key residue is selected from the group consisting
of M (human) to I (mouse) at position 48, R (human) to K (mouse) at
position 67, V (human) to A (mouse) at position 68, I (human) to L
(mouse) at position 70 and R (human) to V (mouse) at position 72 in
the heavy chain variable region; and D (human) to V (mouse) at
position 75 in the light chain variable region. In addition, a few
amino acids have been changed to the consensus amino acids in the
same human variable domain subgroups to eliminate potential
immunogenicity, which includes A to T substitution at position 76
in the heavy chain variable region, and E to D substitution at
position 1 and L to I substitution at position 2 in the light chain
variable region.
[0286] The protein sequences of the CDR grafted variable domains
(VH and VL of Hu2B5.P1) and the variable domains which incorporated
all the back mutations and consensus substitutions (VH and VL of
Hu2B5.P2) based on this humanization analysis are provided below.
It is understood in general that any humanized version with one or
a few such back mutations and consensus substitutions fall within
the scope of this invention. Antibody E comprised VH Hu2B5.P2 and
VL Hu2B5.P2; Antibody F comprised VH Hu2B5.P2 and VL Hu2B5.P1;
Antibody G comprised VH Hu2B5.P1 and VL Hu2B5.P2; and Antibody I
comprised VH Hu2B5.P1 and VL Hu2B5.P1.
TABLE-US-00011 TABLE 11 Humanized Anti-PGE2 Antibodies With CDRs of
2B5-8.0 SSEQ ID Protein Sequence No. region
1234567890123456789012345678901234567890 78 VH Hu2B5.P1
QVQLVQSGAEVKKPGASVKVSCKASGYTFTKYWLGWVRQA
PGQGLEWMGDIYPGYDYTHYNEKFKDRVTITRDTSASTAY
MELSSLRSEDTAVYYCARSDGSSTYWGQGTLVTVSS 79 VL HU2B5.P1
ELVMTQSPLSLPVTPGEPASISCTSSQNIVHSNGNTYLEW
YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKI
SRVEAEDVGVYYCFQVSHVPYTFGQGTKLEIK 80 VH HU2B5.P2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTKYWLGWVRQA
PGQGLEWIGDIYPGYDYTHYNEKFKDKATLTVDTSTSTAY
MELSSLRSEDTAVYYCARSDGSSTYWGQGTLVTVSS 81 VL HU2B5.P2
DIVMTQSPLSLPVTPGEPASISCTSSQNIVHSNGNTYLEW
YLQKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTVFTLKI
SRVEAEDVGVYYCFQVSHVPYTFGQGTKLEIK
Example 4.2.4
Characterization of Humanized Anti PGE.sub.2 Antibodies
[0287] The ability of the purified humanized anti-PGE.sub.2
antibodies to bind PGE.sub.2 was determined by biotin-PGE.sub.2
ELISA or 3H-PGE.sub.2 radioimmunoassay as described in Example
1.1.A, Cross reactivity of the humanized anti-PGE.sub.2 antibodies
was determined by competitive biotin-PGE.sub.2ELISA or 3H-PGE.sub.2
radioimmunoassay as described in Example 1.1.B Inhibition of
PGE.sub.2 activity by the humanized anti-PGE.sub.2 antibodies was
determined using a EP4 bioassay as described in Examples 1.1.C.
[0288] All the humanized anti-PGE.sub.2 antibodies were able to
bind PGE.sub.2 in a biotin-PGE.sub.2 ELISA (FIGS. 6 and 7; Table
12). The humanized anti-PGE.sub.2 antibodies were able to
neutralize and block PGE.sub.2 mediate calcium influx in an EP4
FLIPR assay. The alternative designed humanized anti-PGE.sub.2
antibodies E, F, G and I were also able to bind to PGE.sub.2 in a
.sup.3H-PGE.sub.2 ELISA (Table 13). Hu2B5.7 was selected for
further characterization of prostaglandin binding specificity in a
.sup.3H-PGE.sub.2 competition ELISA and demonstrated specificity to
PGE.sub.2 (Table 13).
TABLE-US-00012 TABLE 12 Characterization of Humanized Antibodies
Humanize Antibody Ch2B5-8.0 Hu2B5.1 Hu2B5.2 Hu2B5.3 Hu2B5.4 Hu2B5.5
Hu2B5.6 Hu2B5.7 Hu2B5.8 Hu2B5.9 EC50 (pM) ELISA 41-60 63 55 59 57
37 89 79 87 NT IC50 (pM)@ PGE.sub.2 25 pm 55 55 55 47 40 47 52 78
78 125 Cellular Potency in EP4 assay Cross Reactivity PGE1 12 16 14
14 14 11 11 9.3 8.5 NT Index (%) PGA2 0.17 0.21 0.18 0.18 0.22 0.18
0.15 0.15 0.20 NT PGD2 <0.01 <0.01 <0.01 <0.01 <0.01
<0.01 <0.01 <0.01 <0.01 NT
TABLE-US-00013 TABLE 13 Characterization of Humanized
Anti-PGE.sub.2 Antibodies (continued) Humanized Antibody ID A E F G
I VH/VL VH Hu2B5.P2/ VH Hu2B5.P2/ VH Hu2B5.P1/ VH Hu2B5.P2/ Hu2B5.9
VL Hu2B5.P2 VL Hu2B5.P1 VL Hu2B5.P2 VL Hu2B5.P2 PGE.sub.2 binding
0.704 0.416 0.955 0.574 1.299 in .sup.3H-PGE.sub.2 ELISA
(IC.sub.50, nM)
TABLE-US-00014 TABLE 14 Characterization of Prostaglandin
Selectivity for Humanized Anti-PGE.sub.2 Antibodies Hu2B5.7
Anti-PGE.sub.2 mAb 2B5 Hu2B5-7.0 PG Selectivity PGE2 100 100 in
.sup.3H-PGE.sub.2 PGE1 12 5.6 competition PGA2 0.17 0.02 ELISA PGD2
0.04 <1 .times. 10.sup.-4 (CRI %) PGF2.alpha. 0.25 0.06 PGI2 NA
0.12 Iloprost NA 0.006 Carbaprostacyclin NA 0.16 Pinane TXA2 NA
0.00016 15R-Pinane TXA2 NA 0.0029 Carbocyclic TXA2 NA 0.00011 TXB2
<0.01 <0.01 6-keto PGF1.alpha. 0.4 0.47 PGB2 0.03 0.0004
8-iso PGF2.alpha. 0.02 0.06 13,14-dihydroxyl-15- <0.01 0.0092
Keto PGE2 2,3-dinor-6-keto- NA 0.19 PGF1.alpha. 15-keto PGE2
<0.01 0.011 19R-hydroxy PGE2 <0.01 0.07 LTE4 <0.01 0.01
5(S)-HETE <0.01 <0.01 Arachidonic acid <0.01 <0.01
Example 4.2.4.a
Humanized Anti PGE.sub.2 Antibodies Block Binding of PGE.sub.2 to
PGE.sub.2 Receptor
[0289] Competitive inhibition of PGE.sub.2 binding to PGE.sub.2
receptors, for example EP4, by an anti-PGE.sub.2 antibody can be
determined by a cell-based or membrane based receptor binding assay
using .sup.3H-PGE.sub.2 as described in Example 1.1.D and a cell
based FACS assay as described in Example 1.1.E.
[0290] For a therapeutic mAb with serum half-life between 10 and 20
days in man, the serum concentration is normally between 5-15
.mu.g/ml, with a weekly or bi-weekly IV or SC 3 mpk or less dosing
regiment. Based on this calculation, hu2B5.1-Hu2B5.9 are likely to
completely (100%) block PGE.sub.2 binding to EP4 in vivo as a
therapeutic mAb, at a serum concentration of 100 nM (or 15 ug/ml),
under a conventional dosing regimen of a monoclonal antibody.
Example 4.2.5
Biophysico-Chemical Characterization of Humanized Anti-PGE.sub.2
Antibodies
[0291] Criteria tested ranged from general drug-like property
parameters such as, parameters indicating intrinsic stability
(differential scanning calorimetry or DSC), and general physical
and chemical stability (e.g., purity including fragmentation and
aggregation monitoring by SEC).
[0292] Analytical methods used for biophysico-chemical
characterization:
Example 4.2.5.1
Size Exclusion Chromatography (SEC)
[0293] Size exclusion chromatography was used to separate proteins
based on size. Proteins are carried in an aqueous mobile phase and
through a porous stationary phase resin packed in a column. The
retention time in the column is a function of the hydrodynamic size
of the protein and the size of the pores in the packed resin bed.
Smaller molecules can penetrate into smaller pores in the resin and
are retained longer than larger molecules. Upon elution from the
column the proteins are detected by UV absorbance. The SEC method
used a TSK gel guard (TOSOH Biosciences, Montgomeryville, Pa., cat.
no. 08543) and a TSK gel G3000SWxL (TOSOH Biosciences,
Montgomeryville, Pa., cat. no. 08541). The mobile phase was 100 mM
Na2HPO4, 200 mM Na2SO4, pH 7.0. The flow rate was 0.3 mL/minute.
Injection volume was 20 .mu.L of 1 mg/mL sample. The column
temperature was room temperature. The autosampler temperature was
2-8.degree. C. The total run time was 50 minutes. The detection was
based on UV absorbance at 214 nm wavelength, with band width set at
8 nm, using reference wavelength at 360 nm with band width 100
nm.
Example 4.2.5.1
Differential Scanning Calorimetry (DSC)
[0294] The thermal stability of the anti-PGE.sub.2 antibodies was
assessed using a DSC instrument. The DSC instrument used was an
automated VP-DSC equipment with Capillary Cell (Microcal, GE
Healthcare Ltd./Microcal, Buckinghamshire, UK). Unfolding of
molecules was studied applying a 1.degree. C./minute scan rate over
a 25.degree. C.-95.degree. C. temperature range for samples at 1
mg/mL. Additional measurement parameters applied were a fitting
period of 16 seconds, a pre-scan wait time of 10 minutes, and
measurements were performed in none-feedback mode. Per individual
measurement, 420 .mu.L of sample/blank were filled into the DSC
measurement sample holder, with a plate fill scheme as provided
below. The thermograms obtained were fitted to a non two state
model to obtain the midpoint temperatures and enthalpies of the
different transitions.
[0295] An additional requirement for successful biologics
development candidate is that the protein remains its native state
and conformation. A protein in aqueous solution is in equilibrium
between the native (folded) conformation and its denatured
(unfolded) conformation. The stability of the native state is based
on the magnitude of the Gibbs free energy (DG) of the system and
the thermodynamic relationship between enthalpy (DH) and entropy
(DS) changes. A positive DG indicates the native state is more
stable than the denatured state--the more positive the DG, the
greater the stability. For a protein to unfold, stabilizing forces
need to be broken. Conformational entropy overcomes stabilizing
forces allowing the protein to unfold at temperatures where entropy
becomes dominant. DSC measures DH of protein unfolding due to heat
denaturation. As a general rule it can be stated that the higher
the transition midpoint (the Tm), the more stable the protein at
lower temperatures. During the same experiment DSC also measures
the change in heat capacity (DCp) for protein denaturation. Heat
capacity changes associated with protein unfolding are primarily
due to changes in hydration of side chains that were buries in the
native state, but become solvent exposed in the denatured state.
DSC has been shown to be a valuable predictor of liquid formulation
stability for proteins and other biological macromolecules
(Remmele, R. L. Jr., Gombotz, W. R., BioPharm 13, 36-46, 2000, and;
Remmele, R. L. Jr., Nightlinger, N. S., Srinivasen, S., Gombotz,
W.R., Pharm. Res. 15, 200-208, 1998).
Example 4.2.6
Stability Of Humanized Anti-PGE.sub.2 Antibod Hu2B5.7 During Clone
Selection Process
Example 4.2.6.A
Stability Of Humanized Anti-PGE.sub.2 Antibod Hu2B5.7 Using DSC and
SEC
[0296] The stability of a series of clones (i.e., anti-PGE.sub.2
antibodies) of the parent anti-PGE.sub.2 antibody was assessed by
using intrinsic thermodynamic clone stability determination using
DSC (0.79 mg/mL clone concentration, formulated at pH 6 in a 10 mM
citrate, 10 mM phosphate buffer) and by accelerated stability
screening (0.79 mg/mL clone concentration, formulated at pH 6 in a
10 mM citrate, 10 mM phosphate buffer, for 7 days at 50.degree. C.)
where clone stability was monitored with SEC (Table 15).
TABLE-US-00015 TABLE 15 Formation Of Aggregates And Fragments In
Hu2B5 humanized antibody variants Hu2B5.1-Hu2B5.9 Clone Samples As
Determined By SEC (Start Of Stability Study) Clone Aggregate (%)
Monomer (%) Fragment (%) Hu2B5.1 1.8071 93.7885 4.4044 Hu2B5.2
1.846 94.8516 3.3025 Hu2B5.3 2.116 94.1987 3.6853 Hu2B5.4 2.234
94.5513 3.2146 Hu2B5.5 1.5906 95.1406 3.2688 Hu2B5.6 1.8265 95.446
2.7275 Hu2B5.7 1.9668 95.5818 2.4514 Hu2B5.8 2.1126 94.5969 3.2904
Hu2B5.9 1.8559 95.6031 2.541
TABLE-US-00016 TABLE 16 Formation Of Aggregates And Fragments In
Hu2B5 humanized antibody variants Hu2B5.1-Hu2B5.9 Clone Samples As
Determined By SEC (7 Days Storage At 50.degree. C.) Clone Aggregate
(%) Monomer (%) Fragment (%) Hu2B5.1 2.5611 91.0715 6.3675 Hu2B5.2
2.1753 93.1491 4.6755 Hu2B5.3 2.6042 92.3518 5.044 Hu2B5.4 2.1689
91.2889 6.5423 Hu2B5.5 1.901 93.7376 4.3614 Hu2B5.6 2.1577 93.817
4.0253 Hu2B5.7 2.205 93.905 3.89 Hu2B5.8 2.5144 93.3016 4.184
Hu2B5.9 1.9559 94.6313 3.4127
[0297] Tables 15 and 16 provide the results of SEC testing for up
to 7 days storage of the humanized anti-PGE.sub.2 antibodies,
showing the levels of monomer at the start and at the end of the
accelerated stability screening. Hu2B5.7 and Hu2B5.9 revealed the
highest monomer levels after 7 days accelerated stability
screening. The results shown in Table 17 demonstrated that hu2B5.7
also had a very favorable intrinsic stability profile (DSC data)
compared to other clones of the panel (e.g., Hu2B5.4).
[0298] An IgG antibody typically shows three unfolding transitions
(Tm): unfolding of the intact antibody is associated with the
melting of the CH2 domain in the Fc fragment, melting of the CH3
domain in the Fc fragment, and melting of the Fab fragment. In
order to select anti-PGE.sub.2 antibodies with desirable drug-like
properties, clones with high Tm values and high intrinsic stability
such as Hu2B5.7 were selected (Table 17)
TABLE-US-00017 TABLE 17 Intrinsic Thermodynamic Clone Stability
Determination Of Humanized Anti-PGE.sub.2 Antibody Clones Via DSC
(0.79 mg/mL Clone Antibody Concentration, Formulated At pH 6 in a
10 mM Citrate, 10 mM Phosphate Buffer) Clone Tm1 (.degree. C.) Tm2
(.degree. C.) Tm3 (.degree. C.) Hu2B5.1 72.835 75.02 82.785 Hu2B5.2
72.845 75.08 82.67 Hu2B5.3 72.67 72.96 82.67 Hu2B5.4 71.095 72.41
82.58 Hu2B5.5 73.31 75.755 82.97 Hu2B5.6 73.035 75.585 82.95
Hu2B5.7 72.67 75.445 82.795 Hu2B5.8 72.96 75.33 82.83 Hu2B5.9
73.235 75.965 83.04
Example 4.2.6.B
Capillary Zone Electrophoresis of Hu2B5.1-Hu2B5.9
[0299] Capillary zone electrophoresis is a capillary
electrophoresis method in which the capillary is filled with buffer
and the separation mechanism is based on differences in
electrophoretic mobility of the analyte through the buffer. The
electrophoretic mobility of a molecule is related to the
charge-to-size ratio. A Beckman-Coulter ProteomeLab PA 800 (Beckman
Coulter, Fullerton, Calif.) was used for the CZE analysis. A
neutral capillary (eCAP neutral, 56 cm total length, 50 .mu.m I. D.
Beckman-Coulter, P/N 477441, Fullerton, Calif.) was used. The
method used a 30.2 cm capillary with a detection window 20.2 cm
from the sample introduction inlet. The running buffer was 100 mM
EACA (6-Aminocaproic acid, Sigma A7824-100 G, St. Louis, Mo.) with
0.1% MC (from 1% Methylcellulose solution, Convergent Bioscience,
cat#101876, Toronto, ON, Canada), pH 5.5.
[0300] The results showed that Hu2B5.1, Hu2B5.3, Hu2B5.5, Hu2B5.7,
Hu2B5.9 migrated more basic (e.g., they had a shorter migration
time) than Hu2B5.2, Hu2B5.4, Hu2B5.6, Hu2B5.8. This may be due to
the fact that Hu2B5.1, Hu2B5.3, Hu2B5.5, Hu2B5.7, Hu2B5.9 all have
R at heavy chain #84 amino acid, while the other four samples have
S at the same position. All nine samples showed a main peak and
minor acidic and basic species, but there was not much difference
among the distribution of the different species.
Example 4.3
Crystallization of Hu2B5.7 Complexed to PGE2
[0301] The Fab portion of Hu2B5.7 is complexed with PGE.sub.2 and
crystals of the complex are generated as follows.
Example 4.3.1
Preparation and Purification of Hu2B5.7 Fab Fragment
[0302] To prepare Hu2B5.7 Fab fragment, Hu2B5.7 IgG in 0.15 M PBS
buffer is first concentrated to 2 mg/ml using an Ultrafree-15
Biomax 10 kDa molecular weight cut-off (MWCO) centrifugal filter
device (Millipore). A papain gel slurry (Pierce) is pre-washed and
charged in 2-3.times. with Buffer A (20 mM Na.sub.2HPO.sub.4, 10 mM
EDTA, 20 mM cysteine) at a 1:1 volume ratio. The concentrated
antibody is then mixed with 50% papain gel slurry and incubated at
37.degree. C. for 24 hours with vigorous shaking. The
antibody/slurry mixture is centrifuged (Beckman 6KR) and the
supernatant is loaded onto a PBS pre-equilibrated Superdex 75. A
major peak is eluted and protein is pooled. A 25 mL Protein A
Sepharose 4 Fast Flow affinity column (Amersham Pharmacia) is
prepared by washing with 100 mL of PBS. The pooled antibody
fragments are applied to the affinity column (2 mL/min flow rate).
Fractions containing Hu2B5.7 Fab fragments (monitored by UV
absorbance at 280 nm) are collected in the flow-thru. Fractions
containing a Hu2B5.7 Fab fragment concentration greater than 0.3
mg/mL (determined by UV absorbance at 280 nm) are pooled and frozen
at -80.degree. C. Sample purity is assessed with SDS-PAGE.
Example 4.3.2
PGE2/Hu2B5.7 Fab Complex Preparation
[0303] PGE2 and Hu2b5.7 Fab protein are mixed at a 1:1 molar ratio
and incubated for 1 hour at 4.degree. C. The complex sample is
loaded onto a pre-equilibrated (20 mM Tris pH 7.5, 150 mM NaCl)
Superdex 200 column at 0.5 ml/minute. The complex is pooled and
concentrated to 24 mg/mL using an Ultrafree-15 Biomax 10 kDa
molecular weight cut-off (MWCO) centrifugal filter device
(Millipore) and frozen at -80.degree. C. Sample purity is assessed
with SDS-PAGE.
Example 4.3.3
Crystallization of PGE2/Hu2B5.7 Fab Complex
[0304] Frozen PGE2/Hu2B5.7 complex stock (.about.24 mg/mL) is
thawed on ice. The complex (1.0 .mu.L) is mixed with 1.0 .mu.L of
reservoir solution (1.75 M Ammonium Sulfate, 100 mM MES pH 6.5, 10
mM CaCl2). The resulting drop is mixed in a sitting drop well
(CrysChem sitting-drop plate) over the reservoir at about
18.degree. C. Diamond-like crystals generally appeared within one
week.
Example 4.3.4
Cryoprotection and Flash Cooling of PGE2/Hu2B5.7 Fab Complex
Crystals
[0305] Crystals of PGE2/Hu2B5.7 Fab complex are harvested using a
fiber loop in mother liquor+20% glycerol. The crystals are
subsequently flash-cooled by plunging into liquid nitrogen.
Example 4.3.5
X-Ray Diffraction Data Collection of PGE2/Hu2B5.7 Fab Complex
[0306] X-ray diffraction data from PGE2/Hu2B5.7 Fab crystals are
collected at the IMCA beamline at the Advanced Photon Source in
Argonne, Ill. The crystals are maintained at a temperature of 100 K
with an Oxford Cryosystems Cryostream cooler during data
collection. A total of 180 frames are collected at an oscillation
range of 1.0.degree.. The data are processed with the HKL2000 suite
of programs (Otwinowski and Minor, 1997). After determining the
crystal orientation, the data are integrated with DENZO and scaled
and merged with SCALEPACK, and placed on an absolute scale and
reduced to structure factor amplitudes with TRUNCATE (French and
Wilson, 1978). Five percent of the unique reflections are assigned,
in a random fashion, to the "free" set, for calculation of the free
R-factor (Rfree) (Brunger, 1992); the remaining 95% of the
reflections constitute the "working" set, for calculation of the
R-factor (R).
Example 4.3.6
Molecular Replacement Solution and Refinement of PGE2/Hu2B5.7 Fab
Complex Crystal Structure
[0307] A maximum likelihood molecular replacement solution is
determined using the program PHASER (Read, 2001). A total of six
PGE2/Hu2B5.7 monomers are solved at 3.0 .ANG. resolution an
appropriate space group. The search model is the crystal structure
of Fab reported previously (Protein Data Bank entry 1BJ1; Muller et
al. 1998). Coordinates are generated based on the molecular
replacement solution.
[0308] The refinement of the PGE2/Hu2B5.7 Fab complex crystal
structure begins with the molecular replacement solution
coordinates, described above, in an appropriate space group.
Refinement begins using rigid-body refinement by the program REFMAC
available in the CCP4 suite of programs (Murshudov et al., 1997,
Collaborative Computational Project, 1994). De novo PGE2 electron
density is observed. Manual building of six PGE2 monomers is guided
by the public PGE2 NMR structure 1IJZ (Moy et al., 2001) using the
molecular graphics program 0 (Jones et al., 1991) and examination
of 2Fo-Fc and Fo-Fc electron-density maps. The refinement program
REFMAC (Murshudov et al., 1997) is used for iterative rounds of
restrained refinement resulting in the following statistics: R of
25.8% (Rfree 30.5%).
Example 5.0
Pharmacokinetic Analyses
Example 5.1
Pharmacokinetic Analysis of Recombinant Mouse Anti-PGE.sub.2
Antibodies
[0309] Pharmacokinetic studies with mouse mAb 2B5.8.0 were carried
out in Sprague Dawley rats and Balb/C mice. Male and female rats
and mice were dosed intravenously or intraperitoneally (mice only)
with a single dose of 4 mg/kg 2B5.8.0 and serum samples were
analyzed using antigen capture based chemiluminescent MSD (Meso
Scale Discovery) method (Meso Scale Discovery, Gaithersburg, Md.).
Pharmacokinetic parameters were calculated by non-compartmental
analysis using WinNonlin.
Example 5.1.1
Assay Used To Quantitate 2B5.8.0 In PK Serum Samples
[0310] The following MSD assay was used to measure antibody
concentration in rat and mouse serum. MSD streptavidin plates (Meso
Scale Discovery, Gaithersburg, Md.) were washed with phosphate
buffered saline containing 0.05% Tween-20 (Sigma, St. Louis, Mo.).
Plates were blocked with 250 .mu.L/well blocking solution (MSD
Block, Meso Scale Discovery, diluted to 3% final concentration in
PBS) for 1 hour, covered, with shaking (600 rpm) at room
temperature. After washing, 70 .mu.L of biotinylated PGE.sub.2
(Prostoglandin E2-biotinamide, Cayman Chemical, Ann Arbor, Mich.,
cat#10006987, lot#190831-191028, 0.01 ug/mL in assay buffer) was
added to each well. The plates were covered and incubated with
shaking (600 rpm) for 1 hour at room temperature
[0311] Prior to analysis, rat and mouse serum samples were thawed
on ice, mixed gently, and centrifuged at 14,000 rpm for 3 minutes
at 4.degree. C. in an eppendorf centrifuge. Standard curve and
control samples were prepared in rat and mouse serum. A Tecan Evo
automated liquid handling station was used to dilute standard
curve, high, medium, and low controls, and serum samples in assay
buffer, keeping 1% final serum concentration constant. MSD plates
were washed again and study samples, standard curve samples and
blanks, as well as high, medium, and low controls were added (70
.mu.L/well). The plates were covered, and incubated for 1 hour with
shaking (600 rpm) at room temperature.
[0312] After incubation, the MSD plates were washed, and 70 .mu.L
of sulfo-tagged goat anti-mouse IgG (Meso Scale Discovery; diluted
to 1 ug/mL in assay buffer) was added to each well. The MSD plates
were covered, and incubated with shaking (600 rpm) for 1 hour at
room temperature, then the plates were washed and developed with
2.times. Read Buffer (Meso Scale Discovery). Chemilumeniscence was
measured within ten minutes on the MSD Sector Imager 6000.
[0313] Standard curves were analyzed using four-parameter logistic
fit and sample concentrations were calculated by XLfit4 software
version 2.2.1 Build 16, (Microsoft Corporation, Redmond, Wash.).
Pharmacokinetic parameters were calculated for each animal using
Winonlin software version 5.0.1 (Pharsight Corporation, Mountain
View, Calif.) by noncompartmental analysis.
Example 5.1.2
Pharmacokinetic Studies of 2B5.8.0 Carried Out in SD Rats and
Balb/C Mice
[0314] Surgically altered (jugular vein cannulated, JVC) and
regular male and female Sprague-Dawley Rats (approximately seven
weeks old, weighing 240-390 grams) were purchased from Charles
River Laboratories (Wilmington, Mass.). The animals were housed in
rooms maintained at constant temperature and humidity under 12
hours light/dark cycle, fed with normal rodent chow and were
allowed food and water ad libitum. Hydration and clinical
conditions of the animals were monitored daily.
[0315] Male and female Balb/c mice (weighing approximately 0.025
kg) were purchased from Charles River Laboratories (Wilmington,
Mass.). The animals were allowed food and water ad libitum. Blood
samples were collected (0.2 mL from the rats via the tail vein and
by cardiac puncture from the mice) at various timepoints (5 mice at
each timepoint) allowed to clot for 30 minutes at room temperature,
centrifuged for 3 minutes at 13,200 rpm, the serum transferred to
eppendorf tubes and stored frozen at -80.degree. C.
[0316] Following intravenous administration in rat, 2B5.8.0
exhibited bi-exponential decay, typical of antibodies. 2B5.8.0
clearance and volumes of distributions were low (Table 18), and
half-life was long, T1/2: 9.1 and 8.9 days (male and female
respectively). Large inter-animal variability was seen among female
rats, however not in males.
[0317] Following IV administration in Balb/C mice, 2B5.8.0 showed
very long half-lives (26.3 and 16.2 days, male and female
respectively) with low clearance and volumes of distributions
(Table 18). Following intraperitoneal administration in mice, at
the early timepoints large inter animal variability was observed in
the females. Absorption was slow, with high Cmax of 37-49 ug/ml
reached by 1-2 days. The half-life was long (13.8-16.1 days) and
the bioavailability was good (65.8-72.0%).
TABLE-US-00018 TABLE 18 Main Pharmacokinetic Parameters Of 2B5.8.0
In Sprague-Dawley Rats And Balb/C Mice IV Species/ T1/2 CL Vz Vss
AUC.sub.0-.quadrature. MRT dose (day) (mL/hr/kg) (mL/kg) (mL/kg)
(mg hr/mL) (day) Rat M (N = 5) 9.1 .+-. 1.3 0.41 .+-. 0.03 127 .+-.
18.9 122 .+-. 18.5 9.9 .+-. 0.7 12.6 .+-. 1.6 (4 mg/kg) F (N = 3)
8.5 .+-. 2.2 0.37 .+-. 0.13 101 .+-. 13.1 88 .+-. 6.2 12.0 .+-. 4.6
10.8 .+-. 3.5 Mouse M 26.3 0.15 135 134 26.9 37.5 (4 mg/kg) F 16.2
0.16 92 89 24.5 22.8 IP Species/ C.sub.max Tmax T1/2 MRT
AUC.sub.0-.quadrature. F dose (.mu.g/mL) (day) (day) (day) (mg
hr/mL) (%) Mouse M 37.3 2 16.1 23.4 19.4 72 (4 mg/kg) F 49.4 1 13.8
19.3 16.1 65.8
Example 5.1.3
Pharmacokinetic Analysis of Recombinant Humanized Anti-PGE.sub.2
hu2B5.7 and hu2B5.4
[0318] Pharmacokinetic studies with hu2B5.7 and hu2B5.4 were
carried out in Sprague Dawley rats. Male rats were dosed
intravenously with a single dose of 4 mg/kg of hu2B5.7 and hu2B5.4
and serum samples were analyzed using antigen capture based
chemiluminescent MSD (Meso Scale Discovery) method. Pharmacokinetic
parameters were calculated by non-compartmental analysis using
WinNonlin.
Example 5.1.3.1
Assay Used to Quantitate Hu2b5.7 in PK Serum Samples
[0319] The following MSD assay was used to measure hu2B5.7 and
hu2B5.4 concentrations in rat serum.
[0320] MSD streptavidin plates (Meso Scale Discovery) were washed
with phosphate buffered saline containing 0.05% Tween-20 (diluted
from 10.times.PBS, Abbott Bioresearch Center, Media Room,
Worcester, Mass. and Tween-20, Sigma, St. Louis, Mo.). Plates were
blocked with 250 .mu.L/well blocking solution (MSD Block, Meso
Scale Discovery, diluted to 3% final concentration in PBS) for 1
hour, covered, with shaking (600 rpm) at room temperature. After
washing, 70 .mu.L of biotinylated PGE2 (Prostoglandin
E2-biotinamide, Cayman Chemical, Ann Arbor, Mich., cat#10006987,
lot#190831-191028; 0.01 ug/mL in assay buffer) was added to each
well. The plates were covered and incubated with shaking (600 rpm)
for 1 hour at room temperature.
[0321] Prior to analysis, rat serum samples were thawed on ice,
mixed gently, and centrifuged at 14,000 rpm for 3 minutes at
4.degree. C. in an eppendorf centrifuge. Standard curve and control
samples were prepared in rat serum. Tecan Evo automated liquid
handling station was used to dilute standard curve, high, medium,
and low controls, and serum samples in assay buffer, keeping 1%
final serum concentration constant. MSD plates were washed again
and study samples, standard curve samples and blanks, as well as
high, medium, and low controls were added (70 .mu.L/well). The
plates were covered, and incubated for 1 hour with shaking (600
rpm) at room temperature.
[0322] After incubation, the MSD plates were washed, and 70 .mu.L
sulfo-tagged goat anti-human IgG (Meso Scale Discovery; diluted to
1 ug/mL in assay buffer) was added to each well. The MSD plates
were covered, and incubated with shaking (600 rpm) for 1 hour at
room temperature, then the plates were washed and developed with
2.times. Read Buffer (Meso Scale Discovery). Chemilumeniscence was
measured within ten minutes on the MSD Sector Imager 6000.
[0323] Standard curves were analyzed using four-parameter logistic
fit and sample concentrations were calculated by XLfit4 software
version 2.2.1 Build 16, (Microsoft Corporation, Redmond, Wash.).
Pharmacokinetic parameters were calculated for each animal using
Winonlin software version 5.0.1 (Pharsight Corporation, Mountain
View, Calif.) by noncompartmental analysis.
Example 5.1.3.2
Pharmacokinetic Studies Carried Out in Sprague-Dawley Rats
[0324] Surgically altered (jugular vein cannulated, JVC) male
Sprague-Dawley Rats (approximately seven weeks old, weighing
240-390 grams) were purchased from Charles River Laboratories
(Wilmington, Mass.). The animals were housed in rooms maintained at
constant temperature and humidity under a 12 hour light/dark cycle,
fed with normal rodent chow and were allowed food and water ad
libitum. Hydration and clinical conditions of the animals were
monitored daily.
[0325] 0.2 mL blood samples were collected from the rats at various
timepoints, allowed to clot for 30 minutes at room temperature,
centrifuged for 3 minutes at 13,200 rpm, the serum transferred to
eppendorf tubes and stored frozen at -80.degree. C.
[0326] Following intravenous administration, hu2B5.7 and hu2B5.4
serum concentrations declined bi-exponentially, typical of
antibodies. Hu2B5.7 and hu2B5.4 clearances and volumes of
distributions were low and half-lives were long; T1/2: 12.4 days
for both antibodies (Table 19). After about 10-14 days, several
animals exhibited unexpected drops in serum hu2B5.7 concentrations.
These sudden drops may have been due to the development of
anti-drug antibodies (ADA); however, this was not confirmed.
Animals with possible ADA responses were omitted from the final
pharmacokinetic calculations.
TABLE-US-00019 TABLE 19 Main Pharmacokinetic Parameters of hu2B5.7
and hu2B5.4 in Male Sprague- Dawley Rats After an Intravenous Dose
of 4 mg/kg IV T1/2 CL Vz Vss AUC.sub.0-.infin. MRT Ab/dose (day)
(mL/hr/kg) (mL/kg) (mL/kg) (mg hr/mL) (day) hu2B5.7/ (N = 3) 12.4
.+-. 0.6 0.41 .+-. 0.07 176 .+-. 30.8 165 .+-. 19.8 9.9 .+-. 1.8
16.8 .+-. 1.0 4 mg/kg hu2B5.4/ (N = 4) 12.4 .+-. 5.1 0.35 .+-. 0.07
143 .+-. 40.2 145 .+-. 31.8 11.8 .+-. 2.0 18.0 .+-. 6.0 4 mg/kg
Example 4.4
In Vivo Efficacy of Recombinant Mouse and Humanized PGE2
Antibodies
[0327] The in vivo efficacy of anti-PGE2 antibodies is assessed as
follows.
Example 4.4.1
In Vivo Efficacy of Mouse and Humanized Anti-PGE2 Antibodies in a
Carrageenan-Induced Footpad Edema Model
[0328] Carrageenan-Induced Footpad Edema is an acute rodent model
of innate immune function. The in vivo efficacy of mouse anti-PGE2
antibody 2B5-8.0 is assessed in a carragenan-induced paw edema
model. The induction of paw inflammation with carrageenan is
performed similarly as previously described (Joseph P. Portanova,
et al. J. Exp. Med. 184: 883-891 (1996)). Intradermal (ID)
injection of an inflammatory agent causes a rapid influx of
neutrophils and fluid edema which peaks at approximately 4 hours,
followed by an influx of macrophages and monocytes which peaks at
approximately 48 hours. C57.BL/6 mice (8-10-week-old, Jackson
Laboratories, Bar Harbor, Me.) were injected ID in the rear footpad
with 30 .mu.L of either PBS (left) or .lamda.-carrageenen (Sigma
Aldrich, St. Louis, Mo.) in PBS (right) at a concentration of 5.0
mg/mL (150 .mu.g/mouse). Rear footpad thickness was measured by
Dyer spring caliper model #310-119 at baseline (time=0), and 4
hours post carrageenan challenge. Significant difference for paw
thickness was determined by comparing mean paw swelling for each
treatment group to vehicle in a Student's two-tailed t-test. Mice
were given a dose titration of an anti-PGE.sub.2 antibody (2B5-8.0)
intraperitonially (IP) 18 hours prior to carrageenan challenge, or
Indomethacin, PO 2 hours prior to challenge. The endpoint measured
was the difference in paw swelling (edema) between right and left
paws 4 hours after treatment. The anti-PGE.sub.2 antibody inhibited
paw edema dose-dependently, and provided a maximal 40-50%
inhibition of paw swelling at 10 mg/kg, comparable to the maximal
inhibition achieved by indomethacin (Table 20).
TABLE-US-00020 TABLE 20 Paw Swelling In Mouse Carrageenan-Indcued
Footpad Edema After Ab Treatment Vehicle Anti-PGE2 Indomethacin
(PBS) (10 mg/kg, i.p.) (3 mg/kg, p.o.) .DELTA. Paw Swelling (mm)
0.857 .+-. 0.06 0.529 .+-. 0.04 0.486 .+-. 0.03 % Inhibition NA 38
.+-. 4.6 57 .+-. 3.5
Example 4.4.2
In Vivo Efficacy of Mouse and Humanized Anti-PGE.sub.2 Antibodies
in a Carrageenan-Induced Hyperalgesia Model
[0329] The in vivo efficacy of mouse anti-PGE2 antibody 2B5-8.0 and
humanized anti-PGE2 antibody Hu2B5.7 is assessed by determining
carragenan-induced hyperalgesia. The induction of paw inflammation
with carrageenan is performed as previously described (Joseph P.
Portanova, et al. J. Exp. Med. 184: 883-891 (1996)). Hyperalgesia
is induced by the injection of 0.1 ml of a 0.1% carrageenan
solution in sterile saline (FMC Corp., Rockland, Me.) into the hind
footpad of a 200-g male Sprague Dawley rat (Charles River
Laboratories, Portage, Me.). A hyperalgesic response to thermal
stimulation is determined in the same animals by the method of
Hargreaves et al. Pain. 32:77-88 (1988)). Hind paws are exposed to
radiant heat emitted from a high intensity projection bulb at
selected times after injection. The amount of time in which each
hind paw remains in contact with the heat source is measured to the
nearest 0.1 s. The hyperalgesic response is expressed as the
difference in the latency withdrawal period between carrageenan-
and saline-injected paws of each animal. In certain experiments,
rats are administered indomethacin by oral gavage in 0.5%
Methocel/0.025% Tween 80 (Sigma Chemical Co., St. Louis, Mo.) 1
hour before carrageenan administration. Other rats are injected
intraperitoneally with mouse anti-PGE2 mAb, 2B5-8.0, or humanized
anti-PGE2 antibody, Hu2B5.7, or isotype-matched antibody 18 hours
before carrageenan injection.
Example 4.4.2
In Vivo Efficacy of Mouse Anti-PGE Antibodies in Collagen-Induced
Arthritis
[0330] Type II bovine collagen (lyophilized) was obtained from the
University of Utah (Salt Lake City, Utah). Male DBA/J mice
(8-10-week-old, Jackson Laboratories, Bar Harbor, Me.) were
immunized intradermally at the base of the tail with 100 .mu.L of
emulsion containing 100 .mu.g of type II bovine collagen dissolved
in 0.1 N of acetic acid and 100 .mu.g of heat-inactivated
Mycobacterium tuberculosis H37Ra (Complete Freund's Adjuvant,
Difco, Laurence, Kans.). Twenty-one days after immunization with
collagen, mice were boosted IP with 1 mg of Zymosan A (Sigma, St.
Louis, Mo.). Following the boost, mice were monitored daily for
arthritis. Each paw was scored by the following criteria: 0=normal;
1=swelling in one site, foot, or ankle; 2=swelling in foot and
ankle; and 3=ankylosis. Scores were summed for each animal, and
total average of all animals in each group was expressed as MAS. In
addition to clinical scores, mice were also evaluated for paw-edema
using Dyer spring calipers model #310-119. Mice were enrolled for
the study between days 24 and 28 at the first clinical signs of
disease. At the termination of the experiment, six paws from each
group were harvested and stored in 10% neutral buffered formalin
for micro CT and histology.
[0331] Micro-computed tomography was performed on a Scanco
.mu.CT-40 unit (Scanco Medical AG) at 60 kVp at 160 .mu.A. The hind
paws (stored in 70% ethanol) were secured in imaging tubes and the
tarsal bone volume was measured for a 1.8-mm section of the mouse
ankle from the base of the tibia to the tarsal/metatarsal joint at
a resolution of 18 .mu.m. The raw micro-computed tomography image
was then analyzed using the Scanco AG .mu.CT Evaluation
program.
[0332] For histopathology analysis, formalin-fixed paws were
sectioned and stained with Gills 3 hematoxylin (Richard-Allan
Scientific, Kalamazoo, Mich.) and eosin with phloxine (Newcomer
Supply, Middleton, Mich.). Severity of disease was evaluated
histologically using the following criteria: 0=normal; 1=minimal
change; 2=mild change; 3=moderate change; and 4=severe change.
Scores were summed for each animal, and the total was expressed as
an average of all animals in each group.
[0333] The therapeutic effects of anti-PGE2 alone was evaluated in
mouse (male DBA/1J) collagen-induced arthritis, a standard
preclinical model for human rheumatoid arthritis. Drug treatment
was initiated after the mice developed signs of arthritic disease
after immunization with bovine type II collagen. Mice were scored
visually for clinical signs of arthritis and the results were
recorded as the mean arthritic score (MAS). Paw swelling and MAS
monitored over time, were also represented as area under the curve
(AUC) (Table 21). After disease onset, treatment with anti-PGE2 mAb
2B5 reduced the AUC for MAS by 22%.
TABLE-US-00021 TABLE 21 Disease Score, Swelling And Bone Volume In
Mouse CIA After Anti-PGE.sub.2 2B5-8.0 Treatment Vehicle Anti-PGE2
(PBS) (8 mg/kg, 2X/week, i.p.) MAS (AUC) 74 .+-. 7.3 57.4 .+-. 6.7
(Score # days) Paw Swelling (AUC) 14.3 .+-. 1.6 8.4 .+-. 1.3 (mm #
days) Bone Volume (mm.sup.3) 1.2 .+-. 0.2 1.6 .+-. 0.1
Example 4.4.2
In Vivo Efficacy of Mouse Anti-PGE2 Antibodies in Adjuvant-Induced
Arthritis
[0334] The in vivo efficacy of mouse anti-PGE2 antibody 2B5-8.0 is
assessed in an adjuvant-induced arthritis model. Arthritis is
induced in male Lewis rats (Harlan, Indianapolis, Ind.) by footpad
injection of Mycobacterium butyricum in mineral oil (Difco
Laboratories, Detroit, Mich.) as described previously.
Dexamethasone and indomethacin (Sigma Chemical Co.) are suspended
in Methocel/Tween and administered twice daily by gavage at dosages
of 0.1 and 2 mg/kg, respectively. 2B5-8.0 and isotype control are
administered daily at a dose of 10 mg/kg by intraperitoneal
injection. Treatments are initiated on day 15 post adjuvant
injection and continued until final assessment of paw volume of
uninjected contralateral paws on day 21. Mice are carefully
examined twice weekly for the visual appearance of arthritis in
peripheral joints, and scores for disease activity are
determined.
INCORPORATION BY REFERENCE
[0335] The contents of all cited references (including literature
references, patents, patent applications, and websites) that maybe
cited throughout this application are hereby expressly incorporated
by reference in their entirety, as are the references cited
therein. The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of immunology,
molecular biology and cell biology, which are well known in the
art.
EQUIVALENTS
[0336] The invention may be embodied in other specific forms
without departing from the spirit or essential characteristics
thereof. The foregoing embodiments are therefore to be considered
in all respects illustrative rather than limiting of the invention
described herein. Scope of the invention is thus indicated by the
appended claims rather than by the foregoing description, and all
changes that come within the meaning and range of equivalency of
the claims are therefore intended to be embraced herein.
Sequence CWU 1
1
831330PRTHomo sapiens 1Ala Ser Thr Lys Gly Pro Ser Val Phe Phe Leu
Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215
220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu
225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys
Ser Leu Ser Leu Ser Pro Gly Lys 325 330 2330PRTHomo sapiens 2Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10
15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu
Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145
150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 330 3106PRTHomo sapiens 3Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65
70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105
4105PRTHomo sapiens 4Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe
Pro Pro Ser Ser Glu 1 5 10 15 Glu Leu Gln Ala Asn Lys Ala Thr Leu
Val Cys Leu Ile Ser Asp Phe 20 25 30 Tyr Pro Gly Ala Val Thr Val
Ala Trp Lys Ala Asp Ser Ser Pro Val 35 40 45 Lys Ala Gly Val Glu
Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 50 55 60 Tyr Ala Ala
Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 65 70 75 80 His
Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 85 90
95 Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 5124PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
5Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Arg Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Asp Ile Asn Trp Val Arg Leu Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Cys Met Asn Pro Thr Thr Gly Lys Thr Gly
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Thr Ile Ala Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Gly
Tyr Ser Pro Gly Tyr Gly Val Ala Tyr Ala Asp 100 105 110 Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 65PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 6Asn
Tyr Asp Ile Asn 1 5 717PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 7Cys Met Asn Pro Thr Thr Gly
Lys Thr Gly Tyr Ala Gln Lys Phe Gln 1 5 10 15 Gly 815PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 8Gly
Arg Gly Tyr Ser Pro Gly Tyr Gly Val Ala Tyr Ala Asp Tyr 1 5 10 15
9108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 9Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Pro Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Thr
Pro Gly Lys Ala Pro Ser Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Pro Pro Pro 85 90
95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105
1011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Arg Ala Ser Gln Ser Ile Ser Thr Tyr Leu Asn 1 5
10 117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Ala Ala Ser Ser Leu Gln Ser 1 5
129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Gln Gln Ser Tyr Ser Pro Pro Pro Thr 1 5
13126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 13Glu Val Gln Leu Val Gln Ser Gly Ala Glu Thr
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Glu Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Glu Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Cys Ile Ser Pro
Tyr Asn Gly Lys Leu His Tyr Ala Gln Glu Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Thr Gly Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Gly Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Phe Ser Phe Tyr Asp Ser Ser Gly Tyr Tyr Tyr Val
100 105 110 Thr Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125 145PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 14Glu Tyr Gly Ile Ser 1 5
1517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Cys Ile Ser Pro Tyr Asn Gly Lys Leu His Tyr Ala
Gln Glu Phe Gln 1 5 10 15 Gly 1617PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 16Gly Gly Phe Ser Phe Tyr
Asp Ser Ser Gly Tyr Tyr Tyr Val Thr Asp 1 5 10 15 His
17108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 17Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Gly Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Ser Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Lys
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Phe
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ser Ala Thr Tyr Tyr Cys Gln Gln Ser Asp Thr Thr Pro Phe 85 90
95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105
1811PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Arg Ala Ser Gln Ser Ile Gly Ser Tyr Leu Asn 1 5
10 197PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Ala Ala Ser Lys Leu Gln Ser 1 5
209PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gln Gln Ser Asp Thr Thr Pro Phe Thr 1 5
21126PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Glu Val Gln Leu Val Gln Ser Gly Ser Glu Leu
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Glu Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Pro Glu Trp Met 35 40 45 Gly Cys Ile Ser Pro
Tyr Asn Gly Lys Leu His Tyr Ala Gln Lys Phe 50 55 60 Leu Gly Arg
Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Arg Ser Leu Lys Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Gly Gly Phe Ser Ser Tyr Asp Ser Ser Gly Tyr Tyr Tyr Val
100 105 110 Thr Asp His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125 2217PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Cys Ile Ser Pro Tyr Asn Gly Lys Leu
His Tyr Ala Gln Lys Phe Leu 1 5 10 15 Gly 2317PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Gly
Gly Phe Ser Ser Tyr Asp Ser Ser Gly Tyr Tyr Tyr Val Thr Asp 1 5 10
15 His 24108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 24Asp Ile Arg Leu Thr Gln Ser Pro
Ser Ser Leu Pro Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Thr Pro Gly Lys Ala Pro Ser Leu Leu Ile 35 40 45 Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Pro
Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 25118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Glu Val Gln Leu Val Gln Ser Gly
Pro Glu Leu Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Leu Thr Thr Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp
Ile Asp Thr Ser Thr Gly Asn Pro Thr Tyr Ala Pro Gly Phe 50 55 60
Leu Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Leu Ser Thr Thr Tyr 65
70 75 80 Leu Gln Ile Ser Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Ser His Thr Arg Pro Gly Asp Phe Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
265PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Thr Tyr Ala Met Asn 1 5 2717PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Trp
Ile Asp Thr Ser Thr Gly Asn Pro Thr Tyr Ala Pro Gly Phe Leu 1 5 10
15 Gly 289PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 28Ser Ser His Thr Arg Pro Gly Asp Phe 1 5
29111PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 29Gln Ser Gly Leu Thr Gln Pro Pro Ser Val Ser
Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser
Glu Ser Asn Val Gly Thr Asn 20 25 30 Ser Val Asn Trp Tyr Gln Gln
Leu Pro Gly Ala Ala Pro Arg Leu Leu 35 40 45 Ile Arg Gly Asn Ser
Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys
Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Arg Leu Gln 65 70 75 80 Ser
Glu Asp Glu Ala Asp Tyr Phe Cys Gly Ala Cys Asp Gly Arg Leu 85 90
95 Ser Gly Leu Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu 100
105 110 3013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 30Ser Gly Ser Glu Ser Asn
Val Gly Thr Asn Ser Val Asn 1 5 10 316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 31Gly
Asn Ser Asp Arg Pro 1 5 3212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 32Gly Ala Cys Asp Gly Arg Leu
Ser Gly Leu Tyr Val 1 5 10 33118PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 33Glu Val Gln Leu Val
Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Thr 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Tyr Thr Leu Thr Thr Tyr 20 25 30 Ala
Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Trp Ile Gly Thr Ser Thr Gly Asn Pro Thr Tyr Ala Gln Gly Phe
50 55 60 Thr Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Asn Thr
Ala His 65 70 75 80 Leu Gln Ile Tyr Ser Leu Lys Ala Glu Asp Thr Ala
Leu Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Leu Thr Arg Pro Ala Asp
Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
3417PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 34Trp Ile Gly Thr Ser Thr Gly Asn Pro Thr Tyr Ala
Gln Gly Phe Thr 1 5 10 15 Gly 359PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 35Ser Ser Leu Thr Arg Pro
Ala Asp Tyr 1 5 36111PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 36Gln Ser Gly Leu Thr Gln
Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile
Ser Cys Phe Gly Ser Ser Ser Asn Ile Gly Ala Gly 20 25 30 Tyr Asp
Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu 35 40 45
Leu Ile Phe Gly Asn Asn Asn Arg Pro Ser Gly Val Pro Asp Arg Phe 50
55 60 Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Thr Gly
Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Cys
Asp Ser Ser 85 90 95 Leu Ser Gly Ala Val Phe Gly Thr Gly Thr Lys
Val Thr Val Leu 100 105 110 3714PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 37Phe Gly Ser Ser Ser Asn
Ile Gly Ala Gly Tyr Asp Val His 1 5 10 386PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 38Gly
Asn Asn Asn Arg Pro 1 5 3911PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 39Gln Ser Cys Asp Ser Ser Leu
Ser Gly Ala Val 1 5 10 40116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 40Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115
41113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 41Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Val Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg 42116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 42Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Ile
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115
43113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 43Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Val Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg 44116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 44Gln Val Gln Leu Gln Gln
Ser Gly Pro Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45
Gly Asp Ile Tyr Pro Tyr Gly Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115
45113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 45Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Val Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg 4625PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 46Gln Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser 20 25 4714PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 47Trp Val Lys Gln Arg Pro Gly His Gly
Leu Glu Trp Ile Gly 1 5 10 4832PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 48Lys Ala Thr Leu Thr Val
Asp Thr Ser Ser Ser Thr Ala Tyr Met Gln 1 5 10 15 Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Ile Tyr Tyr Cys Ala Arg 20 25 30
4911PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala 1 5
10 5023PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro
Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys 20
5115PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Trp Tyr Leu Gln Arg Pro Gly Gln Ser Pro Lys Leu
Leu Ile Tyr 1 5 10 15 5231PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 52Val Pro Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Val Phe Thr Leu 1 5 10 15 Lys Ile Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys 20 25 30
5311PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 1 5
10 5410PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Gly Tyr Thr Phe Thr Lys Tyr Trp Leu Gly 1 5 10
5517PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 55Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn
Glu Lys Phe Lys 1 5 10 15 Asp 567PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 56Ser Asp Gly Ser Ser Thr
Tyr 1 5 5716PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 57Thr Ser Ser Gln Asn Ile Val His Ser
Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 588PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 58Lys
Val Ser Asn Arg Phe Ser Gly 1 5 599PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 59Phe
Gln Val Ser His Val Pro Tyr Thr 1 5 60116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
60Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys
Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His
Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly
Ser Ser Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 61113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 61Asp Val Val Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Thr Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Val 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110 Arg 62116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
62Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys
Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His
Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly
Ser Ser Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 63113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 63Asp Val Val Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Thr Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Val 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105 110 Arg 64116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
64Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys
Tyr 20 25 30 Trp Leu Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His
Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly
Ser Ser Thr Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 65113PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 65Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Thr Ser Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Val
85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 110 Arg 66116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 66Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
67113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 67Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 68116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 68Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
69113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 69Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 70116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 70Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
71113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 71Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 72116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 72Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
73113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 73Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 74116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 74Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Leu Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
75113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 75Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 76116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 76Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
77113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 77Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110 Arg 78116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 78Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu
Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Asp Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50
55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly
Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115
79112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Glu Leu Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser
Ser Gln Asn Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90
95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 80116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 80Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu Gly Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp
Ile Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50 55 60
Lys Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 81112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
81Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Thr Ser Ser Gln Asn Ile Val His
Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg
Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Val Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Phe Gln Val 85 90 95 Ser His Val Pro Tyr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110
82116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic consensus sequence 82Gln Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Lys Tyr 20 25 30 Trp Leu Gly Trp Val
Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile
Tyr Pro Gly Tyr Asp Tyr Thr His Tyr Asn Glu Lys Phe 50 55 60 Lys
Asp Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70
75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe
Cys 85 90 95 Ala Arg Ser Asp Gly Ser Ser Thr Tyr Trp Gly Gln Gly
Thr Leu Val 100 105 110 Thr Val Ser Ala 115 83113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
sequence 83Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser
Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Thr Ser Ser Gln Asn
Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Val Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Val 85 90 95 Ser His
Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100
105 110 Arg
* * * * *
References