U.S. patent application number 14/056116 was filed with the patent office on 2014-05-15 for dual variable domain immunoglobulins and uses thereof.
This patent application is currently assigned to ABBVIE INC.. The applicant listed for this patent is Susan E. Brophy, Tariq Ghayur, Junjian Liu, Sharmila Manoj. Invention is credited to Susan E. Brophy, Tariq Ghayur, Junjian Liu, Sharmila Manoj.
Application Number | 20140134171 14/056116 |
Document ID | / |
Family ID | 43649948 |
Filed Date | 2014-05-15 |
United States Patent
Application |
20140134171 |
Kind Code |
A1 |
Ghayur; Tariq ; et
al. |
May 15, 2014 |
Dual Variable Domain Immunoglobulins and Uses Thereof
Abstract
The present invention relates to engineered multivalent and
multispecific binding proteins, methods of making, and specifically
to their uses in the prevention, diagnosis, and/or treatment of
disease.
Inventors: |
Ghayur; Tariq; (Holliston,
MA) ; Liu; Junjian; (Shrewsbury, MA) ; Manoj;
Sharmila; (Arlington Heights, IL) ; Brophy; Susan
E.; (Lindenhurst, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ghayur; Tariq
Liu; Junjian
Manoj; Sharmila
Brophy; Susan E. |
Holliston
Shrewsbury
Arlington Heights
Lindenhurst |
MA
MA
IL
IL |
US
US
US
US |
|
|
Assignee: |
ABBVIE INC.
North Chicago
IL
|
Family ID: |
43649948 |
Appl. No.: |
14/056116 |
Filed: |
October 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12873926 |
Sep 1, 2010 |
8586714 |
|
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14056116 |
|
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61251946 |
Oct 15, 2009 |
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61238908 |
Sep 1, 2009 |
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Current U.S.
Class: |
424/136.1 ;
435/252.33; 435/254.11; 435/254.2; 435/254.21; 435/320.1; 435/328;
435/419; 435/69.6; 530/387.3; 536/23.53 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
11/16 20180101; A61P 31/18 20180101; A61P 35/00 20180101; C07K
16/2887 20130101; A61P 3/08 20180101; A61P 21/04 20180101; A61P
25/06 20180101; A61P 1/18 20180101; A61P 7/10 20180101; A61P 13/02
20180101; A61P 19/00 20180101; A61P 21/00 20180101; A61P 11/00
20180101; A61P 17/02 20180101; A61P 19/02 20180101; C07K 2317/732
20130101; A61K 45/06 20130101; A61P 39/02 20180101; C07K 16/468
20130101; A61P 1/04 20180101; A61P 31/00 20180101; A61P 33/02
20180101; A61P 25/04 20180101; A61P 25/18 20180101; A61P 9/04
20180101; A61P 31/04 20180101; A61P 31/16 20180101; C07K 2317/33
20130101; A61P 5/14 20180101; A61P 19/04 20180101; A61P 31/14
20180101; A61P 25/14 20180101; A61P 37/02 20180101; A61P 5/50
20180101; A61P 31/10 20180101; C07K 16/2809 20130101; A61P 9/12
20180101; A61P 31/20 20180101; C07K 16/30 20130101; A61P 11/02
20180101; A61P 11/06 20180101; A61P 31/08 20180101; A61P 25/28
20180101; A61P 33/06 20180101; C07K 2317/92 20130101; A61P 17/00
20180101; A61P 17/14 20180101; A61P 25/16 20180101; A61P 27/14
20180101; A61P 3/10 20180101; A61P 19/06 20180101; A61P 19/10
20180101; A61P 33/00 20180101; A61P 1/14 20180101; A61P 3/14
20180101; A61P 13/12 20180101; A61P 25/24 20180101; A61P 37/08
20180101; A61P 1/16 20180101; A61P 29/00 20180101; A61P 7/02
20180101; A61P 7/04 20180101; A61P 25/30 20180101; A61P 27/02
20180101; A61P 5/38 20180101; A61P 7/06 20180101; C07K 16/2803
20130101; C07K 16/2863 20130101; C07K 2317/24 20130101; A61P 1/00
20180101; A61P 5/18 20180101; A61P 9/08 20180101; A61P 9/10
20180101; A61P 37/06 20180101; C07K 2317/31 20130101; C07K 2317/515
20130101; A61P 21/02 20180101; A61P 37/00 20180101; C07K 16/44
20130101; A61P 25/00 20180101; C07K 2317/64 20130101; A61P 9/14
20180101; A61P 15/00 20180101; A61P 17/04 20180101; A61P 17/06
20180101; A61P 43/00 20180101; C07K 16/00 20130101; A61P 15/08
20180101; A61P 9/06 20180101; A61P 25/32 20180101; A61P 31/12
20180101; A61P 35/02 20180101; A61P 39/00 20180101 |
Class at
Publication: |
424/136.1 ;
536/23.53; 435/320.1; 435/252.33; 435/328; 435/419; 435/254.11;
435/254.2; 435/254.21; 435/69.6; 530/387.3 |
International
Class: |
C07K 16/46 20060101
C07K016/46; C07K 16/30 20060101 C07K016/30; C07K 16/44 20060101
C07K016/44 |
Claims
1.-36. (canceled)
37. An isolated nucleic acid encoding a binding protein amino acid
sequence, wherein the binding protein comprises first and second
polypeptide chains, each independently comprising
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain; VD2
is a second variable domain; C is a constant domain; X1 is a
linker; X2 is an Fc region; n is 0 or 1; wherein the VD1 domains on
the first and second polypeptide chains form a first functional
target binding site, and the VD2 domains on the first and second
polypeptide chains forms a second functional target binding site,
wherein the binding protein is capable of binding: (a) methotrexate
(MTX) and CD-3, wherein (i) the variable domains that form a
functional target binding site for MTX comprise three CDRs from SEQ
ID NO: 104 and three CDRs from SEQ ID NO: 105; and (ii) the
variable domains that form a functional target binding site for
CD-3 comprise: three CDRs from SEQ ID NO: 32 and a three CDRs from
SEQ ID NO: 33, three CDRs from SEQ ID NO: 68 and three CDRs from
SEQ ID NO: 69, or three CDRs from SEQ ID NO: 100 and three CDRs
from SEQ ID NO: 101; and (iii) the IC.sub.50 of the binding pair is
at most 4.0 nM as measured in a methotrexate neutralization assay;
(b) MTX and EGFR, wherein (i) the variable domains that form a
functional target binding site for MTX comprise: three CDRs from
SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and (ii) the
variable domains that form a functional target binding site for
EGFR comprise: three CDRs from SEQ ID NO: 34 and three CDRs from
SEQ ID NO: 35, three CDRs from SEQ ID NO: 62 and three CDRs from
SEQ ID NO: 63, or three CDRs from SEQ ID NO: 76 and three CDRs from
SEQ ID NO: 77; and (iii) the IC.sub.50 of the binding pair is at
most 3.9 nM as measured in a methotrexate neutralization assay; or
(c) MTX and HER-2, wherein (i) the variable domains that form a
functional target binding site for MTX comprise: three CDRs from
SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and (ii) the
variable domains that form a functional target binding site for
HER-2 comprise: three CDRs from SEQ ID NO: 36 and three CDRs from
SEQ ID NO: 37; or three CDRs from SEQ ID NO: 98 and three CDRs from
SEQ ID NO: 99; and (iii) the IC.sub.50 of the binding pair is at
most 3.9 nM as measured in a methotrexate neutralization assay.
38. A vector comprising an isolated nucleic acid according to claim
37.
39. The vector according to claim 38, wherein said vector is
selected from the group consisting of pcDNA, pTT, pTT3, pEFBOS,
pBV, pJV, pcDNA3.1 TOPO, pEF6 TOPO, and pBJ.
40. A host cell comprising a vector according to claim 38, wherein
the host cell is optionally selected from the group consisting of a
prokaryotic cell, Escherichia coli, a eukaryotic cell, an animal
cell, a plant cell, a fungal cell, a yeast cell, a Saccharomyces
cervisiae cell, an Sf9 cell, a mammalian cell, an avian cell, an
insect cell, a CHO cell, and a COS cell.
41.-50. (canceled)
51. A method of producing a binding protein, comprising culturing
the host cell of claim 40 under conditions sufficient to produce
the binding protein.
52.-58. (canceled)
59. A method for treating a subject for a disease or a disorder by
administering to the subject a binding protein such that treatment
is achieved, wherein the binding protein comprises first and second
polypeptide chains, each independently comprising
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain; VD2
is a second variable domain; C is a constant domain; X1 is a
linker; X2 is an Fc region; n is 0 or 1; wherein the VD1 domains on
the first and second polypeptide chains form a first functional
target binding site, and the VD2 domains on the first and second
polypeptide chains form a second functional target binding site,
wherein the binding protein is capable of binding: (a) methotrexate
(MTX) and CD3, wherein (i) the variable domains that form a
functional target binding site for MTX comprise three CDRs from SEQ
ID NO: 104 and three CDRs from SEQ ID NO: 105; and (ii) the
variable domains that form a functional target binding site for
CD-3 comprise: three CDRs from SEQ ID NO: 32 and a three CDRs from
SEQ ID NO: 33, three CDRs from SEQ ID NO: 68 and three CDRs from
SEQ ID NO: 69, or three CDRs from SEQ ID NO: 100 and three CDRs
from SEQ ID NO: 101; and (iii) the IC.sub.50 of the binding pair is
at most 4.0 nM as measured in a methotrexate neutralization assay;
(b) MTX and EGFR, wherein (i) the variable domains that form a
functional target binding site for MTX comprise: three CDRs from
SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105; and (ii) the
variable domains that form a functional target binding site for
EGFR comprise: three CDRs from SEQ ID NO: 34 and three CDRs from
SEQ ID NO: 35, three CDRs from SEQ ID NO: 62 and three CDRs from
SEQ ID NO: 61, or three CDRs from SEQ ID NO: 76 and three CDRs from
SEQ ID NO: 77; and (ii) the IC.sub.50 of the binding pair is at
most 3.9 nM as measured in a methotrexate neutralization assay; or
(c) MTX and HER-2, wherein (i) the variable domains that form a
functional target binding site for MTX comprise: three CDRs from
SEQ ID NO: 104 and three CDRs from SEQ ID NO: 105, and (ii) the
variable domains that form a functional target binding site for
HER-2 comprise: three CDRs from SEQ ID NO: 36 and three CDRs from
SEQ ID NO: 37; or three CDRs from SEQ ID NO: 98 and three CDRs from
SEQ ID NO: 99; and (iii) the IC.sub.50 of the binding pair is at
most 3.9 nM as measured in a methotrexate neutralization assay.
60. The method of claim 59, wherein said disorder is selected from
the group comprising 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 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-I 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, cholestatis, 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, 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, Hallervorden-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,
hypersensitivity 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, lymphedema, 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, occlusive
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 ateriosclerotic 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 Supranuclear
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, viral
encephalitis/aseptic meningitis, viral-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 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, keratoconjunctivitis
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, ovarian cancer, 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, prostate and rectal
cancer and hematopoietic malignancies (leukemia and lymphoma),
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, wound healing, yersinia and salmonella associated
arthropathy.
61. The method according to claim 60, wherein said administering to
the subject is 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.
62.-79. (canceled)
80. A binding protein comprising first and second polypeptide
chains, each independently comprising-VD1-(X1)n-VD2-C-(X2)n,
wherein VD1 is a first variable domain; VD2 is a second variable
domain; C is a constant domain; X1 is a linker; and X2 is an Fc
region; wherein the VD1 domains on the first and second polypeptide
chains form a first functional target binding site and the VD2
domains on the first and second polypeptide chains form a second
functional target binding site, and wherein the binding protein is
capable of binding (a) methotrexate (MTX) and mCD-3, wherein the
binding protein comprises: DVD1011 (comprising SEQ ID NOs: 110 and
111), DVD1012 (comprising SEQ ID NOs: 112 and 113), DVD1013
(comprising SEQ ID NOs: 114 and 115), DVD1014 (comprising SEQ ID
NOs: 116 and 117), DVD1015 (comprising SEQ ID NOs: 118 and 119),
DVD1016 (comprising SEQ ID NOs: 120 and 121), DVD1017 (comprising
SEQ ID NOs: 122 and 123), or DVD1018 (comprising SEQ ID NOs: 124
and 125); (b) methotrexate (MTX) and mCD-19, wherein the binding
protein comprises: DVD1019 (comprising SEQ ID NOs: 126 and 127),
DVD1020 (comprising SEQ ID NOs: 128 and 129), DVD1021 (comprising
SEQ ID NOs: 130 and 131), DVD1022 (comprising SEQ ID NOs: 132 and
133), DVD1023 (comprising SEQ ID NOs: 134 and 135), DVD1024
(comprising SEQ ID NOs: 136 and 137), DVD1025 (comprising SEQ ID
NOs: 138 and 139), or DVD1026 (comprising SEQ ID NOs: 140 and 141);
(c) methotrexate (MTX) and hCD-3, wherein the binding protein
comprises: DVD1027 (comprising SEQ ID NOs: 142 and 143), DVD1028
(comprising SEQ ID NOs: 144 and 145), DVD1029 (comprising SEQ ID
NOs: 146 and 147), DVD1030 (comprising SEQ ID NOs: 148 and 149),
DVD1031 (comprising SEQ ID NOs: 150 and 151), DVD1032 (comprising
SEQ ID NOs: 152 and 153), DVD1033 (comprising SEQ ID NOs: 154 and
155), DVD1034 (comprising SEQ ID NOs: 156 and 157), DVD1216
(comprising SEQ ID NOs: 186 and 187), or DVD1217 (comprising SEQ ID
NOs: 188 and 189), (d) methotrexate (MTX) and hCD-19, wherein the
binding protein comprises: DVD1035 (comprising SEQ ID NOs: 158 and
159), DVD1036 (comprising SEQ ID NOs: 160 and 161), DVD1037
(comprising SEQ ID NOs: 162 and 163), DVD1038 (comprising SEQ ID
NOs: 164 and 165), DVD1039 (comprising SEQ ID NOs: 166 and 167),
DVD1040 (comprising SEQ ID NOs: 168 and 169), DVD1041 (comprising
SEQ ID NOs: 170 and 171), or DVD1042 (comprising SEQ ID NOs: 172
and 173); (e) methotrexate (MTX) and hCD-20, wherein the binding
protein comprises: DVD1208 (comprising SEQ ID NOs: 174 and 175), or
DVD1209 (comprising SEQ ID NOs: 176 and 177); (f) methotrexate
(MTX) and EGFR, wherein the binding protein comprises: DVD1210
(comprising SEQ ID NOs: 178 and 179), or DVD1211 (comprising SEQ ID
NOs: 180 and 181); or (g) methotrexate (MTX) and HER-2, wherein the
binding protein comprises DVD1212 (comprising SEQ ID NOs: 182 and
183), or DVD1213 (comprising SEQ ID NOs: 184 and 185).
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. application Ser.
No. 12/873,926, filed Sep. 1, 2010, which claims priority to U.S.
Provisional Patent Application No. 61/238,908, filed Sep. 1, 2009,
and U.S. Provisional Patent Application No. 61/251,946, filed Oct.
15, 2009, which are hereby expressly incorporated herein by
reference in their entirety for any purpose.
FIELD OF THE INVENTION
[0002] The present invention relates to multivalent and
multispecific binding proteins that bind to a prophylactic or
therapeutic agent, methods of making, and specifically to their
uses in the, diagnosis, prevention and/or treatment of acute and
chronic inflammatory diseases, cancer, and other diseases.
BACKGROUND OF THE INVENTION
[0003] Engineered proteins, such as multispecific antibodies that
bind two or more antigens, are known in the art. Such multispecific
binding proteins can be generated using cell fusion, chemical
conjugation, or recombinant DNA techniques.
[0004] Bispecific antibodies have been produced using quadroma
technology (see Milstein, C. and Cuello, A. C. (1983) Nature
305(5934):537-40) based on the somatic fusion of two different
hybridoma cell lines expressing murine monoclonal antibodies (mAbs)
with the desired specificities of the bispecific antibody. Because
of the random pairing of two different immunoglobulin (Ig) heavy
and light chains within the resulting hybrid-hybridoma (or
quadroma) cell line, up to ten different Ig species are generated,
of which only one is the functional bispecific antibody. The
presence of mis-paired by-products, and significantly reduced
production yields, means sophisticated purification procedures are
required.
[0005] Bispecific antibodies can also be produced by chemical
conjugation of two different mAbs (see Staerz, U. D., et al. (1985)
Nature 314(6012): 628-31). This approach does not yield homogeneous
preparation. Other approaches have used chemical conjugation of two
different mAbs or smaller antibody fragments (see Brennan, M., et
al. (1985) Science 229(4708): 81-3).
[0006] Another method used to produce bispecific antibodies is the
coupling of two parental antibodies with a hetero-bifunctional
crosslinker, but the resulting bispecific antibodies suffer from
significant molecular heterogeneity because reaction of the
crosslinker with the parental antibodies is not site-directed. To
obtain more homogeneous preparations of bispecific antibodies two
different Fab fragments have been chemically crosslinked at their
hinge cysteine residues in a site-directed manner (see Glennie, M.
J., et al. (1987) J. Immunol. 139(7): 2367-75). But this method
results in Fab'2 fragments, not full IgG molecule.
[0007] A wide variety of other recombinant bispecific antibody
formats have been developed (see Kriangkum, J., et al. (2001)
Biomol. Engin. 18(2): 31-40). Amongst them tandem single-chain Fv
molecules and diabodies, and various derivatives thereof, are the
most widely used. Routinely, construction of these molecules starts
from two single-chain Fv (scFv) fragments that recognize different
antigens (see Economides, A. N., et al. (2003) Nat. Med. 9(1):
47-52). Tandem scFv molecules (taFv) represent a straightforward
format simply connecting the two scFv molecules with an additional
peptide linker. The two scFv fragments present in these tandem scFv
molecules form separate folding entities. Various linkers can be
used to connect the two scFv fragments and linkers with a length of
up to 63 residues (see Nakanishi, K., et al. (2001) Ann. Rev.
Immunol 19: 423-74). Although the parental scFv fragments can
normally be expressed in soluble form in bacteria, it is, however,
often observed that tandem scFv molecules form insoluble aggregates
in bacteria. Hence, refolding protocols or the use of mammalian
expression systems are routinely applied to produce soluble tandem
scFv molecules. In a recent study, in vivo expression by transgenic
rabbits and cattle of a tandem scFv directed against CD28 and a
melanoma-associated proteoglycan was reported (see Gracie, J. A.,
et al. (1999) J. Clin. Invest. 104(10): 1393-401). In this
construct, the two scFv molecules were connected by a CH1 linker
and serum concentrations of up to 100 mg/L of the bispecific
antibody were found. Various strategies including variations of the
domain order or using middle linkers with varying length or
flexibility were employed to allow soluble expression in bacteria.
A few studies have now reported expression of soluble tandem scFv
molecules in bacteria (see Leung, B. P., et al. (2000) J. Immunol.
164(12): 6495-502; Ito, A., et al. (2003) J. Immunol. 170(9):
4802-9; Karni, A., et al. (2002) J. Neuroimmunol 125(1-2): 134-40)
using either a very short Ala3 linker or long glycine/serine-rich
linkers. In a recent study, phage display of a tandem scFv
repertoire containing randomized middle linkers with a length of 3
or 6 residues was employed to enrich for those molecules that are
produced in soluble and active form in bacteria. This approach
resulted in the isolation of a tandem scFv molecule with a 6 amino
acid residue linker (see Arndt, M. and Krauss, J. (2003) Methods
Mol. Biol. 207: 305-21). It is unclear whether this linker sequence
represents a general solution to the soluble expression of tandem
scFv molecules. Nevertheless, this study demonstrated that phage
display of tandem scFv molecules in combination with directed
mutagenesis is a powerful tool to enrich for these molecules, which
can be expressed in bacteria in an active form.
[0008] Bispecific diabodies (Db) utilize the diabody format for
expression. Diabodies are produced from scFv fragments by reducing
the length of the linker connecting the VH and VL domain to
approximately 5 residues (see Peipp, M. and Valerius, T. (2002)
Biochem. Soc. Trans. 30(4): 507-11). This reduction of linker size
facilitates dimerization of two polypeptide chains by crossover
pairing of the VH and VL domains. Bispecific diabodies are produced
by expressing, two polypeptide chains with, either the structure
VHA-VLB and VHB-VLA (VH-VL configuration), or VLA-VHB and VLB-VHA
(VL-VH configuration) within the same cell. A large variety of
different bispecific diabodies have been produced in the past and
most of them are expressed in soluble form in bacteria. However, a
recent comparative study demonstrates that the orientation of the
variable domains can influence expression and formation of active
binding sites (see Mack, M. et al. (1995) Proc. Natl. Acad. Sci.
USA 92(15): 7021-5). Nevertheless, soluble expression in bacteria
represents an important advantage over tandem scFv molecules.
However, since two different polypeptide chains are expressed
within a single cell inactive homodimers can be produced together
with active heterodimers. This necessitates the implementation of
additional purification steps in order to obtain homogenous
preparations of bispecific diabodies. One approach to force the
generation of bispecific diabodies is the production of
knob-into-hole diabodies (see Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90(14): 6444-8.18). This was demonstrated for
a bispecific diabody directed against HER2 and CD3. A large knob
was introduced in the VH domain by exchanging Val37 with Phe and
Leu45 with Trp and a complementary hole was produced in the VL
domain by mutating Phe98 to Met and Tyr87 to Ala, either in the
anti-HER2 or the anti-CD3 variable domains. By using this approach
the production of bispecific diabodies could be increased from 72%
by the parental diabody to over 90% by the knob-into-hole diabody.
Importantly, production yields did only slightly decrease as a
result of these mutations. However, a reduction in antigen-binding
activity was observed for several analyzed constructs. Thus, this
rather elaborate approach requires the analysis of various
constructs in order to identify those mutations that produce
heterodimeric molecule with unaltered binding activity. In
addition, such approach requires mutational modification of the
immunoglobulin sequence at the constant region, thus creating
non-native and non-natural form of the antibody sequence, which may
result in increased immunogenicity, poor in vivo stability, as well
as undesirable pharmacokinetics.
[0009] Single-chain diabodies (scDb) represent an alternative
strategy for improving the formation of bispecific diabody-like
molecules (see Holliger, P. and Winter, G. (1997) Cancer Immunol
Immunother. 45(3-4): 128-30; Wu, A. M., et al. (1996)
Immunotechnology 2(1): p. 21-36). Bispecific single-chain diabodies
are produced by connecting the two diabody-forming polypeptide
chains with an additional middle linker with a length of
approximately 15 amino acid residues. Consequently, all molecules
with a molecular weight corresponding to monomeric single-chain
diabodies (50-60 kDa) are bispecific. Several studies have
demonstrated that bispecific single chain diabodies are expressed
in bacteria in soluble and active form with the majority of
purified molecules present as monomers (see Holliger, P. and
Winter, G. (1997) Cancer Immunol Immunother. 45(3-4): 128-30; Wu,
A. M., et al. (1996) Immunotechnol. 2(1): 21-36; Pluckthun, A. and
Pack, P. (1997) Immunotechnol. 3(2): 83-105; Ridgway, J. B., et al.
(1996) Protein Engin. 9(7): 617-21). Thus, single-chain diabodies
combine the advantages of tandem scFvs (all monomers are
bispecific) and diabodies (soluble expression in bacteria).
[0010] More recently diabodies have been fused to Fc to generate
more Ig-like molecules, named di-diabodies (see Lu, D., et al.
(2004) J. Biol. Chem. 279(4): 2856-65). In addition, multivalent
antibody constructs comprising two Fab repeats in the heavy chain
of an IgG and that bind four antigen molecules have been described
(see WO 0177342A1, and Miller, K., et al. (2003) J. Immunol.
170(9): 4854-61).
[0011] There is a need in the art for improved multivalent binding
proteins that bind two or more antigens. U.S. Pat. No. 7,612,181
provides a novel family of binding proteins that bind two or more
antigens with high affinity, and which are called dual variable
domain immunoglobulins (DVD-IgTM). The present disclosure provides
further novel binding proteins that bind two or more antigens.
SUMMARY OF THE INVENTION
[0012] This invention pertains to multivalent binding proteins that
bind two or more antigens. The present invention provides a novel
family of binding proteins that bind two or more antigens with high
affinity.
[0013] In one embodiment the invention provides a binding protein
comprising a polypeptide chain, wherein the polypeptide chain
comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable
domain, VD2 is a second variable domain, C is a constant domain, X1
represents an amino acid or polypeptide, X2 represents an Fc region
and n is 0 or 1. In an embodiment the VD1 and VD2 in the binding
protein are heavy chain variable domains. In another embodiment,
the heavy chain variable domain is selected from the group
consisting of a murine heavy chain variable domain, a human heavy
chain variable domain, a CDR grafted heavy chain variable domain,
and a humanized heavy chain variable domain. In yet another,
embodiment VD1 and VD2 bind the same antigen. In another embodiment
VD1 and VD2 bind different antigens. In still another embodiment, C
is a heavy chain constant domain. For example, X1 is a linker with
the proviso that X1 is not CH1. For example, X1 is a linker
selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO:
1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);
SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID
NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8);
RADAAAA(G.sub.4S).sub.4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID
NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP
(SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO:
15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17);
AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);
AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID
NO: 27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an
embodiment, X2 is an Fc region. In another embodiment, X2 is a
variant Fc region.
[0014] In an embodiment, the binding protein disclosed herein
comprises a polypeptide chain, wherein the polypeptide chain
comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first heavy chain
variable domain, VD2 is a second heavy chain variable domain, C is
a heavy chain constant domain, X1 is a linker with the proviso that
it is not CH1, and X2 is an Fc region.
In an embodiment, VD1 and VD2 in the binding protein are light
chain variable domains. In an embodiment, the light chain variable
domain is selected from the group consisting of a murine light
chain variable domain, a human light chain variable domain, a CDR
grafted light chain variable domain, and a humanized light chain
variable domain. In one embodiment VD1 and VD2 bind the same
antigen. In another embodiment VD1 and VD2 bind different antigens.
In an embodiment, C is a light chain constant domain. In another
embodiment, X1 is a linker with the proviso that X1 is not CL1. In
an embodiment, X1 is a linker selected from the group consisting of
AKTTPKLEEGEFSEAR (SEQ ID NO: 1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2);
AKTTPKLGG (SEQ ID NO: 3); SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID
NO: 5); RADAAP (SEQ ID NO: 6); RADAAPTVS (SEQ ID NO: 7);
RADAAAAGGPGS (SEQ ID NO: 8); RADAAAA(G.sub.4S).sub.4 (SEQ ID NO:
9), SAKTTPKLEEGEFSEARV (SEQ ID NO: 10); ADAAP (SEQ ID NO: 11);
ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP (SEQ ID NO: 13); TVAAPSVFIFPP
(SEQ ID NO: 14); QPKAAP (SEQ ID NO: 15); QPKAAPSVTLFPP (SEQ ID NO:
16); AKTTPP (SEQ ID NO: 17); AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP
(SEQ ID NO: 19); AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO:
21); ASTKGPSVFPLAP (SEQ ID NO: 22) GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26); TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID
NO: 27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). In an
embodiment, the binding protein does not comprise X2.
[0015] In an embodiment, both the variable heavy and variable light
chain comprise the same linker. In another embodiment, the variable
heavy and variable light chain comprise different linkers. In
another embodiment, both the variable heavy and variable light
chain comprise a short (about 6 amino acids) linker. In another
embodiment, both the variable heavy and variable light chain
comprise a long (greater than 6 amino acids) linker. In another
embodiment, the variable heavy chain comprises a short linker and
the variable light chain comprises a long linker. In another
embodiment, the variable heavy chain comprises a long linker and
the variable light chain comprises a short linker.
[0016] In an embodiment the binding protein disclosed herein
comprises a polypeptide chain, wherein said polypeptide chain
comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain
variable domain, VD2 is a second light chain variable domain, C is
a light chain constant domain, X1 is a linker with the proviso that
it is not CH1, and X2 does not comprise an Fc region.
[0017] In another embodiment the invention provides a binding
protein comprising two polypeptide chains, wherein said first
polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a
first heavy chain variable domain, VD2 is a second heavy chain
variable domain, C is a heavy chain constant domain, X1 is a linker
with the proviso that it is not CH1, and X2 is an Fc region; and
said second polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n,
wherein VD1 is a first light chain variable domain, VD2 is a second
light chain variable domain, C is a light chain constant domain, X1
is a linker with the proviso that it is not CH1, and X2 does not
comprise an Fc region. In a particular embodiment, the Dual
Variable Domain (DVD) binding protein comprises four polypeptide
chains wherein the first two polypeptide chains comprises
VD1-(X1)n-VD2-C-(X2)n, respectively wherein VD1 is a first heavy
chain variable domain, VD2 is a second heavy chain variable domain,
C is a heavy chain constant domain, X1 is a linker with the proviso
that it is not CH1, and X2 is an Fc region; and the second two
polypeptide chain comprises VD1-(X1)n-VD2-C-(X2)n respectively,
wherein VD1 is a first light chain variable domain, VD2 is a second
light chain variable domain, C is a light chain constant domain, X1
is a linker with the proviso that it is not CH1, and X2 does not
comprise an Fc region. Such a Dual Variable Domain (DVD) protein
has four antigen binding sites.
[0018] In another embodiment the binding proteins disclosed herein
bind one or more targets. In an embodiment, the target is selected
from the group consisting of cytokines, cell surface proteins,
enzymes and receptors. In another embodiment, the binding protein
modulates a biological function of one or more targets. In another
embodiment, the binding protein neutralizes one or more targets.
The binding protein of the invention binds cytokines selected from
the group consisting of lymphokines, monokines, polypeptide
hormones, receptors, and tumor markers. For example, the DVD-Ig of
the invention is capable of binding two or more of the following:
CD-20, human CD-19 (CD-19 or hCD-19), mouse CD-19 (mCD-19), CD-80,
CD-22, CD-40, human CD-3 (CD-3 or hCD-3)), mouse CD-3 (mCD-3),
human epidermal growth factor receptor 2 (HER-2), epidermal growth
factor receptor (EGFR), insulin-like growth factor 1,2 (IGF1,2),
insulin-like growth factor receptor (IGF1R), macrophage stimulating
protein receptor tyrosine kinase (RON), hepatocyte growth factor
(HGF), mesenchymal-epithelial transition factor (c-MET), vascular
endothelial growth factor (VEGF), Drosophila Delta homologue 4
(DLL4), neuropilin 1 (NRP1), placental growth factor (PLGF),
v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 3
(ErbB3), and methotrexate (MTX) (see also Table 2). In a specific
embodiment the binding protein binds pairs of targets selected from
the group consisting of MTX and mCD-3, MTX and mCD-19, MTX and
hCD-3, MTX and hCD-19, MTX and CD-20, MTX and EGFR, and MTX and
HER-2.
[0019] In an embodiment, the binding protein that binds MTX and
mCD-3 comprises a DVD heavy chain amino acid sequence selected from
the group consisting of SEQ ID NO. 110 and SEQ ID NO. 112; and a
DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 111 and SEQ ID NO. 113. In an embodiment,
the binding protein that binds MTX and mCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 110 and a DVD light chain
amino acid sequence of SEQ ID NO: 111. In another embodiment, the
binding protein that binds MTX and mCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
112 and a DVD light chain amino acid sequence of SEQ ID NO:
113.
[0020] In a second embodiment, the binding protein that binds MTX
and mCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 114 and SEQ ID NO. 116; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 115 and SEQ ID NO. 117. In an embodiment,
the binding protein that binds MTX and mCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 114 and a DVD light chain
amino acid sequence of SEQ ID NO: 115. In another embodiment, the
binding protein that binds MTX and mCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
116 and a DVD light chain amino acid sequence of SEQ ID NO:
117.
[0021] In a third embodiment, the binding protein that binds MTX
and mCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 118 and SEQ ID NO. 120; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 119 and SEQ ID NO. 121. In an embodiment,
the binding protein that binds MTX and mCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 118 and a DVD light chain
amino acid sequence of SEQ ID NO: 119. In another embodiment, the
binding protein that binds MTX and mCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
120 and a DVD light chain amino acid sequence of SEQ ID NO:
121.
[0022] In a fourth embodiment, the binding protein that binds MTX
and mCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 122 and SEQ ID NO. 124; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 123 and SEQ ID NO. 125. In an embodiment,
the binding protein that binds MTX and mCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 122 and a DVD light chain
amino acid sequence of SEQ ID NO: 123. In another embodiment, the
binding protein that binds MTX and mCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
124 and a DVD light chain amino acid sequence of SEQ ID NO:
125.
[0023] In an embodiment, the binding protein that binds MTX and
mCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 126 and SEQ ID NO. 128; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 127 and SEQ ID NO. 129. In an embodiment,
the binding protein that binds MTX and mCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 126 and a DVD light chain
amino acid sequence of SEQ ID NO: 127. In another embodiment, the
binding protein that binds MTX and mCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
128 and a DVD light chain amino acid sequence of SEQ ID NO:
129.
[0024] In a second embodiment, the binding protein that binds MTX
and mCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 130 and SEQ ID NO. 132; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 131 and SEQ ID NO. 133. In an embodiment,
the binding protein that binds MTX and mCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 130 and a DVD light chain
amino acid sequence of SEQ ID NO: 131. In another embodiment, the
binding protein that binds MTX and mCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
132 and a DVD light chain amino acid sequence of SEQ ID NO:
133.
[0025] In a third embodiment, the binding protein that binds MTX
and mCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 134 and SEQ ID NO. 136; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 135 and SEQ ID NO. 137. In an embodiment,
the binding protein that binds MTX and mCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 134 and a DVD light chain
amino acid sequence of SEQ ID NO: 135. In another embodiment, the
binding protein that binds MTX and mCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
136 and a DVD light chain amino acid sequence of SEQ ID NO:
137.
[0026] In a fourth embodiment, the binding protein that binds MTX
and mCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 138 and SEQ ID NO. 140; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 139 and SEQ ID NO. 141. In an embodiment,
the binding protein that binds MTX and mCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 138 and a DVD light chain
amino acid sequence of SEQ ID NO: 139. In another embodiment, the
binding protein that binds MTX and mCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
140 and a DVD light chain amino acid sequence of SEQ ID NO:
141.
[0027] In an embodiment, the binding protein that binds MTX and
hCD-3 comprises a DVD heavy chain amino acid sequence selected from
the group consisting of SEQ ID NO. 142 and SEQ ID NO. 144; and a
DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 143 and SEQ ID NO. 145. In an embodiment,
the binding protein that binds MTX and hCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 142 and a DVD light chain
amino acid sequence of SEQ ID NO: 143. In another embodiment, the
binding protein that binds MTX and hCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
144 and a DVD light chain amino acid sequence of SEQ ID NO:
145.
[0028] In a second embodiment, the binding protein that binds MTX
and hCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 146 and SEQ ID NO. 148; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 147 and SEQ ID NO. 149. In an embodiment,
the binding protein that binds MTX and hCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 146 and a DVD light chain
amino acid sequence of SEQ ID NO: 147. In another embodiment, the
binding protein that binds MTX and hCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
148 and a DVD light chain amino acid sequence of SEQ ID NO:
149.
[0029] In a third embodiment, the binding protein that binds MTX
and hCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 150 and SEQ ID NO. 152; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 151 and SEQ ID NO. 153. In an embodiment,
the binding protein that binds MTX and hCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 150 and a DVD light chain
amino acid sequence of SEQ ID NO: 151. In another embodiment, the
binding protein that binds MTX and hCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
152 and a DVD light chain amino acid sequence of SEQ ID NO:
153.
[0030] In a fourth embodiment, the binding protein that binds MTX
and hCD-3 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 154 and SEQ ID NO. 156; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 155 and SEQ ID NO. 157. In an embodiment,
the binding protein that binds MTX and hCD-3 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 154 and a DVD light chain
amino acid sequence of SEQ ID NO: 155. In another embodiment, the
binding protein that binds MTX and hCD-3 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
156 and a DVD light chain amino acid sequence of SEQ ID NO:
157.
[0031] In an embodiment, the binding protein that binds MTX and
hCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 158 and SEQ ID NO. 160; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 159 and SEQ ID NO. 161. In an embodiment,
the binding protein that binds MTX and hCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 158 and a DVD light chain
amino acid sequence of SEQ ID NO: 159. In another embodiment, the
binding protein that binds MTX and hCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
160 and a DVD light chain amino acid sequence of SEQ ID NO:
161.
[0032] In a second embodiment, the binding protein that binds MTX
and hCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 162 and SEQ ID NO. 164; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 163 and SEQ ID NO. 165. In an embodiment,
the binding protein that binds MTX and hCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 162 and a DVD light chain
amino acid sequence of SEQ ID NO: 163. In another embodiment, the
binding protein that binds MTX and hCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
164 and a DVD light chain amino acid sequence of SEQ ID NO:
165.
[0033] In a third embodiment, the binding protein that binds MTX
and hCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 166 and SEQ ID NO. 168; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 167 and SEQ ID NO. 169. In an embodiment,
the binding protein that binds MTX and hCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 166 and a DVD light chain
amino acid sequence of SEQ ID NO: 167. In another embodiment, the
binding protein that binds MTX and hCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
168 and a DVD light chain amino acid sequence of SEQ ID NO:
169.
[0034] In a fourth embodiment, the binding protein that binds MTX
and hCD-19 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 170 and SEQ ID NO. 172; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 171 and SEQ ID NO. 173. In an embodiment,
the binding protein that binds MTX and hCD-19 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 170 and a DVD light chain
amino acid sequence of SEQ ID NO: 171. In another embodiment, the
binding protein that binds MTX and hCD-19 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
172 and a DVD light chain amino acid sequence of SEQ ID NO:
173.
[0035] In an embodiment, the binding protein that binds MTX and
hCD-20 comprises a DVD heavy chain amino acid sequence selected
from the group consisting of SEQ ID NO. 174 and SEQ ID NO. 176; and
a DVD light chain amino acid sequence selected from the group
consisting of SEQ ID NO. 175 and SEQ ID NO. 177. In an embodiment,
the binding protein that binds MTX and hCD-20 comprises a DVD heavy
chain amino acid sequence of SEQ ID NO. 174 and a DVD light chain
amino acid sequence of SEQ ID NO: 175. In another embodiment, the
binding protein that binds MTX and hCD-20 has a reverse orientation
and comprises a DVD heavy chain amino acid sequence of SEQ ID NO.
176 and a DVD light chain amino acid sequence of SEQ ID NO:
177.
[0036] In an embodiment, the binding protein that binds MTX and
EGFR (seq. 3) comprises a DVD heavy chain amino acid sequence
selected from the group consisting of SEQ ID NO. 178 and SEQ ID NO.
180; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 179 and SEQ ID NO. 181. In an
embodiment, the binding protein that binds MTX and EGFR (seq. 3)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 178
and a DVD light chain amino acid sequence of SEQ ID NO: 179. In
another embodiment, the binding protein that binds MTX and EGFR
(seq. 3) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 180 and a DVD light chain amino
acid sequence of SEQ ID NO: 181.
[0037] In an embodiment, the binding protein that binds MTX and
Her-2 (seq. 1) comprises a DVD heavy chain amino acid sequence
selected from the group consisting of SEQ ID NO. 182 and SEQ ID NO.
184; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 183 and SEQ ID NO. 185. In an
embodiment, the binding protein that binds MTX and Her-2 (seq. 1)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 182
and a DVD light chain amino acid sequence of SEQ ID NO: 183. In
another embodiment, the binding protein that binds MTX and Her-2
(seq. 1) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 184 and a DVD light chain amino
acid sequence of SEQ ID NO: 185.
[0038] In an embodiment, the binding protein that binds MTX and
hCD-3 (seq. 1) comprises a DVD heavy chain amino acid sequence
selected from the group consisting of SEQ ID NO. 186 and SEQ ID NO.
188; and a DVD light chain amino acid sequence selected from the
group consisting of SEQ ID NO. 187 and SEQ ID NO. 189. In an
embodiment, the binding protein that binds MTX and hCD-3 (seq. 1)
comprises a DVD heavy chain amino acid sequence of SEQ ID NO. 186
and a DVD light chain amino acid sequence of SEQ ID NO: 187. In
another embodiment, the binding protein that binds MTX and hCD-3
(seq. 1) has a reverse orientation and comprises a DVD heavy chain
amino acid sequence of SEQ ID NO. 188 and a DVD light chain amino
acid sequence of SEQ ID NO: 189.
[0039] In another embodiment the invention provides a binding
protein comprising a polypeptide chain, wherein said polypeptide
chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first
heavy chain variable domain obtained from a first parent antibody,
or antigen binding portion thereof; VD2 is a second heavy chain
variable domain obtained from a second parent antibody, or antigen
binding portion thereof, which can be the same or different from
the first parent antibody; C is a heavy chain constant domain;
(X1)n is a linker with the proviso that it is not CH1, wherein said
(X1)n is either present or absent; and (X2)n is an Fc region,
wherein said (X2)n is either present or absent. In an embodiment,
the Fc region is absent from the binding protein.
[0040] In another embodiment, the invention provides a binding
protein comprising a polypeptide chain, wherein said polypeptide
chain comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first
light chain variable domain obtained from a first parent antibody
or antigen binding portion thereof; VD2 is a second light chain
variable domain obtained from a second parent antibody or antigen
binding portion thereof, which can be the same or different from
the first parent antibody; C is a light chain constant domain;
(X1)n is a linker with the proviso that it is not CH1, wherein said
(X1)n is either present or absent; and (X2)n does not comprise an
Fc region, wherein said (X2)n is either present or absent. In an
embodiment, (X2)n is absent from the binding protein.
[0041] In another embodiment the binding protein of the invention
comprises first and second polypeptide chains, wherein said first
polypeptide chain comprises a first VD1-(X1)n-VD2-C-(X2)n, wherein
VD1 is a first heavy chain variable domain obtained from a first
parent antibody or antigen binding portion thereof; VD2 is a second
heavy chain variable domain obtained from a second parent antibody
or antigen binding portion thereof, which can be the same or
different from the first parent antibody; C is a heavy chain
constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n is
an Fc region, wherein said (X2)n is either present or absent; and
wherein said second polypeptide chain comprises a second
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first light chain variable
domain obtained from a first parent antibody or antigen binding
portion thereof; VD2 is a second light chain variable domain
obtained from a second parent antibody or antigen binding portion
thereof, which can be the same or different from the first parent
antibody; C is a light chain constant domain; (X1)n is a linker
with the proviso that it is not CH1, wherein said (X1)n is either
present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent. In another
embodiment, the binding protein comprises two first polypeptide
chains and two second polypeptide chains. In yet another
embodiment, (X2)n is absent from the second polypeptide. In still
another embodiment, the Fc region, if present in the first
polypeptide is selected from the group consisting of native
sequence Fc region and a variant sequence Fc region. In still
another embodiment, the Fc region is selected from the group
consisting of an Fc region from an IgG1, an Fc region from an IgG2,
an Fc region from an IgG3, an Fc region from an IgG4, an Fc region
from an IgA, an Fc region from an IgM, an Fc region from an IgE,
and an Fc region from an IgD.
[0042] In another embodiment the binding protein of the invention
is a DVD-Ig that binds two antigens comprising four polypeptide
chains, wherein, each of the first and third polypeptide chains
comprise VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy chain
variable domain obtained from a first parent antibody, or antigen
binding portion thereof; VD2 is a second heavy chain variable
domain obtained from a second parent antibody, or antigen binding
portion thereof, which can be the same as or different from the
first parent antibody; C is a heavy chain constant domain; (X1)n is
a linker with the proviso that it is not CH1, wherein said (X1)n is
either present or absent; and (X2)n is an Fc region, wherein said
(X2)n is either present or absent; and wherein each of the second
and fourth polypeptide chains comprise VD1-(X1)n-VD2-C-(X2)n,
wherein VD1 is a first light chain variable domain obtained from a
first parent antibody or antigen binding portion thereof; VD2 is a
second light chain variable domain obtained from a second parent
antibody, or antigen binding portion thereof, which can be the same
as or different from the first parent antibody; C is a light chain
constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n does
not comprise an Fc region, wherein said (X2)n is either present or
absent.
[0043] The invention provides a method of making a DVD-Ig binding
protein by preselecting the parent antibodies. In an embodiment,
the method of making a Dual Variable Domain Immunoglobulin that
binds two antigens comprises the steps of a) obtaining a first
parent antibody, or antigen binding portion thereof, that binds a
first antigen; b) obtaining a second parent antibody or antigen
binding portion thereof, that binds a second antigen; c)
constructing first and third polypeptide chains, each of which
comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first heavy
chain variable domain obtained from said first parent antibody, or
antigen binding portion thereof; VD2 is a second heavy chain
variable domain obtained from said second parent antibody or
antigen binding portion thereof, which can be the same as or
different from the first parent antibody; C is a heavy chain
constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n is
an Fc region, wherein said (X2)n is either present or absent; d)
constructing second and fourth polypeptide chains each of which
comprises VD1-(X1)n-VD2-C-(X2)n, wherein, VD1 is a first light
chain variable domain obtained from said first parent antibody, or
antigen binding portion thereof; VD2 is a second light chain
variable domain obtained from said second parent antibody, or
antigen binding thereof, which can be the same as or different from
the first parent antibody; C is a light chain constant domain;
(X1)n is a linker with the proviso that it is not CH1, wherein said
(X1)n is either present or absent; and (X2)n does not comprise an
Fc region, wherein said (X2)n is either present or absent; and e)
expressing said first, second, third and fourth polypeptide chains;
such that a DVD-Ig binds said first antigen and said second antigen
is generated.
[0044] In still another embodiment, the invention provides a method
of generating a DVD-Ig that binds two antigens with desired
properties comprising the steps of a) obtaining a first parent
antibody, or antigen binding portion thereof, that binds a first
antigen and possessing at least one desired property exhibited by
the DVD-Ig; b) obtaining a second parent antibody, or antigen
binding portion thereof, which can be the same as or different from
the first parent antibody, can bind to a second antigen and
possesses at least one desired property exhibited by the Dual
Variable Domain Immunoglobulin; c) constructing first and third
polypeptide chains comprising VD1-(X1)n-VD2-C-(X2)n, wherein; VD1
is a first heavy chain variable domain obtained from said first
parent antibody, or antigen binding portion thereof; VD2 is a
second heavy chain variable domain obtained from said second parent
antibody, or antigen binding portion thereof; C is a heavy chain
constant domain; (X1)n is a linker with the proviso that it is not
CH1, wherein said (X1)n is either present or absent; and (X2)n is
an Fc region, wherein said (X2)n is either present or absent; d)
constructing second and fourth polypeptide chains comprising
VD1-(X1)n-VD2-C-(X2)n, wherein; VD1 is a first light chain variable
domain obtained from said first parent antibody, or antigen binding
portion thereof; VD2 is a second light chain variable domain
obtained from said second parent antibody, or antigen binding
portion thereof), which can be the same as or different from the
first parent antibody; C is a light chain constant domain; (X1)n is
a linker with the proviso that it is not CH1, wherein said (X1)n is
either present or absent; and (X2)n does not comprise an Fc region,
wherein said (X2)n is either present or absent; e) expressing said
first, second, third and fourth polypeptide chains; such that a
Dual Variable Domain Immunoglobulin capable of binding said first
and said second antigen with desired properties is generated.
[0045] In one embodiment, the VDI of the first and second
polypeptide chains disclosed herein are obtained from the same
parent antibody or antigen binding portion thereof. In another
embodiment, the VDI of the first and second polypeptide chains
disclosed herein are obtained from different parent antibodies or
antigen binding portions thereof. In another embodiment, the VD2 of
the first and second polypeptide chains disclosed herein are
obtained from the same parent antibody or antigen binding portion
thereof. In another embodiment, the VD2 of the first and second
polypeptide chains disclosed herein are obtained from different
parent antibodies or antigen binding portions thereof.
[0046] In one embodiment the first parent antibody or antigen
binding portion thereof, and the second parent antibody or antigen
binding portion thereof, are the same antibody. In another
embodiment the first parent antibody or antigen binding portion
thereof, and the second parent antibody or antigen binding portion
thereof, are different antibodies.
[0047] In one embodiment the first parent antibody or antigen
binding portion thereof, binds a first antigen and the second
parent antibody or antigen binding portion thereof, binds a second
antigen. In a particular embodiment, the first and second antigens
are the same antigen. In another embodiment, the parent antibodies
bind different epitopes on the same antigen. In another embodiment
the first and second antigens are different antigens. In another
embodiment, the first parent antibody or antigen binding portion
thereof, binds the first antigen with a potency different from the
potency with which the second parent antibody or antigen binding
portion thereof, binds the second antigen. In yet another
embodiment, the first parent antibody or antigen binding portion
thereof, binds the first antigen with an affinity different from
the affinity with which the second parent antibody or antigen
binding portion thereof, binds the second antigen.
[0048] In another embodiment the first parent antibody or antigen
binding portion thereof, and the second parent antibody or antigen
binding portion thereof, are selected from the group consisting of,
human antibody, CDR grafted antibody, and humanized antibody. In an
embodiment, the antigen binding portions are selected from the
group consisting of a Fab fragment, a F(ab').sub.2 fragment, a
bivalent fragment comprising two Fab fragments linked by a
disulfide bridge at the hinge region; a Fd fragment consisting of
the VH and CH1 domains; a Fv fragment consisting of the VL and VH
domains of a single arm of an antibody, a dAb fragment, an isolated
complementarity determining region (CDR), a single chain antibody,
and diabodies.
[0049] In another embodiment the binding protein of the invention
possesses at least one desired property exhibited by the first
parent antibody or antigen binding portion thereof, or the second
parent antibody or antigen binding portion thereof. Alternatively,
the first parent antibody or antigen binding portion thereof and
the second parent antibody or antigen binding portion thereof
possess at least one desired property exhibited by the Dual
Variable Domain Immunoglobulin. In an embodiment, the desired
property is selected from one or more antibody parameters. In
another embodiment, the antibody parameters are selected from the
group consisting of antigen specificity, affinity to antigen,
potency, biological function, epitope recognition, stability,
solubility, production efficiency, immunogenicity,
pharmacokinetics, bioavailability, tissue cross reactivity, and
orthologous antigen binding. In an embodiment the binding protein
is multivalent. In another embodiment, the binding protein is
multispecific. The multivalent and or multispecific binding
proteins described herein have desirable properties particularly
from a therapeutic standpoint. For instance, the multivalent and or
multispecific binding protein may (1) be internalized (and/or
catabolized) faster than a bivalent antibody by a cell expressing
an antigen to which the antibodies bind; (2) be an agonist
antibody; and/or (3) induce cell death and/or apoptosis of a cell
expressing an antigen to which the multivalent antibody is binds.
The "parent antibody" which provides at least one antigen binding
specificity of the multivalent and or multispecific binding
proteins may be one which is internalized (and/or catabolized) by a
cell expressing an antigen to which the antibody binds; and/or may
be an agonist, cell death-inducing, and/or apoptosis-inducing
antibody, and the multivalent and or multispecific binding protein
as described herein may display improvement(s) in one or more of
these properties. Moreover, the parent antibody may lack any one or
more of these properties, but may be endowed with them when
constructed as a multivalent binding protein as described
herein.
[0050] In another embodiment the binding protein of the invention
has an on rate constant (Kon) to one or more targets selected from
the group consisting of: at least about 102M-1s-1; at least about
103M-1s-1; at least about 104M-1s-1; at least about 105M-1s-1; and
at least about 106M-1s-1, as measured by surface plasmon resonance.
In an embodiment, the binding protein of the invention has an on
rate constant (Kon) to one or more targets between about 102M-1s-1
and about 103M-1s-1; between about 103M-1s-1 and about 104M-1s-1;
between about 104M-1s-1 and about 105M-1s-1; or between about
105M-1s-1 and about 106M-1s-1, as measured by surface plasmon
resonance.
[0051] In another embodiment the binding protein has an off rate
constant (Koff) for one or more targets selected from the group
consisting of: at most about 10-3s-1; at most about 10-4s-1; at
most about 10-5s-1; and at most about 10-6s-1, as measured by
surface plasmon resonance. In an embodiment, the binding protein of
the invention has an off rate constant (Koff) to one or more
targets of from about 10-3s-1 to about 10-4s-1; of from about
10-4s-1 to about 10-5s-1; or of from about 10-5s-1 to about
10-6s-1, as measured by surface plasmon resonance.
[0052] In another embodiment the binding protein has a dissociation
constant (KD) to one or more targets selected from the group
consisting of: at most about 10-7 M; at most about 10-8 M; at most
about 10-9 M; at most about 10-10 M; at most about 10-11 M; at most
about 10-12 M; and at most about 10-13 M. In an embodiment, the
binding protein of the invention has a dissociation constant (KD)
to its targets of from about 10-7 M to about 10-8 M; of from about
10-8 M to about 10-9 M; of from about 10-9 M to about 10-10 M; of
from about 10-10 to about 10-11 M; of from about 10-11 M to about
10-12 M; or of from about 10-12 M to about 10-13 M.
[0053] In another embodiment, the binding protein described herein
is a conjugate further comprising 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
imaging agent is 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: .sup.3H, .sup.14C, .sup.35S, .sup.90Y,
.sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, and .sup.153Sm. In yet another 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.
[0054] In another embodiment, the binding protein described herein
binds to a cellular protein 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
imaging agent is 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: .sup.3H, .sup.14C, .sup.35S, .sup.90Y,
.sup.99Tc, .sup.111In, .sup.125I, .sup.131I, .sup.177Lu,
.sup.166Ho, and .sup.153Sm. In yet another 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.
[0055] In another embodiment, the binding protein described herein
is a crystallized binding protein and exists as a crystal. In an
embodiment, the crystal is a carrier-free pharmaceutical controlled
release crystal. In yet another embodiment, the crystallized
binding protein has a greater half life in vivo than the soluble
counterpart of said binding protein. In still another embodiment,
the crystallized binding protein retains biological activity.
[0056] In another embodiment, the binding protein described herein
is glycosylated. For example, the glycosylation is a human
glycosylation pattern.
[0057] One aspect of the invention pertains to an isolated nucleic
acid encoding any one of the binding proteins disclosed herein. A
further embodiment provides a vector comprising the isolated
nucleic acid disclosed herein wherein said vector is selected from
the group consisting of pcDNA; pTT (Durocher et al. (2002) Nucl.
Acids Res. 30: 2); pTT3 (pTT with additional multiple cloning site;
pEFBOS (Mizushima, S, and Nagata, S. (1990) Nucl. Acids Res. 18:
17); pBV; pJV; pcDNA3.1 TOPO; pEF6 TOPO; and pBJ. In an embodiment,
the vector is a vector disclosed in U.S. Patent Publication No.
2009/0239259.
[0058] In another aspect a host cell is transformed with the vector
disclosed herein. In an embodiment, the host cell is a prokaryotic
cell. In another embodiment, the host cell is E. coli. In a related
embodiment the host cell is a eukaryotic cell. In another
embodiment, the eukaryotic cell is selected from the group
consisting of a protist cell, an animal cell, a plant cell and a
fungal cell. In yet another embodiment, the host cell is a
mammalian cell including, but not limited to, CHO, COS; NS0, SP2,
PER.C6 or a fungal cell, such as Saccharomyces cerevisiae; or an
insect cell such as Sf9.
[0059] In an embodiment, two or more DVD-Igs, e.g., with different
specificities, are produced in a single recombinant host cell. For
example, the expression of a mixture of antibodies has been called
Oligoclonics.TM. (Merus B. V., The Netherlands); U.S. Pat. Nos.
7,262,028; 7,429,486.
[0060] Another aspect of the invention provides a method of
producing a binding protein disclosed herein comprising culturing
any one of the host cells also disclosed herein in a culture medium
under conditions sufficient to produce the binding protein. In an
embodiment, 50%-75% of the binding protein produced by this method
is a dual specific tetravalent binding protein. In a particular
embodiment, 75%-90% of the binding protein produced by this method
is a dual specific tetravalent binding protein. In a particular
embodiment, 90%-95% of the binding protein produced is a dual
specific tetravalent binding protein.
[0061] One embodiment provides a composition for the release of a
binding protein wherein the composition comprises a formulation
that in turn comprises a crystallized binding protein, as disclosed
herein, and an ingredient, and at least one polymeric carrier. For
example, the polymeric carrier comprises one or more polymers
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 polysaccharides, blends and copolymers
thereof. For example, the ingredient is selected from the group
consisting of albumin, sucrose, trehalose, lactitol, gelatin,
hydroxypropyl-b-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 herein.
[0062] The invention also provides a pharmaceutical composition
comprising a binding protein, as disclosed herein and a
pharmaceutically acceptable carrier. In a further embodiment the
pharmaceutical composition comprises at least one additional
therapeutic agent for treating a disorder. For example, 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 an anti-VEGF
antibody or a VEGF-trap), a kinase inhibitor (including but not
limited to a KDR and a TIE-2 inhibitor), 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 an anti-LFA-1 antibody, an anti-E/L selectin
antibody, a small molecule inhibitor), an anti-cytokine antibody or
functional fragment thereof (including but not limited to an
anti-IL-18, an anti-TNF, and an 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.
[0063] In another aspect, the invention provides a method for
treating a human subject suffering from a disorder in which the
target, or targets, capable of being bound by the binding protein
disclosed herein is/are detrimental, comprising administering to
the human subject a binding protein disclosed herein such that the
activity of the target, or targets in the human subject is
inhibited and one of more symptoms is alleviated or treatment is
achieved. For example, the disorder is selected from the group
comprising 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 hypoglycemia,
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, 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 diseases, 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, occlusive 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 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, ovarian cancer, 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, prostate and rectal
cancer and hematopoietic malignancies (leukemia and lymphoma),
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, wound healing, yersinia and
salmonella associated arthropathy.
[0064] 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).
[0065] In an embodiment, the antibodies of the invention or
antigen-binding portions thereof, are used to treat cancer or in
the prevention or inhibition of metastases from the tumors
described herein either when used alone or in combination with
radiotherapy and/or other chemotherapeutic agents.
[0066] In another aspect the invention provides a method of
treating a patient suffering from a disorder comprising the step of
administering any one of the binding proteins disclosed herein
before, concurrently, or after the administration of a second
agent, as discussed herein. In a particular embodiment the 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. mAbs, anti-IL-6 or IL-6 receptor mAbs,
growth factors, elastase inhibitors, pyridinyl-imidazole compounds,
antibodies or agonists of TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8,
IL-12, IL-13, IL-15, IL-16, IL-18, IL-23, EMAP-II, GM-CSF, FGF, and
PDGF, antibodies of CD2, CD3, CD4, CD8, CD-19, 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..quadrature.converting enzyme inhibitors, T-cell
signalling 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,
antiinflammatory cytokines, IL-4, IL-10, IL-11, IL-13 and
TGF.beta..
[0067] In a particular embodiment the pharmaceutical compositions
disclosed herein are administered to the patient 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.
One aspect of the invention provides at least one anti-idiotypic
antibody to at least one binding protein of the present invention.
The anti-idiotypic 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] FIG. 1A is a schematic representation of Dual Variable
Domain (DVD)-Ig constructs and shows the strategy for generation of
a DVD-Ig from two parent antibodies;
[0069] FIG. 1B, is a schematic representation of constructs
DVD1-Ig, DVD2-Ig, and two chimeric mono-specific antibodies from
hybridoma clones 2D13.E3 (anti-IL-1.alpha.) and 13F5.G5
(anti-IL-1.beta..
DETAILED DESCRIPTION OF THE INVENTION
[0070] This invention pertains to multivalent and/or multispecific
binding proteins that bind two or more antigens. Specifically, the
invention relates to dual variable domain immunoglobulins (DVD-Ig),
and pharmaceutical compositions thereof, as well as nucleic acids,
recombinant expression vectors and host cells for making such
DVD-Igs. Methods of using the DVD-Igs of the invention to detect
specific antigens, either in vitro or in vivo 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" 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. Use of "polypeptide" herein is intended to encompass
polypeptide and fragments and variants (including fragments of
variants) thereof, unless otherwise stated. For an antigenic
polypeptide, a fragment of polypeptide optionally contains at least
one contiguous or nonlinear epitope of polypeptide. The precise
boundaries of the at least one epitope fragment can be confirmed
using ordinary skill in the art. The fragment comprises at least
about 5 contiguous amino acids, such as at least about 10
contiguous amino acids, at least about 15 contiguous amino acids,
or at least about 20 contiguous amino acids. A variant of
polypeptide is as described herein.
[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" 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] "Biological activity" refers to any one or more inherent
biological properties of a molecule (whether present naturally as
found in vivo, or provided or enabled by recombinant means).
Biological properties include but are not limited to binding a
receptor; inducing cell proliferation, inhibiting cell growth,
inducing other cytokines, inducing apoptosis, and enzymatic
activity. Biological activity also includes activity of an Ig
molecule.
[0078] The terms "specific binding" or "specifically binding," 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.
[0079] The term "antibody," 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 antibody formats are known in the art. Nonlimiting
embodiments of which are discussed below.
[0080] 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, and 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.
[0081] The term "Fc region" is used to define the C-terminal region
of an immunoglobulin heavy chain, which may be generated by papain
digestion of an intact antibody. The Fc region may be a native
sequence Fc region or a variant Fc region. The Fc region of an
immunoglobulin generally comprises two constant domains, a CH2
domain and a CH3 domain, and optionally comprises a CH4 domain.
Replacements of amino acid residues in the Fc portion to alter
antibody effector function are known in the art (U.S. Pat. Nos.
5,648,260 and 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
a 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. The dimerization of two identical heavy chains of an
immunoglobulin is mediated by the dimerization of CH3 domains and
is stabilized by the disulfide bonds within the hinge region (Huber
et al. (1976) Nature 264: 415-20; Thies et al. (1999) J. Mol. Biol.
293: 67-79). Mutation of cysteine residues within the hinge regions
to prevent heavy chain-heavy chain disulfide bonds will destabilize
dimeration of CH3 domains. Residues responsible for CH3
dimerization have been identified (Dall'Acqua (1998) Biochem. 37:
9266-73). Therefore, it is possible to generate a monovalent
half-Ig. Interestingly, these monovalent half Ig molecules have
been found in nature for both IgG and IgA subclasses (Seligman
(1978) Ann. Immunol 129: 855-70; Biewenga et al. (1983) Clin. Exp.
Immunol 51: 395-400). The stoichiometry of FcRn: Ig Fc region has
been determined to be 2:1 (West et al. (2000) Biochem. 39:
9698-708), and half Fc is sufficient for mediating FcRn binding
(Kim et al. (1994) Eur. J. Immunol. 24: 542-548). Mutations to
disrupt the dimerization of CH3 domain may not have greater adverse
effect on its FcRn binding as the residues important for CH3
dimerization are located on the inner interface of CH3 b sheet
structure, whereas the region responsible for FcRn binding is
located on the outside interface of CH2-CH3 domains. However, the
half-Ig molecule may have certain advantages in tissue penetration
due to its smaller size in comparison to that of a regular
antibody. In one embodiment at least one amino acid residue is
replaced in the constant region of the binding protein of the
invention, for example the Fc region, such that the dimerization of
the heavy chains is disrupted, resulting in half DVD Ig molecules.
The anti-inflammatory activity of IgG is completely dependent on
sialylation of the N-linked glycan of the IgG Fc fragment. The
precise glycan requirements for anti-inflammatory activity has been
determined, such that an appropriate IgG1 Fc fragment can be
created, thereby generating a fully recombinant, sialylated IgG1 Fc
with greatly enhanced potency (Anthony, R. M., et al. (2008)
Science 320: 373-376).
[0082] The term "antigen-binding portion" of an antibody (or simply
"antibody portion") refers to one or more fragments of an antibody
that retain the ability to bind specifically to an antigen. It has
been shown that the antigen-binding function of an antibody can be
performed by fragments of a full-length antibody. Such antibody
embodiments may also be 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')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 (1989) Nature
341:544-546; PCT Publication No. 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 intended to be
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. p. 790 (ISBN
3-540-41354-5). In addition single chain antibodies also include
"linear antibodies" comprising a pair of tandem Fv segments
(VH-CH1-VH-CH1) which, together with complementary light chain
polypeptides, form a pair of antigen binding regions (Zapata et al.
(1995) Protein Eng. 8(10):1057-1062; and U.S. Pat. No.
5,641,870).
[0083] The term "multivalent binding protein" is used throughout
this specification to denote a binding protein comprising two or
more antigen binding sites. In an embodiment, the multivalent
binding protein is 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 that binds two or more related or unrelated targets. Dual
variable domain (DVD) binding proteins of the invention comprise
two or more antigen binding sites and are tetravalent or
multivalent binding proteins. DVDs 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 referred to as DVD-Ig. 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.
[0084] The term "bispecific antibody" refers to full-length
antibodies that are generated by quadroma technology (see Milstein,
C. and Cuello, A. C. (1983) Nature 305(5934): p. 537-540), by
chemical conjugation of two different monoclonal antibodies (see
Staerz, U. D. et al. (1985) Nature 314(6012): 628-631), or by
knob-into-hole or similar approaches, which introduce mutations in
the Fc region (see Holliger, P. et al. (1993) Proc. Natl. Acad. Sci
USA 90(14): 6444-6448), resulting in multiple different
immunoglobulin species of which only one is the functional
bispecific antibody. By molecular function, a bispecific antibody
binds one antigen (or epitope) on one of its two binding arms (one
pair of HC/LC), and binds a different antigen (or epitope) on its
second arm (a different pair of HC/LC). By this definition, a
bispecific antibody has two distinct antigen binding arms (in both
specificity and CDR sequences), and is monovalent for each antigen
it binds to.
[0085] The term "dual-specific antibody," refers to full-length
antibodies that can bind two different antigens (or epitopes) in
each of its two binding arms (a pair of HC/LC) (see PCT Publication
No. WO 02/02773). Accordingly a dual-specific binding protein has
two identical antigen binding arms, with identical specificity and
identical CDR sequences, and is bivalent for each antigen to which
it binds.
[0086] A "functional antigen binding site" of a binding protein is
one that binds a target antigen. The antigen binding affinity of
the antigen binding site is not necessarily as strong as the parent
antibody from which the antigen binding site is derived, but the
ability to bind antigen must be measurable using any one of a
variety of methods known for evaluating antibody binding to an
antigen. Moreover, the antigen binding affinity of each of the
antigen binding sites of a multivalent antibody herein need not be
quantitatively the same.
[0087] The term "cytokine" is a generic term for proteins released
by one cell population, which act on another cell population as
intercellular mediators. Examples of such cytokines are
lymphokines, monokines, and traditional polypeptide hormones.
Included among the cytokines are growth hormone such as human
growth hormone, N-methionyl human growth hormone, and bovine growth
hormone; parathyroid hormone; thyroxine; insulin; proinsulin;
relaxin; prorelaxin; glycoprotein hormones such as follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
luteinizing hormone (LH); hepatic growth factor; fibroblast growth
factor; prolactin; placental lactogen; tumor necrosis factor-alpha
and -beta; mullerian-inhibiting substance; mouse
gonadotropin-associated peptide; inhibin; activin; vascular
endothelial growth factor; integrin; thrombopoietin (TPO); nerve
growth factors such as NGF-alpha; platelet-growth factor; placental
growth factor, transforming growth factors (TGFs) such as TGF-alpha
and TGF-beta; insulin-like growth factor-1 and -11; erythropoietin
(EPO); osteoinductive factors; interferons such as
interferon-alpha, -beta and -gamma colony stimulating factors
(CSFs) such as macrophage-CSF (M-CSF); granulocyte macrophage-CSF
(GM-CSF); and granulocyte-CSF (G-CSF); interleukins (ILs) such as
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-18, IL-21, IL-22, IL-23, and IL-33; a tumor
necrosis factor such as TNF-alpha or TNF-beta; and other
polypeptide factors including LIF and kit ligand (KL). The term
cytokine includes proteins from natural sources or from recombinant
cell culture and biologically active equivalents of the native
sequence cytokines.
[0088] The term "linker" is used to denote polypeptides comprising
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). Exemplary linkers include,
but are not limited to, AKTTPKLEEGEFSEAR (SEQ ID NO: 1);
AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);
SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID
NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8);
RADAAAA(G.sub.4S).sub.4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID
NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP
(SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO:
15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17);
AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);
AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26), TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID
NO: 27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28).
[0089] 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.
[0090] The term "monoclonal antibody" or "mAb" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each mAb is directed
against a single determinant on the antigen. The modifier
"monoclonal" is not to be construed as requiring production of the
antibody by any particular method.
[0091] The term "human antibody," 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," 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.
[0092] The term "recombinant human antibody," 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) Immunol Today 21: 371-378), antibodies
isolated from an animal (e.g., a mouse) that is transgenic for
human immunoglobulin genes (see, Taylor, L. D., et al. (1992) Nucl.
Acids Res. 20: 6287-6295; Kellermann S-A. and Green L. L. (2002)
Current Opinion in Biotechnol. 13: 593-597; Little M. et al. (2000)
Immunol 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.
[0093] An "affinity matured" antibody is an antibody with one or
more alterations in one or more CDRs thereof which result an
improvement in the affinity of the antibody for antigen, compared
to a parent antibody which does not possess those alteration(s).
Exemplary affinity matured antibodies will have nanomolar or even
picomolar affinities for the target antigen. Affinity matured
antibodies are produced by procedures known in the art. Marks et
al. (1992) Bio/Technology 10: 779-783 describes affinity maturation
by VH and VL domain shuffling. Random mutagenesis of CDR and/or
framework residues is described by Barbas, et al. (1994) Proc Nat.
Acad. Sci. USA 91: 3809-3813; Schier et al. (1995) Gene 169:
147-155; Yelton et al., (1995) J. Immunol. 155: 1994-2004; Jackson
et al. (1995) J. Immunol. 154(7): 3310-9; and Hawkins et al. (1992)
J. Mol. Biol. 226: 889-896; and selective mutation at selective
mutagenesis positions, contact or hypermutation positions with an
activity enhancing amino acid residue is described in U.S. Pat. No.
6,914,128.
[0094] The term "chimeric antibody" refers to antibodies which
comprise heavy and 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.
[0095] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and 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.
[0096] 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 non-human CDR sequences are introduced into
human VH and VL sequences to replace the corresponding human CDR
sequences. Also "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. The
term "substantially" in the context of a CDR refers to a CDR having
an amino acid sequence at least 80%, 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 (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. In an embodiment, 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.
[0097] 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.
[0098] 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" refers to a group of three
CDRs that occur in a single variable region that binds 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 (1987) J. Mol. Biol. 196:901-917 and Chothia et al.
(1989) Nature 342:877-883) 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" designate the light chain and the
heavy chain 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 (1995) FASEB J. 9: 133-139 and
MacCallum (1996) J. Mol. Biol. 262(5): 732-45. Still other CDR
boundary definitions may not strictly follow one of the herein
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 certain embodiments use Kabat or
Chothia defined CDRs.
[0099] 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,
-L2, and -L3 of light chain and CDR-H1, -H2, and -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
FR's within the variable region of a single, naturally occurring
immunoglobulin chain. A FR represents one of the four sub-regions,
and FRs represents two or more of the four sub-regions constituting
a framework region.
[0100] The term "germline antibody gene" or "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. (2002) Crit. Rev. Immunol
22(3): 183-200; Marchalonis et al. (2001) Adv. Exp. Med. Biol. 484:
13-30). 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.
[0101] The term "neutralizing" refers to counteracting the
biological activity of an antigen when a binding protein
specifically binds the antigen. In an embodiment, the neutralizing
binding protein binds the cytokine and reduces its biologically
activity by at least about 20%, 40%, 60%, 80%, 85% or more.
[0102] The term "activity" includes activities such as the binding
specificity and affinity of a DVD-Ig for two or more antigens.
[0103] The term "epitope" includes any polypeptide determinant that
specifically binds 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. An epitope thus consists of
the amino acid residues of a region of an antigen (or fragment
thereof) known to bind to the complementary site on the specific
binding partner. An antigenic fragment can contain more than one
epitope. In certain embodiments, an antibody is said to
specifically bind an antigen when it recognizes its target antigen
in a complex mixture of proteins and/or macromolecules. Antibodies
are said to "bind to the same epitope" if the antibodies
cross-compete (one prevents the binding or modulating effect of the
other). In addition structural definitions of epitopes
(overlapping, similar, identical) are informative, but functional
definitions are often more relevant as they encompass structural
(binding) and functional (modulation, competition) parameters.
[0104] The term "surface plasmon resonance", 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.RTM.
system (BIAcore International AB, a GE Healthcare company, 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.
[0105] The term "Kon" refers to the on rate constant for
association of a binding protein (e.g., an antibody) to the antigen
to form the, e.g., antibody/antigen complex as is known in the art.
The "Kon" also is known by the terms "association rate constant,"
or "ka," as used interchangeably herein. This value indicating the
binding rate of an antibody to its target antigen or the rate of
complex formation between an antibody and antigen also is shown by
the equation:
Antibody ("Ab")+Antigen ("Ag").fwdarw.Ab-Ag.
[0106] The term "Koff" refers to the off rate constant for
dissociation of a binding protein (e.g., an antibody) from the,
e.g., antibody/antigen complex as is known in the art. The "Koff"
also is known by the terms "dissociation rate constant" or "kd" as
used interchangeably herein. This value indicates the dissociation
rate of an antibody from its target antigen or separation of Ab-Ag
complex over time into free antibody and antigen as shown by the
equation below:
Ab+Ag.rarw.Ab-Ag.
[0107] The terms "equilibrium dissociation constant" or "KD," as
used interchangeably herein, refer to the value obtained in a
titration measurement at equilibrium, or by dividing the
dissociation rate constant (koff) by the association rate constant
(kon). The association rate constant, the dissociation rate
constant and the equilibrium dissociation constant are used to
represent the binding affinity of an antibody to an antigen.
Methods for determining association and dissociation rate constants
are well known in the art. Using fluorescence-based techniques
offers high sensitivity and the ability to examine samples in
physiological buffers at equilibrium. Other experimental approaches
and instruments such as a BIAcore.RTM. (biomolecular interaction
analysis) assay can be used (e.g., instrument available from
BIAcore International AB, a GE Healthcare company, Uppsala,
Sweden). Additionally, a KinExA.RTM. (Kinetic Exclusion Assay)
assay, available from Sapidyne Instruments (Boise, Id.) can also be
used.
[0108] "Label" and "detectable label" mean a moiety attached to a
specific binding partner, such as an antibody or an analyte, e.g.,
to render the reaction between members of a specific binding pair,
such as an antibody and an analyte, detectable, and the specific
binding partner, e.g., antibody or analyte, so labeled is referred
to as "detectably labeled." Thus, the term "labeled binding
protein" refers to a protein with a label incorporated that
provides for the identification of the binding protein. In an
embodiment, the label is a detectable marker that can produce a
signal that is detectable by visual or instrumental means, 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, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, .sup.177Lu, .sup.166Ho, and .sup.153Sm);
chromogens, fluorescent labels (e.g., FITC, rhodamine, and
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, and epitope tags); and magnetic agents, such as gadolinium
chelates. Representative examples of labels commonly employed for
immunoassays include moieties that produce light, e.g., acridinium
compounds, and moieties that produce fluorescence, e.g.,
fluorescein. Other labels are described herein. In this regard, the
moiety itself may not be detectably labeled but may become
detectable upon reaction with yet another moiety. Use of
"detectably labeled" is intended to encompass the latter type of
detectable labeling.
[0109] The term "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. In an embodiment, 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. When employed in the context of an
immunoassay, the conjugate antibody is a detectably labeled
antibody used as the detection antibody.
[0110] The terms "crystal" and "crystallized" refer to a binding
protein (e.g., 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 that are well-understood in the field.
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 ea., pp. 20 1-16, Oxford University Press, New York,
N.Y., (1999)."
[0111] The term "polynucleotide" 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.
[0112] The term "isolated polynucleotide" 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.
[0113] The term "vector", 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.
[0114] 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" refers to polynucleotide sequences
which 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 a promoter, a ribosomal binding
site, and a transcription termination sequence; in eukaryotes,
generally, such control sequences include a promoter and a
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.
[0115] "Transformation", 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 which transiently express the inserted DNA
or RNA for limited periods of time.
[0116] The term "recombinant host cell" (or simply "host cell"), is
intended to refer to a cell into which exogenous DNA has been
introduced. In an embodiment, the host cell comprises two or more
(e.g., multiple) nucleic acids encoding antibodies, such as the
host cells described in U.S. Pat. No. 7,262,028, for example. Such
terms are intended to refer not only to the particular subject
cell, but also 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". In an embodiment, host
cells include prokaryotic and eukaryotic cells selected from any of
the Kingdoms of life. In another embodiment, eukaryotic cells
include protist, fungal, plant and animal cells. In another
embodiment, host cells include but are not limited to the
prokaryotic cell line E. coli; mammalian cell lines CHO, HEK 293,
COS, NS0, SP2 and PER.C6; the insect cell line Sf9; and the fungal
cell Saccharomyces cerevisiae.
[0117] 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. (1989) Molecular
Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y.).
[0118] "Transgenic organism", as known in the art, 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.
[0119] The term "regulate" and "modulate" are used interchangeably
and refer to a change or an alteration in the activity of a
molecule of interest (e.g., the biological activity of a cytokine).
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.
[0120] Correspondingly, the term "modulator" is a compound capable
of changing or altering an activity or function of a molecule of
interest (e.g., the biological activity of a cytokine). 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.
[0121] The term "agonist", 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, polypeptides, nucleic acids,
carbohydrates, or any other molecules that bind to the antigen.
[0122] The term "antagonist" or "inhibitor", refers 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 the antigen. Antagonists and inhibitors
of antigens may include, but are not limited to, proteins, nucleic
acids, carbohydrates, and any other molecules, which bind to the
antigen.
[0123] 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,
inhibit or prevent the advancement of a disorder, cause regression
of a disorder, inhibit or 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).
[0124] "Patient" and "subject" may be used interchangeably herein
to refer to an animal, such as a mammal, including a primate (for
example, a human, a monkey, and a chimpanzee), a non-primate (for
example, a cow, a pig, a camel, a llama, a horse, a goat, a rabbit,
a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, and
a whale), a bird (e.g., a duck or a goose), and a shark.
Preferably, the patient or subject is a human, such as a human
being treated or assessed for a disease, disorder or condition, a
human at risk for a disease, disorder or condition, a human having
a disease, disorder or condition, and/or human being treated for a
disease, disorder or condition.
[0125] The term "sample" is used in its broadest sense. A
"biological sample", 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, (e.g., whole blood), plasma, serum,
urine, amniotic fluid, synovial fluid, endothelial cells,
leukocytes, monocytes, other cells, organs, tissues, bone marrow,
lymph nodes and spleen.
[0126] "Component," "components," and "at least one component,"
refer generally to a capture antibody, a detection or conjugate
antibody, a control, a calibrator, a series of calibrators, a
sensitivity panel, a container, a buffer, a diluent, a salt, an
enzyme, a co-factor for an enzyme, a detection reagent, a
pretreatment reagent/solution, a substrate (e.g., as a solution), a
stop solution, and the like that can be included in a kit for assay
of a test sample, such as a patient urine, serum or plasma sample,
in accordance with the methods described herein and other methods
known in the art. Thus, in the context of the present disclosure,
"at least one component," "component," and "components" can include
a polypeptide or other analyte as above, such as a composition
comprising an analyte such as polypeptide, which is optionally
immobilized on a solid support, such as by binding to an
anti-analyte (e.g., anti-polypeptide) antibody. Some components can
be in solution or lyophilized for reconstitution for use in an
assay.
[0127] "Control" refers to a composition known to not contain
analyte ("negative control") or to contain analyte ("positive
control"). A positive control can comprise a known concentration of
analyte. "Control," "positive control," and "calibrator" may be
used interchangeably herein to refer to a composition comprising a
known concentration of analyte. A "positive control" can be used to
establish assay performance characteristics and is a useful
indicator of the integrity of reagents (e.g., analytes).
[0128] "Predetermined cutoff" and "predetermined level" refer
generally to an assay cutoff value that is used to assess
diagnostic/prognostic/therapeutic efficacy results by comparing the
assay results against the predetermined cutoff level, where the
predetermined cutoff/level already has been linked or associated
with various clinical parameters (e.g., severity of disease,
progression/nonprogression/improvement, etc.). While the present
disclosure may provide exemplary predetermined levels, it is
well-known that cutoff values may vary depending on the nature of
the immunoassay (e.g., antibodies employed, etc.). It further is
well within the ordinary skill of one in the art to adapt the
disclosure herein for other immunoassays to obtain
immunoassay-specific cutoff values for those other immunoassays
based on this disclosure. Whereas the precise value of the
predetermined cutoff/level may vary between assays, correlations as
described herein (if any) should be generally applicable.
[0129] "Pretreatment reagent," e.g., lysis, precipitation and/or
solubilization reagent, as used in a diagnostic assay as described
herein is one that lyses any cells and/or solubilizes any analyte
that is/are present in a test sample. Pretreatment is not necessary
for all samples, as described further herein. Among other things,
solubilizing the analyte (e.g., polypeptide of interest) may entail
release of the analyte from any endogenous binding proteins present
in the sample. A pretreatment reagent may be homogeneous (not
requiring a separation step) or heterogeneous (requiring a
separation step). With use of a heterogeneous pretreatment reagent
there is removal of any precipitated analyte binding proteins from
the test sample prior to proceeding to the next step of the
assay.
[0130] "Quality control reagents" in the context of immunoassays
and kits described herein, include, but are not limited to,
calibrators, controls, and sensitivity panels. A "calibrator" or
"standard" typically is used (e.g., one or more, such as a
plurality) in order to establish calibration (standard) curves for
interpolation of the concentration of an analyte, such as an
antibody or an analyte. Alternatively, a single calibrator, which
is near a predetermined positive/negative cutoff, can be used.
Multiple calibrators (i.e., more than one calibrator or a varying
amount of calibrator(s)) can be used in conjunction so as to
comprise a "sensitivity panel."
[0131] "Risk" refers to the possibility or probability of a
particular event occurring either presently or at some point in the
future. "Risk stratification" refers to an array of known clinical
risk factors that allows physicians to classify patients into a
low, moderate, high or highest risk of developing a particular
disease, disorder or condition.
[0132] "Specific" and "specificity" in the context of an
interaction between members of a specific binding pair (e.g., an
antigen (or fragment thereof) and an antibody (or antigenically
reactive fragment thereof)) refer to the selective reactivity of
the interaction. The phrase "specifically binds to" and analogous
phrases refer to the ability of antibodies (or antigenically
reactive fragments thereof) to bind specifically to analyte (or a
fragment thereof) and not bind specifically to other entities.
[0133] "Specific binding partner" is a member of a specific binding
pair. A specific binding pair comprises two different molecules,
which specifically bind to each other through chemical or physical
means. Therefore, in addition to antigen and antibody specific
binding pairs of common immunoassays, other specific binding pairs
can include biotin and avidin (or streptavidin), carbohydrates and
lectins, complementary nucleotide sequences, effector and receptor
molecules, cofactors and enzymes, enzyme inhibitors and enzymes,
and the like. Furthermore, specific binding pairs can include
members that are analogs of the original specific binding members,
for example, an analyte-analog. Immunoreactive specific binding
members include antigens, antigen fragments, and antibodies,
including monoclonal and polyclonal antibodies as well as
complexes, fragments, and variants (including fragments of
variants) thereof, whether isolated or recombinantly produced.
[0134] "Variant" means a polypeptide that differs from a given
polypeptide (e.g., IL-18, BNP, NGAL or HIV polypeptide or
anti-polypeptide antibody) in amino acid sequence by the addition
(e.g., insertion), deletion, or conservative substitution of amino
acids, but that retains the biological activity of the given
polypeptide (e.g., a variant IL-18 can compete with anti-IL-18
antibody for binding to IL-18). A conservative substitution of an
amino acid, i.e., replacing an amino acid with a different amino
acid of similar properties (e.g., hydrophilicity and degree and
distribution of charged regions) is recognized in the art as
typically involving a minor change. These minor changes can be
identified, in part, by considering the hydropathic index of amino
acids, as understood in the art (see, e.g., Kyte et al. (1982) J.
Mol. Biol. 157: 105-132). The hydropathic index of an amino acid is
based on a consideration of its hydrophobicity and charge. It is
known in the art that amino acids of similar hydropathic indexes
can be substituted and still retain protein function. In one
aspect, amino acids having hydropathic indexes of .+-.2 are
substituted. The hydrophilicity of amino acids also can be used to
reveal substitutions that would result in proteins retaining
biological function. A consideration of the hydrophilicity of amino
acids in the context of a peptide permits calculation of the
greatest local average hydrophilicity of that peptide, a useful
measure that has been reported to correlate well with antigenicity
and immunogenicity (see, e.g., U.S. Pat. No. 4,554,101).
Substitution of amino acids having similar hydrophilicity values
can result in peptides retaining biological activity, for example
immunogenicity, as is understood in the art. In one aspect,
substitutions are performed with amino acids having hydrophilicity
values within .+-.2 of each other. Both the hydrophobicity index
and the hydrophilicity value of amino acids are influenced by the
particular side chain of that amino acid. Consistent with that
observation, amino acid substitutions that are compatible with
biological function are understood to depend on the relative
similarity of the amino acids, and particularly the side chains of
those amino acids, as revealed by the hydrophobicity,
hydrophilicity, charge, size, and other properties. "Variant" also
can be used to describe a polypeptide or fragment thereof that has
been differentially processed, such as by proteolysis,
phosphorylation, or other post-translational modification, yet
retains its biological activity or antigen reactivity, e.g., the
ability to bind to IL-18. Use of "variant" herein is intended to
encompass fragments of a variant unless otherwise contradicted by
context.
I. Generation of DVD Binding Protein
[0135] The invention pertains to Dual Variable Domain binding
proteins that bind one or more targets and methods of making the
same. In an embodiment, the binding protein comprises a polypeptide
chain, wherein said polypeptide chain comprises
VD1-(X1)n-VD2-C-(X2)n, wherein VD1 is a first variable domain, VD2
is a second variable domain, C is a constant domain, X1 represents
an amino acid or polypeptide, X2 represents an Fc region and n is 0
or 1. The binding protein of the invention can be generated using
various techniques. The invention provides expression vectors, host
cell and methods of generating the binding protein.
A. Generation of Parent Monoclonal Antibodies
[0136] The variable domains of the DVD binding protein can be
obtained from parent antibodies, including polyclonal and mAbs that
bind antigens of interest. These antibodies may be naturally
occurring or may be generated by recombinant technology.
[0137] MAbs can be prepared using a wide variety of techniques
known in the art including the use of hybridoma, recombinant, and
phage display technologies, or a combination thereof. For example,
mAbs can be produced using hybridoma techniques including those
known in the art and taught, for example, in Harlow et al. (1988)
Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory
Press, 2nd ed.); Hammerling, et al. (1981) in: Monoclonal
Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y.). The term
"monoclonal antibody" 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. Hybridomas are selected, cloned and further
screened for desirable characteristics, including robust hybridoma
growth, high antibody production and desirable antibody
characteristics, as discussed in Example 1 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. In a particular
embodiment, the hybridomas are mouse hybridomas. In another
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 antibody that binds a specific antigen.
[0138] Recombinant mAbs are also 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 No. 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 an immunized animal, are identified, and,
heavy- and light-chain variable region cDNAs are rescued from the
cells by reverse transcriptase-PCR. 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 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 the antigen of interest. 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 Nos. WO 97/29131 and WO
00/56772.
[0139] Monoclonal antibodies are also produced by immunizing a
non-human animal comprising some, or all, of the human
immunoglobulin locus with an antigen of interest. In an embodiment,
the non-human animal is a XENOMOUSE 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. (1994) Nature Genet. 7: 13-21 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 PCT Publication Nos.
WO 91/10741; WO 94/02602; WO 96/34096; WO 96/33735; WO 98/16654; WO
98/24893; WO 98/50433; WO 99/45031; WO 99/53049; WO 00/09560; and
WO 00/037504. The XENOMOUSE transgenic mouse produces an adult-like
human repertoire of fully human antibodies, and generates
antigen-specific human monoclonal antibodies. The XENOMOUSE
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. (1997) Nature Genet. 15:
146-156; Green and Jakobovits (1998) J. Exp. Med. 188: 483-495.
[0140] In vitro methods also can be used to make the parent
antibodies, 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; PCT Publication Nos. WO 92/18619; WO
91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO
92/09690 and WO 97/29131; 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. (1990)
Nature 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) Proc. Natl. Acad.
Sci. USA 89: 3576-3580; Garrad et al. (1991) Bio/Technology 9:
1373-1377; Hoogenboom et al. (1991) Nucl. Acid Res. 19: 4133-4137;
and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88: 7978-7982,
and U.S. Patent Publication No. 2003/0186374.
[0141] Parent 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 which 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. (1995) J. Immunol. Methods 182: 41-50; Ames et al.
(1995) J. Immunol. Methods 184: 177-186; Kettleborough et al.
(1994) Eur. J. Immunol. 24: 952-958; Persic et al. (1997) Gene 187:
9-18; Burton et al. (1994) Advances in Immunol 57: 191-280; PCT
Application No. PCT/GB91/01134; PCT Publication Nos. WO 90/02809;
WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982;
and 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.
[0142] As described in the herein 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 produce recombinantly Fab,
Fab' and F(ab')2 fragments can also be employed using methods known
in the art such as those disclosed in PCT Publication No. WO
92/22324; Mullinax et al. (1992) BioTechniques 12(6): 864-869;
Sawai et al. (1995) AJRI 34: 26-34; and Better et al. (1988)
Science 240: 1041-1043. Examples of techniques, which 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. (1991),
Methods Enzymol. 203:46-88; Shu et al. (1993) Proc. Natl. Acad.
Sci. USA 90: 7995-7999; and Skerra et al. (1988) Science 240:
1038-1040.
[0143] 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 parent antibodies. 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, 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 herein (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 herein.
[0144] In another approach the parent antibodies 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 parent
antibodies include those disclosed in U.S. Pat. No. 6,699,658.
[0145] The antibodies described herein can be further modified to
generate CDR grafted and humanized parent antibodies. CDR-grafted
parent antibodies comprise heavy and light chain variable region
sequences from a human antibody wherein one or more of the CDR
regions of V.sub.H and/or V.sub.L are replaced with CDR sequences
of murine antibodies that bind an antigen of interest. A framework
sequence from any human antibody may serve as the template for CDR
grafting. However, straight chain replacement onto such a framework
often leads to some loss of binding affinity to the antigen. The
more homologous a human antibody is to the original murine
antibody, the less likely the possibility that combining the murine
CDRs with the human framework will introduce distortions in the
CDRs that could reduce affinity. Therefore, in an embodiment, the
human variable framework that is chosen to replace the murine
variable framework apart from the CDRs have at least a 65% sequence
identity with the murine antibody variable region framework. In an
embodiment, the human and murine variable regions apart from the
CDRs have at least 70% sequence identify. In a particular
embodiment, that the human and murine variable regions apart from
the CDRs have at least 75% sequence identity. In another
embodiment, the human and murine variable regions apart from the
CDRs have at least 80% sequence identity. Methods for producing
such antibodies are known in the art (see EP 239,400; PCT
Publication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539;
5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP
519,596; Padlan (1991) Mol. Immunol. 28(4/5): 489-498; Studnicka et
al. (1994) Prot. Engineer. 7(6): 805-814; and Roguska et al. (1994)
Proc. Acad. Sci. USA 91: 969-973), chain shuffling (U.S. Pat. No.
5,565,352), and anti-idiotypic antibodies.
[0146] Humanized antibodies are antibody molecules from non-human
species that bind the desired antigen and have one or more CDRs
from the non-human species and framework regions from a human
immunoglobulin molecule. Known human Ig sequences are disclosed in,
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.
[0147] Framework residues in the human framework regions may be
substituted with the corresponding residue from the CDR donor
antibody to alter, e.g., 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., U.S. Pat. No.
5,585,089; Riechmann et al. (1988) Nature 332:323).
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which 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. In 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.
(1986) Nature 321: 522; Verhoeyen et al. (1988) Science 239: 1534;
Sims et al. (1993) J. Immunol. 151: 2296; Chothia and Lesk (1987)
J. Mol. Biol. 196: 901; Carter et al. (1992) Proc. Natl. Acad. Sci.
USA 89: 4285; Presta et al. (1993) J. Immunol. 151: 2623; Padlan
(1991) Mol. Immunol 28(4/5): 489-498; Studnicka et al. (1994) Prot.
Engineer. 7(6): 805-814; Roguska et al., (1994) Proc. Natl. Acad.
Sci. USA 91: 969-973; PCT Publication No. WO 91/09967: US98/16280;
US96/18978; US91/09630; US91/05939; US94/01234; GB89/01334;
GB91/01134; GB92/01755; WO90/14443; WO90/14424; and WO90/14430;
European Patent Publication Nos. EP 229246; EP 592,106; EP 519,596;
and EP 239,400; and 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; 5,766,886;
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.
B. Criteria for Selecting Parent Monoclonal Antibodies
[0148] An embodiment of the invention pertains to selecting parent
antibodies with at least one or more properties desired in the
DVD-Ig molecule. In an embodiment, the desired property is selected
from one or more antibody parameters. In another embodiment, the
antibody parameters are selected from the group consisting of
antigen specificity, affinity to antigen, potency, biological
function, epitope recognition, stability, solubility, production
efficiency, immunogenicity, pharmacokinetics, bioavailability,
tissue cross reactivity, and orthologous antigen binding.
B1. Affinity to Antigen
[0149] The desired affinity of a therapeutic mAb may depend upon
the nature of the antigen, and the desired therapeutic end-point.
In an embodiment, monoclonal antibodies have higher affinities
(Kd=0.01-0.50 pM) when blocking a cytokine-cytokine receptor
interaction as such interaction are usually high affinity
interactions (e.g., <pM-<nM ranges). In such instances, the
mAb affinity for its target should be equal to or better than the
affinity of the cytokine (ligand) for its receptor. On the other
hand, mAb with lesser affinity (>nM range) could be
therapeutically effective e.g., in clearing circulating potentially
pathogenic proteins e.g., monoclonal antibodies that bind to,
sequester, and clear circulating species of A-.beta. amyloid. In
other instances, reducing the affinity of an existing high affinity
mAb by site-directed mutagenesis or using a mAb with lower affinity
for its target could be used to avoid potential side-effects e.g.,
a high affinity mAb may sequester/neutralize all of its intended
target, thereby completely depleting/eliminating the function(s) of
the targeted protein. In this scenario, a low affinity mAb may
sequester/neutralize a fraction of the target that may be
responsible for the disease symptoms (the pathological or
over-produced levels), thus allowing a fraction of the target to
continue to perform its normal physiological function(s).
Therefore, it may be possible to reduce the Kd to adjust dose
and/or reduce side-effects. The affinity of the parental mAb might
play a role in appropriately targeting cell surface molecules to
achieve desired therapeutic out-come. For example, if a target is
expressed on cancer cells with high density and on normal cells
with low density, a lower affinity mAb will bind a greater number
of targets on tumor cells than normal cells, resulting in tumor
cell elimination via ADCC or CDC, and therefore might have
therapeutically desirable effects. Thus selecting a mAb with
desired affinity may be relevant for both soluble and surface
targets.
[0150] Signaling through a receptor upon interaction with its
ligand may depend upon the affinity of the receptor-ligand
interaction. Similarly, it is conceivable that the affinity of a
mAb for a surface receptor could determine the nature of
intracellular signaling and whether the mAb may deliver an agonist
or an antagonist signal. The affinity-based nature of mAb-mediated
signaling may have an impact of its side-effect profile. Therefore,
the desired affinity and desired functions of therapeutic
monoclonal antibodies need to be determined carefully by in vitro
and in vivo experimentation.
[0151] The desired Kd of a binding protein (e.g., an antibody) may
be determined experimentally depending on the desired therapeutic
outcome. In an embodiment parent antibodies with affinity (Kd) for
a particular antigen equal to, or better than, the desired affinity
of the DVD-Ig for the same antigen are selected. The parent
antibodies for a given DVD-Ig molecule can be the same antibody or
different antibodies. The antigen binding affinity and kinetics are
assessed by Biacore or another similar technique. In one
embodiment, each parent antibody has a dissociation constant (Kd)
to its antigen selected from the group consisting of: 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; and at most 10.sup.-13M. The first parent
antibody from which VD1 is obtained and the second parent antibody
from which VD2 is obtained may have similar or different affinity
(KD) for the respective antigen. Each parent antibody has an on
rate constant (Kon) to the antigen selected from the group
consisting of: at least about 10.sup.2M.sup.-1 s.sup.-1; at least
about 10.sup.3M.sup.-1 s.sup.-1; at least about 10.sup.4M.sup.-1
s.sup.-1; at least about 10.sup.5M.sup.-1 s.sup.-1; and at least
about 10.sup.6M.sup.-1 s.sup.-1, as measured by surface plasmon
resonance. The first parent antibody from which VD1 is obtained and
the second parent antibody from which VD2 is obtained may have
similar or different on rate constant (Kon) for the respective
antigen. In one embodiment, each parent antibody has an off rate
constant (Koff) to the antigen selected from the group consisting
of: at most about 10.sup.-3s.sup.-1; at most about 10.sup.-4
s.sup.-1; at most about 10.sup.-5 s.sup.-1; and at most about
10.sup.-6 s.sup.-1, as measured by surface plasmon resonance. The
first parent antibody from which VD1 is obtained and the second
parent antibody from which VD2 is obtained may have similar or
different off rate constants (Koff) for the respective antigen.
B2. Potency
[0152] The desired affinity/potency of parental monoclonal
antibodies will depend on the desired therapeutic outcome. For
example, for receptor-ligand (R-L) interactions the affinity (kd)
is equal to or better than the R-L kd (pM range). For simple
clearance of a pathologic circulating protein, the kd could be in
low nM range e.g., clearance of various species of circulating
A-.beta. peptide. In addition, the kd will also depend on whether
the target expresses multiple copies of the same epitope e.g., a
mAb targeting conformational epitope in A.beta. oligomers.
[0153] Where VD1 and VD2 bind the same antigen, but distint
epitopes, the DVD-Ig will contain four binding sites for the same
antigen, thus increasing avidity and thereby the apparent kd of the
DVD-Ig. In an embodiment, parent antibodies with equal or lower kd
than that desired in the DVD-Ig are chosen. The affinity
considerations of a parental mAb may also depend upon whether the
DVD-Ig contains four or more identical antigen binding sites (i.e.,
a DVD-Ig from a single mAb). In this case, the apparent kd would be
greater than the mAb due to avidity. Such DVD-Igs can be employed
for cross-linking surface receptor, increase neutralization
potency, enhance clearance of pathological proteins, etc.
[0154] In an embodiment parent antibodies with neutralization
potency for specific antigen equal to or better than the desired
neutralization potential of the DVD-Ig for the same antigen are
selected. The neutralization potency can be assessed by a
target-dependent bioassay where cells of appropriate type produce a
measurable signal (i.e., proliferation or cytokine production) in
response to target stimulation, and target neutralization by the
mAb can reduce the signal in a dose-dependent manner.
B3. Biological Functions
[0155] Monoclonal antibodies can perform potentially several
functions. Some of these functions are listed in Table 1. These
functions can be assessed by both in vitro assays (e.g., cell-based
and biochemical assays) and in vivo animal models.
TABLE-US-00001 TABLE 1 Some Potential Applications For Therapeutic
Antibodies Target (Class) Mechanism of Action (target) Soluble
Neutralization of activity (e.g., a cytokine) (cytokines, other)
Enhance clearance (e.g., A.beta. oligomers) Increase half-life
(e.g., GLP 1) Cell Surface Agonist (e.g., GLP1 R; EPO R; etc.)
(Receptors, other) Antagonist (e.g., integrins; etc.) Cytotoxic (CD
20; etc.) Protein deposits Enhance clearance/degradation (e.g.,
A.beta. plaques, amyloid deposits)
[0156] MAbs with distinct functions described in the examples
herein in Table 1 can be selected to achieve desired therapeutic
outcomes. Two or more selected parent monoclonal antibodies can
then be used in DVD-Ig format to achieve two distinct functions in
a single DVD-Ig molecule. For example, a DVD-Ig can be generated by
selecting a parent mAb that neutralizes function of a specific
cytokine, and selecting a parent mAb that enhances clearance of a
pathological protein. Similarly, two parent monoclonal antibodies
that recognize two different cell surface receptors can be
selected, e.g., one mAb with an agonist function on one receptor
and the other mAb with an antagonist function on a different
receptor. These two selected monoclonal antibodies each with a
distinct function can be used to construct a single DVD-Ig molecule
that will possess the two distinct functions (agonist and
antagonist) of the selected monoclonal antibodies in a single
molecule. Similarly, two antagonistic monoclonal antibodies to cell
surface receptors each blocking binding of respective receptor
ligands (e.g., EGF and IGF) can be used in a DVD-Ig format.
Conversely, an antagonistic anti-receptor mAb (e.g., anti-EGFR) and
a neutralizing anti-soluble mediator (e.g., anti-IGF1/2) mAb can be
selected to make a DVD-Ig.
B4. Epitope Recognition:
[0157] Different regions of proteins may perform different
functions. For example specific regions of a cytokine interact with
the cytokine receptor to bring about receptor activation whereas
other regions of the protein may be required for stabilizing the
cytokine. In this instance one may select a mAb that binds
specifically to the receptor interacting region(s) on the cytokine
and thereby blocks cytokine-receptor interaction. In some cases,
for example, certain chemokine receptors that bind multiple
ligands, a mAb that binds to the epitope (region on chemokine
receptor) that interacts with only one ligand can be selected. In
other instances, monoclonal antibodies can bind to epitopes on a
target that are not directly responsible for physiological
functions of the protein, but binding of a mAb to these regions
could either interfere with physiological functions (steric
hindrance) or alter the conformation of the protein such that the
protein cannot function (mAb to receptors with multiple ligand
which alter the receptor conformation such that none of the ligand
can bind). Anti-cytokine monoclonal antibodies that do not block
binding of the cytokine to its receptor, but block signal
transduction have also been identified (e.g., 125-2H, an anti-IL-18
mAb).
[0158] Examples of epitopes and mAb functions include, but are not
limited to, blocking Receptor-Ligand (R-L) interaction
(neutralizing mAb that binds R-interacting site); steric hindrance
resulting in diminished or no R-binding. An Ab can bind the target
at a site other than a receptor binding site, but still interfere
with receptor binding and functions of the target by inducing
conformational change and eliminating function (e.g., Xolair),
e.g., binding to R but blocking signaling (125-2H).
[0159] In an embodiment, the parental mAb needs to target the
appropriate epitope for maximum efficacy. Such epitope should be
conserved in the DVD-Ig. The binding epitope of a mAb can be
determined by several approaches, including co-crystallography,
limited proteolysis of mAb-antigen complex plus mass spectrometric
peptide mapping (Legros V. et al. (2000) Protein Sci. 9:1002-10),
phage displayed peptide libraries (O'Connor, K. H. et al. (2005) J.
Immunol. Methods. 299:21-35), as well as mutagenesis (Wu C. et al.
(2003) J. Immunol. 170:5571-7).
B5. Physicochemical and Pharmaceutical Properties:
[0160] Therapeutic treatment with antibodies often requires
administration of high doses, often several mg/kg (due to a low
potency on a mass basis as a consequence of a typically large
molecular weight). In order to accommodate patient compliance and
to adequately address chronic disease therapies and outpatient
treatment, subcutaneous (s.c.) or intramuscular (i.m.)
administration of therapeutic mAbs is desirable. For example, the
maximum desirable volume for s.c. administration is .about.1.0 mL,
and therefore, concentrations of >100 mg/mL are desirable to
limit the number of injections per dose. In an embodiment, the
therapeutic antibody is administered in one dose. The development
of such formulations is constrained, however, by protein-protein
interactions (e.g., aggregation, which potentially increases
immunogenicity risks) and by limitations during processing and
delivery (e.g., viscosity). Consequently, the large quantities
required for clinical efficacy and the associated development
constraints limit full exploitation of the potential of antibody
formulation and s.c. administration in high-dose regimens. It is
apparent that the physicochemical and pharmaceutical properties of
a protein molecule and the protein solution are of utmost
importance, e.g., stability, solubility and viscosity features.
B5.1. Stability:
[0161] A "stable" antibody formulation is one in which the antibody
therein essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage. Stability can be
measured at a selected temperature for a selected time period. In
an embodiment, the antibody in the formulation is stable at room
temperature (about 30.degree. C.) or at 40.degree. C. for at least
1 month and/or stable at about 2-8.degree. C. for at least 1 year,
such as, for at least 2 years. Furthermore, in an embodiment, the
formulation is stable following freezing (to, e.g., -70.degree. C.)
and thawing of the formulation, hereinafter referred to as a
"freeze/thaw cycle." In another example, a "stable" formulation may
be one wherein less than about 10% and less than about 5% of the
protein is present as an aggregate in the formulation.
[0162] A DVD-Ig that is stable in vitro at various temperatures for
an extended time period is desirable. One can achieve this by rapid
screening of parental mAbs that are stable in vitro at elevated
temperature, e.g., at 40.degree. C. for 2-4 weeks, and then assess
stability. During storage at 2-8.degree. C., the protein reveals
stability for at least 12 months, e.g., at least 24 months.
Stability (% of monomeric, intact molecule) can be assessed using
various techniques such as cation exchange chromatography, size
exclusion chromatography, SDS-PAGE, as well as bioactivity testing.
For a more comprehensive list of analytical techniques that may be
employed to analyze covalent and conformational modifications see
Jones, A. J. S. (1993) Analytical methods for the assessment of
protein formulations and delivery systems. In: Cleland, J. L.;
Langer, R., editors. Formulation and delivery of peptides and
proteins, 1.sup.st edition, Washington, ACS, pg. 22-45; and
Pearlman, R.; Nguyen, T. H. (1990) Analysis of protein drugs. In:
Lee, V. H., editor. Peptide and protein drug delivery, 1st edition,
New York, Marcel Dekker, Inc., pg. 247-301.
[0163] Heterogeneity and aggregate formation: stability of the
antibody may be such that the formulation may reveal less than
about 10%, such as less than about 5%, such as less than about 2%,
or, within the range of 0.5% to 1.5% or less in the GMP antibody
material that is present as aggregate. Size exclusion
chromatography is a method that is sensitive, reproducible, and
very robust in the detection of protein aggregates.
[0164] In addition to low aggregate levels, the antibody must, in
an embodiment, be chemically stable. Chemical stability may be
determined by ion exchange chromatography (e.g., cation or anion
exchange chromatography), hydrophobic interaction chromatography,
or other methods such as isoelectric focusing or capillary
electrophoresis. For instance, chemical stability of the antibody
may be such that after storage of at least 12 months at 2-8.degree.
C. the peak representing unmodified antibody in a cation exchange
chromatography may increase not more than 20%, such as not more
than 10%, or not more than 5% as compared to the antibody solution
prior to storage testing.
[0165] In an embodiment, the parent antibodies display structural
integrity; correct disulfide bond formation, and correct folding:
Chemical instability due to changes in secondary or tertiary
structure of an antibody may impact antibody activity. For
instance, stability as indicated by activity of the antibody may be
such that after storage of at least 12 months at 2-8.degree. C. the
activity of the antibody may decrease not more than 50%, such as
not more than 30%, not more than 10%, or not more than 5% or 1% as
compared to the antibody solution prior to storage testing.
Suitable antigen-binding assays can be employed to determine
antibody activity.
B5.2. Solubility:
[0166] The "solubility" of a mAb correlates with the production of
correctly folded, monomeric IgG. The solubility of the IgG may
therefore be assessed by HPLC. For example, soluble (monomeric) IgG
will give rise to a single peak on the HPLC chromatograph, whereas
insoluble (e.g., multimeric and aggregated) will give rise to a
plurality of peaks. A person skilled in the art will therefore be
able to detect an increase or decrease in solubility of an IgG
using routine HPLC techniques. For a more comprehensive list of
analytical techniques that may be employed to analyze solubility
(see Jones, A. G. (1993) Dep. Chem. Biochem. Eng., Univ. Coll.
London, London, UK. Editor(s): Shamlou, P. Ayazi. Process.
Solid-Liq. Suspensions, 93-117. Publisher: Butterworth-Heinemann,
Oxford, UK and Pearlman et al. (1990) Adv. in Parenteral Sci. 4
(Pept. Protein Drug Delivery): 247-301). Solubility of a
therapeutic mAb is critical for formulating to high concentration
often required for adequate dosing. As outlined herein,
solubilities of >100 mg/mL may be required to accommodate
efficient antibody dosing. For instance, antibody solubility may be
not less than about 5 mg/mL in early research phase, not less than
about 25 mg/mL in advanced process science stages, or not less than
about 100 mg/mL, or not less than about 150 mg/mL. It is obvious to
a person skilled in the art that the intrinsic properties of a
protein molecule are important the physico-chemical properties of
the protein solution, e.g., stability, solubility, viscosity.
However, a person skilled in the art will appreciate that a broad
variety of excipients exist that may be used as additives to
beneficially impact the characteristics of the final protein
formulation. These excipients may include: (i) liquid solvents,
cosolvents (e.g., alcohols such as ethanol); (ii) buffering agents
(e.g., phosphate, acetate, citrate, and amino acid buffers); (iii)
sugars or sugar alcohols (e.g., sucrose, trehalose, fructose,
raffinose, mannitol, sorbitol, and dextrans); (iv) surfactants
(e.g., polysorbate 20, 40, 60, 80, and poloxamers); (v) isotonicity
modifiers (e.g., salts such as NaCl, sugars, and sugar alcohols);
and (vi) others (e.g., preservatives, chelating agents,
antioxidants, chelating substances (e.g., EDTA), biodegradable
polymers, and carrier molecules (e.g., HSA and PEGs)).
[0167] Viscosity is a parameter of high importance with regard to
antibody manufacture and antibody processing (e.g.,
diafiltration/ultrafiltration), fill-finish processes (pumping
aspects, filtration aspects) and delivery aspects (syringeability,
sophisticated device delivery). Low viscosities enable the liquid
solution of the antibody having a higher concentration. This
enables the same dose may be administered in smaller volumes. Small
injection volumes inhere the advantage of lower pain on injection
sensations, and the solutions not necessarily have to be isotonic
to reduce pain on injection in the patient. The viscosity of the
antibody solution may be such that at shear rates of 100 (1/s)
antibody solution viscosity is below 200 mPa s, below 125 mPa s,
below 70 mPa s, and below 25 mPa s, or even below 10 mPa s.
B 5.3. Production Efficiency
[0168] The generation of a DVD-Ig that is efficiently expressed in
mammalian cells, such as Chinese hamster ovary cells (CHO), will in
an embodiment require two parental monoclonal antibodies which are
themselves expressed efficiently in mammalian cells. The production
yield from a stable mammalian line (i.e. CHO) should be above about
0.5 g/L, above about 1 g/L, or in the range of about 2 to about 5
g/L or more (Kipriyanov, S. M. and Little, M. (1999) Mol.
Biotechnol. 12: 173-201; Carroll, S, and Al-Rubeai, M. (2004)
Expert Opin. Biol. Ther. 4: 1821-9).
[0169] Production of antibodies and Ig fusion proteins in mammalian
cells is influenced by several factors. Engineering of the
expression vector via incorporation of strong promoters, enhancers
and selection markers can maximize transcription of the gene of
interest from an integrated vector copy. The identification of
vector integration sites that are permissive for high levels of
gene transcription can augment protein expression from a vector
(Wurm et al. (2004) Nature Biotechnol. 22(11): 1393-1398).
Furthermore, levels of production are affected by the ratio of
antibody heavy and light chains and various steps in the process of
protein assembly and secretion (Jiang et al. (2006) Biotechnol.
Prog. 22(1): 313-8).
B 6. Immunogenicity
[0170] Administration of a therapeutic mAb may result in certain
incidence of an immune response (i.e., the formation of endogenous
antibodies directed against the therapeutic mAb). Potential
elements that might induce immunogenicity should be analyzed during
selection of the parental monoclonal antibodies, and steps to
reduce such risk can be taken to optimize the parental monoclonal
antibodies prior to DVD-Ig construction. Mouse-derived antibodies
have been found to be highly immunogenic in patients. The
generation of chimeric antibodies comprised of mouse variable and
human constant regions presents a logical next step to reduce the
immunogenicity of therapeutic antibodies (Morrison and Schlom,
1990). Alternatively, immunogenicity can be reduced by transferring
murine CDR sequences into a human antibody framework (reshaping/CDR
grafting/humanization), as described for a therapeutic antibody by
Riechmann et al. (1988) Nature 332: 323-327. Another method is
referred to as "resurfacing" or "veneering," starting with the
rodent variable light and heavy domains, only surface-accessible
framework amino acids are altered to human ones, while the CDR and
buried amino acids remain from the parental rodent antibody
(Roguska et al. (1996) Prot. Engineer 9: 895-904). In another type
of humanization, instead of grafting the entire CDRs, one technique
grafts only the "specificity-determining regions" (SDRs), defined
as the subset of CDR residues that are involved in binding of the
antibody to its target (Kashmiri et al. (2005) Methods 36(1):
25-34). This necessitates identification of the SDRs either through
analysis of available three-dimensional structures of
antibody-target complexes or mutational analysis of the antibody
CDR residues to determine which interact with the target.
Alternatively, fully human antibodies may have reduced
immunogenicity compared to murine, chimeric or humanized
antibodies.
[0171] Another approach to reduce the immunogenicity of therapeutic
antibodies is the elimination of certain specific sequences that
are predicted to be immunogenic. In one approach, after a first
generation biologic has been tested in humans and found to be
unacceptably immunogenic, the B-cell epitopes can be mapped and
then altered to avoid immune detection. Another approach uses
methods to predict and remove potential T-cell epitopes.
Computational methods have been developed to scan and to identify
the peptide sequences of biologic therapeutics with the potential
to bind to MHC proteins (Desmet et al. (2005) Proteins 58: 53-69).
Alternatively a human dendritic cell-based method can be used to
identify CD4+ T-cell epitopes in potential protein allergens
(Stickler et al. (2000) J. Immunother. 23: 654-60; S. L. Morrison
and J. Schlom, (1990) Important Adv. Oncol. 3-18; Riechmann et al.
(1988) Nature 332: 323-327; Roguska et al. (1996) Protein Engineer.
9: 895-904; Kashmiri et al. (2005) Methods 36(1): 25-34; Desmet et
al. (2005) Proteins 58: 53-69; and Stickler et al. (2000) J.
Immunotherapy 23: 654-60.)
B 7. In Vivo Efficacy
[0172] To generate a DVD-Ig molecule with desired in vivo efficacy,
it is important to generate and select mAbs with similarly desired
in vivo efficacy when given in combination. However, in some
instances the DVD-Ig may exhibit in vivo efficacy that cannot be
achieved with the combination of two separate mAbs. For instance, a
DVD-Ig may bring two targets in close proximity leading to an
activity that cannot be achieved with the combination of two
separate mAbs. Additional desirable biological functions are
described herein in section B 3. Parent antibodies with
characteristics desirable in the DVD-Ig molecule may be selected
based on factors such as pharmacokinetic t 1/2; tissue
distribution; soluble versus cell surface targets; and target
concentration-soluble/density-surface.
B 8. In Vivo Tissue Distribution
[0173] To generate a DVD-Ig molecule with desired in vivo tissue
distribution, in an embodiment parent mAbs with similar desired in
vivo tissue distribution profile must be selected. In this regard,
the parent mAbs can be the same antibody or different antibodies.
Alternatively, based on the mechanism of the dual-specific
targeting strategy, it may at other times not be required to select
parent mAbs with the similarly desired in vivo tissue distribution
when given in combination. (e.g., in the case of a DVD-Ig in which
one binding component targets the DVD-Ig to a specific site thereby
bringing the second binding component to the same target site). For
example, one binding specificity of a DVD-Ig could target pancreas
(islet cells) and the other specificity could bring GLP1 to the
pancreas to induce insulin.
B 9. Isotype:
[0174] To generate a DVD-Ig molecule with desired properties
including, but not limited to, isotype, effector functions, and the
circulating half-life, in an embodiment parent mAbs with
appropriate Fc-effector functions depending on the therapeutic
utility and the desired therapeutic end-point are selected. The
parent mAbs can be the same antibody or different antibodies. There
are five main heavy-chain classes or isotypes some of which have
several sub-types and these determine the effector functions of an
antibody molecule. These effector functions reside in the hinge
region, CH2 and CH3 domains of the antibody molecule. However,
residues in other parts of an antibody molecule may have effects on
effector functions as well. The hinge region Fc-effector functions
include: (i) antibody-dependent cellular cytotoxicity, (ii)
complement (C1q) binding, activation and complement-dependent
cytotoxicity (CDC), (iii) phagocytosis/clearance of
antigen-antibody complexes, and (iv) cytokine release in some
instances. These Fc-effector functions of an antibody molecule are
mediated through the interaction of the Fc-region with a set of
class-specific cell surface receptors. Antibodies of the IgG1
isotype are most active while IgG2 and IgG4 having minimal or no
effector functions. The effector functions of the IgG antibodies
are mediated through interactions with three structurally
homologous cellular Fc receptor types (and sub-types) (FcgR1,
FcgRII and FcgRIII). These effector functions of an IgG1 can be
eliminated by mutating specific amino acid residues in the lower
hinge region (e.g., L234A, L235A) that are required for FcgR and
C1q binding Amino acid residues in the Fc region, in particular the
CH2-CH3 domains, also determine the circulating half-life of the
antibody molecule. This Fc function is mediated through the binding
of the Fc-region to the neonatal Fc receptor (FcRn), which is
responsible for recycling of antibody molecules from the acidic
lysosomes back to the general circulation.
[0175] Whether a mAb should have an active or an inactive isotype
will depend on the desired therapeutic end-point for an antibody.
Some examples of usage of isotypes and desired therapeutic outcome
are listed below: [0176] a) If the desired end-point is functional
neutralization of a soluble cytokine, then an inactive isotype may
be used; [0177] b) If the desired out-come is clearance of a
pathological protein, an active isotype may be used; [0178] c) If
the desired out-come is clearance of protein aggregates, an active
isotype may be used; [0179] d) If the desired outcome is to
antagonize a surface receptor, an inactive isotype is used
(Tysabri, IgG4; OKT3, mutated IgG1); [0180] e) If the desired
outcome is to eliminate target cells, an active isotype is used
(Herceptin, IgG1 (and with enhanced effector functions); and [0181]
f) If the desired outcome is to clear proteins from circulation
without entering the CNS, an IgM isotype may be used (e.g.,
clearing circulating Ab peptide species). The Fc effector functions
of a parental mAb can be determined by various in vitro methods
well known in the art.
[0182] As discussed, the selection of isotype, and thereby the
effector functions will depend upon the desired therapeutic
end-point. In cases where simple neutralization of a circulating
target is desired, for example blocking receptor-ligand
interactions, the effector functions may not be required. In such
instances isotypes or mutations in the Fc-region of an antibody
that eliminate effector functions are desirable. In other instances
where elimination of target cells is the therapeutic end-point, for
example elimination of tumor cells, isotypes or mutations or
de-fucosylation in the Fc-region that enhance effector functions
are desirable (Presta, G. L. (2006) Adv. Drug Deliv. Rev.
58:640-656 and Satoh, M. et al. (2006) Expert Opin. Biol. Ther. 6:
1161-1173). Similarly, depending up on the therapeutic utility, the
circulating half-life of an antibody molecule can be
reduced/prolonged by modulating antibody-FcRn interactions by
introducing specific mutations in the Fc region (Dall'Acqua, W. F.
et al. (2006) J. Biol. Chem. 281: 23514-23524; Petkova, S. B.
(2006) et al., Internat. Immunol 18:1759-1769; Vaccaro, C. et al.
(2007) Proc. Natl. Acad. Sci. USA 103: 18709-18714).
[0183] The published information on the various residues that
influence the different effector functions of a normal therapeutic
mAb may need to be confirmed for DVD-Ig. It may be possible that in
a DVD-Ig format additional (different) Fc-region residues, other
than those identified for the modulation of monoclonal antibody
effector functions, may be important.
[0184] Overall, the decision as to which Fc-effector functions
(isotype) will be critical in the final DVD-Ig format will depend
upon the disease indication, therapeutic target, and desired
therapeutic end-point and safety considerations. Listed below are
exemplary appropriate heavy chain and light chain constant regions
including, but not limited to: [0185] IgG1--allotype: G1mz [0186]
IgG1 mutant--A234, A235 [0187] IgG2--allotype: G2m(n-) [0188]
Kappa--Km3 [0189] Lambda
[0190] Fc Receptor and C1q Studies: The possibility of unwanted
antibody-dependent cell-mediated cytotoxicity (ADCC) and
complement-dependent cytotoxicity (CDC) by antibody complexing to
any overexpressed target on cell membranes can be abrogated by the
(for example, L234A, L235A) hinge-region mutations. These
substituted amino acids, present in the IgG1 hinge region of mAb,
are expected to result in diminished binding of mAb to human Fc
receptors (but not FcRn), as FcgR binding is thought to occur
within overlapping sites on the IgG1 hinge region. This feature of
mAb may lead to an improved safety profile over antibodies
containing a wild-type IgG. Binding of mAb to human Fc receptors
can be determined by flow cytometry experiments using cell lines
(e.g., THP-1, K562) and an engineered CHO cell line that expresses
FcgRIIb (or other FcgRs). Compared to IgG1 control monoclonal
antibodies, mAb show reduced binding to FcgRI and FcgRIIa, whereas
binding to FcgRIIb is unaffected. The binding and activation of C1q
by antigen/IgG immune complexes triggers the classical complement
cascade with consequent inflammatory and/or immunoregulatory
responses. The C1q binding site on IgGs has been localized to
residues within the IgG hinge region. C1q binding to increasing
concentrations of mAb was assessed by C1q ELISA. The results
demonstrate that mAb is unable to bind to C1q, as expected when
compared to the binding of a wildtype control IgG1. Overall, the
L234A, L235A hinge region mutation abolishes binding of mAb to
FcgRI, FcgRIIa and C1q but does not impact the interaction of mAb
with FcgRIIb. These data suggest that in vivo, mAb with mutant Fc
will interact normally with the inhibitory FcgRIIb but will likely
fail to interact with the activating FcgRI and FcgRIIa receptors or
C1q.
[0191] Human FcRn binding: The neonatal receptor (FcRn) is
responsible for transport of IgG across the placenta and to control
the catabolic half-life of the IgG molecules. It might be desirable
to increase the terminal half-life of an antibody to improve
efficacy, to reduce the dose or frequency of administration, or to
improve localization to the target. Alternatively, it might be
advantageous to do the converse that is, to decrease the terminal
half-life of an antibody to reduce whole body exposure or to
improve the target-to-non-target binding ratios. Tailoring the
interaction between IgG and its salvage receptor, FcRn, offers a
way to increase or decrease the terminal half-life of IgG. Proteins
in the circulation, including IgG, are taken up in the fluid phase
through micropinocytosis by certain cells, such as those of the
vascular endothelia. IgG can bind FcRn in endosomes under slightly
acidic conditions (pH 6.0-6.5) and can recycle to the cell surface,
where it is released under almost neutral conditions (pH 7.0-7.4).
Mapping of the Fc-region-binding site on FcRn80, 16, 17 showed that
two histidine residues that are conserved across species, His310
and His435, are responsible for the pH dependence of this
interaction. Using phage-display technology, a mouse Fc-region
mutation that increases binding to FcRn and extends the half-life
of mouse IgG was identified (see Victor, G. et al. (1997) Nature
Biotechnol. 15(7): 637-640). Fc-region mutations that increase the
binding affinity of human IgG for FcRn at pH 6.0, but not at pH
7.4, have also been identified (see Dall'Acqua, William F., et al.
(2002) J. Immunol. 169(9): 5171-80). Moreover, in one case, a
similar pH-dependent increase in binding (up to 27-fold) was also
observed for rhesus FcRn, and this resulted in a twofold increase
in serum half-life in rhesus monkeys compared with the parent IgG
(see Hinton, P. R. et al. (2004) J. Biol. Chem. 279(8), 6213-6216).
These findings indicate that it is feasible to extend the plasma
half-life of antibody therapeutics by tailoring the interaction of
the Fc region with FcRn. Conversely, Fc-region mutations that
attenuate interaction with FcRn can reduce antibody half-life.
B.10 Pharmacokinetics (PK):
[0192] To generate a DVD-Ig molecule with desired pharmacokinetic
profile, in an embodiment parent mAbs with the similarly desired
pharmacokinetic profile are selected. One consideration is that
immunogenic response to monoclonal antibodies (i.e., HAHA, human
anti-human antibody response; HACA, human anti-chimeric antibody
response) further complicates the pharmacokinetics of these
therapeutic agents. In an embodiment, monoclonal antibodies with
minimal or no immunogenicity are used for constructing DVD-Ig
molecules such that the resulting DVD-Igs will also have minimal or
no immunogenicity. Some of the factors that determine the PK of a
mAb include, but are not limited to, intrinsic properties of the
mAb (VH amino acid sequence); immunogenicity; FcRn binding and Fc
functions.
[0193] The PK profile of selected parental monoclonal antibodies
can be easily determined in rodents as the PK profile in rodents
correlates well with (or closely predicts) the PK profile of
monoclonal antibodies in cynomolgus monkey and humans. The PK
profile is determined as described in Example section
1.2.2.3.A.
[0194] After the parental monoclonal antibodies with desired PK
characteristics (and other desired functional properties as
discussed herein) are selected, the DVD-Ig is constructed. As the
DVD-Ig molecules contain two antigen-binding domains from two
parental monoclonal antibodies, the PK properties of the DVD-Ig are
assessed as well. Therefore, while determining the PK properties of
the DVD-Ig, PK assays may be employed that determine the PK profile
based on functionality of both antigen-binding domains derived from
the two parent monoclonal antibodies. The PK profile of a DVD-Ig
can be determined as described in Example 1.2.2.3.A. Additional
factors that may impact the PK profile of DVD-Ig include the
antigen-binding domain (CDR) orientation; linker size; and Fc/FcRn
interactions. PK characteristics of parent antibodies can be
evaluated by assessing the following parameters: absorption,
distribution, metabolism and excretion.
[0195] Absorption: To date, administration of therapeutic
monoclonal antibodies is via parenteral routes (e.g., intravenous
[IV], subcutaneous [SC], or intramuscular [IM]). Absorption of a
mAb into the systemic circulation following either SC or IM
administration from the interstitial space is primarily through the
lymphatic pathway. Saturable, presystemic, proteolytic degradation
may result in variable absolute bioavailability following
extravascular administration. Usually, increases in absolute
bioavailability with increasing doses of monoclonal antibodies may
be observed due to saturated proteolytic capacity at higher doses.
The absorption process for a mAb is usually quite slow as the lymph
fluid drains slowly into the vascular system, and the duration of
absorption may occur over hours to several days. The absolute
bioavailability of monoclonal antibodies following SC
administration generally ranges from 50% to 100%. In the case of a
transport-mediating structure at the blood-brain barrier targeted
by the DVD-Ig construct, circulation times in plasma may be reduced
due to enhanced trans-cellular transport at the blood brain barrier
(BBB) into the CNS compartment, where the DVD-Ig is liberated to
enable interaction via its second antigen recognition site.
[0196] Distribution: Following IV administration, monoclonal
antibodies usually follow a biphasic serum (or plasma)
concentration-time profile, beginning with a rapid distribution
phase, followed by a slow elimination phase. In general, a
biexponential pharmacokinetic model best describes this kind of
pharmacokinetic profile. The volume of distribution in the central
compartment (Vc) for a mAb is usually equal to or slightly larger
than the plasma volume (2-3 liters). A distinct biphasic pattern in
serum (plasma) concentration versus time profile may not be
apparent with other parenteral routes of administration, such as IM
or SC, because the distribution phase of the serum (plasma)
concentration-time curve is masked by the long absorption portion.
Many factors, including physicochemical properties, site-specific
and target-oriented receptor mediated uptake, binding capacity of
tissue, and mAb dose can influence biodistribution of a mAb. Some
of these factors can contribute to nonlinearity in biodistribution
for a mAb.
[0197] Metabolism and Excretion: Due to the molecular size, intact
monoclonal antibodies are not excreted into the urine via kidney.
They are primarily inactivated by metabolism (e.g., catabolism).
For IgG-based therapeutic monoclonal antibodies, half-lives
typically ranges from hours or 1-2 days to over 20 days. The
elimination of a mAb can be affected by many factors, including,
but not limited to, affinity for the FcRn receptor, immunogenicity
of the mAb, the degree of glycosylation of the mAb, the
susceptibility for the mAb to proteolysis, and receptor-mediated
elimination.
B.11 Tissue Cross-Reactivity Pattern on Human and Tox Species:
[0198] Identical staining pattern suggests that potential human
toxicity can be evaluated in tox species. Tox species are those
animal in which unrelated toxicity is studied.
[0199] The individual antibodies are selected to meet two criteria:
(1) tissue staining appropriate for the known expression of the
antibody target; and (2) similar staining pattern between human and
tox species tissues from the same organ.
[0200] Criterion 1: Immunizations and/or antibody selections
typically employ recombinant or synthesized antigens (proteins,
carbohydrates or other molecules). Binding to the natural
counterpart and counterscreen against unrelated antigens are often
part of the screening funnel for therapeutic antibodies. However,
screening against a multitude of antigens is often unpractical.
Therefore tissue cross-reactivity studies with human tissues from
all major organs serve to rule out unwanted binding of the antibody
to any unrelated antigens.
[0201] Criterion 2: Comparative tissue cross reactivity studies
with human and tox species tissues (cynomolgus monkey, dog,
possibly rodents and others, the same 36 or 37 tissues are being
tested as in the human study) help to validate the selection of a
tox species. In the typical tissue cross-reactivity studies on
frozen tissue sections therapeutic antibodies may demonstrate the
expected binding to the known antigen and/or to a lesser degree
binding to tissues based either on low level interactions
(unspecific binding, low level binding to similar antigens, low
level charge based interactions etc.). In any case the most
relevant toxicology animal species is the one with the highest
degree of coincidence of binding to human and animal tissue.
[0202] Tissue cross reactivity studies follow the appropriate
regulatory guidelines including EC CPMP Guideline III/5271/94
"Production and quality control of mAbs" and the 1997 U.S. FDA/CBER
"Points to Consider in the Manufacture and Testing of Monoclonal
Antibody Products for Human Use". Cryosections (5 im) of human
tissues obtained at autopsy or biopsy were fixed and dried on
object glass. The peroxidase staining of tissue sections was
performed, using the avidin-biotin system. FDA's Guidance "Points
to Consider in the Manufacture and Testing of Monoclonal Antibody
Products for Human Use".
[0203] Tissue cross reactivity studies are often done in two
stages, with the first stage including cryosections of 32 tissues
(typically: Adrenal Gland, Gastrointestinal Tract, Prostate,
Bladder, Heart, Skeletal Muscle, Blood Cells, Kidney, Skin, Bone
Marrow, Liver, Spinal Cord, Breast, Lung, Spleen, Cerebellum, Lymph
Node, Testes, Cerebral Cortex, Ovary, Thymus, Colon, Pancreas,
Thyroid, Endothelium, Parathyroid, Ureter, Eye, Pituitary, Uterus,
Fallopian Tube and Placenta) from one human donor. In the second
phase a full cross reactivity study is performed with up to 38
tissues (including adrenal, blood, blood vessel, bone marrow,
cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large
intestine, liver, lung, lymph node, breast mammary gland, ovary,
oviduct, pancreas, parathyroid, peripheral nerve, pituitary,
placenta, prostate, salivary gland, skin, small intestine, spinal
cord, spleen, stomach, striated muscle, testis, thymus, thyroid,
tonsil, ureter, urinary bladder, and uterus) from 3 unrelated
adults. Studies are done typically at minimally two dose
levels.
[0204] The therapeutic antibody (i.e., test article) and isotype
matched control antibody may be biotinylated for avidin-biotin
complex (ABC) detection; other detection methods may include
tertiary antibody detection for a FITC (or otherwise) labeled test
article, or precomplexing with a labeled anti-human IgG for an
unlabeled test article.
[0205] Briefly, cryosections (about 5 .mu.m) of human tissues
obtained at autopsy or biopsy are fixed and dried on object glass.
The peroxidase staining of tissue sections is performed, using the
avidin-biotin system. First (in case of a precomplexing detection
system), the test article is incubated with the secondary
biotinylated anti-human IgG and developed into immune complex. The
immune complex at the final concentrations of 2 and 10 .mu.g/mL of
test article is added onto tissue sections on object glass and then
the tissue sections are reacted for 30 minutes with a
avidin-biotin-peroxidase kit. Subsequently, DAB
(3,3'-diaminobenzidine), a substrate for the peroxidase reaction,
is applied for 4 minutes for tissue staining Antigen-Sepharose
beads are used as positive control tissue sections.
[0206] Any specific staining is judged to be either an expected
(e.g., consistent with antigen expression) or unexpected reactivity
based upon known expression of the target antigen in question. Any
staining judged specific is scored for intensity and frequency.
Antigen or serum completion or blocking studies can assist further
in determining whether observed staining is specific or
nonspecific.
[0207] If two selected antibodies are found to meet the selection
criteria--appropriate tissue staining, and matching staining
between human and toxicology animal specific tissue--they can be
selected for DVD-Ig generation.
[0208] The tissue cross reactivity study has to be repeated with
the final DVD-Ig construct, but while these studies follow the same
protocol as outline herein, they are more complex to evaluate
because any binding can come from any of the two parent antibodies,
and any unexplained binding needs to be confirmed with complex
antigen competition studies.
[0209] It is readily apparent that the complex undertaking of
tissue crossreactivity studies with a multispecific molecule like a
DVD-Ig is greatly simplified if the two parental antibodies are
selected for (1) lack of unexpected tissue cross reactivity
findings and (2) for appropriate similarity of tissue cross
reactivity findings between the corresponding human and toxicology
animal species tissues.
B.12 Specificity and Selectivity:
[0210] To generate a DVD-Ig molecule with desired specificity and
selectivity, one needs to generate and select parent mAbs with the
similarly desired specificity and selectivity profile. In this
regard, parent mAbs can be the same antibody or different
antibodies.
[0211] Binding studies for specificity and selectivity with a
DVD-Ig can be complex due to the four or more binding sites, two
each for each antigen. Briefly, binding studies using an enzyme
linked immunosorbent assay (ELISA), BIAcore, KinExA, or other
interaction studies with a DVD-Ig need to monitor the binding of
one, two or more antigens to the DVD-Ig molecule. While BIAcore
technology can resolve the sequential, independent binding of
multiple antigens, more traditional methods, including ELISA, or
more modern techniques, such as KinExA, cannot. Therefore, careful
characterization of each parent antibody is critical. After each
individual antibody has been characterized for specificity,
confirmation of specificity retention of the individual binding
sites in the DVD-Ig molecule is greatly simplified.
[0212] It is readily apparent that the complex undertaking of
determining the specificity of a DVD-Ig is greatly simplified if
the two parental antibodies are selected for specificity prior to
being combined into a DVD-Ig.
[0213] Antigen-antibody interaction studies can take many forms,
including many classical protein protein interaction studies,
ELISA, mass spectrometry, chemical cross-linking, SEC with light
scattering, equilibrium dialysis, gel permeation, ultrafiltration,
gel chromatography, large-zone analytical SEC, micropreparative
ultracentrigugation (sedimentation equilibrium), spectroscopic
methods, titration microcalorimetry, sedimentation equilibrium (in
analytical ultracentrifuge), sedimentation velocity (in analytical
centrifuge), and surface plasmon resonance (including BIAcore).
Relevant references include "Current Protocols in Protein Science,"
Coligan, J. E. et al. (eds.) Volume 3, chapters 19 and 20,
published by John Wiley & Sons Inc., and "Current Protocols in
Immunology," Coligan, J. E. et al. (eds.) published by John Wiley
& Sons Inc., and relevant references included therein.
[0214] Cytokine Release in Whole Blood: The interaction of mAb with
human blood cells can be investigated by a cytokine release (Wing,
M. G. (1995) Therapeut. Immunol 2(4): 183-190; "Current Protocols
in Pharmacology," Enna, S. J. et al. (eds.) published by John Wiley
& Sons Inc; Madhusudan, S. (2004) Clin. Cancer Res. 10(19):
6528-6534; Cox, J. (2006) Methods 38(4): 274-282; Choi, I. (2001)
Eur. J. Immunol. 31(1): 94-106). Briefly, various concentrations of
mAb are incubated with human whole blood for 24 hours. The
concentration tested should cover a wide range including final
concentrations mimicking typical blood levels in patients
(including but not limited to 100 ng/ml-100 .mu.g/ml). Following
the incubation, supernatants and cell lysates were analyzed for the
presence of IL-1R.alpha., TNF-.alpha., IL-1b, IL-6 and IL-8.
Cytokine concentration profiles generated for mAb were compared to
profiles produced by a negative human IgG control and a positive
LPS or PHA control. The cytokine profile displayed by mAb from both
cell supernatants and cell lysates was comparable to control human
IgG. In an embodiment, the monoclonal antibody does not interact
with human blood cells to spontaneously release inflammatory
cytokines.
[0215] Cytokine release studies for a DVD-Ig are complex due to the
four or more binding sites, two each for each antigen. Briefly,
cytokine release studies as described herein measure the effect of
the whole DVD-Ig molecule on whole blood or other cell systems, but
can resolve which portion of the molecule causes cytokine release.
Once cytokine release has been detected, the purity of the DVD-Ig
preparation has to be ascertained, because some co-purifying
cellular components can cause cytokine release on their own. If
purity is not the issue, fragmentation of DVD-Ig (including but not
limited to removal of Fc portion, separation of binding sites
etc.), binding site mutagenesis or other methods may need to be
employed to deconvolute any observations. It is readily apparent
that this complex undertaking is greatly simplified if the two
parental antibodies are selected for lack of cytokine release prior
to being combined into a DVD-Ig.
B.13 Cross Reactivity to Other Species for Toxicological
Studies:
[0216] In an embodiment, the individual antibodies are selected
with sufficient cross-reactivity to appropriate tox species, for
example, cynomolgus monkey. Parental antibodies need to bind to
orthologous species target (i.e., cynomolgus monkey) and elicit
appropriate response (modulation, neutralization, activation). In
an embodiment, the cross-reactivity (affinity/potency) to
orthologous species target should be within 10-fold of the human
target. In practice, the parental antibodies are evaluated for
multiple species, including mouse, rat, dog, monkey (and other
non-human primates), as well as disease model species (i.e., sheep
for asthma model). The acceptable cross-reactivity to tox species
from the perental monoclonal antibodies allows future toxicology
studies of DVD-Ig-Ig in the same species. For that reason, the two
parental monoclonal antibodies should have acceptable
cross-reactivity for a common tox species, thereby allowing
toxicology studies of DVD-Ig in the same species.
[0217] Parent mAbs may be selected from various mAbs that bind
specific targets and are well known in the art. The parent
antibodies can be the same antibody or different antibodies. These
include, but are not limited to anti-TNF antibody (U.S. Pat. No.
6,258,562), anti-IL-12 and/or anti-IL-12p40 antibody (U.S. Pat. No.
6,914,128); anti-IL-18 antibody (U.S. Patent Publication No.
2005/0147610), anti-C5, anti-CBL, anti-CD147, anti-gp120,
anti-VLA-4, anti-CD11a, anti-CD18, anti-VEGF, anti-CD40L, anti
CD-40 (e.g., see PCT Publication No. WO 2007/124299) anti-Id,
anti-ICAM-1, anti-CXCL13, anti-CD2, anti-EGFR, anti-TGF-beta 2,
anti-HGF, anti-cMet, anti DLL-4, anti-NPR1, anti-PLGF, anti-ErbB3,
anti-E-selectin, anti-Fact VII, anti-Her2/neu, anti-F gp,
anti-CD11/18, anti-CD14, anti-ICAM-3, anti-RON, anti-SOST, anti
CD-19, anti-CD80 (e.g., see PCT Publication No. WO 2003/039486,
anti-CD4, anti-CD3, anti-CD23, anti-beta2-integrin,
anti-alpha4beta7, anti-CD52, anti-HLA DR, anti-CD22 (e.g., see U.S.
Pat. No. 5,789,554), anti-CD20, anti-MIF, anti-CD64 (FcR), anti-TCR
alpha beta, anti-CD2, anti-Hep B, anti-CA 125, anti-EpCAM,
anti-gp120, anti-CMV, anti-gpIIbIIIa, anti-IgE, anti-CD25,
anti-CD33, anti-HLA, anti-IGF1,2, anti IGFR, anti-VNRintegrin,
anti-IL-1alpha, anti-IL-1beta, anti-IL-1 receptor, anti-IL-2
receptor, anti-IL-4, anti-IL-4 receptor, anti-IL5, anti-IL-5
receptor, anti-IL-6, anti-IL-8, anti-IL-9, anti-IL-13, anti-IL-13
receptor, anti-IL-17, anti-IL-6R, anti-RANKL, anti-NGF, anti-DKK,
anti-alphaVbeta3, anti-IL-17A, anti-IL23p19 and anti-IL-23 (see
Presta, L. G. (2005) J. Allergy Clin. Immunol 116: 731-6.
[0218] Parent mAbs may also be selected from various therapeutic
antibodies approved for use, in clinical trials, or in development
for clinical use. Such therapeutic antibodies include, but are not
limited to, rituximab (Rituxan.RTM., IDEC/Genentech/Roche) (see,
for example, U.S. Pat. No. 5,736,137), a chimeric anti-CD20
antibody approved to treat Non-Hodgkin's lymphoma; HuMax-CD20, an
anti-CD20 currently being developed by Genmab, an anti-CD20
antibody described in U.S. Pat. No. 5,500,362, AME-133 (Applied
Molecular Evolution), hA20 (Immunomedics, Inc.), HumaLYM
(Intracel), and PRO70769 (PCT Application No. PCT/US2003/040426),
trastuzumab (Herceptin.RTM., Genentech) (see, for example, U.S.
Pat. No. 5,677,171), a humanized anti-Her2/neu antibody approved to
treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg.RTM.),
currently being developed by Genentech; an anti-Her2 antibody (U.S.
Pat. No. 4,753,894; cetuximab (Erbitux.RTM., Imclone) (U.S. Pat.
No. 4,943,533; PCT Publication No. WO 96/40210), a chimeric
anti-EGFR antibody in clinical trials for a variety of cancers;
ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed by
Abgenix-Immunex-Amgen; HuMax-EGFr (U.S. Pat. No. 7,247,301),
currently being developed by Genmab; 425, EMD55900, EMD62000, and
EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864; Murthy, et al.
(1987) Arch. Biochem. Biophys. 252(2): 549-60; Rodeck, et al.
(1987) J. Cell. Biochem. 35(4): 315-20; Kettleborough, et al.
(1991) Protein Eng. 4(7): 773-83); ICR62 (Institute of Cancer
Research) (PCT Publication No. WO 95/20045; Modjtahedi, et al.
(1993) J. Cell. Biophys. 22(1-3): 129-46; Modjtahedi, et al. (1993)
Br. J. Cancer 67(2): 247-53; Modjtahedi, et al. (1996) Br. J.
Cancer, 73(2): 228-35; Modjtahedi, et al. (2003) Int. J. Cancer,
105(2): 273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro de
Immunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.
6,506,883; Mateo, et al. (1997) Immunotechnol. 3(1): 71-81);
mAb-806 (Ludwig Institute for Cancer Research, Memorial
Sloan-Kettering) (Jungbluth, et al. (2003) Proc. Natl. Acad. Sci.
USA. 100(2): 639-44); KSB-102 (KS Biomedix); MR1-1 (WAX, National
Cancer Institute) (PCT Publication No. WO 01/62931A2); and SC100
(Scancell) (PCT Publication No. WO 01/88138); alemtuzumab
(Campath.RTM., Millenium), a humanized mAb currently approved for
treatment of B-cell chronic lymphocytic leukemia; muromonab-CD3
(Orthoclone OKT3.RTM.), an anti-CD3 antibody developed by Ortho
Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin.RTM.),
an anti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab
ozogamicin (Mylotarg.RTM.), an anti-CD33 (p67 protein) antibody
developed by Celltech/Wyeth, alefacept (Amevive.RTM.), an
anti-LFA-3 Fc fusion developed by Biogen), abciximab (ReoPro.RTM.),
developed by Centocor/Lilly, basiliximab (Simulect.RTM.), developed
by Novartis, palivizumab (Synagis.RTM.), developed by Medimmune,
infliximab (Remicade.RTM.), an anti-TNFalpha antibody developed by
Centocor, adalimumab (Humira.RTM.), an anti-TNFalpha antibody
developed by Abbott, Humicade.RTM., an anti-TNFalpha antibody
developed by Celltech, golimumab (CNTO-148), a fully human TNF
antibody developed by Centocor, etanercept (Enbrel.RTM.), an p75
TNF receptor Fc fusion developed by Immunex/Amgen, lenercept, an
p55TNF receptor Fc fusion previously developed by Roche, ABX-CBL,
an anti-CD147 antibody being developed by Abgenix, ABX-IL8, an
anti-IL8 antibody being developed by Abgenix, ABX-MA1, an
anti-MUC18 antibody being developed by Abgenix, Pemtumomab (R1549,
90Y-muHMFG1), an anti-MUC1 in development by Antisoma, Therex
(R1550), an anti-MUC1 antibody being developed by Antisoma,
AngioMab (AS1405), being developed by Antisoma, HuBC-1, being
developed by Antisoma, Thioplatin (AS1407) being developed by
Antisoma, Antegren.RTM. (natalizumab), an anti-alpha-4-beta-1
(VLA-4) and alpha-4-beta-7 antibody being developed by Biogen,
VLA-1 mAb, an anti-VLA-1 integrin antibody being developed by
Biogen, LTBR mAb, an anti-lymphotoxin beta receptor (LTBR) antibody
being developed by Biogen, CAT-152, an anti-TGF-132 antibody being
developed by Cambridge Antibody Technology, ABT 874 (J695), an
anti-IL-12 p40 antibody being developed by Abbott, CAT-192, an
anti-TGF.beta.1 antibody being developed by Cambridge Antibody
Technology and Genzyme, CAT-213, an anti-Eotaxin1 antibody being
developed by Cambridge Antibody Technology, LymphoStat-B.RTM. an
anti-Blys antibody being developed by Cambridge Antibody Technology
and Human Genome Sciences Inc., TRAIL-R1 mAb, an anti-TRAIL-R1
antibody being developed by Cambridge Antibody Technology and Human
Genome Sciences, Inc., Avastin.RTM. bevacizumab, rhuMAb-VEGF), an
anti-VEGF antibody being developed by Genentech, an anti-HER
receptor family antibody being developed by Genentech, Anti-Tissue
Factor (ATF), an anti-Tissue Factor antibody being developed by
Genentech, Xolair.RTM. (Omalizumab), an anti-IgE antibody being
developed by Genentech, Raptiva.RTM. (Efalizumab), an anti-CD11a
antibody being developed by Genentech and Xoma, MLN-02 Antibody
(formerly LDP-02), being developed by Genentech and Millenium
Pharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed by
Genmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab
and Amgen, HuMax-Inflam, being developed by Genmab and Medarex,
HuMax-Cancer, an anti-Heparanase I antibody being developed by
Genmab and Medarex and Oxford GcoSciences, HuMax-Lymphoma, being
developed by Genmab and Amgen, HuMax-TAC, being developed by
Genmab, IDEC-131, and anti-CD40L antibody being developed by IDEC
Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibody
being developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80
antibody being developed by IDEC Pharmaceuticals, IDEC-152, an
anti-CD23 being developed by IDEC Pharmaceuticals, anti-macrophage
migration factor (MIF) antibodies being developed by IDEC
Pharmaceuticals, BEC2, an anti-idiotypic antibody being developed
by Imclone, IMC-1C11, an anti-KDR antibody being developed by
Imclone, DC101, an anti-flk-1 antibody being developed by Imclone,
anti-VE cadherin antibodies being developed by Imclone,
CEA-Cide.RTM. (labetuzumab), an anti-carcinoembryonic antigen (CEA)
antibody being developed by Immunomedics, LymphoCide.RTM.
(Epratuzumab), an anti-CD22 antibody being developed by
Immunomedics, AFP-Cide, being developed by Immunomedics,
MyelomaCide, being developed by Immunomedics, LkoCide, being
developed by Immunomedics, ProstaCide, being developed by
Immunomedics, MDX-010, an anti-CTLA4 antibody being developed by
Medarex, MDX-060, an anti-CD30 antibody being developed by Medarex,
MDX-070 being developed by Medarex, MDX-018 being developed by
Medarex, Osidem.RTM. (IDM-1), and anti-Her2 antibody being
developed by Medarex and Immuno-Designed Molecules, HuMax.RTM.-CD4,
an anti-CD4 antibody being developed by Medarex and Genmab,
HuMax-IL15, an anti-IL15 antibody being developed by Medarex and
Genmab, CNTO 148, an anti-TNF.alpha. antibody being developed by
Medarex and Centocor/J&J, CNTO 1275, an anti-cytokine antibody
being developed by Centocor/J&J, MOR101 and MOR102,
anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies
being developed by MorphoSys, MOR201, an anti-fibroblast growth
factor receptor 3 (FGFR-3) antibody being developed by MorphoSys,
Nuvion.RTM. (visilizumab), an anti-CD3 antibody being developed by
Protein Design Labs, HuZAF.RTM., an anti-gamma interferon antibody
being developed by Protein Design Labs, Anti-.alpha. 5.beta.1
Integrin, being developed by Protein Design Labs, anti-IL-12, being
developed by Protein Design Labs, ING-1, an anti-Ep-CAM antibody
being developed by Xoma, Xolair.RTM. (Omalizumab) a humanized
anti-IgE antibody developed by Genentech and Novartis, and MLN01,
an anti-Beta2 integrin antibody being developed by Xoma. In another
embodiment, the therapeutics include KRN330 (Kirin); huA33 antibody
(A33, Ludwig Institute for Cancer Research); CNTO 95 (alpha V
integrins, Centocor); MEDI-522 (alpha V.beta.3 integrin,
Medimmune); volociximab (alpha V.beta.1 integrin, Biogen/PDL);
Human mAb 216 (B cell glycosolated epitope, NCl); BiTE MT103
(bispecific CD19 x CD3, Medimmune); 4G7xH22 (Bispecific
BcellxFcgammaR1, Medarex/Merck KGa); rM28 (Bispecific CD28 x MAPG,
EP Patent No. EP1444268); MDX447 (EMD 82633) (Bispecific CD64 x
EGFR, Medarex); Catumaxomab (removab) (Bispecific EpCAM x anti-CD3,
Trion/Fres); Ertumaxomab (bispecific HER2/CD3, Fresenius Biotech);
oregovomab (OvaRex) (CA-125, ViRexx); Rencarex.RTM. (WX G250)
(carbonic anhydrase IX, Wilex); CNTO 888 (CCL2, Centocor); TRC105
(CD105 (endoglin), Tracon); BMS-663513 (CD137 agonist, Brystol
Myers Squibb); MDX-1342 (CD19, Medarex); Siplizumab (MEDI-507)
(CD2, Medimmune); Ofatumumab (Humax-CD20) (CD20, Genmab); Rituximab
(Rituxan) (CD20, Genentech); veltuzumab (hA20) (CD20,
Immunomedics); Epratuzumab (CD22, Amgen); lumiliximab (IDEC 152)
(CD23, Biogen); muromonab-CD3 (CD3, Ortho); HuM291 (CD3 fc
receptor, PDL Biopharma); HeFi-1, CD30, NCI); MDX-060 (CD30,
Medarex); MDX-1401 (CD30, Medarex); SGN-30 (CD30, Seattle
Genentics); SGN-33 (Lintuzumab) (CD33, Seattle Genentics);
Zanolimumab (HuMax-CD4) (CD4, Genmab); HCD122 (CD40, Novartis);
SGN-40 (CD40, Seattle Genentics); Campath1h (Alemtuzumab) (CD52,
Genzyme); MDX-1411 (CD70, Medarex); hLL1 (EPB-1) (CD74.38,
Immunomedics); Galiximab (IDEC-144) (CD80, Biogen); MT293
(TRC093/D93) (cleaved collagen, Tracon); HuLuc63 (CS1, PDL Pharma);
ipilimumab (MDX-010) (CTLA4, Brystol Myers Squibb); Tremelimumab
(Ticilimumab, CP-675,2) (CTLA4, Pfizer); HGS-ETR1 (Mapatumumab)
(DR4TRAIL-R1 agonist, Human Genome Science/Glaxo Smith Kline);
AMG-655 (DR5, Amgen); Apomab (DR5, Genentech); CS-1008 (DR5,
Daiichi Sankyo); HGS-ETR2 (lexatumumab) (DR5TRAIL-R2 agonist, HGS);
Cetuximab (Erbitux) (EGFR, Imclone); IMC-11F8, (EGFR, Imclone);
Nimotuzumab (EGFR, YM Bio); Panitumumab (Vectabix) (EGFR, Amgen);
Zalutumumab (HuMaxEGFr) (EGFR, Genmab); CDX-110 (EGFRvIII, AVANT
Immunotherapeutics); adecatumumab (MT201) (Epcam, Merck);
edrecolomab (Panorex, 17-1A) (Epcam, Glaxo/Centocor); MORAb-003
(folate receptor a, Morphotech); KW-2871 (ganglioside GD3, Kyowa);
MORAb-009 (GP-9, Morphotech); CDX-1307 (MDX-1307) (hCGb, Celldex);
Trastuzumab (Herceptin) (HER2, Celldex); Pertuzumab (rhuMAb 2C4)
(HER2 (DI), Genentech); apolizumab (HLA-DR beta chain, PDL Pharma);
AMG-479 (IGF-1R, Amgen); anti-IGF-1R R1507 (IGF1-R, Roche); CP
751871 (IGF1-R, Pfizer); IMC-A12 (IGF1-R, Imclone); BIIB022
(IGF-1R, Biogen); Mik-beta-1 (IL-2Rb (CD122), Hoffman LaRoche);
CNTO 328 (IL6, Centocor); Anti-KIR (1-7F9) (Killer cell Ig-like
Receptor (KIR), Novo); Hu3S193 (Lewis (y), Wyeth, Ludwig Institute
of Cancer Research); hCBE-11 (LT.beta.R, Biogen); HuHMFG1 (MUC1,
Antisoma/NCl); RAV12 (N-linked carbohydrate epitope, Raven); CAL
(parathyroid hormone-related protein (PTH-rP), University of
California); CT-011 (PD1, CureTech); MDX-1106 (ono-4538) (PD1,
Medarex/Ono); MAb CT-011 (PD1, Curetech); IMC-3G3 (PDGFRa,
Imclone); bavituximab (phosphatidylserine, Peregrine); huJ591
(PSMA, Cornell Research Foundation); muJ591 (PSMA, Cornell Research
Foundation); GC1008 (TGFb (pan) inhibitor (IgG4), Genzyme);
Infliximab (Remicade) (TNFa, Centocor); A27.15 (transferrin
receptor, Salk Institute, INSERN WO 2005/111082); E2.3 (transferrin
receptor, Salk Institute); Bevacizumab (Avastin) (VEGF, Genentech);
HuMV833 (VEGF, Tsukuba Research Lab), PCT Publication No.
WO/2000/034337, University of Texas); IMC-18F1 (VEGFR1, Imclone);
IMC-1121 (VEGFR2, Imclone).
B. Construction of DVD Molecules:
[0219] The dual variable domain immunoglobulin (DVD-Ig) molecule is
designed such that two different light chain variable domains (VL)
from the two parent monoclonal antibodies, which can be the same or
different, are linked in tandem directly or via a short linker by
recombinant DNA techniques, followed by the light chain constant
domain, and optionally, an Fc region. Similarly, the heavy chain
comprises two different heavy chain variable domains (VH) linked in
tandem, followed by the constant domain CH1 and Fc region (FIG.
1A).
[0220] The variable domains can be obtained using recombinant DNA
techniques from a parent antibody generated by any one of the
methods described herein. In an embodiment, the variable domain is
a murine heavy or light chain variable domain. In another
embodiment, the variable domain is a CDR grafted or a humanized
variable heavy or light chain domain. In an embodiment, the
variable domain is a human heavy or light chain variable
domain.
[0221] In one embodiment the first and second variable domains are
linked directly to each other using recombinant DNA techniques. In
another embodiment the variable domains are linked via a linker
sequence. In an embodiment, two variable domains are linked. Three
or more variable domains may also be linked directly or via a
linker sequence. The variable domains may bind the same antigen or
may bind different antigens. DVD-Ig molecules of the invention may
include one immunoglobulin variable domain and one
non-immunoglobulin variable domain, such as ligand binding domain
of a receptor, or an active domain of an enzyme. DVD-Ig molecules
may also comprise two or more non-Ig domains.
[0222] The linker sequence may be a single amino acid or a
polypeptide sequence. In an embodiment, the linker sequences are
selected from the group consisting of AKTTPKLEEGEFSEAR (SEQ ID NO:
1); AKTTPKLEEGEFSEARV (SEQ ID NO: 2); AKTTPKLGG (SEQ ID NO: 3);
SAKTTPKLGG (SEQ ID NO: 4); SAKTTP (SEQ ID NO: 5); RADAAP (SEQ ID
NO: 6); RADAAPTVS (SEQ ID NO: 7); RADAAAAGGPGS (SEQ ID NO: 8);
RADAAAA(G.sub.4S).sub.4 (SEQ ID NO: 9); SAKTTPKLEEGEFSEARV (SEQ ID
NO: 10); ADAAP (SEQ ID NO: 11); ADAAPTVSIFPP (SEQ ID NO: 12); TVAAP
(SEQ ID NO: 13); TVAAPSVFIFPP (SEQ ID NO: 14); QPKAAP (SEQ ID NO:
15); QPKAAPSVTLFPP (SEQ ID NO: 16); AKTTPP (SEQ ID NO: 17);
AKTTPPSVTPLAP (SEQ ID NO: 18); AKTTAP (SEQ ID NO: 19);
AKTTAPSVYPLAP (SEQ ID NO: 20); ASTKGP (SEQ ID NO: 21);
ASTKGPSVFPLAP (SEQ ID NO: 22), GGGGSGGGGSGGGGS (SEQ ID NO: 23);
GENKVEYAPALMALS (SEQ ID NO: 24); GPAKELTPLKEAKVS (SEQ ID NO: 25);
GHEAAAVMQVQYPAS (SEQ ID NO: 26), TVAAPSVFIFPPTVAAPSVFIFPP (SEQ ID
NO: 27); and ASTKGPSVFPLAPASTKGPSVFPLAP (SEQ ID NO: 28). The choice
of linker sequences is based on crystal structure analysis of
several Fab molecules. There is a natural flexible linkage between
the variable domain and the CH1/CL constant domain in Fab or
antibody molecular structure. This natural linkage comprises
approximately 10-12 amino acid residues, contributed by 4-6
residues from C-terminus of V domain and 4-6 residues from the
N-terminus of CL/CH1 domain. DVD Igs of the invention were
generated using N-terminal 5-6 amino acid residues, or 11-12 amino
acid residues, of CL or CH1 as linker in light chain and heavy
chain of DVD-Ig, respectively. The N-terminal residues of the CL or
CH1 domain, particularly the first 5-6 amino acid residues, adopt a
loop conformation without strong secondary structure, and,
therefore, can act as a flexible linker between the two variable
domains. The N-terminal residues of the CL or CH1 domain are a
natural extension of the variable domains, as they are part of the
Ig sequences, and, therefore, minimize to a large extent any
immunogenicity potentially arising from the linkers and
junctions.
[0223] Other linker sequences may include any sequence of any
length of the CL/CH1 domain but not all residues of the CL/CH1
domain (for example, the first 5-12 amino acid residues of the
CL/CH1 domains) the light chain linkers can be from Ck or Cl; and
the heavy chain linkers can be derived from CH1 of any isotypes,
including Cg1, Cg2, Cg3, Cg4, Ca1, Ca2, Cd, Ce, and Cm. Linker
sequences may also be derived from other proteins such as Ig-like
proteins, (e.g. TCR, FcR, KIR); G/S based sequences (e.g., G4S
repeats) (SEQ ID NO:29); hinge region-derived sequences; and other
natural sequences from other proteins.
[0224] In an embodiment a constant domain is linked to the two
linked variable domains using recombinant DNA techniques. In an
embodiment, sequence comprising linked heavy chain variable domains
is linked to a heavy chain constant domain and sequence comprising
linked light chain variable domains is linked to a light chain
constant domain. In an embodiment, the constant domains are human
heavy chain constant domain and human light chain constant domain
respectively. In an embodiment, the DVD heavy chain is further
linked to an Fc region. The Fc region may be a native sequence Fc
region, or a variant Fc region. In another embodiment, the Fc
region is a human Fc region. In another embodiment the Fc region
includes Fc region from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, or
IgD.
[0225] In another embodiment two heavy chain DVD polypeptides and
two light chain DVD polypeptides are combined to form a DVD-Ig
molecule. Table 2 lists amino acid sequences of VH and VL regions
of exemplary antibodies for targets useful for treating disease,
e.g., for treating cancer. In an embodiment, the invention provides
a DVD comprising at least two of the VH and/or VL regions listed in
Table 2, in any orientation.
TABLE-US-00002 TABLE 2 List of Amino Acid Sequences of VH and VL
regions of Antibodies for Generating DVD-Igs SEQ ABT Protein
Sequence ID No. Unique ID region
1234567890123456789012345678901234567890 30 AB001VH VH CD20
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT
PGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAY
MQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVS A 31 AB001VL VL CD20
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWFQQKPG
SSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAE
DAATYYCQQWTSNPPTFGGGTKLEIKR 32 AB002VH VH CD3
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR (seq. 1)
PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY
MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS 33 AB002VL VL CD3
QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSG (seq. 1)
TSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAE
DAATYYCQQWSSNPLTFGSGTKLEINR 34 AB003VH VH EGFR
QVQLQESGPGLVKPSETLSLTCTVSGGSVSSGDYYWTWIR (seq. 1)
QSPGKGLEWIGHIYYSGNTNYNPSLKSRLTISIDTSKTQF
SLKLSSVTAADTAIYYCVRDRVTGAFDIWGQGTMVTVSS 35 AB003VL VL EGFR
DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKP (seq. 1)
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP
EDIATYFCQHFDHLPLAFGGGTKVEIKR 36 AB004VH VH HER2
EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA (seq. 1)
PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 37 AB004VL VL HER2
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP (seq. 1)
GKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQP
EDFATYYCQQHYTTPPTFGQGTKVEIKR 38 AB005VH VH RON
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSYAMHWVRQA (seq. 1)
PGKGLEWVAVISYDGSNKYYADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCARFSGWPNNYYYYGMDVWGQGTTV TVSS 39 AB005VL VL RON
DVVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGFNYVDW (seq. 1)
YLQKPGQSPHLLIYFGSYRASGVPDRFSGSGSGTDFTLKI
SRVEAEDVGVYYCMQALQTPPWTFGQGTKVEIRR 40 AB006VH VH CD-19
QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMNWVKQR
PGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAY
MQLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTSV TVSS 41 AB006VL VL CD-19
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDSYLNWY
QQIPGQPPKLLIYDASNLVSGIPPRFSGSGSGTDFTLNIH
PVEKVDAATYHCQQSTEDPWTFGGGTKLEIKR 42 AB007VH VH CD-80
QVQLQESGPGLVKPSETLSLTCAVSGGSISGGYGWGWIRQ
PPGKGLEWIGSFYSSSGNTYYNPSLKSQVTISTDTSKNQF
SLKLNSMTAADTAVYYCVRDRLFSVVGMVYNNWFDVWGPG VLVTVSS 43 AB007VL VL
CD-80 ESALTQPPSVSGAPGQKVTISCTGSTSNIGGYDLHWYQQL
PGTAPKLLIYDINKRPSGISDRFSGSKSGTAASLAITGLQ
TEDEADYYCQSYDSSLNAQVFGGGTRLTVLG 44 AB008VH VH CD-22
QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYWLHWVRQA
PGQGLEWIGYINPRNDYTEYNQNFKDKATITADESTNTAY
MELSSLRSEDTAFYFCARRDITTFYWGQGTTVTVSS 45 AB008VL VL CD-22
DIQLTQSPSSLSASVGDRVTMSCKSSQSVLYSANHKNYLA
WYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTFT
ISSLQPEDIATYYCHQYLSSWTFGGGTKLEIKR 46 AB009VH VH CD-40
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQA
PGKGLEWVAVISYEESNRYHADSVKGRFTISRDNSKITLY
LQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLVTVSS 47 AB009VL VL CD-40
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDW
YLQKPGQSPQVLISLGSNRASGVPDRFSGSGSGTDFTLKI
SRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRR 48 AB010VH VH IGF1,2
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQA
TGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAY
MELSSLRSEDTAVYYCARDPYYYYYGMDVWGQGTTVTVSS 49 AB010VL VL IGF1,2
QSVLTQPPSVSAAPGQKVTISCSGSSSNIENNHVSWYQQL
PGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQ
TGDEADYYCETWDTSLSAGRVFGGGTKLTVLG 50 AB011VH VH IGF1R
EVQLLESGGGLVQPGGSLRLSCTASGFTFSSYAMNWVRQA (seq. 1)
PGKGLEWVSAISGSGGTTFYADSVKGRFTISRDNSRTTLY
LQMNSLRAEDTAVYYCAKDLGWSDSYYYYYGMDVWGQGTT VTVSS 51 AB011VL VL IGF1R
DIQMTQFPSSLSASVGDRVTITCRASQGIRNDLGWYQQKP (seq. 1)
GKAPKRLIYAASRLHRGVPSRFSGSGSGTEFTLTISSLQP
EDFATYYCLQHNSYPCSFGQGTKLEIKR 52 AB012VH VH HGF
QVQLVESGGGLVKPGGSLRLSCAASGFTFSDYYMSWIRQA (seq. 1)
PGKGLEWVSYISSSGSTIYYADSVKGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCARDEYNSGWYVLFDYWGQGTLVTV SS 53 AB012VL VL HGF
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKP (seq. 1)
GKAPNLLIYEASSLQSGVPSRFGGSGSGTDFTLTISSLQP
EDFATYYCQQANGFPWTFGQGTKVEIKR 54 AB013VH VH c-MET
QVQLQQSGPELVRPGASVKWSCPASGYTFTSYWLHWVKKQ
RPGQGLEWIGMIDPSNSDTRFNPPNFKDKATLNVDRSSNT
AYNLLSSLTSADSAVYYCATYGSYVSPLDYWGQGTSVYVS S 55 AB013VL VL c-MET
DIMMSQSPSSLTVSVGEKVTVSCKSSQSLLVTSSQKNYLA
WYQQKPQQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT
ITSVKADDLAVYYCQQYYAYPWTFGDGTKLEIKR 56 AB014VH VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQA (seq. 1)
PGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAY
LQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVT VSS 57 AB014VL VL VEGF
DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKP (seq. 1)
GKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYSTVPWTFGQGTKVEIKR 58 AB015VH VH DLL-4
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDNWISWVRQA (seq. 1)
PGKGLEWVGYISPNSGFTYYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCARDNFGGYFDYWGQGTLVTVSS 59 AB015VL VL DLL-4
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKP (seq. 1)
GKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP
EDFATTYYCQQSYTGTVTFGQGTKVEIKR 60 AB016VH VH NRP1
EVQLVESGGGLVQPGGSLRLSCAASGFSFSSEPISWVRQA (seq. 1)
PGKGLEWVSSITGKNGYTYYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCARWGKKVYGMDVWGQGTLVTVSS 61 AB016VL VL NRP1
DIQMTQSPSSLSASVGDRVTITCRASQSISSYLAWYQQKP (seq. 1)
GKAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYMSVPITFGQGTKVEIKR 62 AB033VH VH EGFR
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQS (seq. 2)
PGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFF
KMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA 63 AB033VL VL EGFR
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRT (seq. 2)
NGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVES
EDIADYYCQQNNNWPTTFGAGTKLELKR 64 AB034VH VH RON
QVQLQESGPGLVKPSEILSLTCTVSGGSISSHYWSWVRQP (seq. 2)
PGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDTSKNQFSL
NLSSVTAADTAVYYCARIPNYYDRSGYYPGYWYFDLWGRG TLVTVSS 65 AB034VL VL RON
QAVLTQPSSLSAPPGASASLTCTLRSGFNVDSYRISWYQQ (seq. 2)
KPGSPPQYLLRYKSDSDKQQGSGVPSRFSGSKDASANAGI
LLISGLQSEDEADYYCMIWHSSAWVFGGGTKLTVLR 66 AB035VH VH NRP1
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQA (seq. 2)
PGKGLEWVSQISPAGGYTNYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCARELPYYRMSKVMDVQGQGTLVTV SS 67 AB035VL VL NRP1
DIQMTQSPSSLSASVGDRVTITCRASQYFSSYLAWYQQKP (seq. 2)
GKAPKLLIYGASSRASGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYLGSPPTFGQGTKVEIKR 68 AB039VH VH CD3
QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMHWVKQR (seq. 2)
PGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAY
MQLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSS 69 AB039VL VL CD3
QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWYQQKSG (seq. 2)
TSPKRWIYDTSKLASGVPAHFRGSGSGTSYSLTISGMEAE
DAATYYCQQWSSNPFTFGSGTKLEINR 70 AB047VH VH PLGF
QVQLQQSGAELVKPGASVKISCKASGYTFTDYYINWVKLA (seq. 1)
PGQGLEWIGWIYPGSGNTKYNEKFKGKATLTIDTSSSTAY
MQLSSLTSEDTAVYFCVRDSPFFDYWGQGTLLTVSS 71 AB047VL VL PLGF
DIVLTQSPDSLAVSLGERVTMNCKSSQSLLNSGMRKSFLA (seq. 1)
WYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLT
ISSVQAEDVAVYYCKQSYHLFTFGSGTKLEIKR 72 AB062VH VH ErbB3
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQP (seq. 1)
PGKGLEWIGEINHSGSTNYNPSLKSRVTISVETSKNQFSL
KLSSVTAADTAVYYCARDKWTWYFDLWGRGTLVTVSS 73 AB062VL VL ErbB3
DIEMTQSPDSLAVSLGERATINCRSSQSVLYSSSNRNYLA (seq. 1)
WYQQNPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLT
ISSLQAEDVAVYYCQQYYSTPRTFGQGTKVEIKR 74 AB063VH VH ErbB3
EVQLVESGGGLVQPGGSLRLSCAASGFTFSIYSMNWVRQA (seq. 2)
PGKGLEWVSYISSSSSTIYYADSVKGRFTISRDNAKNSLY
LQMNSLRDEDTAVYYCARDRGDFDAFDIWGQGTMVTVSS 75 AB063VL VL ErbB3
DIQMTQSPSSLSASVGDRVTITCQASQDITNYLNWYQQKP (seq. 2)
GKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQP
EDIATYNCQQCENFPITFGQGTRLEIKR 76 AB064VH VH EGFR
QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAWNWIRQ (seq. 3)
PPGKGLEWMGYISYSGNTRYQPSLKSRITISRDTSKNQFF
LKLNSVTAADTATYYCVTAGRGFPYWGQGTLVTVSS 77 AB064VL VL EGFR
DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGWLQQKP (seq. 3)
GKSFKGLIYHGTNLDDGVPSRFSGSGSGTDYTLTISSLQP
EDFATYYCVQYAQFPWTFGGGTKLEIKR 78 AB067VH VH ErbB3
EVQLLESGGGLVQPGGSLRLSCAASGFTFSHYVMAWVRQA (seq. 3)
PGKGLEWVSSISSSGGWTLYADSVKGRFTISRDNSKNTLY
LQMNSLRAEDTAVYYCTRGLKMATIFDYWGQGTLVTVSS 79 AB067VL VL ErbB3
QSALTQPASVSGSPGQSITISCTGTSSDVGSYNVVSWYQQ (seq. 3)
HPGKAPKLIIYEVSQRPSGVSNRFSGSKSGNTASLTISGL
QTEDEADYYCCSYAGSSIFVIFGGGTKVTVLG 80 AB069VH VH DLL4
QVQLVQSGAEVKKPGASVKISCKASGYSFTAYYIHWVKQA (seq. 2)
PGQGLEWIGYISSYNGATNYNQKFKGRVTFTTDTSTSTAY
MELRSLRSDDTAVYYCARDYDYDVGMDYWGQGTLVTVSS 81 AB069VL VL DLL4
DIVMTQSPDSLAVSLGERATISCRASESVDNYGISFMKWF (seq. 2)
QQKPGQPPKLLIYAASNQGSGVPDRFSGSGSGTDFTLTIS
SLQAEDVAVYYCQQSKEVPWTFGGGTKVEIKR 82 AB070VH VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGFTISDYWIHWVRQA (seq. 2)
PGKGLEWVAGITPAGGYTYYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCARFVFFLPYAMDYWGQGTLVTVSS 83 AB070VL VL VEGF
DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKP (seq. 2)
GKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQSYTTPPTFGQGTKVEIKR 84 AB071VH VH VEGF
EVQLVESGGGLVQPGGSLRLSCAASGFTINASWIHWVRQA (seq. 3)
PGKGLEWVGAIYPYSGYTNYADSVKGRFTISADTSKNTAY
LQMNSLRAEDTAVYYCARWGHSTSPWAMDYWGQGTLVTVS S 85 AB071VL VL VEGF
DIQMTQSPSSLSASVGDRVTITCRASQVIRRSLAWYQQKP (seq. 3)
GKAPKLLIYAASNLASGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQSNTSPLTFGQGTKVEIKR 86 AB072VH VH DLL4
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVINWVKQK (seq. 3)
PGQGLEWIGLINPYNDGTKYNEKFKVKATLTSDKSSSTAY
MELSSLTSEDSAVYYCASYYYGSRYYFDYWGQGTTLTVSS 87 AB072VL VL DLL4
DIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKP (seq. 3)
GNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQT
EDVATYYCQQYWSIPLTFGAGTKLELKR
88 AB073VH VH DLL4 EVQLQQSGPELEKPGASVKISCKASGYSFTGYNMNWVKQS (seq.
4) NGKSLEWIGNIDPYFGGTNYNQKFKGKATLTVDKSSSTAY
MQLKSLTSEDSAVYYCARNYDYDGGCFDYWGQGTTLTVSS 89 AB073VL VL DLL4
QAVVTQESALTTSPGETVTLTCRSSTGAVTTSNYANWVQE (seq. 4)
KPDHLFTGLIGGTNNRAPGVPARFSGSLIGDKAALTITGA
QTEDEAIYFCALWYSNHWVFGGGTKLTVLG 90 AB074VH VH PLGF
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTTYGIHWVRQS (seq. 2)
PGKGLEWLGVMWSGGDTDYDAAFISRLSISKDNSKSQVFF
KMNSLQANDTGIYYCARYRFYGMDYWGQGTSVTVSS 91 AB074VL VL PLGF
AIQMTQSSSSFSVSLGDRVTITCKASEDIYNRFAWYQQKP (seq. 2)
GNAPRLLISGAASLEAGVPSRFSGSGSGQDYTLSITSLQT
EDVATYYCQQYWSTPWTFGGGTKLEIKR 92 AB075VH VH IGF1R
EVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQA (seq. 2)
PGQGLEWMGGIIPIFGTANYAQKFQGRVTITADKSTSTAY
MELSSLRSEDTAVYYCARAPLRFLEWSTQDHYYYYYMDVW GKGTTVTVSS 93 AB075VL VL
IGF1R SSELTQDPAVSVALGQTVRITCQGDSLRSYYATWYQQKPG (seq. 2)
QAPILVIYGENKRPSGIPDRFSGSSSGNTASLTITGAQAE
DEADYYCKSRDGSGQHLVFGGGTKLTVLG 94 AB077VH VH IGF1R
EVQLVQSGGGLVKPGGSLRLSCAASGFTFSSFAMHWVRQA (seq. 3)
PGKGLEWISVIDTRGATYYADSVKGRFTISRDNAKNSLYL
QMNSLRAEDTAVYYCARLGNFYYGMDVWGQGTTVTVSS 95 AB077VL VL IGF1R
EIVLTQSPGTLSVSPGERATLSCRASQSIGSSLHWYQQKP (seq. 3)
GQAPRLLIKYASQSLSGIPDRFSGSGSGTDFTLTISRLEP
EDFAVYYCHQSSRLPHTFGQGTKVEIKR 96 AB079VH VH HGF
EVQLVQSGAEVKKPGESLKISCKGSGYSFTTYWMHWVRQM (seq. 2)
PGKGLEWMGEINPTNGHTNYNPSFQGQVTISADKSISTAY
LQWSSLKASDTAMYYCARNYVGSIFDYWGQGTLVTVSS 97 AB079VL VL HGF
DIQMTQSPSSLSASVGDRVTITCKASENVVSYVSWYQQKP (seq. 2)
GKAPKLLIYGASNRNTGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCGQSYNYPYTFGQGTKLEIKR 98 AB080VH VH HER2
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQA (seq. 2)
PGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLY
LQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSS 99 AB080VL VL HER2
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKP (seq. 2)
GKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYYTYPYTFGQGTKVEIKR 100 AB110VH VH mCD3
EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQA
PGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLF
LQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS 101 AB110VL VL mCD3
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKP
GKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLES
EDIGSYYCQQYYNYPWTFGPGTKLEIKR 102 AB114VH VH mCD19
EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMHFVKQR
PGQGLEWIGRIDPEDESTKYSEKFKNKATLTADTSSNTAY
LKLSSLTSEDTATYFCIYGGYYFDYWGQGTMVTVSS 103 AB114VL VL mCD19
DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAWYQQKP
GKSPQLLIYGASDLQDGVPSRFSGSGSGTQYSLKITSMQT
EDEGVYFCQQGLTYPRTFGGGTKLELKR 104 AB119VH VH MTX
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAWNWIRQ
FPGNTLEWMGYISYRGSTTHHPSLKSRISITRDTSKNQFF
LQLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 105 AB119VL VL MTX
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEW
YLQKPGQSPKLLIYKVSTRFSGVPDRFSGSGSGTDFTLKI
SRVEAEDLGVYYCFQGSHVPLTFGAGTQLELKR
[0226] Detailed description of specific DVD-Ig molecules that bind
specific targets, and methods of making the same, is provided in
the Examples section below.
C. Production of DVD Proteins
[0227] Binding proteins 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 DVD heavy and DVD light chains is (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 DVD proteins of the
invention in either prokaryotic or eukaryotic host cells, DVD
proteins are expressed in eukaryotic cells, for example, 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 DVD
protein.
[0228] Exemplary 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 Kaufman, R. J.
and Sharp, P. A. (1982) Mol. Biol. 159:601-621), NS0 myeloma cells,
COS cells, SP2 and PER.C6 cells. When recombinant expression
vectors encoding DVD proteins are introduced into mammalian host
cells, the DVD proteins are produced by culturing the host cells
for a period of time sufficient to allow for expression of the DVD
proteins in the host cells or secretion of the DVD proteins into
the culture medium in which the host cells are grown. DVD proteins
can be recovered from the culture medium using standard protein
purification methods.
[0229] In an exemplary system for recombinant expression of DVD
proteins of the invention, a recombinant expression vector encoding
both the DVD heavy chain and the DVD light chain is introduced into
dhfr-CHO cells by calcium phosphate-mediated transfection. Within
the recombinant expression vector, the DVD 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 DVD heavy and light chains and intact DVD protein
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 DVD protein from the culture medium.
Still further the invention provides a method of synthesizing a DVD
protein of the invention by culturing a host cell of the invention
in a suitable culture medium until a DVD protein of the invention
is synthesized. The method can further comprise isolating the DVD
protein from the culture medium.
[0230] An important feature of DVD-Ig is that it can be produced
and purified in a similar way as a conventional antibody. The
production of DVD-Ig results in a homogeneous, single major product
with desired dual-specific activity, without any sequence
modification of the constant region or chemical modifications of
any kind. Other previously described methods to generate
"bi-specific", "multi-specific", and "multi-specific multivalent"
full length binding proteins do not lead to a single primary
product but instead lead to the intracellular or secreted
production of a mixture of assembled inactive, mono-specific,
multi-specific, multivalent, full length binding proteins, and
multivalent full length binding proteins with combination of
different binding sites. As an example, based on the design
described by Miller and Presta (PCT Publication No.
WO2001/077342(A1), there are 16 possible combinations of heavy and
light chains. Consequently only 6.25% of protein is likely to be in
the desired active form, and not as a single major product or
single primary product compared to the other 15 possible
combinations. Separation of the desired, fully active forms of the
protein from inactive and partially active forms of the protein
using standard chromatography techniques, typically used in large
scale manufacturing, is yet to be demonstrated.
[0231] Surprisingly, the design of the "dual-specific multivalent
full length binding proteins" of the present invention leads to a
dual variable domain light chain and a dual variable domain heavy
chain which assemble primarily to the desired "dual-specific
multivalent full length binding proteins".
[0232] At least 50%, at least 75% and at least 90% of the
assembled, and expressed dual variable domain immunoglobulin
molecules are the desired dual-specific tetravalent protein. This
aspect of the invention particularly enhances the commercial
utility of the invention. Therefore, the present invention includes
a method to express a dual variable domain light chain and a dual
variable domain heavy chain in a single cell leading to a single
primary product of a "dual-specific tetravalent full length binding
protein".
[0233] The present invention provides methods of expressing a dual
variable domain light chain and a dual variable domain heavy chain
in a single cell leading to a "primary product" of a "dual-specific
tetravalent full length binding protein," where the "primary
product" is more than 50% of all assembled protein, comprising a
dual variable domain light chain and a dual variable domain heavy
chain.
[0234] The present invention provides methods of expressing a dual
variable domain light chain and a dual variable domain heavy chain
in a single cell leading to a single "primary product" of a
"dual-specific tetravalent full length binding protein," where the
"primary product" is more than 75% of all assembled protein,
comprising a dual variable domain light chain and a dual variable
domain heavy chain.
[0235] The present invention provides methods of expressing a dual
variable domain light chain and a dual variable domain heavy chain
in a single cell leading to a single "primary product" of a
"dual-specific tetravalent full length binding protein," where the
"primary product" is more than 90% of all assembled protein,
comprising a dual variable domain light chain and a dual variable
domain heavy chain.
II. Derivatized DVD Binding Proteins:
[0236] One embodiment provides a labeled binding protein wherein
the binding protein 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 a binding protein of the invention (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 binding protein with
another molecule (such as a streptavidin core region or a
polyhistidine tag).
[0237] Useful detectable agents with which a binding protein 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. A binding protein may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When a binding protein 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. A binding protein may also be derivatized with biotin,
and detected through indirect measurement of avidin or streptavidin
binding.
[0238] Another embodiment of the invention provides a crystallized
binding protein 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.
[0239] Crystallized binding protein of the invention may be
produced according to methods known in the art and as disclosed in
PCT Publication No. WO 02/072636.
[0240] 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 an important
effect on the effector function of the Fc domain, with minimal
effect on antigen binding or half-life of the antibody (R.
Jefferis, R. (2005) Biotechnol. Prog. 21: 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. (1993)
Mol. Immunol. 30:1361-1367), or result in increased affinity for
the antigen (Wallick, S. C., et al. (1988) Exp. Med. 168:1099-1109;
Wright, A., et al. (1991) EMBO J. 10:2717-2723).
[0241] 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 another object of the present invention.
[0242] 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 aglycosylation may increase the affinity of the antibody
for antigen. Such an approach is described in further detail in PCT
Publication WO 2003/016466A2, and U.S. Pat. Nos. 5,714,350 and
6,350,861.
[0243] Additionally or alternatively, a modified binding protein of
the invention can be made that has an altered type of
glycosylation, such as a hypofucosylated antibody having reduced
amounts of fucosyl residues (see Kanda, Y. et al. (2007) J.
Biotech. 130(3): 300-310.) 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 and PCT Publication Nos. WO 03/035835 and WO
99/54342 80.
[0244] 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. In an embodiment, the glycosylated binding protein
comprises glycosyl residues such that the glycosylation pattern is
human.
[0245] 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 choose 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.
[0246] 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 (glycoproteins) produced in these
yeast strains exhibit protein glycosylation identical to that of
animal cells, especially human cells (U.S. Pat. Nos. 7,449,308 and
7,029,872 and PCT Publication No/WO2005/100584).
[0247] In addition to the binding proteins, the present invention
is also directed to anti-idiotypic (anti-Id) antibodies specific
for such binding proteins of the invention. An anti-Id antibody is
an antibody, which 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. It is readily
apparent that it may be easier to generate anti-idiotypic
antibodies to the two or more parent antibodies incorporated into a
DVD-Ig molecule; and confirm binding studies by methods well
recognized in the art (e.g., BIAcore, ELISA) to verify that
anti-idiotypic antibodies specific for the idiotype of each parent
antibody also recognize the idiotype (e.g., antigen binding site)
in the context of the DVD-Ig. The anti-idiotypic antibodies
specific for each of the two or more antigen binding sites of a
DVD-Ig provide ideal reagents to measure DVD-Ig concentrations of a
human DVD-Ig in patient serum; DVD-Ig concentration assays can be
established using a "sandwich assay ELISA format" with an antibody
to a first antigen binding region coated on the solid phase (e.g.,
BIAcore chip, ELISA plate etc.), rinsing with rinsing buffer,
incubating with the serum sample, rinsing again and ultimately
incubating with another anti-idiotypic antibody to the another
antigen binding site, itself labeled with an enzyme for
quantitation of the binding reaction. In an embodiment, for a
DVD-Ig with more than two different binding sites, anti-idiotypic
antibodies to the two outermost binding sites (most distal and
proximal from the constant region) will not only help in
determining the DVD-Ig concentration in human serum but also
document the integrity of the molecule in vivo. Each 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.
[0248] 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. In an embodiment, the
protein having a particularly selected novel glycosylation pattern
exhibits improved or altered biological properties.
III. Uses of DVD-Ig
[0249] Given their ability to bind to two or more antigens the
binding proteins of the invention can be used to detect the
antigens (e.g., in a biological sample, such as serum or plasma),
using a conventional immunoassay, such as an enzyme linked
immunosorbent assays (ELISA), a radioimmunoassay (RIA) or tissue
immunohistochemistry. The DVD-Ig 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,
b-galactosidase, and 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 and
phycoerythrin; an example of a luminescent material includes
luminol; and examples of suitable radioactive material include 3H,
14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, and
153Sm.
[0250] In an embodiment, the binding proteins of the invention
neutralize the activity of the antigens both in vitro and in vivo.
Accordingly, such DVD-Igs can be used to inhibit antigen activity,
e.g., in a cell culture containing the antigens, in human subjects
or in other mammalian subjects having the antigens with which a
binding protein of the invention cross-reacts. In another
embodiment, the invention provides a method for reducing antigen
activity in a subject suffering from a disease or disorder in which
the antigen activity is detrimental. A binding protein of the
invention can be administered to a human subject for therapeutic
purposes.
[0251] The term "a disorder in which antigen activity is
detrimental" is intended to include diseases and other disorders in
which the presence of the antigen 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 antigen activity is detrimental is a disorder in
which reduction of antigen 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 the
antigen in a biological fluid of a subject suffering from the
disorder (e.g., an increase in the concentration of antigen in
serum, plasma, synovial fluid, etc. of the subject). Non-limiting
examples of disorders that can be treated with the binding proteins
of the invention include those disorders discussed below and in the
section pertaining to pharmaceutical compositions of the antibodies
of the invention.
[0252] The DVD-Igs of the invention may bind one antigen or
multiple antigens. Such antigens include, but are not limited to,
the targets listed in the following databases. These target
databases include those listings:
Therapeutic targets (National University of Singapore Therapeutic
Targets Database, Bioinformatics and Drug Design Group); Cytokines
and cytokine receptors (Cytokines & Cells Online Pathfinder
Encyclopedia);
Chemokines (International Union of Basic and Clinical Pharmacology
Database);
[0253] Chemokine receptors and GPCRs (GPCR Data Base);
Olfactory Receptors (Yale University SenseLab Project);
Receptors (International Union of Basic and Clinical Pharmacology
Database);
[0254] Cancer targets (University of Tokyo Human Genome Center,
Cancer Gene Expression DataBase); Secreted proteins as potential
antibody targets (Peking University Secreted Protein Database);
Protein kinases (Peking University Secreted Protein Database), and
Human CD markers (Zola H, (2005) Blood 106: 3123-6).
[0255] DVD-Igs are useful as therapeutic agents to simultaneously
block two different targets to enhance efficacy/safety and/or
increase patient coverage. Such targets may include soluble targets
(e.g., TNF) and cell surface receptor targets (e.g., VEGFR and
EGFR). It can also be used to induce redirected cytotoxicity
between tumor cells and T cells (e.g., Her2 and CD3) for cancer
therapy, or between autoreactive cell and effector cells for
autoimmune disease or transplantation, or between any target cell
and effector cell to eliminate disease-causing cells in any given
disease.
[0256] In addition, DVD-Ig can be used to trigger receptor
clustering and activation when it is designed to target two
different epitopes on the same receptor. This may have benefit in
making agonistic and antagonistic anti-GPCR therapeutics. In this
case, DVD-Ig can be used to target two different epitopes
(including epitopes on both the loop regions and the extracellular
domain) on one cell for clustering/signaling (two cell surface
molecules) or signaling (on one molecule). Similarly, a DVD-Ig
molecule can be designed to triger CTLA-4 ligation, and a negative
signal by targeting two different epitopes (or 2 copies of the same
epitope) of CTLA-4 extracellular domain, leading to down regulation
of the immune response. CTLA-4 is a clinically validated target for
therapeutic treatment of a number of immunological disorders.
CTLA-4/B7 interactions negatively regulate T cell activation by
attenuating cell cycle progression, IL-2 production, and
proliferation of T cells following activation, and CTLA-4 (CD152)
engagement can down-regulate T cell activation and promote the
induction of immune tolerance. However, the strategy of attenuating
T cell activation by agonistic antibody engagement of CTLA-4 has
been unsuccessful since CTLA-4 activation requires ligation. The
molecular interaction of CTLA-4/B7 is in "skewed zipper" arrays, as
demonstrated by crystal structural analysis (Stamper (2001) Nature
410: 608). However none of the currently available CTLA-4 binding
reagents have ligation properties, including anti-CTLA-4 mAbs.
There have been several attempts to address this issue. In one
case, a cell member-bound single chain antibody was generated, and
significantly inhibited allogeneic rejection in mice (Hwang (2002)
J. Immunol. 169:633). In a separate case, artificial APC
surface-linked single-chain antibody to CTLA-4 was generated and
demonstrated to attenuate T cell responses (Griffin (2000) J.
Immunol. 164:4433). In both cases, CTLA-4 ligation was achieved by
closely localized member-bound antibodies in artificial systems.
While these experiments provide proof-of-concept for immune
down-regulation by triggering CTLA-4 negative signaling, the
reagents used in these reports are not suitable for therapeutic
use. To this end, CTLA-4 ligation may be achieved by using a DVD-Ig
molecule, which target two different epitopes (or 2 copies of the
same epitope) of CTLA-4 extracellular domain. The rationale is that
the distance spanning two binding sites of an IgG, approximately
150-170 .ANG., is too large for active ligation of CTLA-4 (30-50
.ANG. between 2 CTLA-4 homodimer). However the distance between the
two binding sites on DVD-Ig (one arm) is much shorter, also in the
range of 30-50 .ANG., allowing proper ligation of CTLA-4.
[0257] Similarly, DVD-Ig can target two different members of a cell
surface receptor complex (e.g., IL-12R alpha and beta).
Furthermore, DVD-Ig can target CR1 and a soluble protein/pathogen
to drive rapid clearance of the target soluble
protein/pathogen.
[0258] Additionally, DVD-Igs of the invention can be employed for
tissue-specific delivery (target a tissue marker and a disease
mediator for enhanced local PK thus higher efficacy and/or lower
toxicity), including intracellular delivery (targeting an
internalizing receptor and a intracellular molecule), and delivery
to the inside of the brain (targeting transferrin receptor and a
CNS disease mediator for crossing the blood-brain barrier). DVD-Ig
can also serve as a carrier protein to deliver an antigen to a
specific location via binding to a non-neutralizing epitope of that
antigen and also to increase the half-life of the antigen.
Furthermore, DVD-Ig can be designed to either be physically linked
to medical devices implanted into patients or target these medical
devices (see Burke, S. E. et al. (2006) Adv. Drug Deliv. Rev.
58(3): 437-446; Hildebrand, H. F. et al. (2006) Surface and
Coatings Technol. 200(22-23): 6318-6324; Wu, P. et al. (2006)
Biomaterials 27(11): 2450-2467; Marques, A. P. et al. (2005)
Biodegrad. Syst. Tissue Eng. and Regen. Med. 377-397). Briefly,
directing appropriate types of cell to the site of medical implant
may promote healing and restoring normal tissue function.
Alternatively, inhibition of mediators (including but not limited
to cytokines), released upon device implantation by a DVD coupled
to or target to a device is also provided. For example, stents have
been used for years in interventional cardiology to clear blocked
arteries and to improve the flow of blood to the heart muscle.
However, traditional bare metal stents have been known to cause
restenosis (re-narrowing of the artery in a treated area) in some
patients and can lead to blood clots. Recently, an anti-CD34
antibody coated stent has been described which reduced restenosis
and prevents blood clots from occurring by capturing endothelial
progenitor cells (EPC) circulating throughout the blood.
Endothelial cells are cells that line blood vessels, allowing blood
to flow smoothly. The EPCs adhere to the hard surface of the stent
forming a smooth layer that not only promotes healing but prevents
restenosis and blood clots, complications previously associated
with the use of stents (Aoji et al. (2005) J. Am. Coll. Cardiol.
45(10):1574-9). In addition to improving outcomes for patients
requiring stents, there are also implications for patients
requiring cardiovascular bypass surgery. For example, a prosthetic
vascular conduit (artificial artery) coated with anti-EPC
antibodies would eliminate the need to use arteries from patients
legs or arms for bypass surgery grafts. This would reduce surgery
and anesthesia times, which in turn will reduce coronary surgery
deaths. DVD-Ig are designed in such a way that it binds to a cell
surface marker (such as CD34) as well as a protein (or an epitope
of any kind, including but not limited to proteins, lipids and
polysaccharides) that has been coated on the implanted device to
facilitate the cell recruitment. Such approaches can also be
applied to other medical implants in general. Alternatively,
DVD-Igs can be coated on medical devices and upon implantation and
releasing all DVDs from the device (or any other need which may
require additional fresh DVD-Ig, including aging and denaturation
of the already loaded DVD-Ig) the device could be reloaded by
systemic administration of fresh DVD-Ig to the patient, where the
DVD-Ig is designed to binds to a target of interest (a cytokine, a
cell surface marker (such as CD34) etc.) with one set of binding
sites and to a target coated on the device (including a protein, an
epitope of any kind, including but not limited to lipids,
polysaccharides and polymers) with the other. This technology has
the advantage of extending the usefulness of coated implants.
A. Use of DVD-Igs in Various Diseases
[0259] DVD-Ig molecules of the invention are also useful as
therapeutic molecules to treat various diseases. Such DVD molecules
may bind one or more targets involved in a specific disease.
Examples of such targets in various diseases are described
below.
1. Human Autoimmune and Inflammatory Response
[0260] Many proteins have been implicated in general autoimmune and
inflammatory responses, including C5, CCL1 (1-309), CCL11
(eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17
(TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21
(MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),
CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3),
CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (1-TAC/IP-9), CXCL12
(SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6
(GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4,
CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2,
IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8,
IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial
Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA,
IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,
CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,
IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3,
TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28,
CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A,
FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5,
TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5,
CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,
CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5,
CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5,
CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2,
XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10
(IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1,
IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB,
IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8,
IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL11RA, IL12A,
IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA,
IL16, IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB,
LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,
TGFB1I1, TGFB2, TGFB3, TGFBI, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,
TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,
TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).
In one aspect, DVD-Igs that bind one or more of the targets listed
herein are provided.
[0261] DVD Igs capable of binding the following pairs of targets to
treat inflammatory disease are contemplated: MTX and mCD-3; MTX and
mCD-19; MTX and hCD-3; MTX and hCD-19; MTX and CD-20; MTX and EGFR;
and MTX and HER-2.
2. Asthma
[0262] Allergic asthma is characterized by the presence of
eosinophilia, goblet cell metaplasia, epithelial cell alterations,
airway hyperreactivity (AHR), and Th2 and Th1 cytokine expression,
as well as elevated serum IgE levels. It is now widely accepted
that airway inflammation is the key factor underlying the
pathogenesis of asthma, involving a complex interplay of
inflammatory cells such as T cells, B cells, eosinophils, mast
cells and macrophages, and of their secreted mediators including
cytokines and chemokines. Corticosteroids are the most important
anti-inflammatory treatment for asthma today, however their
mechanism of action is non-specific and safety concerns exist,
especially in the juvenile patient population. The development of
more specific and targeted therapies is therefore warranted. There
is increasing evidence that IL-13 in mice mimics many of the
features of asthma, including AHR, mucus hypersecretion and airway
fibrosis, independently of eosinophilic inflammation (Finotto et
al. (2005) Int. Immunol 17(8): 993-1007; Padilla et al. (2005) J.
Immunol. 174(12): 8097-8105).
[0263] IL-13 has been implicated as having a pivotal role in
causing pathological responses associated with asthma. The
development of anti-IL-13 mAb therapy to reduce the effects of
IL-13 in the lung is an exciting new approach that offers
considerable promise as a novel treatment for asthma. However other
mediators of differential immunological pathways are also involved
in asthma pathogenesis, and blocking these mediators, in addition
to IL-13, may offer additional therapeutic benefit. Such target
pairs include, but are not limited to, IL-13 and a pro-inflammatory
cytokine, such as tumor necrosis factor-.alpha. (TNF-.alpha.).
TNF-.alpha. may amplify the inflammatory response in asthma and may
be linked to disease severity (McDonnell, et al. (2001) Progr.
Respir. Res. 31: 247-250). This suggests that blocking both IL-13
and TNF-.alpha. may have beneficial effects, particularly in severe
airway disease. In another embodiment the DVD-Ig of the invention
binds the targets IL-13 and TNF.alpha. and is used for treating
asthma.
[0264] Animal models such as OVA-induced asthma mouse model, where
both inflammation and AHR can be assessed, are known in the art and
may be used to determine the ability of various DVD-Ig molecules to
treat asthma. Animal models for studying asthma are disclosed in
Coffman, et al. (2005) J. Exp. Med. 201(12): 1875-1879; Lloyd et
al. (2001) Adv. Immunol 77: 263-295; Boyce et al. (2005) J. Exp.
Med. 201(12): 1869-1873; and Snibson et al. (2005) J. Brit. Soc.
Allerg. Clin. Immunol 35(2): 146-52. In addition to routine safety
assessments of these target pairs, specific tests for the degree of
immunosuppression may be warranted and helpful in selecting the
best target pairs (see Luster et al. (1994) Toxicology 92(1-3):
229-43; Descotes, et al. (1992) Devel. Biol. Stand. 77: 99-102;
Hart et al. (2001) J. Allerg. Clin. Immunol 108(2): 250-257).
[0265] Based on the rationale disclosed herein and using the same
evaluation model for efficacy and safety other pairs of targets
that DVD-Ig molecules can bind and be useful to treat asthma may be
determined. In an embodiment, such targets include, but are not
limited to, IL-13 and IL-1beta, since IL-1beta is also implicated
in inflammatory response in asthma; IL-13 and cytokines and
chemokines that are involved in inflammation, such as IL-13 and
IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13 and
TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-13; IL-13 and LHR
agonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and
IL-13 and ADAM8. The present invention also provides DVD-Igs that
bind one or more targets involved in asthma selected from the group
consisting of mCD-3, mCD-19, hCD-3, hCD-19, CD-20, EGFR, and
HER-2.
3. Rheumatoid Arthritis
[0266] Rheumatoid arthritis (RA), a systemic disease, is
characterized by a chronic inflammatory reaction in the synovium of
joints and is associated with degeneration of cartilage and erosion
of juxta-articular bone. Many pro-inflammatory cytokines including
TNF, chemokines, and growth factors are expressed in diseased
joints. Systemic administration of anti-TNF antibody or sTNFR
fusion protein to mouse models of RA was shown to be
anti-inflammatory and joint protective. Clinical investigations in
which the activity of TNF in RA patients was blocked with
intravenously administered infliximab (Harriman, G. et al. (1999)
Ann. Rheum. Dis. 58 (Suppl 1): 161-4), a chimeric anti-TNF mAb, has
provided evidence that TNF regulates IL-6, IL-8, MCP-1, and VEGF
production, recruitment of immune and inflammatory cells into
joints, angiogenesis, and reduction of blood levels of matrix
metalloproteinases-1 and -3. A better understanding of the
inflammatory pathway in rheumatoid arthritis has led to
identification of other therapeutic targets involved in rheumatoid
arthritis. Promising treatments such as interleukin-6 antagonists
(IL-6 receptor antibody MRA, developed by Chugai, Roche (see
Nishimoto, N. et al. (2004) Arthrit. Rheum. 50(6): 1761-1769),
CTLA4Ig (abatacept, Genovese, M. et al. (2005) N. Engl. J. Med.
353: 1114-23.), and anti-B cell therapy (rituximab; Okamoto, H. and
Kamatani, N. (2004) N. Engl. J. Med. 351: 1909), have already been
tested in randomized controlled trials over the past year. Other
cytokines have been identified and have been shown to be of benefit
in animal models, including interleukin-15 (therapeutic antibody
HuMax-IL.sub.--15, AMG 714 (see Baslund, B. et al. (2005) Arthrit.
Rheum. 52(9): 2686-2692)), interleukin-17, and interleukin-18, and
clinical trials of these agents are currently under way.
Dual-specific antibody therapy, combining anti-TNF and another
mediator, has great potential in enhancing clinical efficacy and/or
patient coverage. For example, blocking both TNF and VEGF can
potentially eradicate inflammation and angiogenesis, both of which
are involved in pathophysiology of RA. Blocking other pairs of
targets involved in RA including, but not limited to: IL-1b and
IL-17. In addition to routine safety assessments of these target
pairs, specific tests for the degree of immunosuppression may be
warranted and helpful in selecting the best target pairs (see
Luster et al. (1994) Toxicol. 92(1-3): 229-43; Descotes et al.
(1992) Devel. Biol. Stand. 77: 99-102; Hart et al. (2001) J.
Allerg. Clin. Immunol 108(2): 250-257). Whether a DVD Ig molecule
will be useful for the treatment of rheumatoid arthritis can be
assessed using pre-clinical animal RA models such as the
collagen-induced arthritis mouse model. Other useful models are
also well known in the art (see Brand, D. D. (2005) Comp. Med.
55(2): 114-22). Based on the cross-reactivity of the parental
antibodies for human and mouse othologues (e.g., reactivity for
human and mouse TNF, human and mouse IL-15, etc.) validation
studies in the mouse CIA model may be conducted with "matched
surrogate antibody" derived DVD-Ig molecules; briefly, a DVD-Ig
based on two (or more) mouse target specific antibodies may be
matched to the extent possible to the characteristics of the
parental human or humanized antibodies used for human DVD-Ig
construction (similar affinity, similar neutralization potency,
similar half-life etc.).
4. SLE
[0267] The immunopathogenic hallmark of SLE is the polyclonal B
cell activation, which leads to hyperglobulinemia, autoantibody
production and immune complex formation. The fundamental
abnormality appears to be the failure of T cells to suppress the
forbidden B cell clones due to generalized T cell dysregulation. In
addition, B and T-cell interaction is facilitated by several
cytokines such as IL-10 as well as co-stimulatory molecules such as
CD40 and CD40L, B7 and CD28 and CTLA-4, which initiate the second
signal. These interactions together with impaired phagocytic
clearance of immune complexes and apoptotic material, perpetuate
the immune response with resultant tissue injury. The following
targets may be involved in SLE and can potentially be used for a
DVD-Ig approach for therapeutic intervention: B cell targeted
therapies: CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10,
IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5,
HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGS1, SLA2, CD81, IFNB1, IL10,
TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5,
HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28,
CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7,
CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, IL1R2, ITGA2,
ITGA3, MS4A1, ST6GAL1, CD1C, CHST10, HLA-A, HLA-DRA, and NT5E.;
co-stimulatory signals: CTLA4 or B7.1/B7.2; inhibition of B cell
survival: BlyS, or BAFF; Complement inactivation: C5; Cytokine
modulation: the key principle is that the net biologic response in
any tissue is the result of a balance between local levels of
proinflammatory or anti-inflammatory cytokines (see Sfikakis, P. P.
et al. (2005) Curr. Opin. Rheumatol. 17:550-7). SLE is considered
to be a Th-2 driven disease with documented elevations in serum
IL-4, IL-6, IL-10. DVD-Igs that bind one or more targets selected
from the group consisting of IL-4, IL-6, IL-10, IFN-.alpha.,
TNF-.alpha., IL-1b and IL-17 are also contemplated. Combination of
targets discussed herein will enhance therapeutic efficacy for SLE
which can be tested in a number of lupus preclinical models (see
Peng, S. L. (2004) Methods Mol. Med.; 102:227-72). Based on the
cross-reactivity of the parental antibodies for human and mouse
othologues (e.g., reactivity for human and mouse CD20, human and
mouse Interferon alpha etc.) validation studies in a mouse lupus
model may be conducted with "matched surrogate antibody" derived
DVD-Ig molecules. Briefly, a DVD-Ig based two (or more) mouse
target specific antibodies may be matched to the extent possible to
the characteristics of the parental human or humanized antibodies
used for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.).
5. Multiple Sclerosis
[0268] Multiple sclerosis (MS) is a complex human autoimmune-type
disease with a predominantly unknown etiology. Immunologic
destruction of myelin basic protein (MBP) throughout the nervous
system is the major pathology of multiple sclerosis. MS is a
disease of complex pathologies, which involves infiltration by CD4+
and CD8+ T cells and of response within the central nervous system.
Expression in the CNS of cytokines, reactive nitrogen species and
costimulator molecules have all been described in MS. Of major
consideration are immunological mechanisms that contribute to the
development of autoimmunity. In particular, antigen expression,
cytokine and leukocyte interactions, and regulatory T-cells, which
help balance/modulate other T-cells such as Th1 and Th2 cells, are
important areas for therapeutic target identification.
[0269] IL-12 is a proinflammatory cytokine that is produced by APC
and promotes differentiation of Th1 effector cells. IL-12 is
produced in the developing lesions of patients with MS as well as
in EAE-affected animals. Previously it was shown that interference
in IL-12 pathways effectively prevents EAE in rodents, and that in
vivo neutralization of IL-12p40 using a anti-IL-12 mAb has
beneficial effects in the myelin-induced EAE model in common
marmosets.
[0270] TWEAK is a member of the TNF family, constitutively
expressed in the central nervous system (CNS), with
pro-inflammatory, proliferative or apoptotic effects depending upon
cell types. Its receptor, Fn14, is expressed in CNS by endothelial
cells, reactive astrocytes and neurons. TWEAK and Fn14 mRNA
expression increased in spinal cord during experimental autoimmune
encephalomyelitis (EAE). Anti-TWEAK antibody treatment in myelin
oligodendrocyte glycoprotein (MOG) induced EAE in C57BL/6 mice
resulted in a reduction of disease severity and leukocyte
infiltration when mice were treated after the priming phase.
[0271] One aspect of the invention pertains to DVD Ig molecules
that bind one or more, for example two, targets selected from the
group consisting of mCD-3, mCD-19, hCD-3, hCD-19, CD-20, EGFR, and
HER-2. An embodiment includes a dual-specific anti-IL-12/TWEAK DVD
Ig as a therapeutic agent beneficial for the treatment of MS.
[0272] Several animal models for assessing the usefulness of the
DVD molecules to treat MS are known in the art (see Steinman. L. et
al. (2005) Trends Immunol 26(11): 565-71; Lublin, F. D. et al.
(1985) Springer Semin. Immunopathol. 8(3): 197-208; Genain, C. P.
et al. (1997) J. Mol. Med. 75(3): 187-97; Tuohy, V. K. et al.
(1999) J. Exp. Med. 189(7): 1033-42; Owens, T. et al. (1995)
Neurol. Clin. 13(1): 51-73; and Hart, B. A. et al. (2005) J.
Immunol. 175(7): 4761-8. Based on the cross-reactivity of the
parental antibodies for human and animal species othologues (e.g.,
reactivity for human and mouse IL-12, human and mouse TWEAK etc.),
validation studies in the mouse EAE model may be conducted with
"matched surrogate antibody" derived DVD-Ig molecules. Briefly, a
DVD-Ig based on two (or more) mouse target specific antibodies may
be matched to the extent possible to the characteristics of the
parental human or humanized antibodies used for human DVD-Ig
construction (similar affinity, similar neutralization potency,
similar half-life etc.). The same concept applies to animal models
in other non-rodent species, where a "matched surrogate antibody"
derived DVD-Ig would be selected for the anticipated pharmacology
and possibly safety studies. In addition to routine safety
assessments of these target pairs specific tests for the degree of
immunosuppression may be warranted and helpful in selecting the
best target pairs (see Luster et al. (1994) Toxicol. 92(1-3):
229-43; Descotes et al. (1992) Devel. Biol. Stand. 77: 99-102;
Jones, R. (2000) IDrugs 3(4): 442-6).
6. Sepsis
[0273] The pathophysiology of sepsis is initiated by the outer
membrane components of both gram-negative organisms
(lipopolysaccharide [LPS], lipid A, endotoxin) and gram-positive
organisms (lipoteichoic acid, peptidoglycan). These outer membrane
components are able to bind to the CD14 receptor on the surface of
monocytes. By virtue of the recently described toll-like receptors,
a signal is then transmitted to the cell, leading to the eventual
production of the proinflammatory cytokines tumor necrosis
factor-alpha (TNF-alpha) and interleukin-1 (IL-1). Overwhelming
inflammatory and immune responses are essential features of septic
shock and play a central part in the pathogenesis of tissue damage,
multiple organ failure, and death induced by sepsis. Cytokines,
especially tumor necrosis factor (TNF) and interleukin (IL-1), have
been shown to be critical mediators of septic shock. These
cytokines have a direct toxic effect on tissues; they also activate
phospholipase A2. These and other effects lead to increased
concentrations of platelet-activating factor, promotion of nitric
oxide synthase activity, promotion of tissue infiltration by
neutrophils, and promotion of neutrophil activity.
[0274] The treatment of sepsis and septic shock remains a clinical
conundrum, and recent prospective trials with biological response
modifiers (i.e., anti-TNF and anti-MIF) aimed at the inflammatory
response have shown only modest clinical benefit. Recently,
interest has shifted toward therapies aimed at reversing the
accompanying periods of immune suppression. Studies in experimental
animals and critically ill patients have demonstrated that
increased apoptosis of lymphoid organs and some parenchymal tissues
contribute to this immune suppression, anergy, and organ system
dysfunction. During sepsis syndromes, lymphocyte apoptosis can be
triggered by the absence of IL-2 or by the release of
glucocorticoids, granzymes, or the so-called `death` cytokines:
tumor necrosis factor alpha or Fas ligand. Apoptosis proceeds via
auto-activation of cytosolic and/or mitochondrial caspases, which
can be influenced by the pro- and anti-apoptotic members of the
Bcl-2 family. In experimental animals, not only can treatment with
inhibitors of apoptosis prevent lymphoid cell apoptosis; it may
also improve outcome. Although clinical trials with anti-apoptotic
agents remain distant due in large part to technical difficulties
associated with their administration and tissue targeting,
inhibition of lymphocyte apoptosis represents an attractive
therapeutic target for the septic patient. Likewise, a
dual-specific agent targeting both inflammatory mediator and an
apoptotic mediator, may have added benefit. One aspect of the
invention pertains to DVD Igs that bind one or more targets
involved in sepsis, in an embodiment two targets, selected from the
group consisting of TNF, IL-1, MIF, IL-6, IL-8, IL-18, IL-12,
IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissue factor, MIP-2,
ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFKB1, PROC, TNFRSF1A, CSF3,
CCR3, IL1RN, MIF, NFKB1, PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine,
IRAK1, NFKB2, SERPINA1, SERPINE1, and TREM1. The efficacy of such
DVD Igs for sepsis can be assessed in preclinical animal models
known in the art (see Buras, J. A., et al. (2005) Nat. Rev. Drug
Discov. 4(10):854-65 and Calandra T, et al. (2000) Nat Med.
6(2):164-70). In an embodiment, the DVD-Ig of the invention
directed to MTX and mCD-3; MTX and mCD-19; MTX and hCD-3; MTX and
hCD-19; MTX and CD-20; MTX and EGFR; and MTX and HER-2 is used to
treat sepsis.
7. Neurological Disorders
7.1. Neurodegenerative Diseases
[0275] Neurodegenerative diseases are either chronic in which case
they are usually age-dependent or acute (e.g., stroke, traumatic
brain injury, spinal cord injury, etc.). They are characterized by
progressive loss of neuronal functions (neuronal cell death,
demyelination), loss of mobility and loss of memory. Emerging
knowledge of the mechanisms underlying chronic neurodegenerative
diseases (e.g., Alzheimer's disease) show a complex etiology and a
variety of factors have been recognized to contribute to their
development and progression e.g., age, glycemic status, amyloid
production and multimerization, accumulation of advanced
glycation-end products (AGE) which bind to their receptor RAGE
(receptor for AGE), increased brain oxidative stress, decreased
cerebral blood flow, neuroinflammation including release of
inflammatory cytokines and chemokines, neuronal dysfunction and
microglial activation. Thus these chronic neurodegenerative
diseases represent a complex interaction between multiple cell
types and mediators. Treatment strategies for such diseases are
limited and mostly constitute either blocking inflammatory
processes with non-specific anti-inflammatory agents (e.g.,
corticosteroids, COX inhibitors) or agents to prevent neuron loss
and/or synaptic functions. These treatments fail to stop disease
progression. Recent studies suggest that more targeted therapies
such as antibodies to soluble A-b peptide (including the A-b
oligomeric forms) can not only help stop disease progression but
may help maintain memory as well. These preliminary observations
suggest that specific therapies targeting more than one disease
mediator (e.g., A-b and a pro-inflammatory cytokine such as TNF)
may provide even better therapeutic efficacy for chronic
neurodegenerative diseases than observed with targeting a single
disease mechanism (e.g., soluble A-a alone) Several animal models
for assessing the usefulness of the DVD molecules to treat MS are
known in the art (see Steinman. L. et al. (2005) Trends Immunol
26(11): 565-71; Lublin, F. D. et al. (1985) Springer Semin.
Immunopathol. 8(3): 197-208; Genain, C. P. et al. (1997) J. Mol.
Med. 75(3): 187-97; Tuohy, V. K. et al. (1999) J. Exp. Med. 189(7):
1033-42; Owens, T. et al. (1995) Neurol. Clin. 13(1): 51-73; and
Hart, B. A. et al. (2005) J. Immunol. 175(7): 4761-8. Based on the
cross-reactivity of the parental antibodies for human and animal
species othologues (e.g., reactivity for human and mouse IL-12,
human and mouse TWEAK etc.), validation studies in the mouse EAE
model may be conducted with "matched surrogate antibody" derived
DVD-Ig molecules. Briefly, a DVD-Ig based on two (or more) mouse
target specific antibodies may be matched to the extent possible to
the characteristics of the parental human or humanized antibodies
used for human DVD-Ig construction (similar affinity, similar
neutralization potency, similar half-life etc.). The same concept
applies to animal models in other non-rodent species, where a
"matched surrogate antibody" derived DVD-Ig would be selected for
the anticipated pharmacology and possibly safety studies. In
addition to routine safety assessments of these target pairs
specific tests for the degree of immunosuppression may be warranted
and helpful in selecting the best target pairs (see Luster et al.
(1994) Toxicol. 92(1-3): 229-43; Descotes et al. (1992) Devel.
Biol. Stand. 77: 99-102; Jones, R. (2000) IDrugs 3(4): 442-6).
[0276] The DVD-Ig molecules of the invention can bind one or more
targets involved in chronic neurodegenerative diseases such as
Alzheimers. Such targets include, but are not limited to, any
mediator, soluble or cell surface, implicated in AD pathogenesis
e.g AGE (S100 A, amphoterin), pro-inflammatory cytokines (e.g.,
IL-1), chemokines (e.g., MCP 1), molecules that inhibit nerve
regeneration (e.g., Nogo, RGM A), molecules that enhance neurite
growth (neurotrophins) and molecules that can mediate transport at
the blood brain barrier (e.g., transferrin receptor, insulin
receptor or RAGE. The efficacy of DVD-Ig molecules can be validated
in pre-clinical animal models such as the transgenic mice that
over-express amyloid precursor protein or RAGE and develop
Alzheimer's disease-like symptoms. In addition, DVD-Ig molecules
can be constructed and tested for efficacy in the animal models and
the best therapeutic DVD-Ig can be selected for testing in human
patients. DVD-Ig molecules can also be employed for treatment of
other neurodegenerative diseases such as Parkinson's disease.
Alpha-Synuclein is involved in Parkinson's pathology. A DVD-Ig
capable of targeting alpha-synuclein and inflammatory mediators
such as TNF, IL-1, MCP-1 can prove effective therapy for
Parkinson's disease and are contemplated in the invention.
7.2 Neuronal Regeneration and Spinal Cord Injury
[0277] Despite an increase in knowledge of the pathologic
mechanisms, spinal cord injury (SCI) is still a devastating
condition and represents a medical indication characterized by a
high medical need. Most spinal cord injuries are contusion or
compression injuries and the primary injury is usually followed by
secondary injury mechanisms (inflammatory mediators e.g., cytokines
and chemokines) that worsen the initial injury and result in
significant enlargement of the lesion area, sometimes more than
10-fold. These primary and secondary mechanisms in SCI are very
similar to those in brain injury caused by other means e.g.,
stroke. No satisfying treatment exists and high dose bolus
injection of methylprednisolone (MP) is the only used therapy
within a narrow time window of 8 h post injury. This treatment,
however, is only intended to prevent secondary injury without
causing any significant functional recovery. It is heavily
criticized for the lack of unequivocal efficacy and severe adverse
effects, like immunosuppression with subsequent infections and
severe histopathological muscle alterations. No other drugs,
biologics or small molecules, stimulating the endogenous
regenerative potential are approved, but promising treatment
principles and drug candidates have shown efficacy in animal models
of SCI in recent years. To a large extent the lack of functional
recovery in human SCI is caused by factors inhibiting neurite
growth, at lesion sites, in scar tissue, in myelin as well as on
injury-associated cells. Such factors are the myelin-associated
proteins NogoA, OMgp and MAG, RGM A, the scar-associated CSPG
(Chondroitin Sulfate Proteoglycans) and inhibitory factors on
reactive astrocytes (some semaphorins and ephrins). However, at the
lesion site not only growth inhibitory molecules are found but also
neurite growth stimulating factors like neurotrophins, laminin, L1
and others. This ensemble of neurite growth inhibitory and growth
promoting molecules may explain that blocking single factors, like
NogoA or RGM A, resulted in significant functional recovery in
rodent SCI models, because a reduction of the inhibitory influences
could shift the balance from growth inhibition to growth promotion.
However, recoveries observed with blocking a single neurite
outgrowth inhibitory molecule were not complete. To achieve faster
and more pronounced recoveries either blocking two neurite
outgrowth inhibitory molecules e.g Nogo and RGM A, or blocking an
neurite outgrowth inhibitory molecule and enhancing functions of a
neurite outgrowth enhancing molecule e.g Nogo and neurotrophins, or
blocking a neurite outgrowth inhibitory moleclule e.g., Nogo and a
pro-inflammatory molecule e.g., TNF, may be desirable (see McGee,
A. W. et al. (2003) Trends Neurosci. 26: 193; Domeniconi, M. et al.
(2005) J. Neurol. Sci. 233: 43; Makwanal, M. et al. (2005) FEBS J.
272: 2628; Dickson, B. J. (2002) Science 298: 1959; Yu, F. and
Teng, H. et al. (2005) J. Neurosci. Res. 79: 273; Karnezis, T. et
al. (2004) Nature Neurosci. 7: 736; Xu, G. et al. (2004) J.
Neurochem. 91: 1018).
[0278] In one aspect, DVD-Igs that bind target pairs such as NgR
and RGM A; NogoA and RGM A; MAG and RGM A; OMGp and RGM A; RGM A
and RGM B; CSPGs and RGM A; aggrecan, midkine, neurocan, versican,
phosphacan, Te38 and TNF-.alpha.; A.beta. globulomer-specific
antibodies combined with antibodies promoting dendrite & axon
sprouting are provided. Dendrite pathology is a very early sign of
AD and it is known that NOGO A restricts dendrite growth. One can
combine one such type of Ab with any of the SCI-candidate
(myelin-proteins) Abs. Other DVD-Ig targets may include any
combination of NgR-p75, NgR-Troy, NgR-Nogo66 (Nogo), NgR-Lingo,
Lingo-Troy, Lingo-p75, MAG and Omgp. Additionally, targets may also
include any mediator, soluble or cell surface, implicated in
inhibition of neurite e.g Nogo, Ompg, MAG, RGM A, semaphorins,
ephrins, soluble A-b, pro-inflammatory cytokines (e.g., IL-1),
chemokines (e.g., MIP 1a), molecules that inhibit nerve
regeneration. The efficacy of anti-nogo/anti-RGM A or similar
DVD-Ig molecules can be validated in pre-clinical animal models of
spinal cord injury. In addition, these DVD-Ig molecules can be
constructed and tested for efficacy in the animal models and the
best therapeutic DVD-Ig can be selected for testing in human
patients. In addition, DVD-Ig molecules can be constructed that
target two distinct ligand binding sites on a single receptor e.g.,
Nogo receptor which binds the three ligand Nogo, Ompg, and MAG and
RAGE that binds A-b and S100 A. Furthermore, neurite outgrowth
inhibitors e.g., nogo and nogo receptor, also play a role in
preventing nerve regeneration in immunological diseases like
multiple sclerosis Inhibition of nogo-nogo receptor interaction has
been shown to enhance recovery in animal models of multiple
sclerosis. Therefore, DVD-Ig molecules that can block the function
of one immune mediator e.g., a cytokine like IL-12, and a neurite
outgrowth inhibitor molecule eg nogo or RGM may offer faster and
greater efficacy than blocking either an immune or an neurite
outgrowth inhibitor molecule alone.
[0279] In general, antibodies do not cross the blood brain barrier
(BBB) in an efficient and relevant manner. However, in certain
neurologic diseases, e.g., stroke, traumatic brain injury, multiple
sclerosis, etc., the BBB may be compromised and allows for
increased penetration of DVD-Igs and antibodies into the brain. In
other neurological conditions, where BBB leakage is not occurring,
one may employ the targeting of endogenous transport systems,
including carrier-mediated transporters such as glucose and amino
acid carriers and receptor-mediated transcytosis-mediating cell
structures/receptors at the vascular endothelium of the BBB, thus
enabling trans-BBB transport of the DVD-Ig. Structures at the BBB
enabling such transport include but are not limited to the insulin
receptor, transferrin receptor, LRP and RAGE. In addition,
strategies enable the use of DVD-Igs also as shuttles to transport
potential drugs into the CNS including low molecular weight drugs,
nanoparticles and nucleic acids (Coloma, M. J. et al. (2000) Pharm
Res. 17(3):266-74; Boado, R. J. et al. (2007) Bioconjug. Chem.
18(2):447-55).
8. Oncological Disorders
[0280] Monoclonal antibody therapy has emerged as an important
therapeutic modality for cancer (von Mehren M, et al (2003) Annu
Rev Med.; 54:343-69). Antibodies may exert antitumor effects by
inducing apoptosis, redirected cytotoxicity, interfering with
ligand-receptor interactions, or preventing the expression of
proteins that are critical to the neoplastic phenotype. In
addition, antibodies can target components of the tumor
microenvironment, perturbing vital structures such as the formation
of tumor-associated vasculature. Antibodies can also target
receptors whose ligands are growth factors, such as the epidermal
growth factor receptor. The antibody thus inhibits natural ligands
that stimulate cell growth from binding to targeted tumor cells.
Alternatively, antibodies may induce an anti-idiotype network,
complement-mediated cytotoxicity, or antibody-dependent cellular
cytotoxicity (ADCC). The use of dual-specific antibody that targets
two separate tumor mediators will likely give additional benefit
compared to a mono-specific therapy. DVD Igs that bind the
following pairs of targets to treat oncological disease are also
contemplated: IGF1 and IGF2; IGF1/2 and HER-2; VEGFR and EGFR; CD20
and CD3; CD138 and CD20; CD38 and CD20; CD38 and CD138; CD40 and
CD20; CD138 and CD40; CD38 and CD40; CD-20 and CD-19; CD-20 and
EGFR; CD-20 and CD-80; CD-20 and CD-22; CD-3 and HER-2; CD-3 and
CD-19; EGFR and HER-2; EGFR and CD-3; EGFR and IGF1,2; EGFR and
IGF1R; EGFR and RON; EGFR and HGF; EGFR and c-MET; HER-2 and
IGF1,2; HER-2 and IGF1R; RON and HGF; VEGF and EGFR; VEGF and
HER-2; VEGF and CD-20; VEGF and IGF1,2; VEGF and DLL4; VEGF and
HGF; VEGF and RON; VEGF and NRP1; CD20 and CD3; VEGF and PLGF; DLL4
and PLGF; ErbB3 and EGFR; HGF and ErbB3, HER-2 and ErbB3; c-Met and
ErbB3; HER-2 and PLGF; HER-2 and HER-2.
[0281] In another embodiment, a DVD-Ig of the invention binds VEGF
and phosphatidylserine; VEGF and ErbB3; VEGF and PLGF; VEGF and
ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF and ANPEP; VEGF and
c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2 and CDCP1; HER-2 and
ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and CDCP1; EGFR and
ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20; CD20 and
CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 and CD52;
CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and
phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and
Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2
and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and
CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and
VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa
and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON
and c-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and
PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and
NRP1; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and
ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF;
CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and
CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL;
CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80
and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and CD3;
CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80 and
DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;
CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22
and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20;
CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30
and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and
VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and
FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4;
CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and
DR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5
and IGF1,2; DR5 and IGFR, DR5 and HER-2, EGFR and DLL4. Other
target combinations include one or more members of the
EGF/erb-2/erb-3 family. Other targets (one or more) involved in
oncological diseases that DVD Igs may bind include, but are not
limited to those selected from the group consisting of: CD52, CD20,
CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, IL2, IL24, INHA, TNF,
TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16,
FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4,
FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B,
IL2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18,
FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C,
CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1,
PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9,
E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4,
MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23,
FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA,
INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3, NR2F6, NR4A3,
ESR1, ESR2, NR0B1, NR0B2, NR1D2, NR1H2, NR1H4, NR1I2, NR2C1, NR2C2,
NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1,
NR5A2, NR6A1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,
BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1,
FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20,
FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,
GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, IL2, IL24,
INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4,
KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL,
PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20,
CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH20, CDH7, CDH8,
CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP,
TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3,
CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584,
FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF,
KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1,
PSCA, SLC2A2, SLC33A1, SLC43A1, STEAP, STEAP2, TPM1, TPM2, TRPC6,
ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1,
KDR, LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB1, VEGF, VEGFC, ANGPTL3,
BAI1, COL4A3, IL8, LAMAS, NRP1, NRP2, STAB1, ANGPTL4, PECAM1, PF4,
PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5,
CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1,
EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK,
TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL18A1, EDG1, ENG, ITGAV,
ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1,
CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p161NK4a),
COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2),
ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2,
IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin),
JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (NGF), NGFR,
NME1 (NM23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1
(PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6
(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1
(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1),
CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1
(fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin),
ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6
(hair-specific type II keratin), MACMARCKS, MT3
(metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2
(p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2),
SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and
TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,
phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,
CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5,
RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2,
EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha,
PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59.
IV. Pharmaceutical Compositions
[0282] The invention also provides pharmaceutical compositions
comprising a binding protein, of the invention and a
pharmaceutically acceptable carrier. The pharmaceutical
compositions comprising binding proteins of the invention are for
use in, but not limited to, diagnosing, detecting, or monitoring a
disorder, in preventing (e.g., inhibiting or delaying the onset of
a disease, disorder or other condition), treating, managing, or
ameliorating of a disorder or one or more symptoms thereof, and/or
in research. In a specific embodiment, a composition comprises one
or more binding proteins of the invention. In another embodiment,
the pharmaceutical composition comprises one or more binding
proteins of the invention and one or more prophylactic or
therapeutic agents other than binding proteins of the invention for
treating a disorder. In an embodiment, the prophylactic or
therapeutic agents are useful for or have been or currently are
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 a carrier, diluent or excipient.
[0283] The binding proteins of the invention can be incorporated
into pharmaceutical compositions suitable for administration to a
subject. Typically, the pharmaceutical composition comprises a
binding protein of the invention and a pharmaceutically acceptable
carrier. The term "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 some embodiments,
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride, are included 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.
[0284] 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 (1987) J. Biol. Chem. 262:4429-4432), 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 a
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, a binding protein 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.
[0285] In an embodiment, specific binding of antibody-coupled
carbon nanotubes (CNTs) to tumor cells in vitro, followed by their
highly specific ablation with near-infrared (NIR) light can be used
to target tumor cells. For example, biotinylated polar lipids can
be used to prepare stable, biocompatible, noncytotoxic CNT
dispersions that are then attached to one or two different
neutralite avidin-derivatized DVD-Igs directed against one or more
tumor antigens (e.g., CD22) (Chakravarty, P. et al. (2008) Proc.
Natl. Acad. Sci. USA 105:8697-8702.
[0286] 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, and not by way of limitation, local infusion, by
injection, or by means of an implant, said 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
antagonists 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 a binding protein of the invention of a subject
to prevent, treat, manage, and/or ameliorate a disorder or one or
more symptoms thereof.
[0287] In another embodiment, the prophylactic or therapeutic agent
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 present disclosure
(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. 71: 105); U.S. Pat. Nos.
5,679,377; 5,916,597; 5,912,015; 5,989,463; and 5,128,326; and PCT
Publication Nos. WO 99/15154; 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 an
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)).
[0288] 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
present disclosure. See, e.g., U.S. Pat. No. 4,526,938; PCT
Publication Nos. WO 91/05548; WO 96/20698, Ning et al. (1996)
Radiotherap. Oncol. 39: 179-189; Song et al. (1995) PDA J. Pharma.
Sci. Tech. 50:372-397; Cleek et al. (1997) Pro. Intl. Symp.
Control. Rel. Bioact. Mater. 24: 853-854, and Lam et al. (1997)
Proc. Intl. Symp. Control Rel. Bioact. Mater. 24:759-760.
[0289] 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.
[0290] 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 lignocamne to
ease pain at the site of the injection.
[0291] 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 well-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). In an embodiment, 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 greater than
water are 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, in an embodiment, 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.
[0292] 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.
[0293] If the method of the invention comprises oral
administration, compositions can be formulated orally 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).
[0294] 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, a binding protein 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.).
[0295] 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.
[0296] The methods of the invention may additionally comprise of
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).
[0297] The methods of the invention encompasse 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.
[0298] 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.
[0299] 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. In an embodiment,
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, 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, e.g., 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. In an
embodiment, the liquid form of the administered composition is
supplied in a hermetically sealed container at least 0.25 mg/ml, 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.
[0300] The binding proteins of the invention can be incorporated
into a pharmaceutical composition suitable for parenteral
administration. In an embodiment, the antibody or antibody-portions
will be prepared as an injectable solution containing 0.1-250 mg/ml
binding protein. 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 and arginine, either of which can be included at a
concentration of 0-0.05%, and polysorbate-80 (optimally included at
a concentration of 0.005-0.01%). Additional surfactants include but
are not limited to polysorbate 20 and BRIJ surfactants. The
pharmaceutical composition comprising the binding proteins 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 PCT Publication No. WO
2004/078140, and U.S. Patent Publication No. 2006/104968).
[0301] 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 form chosen depends on
the intended mode of administration and therapeutic application.
Typical 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 chosen mode of
administration is parenteral (e.g., intravenous, subcutaneous,
intraperitoneal, intramuscular). In an embodiment, the antibody is
administered by intravenous infusion or injection. In another
embodiment, the antibody is administered by intramuscular or
subcutaneous injection.
[0302] 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 herein, 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 herein. In the case of
sterile, lyophilized powders for the preparation of sterile
injectable solutions, the 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.
[0303] The binding proteins of the present invention can be
administered by a variety of methods known in the art, although for
many therapeutic applications, in an embodiment, the 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.
[0304] In certain embodiments, a binding protein 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.
[0305] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, a binding protein of the
invention is coformulated with and/or coadministered with one or
more additional therapeutic agents that are useful for treating
disorders with binding protein of the invention. For example, a
binding protein 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.
[0306] In certain embodiments, a binding protein 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.
Pat. No. 6,660,843 and published PCT Publication No. WO
99/25044.
[0307] In a specific embodiment, nucleic acid sequences encoding a
binding protein 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.
[0308] 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 (1993) Science 260:926-932; 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 U.S. Patent Publication No.
20090297514.
[0309] The binding proteins of the invention are useful in treating
various diseases wherein the targets that are recognized by the
binding proteins are detrimental. Such diseases include, but are
not limited to, 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 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, 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, occlusive
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. (see PCT Publication Nos. WO
2002/097048; WO 95/24918; and WO 00/56772).
[0310] The DVD-Igs of the invention may also treat one or more of
the following diseases: 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 hearingloss, Autoimmune
Lymphoproliferative Syndrome (ALPS), Autoimmune myocarditis,
autoimmune thrombocytopenia (AITP), 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,
malignancies, 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, Ovarian cancer, 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, prostate and rectal
cancer and hematopoietic malignancies (leukemia and lymphoma),
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.
[0311] The binding proteins of the invention can be used to treat
humans suffering from autoimmune diseases, in particular those
associated with inflammation, including, rheumatoid arthritis,
spondylitis, allergy, autoimmune diabetes, autoimmune uveitis. In
an embodiment, the binding proteins of the invention or
antigen-binding portions thereof, are used to treat rheumatoid
arthritis, Crohn's disease, multiple sclerosis, insulin dependent
diabetes mellitus and psoriasis.
[0312] 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).
[0313] In an embodiment, the antibodies of the invention or
antigen-binding portions thereof, are used to treat cancer or in
the prevention or inhibition of metastases from the tumors
described herein either when used alone or in combination with
radiotherapy and/or other chemotherapeutic agents.
[0314] In another embodiment, a DVD-Ig of the invention binds a
prophylactic or therapeutic agent and a cellular protein, thereby
providing for localized drug delivery to a specific target organ,
tissue or cell, or class of tissues or cells. In an embodiment, the
DVD-Ig binds to a cell surface antigen and a prophylactic or
therapeutic agent. The prophylactic agent or therapeutic agent is
useful for preventing, managing, treating, or ameliorating a
disorder or one or more symptoms thereof, e.g., liposomal
particles, microparticles, microcapsules, recombinant cells capable
of expressing the antibody or antibody fragment, stem cells,
receptor-mediated endocytosis (see, e.g., Wu and Wu (1987) J. Biol.
Chem. 262:4429-4432), peptide, nucleic acid (e.g., antisense DND or
RNA or other genetic therapy), peptide nucleic acid (PNA),
nanoparticle, radiotherapeutic agent, retroviral or other vector,
antibacterial, anti-viral, anti-parasitic, or anti-fungal agent,
anti-neoplastic agents, chemotherapeutic agent, such as DNA
alkylating agents, cisplatin, carboplatin, anti-tubulin agents,
paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine, gemzar,
anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin,
irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib,
gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors,
and siRNAs, cytokine suppressive anti-inflammatory drug(s)
(CSAIDs).
[0315] In an embodiment, the DVD-Igs of the invention bind to
methotrexate, 6-MP, azathioprine sulphasalazine, mesalazine,
olsalazine chloroquinine/hydroxychloroquine, pencillamine,
aurothiomalate, azathioprine, cochicine, corticosteroids, beta-2
adrenoreceptor agonists (salbutamol, terbutaline, salmeteral),
xanthines (theophylline, aminophylline), cromoglycate, nedocromil,
ketotifen, ipratropium and oxitropium, cyclosporin, FK506,
rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example,
ibuprofen, corticosteroids such as prednisolone, phosphodiesterase
inhibitors, adensosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, agents which interfere with
signalling by proinflammatory cytokines such as
TNF-.alpha..quadrature. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP
kinase inhibitors), IL-1.beta. converting enzyme inhibitors,
TNF.alpha..quadrature. converting enzyme (TACE) inhibitors, T-cell
signalling inhibitors such as kinase inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g., soluble p55 or p75 TNF
receptors and the derivatives p75TNFRIgG (Enbrel.TM. and p55TNFRIgG
(Lenercept)), sIL-1RI, sIL-1RII, sIL-6R), growth factors,
cytokines, cytotoxi proteins (e.g., TNF), antiinflammatory
cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGF.beta.),
celecoxib, folic acid, hydroxychloroquine sulfate, rofecoxib,
antibodies or a derivative or conjugate thereof (e.g., infliximab
or rituximab), naproxen, valdecoxib, sulfasalazine,
methylprednisolone, meloxicam, methylprednisolone acetate, gold
sodium thiomalate, aspirin, triamcinolone acetonide, propoxyphene
napsylate/apap, folate, nabumetone, diclofenac, piroxicam,
etodolac, diclofenac sodium, oxaprozin, oxycodone hcl, hydrocodone
bitartrate/apap, diclofenac sodium/misoprostol, fentanyl, anakinra,
human recombinant, tramadol hcl, salsalate, sulindac,
cyanocobalamin/fa/pyridoxine, acetaminophen, alendronate sodium,
prednisolone, morphine sulfate, lidocaine hydrochloride,
indomethacin, glucosamine sulf/chondroitin, amitriptyline hcl,
sulfadiazine, oxycodone hcl/acetaminophen, olopatadine hcl,
misoprostol, naproxen sodium, omeprazole, cyclophosphamide,
rituximab, IL-1 TRAP, MRA, CTLA4-IG, IL-18 BP, anti-IL-18,
Anti-IL15, BIRB-796, SCIO-469, VX-702, AMG-548, VX-740,
Roflumilast, IC-485, CDC-801, and Mesopram.
[0316] In another embodiment, the DVD-Ig of the invention binds to
non-steroidal anti-inflammatory drug(s) (NSAIDs); cytokine
suppressive anti-inflammatory drug(s) (CSAIDs); antibodies or
derivatives or conjugates thereof [e.g., CDP-571/BAY-10-3356
(humanized anti-TNF.alpha. antibody; Celltech/Bayer);
cA2/infliximab (chimeric anti-TNF.alpha. antibody; Centocor); 75
kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion protein;
Immunex); 55 kdTNF-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized
anti-CD4 antibody; IDEC/SmithKline; DAB 486-IL-2 and/or DAB
389-IL-2 (IL-2 fusion proteins; Seragen); Anti-Tac (humanized
anti-IL-2R.alpha.; Protein Design Labs/Roche)]; IL-4
(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;
recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering);
IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA
(IL-1 receptor antagonist; Synergen/Amgen); anakinra
(Kineret.RTM./Amgen); TNF-bp/s-TNF (soluble TNF binding protein);
R973401 (phosphodiesterase Type IV inhibitor); MK-966 (COX-2
Inhibitor); Iloprost; methotrexate; thalidomide and
thalidomide-related drugs (e.g., Celgen); leflunomide
(anti-inflammatory and cytokine inhibitor); tranexamic acid
(inhibitor of plasminogen activation); T-614 (cytokine inhibitor);
prostaglandin E1); Tenidap (non-steroidal anti-inflammatory drug);
Naproxen (non-steroidal anti-inflammatory drug); Meloxicam
(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal
anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory
drug); Diclofenac (non-steroidal anti-inflammatory drug);
Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine;
Azathioprine); ICE inhibitor (inhibitor of the enzyme
interleukin-1.beta. converting enzyme); zap-70 and/or lck inhibitor
(inhibitor of the tyrosine kinase zap-70 or lck); VEGF inhibitor
and/or VEGF-R inhibitor (inhibitors of vascular endothelial cell
growth factor or vascular endothelial cell growth factor receptor;
inhibitors of angiogenesis); corticosteroid anti-inflammatory drugs
(e.g., SB203580); TNF-convertase inhibitors; anti-IL-12 or
anti-IL-18 antibodies or derivatives or conjugates thereof;
interleukin-11; interleukin-13; interleukin-17 inhibitors; gold;
penicillamine; chloroquine; chlorambucil; hydroxychloroquine;
cyclosporine; cyclophosphamide; total lymphoid irradiation;
anti-thymocyte globulin or anti-CD4 antibodies or derivates or
conjugates thereof; CD5-toxins; orally-administered peptides and
collagen; lobenzarit disodium; Cytokine Regulating Agents (CRAs)
HP228 and HP466 (Houghten Pharmaceuticals, Inc.); ICAM-1 antisense
phosphorothioate oligo-deoxynucleotides (ISIS 2302; Isis
Pharmaceuticals, Inc.); soluble complement receptor 1 (TP10; T Cell
Sciences, Inc.); prednisone; orgotein; glycosaminoglycan
polysulphate; minocycline; anti-IL2R antibodies or derivates or
conjugates thereof; marine and botanical lipids (fish and plant
seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin.
North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic
acid; flufenamic acid; intravenous immune globulin; zileuton;
azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);
sirolimus (rapamycin); amiprilose (therafectin); cladribine
(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see
Bruncko, Milan et al., Journal of Medicinal Chemistry (2007),
50(4), 641-662); antivirals and immune modulating agents.
[0317] In one embodiment, the DVD-Ig of the invention binds to one
of the following agents for the treatment of rheumatoid arthritis,
for example, small molecule inhibitor of KDR, small molecule
inhibitor of Tie-2; methotrexate; prednisone; celecoxib; folic
acid; hydroxychloroquine sulfate; rofecoxib; etanercept or
infliximab or derivates or conjugates thereof; leflunomide;
naproxen; valdecoxib; sulfasalazine; methylprednisolone; ibuprofen;
meloxicam; methylprednisolone acetate; gold sodium thiomalate;
aspirin; azathioprine; triamcinolone acetonide; propxyphene
napsylate/apap; folate; nabumetone; diclofenac; piroxicam;
etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone
bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra,
human recombinant; tramadol hcl; salsalate; sulindac;
cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium;
prednisolone; morphine sulfate; lidocaine hydrochloride;
indomethacin; glucosamine sulfate/chondroitin; cyclosporine;
amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen;
olopatadine hcl; misoprostol; naproxen sodium; omeprazole;
mycophenolate mofetil; cyclophosphamide; rituximab or derivates or
conjugates thereof; IL-1 TRAP; MRA; CTLA4-Ig or derivates or
conjugates thereof; IL-18 BP; IL-12/23; anti-IL 18 or derivates or
conjugates thereof; anti-IL 15 or derivates or conjugates thereof;
BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;
CDC-801; and mesopram.
[0318] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for inflammatory bowel disease, for example,
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. mAbs or derivates or conjugates
thereof; anti-IL-6 mAbs or derivates or conjugates thereof; growth
factors; elastase inhibitors; pyridinyl-imidazole compounds;
antibodies to or antagonists of other human cytokines or growth
factors, for example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15,
IL-16, IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF or derivates or
conjugates thereof.
[0319] In one embodiment, the DVD-Ig of the invention binds to cell
surface molecules such as CD2, CD3, CD4, CD8, CD25, CD28, CD30,
CD40, CD45, CD69 as methotrexate, cyclosporin, FK506, rapamycin,
mycophenolate mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adenosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines such as TNF.alpha. or IL-1 (e.g., IRAK,
NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta. converting
enzyme inhibitors, TNF.alpha. converting enzyme inhibitors, T-cell
signalling inhibitors such as kinase inhibitors, metalloproteinase
inhibitors, sulfasalazine, azathioprine, 6-mercaptopurines,
angiotensin converting enzyme inhibitors, soluble cytokine
receptors and derivatives thereof (e.g., soluble p55 or p75 TNF
receptors, sIL-1RI, sIL-1RII, sIL-6R) and antiinflammatory
cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and TGF.beta.) and bcl-2
inhibitors.
[0320] In one embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Crohn's disease, for example, TNF
antagonists, for example, anti-TNF antibodies, Adalimumab (PCT
Publication No. WO 97/29131; Humira), CA2 (Remicade), CDP 571,
TNFR-Ig constructs, (p75TNFRIgG (Enbrel) and p55TNFRIgG
(Lenercept)) inhibitors or derivates or conjugates thereof and PDE4
inhibitors. In one embodiment, the DVD-Ig of the invention binds to
corticosteroids, for example, budenoside and dexamethasone. In one
embodiment, the DVD-Ig of the invention binds to sulfasalazine,
5-aminosalicylic acid and olsalazine, and agents which interfere
with synthesis or action of proinflammatory cytokines such as IL-1,
for example, IL-1.beta. converting enzyme inhibitors and IL-1ra. In
one embodiment, the DVD-Ig of the invention binds to T cell
signaling inhibitors, for example, tyrosine kinase inhibitors
6-mercaptopurines. In one embodiment, the DVD-Ig of the invention
binds to IL-11. In one embodiment, the DVD-Ig of the invention
binds to mesalamine, prednisone, azathioprine, mercaptopurine,
infliximab or derivates or conjugates thereof, methylprednisolone
sodium succinate, diphenoxylate/atrop sulfate, loperamide
hydrochloride, methotrexate, omeprazole, folate,
ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap,
tetracycline hydrochloride, fluocinonide, metronidazole,
thimerosal/boric acid, cholestyramine/sucrose, ciprofloxacin
hydrochloride, hyoscyamine sulfate, meperidine hydrochloride,
midazolam hydrochloride, oxycodone hcl/acetaminophen, promethazine
hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim,
celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone,
multivitamins, balsalazide disodium, codeine phosphate/apap,
colesevelam hcl, cyanocobalamin, folic acid, levofloxacin,
methylprednisolone, natalizumab or derivates or conjugates thereof
and interferon-alpha, interferon-beta, and interferon-gamma.
[0321] In one embodiment, the DVD-Ig of the invention binds to
therapeutic agents for multiple sclerosis, for example,
corticosteroids; prednisolone; methylprednisolone; azathioprine;
cyclophosphamide; cyclosporine; methotrexate; 4-aminopyridine;
tizanidine; interferon-.beta.1a (AVONEX; Biogen);
interferon-.beta.1b (BETASERON; Chiron/Berlex); interferon
.alpha.-n3) (Interferon Sciences/Fujimoto), interferon-.alpha.
(Alfa Wassermann/J&J), interferon .beta.1A-1F (Serono/Inhale
Therapeutics), Peginterferon .alpha. 2b (Enzon/Schering-Plough),
Copolymer 1 (Cop-1; COPAXONE; Teva Pharmaceutical Industries,
Inc.); hyperbaric oxygen; intravenous immunoglobulin; clabribine;
antibodies to or antagonists of other human cytokines or growth
factors and their receptors, for example, TNF, LT, IL-1, IL-2,
IL-6, IL-7, IL-8, IL-23, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF,
and PDGF or derivatives or conjugates thereof. In one embodiment,
the DVD-Ig of the invention binds to cell surface molecules such as
CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30, CD40, CD45, CD69,
CD80, CD86, CD90 or their ligands. In one embodiment, the DVD-Ig of
the invention binds to methotrexate, cyclosporine, FK506,
rapamycin, mycophenolate mofetil, leflunomide, NSAIDs, for example,
ibuprofen, corticosteroids such as prednisolone, phosphodiesterase
inhibitors, adensosine agonists, antithrombotic agents, complement
inhibitors, adrenergic agents, agents which interfere with
signalling by proinflammatory cytokines such as TNF.alpha. or IL-1
(e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta.
converting enzyme inhibitors, TACE inhibitors, T-cell signaling
inhibitors such as kinase inhibitors, metalloproteinase inhibitors,
sulfasalazine, azathioprine, 6-mercaptopurines, angiotensin
converting enzyme inhibitors, soluble cytokine receptors and
derivatives thereof (e.g., soluble p55 or p75 TNF receptors,
sIL-1RI, sIL-1RII, sIL-6R), antiinflammatory cytokines (e.g., IL-4,
IL-10, IL-13 and TGF.beta.) and bcl-2 inhibitors.
[0322] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for multiple sclerosis, for example,
interferon-.beta., for example, IFN.beta.1a and IFN.beta.1b;
copaxone, corticosteroids, caspase inhibitors, for example
inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, and
antibodies to CD40 and CD80, and derivates or conjugates
thereof.
[0323] In another embodiment, the DVD-Ig of the invention binds to
the following agents or derivatives or conjugates thereof:
alemtuzumab, dronabinol, Unimed, daclizumab, mitoxantrone,
xaliproden hydrochloride, fampridine, glatiramer acetate,
natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062,
AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine,
CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD
(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,
anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258
(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2,
tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4
Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists,
IL-4 agonists.
[0324] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Angina, for example, nitroglycerin,
isosorbide mononitrate, metoprolol succinate, atenolol, metoprolol
tartrate, amlodipine besylate, diltiazem hydrochloride, isosorbide
dinitrate, clopidogrel bisulfate, nifedipine, atorvastatin calcium,
potassium chloride, furosemide, simvastatin, verapamil hcl,
digoxin, propranolol hydrochloride, carvedilol, lisinopril,
spironolactone, hydrochlorothiazide, enalapril maleate, nadolol,
ramipril, enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
bisoprolol fumarate.
[0325] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Ankylosing Spondylitis, for example,
ibuprofen, diclofenac and misoprostol, naproxen, meloxicam,
indomethacin, diclofenac, celecoxib, rofecoxib, Sulfasalazine,
Methotrexate, azathioprine, minocyclin, prednisone, etanercept,
infliximab, and derivatives or conjugates thereof.
[0326] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Asthma, for example, albuterol,
salmeterol/fluticasone, montelukast sodium, fluticasone propionate,
budesonide, prednisone, salmeterol xinafoate, levalbuterol hcl,
albuterol sulfate/ipratropium, prednisolone sodium phosphate,
triamcinolone acetonide, beclomethasone dipropionate, ipratropium
bromide, azithromycin, pirbuterol acetate, prednisolone,
theophylline anhydrous, methylprednisolone sodium succinate,
clarithromycin, zafirlukast, formoterol fumarate, influenza virus
vaccine, methylprednisolone, amoxicillin trihydrate, flunisolide,
allergy injection, cromolyn sodium, fexofenadine hydrochloride,
flunisolide/menthol, amoxicillin/clavulanate, levofloxacin, inhaler
assist device, guaifenesin, dexamethasone sodium phosphate,
moxifloxacin hcl, doxycycline hyclate, guaifenesin/d-methorphan,
p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine
hydrochloride, mometasone furoate, salmeterol xinafoate,
benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine
hcl/pseudoephed, phenylephrine/cod/promethazine,
codeine/promethazine, cefprozil, dexamethasone,
guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone,
nedocromil sodium, terbutaline sulfate, epinephrine,
methylprednisolone, metaproterenol sulfate.
[0327] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for COPD, for example, albuterol
sulfate/ipratropium, ipratropium bromide, salmeterol/fluticasone,
albuterol, salmeterol xinafoate, fluticasone propionate,
prednisone, theophylline anhydrous, methylprednisolone sodium
succinate, montelukast sodium, budesonide, formoterol fumarate,
triamcinolone acetonide, levofloxacin, guaifenesin, azithromycin,
beclomethasone dipropionate, levalbuterol hcl, flunisolide,
ceftriaxone sodium, amoxicillin trihydrate, gatifloxacin,
zafirlukast, amoxicillin/clavulanate, flunisolide/menthol,
chlorpheniramine/hydrocodone, metaproterenol sulfate,
methylprednisolone, mometasone furoate,
p-ephedrine/cod/chlorphenir, pirbuterol acetate,
p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide,
(R,R)-formoterol, TgAAT, Cilomilast, Roflumilast.
[0328] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for HCV, for example, Interferon-alpha-2a,
Interferon-alpha-2b, Interferon-alpha con1, Interferon-alpha-n1,
Pegylated interferon-alpha-2a, Pegylated interferon-alpha-2b,
ribavirin, Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid,
Glycyrrhizic Acid, Thymalfasin, Maxamine, VX-497 and any compounds
that are used to treat HCV through intervention with the following
targets: HCV polymerase, HCV protease, HCV helicase, HCV IRES
(internal ribosome entry site).
[0329] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Idiopathic Pulmonary Fibrosis, for example,
prednisone, azathioprine, albuterol, colchicine, albuterol sulfate,
digoxin, gamma interferon, methylprednisolone sod succ, lorazepam,
furosemide, lisinopril, nitroglycerin, spironolactone,
cyclophosphamide, ipratropium bromide, actinomycin d, alteplase,
fluticasone propionate, levofloxacin, metaproterenol sulfate,
morphine sulfate, oxycodone hcl, potassium chloride, triamcinolone
acetonide, tacrolimus anhydrous, calcium, interferon-alpha,
methotrexate, mycophenolate mofetil, Interferon-gamma-1
.quadrature..
[0330] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Myocardial Infarction, for example, aspirin,
nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin
sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine
sulfate, metoprolol succinate, warfarin sodium, lisinopril,
isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,
tenecteplase, enalapril maleate, torsemide, retavase, losartan
potassium, quinapril hcl/mag carb, bumetanide, alteplase,
enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate,
diltiazem hydrochloride, captopril, irbesartan, valsartan,
propranolol hydrochloride, fosinopril sodium, lidocaine
hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate,
aminocaproic acid, spironolactone, interferon, sotalol
hydrochloride, potassium chloride, docusate sodium, dobutamine hcl,
alprazolam, pravastatin sodium, atorvastatin calcium, midazolam
hydrochloride, meperidine hydrochloride, isosorbide dinitrate,
epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,
ezetimibe/simvastatin, avasimibe, cariporide, cardiac stem cells,
and growth factors.
[0331] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Psoriasis, for example, a small molecule
inhibitor of KDR, small molecule inhibitor of Tie-2, calcipotriene,
clobetasol propionate, triamcinolone acetonide, halobetasol
propionate, tazarotene, methotrexate, fluocinonide, betamethasone
diprop augmented, fluocinolone acetonide, acitretin, tar shampoo,
betamethasone valerate, mometasone furoate, ketoconazole,
pramoxine/fluocinolone, hydrocortisone valerate, flurandrenolide,
urea, betamethasone, clobetasol propionate/emoll, fluticasone
propionate, azithromycin, hydrocortisone, moisturizing formula,
folic acid, desonide, pimecrolimus, coal tar, diflorasone
diacetate, etanercept folate, lactic acid, methoxsalen, hc/bismuth
subgal/znox/resor, methylprednisolone acetate, prednisone,
sunscreen, halcinonide, salicylic acid, anthralin, clocortolone
pivalate, coal extract, coal tar/salicylic acid, coal tar/salicylic
acid/sulfur, desoximetasone, diazepam, emollient,
fluocinonide/emollient, mineral oil/castor oil/na lact, mineral
oil/peanut oil, petroleum/isopropyl myristate, psoralen, salicylic
acid, soap/tribromsalan, thimerosal/boric acid, celecoxib,
infliximab, cyclosporine, alefacept, efalizumab, tacrolimus,
pimecrolimus, PUVA, UVB, sulfasalazine.
[0332] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Psoriatic Arthritis, for example,
methotrexate, etanercept, rofecoxib, celecoxib, folic acid,
sulfasalazine, naproxen, leflunomide, methylprednisolone acetate,
indomethacin, hydroxychloroquine sulfate, prednisone, sulindac,
betamethasone diprop augmented, infliximab, methotrexate, folate,
triamcinolone acetonide, diclofenac, dimethylsulfoxide, piroxicam,
diclofenac sodium, ketoprofen, meloxicam, methylprednisolone,
nabumetone, tolmetin sodium, calcipotriene, cyclosporine,
diclofenac sodium/misoprostol, fluocinonide, glucosamine sulfate,
gold sodium thiomalate, hydrocodone bitartrate/apap, ibuprofen,
risedronate sodium, sulfadiazine, thioguanine, valdecoxib,
alefacept, efalizumab and bcl-2 inhibitors, or derivatives or
conjugates thereof.
[0333] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Restenosis, for example, sirolimus,
paclitaxel, everolimus, tacrolimus, Zotarolimus, acetaminophen.
[0334] In another embodiment, the DVD-Ig of the invention binds to
therapeutic agents for Sciatica, for example, hydrocodone
bitartrate/apap, rofecoxib, cyclobenzaprine hcl,
methylprednisolone, naproxen, ibuprofen, oxycodone
hcl/acetaminophen, celecoxib, valdecoxib, methylprednisolone
acetate, prednisone, codeine phosphate/apap, tramadol
hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine
hydrochloride, diclofenac sodium, gabapentin, dexamethasone,
carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen,
diazepam, nabumetone, oxycodone hcl, tizanidine hcl, diclofenac
sodium/misoprostol, propoxyphene napsylate/apap,
asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,
etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine
phos/asa, morphine sulfate, multivitamins, naproxen sodium,
orphenadrine citrate, temazepam.
[0335] In one embodiment, the DVD-Ig of the invention binds to
agents for SLE (Lupus), for example, NSAIDS, for example,
diclofenac, naproxen, ibuprofen, piroxicam, indomethacin; COX2
inhibitors, for example, Celecoxib, rofecoxib, valdecoxib;
anti-malarials, for example, hydroxychloroquine; Steroids, for
example, prednisone, prednisolone, budenoside, dexamethasone;
cytotoxics, for example, azathioprine, cyclophosphamide,
mycophenolate mofetil, methotrexate; inhibitors of PDE4 or purine
synthesis inhibitor, for example Cellcept. In one embodiment, the
DVD-Ig of the invention binds to sulfasalazine, 5-aminosalicylic
acid, olsalazine, Imuran and agents which interfere with synthesis,
production or action of proinflammatory cytokines such as IL-1, for
example, caspase inhibitors like IL-1.beta. converting enzyme
inhibitors and IL-1ra. In one embodiment, the DVD-Ig of the
invention binds to T cell signaling inhibitors, for example,
tyrosine kinase inhibitors; or molecules that target T cell
activation molecules, for example, CTLA-4-Ig or B7 family
antibodies, or PD-1 family. In one embodiment, the DVD-Ig of the
invention binds to IL-11 or anti-cytokine antibodies, for example,
fonotolizumab (anti-IFNg antibody), or anti-receptor receptor
antibodies, for example, anti-IL-6 receptor antibody and antibodies
to B-cell surface molecules. In one embodiment, the DVD-Ig of the
invention binds to LJP 394 (abetimus), agents that deplete or
inactivate B-cells, for example, anti-CD20 antibody, and BlyS, TNF
and bcl-2 inhibitors, because bcl-2 overexpression in transgenic
mice has been demonstrated to cause a lupus like phenotype (see
Marquina et al. (2004) J. Immunol. 172(11), 7177-7185), therefore
inhibition is expected to have therapeutic effects.
[0336] In an embodiment, the DVD-Ig of the invention binds MTX and
a cell surface molecule, e.g., MTX and mCD-3; MTX and mCD-19; MTX
and hCD-3; MTX and hCD-19; MTX and CD-20; MTX and EGFR; and MTX and
HER-2.
[0337] The antibodies of the invention, or antigen binding portions
thereof, may be combined with agents that include but are not
limited to, antineoplastic agents, radiotherapy, chemotherapy such
as DNA alkylating agents, cisplatin, carboplatin, anti-tubulin
agents, paclitaxel, docetaxel, taxol, doxorubicin, gemcitabine,
gemzar, anthracyclines, adriamycin, topoisomerase I inhibitors,
topoisomerase II inhibitors, 5-fluorouracil (5-FU), leucovorin,
irinotecan, receptor tyrosine kinase inhibitors (e.g., erlotinib,
gefitinib), COX-2 inhibitors (e.g., celecoxib), kinase inhibitors,
and siRNAs.
[0338] A binding protein of the invention also can be administered
with one or more additional therapeutic agents useful in the
treatment of various diseases.
[0339] A binding protein of the invention can be used alone or in
combination to treat such diseases. It should be understood that
the binding proteins can be used alone or in combination with an
additional agent, e.g., a therapeutic agent, said 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 which
affects the viscosity of the composition.
[0340] It should further be understood that the combinations which
are to be included within this invention are those combinations
useful for their intended purpose. The agents set forth below are
illustrative and are 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.
[0341] Combinations to treat autoimmune and inflammatory diseases
are non-steroidal anti-inflammatory drug(s) also referred to as
NSAIDS which include drugs like ibuprofen. Other combinations are
corticosteroids including prednisolone; the well known side-effects
of steroid use can be reduced or even eliminated by tapering the
steroid dose required when treating patients in combination with
the DVD Igs of this invention. Non-limiting examples of therapeutic
agents for rheumatoid arthritis with which an antibody, or antibody
portion, of the invention can be combined include the following:
cytokine suppressive anti-inflammatory drug(s) (CSAIDs); antibodies
to or antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-15, IL-16, IL-18, IL-21, IL-23, interferons, EMAP-II, GM-CSF,
FGF, and PDGF. Binding proteins 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, and CTLA or their
ligands including CD154 (gp39 or CD40L).
[0342] Combinations of therapeutic agents may interfere at
different points in the autoimmune and subsequent inflammatory
cascade; examples include TNF antagonists like chimeric, humanized
or human TNF antibodies, ADALIMUMAB, (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 TNFa converting enzyme (TACE)
inhibitors; similarly IL-1 inhibitors (Interleukin-1-converting
enzyme inhibitors, IL-1RA etc.) may be effective for the same
reason. Other combinations include Interleukin 11. Yet another
combination includes key players of the autoimmune response which
may act parallel to, dependent on, or in concert with, IL-12
function, especially IL-18 antagonists including IL-18 antibodies,
soluble IL-18 receptors, and IL-18 binding proteins. It has been
shown that IL-12 and IL-18 have overlapping but distinct functions
and a combination of antagonists to both may be most effective. Yet
another combination is non-depleting anti-CD4 inhibitors. Yet other
combinations include antagonists of the co-stimulatory pathway CD80
(B7.1) or CD86 (B7.2) including antibodies, soluble receptors and
antagonistic ligands.
[0343] The binding proteins of the invention may also be combined
with agents, such as methotrexate, 6-MP, azathioprine
sulphasalazine, mesalazine, olsalazine
chloroquinine/hydroxychloroquine, pencillamine, aurothiomalate
(intramuscular and oral), azathioprine, cochicine, corticosteroids
(oral, inhaled and local injection), beta-2 adrenoreceptor agonists
(salbutamol, terbutaline, salmeteral), xanthines (theophylline,
aminophylline), cromoglycate, nedocromil, ketotifen, ipratropium
and oxitropium, cyclosporin, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, for example, ibuprofen,
corticosteroids such as prednisolone, phosphodiesterase inhibitors,
adensosine agonists, antithrombotic agents, complement inhibitors,
adrenergic agents, agents which interfere with signalling by
proinflammatory cytokines, such as TNF-.alpha..quadrature. or IL-1
(e.g., IRAK, NIK, IKK, p38 or MAP kinase inhibitors), IL-1.beta.
converting enzyme inhibitors, TNF-.alpha..quadrature. converting
enzyme (TACE) inhibitors, T-cell signalling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g., soluble p55 or p75 TNF receptors and the derivatives
p75TNFRIgG (Enbrel.TM. and p55TNFRIgG (Lenercept)), sIL-1RI,
sIL-1RII, and sIL-6R), antiinflammatory cytokines (e.g., IL-4,
IL-10, IL-11, IL-13 and TGF.beta.), celecoxib, folic acid,
hydroxychloroquine sulfate, rofecoxib, etanercept, infliximab,
naproxen, valdecoxib, sulfasalazine, methylprednisolone, meloxicam,
methylprednisolone acetate, gold sodium thiomalate, aspirin,
triamcinolone acetonide, propoxyphene napsylate/apap, folate,
nabumetone, diclofenac, piroxicam, etodolac, diclofenac sodium,
oxaprozin, oxycodone hcl, hydrocodone bitartrate/apap, diclofenac
sodium/misoprostol, fentanyl, anakinra, human recombinant, tramadol
hcl, salsalate, sulindac, cyanocobalamin/fa/pyridoxine,
acetaminophen, alendronate sodium, prednisolone, morphine sulfate,
lidocaine hydrochloride, indomethacin, glucosamine
sulf/chondroitin, amitriptyline hcl, sulfadiazine, oxycodone
hcl/acetaminophen, olopatadine hcl, misoprostol, naproxen sodium,
omeprazole, cyclophosphamide, rituximab, IL-1 TRAP, MRA, CTLA4-IG,
IL-18 BP, anti-IL-18, Anti-IL15, BIRB-796, SCIO-469, VX-702,
AMG-548, VX-740, Roflumilast, IC-485, CDC-801, and Mesopram.
Combinations include methotrexate or leflunomide and in moderate or
severe rheumatoid arthritis cases, cyclosporine.
[0344] Nonlimiting additional agents which can also be used in
combination with a binding protein to treat rheumatoid arthritis
include, but are not limited to, the following: non-steroidal
anti-inflammatory drug(s) (NSAIDs); cytokine suppressive
anti-inflammatory drug(s) (CSAIDs); CDP-571/BAY-10-3356 (humanized
anti-TNF.alpha. antibody; Celltech/Bayer); cA2/infliximab (chimeric
anti-TNF.alpha. antibody; Centocor); 75 kdTNFR-IgG/etanercept (75
kD TNF receptor-IgG fusion protein; Immunex; see e.g., (1994)
Arthr. Rheum. 37: S295; (1996) J. Invest. Med. 44: 235A); 55
kdTNF-IgG (55 kD TNF receptor-IgG fusion protein;
Hoffmann-LaRoche); IDEC-CE9.1/SB 210396 (non-depleting primatized
anti-CD4 antibody; IDEC/SmithKline; see e.g., (1995) Arthr. Rheum.
38: S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins;
Seragen; see e.g., (1993) Arthrit. Rheum. 36: 1223); Anti-Tac
(humanized anti-IL-2R.alpha.; Protein Design Labs/Roche); IL-4
(anti-inflammatory cytokine; DNAX/Schering); IL-10 (SCH 52000;
recombinant IL-10, anti-inflammatory cytokine; DNAX/Schering);
IL-4; IL-10 and/or IL-4 agonists (e.g., agonist antibodies); IL-1RA
(IL-1 receptor antagonist; Synergen/Amgen); anakinra
(Kineret.RTM./Amgen); TNF-bp/s-TNF (soluble TNF binding protein;
see e.g., (1996) Arthr. Rheum. 39(9 (supplement)): S284; (1995)
Amer. J. Physiol.--Heart and Circ. Physiol. 268: 37-42); R973401
(phosphodiesterase Type IV inhibitor; see e.g., (1996) Arthr.
Rheum. 39(9 (supplement): S282); MK-966 (COX-2 Inhibitor; see e.g.,
(1996) Arthr. Rheum. 39(9 (supplement): S81); Iloprost (see e.g.,
(1996) Arthr. Rheum. 39(9 (supplement): S82); methotrexate;
thalidomide (see e.g., (1996) Arthr. Rheum. 39(9 (supplement):
S282) and thalidomide-related drugs (e.g., Celgen); leflunomide
(anti-inflammatory and cytokine inhibitor; see e.g., (1996) Arthr.
Rheum. 39(9 (supplement): S131; (1996) Inflamm Res. 45: 103-107);
tranexamic acid (inhibitor of plasminogen activation; see e.g.,
(1996) Arthr. Rheum. 39(9 (supplement): S284); T-614 (cytokine
inhibitor; see e.g., (1996) Arthr. Rheum. 39(9 (supplement): S282);
prostaglandin E1 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement):
S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g.,
(1996) Arthr. Rheum. 39(9 (supplement): S280); Naproxen
(non-steroidal anti-inflammatory drug; see e.g., (1996) Neuro.
Report 7: 1209-1213); Meloxicam (non-steroidal anti-inflammatory
drug); Ibuprofen (non-steroidal anti-inflammatory drug); Piroxicam
(non-steroidal anti-inflammatory drug); Diclofenac (non-steroidal
anti-inflammatory drug); Indomethacin (non-steroidal
anti-inflammatory drug); Sulfasalazine (see e.g., (1996) Arthr.
Rheum. 39(9 (supplement): S281); Azathioprine (see e.g., (1996)
Arthr. Rheum. 39(9 (supplement): S281); ICE inhibitor (inhibitor of
the enzyme interleukin-1.beta. converting enzyme); zap-70 and/or
lck inhibitor (inhibitor of the tyrosine kinase zap-70 or lck);
VEGF inhibitor and/or VEGF-R inhibitor (inhibitors of vascular
endothelial cell growth factor or vascular endothelial cell growth
factor receptor; inhibitors of angiogenesis); corticosteroid
anti-inflammatory drugs (e.g., SB203580); TNF-convertase
inhibitors; anti-IL-12 antibodies; anti-IL-18 antibodies;
interleukin-11 (see e.g., (1996) Arthr. Rheum. 39(9 (supplement):
S296); interleukin-13 (see e.g., (1996) Arthr. Rheum. 39(9
(supplement): S308); interleukin-17 inhibitors (see e.g., (1996)
Arthr. Rheum. 39(9 (supplement): S120); gold; penicillamine;
chloroquine; chlorambucil; hydroxychloroquine; cyclosporine;
cyclophosphamide; total lymphoid irradiation; anti-thymocyte
globulin; anti-CD4 antibodies; CD5-toxins; orally-administered
peptides and collagen; lobenzarit disodium; Cytokine Regulating
Agents (CRAs) HP228 and HP466 (Houghten Pharmaceuticals, Inc.);
ICAM-1 antisense phosphorothioate oligo-deoxynucleotides (ISIS
2302; Isis Pharmaceuticals, Inc.); soluble complement receptor 1
(TP10; T Cell Sciences, Inc.); prednisone; orgotein;
glycosaminoglycan polysulphate; minocycline; anti-IL2R antibodies;
marine and botanical lipids (fish and plant seed fatty acids; see
e.g., DeLuca et al. (1995) Rheum. Dis. Clin. North Am. 21:
759-777); auranofin; phenylbutazone; meclofenamic acid; flufenamic
acid; intravenous immune globulin; zileuton; azaribine;
mycophenolic acid (RS-61443); tacrolimus (FK-506); sirolimus
(rapamycin); amiprilose (therafectin); cladribine
(2-chlorodeoxyadenosine); methotrexate; bcl-2 inhibitors (see
Bruncko, M. et al. (2007) J. Med. Chem. 50(4): 641-662); and
antivirals and immune-modulating agents.
[0345] In one embodiment, the binding protein or antigen-binding
portion thereof, is administered in combination with one of the
following agents for the treatment of rheumatoid arthritis: small
molecule inhibitor of KDR, small molecule inhibitor of Tie-2;
methotrexate; prednisone; celecoxib; folic acid; hydroxychloroquine
sulfate; rofecoxib; etanercept; infliximab; leflunomide; naproxen;
valdecoxib; sulfasalazine; methylprednisolone; ibuprofen;
meloxicam; methylprednisolone acetate; gold sodium thiomalate;
aspirin; azathioprine; triamcinolone acetonide; propxyphene
napsylate/apap; folate; nabumetone; diclofenac; piroxicam;
etodolac; diclofenac sodium; oxaprozin; oxycodone hcl; hydrocodone
bitartrate/apap; diclofenac sodium/misoprostol; fentanyl; anakinra,
human recombinant; tramadol hcl; salsalate; sulindac;
cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate sodium;
prednisolone; morphine sulfate; lidocaine hydrochloride;
indomethacin; glucosamine sulfate/chondroitin; cyclosporine;
amitriptyline hcl; sulfadiazine; oxycodone hcl/acetaminophen;
olopatadine hcl; misoprostol; naproxen sodium; omeprazole;
mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA;
CTLA4-IG; IL-18 BP; IL-12/23; anti-IL 18; anti-IL 15; BIRB-796;
SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485; CDC-801;
and mesopram.
[0346] Non-limiting examples of therapeutic agents for inflammatory
bowel disease with which a binding protein of the invention can be
combined include the following: 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. mAbs; anti-IL-6 mAbs; growth factors; elastase
inhibitors; pyridinyl-imidazole compounds; antibodies to or
antagonists of other human cytokines or growth factors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-15, IL-16,
IL-17, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. 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, CD90 or their ligands. The
antibodies of the invention, or antigen binding portions thereof,
may also be combined with agents, such as methotrexate,
cyclosporin, FK506, rapamycin, mycophenolate mofetil, leflunomide,
NSAIDs, for example, ibuprofen, corticosteroids such as
prednisolone, phosphodiesterase inhibitors, adenosine agonists,
antithrombotic agents, complement inhibitors, adrenergic agents,
agents which interfere with signalling by proinflammatory cytokines
such as TNF.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or MAP kinase
inhibitors), IL-1.beta. converting enzyme inhibitors, TNF.alpha.
converting enzyme inhibitors, T-cell signalling inhibitors such as
kinase inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, sIL-6R)
and antiinflammatory cytokines (e.g., IL-4, IL-10, IL-11, IL-13 and
TGF.beta.) and bcl-2 inhibitors.
[0347] Examples of therapeutic agents for Crohn's disease in which
a binding protein can be combined include the following: TNF
antagonists, for example, anti-TNF antibodies, ADALIMUMAB (PCT
Publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571,
TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG
(LENERCEPT)) inhibitors and PDE4 inhibitors. Antibodies of the
invention, or antigen binding portions thereof, can be combined
with corticosteroids, for example, budenoside and dexamethasone.
Binding proteins of the invention or antigen binding portions
thereof, may also be combined with agents such as sulfasalazine,
5-aminosalicylic acid and olsalazine, and agents which interfere
with synthesis or action of proinflammatory cytokines such as IL-1,
for example, IL-1.beta. converting enzyme inhibitors and IL-1ra.
Antibodies of the invention or antigen binding portion thereof may
also be used with T cell signaling inhibitors, for example,
tyrosine kinase inhibitors 6-mercaptopurines. Binding proteins of
the invention, or antigen binding portions thereof, can be combined
with IL-11. Binding proteins of the invention, or antigen binding
portions thereof, can be combined with mesalamine, prednisone,
azathioprine, mercaptopurine, infliximab, methylprednisolone sodium
succinate, diphenoxylate/atrop sulfate, loperamide hydrochloride,
methotrexate, omeprazole, folate, ciprofloxacin/dextrose-water,
hydrocodone bitartrate/apap, tetracycline hydrochloride,
fluocinonide, metronidazole, thimerosal/boric acid,
cholestyramine/sucrose, ciprofloxacin hydrochloride, hyoscyamine
sulfate, meperidine hydrochloride, midazolam hydrochloride,
oxycodone hcl/acetaminophen, promethazine hydrochloride, sodium
phosphate, sulfamethoxazole/trimethoprim, celecoxib, polycarbophil,
propoxyphene napsylate, hydrocortisone, multivitamins, balsalazide
disodium, codeine phosphate/apap, colesevelam hcl, cyanocobalamin,
folic acid, levofloxacin, methylprednisolone, natalizumab and
interferon-gamma
[0348] Non-limiting examples of therapeutic agents for multiple
sclerosis with which binding proteins of the invention can be
combined include the following: corticosteroids; prednisolone;
methylprednisolone; azathioprine; cyclophosphamide; cyclosporine;
methotrexate; 4-aminopyridine; tizanidine; interferon-.beta.1a
(AVONEX; Biogen); interferon-.beta.1b (BETASERON; Chiron/Berlex);
interferon .alpha.-n3) (Interferon Sciences/Fujimoto),
interferon-.alpha. (Alfa Wassermann/J&J), interferon
.beta.1A-1F (Serono/Inhale Therapeutics), Peginterferon .alpha. 2b
(Enzon/Schering-Plough), Copolymer 1 (Cop-1; COPAXONE; Teva
Pharmaceutical Industries, Inc.); hyperbaric oxygen; intravenous
immunoglobulin; clabribine; and antibodies to or antagonists of
other human cytokines or growth factors and their receptors, for
example, TNF, LT, IL-1, IL-2, IL-6, IL-7, IL-8, IL-23, IL-15,
IL-16, IL-18, EMAP-II, GM-CSF, FGF, and PDGF. Binding proteins of
the invention can be combined with antibodies to cell surface
molecules such as CD2, CD3, CD4, CD8, CD19, CD20, CD25, CD28, CD30,
CD40, CD45, CD69, CD80, CD86, and CD90 and their ligands. Binding
proteins of the invention, may also be combined with agents, such
as methotrexate, cyclosporine, FK506, rapamycin, mycophenolate
mofetil, leflunomide, NSAIDs, such as ibuprofen, corticosteroids
such as prednisolone, phosphodiesterase inhibitors, adensosine
agonists, antithrombotic agents, complement inhibitors, adrenergic
agents, agents which interfere with signalling by proinflammatory
cytokines such as TNF.alpha. or IL-1 (e.g., IRAK, NIK, IKK, p38 or
MAP kinase inhibitors), IL-1.beta. converting enzyme inhibitors,
TACE inhibitors, T-cell signaling inhibitors such as kinase
inhibitors, metalloproteinase inhibitors, sulfasalazine,
azathioprine, 6-mercaptopurines, angiotensin converting enzyme
inhibitors, soluble cytokine receptors and derivatives thereof
(e.g., soluble p55 or p75 TNF receptors, sIL-1RI, sIL-1RII, and
sIL-6R), antiinflammatory cytokines (e.g., IL-4, IL-10, IL-13 and
TGF.beta.), and bcl-2 inhibitors.
[0349] Examples of therapeutic agents for multiple sclerosis in
which binding proteins of the invention can be combined include
interferon-.beta., for example, IFN.beta.1a and IFN.beta.1b;
copaxone, corticosteroids, caspase inhibitors, for example
inhibitors of caspase-1, IL-1 inhibitors, TNF inhibitors, and
antibodies to CD40 ligand and CD80.
[0350] The binding proteins of the invention, may also be combined
with agents, such as alemtuzumab, dronabinol, Unimed, daclizumab,
mitoxantrone, xaliproden hydrochloride, fampridine, glatiramer
acetate, natalizumab, sinnabidol, a-immunokine NNSO3, ABR-215062,
AnergiX.MS, chemokine receptor antagonists, BBR-2778, calagualine,
CPI-1189, LEM (liposome encapsulated mitoxantrone), THC.CBD
(cannabinoid agonist) MBP-8298, mesopram (PDE4 inhibitor), MNA-715,
anti-IL-6 receptor antibody, neurovax, pirfenidone allotrap 1258
(RDP-1258), sTNF-R1, talampanel, teriflunomide, TGF-beta2,
tiplimotide, VLA-4 antagonists (for example, TR-14035, VLA4
Ultrahaler, Antegran-ELAN/Biogen), interferon gamma antagonists,
and IL-4 agonists.
[0351] Non-limiting examples of therapeutic agents for Angina with
which binding proteins of the invention can be combined include the
following: aspirin, nitroglycerin, isosorbide mononitrate,
metoprolol succinate, atenolol, metoprolol tartrate, amlodipine
besylate, diltiazem hydrochloride, isosorbide dinitrate,
clopidogrel bisulfate, nifedipine, atorvastatin calcium, potassium
chloride, furosemide, simvastatin, verapamil hcl, digoxin,
propranolol hydrochloride, carvedilol, lisinopril, spironolactone,
hydrochlorothiazide, enalapril maleate, nadolol, ramipril,
enoxaparin sodium, heparin sodium, valsartan, sotalol
hydrochloride, fenofibrate, ezetimibe, bumetanide, losartan
potassium, lisinopril/hydrochlorothiazide, felodipine, captopril,
and bisoprolol fumarate.
[0352] Non-limiting examples of therapeutic agents for Ankylosing
Spondylitis with which binding proteins of the invention can be
combined include the following: ibuprofen, diclofenac and
misoprostol, naproxen, meloxicam, indomethacin, diclofenac,
celecoxib, rofecoxib, Sulfasalazine, Methotrexate, azathioprine,
minocyclin, prednisone, etanercept, and infliximab.
[0353] Non-limiting examples of therapeutic agents for Asthma with
which binding proteins of the invention can be combined include the
following: albuterol, salmeterol/fluticasone, montelukast sodium,
fluticasone propionate, budesonide, prednisone, salmeterol
xinafoate, levalbuterol hcl, albuterol sulfate/ipratropium,
prednisolone sodium phosphate, triamcinolone acetonide,
beclomethasone dipropionate, ipratropium bromide, azithromycin,
pirbuterol acetate, prednisolone, theophylline anhydrous,
methylprednisolone sodium succinate, clarithromycin, zafirlukast,
formoterol fumarate, influenza virus vaccine, methylprednisolone,
amoxicillin trihydrate, flunisolide, allergy injection, cromolyn
sodium, fexofenadine hydrochloride, flunisolide/menthol,
amoxicillin/clavulanate, levofloxacin, inhaler assist device,
guaifenesin, dexamethasone sodium phosphate, moxifloxacin hcl,
doxycycline hyclate, guaifenesin/d-methorphan,
p-ephedrine/cod/chlorphenir, gatifloxacin, cetirizine
hydrochloride, mometasone furoate, salmeterol xinafoate,
benzonatate, cephalexin, pe/hydrocodone/chlorphenir, cetirizine
hcl/pseudoephed, phenylephrine/cod/promethazine,
codeine/promethazine, cefprozil, dexamethasone,
guaifenesin/pseudoephedrine, chlorpheniramine/hydrocodone,
nedocromil sodium, terbutaline sulfate, epinephrine,
methylprednisolone, and metaproterenol sulfate.
[0354] Non-limiting examples of therapeutic agents for COPD with
which binding proteins of the invention can be combined include the
following: albuterol sulfate/ipratropium, ipratropium bromide,
salmeterol/fluticasone, albuterol, salmeterol xinafoate,
fluticasone propionate, prednisone, theophylline anhydrous,
methylprednisolone sodium succinate, montelukast sodium,
budesonide, formoterol fumarate, triamcinolone acetonide,
levofloxacin, guaifenesin, azithromycin, beclomethasone
dipropionate, levalbuterol hcl, flunisolide, ceftriaxone sodium,
amoxicillin trihydrate, gatifloxacin, zafirlukast,
amoxicillin/clavulanate, flunisolide/menthol,
chlorpheniramine/hydrocodone, metaproterenol sulfate,
methylprednisolone, mometasone furoate,
p-ephedrine/cod/chlorphenir, pirbuterol acetate,
p-ephedrine/loratadine, terbutaline sulfate, tiotropium bromide,
(R,R)-formoterol, TgAAT, Cilomilast, and Roflumilast.
[0355] Non-limiting examples of therapeutic agents for HCV with
which binding proteins of the invention can be combined include the
following: Interferon-alpha-2a, Interferon-alpha-2b,
Interferon-alpha con1, Interferon-alpha-n1, Pegylated
interferon-alpha-2a, Pegylated interferon-alpha-2b, ribavirin,
Peginterferon alfa-2b+ribavirin, Ursodeoxycholic Acid, Glycyrrhizic
Acid, Thymalfasin, Maxamine, VX-497 and any compounds that are used
to treat HCV through intervention with the following targets: HCV
polymerase, HCV protease, HCV helicase, and HCV IRES (internal
ribosome entry site).
[0356] Non-limiting examples of therapeutic agents for Idiopathic
Pulmonary Fibrosis with which binding proteins of the invention can
be combined include the following: prednisone, azathioprine,
albuterol, colchicine, albuterol sulfate, digoxin, gamma
interferon, methylprednisolone sod succ, lorazepam, furosemide,
lisinopril, nitroglycerin, spironolactone, cyclophosphamide,
ipratropium bromide, actinomycin d, alteplase, fluticasone
propionate, levofloxacin, metaproterenol sulfate, morphine sulfate,
oxycodone hcl, potassium chloride, triamcinolone acetonide,
tacrolimus anhydrous, calcium, interferon-alpha, methotrexate,
mycophenolate mofetil, and Interferon-gamma-1.beta..
[0357] Non-limiting examples of therapeutic agents for Myocardial
Infarction with which binding proteins of the invention can be
combined include the following: aspirin, nitroglycerin, metoprolol
tartrate, enoxaparin sodium, heparin sodium, clopidogrel bisulfate,
carvedilol, atenolol, morphine sulfate, metoprolol succinate,
warfarin sodium, lisinopril, isosorbide mononitrate, digoxin,
furosemide, simvastatin, ramipril, tenecteplase, enalapril maleate,
torsemide, retavase, losartan potassium, quinapril hcl/mag carb,
bumetanide, alteplase, enalaprilat, amiodarone hydrochloride,
tirofiban hcl m-hydrate, diltiazem hydrochloride, captopril,
irbesartan, valsartan, propranolol hydrochloride, fosinopril
sodium, lidocaine hydrochloride, eptifibatide, cefazolin sodium,
atropine sulfate, aminocaproic acid, spironolactone, interferon,
sotalol hydrochloride, potassium chloride, docusate sodium,
dobutamine hcl, alprazolam, pravastatin sodium, atorvastatin
calcium, midazolam hydrochloride, meperidine hydrochloride,
isosorbide dinitrate, epinephrine, dopamine hydrochloride,
bivalirudin, rosuvastatin, ezetimibe/simvastatin, avasimibe, and
cariporide.
[0358] Non-limiting examples of therapeutic agents for Psoriasis
with which binding proteins of the invention can be combined
include the following: small molecule inhibitor of KDR, small
molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate,
triamcinolone acetonide, halobetasol propionate, tazarotene,
methotrexate, fluocinonide, betamethasone diprop augmented,
fluocinolone acetonide, acitretin, tar shampoo, betamethasone
valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone,
hydrocortisone valerate, flurandrenolide, urea, betamethasone,
clobetasol propionate/emoll, fluticasone propionate, azithromycin,
hydrocortisone, moisturizing formula, folic acid, desonide,
pimecrolimus, coal tar, diflorasone diacetate, etanercept folate,
lactic acid, methoxsalen, hc/bismuth subgal/znox/resor,
methylprednisolone acetate, prednisone, sunscreen, halcinonide,
salicylic acid, anthralin, clocortolone pivalate, coal extract,
coal tar/salicylic acid, coal tar/salicylic acid/sulfur,
desoximetasone, diazepam, emollient, fluocinonide/emollient,
mineral oil/castor oil/na lact, mineral oil/peanut oil,
petroleum/isopropyl myristate, psoralen, salicylic acid,
soap/tribromsalan, thimerosal/boric acid, celecoxib, infliximab,
cyclosporine, alefacept, efalizumab, tacrolimus, pimecrolimus,
PUVA, UVB, and sulfasalazine.
[0359] Non-limiting examples of therapeutic agents for Psoriatic
Arthritis with which binding proteins of the invention can be
combined include the following: methotrexate, etanercept,
rofecoxib, celecoxib, folic acid, sulfasalazine, naproxen,
leflunomide, methylprednisolone acetate, indomethacin,
hydroxychloroquine sulfate, prednisone, sulindac, betamethasone
diprop augmented, infliximab, methotrexate, folate, triamcinolone
acetonide, diclofenac, dimethylsulfoxide, piroxicam, diclofenac
sodium, ketoprofen, meloxicam, methylprednisolone, nabumetone,
tolmetin sodium, calcipotriene, cyclosporine, diclofenac
sodium/misoprostol, fluocinonide, glucosamine sulfate, gold sodium
thiomalate, hydrocodone bitartrate/apap, ibuprofen, risedronate
sodium, sulfadiazine, thioguanine, valdecoxib, alefacept,
efalizumab and bcl-2 inhibitors.
[0360] Non-limiting examples of therapeutic agents for Restenosis
with which binding proteins of the invention can be combined
include the following: sirolimus, paclitaxel, everolimus,
tacrolimus, Zotarolimus, and acetaminophen.
[0361] Non-limiting examples of therapeutic agents for Sciatica
with which binding proteins of the invention can be combined
include the following: hydrocodone bitartrate/apap, rofecoxib,
cyclobenzaprine hcl, methylprednisolone, naproxen, ibuprofen,
oxycodone hcl/acetaminophen, celecoxib, valdecoxib,
methylprednisolone acetate, prednisone, codeine phosphate/apap,
tramadol hcl/acetaminophen, metaxalone, meloxicam, methocarbamol,
lidocaine hydrochloride, diclofenac sodium, gabapentin,
dexamethasone, carisoprodol, ketorolac tromethamine, indomethacin,
acetaminophen, diazepam, nabumetone, oxycodone hcl, tizanidine hcl,
diclofenac sodium/misoprostol, propoxyphene napsylate/apap,
asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,
etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine
phos/asa, morphine sulfate, multivitamins, naproxen sodium,
orphenadrine citrate, and temazepam.
[0362] Examples of therapeutic agents for SLE (Lupus) in which
binding proteins of the invention can be combined include the
following: NSAIDS, for example, diclofenac, naproxen, ibuprofen,
piroxicam, indomethacin; COX2 inhibitors, for example, Celecoxib,
rofecoxib, valdecoxib; anti-malarials, for example,
hydroxychloroquine; Steroids, for example, prednisone,
prednisolone, budenoside, dexamethasone; Cytotoxics, for example,
azathioprine, cyclophosphamide, mycophenolate mofetil,
methotrexate; and inhibitors of PDE4 or purine synthesis inhibitor,
for example Cellcept. Binding proteins of the invention, may also
be combined with agents such as sulfasalazine, 5-aminosalicylic
acid, olsalazine, Imuran and agents which interfere with synthesis,
production or action of proinflammatory cytokines such as IL-1, for
example, caspase inhibitors like IL-1.beta. converting enzyme
inhibitors and IL-1ra. Binding proteins of the invention may also
be used with T cell signaling inhibitors, for example, tyrosine
kinase inhibitors; or molecules that target T cell activation
molecules, for example, CTLA-4-IgG or anti-B7 family antibodies,
and anti-PD-1 family antibodies. Binding proteins of the invention,
can be combined with IL-11 or anti-cytokine antibodies, for
example, fonotolizumab (anti-IFNg antibody), or anti-receptor
receptor antibodies, for example, anti-IL-6 receptor antibody and
antibodies to B-cell surface molecules. Antibodies of the invention
or antigen binding portion thereof may also be used with LJP 394
(abetimus), agents that deplete or inactivate B-cells, for example,
Rituximab (anti-CD20 antibody), lymphostat-B (anti-BlyS antibody),
TNF antagonists, for example, anti-TNF antibodies, Adalimumab (PCT
Publication No. WO 97/29131; HUMIRA), CA2 (REMICADE), CDP 571,
TNFR-Ig constructs, (p75TNFRIgG (ENBREL) and p55TNFRIgG
(LENERCEPT)) and bcl-2 inhibitors, because bcl-2 overexpression in
transgenic mice has been demonstrated to cause a lupus like
phenotype (see Marquina, R. et al. (2004) J. Immunol. 172(11):
7177-7185), therefore inhibition is expected to have therapeutic
effects.
[0363] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of a binding protein 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 binding protein 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
binding protein 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.
[0364] 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. The term "dosage unit
form" 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.
[0365] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of a binding protein of the
invention is 0.1-20 mg/kg, for example, 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.
[0366] 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.
V. Diagnostics
[0367] The disclosure herein also provides diagnostic applications.
This is further elucidated below.
I. Method of Assay
[0368] The present disclosure also provides a method for
determining the presence, amount or concentration of an analyte (or
a fragment thereof) in a test sample using at least one DVD-Ig as
described herein. Any suitable assay as is known in the art can be
used in the method. Examples include, but are not limited to,
immunoassay, such as sandwich immunoassay (e.g., monoclonal,
polyclonal and/or DVD-Ig sandwich immunoassays or any variation
thereof (e.g., monoclonal/DVD-Ig, DVD-Ig/polyclonal, etc.),
including radioisotope detection (radioimmunoassay (RIA)) and
enzyme detection (enzyme immunoassay (EIA) or enzyme-linked
immunosorbent assay (ELISA) (e.g., Quantikine ELISA assays, R&D
Systems, Minneapolis, Minn.))), competitive inhibition immunoassay
(e.g., forward and reverse), fluorescence polarization immunoassay
(FPIA), enzyme multiplied immunoassay technique (EMIT),
bioluminescence resonance energy transfer (BRET), and homogeneous
chemiluminescent assay, etc. In a SELDI-based immunoassay, a
capture reagent that specifically binds an analyte (or a fragment
thereof) of interest is attached to the surface of a mass
spectrometry probe, such as a pre-activated protein chip array. The
analyte (or a fragment thereof) is then specifically captured on
the biochip, and the captured analyte (or a fragment thereof) is
detected by mass spectrometry. Alternatively, the analyte (or a
fragment thereof) can be eluted from the capture reagent and
detected by traditional MALDI (matrix-assisted laser
desorption/ionization) or by SELDI. A chemiluminescent
microparticle immunoassay, in particular one employing the
ARCHITECT.RTM. automated analyzer (Abbott Laboratories, Abbott
Park, Ill.), is an example of a preferred immunoassay.
[0369] Methods well-known in the art for collecting, handling and
processing urine, blood, serum and plasma, and other body fluids,
are used in the practice of the present disclosure, for instance,
when a DVD-Ig as described herein is employed as an
immunodiagnostic reagent and/or in an analyte immunoassay kit. The
test sample can comprise further moieties in addition to the
analyte of interest, such as antibodies, antigens, haptens,
hormones, drugs, enzymes, receptors, proteins, peptides,
polypeptides, oligonucleotides and/or polynucleotides. For example,
the sample can be a whole blood sample obtained from a subject. It
can be necessary or desired that a test sample, particularly whole
blood, be treated prior to immunoassay as described herein, e.g.,
with a pretreatment reagent. Even in cases where pretreatment is
not necessary (e.g., most urine samples), pretreatment optionally
can be done (e.g., as part of a regimen on a commercial
platform).
[0370] The pretreatment reagent can be any reagent appropriate for
use with the immunoassay and kits of the invention. The
pretreatment optionally comprises: (a) one or more solvents (e.g.,
methanol and ethylene glycol) and optionally, salt, (b) one or more
solvents and salt, and optionally, detergent, (c) detergent, or (d)
detergent and salt. Pretreatment reagents are known in the art, and
such pretreatment can be employed, e.g., as used for assays on
Abbott TDx, AxSYM.RTM., and ARCHITECT.RTM. analyzers (Abbott
Laboratories, Abbott Park, Ill.), as described in the literature
(see, e.g., Yatscoff et al., (1990) Clin. Chem. 36: 1969-1973 and
Wallemacq et al. (1999) Clin. Chem. 45: 432-435), and/or as
commercially available. Additionally, pretreatment can be done as
described in U.S. Pat. No. 5,135,875, EU Patent Publication No.
EU0471293, U.S. Pat. No. 6,660,843, and U.S. Patent Application No.
20080020401. The pretreatment reagent can be a heterogeneous agent
or a homogeneous agent.
[0371] With use of a heterogeneous pretreatment reagent, the
pretreatment reagent precipitates analyte binding protein (e.g.,
protein that can bind to an analyte or a fragment thereof) present
in the sample. Such a pretreatment step comprises removing any
analyte binding protein by separating from the precipitated analyte
binding protein the supernatant of the mixture formed by addition
of the pretreatment agent to sample. In such an assay, the
supernatant of the mixture absent any binding protein is used in
the assay, proceeding directly to the antibody capture step.
[0372] With use of a homogeneous pretreatment reagent there is no
such separation step. The entire mixture of test sample and
pretreatment reagent are contacted with a labeled specific binding
partner for analyte (or a fragment thereof), such as a labeled
anti-analyte antibody (or an antigenically reactive fragment
thereof). The pretreatment reagent employed for such an assay
typically is diluted in the pretreated test sample mixture, either
before or during capture by the first specific binding partner.
Despite such dilution, a certain amount of the pretreatment reagent
is still present (or remains) in the test sample mixture during
capture. According to the invention, the labeled specific binding
partner can be a DVD-Ig (or a fragment, a variant, or a fragment of
a variant thereof).
[0373] In a heterogeneous format, after the test sample is obtained
from a subject, a first mixture is prepared. The mixture contains
the test sample being assessed for an analyte (or a fragment
thereof) and a first specific binding partner, wherein the first
specific binding partner and any analyte contained in the test
sample form a first specific binding partner-analyte complex.
Preferably, the first specific binding partner is an anti-analyte
antibody or a fragment thereof. The first specific binding partner
can be a DVD-Ig (or a fragment, a variant, or a fragment of a
variant thereof) as described herein. The order in which the test
sample and the first specific binding partner are added to form the
mixture is not critical. Preferably, the first specific binding
partner is immobilized on a solid phase. The solid phase used in
the immunoassay (for the first specific binding partner and,
optionally, the second specific binding partner) can be any solid
phase known in the art, such as, but not limited to, a magnetic
particle, a bead, a test tube, a microtiter plate, a cuvette, a
membrane, a scaffolding molecule, a film, a filter paper, a disc
and a chip.
[0374] After the mixture containing the first specific binding
partner-analyte complex is formed, any unbound analyte is removed
from the complex using any technique known in the art. For example,
the unbound analyte can be removed by washing. Desirably, however,
the first specific binding partner is present in excess of any
analyte present in the test sample, such that all analyte that is
present in the test sample is bound by the first specific binding
partner.
[0375] After any unbound analyte is removed, a second specific
binding partner is added to the mixture to form a first specific
binding partner-analyte-second specific binding partner complex.
The second specific binding partner is preferably an anti-analyte
antibody that binds to an epitope on analyte that differs from the
epitope on analyte bound by the first specific binding partner.
Moreover, also preferably, the second specific binding partner is
labeled with or contains a detectable label as described above. The
second specific binding partner can be a DVD-Ig (or a fragment, a
variant, or a fragment of a variant thereof) as described
herein.
[0376] Any suitable detectable label as is known in the art can be
used. For example, the detectable label can be a radioactive label
(such as .sup.3H, .sup.125I, .sup.35S, .sup.14C, .sup.32P, and
.sup.33P), an enzymatic label (such as horseradish peroxidase,
alkaline peroxidase, glucose 6-phosphate dehydrogenase, and the
like), a chemiluminescent label (such as acridinium esters,
thioesters, or sulfonamides; luminol, isoluminol, phenanthridinium
esters, and the like), a fluorescent label (such as fluorescein
(e.g., 5-fluorescein, 6-carboxyfluorescein, 3'6-carboxyfluorescein,
5(6)-carboxyfluorescein, 6-hexachloro-fluorescein,
6-tetrachlorofluorescein, fluorescein isothiocyanate, and the
like)), rhodamine, phycobiliproteins, R-phycoerythrin, quantum dots
(e.g., zinc sulfide-capped cadmium selenide), a thermometric label,
or an immuno-polymerase chain reaction label. An introduction to
labels, labeling procedures and detection of labels is found in
Polak and Van Noorden, Introduction to Immunocytochemistry,
2.sup.nd ed., Springer Verlag, N.Y. (1997), and in Haugland,
Handbook of Fluorescent Probes and Research Chemicals (1996), which
is a combined handbook and catalogue published by Molecular Probes,
Inc., Eugene, Oreg. A fluorescent label can be used in FPIA (see,
e.g., U.S. Pat. Nos. 5,593,896; 5,573,904; 5,496,925; 5,359,093;
and 5,352,803. An acridinium compound can be used as a detectable
label in a homogeneous or heterogeneous chemiluminescent assay
(see, e.g., Adamczyk et al. (2006) Bioorg. Med. Chem. Lett. 16:
1324-1328; Adamczyk et al. (2004) Bioorg. Med. Chem. Lett. 4:
2313-2317; Adamczyk et al. (2004) Biorg. Med. Chem. Lett. 14:
3917-3921; and Adamczyk et al. (2003) Org. Lett. 5: 3779-3782).
[0377] A preferred acridinium compound is an
acridinium-9-carboxamide. Methods for preparing acridinium
9-carboxamides are described in Mattingly (1991) J. Biolumin.
Chemilumin. 6: 107-114; Adamczyk et al. (1998) J. Org. Chem. 63:
5636-5639; Adamczyk et al. (1999) Tetrahedron 55: 10899-10914;
Adamczyk et al. (1999) Org. Lett. 1: 779-781; Adamczyk et al.
(2000) Biocon. Chem. 11: 714-724; Mattingly et al., In Luminescence
Biotechnology: Instruments and Applications; Dyke, K. V. Ed.; CRC
Press: Boca Raton, pp. 77-105 (2002); Adamczyk et al. (2003) Org.
Lett. 5: 3779-3782; and U.S. Pat. Nos. 5,468,646; 5,543,524; and
5,783,699. Another preferred acridinium compound is an
acridinium-9-carboxylate aryl ester. An example of an
acridinium-9-carboxylate aryl ester is
10-methyl-9-(phenoxycarbonyl)acridinium fluorosulfonate (available
from Cayman Chemical, Ann Arbor, Mich.). Methods for preparing
acridinium 9-carboxylate aryl esters are described in McCapra et
al. (1965) Photochem. Photobiol. 4: 1111-21; Razavi et al. (2000)
Luminescence 15: 245-249; Razavi et al. (2000) Luminescence 15:
239-244; and U.S. Pat. No. 5,241,070. Further details regarding
acridinium-9-carboxylate aryl ester and its use are set forth in US
Patent Publication No. 20080248493.
[0378] Chemiluminescent assays (e.g., using acridinium as described
above or other chemiluminescent agents) can be performed in
accordance with the methods described in Adamczyk et al. (2006)
Anal. Chim. Acta 579(1): 61-67. While any suitable assay format can
be used, a microplate chemiluminometer (Mithras LB-940, Berthold
Technologies U.S.A., LLC, Oak Ridge, Tenn.) enables the assay of
multiple samples of small volumes rapidly.
[0379] The order in which the test sample and the specific binding
partner(s) are added to form the mixture for chemiluminescent assay
is not critical. If the first specific binding partner is
detectably labeled with a chemiluminescent agent such as an
acridinium compound, detectably labeled first specific binding
partner-analyte complexes form. Alternatively, if a second specific
binding partner is used and the second specific binding partner is
detectably labeled with a chemiluminescent agent such as an
acridinium compound, detectably labeled first specific binding
partner-analyte-second specific binding partner complexes form. Any
unbound specific binding partner, whether labeled or unlabeled, can
be removed from the mixture using any technique known in the art,
such as washing.
[0380] Hydrogen peroxide can be generated in situ in the mixture or
provided or supplied to the mixture (e.g., the source of the
hydrogen peroxide being one or more buffers or other solutions that
are known to contain hydrogen peroxide) before, simultaneously
with, or after the addition of an above-described acridinium
compound. Hydrogen peroxide can be generated in situ in a number of
ways such as would be apparent to one skilled in the art.
[0381] Upon the simultaneous or subsequent addition of at least one
basic solution to the sample, a detectable signal, namely, a
chemiluminescent signal, indicative of the presence of analyte is
generated. The basic solution contains at least one base and has a
pH greater than or equal to 10, preferably, greater than or equal
to 12. Examples of basic solutions include, but are not limited to,
sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium
hydroxide, magnesium hydroxide, sodium carbonate, sodium
bicarbonate, calcium hydroxide, calcium carbonate, and calcium
bicarbonate. The amount of basic solution added to the sample
depends on the concentration of the basic solution. Based on the
concentration of the basic solution used, one skilled in the art
can easily determine the amount of basic solution to add to the
sample.
[0382] The chemiluminescent signal that is generated can be
detected using routine techniques known to those skilled in the
art. Based on the intensity of the signal generated, the amount of
analyte in the sample can be quantified. Specifically, the amount
of analyte in the sample is proportional to the intensity of the
signal generated. The amount of analyte present can be quantified
by comparing the amount of light generated to a standard curve for
analyte or by comparison to a reference standard. The standard
curve can be generated using serial dilutions or solutions of known
concentrations of analyte by mass spectroscopy, gravimetric
methods, and other techniques known in the art. While the above is
described with emphasis on use of an acridinium compound as the
chemiluminescent agent, one of ordinary skill in the art can
readily adapt this description for use of other chemiluminescent
agents.
[0383] Analyte immunoassays generally can be conducted using any
format known in the art, such as, but not limited to, a sandwich
format. Specifically, in one immunoassay format, at least two
antibodies are employed to separate and quantify analyte, such as
human analyte, or a fragment thereof in a sample. More
specifically, the at least two antibodies bind to different
epitopes on an analyte (or a fragment thereof) forming an immune
complex, which is referred to as a "sandwich." Generally, in the
immunoassays one or more antibodies can be used to capture the
analyte (or a fragment thereof) in the test sample (these
antibodies are frequently referred to as a "capture" antibody or
"capture" antibodies) and one or more antibodies can be used to
bind a detectable (namely, quantifiable) label to the sandwich
(these antibodies are frequently referred to as the "detection
antibody," the "detection antibodies," the "conjugate," or the
"conjugates"). Thus, in the context of a sandwich immunoassay
format, a binding protein or a DVD-Ig (or a fragment, a variant, or
a fragment of a variant thereof) as described herein can be used as
a capture antibody, a detection antibody, or both. For example, one
binding protein or DVD-Ig having a domain that can bind a first
epitope on an analyte (or a fragment thereof) can be used as a
capture agent and/or another binding protein or DVD-Ig having a
domain that can bind a second epitope on an analyte (or a fragment
thereof) can be used as a detection agent. In this regard, a
binding protein or a DVD-Ig having a first domain that can bind a
first epitope on an analyte (or a fragment thereof) and a second
domain that can bind a second epitope on an analyte (or a fragment
thereof) can be used as a capture agent and/or a detection agent.
Alternatively, one binding protein or DVD-Ig having a first domain
that can bind an epitope on a first analyte (or a fragment thereof)
and a second domain that can bind an epitope on a second analyte
(or a fragment thereof) can be used as a capture agent and/or a
detection agent to detect, and optionally quantify, two or more
analytes. In the event that an analyte can be present in a sample
in more than one form, such as a monomeric form and a
dimeric/multimeric form, which can be homomeric or heteromeric, one
binding protein or DVD-Ig having a domain that can bind an epitope
that is only exposed on the monomeric form and another binding
protein or DVD-Ig having a domain that can bind an epitope on a
different part of a dimeric/multimeric form can be used as capture
agents and/or detection agents, thereby enabling the detection, and
optional quantification, of different forms of a given analyte.
Furthermore, employing binding proteins or DVD-Igs with
differential affinities within a single binding protein or DVD-Ig
and/or between binding proteins or DVD-Igs can provide an avidity
advantage. In the context of immunoassays as described herein, it
generally may be helpful or desired to incorporate one or more
linkers within the structure of a binding protein or a DVD-Ig. When
present, optimally the linker should be of sufficient length and
structural flexibility to enable binding of an epitope by the inner
domains as well as binding of another epitope by the outer domains.
In this regard, when a binding protein or a DVD-Ig can bind two
different analytes and one analyte is larger than the other,
desirably the larger analyte is bound by the outer domains.
[0384] Generally speaking, a sample being tested for (for example,
suspected of containing) analyte (or a fragment thereof) can be
contacted with at least one capture agent (or agents) and at least
one detection agent (which can be a second detection agent or a
third detection agent or even a successively numbered agent, e.g.,
as where the capture and/or detection agent comprises multiple
agents) either simultaneously or sequentially and in any order. For
example, the test sample can be first contacted with at least one
capture agent and then (sequentially) with at least one detection
agent. Alternatively, the test sample can be first contacted with
at least one detection agent and then (sequentially) with at least
one capture agent. In yet another alternative, the test sample can
be contacted simultaneously with a capture agent and a detection
agent.
[0385] In the sandwich assay format, a sample suspected of
containing analyte (or a fragment thereof) is first brought into
contact with at least one first capture agent under conditions that
allow the formation of a first agent/analyte complex. If more than
one capture agent is used, a first capture agent/analyte complex
comprising two or more capture agents is formed. In a sandwich
assay, the agents, i.e., preferably, the at least one capture
agent, are used in molar excess amounts of the maximum amount of
analyte (or a fragment thereof) expected in the test sample. For
example, from about 5 .mu.g to about 1 mg of agent per mL of buffer
(e.g., microparticle coating buffer) can be used.
[0386] Competitive inhibition immunoassays, which are often used to
measure small analytes because binding by only one antibody (i.e.,
a binding protein and/or a DVD-Ig in the context of the present
disclosure) is required, comprise sequential and classic formats.
In a sequential competitive inhibition immunoassay a capture agent
to an analyte of interest is coated onto a well of a microtiter
plate or other solid support. When the sample containing the
analyte of interest is added to the well, the analyte of interest
binds to the capture agent. After washing, a known amount of
labeled (e.g., biotin or horseradish peroxidase (HRP)) analyte
capable of binding the capture antibody is added to the well. A
substrate for an enzymatic label is necessary to generate a signal.
An example of a suitable substrate for HRP is
3,3',5,5'-tetramethylbenzidine (TMB). After washing, the signal
generated by the labeled analyte is measured and is inversely
proportional to the amount of analyte in the sample. In a classic
competitive inhibition immunoassay typically an antibody (i.e., a
binding protein and/or a DVD-Ig in the context of the present
disclosure) to an analyte of interest is coated onto a solid
support (e.g., a well of a microtiter plate). However, unlike the
sequential competitive inhibition immunoassay, the sample and the
labeled analyte are added to the well at the same time. Any analyte
in the sample competes with labeled analyte for binding to the
capture agent. After washing, the signal generated by the labeled
analyte is measured and is inversely proportional to the amount of
analyte in the sample. Of course, there are many variations of
these formats--e.g., such as when binding to the solid substrate
takes place, whether the format is one-step, two-step, delayed
two-step, and the like--and these would be recognized by one of
ordinary skill in the art.
[0387] Optionally, prior to contacting the test sample with the at
least one capture agent (for example, the first capture agent), the
at least one capture agent can be bound to a solid support, which
facilitates the separation of the first agent/analyte (or a
fragment thereof) complex from the test sample. The substrate to
which the capture agent is bound can be any suitable solid support
or solid phase that facilitates separation of the capture
agent-analyte complex from the sample.
[0388] Examples include a well of a plate, such as a microtiter
plate, a test tube, a porous gel (e.g., silica gel, agarose,
dextran, or gelatin), a polymeric film (e.g., polyacrylamide),
beads (e.g., polystyrene beads or magnetic beads), a strip of a
filter/membrane (e.g., nitrocellulose or nylon), microparticles
(e.g., latex particles, magnetizable microparticles (e.g.,
microparticles having ferric oxide or chromium oxide cores and
homo- or hetero-polymeric coats and radii of about 1-10 microns).
The substrate can comprise a suitable porous material with a
suitable surface affinity to bind antigens and sufficient porosity
to allow access by detection antibodies. A microporous material is
generally preferred, although a gelatinous material in a hydrated
state can be used. Such porous substrates are preferably in the
form of sheets having a thickness of about 0.01 to about 0.5 mm,
preferably about 0.1 mm. While the pore size may vary quite a bit,
preferably the pore size is from about 0.025 to about 15 microns,
more preferably from about 0.15 to about 15 microns. The surface of
such substrates can be passively coated or activated by chemical
processes that cause covalent linkage of an antibody to the
substrate. Irreversible binding, generally by adsorption through
hydrophobic forces, of the antigen or the antibody to the substrate
results; alternatively, a chemical coupling agent or other means
can be used to bind covalently the antibody to the substrate,
provided that such binding does not interfere with the ability of
the antibody to bind to analyte. Alternatively, the antibody (i.e.,
binding protein and/or DVD-Ig in the context of the present
disclosure) can be bound with microparticles, which have been
previously coated with streptavidin (e.g., DYNAL.RTM. Magnetic
Beads, Invitrogen, Carlsbad, Calif.) or biotin (e.g., using
Power-BindTM-SA-MP streptavidin-coated microparticles (Seradyn,
Indianapolis, Ind.)) or anti-species-specific monoclonal antibodies
(i.e., binding proteins and/or DVD-Igs in the context of the
present disclosure). If necessary or desired, the substrate (e.g.,
for the label) can be derivatized to allow reactivity with various
functional groups on the antibody (i.e., binding protein or DVD-Ig
in the context of the present disclosure). Such derivatization
requires the use of certain coupling agents, examples of which
include, but are not limited to, maleic anhydride,
N-hydroxysuccinimide, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. If desired, one or
more capture agents, such as antibodies (or fragments thereof)
(i.e., binding proteins and/or DVD-Igs in the context of the
present disclosure), each of which is specific for analyte(s) can
be attached to solid phases in different physical or addressable
locations (e.g., such as in a biochip configuration (see, e.g.,
U.S. Pat. No. 6,225,047; PCT Publication No. WO 99/51773; U.S. Pat.
No. 6,329,209; PCT Publication No. WO 00/56934, and U.S. Pat. No.
5,242,828). If the capture agent is attached to a mass spectrometry
probe as the solid support, the amount of analyte bound to the
probe can be detected by laser desorption ionization mass
spectrometry. Alternatively, a single column can be packed with
different beads, which are derivatized with the one or more capture
agents, thereby capturing the analyte in a single place (see,
antibody-derivatized, bead-based technologies, e.g., the xMAP
technology of Luminex (Austin, Tex.)).
[0389] After the test sample being assayed for analyte (or a
fragment thereof) is brought into contact with the at least one
capture agent (for example, the first capture agent), the mixture
is incubated in order to allow for the formation of a first capture
agent (or multiple capture agent)-analyte (or a fragment thereof)
complex. The incubation can be carried out at a pH of from about
4.5 to about 10.0, at a temperature of from about 2.degree. C. to
about 45.degree. C., and for a period from at least about one (1)
minute to about eighteen (18) hours, preferably from about 1 to
about 24 minutes, most preferably for about 4 to about 18 minutes.
The immunoassay described herein can be conducted in one step
(meaning the test sample, at least one capture agent and at least
one detection agent are all added sequentially or simultaneously to
a reaction vessel) or in more than one step, such as two steps,
three steps, etc.
[0390] After formation of the (first or multiple) capture
agent/analyte (or a fragment thereof) complex, the complex is then
contacted with at least one detection agent under conditions which
allow for the formation of a (first or multiple) capture
agent/analyte (or a fragment thereof)/second detection agent
complex). While captioned for clarity as the "second" agent (e.g.,
second detection agent), in fact, where multiple agents are used
for capture and/or detection, the at least one detection agent can
be the second, third, fourth, etc., agents used in the immunoassay.
If the capture agent/analyte (or a fragment thereof) complex is
contacted with more than one detection agent, then a (first or
multiple) capture agent/analyte (or a fragment thereof)/(multiple)
detection agent complex is formed. As with the capture agent (e.g.,
the first capture agent), when the at least one (e.g., second and
any subsequent) detection agent is brought into contact with the
capture agent/analyte (or a fragment thereof) complex, a period of
incubation under conditions similar to those described above is
required for the formation of the (first or multiple) capture
agent/analyte (or a fragment thereof)/(second or multiple)
detection agent complex. Preferably, at least one detection agent
contains a detectable label. The detectable label can be bound to
the at least one detection agent (e.g., the second detection agent)
prior to, simultaneously with, or after the formation of the (first
or multiple) capture agent/analyte (or a fragment thereof)/(second
or multiple) detection agent complex. Any detectable label known in
the art can be used (see discussion above, including of the Polak
and Van Noorden (1997) and Haugland (1996) references).
[0391] The detectable label can be bound to the agents either
directly or through a coupling agent. An example of a coupling
agent that can be used is EDAC (1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide, hydrochloride), which is commercially available from
Sigma-Aldrich, St. Louis, Mo. Other coupling agents that can be
used are known in the art. Methods for binding a detectable label
to an antibody are known in the art. Additionally, many detectable
labels can be purchased or synthesized that already contain end
groups that facilitate the coupling of the detectable label to the
agent, such as CPSP-Acridinium Ester (i.e.,
9-[N-tosyl-N-(3-carboxypropyl)]-10-(3-sulfopropyl)acridinium
carboxamide) or SPSP-Acridinium Ester (i.e.,
N10-(3-sulfopropyl)-N-(3-sulfopropyl)-acridinium-9-carboxamide).
[0392] The (first or multiple) capture agent/analyte/(second or
multiple) detection agent complex can be, but does not have to be,
separated from the remainder of the test sample prior to
quantification of the label. For example, if the at least one
capture agent (e.g., the first capture agent, such as a binding
protein and/or a DVD-Ig in accordance with the present disclosure)
is bound to a solid support, such as a well or a bead, separation
can be accomplished by removing the fluid (of the test sample) from
contact with the solid support. Alternatively, if the at least
first capture agent is bound to a solid support, it can be
simultaneously contacted with the analyte-containing sample and the
at least one second detection agent to form a first (multiple)
agent/analyte/second (multiple) agent complex, followed by removal
of the fluid (test sample) from contact with the solid support. If
the at least one first capture agent is not bound to a solid
support, then the (first or multiple) capture agent/analyte/(second
or multiple) detection agent complex does not have to be removed
from the test sample for quantification of the amount of the
label.
[0393] After formation of the labeled capture
agent/analyte/detection agent complex (e.g., the first capture
agent/analyte/second detection agent complex), the amount of label
in the complex is quantified using techniques known in the art. For
example, if an enzymatic label is used, the labeled complex is
reacted with a substrate for the label that gives a quantifiable
reaction such as the development of color. If the label is a
radioactive label, the label is quantified using appropriate means,
such as a scintillation counter. If the label is a fluorescent
label, the label is quantified by stimulating the label with a
light of one color (which is known as the "excitation wavelength")
and detecting another color (which is known as the "emission
wavelength") that is emitted by the label in response to the
stimulation. If the label is a chemiluminescent label, the label is
quantified by detecting the light emitted either visually or by
using luminometers, x-ray film, high speed photographic film, a CCD
camera, etc. Once the amount of the label in the complex has been
quantified, the concentration of analyte or a fragment thereof in
the test sample is determined by appropriate means, such as by use
of a standard curve that has been generated using serial dilutions
of analyte or a fragment thereof of known concentration. Other than
using serial dilutions of analyte or a fragment thereof, the
standard curve can be generated gravimetrically, by mass
spectroscopy and by other techniques known in the art.
[0394] In a chemiluminescent microparticle assay employing the
ARCHITECT.RTM. analyzer, the conjugate diluent pH should be about
6.0+/-0.2, the microparticle coating buffer should be maintained at
about room temperature (i.e., at from about 17 to about 27.degree.
C.), the microparticle coating buffer pH should be about 6.5+/-0.2,
and the microparticle diluent pH should be about 7.8+/-0.2. Solids
preferably are less than about 0.2%, such as less than about 0.15%,
less than about 0.14%, less than about 0.13%, less than about
0.12%, or less than about 0.11%, such as about 0.10%.
[0395] FPIAs are based on competitive binding immunoassay
principles. A fluorescently labeled compound, when excited by a
linearly polarized light, will emit fluorescence having a degree of
polarization inversely proportional to its rate of rotation. When a
fluorescently labeled tracer-antibody complex is excited by a
linearly polarized light, the emitted light remains highly
polarized because the fluorophore is constrained from rotating
between the time light is absorbed and the time light is emitted.
When a "free" tracer compound (i.e., a compound that is not bound
to an antibody) is excited by linearly polarized light, its
rotation is much faster than the corresponding tracer-antibody
conjugate (or tracer-binding protein and/or tracer-DVD-Ig in
accordance with the present disclosure) produced in a competitive
binding immunoassay. FPIAs are advantageous over RIAs inasmuch as
there are no radioactive substances requiring special handling and
disposal. In addition, FPIAs are homogeneous assays that can be
easily and rapidly performed.
[0396] In view of the above, a method of determining the presence,
amount, or concentration of analyte (or a fragment thereof) in a
test sample is provided. The method comprises assaying the test
sample for an analyte (or a fragment thereof) by an assay (i)
employing (i') at least one of an antibody, a fragment of an
antibody that can bind to an analyte, a variant of an antibody that
can bind to an analyte, a fragment of a variant of an antibody that
can bind to an analyte, a binding protein as disclosed herein, and
a DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof) that can bind to an analyte, and (ii') at least one
detectable label and (ii) comprising comparing a signal generated
by the detectable label as a direct or indirect indication of the
presence, amount or concentration of analyte (or a fragment
thereof) in the test sample to a signal generated as a direct or
indirect indication of the presence, amount or concentration of
analyte (or a fragment thereof) in a control or calibrator. The
calibrator is optionally part of a series of calibrators, in which
each of the calibrators differs from the other calibrators by the
concentration of analyte.
[0397] The method can comprise (i) contacting the test sample with
at least one first specific binding partner for analyte (or a
fragment thereof) selected from the group consisting of an
antibody, a fragment of an antibody that can bind to an analyte, a
variant of an antibody that can bind to an analyte, a fragment of a
variant of an antibody that can bind to an analyte, a binding
protein as disclosed herein, and a DVD-Ig (or a fragment, a
variant, or a fragment of a variant thereof) that can bind to an
analyte so as to form a first specific binding partner/analyte (or
fragment thereof) complex, (ii) contacting the first specific
binding partner/analyte (or fragment thereof) complex with at least
one second specific binding partner for analyte (or fragment
thereof) selected from the group consisting of a detectably labeled
anti-analyte antibody, a detectably labeled fragment of an
anti-analyte antibody that can bind to analyte, a detectably
labeled variant of an anti-analyte antibody that can bind to
analyte, a detectably labeled fragment of a variant of an
anti-analyte antibody that can bind to analyte, a detectably
labeled binding protein as disclosed herein that can bind to
analyte, and a detectably labeled DVD-Ig (or a fragment, a variant,
or a fragment of a variant thereof) so as to form a first specific
binding partner/analyte (or fragment thereof)/second specific
binding partner complex, and (iii) determining the presence, amount
or concentration of analyte in the test sample by detecting or
measuring the signal generated by the detectable label in the first
specific binding partner/analyte (or fragment thereof)/second
specific binding partner complex formed in (ii). A method in which
at least one first specific binding partner for analyte (or a
fragment thereof) and/or at least one second specific binding
partner for analyte (or a fragment thereof) is a binding protein as
disclosed herein or a DVD-Ig (or a fragment, a variant, or a
fragment of a variant thereof) as described herein can be
preferred.
[0398] Alternatively, the method can comprise contacting the test
sample with at least one first specific binding partner for analyte
(or a fragment thereof) selected from the group consisting of an
antibody, a fragment of an antibody that can bind to an analyte, a
variant of an antibody that can bind to an analyte, a fragment of a
variant of an antibody that can bind to an analyte, a binding
protein as disclosed herein, and a DVD-Ig (or a fragment, a
variant, or a fragment of a variant thereof) and simultaneously or
sequentially, in either order, contacting the test sample with at
least one second specific binding partner, which can compete with
analyte (or a fragment thereof) for binding to the at least one
first specific binding partner and which is selected from the group
consisting of a detectably labeled analyte, a detectably labeled
fragment of analyte that can bind to the first specific binding
partner, a detectably labeled variant of analyte that can bind to
the first specific binding partner, and a detectably labeled
fragment of a variant of analyte that can bind to the first
specific binding partner. Any analyte (or a fragment thereof)
present in the test sample and the at least one second specific
binding partner compete with each other to form a first specific
binding partner/analyte (or fragment thereof) complex and a first
specific binding partner/second specific binding partner complex,
respectively. The method further comprises determining the
presence, amount or concentration of analyte in the test sample by
detecting or measuring the signal generated by the detectable label
in the first specific binding partner/second specific binding
partner complex formed in (ii), wherein the signal generated by the
detectable label in the first specific binding partner/second
specific binding partner complex is inversely proportional to the
amount or concentration of analyte in the test sample.
[0399] The above methods can further comprise diagnosing,
prognosticating, or assessing the efficacy of a
therapeutic/prophylactic treatment of a patient from whom the test
sample was obtained. If the method further comprises assessing the
efficacy of a therapeutic/prophylactic treatment of the patient
from whom the test sample was obtained, the method optionally
further comprises modifying the therapeutic/prophylactic treatment
of the patient as needed to improve efficacy. The method can be
adapted for use in an automated system or a semi-automated
system.
[0400] More specifically, a method of determining the presence,
amount or concentration of an antigen (or a fragment thereof) in a
test sample is provided. The method comprises assaying the test
sample for the antigen (or a fragment thereof) by an immunoassay.
The immunoassay (i) employs at least one binding protein and at
least one detectable label and (ii) comprises comparing a signal
generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of the antigen
(or a fragment thereof) in the test sample to a signal generated as
a direct or indirect indication of the presence, amount or
concentration of the antigen (or a fragment thereof) in a control
or a calibrator. The calibrator is optionally part of a series of
calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of the antigen (or a
fragment thereof). One of the at least one binding protein (i')
comprises a polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, in
which VD1 is a first heavy chain variable domain obtained from a
first parent antibody (or antigen binding portion thereof), VD2 is
a second heavy chain variable domain obtained from a second parent
antibody (or antigen binding portion thereof), which can be the
same as or different from the first parent antibody, C is a heavy
chain constant domain, (X1)n is a linker, which is optionally
present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and (ii') can bind a pair of
antigens. The method can comprise (i) contacting the test sample
with at least one capture agent, which binds to an epitope on the
antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, (ii) contacting the
capture agent/antigen (or a fragment thereof) complex with at least
one detection agent, which comprises a detectable label and binds
to an epitope on the antigen (or a fragment thereof) that is not
bound by the capture agent, to form a capture agent/antigen (or a
fragment thereof)/detection agent complex, and (iii) determining
the presence, amount or concentration of the antigen (or a fragment
thereof) in the test sample based on the signal generated by the
detectable label in the capture agent/antigen (or a fragment
thereof)/detection agent complex formed in (ii), wherein at least
one capture agent and/or at least one detection agent is the at
least one binding protein. Alternatively, the method can comprise
(i) contacting the test sample with at least one capture agent,
which binds to an epitope on the antigen (or a fragment thereof) so
as to form a capture agent/antigen (or a fragment thereof) complex,
and simultaneously or sequentially, in either order, contacting the
test sample with detectably labeled antigen (or a fragment
thereof), which can compete with any antigen (or a fragment
thereof) in the test sample for binding to the at least one capture
agent, wherein any antigen (or a fragment thereof) present in the
test sample and the detectably labeled antigen compete with each
other to form a capture agent/antigen (or a fragment thereof)
complex and a capture agent/detectably labeled antigen (or a
fragment thereof) complex, respectively, and (ii) determining the
presence, amount or concentration of the antigen (or a fragment
thereof) in the test sample based on the signal generated by the
detectable label in the capture agent/detectably labeled antigen
(or a fragment thereof) complex formed in (ii), wherein at least
one capture agent is the at least one binding protein and wherein
the signal generated by the detectable label in the capture
agent/detectably labeled antigen (or a fragment thereof) complex is
inversely proportional to the amount or concentration of antigen
(or a fragment thereof) in the test sample. The test sample can be
from a patient, in which case the method can further comprise
diagnosing, prognosticating, or assessing the efficacy of
therapeutic/prophylactic treatment of the patient. If the method
further comprises assessing the efficacy of
therapeutic/prophylactic treatment of the patient, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy. The method
can be adapted for use in an automated system or a semi-automated
system.
[0401] Another method of determining the presence, amount or
concentration of an antigen (or a fragment thereof) in a test
sample is provided. The method comprises assaying the test sample
for the antigen (or a fragment thereof) by an immunoassay. The
immunoassay (i) employs at least one binding protein and at least
one detectable label and (ii) comprises comparing a signal
generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of the antigen
(or a fragment thereof) in the test sample to a signal generated as
a direct or indirect indication of the presence, amount or
concentration of the antigen (or a fragment thereof) in a control
or a calibrator. The calibrator is optionally part of a series of
calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of the antigen (or a
fragment thereof). One of the at least one binding protein (i')
comprises a polypeptide chain comprising VD1-(X1)n-VD2-C-(X2)n, in
which VD1 is a first light chain variable domain obtained from a
first parent antibody (or antigen binding portion thereof), VD2 is
a second light chain variable domain obtained from a second parent
antibody (or antigen binding portion thereof), which can be the
same as or different from the first parent antibody, C is a light
chain constant domain, (X1)n is a linker, which is optionally
present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and (ii') can bind a pair of
antigens. The method can comprise (i) contacting the test sample
with at least one capture agent, which binds to an epitope on the
antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, (ii) contacting the
capture agent/antigen (or a fragment thereof) complex with at least
one detection agent, which comprises a detectable label and binds
to an epitope on the antigen (or a fragment thereof) that is not
bound by the capture agent, to form a capture agent/antigen (or a
fragment thereof)/detection agent complex, and (iii) determining
the presence, amount or concentration of the antigen (or a fragment
thereof) in the test sample based on the signal generated by the
detectable label in the capture agent/antigen (or a fragment
thereof)/detection agent complex formed in (ii), wherein at least
one capture agent and/or at least one detection agent is the at
least one binding protein. Alternatively, the method can comprise
(i) contacting the test sample with at least one capture agent,
which binds to an epitope on the antigen (or a fragment thereof) so
as to form a capture agent/antigen (or a fragment thereof) complex,
and simultaneously or sequentially, in either order, contacting the
test sample with detectably labeled antigen (or a fragment
thereof), which can compete with any antigen (or a fragment
thereof) in the test sample for binding to the at least one capture
agent, wherein any antigen (or a fragment thereof) present in the
test sample and the detectably labeled antigen compete with each
other to form a capture agent/antigen (or a fragment thereof)
complex and a capture agent/detectably labeled antigen (or a
fragment thereof) complex, respectively, and
[0402] (ii) determining the presence, amount or concentration of
the antigen (or a fragment thereof) in the test sample based on the
signal generated by the detectable label in the capture
agent/detectably labeled antigen (or a fragment thereof) complex
formed in (ii), wherein at least one capture agent is the at least
one binding protein and wherein the signal generated by the
detectable label in the capture agent/detectably labeled antigen
(or a fragment thereof) complex is inversely proportional to the
amount or concentration of antigen (or a fragment thereof) in the
test sample. If the test sample is from a patient, the method can
further comprise diagnosing, prognosticating, or assessing the
efficacy of therapeutic/prophylactic treatment of the patient. If
the method further comprises assessing the efficacy of
therapeutic/prophylactic treatment of the patient, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy. The method
can be adapted for use in an automated system or a semi-automated
system.
[0403] Yet another method of determining the presence, amount or
concentration of an antigen (or a fragment thereof) in a test
sample is provided. The method comprises assaying the test sample
for the antigen (or a fragment thereof) by an immunoassay. The
immunoassay (i) employs at least one binding protein and at least
one detectable label and (ii) comprises comparing a signal
generated by the detectable label as a direct or indirect
indication of the presence, amount or concentration of the antigen
(or a fragment thereof) in the test sample to a signal generated as
a direct or indirect indication of the presence, amount or
concentration of the antigen (or a fragment thereof) in a control
or a calibrator. The calibrator is optionally part of a series of
calibrators in which each of the calibrators differs from the other
calibrators in the series by the concentration of the antigen (or a
fragment thereof). One of the at least one binding protein (i')
comprises a first polypeptide chain and a second polypeptide chain,
wherein the first polypeptide chain comprises a first
VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavy chain variable
domain obtained from a first parent antibody (or antigen binding
portion thereof), VD2 is a second heavy chain variable domain
obtained from a second parent antibody (or antigen binding portion
thereof), which can be the same as or different from the first
parent antibody, C is a heavy chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and wherein the second polypeptide chain comprises a second
VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain variable
domain obtained from a first parent antibody (or antigen binding
portion thereof), VD2 is a second light chain variable domain
obtained from a second parent antibody (or antigen binding portion
thereof), which can be the same as or different from the first
parent antibody, C is a light chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and (ii') can bind a pair of antigens. The method can comprise (i)
contacting the test sample with at least one capture agent, which
binds to an epitope on the antigen (or a fragment thereof) so as to
form a capture agent/antigen (or a fragment thereof) complex, (ii)
contacting the capture agent/antigen (or a fragment thereof)
complex with at least one detection agent, which comprises a
detectable label and binds to an epitope on the antigen (or a
fragment thereof) that is not bound by the capture agent, to form a
capture agent/antigen (or a fragment thereof)/detection agent
complex, and (iii) determining the presence, amount or
concentration of the antigen (or a fragment thereof) in the test
sample based on the signal generated by the detectable label in the
capture agent/antigen (or a fragment thereof)/detection agent
complex formed in (ii), wherein at least one capture agent and/or
at least one detection agent is the at least one binding protein.
Alternatively, the method can comprise (i) contacting the test
sample with at least one capture agent, which binds to an epitope
on the antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, and simultaneously
or sequentially, in either order, contacting the test sample with
detectably labeled antigen (or a fragment thereof), which can
compete with any antigen (or a fragment thereof) in the test sample
for binding to the at least one capture agent, wherein any antigen
(or a fragment thereof) present in the test sample and the
detectably labeled antigen compete with each other to form a
capture agent/antigen (or a fragment thereof) complex and a capture
agent/detectably labeled antigen (or a fragment thereof) complex,
respectively, and (ii) determining the presence, amount or
concentration of the antigen (or a fragment thereof) in the test
sample based on the signal generated by the detectable label in the
capture agent/detectably labeled antigen (or a fragment thereof)
complex formed in (ii), wherein at least one capture agent is the
at least one binding protein and wherein the signal generated by
the detectable label in the capture agent/detectably labeled
antigen (or a fragment thereof) complex is inversely proportional
to the amount or concentration of antigen (or a fragment thereof)
in the test sample. If the test sample is from a patient, the
method can further comprise diagnosing, prognosticating, or
assessing the efficacy of therapeutic/prophylactic treatment of the
patient. If the method further comprises assessing the efficacy of
therapeutic/prophylactic treatment of the patient, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy. The method
can be adapted for use in an automated system or a semi-automated
system.
[0404] Still yet another method of determining the presence, amount
or concentration of an antigen (or a fragment thereof) in a test
sample is provided. The method comprises assaying the test sample
for the antigen (or a fragment thereof) by an immunoassay. The
immunoassay (i) employs at least one DVD-Ig that can bind two
antigens and at least one detectable label and (ii) comprises
comparing a signal generated by the detectable label as a direct or
indirect indication of the presence, amount or concentration of the
antigen (or a fragment thereof) in the test sample to a signal
generated as a direct or indirect indication of the presence,
amount or concentration of the antigen (or a fragment thereof) in a
control or a calibrator. The calibrator is optionally part of a
series of calibrators in which each of the calibrators differs from
the other calibrators in the series by the concentration of the
antigen (or a fragment thereof). One of the at least one DVD-Ig
(i') comprises four polypeptide chains, wherein the first and third
polypeptide chains comprise a first VD1-(X1)n-VD2-C-(X2)n, in which
VD1 is a first heavy chain variable domain obtained from a first
parent antibody (or antigen binding portion thereof), VD2 is a
second heavy chain variable domain obtained from a second parent
antibody (or antigen binding portion thereof), which can be the
same as or different from the first parent antibody, C is a heavy
chain constant domain, (X1)n is a linker, which is optionally
present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and wherein the second and
fourth polypeptide chains comprise a second VD1-(X1)n-VD2-C-(X2)n,
in which VD1 is a first light chain variable domain obtained from a
first parent antibody (or antigen binding portion thereof), VD2 is
a second light chain variable domain obtained from a second parent
antibody (or antigen binding portion thereof), which can be the
same as or different from the first parent antibody, C is a light
chain constant domain, (X1)n is a linker, which is optionally
present and, when present, is other than CH1, and (X2)n is an Fc
region, which is optionally present, and (ii') can bind two
antigens (or fragments thereof). The method can comprise (i)
contacting the test sample with at least one capture agent, which
binds to an epitope on the antigen (or a fragment thereof) so as to
form a capture agent/antigen (or a fragment thereof) complex, (ii)
contacting the capture agent/antigen (or a fragment thereof)
complex with at least one detection agent, which comprises a
detectable label and binds to an epitope on the antigen (or a
fragment thereof) that is not bound by the capture agent, to form a
capture agent/antigen (or a fragment thereof)/detection agent
complex, and (iii) determining the presence, amount or
concentration of the antigen (or a fragment thereof) in the test
sample based on the signal generated by the detectable label in the
capture agent/antigen (or a fragment thereof)/detection agent
complex formed in (ii), wherein at least one capture agent and/or
at least one detection agent is the at least one DVD-Ig.
Alternatively, the method can comprise (i) contacting the test
sample with at least one capture agent, which binds to an epitope
on the antigen (or a fragment thereof) so as to form a capture
agent/antigen (or a fragment thereof) complex, and simultaneously
or sequentially, in either order, contacting the test sample with
detectably labeled antigen (or a fragment thereof), which can
compete with any antigen (or a fragment thereof) in the test sample
for binding to the at least one capture agent, wherein any antigen
(or a fragment thereof) present in the test sample and the
detectably labeled antigen compete with each other to form a
capture agent/antigen (or a fragment thereof) complex and a capture
agent/detectably labeled antigen (or a fragment thereof) complex,
respectively, and (ii) determining the presence, amount or
concentration of the antigen (or a fragment thereof) in the test
sample based on the signal generated by the detectable label in the
capture agent/detectably labeled antigen (or a fragment thereof)
complex formed in (ii), wherein at least one capture agent is the
at least one DVD-Ig and wherein the signal generated by the
detectable label in the capture agent/detectably labeled antigen
(or a fragment thereof) complex is inversely proportional to the
amount or concentration of antigen (or a fragment thereof) in the
test sample. If the test sample is from a patient, the method can
further comprise diagnosing, prognosticating, or assessing the
efficacy of therapeutic/prophylactic treatment of the patient. If
the method further comprises assessing the efficacy of
therapeutic/prophylactic treatment of the patient, the method
optionally further comprises modifying the therapeutic/prophylactic
treatment of the patient as needed to improve efficacy. The method
can be adapted for use in an automated system or a semi-automated
system.
[0405] With regard to the methods of assay (and kit therefor), it
may be possible to employ commercially available anti-analyte
antibodies or methods for production of anti-analyte as described
in the literature. Commercial supplies of various antibodies
include, but are not limited to, Santa Cruz Biotechnology Inc.
(Santa Cruz, Calif.), GenWay Biotech, Inc. (San Diego, Calif.), and
R&D Systems (RDS; Minneapolis, Minn.).
[0406] Generally, a predetermined level can be employed as a
benchmark against which to assess results obtained upon assaying a
test sample for analyte or a fragment thereof, e.g., for detecting
disease or risk of disease. Generally, in making such a comparison,
the predetermined level is obtained by running a particular assay a
sufficient number of times and under appropriate conditions such
that a linkage or association of analyte presence, amount or
concentration with a particular stage or endpoint of a disease,
disorder or condition or with particular clinical indicia can be
made. Typically, the predetermined level is obtained with assays of
reference subjects (or populations of subjects). The analyte
measured can include fragments thereof, degradation products
thereof, and/or enzymatic cleavage products thereof.
[0407] In particular, with respect to a predetermined level as
employed for monitoring disease progression and/or treatment, the
amount or concentration of analyte or a fragment thereof may be
"unchanged," "favorable" (or "favorably altered"), or "unfavorable"
(or "unfavorably altered"). "Elevated" or "increased" refers to an
amount or a concentration in a test sample that is higher than a
typical or normal level or range (e.g., predetermined level), or is
higher than another reference level or range (e.g., earlier or
baseline sample). The term "lowered" or "reduced" refers to an
amount or a concentration in a test sample that is lower than a
typical or normal level or range (e.g., predetermined level), or is
lower than another reference level or range (e.g., earlier or
baseline sample). The term "altered" refers to an amount or a
concentration in a sample that is altered (increased or decreased)
over a typical or normal level or range (e.g., predetermined
level), or over another reference level or range (e.g., earlier or
baseline sample).
[0408] The typical or normal level or range for analyte is defined
in accordance with standard practice. Because the levels of analyte
in some instances will be very low, a so-called altered level or
alteration can be considered to have occurred when there is any net
change as compared to the typical or normal level or range, or
reference level or range, that cannot be explained by experimental
error or sample variation. Thus, the level measured in a particular
sample will be compared with the level or range of levels
determined in similar samples from a so-called normal subject. In
this context, a "normal subject" is an individual with no
detectable disease, for example, and a "normal" (sometimes termed
"control") patient or population is/are one(s) that exhibit(s) no
detectable disease, respectively, for example. Furthermore, given
that analyte is not routinely found at a high level in the majority
of the human population, a "normal subject" can be considered an
individual with no substantial detectable increased or elevated
amount or concentration of analyte, and a "normal" (sometimes
termed "control") patient or population is/are one(s) that
exhibit(s) no substantial detectable increased or elevated amount
or concentration of analyte. An "apparently normal subject" is one
in which analyte has not yet been or currently is being assessed.
The level of an analyte is said to be "elevated" when the analyte
is normally undetectable (e.g., the normal level is zero, or within
a range of from about 25 to about 75 percentiles of normal
populations), but is detected in a test sample, as well as when the
analyte is present in the test sample at a higher than normal
level. Thus, inter alia, the disclosure provides a method of
screening for a subject having, or at risk of having, a particular
disease, disorder, or condition. The method of assay can also
involve the assay of other markers and the like.
[0409] Accordingly, the methods described herein also can be used
to determine whether or not a subject has or is at risk of
developing a given disease, disorder or condition. Specifically,
such a method can comprise the steps of:
[0410] (a) determining the concentration or amount in a test sample
from a subject of analyte (or a fragment thereof) (e.g., using the
methods described herein, or methods known in the art); and
[0411] (b) comparing the concentration or amount of analyte (or a
fragment thereof) determined in step (a) with a predetermined
level, wherein, if the concentration or amount of analyte
determined in step (a) is favorable with respect to a predetermined
level, then the subject is determined not to have or be at risk for
a given disease, disorder or condition. However, if the
concentration or amount of analyte determined in step (a) is
unfavorable with respect to the predetermined level, then the
subject is determined to have or be at risk for a given disease,
disorder or condition.
[0412] Additionally, provided herein is method of monitoring the
progression of disease in a subject. Optimally the method
comprising the steps of:
[0413] (a) determining the concentration or amount in a test sample
from a subject of analyte;
[0414] (b) determining the concentration or amount in a later test
sample from the subject of analyte; and
[0415] (c) comparing the concentration or amount of analyte as
determined in step (b) with the concentration or amount of analyte
determined in step (a), wherein if the concentration or amount
determined in step (b) is unchanged or is unfavorable when compared
to the concentration or amount of analyte determined in step (a),
then the disease in the subject is determined to have continued,
progressed or worsened. By comparison, if the concentration or
amount of analyte as determined in step (b) is favorable when
compared to the concentration or amount of analyte as determined in
step (a), then the disease in the subject is determined to have
discontinued, regressed or improved.
[0416] Optionally, the method further comprises comparing the
concentration or amount of analyte as determined in step (b), for
example, with a predetermined level. Further, optionally the method
comprises treating the subject with one or more pharmaceutical
compositions for a period of time if the comparison shows that the
concentration or amount of analyte as determined in step (b), for
example, is unfavorably altered with respect to the predetermined
level.
[0417] Still further, the methods can be used to monitor treatment
in a subject receiving treatment with one or more pharmaceutical
compositions. Specifically, such methods involve providing a first
test sample from a subject before the subject has been administered
one or more pharmaceutical compositions. Next, the concentration or
amount in a first test sample from a subject of analyte is
determined (e.g., using the methods described herein or as known in
the art). After the concentration or amount of analyte is
determined, optionally the concentration or amount of analyte is
then compared with a predetermined level. If the concentration or
amount of analyte as determined in the first test sample is lower
than the predetermined level, then the subject is not treated with
one or more pharmaceutical compositions. However, if the
concentration or amount of analyte as determined in the first test
sample is higher than the predetermined level, then the subject is
treated with one or more pharmaceutical compositions for a period
of time. The period of time that the subject is treated with the
one or more pharmaceutical compositions can be determined by one
skilled in the art (for example, the period of time can be from
about seven (7) days to about two years, preferably from about
fourteen (14) days to about one (1) year).
[0418] During the course of treatment with the one or more
pharmaceutical compositions, second and subsequent test samples are
then obtained from the subject. The number of test samples and the
time in which said test samples are obtained from the subject are
not critical. For example, a second test sample could be obtained
seven (7) days after the subject is first administered the one or
more pharmaceutical compositions, a third test sample could be
obtained two (2) weeks after the subject is first administered the
one or more pharmaceutical compositions, a fourth test sample could
be obtained three (3) weeks after the subject is first administered
the one or more pharmaceutical compositions, a fifth test sample
could be obtained four (4) weeks after the subject is first
administered the one or more pharmaceutical compositions, etc.
[0419] After each second or subsequent test sample is obtained from
the subject, the concentration or amount of analyte is determined
in the second or subsequent test sample is determined (e.g., using
the methods described herein or as known in the art). The
concentration or amount of analyte as determined in each of the
second and subsequent test samples is then compared with the
concentration or amount of analyte as determined in the first test
sample (e.g., the test sample that was originally optionally
compared to the predetermined level). If the concentration or
amount of analyte as determined in step (c) is favorable when
compared to the concentration or amount of analyte as determined in
step (a), then the disease in the subject is determined to have
discontinued, regressed or improved, and the subject should
continue to be administered the one or pharmaceutical compositions
of step (b). However, if the concentration or amount determined in
step (c) is unchanged or is unfavorable when compared to the
concentration or amount of analyte as determined in step (a), then
the disease in the subject is determined to have continued,
progressed or worsened, and the subject should be treated with a
higher concentration of the one or more pharmaceutical compositions
administered to the subject in step (b) or the subject should be
treated with one or more pharmaceutical compositions that are
different from the one or more pharmaceutical compositions
administered to the subject in step (b). Specifically, the subject
can be treated with one or more pharmaceutical compositions that
are different from the one or more pharmaceutical compositions that
the subject had previously received to decrease or lower said
subject's analyte level.
[0420] Generally, for assays in which repeat testing may be done
(e.g., monitoring disease progression and/or response to
treatment), a second or subsequent test sample is obtained at a
period in time after the first test sample has been obtained from
the subject. Specifically, a second test sample from the subject
can be obtained minutes, hours, days, weeks or years after the
first test sample has been obtained from the subject. For example,
the second test sample can be obtained from the subject at a time
period of about 1 minute, about 5 minutes, about 10 minutes, about
15 minutes, about 30 minutes, about 45 minutes, about 60 minutes,
about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6
hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours,
about 11 hours, about 12 hours, about 13 hours, about 14 hours,
about 15 hours, about 16 hours, about 17 hours, about 18 hours,
about 19 hours, about 20 hours, about 21 hours, about 22 hours,
about 23 hours, about 24 hours, about 2 days, about 3 days, about 4
days, about 5 days, about 6 days, about 7 days, about 2 weeks,
about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7
weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11
weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15
weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19
weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23
weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27
weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31
weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35
weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39
weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43
weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47
weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51
weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5
years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5
years, about 5.0 years, about 5.5. years, about 6.0 years, about
6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about
8.5 years, about 9.0 years, about 9.5 years or about 10.0 years
after the first test sample from the subject is obtained.
[0421] When used to monitor disease progression, the above assay
can be used to monitor the progression of disease in subjects
suffering from acute conditions. Acute conditions, also known as
critical care conditions, refer to acute, life-threatening diseases
or other critical medical conditions involving, for example, the
cardiovascular system or excretory system. Typically, critical care
conditions refer to those conditions requiring acute medical
intervention in a hospital-based setting (including, but not
limited to, the emergency room, intensive care unit, trauma center,
or other emergent care setting) or administration by a paramedic or
other field-based medical personnel. For critical care conditions,
repeat monitoring is generally done within a shorter time frame,
namely, minutes, hours or days (e.g., about 1 minute, about 5
minutes, about 10 minutes, about 15 minutes, about 30 minutes,
about 45 minutes, about 60 minutes, about 2 hours, about 3 hours,
about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8
hours, about 9 hours, about 10 hours, about 11 hours, about 12
hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, about 24
hours, about 2 days, about 3 days, about 4 days, about 5 days,
about 6 days or about 7 days), and the initial assay likewise is
generally done within a shorter timeframe, e.g., about minutes,
hours or days of the onset of the disease or condition.
[0422] The assays also can be used to monitor the progression of
disease in subjects suffering from chronic or non-acute conditions.
Non-critical care or, non-acute conditions, refers to conditions
other than acute, life-threatening disease or other critical
medical conditions involving, for example, the cardiovascular
system and/or excretory system. Typically, non-acute conditions
include those of longer-term or chronic duration. For non-acute
conditions, repeat monitoring generally is done with a longer
timeframe, e.g., hours, days, weeks, months or years (e.g., about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
10 hours, about 11 hours, about 12 hours, about 13 hours, about 14
hours, about 15 hours, about 16 hours, about 17 hours, about 18
hours, about 19 hours, about 20 hours, about 21 hours, about 22
hours, about 23 hours, about 24 hours, about 2 days, about 3 days,
about 4 days, about 5 days, about 6 days, about 7 days, about 2
weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks,
about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about
11 weeks, about 12 weeks, about 13 weeks, about 14 weeks, about 15
weeks, about 16 weeks, about 17 weeks, about 18 weeks, about 19
weeks, about 20 weeks, about 21 weeks, about 22 weeks, about 23
weeks, about 24 weeks, about 25 weeks, about 26 weeks, about 27
weeks, about 28 weeks, about 29 weeks, about 30 weeks, about 31
weeks, about 32 weeks, about 33 weeks, about 34 weeks, about 35
weeks, about 36 weeks, about 37 weeks, about 38 weeks, about 39
weeks, about 40 weeks, about 41 weeks, about 42 weeks, about 43
weeks, about 44 weeks, about 45 weeks, about 46 weeks, about 47
weeks, about 48 weeks, about 49 weeks, about 50 weeks, about 51
weeks, about 52 weeks, about 1.5 years, about 2 years, about 2.5
years, about 3.0 years, about 3.5 years, about 4.0 years, about 4.5
years, about 5.0 years, about 5.5. years, about 6.0 years, about
6.5 years, about 7.0 years, about 7.5 years, about 8.0 years, about
8.5 years, about 9.0 years, about 9.5 years or about 10.0 years),
and the initial assay likewise generally is done within a longer
time frame, e.g., about hours, days, months or years of the onset
of the disease or condition.
[0423] Furthermore, the above assays can be performed using a first
test sample obtained from a subject where the first test sample is
obtained from one source, such as urine, serum or plasma.
Optionally, the above assays can then be repeated using a second
test sample obtained from the subject where the second test sample
is obtained from another source. For example, if the first test
sample was obtained from urine, the second test sample can be
obtained from serum or plasma. The results obtained from the assays
using the first test sample and the second test sample can be
compared. The comparison can be used to assess the status of a
disease or condition in the subject.
[0424] Moreover, the present disclosure also relates to methods of
determining whether a subject predisposed to or suffering from a
given disease, disorder or condition will benefit from treatment.
In particular, the disclosure relates to analyte companion
diagnostic methods and products. Thus, the method of "monitoring
the treatment of disease in a subject" as described herein further
optimally also can encompass selecting or identifying candidates
for therapy.
[0425] Thus, in particular embodiments, the disclosure also
provides a method of determining whether a subject having, or at
risk for, a given disease, disorder or condition is a candidate for
therapy. Generally, the subject is one who has experienced some
symptom of a given disease, disorder or condition or who has
actually been diagnosed as having, or being at risk for, a given
disease, disorder or condition, and/or who demonstrates an
unfavorable concentration or amount of analyte or a fragment
thereof, as described herein.
[0426] The method optionally comprises an assay as described
herein, where analyte is assessed before and following treatment of
a subject with one or more pharmaceutical compositions (e.g.,
particularly with a pharmaceutical related to a mechanism of action
involving analyte), with immunosuppressive therapy, or by
immunoabsorption therapy, or where analyte is assessed following
such treatment and the concentration or the amount of analyte is
compared against a predetermined level. An unfavorable
concentration of amount of analyte observed following treatment
confirms that the subject will not benefit from receiving further
or continued treatment, whereas a favorable concentration or amount
of analyte observed following treatment confirms that the subject
will benefit from receiving further or continued treatment. This
confirmation assists with management of clinical studies, and
provision of improved patient care.
[0427] It goes without saying that, while certain embodiments
herein are advantageous when employed to assess a given disease,
disorder or condition as discussed herein, the assays and kits can
be employed to assess analyte in other diseases, disorders and
conditions. The method of assay can also involve the assay of other
markers and the like.
[0428] The method of assay also can be used to identify a compound
that ameliorates a given disease, disorder or condition. For
example, a cell that expresses analyte can be contacted with a
candidate compound. The level of expression of analyte in the cell
contacted with the compound can be compared to that in a control
cell using the method of assay described herein.
B. Kit
[0429] A kit for assaying a test sample for the presence, amount or
concentration of an analyte (or a fragment thereof) in a test
sample is also provided. The kit comprises at least one component
for assaying the test sample for the analyte (or a fragment
thereof) and instructions for assaying the test sample for the
analyte (or a fragment thereof). The at least one component for
assaying the test sample for the analyte (or a fragment thereof)
can include a composition comprising a binding protein as disclosed
herein and/or an anti-analyte DVD-Ig (or a fragment, a variant, or
a fragment of a variant thereof), which is optionally immobilized
on a solid phase.
[0430] The kit can comprise at least one component for assaying the
test sample for an analyte by immunoassay, e.g., chemiluminescent
microparticle immunoassay, and instructions for assaying the test
sample for an analyte by immunoassay, e.g., chemiluminescent
microparticle immunoassay. For example, the kit can comprise at
least one specific binding partner for an analyte, such as an
anti-analyte, monoclonal/polyclonal antibody (or a fragment thereof
that can bind to the analyte, a variant thereof that can bind to
the analyte, or a fragment of a variant that can bind to the
analyte) a binding protein as disclosed herein or an anti-analyte
DVD-Ig (or a fragment, a variant, or a fragment of a variant
thereof), either of which can be detectably labeled. Alternatively
or additionally, the kit can comprise detectably labeled analyte
(or a fragment thereof that can bind to an anti-analyte,
monoclonal/polyclonal antibody a binding protein as disclosed
herein, or an anti-analyte DVD-Ig (or a fragment, a variant, or a
fragment of a variant thereof)), which can compete with any analyte
in a test sample for binding to an anti-analyte,
monoclonal/polyclonal antibody (or a fragment thereof that can bind
to the analyte, a variant thereof that can bind to the analyte, or
a fragment of a variant that can bind to the analyte), a binding
protein as disclosed herein, or an anti-analyte DVD-Ig (or a
fragment, a variant, or a fragment of a variant thereof), either of
which can be immobilized on a solid support. The kit can comprise a
calibrator or control, e.g., isolated or purified analyte. The kit
can comprise at least one container (e.g., tube, microtiter plates
or strips, which can be already coated with a first specific
binding partner, for example) for conducting the assay, and/or a
buffer, such as an assay buffer or a wash buffer, either one of
which can be provided as a concentrated solution, a substrate
solution for the detectable label (e.g., an enzymatic label), or a
stop solution. Preferably, the kit comprises all components, i.e.,
reagents, standards, buffers, diluents, etc., which are necessary
to perform the assay. The instructions can be in paper form or
computer-readable form, such as a disk, CD, DVD, or the like.
[0431] More specifically, provided is a kit for assaying a test
sample for an antigen (or a fragment thereof). The kit comprises at
least one component for assaying the test sample for an antigen (or
a fragment thereof) and instructions for assaying the test sample
for an antigen (or a fragment thereof), wherein the at least one
component includes at least one composition comprising a binding
protein, which (i') comprises a polypeptide chain comprising
VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavy chain variable
domain obtained from a first parent antibody (or antigen binding
portion thereof), VD2 is a second heavy chain variable domain
obtained from a second parent antibody (or antigen binding portion
thereof), which can be same as or different from the first parent
antibody, C is a heavy chain constant domain, (X1)n is a linker,
which is optionally present and, when present, is other than CH1,
and (X2)n is an Fc region, which is optionally present, and (ii')
can bind a pair of antigens, wherein the binding protein is
optionally detectably labeled.
[0432] Further provided is another kit for assaying a test sample
for an antigen (or a fragment thereof). The kit comprises at least
one component for assaying the test sample for an antigen (or a
fragment thereof) and instructions for assaying the test sample for
an antigen (or a fragment thereof), wherein the at least one
component includes at least one composition comprising a binding
protein, which (i') comprises a polypeptide chain comprising
VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain variable
domain obtained from a first parent antibody (or antigen binding
portion thereof), VD2 is a second light chain variable domain
obtained from a second parent antibody (or antigen binding portion
thereof), which can be the same as or different from the first
parent antibody, C is a light chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and (ii') can bind a pair of antigens, wherein the binding protein
is optionally detectably labeled.
[0433] Still further provided is another kit for assaying a test
sample for an antigen (or a fragment thereof). The kit comprises at
least one component for assaying the test sample for an antigen (or
a fragment thereof) and instructions for assaying the test sample
for an antigen (or a fragment thereof), wherein the at least one
component includes at least one composition comprising a binding
protein, which (i') comprises a first polypeptide chain and a
second polypeptide chain, wherein the first polypeptide chain
comprises a first VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first
heavy chain variable domain obtained from a first parent antibody
(or antigen binding portion thereof), VD2 is a second heavy chain
variable domain obtained from a second parent antibody (or antigen
binding portion thereof), which can be the same as or different
from the first parent antibody, C is a heavy chain constant domain,
(X1)n is a linker, which is optionally present and, when present,
is other than CH1, and (X2)n is an Fc region, which is optionally
present, and wherein the second polypeptide chain comprises a
second VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain
variable domain obtained from a first parent antibody (or antigen
binding portion thereof), VD2 is a second light chain variable
domain obtained from a second parent antibody (or antigen binding
portion thereof), which can be the same as or different from the
first parent antibody, C is a light chain constant domain, (X1)n is
a linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and (ii') can bind a pair of antigens, wherein the binding protein
is optionally detectably labeled.
[0434] Even still further provided is another kit for assaying a
test sample for an antigen (or a fragment thereof). The kit
comprises at least one component for assaying the test sample for
an antigen (or a fragment thereof) and instructions for assaying
the test sample for an antigen (or a fragment thereof), wherein the
at least one component includes at least one composition comprising
a DVD-Ig, which (i') comprises four polypeptide chains, wherein the
first and third polypeptide chains comprise a first
VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first heavy chain variable
domain obtained from a first parent antibody (or antigen binding
portion thereof), VD2 is a second heavy chain variable domain
obtained from a second parent antibody (or antigen binding portion
thereof), which can be the same as or different from the first
parent antibody, C is a heavy chain constant domain, (X1)n is a
linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and wherein the second and fourth polypeptide chains comprise a
second VD1-(X1)n-VD2-C-(X2)n, in which VD1 is a first light chain
variable domain obtained from a first parent antibody (or antigen
binding portion thereof), VD2 is a second light chain variable
domain obtained from a second parent antibody (or antigen binding
portion thereof), which can be the same as or different from the
first parent antibody, C is a light chain constant domain, (X1)n is
a linker, which is optionally present and, when present, is other
than CH1, and (X2)n is an Fc region, which is optionally present,
and (ii') can bind two antigens (or fragments thereof), wherein the
DVD-Ig is optionally detectably labeled.
[0435] Any antibodies, such as an anti-analyte antibody, any
binding proteins as disclosed herein, any anti-analyte DVD-Igs, or
tracers can incorporate a detectable label as described herein,
such as a fluorophore, a radioactive moiety, an enzyme, a
biotin/avidin label, a chromophore, a chemiluminescent label, or
the like, or the kit can include reagents for carrying out
detectable labeling. The antibodies, calibrators and/or controls
can be provided in separate containers or pre-dispensed into an
appropriate assay format, for example, into microtiter plates.
[0436] Optionally, the kit includes quality control components (for
example, sensitivity panels, calibrators, and positive controls).
Preparation of quality control reagents is well-known in the art
and is described on insert sheets for a variety of immunodiagnostic
products. Sensitivity panel members optionally are used to
establish assay performance characteristics, and further optionally
are useful indicators of the integrity of the immunoassay kit
reagents, and the standardization of assays.
[0437] The kit can also optionally include other reagents required
to conduct a diagnostic assay or facilitate quality control
evaluations, such as buffers, salts, enzymes, enzyme co-factors,
enzyme substrates, detection reagents, and the like. Other
components, such as buffers and solutions for the isolation and/or
treatment of a test sample (e.g., pretreatment reagents), also can
be included in the kit. The kit can additionally include one or
more other controls. One or more of the components of the kit can
be lyophilized, in which case the kit can further comprise reagents
suitable for the reconstitution of the lyophilized components.
[0438] The various components of the kit optionally are provided in
suitable containers as necessary, e.g., a microtiter plate. The kit
can further include containers for holding or storing a sample
(e.g., a container or cartridge for a urine sample). Where
appropriate, the kit optionally also can contain reaction vessels,
mixing vessels, and other components that facilitate the
preparation of reagents or the test sample. The kit can also
include one or more instruments for assisting with obtaining a test
sample, such as a syringe, pipette, forceps, measured spoon, or the
like.
[0439] If the detectable label is at least one acridinium compound,
the kit can comprise at least one acridinium-9-carboxamide, at
least one acridinium-9-carboxylate aryl ester, or any combination
thereof. If the detectable label is at least one acridinium
compound, the kit also can comprise a source of hydrogen peroxide,
such as a buffer, a solution, and/or at least one basic solution.
If desired, the kit can contain a solid phase, such as a magnetic
particle, bead, test tube, microtiter plate, cuvette, membrane,
scaffolding molecule, film, filter paper, disc or chip.
C. Adaptation of Kit and Method
[0440] The kit (or components thereof), as well as the method of
determining the presence, amount or concentration of an analyte in
a test sample by an assay, such as an immunoassay as described
herein, can be adapted for use in a variety of automated and
semi-automated systems (including those wherein the solid phase
comprises a microparticle), as described, e.g., in U.S. Pat. Nos.
5,089,424 and 5,006,309, and as commercially marketed, e.g., by
Abbott Laboratories (Abbott Park, Ill.) as ARCHITECT.RTM..
[0441] Some of the differences between an automated or
semi-automated system as compared to a non-automated system (e.g.,
ELISA) include the substrate to which the first specific binding
partner (e.g., an anti-analyte, monoclonal/polyclonal antibody (or
a fragment thereof, a variant thereof, or a fragment of a variant
thereof), a binding protein as disclosed herein, or an anti-analyte
DVD-Ig (or a fragment thereof, a variant thereof, or a fragment of
a variant thereof) is attached; either way, sandwich formation and
analyte reactivity can be impacted), and the length and timing of
the capture, detection and/or any optional wash steps. Whereas a
non-automated format, such as an ELISA, may require a relatively
longer incubation time with sample and capture reagent (e.g., about
2 hours), an automated or semi-automated format (e.g.,
ARCHITECT.RTM., Abbott Laboratories) may have a relatively shorter
incubation time (e.g., approximately 18 minutes for
ARCHITECT.RTM.). Similarly, whereas a non-automated format, such as
an ELISA, may incubate a detection antibody, such as the conjugate
reagent, for a relatively longer incubation time (e.g., about 2
hours), an automated or semi-automated format (e.g.,
ARCHITECT.RTM.) may have a relatively shorter incubation time
(e.g., approximately 4 minutes for the ARCHITECT.RTM.).
[0442] Other platforms available from Abbott Laboratories include,
but are not limited to, AxSYM.RTM., IMx.RTM. (see, e.g., U.S. Pat.
No. 5,294,404), PRISM.RTM., EIA (bead), and Quantum.TM. II, as well
as other platforms. Additionally, the assays, kits and kit
components can be employed in other formats, for example, on
electrochemical or other hand-held or point-of-care assay systems.
The present disclosure is, for example, applicable to the
commercial Abbott Point of Care (i-STAT.RTM., Abbott Laboratories)
electrochemical immunoassay system that performs sandwich
immunoassays. Immunosensors and their methods of manufacture and
operation in single-use test devices are described, for example in,
U.S. Pat. Nos. 5,063,081; 7,419,821; and 7,682,833; and U.S. Patent
Publication Nos. 20040018577 and 20060160164.
[0443] In particular, with regard to the adaptation of an analyte
assay to the I-STAT.RTM. system, the following configuration is
preferred. A microfabricated silicon chip is manufactured with a
pair of gold amperometric working electrodes and a silver-silver
chloride reference electrode. On one of the working electrodes,
polystyrene beads (0.2 mm diameter) with immobilized anti-analyte,
monoclonal/polyclonal antibody (or a fragment thereof, a variant
thereof, or a fragment of a variant thereof), a binding protein as
disclosed herein, or anti-analyte DVD-Ig (or a fragment thereof, a
variant thereof, or a fragment of a variant thereof), are adhered
to a polymer coating of patterned polyvinyl alcohol over the
electrode. This chip is assembled into an I-STAT.RTM. cartridge
with a fluidics format suitable for immunoassay. On a portion of
the wall of the sample-holding chamber of the cartridge there is a
layer comprising a specific binding partner for an analyte, such as
an anti-analyte, monoclonal/polyclonal antibody (or a fragment
thereof, a variant thereof, or a fragment of a variant thereof that
can bind the analyte), a binding protein as disclosed herein, or an
anti-analyte DVD-Ig (or a fragment thereof, a variant thereof, or a
fragment of a variant thereof that can bind the analyte), either of
which can be detectably labeled. Within the fluid pouch of the
cartridge is an aqueous reagent that includes p-aminophenol
phosphate.
[0444] In operation, a sample suspected of containing an analyte is
added to the holding chamber of the test cartridge, and the
cartridge is inserted into the I-STAT.RTM. reader. After the
specific binding partner for an analyte has dissolved into the
sample, a pump element within the cartridge forces the sample into
a conduit containing the chip. Here it is oscillated to promote
formation of the sandwich. In the penultimate step of the assay,
fluid is forced out of the pouch and into the conduit to wash the
sample off the chip and into a waste chamber. In the final step of
the assay, the alkaline phosphatase label reacts with p-aminophenol
phosphate to cleave the phosphate group and permit the liberated
p-aminophenol to be electrochemically oxidized at the working
electrode. Based on the measured current, the reader is able to
calculate the amount of analyte in the sample by means of an
embedded algorithm and factory-determined calibration curve.
[0445] It further goes without saying that the methods and kits as
described herein necessarily encompass other reagents and methods
for carrying out the immunoassay. For instance, encompassed are
various buffers such as are known in the art and/or which can be
readily prepared or optimized to be employed, e.g., for washing, as
a conjugate diluent, microparticle diluent, and/or as a calibrator
diluent. An exemplary conjugate diluent is ARCHITECT.RTM. conjugate
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), a
salt, a protein blocker, an antimicrobial agent, and a detergent.
An exemplary calibrator diluent is ARCHITECT.RTM. human calibrator
diluent employed in certain kits (Abbott Laboratories, Abbott Park,
Ill.), which comprises a buffer containing MES, other salt, a
protein blocker, and an antimicrobial agent. Additionally, as
described in U.S. Patent Application No. 61/142,048 filed Dec. 31,
2008, improved signal generation may be obtained, e.g., in an
I-Stat cartridge format, using a nucleic acid sequence linked to
the signal antibody as a signal amplifier.
EXEMPLIFICATION
[0446] It will be readily apparent to those skilled in the art that
other suitable modifications and adaptations of the methods
described herein are obvious and may be made using suitable
equivalents without departing from the scope of the claimed
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 claimed invention.
Example 1
Design, Construction, and Analysis of a DVD-Ig
Example 1.1
Assays Used to Identify and Characterize Parent Antibodies and
DVD-Ig
[0447] The following assays were used throughout the Examples to
identify and characterize parent antibodies and DVD-Ig, unless
otherwise stated.
Example 1.1.1
Assays Used to Determine Binding and Affinity of Parent Antibodies
and DVD-Ig for their Target Antigen(s)
Example 1.1.1A
Direct Bind ELISA
[0448] Enzyme Linked Immunosorbent Assays to screen for antibodies
that bind a desired target antigen are performed as follows. High
bind ELISA plates (Corning Costar #3369, Acton, Mass.) are coated
with 100 .mu.L/well of 10 .mu.g/ml of desired target antigen
(R&D Systems, Minneapolis, Minn.) or desired target antigen
extra-cellular domain/FC fusion protein (R&D Systems,
Minneapolis, Minn.) or monoclonal mouse anti-polyHistidine antibody
(R&D Systems #MAB050, Minneapolis, Minn.) in phosphate buffered
saline (10.times.PBS, Abbott Bioresearch Center, Media
Prep#MPS-073, Worcester, Mass.) overnight at 4.degree. C. Plates
are washed four times with PBS containing 0.02% Tween 20. Plates
are blocked by the addition of 300 .mu.L/well blocking solution
(non-fat dry milk powder, various retail suppliers, diluted to 2%
in PBS) for 1/2 hour at room temperature. Plates are washed four
times after blocking with PBS containing 0.02% Tween 20.
[0449] Alternatively, one hundred microliters per well of 10
.mu.g/ml of Histidine (His) tagged desired target antigen (R&D
Systems, Minneapolis, Minn.) is added to ELISA plates coated with
monoclonal mouse anti-polyHistidine antibody as described above and
incubated for 1 hour at room temperature. Wells are washed four
times with PBS containing 0.02% Tween 20.
[0450] One hundred microliters of antibody or DVD-Ig preparations
diluted in blocking solution as described above is added to the
desired target antigen plate or desired target antigen/FC fusion
plate or the anti-polyHistidine antibody/His tagged desired target
antigen plate prepared as described above and incubated for 1 hour
at room temperature. Wells are washed four times with PBS
containing 0.02% Tween 20.
[0451] One hundred microliters of 10 ng/mL goat anti-human IgG--FC
specific HRP conjugated antibody (Southern Biotech #2040-05,
Birmingham, Ala.) is added to each well of the desired target
antigen plate or anti-polyHistidine antibody/Histidine tagged
desired target antigen plate. Alternatively, one hundred
microliters of 10 ng/mL goat anti-human IgG--kappa light chain
specific HRP conjugated antibody (Southern Biotech #2060-05
Birmingham, Ala.) is added to each well of the desired target
antigen/FC fusion plate and incubated for 1 hour at room
temperature. Plates are washed 4 times with PBS containing 0.02%
Tween 20.
[0452] One hundred microliters of enhanced TMB solution (Neogen
Corp. #308177, K Blue, Lexington, Ky.) is added to each well and
incubated for 10 minutes at room temperature. The reaction is
stopped by the addition of 50 .mu.L 1N sulphuric acid. Plates are
read spectrophotometrically at a wavelength of 450 nm
[0453] In the Direct Bind ELISA, binding is sometimes not observed,
probably because the antibody binding site on the target antigen is
either "masked" or the antigen is "distorted" when coated to the
plastic surface. The inability of a DVD-Ig to bind its target may
also be due to steric limitation imposed on DVD-Ig by the Direct
Bind ELISA format. The parent antibodies and DVD-Igs that do not
bind in the Direct Bind ELISA format can bind to target antigen in
other ELISA formats, such as FACS, Biacore or bioassay. Non-binding
of a DVD-Ig can also be restored by adjusting the linker length
between the two variable domains of the DVD-Ig.
Example 1.1.1.B
Capture ELISA
[0454] ELISA plates (Nunc, MaxiSorp, Rochester, N.Y.) are incubated
overnight at 4.degree. C. with anti-human Fc antibody (5 .mu.g/ml
in PBS, Jackson Immunoresearch, West Grove, Pa.). Plates are washed
three times in washing buffer (PBS containing 0.05% Tween 20), and
blocked for 1 hour at 25.degree. C. in blocking buffer (PBS
containing 1% BSA). Wells are washed three times, and serial
dilutions of each antibody or DVD-Ig in PBS containing 0.1% BSA are
added to the wells and incubated at 25.degree. C. for 1 hour. The
wells are washed three times, and biotinylated antigen (2 nM) os
added to the plates and incubated for 1 hour at 25.degree. C. The
wells are three times, and then incubated for 1 hour at 25.degree.
C. with streptavidin-HRP (KPL #474-3000, Gaithersburg, Md.). The
wells are washed three times, and 100 .mu.l of ULTRA-TMB ELISA
(Pierce, Rockford, Ill.) are added per well. Following color
development the reaction is stopped with 1N HCL and absorbance at
450 nM is measured.
Example 1.1.1.C
Affinity Determination Using BIACORE Technology
BIACORE Methods:
[0455] The BIACORE assay (Biacore, Inc, Piscataway, N.J.)
determines the affinity of antibodies or DVD-Ig with kinetic
measurements of on-rate and off-rate constants. Binding of
antibodies or DVD-Ig to a target antigen (for example, a purified
recombinant target antigen) is determined by surface plasmon
resonance-based measurements with a Biacore.RTM. 1000 or 3000
instrument (Biacore.RTM. AB, Uppsala, Sweden) using running HBS-EP
(10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005%
surfactant P20) at 25.degree. C. All chemicals are obtained from
Biacore.RTM. AB (Uppsala, Sweden) or otherwise from a different
source as described in the text. For example, approximately 5000 RU
of goat anti-mouse IgG, (Fc.gamma.), fragment specific polyclonal
antibody (Pierce Biotechnology Inc, Rockford, Ill.) diluted in 10
mM sodium acetate (pH 4.5) is directly immobilized across a CM5
research grade biosensor chip using a standard amine coupling kit
according to manufacturer's instructions and procedures at 25
.mu.g/ml. Unreacted moieties on the biosensor surface are blocked
with ethanolamine. Modified carboxymethyl dextran surface in
flowcell 2 and 4 is used as a reaction surface. Unmodified
carboxymethyl dextran without goat anti-mouse IgG in flow cell 1
and 3 is used as the reference surface. For kinetic analysis, rate
equations derived from the 1:1 Langmuir binding model are fitted
simultaneously to association and dissociation phases of all eight
injections (using global fit analysis) with the use of
Biaevaluation 4.0.1 software. Purified antibodies or DVD-Ig are
diluted in HEPES-buffered saline for capture across goat anti-mouse
IgG specific reaction surfaces. Antibodies or DVD-Ig to be captured
as a ligand (25 .mu.g/ml) are injected over reaction matrices at a
flow rate of 5 .mu.l/min. The association and dissociation rate
constants, k.sub.on (M.sup.-1 s.sup.-1) and k.sub.off (s.sup.-1)
are determined under a continuous flow rate of 25 .mu.l/min. Rate
constants are derived by making kinetic binding measurements at
different antigen concentrations ranging from 10-200 nM. The
equilibrium dissociation constant (M) of the reaction between
antibodies or DVD-Igs and the target antigen is then calculated
from the kinetic rate constants by the following formula:
K.sub.D=k.sub.off/k.sub.on. Binding is recorded as a function of
time and kinetic rate constants are calculated. In this assay,
on-rates as fast as 10.sup.6M.sup.-1 s.sup.-1 and off-rates as slow
as 10.sup.-6 s.sup.-1 can be measured.
Example 1.1.2
Assays Used to Determine the Functional Activity of Parent
Antibodies and DVD-Ig
Example 1.1.2.A
Cytokine Bioassay
[0456] The ability of an anti-cytokine or an anti-growth factor
parent antibody or DVD-Ig containing anti-cytokine or anti-growth
factor sequences to inhibit or neutralize a target cytokine or
growth factor bioactivity is analyzed by determining the inhibitory
potential of the antibody or DVD-Ig. For example, the ability of an
anti-IL-4 antibody to inhibit IL-4 mediated IgE production may be
used. For example, human naive B cells are isolated from peripheral
blood, respectively, buffy coats by Ficoll-paque density
centrifugation, followed by magnetic separation with MACS beads
(Miltenyi Biotec, Bergisch Gladbach, Germany) specific for human
sIgD FITC labeled goat F(ab)2 antibodies followed by anti-FITC MACS
beads. Magnetically sorted naive B cells are adjusted to
3.times.10.sup.5 cells per ml in XV15 and plated out in 100 .mu.l
per well of 96-well plates in a 6.times.6 array in the center of
the plate, surrounded by PBS filled wells during the 10 days of
culture at 37.degree. C. in the presence of 5% CO.sub.2. One plate
each is prepared per antibody to be tested, consisting of 3 wells
each of un-induced and induced controls and quintuplicate repeats
of antibody titrations starting at 7 .mu.g/ml and running in 3-fold
dilution down to 29 ng/ml final concentrations added in 50 .mu.l
four times concentrated pre-dilution. To induce IgE production,
rhIL-4 at 20 ng/ml plus anti-CD40 monoclonal antibody (Novartis,
Basel, Switzerland) at 0.5 .mu.g/ml final concentrations in 50
.mu.l each are added to each well, and IgE concentrations are
determined at the end of the culture period by a standard sandwich
ELISA method.
Example 1.1.2.B
Cytokine Release Assay
[0457] The ability of a parent antibody or DVD-Ig to cause cytokine
release is analyzed. Peripheral blood is withdrawn from three
healthy donors by venipuncture into heparized vacutainer tubes.
Whole blood is diluted 1:5 with RPMI-1640 medium and placed in
24-well tissue culture plates at 0.5 mL per well. The anti-cytokine
antibodies (e.g., anti-IL-4) are diluted into RPMI-1640 and placed
in the plates at 0.5 mL/well to give final concentrations of 200,
100, 50, 10, and 1 .mu.g/mL. The final dilution of whole blood in
the culture plates is 1:10. LPS and PHA are added to separate wells
at 2 .mu.g/mL and 5 .mu.g/mL final concentration as a positive
control for cytokine release. Polyclonal human IgG is used as
negative control antibody. The experiment is performed in
duplicate. Plates are incubated at 37.degree. C. at 5% CO.sub.2.
Twenty-four hours later the contents of the wells are transferred
into test tubes and spun for 5 minutes at 1200 rpm. Cell-free
supernatants are collected and frozen for cytokine assays. Cells
left over on the plates and in the tubes are lysed with 0.5 mL of
lysis solution, and placed at -20.degree. C. and thawed. 0.5 mL of
medium is added (to bring the volume to the same level as the
cell-free supernatant samples) and the cell preparations are
collected and frozen for cytokine assays. Cell-free supernatants
and cell lysates are assayed for cytokine levels by ELISA, for
example, for levels of IL-8, IL-6, IL-113, IL-1RA, or
TNF-.alpha..
Example 1.1.2.C
Cytokine Cross-Reactivity Study
[0458] The ability of an anti-cytokine parent antibody or DVD-Ig
directed to a cytokine(s) of interest to cross react with other
cytokines is analyzed. Parent antibodies or DVD-Ig are immobilized
on a Biacore biosensor matrix. An anti-human Fc mAb is covalently
linked via free amine groups to the dextran matrix by first
activating carboxyl groups on the matrix with 100 mM
N-hydroxysuccinimide (NHS) and 400 mM
N-Ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride
(EDC). Approximately 50 .mu.L of each antibody or DVD-Ig
preparation at a concentration of 25 .mu.g/mL, diluted in sodium
acetate, pH 4.5, is injected across the activated biosensor and
free amines on the protein are bound directly to the activated
carboxyl groups. Typically, 5000 Resonance Units (RU's) are
immobilized. Unreacted matrix EDC-esters are deactivated by an
injection of 1 M ethanolamine. A second flow cell is prepared as a
reference standard by immobilizing human IgG1/K using the standard
amine coupling kit. SPR measurements are performed using the CM
biosensor chip. All antigens to be analyzed on the biosensor
surface are diluted in HBS-EP running buffer containing 0.01%
P20.
[0459] To examine the cytokine binding specificity, excess cytokine
of interest (100 nM, e.g., soluble recombinant human) is injected
across the anti-cytokine parent antibody or DVD-Ig immobilized
biosensor surface (5 minute contact time). Before injection of the
cytokine of interest and immediately afterward, HBS-EP buffer alone
flows through each flow cell. The net difference in the signals
between the baseline and the point corresponding to approximately
30 seconds after completion of cytokine injection are taken to
represent the final binding value. Again, the response is measured
in Resonance Units. Biosensor matrices are regenerated using 10 mM
HCl before injection of the next sample where a binding event is
observed, otherwise running buffer is injected over the matrices.
Human cytokines (e.g., 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, IL-13, IL-15,
IL-16, IL-17, IL-18, IL-19, IL-20, IL-22, IL-23, IL-27,
TNF-.alpha., TNF-.beta., and IFN-.gamma., for example) are also
simultaneously injected over the immobilized mouse IgG1/K reference
surface to record any nonspecific binding background. By preparing
a reference and reaction surface, Biacore can automatically
subtract the reference surface data from the reaction surface data
in order to eliminate the majority of the refractive index change
and injection noise. Thus, it is possible to ascertain the true
binding response attributed to an anti-cytokine antibody or DVD-Ig
binding reaction.
[0460] When a cytokine of interest is injected across immobilized
anti-cytokine antibody, significant binding is observed. 10 mM HCl
regeneration completely removes all non-covalently associated
proteins. Examination of the sensorgram shows that immobilized
anti-cytokine antibody or DVD-Ig binding to soluble cytokine is
strong and robust. After confirming the expected result with the
cytokine of interest, the panel of remaining recombinant human
cytokines is tested, for each antibody or DVD-Ig separately. The
amount of anti-cytokine antibody or DVD-Ig bound or unbound
cytokine for each injection cycle is recorded. The results from
three independent experiments are used to determine the specificity
profile of each antibody or DVD-Ig. Antibodies or DVD-Ig with the
expected binding to the cytokine of interest and no binding to any
other cytokine are selected.
Example 1.1.2.D
Tissue Cross Reactivity
[0461] Tissue cross reactivity studies are done in three stages,
with the first stage including cryosections of 32 tissues, second
stage including up to 38 tissues, and the 3.sup.rd stage including
additional tissues from 3 unrelated adults as described below.
Studies are done typically at two dose levels.
[0462] Stage 1: Cryosections (about 5 .mu.m) of human tissues (32
tissues (typically: Adrenal Gland, Gastrointestinal Tract,
Prostate, Bladder, Heart, Skeletal Muscle, Blood Cells, Kidney,
Skin, Bone Marrow, Liver, Spinal Cord, Breast, Lung, Spleen,
Cerebellum, Lymph Node, Testes, Cerebral Cortex, Ovary, Thymus,
Colon, Pancreas, Thyroid, Endothelium, Parathyroid, Ureter, Eye,
Pituitary, Uterus, Fallopian Tube and Placenta) from one human
donor obtained at autopsy or biopsy) are fixed and dried on object
glass. The peroxidase staining of tissue sections is performed,
using the avidin-biotin system.
[0463] Stage 2:Cryosections (about 5 .mu.m) of human tissues 38
tissues (including adrenal, blood, blood vessel, bone marrow,
cerebellum, cerebrum, cervix, esophagus, eye, heart, kidney, large
intestine, liver, lung, lymph node, breast mammary gland, ovary,
oviduct, pancreas, parathyroid, peripheral nerve, pituitary,
placenta, prostate, salivary gland, skin, small intestine, spinal
cord, spleen, stomach, striated muscle, testis, thymus, thyroid,
tonsil, ureter, urinary bladder, and uterus) from 3 unrelated
adults obtained at autopsy or biopsy) are fixed and dried on object
glass. The peroxidase staining of tissue sections is performed,
using the avidin-biotin system.
[0464] Stage 3: Cryosections (about 5 .mu.m) of cynomolgus monkey
tissues (38 tissues (including adrenal, blood, blood vessel, bone
marrow, cerebellum, cerebrum, cervix, esophagus, eye, heart,
kidney, large intestine, liver, lung, lymph node, breast mammary
gland, ovary, oviduct, pancreas, parathyroid, peripheral nerve,
pituitary, placenta, prostate, salivary gland, skin, small
intestine, spinal cord, spleen, stomach, striated muscle, testis,
thymus, thyroid, tonsil, ureter, urinary bladder, and uterus) from
3 unrelated adult monkeys obtained at autopsy or biopsy) are fixed
and dried on object glass. The peroxidase staining of tissue
sections is performed, using the avidin-biotin system.
[0465] The antibody or DVD-Ig is incubated with the secondary
biotinylated anti-human IgG and developed into immune complex. The
immune complex at the final concentrations of 2 and 10 .mu.g/mL of
antibody or DVD-Ig is added onto tissue sections on object glass
and then the tissue sections are reacted for 30 minutes with a
avidin-biotin-peroxidase kit. Subsequently, DAB
(3,3'-diaminobenzidine), a substrate for the peroxidase reaction,
is applied for 4 minutes for tissue staining Antigen-Sepharose
beads are used as positive control tissue sections. Target antigen
and human serum blocking studies serve as additional controls. The
immune complex at the final concentrations of 2 and 10 .mu.g/mL of
antibody or DVD-Ig is pre-incubated with target antigen (final
concentration of 100 .mu.g/ml) or human serum (final concentration
10%) for 30 minutes, and then added onto the tissue sections on
object glass and then the tissue sections are reacted for 30
minutes with a avidin-biotin-peroxidase kit. Subsequently, DAB
(3,3'-diaminobenzidine), a substrate for the peroxidase reaction,
is applied for 4 minutes for tissue staining
[0466] Any specific staining is judged to be either an expected
(e.g., consistent with antigen expression) or unexpected reactivity
based upon known expression of the target antigen in question. Any
staining judged specific is scored for intensity and frequency. The
tissue staining between stage 2 (human tissue) and stage 3
(cynomolgus monkey tissue) is either judged to be similar or
different.
Example 1.1.2.E
Neutralization of Methotrexate
[0467] 4T1 cells were grown to a semi-confluent density and
harvested using 0.05% tryspin (Gibco#25300). The cells were washed
with PBS, counted and resuspended at 2E5 cells/mL in assay media.
The cells were seeded in a 96-well plate (Costar#3599) at a volume
of 100 .mu.L and 2E4 cells/well. The DVD-Ig.TM. and control IgG
were diluted to a 4.times. concentration in assay media and serial
1:3 dilutions were performed. The methotrexate was diluted to a
4.times. concentration in assay media to 0.1 uM. The DVD-Ig.TM. was
added to the plated cells at 50 .mu.L for a final concentration of
150 nM-0.0025 nM. The methotrexate was added to the plated cells at
50 .mu.L for a final concentration of 25 nM MTX. The plates were
incubated for 48 hours at 37.degree. C., 5% CO.sub.2. To quantitate
viability, 20 .mu.L of WST-1 reagent (Roche cat#11644807001) was
added to each well. Plates were incubated under assay conditions
for 3.5 hours, and were read at OD 420-600 nm on a Spectromax 190
ELISA plate reader. An average IC50 from several assays is included
in Table 3.
TABLE-US-00003 TABLE 3 Methotrexate Neutralization Assay With MTX
Parent Antibody and DVD-Ig Constructs N-terminal C-terminal
N-terminal C-terminal VD MTX VD MTX Parent Variable Variable
Neutrali- Neutrali- Antibody or Domain Domain zationAssay
zationAssay DVD-Ig ID (VD) (VD) IC50 nM IC50 nM AB119 MTX 10.51
DVD1211 EGFR (seq 3) MTX 3.9 DVD1213 Her2 (seq1) MTX 3.9 DVD1217
CD3 (seq1) MTX 4.0 All DVD-Igs containing VDs from AB119 in the
C-terminal position showed neutralization in the methotrexate
neutralization assay.
Example 1.1.2.F
Tumor Cell Growth Inhibitory Effect of Monoclonal Antibodies or
DVD-Igs In Vitro
[0468] Monoclonal antibodies or DVD-Igs diluted in D-PBS-BSA
(Dulbecco's phosphate buffered saline with 0.1% BSA) 20 .mu.L are
added to human tumor cells at final concentrations of 0.01
.mu.g/mL-100 .mu.g/mL in 200 .mu.L. The plates are incubated at
37.degree. C. in a humidified, 5% CO.sub.2 atmosphere for three
days. The number of live cells in each well is quantified using MTS
reagents according to the manufacturer's instructions (Promega,
Madison, Wis.) to determine the percent of tumor growth inhibition.
Wells without antibody treatment are used as controls of 0%
inhibition whereas wells without cells are considered to show 100%
inhibition.
Example 1.1.2.G
Tumoricidal Effect of A Parent or DVD-12 Antibody In Vitro
[0469] Parent antibodies or DVD-Ig that bind to target antigens on
tumor cells may be analyzed for tumoricidal activity. Briefly,
parent antibodies or DVD-Ig are diluted in D-PBS-BSA (Dulbecco's
phosphate buffered saline with 0.1% BSA) and added to human tumor
cells at final concentrations of 0.01 .mu.g/mL to 100 .mu.g/mL in
200 .mu.L. The plates are incubated at 37.degree. C. in a
humidified, 5% CO.sub.2 atmosphere for 3 days. The number of live
cells in each well is quantified using MTS reagents according to
the manufacturer's instructions (Promega, Madison, Wis.) to
determine the percent of tumor growth inhibition. Wells without
antibody treatment are used as controls of 0% inhibition whereas
wells without cells are considered to show 100% inhibition.
[0470] For assessment of apoptosis, caspase-3 activation is
determined by the following protocol: antibody-treated cells in 96
well plates are lysed in 120 ml of 1.times. lysis buffer (1.67 mM
Hepes, pH 7.4, 7 mM KCl, 0.83 mM MgCl.sub.2, 0.11 mM EDTA, 0.11 mM
EGTA, 0.57% CHAPS, 1 mM DTT, 1.times. protease inhibitor cocktail
tablet; EDTA-free; Roche Pharmaceuticals, Nutley, N.J.) at room
temperature with shaking for 20 minutes. After cell lysis, 80 ml of
a caspase-3 reaction buffer (48 mM Hepes, pH 7.5, 252 mM sucrose,
0.1% CHAPS, 4 mM DTT, and 20 .mu.M Ac-DEVD-AMC substrate; Biomol
Research Labs, Inc., Plymouth Meeting, Pa.) is added and the plates
are incubated for 2 hours at 37.degree. C. The plates are read on a
1420 VICTOR Multilabel Counter (Perkin Elmer Life Sciences, Downers
Grove, Ill.) using the following settings: excitation=360/40,
emission=460/40. An increase of fluorescence units from
antibody-treated cells relative to the isotype antibody
control-treated cells is seen, which is indicative of
apoptosis.
Example 1.1.2.H
Inhibition of Antigen-Induced Receptor Phosphorylation by Parent
Antibodies or DVD-Ig Constructs In Vitro
[0471] Parent antibodies or DVD-Ig that bind to cell receptors or
their ligands may be tested for inhibition of receptor activation.
Parent antibodies or DVD-Ig diluted in D-PBS-BSA (Dulbecco's
phosphate buffered saline with 0.1% BSA) are added to human
carcinoma cells at final concentrations of 0.01 mg/mL to 100 mg/mL
(180 mL). The plates are incubated at 37.degree. C. in a
humidified, 5% CO2 atmosphere for 1 hour. Growth factors (e.g.,
EGF) at a final concentration of 1-100 ng/mL (20 mL) are added to
the cells for 5-15 minutes to stimulate receptor (e.g., EGFR)
autophosphorylation. Wells without antibody treatment are used as
controls of 0% inhibition whereas wells without growth factor
stimulation are considered to show 100% inhibition. Cell lysates
are made by incubation with cell extraction buffer (10 mM Tris, pH
7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 1 mM sodium
orthovanadate, 1% Triton X-100, 10% Glycerol, 0.1% SDS, and
protease inhibitor cocktail). For example, phospho-EGFR in these
cell lysates is determined using the p-EGFR ELISA kit from R&D
Systems (#DYC1095, Minneapolis, Minn.) according to the
manufacturer's instructions.
Example 1.1.2.I
Efficacy of an Anti-Tumor Cell Antigen Antibody or DVD-Ig by Itself
or in Combination with Chemotherapy on the Growth of Human
Carcinoma Xenografts (Subcutaneous Flank, Orthotopic, or
Spontaneous Metastases)
[0472] Human cancer cells are grown in vitro to 99% viability, 85%
confluence in tissue culture flasks. SCID mice (Charles Rivers
Labs) at 19-25 grams are ear tagged and shaved. Mice are then
inoculated subcutaneously into the right flank with 0.2 ml of
2.times.106 human tumor cells (1:1 matrigel) on study day 0.
Administration (IP, Q3D/week) of vehicle (PBS), antibody or DVD-Ig,
and/or chemotherapy is initiated after mice are size matched into
separate ages of mice with mean tumor volumes of approximately 150
to 200 mm3. The tumors are measured by a pair of calipers twice a
week starting on approximately day 10 post inoculation and the
tumor volumes calculated according to the formula V=L.times.W2/2
(V: volume, mm3; L: length, mm; W: width, mm). Reduction in tumor
volume is seen in animals treated with the antibody or DVD-Ig alone
or in combination with chemotherapy relative to tumors in animals
that received only vehicle or an isotype control mAb.
Example 1.1.2.J
Binding of Monoclonal Antibodies to the Surface of Human Tumor Cell
Lines as Assessed by Flow Cytometry
[0473] Stable cell lines overexpressing a cell-surface antigen of
interest or human tumor cell lines were harvested from tissue
culture flasks and resuspended in phosphate buffered saline (PBS)
containing 5% fetal bovine serum (PBS/FBS). Prior to staining,
human tumor cells were incubated on ice with (100 .mu.l) human IgG
at 5 .mu.g/ml in PBS/FCS. 1-5.times.10.sup.5 cells were incubated
with antibody or DVD-Ig (2 .mu.g/mL) in PBS/FBS for 30-60 minutes
on ice. Cells were washed twice and 100 .mu.l of F(ab')2 goat anti
human IgG, Fc.gamma.-phycoerythrin (1:200 dilution in PBS) (Jackson
ImmunoResearch, West Grove, Pa., Cat.#109-116-170) was added. After
30 minutes incubation on ice, cells were washed twice and
resuspended in PBS/FBS. Fluorescence was measured using a Becton
Dickinson FACSCalibur (Becton Dickinson, San Jose, Calif.).
[0474] Table 4 shows the FACS data for the DVD-Ig constructs. The
geometric mean is the n root of the multiplication product of n
fluorescent signals (a1.times.a2.times.a3 . . . an). With
log-transformed data the geometric mean is used to normalize the
weighting of the data distribution. Table 4 contains the FACS
geometric mean of parent antibodies and DVD-Ig constructs.
TABLE-US-00004 TABLE 4 Fluorescent Activated Cell Sorting of DVD-Ig
Constructs FACS FACS Parent N-terminal C-terminal Geometric
Geometric Antibody or Variable Variable Mean Mean DVD-Ig ID Domain
(VD) Domain (VD) N-terminal C-terminal AB002 CD3 (seq1) 254 DVD1217
CD3 (seq 1) MTX 291 AB004 Her2 (seq 1) 88.3 DVD1213 Her2 (seq 1)
MTX 100.7 AB064 EGFR (seq3) 817.2 DVD1211 EGFR (seq3) MTX 739.2 All
DVD-Igs showed binding to their cell surface targets. The
N-terminal domains of DVD-Igs bound their targets on the cell
surface as well as or better than the parent antibody. Binding can
be restored or improved by adjusting linker length.
Example 1.1.2.K
Localization of Methotrexate by DVD-Ig Constructs to the Surface of
Human Tumor Cell Lines as Assessed by Flow Cytometry
[0475] Stable cell lines overexpressing a cell-surface antigen of
interest or human tumor cell lines were harvested from tissue
culture flasks and resuspended in phosphate buffered saline (PBS)
containing 5% fetal bovine serum (PBS/FBS). Prior to staining,
human tumor cells were incubated on ice with (100 .mu.l) human IgG
at 5 .mu.g/ml in PBS/FCS. 1-5.times.10.sup.5 cells were incubated
with antibody or DVD-Ig (2 .mu.g/mL) in PBS/FBS for 30-60 minutes
on ice. Cells were washed twice and 100 .mu.l of Alexa
488-Methotrexate (1:200 dilution in PBS) (Invitrogen, Carlsbad,
Calif., Cat.#M23271) was added. After 30 minutes incubation on ice,
cells were washed twice and resuspended in PBS/FBS. Fluorescence
was measured using a Becton Dickinson FACSCalibur (Becton
Dickinson, San Jose, Calif.).
[0476] Table 5 shows the FACS data for the DVD-Ig constructs. The
geometric mean is the n root of the multiplication product of n
fluorescent signals (a1.times.a2.times.a3 . . . an). With
log-transformed data the geometric mean is used to normalize the
weighting of the data distribution. The following table contains
the FACS geometric mean of parent antibodies and DVD-Ig
constructs.
TABLE-US-00005 TABLE 5 Methotrexate Localization by Fluorescent
Activated Cell Sorting of DVD-Ig Constructs Parent N-terminal
C-terminal FACS Antibody or Variable Variable Geometric DVD-Ig ID
Domain (VD) Domain (VD) Mean AB119 MTX 0.16 AB004 Her2 (seq 1) 0
DVD1213 Her2 (seq 1) MTX 1.94 AB064 EGFR(seq3) 0 AB119 MTX 0
DVD1211 EGFR(seq3) MTX 12.25 All DVD-Igs showed localization of
methotrexate to their cell surface. Binding can be restored or
improved by adjusting linker length.
Example 1.1.2.L
Localization of Methotrexate by DVD-Ig Constructs to the Surface of
Human Tumor Cell Lines as Assessed by Confocal Microscopy
[0477] Stable cell lines overexpressing a cell-surface antigen of
interest or human tumor cell lines are harvested from tissue
culture flasks and resuspended in phosphate buffered saline (PBS)
containing 5% fetal bovine serum (PBS/FBS). Prior to staining,
human tumor cells are incubated on ice with (100 .mu.l) human IgG
at 5 .mu.g/ml in PBS/FCS. 1-5.times.10.sup.5 cells are incubated
with antibody or DVD-Ig (2 .mu.g/mL) in PBS/FBS for 30-60 minutes
on ice. Cells are washed twice and 100 .mu.l of Alexa
488-Methotrexate (1:200 dilution in PBS) (Invitrogen, Carlsbad,
Calif., Cat.#M23271) is added. After 30 minutes incubation on ice,
cells are washed twice and resuspended in PBS.
Example 1.2
Generation of Parent Monoclonal Antibodies to a Human Antigen of
Interest
[0478] Parent mouse mAbs able to bind to and neutralize a human
antigen of interest and a variant thereof are obtained as
follows:
Example 1.2.A
Immunization of Mice with a Human Antigen of Interest
[0479] Twenty micrograms of recombinant purified human antigen
(e.g., IGF1,2) mixed with complete Freund's adjuvant or Immunoeasy
adjuvant (Qiagen, Valencia, Calif.) is injected subcutaneously into
five 6-8 week-old Balb/C, five C57B/6 mice, and five AJ mice on Day
1. On days 24, 38, and 49, twenty micrograms of recombinant
purified human antigen variant mixed with incomplete Freund's
adjuvant or Immunoeasy adjuvant is injected subcutaneously into the
same mice. On day 84 or day 112 or day 144, mice are injected
intravenously with 1 .mu.g recombinant purified human antigen of
interest.
Example 1.2.B
Generation of a Hybridoma
[0480] Splenocytes obtained from the immunized mice described in
Example 1.2.A are fused with SP2/O-Ag-14 cells at a ratio of 5:1
according to the established method described in Kohler, G. and
Milstein (1975) Nature, 256:495 to generate hybridomas. Fusion
products are plated in selection media containing azaserine and
hypoxanthine 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 are observed. Supernatant from each well
containing hybridoma colonies is tested by ELISA for the presence
of antibody to the antigen of interest (as described in Example
1.1.1). Supernatants displaying antigen-specific activity are then
tested for activity (as described in the assays of Example 1.1.2),
for example, the ability to neutralize the antigen of interest in a
bioassay such as that described in Example 1.1.2).
Example 1.2.C
Identification and Characterization of Parent Monoclonal Antibodies
to a Human Target Antigen of Interest
Example 1.2.C.1
Analyzing Parent Monoclonal Antibody Neutralizing Activity
[0481] Hybridoma supernatants are assayed for the presence of
parent antibodies that bind an antigen of interest, generated
according to Examples 1.2.A and 1.2.B, and also bind a variant of
the antigen of interest ("antigen variant"). Supernatants with
antibodies positive in both assays are then tested for their
antigen neutralization potency, for example, in the cytokine
bioassay of Example 1.1.2. The hybridomas producing antibodies with
IC.sub.50 values in the bioassay less than 1,000 pM, in an
embodiment, less than 100 pM are scaled up and cloned by limiting
dilution. Hybridoma cells are expanded into media containing 10%
low IgG fetal bovine serum (Hyclone #SH30151, Logan, Utah). On
average, 250 mL of each hybridoma supernatant (derived from a
clonal population) is harvested, concentrated and purified by
protein A affinity chromatography, as described in Harlow, E. and
Lane, D. 1988 "Antibodies: A Laboratory Manual". The ability of
purified mAbs to inhibit the activity of its target antigen is
determined, for example, using the cytokine bioassay as described
in Example 1.1.2.
Example 1.2.C.2
Analyzing Parent Monoclonal Antibody Cross-Reactivity to Cynomolgus
Target Antigen of Interest
[0482] To determine whether the selected mAbs described herein
recognize cynomolgus antigen of interest, BIACORE analysis is
conducted as described herein (Example 1.1.1.B) using recombinant
cynomolgus target antigen. In addition, neutralization potencies of
mAbs against recombinant cynomolgus antigen of interest may also be
measured in the cytokine bioassay (Example 1.1.2.A). MAbs with good
cyno cross-reactivity (in an embodiment, within 5-fold of
reactivity for human antigen) are selected for future
characterization.
Example 1.2.D
Determination of the Amino Acid Sequence of the Variable Region for
Each Murine Anti-Human Monoclonal Antibody
[0483] Isolation of the cDNAs, expression and characterization of
the recombinant anti-human mouse mAbs is conducted as follows. For
each amino acid sequence determination, approximately
1.times.10.sup.6 hybridoma cells are isolated by centrifugation and
processed to isolate total RNA with Trizol (Gibco BRL/Invitrogen,
Carlsbad, Calif.) following manufacturer's instructions. Total RNA
is subjected to first strand DNA synthesis using the SuperScript
First-Strand Synthesis System (Invitrogen, Carlsbad, Calif.) per
the manufacturer's instructions. Oligo(dT) is used to prime
first-strand synthesis to select for poly(A)+ RNA. The first-strand
cDNA product is then amplified by PCR with primers designed for
amplification of murine immunoglobulin variable regions (Ig-Primer
Sets, Novagen, Madison, Wis.). PCR products are resolved on an
agarose gel, excised, purified, and then subcloned with the TOPO
Cloning kit into pCR2.1-TOPO vector (Invitrogen, Carlsbad, Calif.)
and transformed into TOP10 chemically competent E. coli
(Invitrogen, Carlsbad, Calif.). Colony PCR is performed on the
transformants to identify clones containing insert. Plasmid DNA is
isolated from clones containing insert using a QIAprep Miniprep kit
(Qiagen, Valencia, Calif.). Inserts in the plasmids are sequenced
on both strands to determine the variable heavy or variable light
chain DNA sequences using M13 forward and M13 reverse primers
(Fermentas Life Sciences, Hanover Md.). Variable heavy and variable
light chain sequences of the mAbs are identified. In an embodiment,
the selection criteria for a panel of lead mAbs for next step
development (humanization) includes the following: [0484] The
antibody does not contain any N-linked glycosylation sites (NXS),
except from the standard one in CH2 [0485] The antibody does not
contain any extra cysteines in addition to the normal cysteines in
every antibody [0486] The antibody sequence is aligned with the
closest human germline sequences for VH and VL and any unusual
amino acids should be checked for occurrence in other natural human
antibodies [0487] N-terminal Glutamine (Q) is changed to Glutamic
acid (E) if it does not affect the activity of the antibody. This
will reduce heterogeneity due to cyclization of Q [0488] Efficient
signal sequence cleavage is confirmed by Mass Spectrophotometry.
This can be done with COS cell or 293 cell material [0489] The
protein sequence is checked for the risk of deamidation of Asn that
could result in loss of activity [0490] The antibody has a low
level of aggregation [0491] The antibody has solubility >5-10
mg/ml (in research phase); >25 mg/ml [0492] The antibody has a
normal size (5-6 nm) by Dynamic Light Scattering (DLS) [0493] The
antibody has a low charge heterogeneity [0494] The antibody lacks
cytokine release (see Example 1.1.2.B) [0495] The antibody has
specificity for the intended cytokine (see Example 1.1.2.C) [0496]
The antibody lacks unexpected tissue cross reactivity (see Example
1.1.2.D) [0497] The antibody has similarity between human and
cynomolgus tissue cross reactivity (see Example 1.1.2.D)
Example 1.2.2
Recombinant Humanized Parent Antibodies
Example 1.2.2.1
Construction And Expression Of Recombinant Chimeric Anti Human
Parent Antibodies
[0498] The DNA encoding the heavy chain constant region of murine
anti-human parent mAbs is replaced by a cDNA fragment encoding the
human IgG1 constant region containing 2 hinge-region amino acid
mutations by homologous recombination in bacteria. These mutations
are a leucine to alanine change at position 234 (EU numbering) and
a leucine to alanine change at position 235 (Lund et al. (1991) J.
Immunol.: 147:2657). The light chain constant region of each of
these antibodies is replaced by a human kappa constant region.
Full-length chimeric antibodies are transiently expressed in COS
cells by co-transfection of chimeric heavy and light chain cDNAs
ligated into the pBOS expression plasmid (Mizushima and Nagata
(1990) Nucl. Acids Res. 18: 5322). Cell supernatants containing
recombinant chimeric antibody are purified by Protein A Sepharose
chromatography and bound antibody is eluted by addition of acid
buffer. Antibodies are neutralized and dialyzed into PBS.
[0499] The heavy chain cDNA encoding a chimeric mAb is
co-transfected with its chimeric light chain cDNA (both ligated in
the pBOS vector) into COS cells. Cell supernatant containing
recombinant chimeric antibody is purified by Protein A Sepharose
chromatography and bound antibody is eluted by addition of acid
buffer. Antibodies are neutralized and dialyzed into PBS.
[0500] The purified chimeric anti-human parent mAbs are then tested
for their ability to bind (by Biacore) and for functional activity,
e.g., to inhibit the cytokine induced production of IgE as
described in Examples 1.1.1 and 1.1.2. Chimeric mAbs that maintain
the activity of the parent hybridoma mAbs are selected for future
development.
Example 1.2.2.2
Construction And Expression Of Humanized Anti Human Parent
Antibodies
Example 1.2.2.2.A
Selection of Human Antibody Frameworks
[0501] Each murine variable heavy and variable light chain gene
sequence is separately aligned against 44 human immunoglobulin
germline variable heavy chain or 46 germline variable light chain
sequences (derived from NCBI Ig Blast website) using Vector NTI
software.
[0502] Humanization is 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 are
mutated to human residues where murine and human framework residues
are different, with a few exceptions. Additional humanization
strategies are 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.
[0503] Homology modeling is used to identify residues unique to the
murine antibody sequences that are predicted to be critical to the
structure of the antibody combining site, the CDRs. Homology
modeling is a computational method whereby approximate three
dimensional coordinates are generated for a protein. The source of
initial coordinates and guidance for their further refinement is a
second protein, the reference protein, for which the three
dimensional coordinates are known and the sequence of which is
related to the sequence of the first protein. The relationship
among the sequences of the two proteins is used to generate a
correspondence between the reference protein and the protein for
which coordinates are desired, the target protein. The primary
sequences of the reference and target proteins are aligned with
coordinates of identical portions of the two proteins transferred
directly from the reference protein to the target protein.
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 transferred model coordinates. This
computational protein structure may be further refined or employed
directly in modeling studies. The quality of the model structure is
determined by the accuracy of the contention that the reference and
target proteins are related and the precision with which the
sequence alignment is constructed.
[0504] For the murine mAbs, a combination of BLAST searching and
visual inspection is 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 are
constructed manually and model coordinates are generated with the
program Jackal (see Petrey, D. et al. (2003) Proteins 53 (Suppl.
6): 430-435).
[0505] The primary sequences of the murine and human framework
regions of the selected antibodies share significant identity.
Residue positions that differ are candidates for inclusion of the
murine residue in the humanized sequence in order to retain the
observed binding potency of the murine antibody. A list of
framework residues that differ between the human and murine
sequences is constructed manually. Table 7 shows the framework
sequences chosen for this study.
TABLE-US-00006 TABLE 6 Sequence Of Human IgG Heavy Chain Constant
Domain And Light Chain Constant Domain SEQ Sequence Protein ID NO
12345678901234567890123456789012345678901 Wild type hIgG1 106
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW constant region
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Mutant hIgG1 constant
107 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW region
NSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI
CNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSV
FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK Ig kappa constant 108
TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK region
VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHK VYACEVTHQGLSSPVTKSFNRGEC
Ig Lambda 109 QPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAW constant
region KADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR
SYSCQVTHEGSTVEKTVAPTECS
[0506] 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 are
ranked according to their distance from any atom in the CDRs. Those
residues that fell within 4.5 .ANG. of any CDR atom are identified
as most important and are recommended to be candidates for
retention of the murine residue in the humanized antibody (i.e.,
back mutation).
[0507] In silico constructed humanized antibodies are constructed
using oligonucleotides. For each variable region cDNA, 6
oligonucleotides of 60-80 nucleotides each are designed to overlap
each other by 20 nucleotides at the 5' and/or 3' end of each
oligonucleotide. In an annealing reaction, all 6 oligonucleotides
are combined, boiled, and annealed in the presence of dNTPs. DNA
polymerase I, Large (Klenow) fragment (New England Biolabs #M0210,
Beverley, Mass.) is added to fill-in the approximately 40 bp gaps
between the overlapping oligonucleotides. PCR is performed to
amplify the entire variable region gene using two outermost primers
containing overhanging sequences complementary to the multiple
cloning site in a modified pBOS vector (Mizushima, S. and Nagata,
S. (1990) Nucleic Acids Res. 18: 17). The PCR products derived from
each cDNA assembly are separated on an agarose gel and the band
corresponding to the predicted variable region cDNA size is excised
and purified. The variable heavy region is inserted in-frame onto a
cDNA fragment encoding the human IgG1 constant region containing 2
hinge-region amino acid mutations by homologous recombination in
bacteria. These mutations are a leucine to alanine change at
position 234 (EU numbering) and a leucine to alanine change at
position 235 (Lund et al. (1991) J. Immunol. 147:2657). The
variable light chain region is inserted in-frame with the human
kappa constant region by homologous recombination. Bacterial
colonies are isolated and plasmid DNA extracted. cDNA inserts are
sequenced in their entirety. Correct humanized heavy and light
chains corresponding to each antibody are co-transfected into COS
cells to transiently produce full-length humanized anti-human
antibodies. Cell supernatants containing recombinant chimeric
antibody are purified by Protein A Sepharose chromatography and
bound antibody is eluted by addition of acid buffer. Antibodies are
neutralized and dialyzed into PBS.
Example 1.2.2.3
Characterization of Humanized Antibodies
[0508] The ability of purified humanized antibodies to inhibit a
functional activity is determined, e.g., using the cytokine
bioassay as described in Examples 1.1.2.A. The binding affinities
of the humanized antibodies to recombinant human antigen are
determined using surface plasmon resonance (Biacore.RTM.)
measurement as described in Example 1.1.1.B. The IC.sub.50 values
from the bioassays and the affinity of the humanized antibodies are
ranked. The humanized mAbs that fully maintain the activity of the
parent hybridoma mAbs are selected as candidates for future
development. The top 2-3 most favorable humanized mAbs are further
characterized.
Example 1.2.2.3.A
Pharmacokinetic Analysis of Humanized Antibodies
[0509] Pharmacokinetic studies are carried out in Sprague-Dawley
rats and cynomolgus monkeys. Male and female rats and cynomolgus
monkeys are dosed intravenously or subcutaneously with a single
dose of 4 mg/kg mAb and samples are analyzed using antigen capture
ELISA, and pharmacokinetic parameters are determined by
noncompartmental analysis. Briefly, ELISA plates are coated with
goat anti-biotin antibody (5 mg/ml, 4.degree. C., overnight),
blocked with Superblock (Pierce), and incubated with biotinylated
human antigen at 50 ng/ml in 10% Superblock TTBS at room
temperature for 2 hours. Serum samples are serially diluted (0.5%
serum, 10% Superblock in TTBS) and incubated on the plate for 30
minutes at room temperature. Detection is carried out with
HRP-labeled goat anti human antibody and concentrations are
determined with the help of standard curves using the four
parameter logistic fit. Values for the pharmacokinetic parameters
are determined by non-compartmental model using WinNonlin software
(Pharsight Corporation, Mountain View, Calif.). Humanized mAbs with
good pharmacokinetics profile (T1/2 is 8-13 days or better, with
low clearance and excellent bioavailability 50-100%) are
selected.
Example 1.2.2.3.B
Physicochemical and In Vitro Stability Analysis of Humanized
Monoclonal Antibodies
Size Exclusion Chromatography
[0510] Antibodies are diluted to 2.5 mg/mL with water and 20 mL is
analyzed on a Shimadzu HPLC system using a TSK gel G3000 SWXL
column (Tosoh Bioscience, cat#k5539-05k). Samples are eluted from
the column with 211 mM sodium sulfate, 92 mM sodium phosphate, pH
7.0, at a flow rate of 0.3 mL/minutes. The HPLC system operating
conditions are the following:
[0511] Mobile phase: 211 mM Na.sub.2SO.sub.4, 92 mM
Na.sub.2HPO.sub.4*7H.sub.2O, pH 7.0
[0512] Gradient: Isocratic
[0513] Flow rate: 0.3 mL/minute
[0514] Detector wavelength: 280 nm
[0515] Autosampler cooler temp: 4.degree. C.
[0516] Column oven temperature: Ambient
[0517] Run time: 50 minutes
[0518] Table 7 contains purity data of parent antibodies and DVD-Ig
constructs expressed as percent monomer (unaggregated protein of
the expected molecular weight) as determined by the above
protocol.
TABLE-US-00007 TABLE 7 Purity of Parent Antibodies and DVD-Ig
Constructs as Determined by Size Exclusion Chromatography Parent
N-terminal C-terminal Antibody or Variable Variable % Monomer
DVD-Ig ID Domain (VD) Domain (VD) (purity) AB119 MTX 100 DVD1209
CD20 MTX 37.4 DVD1211 EGFR(seq3) MTX 99.6 DVD1213 Her2 (seq1) MTX
98.5 DVD1217 CD-3 (seq1) MTX 93.2 DVD-Igs showed an excellent SEC
profile with most DVD-Ig showing >90% monomer. This DVD-Ig
profile is similar to that observed for parent antibodies.
SDS-PAGE
[0519] Antibodies are analyzed by sodium dodecyl
sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) under both
reducing and non-reducing conditions. Adalimumab lot AFP04C is used
as a control. For reducing conditions, the samples are mixed 1:1
with 2.times. tris glycine SDS-PAGE sample buffer (Invitrogen,
cat#LC2676, lot#1323208) with 100 mM DTT, and heated at 60.degree.
C. for 30 minutes. For non-reducing conditions, the samples are
mixed 1:1 with sample buffer and heated at 100.degree. C. for 5
minutes. The reduced samples (10 mg per lane) are loaded on a 12%
pre-cast tris-glycine gel (Invitrogen, cat#EC6005box, lot#6111021),
and the non-reduced samples (10 mg per lane) are loaded on an
8%-16% pre-cast tris-glycine gel (Invitrogen, cat#EC6045box,
lot#6111021). SeeBlue Plus 2 (Invitrogen, cat#LC5925, lot#1351542)
is used as a molecular weight marker. The gels are run in a XCell
SureLock mini cell gel box (Invitrogen, cat#EI0001) and the
proteins are separated by first applying a voltage of 75 to stack
the samples in the gel, followed by a constant voltage of 125 until
the dye front reached the bottom of the gel. The running buffer
used is 1.times. tris glycine SDS buffer, prepared from a 10.times.
tris glycine SDS buffer (ABC, MPS-79-080106)). The gels are stained
overnight with colloidal blue stain (Invitrogen cat#46-7015,
46-7016) and destained with Milli-Q water until the background is
clear. The stained gels are then scanned using an Epson Expression
scanner (model 1680, S/N DASX003641).
Sedimentation Velocity Analysis
[0520] Antibodies are loaded into the sample chamber of each of
three standard two-sector carbon epon centerpieces. These
centerpieces have a 1.2 cm optical path length and are built with
sapphire windows. PBS is used for a reference buffer and each
chamber contained 140 .mu.L. All samples are examined
simultaneously using a 4-hole (AN-60Ti) rotor in a Beckman
ProteomeLab XL-I analytical ultracentrifuge (serial #PL106C01).
[0521] Run conditions are programmed and centrifuge control is
performed using ProteomeLab (v5.6). The samples and rotor are
allowed to thermally equilibrate for one hour prior to analysis
(20.0.+-.0.1.degree. C.). Confirmation of proper cell loading is
performed at 3000 rpm and a single scan is recorded for each cell.
The sedimentation velocity conditions are the following:
[0522] Sample Cell Volume: 420 mL
[0523] Reference Cell Volume: 420 mL
[0524] Temperature: 20.degree. C.
[0525] Rotor Speed: 35,000 rpm
[0526] Time: 8:00 hours
[0527] UV Wavelength: 280 nm
[0528] Radial Step Size: 0.003 cm
[0529] Data Collection One data point per step without signal
averaging.
[0530] Total Number of Scans: 100
LC-MS Molecular Weight Measurement of Intact Antibodies
[0531] Molecular weights of intact antibodies are analyzed by
LC-MS. Each antibody is diluted to approximately 1 mg/mL with
water. An 1100 HPLC (Agilent) system with a protein microtrap
(Michrom Bioresources, Inc, cat#004/25109/03) is used to desalt and
introduce 5 mg of the sample into an API Qstar pulsar i mass
spectrometer (Applied Biosystems). A short gradient is used to
elute the samples. The gradient is run with mobile phase A (0.08%
FA, 0.02% TFA in HPLC water) and mobile phase B (0.08% FA and 0.02%
TFA in acetonitrile) at a flow rate of 50 mL/minute. The mass
spectrometer is operated at 4.5 kvolts spray voltage with a scan
range from 2000 to 3500 mass to charge ratio.
LC-MS Molecular Weight Measurement of Antibody Light and Heavy
Chains
[0532] Molecular weight measurement of antibody light chain (LC),
heavy chain (HC) and deglycosylated HC are analyzed by LC-MS.
Antibody is diluted to 1 mg/mL with water and the sample is reduced
to LC and HC with a final concentration of 10 mM DTT for 30 minutes
at 37.degree. C. To deglycosylate the antibody, 100 mg of the
antibody is incubated with 2 mL of PNGase F, 5 mL of 10%
N-octylglucoside in a total volume of 100 mL overnight at
37.degree. C. After deglycosylation the sample is reduced with a
final concentration of 10 mM DTT for 30 minutes at 37.degree. C. An
Agilent 1100 HPLC system with a C4 column (Vydac, cat#214TP5115,
S/N 060206537204069) is used to desalt and introduce the sample (5
mg) into an API Qstar pulsar i mass spectrometer (Applied
Biosystems). A short gradient is used to elute the sample. The
gradient is run with mobile phase A (0.08% FA, 0.02% TFA in HPLC
water) and mobile phase B (0.08% FA and 0.02% TFA in acetonitrile)
at a flow rate of 50 mL/minute. The mass spectrometer is operated
at 4.5 kvolts spray voltage with a scan range from 800 to 3500 mass
to charge ratio.
Peptide Mapping
[0533] Antibody is denatured for 15 minutes at room temperature
with a final concentration of 6 M guanidine hydrochloride in 75 mM
ammonium bicarbonate. The denatured samples are reduced with a
final concentration of 10 mM DTT at 37.degree. C. for 60 minutes,
followed by alkylation with 50 mM iodoacetic acid (IAA) in the dark
at 37.degree. C. for 30 minutes. Following alkylation, the sample
is dialyzed overnight against four liters of 10 mM ammonium
bicarbonate at 4.degree. C. The dialyzed sample is diluted to 1
mg/mL with 10 mM ammonium bicarbonate, pH 7.8 and 100 mg of
antibody is either digested with trypsin (Promega, cat#V5111) or
Lys-C (Roche, cat#11 047 825 001) at a 1:20 (w/w)
trypsin/Lys-C:antibody ratio at 37.degree. C. for 4 hrs. Digests
are quenched with 1 mL of 1 N HCl. For peptide mapping with mass
spectrometer detection, 40 mL of the digests are separated by
reverse phase high performance liquid chromatography (RPHPLC) on a
C18 column (Vydac, cat#218TP51, S/N NE9606 10.3.5) with an Agilent
1100 HPLC system. The peptide separation is run with a gradient
using mobile phase A (0.02% TFA and 0.08% FA in HPLC grade water)
and mobile phase B (0.02% TFA and 0.08% FA in acetonitrile) at a
flow rate of 50 mL/minutes. The API QSTAR Pulsar i mass spectromer
is operated in positive mode at 4.5 kvolts spray voltage and a scan
range from 800 to 2500 mass to charge ratio.
Disulfide Bond Mapping
[0534] To denature the antibody, 100 mL of the antibody is mixed
with 300 mL of 8 M guanidine HCl in 100 mM ammonium bicarbonate.
The pH is checked to ensure that it is between 7 and 8 and the
samples are denatured for 15 minutes at room temperature in a final
concentration of 6 M guanidine HCl. A portion of the denatured
sample (100 mL) is diluted to 600 mL with Milli-Q water to give a
final guanidine-HCl concentration of 1 M. The sample (220 mg) is
digested with either trypsin (Promega, cat #V5111, lot#22265901) or
Lys-C (Roche, cat#11047825001, lot#12808000) at a 1:50 trypsin or
1:50 Lys-C: antibody (w/w) ratios (4.4 mg enzyme: 220 mg sample) at
37.degree. C. for approximately 16 hours. An additional 5 mg of
trypsin or Lys-C is added to the samples and digestion is allowed
to proceed for an additional 2 hours at 37.degree. C. Digestions
are stopped by adding 1 mL of TFA to each sample. Digested samples
are separated by RPHPLC using a C18 column (Vydac, cat#218TP51 S/N
NE020630-4-1A) on an Agilent HPLC system. The separation is run
with the same gradient used for peptide mapping using mobile phase
A (0.02% TFA and 0.08% FA in HPLC grade water) and mobile phase B
(0.02% TFA and 0.08% FA in acetonitrile) at a flow rate of 50
mL/minute. The HPLC operating conditions are the same as those used
for peptide mapping. The API QSTAR Pulsar i mass spectromer is
operated in positive mode at 4.5 kvolts spray voltage and a scan
range from 800 to 2500 mass-to-charge ratio. Disulfide bonds are
assigned by matching the observed MWs of peptides with the
predicted MWs of tryptic or Lys-C peptides linked by disulfide
bonds.
Free Sulfhydryl Determination
[0535] The method used to quantify free cysteines in an antibody is
based on the reaction of Ellman's reagent, 5,5
-dithio-bis(2-nitrobenzoic acid) (DTNB), with sulfhydryl groups
(SH) which gives rise to a characteristic chromophoric product,
5-thio-(2-nitrobenzoic acid) (TNB). The reaction is illustrated in
the formula:
DTNB+RSH.RTM.RS-TNB+TNB-+H+
[0536] The absorbance of the TNB- is measured at 412 nm using a
Cary 50 spectrophotometer. An absorbance curve is plotted using
dilutions of 2 mercaptoethanol (b-ME) as the free SH standard and
the concentrations of the free sulfhydryl groups in the protein are
determined from absorbance at 412 nm of the sample.
[0537] The b-ME standard stock is prepared by a serial dilution of
14.2 M b-ME with HPLC grade water to a final concentration of 0.142
mM. Then standards in triplicate for each concentration are
prepared. Antibody is concentrated to 10 mg/mL using an amicon
ultra 10,000 MWCO centrifugal filter (Millipore, cat#UFC801096,
lot#L3KN5251) and the buffer is changed to the formulation buffer
used for adalimumab (5.57 mM sodium phosphate monobasic, 8.69 mM
sodium phosphate dibasic, 106.69 mM NaCl, 1.07 mM sodium citrate,
6.45 mM citric acid, 66.68 mM mannitol, pH 5.2, 0.1% (w/v) Tween).
The samples are mixed on a shaker at room temperature for 20
minutes. Then 180 mL of 100 mM Tris buffer, pH 8.1 is added to each
sample and standard followed by the addition of 300 mL of 2 mM DTNB
in 10 mM phosphate buffer, pH 8.1. After thorough mixing, the
samples and standards are measured for absorption at 412 nm on a
Cary 50 spectrophotometer. The standard curve is obtained by
plotting the amount of free SH and OD.sub.412 nm of the b-ME
standards. Free SH content of samples are calculated based on this
curve after subtraction of the blank.
Weak Cation Exchange Chromatography
[0538] Antibody is diluted to 1 mg/mL with 10 mM sodium phosphate,
pH 6.0. Charge heterogeneity is analyzed using a Shimadzu HPLC
system with a WCX-10 ProPac analytical column (Dionex, cat#054993,
S/N 02722). The samples are loaded on the column in 80% mobile
phase A (10 mM sodium phosphate, pH 6.0) and 20% mobile phase B (10
mM sodium phosphate, 500 mM NaCl, pH 6.0) and eluted at a flow rate
of 1.0 mL/minute.
Oligosaccharide Profiling
[0539] Oligosaccharides released after PNGase F treatment of
antibody are derivatized with 2-aminobenzamide (2-AB) labeling
reagent. The fluorescent-labeled oligosaccharides are separated by
normal phase high performance liquid chromatography (NPHPLC) and
the different forms of oligosaccharides are characterized based on
retention time comparison with known standards.
[0540] The antibody is first digested with PNGaseF to cleave
N-linked oligosaccharides from the Fc portion of the heavy chain.
The antibody (200 mg) is placed in a 500 mL Eppendorf tube along
with 2 mL PNGase F and 3 mL of 10% N-octylglucoside. Phosphate
buffered saline is added to bring the final volume to 60 mL. The
sample is incubated overnight at 37.degree. C. in an Eppendorf
thermomixer set at 700 RPM. Adalimumab lot AFP04C is also digested
with PNGase F as a control.
[0541] After PNGase F treatment, the samples are incubated at
95.degree. C. for 5 minutes in an Eppendorf thermomixer set at 750
RPM to precipitate out the proteins, then the samples are placed in
an Eppendorf centrifuge for 2 minutes at 10,000 RPM to spin down
the precipitated proteins. The supernatent containing the
oligosaccharides are transferred to a 500 mL Eppendorf tube and
dried in a speed-vac at 65.degree. C.
[0542] The oligosaccharides are labeled with 2AB using a 2AB
labeling kit purchased from Prozyme (cat#GKK-404, lot#132026). The
labeling reagent is prepared according to the manufacturer's
instructions. Acetic acid (150 mL, provided in kit) is added to the
DMSO vial (provided in kit) and mixed by pipeting the solution up
and down several times. The acetic acid/DMSO mixture (100 mL) is
transferred to a vial of 2-AB dye (just prior to use) and mixed
until the dye is fully dissolved. The dye solution is then added to
a vial of reductant (provided in kit) and mixed well (labeling
reagent). The labeling reagent (5 mL) is added to each dried
oligosaccharide sample vial, and mixed thoroughly. The reaction
vials are placed in an Eppendorf thermomixer set at 65.degree. C.
and 700-800 RPM for 2 hours of reaction.
[0543] After the labeling reaction, the excess fluorescent dye is
removed using GlycoClean S Cartridges from Prozyme (cat#GKI-4726).
Prior to adding the samples, the cartridges are washed with 1 mL of
milli-Q water followed with 5 washes of 1 mL 30% acetic acid
solution. Just prior to adding the samples, 1 mL of acetonitrile
(Burdick and Jackson, cat#AH015-4) is added to the cartridges.
[0544] After all of the acetonitrile passed through the cartridge,
the sample is spotted onto the center of the freshly washed disc
and allowed to adsorb onto the disc for 10 minutes. The disc is
washed with 1 mL of acetonitrile followed by five washes of 1 mL of
96% acetonitrile. The cartridges are placed over a 1.5 mL Eppendorf
tube and the 2-AB labeled oligosaccharides are eluted with 3 washes
(400 mL each wash) of milli Q water.
[0545] The oligosaccharides are separated using a Glycosep N HPLC
(cat#GKI-4728) column connected to a Shimadzu HPLC system. The
Shimadzu HPLC system consisted of a system controller, degasser,
binary pumps, autosampler with a sample cooler, and a fluorescent
detector.
Stability at Elevated Temperatures
[0546] The buffer of antibody is either 5.57 mM sodium phosphate
monobasic, 8.69 mM sodium phosphate dibasic, 106.69 mM NaCl, 1.07
mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol, 0.1%
(w/v) Tween, pH 5.2; or 10 mM histidine, 10 mM methionine, 4%
mannitol, pH 5.9 using Amicon ultra centrifugal filters. The final
concentration of the antibodies is adjusted to 2 mg/mL with the
appropriate buffers. The antibody solutions are then filter
sterized and 0.25 mL aliquots are prepared under sterile
conditions. The aliquots are left at either -80.degree. C.,
5.degree. C., 25.degree. C., or 40.degree. C. for 1, 2 or 3 weeks.
At the end of the incubation period, the samples are analyzed by
size exclusion chromatography and SDS-PAGE.
[0547] The stability samples are analyzed by SDS-PAGE under both
reducing and non-reducing conditions. The procedure used is the
same as described herein. The gels are stained overnight with
colloidal blue stain (Invitrogen cat#46-7015, 46-7016) and
destained with Milli-Q water until the background is clear. The
stained gels are then scanned using an Epson Expression scanner
(model 1680, S/N DASX003641). To obtain more sensitivity, the same
gels are silver stained using silver staining kit (Owl Scientific)
and the recommended procedures given by the manufacturer is
used.
Example 1.2.2.3.C
Efficacy of a Humanized Monoclonal Antibody by Itself or in
Combination with Chemotherapy on the Growth of Human Carcinoma
Xenografts
[0548] Human cancer cells are grown in vitro to 99% viability, 85%
confluence in tissue culture flasks. SCID female or male mice
(Charles Rivers Labs) at 19-25 grams, are ear tagged and shaved.
Mice are then inoculated subcutaneously into the right flank with
0.2 ml of 2.times.10.sup.6 human tumor cells (1:1 matrigel) on
study day 0. Administration (IP, Q3D/week) of vehicle (PBS),
humanized antibody, and/or chemotherapy is initiated after mice are
size matched into separate cages of mice with mean tumor volumes of
approximately 150 to 200 mm.sup.3. The tumors are measured by a
pair of calipers twice a week starting on approximately day 10 post
inoculation and the tumor volumes calculated according to the
formula V=L.times.W.sup.2/2 (V: volume, mm.sup.3; L: length, mm; W:
width, mm). Reduction in tumor volume is seen in animals treated
with mAb alone or in combination with chemotherapy relative to
tumors in animals that received only vehicle or an isotype control
mAb.
Example 1.2.2.3.D
FACS Based Redirected Cytotoxicity (rCTL) Assay
[0549] Human CD3+ T cells are isolated from previously frozen
isolated peripheral blood mononuclear cells (PBMC) by a negative
selection enrichment column (R&D Systems, Minneapolis, Minn.;
Cat.#HTCC-525). T cells are stimulated for 4 days in flasks (vent
cap, Corning, Acton, Mass.) coated with 10 m/mL anti-CD3 (OKT-3,
eBioscience, Inc., San Diego, Calif.) and 2 .mu.g/mL anti-CD28
(CD28.2, eBioscience, Inc., San Diego, Calif.) in D-PBS
(Invitrogen, Carlsbad, Calif.) and cultured in 30 U/mL IL-2 (Roche)
in complete RPMI 1640 media (Invitrogen, Carlsbad, Calif.) with
L-glutamine, 55 mM .beta.-ME, Pen/Strep, 10% FBS). T cells are then
rested overnight in 30 U/mL IL-2 before using in assay. DoHH2 or
Raji target cells are labeled with PKH26 (Sigma-Aldrich, St. Louis,
Mo.) according to manufacturer's instructions. RPMI 1640 media (no
phenol, Invitrogen, Carlsbad, Calif.) containing L-glutamine and
10% FBS (Hyclone, Logan, Utah) is used throughout the rCTL assay.
(See Dreier et al. (2002) Int J Cancer 100:690).
[0550] Effector T cells (E) and targets (T) are plated at a final
cell concentration of 10.sup.5 and 10.sup.4 cells/well in 96-well
plates (Costar #3799, Acton, Mass.), respectively to give an E:T
ratio of 10:1. DVD-Ig molecules are diluted to obtain
concentration-dependent titration curves. After an overnight
incubation cells are pelleted and washed with D-PBS once before
resuspending in FACS buffer containing 0.1% BSA (Invitrogen,
Carlsbad, Calif.), 0.1% sodium azide and 0.5 .mu.g/mL propidium
iodide (BD) in D-PBS. FACS data is collected on a FACS Canto II
machine (Becton Dickinson, San Jose, Calif.) and analyzed in Flowjo
(Treestar). The percent live targets in the DVD-Ig treated samples
divided by the percent total targets (control, no treatment) is
calculated to determine percent specific lysis. IC50s are
calculated in Prism (Graphpad).
Example 1.2.2.3.E
Antibody-Dependent Cell Mediated Cytotoxicity (ADCC) Method
[0551] Target (e.g., DoHH2, A431, and U87MGde2-7) cells are
harvested and washed with 10 mL RPMI no phenol red medium
(Invitrogen, Carlsbad, Calif., Cat.#11835) and incubated in
calcein-AM (eBioscience, San Diego, Calif., Cat.#65-0853) at a
concentration of 4.times.106 cells/mL for 30 minutes. Target cells
are washed 3 times with 10 mL RPMI no phenol red 10% FBS (Thermo
Scientific HyClone, Logan, Utah, Cat.#SH30070.03) and aliquoted at
180,000 cells/well in a 96-well round bottom plate. Antibodies and
DVD-Igs are diluted in RPMI no phenol red 10% FBS to 10 mg/mL. The
supernatant is removed from the target cells, and 30 mL/well of the
diluted antibodies and DVD-Igs are added. Cells are incubated on
ice for 1 hour and then washed 3 times with 150 mL/well RPMI no
phenol red 10% FBS. Cells are transferred to a 2 mL assay block and
resuspended at a concentration of 1.33.times.105 cells/mL.
Unactivated human NK cells (Astarte Biologics, Redmond, Wash.,
Cat.#1027) or activated human NK cells (In-house blood donor
program PBMCs activated 2 weeks using kit, Myltenyi Biotech,
Auburn, Calif., Cat.#130-094-483) are thawed, washed with 10 mL
RPMI no phenol red 10% FBS twice, and resuspended at 1.2.times.106
cells/mL. NK cells and target cells are then aliquoted at a ratio
of 9:1 onto 96-well V bottom plate by transferring 75 mL of NK
cells and 75 mL of target cells to the same well. Media is added
instead of NK cells for wells that are used to determine
spontaneous calcein-AM release. 2% triton (Sigma-Aldrich, St.
Louis, Mo., Cat.#93443) is added instead of NK cells to wells that
are used to determine total lysis. All conditions are plated in
triplicate. Cells are incubated for 2-2.5 hours at 37.degree. C.
and then spun down at 1300 rpm for 5 minutes. 100 mL/well of
supernatant is transferred to black cliniplates. Plates are read on
a 2103 EnVision Multilabel Reader (Perkin Elmer, Waltham,
Mass.)
Example 1.2.2.3.F
FcR Binding Method
[0552] Cells (e.g., FcRn: FcRnGPI-CHO, FcgRI: THP-1, FcgRIIa: K562,
FcgRIIb: CHO-FcgRII-b-1) are plated at 1.times.105 cells/well on a
96 well round bottom plate. Antibodies and DVD-Igs are diluted 100
mg/ml in FACS buffer (1% FBS in PBS pH6.4 for FcRn samples, pH7.4
for the rest). The supernatant is removed from the cells and 30 mL
of diluted antibodies and DVD-Igs was added to well. Cells are
incubated with the antibodies at 4.degree. C. for 2 hours.
Following incubation, the cells are washed three times with 150
.mu.L FACS buffer (pH 6.4 for FcRn samples, pH 7.4 for the rest).
The cells are resuspended in 50 .mu.L FACS buffer (pH 6.4 for FcRn
samples, pH 7.4 for the rest) with 1:125 diluted R-PE conjugated
anti-human IgG F(Ab')2 (Jackson ImmunoResearch, West Grove, Pa.,
Cat.#109-116-170) and incubated at 4.degree. C. for 40 minutes.
Cells are washed three times, and finally resuspended in 100 mL
FACS buffer (pH 6.4 for FcRn samples, pH 7.4 for the rest). Samples
are run on a FACSCalibur machine (Becton Dickinson, San Jose,
Calif.). FACSCalibur settings for FL2 are adjusted such that a
non-antibody-treated control sample had a GMFI of 3. Experimental
samples were run subsequently. FlowJo software (Treestar, Inc,
Ashland, Oreg.) was used to analyze the data and determine R-PE
GMFI on live cells as designated by a forward and side scatter
gate.
Example 1.4
Generation of a DVD-Ig
[0553] DVD-Ig molecules that bind two antigens are constructed
using two parent monoclonal antibodies, one against human antigen
A, and the other against human antigen B, selected as described
herein.
Example 1.4.1
Generation of a DVD-Ig Having Two Linker Lengths
[0554] A constant region containing .mu.1 Fc with mutations at 234,
and 235 to eliminate ADCC/CDC effector functions is used. Four
different anti-A/B DVD-Ig constructs are generated: 2 with short
linker (SL) and 2 with long linker (LL), each in two different
domain orientations: V.sub.A-V.sub.B-C and V.sub.B-V.sub.A-C (see
Table 8). The linker sequences, derived from the N-terminal
sequence of human Cl/Ck or CH1 domain, are as follows:
TABLE-US-00008 For DVDAB constructs: light chain (if anti-A has
.lamda.): Short linker: (SEQ ID NO: 15) QPKAAP; Long linker: (SEQ
ID NO: 16) QPKAAPSVTLFPP light chain (if anti-A has .kappa.): Short
linker: (SEQ ID NO: 13) TVAAP; Long linker: (SEQ ID NO: 14)
TVAAPSVFIFPP heavy chain (.gamma.1): Short linker: (SEQ ID NO: 21)
ASTKGP; Long linker: (SEQ ID NO: 22) ASTKGPSVFPLAP For DVDBA
constructs: light chain (if anti-B has .lamda.): Short linker: (SEQ
ID NO: 15) QPKAAP; Long linker: (SEQ ID NO: 16) QPKAAPSVTLFPP light
chain (if anti-B has k): Short linker: (SEQ ID NO: 13) TVAAP; Long
linker: (SEQ ID NO: 14) TVAAPSVFIFPP heavy chain (.gamma.1): Short
linker: (SEQ ID NO: 21) ASTKGP; Long linker: (SEQ ID NO: 22)
ASTKGPSVFPLAP
[0555] Heavy and light chain constructs are subcloned into the pBOS
expression vector, and expressed in COS cells, followed by
purification by Protein A chromatography. The purified materials
are subjected to SDS-PAGE and SEC analysis.
[0556] Table 8 describes the heavy chain and light chain constructs
used to express each anti-A/B DVD-Ig protein.
TABLE-US-00009 TABLE 8 Anti-A/B DVD-Ig Constructs Heavy Light
DVD-Ig protein chain construct chain construct DVDABSL DVDABHC-SL
DVDABLC-SL DVDABLL DVDABHC-LL DVDABLC-LL DVDBASL DVDBAHC-SL
DVDBALC-SL DVDBALL DVDBAHC-LL DVDBALC-LL
Example 1.4.2
Molecular cloning of DNA constructs for DVDABSL and DVDABLL
[0557] To generate heavy chain constructs DVDABHC-LL and
DVDABHC-SL, VH domain of A antibody is PCR amplified using specific
primers (3' primers contain short/long linker sequence for SL/LL
constructs, respectively); meanwhile VH domain of B antibody is
amplified using specific primers (5' primers contains short/long
linker sequence for SL/LL constructs, respectively). Both PCR
reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used
together as overlapping template for the subsequent overlapping PCR
reaction. The overlapping PCR products are subcloned into Srf I and
Sal I double digested pBOS-hC.gamma.1,z non-a mammalian expression
vector (Abbott) by using standard homologous recombination
approach.
[0558] To generate light chain constructs DVDABLC-LL and
DVDABLC-SL, VL domain of A antibody is PCR amplified using specific
primers (3' primers contain short/long linker sequence for SL/LL
constructs, respectively); meanwhile VL domain of B antibody is
amplified using specific primers (5' primers contains short/long
linker sequence for SL/LL constructs, respectively). Both PCR
reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used
together as overlapping template for the subsequent overlapping PCR
reaction using standard PCR conditions. The overlapping PCR
products are subcloned into Srf I and Not I double digested
pBOS-hCk mammalian expression vector (Abbott) by using standard
homologous recombination approach. Similar approach has been used
to generate DVDBASL and DVDBALL as described below:
Example 1.4.3
Molecular cloning of DNA constructs for DVDBASL and DVDBALL
[0559] To generate heavy chain constructs DVDBAHC-LL and
DVDBAHC-SL, VH domain of antibody B is PCR amplified using specific
primers (3' primers contain short/long linker sequence for SL/LL
constructs, respectively); meanwhile VH domain of antibody A is
amplified using specific primers (5' primers contains short/long
linker sequence for SL/LL constructs, respectively). Both PCR
reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used
together as overlapping template for the subsequent overlapping PCR
reaction using standard PCR conditions. The overlapping PCR
products are subcloned into Srf I and Sal I double digested
pBOS-hC.gamma.1,z non-a mammalian expression vector (Abbott) by
using standard homologous recombination approach.
[0560] To generate light chain constructs DVDBALC-LL and
DVDBALC-SL, VL domain of antibody B is PCR amplified using specific
primers (3' primers contain short/long linker sequence for SL/LL
constructs, respectively); meanwhile VL domain of antibody A is
amplified using specific primers (5' primers contains short/long
linker sequence for SL/LL constructs, respectively). Both PCR
reactions are performed according to standard PCR techniques and
procedures. The two PCR products are gel-purified, and used
together as overlapping template for the subsequent overlapping PCR
reaction using standard PCR conditions. The overlapping PCR
products are subcloned into Srf I and Not I double digested
pBOS-hCk mammalian expression vector (Abbott) by using standard
homologous recombination approach.
Example 1.4.4
Construction and Expression of Additional DVD-Ig
Example 1.4.4.1
Preparation of DVD-Ig Vector Constructs
[0561] Parent antibody amino acid sequences for specific
antibodies, which recognize specific antigens or epitopes thereof,
for incorporation into a DVD-Ig can be obtained by preparation of
hybridomas as described above or can be obtained by sequencing
known antibody proteins or nucleic acids. In addition, known
sequences can be obtained from the literature. The sequences can be
used to synthesize nucleic acids using standard DNA synthesis or
amplification technologies and assembling the desired antibody
fragments into expression vectors, using standard recombinant DNA
technology, for expression in cells.
[0562] For example, nucleic acid codons were determined from amino
acids sequences and oligonucleotide DNA is synthesized by Blue
Heron Biotechnology, Inc., Bothell, Wash. USA. The oligonucleotides
were assembled into 300-2,000 base pair double-stranded DNA
fragments, cloned into a plasmid vector and sequence-verified.
Cloned fragments were assembled using an enzymatic process to yield
the complete gene and subcloned into an expression vector. (See
U.S. Pat. Nos. 7,306,914; 7,297,541; 7,279,159; 7,150,969;
20080115243; 20080102475; 20080081379; 20080075690; 20080063780;
20080050506; 20080038777; 20080022422; 20070289033; 20070287170;
20070254338; 20070243194; 20070225227; 20070207171; 20070150976;
20070135620; 20070128190; 20070104722; 20070092484; 20070037196;
20070028321; 20060172404; 20060162026; 20060153791; 20030215458;
20030157643).
[0563] A group of pHybE vectors (U.S. Patent Application Ser. No.
61/021,282) were used for parental antibody and DVD-Ig cloning. V1,
derived from pJP183; pHybE-hCg1,z,non-a V2, was used for cloning of
antibody and DVD heavy chains with a wildtype constant region. V2,
derived from pJP191; pHybE-hCk V2, was used for cloning of antibody
and DVD light chains with a kappa constant region. V3, derived from
pJP192; pHybE-hCl V2, was used for cloning of antibody and DVDs
light chains with a lambda constant region. V4, built with a lambda
signal peptide and a kappa constant region, was used for cloning of
DVD light chains with a lambda-kappa hybrid V domain. V5, built
with a kappa signal peptide and a lambda constant region, was used
for cloning of DVD light chains with a kappa-lambda hybrid V
domain. V7, derived from pJP183; pHybE-hCg1,z,non-a V2, was used
for cloning of antibody and DVD heavy chains with a (234,235 AA)
mutant constant region.
[0564] Table 9 shows the vectors that were used in the cloning of
the parent antibodies and DVD-Ig VH and VL chains.
TABLE-US-00010 TABLE 9 Vectors Used to Clone Parent Antibodies and
DVD-Igs ID Heavy chain vector Light chain vector AB001 V1 V2 AB002
V1 V2 AB004 V1 V2 AB006 V1 V2 AB039 V1 V2 AB064 V1 V2 AB110 V1 V2
AB114 V1 V2 AB119 V1 V2 DVD1011 V1 V2 DVD1012 V1 V2 DVD1013 V1 V2
DVD1014 V1 V2 DVD1015 V1 V2 DVD1016 V1 V2 DVD1017 V1 V2 DVD1018 V1
V2 DVD1019 V1 V2 DVD1020 V1 V2 DVD1021 V1 V2 DVD1022 V1 V2 DVD1023
V1 V2 DVD1024 V1 V2 DVD1025 V1 V2 DVD1026 V1 V2 DVD1027 V1 V2
DVD1028 V1 V2 DVD1029 V1 V2 DVD1030 V1 V2 DVD1031 V1 V2 DVD1032 V1
V2 DVD1033 V1 V2 DVD1034 V1 V2 DVD1035 V1 V2 DVD1036 V1 V2 DVD1037
V1 V2 DVD1038 V1 V2 DVD1039 V1 V2 DVD1040 V1 V2 DVD1041 V1 V2
DVD1042 V1 V2 DVD1208 V1 V2 DVD1209 V1 V2 DVD1210 V1 V2 DVD1211 V1
V2 DVD1212 V1 V2 DVD1213 V1 V2 DVD1216 V1 V2 DVD1217 V1 V2
Example 1.4.4.2
Transfection and Expression in 293 Cells
[0565] Expression of the reference antibodies and DVD-Igs was
accomplished by transiently cotransfecting HEK293 (EBNA) cells with
plasmids containing the corresponding light-chain (LC) and
heavy-chain (HC) nucleic acids. HEK293 (EBNA) cells were propagated
in Freestyle 293 media (Invitrogen, Carlsbad Calif.) at a 0.5
L-scale in flasks (2 L Corning Cat#431198) shaking in a CO2
incubator (8% CO2, 125 RPM, 37.degree. C.). When the cultures
reached a density of 1.times.106 cells/ml, cells were transfected
with transfection complex. Transfection complex was prepared by
first mixing 150 .mu.g LC-plasmid and 100m HC-plasmid together in
25 ml of Freestyle media, followed by the addition of 500 ul PEI
stock solution [stock solution: 1 mg/ml (pH 7.0) Linear 25 kDa PEI,
Polysciences Cat#23966]. The transfection complex was mixed by
inversion and allowed to incubate at room temperature for 10
minutes prior to being added to the cell culture. Following
transfection, cultures continued to be grown in the CO2 incubator
(8% CO2, 125 RPM, 37.degree. C.). Twenty-four hours after
transfection, the culture was supplemented with 25 ml of a 10%
Tryptone N1 solution (Organo Technie, La Courneuve France
Cat#19553). Nine days after transfection, cells were removed from
the cultures by centrifugation (16,000 g, 10 minutes), and the
retained supernatant was sterile filtered (Millipore HV Durapore
Stericup, 0.45 um) and placed at 4.degree. C. until initiation of
the purification step.
[0566] Each antibody or DVD-Ig was individually purified using a
disposable 1 ml packed column (packed by Orochem Technologies)
containing MabSelect SuRe resin (GE Healthcare). Columns were
pre-equilibriated in PBS and then loaded with the harvested 0.55 L
samples overnight (15 hours) at 1 ml/minute with the flow-through
being recirculated back into the feed container. Following the
loading step, columns were washed with 20 ml PBS and protein was
eluted by feeding elution buffer [50 mM Citric acid pH 3.5] at 4
ml/min and collecting fractions (1 ml) in tubes already containing
0.2 ml of 1.5M Tris pH 8.2 (bringing the final pH to approximately
6.0). Fractions containing antibody were pooled based on the
chromatograms and dialyzed into the final storage buffer [10 mM
citric acid, 10 mM Na2HPO4, pH 6.0]. Following dialysis, samples
were filtered through a 0.22 um Steriflip (Millipore) and the
protein concentration was determined by absorbance [Hewlett Packard
8453 diode array spectrophotometer]. SDS-PAGE analysis was
performed on analytical samples (both reduced and non-reduced) to
assess final purity, verify the presence of appropriately sized
heavy- and light-chain bands, and confirm the absence of
significant amounts of free (e.g., uncomplexed) light chain (in the
non-reduced samples).
[0567] Table 10 contains the yield data for parent antibodies or
DVD-Ig constructs expressed as milligrams per liter in 293
cells.
TABLE-US-00011 TABLE 10 Transient Expression in Yields of Parent
Antibodies and DVD-Ig Constructs in 293 Cells Parent N-terminal
C-terminal Antibody or Variable Variable Expression DVD-Ig ID
Domain (VD) Domain (VD) Yield (mg/L) AB119 MTX 1.6 DVD1208 MTX CD20
0 DVD1209 CD20 MTX 0.12 DVD1210 MTX EGFR (seq3) 0 DVD1211 EGFR (seq
3) MTX 1.94 DVD1212 MTX Her2 (seq1) 0 DVD1213 Her2 (seq1) MTX 2.6
DVD1216 MTX CD-3 (seq 1) 0 DVD1217 CD-3 (seq 1) MTX 0.58 All
DVD-Igs expressed well in 293 cells. DVD-Igs could be easily
purified over a protein A column. In most cases >5 mg/L purified
DVD-Ig could be obtained easily from supernatants of 293 cells.
Example 1.4.5
Characterization and Lead Selection of A/B DVD-Igs
[0568] The binding affinities of anti-A/B DVD-Igs are analyzed on
Biacore against both protein A and protein B. The tetravalent
property of the DVD-Ig is examined by multiple binding studies on
Biacore. Meanwhile, the neutralization potency of the DVD-Igs for
protein A and protein B are assessed by bioassays, respectively, as
described herein. The DVD-Ig molecules that best retain the
affinity and potency of the original parent mAbs are selected for
in-depth physicochemical and bio-analytical (rat PK)
characterizations as described herein for each mAb. Based on the
collection of analyses, the final lead DVD-Ig is advanced into CHO
stable cell line development, and the CHO-derived material is
employed in stability, pharmacokinetic and efficacy studies in
cynomolgus monkey, and preformulation activities.
Example 2
Generation and Characterization of Dual Variable Domain
Immunoglobulins (DVD-Ig)
[0569] Dual variable domain immunoglobulins (DVD-Ig) using parent
antibodies with known amino acid sequences were generated by
synthesizing polynucleotide fragments encoding DVD-Ig variable
heavy and DVD-Ig variable light chain sequences and cloning the
fragments into a pHybC-D2 vector according to Example 1.4.4.1. The
DVD-Ig constructs were cloned into and expressed in 293 cells as
described in Example 1,4.4.2. The DVD-Ig protein was purified
according to standard methods. Functional characteristics were
determined according to the methods described in Example 1.1.1 and
1.1.2 as indicated. DVD-Ig VH and VL chains for the DVD-Igs of the
invention are provided below.
Example 2.1
Generation of Methotrexate and Mouse CD-3 DVD-Igs with Short-Short
Linkers
TABLE-US-00012 [0570] TABLE 11 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 110 DVD1011H AB119VH
AB110VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPEVQLVESGGGLVQPGKSL
KLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITS
SSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKS EDTAMYYCARFDWDKNYWGQGTMVTVSS
111 DVD1011L AB119VL AB110VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPSSLPASLGDRVTIN
CQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG
VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYY NYPWTFGPGTKLEIKR 112 DVD1012H
AB110VH AB119VH EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMH
WVRQAPGRGLESVAYITSSSINIKYADAVKGRFTV
SRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNY
WGQGTMVTVSSASTKGPDVQLQESGPGLVKPSQSL
SLTCTVTGFSITSPYAWNWIRQFPGNTLEWMGYIS
YRGSTTHHPSLKSRISITRDTSKNQFFLQLNSVTT EDTATYFCSSYGNYGAYSGQGTLVTVSA
113 DVD1012L AB110VL AB119VL DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNW
YQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRD
SSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLE
IKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.2
Generation of Methotrexate and Mouse CD-3 DVD-Igs with Long-Long
Linkers
TABLE-US-00013 [0571] TABLE 12 DVD Outer Inner Variable Variable
Variable SEQ Domain Domain Domain Sequence ID NO Name Name Name
12345678901234567890123456789012345 114 DVD1013H AB119VH AB110VH
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPEVQLVESGGGL
VQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLE
SVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFL
QMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS 115 DVD1013L AB119VL AB110VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDIQMTQSPSSLPASL
GDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYY
TNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGS YYCQQYYNYPWTFGPGTKLEIKR 116
DVD1014H AB110VH AB119VH EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMH
WVRQAPGRGLESVAYITSSSINIKYADAVKGRFTV
SRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNY
WGQGTMVTVSSASTKGPSVFPLAPDVQLQESGPGL
VKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNTL
EWMGYISYRGSTTHHPSLKSRISITRDTSKNQFFL
QLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 117 DVD1014L AB110VL AB119VL
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNW
YQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRD
SSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLE
IKRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYK
VSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQGSHVPLTFGAGTQLELKR
Example 2.3
Generation of Methotrexate and Mouse CD-3 DVD-Igs with Long-Short
Linkers
TABLE-US-00014 [0572] TABLE 13 DVD Outer Inner Variable Variable
Variable SEQ Domain Domain Domain Sequence ID NO Name Name Name
12345678901234567890123456789012345 118 DVD1015H AB119VH AB110VH
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPEVQLVESGGGL
VQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLE
SVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFL
QMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSS 119 DVD1015L AB119VL AB110VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPSSLPASLGDRVTIN
CQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADG
VPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYY NYPWTFGPGTKLEIKR 120 DVD1016H
AB110VH AB119VH EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMH
WVRQAPGRGLESVAYITSSSINIKYADAVKGRFTV
SRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNY
WGQGTMVTVSSASTKGPSVFPLAPDVQLQESGPGL
VKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNTL
EWMGYISYRGSTTHHPSLKSRISITRDTSKNQFFL
QLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 121 DVD1016L AB110VL AB119VL
DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNW
YQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRD
SSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLE
IKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.4
Generation of Methotrexate and Mouse CD-3 DVD-Igs with Short-Long
Linkers
TABLE-US-00015 [0573] TABLE 14 DVD Outer Inner Variable Variable
Variable SEQ Domain Domain Domain Sequence ID NO Name Name Name
12345678901234567890123456789012345 122 DVD1017H AB119VH AB110VH
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPEVQLVESGGGLVQPGKSL
KLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITS
SSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKS EDTAMYYCARFDWDKNYWGQGTMVTVSS
123 DVD1017L AB119VL AB110VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDIQMTQSPSSLPASL
GDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYY
TNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGS YYCQQYYNYPWTFGPGTKLEIKR 124
DVD1018H AB110VH AB119VH EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMH
WVRQAPGRGLESVAYITSSSINIKYADAVKGRFTV
SRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNY
WGQGTMVTVSSASTKGPDVQLQESGPGLVKPSQSL
SLTCTVTGFSITSPYAWNWIRQFPGNTLEWMGYIS
YRGSTTHHPSLKSRISITRDTSKNQFFLQLNSVTT EDTATYFCSSYGNYGAYSGQGTLVTVSA
125 DVD1018L AB110VL AB119VL DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNW
YQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRD
SSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLE
IKRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYK
VSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQGSHVPLTFGAGTQLELKR
Example 2.5
Generation of Methotrexate and Mouse CD-19 DVD-Igs with Short-Short
Linkers
TABLE-US-00016 [0574] TABLE 15 DVD Outer Inner Variable Variable
Variable SEQ Domain Domain Domain Sequence ID NO Name Name Name
12345678901234567890123456789012345 126 DVD1019H AB119VH AB114VH
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPEVQLQQSGAELVRPGTSV
KLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDP
EDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTS EDTATYFCIYGGYYFDYWGQGVMVTVSS
127 DVD1019L AB119VL AB114VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPASLSTSLGETVTIQ
CQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDG
VPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGL TYPRTFGGGTKLELKR 128 DVD1020H
AB114VH AB119VH EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMH
FVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATL
TADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDY
WGQGVMVTVSSASTKGPDVQLQESGPGLVKPSQSL
SLTCTVTGFSITSPYAWNWIRQFPGNTLEWMGYIS
YRGSTTHHPSLKSRISITRDTSKNQFFLQLNSVTT EDTATYFCSSYGNYGAYSGQGTLVTVSA
129 DVD1020L AB114VL AB119VL DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAW
YQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQ
YSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLE
LKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.6
Generation of Methotrexate and Mouse CD-19 DVD-Igs with Long-Long
Linkers
TABLE-US-00017 [0575] TABLE 16 DVD Outer Inner Variable Variable
Variable SEQ Domain Domain Domain Sequence ID NO Name Name Name
12345678901234567890123456789012345 130 DVD1021H AB119VH AB114VH
DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPEVQLQQSGAEL
VRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLE
WIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYL
KLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSS 131 DVD1021L AB119VL AB114VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDIQMTQSPASLSTSL
GETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYG
ASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGV YFCQQGLTYPRTFGGGTKLELKR 132
DVD1022H AB114VH AB119VH EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMH
FVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATL
TADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDY
WGQGVMVTVSSASTKGPSVFPLAPDVQLQESGPGL
VKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNTL
EWMGYISYRGSTTHHPSLKSRISITRDTSKNQFFL
QLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 133 DVD1022L AB114VL AB119VL
DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAW
YQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQ
YSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLE
LKRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYK
VSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQGSHVPLTFGAGTQLELKR
Example 2.7
Generation of Methotrexate and Mouse CD-19 DVD-Igs with Long-Short
Linkers
TABLE-US-00018 [0576] TABLE 17 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 134 DVD1023H AB119VH
AB114VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPEVQLQQSGAEL
VRPGTSVKLSCKVSGDTITFYYMHFVKQRPGQGLE
WIGRIDPEDESTKYSEKFKNKATLTADTSSNTAYL
KLSSLTSEDTATYFCIYGGYYFDYWGQGVMVTVSS 135 DVD1023L AB119VL AB114VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPASLSTSLGETVTIQ
CQASEDIYSGLAWYQQKPGKSPQLLIYGASDLQDG
VPSRFSGSGSGTQYSLKITSMQTEDEGVYFCQQGL TYPRTFGGGTKLELKR 136 DVD1024H
AB114VH AB119VH EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMH
FVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATL
TADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDY
WGQGVMVTVSSASTKGPSVFPLAPDVQLQESGPGL
VKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNTL
EWMGYISYRGSTTHHPSLKSRISITRDTSKNQFFL
QLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 137 DVD1024L AB114VL AB119VL
DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAW
YQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQ
YSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLE
LKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.8
Generation of Methotrexate and Mouse CD-19 DVD-Igs with Short-Long
Linkers
TABLE-US-00019 [0577] TABLE 18 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 138 DVD1025H AB119VH
AB114VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPEVQLQQSGAELVRPGTSV
KLSCKVSGDTITFYYMHFVKQRPGQGLEWIGRIDP
EDESTKYSEKFKNKATLTADTSSNTAYLKLSSLTS EDTATYFCIYGGYYFDYWGQGVMVTVSS
139 DVD1025L AB119VL AB114VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDIQMTQSPASLSTSL
GETVTIQCQASEDIYSGLAWYQQKPGKSPQLLIYG
ASDLQDGVPSRFSGSGSGTQYSLKITSMQTEDEGV YFCQQGLTYPRTFGGGTKLELKR 140
DVD1026H AB114VH AB119VH EVQLQQSGAELVRPGTSVKLSCKVSGDTITFYYMH
FVKQRPGQGLEWIGRIDPEDESTKYSEKFKNKATL
TADTSSNTAYLKLSSLTSEDTATYFCIYGGYYFDY
WGQGVMVTVSSASTKGPDVQLQESGPGLVKPSQSL
SLTCTVTGFSITSPYAWNWIRQFPGNTLEWMGYIS
YRGSTTHHPSLKSRISITRDTSKNQFFLQLNSVTT EDTATYFCSSYGNYGAYSGQGTLVTVSA
141 DVD1026L AB114VL AB119VL DIQMTQSPASLSTSLGETVTIQCQASEDIYSGLAW
YQQKPGKSPQLLIYGASDLQDGVPSRFSGSGSGTQ
YSLKITSMQTEDEGVYFCQQGLTYPRTFGGGTKLE
LKRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQAS
ISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYK
VSTRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGV YYCFQGSHVPLTFGAGTQLELKR
Example 2.9
Generation of Methotrexate and Human CD-3 (Seq. 2) DVD-Igs with
Short-Short Linkers
TABLE-US-00020 [0578] TABLE 19 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 142 DVD1027H AB119VH
AB039VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQSGAELARPGASV
KMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINP
SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTS EDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
143 DVD1027L AB119VL AB039VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPQIVLTQSPAIMSASPGEKVTMT
CSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGV
PAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSS NPFTFGSGTKLEINR 144 DVD1028H
AB039VH AB119VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL
TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSSASTKGPDVQLQESGPGLVKPS
QSLSLTCTVTGFSITSPYAWNWIRQFPGNTLEWMG
YISYRGSTTHHPSLKSRISITRDTSKNQFFLQLNS VTTEDTATYFCSSYGNYGAYSGQGTLVTVSA
145 DVD1028L AB039VL AB119VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY
QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY
SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI
NRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQS
IVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSGV
PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGAGTQLELKR
Example 2.10
Generation of Methotrexate and Human CD-3 (Seq. 2) DVD-Igs with
Long-Long-Short Linkers
TABLE-US-00021 [0579] TABLE 20 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 146 DVD1029H AB119VH
AB039VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPQVQLQQSGAEL
ARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLE
WIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYM
QLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLT VSS 147 DVD1029L AB119VL
AB039VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPQIVLTQSPAIMSASP
GEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDT
SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATY YCQQWSSNPFTFGSGTKLEINR 148
DVD1030H AB039VH AB119VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL
TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSSASTKGPSVFPLAPDVQLQESG
PGLVKPSQSLSLTCTVTGFSITSPYAWNWIRQFPG
NTLEWMGYISYRGSTTHHPSLKSRISITRDTSKNQ
FFLQLNSVTTEDTATYFCSSYGNYGAYSGQGTLVT VSA 149 DVD1030L AB039VL
AB119VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY
QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY
SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI
NRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQASI
SCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKV
STRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPLTFGAGTQLELKR
Example 2.11
Generation of Methotrexate and Human CD-3 (Seq. 2) DVD-Igs with
Long-Short Linkers
TABLE-US-00022 [0580] TABLE 21 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 150 DVD1031H AB119VH
AB039VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPQVQLQQSGAEL
ARPGASVKMSCKASGYTFTRYTMHWVKQRPGQGLE
WIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYM
QLSSLTSEDSAVYYCARYYDDHYCLDYWGQGTTLT VSS 151 DVD1031L AB119VL
AB039VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPQIVLTQSPAIMSASPGEKVTMT
CSASSSVSYMNWYQQKSGTSPKRWIYDTSKLASGV
PAHFRGSGSGTSYSLTISGMEAEDAATYYCQQWSS NPFTFGSGTKLEINR 152 DVD1032H
AB039VH AB119VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL
TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSSASTKGPSVFPLAPDVQLQESG
PGLVKPSQSLSLTCTVTGFSITSPYAWNWIRQFPG
NTLEWMGYISYRGSTTHHPSLKSRISITRDTSKNQ
FFLQLNSVTTEDTATYFCSSYGNYGAYSGQGTLVT VSA 153 DVD1032L AB039VL
AB119VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY
QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY
SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI
NRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQS
IVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSGV
PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGAGTQLELKR
Example 2.12
Generation of Methotrexate and Human CD-3 (Seq. 2) DVD-Igs with
Short-Long Linkers
TABLE-US-00023 [0581] TABLE 22 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 154 DVD1033H AB119VH
AB039VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQSGAELARPGASV
KMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINP
SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTS EDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
155 DVD1033L AB119VL AB039VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPQIVLTQSPAIMSASP
GEKVTMTCSASSSVSYMNWYQQKSGTSPKRWIYDT
SKLASGVPAHFRGSGSGTSYSLTISGMEAEDAATY YCQQWSSNPFTFGSGTKLEINR 156
DVD1034H AB039VH AB119VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL
TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSSASTKGPDVQLQESGPGLVKPS
QSLSLTCTVTGFSITSPYAWNWIRQFPGNTLEWMG
YISYRGSTTHHPSLKSRISITRDTSKNQFFLQLNS VTTEDTATYFCSSYGNYGAYSGQGTLVTVSA
157 DVD1034L AB039VL AB119VL QIVLTQSPAIMSASPGEKVTMTCSASSSVSYMNWY
QQKSGTSPKRWIYDTSKLASGVPAHFRGSGSGTSY
SLTISGMEAEDAATYYCQQWSSNPFTFGSGTKLEI
NRTVAAPSVFIFPPDVLLTQIPLSLPVSLGDQASI
SCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKV
STRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVY YCFQGSHVPLTFGAGTQLELKR
Example 2.13
Generation of Methotrexate and Human CD-19 DVD-Igs with Short-Short
Linkers
TABLE-US-00024 [0582] TABLE 23 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 158 DVD1035H AB119VH
AB006VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQSGAELVRPGSSV
KISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWP
GDGDTNYNGKFKGKATLTADESSSTAYMQLSSLAS
EDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVS S 159 DVD1035L AB119VL AB006VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDILLTQTPASLAVSLGQRATIS
CKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASN
LVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWTFGGGTKLEIKR 169
DVD1036H AB006VH AB119VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN
WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL
TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG
RYYYAMDYWGQGTSVTVSSASTKGPDVQLQESGPG
LVKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNT
LEWMGYISYRGSTTHHPSLKSRISITRDTSKNQFF
LQLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVS A 161 DVD1036L AB006VL AB119VL
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS
YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG
SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG
TKLEIKRTVAAPDVLLTQIPLSLPVSLGDQASISC
RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVST
RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPLTFGAGTQLELKR
Example 2.14
Generation of Methotrexate and Human CD-19 DVD-Igs with Long-Long
Linkers
TABLE-US-00025 [0583] TABLE 24 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 162 DVD1037H AB119VH
AB006VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPQVQLQQSGAEL
VRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLE
WIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYM
QLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQ GTSVTVSS 163 DVD1037L AB119VL
AB006VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDILLTQTPASLAVSL
GQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKL
LIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKV DAATYHCQQSTEDPWTFGGGTKLEIKR 164
DVD1038H AB006VH AB119VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN
WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL
TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG
RYYYAMDYWGQGTSVTVSSASTKGPSVFPLAPDVQ
LQESGPGLVKPSQSLSLTCTVTGFSITSPYAWNWI
RQFPGNTLEWMGYISYRGSTTHHPSLKSRISITRD
TSKNQFFLQLNSVTTEDTATYFCSSYGNYGAYSGQ GTLVTVSA 165 DVD1038L AB006VL
AB119VL DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS
YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG
SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG
TKLEIKRTVAAPSVFIFPPDVLLTQIPLSLPVSLG
DQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKL
LIYKVSTRFSGVPDRFSGSGSGTDFTLKISRVEAE DLGVYYCFQGSHVPLTFGAGTQLELKR
Example 2.15
Generation of Methotrexate and Human CD-19 DVD-Igs with Long-Short
Linkers
TABLE-US-00026 [0584] TABLE 25 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 166 DVD1039H AB119VH
AB006VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPSVFPLAPQVQLQQSGAEL
VRPGSSVKISCKASGYAFSSYWMNWVKQRPGQGLE
WIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYM
QLSSLASEDSAVYFCARRETTTVGRYYYAMDYWGQ GTSVTVSS 167 DVD1039L AB119VL
AB006VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDILLTQTPASLAVSLGQRATIS
CKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASN
LVSGIPPRFSGSGSGTDFTLNIHPVEKVDAATYHC QQSTEDPWTFGGGTKLEIKR 168
DVD1040H AB006VH AB119VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN
WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL
TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG
RYYYAMDYWGQGTSVTVSSASTKGPSVFPLAPDVQ
LQESGPGLVKPSQSLSLTCTVTGFSITSPYAWNWI
RQFPGNTLEWMGYISYRGSTTHHPSLKSRISITRD
TSKNQFFLQLNSVTTEDTATYFCSSYGNYGAYSGQ GTLVTVSA 169 DVD1040L AB006VL
AB119VL DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS
YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG
SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG
TKLEIKRTVAAPDVLLTQIPLSLPVSLGDQASISC
RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVST
RFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC FQGSHVPLTFGAGTQLELKR
Example 2.16
Generation of Methotrexate and Human CD-19 DVD-Igs with Short-Long
Linkers
TABLE-US-00027 [0585] TABLE 26 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 170 DVD1041H AB119VH
AB006VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQSGAELVRPGSSV
KISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWP
GDGDTNYNGKFKGKATLTADESSSTAYMQLSSLAS
EDSAVYFCARRETTTVGRYYYAMDYWGQGTSVTVS S 171 DVD1041L AB119VL AB006VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPSVFIFPPDILLTQTPASLAVSL
GQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKL
LIYDASNLVSGIPPRFSGSGSGTDFTLNIHPVEKV DAATYHCQQSTEDPWTFGGGTKLEIKR 172
DVD1042H AB006VH AB119VH QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN
WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATL
TADESSSTAYMQLSSLASEDSAVYFCARRETTTVG
RYYYAMDYWGQGTSVTVSSASTKGPDVQLQESGPG
LVKPSQSLSLTCTVTGFSITSPYAWNWIRQFPGNT
LEWMGYISYRGSTTHHPSLKSRISITRDTSKNQFF
LQLNSVTTEDTATYFCSSYGNYGAYSGQGTLVTVS A 173 DVD1042L AB006VL AB119VL
DILLTQTPASLAVSLGQRATISCKASQSVDYDGDS
YLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG
SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG
TKLEIKRTVAAPSVFIFPPDVLLTQIPLSLPVSLG
DQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKL
LIYKVSTRFSGVPDRFSGSGSGTDFTLKISRVEAE DLGVYYCFQGSHVPLTFGAGTQLELKR
Example 2.17
Generation of Methotrexate and Human CD-20 DVD-Igs with Short-Short
Linkers
TABLE-US-00028 [0586] TABLE 27 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 174 DVD1208H AB119VH
AB001VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQPGAELVKPGASV
KMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYP
GNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTS
EDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSA 175 DVD1208L AB119VL AB001VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPQIVLSQSPAILSPSPGEKVTMT
CRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGV
PVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTS NPPTFGGGTKLEIKR 176 DVD1209H
AB001VH AB119VH QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMH
WVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATL
TADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGD
WYFNVWGAGTTVTVSAASTKGPDVQLQESGPGLVK
PSQSLSLTCTVTGFSITSPYAWNWIRQFPGNTLEW
MGYISYRGSTTHHPSLKSRISITRDTSKNQFFLQL
NSVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 177 DVD1209L AB001VL AB119VL
QIVLSQSPAILSPSPGEKVTMTCRASSSVSYIHWF
QQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSY
SLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEI
KRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQS
IVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSGV
PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGAGTQLELKR
Example 2.18
Generation of Methotrexate and Human EGFR (Seq. 3) DVD-Igs with
Short-Short Linkers
TABLE-US-00029 [0587] TABLE 28 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 178 DVD1210H AB119VH
AB064VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQESGPGLVKPSQTL
SLTCTVSGYSISSDFAWNWIRQPPGKGLEWMGYIS
YSGNTRYQPSLKSRITISRDTSKNQFFLKLNSVTA ADTATYYCVTAGRGFPYWGQGTLVTVSS
179 DVD1210L AB119VL AB064VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPSSMSVSVGDRVTIT
CHSSQDINSNIGWLQQKPGKSFKGLIYHGTNLDDG
VPSRFSGSGSGTDYTLTISSLQPEDFATYYCVQYA QFPWTFGGGTKLEIKR 180 DVD1211H
AB064VH AB119VH QVQLQESGPGLVKPSQTLSLTCTVSGYSISSDFAW
NWIRQPPGKGLEWMGYISYSGNTRYQPSLKSRITI
SRDTSKNQFFLKLNSVTAADTATYYCVTAGRGFPY
WGQGTLVTVSSASTKGPDVQLQESGPGLVKPSQSL
SLTCTVTGFSITSPYAWNWIRQFPGNTLEWMGYIS
YRGSTTHHPSLKSRISITRDTSKNQFFLQLNSVTT EDTATYFCSSYGNYGAYSGQGTLVTVSA
181 DVD1211L AB064VL AB119VL DIQMTQSPSSMSVSVGDRVTITCHSSQDINSNIGW
LQQKPGKSFKGLIYHGTNLDDGVPSRFSGSGSGTD
YTLTISSLQPEDFATYYCVQYAQFPWTFGGGTKLE
IKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.19
Generation of Methotrexate and Human HER-2 (Seq. 1) DVD-Igs with
Short-Short Linkers
TABLE-US-00030 [0588] TABLE 29 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 182 DVD1212H AB119VH
AB004VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPEVQLVESGGGLVQPGGSL
RLSCAASGFNIKDTYIHWVRQAPGKGLEWVARTYP
TNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRA
EDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS 183 DVD1212L AB119VL AB004VL
DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPDIQMTQSPSSLSASVGDRVTIT
CRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSG
VPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHY TTPPTFGQGTKVEIKR 184 DVD1213H
AB004VH AB119VH EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIH
WVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI
SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY
AMDYWGQGTLVTVSSASTKGPDVQLQESGPGLVKP
SQSLSLTCTVTGFSITSPYAWNWIRQFPGNTLEWM
GYISYRGSTTHHPSLKSRISITRDTSKNQFFLQLN
SVTTEDTATYFCSSYGNYGAYSGQGTLVTVSA 185 DVD1213L AB004VL AB119VL
DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW
YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD
FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVE
IKRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQ
SIVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSG
VPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGS HVPLTFGAGTQLELKR
Example 2.20
Generation of Methotrexate and Human CD-3 (Seq. 1) DVD-Igs with
Short-Short Linkers
TABLE-US-00031 [0589] TABLE 30 DVD Outer Inner SEQ Variable
Variable Variable ID Domain Domain Domain Sequence NO Name Name
Name 12345678901234567890123456789012345 186 DVD1216H AB119VH
AB002VH DVQLQESGPGLVKPSQSLSLTCTVTGFSITSPYAW
NWIRQFPGNTLEWMGYISYRGSTTHHPSLKSRISI
TRDTSKNQFFLQLNSVTTEDTATYFCSSYGNYGAY
SGQGTLVTVSAASTKGPQVQLQQSGAELARPGASV
KMSCKASGYTFTRYTMHWVKQRPGQGLEWIGYINP
SRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTS EDSAVYYCARYYDDHYCLDYWGQGTTLTVSS
187 DVD1216L AB119VL AB002VL DVLLTQIPLSLPVSLGDQASISCRSSQSIVHSNGN
TYLEWYLQKPGQSPKLLIYKVSTRFSGVPDRFSGS
GSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGA
GTQLELKRTVAAPQIVLTQSPAIMSASPGEKVTMT
CRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGV
PYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSS NPLTFGSGTKLEINR 188 DVD1217H
AB002VH AB119VH QVQLQQSGAELARPGASVKMSCKASGYTFTRYTMH
WVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATL
TTDKSSSTAYMQLSSLTSEDSAVYYCARYYDDHYC
LDYWGQGTTLTVSSASTKGPDVQLQESGPGLVKPS
QSLSLTCTVTGFSITSPYAWNWIRQFPGNTLEWMG
YISYRGSTTHHPSLKSRISITRDTSKNQFFLQLNS VTTEDTATYFCSSYGNYGAYSGQGTLVTVSA
189 DVD1217L AB002VL AB119VL QIVLTQSPAIMSASPGEKVTMTCRASSSVSYMNWY
QQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSY
SLTISSMEAEDAATYYCQQWSSNPLTFGSGTKLEI
NRTVAAPDVLLTQIPLSLPVSLGDQASISCRSSQS
IVHSNGNTYLEWYLQKPGQSPKLLIYKVSTRFSGV
PDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSH VPLTFGAGTQLELKR
Example 2.21
Cloning Vector Sequences Used to Clone Parent Antibody and DVD-Ig
Sequences
TABLE-US-00032 [0590] TABLE 31 Vector Nucleotide sequences name SEQ
ID NO 123456789012345678901234567890123456789012345678901 V1 190
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG
GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGC
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGG
ATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA
TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGG
CAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCC
CCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCG
GGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGC
CCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGA
CTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTG
GCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTG
CCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACAT
GTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATC
AGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCA
ATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTC
CCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGT
TACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTA
AGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATG
GGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGG
CTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCT
TCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAA
GGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATA
AAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAA
CCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCT
GAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACT
GGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGT
GCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACA
GGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGAC
GCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAA
CGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTT
TTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTG
CGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCC
GCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCC
GGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGC
TGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAG
GGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATG
TTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC
TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCAT
ATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCT
GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA
TCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATG
CTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGG
TAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATAT
CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCT
AATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATA
TGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG
GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT
CTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGA
ATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA
TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAA
TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA
TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG
GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC
GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA
AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT
CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT
GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA
CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT
AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA
CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA
CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAA
TGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGC
AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG
GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT
GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG
TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC
CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA
ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA
TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC
CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA
AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG
CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA
AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT
ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA
CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC
TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA
GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA
GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA
GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC
GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG
AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA
GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGC
CAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA
CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC
CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA
GTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC
CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG
GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA
GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT
TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGC
CAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAA
GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA
TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGAC
TAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT
TCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGC
TTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATG
GACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTG
GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGG
CAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA
TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATAT
AAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG
AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGG
TTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGAT
TCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG
CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCG
CTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGC
TTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCT
TTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGG
TATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCG
CACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACG
GGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCC
GTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGC
GTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATG
GAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAA
AAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCG
GGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTC
TTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG
GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGA
ATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT
GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAG
ATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGG
CTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC V2 191
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA
GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC
TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGA
CCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT
GGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATT
TGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGG
ACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGC
ATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCA
CATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTG
ACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATC
CTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGT
GTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCC
AGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCC
TGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTT
ATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGT
AGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACG
GGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGC
GATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGA
TTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATC
AAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCC
TTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGT
GAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGA
CGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAA
ACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTT
TAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCA
TCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGA
TACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCA
TGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGC
AGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCC
ACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAA
TTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGG
ACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCAC
TGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATAC
CTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTG
GAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGG
TCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGG
GTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATA
TCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCC
TAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCAT
AGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCT
GGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAA
TAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATA
CTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGC
ATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATAT
CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCT
AATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATA
TGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCG
GGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTG
AAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT
AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG
AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT
GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT
GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA
AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG
TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC
TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCA
AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA
CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT
ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG
GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT
ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTT
GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT
AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC
CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG
GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTAT
CGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG
ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA
CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT
TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC
TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA
AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC
AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC
AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC
TGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC
ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA
TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT
GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA
AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG
GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT
TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC
GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT
TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG
AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG
CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG
GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG
CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA
GGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTA
GCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC
TCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC
TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTT
TTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT
AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAG
ATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTA
GGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCG
CACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC
CGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA
CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT
AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT
AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCC
TTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCC
CGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGG
AGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG
CCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAA
GTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG
GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT
TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTC
GGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC
TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGC
CCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGA
AAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG
GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT
TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC
CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG
GGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGAC
TGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT
TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG
TTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGA
CCTCGAGATCCATTGTGCCCGGGCGCACCATGGACATGCGCGTGCCCGCCC
AGCTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC V3 192
CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG
CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCG
GGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGA
GTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGC
AGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGC
TGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA
GAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCT
CGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA
ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGG
TCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACG
AACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATG
TAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACAT
GTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACA
TCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTG
GAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTA
ACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGG
GGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGT
GTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATA
AGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGT
ATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGA
AGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGAT
ATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTC
CACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAG
GAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTC
GTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAG
GTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGG
GGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACC
CCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAA
CAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCT
CACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATAC
TGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGT
TGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGC
GGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACG
CCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTG
TGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACT
GTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGC
GGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTG
CATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAG
GACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCC
TCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTA
GCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCT
GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA
TCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGG
CTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGG
TAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAG
AGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTA
CCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATA
TGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG
GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT
TTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGC
TATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGT
AGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAG
ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT
AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC
CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG
ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG
TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT
TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA
GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT
TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA
GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC
ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG
CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC
AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA
TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC
AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCG
CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT
AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT
TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC
TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT
AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA
GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA
AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA
AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA
ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG
ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA
AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC
TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT
TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC
GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG
CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA
GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA
GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG
CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG
GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA
TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT
GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGC
CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC
TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC
TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA
GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC
GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAG
TGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC
TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA
ACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCT
AGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCA
ATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAA
CTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA
TTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT
GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATG
GATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGT
CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCAC
ATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGG
TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTG
GCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT
CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAG
TGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTG
CGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA
GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGC
CCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG
CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC
TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCA
AGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT
TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC
GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA
AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCC
GCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG
ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCG
CTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC
GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAG
GCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGG
GGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA
AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT
TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTT
TTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCT
CGAGATCCATTGTGCCCGGGCGCCACCATGACTTGGACCCCACTCCTCTTC
CTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG V4 193
ACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGA
GAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC
CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGC
AGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCC
TGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGTTGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGA
CCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTT
GGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATT
TGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGG
ACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGC
ATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCA
CATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTG
ACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATC
CTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGT
GTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCC
AGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCC
TGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTT
ATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGT
AGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACG
GGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGC
GATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGA
TTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATC
AAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCC
TTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGT
GAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGA
CGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAA
ACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTT
TAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCA
TCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGA
TACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCA
TGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGC
AGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCC
ACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAA
TTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGG
ACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCAC
TGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATAC
CTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTG
GAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGG
TCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGG
GTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATA
TCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCC
TAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCAT
AGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCT
GGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAA
TAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATA
CTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGC
ATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATAT
CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCT
AATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATA
TGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCG
GGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTG
AAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGAT
AATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGG
AACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCAT
GAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT
GAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTG
CCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA
AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGG
TAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCAC
TTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCA
AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTA
CTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATT
ATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCT
GACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGG
GGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCAT
ACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTT
GCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATT
AATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGC
CCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGG
GTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTAT
CGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAG
ACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGA
CCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATT
TAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCC
TTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAA
AGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAAC
AAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACC
AACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATAC
TGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGC
ACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAG
TGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA
TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTT
GGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGA
AAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG
CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTG
GTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATT
TTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGC
GGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTT
TCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTG
AGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAG
CGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTG
GCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGG
CAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCA
GGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGG
ATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTA
GCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATC
TCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCC
TAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTT
TTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT
AGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAG
ATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTA
GGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCG
CACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC
CGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA
CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT
AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGT
AAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCC
TTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCC
CGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGG
AGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG
CCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAA
GTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTG
GCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT
TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTC
GGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTC
TCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGC
CCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGA
AAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCG
GCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTT
TCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTC
CAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTG
GGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGAC
TGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCT
TTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG
TTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGA
CCTCGAGATCCATTGTGCCCGGGCGCACCATGACTTGGACCCCACTCCTCT
TCCTCACCCTCCTCCTCCACTGCACAGGAAGCTTATCG V5 194
CAACCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCGCCCTCCTCTGAGGAG
CTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCG
GGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGA
GTGGAGACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGC
AGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAGAAGCTACAGC
TGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACA
GAATGTTCATGAGCGGCCGCTCGAGGCCGGCAAGGCCGGATCCCCCGACCT
CGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGA
ATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTGG
TCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCCCCGCCCCGGACG
AACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCGGGGCAGTGCATG
TAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGCCCTGTTCCACAT
GTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGACTGTAGTTGACA
TCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTGGCTTTCATCCTG
GAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTGCCTTTATGTGTA
ACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACATGTACCTCCCAGG
GGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATCAGAGGGGCCTGT
GTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCAATAGTGTTTATA
AGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTCCCGGGTAGTAGT
ATATACTATCCAGACTAACCCTAATTCAATAGCATATGTTACCCAACGGGA
AGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTAAGGAACAGCGAT
ATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATGGGGTCAGGATTC
CACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGGCTGAAGATCAAG
GAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCTTCATTCTCCTTC
GTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAAGGTGTATGTGAG
GTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATAAAATTTGGACGG
GGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAACCCTCACAAACC
CCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCTGAATATCTTTAA
CAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACTGGATGTCCATCT
CACACGAATTTATGGCTATGGGCAACACATAATCCTAGTGCAATATGATAC
TGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACAGGTGAACCATGT
TGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGACGCCGACAGCAGC
GGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAACGGGGCTCCACG
CCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTTTTTTTGAAATTG
TGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTGCGGTTTTGGACT
GTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCCGCTAACCACTGC
GGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCCGGGGAATACCTG
CATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGCTGCGATCTGGAG
GACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAGGGTTGTTGGTCC
TCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATGTTGCCATGGGTA
GCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCT
GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA
TCTATATCTGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATAGG
CTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGG
TAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATGCTATCCTAATAG
AGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGGTAGCATATACTA
CCCAAATATCTGGATAGCATATGCTATCCTAATCTATATCTGGGTAGCATA
TGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG
GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT
TTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGC
TATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGT
AGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGAATTTTCTTGAAG
ACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAATGTCATGATAAT
AATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAAC
CCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG
ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAG
TATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCT
TCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGA
TCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAA
GATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTT
TAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA
GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTC
ACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATG
CAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGAC
AACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGA
TCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACC
AAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGCAACAACGTTGCG
CAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAAT
AGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCT
TCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTC
TCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGT
AGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACA
GATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA
AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAA
AAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTA
ACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGG
ATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAA
AAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAAC
TCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGT
TCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACC
GCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGG
CGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAA
GGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGA
GCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAG
CGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAG
GGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTA
TCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTT
GTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGC
CTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCC
TGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGC
TGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGA
GGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCC
GATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAG
TGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGC
TTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATA
ACAATTTCACACAGGAAACAGCTATGACCATGATTACGCCAAGCTCTAGCT
AGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCA
ATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAA
CTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA
TTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGT
GAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTTGCAAAGATG
GATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATGGACCTTCTAGGT
CTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCAC
ATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGG
TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTG
GCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGT
CGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAG
TGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTG
CGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGA
GCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGC
CCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCG
CGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTC
TCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCA
AGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTT
TGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGC
GAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCA
AGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCC
GCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG
ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCG
CTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCC
GTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAG
GCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGG
GGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGA
AGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTT
TGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTT
TTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAGATCCCTCGACCT
CGAGATCCATTGTGCCCGGGCGCCACCATGGACATGCGCGTGCCCGCCCAG
CTGCTGGGCCTGCTGCTGCTGTGGTTCCCCGGCTCGCGATGC V7 195
GCGTCGACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC
ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCC
GAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTG
GTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTG
AATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCT
TGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAAGCCGCGGGG
GGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATC
TCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCC
AAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC
GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGC
AAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAA
GCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGC
GAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAAC
AACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGC
CTCTCCCTGTCTCCGGGTAAATGAGCGGCCGCTCGAGGCCGGCAAGGCCGG
ATCCCCCGACCTCGACCTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA
TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGG
CAAATCATTTGGTCGAGATCCCTCGGAGATCTCTAGCTAGAGGATCGATCC
CCGCCCCGGACGAACTAAACCTGACTACGACATCTCTGCCCCTTCTTCGCG
GGGCAGTGCATGTAATCCCTTCAGTTGGTTGGTACAACTTGCCAACTGGGC
CCTGTTCCACATGTGACACGGGGGGGGACCAAACACAAAGGGGTTCTCTGA
CTGTAGTTGACATCCTTATAAATGGATGTGCACATTTGCCAACACTGAGTG
GCTTTCATCCTGGAGCAGACTTTGCAGTCTGTGGACTGCAACACAACATTG
CCTTTATGTGTAACTCTTGGCTGAAGCTCTTACACCAATGCTGGGGGACAT
GTACCTCCCAGGGGCCCAGGAAGACTACGGGAGGCTACACCAACGTCAATC
AGAGGGGCCTGTGTAGCTACCGATAAGCGGACCCTCAAGAGGGCATTAGCA
ATAGTGTTTATAAGGCCCCCTTGTTAACCCTAAACGGGTAGCATATGCTTC
CCGGGTAGTAGTATATACTATCCAGACTAACCCTAATTCAATAGCATATGT
TACCCAACGGGAAGCATATGCTATCGAATTAGGGTTAGTAAAAGGGTCCTA
AGGAACAGCGATATCTCCCACCCCATGAGCTGTCACGGTTTTATTTACATG
GGGTCAGGATTCCACGAGGGTAGTGAACCATTTTAGTCACAAGGGCAGTGG
CTGAAGATCAAGGAGCGGGCAGTGAACTCTCCTGAATCTTCGCCTGCTTCT
TCATTCTCCTTCGTTTAGCTAATAGAATAACTGCTGAGTTGTGAACAGTAA
GGTGTATGTGAGGTGCTCGAAAACAAGGTTTCAGGTGACGCCCCCAGAATA
AAATTTGGACGGGGGGTTCAGTGGTGGCATTGTGCTATGACACCAATATAA
CCCTCACAAACCCCTTGGGCAATAAATACTAGTGTAGGAATGAAACATTCT
GAATATCTTTAACAATAGAAATCCATGGGGTGGGGACAAGCCGTAAAGACT
GGATGTCCATCTCACACGAATTTATGGCTATGGGCAACACATAATCCTAGT
GCAATATGATACTGGGGTTATTAAGATGTGTCCCAGGCAGGGACCAAGACA
GGTGAACCATGTTGTTACACTCTATTTGTAACAAGGGGAAAGAGAGTGGAC
GCCGACAGCAGCGGACTCCACTGGTTGTCTCTAACACCCCCGAAAATTAAA
CGGGGCTCCACGCCAATGGGGCCCATAAACAAAGACAAGTGGCCACTCTTT
TTTTTGAAATTGTGGAGTGGGGGCACGCGTCAGCCCCCACACGCCGCCCTG
CGGTTTTGGACTGTAAAATAAGGGTGTAATAACTTGGCTGATTGTAACCCC
GCTAACCACTGCGGTCAAACCACTTGCCCACAAAACCACTAATGGCACCCC
GGGGAATACCTGCATAAGTAGGTGGGCGGGCCAAGATAGGGGCGCGATTGC
TGCGATCTGGAGGACAAATTACACACACTTGCGCCTGAGCGCCAAGCACAG
GGTTGTTGGTCCTCATATTCACGAGGTCGCTGAGAGCACGGTGGGCTAATG
TTGCCATGGGTAGCATATACTACCCAAATATCTGGATAGCATATGCTATCC
TAATCTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCAT
ATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAATTTATATCT
GGGTAGCATAGGCTATCCTAATCTATATCTGGGTAGCATATGCTATCCTAA
TCTATATCTGGGTAGTATATGCTATCCTAATCTGTATCCGGGTAGCATATG
CTATCCTAATAGAGATTAGGGTAGTATATGCTATCCTAATTTATATCTGGG
TAGCATATACTACCCAAATATCTGGATAGCATATGCTATCCTAATCTATAT
CTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGCATAGGCTATCCT
AATCTATATCTGGGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATA
TGCTATCCTAATTTATATCTGGGTAGCATAGGCTATCCTAATCTATATCTG
GGTAGCATATGCTATCCTAATCTATATCTGGGTAGTATATGCTATCCTAAT
CTGTATCCGGGTAGCATATGCTATCCTCATGATAAGCTGTCAAACATGAGA
ATTTTCTTGAAGACGAAAGGGCCTCGTGATACGCCTATTTTTATAGGTTAA
TGTCATGATAATAATGGTTTCTTAGACGTCAGGTGGCACTTTTCGGGGAAA
TGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTA
TCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAG
GAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGC
GGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAA
AGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCT
CAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAAT
GATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGA
CGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT
GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGT
AAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAA
CTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCA
CAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAA
TGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGCAGCAATGGC
AACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCG
GCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCT
GCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGG
TGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCC
CTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGA
ACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA
ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCA
TTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGAC
CAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGA
AAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTG
CTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCA
AGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGAT
ACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAA
CTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGC
TGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATA
GTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACA
GCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGA
GCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCC
GGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGG
AAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGA
GCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGC
CAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCA
CATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGC
CTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGA
GTCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCC
CGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTG
GAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTA
GGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAAT
TGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTACGC
CAAGCTCTAGCTAGAGGTCGAGTCCCTCCCCAGCAGGCAGAAGTATGCAAA
GCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCA
TCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGAC
TAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTAT
TCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGC
TTTGCAAAGATGGATAAAGTTTTAAACAGAGAGGAATCTTTGCAGCTAATG
GACCTTCTAGGTCTTGAAAGGAGTGGGAATTGGCTCCGGTGCCCGTCAGTG
GGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGG
CAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA
TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATAT
AAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG
AACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGG
TTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGAT
TCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTG
CGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCG
CTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGC
TTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCT
TTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGG
TATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCG
CACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACG
GGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCC
GTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGC
GTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATG
GAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAA
AAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCG
GGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTC
TTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG
GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGA
ATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT
GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTCTCTAGAG
ATCCCTCGACCTCGAGATCCATTGTGCCCGGGCGCCACCATGGAGTTTGGG
CTGAGCTGGCTTTTTCTTGTCGCGATTTTAAAAGGTGTCCAGTGC
[0591] The present invention incorporates by reference in their
entirety techniques well known in the field of molecular biology
and drug delivery. These techniques include, but are not limited
to, techniques described in the following publications: [0592]
Ausubel et al. (eds.), Current Protocols in Molecular Biology, John
Wiley &Sons, NY (1993); [0593] Ausubel, F. M. et al. eds.,
Short Protocols In Molecular Biology (4th Ed. 1999) John Wiley
& Sons, NY. (ISBN 0-471-32938-X). [0594] Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and
Ball (eds.), Wiley, New York (1984); [0595] Giege, R. and Ducruix,
A. Barrett, Crystallization of Nucleic Acids and Proteins, a
Practical Approach, 2nd ea., pp. 20 1-16, Oxford University Press,
New York, N.Y., (1999); [0596] Goodson, in Medical Applications of
Controlled Release, vol. 2, pp. 115-138 (1984); [0597] Hammerling,
et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681
(Elsevier, N.Y., 1981; [0598] Harlow et al., Antibodies: A
Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.
1988); [0599] Kabat et al., Sequences of Proteins of Immunological
Interest (National Institutes of Health, Bethesda, Md. (1987) and
(1991); [0600] 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; [0601] Kontermann
and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New
York. 790 pp. (ISBN 3-540-41354-5). [0602] Kriegler, Gene Transfer
and Expression, A Laboratory Manual, Stockton Press, NY (1990);
[0603] Lu and Weiner eds., Cloning and Expression Vectors for Gene
Function Analysis (2001) BioTechniques Press. Westborough, Mass.
298 pp. (ISBN 1-881299-21-X). [0604] Medical Applications of
Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton,
Fla. (1974); [0605] Old, R. W. & S. B. Primrose, Principles of
Gene Manipulation: An Introduction To Genetic Engineering (3d Ed.
1985) Blackwell Scientific Publications, Boston. Studies in
Microbiology; V.2:409 pp. (ISBN 0-632-01318-4). [0606] Sambrook, J.
et al. eds., Molecular Cloning: A Laboratory Manual (2d Ed. 1989)
Cold Spring Harbor Laboratory Press, NY. Vols. 1-3. (ISBN
0-87969-309-6). [0607] Sustained and Controlled Release Drug
Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New
York, 1978 [0608] Winnacker, E. L. From Genes To Clones:
Introduction To Gene Technology (1987) VCH Publishers, NY
(translated by Horst Ibelgaufts). 634 pp. (ISBN 0-89573-614-4).
INCORPORATION BY REFERENCE
[0609] 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 for any purpose, 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
[0610] 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
195116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ala Lys Thr Thr Pro Lys Leu Glu Glu Gly Glu Phe
Ser Glu Ala Arg 1 5 10 15 217PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 2Ala Lys Thr Thr Pro Lys Leu
Glu Glu Gly Glu Phe Ser Glu Ala Arg 1 5 10 15 Val 39PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 3Ala
Lys Thr Thr Pro Lys Leu Gly Gly 1 5 410PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Ser
Ala Lys Thr Thr Pro Lys Leu Gly Gly 1 5 10 56PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 5Ser
Ala Lys Thr Thr Pro 1 5 66PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 6Arg Ala Asp Ala Ala Pro 1 5
79PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Arg Ala Asp Ala Ala Pro Thr Val Ser 1 5
812PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Arg Ala Asp Ala Ala Ala Ala Gly Gly Pro Gly Ser
1 5 10 927PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Arg Ala Asp Ala Ala Ala Ala Gly Gly Gly Gly Ser
Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
Ser 20 25 1018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 10Ser Ala Lys Thr Thr Pro Lys Leu Glu
Glu Gly Glu Phe Ser Glu Ala 1 5 10 15 Arg Val 115PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 11Ala
Asp Ala Ala Pro 1 5 1212PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Ala Asp Ala Ala Pro Thr Val
Ser Ile Phe Pro Pro 1 5 10 135PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 13Thr Val Ala Ala Pro 1 5
1412PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
1 5 10 156PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Gln Pro Lys Ala Ala Pro 1 5 1613PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Gln
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro 1 5 10
176PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ala Lys Thr Thr Pro Pro 1 5 1813PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Ala
Lys Thr Thr Pro Pro Ser Val Thr Pro Leu Ala Pro 1 5 10
196PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Ala Lys Thr Thr Ala Pro 1 5 2013PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 20Ala
Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro 1 5 10
216PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Ala Ser Thr Lys Gly Pro 1 5 2213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 1 5 10
2315PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 23Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser 1 5 10 15 2415PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 24Gly Glu Asn Lys Val Glu Tyr
Ala Pro Ala Leu Met Ala Leu Ser 1 5 10 15 2515PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 25Gly
Pro Ala Lys Glu Leu Thr Pro Leu Lys Glu Ala Lys Val Ser 1 5 10 15
2615PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 26Gly His Glu Ala Ala Ala Val Met Gln Val Gln Tyr
Pro Ala Ser 1 5 10 15 2724PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Thr Val Ala Ala 1 5 10 15 Pro Ser Val Phe Ile
Phe Pro Pro 20 2826PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 28Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Ala Ser Thr 1 5 10 15 Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro 20 25 295PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 29Gly Gly Gly Gly Ser 1 5
30121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 30Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln
Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala 115 120
31107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu
Ser Pro Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala
Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro
Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu
Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly
Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp
Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90
95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105
32119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 32Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys
Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
100 105 110 Thr Thr Leu Thr Val Ser Ser 115 33107PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
33Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg
Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ser Gly Thr
Lys Leu Glu Ile Asn Arg 100 105 34119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
34Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser
Gly 20 25 30 Asp Tyr Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Ile Gly His Ile Tyr Tyr Ser Gly Asn Thr
Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Leu Thr Ile Ser Ile
Asp Thr Ser Lys Thr Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Ile Tyr Tyr 85 90 95 Cys Val Arg Asp Arg
Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val
Thr Val Ser Ser 115 35108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 35Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His
Leu Pro Leu 85 90 95 Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
Arg 100 105 36120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 36Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg
Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
37108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Asp Ile Gln Met 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 Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe
Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg 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 His Tyr Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
38124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 38Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu
Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr
Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Phe Ser Gly Trp Pro Asn Asn Tyr Tyr Tyr Tyr Gly Met Asp
100 105 110 Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
39114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 39Asp Val 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 Arg Ser
Ser Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Phe Asn Tyr Val Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro His Leu Leu Ile
Tyr Phe Gly Ser Tyr Arg Ala 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 Met Gln Ala 85 90
95 Leu Gln Thr Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110 Arg Arg 40124PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 40Gln Val Gln Leu Gln Gln
Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile
Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45
Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50
55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala
Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val
Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr
Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr
Val Ser Ser 115 120 41112PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 41Asp Ile Leu Leu Thr Gln
Thr Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr
Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly Asp
Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40 45
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro 50
55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile
His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln
Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 110 42127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
42Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Gly Ser Ile Ser Gly
Gly 20 25 30 Tyr Gly Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp 35 40 45 Ile Gly Ser Phe Tyr Ser Ser Ser Gly Asn Thr
Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Gln Val Thr Ile Ser Thr
Asp Thr Ser Lys Asn Gln Phe 65
70 75 80 Ser Leu Lys Leu Asn Ser Met Thr Ala Ala Asp Thr Ala Val
Tyr Tyr 85 90 95 Cys Val Arg Asp Arg Leu Phe Ser Val Val Gly Met
Val Tyr Asn Asn 100 105 110 Trp Phe Asp Val Trp Gly Pro Gly Val Leu
Val Thr Val Ser Ser 115 120 125 43111PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
43Glu Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln 1
5 10 15 Lys Val Thr Ile Ser Cys Thr Gly Ser Thr Ser Asn Ile Gly Gly
Tyr 20 25 30 Asp Leu His Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Asp Ile Asn Lys Arg Pro Ser Gly Ile
Ser Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ala Ala Ser
Leu Ala Ile Thr Gly Leu Gln 65 70 75 80 Thr Glu Asp Glu Ala Asp Tyr
Tyr Cys Gln Ser Tyr Asp Ser Ser Leu 85 90 95 Asn Ala Gln Val Phe
Gly Gly Gly Thr Arg Leu Thr Val Leu Gly 100 105 110
44116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 44Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro
Arg Asn Asp Tyr Thr Glu Tyr Asn Gln Asn Phe 50 55 60 Lys Asp Lys
Ala Thr Ile Thr Ala Asp Glu Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Phe Cys 85 90
95 Ala Arg Arg Asp Ile Thr Thr Phe Tyr Trp Gly Gln Gly Thr Thr Val
100 105 110 Thr Val Ser Ser 115 45113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
45Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30 Ala Asn His Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu
Asp Ile Ala Thr Tyr Tyr Cys His Gln 85 90 95 Tyr Leu Ser Ser Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg
46120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 46Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Tyr
Glu Glu Ser Asn Arg Tyr His Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Ile Thr Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Gly Gly Ile Ala Ala Pro Gly Pro Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
47113PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 47Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu
Thr Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Val Leu Ile
Ser Leu Gly Ser Asn Arg Ala 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 Met Gln Ala 85 90
95 Arg Gln Thr Pro Phe Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Arg
100 105 110 Arg 48120PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 48Gln 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 Ser Tyr 20 25 30 Asp Ile
Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Trp Met Asn Pro Asn Ser Gly Asn Thr Gly Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile 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 Asp Pro Tyr Tyr Tyr Tyr Tyr Gly Met
Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115
120 49112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 49Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser
Ser Ser Asn Ile Glu Asn Asn 20 25 30 His Val Ser Trp Tyr Gln Gln
Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn
Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys
Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr
Gly Asp Glu Ala Asp Tyr Tyr Cys Glu Thr Trp Asp Thr Ser Leu 85 90
95 Ser Ala Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105 110 50125PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 50Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Thr Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Asn Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Ser Gly Ser Gly Gly Thr Thr Phe Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Arg Thr Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Leu Gly Trp Ser Asp Ser Tyr Tyr Tyr
Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 125 51108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 51Asp Ile Gln Met Thr Gln
Phe Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30 Leu Gly
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40 45
Tyr Ala Ala Ser Arg Leu His Arg Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser
Tyr Pro Cys 85 90 95 Ser Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
Arg 100 105 52122PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 52Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Ser Trp
Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr
Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Glu Tyr Asn Ser Gly Trp Tyr Val Leu
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 53108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 53Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Val Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile 35 40 45 Tyr Glu
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Gly 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 Ala Asn Gly Phe
Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105 54121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 54Gln Val Gln Leu Gln Gln Ser Gly
Pro Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Trp Ser Cys
Pro Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Trp Leu His Trp
Val Lys Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 35 40 45 Ile Gly
Met Ile Asp Pro Ser Asn Ser Asp Thr Arg Phe Asn Pro Pro 50 55 60
Asn Phe Lys Asp Lys Ala Thr Leu Asn Val Asp Arg Ser Ser Asn Thr 65
70 75 80 Ala Tyr Asn Leu Leu Ser Ser Leu Thr Ser Ala Asp Ser Ala
Val Tyr 85 90 95 Tyr Cys Ala Thr Tyr Gly Ser Tyr Val Ser Pro Leu
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Tyr Val Ser Ser 115
120 55114PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 55Asp Ile Met Met Ser Gln Ser Pro Ser Ser Leu
Thr Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Val Ser Cys Lys Ser
Ser Gln Ser Leu Leu Val Thr 20 25 30 Ser Ser Gln Lys Asn Tyr Leu
Ala Trp Tyr Gln Gln Lys Pro Gln Gln 35 40 45 Ser Pro Lys Leu Leu
Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile
Thr Ser Val Lys Ala Asp Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90
95 Tyr Tyr Ala Tyr Pro Trp Thr Phe Gly Asp Gly Thr Lys Leu Glu Ile
100 105 110 Lys Arg 56123PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 56Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met
Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50
55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His
Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser 115 120 57108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 57Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45
Tyr Phe Thr Ser Ser Leu His 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 Tyr Ser Thr
Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 58118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 58Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr Asp Asn 20 25 30 Trp Ile Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Tyr
Ile Ser Pro Asn Ser Gly Phe Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Asn Phe Gly Gly Tyr Phe Asp Tyr Trp
Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
59109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 59Asp Ile Gln Met 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 Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe
Leu Tyr 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 Thr Tyr Tyr Cys Gln Gln Ser Tyr Thr Gly Thr 85 90
95 Val Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
60119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 60Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Ser Phe Ser Ser Glu 20 25 30 Pro Ile Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Thr Gly
Lys Asn Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Trp Gly Lys Lys Val Tyr Gly Met Asp Val
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
61108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 61Asp Ile Gln Met 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 Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser
Arg Ala 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 Tyr Met Ser Val Pro Ile 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
62119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 62Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu
Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Asn Tyr 20 25 30 Gly Val His Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Ile Trp Ser
Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr 50 55 60 Ser Arg Leu
Ser Ile Asn Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys
Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90
95 Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly
100 105 110 Thr Leu Val Thr Val Ser Ala 115 63108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
63Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly 1
5 10 15 Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr
Asn 20 25 30 Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg
Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Ser Ile Asn Ser Val Glu Ser 65 70 75 80 Glu Asp Ile Ala Asp Tyr Tyr
Cys Gln Gln Asn Asn Asn Trp Pro Thr 85 90 95 Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg 100 105 64127PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
64Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Ile Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
His 20 25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr
Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Asn Leu Ser Ser Val Thr Ala
Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Ile Pro Asn Tyr
Tyr Asp Arg Ser Gly Tyr Tyr Pro Gly Tyr Trp 100 105 110 Tyr Phe Asp
Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser 115 120 125
65116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 65Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser
Ala Pro Pro Gly Ala 1 5 10 15 Ser Ala Ser Leu Thr Cys Thr Leu Arg
Ser Gly Phe Asn Val Asp Ser 20 25 30 Tyr Arg Ile Ser Trp Tyr Gln
Gln Lys Pro Gly Ser Pro Pro Gln Tyr 35 40 45 Leu Leu Arg Tyr Lys
Ser Asp Ser Asp Lys Gln Gln Gly Ser Gly Val 50 55 60 Pro Ser Arg
Phe Ser Gly Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile 65 70 75 80 Leu
Leu Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys 85 90
95 Met Ile Trp His Ser Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu
100 105 110 Thr Val Leu Arg 115 66122PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
66Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Gln Ile Ser Pro Ala Gly Gly Tyr Thr Asn
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Leu Pro
Tyr Tyr Arg Met Ser Lys Val Met Asp Val Gln 100 105 110 Gly Gln Gly
Thr Leu Val Thr Val Ser Ser 115 120 67108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
67Asp Ile Gln Met 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 Tyr Phe Ser Ser
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Ser Arg Ala 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 Tyr Leu Gly Ser Pro Pro 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 68119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
68Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg
Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn
Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp
Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu
Thr Val Ser Ser 115 69107PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 69Gln Ile Val Leu Thr Gln
Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr
Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp
Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50
55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala
Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn
Pro Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg
100 105 70116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 70Gln Val Gln Leu Gln Gln Ser Gly
Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Ile Asn Trp
Val Lys Leu Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp
Ile Tyr Pro Gly Ser Gly Asn Thr Lys Tyr Asn Glu Lys Phe 50 55 60
Lys Gly Lys Ala Thr Leu Thr Ile Asp Thr Ser Ser Ser Thr Ala Tyr 65
70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr
Phe Cys 85 90 95 Val Arg Asp Ser Pro Phe Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Leu 100 105 110 Thr Val Ser Ser 115 71113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
71Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Val Thr Met Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn
Ser 20 25 30 Gly Met Arg Lys Ser Phe Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys Lys Gln 85 90 95 Ser Tyr His Leu Phe
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg
72117PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 72Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu
Leu Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Tyr
Gly Gly Ser Phe Ser Gly Tyr 20 25 30 Tyr Trp Ser Trp Ile Arg Gln
Pro Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Asn His
Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Val
Thr Ile Ser Val Glu Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90
95 Arg Asp Lys Trp Thr Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu
100 105 110 Val Thr Val Ser Ser 115 73114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
73Asp Ile Glu Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1
5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ser Ser Gln Ser Val Leu Tyr
Ser 20 25 30 Ser Ser Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Asn
Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr
Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu
Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 110 Lys Arg
74119PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 74Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser
Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Arg Gly Asp Phe Asp Ala Phe Asp Ile Trp Gly Gln Gly
100 105 110 Thr Met Val Thr Val Ser Ser 115 75108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
75Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Asn
Cys Gln Gln Cys Glu Asn Phe Pro Ile 85 90 95 Thr Phe Gly Gln Gly
Thr Arg Leu Glu Ile Lys Arg 100 105 76116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
76Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser Ser
Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr Arg
Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp
Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr
Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly Arg
Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser
Ser 115 77108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 77Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys His Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu
Gln Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His
Gly Thr Asn Leu Asp Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe
Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 78119PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 78Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Val Met Ala Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser
Ile Ser Ser Ser Gly Gly Trp Thr Leu Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Leu Lys Met Ala Thr Ile
Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 79112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 79Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser
Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr
Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Val Val Ser Trp Tyr Gln
Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Ile Ile Tyr Glu Val
Ser Gln Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser
Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln
Thr Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90
95 Ser Ile Phe Val Ile Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly
100 105 110 80119PRTArtificial 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 Ile Ser Cys
Lys Ala Ser Gly Tyr Ser Phe Thr Ala Tyr 20 25 30 Tyr Ile His Trp
Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr
Ile Ser Ser Tyr Asn Gly Ala Thr Asn Tyr Asn Gln Lys Phe 50 55 60
Lys Gly Arg Val Thr Phe 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 Asp Tyr Asp Tyr Asp Val Gly Met Asp Tyr
Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115
81112PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 81Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Arg Ala
Ser Glu Ser Val Asp Asn Tyr 20 25 30 Gly Ile Ser Phe Met Lys Trp
Phe Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr
Ala Ala Ser Asn Gln Gly Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser
Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Ser Lys 85 90
95 Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg
100 105 110 82120PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 82Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Ile Ser Asp Tyr 20 25 30 Trp Ile His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Gly
Ile Thr Pro Ala Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Phe Val Phe Phe Leu Pro Tyr Ala Met Asp
Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120
83108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 83Asp Ile Gln Met 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 Asp Val Ser Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe
Leu Tyr 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 Thr Thr Pro Pro 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
84121PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 84Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Ile Asn Ala Ser 20 25 30 Trp Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Ala Ile Tyr Pro
Tyr Ser Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Trp Gly His Ser Thr Ser Pro Trp Ala Met Asp Tyr Trp Gly
100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120
85108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 85Asp Ile Gln Met 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 Val Ile Arg Arg Ser 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Asn
Leu Ala 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 Asn Thr Ser Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
86120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 86Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Val Ile Asn Trp Val Lys Gln
Lys Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro
Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Val Lys
Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Ser Tyr Tyr Tyr Gly Ser Arg Tyr Tyr Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120
87108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 87Asp Ile Gln Met Thr Gln Ser Ser Ser Tyr Leu
Ser Val Ser Leu Gly 1 5 10 15 Gly Arg Val Thr Ile Thr Cys Lys Ala
Ser Asp His Ile Asn Asn Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Gly Ala Thr Ser
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu
Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Ile Pro Leu 85 90
95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg 100 105
88120PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 88Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu
Glu Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser
Gly Tyr Ser Phe Thr Gly Tyr 20 25 30 Asn Met Asn Trp Val Lys Gln
Ser Asn Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Asn Ile Asp Pro
Tyr Phe Gly Gly Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asn Tyr Asp Tyr Asp Gly Gly Cys Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Thr Leu Thr Val Ser Ser 115 120
89110PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 89Gln Ala Val Val Thr Gln Glu Ser Ala Leu Thr
Thr Ser Pro Gly Glu 1 5 10 15 Thr Val Thr Leu Thr Cys Arg Ser Ser
Thr Gly Ala Val Thr Thr Ser 20 25 30 Asn Tyr Ala Asn Trp Val Gln
Glu Lys Pro Asp His Leu Phe Thr Gly 35 40 45 Leu Ile Gly Gly Thr
Asn Asn Arg Ala Pro Gly Val Pro Ala Arg Phe 50 55 60 Ser Gly Ser
Leu Ile Gly Asp Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 Gln
Thr Glu Asp Glu Ala Ile Tyr Phe Cys Ala Leu Trp Tyr Ser Asn 85 90
95 His Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 100 105
110 90116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 90Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu
Val Gln Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser
Gly Phe Ser Leu Thr Thr Tyr 20 25 30 Gly Ile His Trp Val Arg Gln
Ser Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val Met Trp Ser
Gly Gly Asp Thr Asp Tyr Asp Ala Ala Phe Ile 50 55 60 Ser Arg Leu
Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Phe 65 70 75 80 Lys
Met Asn Ser Leu Gln Ala Asn Asp Thr Gly Ile Tyr Tyr Cys Ala 85 90
95 Arg Tyr Arg Phe Tyr Gly Met Asp Tyr Trp Gly Gln Gly Thr Ser Val
100 105 110 Thr Val Ser Ser 115 91108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
91Ala Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn
Arg 20 25 30 Phe Ala Trp Tyr Gln Gln Lys Pro Gly Asn Ala Pro Arg
Leu Leu Ile 35 40 45 Ser Gly Ala Ala Ser Leu Glu Ala Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Thr Leu
Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Gln Tyr Trp Ser Thr Pro Trp 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 92130PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
92Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Lys 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 Ala Pro Leu
Arg Phe Leu Glu Trp Ser Thr Gln Asp His Tyr 100 105 110 Tyr Tyr Tyr
Tyr Met Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val 115 120 125 Ser
Ser 130 93109PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 93Ser Ser Glu Leu Thr Gln Asp Pro
Ala Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Val Arg Ile Thr Cys
Gln Gly Asp Ser Leu Arg Ser Tyr Tyr Ala 20 25 30 Thr Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Ile Leu Val Ile Tyr 35 40 45 Gly Glu
Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser 50 55 60
Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu 65
70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Lys Ser Arg Asp Gly Ser Gly
Gln His 85 90 95 Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
Gly 100 105 94118PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 94Glu Val Gln Leu Val Gln Ser Gly
Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe 20 25 30 Ala Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Ser Val
Ile Asp Thr Arg Gly Ala Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Ala 85 90 95 Arg Leu Gly Asn Phe Tyr Tyr Gly Met Asp Val Trp
Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115
95108PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 95Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu His Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln
Ser Leu Ser Gly Ile Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys His Gln Ser Ser Arg Leu Pro His 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
96118PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 96Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe Thr Thr Tyr 20 25 30 Trp Met His Trp Val Arg Gln
Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Glu Ile Asn Pro
Thr Asn Gly His Thr Asn Tyr Asn Pro Ser Phe 50 55 60 Gln Gly Gln
Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys
85 90 95 Ala Arg Asn Tyr Val Gly Ser Ile Phe Asp Tyr Trp Gly Gln
Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 97108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
97Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asn Val Val Ser
Tyr 20 25 30 Val Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asn Arg Asn Thr 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 Gly Gln Ser Tyr Asn Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 98119PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
98Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp
Tyr 20 25 30 Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile
Tyr Asn Gln Arg Phe 50 55 60 Lys Gly Arg Phe Thr Leu Ser Val Asp
Arg Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asn Leu Gly
Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 99108PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 99Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly 20 25 30 Val Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr 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 Tyr Tyr Ile
Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg 100 105 100116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 100Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys
Glu Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp
Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr
Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65
70 75 80 Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr
Tyr Cys 85 90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln
Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 101108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
101Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly
1 5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser
Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser
Phe Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr
Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro
Gly Thr Lys Leu Glu Ile Lys Arg 100 105 102116PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
102Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val Ser Gly Asp Thr Ile Thr
Phe Tyr 20 25 30 Tyr Met His Phe Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu Asp Glu Ser Thr
Lys Tyr Ser Glu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Ala
Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Lys Leu Ser Ser Leu
Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ile Tyr Gly Gly
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met Val 100 105 110 Thr Val
Ser Ser 115 103108PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 103Asp Ile Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Thr Ser Leu Gly 1 5 10 15 Glu Thr Val Thr Ile Gln
Cys Gln Ala Ser Glu Asp Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly
Ala Ser Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr 65
70 75 80 Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr
Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
100 105 104116PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 104Asp Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys
Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly
Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65
70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr
Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln
Gly Thr Leu Val 100 105 110 Thr Val Ser Ala 115 105113PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
105Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg
106330PRTHomo sapiens 106Ala 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 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 107330PRTHomo sapiens
107Ala 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 108106PRTHomo sapiens 108Thr 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 109105PRTHomo sapiens 109Gln 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
110238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 110Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Glu Val Gln
Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Lys Ser
Leu Lys Leu Ser Cys 130 135 140 Glu Ala Ser Gly Phe Thr Phe Ser Gly
Tyr Gly Met His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Arg Gly
Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser 165 170 175 Ser Ile Asn Ile
Lys Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Val 180 185 190 Ser Arg
Asp Asn Ala Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu 195 200 205
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp 210
215 220 Lys Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 225
230 235 111226PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 111Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10
15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu
Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr
Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala
Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 115 120 125 Leu Pro
Ala Ser Leu Gly Asp Arg Val Thr Ile Asn Cys Gln Ala Ser 130 135 140
Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys 145
150 155 160 Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn Lys Leu Ala Asp
Gly Val 165 170 175 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp
Ser Ser Phe Thr 180 185 190 Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly
Ser Tyr Tyr Cys Gln Gln 195 200 205 Tyr Tyr Asn Tyr Pro Trp Thr Phe
Gly Pro Gly Thr Lys Leu Glu Ile 210 215 220 Lys Arg 225
112238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 112Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Thr
Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70 75 80
Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln
Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser
Leu Ser Leu Thr Cys 130 135 140 Thr Val Thr Gly Phe Ser Ile Thr Ser
Pro Tyr Ala Trp Asn Trp Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr 165 170 175 Arg Gly Ser Thr
Thr His His Pro Ser Leu Lys Ser Arg Ile Ser Ile 180 185 190 Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val 195 200 205
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr 210
215 220 Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225
230 235 113226PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 113Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Pro Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Asn
Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr
Thr Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser 65
70 75 80 Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr
Pro Trp 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Asp Val Leu Leu Thr Gln Ile Pro Leu
Ser Leu Pro Val Ser Leu 115 120 125 Gly Asp Gln Ala Ser Ile Ser Cys
Arg Ser Ser Gln Ser Ile Val His 130 135 140 Ser Asn Gly Asn Thr Tyr
Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln 145 150 155 160 Ser Pro Lys
Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly Val 165 170 175 Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 180 185
190 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln
195 200 205 Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu
Glu Leu 210 215 220 Lys Arg 225 114245PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
114Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr
Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr
Thr His His Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly
Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 130
135 140 Lys Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser
Gly 145 150 155 160 Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Arg
Gly Leu Glu Ser 165 170 175 Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn
Ile Lys Tyr Ala Asp Ala 180 185 190 Val Lys Gly Arg Phe Thr Val Ser
Arg Asp Asn Ala Lys Asn Leu Leu 195 200 205 Phe Leu Gln Met Asn Ile
Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr 210 215 220 Cys Ala Arg Phe
Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met 225 230 235 240 Val
Thr Val Ser Ser 245 115233PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 115Asp Val Leu Leu Thr
Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly
Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser
Ser Leu Pro Ala Ser Leu Gly Asp Arg Val 130 135 140 Thr Ile Asn Cys
Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp 145 150 155 160 Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr 165 170
175 Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser
180 185 190 Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu
Asp Ile 195 200 205 Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro
Trp Thr Phe Gly 210 215 220 Pro Gly Thr Lys Leu Glu Ile Lys Arg 225
230 116245PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 116Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Thr
Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70 75 80
Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser 130 135 140 Gln Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser 145 150 155 160 Pro Tyr Ala Trp Asn Trp
Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu 165 170 175 Trp Met Gly Tyr
Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser 180 185 190 Leu Lys
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 195 200 205
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe 210
215 220 Cys Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr
Leu 225 230 235 240 Val Thr Val Ser Ala 245 117233PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
117Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly
1 5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser
Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser
Phe Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr
Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser
Val Phe Ile Phe Pro Pro Asp Val Leu Leu Thr Gln Ile Pro 115 120 125
Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg 130
135 140 Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
Trp 145 150 155 160 Tyr Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile Tyr Lys Val 165 170 175 Ser Thr Arg Phe Ser Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Leu 195 200 205 Gly Val Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Leu Thr Phe Gly 210 215 220 Ala Gly Thr Gln
Leu Glu Leu Lys Arg 225 230 118245PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 118Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly 130 135 140 Lys Ser Leu Lys
Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Gly 145 150 155 160 Tyr
Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser 165 170
175 Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala
180 185 190 Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn
Leu Leu 195 200 205 Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr
Ala Met Tyr Tyr 210 215 220 Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr
Trp Gly Gln Gly Thr Met 225 230 235 240 Val Thr Val Ser Ser 245
119226PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 119Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser 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 Lys Leu Leu
Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu
Lys 100 105 110 Arg Thr Val Ala Ala Pro Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser 115 120 125 Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile
Asn Cys Gln Ala Ser 130 135 140 Gln Asp Ile Ser Asn Tyr Leu Asn Trp
Tyr Gln Gln Lys Pro Gly Lys 145 150 155 160 Ala Pro Lys Leu Leu Ile
Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val 165 170 175 Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Arg Asp Ser Ser Phe Thr 180 185 190 Ile Ser
Ser Leu Glu Ser Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln 195 200 205
Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile 210
215 220 Lys Arg 225 120245PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 120Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys
Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly
Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40
45 Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val
50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu
Leu Phe 65 70 75 80 Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala
Met Tyr Tyr Cys
85 90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr
Met Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala 115 120 125 Pro Asp Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser 130 135 140 Gln Ser Leu Ser Leu Thr Cys Thr
Val Thr Gly Phe Ser Ile Thr Ser 145 150 155 160 Pro Tyr Ala Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu 165 170 175 Trp Met Gly
Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser 180 185 190 Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 195 200
205 Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe
210 215 220 Cys Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly
Thr Leu 225 230 235 240 Val Thr Val Ser Ala 245 121226PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
121Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly
1 5 10 15 Asp Arg Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser
Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro
Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Asn Lys Leu Ala Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Ser Ser
Phe Thr Ile Ser Ser Leu Glu Ser 65 70 75 80 Glu Asp Ile Gly Ser Tyr
Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp 85 90 95 Thr Phe Gly Pro
Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Asp
Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu 115 120 125
Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His 130
135 140 Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly
Gln 145 150 155 160 Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg
Phe Ser Gly Val 165 170 175 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys 180 185 190 Ile Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys Phe Gln 195 200 205 Gly Ser His Val Pro Leu
Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu 210 215 220 Lys Arg 225
122238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 122Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Glu Val Gln
Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val Gln Pro Gly Lys Ser
Leu Lys Leu Ser Cys 130 135 140 Glu Ala Ser Gly Phe Thr Phe Ser Gly
Tyr Gly Met His Trp Val Arg 145 150 155 160 Gln Ala Pro Gly Arg Gly
Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser 165 170 175 Ser Ile Asn Ile
Lys Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Val 180 185 190 Ser Arg
Asp Asn Ala Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu 195 200 205
Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp 210
215 220 Lys Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 225
230 235 123233PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 123Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ser Ser Leu
Pro Ala Ser Leu Gly Asp Arg Val 130 135 140 Thr Ile Asn Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp 145 150 155 160 Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr 165 170 175 Asn
Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 180 185
190 Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile
195 200 205 Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr
Phe Gly 210 215 220 Pro Gly Thr Lys Leu Glu Ile Lys Arg 225 230
124238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 124Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Lys 1 5 10 15 Ser Leu Lys Leu Ser Cys Glu Ala
Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Arg Gly Leu Glu Ser Val 35 40 45 Ala Tyr Ile Thr
Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val 50 55 60 Lys Gly
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe 65 70 75 80
Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85
90 95 Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln
Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser
Leu Ser Leu Thr Cys 130 135 140 Thr Val Thr Gly Phe Ser Ile Thr Ser
Pro Tyr Ala Trp Asn Trp Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr 165 170 175 Arg Gly Ser Thr
Thr His His Pro Ser Leu Lys Ser Arg Ile Ser Ile 180 185 190 Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val 195 200 205
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr 210
215 220 Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225
230 235 125233PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 125Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Pro Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Asn
Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr
Thr Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser 65
70 75 80 Glu Asp Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr
Pro Trp 85 90 95 Thr Phe Gly Pro Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Asp Val
Leu Leu Thr Gln Ile Pro 115 120 125 Leu Ser Leu Pro Val Ser Leu Gly
Asp Gln Ala Ser Ile Ser Cys Arg 130 135 140 Ser Ser Gln Ser Ile Val
His Ser Asn Gly Asn Thr Tyr Leu Glu Trp 145 150 155 160 Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val 165 170 175 Ser
Thr Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 180 185
190 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu
195 200 205 Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Leu Thr
Phe Gly 210 215 220 Ala Gly Thr Gln Leu Glu Leu Lys Arg 225 230
126238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 126Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Glu Val Gln
Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser
Val Lys Leu Ser Cys 130 135 140 Lys Val Ser Gly Asp Thr Ile Thr Phe
Tyr Tyr Met His Phe Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu 165 170 175 Asp Glu Ser Thr
Lys Tyr Ser Glu Lys Phe Lys Asn Lys Ala Thr Leu 180 185 190 Thr Ala
Asp Thr Ser Ser Asn Thr Ala Tyr Leu Lys Leu Ser Ser Leu 195 200 205
Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ile Tyr Gly Gly Tyr Tyr 210
215 220 Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser 225
230 235 127226PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 127Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Asp Ile Gln Met
Thr Gln Ser Pro Ala Ser 115 120 125 Leu Ser Thr Ser Leu Gly Glu Thr
Val Thr Ile Gln Cys Gln Ala Ser 130 135 140 Glu Asp Ile Tyr Ser Gly
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 145 150 155 160 Ser Pro Gln
Leu Leu Ile Tyr Gly Ala Ser Asp Leu Gln Asp Gly Val 165 170 175 Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys 180 185
190 Ile Thr Ser Met Gln Thr Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln
195 200 205 Gly Leu Thr Tyr Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu
Glu Leu 210 215 220 Lys Arg 225 128238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
128Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr
1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val Ser Gly Asp Thr Ile Thr
Phe Tyr 20 25 30 Tyr Met His Phe Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp Pro Glu Asp Glu Ser Thr
Lys Tyr Ser Glu Lys Phe 50 55 60 Lys Asn Lys Ala Thr Leu Thr Ala
Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Leu Lys Leu Ser Ser Leu
Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ile Tyr Gly Gly
Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met Val 100 105 110 Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln Leu Gln Glu 115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys 130
135 140 Thr Val Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp Asn Trp
Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly
Tyr Ile Ser Tyr 165 170 175 Arg Gly Ser Thr Thr His His Pro Ser Leu
Lys Ser Arg Ile Ser Ile 180 185 190 Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe Leu Gln Leu Asn Ser Val 195 200 205 Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr 210 215 220 Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225 230 235
129226PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 129Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Thr Ser Leu Gly 1 5 10 15 Glu Thr Val Thr Ile Gln Cys Gln
Ala Ser Glu Asp Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Ser
Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr 65 70 75 80
Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala
Ala 100 105 110 Pro Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro
Val Ser Leu 115 120 125 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His 130 135 140 Ser Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly Gln 145 150 155 160 Ser Pro Lys Leu Leu Ile
Tyr Lys Val Ser Thr
Arg Phe Ser Gly Val 165 170 175 Pro Asp Arg Phe Ser Gly Ser Gly Ser
Gly Thr Asp Phe Thr Leu Lys 180 185 190 Ile Ser Arg Val Glu Ala Glu
Asp Leu Gly Val Tyr Tyr Cys Phe Gln 195 200 205 Gly Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu 210 215 220 Lys Arg 225
130245PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 130Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Leu Ser Cys Lys Val
Ser Gly Asp Thr Ile Thr Phe 145 150 155 160 Tyr Tyr Met His Phe Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170 175 Ile Gly Arg Ile
Asp Pro Glu Asp Glu Ser Thr Lys Tyr Ser Glu Lys 180 185 190 Phe Lys
Asn Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala 195 200 205
Tyr Leu Lys Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe 210
215 220 Cys Ile Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val
Met 225 230 235 240 Val Thr Val Ser Ser 245 131233PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
131Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Asp Ile Gln 115 120 125
Met Thr Gln Ser Pro Ala Ser Leu Ser Thr Ser Leu Gly Glu Thr Val 130
135 140 Thr Ile Gln Cys Gln Ala Ser Glu Asp Ile Tyr Ser Gly Leu Ala
Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu
Ile Tyr Gly Ala 165 170 175 Ser Asp Leu Gln Asp Gly Val Pro Ser Arg
Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr Gln Tyr Ser Leu Lys Ile
Thr Ser Met Gln Thr Glu Asp Glu 195 200 205 Gly Val Tyr Phe Cys Gln
Gln Gly Leu Thr Tyr Pro Arg Thr Phe Gly 210 215 220 Gly Gly Thr Lys
Leu Glu Leu Lys Arg 225 230 132245PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 132Glu Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys
Leu Ser Cys Lys Val Ser Gly Asp Thr Ile Thr Phe Tyr 20 25 30 Tyr
Met His Phe Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Arg Ile Asp Pro Glu Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe
50 55 60 Lys Asn Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr
Ala Tyr 65 70 75 80 Leu Lys Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ile Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp
Gly Gln Gly Val Met Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser 130 135 140 Gln Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser 145 150 155 160 Pro
Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu 165 170
175 Trp Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser
180 185 190 Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn
Gln Phe 195 200 205 Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr
Ala Thr Tyr Phe 210 215 220 Cys Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr
Ser Gly Gln Gly Thr Leu 225 230 235 240 Val Thr Val Ser Ala 245
133233PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 133Asp Ile Gln Met Thr Gln Ser Pro Ala Ser
Leu Ser Thr Ser Leu Gly 1 5 10 15 Glu Thr Val Thr Ile Gln Cys Gln
Ala Ser Glu Asp Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Ser
Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr 65 70 75 80
Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala
Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Asp Val Leu Leu Thr
Gln Ile Pro 115 120 125 Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala
Ser Ile Ser Cys Arg 130 135 140 Ser Ser Gln Ser Ile Val His Ser Asn
Gly Asn Thr Tyr Leu Glu Trp 145 150 155 160 Tyr Leu Gln Lys Pro Gly
Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val 165 170 175 Ser Thr Arg Phe
Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu 195 200 205
Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe Gly 210
215 220 Ala Gly Thr Gln Leu Glu Leu Lys Arg 225 230
134245PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 134Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly 130 135 140 Thr Ser Val Lys Leu Ser Cys Lys Val
Ser Gly Asp Thr Ile Thr Phe 145 150 155 160 Tyr Tyr Met His Phe Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170 175 Ile Gly Arg Ile
Asp Pro Glu Asp Glu Ser Thr Lys Tyr Ser Glu Lys 180 185 190 Phe Lys
Asn Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala 195 200 205
Tyr Leu Lys Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe 210
215 220 Cys Ile Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val
Met 225 230 235 240 Val Thr Val Ser Ser 245 135226PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
135Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr
Val Ala Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ala Ser 115 120 125
Leu Ser Thr Ser Leu Gly Glu Thr Val Thr Ile Gln Cys Gln Ala Ser 130
135 140 Glu Asp Ile Tyr Ser Gly Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Lys 145 150 155 160 Ser Pro Gln Leu Leu Ile Tyr Gly Ala Ser Asp Leu
Gln Asp Gly Val 165 170 175 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly
Thr Gln Tyr Ser Leu Lys 180 185 190 Ile Thr Ser Met Gln Thr Glu Asp
Glu Gly Val Tyr Phe Cys Gln Gln 195 200 205 Gly Leu Thr Tyr Pro Arg
Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu 210 215 220 Lys Arg 225
136245PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 136Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val
Ser Gly Asp Thr Ile Thr Phe Tyr 20 25 30 Tyr Met His Phe Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp
Pro Glu Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe 50 55 60 Lys Asn
Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80
Leu Lys Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ile Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser 130 135 140 Gln Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser 145 150 155 160 Pro Tyr Ala Trp Asn Trp
Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu 165 170 175 Trp Met Gly Tyr
Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser 180 185 190 Leu Lys
Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe 195 200 205
Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe 210
215 220 Cys Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr
Leu 225 230 235 240 Val Thr Val Ser Ala 245 137226PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
137Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Thr Ser Leu Gly
1 5 10 15 Glu Thr Val Thr Ile Gln Cys Gln Ala Ser Glu Asp Ile Tyr
Ser Gly 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ser Pro
Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Asp Leu Gln Asp Gly Val
Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Gln Tyr Ser
Leu Lys Ile Thr Ser Met Gln Thr 65 70 75 80 Glu Asp Glu Gly Val Tyr
Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg 85 90 95 Thr Phe Gly Gly
Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala 100 105 110 Pro Asp
Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu 115 120 125
Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His 130
135 140 Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly
Gln 145 150 155 160 Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg
Phe Ser Gly Val 165 170 175 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys 180 185 190 Ile Ser Arg Val Glu Ala Glu Asp
Leu Gly Val Tyr Tyr Cys Phe Gln 195 200 205 Gly Ser His Val Pro Leu
Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu 210 215 220 Lys Arg 225
138238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 138Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Glu Val Gln
Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Val Arg Pro Gly Thr Ser
Val Lys Leu Ser Cys 130 135 140 Lys Val Ser Gly Asp Thr Ile Thr Phe
Tyr Tyr Met His Phe Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu 165 170 175 Asp Glu Ser Thr
Lys Tyr Ser Glu Lys Phe Lys Asn Lys Ala Thr Leu 180 185 190 Thr Ala
Asp Thr Ser Ser Asn Thr Ala Tyr Leu Lys Leu Ser Ser Leu 195 200 205
Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys Ile Tyr Gly Gly Tyr Tyr 210
215 220 Phe Asp Tyr Trp Gly Gln Gly Val Met Val Thr Val Ser Ser 225
230
235 139233PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 139Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser 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 Lys Leu Leu
Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu
Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Asp Ile Gln 115 120 125 Met Thr Gln Ser Pro Ala Ser Leu Ser Thr Ser
Leu Gly Glu Thr Val 130 135 140 Thr Ile Gln Cys Gln Ala Ser Glu Asp
Ile Tyr Ser Gly Leu Ala Trp 145 150 155 160 Tyr Gln Gln Lys Pro Gly
Lys Ser Pro Gln Leu Leu Ile Tyr Gly Ala 165 170 175 Ser Asp Leu Gln
Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 180 185 190 Gly Thr
Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr Glu Asp Glu 195 200 205
Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr Pro Arg Thr Phe Gly 210
215 220 Gly Gly Thr Lys Leu Glu Leu Lys Arg 225 230
140238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 140Glu Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Val
Ser Gly Asp Thr Ile Thr Phe Tyr 20 25 30 Tyr Met His Phe Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Asp
Pro Glu Asp Glu Ser Thr Lys Tyr Ser Glu Lys Phe 50 55 60 Lys Asn
Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80
Leu Lys Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ile Tyr Gly Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Val Met
Val 100 105 110 Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln
Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser
Leu Ser Leu Thr Cys 130 135 140 Thr Val Thr Gly Phe Ser Ile Thr Ser
Pro Tyr Ala Trp Asn Trp Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr 165 170 175 Arg Gly Ser Thr
Thr His His Pro Ser Leu Lys Ser Arg Ile Ser Ile 180 185 190 Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu Asn Ser Val 195 200 205
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr 210
215 220 Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225
230 235 141233PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 141Asp Ile Gln Met Thr Gln Ser Pro
Ala Ser Leu Ser Thr Ser Leu Gly 1 5 10 15 Glu Thr Val Thr Ile Gln
Cys Gln Ala Ser Glu Asp Ile Tyr Ser Gly 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly
Ala Ser Asp Leu Gln Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Gln Tyr Ser Leu Lys Ile Thr Ser Met Gln Thr 65
70 75 80 Glu Asp Glu Gly Val Tyr Phe Cys Gln Gln Gly Leu Thr Tyr
Pro Arg 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Asp Val
Leu Leu Thr Gln Ile Pro 115 120 125 Leu Ser Leu Pro Val Ser Leu Gly
Asp Gln Ala Ser Ile Ser Cys Arg 130 135 140 Ser Ser Gln Ser Ile Val
His Ser Asn Gly Asn Thr Tyr Leu Glu Trp 145 150 155 160 Tyr Leu Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val 165 170 175 Ser
Thr Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser 180 185
190 Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu
195 200 205 Gly Val Tyr Tyr Cys Phe Gln Gly Ser His Val Pro Leu Thr
Phe Gly 210 215 220 Ala Gly Thr Gln Leu Glu Leu Lys Arg 225 230
142241PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 142Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Gln Val Gln
Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser
Val Lys Met Ser Cys 130 135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Arg
Tyr Thr Met His Trp Val Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 165 170 175 Arg Gly Tyr Thr
Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu 180 185 190 Thr Thr
Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Ser Leu 195 200 205
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp 210
215 220 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val
Ser 225 230 235 240 Ser 143225PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 143Asp Val Leu Leu Thr
Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly
Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Gln Ile
Val Leu Thr Gln Ser Pro Ala Ile 115 120 125 Met Ser Ala Ser Pro Gly
Glu Lys Val Thr Met Thr Cys Ser Ala Ser 130 135 140 Ser Ser Val Ser
Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 145 150 155 160 Pro
Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 165 170
175 Ala His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile
180 185 190 Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln
Gln Trp 195 200 205 Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys
Leu Glu Ile Asn 210 215 220 Arg 225 144241PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
144Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala
1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr
Arg Tyr 20 25 30 Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr
Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr
Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr
Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln 115 120 125
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser 130
135 140 Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala
Trp 145 150 155 160 Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu
Trp Met Gly Tyr 165 170 175 Ile Ser Tyr Arg Gly Ser Thr Thr His His
Pro Ser Leu Lys Ser Arg 180 185 190 Ile Ser Ile Thr Arg Asp Thr Ser
Lys Asn Gln Phe Phe Leu Gln Leu 195 200 205 Asn Ser Val Thr Thr Glu
Asp Thr Ala Thr Tyr Phe Cys Ser Ser Tyr 210 215 220 Gly Asn Tyr Gly
Ala Tyr Ser Gly Gln Gly Thr Leu Val Thr Val Ser 225 230 235 240 Ala
145225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 145Gln Ile Val Leu Thr Gln Ser Pro Ala Ile
Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser
Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys
Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys
Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60 Gly Ser
Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85
90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala
Pro 100 105 110 Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val
Ser Leu Gly 115 120 125 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 130 135 140 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 145 150 155 160 Pro Lys Leu Leu Ile Tyr
Lys Val Ser Thr Arg Phe Ser Gly Val Pro 165 170 175 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 180 185 190 Ser Arg
Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 195 200 205
Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 210
215 220 Arg 225 146248PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 146Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly 130 135 140 Ala Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg 145 150 155 160 Tyr
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170
175 Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys
180 185 190 Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser
Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr 210 215 220 Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys
Leu Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr Thr Leu Thr Val Ser
Ser 245 147232PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 147Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Gln Ile Val 115 120 125 Leu Thr Gln Ser Pro Ala Ile Met
Ser Ala Ser Pro Gly Glu Lys Val 130 135 140 Thr Met Thr Cys Ser Ala
Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr 145 150 155 160 Gln Gln Lys
Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser 165 170 175 Lys
Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser Gly Ser Gly 180 185
190 Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu Asp Ala Ala
195 200 205 Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr Phe
Gly Ser 210 215 220 Gly Thr Lys Leu Glu Ile Asn Arg 225 230
148248PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 148Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr Met His Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Asn
Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Asp
Lys Ala Thr Leu Thr Thr
Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr
Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr
Leu Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125
Pro Leu Ala Pro Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 130
135 140 Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe
Ser 145 150 155 160 Ile Thr Ser Pro Tyr Ala Trp Asn Trp Ile Arg Gln
Phe Pro Gly Asn 165 170 175 Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr
Arg Gly Ser Thr Thr His 180 185 190 His Pro Ser Leu Lys Ser Arg Ile
Ser Ile Thr Arg Asp Thr Ser Lys 195 200 205 Asn Gln Phe Phe Leu Gln
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala 210 215 220 Thr Tyr Phe Cys
Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln 225 230 235 240 Gly
Thr Leu Val Thr Val Ser Ala 245 149232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
149Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly
Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro 100 105 110 Ser Val
Phe Ile Phe Pro Pro Asp Val Leu Leu Thr Gln Ile Pro Leu 115 120 125
Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser 130
135 140 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr 145 150 155 160 Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
Tyr Lys Val Ser 165 170 175 Thr Arg Phe Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly 180 185 190 Thr Asp Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly 195 200 205 Val Tyr Tyr Cys Phe Gln
Gly Ser His Val Pro Leu Thr Phe Gly Ala 210 215 220 Gly Thr Gln Leu
Glu Leu Lys Arg 225 230 150248PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 150Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala 115 120 125 Pro Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly 130 135 140 Ala Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg 145 150 155 160 Tyr
Thr Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170
175 Ile Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys
180 185 190 Phe Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser
Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Tyr 210 215 220 Cys Ala Arg Tyr Tyr Asp Asp His Tyr Cys
Leu Asp Tyr Trp Gly Gln 225 230 235 240 Gly Thr Thr Leu Thr Val Ser
Ser 245 151225PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 151Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Gln Ile Val Leu
Thr Gln Ser Pro Ala Ile 115 120 125 Met Ser Ala Ser Pro Gly Glu Lys
Val Thr Met Thr Cys Ser Ala Ser 130 135 140 Ser Ser Val Ser Tyr Met
Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 145 150 155 160 Pro Lys Arg
Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser Gly Val Pro 165 170 175 Ala
His Phe Arg Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 180 185
190 Ser Gly Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp
195 200 205 Ser Ser Asn Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
Ile Asn 210 215 220 Arg 225 152248PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 152Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Asp Val
Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 130 135 140 Lys Pro Ser Gln
Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser 145 150 155 160 Ile
Thr Ser Pro Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn 165 170
175 Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His
180 185 190 His Pro Ser Leu Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr
Ser Lys 195 200 205 Asn Gln Phe Phe Leu Gln Leu Asn Ser Val Thr Thr
Glu Asp Thr Ala 210 215 220 Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr
Gly Ala Tyr Ser Gly Gln 225 230 235 240 Gly Thr Leu Val Thr Val Ser
Ala 245 153225PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 153Gln Ile Val Leu Thr Gln Ser Pro
Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr
Cys Ser Ala Ser Ser Ser Val Ser Tyr Met 20 25 30 Asn Trp Tyr Gln
Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr
Ser Lys Leu Ala Ser Gly Val Pro Ala His Phe Arg Gly Ser 50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Gly Met Glu Ala Glu 65
70 75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro
Phe Thr 85 90 95 Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn Arg Thr
Val Ala Ala Pro 100 105 110 Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 115 120 125 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Ile Val His Ser 130 135 140 Asn Gly Asn Thr Tyr Leu
Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 145 150 155 160 Pro Lys Leu
Leu Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly Val Pro 165 170 175 Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 180 185
190 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly
195 200 205 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu
Leu Lys 210 215 220 Arg 225 154241PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 154Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Ala
Arg Pro Gly Ala Ser Val Lys Met Ser Cys 130 135 140 Lys Ala Ser Gly
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys 145 150 155 160 Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 165 170
175 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu
180 185 190 Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu 195 200 205 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
Tyr Tyr Asp Asp 210 215 220 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val Ser 225 230 235 240 Ser 155232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
155Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Gln Ile Val 115 120 125
Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val 130
135 140 Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met Asn Trp
Tyr 145 150 155 160 Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile
Tyr Asp Thr Ser 165 170 175 Lys Leu Ala Ser Gly Val Pro Ala His Phe
Arg Gly Ser Gly Ser Gly 180 185 190 Thr Ser Tyr Ser Leu Thr Ile Ser
Gly Met Glu Ala Glu Asp Ala Ala 195 200 205 Thr Tyr Tyr Cys Gln Gln
Trp Ser Ser Asn Pro Phe Thr Phe Gly Ser 210 215 220 Gly Thr Lys Leu
Glu Ile Asn Arg 225 230 156241PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 156Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Asp Val Gln 115 120 125 Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln Ser Leu Ser 130 135 140 Leu Thr Cys Thr
Val Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp 145 150 155 160 Asn
Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly Tyr 165 170
175 Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu Lys Ser Arg
180 185 190 Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu
Gln Leu 195 200 205 Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe
Cys Ser Ser Tyr 210 215 220 Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly
Thr Leu Val Thr Val Ser 225 230 235 240 Ala 157232PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
157Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro
Ala His Phe Arg Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Gly Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn Pro Phe Thr 85 90 95 Phe Gly Ser Gly
Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro 100 105 110 Ser Val
Phe Ile Phe Pro Pro Asp Val Leu Leu Thr Gln Ile Pro Leu 115 120
125 Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser
130 135 140 Ser Gln Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu
Trp Tyr 145 150 155 160 Leu Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu
Ile Tyr Lys Val Ser 165 170 175 Thr Arg Phe Ser Gly Val Pro Asp Arg
Phe Ser Gly Ser Gly Ser Gly 180 185 190 Thr Asp Phe Thr Leu Lys Ile
Ser Arg Val Glu Ala Glu Asp Leu Gly 195 200 205 Val Tyr Tyr Cys Phe
Gln Gly Ser His Val Pro Leu Thr Phe Gly Ala 210 215 220 Gly Thr Gln
Leu Glu Leu Lys Arg 225 230 158246PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 158Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Val
Arg Pro Gly Ser Ser Val Lys Ile Ser Cys 130 135 140 Lys Ala Ser Gly
Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys 145 150 155 160 Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly 165 170
175 Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
180 185 190 Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu 195 200 205 Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
Arg Glu Thr Thr 210 215 220 Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser 245
159230PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 159Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser 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 Lys Leu Leu
Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu
Lys 100 105 110 Arg Thr Val Ala Ala Pro Asp Ile Leu Leu Thr Gln Thr
Pro Ala Ser 115 120 125 Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile
Ser Cys Lys Ala Ser 130 135 140 Gln Ser Val Asp Tyr Asp Gly Asp Ser
Tyr Leu Asn Trp Tyr Gln Gln 145 150 155 160 Ile Pro Gly Gln Pro Pro
Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu 165 170 175 Val Ser Gly Ile
Pro Pro Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 180 185 190 Phe Thr
Leu Asn Ile His Pro Val Glu Lys Val Asp Ala Ala Thr Tyr 195 200 205
His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr 210
215 220 Lys Leu Glu Ile Lys Arg 225 230 160246PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
160Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser
1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser
Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly
Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr
Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala
Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu
Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu
Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp
Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125
Gly Pro Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro 130
135 140 Ser Gln Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile
Thr 145 150 155 160 Ser Pro Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro
Gly Asn Thr Leu 165 170 175 Glu Trp Met Gly Tyr Ile Ser Tyr Arg Gly
Ser Thr Thr His His Pro 180 185 190 Ser Leu Lys Ser Arg Ile Ser Ile
Thr Arg Asp Thr Ser Lys Asn Gln 195 200 205 Phe Phe Leu Gln Leu Asn
Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 210 215 220 Phe Cys Ser Ser
Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr 225 230 235 240 Leu
Val Thr Val Ser Ala 245 161230PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 161Asp Ile Leu Leu Thr
Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala
Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp 20 25 30 Gly
Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro 35 40
45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro
50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn
Ile His 65 70 75 80 Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys
Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Asp Val Leu
Leu Thr Gln Ile Pro Leu Ser Leu 115 120 125 Pro Val Ser Leu Gly Asp
Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln 130 135 140 Ser Ile Val His
Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln 145 150 155 160 Lys
Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg 165 170
175 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
180 185 190 Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly
Val Tyr 195 200 205 Tyr Cys Phe Gln Gly Ser His Val Pro Leu Thr Phe
Gly Ala Gly Thr 210 215 220 Gln Leu Glu Leu Lys Arg 225 230
162253PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 162Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala 115 120 125 Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly 130 135 140 Ser Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ala Phe Ser Ser 145 150 155 160 Tyr Trp Met Asn Trp Val
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp 165 170 175 Ile Gly Gln Ile
Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys 180 185 190 Phe Lys
Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala 195 200 205
Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe 210
215 220 Cys Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala
Met 225 230 235 240 Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser 245 250 163237PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 163Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Asp Ile Leu 115 120 125 Leu Thr Gln Thr Pro Ala Ser Leu
Ala Val Ser Leu Gly Gln Arg Ala 130 135 140 Thr Ile Ser Cys Lys Ala
Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser 145 150 155 160 Tyr Leu Asn
Trp Tyr Gln Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu 165 170 175 Ile
Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser 180 185
190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu
195 200 205 Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu
Asp Pro 210 215 220 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
Arg 225 230 235 164253PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 164Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp
Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala
Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg
Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Asp Val Gln Leu Gln Glu Ser 130 135 140 Gly Pro Gly Leu
Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr 145 150 155 160 Val
Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp Asn Trp Ile Arg 165 170
175 Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr Arg
180 185 190 Gly Ser Thr Thr His His Pro Ser Leu Lys Ser Arg Ile Ser
Ile Thr 195 200 205 Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu
Asn Ser Val Thr 210 215 220 Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser
Ser Tyr Gly Asn Tyr Gly 225 230 235 240 Ala Tyr Ser Gly Gln Gly Thr
Leu Val Thr Val Ser Ala 245 250 165237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
165Asp Ile Leu Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro
Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu
Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp
Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Asp Val Leu Leu 115 120 125
Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser 130
135 140 Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn
Thr 145 150 155 160 Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu 165 170 175 Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly
Val Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser Arg Val Glu 195 200 205 Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Phe Gln Gly Ser His Val Pro 210 215 220 Leu Thr Phe Gly
Ala Gly Thr Gln Leu Glu Leu Lys Arg 225 230 235 166253PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
166Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr
Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr
Thr His His Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly
Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala 115 120 125
Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly 130
135 140 Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser
Ser 145 150 155
160 Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp
165 170 175 Ile Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn
Gly Lys 180 185 190 Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser
Ser Ser Thr Ala 195 200 205 Tyr Met Gln Leu Ser Ser Leu Ala Ser Glu
Asp Ser Ala Val Tyr Phe 210 215 220 Cys Ala Arg Arg Glu Thr Thr Thr
Val Gly Arg Tyr Tyr Tyr Ala Met 225 230 235 240 Asp Tyr Trp Gly Gln
Gly Thr Ser Val Thr Val Ser Ser 245 250 167230PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
167Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr
Val Ala Ala Pro Asp Ile Leu Leu Thr Gln Thr Pro Ala Ser 115 120 125
Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser 130
135 140 Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn Trp Tyr Gln
Gln 145 150 155 160 Ile Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp
Ala Ser Asn Leu 165 170 175 Val Ser Gly Ile Pro Pro Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp 180 185 190 Phe Thr Leu Asn Ile His Pro Val
Glu Lys Val Asp Ala Ala Thr Tyr 195 200 205 His Cys Gln Gln Ser Thr
Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr 210 215 220 Lys Leu Glu Ile
Lys Arg 225 230 168253PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 168Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys
Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp
Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Gln Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe
50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala
Val Tyr Phe Cys 85 90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg
Tyr Tyr Tyr Ala Met Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Asp Val Gln Leu Gln Glu Ser 130 135 140 Gly Pro Gly Leu
Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys Thr 145 150 155 160 Val
Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp Asn Trp Ile Arg 165 170
175 Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly Tyr Ile Ser Tyr Arg
180 185 190 Gly Ser Thr Thr His His Pro Ser Leu Lys Ser Arg Ile Ser
Ile Thr 195 200 205 Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln Leu
Asn Ser Val Thr 210 215 220 Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser
Ser Tyr Gly Asn Tyr Gly 225 230 235 240 Ala Tyr Ser Gly Gln Gly Thr
Leu Val Thr Val Ser Ala 245 250 169230PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
169Asp Ile Leu Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro
Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu
Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp
Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val
Ala Ala Pro Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu 115 120 125
Pro Val Ser Leu Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln 130
135 140 Ser Ile Val His Ser Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu
Gln 145 150 155 160 Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Lys
Val Ser Thr Arg 165 170 175 Phe Ser Gly Val Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp 180 185 190 Phe Thr Leu Lys Ile Ser Arg Val
Glu Ala Glu Asp Leu Gly Val Tyr 195 200 205 Tyr Cys Phe Gln Gly Ser
His Val Pro Leu Thr Phe Gly Ala Gly Thr 210 215 220 Gln Leu Glu Leu
Lys Arg 225 230 170246PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 170Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Gln Val Gln Leu Gln Gln 115 120 125 Ser Gly Ala Glu Leu Val
Arg Pro Gly Ser Ser Val Lys Ile Ser Cys 130 135 140 Lys Ala Ser Gly
Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys 145 150 155 160 Gln
Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Trp Pro Gly 165 170
175 Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Ala Thr Leu
180 185 190 Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu 195 200 205 Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
Arg Glu Thr Thr 210 215 220 Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp
Tyr Trp Gly Gln Gly Thr 225 230 235 240 Ser Val Thr Val Ser Ser 245
171237PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 171Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser 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 Lys Leu Leu
Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu
Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro
Asp Ile Leu 115 120 125 Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser
Leu Gly Gln Arg Ala 130 135 140 Thr Ile Ser Cys Lys Ala Ser Gln Ser
Val Asp Tyr Asp Gly Asp Ser 145 150 155 160 Tyr Leu Asn Trp Tyr Gln
Gln Ile Pro Gly Gln Pro Pro Lys Leu Leu 165 170 175 Ile Tyr Asp Ala
Ser Asn Leu Val Ser Gly Ile Pro Pro Arg Phe Ser 180 185 190 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His Pro Val Glu 195 200 205
Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro 210
215 220 Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 225 230
235 172246PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 172Gln Val Gln Leu Gln Gln Ser Gly Ala Glu
Leu Val Arg Pro Gly Ser 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala
Ser Gly Tyr Ala Phe Ser Ser Tyr 20 25 30 Trp Met Asn Trp Val Lys
Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gln Ile Trp
Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85
90 95 Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met
Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala
Ser Thr Lys 115 120 125 Gly Pro Asp Val Gln Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro 130 135 140 Ser Gln Ser Leu Ser Leu Thr Cys Thr
Val Thr Gly Phe Ser Ile Thr 145 150 155 160 Ser Pro Tyr Ala Trp Asn
Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu 165 170 175 Glu Trp Met Gly
Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro 180 185 190 Ser Leu
Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln 195 200 205
Phe Phe Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr 210
215 220 Phe Cys Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly
Thr 225 230 235 240 Leu Val Thr Val Ser Ala 245 173237PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
173Asp Ile Leu Leu Thr Gln Thr Pro Ala Ser Leu Ala Val Ser Leu Gly
1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp
Tyr Asp 20 25 30 Gly Asp Ser Tyr Leu Asn Trp Tyr Gln Gln Ile Pro
Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu
Val Ser Gly Ile Pro Pro 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Lys Val Asp
Ala Ala Thr Tyr His Cys Gln Gln Ser Thr 85 90 95 Glu Asp Pro Trp
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Asp Val Leu Leu 115 120 125
Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly Asp Gln Ala Ser 130
135 140 Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser Asn Gly Asn
Thr 145 150 155 160 Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu 165 170 175 Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly
Val Pro Asp Arg Phe Ser 180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile Ser Arg Val Glu 195 200 205 Ala Glu Asp Leu Gly Val
Tyr Tyr Cys Phe Gln Gly Ser His Val Pro 210 215 220 Leu Thr Phe Gly
Ala Gly Thr Gln Leu Glu Leu Lys Arg 225 230 235 174243PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
174Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr
Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr
Thr His His Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly
Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Gln Val Gln Leu Gln Gln 115 120 125
Pro Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Met Ser Cys 130
135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Asn Met His Trp Val
Lys 145 150 155 160 Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile Gly Ala
Ile Tyr Pro Gly 165 170 175 Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
Lys Gly Lys Ala Thr Leu 180 185 190 Thr Ala Asp Lys Ser Ser Ser Thr
Ala Tyr Met Gln Leu Ser Ser Leu 195 200 205 Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr 210 215 220 Gly Gly Asp Trp
Tyr Phe Asn Val Trp Gly Ala Gly Thr Thr Val Thr 225 230 235 240 Val
Ser Ala 175225PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 175Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Gln Ile Val Leu
Ser Gln Ser Pro Ala Ile 115 120 125 Leu Ser Pro Ser Pro Gly Glu Lys
Val Thr Met Thr Cys Arg Ala Ser 130 135 140 Ser Ser Val Ser Tyr Ile
His Trp Phe Gln Gln Lys Pro Gly Ser Ser 145 150 155 160 Pro Lys Pro
Trp Ile Tyr Ala Thr Ser Asn Leu Ala Ser Gly Val Pro 165 170 175 Val
Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr Ser Leu Thr Ile 180 185 190 Ser Arg Val Glu Ala Glu Asp Ala
Ala Thr Tyr Tyr Cys Gln Gln Trp 195 200 205 Thr Ser Asn Pro Pro Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 210 215 220 Arg 225
176243PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 176Gln Val Gln Leu Gln Gln Pro Gly Ala Glu
Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala
Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys
Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr
Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly
Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp
Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Asp 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln Ser 130 135 140 Leu Ser Leu Thr Cys Thr Val Thr Gly
Phe Ser Ile Thr Ser Pro Tyr 145 150 155 160 Ala Trp Asn Trp Ile Arg
Gln Phe Pro Gly Asn Thr Leu Glu Trp Met 165 170 175 Gly Tyr Ile Ser
Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu Lys 180 185 190 Ser Arg
Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu 195 200 205
Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser 210
215 220 Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val
Thr 225 230 235 240 Val Ser Ala 177225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
177Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Pro Ser Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser
Tyr Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys
Pro Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro
Val Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Asp Val
Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 115 120 125
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 130
135 140 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 145 150 155 160 Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe
Ser Gly Val Pro 165 170 175 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 180 185 190 Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Phe Gln Gly 195 200 205 Ser His Val Pro Leu Thr
Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 210 215 220 Arg 225
178238PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 178Asp Val Gln Leu Gln Glu Ser Gly Pro Gly
Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val
Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile
Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile
Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu 50 55 60 Lys Ser
Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80
Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85
90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu
Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Gln Val Gln
Leu Gln Glu 115 120 125 Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Thr
Leu Ser Leu Thr Cys 130 135 140 Thr Val Ser Gly Tyr Ser Ile Ser Ser
Asp Phe Ala Trp Asn Trp Ile 145 150 155 160 Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp Met Gly Tyr Ile Ser Tyr 165 170 175 Ser Gly Asn Thr
Arg Tyr Gln Pro Ser Leu Lys Ser Arg Ile Thr Ile 180 185 190 Ser Arg
Asp Thr Ser Lys Asn Gln Phe Phe Leu Lys Leu Asn Ser Val 195 200 205
Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys Val Thr Ala Gly Arg Gly 210
215 220 Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 225
230 235 179226PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 179Asp Val Leu Leu Thr Gln Ile Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser 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 Lys
Leu Leu Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln
Leu Glu Leu Lys 100 105 110 Arg Thr Val Ala Ala Pro Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser 115 120 125 Met Ser Val Ser Val Gly Asp Arg
Val Thr Ile Thr Cys His Ser Ser 130 135 140 Gln Asp Ile Asn Ser Asn
Ile Gly Trp Leu Gln Gln Lys Pro Gly Lys 145 150 155 160 Ser Phe Lys
Gly Leu Ile Tyr His Gly Thr Asn Leu Asp Asp Gly Val 165 170 175 Pro
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr 180 185
190 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln
195 200 205 Tyr Ala Gln Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
Glu Ile 210 215 220 Lys Arg 225 180238PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
180Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Ser
Ser Asp 20 25 30 Phe Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys
Gly Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Ser Gly Asn Thr
Arg Tyr Gln Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg
Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Lys Leu Asn Ser Val
Thr Ala Ala Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Thr Ala Gly
Arg Gly Phe Pro Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Asp Val Gln Leu Gln Glu 115 120 125
Ser Gly Pro Gly Leu Val Lys Pro Ser Gln Ser Leu Ser Leu Thr Cys 130
135 140 Thr Val Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp Asn Trp
Ile 145 150 155 160 Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly
Tyr Ile Ser Tyr 165 170 175 Arg Gly Ser Thr Thr His His Pro Ser Leu
Lys Ser Arg Ile Ser Ile 180 185 190 Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe Leu Gln Leu Asn Ser Val 195 200 205 Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys Ser Ser Tyr Gly Asn Tyr 210 215 220 Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val Thr Val Ser Ala 225 230 235
181226PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 181Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Met Ser Val Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys His
Ser Ser Gln Asp Ile Asn Ser Asn 20 25 30 Ile Gly Trp Leu Gln Gln
Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly Thr
Asn Leu Asp Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Val Gln Tyr Ala Gln Phe Pro Trp 85
90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala
Ala 100 105 110 Pro Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro
Val Ser Leu 115 120 125 Gly Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His 130 135 140 Ser Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly Gln 145 150 155 160 Ser Pro Lys Leu Leu Ile
Tyr Lys Val Ser Thr Arg Phe Ser Gly Val 165 170 175 Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 180 185 190 Ile Ser
Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln 195 200 205
Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu 210
215 220 Lys Arg 225 182242PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 182Asp Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser
Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr Ser Pro 20 25 30 Tyr
Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp 35 40
45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu
50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln
Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala
Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly Asn Tyr Gly Ala Tyr Ser
Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ala Ala Ser Thr Lys
Gly Pro Glu Val Gln Leu Val Glu 115 120 125 Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 130 135 140 Ala Ala Ser Gly
Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val Arg 145 150 155 160 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro Thr 165 170
175 Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile
180 185 190 Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn
Ser Leu 195 200 205 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg
Trp Gly Gly Asp 210 215 220 Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val 225 230 235 240 Ser Ser 183226PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
183Asp Val Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly
1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser 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 Lys Leu Leu Ile Tyr Lys Val Ser Thr
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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro
Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 100 105 110 Arg Thr
Val Ala Ala Pro Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 115 120 125
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser 130
135 140 Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys 145 150 155 160 Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu
Tyr Ser Gly Val 165 170 175 Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly
Thr Asp Phe Thr Leu Thr 180 185 190 Ile Ser Ser Leu Gln Pro Glu Asp
Phe Ala Thr Tyr Tyr Cys Gln Gln 195 200 205 His Tyr Thr Thr Pro Pro
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile 210 215 220 Lys Arg 225
184242PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 184Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr
Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly
Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Asp Val 115 120 125 Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys
Pro Ser Gln Ser Leu 130 135 140 Ser Leu Thr Cys Thr Val Thr Gly Phe
Ser Ile Thr Ser Pro Tyr Ala 145 150 155 160 Trp Asn Trp Ile Arg Gln
Phe Pro Gly Asn Thr Leu Glu Trp Met Gly 165 170 175 Tyr Ile Ser Tyr
Arg Gly Ser Thr Thr His His Pro Ser Leu Lys Ser 180 185 190 Arg Ile
Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu Gln 195 200 205
Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ser Ser 210
215 220 Tyr Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val Thr
Val 225 230 235 240 Ser Ala 185226PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 185Asp Ile Gln Met 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 Asp Val Asn Thr Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Arg 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 His
Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Asp Val Leu Leu Thr Gln
Ile Pro Leu Ser Leu Pro Val Ser Leu 115 120 125 Gly Asp Gln Ala Ser
Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His 130 135 140 Ser Asn Gly
Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln 145 150 155 160
Ser Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe Ser Gly Val 165
170 175 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys 180 185 190 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr
Cys Phe Gln 195 200 205 Gly Ser His Val Pro Leu Thr Phe Gly Ala Gly
Thr Gln Leu Glu Leu 210 215 220 Lys Arg 225 186241PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
186Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln
1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Phe Ser Ile Thr
Ser Pro 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn
Thr Leu Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ser Thr
Thr His His Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg
Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Gln Leu Asn Ser Val
Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ser Ser Tyr Gly
Asn Tyr Gly Ala Tyr Ser Gly Gln Gly Thr Leu Val 100 105 110 Thr Val
Ser Ala Ala Ser Thr Lys Gly Pro Gln Val Gln Leu Gln Gln 115 120 125
Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys 130
135 140 Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val
Lys 145 150 155 160 Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr
Ile Asn Pro Ser 165 170 175 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
Lys Asp Lys Ala Thr Leu 180 185 190 Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr Met Gln Leu Ser Ser Leu 195 200 205 Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys Ala Arg Tyr Tyr Asp Asp 210 215 220 His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser 225 230 235 240 Ser
187225PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 187Asp Val Leu Leu Thr Gln Ile Pro Leu Ser
Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser 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 Lys Leu Leu
Ile Tyr Lys Val Ser Thr 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 Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85
90 95 Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Gln Leu Glu Leu
Lys 100 105 110 Arg Thr Val Ala Ala Pro Gln Ile Val Leu Thr Gln Ser
Pro Ala Ile 115 120 125 Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met
Thr Cys Arg Ala Ser 130 135 140 Ser Ser Val Ser Tyr Met Asn Trp Tyr
Gln Gln Lys Ser Gly Thr Ser 145 150 155 160 Pro Lys Arg Trp Ile Tyr
Asp Thr Ser Lys Val Ala Ser Gly Val Pro 165 170 175 Tyr Arg Phe Ser
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile 180 185 190 Ser Ser
Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp 195 200 205
Ser Ser Asn Pro Leu Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Asn 210
215 220 Arg 225 188241PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 188Gln Val Gln Leu Gln
Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys
Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30 Thr
Met His Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40
45 Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe
50 55 60 Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Ser Thr
Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Tyr Tyr Asp Asp His Tyr Cys Leu
Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Asp Val Gln 115 120 125 Leu Gln Glu Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln Ser Leu Ser 130 135 140 Leu Thr Cys Thr
Val Thr Gly Phe Ser Ile Thr Ser Pro Tyr Ala Trp 145 150 155 160 Asn
Trp Ile Arg Gln Phe Pro Gly Asn Thr Leu Glu Trp Met Gly Tyr 165 170
175 Ile Ser Tyr Arg Gly Ser Thr Thr His His Pro Ser Leu Lys Ser Arg
180 185 190 Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe Leu
Gln Leu 195 200 205 Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Phe
Cys Ser Ser Tyr 210 215 220 Gly Asn Tyr Gly Ala Tyr Ser Gly Gln Gly
Thr Leu Val Thr Val Ser 225 230 235 240 Ala 189225PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
189Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser
Tyr Met 20 25 30 Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys
Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Val Ala Ser Gly Val Pro
Tyr Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu
Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr
Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Ser Gly
Thr Lys Leu Glu Ile Asn Arg Thr Val Ala Ala Pro 100 105 110 Asp Val
Leu Leu Thr Gln Ile Pro Leu Ser Leu Pro Val Ser Leu Gly 115 120 125
Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 130
135 140 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln
Ser 145 150 155 160 Pro Lys Leu Leu Ile Tyr Lys Val Ser Thr Arg Phe
Ser Gly Val Pro 165 170 175 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 180 185 190 Ser Arg Val Glu Ala Glu Asp Leu
Gly Val Tyr Tyr Cys Phe Gln Gly 195 200 205 Ser His Val Pro Leu Thr
Phe Gly Ala Gly Thr Gln Leu Glu Leu Lys 210 215 220 Arg 225
1907185DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 190gcgtcgacca agggcccatc ggtcttcccc
ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
240tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 300aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaact cctgggggga 360ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 420gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgcgaggag 720atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
840ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc tccgggtaaa
tgagcggccg ctcgaggccg gcaaggccgg 1020atcccccgac ctcgacctct
ggctaataaa ggaaatttat tttcattgca atagtgtgtt 1080ggaatttttt
gtgtctctca ctcggaagga catatgggag ggcaaatcat ttggtcgaga
1140tccctcggag atctctagct agaggatcga tccccgcccc ggacgaacta
aacctgacta 1200cgacatctct gccccttctt cgcggggcag tgcatgtaat
cccttcagtt ggttggtaca 1260acttgccaac tgggccctgt tccacatgtg
acacgggggg ggaccaaaca caaaggggtt 1320ctctgactgt agttgacatc
cttataaatg gatgtgcaca tttgccaaca ctgagtggct 1380ttcatcctgg
agcagacttt gcagtctgtg gactgcaaca caacattgcc tttatgtgta
1440actcttggct gaagctctta caccaatgct gggggacatg tacctcccag
gggcccagga 1500agactacggg aggctacacc aacgtcaatc agaggggcct
gtgtagctac cgataagcgg 1560accctcaaga gggcattagc aatagtgttt
ataaggcccc cttgttaacc ctaaacgggt 1620agcatatgct tcccgggtag
tagtatatac tatccagact aaccctaatt caatagcata 1680tgttacccaa
cgggaagcat atgctatcga attagggtta gtaaaagggt cctaaggaac
1740agcgatatct cccaccccat gagctgtcac ggttttattt acatggggtc
aggattccac 1800gagggtagtg aaccatttta gtcacaaggg cagtggctga
agatcaagga gcgggcagtg 1860aactctcctg aatcttcgcc tgcttcttca
ttctccttcg tttagctaat agaataactg 1920ctgagttgtg aacagtaagg
tgtatgtgag gtgctcgaaa acaaggtttc aggtgacgcc 1980cccagaataa
aatttggacg gggggttcag tggtggcatt gtgctatgac accaatataa
2040ccctcacaaa ccccttgggc aataaatact agtgtaggaa tgaaacattc
tgaatatctt 2100taacaataga aatccatggg gtggggacaa gccgtaaaga
ctggatgtcc atctcacacg 2160aatttatggc tatgggcaac acataatcct
agtgcaatat gatactgggg ttattaagat 2220gtgtcccagg cagggaccaa
gacaggtgaa ccatgttgtt acactctatt tgtaacaagg 2280ggaaagagag
tggacgccga cagcagcgga ctccactggt tgtctctaac acccccgaaa
2340attaaacggg gctccacgcc aatggggccc ataaacaaag acaagtggcc
actctttttt 2400ttgaaattgt ggagtggggg cacgcgtcag cccccacacg
ccgccctgcg gttttggact 2460gtaaaataag ggtgtaataa cttggctgat
tgtaaccccg ctaaccactg cggtcaaacc 2520acttgcccac aaaaccacta
atggcacccc ggggaatacc tgcataagta ggtgggcggg 2580ccaagatagg
ggcgcgattg ctgcgatctg gaggacaaat tacacacact tgcgcctgag
2640cgccaagcac agggttgttg gtcctcatat tcacgaggtc gctgagagca
cggtgggcta 2700atgttgccat gggtagcata tactacccaa atatctggat
agcatatgct atcctaatct 2760atatctgggt agcataggct atcctaatct
atatctgggt agcatatgct atcctaatct 2820atatctgggt agtatatgct
atcctaattt atatctgggt agcataggct atcctaatct 2880atatctgggt
agcatatgct atcctaatct atatctgggt agtatatgct atcctaatct
2940gtatccgggt agcatatgct atcctaatag agattagggt agtatatgct
atcctaattt 3000atatctgggt agcatatact acccaaatat ctggatagca
tatgctatcc taatctatat 3060ctgggtagca tatgctatcc taatctatat
ctgggtagca taggctatcc taatctatat 3120ctgggtagca tatgctatcc
taatctatat ctgggtagta tatgctatcc taatttatat 3180ctgggtagca
taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
3240ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc
tcatgataag 3300ctgtcaaaca tgagaatttt cttgaagacg aaagggcctc
gtgatacgcc tatttttata 3360ggttaatgtc atgataataa tggtttctta
gacgtcaggt ggcacttttc ggggaaatgt 3420gcgcggaacc cctatttgtt
tatttttcta aatacattca aatatgtatc cgctcatgag 3480acaataaccc
tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
3540tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
ttgctcaccc 3600agaaacgctg gtgaaagtaa aagatgctga agatcagttg
ggtgcacgag tgggttacat 3660cgaactggat ctcaacagcg gtaagatcct
tgagagtttt cgccccgaag aacgttttcc 3720aatgatgagc acttttaaag
ttctgctatg tggcgcggta ttatcccgtg ttgacgccgg 3780gcaagagcaa
ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
3840agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
gtgctgccat 3900aaccatgagt gataacactg cggccaactt acttctgaca
acgatcggag gaccgaagga 3960gctaaccgct tttttgcaca acatggggga
tcatgtaact cgccttgatc gttgggaacc 4020ggagctgaat gaagccatac
caaacgacga gcgtgacacc acgatgcctg cagcaatggc 4080aacaacgttg
cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
4140aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
cccttccggc 4200tggctggttt attgctgata aatctggagc cggtgagcgt
gggtctcgcg gtatcattgc 4260agcactgggg ccagatggta agccctcccg
tatcgtagtt atctacacga cggggagtca 4320ggcaactatg gatgaacgaa
atagacagat cgctgagata ggtgcctcac tgattaagca 4380ttggtaactg
tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
4440ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
aaatccctta 4500acgtgagttt tcgttccact gagcgtcaga ccccgtagaa
aagatcaaag gatcttcttg 4560agatcctttt tttctgcgcg taatctgctg
cttgcaaaca aaaaaaccac cgctaccagc 4620ggtggtttgt ttgccggatc
aagagctacc aactcttttt ccgaaggtaa ctggcttcag 4680cagagcgcag
ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
4740gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
tggctgctgc 4800cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga
cgatagttac cggataaggc 4860gcagcggtcg ggctgaacgg ggggttcgtg
cacacagccc agcttggagc gaacgaccta 4920caccgaactg agatacctac
agcgtgagct atgagaaagc gccacgcttc ccgaagggag 4980aaaggcggac
aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
5040tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
tctgacttga 5100gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta
tggaaaaacg ccagcaacgc 5160ggccttttta cggttcctgg ccttttgctg
gccttttgct cacatgttct ttcctgcgtt 5220atcccctgat tctgtggata
accgtattac cgcctttgag tgagctgata ccgctcgccg 5280cagccgaacg
accgagcgca gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg
5340caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
acaggtttcc 5400cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg
agttagctca ctcattaggc 5460accccaggct ttacacttta tgcttccggc
tcgtatgttg tgtggaattg tgagcggata 5520acaatttcac acaggaaaca
gctatgacca tgattacgcc aagctctagc tagaggtcga 5580gtccctcccc
agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccatag
5640tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
cattctccgc 5700cccatggctg actaattttt tttatttatg cagaggccga
ggccgcctcg gcctctgagc 5760tattccagaa gtagtgagga ggcttttttg
gaggcctagg cttttgcaaa aagctttgca 5820aagatggata aagttttaaa
cagagaggaa tctttgcagc taatggacct tctaggtctt 5880gaaaggagtg
ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt
5940ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa
ggtggcgcgg 6000ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt
tttcccgagg gtgggggaga 6060accgtatata agtgcagtag tcgccgtgaa
cgttcttttt cgcaacgggt ttgccgccag 6120aacacaggta agtgccgtgt
gtggttcccg cgggcctggc ctctttacgg gttatggccc 6180ttgcgtgcct
tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg
6240ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt
cgcctcgtgc 6300ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
cgaatctggt ggcaccttcg 6360cgcctgtctc gctgctttcg ataagtctct
agccatttaa aatttttgat gacctgctgc 6420gacgcttttt ttctggcaag
atagtcttgt aaatgcgggc caagatctgc acactggtat 6480ttcggttttt
ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc
6540gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc
aagctggccg 6600gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
ccgccctggg cggcaaggct 6660ggcccggtcg gcaccagttg cgtgagcgga
aagatggccg cttcccggcc ctgctgcagg 6720gagctcaaaa tggaggacgc
ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag 6780gaaaagggcc
tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc
6840gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag
gttgggggga 6900ggggttttat gcgatggagt ttccccacac tgagtgggtg
gagactgaag ttaggccagc 6960ttggcacttg atgtaattct ccttggaatt
tgcccttttt gagtttggat cttggttcat 7020tctcaagcct cagacagtgg
ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat 7080tctctagaga
tccctcgacc tcgagatcca ttgtgcccgg gcgccaccat ggagtttggg
7140ctgagctggc tttttcttgt cgcgatttta aaaggtgtcc agtgc
71851916521DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide
191acggtggctg caccatctgt cttcatcttc ccgccatctg atgagcagtt
gaaatctgga 60actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa
agtacagtgg 120aaggtggata acgccctcca atcgggtaac tcccaggaga
gtgtcacaga gcaggacagc 180aaggacagca cctacagcct cagcagcacc
ctgacgctga gcaaagcaga ctacgagaaa 240cacaaagtct acgcctgcga
agtcacccat cagggcctga gctcgcccgt cacaaagagc 300ttcaacaggg
gagagtgttg agcggccgct cgaggccggc aaggccggat cccccgacct
360cgacctctgg ctaataaagg aaatttattt tcattgcaat agtgtgttgg
aattttttgt 420gtctctcact cggaaggaca tatgggaggg caaatcattt
ggtcgagatc cctcggagat 480ctctagctag aggatcgatc cccgccccgg
acgaactaaa cctgactacg acatctctgc 540cccttcttcg cggggcagtg
catgtaatcc cttcagttgg ttggtacaac ttgccaactg 600ggccctgttc
cacatgtgac acgggggggg accaaacaca aaggggttct ctgactgtag
660ttgacatcct tataaatgga tgtgcacatt tgccaacact gagtggcttt
catcctggag 720cagactttgc agtctgtgga ctgcaacaca acattgcctt
tatgtgtaac tcttggctga 780agctcttaca ccaatgctgg gggacatgta
cctcccaggg gcccaggaag actacgggag 840gctacaccaa cgtcaatcag
aggggcctgt gtagctaccg ataagcggac cctcaagagg 900gcattagcaa
tagtgtttat aaggccccct tgttaaccct aaacgggtag catatgcttc
960ccgggtagta gtatatacta tccagactaa ccctaattca atagcatatg
ttacccaacg 1020ggaagcatat gctatcgaat tagggttagt aaaagggtcc
taaggaacag cgatatctcc 1080caccccatga gctgtcacgg ttttatttac
atggggtcag gattccacga gggtagtgaa 1140ccattttagt cacaagggca
gtggctgaag atcaaggagc gggcagtgaa ctctcctgaa 1200tcttcgcctg
cttcttcatt ctccttcgtt tagctaatag aataactgct gagttgtgaa
1260cagtaaggtg tatgtgaggt gctcgaaaac aaggtttcag gtgacgcccc
cagaataaaa 1320tttggacggg gggttcagtg gtggcattgt gctatgacac
caatataacc ctcacaaacc 1380ccttgggcaa taaatactag tgtaggaatg
aaacattctg aatatcttta acaatagaaa 1440tccatggggt ggggacaagc
cgtaaagact ggatgtccat ctcacacgaa tttatggcta 1500tgggcaacac
ataatcctag tgcaatatga tactggggtt attaagatgt gtcccaggca
1560gggaccaaga caggtgaacc atgttgttac actctatttg taacaagggg
aaagagagtg 1620gacgccgaca gcagcggact ccactggttg tctctaacac
ccccgaaaat taaacggggc 1680tccacgccaa tggggcccat aaacaaagac
aagtggccac tctttttttt gaaattgtgg 1740agtgggggca cgcgtcagcc
cccacacgcc gccctgcggt tttggactgt aaaataaggg 1800tgtaataact
tggctgattg taaccccgct aaccactgcg gtcaaaccac ttgcccacaa
1860aaccactaat ggcaccccgg ggaatacctg cataagtagg tgggcgggcc
aagatagggg 1920cgcgattgct gcgatctgga ggacaaatta cacacacttg
cgcctgagcg ccaagcacag 1980ggttgttggt cctcatattc acgaggtcgc
tgagagcacg gtgggctaat gttgccatgg 2040gtagcatata ctacccaaat
atctggatag catatgctat cctaatctat atctgggtag 2100cataggctat
cctaatctat atctgggtag catatgctat cctaatctat atctgggtag
2160tatatgctat cctaatttat atctgggtag cataggctat cctaatctat
atctgggtag 2220catatgctat cctaatctat atctgggtag tatatgctat
cctaatctgt atccgggtag 2280catatgctat cctaatagag attagggtag
tatatgctat cctaatttat atctgggtag 2340catatactac ccaaatatct
ggatagcata tgctatccta atctatatct gggtagcata 2400tgctatccta
atctatatct gggtagcata ggctatccta atctatatct gggtagcata
2460tgctatccta atctatatct gggtagtata tgctatccta atttatatct
gggtagcata 2520ggctatccta atctatatct gggtagcata tgctatccta
atctatatct gggtagtata 2580tgctatccta atctgtatcc gggtagcata
tgctatcctc atgataagct gtcaaacatg 2640agaattttct tgaagacgaa
agggcctcgt gatacgccta tttttatagg ttaatgtcat 2700gataataatg
gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
2760tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
aataaccctg 2820ataaatgctt caataatatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 2880ccttattccc ttttttgcgg cattttgcct
tcctgttttt gctcacccag aaacgctggt 2940gaaagtaaaa gatgctgaag
atcagttggg tgcacgagtg ggttacatcg aactggatct 3000caacagcggt
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
3060ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt gacgccgggc
aagagcaact 3120cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 3180gcatcttacg gatggcatga cagtaagaga
attatgcagt gctgccataa ccatgagtga 3240taacactgcg gccaacttac
ttctgacaac gatcggagga ccgaaggagc taaccgcttt 3300tttgcacaac
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
3360agccatacca aacgacgagc gtgacaccac gatgcctgca gcaatggcaa
caacgttgcg 3420caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 3480ggaggcggat aaagttgcag gaccacttct
gcgctcggcc cttccggctg gctggtttat 3540tgctgataaa tctggagccg
gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 3600agatggtaag
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
3660tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
ggtaactgtc 3720agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 3780gatctaggtg aagatccttt ttgataatct
catgaccaaa atcccttaac gtgagttttc 3840gttccactga gcgtcagacc
ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 3900tctgcgcgta
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
3960gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
gagcgcagat 4020accaaatact gttcttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 4080accgcctaca tacctcgctc tgctaatcct
gttaccagtg gctgctgcca gtggcgataa 4140gtcgtgtctt accgggttgg
actcaagacg atagttaccg gataaggcgc agcggtcggg 4200ctgaacgggg
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
4260atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
aggcggacag 4320gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 4380cgcctggtat ctttatagtc ctgtcgggtt
tcgccacctc tgacttgagc gtcgattttt 4440gtgatgctcg tcaggggggc
ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 4500gttcctggcc
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
4560tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
gccgaacgac 4620cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 4680ccccgcgcgt tggccgattc attaatgcag
ctggcacgac aggtttcccg actggaaagc 4740gggcagtgag cgcaacgcaa
ttaatgtgag ttagctcact cattaggcac cccaggcttt 4800acactttatg
cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
4860aggaaacagc tatgaccatg attacgccaa gctctagcta gaggtcgagt
ccctccccag 4920caggcagaag tatgcaaagc atgcatctca attagtcagc
aaccatagtc ccgcccctaa 4980ctccgcccat cccgccccta actccgccca
gttccgccca ttctccgccc catggctgac 5040taattttttt tatttatgca
gaggccgagg ccgcctcggc ctctgagcta ttccagaagt 5100agtgaggagg
cttttttgga ggcctaggct tttgcaaaaa gctttgcaaa gatggataaa
5160gttttaaaca gagaggaatc tttgcagcta atggaccttc taggtcttga
aaggagtggg 5220aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg
cccacagtcc ccgagaagtt 5280ggggggaggg gtcggcaatt gaaccggtgc
ctagagaagg tggcgcgggg taaactggga 5340aagtgatgtc gtgtactggc
tccgcctttt tcccgagggt gggggagaac cgtatataag 5400tgcagtagtc
gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtaag
5460tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt tatggccctt
gcgtgccttg 5520aattacttcc acctggctgc agtacgtgat tcttgatccc
gagcttcggg ttggaagtgg 5580gtgggagagt tcgaggcctt gcgcttaagg
agccccttcg cctcgtgctt gagttgaggc 5640ctggcctggg cgctggggcc
gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc 5700tgctttcgat
aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
5760ctggcaagat agtcttgtaa atgcgggcca agatctgcac actggtattt
cggtttttgg 5820ggccgcgggc ggcgacgggg cccgtgcgtc ccagcgcaca
tgttcggcga ggcggggcct 5880gcgagcgcgg ccaccgagaa tcggacgggg
gtagtctcaa gctggccggc ctgctctggt 5940gcctggcctc gcgccgccgt
gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc 6000accagttgcg
tgagcggaaa gatggccgct tcccggccct gctgcaggga gctcaaaatg
6060gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc acacaaagga
aaagggcctt 6120tccgtcctca gccgtcgctt catgtgactc cacggagtac
cgggcgccgt ccaggcacct 6180cgattagttc tcgagctttt ggagtacgtc
gtctttaggt tggggggagg ggttttatgc 6240gatggagttt ccccacactg
agtgggtgga gactgaagtt aggccagctt ggcacttgat 6300gtaattctcc
ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
6360gacagtggtt caaagttttt ttcttccatt tcaggtgtcg tgaggaattc
tctagagatc 6420cctcgacctc gagatccatt gtgcccgggc gcaccatgga
catgcgcgtg cccgcccagc 6480tgctgggcct gctgctgctg tggttccccg
gctcgcgatg c 65211926513DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 192caacccaagg
ctgccccctc ggtcactctg ttcccgccct cctctgagga gcttcaagcc 60aacaaggcca
cactggtgtg tctcataagt gacttctacc cgggagccgt gacagtggcc
120tggaaggcag atagcagccc cgtcaaggcg ggagtggaga ccaccacacc
ctccaaacaa 180agcaacaaca agtacgcggc cagcagctac ctgagcctga
cgcctgagca gtggaagtcc 240cacagaagct acagctgcca ggtcacgcat
gaagggagca ccgtggagaa gacagtggcc 300cctacagaat gttcatgagc
ggccgctcga ggccggcaag gccggatccc ccgacctcga 360cctctggcta
ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
420tctcactcgg aaggacatat gggagggcaa atcatttggt cgagatccct
cggagatctc 480tagctagagg atcgatcccc gccccggacg aactaaacct
gactacgaca tctctgcccc 540ttcttcgcgg ggcagtgcat gtaatccctt
cagttggttg gtacaacttg ccaactgggc 600cctgttccac atgtgacacg
gggggggacc aaacacaaag gggttctctg actgtagttg 660acatccttat
aaatggatgt gcacatttgc caacactgag tggctttcat cctggagcag
720actttgcagt ctgtggactg caacacaaca ttgcctttat gtgtaactct
tggctgaagc 780tcttacacca atgctggggg acatgtacct cccaggggcc
caggaagact acgggaggct 840acaccaacgt caatcagagg ggcctgtgta
gctaccgata agcggaccct caagagggca 900ttagcaatag tgtttataag
gcccccttgt taaccctaaa cgggtagcat atgcttcccg 960ggtagtagta
tatactatcc agactaaccc taattcaata gcatatgtta cccaacggga
1020agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga
tatctcccac 1080cccatgagct gtcacggttt tatttacatg gggtcaggat
tccacgaggg tagtgaacca 1140ttttagtcac aagggcagtg gctgaagatc
aaggagcggg cagtgaactc tcctgaatct 1200tcgcctgctt cttcattctc
cttcgtttag ctaatagaat aactgctgag ttgtgaacag 1260taaggtgtat
gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag aataaaattt
1320ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc
acaaacccct 1380tgggcaataa atactagtgt aggaatgaaa cattctgaat
atctttaaca atagaaatcc 1440atggggtggg gacaagccgt aaagactgga
tgtccatctc acacgaattt atggctatgg 1500gcaacacata atcctagtgc
aatatgatac tggggttatt aagatgtgtc ccaggcaggg 1560accaagacag
gtgaaccatg ttgttacact ctatttgtaa caaggggaaa gagagtggac
1620gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa
acggggctcc 1680acgccaatgg ggcccataaa caaagacaag tggccactct
tttttttgaa attgtggagt 1740gggggcacgc gtcagccccc acacgccgcc
ctgcggtttt ggactgtaaa ataagggtgt 1800aataacttgg ctgattgtaa
ccccgctaac cactgcggtc aaaccacttg cccacaaaac 1860cactaatggc
accccgggga atacctgcat aagtaggtgg gcgggccaag ataggggcgc
1920gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca
agcacagggt 1980tgttggtcct catattcacg aggtcgctga gagcacggtg
ggctaatgtt gccatgggta 2040gcatatacta cccaaatatc tggatagcat
atgctatcct aatctatatc tgggtagcat 2100aggctatcct aatctatatc
tgggtagcat atgctatcct aatctatatc tgggtagtat 2160atgctatcct
aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat
2220atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc
cgggtagcat 2280atgctatcct aatagagatt agggtagtat atgctatcct
aatttatatc tgggtagcat 2340atactaccca aatatctgga tagcatatgc
tatcctaatc tatatctggg tagcatatgc 2400tatcctaatc tatatctggg
tagcataggc tatcctaatc tatatctggg tagcatatgc 2460tatcctaatc
tatatctggg tagtatatgc tatcctaatt tatatctggg tagcataggc
2520tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg
tagtatatgc 2580tatcctaatc tgtatccggg tagcatatgc tatcctcatg
ataagctgtc aaacatgaga 2640attttcttga agacgaaagg gcctcgtgat
acgcctattt ttataggtta atgtcatgat 2700aataatggtt tcttagacgt
caggtggcac ttttcgggga aatgtgcgcg gaacccctat 2760ttgtttattt
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
2820aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
gtgtcgccct 2880tattcccttt tttgcggcat tttgccttcc tgtttttgct
cacccagaaa cgctggtgaa 2940agtaaaagat gctgaagatc agttgggtgc
acgagtgggt tacatcgaac tggatctcaa 3000cagcggtaag atccttgaga
gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3060taaagttctg
ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
3120tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
cagaaaagca 3180tcttacggat ggcatgacag taagagaatt atgcagtgct
gccataacca tgagtgataa 3240cactgcggcc aacttacttc tgacaacgat
cggaggaccg aaggagctaa ccgctttttt 3300gcacaacatg ggggatcatg
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3360cataccaaac
gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
3420actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
actggatgga 3480ggcggataaa gttgcaggac cacttctgcg ctcggccctt
ccggctggct ggtttattgc 3540tgataaatct ggagccggtg agcgtgggtc
tcgcggtatc attgcagcac tggggccaga 3600tggtaagccc tcccgtatcg
tagttatcta cacgacgggg agtcaggcaa ctatggatga 3660acgaaataga
cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
3720ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
ttaaaaggat 3780ctaggtgaag atcctttttg ataatctcat gaccaaaatc
ccttaacgtg agttttcgtt 3840ccactgagcg tcagaccccg tagaaaagat
caaaggatct tcttgagatc ctttttttct 3900gcgcgtaatc tgctgcttgc
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 3960ggatcaagag
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
4020aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
ctgtagcacc 4080gcctacatac ctcgctctgc taatcctgtt accagtggct
gctgccagtg gcgataagtc 4140gtgtcttacc gggttggact caagacgata
gttaccggat aaggcgcagc ggtcgggctg 4200aacggggggt tcgtgcacac
agcccagctt ggagcgaacg acctacaccg aactgagata 4260cctacagcgt
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
4320tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc 4380ctggtatctt tatagtcctg tcgggtttcg ccacctctga
cttgagcgtc gatttttgtg 4440atgctcgtca ggggggcgga gcctatggaa
aaacgccagc aacgcggcct ttttacggtt 4500cctggccttt tgctggcctt
ttgctcacat gttctttcct gcgttatccc ctgattctgt 4560ggataaccgt
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
4620gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
cgcctctccc 4680cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
tttcccgact ggaaagcggg 4740cagtgagcgc aacgcaatta atgtgagtta
gctcactcat taggcacccc aggctttaca 4800ctttatgctt ccggctcgta
tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 4860aaacagctat
gaccatgatt acgccaagct ctagctagag gtcgagtccc tccccagcag
4920gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
cccctaactc 4980cgcccatccc gcccctaact ccgcccagtt ccgcccattc
tccgccccat ggctgactaa 5040ttttttttat ttatgcagag gccgaggccg
cctcggcctc tgagctattc cagaagtagt 5100gaggaggctt ttttggaggc
ctaggctttt gcaaaaagct ttgcaaagat ggataaagtt 5160ttaaacagag
aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
5220tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
agaagttggg 5280gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
cgcggggtaa actgggaaag 5340tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt atataagtgc 5400agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 5460cgtgtgtggt
tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
5520tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
gaagtgggtg 5580ggagagttcg aggccttgcg cttaaggagc cccttcgcct
cgtgcttgag ttgaggcctg 5640gcctgggcgc tggggccgcc gcgtgcgaat
ctggtggcac cttcgcgcct gtctcgctgc 5700tttcgataag tctctagcca
tttaaaattt ttgatgacct gctgcgacgc tttttttctg 5760gcaagatagt
cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
5820cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
ggggcctgcg 5880agcgcggcca ccgagaatcg gacgggggta gtctcaagct
ggccggcctg ctctggtgcc 5940tggcctcgcg ccgccgtgta tcgccccgcc
ctgggcggca aggctggccc ggtcggcacc 6000agttgcgtga gcggaaagat
ggccgcttcc cggccctgct gcagggagct caaaatggag 6060gacgcggcgc
tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
6120gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
ggcacctcga 6180ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
ggggaggggt tttatgcgat 6240ggagtttccc cacactgagt gggtggagac
tgaagttagg ccagcttggc acttgatgta 6300attctccttg gaatttgccc
tttttgagtt tggatcttgg ttcattctca agcctcagac 6360agtggttcaa
agtttttttc ttccatttca ggtgtcgtga ggaattctct agagatccct
6420cgacctcgag atccattgtg cccgggcgcc accatgactt ggaccccact
cctcttcctc 6480accctcctcc tccactgcac aggaagctta tcg
65131936515DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 193acggtggctg caccatctgt cttcatcttc
ccgccatctg atgagcagtt gaaatctgga 60actgcctctg ttgtgtgcct gctgaataac
ttctatccca gagaggccaa agtacagtgg 120aaggtggata acgccctcca
atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 180aaggacagca
cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa
240cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt
cacaaagagc 300ttcaacaggg gagagtgttg agcggccgct cgaggccggc
aaggccggat cccccgacct 360cgacctctgg ctaataaagg aaatttattt
tcattgcaat agtgtgttgg aattttttgt 420gtctctcact cggaaggaca
tatgggaggg caaatcattt ggtcgagatc cctcggagat 480ctctagctag
aggatcgatc cccgccccgg acgaactaaa cctgactacg acatctctgc
540cccttcttcg cggggcagtg catgtaatcc cttcagttgg ttggtacaac
ttgccaactg 600ggccctgttc cacatgtgac acgggggggg accaaacaca
aaggggttct ctgactgtag 660ttgacatcct tataaatgga tgtgcacatt
tgccaacact gagtggcttt catcctggag 720cagactttgc agtctgtgga
ctgcaacaca acattgcctt tatgtgtaac tcttggctga 780agctcttaca
ccaatgctgg gggacatgta cctcccaggg gcccaggaag actacgggag
840gctacaccaa cgtcaatcag aggggcctgt gtagctaccg ataagcggac
cctcaagagg 900gcattagcaa tagtgtttat aaggccccct tgttaaccct
aaacgggtag catatgcttc 960ccgggtagta gtatatacta tccagactaa
ccctaattca atagcatatg ttacccaacg 1020ggaagcatat gctatcgaat
tagggttagt aaaagggtcc taaggaacag cgatatctcc 1080caccccatga
gctgtcacgg ttttatttac atggggtcag gattccacga gggtagtgaa
1140ccattttagt cacaagggca gtggctgaag atcaaggagc gggcagtgaa
ctctcctgaa 1200tcttcgcctg cttcttcatt ctccttcgtt tagctaatag
aataactgct gagttgtgaa 1260cagtaaggtg tatgtgaggt gctcgaaaac
aaggtttcag gtgacgcccc cagaataaaa 1320tttggacggg gggttcagtg
gtggcattgt gctatgacac caatataacc ctcacaaacc 1380ccttgggcaa
taaatactag tgtaggaatg aaacattctg aatatcttta acaatagaaa
1440tccatggggt ggggacaagc cgtaaagact ggatgtccat ctcacacgaa
tttatggcta 1500tgggcaacac ataatcctag tgcaatatga tactggggtt
attaagatgt gtcccaggca 1560gggaccaaga caggtgaacc atgttgttac
actctatttg taacaagggg aaagagagtg 1620gacgccgaca gcagcggact
ccactggttg tctctaacac ccccgaaaat taaacggggc 1680tccacgccaa
tggggcccat aaacaaagac aagtggccac tctttttttt gaaattgtgg
1740agtgggggca cgcgtcagcc cccacacgcc gccctgcggt tttggactgt
aaaataaggg
1800tgtaataact tggctgattg taaccccgct aaccactgcg gtcaaaccac
ttgcccacaa 1860aaccactaat ggcaccccgg ggaatacctg cataagtagg
tgggcgggcc aagatagggg 1920cgcgattgct gcgatctgga ggacaaatta
cacacacttg cgcctgagcg ccaagcacag 1980ggttgttggt cctcatattc
acgaggtcgc tgagagcacg gtgggctaat gttgccatgg 2040gtagcatata
ctacccaaat atctggatag catatgctat cctaatctat atctgggtag
2100cataggctat cctaatctat atctgggtag catatgctat cctaatctat
atctgggtag 2160tatatgctat cctaatttat atctgggtag cataggctat
cctaatctat atctgggtag 2220catatgctat cctaatctat atctgggtag
tatatgctat cctaatctgt atccgggtag 2280catatgctat cctaatagag
attagggtag tatatgctat cctaatttat atctgggtag 2340catatactac
ccaaatatct ggatagcata tgctatccta atctatatct gggtagcata
2400tgctatccta atctatatct gggtagcata ggctatccta atctatatct
gggtagcata 2460tgctatccta atctatatct gggtagtata tgctatccta
atttatatct gggtagcata 2520ggctatccta atctatatct gggtagcata
tgctatccta atctatatct gggtagtata 2580tgctatccta atctgtatcc
gggtagcata tgctatcctc atgataagct gtcaaacatg 2640agaattttct
tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
2700gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
gcggaacccc 2760tatttgttta tttttctaaa tacattcaaa tatgtatccg
ctcatgagac aataaccctg 2820ataaatgctt caataatatt gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc 2880ccttattccc ttttttgcgg
cattttgcct tcctgttttt gctcacccag aaacgctggt 2940gaaagtaaaa
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
3000caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
tgatgagcac 3060ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
gacgccgggc aagagcaact 3120cggtcgccgc atacactatt ctcagaatga
cttggttgag tactcaccag tcacagaaaa 3180gcatcttacg gatggcatga
cagtaagaga attatgcagt gctgccataa ccatgagtga 3240taacactgcg
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
3300tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
agctgaatga 3360agccatacca aacgacgagc gtgacaccac gatgcctgca
gcaatggcaa caacgttgcg 3420caaactatta actggcgaac tacttactct
agcttcccgg caacaattaa tagactggat 3480ggaggcggat aaagttgcag
gaccacttct gcgctcggcc cttccggctg gctggtttat 3540tgctgataaa
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
3600agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
caactatgga 3660tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 3720agaccaagtt tactcatata tactttagat
tgatttaaaa cttcattttt aatttaaaag 3780gatctaggtg aagatccttt
ttgataatct catgaccaaa atcccttaac gtgagttttc 3840gttccactga
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
3900tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
tggtttgttt 3960gccggatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 4020accaaatact gttcttctag tgtagccgta
gttaggccac cacttcaaga actctgtagc 4080accgcctaca tacctcgctc
tgctaatcct gttaccagtg gctgctgcca gtggcgataa 4140gtcgtgtctt
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
4200ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
ccgaactgag 4260atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 4320gtatccggta agcggcaggg tcggaacagg
agagcgcacg agggagcttc cagggggaaa 4380cgcctggtat ctttatagtc
ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 4440gtgatgctcg
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
4500gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
cccctgattc 4560tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 4620cgagcgcagc gagtcagtga gcgaggaagc
ggaagagcgc ccaatacgca aaccgcctct 4680ccccgcgcgt tggccgattc
attaatgcag ctggcacgac aggtttcccg actggaaagc 4740gggcagtgag
cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
4800acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
aatttcacac 4860aggaaacagc tatgaccatg attacgccaa gctctagcta
gaggtcgagt ccctccccag 4920caggcagaag tatgcaaagc atgcatctca
attagtcagc aaccatagtc ccgcccctaa 4980ctccgcccat cccgccccta
actccgccca gttccgccca ttctccgccc catggctgac 5040taattttttt
tatttatgca gaggccgagg ccgcctcggc ctctgagcta ttccagaagt
5100agtgaggagg cttttttgga ggcctaggct tttgcaaaaa gctttgcaaa
gatggataaa 5160gttttaaaca gagaggaatc tttgcagcta atggaccttc
taggtcttga aaggagtggg 5220aattggctcc ggtgcccgtc agtgggcaga
gcgcacatcg cccacagtcc ccgagaagtt 5280ggggggaggg gtcggcaatt
gaaccggtgc ctagagaagg tggcgcgggg taaactggga 5340aagtgatgtc
gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
5400tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa
cacaggtaag 5460tgccgtgtgt ggttcccgcg ggcctggcct ctttacgggt
tatggccctt gcgtgccttg 5520aattacttcc acctggctgc agtacgtgat
tcttgatccc gagcttcggg ttggaagtgg 5580gtgggagagt tcgaggcctt
gcgcttaagg agccccttcg cctcgtgctt gagttgaggc 5640ctggcctggg
cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc
5700tgctttcgat aagtctctag ccatttaaaa tttttgatga cctgctgcga
cgcttttttt 5760ctggcaagat agtcttgtaa atgcgggcca agatctgcac
actggtattt cggtttttgg 5820ggccgcgggc ggcgacgggg cccgtgcgtc
ccagcgcaca tgttcggcga ggcggggcct 5880gcgagcgcgg ccaccgagaa
tcggacgggg gtagtctcaa gctggccggc ctgctctggt 5940gcctggcctc
gcgccgccgt gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc
6000accagttgcg tgagcggaaa gatggccgct tcccggccct gctgcaggga
gctcaaaatg 6060gaggacgcgg cgctcgggag agcgggcggg tgagtcaccc
acacaaagga aaagggcctt 6120tccgtcctca gccgtcgctt catgtgactc
cacggagtac cgggcgccgt ccaggcacct 6180cgattagttc tcgagctttt
ggagtacgtc gtctttaggt tggggggagg ggttttatgc 6240gatggagttt
ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat
6300gtaattctcc ttggaatttg ccctttttga gtttggatct tggttcattc
tcaagcctca 6360gacagtggtt caaagttttt ttcttccatt tcaggtgtcg
tgaggaattc tctagagatc 6420cctcgacctc gagatccatt gtgcccgggc
gcaccatgac ttggacccca ctcctcttcc 6480tcaccctcct cctccactgc
acaggaagct tatcg 65151946519DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 194caacccaagg
ctgccccctc ggtcactctg ttcccgccct cctctgagga gcttcaagcc 60aacaaggcca
cactggtgtg tctcataagt gacttctacc cgggagccgt gacagtggcc
120tggaaggcag atagcagccc cgtcaaggcg ggagtggaga ccaccacacc
ctccaaacaa 180agcaacaaca agtacgcggc cagcagctac ctgagcctga
cgcctgagca gtggaagtcc 240cacagaagct acagctgcca ggtcacgcat
gaagggagca ccgtggagaa gacagtggcc 300cctacagaat gttcatgagc
ggccgctcga ggccggcaag gccggatccc ccgacctcga 360cctctggcta
ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc
420tctcactcgg aaggacatat gggagggcaa atcatttggt cgagatccct
cggagatctc 480tagctagagg atcgatcccc gccccggacg aactaaacct
gactacgaca tctctgcccc 540ttcttcgcgg ggcagtgcat gtaatccctt
cagttggttg gtacaacttg ccaactgggc 600cctgttccac atgtgacacg
gggggggacc aaacacaaag gggttctctg actgtagttg 660acatccttat
aaatggatgt gcacatttgc caacactgag tggctttcat cctggagcag
720actttgcagt ctgtggactg caacacaaca ttgcctttat gtgtaactct
tggctgaagc 780tcttacacca atgctggggg acatgtacct cccaggggcc
caggaagact acgggaggct 840acaccaacgt caatcagagg ggcctgtgta
gctaccgata agcggaccct caagagggca 900ttagcaatag tgtttataag
gcccccttgt taaccctaaa cgggtagcat atgcttcccg 960ggtagtagta
tatactatcc agactaaccc taattcaata gcatatgtta cccaacggga
1020agcatatgct atcgaattag ggttagtaaa agggtcctaa ggaacagcga
tatctcccac 1080cccatgagct gtcacggttt tatttacatg gggtcaggat
tccacgaggg tagtgaacca 1140ttttagtcac aagggcagtg gctgaagatc
aaggagcggg cagtgaactc tcctgaatct 1200tcgcctgctt cttcattctc
cttcgtttag ctaatagaat aactgctgag ttgtgaacag 1260taaggtgtat
gtgaggtgct cgaaaacaag gtttcaggtg acgcccccag aataaaattt
1320ggacgggggg ttcagtggtg gcattgtgct atgacaccaa tataaccctc
acaaacccct 1380tgggcaataa atactagtgt aggaatgaaa cattctgaat
atctttaaca atagaaatcc 1440atggggtggg gacaagccgt aaagactgga
tgtccatctc acacgaattt atggctatgg 1500gcaacacata atcctagtgc
aatatgatac tggggttatt aagatgtgtc ccaggcaggg 1560accaagacag
gtgaaccatg ttgttacact ctatttgtaa caaggggaaa gagagtggac
1620gccgacagca gcggactcca ctggttgtct ctaacacccc cgaaaattaa
acggggctcc 1680acgccaatgg ggcccataaa caaagacaag tggccactct
tttttttgaa attgtggagt 1740gggggcacgc gtcagccccc acacgccgcc
ctgcggtttt ggactgtaaa ataagggtgt 1800aataacttgg ctgattgtaa
ccccgctaac cactgcggtc aaaccacttg cccacaaaac 1860cactaatggc
accccgggga atacctgcat aagtaggtgg gcgggccaag ataggggcgc
1920gattgctgcg atctggagga caaattacac acacttgcgc ctgagcgcca
agcacagggt 1980tgttggtcct catattcacg aggtcgctga gagcacggtg
ggctaatgtt gccatgggta 2040gcatatacta cccaaatatc tggatagcat
atgctatcct aatctatatc tgggtagcat 2100aggctatcct aatctatatc
tgggtagcat atgctatcct aatctatatc tgggtagtat 2160atgctatcct
aatttatatc tgggtagcat aggctatcct aatctatatc tgggtagcat
2220atgctatcct aatctatatc tgggtagtat atgctatcct aatctgtatc
cgggtagcat 2280atgctatcct aatagagatt agggtagtat atgctatcct
aatttatatc tgggtagcat 2340atactaccca aatatctgga tagcatatgc
tatcctaatc tatatctggg tagcatatgc 2400tatcctaatc tatatctggg
tagcataggc tatcctaatc tatatctggg tagcatatgc 2460tatcctaatc
tatatctggg tagtatatgc tatcctaatt tatatctggg tagcataggc
2520tatcctaatc tatatctggg tagcatatgc tatcctaatc tatatctggg
tagtatatgc 2580tatcctaatc tgtatccggg tagcatatgc tatcctcatg
ataagctgtc aaacatgaga 2640attttcttga agacgaaagg gcctcgtgat
acgcctattt ttataggtta atgtcatgat 2700aataatggtt tcttagacgt
caggtggcac ttttcgggga aatgtgcgcg gaacccctat 2760ttgtttattt
ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
2820aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
gtgtcgccct 2880tattcccttt tttgcggcat tttgccttcc tgtttttgct
cacccagaaa cgctggtgaa 2940agtaaaagat gctgaagatc agttgggtgc
acgagtgggt tacatcgaac tggatctcaa 3000cagcggtaag atccttgaga
gttttcgccc cgaagaacgt tttccaatga tgagcacttt 3060taaagttctg
ctatgtggcg cggtattatc ccgtgttgac gccgggcaag agcaactcgg
3120tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
cagaaaagca 3180tcttacggat ggcatgacag taagagaatt atgcagtgct
gccataacca tgagtgataa 3240cactgcggcc aacttacttc tgacaacgat
cggaggaccg aaggagctaa ccgctttttt 3300gcacaacatg ggggatcatg
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc 3360cataccaaac
gacgagcgtg acaccacgat gcctgcagca atggcaacaa cgttgcgcaa
3420actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
actggatgga 3480ggcggataaa gttgcaggac cacttctgcg ctcggccctt
ccggctggct ggtttattgc 3540tgataaatct ggagccggtg agcgtgggtc
tcgcggtatc attgcagcac tggggccaga 3600tggtaagccc tcccgtatcg
tagttatcta cacgacgggg agtcaggcaa ctatggatga 3660acgaaataga
cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
3720ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
ttaaaaggat 3780ctaggtgaag atcctttttg ataatctcat gaccaaaatc
ccttaacgtg agttttcgtt 3840ccactgagcg tcagaccccg tagaaaagat
caaaggatct tcttgagatc ctttttttct 3900gcgcgtaatc tgctgcttgc
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc 3960ggatcaagag
ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
4020aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
ctgtagcacc 4080gcctacatac ctcgctctgc taatcctgtt accagtggct
gctgccagtg gcgataagtc 4140gtgtcttacc gggttggact caagacgata
gttaccggat aaggcgcagc ggtcgggctg 4200aacggggggt tcgtgcacac
agcccagctt ggagcgaacg acctacaccg aactgagata 4260cctacagcgt
gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
4320tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
ggggaaacgc 4380ctggtatctt tatagtcctg tcgggtttcg ccacctctga
cttgagcgtc gatttttgtg 4440atgctcgtca ggggggcgga gcctatggaa
aaacgccagc aacgcggcct ttttacggtt 4500cctggccttt tgctggcctt
ttgctcacat gttctttcct gcgttatccc ctgattctgt 4560ggataaccgt
attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
4620gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
cgcctctccc 4680cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
tttcccgact ggaaagcggg 4740cagtgagcgc aacgcaatta atgtgagtta
gctcactcat taggcacccc aggctttaca 4800ctttatgctt ccggctcgta
tgttgtgtgg aattgtgagc ggataacaat ttcacacagg 4860aaacagctat
gaccatgatt acgccaagct ctagctagag gtcgagtccc tccccagcag
4920gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
cccctaactc 4980cgcccatccc gcccctaact ccgcccagtt ccgcccattc
tccgccccat ggctgactaa 5040ttttttttat ttatgcagag gccgaggccg
cctcggcctc tgagctattc cagaagtagt 5100gaggaggctt ttttggaggc
ctaggctttt gcaaaaagct ttgcaaagat ggataaagtt 5160ttaaacagag
aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat
5220tggctccggt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
agaagttggg 5280gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg
cgcggggtaa actgggaaag 5340tgatgtcgtg tactggctcc gcctttttcc
cgagggtggg ggagaaccgt atataagtgc 5400agtagtcgcc gtgaacgttc
tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc 5460cgtgtgtggt
tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat
5520tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
gaagtgggtg 5580ggagagttcg aggccttgcg cttaaggagc cccttcgcct
cgtgcttgag ttgaggcctg 5640gcctgggcgc tggggccgcc gcgtgcgaat
ctggtggcac cttcgcgcct gtctcgctgc 5700tttcgataag tctctagcca
tttaaaattt ttgatgacct gctgcgacgc tttttttctg 5760gcaagatagt
cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc
5820cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
ggggcctgcg 5880agcgcggcca ccgagaatcg gacgggggta gtctcaagct
ggccggcctg ctctggtgcc 5940tggcctcgcg ccgccgtgta tcgccccgcc
ctgggcggca aggctggccc ggtcggcacc 6000agttgcgtga gcggaaagat
ggccgcttcc cggccctgct gcagggagct caaaatggag 6060gacgcggcgc
tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc
6120gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
ggcacctcga 6180ttagttctcg agcttttgga gtacgtcgtc tttaggttgg
ggggaggggt tttatgcgat 6240ggagtttccc cacactgagt gggtggagac
tgaagttagg ccagcttggc acttgatgta 6300attctccttg gaatttgccc
tttttgagtt tggatcttgg ttcattctca agcctcagac 6360agtggttcaa
agtttttttc ttccatttca ggtgtcgtga ggaattctct agagatccct
6420cgacctcgag atccattgtg cccgggcgcc accatggaca tgcgcgtgcc
cgcccagctg 6480ctgggcctgc tgctgctgtg gttccccggc tcgcgatgc
65191957185DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 195gcgtcgacca agggcccatc ggtcttcccc
ctggcaccct cctccaagag cacctctggg 60ggcacagcgg ccctgggctg cctggtcaag
gactacttcc ccgaaccggt gacggtgtcg 120tggaactcag gcgccctgac
cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180ggactctact
ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc
240tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa
agttgagccc 300aaatcttgtg acaaaactca cacatgccca ccgtgcccag
cacctgaagc cgcgggggga 360ccgtcagtct tcctcttccc cccaaaaccc
aaggacaccc tcatgatctc ccggacccct 420gaggtcacat gcgtggtggt
ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480tacgtggacg
gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac
540agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct
gaatggcaag 600gagtacaagt gcaaggtctc caacaaagcc ctcccagccc
ccatcgagaa aaccatctcc 660aaagccaaag ggcagccccg agaaccacag
gtgtacaccc tgcccccatc ccgcgaggag 720atgaccaaga accaggtcag
cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780gccgtggagt
gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg
840ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa
gagcaggtgg 900cagcagggga acgtcttctc atgctccgtg atgcatgagg
ctctgcacaa ccactacacg 960cagaagagcc tctccctgtc tccgggtaaa
tgagcggccg ctcgaggccg gcaaggccgg 1020atcccccgac ctcgacctct
ggctaataaa ggaaatttat tttcattgca atagtgtgtt 1080ggaatttttt
gtgtctctca ctcggaagga catatgggag ggcaaatcat ttggtcgaga
1140tccctcggag atctctagct agaggatcga tccccgcccc ggacgaacta
aacctgacta 1200cgacatctct gccccttctt cgcggggcag tgcatgtaat
cccttcagtt ggttggtaca 1260acttgccaac tgggccctgt tccacatgtg
acacgggggg ggaccaaaca caaaggggtt 1320ctctgactgt agttgacatc
cttataaatg gatgtgcaca tttgccaaca ctgagtggct 1380ttcatcctgg
agcagacttt gcagtctgtg gactgcaaca caacattgcc tttatgtgta
1440actcttggct gaagctctta caccaatgct gggggacatg tacctcccag
gggcccagga 1500agactacggg aggctacacc aacgtcaatc agaggggcct
gtgtagctac cgataagcgg 1560accctcaaga gggcattagc aatagtgttt
ataaggcccc cttgttaacc ctaaacgggt 1620agcatatgct tcccgggtag
tagtatatac tatccagact aaccctaatt caatagcata 1680tgttacccaa
cgggaagcat atgctatcga attagggtta gtaaaagggt cctaaggaac
1740agcgatatct cccaccccat gagctgtcac ggttttattt acatggggtc
aggattccac 1800gagggtagtg aaccatttta gtcacaaggg cagtggctga
agatcaagga gcgggcagtg 1860aactctcctg aatcttcgcc tgcttcttca
ttctccttcg tttagctaat agaataactg 1920ctgagttgtg aacagtaagg
tgtatgtgag gtgctcgaaa acaaggtttc aggtgacgcc 1980cccagaataa
aatttggacg gggggttcag tggtggcatt gtgctatgac accaatataa
2040ccctcacaaa ccccttgggc aataaatact agtgtaggaa tgaaacattc
tgaatatctt 2100taacaataga aatccatggg gtggggacaa gccgtaaaga
ctggatgtcc atctcacacg 2160aatttatggc tatgggcaac acataatcct
agtgcaatat gatactgggg ttattaagat 2220gtgtcccagg cagggaccaa
gacaggtgaa ccatgttgtt acactctatt tgtaacaagg 2280ggaaagagag
tggacgccga cagcagcgga ctccactggt tgtctctaac acccccgaaa
2340attaaacggg gctccacgcc aatggggccc ataaacaaag acaagtggcc
actctttttt 2400ttgaaattgt ggagtggggg cacgcgtcag cccccacacg
ccgccctgcg gttttggact 2460gtaaaataag ggtgtaataa cttggctgat
tgtaaccccg ctaaccactg cggtcaaacc 2520acttgcccac aaaaccacta
atggcacccc ggggaatacc tgcataagta ggtgggcggg 2580ccaagatagg
ggcgcgattg ctgcgatctg gaggacaaat tacacacact tgcgcctgag
2640cgccaagcac agggttgttg gtcctcatat tcacgaggtc gctgagagca
cggtgggcta 2700atgttgccat gggtagcata tactacccaa atatctggat
agcatatgct atcctaatct 2760atatctgggt agcataggct atcctaatct
atatctgggt agcatatgct atcctaatct 2820atatctgggt agtatatgct
atcctaattt atatctgggt agcataggct atcctaatct 2880atatctgggt
agcatatgct atcctaatct atatctgggt agtatatgct atcctaatct
2940gtatccgggt agcatatgct atcctaatag agattagggt agtatatgct
atcctaattt 3000atatctgggt agcatatact acccaaatat ctggatagca
tatgctatcc taatctatat 3060ctgggtagca tatgctatcc taatctatat
ctgggtagca taggctatcc taatctatat 3120ctgggtagca tatgctatcc
taatctatat ctgggtagta tatgctatcc taatttatat 3180ctgggtagca
taggctatcc taatctatat ctgggtagca tatgctatcc taatctatat
3240ctgggtagta tatgctatcc taatctgtat ccgggtagca tatgctatcc
tcatgataag 3300ctgtcaaaca tgagaatttt cttgaagacg aaagggcctc
gtgatacgcc tatttttata 3360ggttaatgtc atgataataa tggtttctta
gacgtcaggt ggcacttttc ggggaaatgt 3420gcgcggaacc cctatttgtt
tatttttcta aatacattca aatatgtatc cgctcatgag 3480acaataaccc
tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
3540tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
ttgctcaccc
3600agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
tgggttacat 3660cgaactggat ctcaacagcg gtaagatcct tgagagtttt
cgccccgaag aacgttttcc 3720aatgatgagc acttttaaag ttctgctatg
tggcgcggta ttatcccgtg ttgacgccgg 3780gcaagagcaa ctcggtcgcc
gcatacacta ttctcagaat gacttggttg agtactcacc 3840agtcacagaa
aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
3900aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
gaccgaagga 3960gctaaccgct tttttgcaca acatggggga tcatgtaact
cgccttgatc gttgggaacc 4020ggagctgaat gaagccatac caaacgacga
gcgtgacacc acgatgcctg cagcaatggc 4080aacaacgttg cgcaaactat
taactggcga actacttact ctagcttccc ggcaacaatt 4140aatagactgg
atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
4200tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
gtatcattgc 4260agcactgggg ccagatggta agccctcccg tatcgtagtt
atctacacga cggggagtca 4320ggcaactatg gatgaacgaa atagacagat
cgctgagata ggtgcctcac tgattaagca 4380ttggtaactg tcagaccaag
tttactcata tatactttag attgatttaa aacttcattt 4440ttaatttaaa
aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
4500acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
gatcttcttg 4560agatcctttt tttctgcgcg taatctgctg cttgcaaaca
aaaaaaccac cgctaccagc 4620ggtggtttgt ttgccggatc aagagctacc
aactcttttt ccgaaggtaa ctggcttcag 4680cagagcgcag ataccaaata
ctgttcttct agtgtagccg tagttaggcc accacttcaa 4740gaactctgta
gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
4800cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
cggataaggc 4860gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc
agcttggagc gaacgaccta 4920caccgaactg agatacctac agcgtgagct
atgagaaagc gccacgcttc ccgaagggag 4980aaaggcggac aggtatccgg
taagcggcag ggtcggaaca ggagagcgca cgagggagct 5040tccaggggga
aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
5100gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
ccagcaacgc 5160ggccttttta cggttcctgg ccttttgctg gccttttgct
cacatgttct ttcctgcgtt 5220atcccctgat tctgtggata accgtattac
cgcctttgag tgagctgata ccgctcgccg 5280cagccgaacg accgagcgca
gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg 5340caaaccgcct
ctccccgcgc gttggccgat tcattaatgc agctggcacg acaggtttcc
5400cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
ctcattaggc 5460accccaggct ttacacttta tgcttccggc tcgtatgttg
tgtggaattg tgagcggata 5520acaatttcac acaggaaaca gctatgacca
tgattacgcc aagctctagc tagaggtcga 5580gtccctcccc agcaggcaga
agtatgcaaa gcatgcatct caattagtca gcaaccatag 5640tcccgcccct
aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc
5700cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
gcctctgagc 5760tattccagaa gtagtgagga ggcttttttg gaggcctagg
cttttgcaaa aagctttgca 5820aagatggata aagttttaaa cagagaggaa
tctttgcagc taatggacct tctaggtctt 5880gaaaggagtg ggaattggct
ccggtgcccg tcagtgggca gagcgcacat cgcccacagt 5940ccccgagaag
ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg
6000ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg
gtgggggaga 6060accgtatata agtgcagtag tcgccgtgaa cgttcttttt
cgcaacgggt ttgccgccag 6120aacacaggta agtgccgtgt gtggttcccg
cgggcctggc ctctttacgg gttatggccc 6180ttgcgtgcct tgaattactt
ccacctggct gcagtacgtg attcttgatc ccgagcttcg 6240ggttggaagt
gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc
6300ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt
ggcaccttcg 6360cgcctgtctc gctgctttcg ataagtctct agccatttaa
aatttttgat gacctgctgc 6420gacgcttttt ttctggcaag atagtcttgt
aaatgcgggc caagatctgc acactggtat 6480ttcggttttt ggggccgcgg
gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc 6540gaggcggggc
ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg
6600gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg
cggcaaggct 6660ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
cttcccggcc ctgctgcagg 6720gagctcaaaa tggaggacgc ggcgctcggg
agagcgggcg ggtgagtcac ccacacaaag 6780gaaaagggcc tttccgtcct
cagccgtcgc ttcatgtgac tccacggagt accgggcgcc 6840gtccaggcac
ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga
6900ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag
ttaggccagc 6960ttggcacttg atgtaattct ccttggaatt tgcccttttt
gagtttggat cttggttcat 7020tctcaagcct cagacagtgg ttcaaagttt
ttttcttcca tttcaggtgt cgtgaggaat 7080tctctagaga tccctcgacc
tcgagatcca ttgtgcccgg gcgccaccat ggagtttggg 7140ctgagctggc
tttttcttgt cgcgatttta aaaggtgtcc agtgc 7185
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