U.S. patent application number 12/718147 was filed with the patent office on 2010-09-30 for human epo mimetic hinge core mimetibodies, compositions, methods and uses.
Invention is credited to John Ghrayeb, George A. Heavner, Chichi Huang, David M. Knight, Thomas C. Nesspor, Bernard J. Scallon.
Application Number | 20100249032 12/718147 |
Document ID | / |
Family ID | 35996746 |
Filed Date | 2010-09-30 |
United States Patent
Application |
20100249032 |
Kind Code |
A1 |
Heavner; George A. ; et
al. |
September 30, 2010 |
Human EPO Mimetic Hinge Core Mimetibodies, Compositions, Methods
and Uses
Abstract
The present invention relates to at least one novel human EPO
mimetic hinge core mimetibody or specified portion or variant,
including isolated nucleic acids that encode at least one EPO
mimetic hinge core mimetibody or specified portion or variant, EPO
mimetic hinge core mimetibody or specified portion or variants,
vectors, host cells, transgenic animals or plants, and methods of
making and using thereof, including therapeutic compositions,
methods and devices.
Inventors: |
Heavner; George A.; (Malvem,
PA) ; Knight; David M.; (Berwyn, PA) ;
Ghrayeb; John; (Downingtown, PA) ; Scallon; Bernard
J.; (Wayne, PA) ; Nesspor; Thomas C.;
(Collegeville, PA) ; Huang; Chichi; (Berwyn,
PA) |
Correspondence
Address: |
PHILIP S. JOHNSON;JOHNSON & JOHNSON
ONE JOHNSON & JOHNSON PLAZA
NEW BRUNSWICK
NJ
08933-7003
US
|
Family ID: |
35996746 |
Appl. No.: |
12/718147 |
Filed: |
March 5, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12031849 |
Mar 27, 2009 |
7718176 |
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12718147 |
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Current U.S.
Class: |
514/7.7 ;
530/324; 530/326; 530/327; 530/328; 530/387.3; 536/23.5 |
Current CPC
Class: |
A61P 15/00 20180101;
A61P 31/00 20180101; A61P 11/00 20180101; A61P 9/00 20180101; A61P
13/00 20180101; A61P 3/00 20180101; A61P 1/00 20180101; A61P 5/00
20180101; C07K 14/505 20130101; A61P 19/00 20180101; A61P 25/00
20180101; A61P 17/00 20180101; A61P 27/16 20180101; A61P 7/00
20180101; A61P 37/00 20180101; A61P 21/00 20180101; Y10S 435/972
20130101 |
Class at
Publication: |
514/13 ;
536/23.5; 530/324; 530/326; 530/327; 530/387.3; 530/328; 514/14;
514/15 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C07H 21/04 20060101 C07H021/04; C07K 7/00 20060101
C07K007/00; C07K 16/00 20060101 C07K016/00; A61K 38/08 20060101
A61K038/08; A61P 19/00 20060101 A61P019/00; A61P 9/00 20060101
A61P009/00; A61P 5/00 20060101 A61P005/00; A61P 3/00 20060101
A61P003/00; A61P 1/00 20060101 A61P001/00; A61P 15/00 20060101
A61P015/00; A61P 17/00 20060101 A61P017/00; A61P 21/00 20060101
A61P021/00; A61P 13/00 20060101 A61P013/00; A61P 25/00 20060101
A61P025/00; A61P 11/00 20060101 A61P011/00; A61P 27/16 20060101
A61P027/16; A61P 31/00 20060101 A61P031/00; A61P 37/00 20060101
A61P037/00 |
Claims
1. A nucleic acid encoding an EPO mimetic hinge core mimetibody
comprising a polynucleotide encoding a sequence selected from the
group consisting of SEQ ID NOS:82-87 and 89.
2. A nucleic acid complement to the nucleic acid of claim 1.
3. A nucleic acid encoding an EPO mimetic hinge core mimetibody
comprising a polynucleotide encoding an amino acid sequence
selected from the group consisting of SEQ ID NOS:1-30, or a
polynucleotide complementary thereto.
4. A nucleic acid encoding an EPO mimetic hinge core mimetibody
comprising a polynucleotide encoding a polypeptide according to
Formula (I): ((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is a
portion of the N-terminus of an immunoglobulin variable region, P
is a bioactive EPO mimetic peptide, L is a linker sequence, H is a
portion of an immunoglobulin variable hinge core region, CH2 is a
portion of an immunoglobulin CH2 constant region, CH3 is a portion
of an immunoglobulin CH3 constant region, m, n, o, p, q, r, and s
can independently be independently an integer between 0, 1 or 2 and
10.
5. An EPO mimetic hinge core mimetibody polypeptide, comprising a
sequence selected from the group consisting of SEQ ID NOS:82 and
84.
6. (canceled)
7. An EPO mimetic hinge core mimetibody polypeptide, comprising a
sequence selected from the group consisting of SEQ ID NOS:1-30.
8. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is selected from the group consisting of GS, GGGS (SEQ
ID NO:73) and GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75), CH2
is APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAK (SEQ ID
NO:76), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID
NO:78), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
9. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is selected from the group consisting of GS, GGGS (SEQ
ID NO:73) and GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75), CH2
is APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAK (SEQ ID
NO:77), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGK (SEQ ID
NO:78), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
10. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is selected from the group consisting of GS, GGGS (SEQ
ID NO:73) and GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75), CH2
is APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAK (SEQ ID
NO:79), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID
NO:81), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
11. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is selected from the group consisting of GS, GGGS (SEQ
ID NO:73) and GSGGGS (SEQ ID NO:74), H is CPPCP (SEQ ID NO:75), CH2
is APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAK (SEQ ID
NO:80), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID
NO:81), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
12. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is the N-terminal
portion of a human variable region, P is a bioactive peptide
selected from the group consisting of SEQ ID NOS:1-30, L is linker
polypeptide, H is a portion of an immunoglobulin variable hinge
core region, CH2 is
APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAK (SEQ ID
NO:76), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ
ID NO:78), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
13. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is the N-terminal
portion of a human variable region, P is a bioactive peptide
selected from the group consisting of SEQ ID NOS:1-30, L is a
linker polypeptide, H is a portion of an immunoglobulin variable
hinge core region, CH2 is
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAK (SEQ ID
NO:77), CH3 is GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGK (SEQ
ID NO:78), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
14. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is the N-terminal
portion of a human variable region, P is a bioactive peptide
selected from the group consisting of SEQ ID NOS:1-30, L is a
linker polypeptide, H is a portion of an immunoglobulin variable
hinge core region, CH2 is
APEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVE
VHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAK (SEQ ID
NO:79), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID
NO:81), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
15. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is the N-terminal
portion of a human variable region, P is a bioactive peptide
selected from the group consisting of SEQ ID NOS:1-30, L is a
linker polypeptide, H is a portion of an immunoglobulin variable
hinge core region, CH2 is
APEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGV EVH
NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAK (SEQ ID
NO:80), CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLS LGK (SEQ ID
NO:81), and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
16. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is QIQ, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is a linker polypeptide, H is a portion of an
immunoglobulin variable hinge core region, CH2 is a portion of an
immunoglobulin CH2 constant region, CH3 is a portion of an
immunoglobulin CH3 constant region, and m, n, o, p, q, r, s are
independently an integer between 0, 1 or 2 and 10.
17. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is a portion of
the N-terminus of an immunoglobulin variable region, P is a
bioactive EPO mimetic peptide, L is selected from the group
consisting of GS, GGGS (SEQ ID NO:73) and GSGGGS (SEQ ID NO:74), H
is a portion of an immunoglobulin variable hinge core region, CH2
is a portion of an immunoglobulin CH2 constant region, CH3 is a
portion of an immunoglobulin CH3 constant region, and m, n, o, p,
q, r, s are independently an integer between 0, 1 or 2 and 10.
18. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is a portion of
the N-terminus of an immunoglobulin variable region, P is a
bioactive EPO mimetic peptide, L is a linker polypeptide, H is a
portion of an immunoglobulin variable hinge core region, CH2 is a
portion of an immunoglobulin CH2 constant region, CH3 is a portion
of an immunoglobulin CH3 constant region, and m, n, o, p, q, r, s
are independently an integer between 0, 1 or 2 and 10.
19. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is a portion of
the N-terminus of an immunoglobulin variable region, P is a
bioactive EPO mimetic peptide, L is a linker polypeptide, H is
CPPCP (SEQ ID NO:75), CH2 is a portion of an immunoglobulin CH2
constant region, CH3 is a portion of an immunoglobulin CH3 constant
region, and m, n, o, p, q, r, s are independently an integer
between 0, 1 or 2 and 10.
20. An EPO mimetic hinge core mimetibody polypeptide, comprising a
polypeptide according to Formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s), where V is a portion of
the N-terminus of an immunoglobulin variable region, P is a
bioactive peptide selected from the group consisting of SEQ ID
NOS:1-30, L is a linker polypeptide, H is a portion of an
immunoglobulin variable hinge core region, CH2 is a portion of an
immunoglobulin CH2 constant region, CH3 is a portion of an
immunoglobulin CH3 constant region, and m, n, o, p, q, r, s are
independently an integer between 0, 1 or 2 and 10.
21-40. (canceled)
41. A method for treating a disorder associated with erythropoietin
receptor activity comprising administering an effective amount of
the EPO mimetic hinge core mimetibody polypeptide of claim 7 to a
patient in need thereof.
42. The method of claim 41 wherein said disorder is selected from
the group consisting of, bone and joint disorders, cardiovascular
disorders, a dental or oral disorder, a dermatologic disorder, an
ear, nose or throat disorder, an endocrine or metabolic disorder, a
gastrointestinal disorder, a gynecologic disorder, a hepatic or
biliary disorder, an obstetric disorder, a hematologic disorder, an
immunologic or allergic disorder, an infectious disease disorder, a
musculoskeletal disorder, an oncologic disorder, a neurologic
disorder, a nutritional disorder, an ophthalmologic disorder, a
pediatric disorder, a poisoning disorder, a psychiatric disorder, a
renal disorder, and a pulmonary disorder.
43. The method of claim 42 wherein said hematologic disorder is
selected from the group consisting of cancer treatment related
anemia, radiotherapy or chemotherapy related anemia, viral or
bacterial infection treatment related anemia, renal anemia, anemia
of prematurity, pediatric and/or adult cancer-associated anemia,
anemia associated with lymphoma, myeloma, multiple myeloma,
AIDS-associated anemia, cyclic neutropenia or Kostmann syndrome
(congenital agranulocytosis), end-stage renal disease, anemia
associated with dialysis, chronic renal insufficiency, congenital
hypoplastic anemia, thalassemia major, sickle cell disease, and
vaso-occlusive complications of sickle cell disease.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of U.S. application Ser.
No. 12/031,849, filed Feb. 15, 2008, currently allowed, which is a
divisional of U.S. Pat. No. 7,393,663 issued Jul. 1, 2008, which
claims the benefit of U.S. provisional application No. 60/507,349
filed Sep. 30, 2003 which are hereby incorporated by reference
herein.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to mammalian EPO mimetic hinge
core mimetibodies, specified portions and variants specific for
biologically active proteins, fragment or ligands, EPO mimetic
hinge core mimetibody encoding and complementary nucleic acids,
host cells, and methods of making and using thereof, including
therapeutic formulations, administration and devices.
[0004] 2. Related Art
[0005] Recombinant proteins are an emerging class of therapeutic
agents. Such recombinant therapeutics have engendered advances in
protein formulation and chemical modification. Such modifications
can potentially enhance the therapeutic utility of therapeutic
proteins, such as by increasing half lives (e.g., by blocking their
exposure to proteolytic enzymes), enhancing biological activity, or
reducing unwanted side effects. One such modification is the use of
immunoglobulin fragments fused to receptor proteins, such as
enteracept. Therapeutic proteins have also been constructed using
the Fc domain to attempt to provide a longer half-life or to
incorporate functions such as Fc receptor binding, protein A
binding, and complement fixation. One specific and vital role of
the mammalian hematopoietic system is the production of
erythrocytes, or red blood cells, which transport oxygen to the
various tissues of the animal's body. The process of producing
erythrocytes ("erythropoiesis") occurs continuously throughout an
animal's life span to offset erythrocyte destruction. The typical
red blood cell has a relatively short life-span, usually 100 to 120
days. Erythropoiesis is a precisely controlled physiological
mechanism whereby sufficient numbers of erythrocytes are produced
to enable proper tissue oxygenation, but not so many as to impede
circulation.
[0006] Erythropoiesis is now known to be primarily controlled by
the polypeptide erythropoietin (EPO), an acidic glycoprotein.
Erythropoietin is produced as the result of the expression of a
single copy gene located in a chromosome of a mammal. The amino
acid sequence for recombinant human EPO ("rHuEPO") is substantially
identical to the amino acid sequence for EPO obtained from human
urinary sources. However, the glycosylation of rHuEPO differs from
that of urinary EPO and human serum EPO.
[0007] In a healthy mammal, EPO is present in the blood plasma in
very low concentrations, as the tissues are being sufficiently
oxygenated by the existing number of circulating erythrocytes. The
EPO present stimulates the production of new erythrocytes to
replace those lost to the aging process. Additionally, EPO
production is stimulated under conditions of hypoxia, wherein the
oxygen supply to the body's tissues is reduced below normal
physiological levels despite adequate perfusion of the tissue by
blood. Hypoxia may be caused by hemorrhaging, radiation-induced
erythrocyte destruction, various anemias, high altitude, or long
periods of unconsciousness. In contrast, should the number of red
blood cells in circulation exceed what is needed for normal tissue
oxygenation, EPO production is reduced.
[0008] However, certain disease states involve abnormal
erythropoiesis. Recombinant human EPO (rHuEPO) is being used
therapeutically in a number of countries. In the United States, the
U.S. Food and Drug Administration (FDA) has approved rHuEPO's use
in treating anemia associated with end-stage renal disease.
Patients undergoing hemodialysis to treat this disorder typically
suffer severe anemia, caused by the rupture and premature death of
erythrocytes as a result of the dialysis treatment. EPO is also
useful in the treatment of other types of anemia. For instance,
chemotherapy-induced anemia, anemia associated with myelodysplasia,
those associated with various congenital disorders, AIDS-related
anemia, and prematurity-associated anemia, may be treated with EPO.
Additionally, EPO may play a role in other areas, such as helping
to more quickly restore a normal hematocrit in bone marrow
transplantation patients, in patients preparing for autologous
blood transfusions, and in patients suffering from iron overload
disorders.
[0009] Erythropoietin (EPO) is a glycoprotein hormone composed of
165 amino acids and four carbohydrate chains that functions as the
primary regulator of erythropoiesis by binding to a specific
receptor on the surface of erythrocyte precursor cells. This
binding signals their proliferation and differentiation into mature
red blood cells. The erythropoietin receptor is a 484-amino acid
glycoprotein with high affinity for erythropoietin. For the
erythropoietin receptor, ligand-induced homodimerization may be one
of the key event that governs activation.
[0010] Erythropoietin has a relatively short half-life.
Intravenously administered erythropoietin is eliminated at a rate
consistent with first order kinetics with a circulating half-life
ranging from approximately 3 to 4 hours in patients with CRF.
Within the therapeutic dose range, detectable levels of plasma
erythropoietin are maintained for at least 24 hours. After
subcutaneous administration of erythropoietin, peak serum levels
are achieved within 5-24 hours and decline slowly thereafter.
[0011] Small peptidomimetics of erythropoietin were identified by
several groups through screening of random phage display peptide
libraries for affinity to the erythropoietin receptor. These
sequences have no homology with erythropoietin. In functional
assays several of these peptides showed activity, but only
1/100,000.sup.th that of recombinant erythropoietin. Although
several attempts have been made to increase the potency of these
peptides by preparing covalent dimers or multimers of
peptidomimetics, these compounds are still 1,000-10,000 fold less
active than erythropoietin on a molar basis and have very short
half lives that has made them not suitable for use as
therapeutics.
[0012] Accordingly, there is a need to provide improved and/or
modified versions of EPO therapeutic proteins, which overcome one
more of these and other problems known in the art.
SUMMARY OF THE INVENTION
[0013] The present invention provides human EPO mimetic hinge core
mimetibodies, including modified immunoglobulins, cleavage products
and other specified portions and variants thereof, as well as EPO
mimetic hinge core mimetibody compositions, encoding or
complementary nucleic acids, vectors, host cells, compositions,
formulations, devices, transgenic animals, transgenic plants, and
methods of making and using thereof, as described and/or enabled
herein, in combination with what is known in the art.
[0014] The present invention also provides at least one isolated
EPO mimetic hinge core mimetibody or specified portion or variant
as described herein and/or as known in the art. The EPO mimetic
hinge core mimetibody can optionally comprise at least one CH3
region directly linked with at least one CH2 region directly linked
with at least one portion of at least one hinge region or fragment
thereof (H), directly linked with an optional linker sequence (L),
directly linked to at least one EPO mimetic therapeutic peptide
(P), optionally further directly linked with at least a portion of
at least one variable antibody sequence (V). In a preferred
embodiment a pair of a CH3-CH2-hinge-linker-therapeutic peptide
with an optional N-terminal antibody sequence, the pair optionally
linked by association or covalent linkage, such as, but not limited
to, at least one Cys-Cys disulfide bond or at least one CH4 or
other immunoglobulin sequence. In one embodiment, an EPO mimetic
hinge core mimetibody comprises formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),
where V is at least one portion of an N-terminus of an
immunoglobulin variable region, P is at least one bioactive EPO
mimetic polypeptide, L is at least one linker sequence, H is least
one portion of an immunoglobulin variable region, CH2 is at least a
portion of an immunoglobulin CH2 constant region, CH3 is at least a
portion of an immunoglobulin CH3 constant region, m, n, o p, q, r,
and s can be independently an integer between 0, 1 or 2 and 10,
mimicking different types of immunoglobulin molecules, e.g., but
not limited to IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE,
or any subclass thereof, and the like, or any combination
thereof.
[0015] Thus, an EPO mimetic hinge core mimetibody of the present
invention mimics at least a portion of an antibody or
immunoglobulin structure or function with its inherent properties
and functions, while providing a therapeutic peptide and its
inherent or acquired in vitro, in vivo or in situ properties or
activities. The various portions of the antibody and therapeutic
peptide portions of at least one EPO mimetic hinge core mimetibody
of the present invention can vary as described herein in
combination with what is known in the art.
[0016] The present invention provides, in one aspect, isolated
nucleic acid molecules comprising, complementary, having
significant identity or hybridizing to, a polynucleotide encoding
specific mimetibodies or specified portions or variants thereof,
comprising at least one specified sequence, domain, portion or
variant thereof. The present invention further provides recombinant
vectors comprising at least one of said isolated EPO mimetic hinge
core mimetibody nucleic acid molecules, host cells containing such
nucleic acids and/or recombinant vectors, as well as methods of
making and/or using such EPO mimetic hinge core mimetibody nucleic
acids, vectors and/or host cells.
[0017] At least one EPO mimetic hinge core mimetibody or specified
portion or variant of the invention mimics the binding of the P
portion of the mimetibody to at least one ligand, or has at least
one biological activity of, at least one protein, subunit,
fragment, portion or any combination thereof.
[0018] The present invention also provides at least one isolated
EPO mimetic hinge core mimetibody or specified portion or variant
as described herein and/or as known in the art, wherein the EPO
mimetic hinge core mimetibody or specified portion or variant has
at least one activity, such as, but not limited to known biological
activities of at least one bioactive peptide or polypeptide
corresponding to the P portion of Formula I. An EPO mimetic hinge
core mimetibody can thus be screened for a corresponding activity
according to known methods, such as at least one neutralizing
activity towards a protein or fragment thereof.
[0019] The present invention also provides at least one composition
comprising (a) at least one isolated EPO mimetic hinge core
mimetibody or specified portion or variant encoding nucleic acid
and/or EPO mimetic hinge core mimetibody as described herein; and
(b) a suitable carrier or diluent. The carrier or diluent can
optionally be pharmaceutically acceptable, according to known
methods. The composition can optionally further comprise at least
one further compound, protein or composition.
[0020] The present invention also provides at least one method for
expressing at least one EPO mimetic hinge core mimetibody or
specified portion or variant in a host cell, comprising culturing a
host cell as described herein and/or as known in the art under
conditions wherein at least one EPO mimetic hinge core mimetibody
or specified portion or variant is expressed in detectable and/or
recoverable amounts.
[0021] The present invention further provides at least one EPO
mimetic hinge core mimetibody, specified portion or variant in a
method or composition, when administered in a therapeutically
effective amount, for modulation, for treating or reducing the
symptoms of at least one of a bone and joint disorder,
cardiovascular disorder, a dental or oral disorder, a dermatologic
disorder, an ear, nose or throat disorder, an endocrine or
metabolic disorder, a gastrointestinal disorder, a gynecologic
disorder, a hepatic or biliary disorder, a an obstetric disorder, a
hematologic disorder, an immunologic or allergic disorder, an
infectious disease, a musculoskeletal disorder, a oncologic
disorder, a neurologic disorder, a nutritional disorder, an
ophthalmologic disorder, a pediatric disorder, a poisoning
disorder, a psychiatric disorder, a renal disorder, a pulmonary
disorder, or any other known disorder. (See, e.g., The Merck
Manual, 17th ed., Merck Research Laboratories, Merck and Co.,
Whitehouse Station, N.J. (1999), entirely incorporated herein by
reference), as needed in many different conditions, such as but not
limited to, prior to, subsequent to, or during a related disease or
treatment condition, as known in the art.
[0022] The present invention further provides at least one EPO
mimetic hinge core mimetibody, specified portion or variant in a
method or composition, when administered in a therapeutically
effective amount, for modulation, for treating or reducing the
symptoms of, at least one immune, cardiovascular, infectious,
malignant, and/or neurologic disease in a cell, tissue, organ,
animal or patient and/or, as needed in many different conditions,
such as but not limited to, prior to, subsequent to, or during a
related disease or treatment condition, as known in the art and/or
as described herein.
[0023] The present invention also provides at least one
composition, device and/or method of delivery of a therapeutically
or prophylactically effective amount of at least one EPO mimetic
hinge core mimetibody or specified portion or variant, according to
the present invention.
[0024] The present invention further provides at least one
anti-idiotype antibody to at least one EPO mimetic hinge core
mimetibody of the present invention. The anti-idiotype antibody
includes any protein or peptide containing molecule that comprises
at least a portion of an immunoglobulin molecule, such as but not
limited to at least one complimentarity 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
competitively binds an EPO receptor binding region of at least one
EPO mimetic hinge core mimetibody of the present invention. Such
idiotype antibodies of the invention can include or be derived from
any mammal, such as but not limited to a human, a mouse, a rabbit,
a rat, a rodent, a primate, and the like.
[0025] The present invention also provides at least one isolated
nucleic acid molecule comprising, complementary, or hybridizing to,
a polynucleotide encoding at least one EPO mimetic hinge core
mimetibody anti-idiotype antibody, comprising at least one
specified sequence, domain, portion or variant thereof. The present
invention further provides recombinant vectors comprising said EPO
mimetic hinge core mimetibody anti-idiotype antibody encoding
nucleic acid molecules, host cells containing such nucleic acids
and/or recombinant vectors, as well as methods of making and/or
using such anti-idiotype antibody nucleic acids, vectors and/or
host cells.
[0026] The present invention also provides at least one method for
expressing at least one EPO mimetic hinge core mimetibody, or EPO
mimetic hinge core mimetibody anti-idiotype antibody, in a host
cell, comprising culturing a host cell as described herein under
conditions wherein at least one EPO mimetic hinge core mimetibody
or anti-idiotype antibody is expressed in detectable and/or
recoverable amounts.
[0027] The present invention also provides at least one composition
comprising (a) an isolated EPO mimetic hinge core mimetibody
encoding nucleic acid and/or EPO mimetic hinge core mimetibody as
described herein; and (b) a suitable carrier or diluent. The
carrier or diluent can optionally be pharmaceutically acceptable,
according to known carriers or diluents. The composition can
optionally further comprise at least one further compound, protein
or composition.
[0028] The present invention further provides at least one EPO
mimetic hinge core mimetibody method or composition, for
administering a therapeutically effective amount to modulate or
treat at least one protein related condition in a cell, tissue,
organ, animal or patient and/or, prior to, subsequent to, or during
a related condition, as known in the art and/or as described
herein.
[0029] The present invention also provides at least one
composition, device and/or method of delivery of a therapeutically
or prophylactically effective amount of at least one EPO mimetic
hinge core mimetibody, according to the present invention.
[0030] The present invention further provides at least one EPO
mimetic hinge core mimetibody method or composition, for diagnosing
at least one EPO related condition in a cell, tissue, organ, animal
or patient and/or, prior to, subsequent to, or during a related
condition, as known in the art and/or as described herein.
[0031] The present invention also provides at least one
composition, device and/or method of delivery for diagnosing of at
least one EPO mimetic hinge core mimetibody, according to the
present invention.
[0032] In one aspect, the present invention provides at least one
isolated human EPO mimetic hinge core mimetibody, comprising at
least one P(n) region comprising at least a portion of at least one
of SEQ ID NOS:1-30, e.g., as presented in Table 1 below, or
optionally with one or more substitutions, deletions or insertions
as described herein or as known in the art. In other aspect the
present invention provides at least one isolated human EPO mimetic
hinge core mimetibody, wherein the EPO mimetic hinge core
mimetibody specifically binds at least one epitope comprising at
least 1-3 of at least one ligand or binding region which ligand
binds to at least a portion of at least one of SEQ ID NOS:1-30 as
presented in Table 1 below, or optionally with one or more
substitutions, deletions or insertions as described herein or as
known in the art.
[0033] The at least one EPO mimetic hinge core mimetibody can
optionally further at least one of: bind protein with an affinity
of at least one selected from at least 10.sup.-9 M, at least
10.sup.-10 M, at least 10.sup.-11 M, or at least 10.sup.-12 M;
substantially neutralize at least one activity of at least one
protein or portion thereof. Also provided is an isolated nucleic
acid encoding at least one isolated human EPO mimetic hinge core
mimetibody; an isolated nucleic acid vector comprising the isolated
nucleic acid, and/or a prokaryotic or eukaryotic host cell
comprising the isolated nucleic acid. The host cell can optionally
be at least one selected from COS-1, COS-7, HEK293, BHK21, CHO,
BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells,
or any derivative, immortalized or transformed cell thereof. Also
provided is a method for producing at least one EPO mimetic hinge
core mimetibody, comprising translating the EPO mimetic hinge core
mimetibody encoding nucleic acid under conditions in vitro, in vivo
or in situ, such that the EPO mimetic hinge core mimetibody is
expressed in detectable or recoverable amounts.
[0034] Also provided is a composition comprising at least one
isolated human EPO mimetic hinge core mimetibody and at least one
pharmaceutically acceptable carrier or diluent. The composition can
optionally further comprise an effective amount of at least one
compound or protein selected from at least one of a detectable
label or reporter, an anti-infective drug, a cardiovascular (CV)
system drug, a central nervous system (CNS) drug, an autonomic
nervous system (ANS) drug, a respiratory tract drug, a
gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid
or electrolyte balance, a hematologic drug, an antineoplastic, an
immunomodulation drug, an ophthalmic, optic or nasal drug, a
topical drug, a nutritional drug, a TNF antagonist, an
antirheumatic, a muscle relaxant, a narcotic, a non-steroid
anti-inflammatory drug (NTHE), 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, or a cytokine
antagonist.
[0035] The present invention further provides an anti-idiotype
antibody or fragment that specifically binds at least one EPO
mimetic hinge core mimetibody of the present invention.
[0036] Also provided is a method for diagnosing or treating a
disease condition in a cell, tissue, organ or animal,
comprising
[0037] (a) contacting or administering a composition comprising an
effective amount of at least one isolated human EPO mimetic hinge
core mimetibody of the invention with, or to, the cell, tissue,
organ or animal. The method can optionally further comprise using
an effective amount of 0.001-50 mg/kilogram of the cells, tissue,
organ or animal. The method can optionally further comprise using
the contacting or the administrating by at least one mode selected
from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
The method can optionally further comprise administering, prior,
concurrently or after the (a) contacting or administering, at least
one composition comprising an effective amount of at least one
compound or protein selected from at least one of a detectable
label or reporter, an anti-infective drug, a cardiovascular (CV)
system drug, a central nervous system (CNS) drug, an autonomic
nervous system (ANS) drug, a respiratory tract drug, a
gastrointestinal (GI) tract drug, a hormonal drug, a drug for fluid
or electrolyte balance, a hematologic drug, an antineoplastic, an
immunomodulation drug, an ophthalmic, optic or nasal drug, a
topical drug, a nutritional drug, 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, or a cytokine
antagonist.
[0038] Also provided is a medical device, comprising at least one
isolated human EPO mimetic hinge core mimetibody of the invention,
wherein the device is suitable to contacting or administering the
at least one EPO mimetic hinge core mimetibody by at least one mode
selected from parenteral, subcutaneous, intramuscular, intravenous,
intrarticular, intrabronchial, intraabdominal, intracapsular,
intracartilaginous, intracavitary, intracelial, intracelebellar,
intracerebroventricular, intracolic, intracervical, intragastric,
intrahepatic, intramyocardial, intraosteal, intrapelvic,
intrapericardiac, intraperitoneal, intrapleural, intraprostatic,
intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,
intrasynovial, intrathoracic, intrauterine, intravesical, bolus,
vaginal, rectal, buccal, sublingual, intranasal, or
transdermal.
[0039] Also provided is an article of manufacture for human
pharmaceutical or diagnostic use, comprising packaging material and
a container comprising a solution or a lyophilized form of at least
one isolated human EPO mimetic hinge core mimetibody of the present
invention. The article of manufacture can optionally comprise
having the container as a component of a parenteral, subcutaneous,
intramuscular, intravenous, intrarticular, intrabronchial,
intraabdominal, intracapsular, intracartilaginous, intracavitary,
intracelial, intracelebellar, intracerebroventricular, intracolic,
intracervical, intragastric, intrahepatic, intramyocardial,
intraosteal, intrapelvic, intrapericardiac, intraperitoneal,
intrapleural, intraprostatic, intrapulmonary, intrarectal,
intrarenal, intraretinal, intraspinal, intrasynovial,
intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal,
buccal, sublingual, intranasal, or transdermal delivery device or
system.
[0040] Also provided is a method for producing at least one
isolated human EPO mimetic hinge core mimetibody of the present
invention, comprising providing a host cell or transgenic animal or
transgenic plant or plant cell capable of expressing in recoverable
amounts the EPO mimetic hinge core mimetibody. Further provided in
the present invention is at least one EPO mimetic hinge core
mimetibody produced by the above method.
[0041] The present invention also provides at least one method for
expressing at least one EPO mimetic hinge core mimetibody, or
anti-idiotype antibody, in a host cell, comprising culturing a host
cell as described herein under conditions wherein at least one EPO
mimetic hinge core mimetibody is expressed in detectable and/or
recoverable amounts.
[0042] The present invention further provides any invention
described herein.
DESCRIPTION OF THE FIGURES
[0043] FIGS. 1-42 (SEQ ID NOS:31-72) show examples of heavy/light
chain variable/constant region sequences, frameworks/subdomains and
substitutions, portions of which can be used in Ig derived proteins
of the present invention, as taught herein. Framework, CDR and
hinge regions are labeled in boxes. Sequence residues are numbered
for each amino acid position. A list of amino acid substitutions or
gaps (denoted by a "-") observed at each position in the aligned
sequences are shown below each sequence residue.
[0044] FIG. 1 depicts Vh1 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:31).
[0045] FIG. 2 depicts Vh2 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:32).
[0046] FIG. 3 depicts Vh3a heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:33).
[0047] FIG. 4 depicts Vh3b heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:34).
[0048] FIG. 5 depicts Vh3c heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:35).
[0049] FIG. 6 depicts Vh4 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:36).
[0050] FIG. 7 depicts Vh5 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:37).
[0051] FIG. 8 depicts Vh6 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:38).
[0052] FIG. 9 depicts Vh7 heavy chain variable region sequences,
frameworks and substitutions (SEQ ID NO:39).
[0053] FIG. 10 depicts .kappa.1.sub.--4 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:40).
[0054] FIG. 11 depicts .kappa.2 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:41).
[0055] FIG. 12 depicts .kappa.3 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:42).
[0056] FIG. 13 depicts .kappa.5 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:43).
[0057] FIG. 14 depicts .kappa.New1 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:44).
[0058] FIG. 15 depicts .kappa.New2 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:45).
[0059] FIG. 16 depicts .kappa.New3 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:46).
[0060] FIG. 17 depicts .lamda.1a light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:47).
[0061] FIG. 18 depicts .lamda.1b light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:48).
[0062] FIG. 19 depicts .lamda.2 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:49).
[0063] FIG. 20 depicts .lamda.3a light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:50).
[0064] FIG. 21 depicts .lamda.3b light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:51).
[0065] FIG. 22 depicts .lamda.3c light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:52).
[0066] FIG. 23 depicts .lamda.3e light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:53).
[0067] FIG. 24 depicts .lamda.4a light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:54).
[0068] FIG. 25 depicts .lamda.4b light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:55).
[0069] FIG. 26 depicts .lamda.5 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:56).
[0070] FIG. 27 depicts .lamda.6 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:57).
[0071] FIG. 28 depicts .lamda.7 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:58).
[0072] FIG. 29 depicts .lamda.8 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:59).
[0073] FIG. 30 depicts .lamda.9 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:60).
[0074] FIG. 31 depicts .lamda.10 light chain variable region
sequences, frameworks and substitutions (SEQ ID NO:61).
[0075] FIG. 32 depicts IgA1 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:62).
[0076] FIG. 33 depicts IgA2 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:63).
[0077] FIG. 34 depicts IgD heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:64).
[0078] FIG. 35 depicts IgE heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:65).
[0079] FIG. 36 depicts IgG1 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:66).
[0080] FIG. 37 depicts IgG2 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:67).
[0081] FIG. 38 depicts IgG3 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:68).
[0082] FIG. 39 depicts IgG4 heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:69).
[0083] FIG. 40 depicts IgM heavy chain constant region sequences,
subdomains and substitutions (SEQ ID NO:70).
[0084] FIG. 41 depicts Ig.kappa.c light chain constant region
sequences and substitutions (SEQ ID NO:71).
[0085] FIG. 42 depicts Ig.lamda.c light chain constant region
sequences and substitutions (SEQ ID NO:72).
DESCRIPTION OF THE INVENTION
[0086] The present invention provides isolated, recombinant and/or
synthetic mimetibodies or specified portions or variants, as well
as compositions and encoding nucleic acid molecules comprising at
least one polynucleotide encoding at least one EPO mimetic hinge
core mimetibody. Such mimetibodies or specified portions or
variants of the present invention comprise specific EPO mimetic
hinge core mimetibody sequences, domains, fragments and specified
variants thereof, and methods of making and using said nucleic
acids and mimetibodies or specified portions or variants, including
therapeutic compositions, methods and devices.
[0087] The present invention also provides at least one isolated
EPO mimetic hinge core mimetibody or specified portion or variant
as described herein and/or as known in the art. The EPO mimetic
hinge core mimetibody can optionally comprise at least one CH3
region directly linked with at least one CH2 region directly linked
with at least one hinge region or fragment thereof (H), directly
linked with an optional linker sequence (L), directly linked to at
least one therapeutic peptide (P), optionally further directly
linked with at least a portion of at least one variable (V)
antibody sequence.
[0088] In a preferred embodiment an EPO mimetic hinge core
mimetibody comprises formula (I):
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),
where V is at least one portion of an N-terminus of an
immunoglobulin variable region, P is at least one bioactive
peptide, L is polypeptide that provides structural flexibility by
allowing the mimetibody to have alternative orientations and
binding properties, H is at least a portion of an immunoglobulin
variable hinge region, CH2 is at least a portion of an
immunoglobulin CH2 constant region, CH3 is at least a portion of an
immunoglobulin CH3 constant region, m, n, o, p, q, r, and s can be
independently an integer between 0, 1 or 2 and 10, mimicking
different types of immunoglobulin molecules, e.g., but not limited
to IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, and the like, or
combination thereof. The monomer where m=1 can be linked to other
monomers by association or covalent linkage, such as, but not
limited to, a Cys-Cys disulfide bond or other immunoglobulin
sequence. EPO mimetic hinge core mimetibody of the present
invention mimics an antibody structure with its inherent properties
and functions, while providing a therapeutic peptide and its
inherent or acquired in vitro, in vivo or in situ properties or
activities. The various portions of the antibody and therapeutic
peptide portions of at least one EPO mimetic hinge core mimetibody
of the present invention can vary as described herein in
combination with what is known in the art.
[0089] As used herein, a "EPO mimetic hinge core mimetibody," "EPO
mimetic hinge core mimetibody portion," or "EPO mimetic hinge core
mimetibody fragment" and/or "EPO mimetic hinge core mimetibody
variant" and the like mimics, has or simulates at least one ligand
binding or at least one biological activity of at least one
protein, such as ligand binding or activity in vitro, in situ
and/or preferably in vivo, such as but not limited to at least one
of SEQ ID NOS:1-30. For example, a suitable EPO mimetic hinge core
mimetibody, specified portion or variant of the present invention
can bind at least one protein ligand and includes at least one
protein ligand, receptor, soluble receptor, and the like. A
suitable EPO mimetic hinge core mimetibody, specified portion, or
variant can also modulate, increase, modify, activate, at least one
protein receptor signaling or other measurable or detectable
activity.
[0090] Mimetibodies useful in the methods and compositions of the
present invention are characterized by suitable affinity binding to
protein ligands or receptors and optionally and preferably having
low toxicity. In particular, an EPO mimetic hinge core mimetibody,
where the individual components, such as the portion of variable
region, constant region (without a CH1 portion) and framework, or
any portion thereof (e.g., a portion of the J, D or V regions of
the variable heavy or light chain; at least a portion of at least
one hinge region, the constant heavy chain or light chain, and the
like) individually and/or collectively optionally and preferably
possess low immunogenicity, is useful in the present invention. The
mimetibodies that can be used in the invention are optionally
characterized by their ability to treat patients for extended
periods with good to excellent alleviation of symptoms and low
toxicity. Low immunogenicity and/or high affinity, as well as other
undefined properties, may contribute to the therapeutic results
achieved. "Low immunogenicity" is defined herein as raising
significant HAMA, HACA or HAHA responses in less than about 75%, or
preferably less than about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9,
8, 7, 6, 5, 4, 3, 2, and/or 1% of the patients treated and/or
raising low titers in the patient treated (less than about 300,
preferably less than about 100 measured with a double antigen
enzyme immunoassay) (see, e.g., Elliott et al., Lancet
344:1125-1127 (1994)).
Utility
[0091] The isolated nucleic acids of the present invention can be
used for production of at least one EPO mimetic hinge core
mimetibody, fragment or specified variant thereof, which can be
used to effect in an cell, tissue, organ or animal (including
mammals and humans), to modulate, treat, alleviate, help prevent
the incidence of, or reduce the symptoms of, at least one protein
related condition, selected from, but not limited to, at least one
of an immune disorder or disease, a cardiovascular disorder or
disease, an infectious, malignant, and/or neurologic disorder or
disease, a(n) anemia; a(n) immune/autoimmune; and/or a(n)
cancer/infectious, as well as other known or specified protein
related conditions.
[0092] Such a method can comprise administering an effective amount
of a composition or a pharmaceutical composition comprising at
least one EPO mimetic hinge core mimetibody or specified portion or
variant to a cell, tissue, organ, animal or patient in need of such
modulation, treatment, alleviation, prevention, or reduction in
symptoms, effects or mechanisms. The effective amount can comprise
an amount of about 0.0001 to 500 mg/kg per single or multiple
administration, or to achieve a serum concentration of 0.0001-5000
.mu.g/ml serum concentration per single or multiple administration,
or any effective range or value therein, as done and determined
using known methods, as described herein or known in the relevant
arts.
CITATIONS
[0093] All publications or patents cited herein are entirely
incorporated herein by reference as they show the state of the art
at the time of the present invention and/or to provide description
and enablement of the present invention. Publications refer to any
scientific or patent publications, or any other information
available in any media format, including all recorded, electronic
or printed formats. The following references are entirely
incorporated herein by reference: Ausubel, et al., ed., Current
Protocols in Molecular Biology, John Wiley & Sons, Inc., NY,
N.Y. (1987-2003); Sambrook, et al., Molecular Cloning: A Laboratory
Manual, 2.sup.nd Edition, Cold Spring Harbor, N.Y. (1989); Harlow
and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, N.Y.
(1989); Colligan, et al., eds., Current Protocols in Immunology,
John Wiley & Sons, Inc., NY (1994-2003); Colligan et al.,
Current Protocols in Protein Science, John Wiley & Sons, NY,
N.Y., (1997-2003).
Mimetibodies of the Present Invention
[0094] The EPO mimetic hinge core mimetibody can optionally
comprise at least one CH3 region directly linked with at least one
CH2 region directly linked with at least one portion of at least
one hinge region fragment (H) such as comprising at least one core
hinge region, directly linked with an optional linker sequence (L),
directly linked to at least one therapeutic peptide (P), optionally
further directly linked with at least a portion of at least one
variable antibody sequence (V). In a preferred embodiment a pair of
a CH3-CH2-H-L-V, the pair linked by association or covalent linkage
Thus, an EPO mimetic hinge core mimetibody of the present invention
mimics an antibody structure with its inherent properties and
functions, while providing a therapeutic peptide and its inherent
or acquired in vitro, in vivo or in situ properties or activities.
The various portions of the antibody and therapeutic peptide
portions of at least one EPO mimetic hinge core mimetibody of the
present invention can vary as described herein in combination with
what is known in the art.
[0095] Mimetibodies of the present invention thus provide at least
one suitable property as compared to known proteins, such as, but
not limited to, at least one of increased half-life, increased
activity, more specific activity, increased avidity, increased or
decrease off rate, a selected or more suitable subset of
activities, less immunogenicity, increased quality or duration of
at least one desired therapeutic effect, less side effects, and the
like.
[0096] Fragments of mimetibodies according to Formula (I) can be
produced by enzymatic cleavage, synthetic or recombinant
techniques, as known in the art and/or as described herein.
Mimetibodies can also be produced in a variety of truncated forms
using antibody genes in which one or more stop codons have been
introduced upstream of the natural stop site. The various portions
of mimetibodies can be joined together chemically by conventional
techniques, or can be prepared as a contiguous protein using
genetic engineering techniques. For example, a nucleic acid
encoding at least one of the constant regions of a human antibody
chain can be expressed to produce a contiguous protein for use in
mimetibodies of the present invention. See, e.g., Ladner et al.,
U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science, 242:
423-426 (1988), regarding single chain antibodies.
[0097] As used herein, the term "human mimetibody" refers to an
antibody in which substantially every part of the protein (e.g.,
EPO mimetic peptide, framework, C.sub.L, C.sub.H domains (e.g.,
C.sub.H2, C.sub.H3), hinge, (V.sub.L, V.sub.H)) is expected to be
substantially non-immunogenic in humans with only minor sequence
changes or variations. Such changes or variations optionally and
preferably retain or reduce the immunogenicity in humans relative
to non-modified human antibodies, or mimetibodies of the present
invention. Thus, a human antibody and corresponding EPO mimetic
hinge core mimetibody of the present invention is distinct from a
chimeric or humanized antibody. It is pointed out that a human
antibody and EPO mimetic hinge core mimetibody can be produced by a
non-human animal or cell that is capable of expressing human
immunoglobulins (e.g., heavy chain and/or light chain) genes.
[0098] Human mimetibodies that are specific for at least one
protein ligand or receptor thereof can be designed against an
appropriate ligand, such as isolated and/or EPO protein receptor or
ligand, or a portion thereof (including synthetic molecules, such
as synthetic peptides). Preparation of such mimetibodies are
performed using known techniques to identify and characterize
ligand binding regions or sequences of at least one protein or
portion thereof.
[0099] In a preferred embodiment, at least one EPO mimetic hinge
core mimetibody or specified portion or variant of the present
invention is produced by at least one cell line, mixed cell line,
immortalized cell or clonal population of immortalized and/or
cultured cells. Immortalized protein producing cells can be
produced using suitable methods. Preferably, the at least one EPO
mimetic hinge core mimetibody or specified portion or variant is
generated by providing nucleic acid or vectors comprising DNA
derived or having a substantially similar sequence to, at least one
human immunoglobulin locus that is functionally rearranged, or
which can undergo functional rearrangement, and which further
comprises a mimetibody structure as described herein, e.g., but not
limited to Formula (I), wherein portions of C- and N-terminal
variable regions can be used for V, hinge regions for H, CH2 for
CH2 and CH3 for CH3, as known in the art.
[0100] The term "functionally rearranged," as used herein refers to
a segment of nucleic acid from an immunoglobulin locus that has
undergone V(D)J recombination, thereby producing an immunoglobulin
gene that encodes an immunoglobulin chain (e.g., heavy chain), or
any portion thereof. A functionally rearranged immunoglobulin gene
can be directly or indirectly identified using suitable methods,
such as, for example, nucleotide sequencing, hybridization (e.g.,
Southern blotting, Northern blotting) using probes that can anneal
to coding joints between gene segments or enzymatic amplification
of immunoglobulin genes (e.g., polymerase chain reaction) with
primers that can anneal to coding joints between gene segments.
Whether a cell produces an EPO mimetic hinge core mimetibody or
portion or variant comprising a particular variable region or a
variable region comprising a particular sequence (e.g., at least
one P sequence) can also be determined using suitable methods.
[0101] Mimetibodies, specified portions and variants of the present
invention can also be prepared using at least one EPO mimetic hinge
core mimetibody or specified portion or variant encoding nucleic
acid to provide transgenic animals or mammals, such as goats, cows,
horses, sheep, and the like, that produce such mimetibodies or
specified portions or variants in their milk. Such animals can be
provided using known methods as applied for antibody encoding
sequences. See, e.g., but not limited to, U.S. Pat. Nos. 5,827,690;
5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489,
and the like, each of which is entirely incorporated herein by
reference.
[0102] Mimetibodies, specified portions and variants of the present
invention can additionally be prepared using at least one EPO
mimetic hinge core mimetibody or specified portion or variant
encoding nucleic acid to provide transgenic plants and cultured
plant cells (e.g., but not limited to tobacco and maize) that
produce such mimetibodies, specified portions or variants in the
plant parts or in cells cultured therefrom. As a non-limiting
example, transgenic tobacco leaves expressing recombinant proteins
have been successfully used to provide large amounts of recombinant
proteins, e.g., using an inducible promoter. See, e.g., Cramer et
al., Curr. Top. Microbol. Immunol 240:95-118 (1999) and references
cited therein. Also, transgenic maize or corn have been used to
express mammalian proteins at commercial production levels, with
biological activities equivalent to those produced in other
recombinant systems or purified from natural sources. See, e.g.,
Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references
cited therein. Antibodies have also been produced in large amounts
from transgenic plant seeds including antibody fragments, such as
single chain mimetibodies (scFv's), including tobacco seeds and
potato tubers. See, e.g., Conrad et al., Plant Mol. Biol.
38:101-109 (1998) and references cited therein. Thus, mimetibodies,
specified portions and variants of the present invention can also
be produced using transgenic plants, according to know methods. See
also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108
(October, 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma
et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem.
Soc. Trans. 22:940-944 (1994); and references cited therein. The
above references are entirely incorporated herein by reference.
[0103] The mimetibodies of the invention can bind human protein
ligands with a wide range of affinities (K.sub.D). In a preferred
embodiment, at least one human EPO mimetic hinge core mimetibody of
the present invention can optionally bind at least one protein
ligand with high affinity. For example, at least one EPO mimetic
hinge core mimetibody of the present invention can bind at least
one protein ligand with a K.sub.D equal to or less than about
10.sup.-7 M or, more preferably, with a K.sub.D equal to or less
than about 0.1-9.9 (or any range or value therein).times.10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12, or
10.sup.-13 M, or any range or value therein.
[0104] The affinity or avidity of an EPO mimetic hinge core
mimetibody for at least one protein ligand can be determined
experimentally using any suitable method, e.g., as used for
determining antibody-antigen binding affinity or avidity. (See, for
example, Berzofsky, et al., "Antibody-Antigen Interactions," In
Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York,
N.Y. (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New
York, N.Y. (1992); and methods described herein). The measured
affinity of a particular EPO mimetic hinge core mimetibody-ligand
interaction can vary if measured under different conditions (e.g.,
salt concentration, pH). Thus, measurements of affinity and other
ligand-binding parameters (e.g., K.sub.D, K.sub.a, K.sub.d) are
preferably made with standardized solutions of EPO mimetic hinge
core mimetibody and ligand, and a standardized buffer, such as the
buffer described herein.
Nucleic Acid Molecules
[0105] Using the information provided herein, such as the
nucleotide sequences encoding at least 90-100% of the contiguous
amino acids of at least one of SEQ ID NOS:1-30 as well as at least
one portion of an antibody, wherein the above sequences are
inserted as the P sequence of Formula (I) to provide an EPO mimetic
hinge core mimetibody of the present invention, further comprising
specified fragments, variants or consensus sequences thereof, or a
deposited vector comprising at least one of these sequences, a
nucleic acid molecule of the present invention encoding at least
one EPO mimetic hinge core mimetibody or specified portion or
variant can be obtained using methods described herein or as known
in the art.
[0106] Nucleic acid molecules of the present invention can be in
the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in
the form of DNA, including, but not limited to, cDNA and genomic
DNA obtained by cloning or produced synthetically, or any
combination thereof. The DNA can be triple-stranded,
double-stranded or single-stranded, or any combination thereof. Any
portion of at least one strand of the DNA or RNA can be the coding
strand, also known as the sense strand, or it can be the non-coding
strand, also referred to as the anti-sense strand.
[0107] Isolated nucleic acid molecules of the present invention can
include nucleic acid molecules comprising an open reading frame
(ORF), optionally with one or more introns, nucleic acid molecules
comprising the coding sequence for an EPO mimetic hinge core
mimetibody or specified portion or variant; and nucleic acid
molecules which comprise a nucleotide sequence substantially
different from those described above but which, due to the
degeneracy of the genetic code, still encode at least one EPO
mimetic hinge core mimetibody as described herein and/or as known
in the art. Of course, the genetic code is well known in the art.
Thus, it would be routine for one skilled in the art to generate
such degenerate nucleic acid variants that code for specific EPO
mimetic hinge core mimetibody or specified portion or variants of
the present invention. See, e.g., Ausubel, et al., supra, and such
nucleic acid variants are included in the present invention.
[0108] As indicated herein, nucleic acid molecules of the present
invention which comprise a nucleic acid encoding an EPO mimetic
hinge core mimetibody or specified portion or variant can include,
but are not limited to, those encoding the amino acid sequence of
an EPO mimetic hinge core mimetibody fragment, by itself; the
coding sequence for the entire EPO mimetic hinge core mimetibody or
a portion thereof; the coding sequence for an EPO mimetic hinge
core mimetibody, fragment or portion, as well as additional
sequences, such as the coding sequence of at least one signal
leader or fusion peptide, with or without the aforementioned
additional coding sequences, such as at least one intron, together
with additional, non-coding sequences, including but not limited
to, non-coding 5' and 3' sequences, such as the transcribed,
non-translated sequences that play a role in transcription, mRNA
processing, including splicing and polyadenylation signals (for
example--ribosome binding and stability of mRNA); an additional
coding sequence that codes for additional amino acids, such as
those that provide additional functionalities. Thus, the sequence
encoding an EPO mimetic hinge core mimetibody or specified portion
or variant can be fused to a marker sequence, such as a sequence
encoding a peptide that facilitates purification of the fused EPO
mimetic hinge core mimetibody or specified portion or variant
comprising an EPO mimetic hinge core mimetibody fragment or
portion.
Polynucleotides which Selectively Hybridize to a Polynucleotide as
Described Herein
[0109] The present invention provides isolated nucleic acids that
hybridize under selective hybridization conditions to a
polynucleotide disclosed herein, or others disclosed herein,
including specified variants or portions thereof. Thus, the
polynucleotides of this embodiment can be used for isolating,
detecting, and/or quantifying nucleic acids comprising such
polynucleotides.
[0110] Low or moderate stringency hybridization conditions are
typically, but not exclusively, employed with sequences having a
reduced sequence identity relative to complementary sequences.
Moderate and high stringency conditions can optionally be employed
for sequences of greater identity. Low stringency conditions allow
selective hybridization of sequences having about 40-99% sequence
identity and can be employed to identify orthologous or paralogous
sequences.
[0111] Optionally, polynucleotides of this invention will encode at
least a portion of an EPO mimetic hinge core mimetibody or
specified portion or variant encoded by the polynucleotides
described herein. The polynucleotides of this invention embrace
nucleic acid sequences that can be employed for selective
hybridization to a polynucleotide encoding an EPO mimetic hinge
core mimetibody or specified portion or variant of the present
invention. See, e.g., Ausubel, supra; Colligan, supra, each
entirely incorporated herein by reference.
[0112] Construction of Nucleic Acids
[0113] The isolated nucleic acids of the present invention can be
made using (a) recombinant methods, (b) synthetic techniques, (c)
purification techniques, or combinations thereof, as well-known in
the art.
[0114] The nucleic acids can conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites can be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences can
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the coding sequence--is optionally
a vector, adapter, or linker for cloning and/or expression of a
polynucleotide of the present invention.
[0115] Additional sequences can be added to such cloning and/or
expression sequences to optimize their function in cloning and/or
expression, to aid in isolation of the polynucleotide, or to
improve the introduction of the polynucleotide into a cell. Use of
cloning vectors, expression vectors, adapters, and linkers is well
known in the art. See, e.g., Ausubel, supra; or Sambrook,
supra.
Recombinant Methods for Constructing Nucleic Acids
[0116] The isolated nucleic acid compositions of this invention,
such as RNA, cDNA, genomic DNA, or any combination thereof, can be
obtained from biological sources using any number of cloning
methodologies known to those of skill in the art. In some
embodiments, oligonucleotide probes that selectively hybridize,
under suitable stringency conditions, to the polynucleotides of the
present invention are used to identify the desired sequence in a
cDNA or genomic DNA library. The isolation of RNA, and construction
of cDNA and genomic libraries, is well known to those of ordinary
skill in the art. (See, e.g., Ausubel, supra; or Sambrook,
supra).
Synthetic Methods for Constructing Nucleic Acids
[0117] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by known methods (see,
e.g., Ausubel, et al., supra). Chemical synthesis generally
produces a single-stranded oligonucleotide, which can be converted
into double-stranded DNA by hybridization with a complementary
sequence, or by polymerization with a DNA polymerase using the
single strand as a template. One of skill in the art will recognize
that while chemical synthesis of DNA can be limited to sequences of
about 100 or more bases, longer sequences can be obtained by the
ligation of shorter sequences.
Recombinant Expression Cassettes
[0118] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence of the present invention, for
example a cDNA or a genomic sequence encoding an EPO mimetic hinge
core mimetibody or specified portion or variant of the present
invention, can be used to construct a recombinant expression
cassette that can be introduced into at least one desired host
cell. A recombinant expression cassette will typically comprise a
polynucleotide of the present invention operably linked to
transcriptional initiation regulatory sequences that will direct
the transcription of the polynucleotide in the intended host cell.
Both heterologous and non-heterologous (i.e., endogenous) promoters
can be employed to direct expression of the nucleic acids of the
present invention.
[0119] In some embodiments, isolated nucleic acids that serve as
promoter, enhancer, or other elements can be introduced in the
appropriate position (upstream, downstream or in intron) of a
non-heterologous form of a polynucleotide of the present invention
so as to up or down regulate expression of a polynucleotide of the
present invention. For example, endogenous promoters can be altered
in vivo or in vitro by mutation, deletion and/or substitution, as
known in the art. A polynucleotide of the present invention can be
expressed in either sense or anti-sense orientation as desired. It
will be appreciated that control of gene expression in either sense
or anti-sense orientation can have a direct impact on the
observable characteristics. Another method of suppression is sense
suppression. Introduction of nucleic acid configured in the sense
orientation has been shown to be an effective means by which to
block the transcription of target genes.
Vectors and Host Cells
[0120] The present invention also relates to vectors that include
isolated nucleic acid molecules of the present invention, host
cells that are genetically engineered with the recombinant vectors,
and the production of at least one EPO mimetic hinge core
mimetibody or specified portion or variant by recombinant
techniques, as is well known in the art. See, e.g., Sambrook, et
al., supra; Ausubel, et al., supra, each entirely incorporated
herein by reference.
[0121] The polynucleotides can optionally be joined to a vector
containing a selectable marker for propagation in a host.
Generally, a plasmid vector is introduced into a cell using
suitable known methods, such as electroporation and the like, other
known methods include the use of the vector as a precipitate, such
as a calcium phosphate precipitate, or in a complex with a charged
lipid. If the vector is a virus, it can be packaged in vitro using
an appropriate packaging cell line and then transduced into host
cells.
[0122] The DNA insert should be operatively linked to an
appropriate promoter. The expression constructs will further
contain sites optionally for at least one of transcription
initiation, termination and, in the transcribed region, a ribosome
binding site for translation. The coding portion of the mature
transcripts expressed by the constructs will preferably include a
translation initiating at the beginning and a termination codon
(e.g., UAA, UGA or UAG) appropriately positioned at the end of the
mRNA to be translated, with UAA and UAG preferred for mammalian or
eukaryotic cell expression.
[0123] Expression vectors will preferably but optionally include at
least one selectable marker. Such markers include, e.g., but not
limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S.
Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636;
5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or
glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359;
5,827,739) resistance for eukaryotic cell culture, and tetracycline
or ampicillin resistance genes for culturing in E. coli and other
bacteria or prokaryotics (the above patents are entirely
incorporated hereby by reference). Appropriate culture mediums and
conditions for the above-described host cells are known in the art.
Suitable vectors will be readily apparent to the skilled artisan.
Introduction of a vector construct into a host cell can be effected
by calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection or other known methods.
Such methods are described in the art, such as Sambrook, supra,
Chapters 1-4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15,
16.
[0124] At least one EPO mimetic hinge core mimetibody or specified
portion or variant of the present invention can be expressed in a
modified form, such as a fusion protein, and can include not only
secretion signals, but also additional heterologous functional
regions. For instance, a region of additional amino acids,
particularly charged amino acids, can be added to the N-terminus of
an EPO mimetic hinge core mimetibody or specified portion or
variant to improve stability and persistence in the host cell,
during purification, or during subsequent handling and storage.
Also, peptide moieties can be added to an EPO mimetic hinge core
mimetibody or specified portion or variant of the present invention
to facilitate purification. Such regions can be removed prior to
final preparation of an EPO mimetic hinge core mimetibody or at
least one fragment thereof. Such methods are described in many
standard laboratory manuals, such as Sambrook, supra, Chapters
17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and
18.
[0125] Those of ordinary skill in the art are knowledgeable in the
numerous expression systems available for expression of a nucleic
acid encoding a protein of the present invention.
[0126] Illustrative of cell cultures useful for the production of
the mimetibodies, specified portions or variants thereof, are
mammalian cells. Mammalian cell systems often will be in the form
of monolayers of cells although mammalian cell suspensions or
bioreactors can also be used. A number of suitable host cell lines
capable of expressing intact glycosylated proteins have been
developed in the art, and include the COS-1 (e.g., ATCC CRL 1650),
COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO
(e.g., ATCC CRL 1610, DG-44) and BSC-1 (e.g., ATCC CRL-26) cell
lines, hepG2 cells, P3X63Ag8.653, SP2/0-Ag14, 293 cells, HeLa cells
and the like, which are readily available from, for example,
American Type Culture Collection, Manassas, Va. Preferred host
cells include cells of lymphoid origin such as myeloma and lymphoma
cells. Particularly preferred host cells are P3X63Ag8.653 cells
(ATCC Accession Number CRL-1580) and SP2/0-Ag14 cells (ATCC
Accession Number CRL-1851).
[0127] Expression vectors for these cells can include one or more
of the following expression control sequences, such as, but not
limited to an origin of replication; a promoter (e.g., late or
early SV40 promoters, the CMV promoter (e.g., U.S. Pat. Nos.
5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate
kinase) promoter, an EF-1 alpha promoter (e.g, U.S. Pat. No.
5,266,491), at least one human immunoglobulin promoter; an
enhancer, and/or processing information sites, such as ribosome
binding sites, RNA splice sites, polyadenylation sites (e.g., an
SV40 large T Ag poly A addition site), and transcriptional
terminator sequences. See, e.g., Ausubel et al., supra; Sambrook,
et al., supra. Other cells useful for production of nucleic acids
or proteins of the present invention are known and/or available,
for instance, from the American Type Culture Collection Catalogue
of Cell Lines and Hybridomas (www.atcc.org) or other known or
commercial sources.
[0128] When eukaryotic host cells are employed, polyadenylation or
transcription terminator sequences are typically incorporated into
the vector. An example of a terminator sequence is the
polyadenylation sequence from the bovine growth hormone gene.
Sequences for accurate splicing of the transcript can also be
included. An example of a splicing sequence is the VP1 intron from
SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally,
gene sequences to control replication in the host cell can be
incorporated into the vector, as known in the art.
Purification of an EPO Mimetic Hinge Core Mimetibody or Specified
Portion or Variant Thereof
[0129] An EPO mimetic hinge core mimetibody or specified portion or
variant can be recovered and purified from recombinant cell
cultures by well-known methods including, but not limited to,
protein A purification, ammonium sulfate or ethanol precipitation,
acid extraction, anion or cation exchange chromatography,
phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography, hydroxylapatite
chromatography and lectin chromatography. High performance liquid
chromatography ("HPLC") can also be employed for purification. See,
e.g., Colligan, Current Protocols in Immunology, or Current
Protocols in Protein Science, John Wiley & Sons, NY, N.Y.,
(1997-2003), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely
incorporated herein by reference.
[0130] Mimetibodies or specified portions or variants of the
present invention include naturally purified products, products of
chemical synthetic procedures, and products produced by recombinant
techniques from a eukaryotic host, including, for example, yeast,
higher plant, insect and mammalian cells. Depending upon the host
employed in a recombinant production procedure, the EPO mimetic
hinge core mimetibody or specified portion or variant of the
present invention can be glycosylated or can be non-glycosylated,
with glycosylated preferred. Such methods are described in many
standard laboratory manuals, such as Sambrook, supra, Sections
17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20,
Colligan, Protein Science, supra, Chapters 12-14, all entirely
incorporated herein by reference.
Mimetibodies, Specified Fragments and/or Variants
[0131] The isolated mimetibodies of the present invention comprise
an EPO mimetic hinge core mimetibody or specified portion or
variant encoded by any one of the polynucleotides of the present
invention as discussed more fully herein, or any isolated or
prepared EPO mimetic hinge core mimetibody or specified portion or
variant thereof.
[0132] Preferably, the EPO mimetic hinge core mimetibody or
ligand-binding portion or variant binds at least one EPO protein
ligand or receptor, and, thereby provides at least one EPO
biological activity of the corresponding protein or a fragment
thereof. Different therapeutically or diagnostically significant
proteins are well known in the art and suitable assays or
biological activities of such proteins are also well known in the
art.
[0133] Non-limiting examples of suitable EPO mimetic peptides for
this invention appear in Table 1 below. These peptides can be
prepared by methods disclosed and/or known in the art. Single
letter amino acid abbreviations are used in most cases. The X in
these sequences (and throughout this specification, unless
specified otherwise in a particular instance) means that any of the
20 naturally occurring or known amino acid residues or know
derivatives thereof may be present, or any know modified amino acid
thereof. Any of these peptides may be linked in tandem (i.e.,
sequentially), with or without linkers, and a few tandemlinked
examples are provided in the table. Linkers are listed as "A" and
may be any of the linkers described herein. Tandem repeats and
linkers are shown separated by dashes for clarity. Any peptide
containing a cysteinyl residue may optionally be cross-linked with
another Cys-containing peptide, either or both of which may be
linked to a vehicle. A few crosslinked examples are provided in the
table. Any peptide having more than one Cys residue may form an
intrapeptide disulfide bond, as well; see, for example, EPO-mimetic
peptides in Table 1. A few examples of intrapeptide
disulfide-bonded peptides are specified in the table. Any of these
peptides may be derivatized as described herein, and a few
derivatized examples are provided in the table. For derivatives in
which the carboxyl terminus may be capped with an amino group, the
capping amino group is shown as --NH.sub.2. For derivatives in
which amino acid residues are substituted by moieties other than
amino acid residues, the substitutions are denoted by a .delta.,
which signifies any of the moieties known in the art, e.g., as
described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and
Cuthbertson et al. (1997), J. Med. Chem. 40:2876-82, which are
entirely incorporated by reference. The J substituent and the Z
substituents (Z.sub.5, Z.sub.6, . . . Z.sub.40) are as defined in
U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which are
entirely incorporated herein by reference. For the EPO-mimetic
sequences (Table 1), the substituents X.sub.2 through X.sub.11 and
the integer "n" are as defined in WO 96/40772, which is entirely
incorporated by reference. Residues appearing in boldface are
D-amino acids, but can be optionally L-amino acids. All peptides
are linked through peptide bonds unless otherwise noted.
Abbreviations are listed at the end of this specification. In the
"SEQ ID NO." column, "NR" means that no sequence listing is
required for the given sequence.
TABLE-US-00001 TABLE 1 EPO-mimetic peptide sequences
Sequence/structure SEQ ID NO: YXCXXGPXTWXCXP 1
YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 1
YXCXXGPXTWXCXP-.LAMBDA.-YXCXXGPXTWXCXP 1 ##STR00001## 1
GGTYSCHFGPLTWVCKPQGG 2 GGDYHCRMGPLTWVCKPLGG 3 GGVYACRMGPITWVCSPLGG
4 VGNYMCHFGPITWVCRPGGG 5 GGLYLCRFGPVTWDCGYKGG 6
GGTYSCHFGPLTWVCKPQGG 7 GGTYSCHFGPLTWVCKPQGG-GGTYSCHFGPLTWVCKPQGG 7
GGTYSCHFGPLTWVCKPQGG-.LAMBDA.-GGTYSCHFGPLTWVCKPQGG 7
GGTYSCHFGPLTWVCKPQGGSSK 8
GGTYSCHFGPLTWVCKPQGGSSK-GGTYSCHFGPLTWVCKPQGGSSK 8
GGTYSCHFGPLTWVCKPQGGSSK-.LAMBDA.-GGTYSCHFGPLTWVCKPQGGSSK 8
##STR00002## 8 GGTYSCHFGPLTWVCKPWGGSSK(-.LAMBDA.-biotin) 8
CX.sub.4X.sub.5GPX.sub.6TWX.sub.7C 9 GGTYSCHGPLTWVCKPQGG 10
VGNYMAHMGPITWVCRPGG 11 GGPHHVYACRMGPLTWIC 12 GGTYSCHFGPLTWVCKPQ 13
GGLYACHMGPMTWVCQPLRG 14 TIAQYICYMGPETWECRPSPKA 15 YSCHFGPLTWVCK 16
YCHFGPLTWVC 17 X.sub.3X.sub.4X.sub.5GPX.sub.6TWX.sub.7X.sub.8 18
YX.sub.2X.sub.3X.sub.4X.sub.5GPX.sub.6TWX.sub.7X.sub.8 19
X.sub.1YX.sub.2X.sub.3X.sub.4X.sub.5GPX.sub.6X.sub.7X.sub.8X.sub.9X.sub.10-
X.sub.11 20
X.sub.1YX.sub.2CX.sub.4X.sub.5GPX.sub.6TWX.sub.7CX.sub.9X.sub.10X.sub.11
21 GGLYLCRFGPVTWDCGYKGG 22 GGTYSCHFGPLTWVCKPQGG 23
VGNYMCHFGPITWVCRPGGG 24 GGVYACRMGPITWVCSPLGG 25
TIAQYICYMGPETWECRPSPKA 26 YSCHFGPLTWVCK 27 YCHFGPLTWVC 28
SCHFGPLTWVCK 29
(AX.sub.2).sub.nX.sub.3X.sub.4X.sub.5GPX.sub.6TWX.sub.7X.sub.8
30
[0134] EPO biological activities are well known in the art. See,
e.g., Anagnostou A et al Erythropoietin has a mitogenic and
positive chemotactic effect on endothelial cells. Proceedings of
the National Academy of Science (USA) 87: 5978-82 (1990); Fandrey J
and Jelkman W E Interleukin 1 and tumor necrosis factor-alpha
inhibit erythropoietin production in vitro. Annals of the New York
Academy of Science 628: 250-5 (1991); Geissler K et al Recombinant
human erythropoietin: A multipotential hemopoietic growth factor in
vivo and in vitro. Contrib. Nephrol. 87: 1-10 (1990); Gregory C J
Erythropoietin sensitivity as a differentiation marker in the
hemopoietic system. Studies of three erythropoietic colony
responses in culture. Journal of Cellular Physiology 89: 289-301
(1976); Jelkman W et al Monokines inhibiting erythropoietin
production in human hepatoma cultures and in isolated perfused rat
kidneys. Life Sci. 50: 301-8 (1992); Kimata H et al Human
recombinant erythropoietin directly stimulates B cell
immunoglobulin production and proliferation in serum-free medium.
Clinical and Experimental Immunology 85: 151-6 (1991); Kimata H et
al Erythropoietin enhances immunoglobulin production and
proliferation by human plasma cells in a serum-free medium. Clin.
Immunology Immunopathol. 59: 495-501 (1991); Kimata H et al Effect
of recombinant human erythropoietin on human IgE production in
vitro Clinical and Experimental Immunology 83: 483-7 (1991); Koury
M J and Bondurant M C Erythropoietin retards DNA breakdown and
prevents programmed cell death in erythroid progenitor cells.
Science 248: 378-81 (1990); Lim V S et al Effect of recombinant
human erythropoietin on renal function in humans. Kidney
International 37: 131-6 (1990); Mitjavila M T et al Autocrine
stimulation by erythropoietin and autonomous growth of human
erythroid leukemic cells in vitro. Journal of Clinical
Investigation 88: 789-97 (1991); Andre M et al Performance of an
immunoradiometric assay of erythropoietin and results for specimens
from anemic and polycythemic patients. Clinical Chemistry 38:
758-63 (1992); Hankins W D et al Erythropoietin-dependent and
erythropoietin-producing cell lines. Implications for research and
for leukemia therapy. Annals of the New York Academy of Science
554: 21-8 (1989); Kendall R G T et al Storage and preparation of
samples for erythropoietin radioimmunoassay. Clin. Lab. Haematology
13: 189-96 (1991); Krumvieh D et al Comparison of relevant
biological assays for the determination of biological active
erythropoietin. Dev. Biol. Stand. 69: 15-22 (1988); Ma D D et al
Assessment of an EIA for measuring human serum erythropoietin as
compared with RIA and an in-vitro bioassay. British Journal of
Haematology 80: 431-6 (1992); Noe G et al A sensitive sandwich
ELISA for measuring erythropoietin in human serum British Journal
of Haematology 80: 285-92 (1992); Pauly J U et al Highly specific
and highly sensitive enzyme immunoassays for antibodies to human
interleukin 3 (IL3) and human erythropoietin (EPO) in serum.
Behring Institut Mitteilungen 90: 112-25 (1991); Sakata S and Enoki
Y Improved microbioassay for plasma erythropoietin based on CFU-E
colony formation. Ann Hematology 64: 224-30 (1992); Sanengen T et
al Immunoreactive erythropoietin and erythropoiesis stimulating
factor(s) in plasma from hypertransfused neonatal and adult mice.
Studies with a radioimmunoassay and a cell culture assay for
erythropoietin. Acta Physiol. Scand. 135: 11-6 (1989); Widness J A
et al A sensitive and specific erythropoietin immunoprecipitation
assay: application to pharmacokinetic studies. Journal of Lab.
Clin. Med. 119: 285-94 (1992); for further information see also
individual cell lines used in individual bioassays. Each of the
above references are entirely incorporated herein by reference. EPO
can be assayed by employing cell lines such as HCD57, NFS-60, TF-1
and UT-7, which respond to the factor. EPO activity can be assessed
also in a Colony formation assay by determining the number of CFU-E
from bone marrow cells. An alternative and entirely different
detection method is RT-PCR quantitation of cytokines.
[0135] An EPO mimetic hinge core mimetibody, or specified portion
or variant thereof, that partially or preferably substantially
provides at least one biological activity of at least one protein
or fragment, can bind the protein or fragment ligand and thereby
provide at least one activity that is otherwise mediated through
the binding of protein to at least one protein ligand or receptor
or through other protein-dependent or mediated mechanisms. As used
herein, the term "EPO mimetic hinge core mimetibody activity"
refers to an EPO mimetic hinge core mimetibody that can modulate or
cause at least one protein-dependent activity by about 20-10,000%,
preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000,
2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000% or more depending
on the assay.
[0136] The capacity of an EPO mimetic hinge core mimetibody or
specified portion or variant to provide at least one
protein-dependent activity is preferably assessed by at least one
suitable protein biological assay, as described herein and/or as
known in the art. A human EPO mimetic hinge core mimetibody or
specified portion or variant of the invention can be similar to any
class (IgG, IgA, IgM, etc.) or isotype and can comprise at least a
portion of a kappa or lambda light chain. In one embodiment, the
human EPO mimetic hinge core mimetibody or specified portion or
variant comprises an IgG heavy chain variable fragment, hinge
region, CH2 and CH3, for example, at least one of isotypes, IgG1,
IgG2, IgG3 or IgG4.
[0137] At least one EPO mimetic hinge core mimetibody or specified
portion or variant of the invention binds at least one specified
ligand specific to at least one protein, subunit, fragment, portion
or any combination thereof. The at least one EPO mimetic peptide of
at least one EPO mimetic hinge core mimetibody, specified portion
or variant of the present invention can optionally bind at least
one specified ligand epitope of the ligand. The binding epitope can
comprise any combination of at least one amino acid sequence of at
least 1-3 amino acids to the entire specified portion of contiguous
amino acids of the sequences selected from the group consisting of
a protein ligand, such as an EPO receptors or portion thereof.
[0138] Such mimetibodies can be prepared by joining together the
various portions of Formula (I) of the EPO mimetic hinge core
mimetibody using known techniques, by preparing and expressing at
least one (i.e., one or more) nucleic acid molecules that encode
the EPO mimetic hinge core mimetibody, using known techniques of
recombinant DNA technology or by using any other suitable method,
such as chemical synthesis.
[0139] Mimetibodies that bind to human EPO ligands or receptors and
that comprise at least a one portion defined heavy or light chain
variable region can be prepared using suitable methods, such as
phage display (Katsube, Y., et al., Int J Mol. Med, 1(5):863-868
(1998)) or methods that employ transgenic animals, as known in the
art and/or as described herein. The EPO mimetic hinge core
mimetibody, specified portion or variant can be expressed using the
encoding nucleic acid or portion thereof in a suitable host
cell.
[0140] Preferably, such mimetibodies or ligand-binding fragments
thereof can bind human EPO ligands or receptors with high affinity
(e.g., K.sub.D less than or equal to about 10.sup.-7 M). Amino acid
sequences that are substantially the same as the sequences
described herein include sequences comprising conservative amino
acid substitutions, as well as amino acid deletions and/or
insertions. A conservative amino acid substitution refers to the
replacement of a first amino acid by a second amino acid that has
chemical and/or physical properties (e.g, charge, structure,
polarity, hydrophobicity/hydrophilicity) that are similar to those
of the first amino acid. Conservative substitutions include
replacement of one amino acid by another within the following
groups: lysine (K), arginine (R) and histidine (H); aspartate (D)
and glutamate (E); asparagine (N), glutamine (Q), serine (S),
threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine
(V), leucine (L), isoleucine (I), proline (P), phenylalanine (F),
tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W
and Y; C, S and T.
Amino Acid Codes
[0141] The amino acids that make up mimetibodies or specified
portions or variants of the present invention are often
abbreviated. The amino acid designations can be indicated by
designating the amino acid by its single letter code, its three
letter code, name, or three nucleotide codon(s) as is well
understood in the art (see Alberts, B., et al., Molecular Biology
of The Cell, Third Ed., Garland Publishing, Inc., New York, 1994),
as presented in Table
TABLE-US-00002 TABLE 2 SINGLE THREE LETTER LETTER THREE NUCLEOTIDE
CODE CODE NAME CODON(S) A Ala Alanine GCA, GCC, GCG, GCU C Cys
Cysteine UGC, UGU D Asp Aspartic acid GAC, GAU E Glu Glutamic acid
GAA, GAG F Phe Phenylanine UUC, UUU G Gly Glycine GGA, GGC, GGG,
GGU H His Histidine CAC, CAU I Ile Isoleucine AUA, AUC, AUU K Lys
Lysine AAA, AAG L Leu Leucine UUA, UUG, CUA, CUC, CUG, CUU M Met
Methionine AUG N Asn Asparagine AAC, AAU P Pro Proline CCA, CCC,
CCG, CCU Q Gln Glutamine CAA, CAG R Arg Arginine AGA, AGG, CGA,
CGC, CGG, CGU S Ser Serine AGC, AGU, UCA, UCC, UCG, UCU T Thr
Threonine ACA, ACC, ACG, ACU V Val Valine GUA, GUC, GUG, GUU W Trp
Tryptophan UGG Y Tyr Tyrosine UAC, UAU
[0142] An EPO mimetic hinge core mimetibody or specified portion or
variant of the present invention can include one or more amino acid
substitutions, deletions or additions, either from natural
mutations or human manipulation, as specified herein. Such or other
sequences that can be used in the present invention, include, but
are not limited to the following sequences presented in Table 3, as
further described in FIGS. 1-42, with corresponding SEQ ID
NOS31-72.
TABLE-US-00003 TABLE 3 SEQ ID AA REGIONS NO NO FR1 CDR1 FR2 CDR2
FR3 CDR3 FR4 31 Heavy chain Vh1 125 1-31 32 33-46 47 48-79 80
81-125 32 variable Vh2 124 1-30 31 32-45 46 47-78 79 80-124 33
region Vh3a 100 1-31 32 33-46 47 48-79 80 81-100 34 Vh3b 102 1-30
31 32-45 46 47-78 79 80-102 35 Vh3c 101 1-30 31 32-45 46 47-79 80
81-101 36 Vh4 108 1-33 34 35-48 49 50-81 82 83-108 37 Vh5 132 1-31
32 33-46 47 48-79 80 81-132 38 Vh6 125 1-30 31 32-45 46 47-78 79
80-125 39 Vh7 91 1-30 31 32-45 46 47-78 79 80-91 40 Light chain
.kappa.1-4 93 1-24 25 26-40 41 42-73 74 75-93 41 variable .kappa.2
92 1-23 24 25-39 40 41-72 73 74-92 42 region .kappa.3 91 1-23 24
25-39 40 41-72 73 74-91 43 .kappa.5 85 1-23 24 25-39 40 41-72 73
74-85 44 .kappa. new1 79 1-17 18 19-33 34 35-66 67 68-79 45 .kappa.
new2 77 1-15 16 17-31 32 33-64 65 66-77 46 .kappa. new3 95 1-24 25
26-40 41 42-73 74 75-95 47 .lamda.1a 98 1-22 23 24-38 39 40-71 72
73-98 48 .lamda.1b 99 1-23 24 25-39 40 41-72 73 74-99 49 .lamda.2
99 1-22 23 24-38 39 40-71 72 73-99 50 .lamda.3a 107 1-22 23 24-38
39 40-71 72 73-107 51 .lamda.3b 93 1-22 23 24-39 40 41-72 73 74-93
52 .lamda.3c 98 1-22 23 24-38 39 40-71 72 73-98 53 .lamda.3e 98
1-22 23 24-38 39 40-71 72 73-98 54 .lamda.4a 94 1-22 23 24-38 39
40-71 72 73-94 55 .lamda.4b 95 1-22 23 24-38 39 40-71 72 73-95 56
.lamda.5 88 1-22 23 24-39 40 41-74 75 76-88 57 .lamda.6 101 1-22 23
24-38 39 40-73 74 75-101 58 .lamda.7 89 1-22 23 24-38 39 40-71 72
73-89 59 .lamda.8 89 1-22 23 24-38 39 40-71 72 73-89 60 .lamda.9 91
1-22 23 24-38 39 40-79 80 81-91 61 .lamda.10 87 1-22 23 24-38 39
40-71 72 73-87 SEQ ID AA REGIONS NO NO CH1 hinge1 hinge2 hinge3
hinge4 CH2 CH3 62 Heavy chain IgA1 354 1-102 103-121 122-222
223-354 63 constant IgA2 340 1-102 103-108 109-209 210-340 64
region IgD 384 1-101 102-135 136-159 160-267 268-384 65 IgE 497
1-103 104-210 211-318 66 IgG1 339 1-98 99-113 114-223 224-339 67
IgG2 326 1-98 99-110 111-219 220-326 68 IgG3 377 1-98 99-115
116-130 131-145 146-160 161-270 271-377 69 IgG4 327 1-98 99-110
111-220 221-327 70 IgM 476 1-104 105-217 218-323 71 Light chain
Ig.kappa.c 107 72 constant Ig.lamda.c 107 region
[0143] Of course, the number of amino acid substitutions a skilled
artisan would make depends on many factors, including those
described above. Generally speaking, the number of amino acid
substitutions, insertions or deletions for at least one of an EPO
mimetic hinge core mimetibody will not be more than 40, 30, 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino
acids, such as 1-30 or any range or value therein, as specified
herein.
[0144] The following description of the components of an EPO hinge
core mimetibody of the present invention is based on the use of the
formula I of the present invention,
((V(m)-P(n)-L(o)-H(p)-CH2(q)-CH3(r))(s),
[0145] where V is at least one portion of an N-terminus of an
immunoglobulin variable region, P is at least one bioactive
peptide, L is at least one linker polypeptide H is at least one
portion of at least one immunoglobulin hinge region, CH2 is at
least a portion of an immunoglobulin CH2 constant region, CH3 is at
least a portion of an immunoglobulin CH3 constant region, m, n, o,
p, q, r and s are independently an integer between 0, 1 or 2 and
10, mimicking different types of immunoglobulin molecules, e.g.,
but not limited to IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, IgE, and
the like, or any subclass thereof, or any combination thereof.
[0146] In hinge core mimetibodies of the present invention, the
optional N-terminal V portion can comprise 1-20 amino acids of at
least one heavy chain variable framework 1 (FR1) region, e.g., as
presented in FIGS. 1-9 (SEQ ID NOS:31-39) or at least one LC
variable region, e.g., as presented in FIGS. 10-31 (SEQ ID
NOS:40-61), including substitutions, deletions or insertions as
presented in these Figures, with those of FIGS. 5, 6, and 8
preferred. Also preferred are variable sequences that comprise the
sequence Q-X-Q.
[0147] The P portion can comprise at least one any therapeutic
peptide as known in the art or as described herein, such as, but
not limited to those presented in Table 1, SEQ ID NOS:1-30, or as
known in the art, or any combination or consensus sequence thereof,
or any fusion protein thereof.
[0148] The optional linker sequence can be any suitable peptide
linker as known in the art. Preferred sequence include any
combination of G and S, e.g., X1-X2-X3-X4-Xn, where X can be G or
S, and n can be 5-30. Non-limiting examples include, GS, GGGS,
GSGGGS, GSGGGSGG, and the like.
[0149] In the present invention, the CH1 portion is not used and a
variable number of amino acids from the N-terminus of the hinge
region are deleted, e.g., as referenced to FIGS. 1-42 and Table 3.
The variable number of amino acids used for the hinge core portion
of a mimetibody of the present invention include, but are not
limited to, deletion of any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, or 1-3, 2-5, 2-7,
2-8, 3-9, 4-10, 5-9, 5-10, 5-15, 10-20, 2-30, 20-40, 10-50, or any
range or value therein, of the N-terminal amino acids of at least
one hinge region, e.g., as presented in FIGS. 32-40, or Table 3
above, e.g., but not limited to, deletion of any to all of the
amino acids 99-101 to 105-157 of amino acids 99-105, 99-108,
99-111, 99-112, 99-113, 99-114, 99-115, 99-119, 99-125, 99-128,
99-134, 99-140, 99-143, 99-149, 99-155 and 99-158 of FIGS. 32-40,
corresponding to SEQ ID NOS:62-70, including the substitutions,
insertions or deletions described in FIGS. 32-40. In preferred
embodiments, a hinge core regions of the present invention includes
a deletion of the N-terminus of the hinge region to provide a hinge
core region that includes a deletion up to but not including a Cys
residue or up to but not including a sequence Cys-Pro-Xaa-Cys. In
further preferred embodiment, such hinge core sequences used in a
hinge core mimetibody of the present invention include amino acids
109-113 or 112-113 of FIG. 36 (SEQ ID NO:66) (IgG1); 105-110 or
109-110 of FIG. 37 (SEQ ID NO:67) (IgG2); 111-160, 114-160,
120-160, 126-160, 129-160, 135-160, 141-160, 144-160, 150-160,
156-160 and 159-160 of FIG. 38 (SEQ ID NO:68) (IgG3); or 106-110 or
109-110 of FIG. 39 (SEQ ID NO:69) (IgG4).
[0150] The CH2, CH3 and optional CH4 sequence can be any suitable
human or human compatable sequence, e.g., as presented in FIGS.
1-42 and Table 3, or as known in the art, or any combination or
consensus sequence thereof, or any fusion protein thereof.
[0151] Amino acids in an EPO mimetic hinge core mimetibody or
specified portion or variant of the present invention that are
essential for function can be identified by methods known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and
Wells, Science 244:1081-1085 (1989)). The latter procedure
introduces single alanine mutations at every residue in the
molecule. The resulting mutant molecules are then tested for
biological activity, such as, but not limited to at least one
protein related activity, as specified herein or as known in the
art. Sites that are critical for EPO mimetic hinge core mimetibody
or specified portion or variant binding can also be identified by
structural analysis such as crystallization, nuclear magnetic
resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol.
224:899-904 (1992) and de Vos, et al., Science 255:306-312
(1992)).
[0152] Mimetibodies or specified portions or variants of the
present invention can comprise as the P portion of Formula (I), but
are not limited to, at least one portion, sequence or combination
selected from 3 to all the of at least one of SEQ ID NOS:1-30.
Non-limiting variants that can enhance or maintain at least one of
the listed activities above include, but are not limited to, any of
the above polypeptides, further comprising at least one mutation
corresponding to at least one substitution, insertion or deletion
that does not significantly affect the suitable biological
activities or functions of said EPO mimetic hinge core
mimetibody.
[0153] An EPO mimetic hinge core mimetibody or specified portion or
variant can further optionally comprise at least one functional
portion of at least one polypeptide as P portion of Formula (I), at
least one of 90-100% of SEQ ID NOS:1-30. An EPO mimetic hinge core
mimetibody can further optionally comprise an amino acid sequence
for the P portion of Formula (I), selected from one or more of SEQ
ID NOS:1-30.
[0154] In one embodiment, the P amino acid sequence, or portion
thereof has about 90-100% identity (i.e., 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100 or any range or value therein) to the
corresponding amino acid sequence of the corresponding portion of
at least one of SEQ ID NOS:1-30. Preferably, 90-100% amino acid
identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any
range or value therein) is determined using a suitable computer
algorithm, as known in the art.
[0155] Mimetibodies or specified portions or variants of the
present invention can comprise any number of contiguous amino acid
residues from an EPO mimetic hinge core mimetibody or specified
portion or variant of the present invention, wherein that number is
selected from the group of integers consisting of from 10-100% of
the number of contiguous residues in an EPO mimetic hinge core
mimetibody. Optionally, this subsequence of contiguous amino acids
is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50,
60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190,
200, 210, 220, 230, 240, 250 or more amino acids in length, or any
range or value therein. Further, the number of such subsequences
can be any integer selected from the group consisting of from 1 to
20, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, or more.
[0156] As those of skill will appreciate, the present invention
includes at least one biologically active EPO mimetic hinge core
mimetibody or specified portion or variant of the present
invention. Biologically active mimetibodies or specified portions
or variants have a specific activity at least 20%, 30%, or 40%, and
preferably at least 50%, 60%, or 70%, and most preferably at least
80%, 90%, or 95%-1000% of that of the native (non-synthetic),
endogenous or related and known inserted or fused protein or
specified portion or variant. Methods of assaying and quantifying
measures of enzymatic activity and substrate specificity, are well
known to those of skill in the art.
[0157] In another aspect, the invention relates to human
mimetibodies and ligand-binding fragments, as described herein,
which are modified by the covalent attachment of an organic moiety.
Such modification can produce an EPO mimetic hinge core mimetibody
or ligand-binding fragment with improved pharmacokinetic properties
(e.g., increased in vivo serum half-life). The organic moiety can
be a linear or branched hydrophilic polymeric group, fatty acid
group, or fatty acid ester group. In particular embodiments, the
hydrophilic polymeric group can have a molecular weight of about
800 to about 120,000 Daltons and can be a polyalkane glycol (e.g.,
polyethylene glycol (PEG), polypropylene glycol (PPG)),
carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone,
and the fatty acid or fatty acid ester group can comprise from
about eight to about forty carbon atoms.
[0158] The modified mimetibodies and ligand-binding fragments of
the invention can comprise one or more organic moieties that are
covalently bonded, directly or indirectly, to the EPO mimetic hinge
core mimetibody or specified portion or variant. Each organic
moiety that is bonded to an EPO mimetic hinge core mimetibody or
ligand-binding fragment of the invention can independently be a
hydrophilic polymeric group, a fatty acid group or a fatty acid
ester group. As used herein, the term "fatty acid" encompasses
mono-carboxylic acids and di-carboxylic acids. A "hydrophilic
polymeric group," as the term is used herein, refers to an organic
polymer that is more soluble in water than in octane. For example,
polylysine is more soluble in water than in octane. Thus, an EPO
mimetic hinge core mimetibody modified by the covalent attachment
of polylysine is encompassed by the invention. Hydrophilic polymers
suitable for modifying mimetibodies of the invention can be linear
or branched and include, for example, polyalkane glycols (e.g.,
PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like),
carbohydrates (e.g., dextran, cellulose, oligosaccharides,
polysaccharides and the like), polymers of hydrophilic amino acids
(e.g., polylysine, polyarginine, polyaspartate and the like),
polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide
and the like) and polyvinyl pyrolidone. Preferably, the hydrophilic
polymer that modifies the EPO mimetic hinge core mimetibody of the
invention has a molecular weight of about 800 to about 150,000
Daltons as a separate molecular entity. For example, PEG.sub.2500,
PEG.sub.5000, PEG.sub.7500, PEG.sub.9000, PEG.sub.10000,
PEG.sub.12500, PEG.sub.15000, and PEG.sub.20,000, wherein the
subscript is the average molecular weight of the polymer in
Daltons, can be used.
[0159] The hydrophilic polymeric group can be substituted with one
to about six alkyl, fatty acid or fatty acid ester groups.
Hydrophilic polymers that are substituted with a fatty acid or
fatty acid ester group can be prepared by employing suitable
methods. For example, a polymer comprising an amine group can be
coupled to a carboxylate of the fatty acid or fatty acid ester, and
an activated carboxylate (e.g., activated with N,N-carbonyl
diimidazole) on a fatty acid or fatty acid ester can be coupled to
a hydroxyl group on a polymer.
[0160] Fatty acids and fatty acid esters suitable for modifying
mimetibodies of the invention can be saturated or can contain one
or more units of unsaturation. Fatty acids that are suitable for
modifying mimetibodies of the invention include, for example,
n-dodecanoate (C.sub.12, laurate), n-tetradecanoate (C.sub.14,
myristate), n-octadecanoate (C.sub.18, stearate), n-eicosanoate
(C.sub.20, arachidate), n-docosanoate (C.sub.22, behenate),
n-triacontanoate (C.sub.30), n-tetracontanoate (C.sub.40),
cis-.DELTA.9-octadecanoate (C.sub.18, oleate), all
cis-.DELTA.5,8,11,14-eicosatetraenoate (C.sub.20, arachidonate),
octanedioic acid, tetradecanedioic acid, octadecanedioic acid,
docosanedioic acid, and the like. Suitable fatty acid esters
include mono-esters of dicarboxylic acids that comprise a linear or
branched lower alkyl group. The lower alkyl group can comprise from
one to about twelve, preferably one to about six, carbon atoms.
[0161] The modified human mimetibodies and ligand-binding fragments
can be prepared using suitable methods, such as by reaction with
one or more modifying agents. A "modifying agent" as the term is
used herein, refers to a suitable organic group (e.g., hydrophilic
polymer, a fatty acid, a fatty acid ester) that comprises an
activating group. An "activating group" is a chemical moiety or
functional group that can, under appropriate conditions, react with
a second chemical group thereby forming a covalent bond between the
modifying agent and the second chemical group. For example,
amine-reactive activating groups include electrophilic groups such
as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo),
N-hydroxysuccinimidyl esters (NHS), and the like. Activating groups
that can react with thiols include, for example, maleimide,
iodoacetyl, acryloyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic
acid thiol (TNB-thiol), and the like. An aldehyde functional group
can be coupled to amine- or hydrazide-containing molecules, and an
azide group can react with a trivalent phosphorous group to form
phosphoramidate or phosphorimide linkages. Suitable methods to
introduce activating groups into molecules are known in the art
(see for example, Hermanson, G. T., Bioconjugate Techniques,
Academic Press: San Diego, Calif. (1996)). An activating group can
be bonded directly to the organic group (e.g., hydrophilic polymer,
fatty acid, fatty acid ester), or through a linker moiety, for
example a divalent C.sub.1-C.sub.12 group wherein one or more
carbon atoms can be replaced by a heteroatom such as oxygen,
nitrogen or sulfur. Suitable linker moieties include, for example,
tetraethylene glycol, --(CH.sub.2).sub.3--,
--NH--(CH.sub.2).sub.6--NH--, --(CH.sub.2).sub.2--NH-- and
--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH.sub.2--CH.sub.2--O--CH--NH--.
Modifying agents that comprise a linker moiety can be produced, for
example, by reacting a mono-Boc-alkyldiamine (e.g.,
mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid
in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
(EDC) to form an amide bond between the free amine and the fatty
acid carboxylate. The Boc protecting group can be removed from the
product by treatment with trifluoroacetic acid (TFA) to expose a
primary amine that can be coupled to another carboxylate as
described, or can be reacted with maleic anhydride and the
resulting product cyclized to produce an activated maleimido
derivative of the fatty acid. (See, for example, Thompson, et al.,
WO 92/16221 the entire teachings of which are incorporated herein
by reference.)
[0162] The modified mimetibodies of the invention can be produced
by reacting an human EPO mimetic hinge core mimetibody or
ligand-binding fragment with a modifying agent. For example, the
organic moieties can be bonded to the EPO mimetic hinge core
mimetibody in a non-site specific manner by employing an
amine-reactive modifying agent, for example, an NHS ester of PEG.
Modified human mimetibodies or ligand-binding fragments can also be
prepared by reducing disulfide bonds (e.g., intra-chain disulfide
bonds) of an EPO mimetic hinge core mimetibody or ligand-binding
fragment. The reduced EPO mimetic hinge core mimetibody or
ligand-binding fragment can then be reacted with a thiol-reactive
modifying agent to produce the modified EPO mimetic hinge core
mimetibody of the invention. Modified human mimetibodies and
ligand-binding fragments comprising an organic moiety that is
bonded to specific sites of an EPO mimetic hinge core mimetibody or
specified portion or variant of the present invention can be
prepared using suitable methods, such as reverse proteolysis (Fisch
et al., Bioconjugate Chem., 3:147-153 (1992); Werlen et al.,
Bioconjugate Chem., 5:411-417 (1994); Kumaran et al., Protein Sci.
6(10):2233-2241 (1997); Itoh et al., Bioorg. Chem., 24(1): 59-68
(1996); Capellas et al., Biotechnol. Bioeng., 56(4):456-463
(1997)), and the methods described in Hermanson, G. T.,
Bioconjugate Techniques, Academic Press: San Diego, Calif.
(1996).
EPO Mimetic Hinge Core Mimetibody Compositions
[0163] The present invention also provides at least one EPO mimetic
hinge core mimetibody or specified portion or variant composition
comprising at least one, at least two, at least three, at least
four, at least five, at least six or more mimetibodies or specified
portions or variants thereof, as described herein and/or as known
in the art that are provided in a non-naturally occurring
composition, mixture or form. Such composition percentages are by
weight, volume, concentration, molarity, or molality as liquid or
dry solutions, mixtures, suspension, emulsions or colloids, as
known in the art or as described herein.
[0164] Such compositions can comprise 0.00001-99.9999 percent by
weight, volume, concentration, molarity, or molality as liquid,
gas, or dry solutions, mixtures, suspension, emulsions or colloids,
as known in the art or as described herein, on any range or value
therein, such as but not limited to 0.00001, 0.00003, 0.00005,
0.00009, 0.0001, 0.0003, 0.0005, 0.0009, 0.001, 0.003, 0.005,
0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8,
4.9, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2,
99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9%. Such compositions of the
present invention thus include but are not limited to 0.00001-100
mg/ml and/or 0.00001-100 mg/g.
[0165] The composition can optionally further comprise an effective
amount of at least one compound or protein selected from at least
one of an anti-infective drug, a cardiovascular (CV) system drug, a
central nervous system (CNS) drug, an autonomic nervous system
(ANS) drug, a respiratory tract drug, a gastrointestinal (GI) tract
drug, a hormonal drug, a drug for fluid or electrolyte balance, a
hematologic drug, an antineoplastic, an immunomodulation drug, an
ophthalmic, optic or nasal drug, a topical drug, a nutritional drug
or the like. Such drugs are well known in the art, including
formulations, indications, dosing and administration for each
presented herein (see, e.g., Nursing 2001 Handbook of Drugs,
21.sup.st edition, Springhouse Corp., Springhouse, Pa., 2001;
Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang,
Prentice-Hall, Inc, Upper Saddle River, N.J.; Pharmcotherapy
Handbook, Wells et al., ed., Appleton & Lange, Stamford, Conn.,
each entirely incorporated herein by reference).
[0166] The anti-infective drug can be at least one selected from
amebicides or at least one antiprotozoals, anthelmintics,
antifungals, antimalarials, antituberculotics or at least one
antileprotics, aminoglycosides, penicillins, cephalosporins,
tetracyclines, sulfonamides, fluoroquinolones, antivirals,
macrolide anti-infectives, miscellaneous anti-infectives. The CV
drug can be at least one selected from inotropics, antiarrhythmics,
antianginals, antihypertensives, antilipemics, miscellaneous
cardiovascular drugs. The CNS drug can be at least one selected
from normarcotic analgesics or at least one selected from
antipyretics, nonsteroidal anti-inflammatory drugs, narcotic or at
least one opiod analgesics, sedative-hypnotics, anticonvulsants,
antidepressants, antianxiety drugs, antipsychotics, central nervous
system stimulants, antiparkinsonians, miscellaneous central nervous
system drugs. The ANS drug can be at least one selected from
cholinergics (parasympathomimetics), anticholinergics, adrenergics
(sympathomimetics), adrenergic blockers (sympatholytics), skeletal
muscle relaxants, neuromuscular blockers. The respiratory tract
drug can be at least one selected from antihistamines,
bronchodilators, expectorants or at least one antitussives,
miscellaneous respiratory drugs. The GI tract drug can be at least
one selected from antacids or at least one adsorbents or at least
one antiflatulents, digestive enzymes or at least one gallstone
solubilizers, antidiarrheals, laxatives, antiemetics, antiulcer
drugs. The hormonal drug can be at least one selected from
corticosteroids, androgens or at least one anabolic steroids,
estrogens or at least one progestins, gonadotropins, antidiabetic
drugs or at least one glucagon, thyroid hormones, thyroid hormone
antagonists, pituitary hormones, parathyroid-like drugs. The drug
for fluid and electrolyte balance can be at least one selected from
diuretics, electrolytes or at least one replacement solutions,
acidifiers or at least one alkalinizers. The hematologic drug can
be at least one selected from hematinics, anticoagulants, blood
derivatives, thrombolytic enzymes. The antineoplastics can be at
least one selected from alkylating drugs, antimetabolites,
antibiotic antineoplastics, antineoplastics that alter hormone
balance, miscellaneous antineoplastics. The immunomodulation drug
can be at least one selected from immunosuppressants, vaccines or
at least one toxoids, antitoxins or at least one antivenins, immune
serums, biological response modifiers. The ophthalmic, optic, and
nasal drugs can be at least one selected from ophthalmic
anti-infectives, ophthalmic anti-inflammatories, miotics,
mydriatics, ophthalmic vasoconstrictors, miscellaneous ophthalmics,
optics, nasal drugs. The topical drug can be at least one selected
from local anti-infectives, scabicides or at least one
pediculicides, topical corticosteroids. The nutritional drug can be
at least one selected from vitamins, minerals, or calorics. See,
e.g., contents of Nursing 2001 Drug Handbook, supra.
[0167] The at least one amebicide or antiprotozoal can be at least
one selected from atovaquone, chloroquine hydrochloride,
chloroquine phosphate, metronidazole, metronidazole hydrochloride,
pentamidine isethionate. The at least one anthelmintic can be at
least one selected from mebendazole, pyrantel pamoate,
thiabendazole. The at least one antifungal can be at least one
selected from amphotericin B, amphotericin B cholesteryl sulfate
complex, amphotericin B lipid complex, amphotericin B liposomal,
fluconazole, flucytosine, griseofulvin microsize, griseofulvin
ultramicrosize, itraconazole, ketoconazole, nystatin, terbinafine
hydrochloride. The at least one antimalarial can be at least one
selected from chloroquine hydrochloride, chloroquine phosphate,
doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride,
primaquine phosphate, pyrimethamine, pyrimethamine with
sulfadoxine. The at least one antituberculotic or antileprotic can
be at least one selected from clofazimine, cycloserine, dapsone,
ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin,
rifampin, rifapentine, streptomycin sulfate. The at least one
aminoglycoside can be at least one selected from amikacin sulfate,
gentamicin sulfate, neomycin sulfate, streptomycin sulfate,
tobramycin sulfate. The at least one penicillin can be at least one
selected from amoxicillin/clavulanate potassium, amoxicillin
trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate,
ampicillin sodium/sulbactam sodium, cloxacillin sodium,
dicloxacillin sodium, mezlocillin sodium, nafcillin sodium,
oxacillin sodium, penicillin G benzathine, penicillin G potassium,
penicillin G procaine, penicillin G sodium, penicillin V potassium,
piperacillin sodium, piperacillin sodium/tazobactam sodium,
ticarcillin disodium, ticarcillin disodium/clavulanate potassium.
The at least one cephalosporin can be at least one selected from at
least one of cefaclor, cefadroxil, cefazolin sodium, cefdinir,
cefepime hydrochloride, cefixime, cefinetazole sodium, cefonicid
sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium,
cefoxitin sodium, cefpodoxime proxetil, cefprozil, ceftazidime,
ceftibuten, ceftizoxime sodium, ceftriaxone sodium, cefuroxime
axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin
monohydrate, cephradine, loracarbef. The at least one tetracycline
can be at least one selected from demeclocycline hydrochloride,
doxycycline calcium, doxycycline hyclate, doxycycline
hydrochloride, doxycycline monohydrate, minocycline hydrochloride,
tetracycline hydrochloride. The at least one sulfonamide can be at
least one selected from co-trimoxazole, sulfadiazine,
sulfamethoxazole, sulfisoxazole, sulfisoxazole acetyl. The at least
one fluoroquinolone can be at least one selected from
alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin,
lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin,
sparfloxacin, trovafloxacin mesylate. The at least one
fluoroquinolone can be at least one selected from alatrofloxacin
mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin
hydrochloride, nalidixic acid, norfloxacin, ofloxacin,
sparfloxacin, trovafloxacin mesylate. The at least one antiviral
can be at least one selected from abacavir sulfate, acyclovir
sodium, amantadine hydrochloride, amprenavir, cidofovir,
delavirdine mesylate, didanosine, efavirenz, famciclovir,
fomivirsen sodium, foscarnet sodium, ganciclovir, indinavir
sulfate, lamivudine, lamivudine/zidovudine, nelfinavir mesylate,
nevirapine, oseltamivir phosphate, ribavirin, rimantadine
hydrochloride, ritonavir, saquinavir, saquinavir mesylate,
stavudine, valacyclovir hydrochloride, zalcitabine, zanamivir,
zidovudine. The at least one macroline anti-infective can be at
least one selected from azithromycin, clarithromycin,
dirithromycin, erythromycin base, erythromycin estolate,
erythromycin ethylsuccinate, erythromycin lactobionate,
erythromycin stearate. The at least one miscellaneous
anti-infective can be at least one selected from aztreonam,
bacitracin, chloramphenicol sodium sucinate, clindamycin
hydrochloride, clindamycin palmitate hydrochloride, clindamycin
phosphate, imipenem and cilastatin sodium, meropenem,
nitrofurantoin macrocrystals, nitrofurantoin microcrystals,
quinupristin/dalfopristin, spectinomycin hydrochloride,
trimethoprim, vancomycin hydrochloride. (See, e.g., pp. 24-214 of
Nursing 2001 Drug Handbook.)
[0168] The at least one inotropic can be at least one selected from
aminone lactate, digoxin, milrinone lactate. The at least one
antiarrhythmic can be at least one selected from adenosine,
amiodarone hydrochloride, atropine sulfate, bretylium tosylate,
diltiazem hydrochloride, diisopyramide, diisopyramide phosphate,
esmolol hydrochloride, flecaimide acetate, ibutilide fumarate,
lidocaine hydrochloride, mexiletine hydrochloride, moricizine
hydrochloride, phenyloin, phenyloin sodium, procainamide
hydrochloride, propafenone hydrochloride, propranolol
hydrochloride, quinidine bisulfate, quinidine gluconate, quinidine
polygalacturonate, quinidine sulfate, sotalol, tocamide
hydrochloride, verapamil hydrochloride. The at least one
antianginal can be at least one selected from amlodipidine
besylate, amyl nitrite, bepridil hydrochloride, diltiazem
hydrochloride, isosorbide dinitrate, isosorbide mononitrate,
nadolol, nicardipine hydrochloride, nifedipine, nitroglycerin,
propranolol hydrochloride, verapamil, verapamil hydrochloride. The
at least one antihypertensive can be at least one selected from
acebutolol hydrochloride, amlodipine besylate, atenolol, benazepril
hydrochloride, betaxolol hydrochloride, bisoprolol fumarate,
candesartan cilexetil, captopril, carteolol hydrochloride,
carvedilol, clonidine, clonidine hydrochloride, diazoxide,
diltiazem hydrochloride, doxazocin mesylate, enelaprilat, enalapril
maleate, eprosartan mesylate, felodipine, fenoldopam mesylate,
fosinopril sodium, guanabenz acetate, guanadrel sulfate, guanfacine
hydrochloride, hydralazine hydrochloride, irbesartan, isradipine,
labetalol hydrochloride, lisinopril, losartan potassium,
methyldopa, methyldopate hydrochloride, metoprolol succinate,
metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol,
nicardipine hydrochloride, nifedipine, nisoldipine, nitroprusside
sodium, penbutolol sulfate, perindopril erbumine, phentolamine
mesylate, pindolol, prazosin hydrochloride, propranolol
hydrochloride, quinapril hydrochloride, ramipril, telmisartan,
terazosin hydrochloride, timolol maleate, trandolapril, valsartan,
verapamil hydrochloride The at least one antilipemic can be at
least one selected from atorvastatin calcium, cerivastatin sodium,
cholestyramine, colestipol hydrochloride, fenofibrate (micronized),
fluvastatin sodium, gemfibrozil, lovastatin, niacin, pravastatin
sodium, simvastatin. The at least one miscellaneous CV drug can be
at least one selected from abciximab, alprostadil, arbutamine
hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole,
eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine
hydrochloride, tirofiban hydrochloride. (See, e.g., pp. 215-336 of
Nursing 2001 Drug Handbook.)
[0169] The at least one normarcotic analgesic or antipyretic can be
at least one selected from acetaminophen, aspirin, choline
magnesium trisalicylate, diflunisal, magnesium salicylate. The at
least one nonsteroidal anti-inflammatory drug can be at least one
selected from celecoxib, diclofenac potassium, diclofenac sodium,
etodolac, fenoprofen calcium, flurbiprofen, ibuprofen,
indomethacin, indomethacin sodium trihydrate, ketoprofen, ketorolac
tromethamine, nabumetone, naproxen, naproxen sodium, oxaprozin,
piroxicam, rofecoxib, sulindac. The at least one narcotic or opiod
analgesic can be at least one selected from alfentanil
hydrochloride, buprenorphine hydrochloride, butorphanol tartrate,
codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl
transdermal system, fentanyl transmucosal, hydromorphone
hydrochloride, meperidine hydrochloride, methadone hydrochloride,
morphine hydrochloride, morphine sulfate, morphine tartrate,
nalbuphine hydrochloride, oxycodone hydrochloride, oxycodone
pectinate, oxymorphone hydrochloride, pentazocine hydrochloride,
pentazocine hydrochloride and naloxone hydrochloride, pentazocine
lactate, propoxyphene hydrochloride, propoxyphene napsylate,
remifentanil hydrochloride, sufentanil citrate, tramadol
hydrochloride. The at least one sedative-hypnotic can be at least
one selected from chloral hydrate, estazolam, flurazepam
hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital
sodium, secobarbital sodium, temazepam, triazolam, zaleplon,
zolpidem tartrate. The at least one anticonvulsant can be at least
one selected from acetazolamide sodium, carbamazepine, clonazepam,
clorazepate dipotassium, diazepam, divalproex sodium, ethosuximide,
fosphenyloin sodium, gabapentin, lamotrigine, magnesium sulfate,
phenobarbital, phenobarbital sodium, phenyloin, phenyloin sodium,
phenyloin sodium (extended), primidone, tiagabine hydrochloride,
topiramate, valproate sodium, valproic acid. The at least one
antidepressant can be at least one selected from amitriptyline
hydrochloride, amitriptyline pamoate, amoxapine, bupropion
hydrochloride, citalopram hydrobromide, clomipramine hydrochloride,
desipramine hydrochloride, doxepin hydrochloride, fluoxetine
hydrochloride, imipramine hydrochloride, imipramine pamoate,
mirtazapine, nefazodone hydrochloride, nortriptyline hydrochloride,
paroxetine hydrochloride, phenelzine sulfate, sertraline
hydrochloride, tranylcypromine sulfate, trimipramine maleate,
venlafaxine hydrochloride. The at least one antianxiety drug can be
at least one selected from alprazolam, buspirone hydrochloride,
chlordiazepoxide, chlordiazepoxide hydrochloride, clorazepate
dipotassium, diazepam, doxepin hydrochloride, hydroxyzine ebonite,
hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam,
meprobamate, midazolam hydrochloride, oxazepam. The at least one
antipsychotic drug can be at least one selected from chlorpromazine
hydrochloride, clozapine, fluphenazine decanoate, fluphenazine
enanthate, fluphenazine hydrochloride, haloperidol, haloperidol
decanoate, haloperidol lactate, loxapine hydrochloride, loxapine
succinate, mesoridazine besylate, molindone hydrochloride,
olanzapine, perphenazine, pimozide, proclorperazine, quetiapine
fumarate, risperidone, thioridazine hydrochloride, thiothixene,
thiothixene hydrochloride, trifluoperazine hydrochloride. The at
least one central nervous system stimulant can be at least one
selected from amphetamine sulfate, caffeine, dextroamphetamine
sulfate, doxapram hydrochloride, methamphetamine hydrochloride,
methylphenidate hydrochloride, modafinil, pemoline, phentermine
hydrochloride. The at least one antiparkinsonian can be at least
one selected from amantadine hydrochloride, benztropine mesylate,
biperiden hydrochloride, biperiden lactate, bromocriptine mesylate,
carbidopa-levodopa, entacapone, levodopa, pergolide mesylate,
pramipexole dihydrochloride, ropinirole hydrochloride, selegiline
hydrochloride, tolcapone, trihexyphenidyl hydrochloride. The at
least one miscellaneous central nervous system drug can be at least
one selected from bupropion hydrochloride, donepezil hydrochloride,
droperidol, fluvoxamine maleate, lithium carbonate, lithium
citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine
transdermal system, propofol, rizatriptan benzoate, sibutramine
hydrochloride monohydrate, sumatriptan succinate, tacrine
hydrochloride, zolmitriptan. (See, e.g., pp. 337-530 of Nursing
2001 Drug Handbook.)
[0170] The at least one cholinergic (e.g., parasymathomimetic) can
be at least one selected from bethanechol chloride, edrophonium
chloride, neostigmine bromide, neostigmine methylsulfate,
physostigmine salicylate, pyridostigmine bromide. The at least one
anticholinergics can be at least one selected from atropine
sulfate, dicyclomine hydrochloride, glycopyrrolate, hyoscyamine,
hyoscyamine sulfate, propantheline bromide, scopolamine,
scopolamine butylbromide, scopolamine hydrobromide. The at least
one adrenergics (sympathomimetics) can be at least one selected
from dobutamine hydrochloride, dopamine hydrochloride, metaraminol
bitartrate, norepinephrine bitartrate, phenylephrine hydrochloride,
pseudoephedrine hydrochloride, pseudoephedrine sulfate. The at
least one adrenergic blocker (sympatholytic) can be at least one
selected from dihydroergotamine mesylate, ergotamine tartrate,
methysergide maleate, propranolol hydrochloride. The at least one
skeletal muscle relaxant can be at least one selected from
baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine
hydrochloride, dantrolene sodium, methocarbamol, tizanidine
hydrochloride. The at least one neuromuscular blockers can be at
least one selected from atracurium besylate, cisatracurium
besylate, doxacurium chloride, mivacurium chloride, pancuronium
bromide, pipecuronium bromide, rapacuronium bromide, rocuronium
bromide, succinylcholine chloride, tubocurarine chloride,
vecuronium bromide. (See, e.g., pp. 531-84 of Nursing 2001 Drug
Handbook.)
[0171] The at least one antihistamine can be at least one selected
from brompheniramine maleate, cetirizine hydrochloride,
chlorpheniramine maleate, clemastine fumarate, cyproheptadine
hydrochloride, diphenhydramine hydrochloride, fexofenadine
hydrochloride, loratadine, promethazine hydrochloride, promethazine
theoclate, triprolidine hydrochloride. The at least one
bronchodilators can be at least one selected from albuterol,
albuterol sulfate, aminophylline, atropine sulfate, ephedrine
sulfate, epinephrine, epinephrine bitartrate, epinephrine
hydrochloride, ipratropium bromide, isoproterenol, isoproterenol
hydrochloride, isoproterenol sulfate, levalbuterol hydrochloride,
metaproterenol sulfate, oxtriphylline, pirbuterol acetate,
salmeterol xinofoate, terbutaline sulfate, theophylline. The at
least one expectorants or antitussives can be at least one selected
from benzonatate, codeine phosphate, codeine sulfate,
dextramethorphan hydrobromide, diphenhydramine hydrochloride,
guaifenesin, hydromorphone hydrochloride. The at least one
miscellaneous respiratory drug can be at least one selected from
acetylcysteine, beclomethasone dipropionate, beractant, budesonide,
calfactant, cromolyn sodium, dornase alfa, epoprostenol sodium,
flunisolide, fluticasone propionate, montelukast sodium, nedocromil
sodium, palivizumab, triamcinolone acetonide, zafirlukast,
zileuton. (See, e.g., pp. 585-642 of Nursing 2001 Drug
Handbook.)
[0172] The at least one antacid, adsorbents, or antiflatulents can
be at least one selected from aluminum carbonate, aluminum
hydroxide, calcium carbonate, magaldrate, magnesium hydroxide,
magnesium oxide, simethicone, sodium bicarbonate. The at least one
digestive enzymes or gallstone solubilizers can be at least one
selected from pancreatin, pancrelipase, ursodiol. The at least one
antidiarrheal can be at least one selected from attapulgite,
bismuth subsalicylate, calcium polycarbophil, diphenoxylate
hydrochloride or atropine sulfate, loperamide, octreotide acetate,
opium tincture, opium tincure (camphorated). The at least one
laxative can be at least one selected from bisacodyl, calcium
polycarbophil, cascara sagrada, cascara sagrada aromatic
fluidextract, cascara sagrada fluidextract, castor oil, docusate
calcium, docusate sodium, glycerin, lactulose, magnesium citrate,
magnesium hydroxide, magnesium sulfate, methylcellulose, mineral
oil, polyethylene glycol or electrolyte solution, psyllium, senna,
sodium phosphates. The at least one antiemetic can be at least one
selected from chlorpromazine hydrochloride, dimenhydrinate,
dolasetron mesylate, dronabinol, granisetron hydrochloride,
meclizine hydrochloride, metoclopramide hydrochloride, ondansetron
hydrochloride, perphenazine, proclorperazine, proclorperazine
edisylate, proclorperazine maleate, promethazine hydrochloride,
scopolamine, thiethylperazine maleate, trimethobenzamide
hydrochloride. The at least one antiulcer drug can be at least one
selected from cimetidine, cimetidine hydrochloride, famotidine,
lansoprazole, misoprostol, nizatidine, omeprazole, rabeprazole
sodium, rantidine bismuth citrate, ranitidine hydrochloride,
sucralfate. (See, e.g., pp. 643-95 of Nursing 2001 Drug Handbook.)
The at least one corticosteroids can be at least one selected from
betamethasone, betamethasone acetate or betamethasone sodium
phosphate, betamethasone sodium phosphate, cortisone acetate,
dexamethasone, dexamethasone acetate, dexamethasone sodium
phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone
acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate,
hydrocortisone sodium succinate, methylprednisolone,
methylprednisolone acetate, methylprednisolone sodium succinate,
prednisolone, prednisolone acetate, prednisolone sodium phosphate,
prednisolone tebutate, prednisone, triamcinolone, triamcinolone
acetonide, triamcinolone diacetate. The at least one androgen or
anabolic steroids can be at least one selected from danazol,
fluoxymesterone, methyltestosterone, nandrolone decanoate,
nandrolone phenpropionate, testosterone, testosterone cypionate,
testosterone enanthate, testosterone propionate, testosterone
transdermal system. The at least one estrogen or progestin can be
at least one selected from esterified estrogens, estradiol,
estradiol cypionate, estradiol/norethindrone acetate transdermal
system, estradiol valerate, estrogens (conjugated), estropipate,
ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl
estradiol and ethynodiol diacetate, ethinyl estradiol and
desogestrel, ethinyl estradiol and ethynodiol diacetate, ethinyl
estradiol and levonorgestrel, ethinyl estradiol and norethindrone,
ethinyl estradiol and norethindrone acetate, ethinyl estradiol and
norgestimate, ethinyl estradiol and norgestrel, ethinyl estradiol
and norethindrone and acetate and ferrous fumarate, levonorgestrel,
medroxyprogesterone acetate, mestranol and norethindrone,
norethindrone, norethindrone acetate, norgestrel, progesterone. The
at least one gonadroptropin can be at least one selected from
ganirelix acetate, gonadoreline acetate, histrelin acetate,
menotropins. The at least one antidiabetic or glucaon can be at
least one selected from acarbose, chlorpropamide, glimepiride,
glipizide, glucagon, glyburide, insulins, metformin hydrochloride,
miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazone
maleate, troglitazone. The at least one thyroid hormone can be at
least one selected from levothyroxine sodium, liothyronine sodium,
liotrix, thyroid. The at least one thyroid hormone antagonist can
be at least one selected from methimazole, potassium iodide,
potassium iodide (saturated solution), propylthiouracil,
radioactive iodine (sodium iodide .sup.131I), strong iodine
solution. The at least one pituitary hormone can be at least one
selected from corticotropin, cosyntropin, desmopressin acetate,
leuprolide acetate, repository corticotropin, somatrem, somatropin,
vasopressin. The at least one parathyroid-like drug can be at least
one selected from calcifediol, calcitonin (human), calcitonin
(salmon), calcitriol, dihydrotachysterol, etidronate disodium.
(See, e.g., pp. 696-796 of Nursing 2001 Drug Handbook.)
[0173] The at least one diuretic can be at least one selected from
acetazolamide, acetazolamide sodium, amiloride hydrochloride,
bumetamide, chlorthalidone, ethacrylate sodium, ethacrynic acid,
furosemide, hydrochlorothiazide, indapamide, mannitol, metolazone,
spironolactone, torsemide, triamteren, urea. The at least one
electrolyte or replacement solution can be at least one selected
from calcium acetate, calcium carbonate, calcium chloride, calcium
citrate, calcium glubionate, calcium gluceptate, calcium gluconate,
calcium lactate, calcium phosphate (dibasic), calcium phosphate
(tribasic), dextran (high-molecular-weight), dextran
(low-molecular-weight), hetastarch, magnesium chloride, magnesium
sulfate, potassium acetate, potassium bicarbonate, potassium
chloride, potassium gluconate, Ringer's injection, Ringer's
injection (lactated), sodium chloride. The at least one acidifier
or alkalinizer can be at least one selected from sodium
bicarbonate, sodium lactate, tromethamine (See, e.g., pp. 797-833
of Nursing 2001 Drug Handbook.)
[0174] The at least one hematinic can be at least one selected from
ferrous fumarate, ferrous gluconate, ferrous sulfate, ferrous
sulfate (dried), iron dextran, iron sorbitol, polysaccharide-iron
complex, sodium ferric gluconate complex. The at least one
anticoagulant can be at least one selected from ardeparin sodium,
dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin
calcium, heparin sodium, warfarin sodium. The at least one blood
derivative can be at least one selected from albumin 5%, albumin
25%, antihemophilic factor, anti-inhibitor coagulant complex,
antithrombin III (human), factor IX (human), factor IX complex,
plasma protein fractions. The at least one thrombolytic enzyme can
be at least one selected from alteprase, antistreplase, reteplase
(recombinant), streptokinase, urokinase. (See, e.g., pp. 834-66 of
Nursing 2001 Drug Handbook.)
[0175] The at least one alkylating drug can be at least one
selected from busulfan, carboplatin, carmustine, chlorambucil,
cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine
hydrochloride, melphalan, melphalan hydrochloride, streptozocin,
temozolomide, thiotepa. The at least one antimetabolite can be at
least one selected from capecitabine, cladribine, cytarabine,
floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea,
mercaptopurine, methotrexate, methotrexate sodium, thioguanine. The
at least one antibiotic antineoplastic can be at least one selected
from bleomycin sulfate, dactinomycin, daunorubicin citrate
liposomal, daunorubicin hydrochloride, doxorubicin hydrochloride,
doxorubicin hydrochloride liposomal, epirubicin hydrochloride,
idarubicin hydrochloride, mitomycin, pentostatin, plicamycin,
valrubicin. The at least one antineoplastics that alter hormone
balance can be at least one selected from anastrozole,
bicalutamide, estramustine phosphate sodium, exemestane, flutamide,
goserelin acetate, letrozole, leuprolide acetate, megestrol
acetate, nilutamide, tamoxifen citrate, testolactone, toremifene
citrate. The at least one miscellaneous antineoplastic can be at
least one selected from asparaginase, bacillus Calmette-Guerin
(BCG) (live intravesical), dacarbazine, docetaxel, etoposide,
etoposide phosphate, gemcitabine hydrochloride, irinotecan
hydrochloride, mitotane, mitoxantrone hydrochloride, paclitaxel,
pegaspargase, porfimer sodium, procarbazine hydrochloride,
rituximab, teniposide, topotecan hydrochloride, trastuzumab,
tretinoin, vinblastine sulfate, vincristine sulfate, vinorelbine
tartrate. (See, e.g., pp. 867-963 of Nursing 2001 Drug
Handbook.)
[0176] The at least one immunosuppressant can be at least one
selected from azathioprine, basiliximab, cyclosporine, daclizumab,
lymphocyte immune globulin, muromonab-CD3, mycophenolate mofetil,
mycophenolate mofetil hydrochloride, sirolimus, tacrolimus. The at
least one vaccine or toxoid can be at least one selected from BCG
vaccine, cholera vaccine, diphtheria and tetanus toxoids
(adsorbed), diphtheria and tetanus toxoids and acellular pertussis
vaccine adsorbed, diphtheria and tetanus toxoids and whole-cell
pertussis vaccine, Haemophilus b conjugate vaccines, hepatitis A
vaccine (inactivated), hepatitis B vaccine (recombinant), influenza
virus vaccine 1999-2000 trivalent types A & B (purified surface
antigen), influenza virus vaccine 1999-2000 trivalent types A &
B (subvirion or purified subvirion), influenza virus vaccine
1999-2000 trivalent types A & B (whole virion), Japanese
encephalitis virus vaccine (inactivated), Lyme disease vaccine
(recombinant OspA), measles and mumps and rubella virus vaccine
(live), measles and mumps and rubella virus vaccine (live
attenuated), measles virus vaccine (live attenuated), meningococcal
polysaccharide vaccine, mumps virus vaccine (live), plague vaccine,
pneumococcal vaccine (polyvalent), poliovirus vaccine
(inactivated), poliovirus vaccine (live, oral, trivalent), rabies
vaccine (adsorbed), rabies vaccine (human diploid cell), rubella
and mumps virus vaccine (live), rubella virus vaccine (live,
attenuated), tetanus toxoid (adsorbed), tetanus toxoid (fluid),
typhoid vaccine (oral), typhoid vaccine (parenteral), typhoid Vi
polysaccharide vaccine, varicella virus vaccine, yellow fever
vaccine. The at least one antitoxin or antivenin can be at least
one selected from black widow spider antivenin, Crotalidae
antivenom (polyvalent), diphtheria antitoxin (equine), Micrurus
fulvius antivenin). The at least one immune serum can be at least
one selected from cytomegalovirus immune globulin (intravenous),
hepatitis B immune globulin (human), immune globulin intramuscular,
immune globulin intravenous, rabies immune globulin (human),
respiratory syncytial virus immune globulin intravenous (human),
Rh.sub.0(D) immune globulin (human), Rh.sub.0(D) immune globulin
intravenous (human), tetanus immune globulin (human),
varicella-zoster immune globulin. The at least one biological
response modifiers can be at least one selected from aldesleukin,
epoetin alfa, filgrastim, glatiramer acetate for injection,
interferon alfacon-1, interferon alfa-2a (recombinant), interferon
alfa-2b (recombinant), interferon beta-1a, interferon beta-1b
(recombinant), interferon gamma-1b, levamisole hydrochloride,
oprelvekin, sargramostim. (See, e.g., pp. 964-1040 of Nursing 2001
Drug Handbook.)
[0177] The at least one ophthalmic anti-infectives can be selected
form bacitracin, chloramphenicol, ciprofloxacin hydrochloride,
erythromycin, gentamicin sulfate, ofloxacin 0.3%, polymyxin B
sulfate, sulfacetamide sodium 10%, sulfacetamide sodium 15%,
sulfacetamide sodium 30%, tobramycin, vidarabine. The at least one
ophthalmic anti-inflammatories can be at least one selected from
dexamethasone, dexamethasone sodium phosphate, diclofenac sodium
0.1%, fluorometholone, flurbiprofen sodium, ketorolac tromethamine,
prednisolone acetate (suspension) prednisolone sodium phosphate
(solution). The at least one mitotic can be at least one selected
from acetylcholine chloride, carbachol (intraocular), carbachol
(topical), echothiophate iodide, pilocarpine, pilocarpine
hydrochloride, pilocarpine nitrate. The at least one mydriatic can
be at least one selected from atropine sulfate, cyclopentolate
hydrochloride, epinephrine hydrochloride, epinephryl borate,
homatropine hydrobromide, phenylephrine hydrochloride, scopolamine
hydrobromide, tropicamide. The at least one ophthalmic
vasoconstrictors can be at least one selected from naphazoline
hydrochloride, oxymetazoline hydrochloride, tetrahydrozoline
hydrochloride. The at least one miscellaneous ophthalmics can be at
least one selected from apraclonidine hydrochloride, betaxolol
hydrochloride, brimonidine tartrate, carteolol hydrochloride,
dipivefrine hydrochloride, dorzolamide hydrochloride, emedastine
difumarate, fluorescein sodium, ketotifen fumarate, latanoprost,
levobunolol hydrochloride, metipranolol hydrochloride, sodium
chloride (hypertonic), timolol maleate. The at least one optic can
be at least one selected from boric acid, carbamide peroxide,
chloramphenicol, triethanolamine polypeptide oleate-condensate. The
at least one nasal drug can be at least one selected from
beclomethasone dipropionate, budesonide, ephedrine sulfate,
epinephrine hydrochloride, flunisolide, fluticasone propionate,
naphazoline hydrochloride, oxymetazoline hydrochloride,
phenylephrine hydrochloride, tetrahydrozoline hydrochloride,
triamcinolone acetonide, xylometazoline hydrochloride. (See, e.g.,
pp. 1041-97 of Nursing 2001 Drug Handbook.)
[0178] The at least one local anti-infectives can be at least one
selected from acyclovir, amphotericin B, azelaic acid cream,
bacitracin, butoconazole nitrate, clindamycin phosphate,
clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate,
ketoconazole, mafenide acetate, metronidazole (topical), miconazole
nitrate, mupirocin, naftifine hydrochloride, neomycin sulfate,
nitrofurazone, nystatin, silver sulfadiazine, terbinafine
hydrochloride, terconazole, tetracycline hydrochloride,
tioconazole, tolnaftate. The at least one scabicide or pediculicide
can be at least one selected from crotamiton, lindane, permethrin,
pyrethrins. The at least one topical corticosteroid can be at least
one selected from betamethasone dipropionate, betamethasone
valerate, clobetasol propionate, desonide, desoximetasone,
dexamethasone, dexamethasone sodium phosphate, diflorasone
diacetate, fluocinolone acetonide, fluocinonide, flurandrenolide,
fluticasone propionate, halcinonide, hydrocortisone, hydrocortisone
acetate, hydrocortisone butyrate, hydrocortisone valerate,
mometasone furoate, triamcinolone acetonide. (See, e.g., pp.
1098-1136 of Nursing 2001 Drug Handbook.)
[0179] The at least one vitamin or mineral can be at least one
selected from vitamin A, vitamin B complex, cyanocobalamin, folic
acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide,
pyridoxine hydrochloride, riboflavin, thiamine hydrochloride,
vitamin C, vitamin D, cholecalciferol, ergocalciferol, vitamin D
analogue, doxercalciferol, paricalcitol, vitamin E, vitamin K
analogue, phytonadione, sodium fluoride, sodium fluoride (topical),
trace elements, chromium, copper, iodine, manganese, selenium,
zinc. The at least one calorics can be at least one selected from
amino acid infusions (crystalline), amino acid infusions in
dextrose, amino acid infusions with electrolytes, amino acid
infusions with electrolytes in dextrose, amino acid infusions for
hepatic failure, amino acid infusions for high metabolic stress,
amino acid infusions for renal failure, dextrose, fat emulsions,
medium-chain triglycerides. (See, e.g., pp. 1137-63 of Nursing 2001
Drug Handbook.)
[0180] EPO mimetic hinge core mimetibody antibody or polypeptide
compositions of the present invention can further comprise at least
one of any suitable and/or effective amount of a composition or
pharmaceutical composition comprising at least one EPO mimetic
hinge core mimetibody protein or antibody to a cell, tissue, organ,
animal or patient in need of such modulation, treatment or therapy,
optionally further comprising at least one selected from at least
one TNF antagonist (e.g., but not limited to a TNF chemical or
protein antagonist, TNF monoclonal or polyclonal antibody or
fragment, a soluble TNF receptor (e.g., p55, p70 or p85) or
fragment, fusion polypeptides thereof, or a small molecule TNF
antagonist, e.g., TNF binding protein I or II (TBP-1 or TBP-II),
nerelimonmab, infliximab, enteracept, CDP-571, CDP-870, afelimomab,
lenercept, and the like), an antirheumatic (e.g., methotrexate,
auranofin, aurothioglucose, azathioprine, etanercept, gold sodium
thiomalate, hydroxychloroquine sulfate, leflunomide,
sulfasalazine), a muscle relaxant, a narcotic, a non-steroid
inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative,
a local anesthetic, a neuromuscular blocker, an antimicrobial
(e.g., aminoglycoside, an antifungal, an antiparasitic, an
antiviral, a carbapenem, cephalosporin, a fluoroquinolone, a
macrolide, a penicillin, a sulfonamide, a tetracycline, another
antimicrobial), an antipsoriatic, a corticosteriod, an anabolic
steroid, a diabetes related agent, a mineral, a nutritional, a
thyroid agent, a vitamin, a calcium related hormone, an
antidiarrheal, an antitussive, an antiemetic, an antiulcer, a
laxative, an anticoagulant, an erythropieitin (e.g., epoetin
alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim
(GM-CSF, Leukine), an immunization, an immunoglobulin, an
immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a
growth hormone, a hormone replacement drug, an estrogen receptor
modulator, a mydriatic, a cycloplegic, an alkylating agent, an
antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an
antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a
hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an
asthma medication, a beta agonist, an inhaled steroid, a
leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine
or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine
antagonist. Non-limiting examples of such cytokines include, but
are not limited to, any of IL-1 to IL-23. Suitable dosages are well
known in the art. See, e.g., Wells et al., eds., Pharmacotherapy
Handbook, 2.sup.nd Edition, Appleton and Lange, Stamford, Conn.
(2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000,
Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000),
each of which references are entirely incorporated herein by
reference.
[0181] Such compositions can also include toxin molecules that are
associated, bound, co-formulated or co-administered with at least
one antibody or polypeptide of the present invention. The toxin can
optionally act to selectively kill the pathologic cell or tissue.
The pathologic cell can be a cancer or other cell. Such toxins can
be, but are not limited to, purified or recombinant toxin or toxin
fragment comprising at least one functional cytotoxic domain of
toxin, e.g., selected from at least one of ricin, diphtheria toxin,
a venom toxin, or a bacterial toxin. The term toxin also includes
both endotoxins and exotoxins produced by any naturally occurring,
mutant or recombinant bacteria or viruses which may cause any
pathological condition in humans and other mammals, including toxin
shock, which can result in death. Such toxins may include, but are
not limited to, enterotoxigenic E. coli heat-labile enterotoxin
(LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas
enterotoxins, toxic shock syndrome toxin-1 (TSST-1), Staphylococcal
enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal
enterotoxins and the like. Such bacteria include, but are not
limited to, strains of a species of enterotoxigenic E. coli (ETEC),
enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7),
Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus
pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella
flexneri, Shigella boydii, and Shigella sonnei), Salmonella species
(e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella
enteritidis), Clostridium species (e.g., Clostridium perfringens,
Clostridium difficile, Clostridium botulinum), Camphlobacter
species (e.g., Camphlobacter jejuni, Camphlobacter fetus),
Heliobacter species, (e.g., Heliobacter pylori), Aeromonas species
(e.g., Aeromonas sobria, Aeromonas hydrophila, Aeromonas caviae),
Pleisomonas shigelloides, Yersina enterocolitica, Vibrios species
(e.g., Vibrios cholerae, Vibrios parahemolyticus), Klebsiella
species, Pseudomonas aeruginosa, and Streptococci. See, e.g.,
Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and
Co., Boston, (1990); Evans et al., eds., Bacterial Infections of
Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum
Medical Book Co., New York (1991); Mandell et al, Principles and
Practice of Infectious Diseases, 3d. Ed., Churchill Livingstone,
New York (1990); Berkow et al, eds., The Merck Manual, 16th
edition, Merck and Co., Rahway, N.J., 1992; Wood et al, FEMS
Microbiology Immunology, 76:121-134 (1991); Marrack et al, Science,
248:705-711 (1990), the contents of which references are
incorporated entirely herein by reference.
[0182] EPO mimetic hinge core mimetibody or specified portion or
variant compositions of the present invention can further comprise
at least one of any suitable auxiliary, such as, but not limited
to, diluent, binder, stabilizer, buffers, salts, lipophilic
solvents, preservative, adjuvant or the like. Pharmaceutically
acceptable auxiliaries are preferred. Non-limiting examples of, and
methods of preparing such sterile solutions are well known in the
art, such as, but limited to, Gennaro, Ed., Remington's
Pharmaceutical Sciences, 18.sup.th Edition, Mack Publishing Co.
(Easton, Pa.) 1990. Pharmaceutically acceptable carriers can be
routinely selected that are suitable for the mode of
administration, solubility and/or stability of the EPO mimetic
hinge core mimetibody composition as well known in the art or as
described herein.
[0183] Pharmaceutical excipients and additives useful in the
present composition include but are not limited to proteins,
peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides;
derivatized sugars such as alditols, aldonic acids, esterified
sugars and the like; and polysaccharides or sugar polymers), which
can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein
excipients include serum albumin such as human serum albumin (HSA),
recombinant human albumin (rHA), gelatin, casein, and the like.
Representative amino acid/EPO mimetic hinge core mimetibody or
specified portion or variant components, which can also function in
a buffering capacity, include alanine, glycine, arginine, betaine,
histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,
isoleucine, valine, methionine, phenylalanine, aspartame, and the
like. One preferred amino acid is glycine.
[0184] Carbohydrate excipients suitable for use in the invention
include, for example, monosaccharides such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol
(glucitol), myoinositol and the like. Preferred carbohydrate
excipients for use in the present invention are mannitol,
trehalose, and raffinose.
[0185] EPO mimetic hinge core mimetibody compositions can also
include a buffer or a pH adjusting agent; typically, the buffer is
a salt prepared from an organic acid or base. Representative
buffers include organic acid salts such as salts of citric acid,
ascorbic acid, gluconic acid, carbonic acid, tartaric acid,
succinic acid, acetic acid, or phthalic acid; Tris, tromethamine
hydrochloride, or phosphate buffers. Preferred buffers for use in
the present compositions are organic acid salts such as
citrate.
[0186] Additionally, the EPO mimetic hinge core mimetibody or
specified portion or variant compositions of the invention can
include polymeric excipients/additives such as
polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates
(e.g., cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin),
polyethylene glycols, flavoring agents, antimicrobial agents,
sweeteners, antioxidants, antistatic agents, surfactants (e.g.,
polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g.,
phospholipids, fatty acids), steroids (e.g., cholesterol), and
chelating agents (e.g., EDTA).
[0187] These and additional known pharmaceutical excipients and/or
additives suitable for use in the EPO mimetic hinge core mimetibody
compositions according to the invention are known in the art, e.g.,
as listed in "Remington: The Science & Practice of Pharmacy",
19.sup.th ed., Williams & Williams, (1995), and in the
"Physician's Desk Reference", 52.sup.nd ed., Medical Economics,
Montvale, N.J. (1998), the disclosures of which are entirely
incorporated herein by reference. Preferred carrier or excipient
materials are carbohydrates (e.g., saccharides and alditols) and
buffers (e.g., citrate) or polymeric agents.
Formulations
[0188] As noted above, the invention provides for stable
formulations, which can preferably include a suitable buffer with
saline or a chosen salt, as well as optional preserved solutions
and formulations containing a preservative as well as multi-use
preserved formulations suitable for pharmaceutical or veterinary
use, comprising at least one EPO mimetic hinge core mimetibody or
specified portion or variant in a pharmaceutically acceptable
formulation. Preserved formulations contain at least one known
preservative or optionally selected from the group consisting of at
least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol,
benzyl alcohol, phenylmercuric nitrite, phenoxyethanol,
formaldehyde, chlorobutanol, magnesium chloride (e.g.,
hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the
like), benzalkonium chloride, benzethonium chloride, sodium
dehydroacetate and thimerosal, or mixtures thereof in an aqueous
diluent. Any suitable concentration or mixture can be used as known
in the art, such as 0.001-5%, or any range or value therein, such
as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02,
0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0,
1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3,
2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or
value therein. Non-limiting examples include, no preservative,
0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3%
benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%),
0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g.,
0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkylparaben(s)
(e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01,
0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and
the like.
[0189] As noted above, the invention provides an article of
manufacture, comprising packaging material and at least one vial
comprising a solution of at least one EPO mimetic hinge core
mimetibody or specified portion or variant with the prescribed
buffers and/or preservatives, optionally in an aqueous diluent,
wherein said packaging material comprises a label that indicates
that such solution can be held over a period of 1, 2, 3, 4, 5, 6,
9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
The invention further comprises an article of manufacture,
comprising packaging material, a first vial comprising lyophilized
at least one EPO mimetic hinge core mimetibody or specified portion
or variant, and a second vial comprising an aqueous diluent of
prescribed buffer or preservative, wherein said packaging material
comprises a label that instructs a patient to reconstitute the at
least one EPO mimetic hinge core mimetibody or specified portion or
variant in the aqueous diluent to form a solution that can be held
over a period of twenty-four hours or greater.
[0190] The at least one EPO mimetic hinge core mimetibody or
specified portion or variant used in accordance with the present
invention can be produced by recombinant means, including from
mammalian cell or transgenic preparations, or can be purified from
other biological sources, as described herein or as known in the
art.
[0191] The range of amounts of at least one EPO mimetic hinge core
mimetibody or specified portion or variant in the product of the
present invention includes amounts yielding upon reconstitution, if
in a wet/dry system, concentrations from about 1.0 .mu.g/ml to
about 1000 mg/ml, although lower and higher concentrations are
operable and are dependent on the intended delivery vehicle, e.g.,
solution formulations will differ from transdermal patch,
pulmonary, transmucosal, or osmotic or micro pump methods.
[0192] Preferably, the aqueous diluent optionally further comprises
a pharmaceutically acceptable preservative. Preferred preservatives
include those selected from the group consisting of phenol,
m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol,
alkylparaben (methyl, ethyl, propyl, butyl and the like),
benzalkonium chloride, benzethonium chloride, sodium dehydroacetate
and thimerosal, or mixtures thereof. The concentration of
preservative used in the formulation is a concentration sufficient
to yield an anti-microbial effect. Such concentrations are
dependent on the preservative selected and are readily determined
by the skilled artisan.
[0193] Other excipients, e.g. isotonicity agents, buffers,
antioxidants, preservative enhancers, can be optionally and
preferably added to the diluent. An isotonicity agent, such as
glycerin, is commonly used at known concentrations. A
physiologically tolerated buffer is preferably added to provide
improved pH control. The formulations can cover a wide range of
pHs, such as from about pH 4 to about pH 10, and preferred ranges
from about pH 5 to about pH 9, and a most preferred range of about
6.0 to about 8.0. Preferably the formulations of the present
invention have pH between about 6.8 and about 7.8. Preferred
buffers include phosphate buffers, most preferably sodium
phosphate, particularly phosphate buffered saline (PBS).
[0194] Other additives, such as a pharmaceutically acceptable
solubilizers like Tween 20 (polyoxyethylene (20) sorbitan
monolaurate), Tween 40 (polyoxyethylene (20) sorbitan
monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan
monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block
copolymers), and PEG (polyethylene glycol) or non-ionic surfactants
such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic.RTM.
polyls, other block co-polymers, and chelators such as EDTA and
EGTA can optionally be added to the formulations or compositions to
reduce aggregation. These additives are particularly useful if a
pump or plastic container is used to administer the formulation.
The presence of pharmaceutically acceptable surfactant mitigates
the propensity for the protein to aggregate.
[0195] The formulations of the present invention can be prepared by
a process which comprises mixing at least one EPO mimetic hinge
core mimetibody or specified portion or variant and a preservative
selected from the group consisting of phenol, m-cresol, p-cresol,
o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl,
ethyl, propyl, butyl and the like), benzalkonium chloride,
benzethonium chloride, sodium dehydroacetate and thimerosal or
mixtures thereof in an aqueous diluent. Mixing the at least one EPO
mimetic hinge core mimetibody or specified portion or variant and
preservative in an aqueous diluent is carried out using
conventional dissolution and mixing procedures. To prepare a
suitable formulation, for example, a measured amount of at least
one EPO mimetic hinge core mimetibody or specified portion or
variant in buffered solution is combined with the desired
preservative in a buffered solution in quantities sufficient to
provide the protein and preservative at the desired concentrations.
Variations of this process would be recognized by one of ordinary
skill in the art. For example, the order the components are added,
whether additional additives are used, the temperature and pH at
which the formulation is prepared, are all factors that may be
optimized for the concentration and means of administration
used.
[0196] The claimed formulations can be provided to patients as
clear solutions or as dual vials comprising a vial of lyophilized
at least one EPO mimetic hinge core mimetibody or specified portion
or variant that is reconstituted with a second vial containing
water, a preservative and/or excipients, preferably a phosphate
buffer and/or saline and a chosen salt, in an aqueous diluent.
Either a single solution vial or dual vial requiring reconstitution
can be reused multiple times and can suffice for a single or
multiple cycles of patient treatment and thus can provide a more
convenient treatment regimen than currently available.
[0197] The present claimed articles of manufacture are useful for
administration over a period of immediately to twenty-four hours or
greater. Accordingly, the presently claimed articles of manufacture
offer significant advantages to the patient. Formulations of the
invention can optionally be safely stored at temperatures of from
about 2 to about 40.degree. C. and retain the biologically activity
of the protein for extended periods of time, thus, allowing a
package label indicating that the solution can be held and/or used
over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater.
If preserved diluent is used, such label can include use up to at
least one of 1-12 months, one-half, one and a half, and/or two
years.
[0198] The solutions of at least one EPO mimetic hinge core
mimetibody or specified portion or variant in the invention can be
prepared by a process that comprises mixing at least one EPO
mimetic hinge core mimetibody or specified portion or variant in an
aqueous diluent. Mixing is carried out using conventional
dissolution and mixing procedures. To prepare a suitable diluent,
for example, a measured amount of at least one EPO mimetic hinge
core mimetibody or specified portion or variant in water or buffer
is combined in quantities sufficient to provide the protein and
optionally a preservative or buffer at the desired concentrations.
Variations of this process would be recognized by one of ordinary
skill in the art. For example, the order the components are added,
whether additional additives are used, the temperature and pH at
which the formulation is prepared, are all factors that may be
optimized for the concentration and means of administration
used.
[0199] The claimed products can be provided to patients as clear
solutions or as dual vials comprising a vial of lyophilized at
least one EPO mimetic hinge core mimetibody or specified portion or
variant that is reconstituted with a second vial containing the
aqueous diluent. Either a single solution vial or dual vial
requiring reconstitution can be reused multiple times and can
suffice for a single or multiple cycles of patient treatment and
thus provides a more convenient treatment regimen than currently
available.
[0200] The claimed products can be provided indirectly to patients
by providing to pharmacies, clinics, or other such institutions and
facilities, clear solutions or dual vials comprising a vial of
lyophilized at least one EPO mimetic hinge core mimetibody or
specified portion or variant that is reconstituted with a second
vial containing the aqueous diluent. The clear solution in this
case can be up to one liter or even larger in size, providing a
large reservoir from which smaller portions of the at least one EPO
mimetic hinge core mimetibody or specified portion or variant
solution can be retrieved one or multiple times for transfer into
smaller vials and provided by the pharmacy or clinic to their
customers and/or patients.
[0201] Recognized devices comprising these single vial systems
include those pen-injector devices for delivery of a solution such
as Humaject.RTM., NovoPen.RTM., B-D.RTM. Pen, AutoPen.RTM., and
OptiPen.RTM.. Recognized devices comprising a dual vial system
include those pen-injector systems for reconstituting a lyophilized
drug in a cartridge for delivery of the reconstituted solution such
as the HumatroPen.RTM..
[0202] The products presently claimed include packaging material.
The packaging material provides, in addition to the information
required by the regulatory agencies, the conditions under which the
product can be used. The packaging material of the present
invention provides instructions to the patient to reconstitute the
at least one EPO mimetic hinge core mimetibody or specified portion
or variant in the aqueous diluent to form a solution and to use the
solution over a period of 2-24 hours or greater for the two vial,
wet/dry, product. For the single vial, solution product, the label
indicates that such solution can be used over a period of 2-24
hours or greater. The presently claimed products are useful for
human pharmaceutical product use.
[0203] The formulations of the present invention can be prepared by
a process that comprises mixing at least one EPO mimetic hinge core
mimetibody or specified portion or variant and a selected buffer,
preferably a phosphate buffer containing saline or a chosen salt.
Mixing the at least one EPO mimetic hinge core mimetibody or
specified portion or variant and buffer in an aqueous diluent is
carried out using conventional dissolution and mixing procedures.
To prepare a suitable formulation, for example, a measured amount
of at least one EPO mimetic hinge core mimetibody or specified
portion or variant in water or buffer is combined with the desired
buffering agent in water in quantities sufficient to provide the
protein and buffer at the desired concentrations. Variations of
this process would be recognized by one of ordinary skill in the
art. For example, the order the components are added, whether
additional additives are used, the temperature and pH at which the
formulation is prepared, are all factors that can be optimized for
the concentration and means of administration used.
[0204] The claimed stable or preserved formulations can be provided
to patients as clear solutions or as dual vials comprising a vial
of lyophilized at least one EPO mimetic hinge core mimetibody or
specified portion or variant that is reconstituted with a second
vial containing a preservative or buffer and excipients in an
aqueous diluent. Either a single solution vial or dual vial
requiring reconstitution can be reused multiple times and can
suffice for a single or multiple cycles of patient treatment and
thus provides a more convenient treatment regimen than currently
available.
[0205] At least one EPO mimetic hinge core mimetibody or specified
portion or variant in either the stable or preserved formulations
or solutions described herein, can be administered to a patient in
accordance with the present invention via a variety of delivery
methods including SC or IM injection; transdermal, pulmonary,
transmucosal, implant, osmotic pump, cartridge, micro pump, or
other means appreciated by the skilled artisan, as well-known in
the art.
Therapeutic Applications
[0206] The present invention for mimetibodies also provides a
method for modulating or treating anemia, in a cell, tissue, organ,
animal, or patient including, but not limited to, at least one of
any anemia, cancer treatment related anemia, radiotherapy or
chemotherapy related anemia, viral or bacterial infection treatment
related anemia, renal anemia, anemia of prematurity, pediatric
and/or adult cancer-associated anemia, anemia associated with
lymphoma, myeloma, multiple myeloma, AIDS-associated anemia,
concomitant treatment for patients with or without autologous blood
donation awaiting elective surgery, preoperative and post operative
for surgery, autologous blood donation or transfusion,
perioperative management, cyclic neutropenia or Kostmann syndrome
(congenital agranulocytosis), end-stage renal disease, anemia
associated with dialysis, chronic renal insufficiency, primary
hemopoietic diseases, such as congenital hypoplastic anemia,
thalassemia major, or sickle cell disease, vaso-occlusive
complications of sickle cell disease. Furman et al., Pediatrics
1992; 90: 716-728, Goldberg Science. 1988; 242:1412-1415; Paul et
al., Exp Hematol. 1984; 12:825-830; Erslev et al., Arch Intern Med.
1968; 122:230-235; Ersley et al., Ann Clin Lab Sci. 1980;
10:250-257; Jacobs et al., Nature. 1985; 313:806-810; Lin et al.,
Proc Natl Acad Sci USA. 1985; 82:7580-7584; Law et al., Proc Natl
Acad Sci USA. 1986; 83:6920-6924; Goldwasser et al., J Biol Chem.
1974; 249:4202-4206; Eaves et al., Blood. 1978; 52:1196-1210;
Sawyer et al., Blood. 1989; 74:103-109; Winearls et al., Lancet.
1986; 2:1175-1178; Eschbach et al., N Engl J Med. 1987; 316:73-78;
Eschbach et al., Ann Intern Med. 1989; 111:992-1000, each reference
entirely incorporated herein by reference.
[0207] Mimetibodies of the present invention can also be used for
non-renal forms of anemia induced, for example, by chronic
infections, inflammatory processes, radiation therapy, and
cytostatic drug treatment, and encouraging results in patients with
non-renal anemia have been reported. See, e.g., Abels R I and
Rudnick S A Erythropoietin: evolving clinical applications.
Experimental Hematology 19: 842-50 (1991); Graber S E and Krantz S
B Erythropoietin: biology and clinical use. Hematology/Oncol. Clin.
North Amer. 3: 369-400 (1989); Jelkman W and Gross A J (eds)
Erythropoietin. Springer, Berlin 1989; Koury M J and Bondurant M C
The molecular mechanism of erythropoietin action. European Journal
of Biochemistry 210: 649-63 (1992); Krantz S B Erythropoietin.
Blood 77: 419-34 (1991); Tabbara I A Erythropoietin. Biology and
clinical applications. Archives of Internal Medicine 153: 298-304
(1993), each entirely incorporated herein by reference.
[0208] The present invention also provides a method for modulating
or treating an anemia or blood cell related condition, in a cell,
tissue, organ, animal, or patient, wherein said anemia or blood
cell related condition is associated with at least one including,
but not limited to, at least one of immune related disease,
cardiovascular disease, infectious, malignant and/or neurologic
disease. Such a method can optionally comprise administering an
effective amount of at least one composition or pharmaceutical
composition comprising at least one EPO mimetic hinge core
mimetibody or specified portion or variant to a cell, tissue,
organ, animal or patient in need of such modulation, treatment or
therapy.
[0209] The present invention also provides a method for modulating
or treating cancer/infectious disease in a cell, tissue, organ,
animal or patient, including, but not limited to, at least one of
acute or chronic bacterial infection, acute and chronic parasitic
or infectious processes, including bacterial, viral and fungal
infections, HIV infection/HIV neuropathy, meningitis, hepatitis,
septic arthritis, peritonitis, pneumonia, epiglottis, e. coli
0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenic
purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy,
toxic shock syndrome, streptococcal myositis, gas gangrene,
mycobacterium tuberculosis, mycobacterium avium intracellular,
pneumocystis carinii pneumonia, pelvic inflammatory disease,
orchitis/epidydimitis, legionella, lyme disease, influenza a,
epstein-barr virus, vital-associated hemaphagocytic syndrome, vital
encephalitis/aseptic meningitis, and the like; (ii) leukemia, acute
leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB
ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia
(CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia,
myelodysplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a
malignant lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma,
multiple myeloma, Kaposi's sarcoma, colorectal carcinoma,
pancreatic carcinoma, nasopharyngeal carcinoma, malignant
histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy,
solid tumors, adenocarcinomas, sarcomas, malignant melanoma, and
the like; or (iii) neurodegenerative diseases, multiple sclerosis,
migraine headache, AIDS dementia complex, demyelinating diseases,
such as multiple sclerosis and acute transverse myelitis;
extrapyramidal and cerebellar disorders' such as lesions of the
corticospinal system; disorders of the basal ganglia or cerebellar
disorders; hyperkinetic movement disorders such as Huntington's
Chorea and senile chorea; drug-induced movement disorders, such as
those induced by drugs which block CNS dopamine receptors;
hypokinetic movement disorders, such as Parkinson's disease;
Progressive supranucleo Palsy; structural lesions of the
cerebellum; spinocerebellar degenerations, such as spinal ataxia,
Friedreich's ataxia, cerebellar cortical degenerations, multiple
systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and
Machado-Joseph); systemic disorders (Refsum's disease,
abetalipoprotemia, ataxia, telangiectasia, and mitochondrial
multi.system disorder); demyelinating core disorders, such as
multiple sclerosis, acute transverse myelitis; and disorders of the
motor unit' such as neurogenic muscular atrophies (anterior horn
cell degeneration, such as amyotrophic lateral sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy);
Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy
body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff
syndrome; chronic alcoholism; Creutzfeldt-Jakob disease; Subacute
sclerosing panencephalitis, Hallerrorden-Spatz disease; and
Dementia pugilistica, and the like. Such a method can optionally
comprise administering an effective amount of a composition or
pharmaceutical composition comprising at least one TNF antibody or
specified portion or variant to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy. See,
e.g., the Merck Manual, 16.sup.th Edition, Merck & Company,
Rahway, N.J. (1992)
[0210] Such a method can optionally comprise administering an
effective amount of at least one composition or pharmaceutical
composition comprising at least one EPO mimetic hinge core
mimetibody or specified portion or variant to a cell, tissue,
organ, animal or patient in need of such modulation, treatment or
therapy.
[0211] The present invention also provides a method for modulating
or treating at least one cardiovascular disease in a cell, tissue,
organ, animal, or patient, including, but not limited to, at least
one of cardiac stun syndrome, myocardial infarction, congestive
heart failure, stroke, ischemic stroke, hemorrhage,
arteriosclerosis, atherosclerosis, diabetic arteriosclerotic
disease, hypertension, arterial hypertension, renovascular
hypertension, syncope, shock, syphilis of the cardiovascular
system, heart failure, cor pulmonale, primary pulmonary
hypertension, cardiac arrhythmias, atrial ectopic beats, atrial
flutter, atrial fibrillation (sustained or paroxysmal), chaotic or
multifocal atrial tachycardia, regular narrow QRS tachycardia,
specific arrythmias, ventricular fibrillation, His bundle
arrythmias, atrioventricular block, bundle branch block, myocardial
ischemic disorders, coronary artery disease, angina pectoris,
myocardial infarction, cardiomyopathy, dilated congestive
cardiomyopathy, restrictive cardiomyopathy, valvular heart
diseases, endocarditis, pericardial disease, cardiac tumors, aortic
and peripheral aneurysms, aortic dissection, inflammation of the
aorta, occlusion of the abdominal aorta and its branches,
peripheral vascular disorders, occlusive arterial disorders,
peripheral atherosclerotic disease, thromboangiitis obliterans,
functional peripheral arterial disorders, Raynaud's phenomenon and
disease, acrocyanosis, erythromelalgia, venous diseases, venous
thrombosis, varicose veins, arteriovenous fistula, lymphedema,
lipedema, unstable angina, reperfusion injury, post pump syndrome,
ischemia-reperfusion injury, and the like. Such a method can
optionally comprise administering an effective amount of a
composition or pharmaceutical composition comprising at least one
EPO mimetic hinge core mimetibody or specified portion or variant
to a cell, tissue, organ, animal or patient in need of such
modulation, treatment or therapy.
[0212] Any method of the present invention can comprise
administering an effective amount of a composition or
pharmaceutical composition comprising at least one EPO mimetic
hinge core mimetibody or specified portion or variant to a cell,
tissue, organ, animal or patient in need of such modulation,
treatment or therapy. Such a method can optionally further comprise
co-administration or combination therapy for treating such immune
diseases, wherein the administering of said at least one EPO
mimetic hinge core mimetibody, specified portion or variant
thereof, further comprises administering, before concurrently,
and/or after, at least one selected from at least one TNF
antagonist (e.g., but not limited to a TNF antibody or fragment, a
soluble TNF receptor or fragment, fusion proteins thereof, or a
small molecule 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 (e.g., aminoglycoside, an
antifungal, an antiparasitic, an antiviral, a carbapenem,
cephalosporin, a fluoroquinolone, a macrolide, a penicillin, a
sulfonamide, a tetracycline, another antimicrobial), an
antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes
related agent, a mineral, a nutritional, a thyroid agent, a
vitamin, a calcium related hormone, an antidiarrheal, an
antitussive, an antiemetic, an antiulcer, a laxative, an
anticoagulant, an erythropieitin (e.g., epoetin alpha), a
filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF,
Leukine), an immunization, an immunoglobulin, an immunosuppressive
(e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a
hormone replacement drug, an estrogen receptor modulator, a
mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a
mitotic inhibitor, a radiopharmaceutical, an antidepressant,
antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a
sympathomimetic, a stimulant, donepezil, tacrine, an asthma
medication, a beta agonist, an inhaled steroid, a leukotriene
inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog,
dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist.
Suitable dosages are well known in the art. See, e.g., Wells et
al., eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and
Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket
Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma
Linda, Calif. (2000), each of which references are entirely
incorporated herein by reference.
[0213] Mimetibodies can also be used ex vivo, such as in autologous
marrow culture. Briefly, bone marrow is removed from a patient
prior to chemotherapy and treated with TPO and/or EPO, optionally
in combination with mimetibodies, optionally in combination with
one or more additional cytokines. The treated marrow is then
returned to the patient after chemotherapy to speed the recovery of
the marrow. In addition, TPO, alone and in combination with EPO
mimetibodies and/or EPO, can also be used for the ex vivo expansion
of marrow or peripheral blood progenitor (PBPC) cells. Prior to
chemotherapy treatment, marrow can be stimulated with stem cell
factor (SCF) or G-CSF to release early progenitor cells into
peripheral circulation. These progenitors are optionally collected
and concentrated from peripheral blood and then treated in culture
with TPO and mimetibodies, optionally in combination with one or
more other cytokines, including but not limited to SCF, G-CSF,
IL-3, GM-CSF, IL-6 or IL-11, to differentiate and proliferate into
high-density megakaryocyte cultures, which are optionally then be
returned to the patient following high-dose chemotherapy. Doses of
TPO for ex vivo treatment of bone marrow will be in the range of
100 pg/ml to 10 ng/ml, preferably 500 pg/ml to 3 ng/ml. Doses of
mimetibodies will be equivalent in activity to EPO which can be
used from 0.1 units/ml to 20 units/ml, preferably from 0.5 units/ml
to 2 units/ml, or any range or value therein.
[0214] TNF antagonists suitable for compositions, combination
therapy, co-administration, devices and/or methods of the present
invention (further comprising at least one anti body, specified
portion and variant thereof, of the present invention), include,
but are not limited to, anti-TNF antibodies, ligand-binding
fragments thereof, and receptor molecules which bind specifically
to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF
release or its action on target cells, such as thalidomide,
tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and
rolipram), A2b adenosine receptor agonists and A2b adenosine
receptor enhancers; compounds which prevent and/or inhibit TNF
receptor signalling, such as mitogen activated protein (MAP) kinase
inhibitors; compounds which block and/or inhibit membrane TNF
cleavage, such as metalloproteinase inhibitors; compounds which
block and/or inhibit TNF activity, such as angiotensin converting
enzyme (ACE) inhibitors (e.g., captopril); and compounds which
block and/or inhibit TNF production and/or synthesis, such as MAP
kinase inhibitors.
[0215] As used herein, a "tumor necrosis factor antibody," "TNF
antibody," "TNF.alpha. antibody," or fragment and the like
decreases, blocks, inhibits, abrogates or interferes with
TNF.alpha. activity in vitro, in situ and/or preferably in vivo.
For example, a suitable TNF human antibody of the present invention
can bind TNF.alpha. and includes anti-TNF antibodies,
antigen-binding fragments thereof, and specified mutants or domains
thereof that bind specifically to TNF.alpha.. A suitable TNF
antibody or fragment can also decrease block, abrogate, interfere,
prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF
release, TNF receptor signaling, membrane TNF cleavage, TNF
activity, TNF production and/or synthesis.
[0216] Chimeric antibody cA2 consists of the antigen binding
variable region of the high-affinity neutralizing mouse anti-human
TNF.alpha. IgG1 antibody, designated A2, and the constant regions
of a human IgG1, kappa immunoglobulin. The human IgG1 Fc region
improves allogeneic antibody effector function, increases the
circulating serum half-life and decreases the immunogenicity of the
antibody. The avidity and epitope specificity of the chimeric
antibody cA2 is derived from the variable region of the murine
antibody A2. In a particular embodiment, a preferred source for
nucleic acids encoding the variable region of the murine antibody
A2 is the A2 hybridoma cell line.
[0217] Chimeric A2 (cA2) neutralizes the cytotoxic effect of both
natural and recombinant human TNF.alpha. in a dose dependent
manner. From binding assays of chimeric antibody cA2 and
recombinant human TNF.alpha., the affinity constant of chimeric
antibody cA2 was calculated to be 1.04.times.10.sup.10M.sup.-1.
Preferred methods for determining monoclonal antibody specificity
and affinity by competitive inhibition can be found in Harlow, et
al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, N.Y., 1988; Colligan et al., eds.,
Current Protocols in Immunology, Greene Publishing Assoc. and Wiley
Interscience, New York, (1992-2003); Kozbor et al., Immunol. Today,
4:72-79 (1983); Ausubel et al., eds. Current Protocols in Molecular
Biology, Wiley Interscience, New York (1987-2003); and Muller,
Meth. Enzymol., 92:589-601 (1983), which references are entirely
incorporated herein by reference.
[0218] In a particular embodiment, murine monoclonal antibody A2 is
produced by a cell line designated c134A. Chimeric antibody cA2 is
produced by a cell line designated c168A.
[0219] Additional examples of monoclonal anti-TNF antibodies that
can be used in the present invention are described in the art (see,
e.g., U.S. Pat. No. 5,231,024; Moller, A. et al., Cytokine
2(3):162-169 (1990); U.S. application Ser. No. 07/943,852 (filed
Sep. 11, 1992); Rathjen et al., International Publication No. WO
91/02078 (published Feb. 21, 1991); Rubin et al., EPO Patent
Publication No. 0 218 868 (published Apr. 22, 1987); Yone et al.,
EPO Patent Publication No. 0 288 088 (Oct. 26, 1988); Liang, et
al., Biochem. Biophys. Res. Comm. 137:847-854 (1986); Meager, et
al., Hybridoma 6:305-311 (1987); Fendly et al., Hybridoma 6:359-369
(1987); Bringman, et al., Hybridoma 6:489-507 (1987); and Hirai, et
al., J. Immunol. Meth. 96:57-62 (1987), which references are
entirely incorporated herein by reference).
TNF Receptor Molecules
[0220] Preferred TNF receptor molecules useful in the present
invention are those that bind TNF.alpha. with high affinity (see,
e.g., Feldmann et al., International Publication No. WO 92/07076
(published Apr. 30, 1992); Schall et al., Cell 61:361-370 (1990);
and Loetscher et al., Cell 61:351-359 (1990), which references are
entirely incorporated herein by reference) and optionally possess
low immunogenicity. In particular, the 55 kDa (p55 TNF-R) and the
75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the
present invention. Truncated forms of these receptors, comprising
the extracellular domains (ECD) of the receptors or functional
portions thereof (see, e.g., Corcoran et al., Eur. J. Biochem.
223:831-840 (1994)), are also useful in the present invention.
Truncated forms of the TNF receptors, comprising the ECD, have been
detected in urine and serum as 30 kDa and 40 kDa TNF.alpha.
inhibitory binding proteins (Engelmann, H. et al., J. Biol. Chem.
265:1531-1536 (1990)). TNF receptor multimeric molecules and TNF
immunoreceptor fusion molecules, and derivatives and fragments or
portions thereof, are additional examples of TNF receptor molecules
which are useful in the methods and compositions of the present
invention. The TNF receptor molecules which can be used in the
invention are characterized by their ability to treat patients for
extended periods with good to excellent alleviation of symptoms and
low toxicity. Low immunogenicity and/or high affinity, as well as
other undefined properties, may contribute to the therapeutic
results achieved.
[0221] TNF receptor multimeric molecules useful in the present
invention comprise all or a functional portion of the ECD of two or
more TNF receptors linked via one or more polypeptide linkers or
other nonpeptide linkers, such as polyethylene glycol (PEG). The
multimeric molecules can further comprise a signal peptide of a
secreted protein to direct expression of the multimeric molecule.
These multimeric molecules and methods for their production have
been described in U.S. application Ser. No. 08/437,533 (filed May
9, 1995), the content of which is entirely incorporated herein by
reference.
[0222] TNF immunoreceptor fusion molecules useful in the methods
and compositions of the present invention comprise at least one
portion of one or more immunoglobulin molecules and all or a
functional portion of one or more TNF receptors. These
immunoreceptor fusion molecules can be assembled as monomers, or
hetero- or homo-multimers. The immunoreceptor fusion molecules can
also be monovalent or multivalent. An example of such a TNF
immunoreceptor fusion molecule is TNF receptor/IgG fusion protein.
TNF immunoreceptor fusion molecules and methods for their
production have been described in the art (Lesslauer et al., Eur.
J. Immunol. 21:2883-2886 (1991); Ashkenazi et al., Proc. Natl.
Acad. Sci. USA 88:10535-10539 (1991); Peppel et al., J. Exp. Med.
174:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA
91:215-219 (1994); Butler et al., Cytokine 6(6):616-623 (1994);
Baker et al., Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al.,
U.S. Pat. No. 5,447,851; and U.S. application Ser. No. 08/442,133
(filed May 16, 1995), each of which references are entirely
incorporated herein by reference). Methods for producing
immunoreceptor fusion molecules can also be found in Capon et al.,
U.S. Pat. No. 5,116,964; Capon et al., U.S. Pat. No. 5,225,538; and
Capon et al., Nature 337:525-531 (1989), which references are
entirely incorporated herein by reference.
[0223] A functional equivalent, derivative, fragment or region of
TNF receptor molecule refers to the portion of the TNF receptor
molecule, or the portion of the TNF receptor molecule sequence
which encodes TNF receptor molecule, that is of sufficient size and
sequences to functionally resemble TNF receptor molecules that can
be used in the present invention (e.g., bind TNF.alpha. with high
affinity and possess low immunogenicity). A functional equivalent
of TNF receptor molecule also includes modified TNF receptor
molecules that functionally resemble TNF receptor molecules that
can be used in the present invention (e.g., bind TNF.alpha. with
high affinity and possess low immunogenicity). For example, a
functional equivalent of TNF receptor molecule can contain a
"SILENT" codon or one or more amino acid substitutions, deletions
or additions (e.g., substitution of one acidic amino acid for
another acidic amino acid; or substitution of one codon encoding
the same or different hydrophobic amino acid for another codon
encoding a hydrophobic amino acid). See Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Assoc. and
Wiley-Interscience, New York (1987-2003).
[0224] Cytokines include, but are not limited to all known
cytokines. See, e.g., CopewithCytokines.com. Cytokine antagonists
include, but are not limited to, any antibody, fragment or mimetic,
any soluble receptor, fragment or mimetic, any small molecule
antagonist, or any combination thereof.
[0225] Any method of the present invention can comprise a method
for treating a protein mediated disorder, comprising administering
an effective amount of a composition or pharmaceutical composition
comprising at least one EPO mimetic hinge core mimetibody or
specified portion or variant to a cell, tissue, organ, animal or
patient in need of such modulation, treatment or therapy. Such a
method can optionally further comprise co-administration or
combination therapy for treating such immune diseases, wherein the
administering of said at least one EPO mimetic hinge core
mimetibody, specified portion or variant thereof, further comprises
administering, before concurrently, and/or after, at least one
selected from at least one other cytokines such as IL-3, -6 and
-11; stem cell factor; G-CSF and GM-CSF.
[0226] Typically, treatment of pathologic conditions is effected by
administering an effective amount or dosage of at least one EPO
mimetic hinge core mimetibody composition that total, on average, a
range from at least about 0.01 to 500 milligrams of at least one
EPO mimetic hinge core mimetibody or specified portion or
variant/kilogram of patient per dose, and preferably from at least
about 0.1 to 100 milligrams EPO mimetic hinge core mimetibody or
specified portion or variant/kilogram of patient per single or
multiple administration, depending upon the specific activity of
contained in the composition. Alternatively, the effective serum
concentration can comprise 0.1-5000 mg/ml serum concentration per
single or multiple administration. Suitable dosages are known to
medical practitioners and will, of course, depend upon the
particular disease state, specific activity of the composition
being administered, and the particular patient undergoing
treatment. In some instances, to achieve the desired therapeutic
amount, it can be necessary to provide for repeated administration,
i.e., repeated individual administrations of a particular monitored
or metered dose, where the individual administrations are repeated
until the desired daily dose or effect is achieved.
[0227] Preferred doses can optionally include 0.01, 0.02, 0.03,
0.04, 0.05. 0.06, 0.07, 0.08, 009, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,
0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30
mg/kg/administration, or any range, value or fraction thereof, or
to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2,
1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5,
5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9,
10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9,
14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0,
8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5,
12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9, 16, 16.5, 16.9,
17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900,
1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000
.mu.g/ml serum concentration per single or multiple administration,
or any range, value or fraction thereof.
[0228] Alternatively, the dosage administered can vary depending
upon known factors, such as the pharmacodynamic characteristics of
the particular agent, and its mode and route of administration;
age, health, and weight of the recipient; nature and extent of
symptoms, kind of concurrent treatment, frequency of treatment, and
the effect desired. Usually a dosage of active ingredient can be
about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily
0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per
administration or in sustained release form is effective to obtain
desired results.
[0229] As a non-limiting example, treatment of humans or animals
can be provided as a one-time or periodic dosage of at least one
EPO mimetic hinge core mimetibody or specified portion or variant
of the present invention 0.01 to 100 mg/kg, such as 0.5, 0.9, 1.0,
1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60,
70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,
22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,
39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any
combination thereof, using single, infusion or repeated doses.
[0230] Dosage forms (composition) suitable for internal
administration generally contain from about 0.0001 milligram to
about 500 milligrams of active ingredient per unit or container. In
these pharmaceutical compositions the active ingredient will
ordinarily be present in an amount of about 0.5-95% by weight based
on the total weight of the composition.
[0231] For parenteral administration, the EPO mimetic hinge core
mimetibody or specified portion or variant can be formulated as a
solution, suspension, emulsion or lyophilized powder in
association, or separately provided, with a pharmaceutically
acceptable parenteral vehicle. Examples of such vehicles are water,
saline, Ringer's solution, dextrose solution, and 5% human serum
albumin. Liposomes and nonaqueous vehicles such as fixed oils may
also be used. The vehicle or lyophilized powder may contain
additives that maintain isotonicity (e.g., sodium chloride,
mannitol) and chemical stability (e.g., buffers and preservatives).
The formulation is sterilized by known or suitable techniques.
[0232] Suitable pharmaceutical carriers are described in the most
recent edition of Remington's Pharmaceutical Sciences, A. Osol, a
standard reference text in this field.
Therapeutic Administration
[0233] Many known and developed modes of can be used according to
the present invention for administering pharmaceutically effective
amounts of at least one EPO mimetic hinge core mimetibody or
specified portion or variant according to the present invention.
While pulmonary administration is used in the following
description, other modes of administration can be used according to
the present invention with suitable results.
[0234] An EPO mimetic hinge core mimetibody of the present
invention can be delivered in a carrier, as a solution, emulsion,
colloid, or suspension, or as a powder, using any of a variety of
devices and methods suitable for administration by inhalation or
other modes described here within or known in the art.
Parenteral Formulations and Administration
[0235] Formulations for parenteral administration can contain as
common excipients sterile water or saline, polyalkylene glycols
such as polyethylene glycol, oils of vegetable origin, hydrogenated
naphthalenes and the like. Aqueous or oily suspensions for
injection can be prepared by using an appropriate emulsifier or
humidifier and a suspending agent, according to known methods.
Agents for injection can be a non-toxic, non-orally administrable
diluting agent such as aqueous solution or a sterile injectable
solution or suspension in a solvent. As the usable vehicle or
solvent, water, Ringer's solution, isotonic saline, etc. are
allowed; as an ordinary solvent, or suspending solvent, sterile
involatile oil can be used. For these purposes, any kind of
involatile oil and fatty acid can be used, including natural or
synthetic or semisynthetic fatty oils or fatty acids; natural or
synthetic or semisynthetic mono- or di- or tri-glycerides. Parental
administration is known in the art and includes, but is not limited
to, conventional means of injections, a gas pressured needle-less
injection device as described in U.S. Pat. No. 5,851,198, and a
laser perforator device as described in U.S. Pat. No. 5,839,446
entirely incorporated herein by reference.
Alternative Delivery
[0236] The invention further relates to the administration of at
least one EPO mimetic hinge core mimetibody or specified portion or
variant by parenteral, subcutaneous, intramuscular, intravenous,
bolus, vaginal, rectal, buccal, sublingual, intranasal, or
transdermal means. Protein, EPO mimetic hinge core mimetibody or
specified portion or variant compositions can be prepared for use
for parenteral (subcutaneous, intramuscular or intravenous)
administration particularly in the form of liquid solutions or
suspensions; for use in vaginal or rectal administration
particularly in semisolid forms such as creams and suppositories;
for buccal, or sublingual administration particularly in the form
of tablets or capsules; or intranasally particularly in the form of
powders, nasal drops or aerosols or certain agents; or
transdermally particularly in the form of a gel, ointment, lotion,
suspension or patch delivery system with chemical enhancers such as
dimethyl sulfoxide to either modify the skin structure or to
increase the drug concentration in the transdermal patch
(Junginger, et al. In "Drug Permeation Enhancement"; Hsieh, D. S.,
Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely
incorporated herein by reference), or with oxidizing agents that
enable the application of formulations containing proteins and
peptides onto the skin (WO 98/53847), or applications of electric
fields to create transient transport pathways such as
electroporation, or to increase the mobility of charged drugs
through the skin such as iontophoresis, or application of
ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and
4,767,402) (the above publications and patents being entirely
incorporated herein by reference).
Pulmonary/Nasal Administration
[0237] For pulmonary administration, preferably at least one EPO
mimetic hinge core mimetibody or specified portion or variant
composition is delivered in a particle size effective for reaching
the lower airways of the lung or sinuses. According to the
invention, at least one EPO mimetic hinge core mimetibody or
specified portion or variant can be delivered by any of a variety
of inhalation or nasal devices known in the art for administration
of a therapeutic agent by inhalation. These devices capable of
depositing aerosolized formulations in the sinus cavity or alveoli
of a patient include metered dose inhalers, nebulizers, dry powder
generators, sprayers, and the like. Other devices suitable for
directing the pulmonary or nasal administration of EPO mimetic
hinge core mimetibody or specified portion or variants are also
known in the art. All such devices can use of formulations suitable
for the administration for the dispensing of EPO mimetic hinge core
mimetibody or specified portion or variant in an aerosol. Such
aerosols can be comprised of either solutions (both aqueous and non
aqueous) or solid particles. Metered dose inhalers like the
Ventolin.RTM. metered dose inhaler, typically use a propellent gas
and require actuation during inspiration (See, e.g., WO 94/16970,
WO 98/35888). Dry powder inhalers like Turbuhaler.TM. (Astra),
Rotahaler.RTM. (Glaxo), Diskus.RTM. (Glaxo), Spiros.TM. inhaler
(Dura), devices marketed by Inhale Therapeutics, and the
Spinhaler.RTM. powder inhaler (Fisons), use breath-actuation of a
mixed powder (U.S. Pat. No. 4,668,218 Astra, EP 237507 Astra, WO
97/25086 Glaxo, WO 94/08552 Dura, U.S. Pat. No. 5,458,135 Inhale,
WO 94/06498 Fisons, entirely incorporated herein by reference).
Nebulizers like AERx.TM. Aradigm, the Ultravent.RTM. nebulizer
(Mallinckrodt), and the Acorn nebulizer (Marquest Medical Products)
(U.S. Pat. No. 5,404,871 Aradigm, WO 97/22376), the above
references entirely incorporated herein by reference, produce
aerosols from solutions, while metered dose inhalers, dry powder
inhalers, etc. generate small particle aerosols. These specific
examples of commercially available inhalation devices are intended
to be a representative of specific devices suitable for the
practice of this invention, and are not intended as limiting the
scope of the invention. Preferably, a composition comprising at
least one EPO mimetic hinge core mimetibody or specified portion or
variant is delivered by a dry powder inhaler or a sprayer. There
are a several desirable features of an inhalation device for
administering at least one EPO mimetic hinge core mimetibody or
specified portion or variant of the present invention. For example,
delivery by the inhalation device is advantageously reliable,
reproducible, and accurate. The inhalation device can optionally
deliver small dry particles, e.g. less than about 10 .mu.m,
preferably about 1-5 .mu.m, for good respirability.
Administration of EPO Mimetic Hinge Core Mimetibody or Specified
Portion or Variant Compositions as a Spray
[0238] A spray including EPO mimetic hinge core mimetibody or
specified portion or variant composition protein can be produced by
forcing a suspension or solution of at least one EPO mimetic hinge
core mimetibody or specified portion or variant through a nozzle
under pressure. The nozzle size and configuration, the applied
pressure, and the liquid feed rate can be chosen to achieve the
desired output and particle size. An electrospray can be produced,
for example, by an electric field in connection with a capillary or
nozzle feed. Advantageously, particles of at least one EPO mimetic
hinge core mimetibody or specified portion or variant composition
protein delivered by a sprayer have a particle size less than about
10 .mu.m, preferably in the range of about 1 .mu.m to about 5
.mu.m, and most preferably about 2 .mu.m to about 3 .mu.m.
[0239] Formulations of at least one EPO mimetic hinge core
mimetibody or specified portion or variant composition protein
suitable for use with a sprayer typically include EPO mimetic hinge
core mimetibody or specified portion or variant composition protein
in an aqueous solution at a concentration of about 1 mg to about 20
mg of at least one EPO mimetic hinge core mimetibody or specified
portion or variant composition protein per ml of solution. The
formulation can include agents such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for
stabilization of the EPO mimetic hinge core mimetibody or specified
portion or variant composition protein, such as a buffer, a
reducing agent, a bulk protein, or a carbohydrate. Bulk proteins
useful in formulating EPO mimetic hinge core mimetibody or
specified portion or variant composition proteins include albumin,
protamine, or the like. Typical carbohydrates useful in formulating
EPO mimetic hinge core mimetibody or specified portion or variant
composition proteins include sucrose, mannitol, lactose, trehalose,
glucose, or the like. The EPO mimetic hinge core mimetibody or
specified portion or variant composition protein formulation can
also include a surfactant, which can reduce or prevent
surface-induced aggregation of the EPO mimetic hinge core
mimetibody or specified portion or variant composition protein
caused by atomization of the solution in forming an aerosol.
Various conventional surfactants can be employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene
sorbitol fatty acid esters. Amounts will generally range between
0.001 and 14% by weight of the formulation. Especially preferred
surfactants for purposes of this invention are polyoxyethylene
sorbitan monooleate, polysorbate 80, polysorbate 20, or the like.
Additional agents known in the art for formulation of a protein
such as mimetibodies, or specified portions or variants, can also
be included in the formulation.
Administration of EPO Mimetic Hinge Core Mimetibody or Specified
Portion or Variant Compositions by a Nebulizer
[0240] EPO mimetic hinge core mimetibody or specified portion or
variant composition protein can be administered by a nebulizer,
such as jet nebulizer or an ultrasonic nebulizer. Typically, in a
jet nebulizer, a compressed air source is used to create a
high-velocity air jet through an orifice. As the gas expands beyond
the nozzle, a low-pressure region is created, which draws a
solution of EPO mimetic hinge core mimetibody or specified portion
or variant composition protein through a capillary tube connected
to a liquid reservoir. The liquid stream from the capillary tube is
sheared into unstable filaments and droplets as it exits the tube,
creating the aerosol. A range of configurations, flow rates, and
baffle types can be employed to achieve the desired performance
characteristics from a given jet nebulizer. In an ultrasonic
nebulizer, high-frequency electrical energy is used to create
vibrational, mechanical energy, typically employing a piezoelectric
transducer. This energy is transmitted to the formulation of EPO
mimetic hinge core mimetibody or specified portion or variant
composition protein either directly or through a coupling fluid,
creating an aerosol including the EPO mimetic hinge core mimetibody
or specified portion or variant composition protein.
Advantageously, particles of EPO mimetic hinge core mimetibody or
specified portion or variant composition protein delivered by a
nebulizer have a particle size less than about 10 .mu.m, preferably
in the range of about 1 .mu.m to about 5 .mu.m, and most preferably
about 2 .mu.m to about 3 .mu.m.
[0241] Formulations of at least one EPO mimetic hinge core
mimetibody or specified portion or variant suitable for use with a
nebulizer, either jet or ultrasonic, typically include EPO mimetic
hinge core mimetibody or specified portion or variant composition
protein in an aqueous solution at a concentration of about 1 mg to
about 20 mg of at least one EPO mimetic hinge core mimetibody or
specified portion or variant protein per ml of solution. The
formulation can include agents such as an excipient, a buffer, an
isotonicity agent, a preservative, a surfactant, and, preferably,
zinc. The formulation can also include an excipient or agent for
stabilization of the at least one EPO mimetic hinge core mimetibody
or specified portion or variant composition protein, such as a
buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk
proteins useful in formulating at least one EPO mimetic hinge core
mimetibody or specified portion or variant composition proteins
include albumin, protamine, or the like. Typical carbohydrates
useful in formulating at least one EPO mimetic hinge core
mimetibody or specified portion or variant include sucrose,
mannitol, lactose, trehalose, glucose, or the like. The at least
one EPO mimetic hinge core mimetibody or specified portion or
variant formulation can also include a surfactant, which can reduce
or prevent surface-induced aggregation of the at least one EPO
mimetic hinge core mimetibody or specified portion or variant
caused by atomization of the solution in forming an aerosol.
Various conventional surfactants can be employed, such as
polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene
sorbital fatty acid esters. Amounts will generally range between
0.001 and 4% by weight of the formulation. Especially preferred
surfactants for purposes of this invention are polyoxyethylene
sorbitan mono-oleate, polysorbate 80, polysorbate 20, or the like.
Additional agents known in the art for formulation of a protein
such as at least one EPO mimetic hinge core mimetibody or specified
portion or variant protein can also be included in the
formulation.
Administration of EPO Mimetic Hinge Core Mimetibody or Specified
Portion or Variant Compositions by a Metered Dose Inhaler
[0242] In a metered dose inhaler (MDI), a propellant, at least one
EPO mimetic hinge core mimetibody or specified portion or variant,
and any excipients or other additives are contained in a canister
as a mixture including a liquefied compressed gas. Actuation of the
metering valve releases the mixture as an aerosol, preferably
containing particles in the size range of less than about 10 .mu.m,
preferably about 1 .mu.m to about 5 .mu.m, and most preferably
about 2 .mu.m to about 3 .mu.m. The desired aerosol particle size
can be obtained by employing a formulation of EPO mimetic hinge
core mimetibody or specified portion or variant composition protein
produced by various methods known to those of skill in the art,
including jet-milling, spray drying, critical point condensation,
or the like. Preferred metered dose inhalers include those
manufactured by 3M or Glaxo and employing a hydrofluorocarbon
propellant.
[0243] Formulations of at least one EPO mimetic hinge core
mimetibody or specified portion or variant for use with a
metered-dose inhaler device will generally include a finely divided
powder containing at least one EPO mimetic hinge core mimetibody or
specified portion or variant as a suspension in a non-aqueous
medium, for example, suspended in a propellant with the aid of a
surfactant. The propellant can be any conventional material
employed for this purpose, such as chlorofluorocarbon, a
hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon,
including trichlorofluoromethane, dichlorodifluoromethane,
dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a
(hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227), or the
like. Preferably the propellant is a hydrofluorocarbon. The
surfactant can be chosen to stabilize the at least one EPO mimetic
hinge core mimetibody or specified portion or variant as a
suspension in the propellant, to protect the active agent against
chemical degradation, and the like. Suitable surfactants include
sorbitan trioleate, soya lecithin, oleic acid, or the like. In some
cases solution aerosols are preferred using solvents such as
ethanol. Additional agents known in the art for formulation of a
protein such as protein can also be included in the
formulation.
[0244] One of ordinary skill in the art will recognize that the
methods of the current invention can be achieved by pulmonary
administration of at least one EPO mimetic hinge core mimetibody or
specified portion or variant compositions via devices not described
herein.
Mucosal Formulations and Administration
[0245] For absorption through mucosal surfaces, compositions and
methods of administering at least one EPO mimetic hinge core
mimetibody or specified portion or variant include an emulsion
comprising a plurality of submicron particles, a mucoadhesive
macromolecule, a bioactive peptide, and an aqueous continuous
phase, which promotes absorption through mucosal surfaces by
achieving mucoadhesion of the emulsion particles (U.S. Pat. No.
5,514,670). Mucous surfaces suitable for application of the
emulsions of the present invention can include corneal,
conjunctival, buccal, sublingual, nasal, vaginal, pulmonary,
stomachic, intestinal, and rectal routes of administration.
Formulations for vaginal or rectal administration, e.g.
suppositories, can contain as excipients, for example,
polyalkyleneglycols, vaseline, cocoa butter, and the like.
Formulations for intranasal administration can be solid and contain
as excipients, for example, lactose or can be aqueous or oily
solutions of nasal drops. For buccal administration excipients
include sugars, calcium stearate, magnesium stearate,
pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
Oral Formulations and Administration
[0246] Formulations for oral rely on the co-administration of
adjuvants (e.g., resorcinols and nonionic surfactants such as
polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to
increase artificially the permeability of the intestinal walls, as
well as the co-administration of enzymatic inhibitors (e.g.,
pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and
trasylol) to inhibit enzymatic degradation. The active constituent
compound of the solid-type dosage form for oral administration can
be mixed with at least one additive, including sucrose, lactose,
cellulose, mannitol, trehalose, raffinose, maltitol, dextran,
starches, agar, arginates, chitins, chitosans, pectins, gum
tragacanth, gum arabic, gelatin, collagen, casein, albumin,
synthetic or semisynthetic polymer, and glyceride. These dosage
forms can also contain other type(s) of additives, e.g., inactive
diluting agent, lubricant such as magnesium stearate, paraben,
preserving agent such as sorbic acid, ascorbic acid,
.alpha.-tocopherol, antioxidant such as cysteine, disintegrator,
binder, thickener, buffering agent, sweetening agent, flavoring
agent, perfuming agent, etc.
[0247] Tablets and pills can be further processed into
enteric-coated preparations. The liquid preparations for oral
administration include emulsion, syrup, elixir, suspension and
solution preparations allowable for medical use. These preparations
may contain inactive diluting agents ordinarily used in said field,
e.g., water. Liposomes have also been described as drug delivery
systems for insulin and heparin (U.S. Pat. No. 4,239,754). More
recently, microspheres of artificial polymers of mixed amino acids
(proteinoids) have been used to deliver pharmaceuticals (U.S. Pat.
No. 4,925,673). Furthermore, carrier compounds described in U.S.
Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to
deliver biologically active agents orally are known in the art.
Transdermal Formulations and Administration
[0248] For transdermal administration, the at least one EPO mimetic
hinge core mimetibody or specified portion or variant is
encapsulated in a delivery device such as a liposome or polymeric
nanoparticles, microparticle, microcapsule, or microspheres
(referred to collectively as microparticles unless otherwise
stated). A number of suitable devices are known, including
microparticles made of synthetic polymers such as polyhydroxy acids
such as polylactic acid, polyglycolic acid and copolymers thereof,
polyorthoesters, polyanhydrides, and polyphosphazenes, and natural
polymers such as collagen, polyamino acids, albumin and other
proteins, alginate and other polysaccharides, and combinations
thereof (U.S. Pat. Nos. 5,814,599).
Prolonged Administration and Formulations
[0249] It can be sometimes desirable to deliver the compounds of
the present invention to the subject over prolonged periods of
time, for example, for periods of one week to one year from a
single administration. Various slow release, depot or implant
dosage forms can be utilized. For example, a dosage form can
contain a pharmaceutically acceptable non-toxic salt of the
compounds that has a low degree of solubility in body fluids, for
example, (a) an acid addition salt with a polybasic acid such as
phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic
acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene
mono- or di-sulfonic acids, polygalacturonic acid, and the like;
(b) a salt with a polyvalent metal cation such as zinc, calcium,
bismuth, barium, magnesium, aluminum, copper, cobalt, nickel,
cadmium and the like, or with an organic cation formed from e.g.,
N,N'-dibenzyl-ethylenediamine or ethylenediamine; or (c)
combinations of (a) and (b) e.g. a zinc tannate salt. Additionally,
the compounds of the present invention or, preferably, a relatively
insoluble salt such as those just described, can be formulated in a
gel, for example, an aluminum monostearate gel with, e.g. sesame
oil, suitable for injection. Particularly preferred salts are zinc
salts, zinc tannate salts, pamoate salts, and the like. Another
type of slow release depot formulation for injection would contain
the compound or salt dispersed for encapsulated in a slow
degrading, non-toxic, non-antigenic polymer such as a polylactic
acid/polyglycolic acid polymer for example as described in U.S.
Pat. No. 3,773,919. The compounds or, preferably, relatively
insoluble salts such as those described above can also be
formulated in cholesterol matrix silastic pellets, particularly for
use in animals. Additional slow release, depot or implant
formulations, e.g. gas or liquid liposomes are known in the
literature (U.S. Pat. Nos. 5,770,222 and "Sustained and Controlled
Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker,
Inc., N.Y., 1978).
[0250] Having generally described the invention, the same will be
more readily understood by reference to the following examples,
which are provided by way of illustration and are not intended as
limiting.
Example 1
Cloning and Expression of an EPO Mimetic Hinge Core Mimetibody in
Mammalian Cells
[0251] A typical mammalian expression vector contains at least one
promoter element, which mediates the initiation of transcription of
mRNA, the EPO mimetic hinge core mimetibody or specified portion or
variant coding sequence, and signals required for the termination
of transcription and polyadenylation of the transcript. Additional
elements include enhancers, Kozak sequences and intervening
sequences flanked by donor and acceptor sites for RNA splicing.
Highly efficient transcription can be achieved with the early and
late promoters from SV40, the long terminal repeats (LTRS) from
Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the
cytomegalovirus (CMV). However, cellular elements can also be used
(e.g., the human actin promoter). Suitable expression vectors for
use in practicing the present invention include, for example,
vectors such as pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX
(Clonetech Labs, Palo Alto, Calif.), pcDNA3.1 (+/-), pcDNA/Zeo
(+/-) or pcDNA3.1/Hygro (+/-) (Invitrogen), PSVL and PMSG
(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC
37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be
used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and
C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells
and Chinese hamster ovary (CHO) cells.
[0252] Alternatively, the gene can be expressed in stable cell
lines that contain the gene integrated into a chromosome. The
co-transfection with a selectable marker such as dhfr, gpt,
neomycin, or hygromycin allows the identification and isolation of
the transfected cells.
[0253] The transfected gene can also be amplified to express large
amounts of the encoded EPO mimetic hinge core mimetibody or
specified portion or variant. The DHFR (dihydrofolate reductase)
marker is useful to develop cell lines that carry several hundred
or even several thousand copies of the gene of interest. Another
useful selection marker is the enzyme glutamine synthase (GS)
(Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et
al., Bio/Technology 10:169-175 (1992)). Using these markers, the
mammalian cells are grown in selective medium and the cells with
the highest resistance are selected. These cell lines contain the
amplified gene(s) integrated into a chromosome. Chinese hamster
ovary (CHO) and NSO cells are often used for the production of EPO
mimetic hinge core mimetibody or specified portion or variants.
[0254] The expression vectors pC1 and pC4 contain the strong
promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec.
Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer
(Boshart, et al., Cell 41:521-530 (1985)). Multiple cloning sites,
e.g., with the restriction enzyme cleavage sites BamHI, XbaI and
Asp718, facilitate the cloning of the gene of interest. The vectors
contain in addition the 3' intron, the polyadenylation and
termination signal of the rat preproinsulin gene.
Cloning and Expression in CHO Cells
[0255] The vector pC4 is used for the expression of EPO mimetic
hinge core mimetibody or specified portion or variant. Plasmid pC4
is a derivative of the plasmid pSV2-dhfr (ATCC Accession No.
37146). The plasmid contains the mouse DHFR gene under control of
the SV40 early promoter. Chinese hamster ovary- or other cells
lacking dihydrofolate activity that are transfected with these
plasmids can be selected by growing the cells in a selective medium
(e.g., alpha minus MEM, Life Technologies, Gaithersburg, Md.)
supplemented with the chemotherapeutic agent methotrexate. The
amplification of the DHFR genes in cells resistant to methotrexate
(MTX) has been well documented (see, e.g., F. W. Alt, et al., J.
Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem.
et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A.
Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing
concentrations of MTX develop resistance to the drug by
overproducing the target enzyme, DHFR, as a result of amplification
of the DHFR gene. If a second gene is linked to the DHFR gene, it
is usually co-amplified and over-expressed. It is known in the art
that this approach can be used to develop cell lines carrying more
than 1,000 copies of the amplified gene(s). Subsequently, when the
methotrexate is withdrawn, cell lines are obtained that contain the
amplified gene integrated into one or more chromosome(s) of the
host cell.
[0256] Plasmid pC4 contains for expressing the gene of interest the
strong promoter of the long terminal repeat (LTR) of the Rous
Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985))
plus a fragment isolated from the enhancer of the immediate early
gene of human cytomegalovirus (CMV) (Boshart, et al., Cell
41:521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and
Asp718 restriction enzyme cleavage sites that allow integration of
the genes. Behind these cloning sites the plasmid contains the 3'
intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the
expression, e.g., the human b-actin promoter, the SV40 early or
late promoters or the long terminal repeats from other
retroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On
gene expression systems and similar systems can be used to express
the EPO in a regulated way in mammalian cells (M. Gossen, and H.
Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the
polyadenylation of the mRNA other signals, e.g., from the human
growth hormone or globin genes can be used as well. Stable cell
lines carrying a gene of interest integrated into the chromosomes
can also be selected upon co-transfection with a selectable marker
such as gpt, G418 or hygromycin. It is advantageous to use more
than one selectable marker in the beginning, e.g., G418 plus
methotrexate.
[0257] The plasmid pC4 is digested with restriction enzymes and
then dephosphorylated using calf intestinal phosphatase by
procedures known in the art. The vector is then isolated from a 1%
agarose gel.
[0258] The DNA sequence encoding the complete EPO mimetic hinge
core mimetibody or specified portion or variant is used,
corresponding to HC and LC variable regions of an EPO mimetic hinge
core mimetibody of the present invention, according to known method
steps. Isolated nucleic acid encoding a suitable human constant
region (i.e., HC and LC regions) is also used in this
construct.
[0259] The isolated variable and constant region encoding DNA and
the dephosphorylated vector are then ligated with T4 DNA ligase. E.
coli HB101 or XL-1 Blue cells are then transformed and bacteria are
identified that contain the fragment inserted into plasmid pC4
using, for instance, restriction enzyme analysis.
[0260] Chinese hamster ovary (CHO) cells lacking an active DHFR
gene are used for transfection. 5 .mu.g of the expression plasmid
pC4 is cotransfected with 0.5 .mu.g of the plasmid pSV2-neo using
lipofectin. The plasmid pSV2neo contains a dominant selectable
marker, the neo gene from Tn5 encoding an enzyme that confers
resistance to a group of antibiotics including G418. The cells are
seeded in alpha minus MEM supplemented with 1 .mu.g/ml G418. After
2 days, the cells are trypsinized and seeded in hybridoma cloning
plates (Greiner, Germany) in alpha minus MEM supplemented with 10,
25, or 50 ng/ml of methotrexate plus 1 .mu.g/ml G418. After about
10-14 days single clones are trypsinized and then seeded in 6-well
petri dishes or 10 ml flasks using different concentrations of
methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones
growing at the highest concentrations of methotrexate are then
transferred to new 6-well plates containing even higher
concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM).
The same procedure is repeated until clones are obtained that grow
at a concentration of 100-200 mM. Expression of the desired gene
product is analyzed, for instance, by SDS-PAGE and Western blot or
by reverse phase HPLC analysis.
Example 2
Non-Limiting Example of an EPO Mimetic Hinge Core Mimetibody of the
Invention
[0261] Background: EMP-1 (EPO mimetic peptide-1) is a 20 amino acid
peptide with no sequence homology to human erythropoietin (HuEPO),
but with the ability (as a dimer) to activate the EPO receptor
(Wrighton et al, 1996, Science, vol. 273, 458-463). However, its
relatively low activity (10,000 to 100,000 fold less than HuEPO)
and short half-life (ex-vivo half-life of 8 hours in 50% serum, in
vivo half-life unknown), compromise its utility as a therapeutic.
Therefore, a way was needed to confer upon the peptide a longer
half-life, without disturbing, and possibly improving its potency.
To this end, several attempts have been made to increase the
activity of EMP-1 by stabilizing the dimerization of the peptide or
by incorporating the peptide into larger structures to increase
half-life. Wrighten et al. (1997, Nature Biotechnology, vol. 15,
1261-65) combined biotin labeled EMP-1 with streptavidin to
stabilize dimerization. They saw a 100 fold increase in activity in
an in vitro cell proliferation assay. They also used anti-biotin
antibodies to stabilize the peptide dimer, however only a 10-fold
increase in activity was seen. The same authors prepared a
chemically defined dimeric form of EMP-1. In this case an 100-fold
increase in activity was seen in vivo. Another group sought to
improve the activity of EMP-1 through covalent linkage to
polyethylene glycol (PEG) (Johnson et al., 1997, Chem. & Bio.,
vol. 4(12), 939-50). They reported an increase in potency of up to
1000 fold, however the construct was found to be immunogenic in
mice (the antibodies were directed to the peptide) (Dana Johnson,
Personal communications). Kuai et al. (2000, J. Peptide Res., vol.
56, 59-62) inserted the EMP-1 peptide into the sequence of
plasminogen activator inhibitor-1, (PAI-1). It was thought that the
insertion of EMP-1 into this scaffold would both stabilize
dimerization and increase half-life. In an in vivo assay the
potency of this construct was seen to be significantly higher, such
as more than 2500 fold higher than EMP-1 alone. It should be noted
that different in vitro assays and in vivo models were used in
these studies and the reported potencies may not be comparable to
each other or to results presented herein.
EPO Mimetic Hinge Core Mimetibody of the Present Invention
[0262] A specific, non-limiting, example of this invention is the
EMP-hinge core mimetibody construct where V is the first several
N-terminal amino acids of a naturally occurring HC or LC antibody,
P is a single copy of the bioactive EMP-1 peptide and L is a tandem
repeat of either Gly-Ser or Gly-Gly-Gly-Ser flexible linker, H is a
hinge core region and CH2 & CH3 are of the IgG1 or IgG4 isotype
subclass. It is thought that this structure will constrain the
EMP-1 peptide, but allow sufficient flexibility such that the
dimerization of the peptides as part of the assembled homodimer is
stabilized. In support of this, the activity of EMP-hinge core
mimetibody in an in vitro cell proliferation assay is more than 500
fold greater than the EMP-1 peptide and only substantially similar
to recombinant HuEPO (rHuEPO). In addition, it is expected that the
half-life of this construct will be many times that of rHuEPO or
the EMP-1 peptide alone and similar to that of an IgG.
Consistently, normal mice treated with EMP-hinge core mimetibody
attain a significantly higher maximal hematocrit compared to mice
treated with rHuEPO, when equal activity units are given, and
elevated levels are maintained for a longer period. This construct
is efficiently secreted from cells and appears to be properly
folded; overcoming problems associated with 1.sup.st generation
mimetibodies.
[0263] In addition to the basic structure described above, variants
with potentially favorable biological characteristics are
described. These include constructs that may have a decreased
tendency to self-associate, reduced immune effector functions or
decreased immunogenicity. Other modifications that confer desired
characteristics such as improved conformation of the biologically
active peptide, and transfer across the blood-brain barrier are
envisioned. The proposed variants and modifications may be combined
in any fashion to yield constructs with desired activities.
[0264] Using recombinant DNA methods, the EMP-1 peptide was
inserted into an intermediate vector between an immunoglobulin
signal peptide and a human J sequence. This was done using
complementary synthetic oligonucletides with ends compatible with
the restriction sites present in the vector These oligonucleotides
comprised coding sequences for the signal peptidase consensus site
(QIQ), the EMP-1 peptide (SEQ ID NO:2), and a flexible linker
composed of either GS or GGGS. A restriction fragment containing
the above-mentioned functional elements was then transferred into
an expression vector. This vector contained the anti-CD4
immunoglobulin promoter and enhancer, and the coding sequence for a
human IgG1 hinge core sequence, and a portion of an IgG1 hinge core
region, CPPCP (109-113 of SEQ ID NO:66, as shown in FIG. 36C), an
HC constant region 2 (CH2) and constant region 3 (CH3) as well as
the necessary elements for plasmid replication and selection in
bacteria and selection for stable expressers in mammalian
cells.
[0265] This plasmid was linearized and introduced into the NSO
mouse myeloma cell line via electroporation. Resistant cells were
selected and high expressers of EMP-hinge core mimetibody were
identified by ELISA assay of culture supernatants. Purification of
the construct from cell culture supernatants was accomplished by
standard proteinA affinity chromatography. Passage of the purified
product through SDS-containing polyacrylamide gels under both
denaturing and reducing conditions confirmed the expected size of
the purified product. The identity of the purified protein was
further confirmed by mass spectrometry and N-terminal
sequencing.
[0266] The amino acid sequences of EMP-hinge core mimetibodes are
shown below. Functional domains are annotated above the peptide
coding sequence. The three amino acid signal peptide consensus
sequence corresponds to the first three amino acids of a naturally
occurring immunoglobulin. These amino acids are thought to
contribute to the efficient removal of the signal peptide by signal
peptidase in the endoplasmic reticulum. This sequence is
immediately followed by the EMP-1 coding sequence. The two
C-terminal amino acids of the EMP-1 sequence combined with the next
six amino acids form a flexible linker characterized by the
Gly-Gly-Gly-Ser repeat. A human joining (J) region sequence
follows. It is thought that the J sequence will provide even more
flexibility to allow the EMP-1 dimmer to assume the proper
conformation, and allow the dimmer to protrude from the globular
structure of the immunoglobulin and penetrate into the cleft
between two EPO receptors. The HC hinge region is also included in
the construct immediately following the J region. There are three
cysteines in the IgG1 hinge region (highlighted). The first would
normally pair to the immunoglobulin light chain (LC) and the second
two participate in interchain bonds between two HCs. The remainder
of the sequence is composed of the CH2 & CH3 regions, which
constitute the bulk of the protein. One of the reasons that
immunoglobulins are believed to have a long serum half-life is
their ability to bind the FcRn that extends the serum half-life by
returning pinocytosed immunoglobulin back to the extracellular
space. The binding site of the FcRn overlaps the junction of the
CH2 and CH3 regions (Sheilds et al, 2001, J. Biol. Chem., vol. 276
(9), 6591-6604).
[0267] The peptide sequence of EMP-hinge core mimetibody showing
important functional domains.
TABLE-US-00004 V EMP-1 Peptide Linker Hinge Ia-G1 CH2 1
QIQGGTYSCHFGPLTWVCKPQGG GS CPPCP APELLGGP (SEQ ID NO: 82) IgG1 CH2
----------------------------------------------------- 61
SVFLFPFKPKDTLMISKTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG1 CH3 122
TYRVVSVLTVLHQDWLNGKEYKCKVENKALPAPIEKTISKAKGQPREPQVYTLPPERDEL IgG1
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ IgG1
CH3 241 QGNVFSCSVMHEALHNHYTQKSLSLSPGK V EMP-1 Peptide Linker Hinge
IgG1 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GGGS CPPCP APELLGGP (SEQ ID NO:
83) IgG1 CH2 -----------------------------------------------------
61 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IcTG1 CH3 122
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL IgG1
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ IgG1
CH3 241 QGNVFSCSVMHEALHNHYTQKSLSLSPGK V EMP-1 Peptide Linker Hinge
IgG1 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GSGGGS CPPCP APELLGGP (SEQ ID
NO: 84) IgG1 CH2
----------------------------------------------------- 61
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG1 CH3 122
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL IgG1
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ IgG1
CH3 241 QGNVFSCSVMHEALHNHYTQKSLSLSPGK V EMP-1 Peptide Linker Hinge
IgG1 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GS CPPCP APEAAGGP (SEQ ID NO:
85) IgG1 CH2 -----------------------------------------------------
61 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG1 CH3 122
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL IgG1
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ IgG1
CH3 241 QGNVFSCSVMHEALHNHYTQKSLSLSPGK V EMP-1 Peptide Linker Hinge
IgG1 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GGGS CPPCP APEAAGGP (SEQ ID NO:
86) IgG1 CH2 -----------------------------------------------------
61 SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG1 CH3 122
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL IgG1
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ IgG1
CH3 241 QGNVFSCSVMHEALHNHYTQKSLSLSPGK V EMP-1 Peptide Linker Hinge
IgG4 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GS CPPCP APEFLGGP (SEQ ID NO:
87) IgG 4 CH2 --------------------------------------- 61
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG4 CH3 121
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM IgG4
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ IgG4
CH3 241 EGNVFSCSVMHEALHNHYTQKSLSLSLGK V EMP-1 Peptide Linker Hinge
IgG4 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GS CPPCP APEAAGGP (SEQ ID NO:
88) IgG 4 CH2 --------------------------------------- 61
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG4 CH3 121
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM IgG4
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ IgG4
CH3 241 EGNVFSCSVMHEALHNHYTQKSLSLSLGK V EMP--1 Peptide Linker Hinge
IgG4 CH2 1 QIQGGTYSCHFGPLTWVCKPQGG GGGS CPPCP APEAAGGP (SEQ ID NO:
89) IgG 4 CH2 --------------------------------------- 61
SVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ IgG4 CH3 121
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEM IgG4
CH3 183
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQ IgG4
CH3 241 EGNVFSCSVMHEALHNHYTQKSLSLSLGK
[0268] It is well known that two IgG heavy chains are assembled
during cellular processing via disulfide bonds between cysteines
located in the hinge region to form a homodimer. It is expected
that this will also occur between the modified peptides to form the
assembled EMP-hinge core mimetibody construct. In addition, it is
expected that the intrachain disulfide bond between the two
cysteines in the EMP-1 peptide will also form. The expected
structure of EMP-Hinge core mimetibody contains two EMP-1 peptides.
The spatial arrangement of the peptides at the N-terminus along
with the flexibility of adjoining sequences should allow the
peptides to form the bioactive dimer.
[0269] The activity of EMP-Hinge core mimetibody was first tested
in an in vitro bioactivity assay. For this assay, the EPO dependent
UT-7/EPO cell line, derived from a patient with acute
megakaryoblastic leukemia, was used (Komatsu et al., 1993, Blood,
vol. 82 (2), 456-464). These cells undergo programmed cell death 48
to 72 hours after withdraw from media supplemented with rHuEPO.
Cells that have been incubated in the absence of rHuEPO for 24
hours can be saved if treated with rHuEPO or an EPO agonist.
EMP-Hinge core mimetibody was added to cells starved without rHuEPO
and cell viability was determined 48 hours after treatment using
the tetrazolium compound MTS (CellTiter 96 Aq.sub.ueous One
Solution, Promega) that is metabolized by living cells to yield a
product with an absorbance that can be measured. Results of a
typical assay showed the potency of EMP-Hinge core mimetibody on a
molar basis to be 500 fold greater than the EMP-1 peptide and 5
fold less than rHuEPO. In addition, these same cells were
stimulated with EMP-Hinge core mimetibody and tyrosine
phosphorylation patterns visualized by running cell lysate through
a polyacrylamide gel. The pattern exhibited by EMP-Hinge core
mimetibody was similar to that of rHuEPO, indicating that the
mechanism by which EMP-Hinge core mimetibody acts on these cells is
like that of rHuEPO.
[0270] In vivo studies were done in normal mice to compare the
half-life of EMP-Hinge core mimetibody to that of rHuEPO and to
compare their effects on erythropoiesis. When mice were dosed
equally, EMP-Hinge core mimetibody gave a higher maximal response
and the response was prolonged compared to rHuEPO.
[0271] The serum concentrations of both rHuEPO and EMP-Hinge core
mimetibody were measured by ELISA. The approximate half-life of EMP
hinge core mimetibodies was at least several times that of
rHuEPO.
[0272] It has been shown that mutation of two lysine (L) residues,
L234 & L235, in the IgG1 lower hinge region to alanine (A) will
abrogate the ability of the immunoglobulin to mediate complement
dependent cytotoxicity (CDC) and antibody dependant cellular
cytotoxicity (ADCC) (Hezereh et al., 2001, J. Virol., vol. 75 (24),
12161-68). Preliminary studies have shown that EMP-Hinge core
mimetibody does not mediate complement lysis of cells that express
the EPO receptor. This may be due to the low number of receptors
that are found on erythroid progenitor cells. In addition the in
vivo expansion of erythroid progenitors as evidenced by significant
increases in hematocrit supports the possible functional
irrelevance of immune effector functions. However, while no
effector function associated affects have been observed, there
remains an interest in introducing these mutations as a
precautionary step.
[0273] Another modification that would result in a decrease in
mediation of immune effector functions is the removal of the
glycosylation attachment site. This can be accomplished by mutation
of the asparagine at position 297 (N297) to glutamine (Q).
Additional changes can optionally include replacing the threonine
(T) with an alternative amino acid to reduce or modify
O-glycosylation, e.g., T34 or T47 with Aglycosylated versions of
the IgG1 subclass are known to be poor mediators of immune effector
function (Jefferis et al. 1998, Immol Rev., vol. 163, 50-76).
[0274] Advantages: The novel construct, EMP-Hinge core mimetibody
described above offers an alternative way of displaying the
bioactive peptide EMP-1. The activity of this construct is in the
range of rHuEPO and the in vivo half-life is similar to that of an
IgG. In addition, proposed modifications are expected to, in
combination and in addition to the novel features of EMP-Hinge core
mimetibody, enhance the utility of the EMP-Hinge core mimetibody
construct.
[0275] It will be clear that the invention can be practiced
otherwise than as particularly described in the foregoing
description and examples.
[0276] Numerous modifications and variations of the present
invention are possible in light of the above teachings and,
therefore, are within the scope of the present invention
Sequence CWU 1
1
89114PRTArtificialsynthetic peptide 1Tyr Xaa Cys Xaa Xaa Gly Pro
Xaa Thr Trp Xaa Cys Xaa Pro1 5 10220PRTArtificialsynthetic peptide
2Gly Gly Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp Val Cys Lys1 5
10 15Pro Gln Gly Gly 20320PRTArtificialsynthetic peptide 3Gly Gly
Asp Tyr His Cys Arg Met Gly Pro Leu Thr Trp Val Cys Lys1 5 10 15Pro
Leu Gly Gly 20420PRTArtificialsynthetic peptide 4Gly Gly Val Tyr
Ala Cys Arg Met Gly Pro Ile Thr Trp Val Cys Ser1 5 10 15Pro Leu Gly
Gly 20520PRTArtificialsynthetic peptide 5Val Gly Asn Tyr Met Cys
His Phe Gly Pro Ile Thr Trp Val Cys Arg1 5 10 15Pro Gly Gly Gly
20620PRTArtificialsynthetic peptide 6Gly Gly Leu Tyr Leu Cys Arg
Phe Gly Pro Val Thr Trp Asp Cys Gly1 5 10 15Tyr Lys Gly Gly
20720PRTArtificialsynthetic peptide 7Gly Gly Thr Tyr Ser Cys His
Phe Gly Pro Leu Thr Trp Val Cys Lys1 5 10 15Pro Gln Gly Gly
20823PRTArtificialsynthetic peptide 8Gly Gly Thr Tyr Ser Cys His
Phe Gly Pro Leu Thr Trp Val Cys Lys1 5 10 15Pro Gln Gly Gly Ser Ser
Lys 20910PRTArtificialsynthetic peptide 9Cys Xaa Xaa Gly Pro Xaa
Thr Trp Xaa Cys1 5 101019PRTArtificialsynthetic peptide 10Gly Gly
Thr Tyr Ser Cys His Gly Pro Leu Thr Trp Val Cys Lys Pro1 5 10 15Gln
Gly Gly1119PRTArtificialsynthetic peptide 11Val Gly Asn Tyr Met Ala
His Met Gly Pro Ile Thr Trp Val Cys Arg1 5 10 15Pro Gly
Gly1218PRTArtificialsynthetic peptide 12Gly Gly Pro His His Val Tyr
Ala Cys Arg Met Gly Pro Leu Thr Trp1 5 10 15Ile
Cys1318PRTArtificialsynthetic peptide 13Gly Gly Thr Tyr Ser Cys His
Phe Gly Pro Leu Thr Trp Val Cys Lys1 5 10 15Pro
Gln1420PRTArtificialsynthetic peptide 14Gly Gly Leu Tyr Ala Cys His
Met Gly Pro Met Thr Trp Val Cys Gln1 5 10 15Pro Leu Arg Gly
201522PRTArtificialsynthetic peptide 15Thr Ile Ala Gln Tyr Ile Cys
Tyr Met Gly Pro Glu Thr Trp Glu Cys1 5 10 15Arg Pro Ser Pro Lys Ala
201613PRTArtificialsynthetic peptide 16Tyr Ser Cys His Phe Gly Pro
Leu Thr Trp Val Cys Lys1 5 101711PRTArtificialsynthetic peptide
17Tyr Cys His Phe Gly Pro Leu Thr Trp Val Cys1 5
101810PRTArtificialsynthetic peptide 18Xaa Xaa Xaa Gly Pro Xaa Thr
Trp Xaa Xaa1 5 101912PRTArtificialsynthetic peptide 19Tyr Xaa Xaa
Xaa Xaa Gly Pro Xaa Thr Trp Xaa Xaa1 5 102014PRTArtificialsynthetic
peptide 20Xaa Tyr Xaa Xaa Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa Xaa1
5 102118PRTArtificialsynthetic peptide 21Xaa Tyr Xaa Cys Xaa Xaa
Gly Pro Xaa Thr Trp Xaa Cys Xaa Xaa Xaa1 5 10 15Leu
Leu2220PRTArtificialsynthetic peptide 22Gly Gly Leu Tyr Leu Cys Arg
Phe Gly Pro Val Thr Trp Asp Cys Gly1 5 10 15Tyr Lys Gly Gly
202320PRTArtificialsynthetic peptide 23Gly Gly Thr Tyr Ser Cys His
Phe Gly Pro Leu Thr Trp Val Cys Lys1 5 10 15Pro Gln Gly Gly
202420PRTArtificialsynthetic peptide 24Val Gly Asn Tyr Met Cys His
Phe Gly Pro Ile Thr Trp Val Cys Arg1 5 10 15Pro Gly Gly Gly
202520PRTArtificialsynthetic peptide 25Gly Gly Val Tyr Ala Cys Arg
Met Gly Pro Ile Thr Trp Val Cys Ser1 5 10 15Pro Leu Gly Gly
202622PRTArtificialsynthetic peptide 26Thr Ile Ala Gln Tyr Ile Cys
Tyr Met Gly Pro Glu Thr Trp Glu Cys1 5 10 15Arg Pro Ser Pro Lys Ala
202713PRTArtificialsynthetic peptide 27Tyr Ser Cys His Phe Gly Pro
Leu Thr Trp Val Cys Lys1 5 102811PRTArtificialsynthetic peptide
28Tyr Cys His Phe Gly Pro Leu Thr Trp Val Cys1 5
102912PRTArtificialsynthetic peptide 29Ser Cys His Phe Gly Pro Leu
Thr Trp Val Cys Lys1 5 103012PRTArtificialsynthetic peptide 30Ala
Xaa Xaa Xaa Xaa Gly Pro Xaa Thr Trp Xaa Xaa1 5 1031125PRTHomo
sapiensmisc_feature(1)..(125)Vh1 heavy chain variable region 31Gln
Val Gln Leu Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly1 5 10
15Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Xaa
20 25 30Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly Xaa
Arg 35 40 45Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr Met
Glu Leu 50 55 60Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Xaa65 70 75 80Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Ser Thr Lys Gly 85 90 95Pro Ser Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly 100 105 110Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro 115 120 12532124PRTHomo
sapiensmisc_feature(1)..(124)Vh2 heavy chain variable region 32Gln
Ile Thr Leu Lys Glu Ser Gly Pro Ala Leu Val Lys Pro Thr Gln1 5 10
15Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ser Xaa Trp
20 25 30Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala Xaa Arg
Leu 35 40 45Thr Ile Thr Lys Asp Thr Ser Lys Asn Gln Val Val Leu Thr
Met Thr 50 55 60Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
Arg Xaa Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Pro Thr Ser Pro 85 90 95Lys Val Phe Pro Leu Ser Leu Ser Ser Lys
Ser Thr Ser Gly Gly Thr 100 105 110Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro 115 12033100PRTHomo
sapiensmisc_feature(1)..(100)Vh3a heavy chain variable region 33Glu
Val Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly1 5 10
15Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa
20 25 30Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Xaa
Arg 35 40 45Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
Gln Met 50 55 60Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
Ala Arg Xaa65 70 75 80Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Ser Thr Lys Ala 85 90 95Pro Ser Val Phe 10034102PRTHomo
sapiensmisc_feature(1)..(102)Vh3b heavy chain variable region 34Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Xaa Trp
20 25 30Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Arg
Phe 35 40 45Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Leu Tyr Leu Gln
Met Asn 50 55 60Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Thr Xaa Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro 85 90 95Ser Val Phe Pro Leu Ala 10035101PRTHomo
sapiensmisc_feature(1)..(101)Vh3c heavy chain variable region 35Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Phe Thr Phe Gly Xaa Trp
20 25 30Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Xaa Arg
Phe 35 40 45Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile Ala Tyr Leu Gln
Met Asn 50 55 60Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Thr
Arg Asn Xaa65 70 75 80Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Gly Ser Thr Lys Gly 85 90 95Pro Ser Val Leu Pro 10036108PRTHomo
sapiensmisc_feature(1)..(108)Vh4 heavy chain variable region 36Gln
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10
15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ser Ile Ser Ser
20 25 30Ser Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
Gly 35 40 45Xaa Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe
Ser Leu 50 55 60Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys Ala65 70 75 80Arg Xaa Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala Pro Thr 85 90 95Lys Ala Pro Asp Val Phe Pro Ile Ile Ser
Gly Cys 100 10537132PRTHomo sapiensmisc_feature(1)..(132)Vh5 heavy
chain variable region 37Glu Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly1 5 10 15Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe Thr Xaa 20 25 30Trp Val Arg Gln Met Pro Gly Lys Gly
Leu Glu Trp Met Gly Xaa Gln 35 40 45Val Thr Ile Ser Ala Asp Lys Ser
Ile Ser Thr Ala Tyr Leu Gln Trp 50 55 60Ser Ser Leu Lys Ala Ser Asp
Thr Ala Met Tyr Tyr Cys Ala Arg Xaa65 70 75 80Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Ala 85 90 95Pro Ser Val Phe
Pro Leu Val Ser Cys Glu Asn Ser Pro Ser Asp Thr 100 105 110Ser Ser
Val Ala Val Gly Cys Leu Ala Gln Asp Phe Leu Pro Asp Ser 115 120
125Ile Thr Phe Ser 13038125PRTHomo sapiensmisc_feature(1)..(125)Vh6
heavy chain variable region 38Gln Val Gln Leu Gln Gln Ser Gly Pro
Gly Leu Val Lys Pro Ser Gln1 5 10 15Thr Leu Ser Leu Thr Cys Ala Ile
Ser Gly Asp Ser Val Ser Xaa Trp 20 25 30Ile Arg Gln Ser Pro Ser Arg
Gly Leu Glu Trp Leu Gly Xaa Arg Ile 35 40 45Thr Ile Asn Pro Asp Thr
Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn 50 55 60Ser Val Thr Pro Glu
Asp Thr Ala Val Tyr Tyr Cys Ala Arg Xaa Trp65 70 75 80Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Gly Ser Ala Ser Ala Pro 85 90 95Thr Leu
Phe Pro Leu Val Ser Cys Glu Asn Ser Pro Ser Asp Thr Ser 100 105
110Ser Val Ala Val Gly Cys Leu Ala Gln Asp Phe Leu Pro 115 120
1253991PRTHomo sapiensmisc_feature(1)..(91)Vh7 heavy chain variable
region 39Gln Val Gln Leu Val Gln Ser Gly Ser Glu Leu Lys Lys Pro
Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe
Thr Xaa Trp 20 25 30Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
Gly Xaa Arg Phe 35 40 45Val Phe Ser Leu Asp Thr Ser Val Ser Thr Ala
Tyr Leu Gln Ile Ser 50 55 60Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr
Tyr Cys Ala Arg Xaa Trp65 70 75 80Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ser 85 904093PRTHomo sapiensmisc_feature(1)..(93)Kappa 1_4
light chain variable region 40Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Arg Val Thr Ile Thr Cys
Xaa Trp Tyr Gln Gln Lys Pro Gly 20 25 30Lys Ala Pro Lys Leu Leu Ile
Tyr Xaa Gly Val Pro Ser Arg Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60Pro Glu Asp Phe Ala
Thr Tyr Tyr Cys Xaa Phe Gly Gln Gly Thr Lys65 70 75 80Val Glu Ile
Lys Arg Thr Val Ala Ala Pro Ser Val Phe 85 904192PRTHomo
sapiensmisc_feature(1)..(92)Kappa2 light chain variable region
41Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly1
5 10 15Gln Pro Ala Ser Ile Ser Cys Xaa Trp Tyr Leu Gln Lys Pro Gly
Gln 20 25 30Ser Pro Gln Leu Leu Ile Tyr Xaa Gly Val Pro Asp Arg Phe
Ser Gly 35 40 45Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg
Val Glu Ala 50 55 60Glu Asp Val Gly Val Tyr Tyr Cys Xaa Phe Gly Gln
Gly Thr Lys Val65 70 75 80Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
Val Phe 85 904291PRTHomo sapiensmisc_feature(1)..(91)Kappa3 light
chain variable region 42Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Xaa Trp Tyr
Gln Gln Lys Pro Gly Gln 20 25 30Ala Pro Arg Leu Leu Ile Tyr Xaa Gly
Ile Pro Asp Arg Phe Ser Gly 35 40 45Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu Pro 50 55 60Glu Asp Phe Ala Val Tyr Tyr
Cys Xaa Phe Gly Gln Gly Thr Lys Val65 70 75 80Glu Ile Lys Arg Thr
Val Ala Ala Pro Ser Val 85 904385PRTHomo
sapiensmisc_feature(1)..(85)Kappa5 light chain variable region
43Glu Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly1
5 10 15Asp Lys Val Asn Ile Ser Cys Xaa Trp Tyr Gln Gln Lys Pro Gly
Glu 20 25 30Ala Ala Ile Phe Ile Ile Gln Xaa Gly Ile Pro Pro Arg Phe
Ser Gly 35 40 45Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Asn Asn
Ile Glu Ser 50 55 60Glu Asp Ala Ala Tyr Tyr Phe Cys Xaa Leu Arg His
Phe Trp Pro Gly65 70 75 80Asp Gln Ala Ala Gly 854479PRTHomo
sapiensmisc_feature(1)..(67)KappaNew1 light chain variable region
44Glu Ile Val Met Thr Gln Ser Pro Val Asn Leu Ser Met Ser Ala Gly1
5 10 15Glu Xaa Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Phe
Ile 20 25 30Tyr Xaa Gly Ile Ser Ala Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp 35 40 45Phe Thr Leu Thr Ile Thr Ser Leu Gln Ser Glu Asp Phe
Ala Val Tyr 50 55 60Tyr Cys Xaa Phe Gly Gln Gly Thr Lys Leu Asp Ile
Lys Arg Thr65 70 754577PRTHomo
sapiensmisc_feature(1)..(65)KappaNew2 light chain variable region
45Glu Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Xaa1
5 10 15Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val Ile His
Xaa 20 25 30Gly Ile Ser Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp
Phe Thr 35 40 45Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Leu
Tyr Tyr Cys 50 55 60Xaa Phe Gly Gln Gly Thr Lys Leu Asp Phe Lys Arg
Thr65 70 754695PRTHomo sapiensmisc_feature(1)..(95)KappaNew3 light
chain variable region 46Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu
Ala Val Ser Leu Gly1 5 10 15Gly Arg Arg Ala Thr Ile Asn Cys Xaa Trp
Tyr Gln Gln Lys Pro Gly 20 25 30Gln Pro Pro Lys Leu Leu Ile Tyr Xaa
Gly Val Pro Asp Arg Phe Ser 35 40 45Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Gln 50 55 60Ala Glu Asp Val Ala Val Tyr
Tyr Cys Xaa Phe Gly Gly Gly Thr Lys65 70 75 80Val Glu Ile Lys Arg
Thr Val Ala Ala Pro Ser Val Phe Lys Phe 85 90 954798PRTHomo
sapiensmisc_feature(1)..(98)Lambda1a light chain variable region
47Gln
Ser Val Leu Thr Gln Pro Pro Ser Val Ser Gly Ala Pro Gly Gln1 5 10
15Arg Val Thr Ile Ser Cys Xaa Trp Tyr Gln Gln Leu Pro Gly Thr Ala
20 25 30Pro Lys Leu Leu Ile Tyr Xaa Gly Val Pro Asp Arg Phe Ser Gly
Ser 35 40 45Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln
Ser Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly Thr
Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val
Thr Leu Phe Pro Pro 85 90 95Ser Ser4899PRTHomo
sapiensmisc_feature(1)..(99)Lambda1b light chain variable region
48Ala Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly1
5 10 15Gln Lys Val Thr Ile Ser Cys Xaa Trp Tyr Gln Gln Leu Pro Gly
Thr 20 25 30Ala Pro Lys Leu Leu Ile Tyr Xaa Gly Ile Pro Asp Arg Phe
Ser Gly 35 40 45Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly
Leu Gln Thr 50 55 60Gly Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly
Gly Thr Lys Leu65 70 75 80Thr Val Leu Gly Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro 85 90 95Pro Ser Ser4999PRTHomo
sapiensmisc_feature(1)..(72)Lambda2 light chain variable region
49Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln1
5 10 15Ser Ile Thr Ile Ser Cys Xaa Trp Tyr Gln Gln His Pro Gly Lys
Ala 20 25 30Pro Lys Leu Met Ile Tyr Xaa Gly Val Ser Asn Arg Phe Ser
Gly Ser 35 40 45Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu
Gln Ala Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Thr Lys Leu65 70 75 80Thr Val Leu Gly Gln Pro Lys Ala Ala Pro
Ser Val Thr Leu Phe Pro 85 90 95Pro Ser Ser50107PRTHomo
sapiensmisc_feature(1)..(107)Lambda3a light chain variable region
50Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1
5 10 15Thr Ala Arg Ile Thr Cys Xaa Trp Tyr Gln Gln Lys Pro Gly Gln
Ala 20 25 30Pro Val Leu Val Ile Tyr Xaa Gly Ile Pro Glu Arg Phe Ser
Gly Ser 35 40 45Ser Ser Gly Thr Thr Ala Thr Leu Thr Ile Ser Gly Val
Gln Ala Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro 85 90 95Ser Ser Glu Glu Leu Gln Ala Asn Lys
Ala Thr 100 1055193PRTHomo sapiensmisc_feature(1)..(93)Lambda3b
light chain variable region 51Ser Tyr Val Leu Thr Gln Pro Pro Ser
Val Ser Val Ala Pro Gly Gln1 5 10 15Thr Ala Arg Ile Thr Cys Xaa Trp
Tyr Gln Gln Lys Pro Gly Gln Ala 20 25 30Pro Val Leu Val Val Tyr Asp
Xaa Gly Ile Pro Glu Arg Phe Ser Gly 35 40 45Ser Asn Ser Gly Asn Thr
Ala Thr Leu Thr Ile Ser Arg Val Glu Ala 50 55 60Gly Asp Glu Ala Asp
Tyr Tyr Cys Xaa Phe Gly Gly Gly Thr Lys Leu65 70 75 80Thr Val Leu
Gly Gln Pro Lys Ala Ala Pro Thr Val Thr 85 905298PRTHomo
sapiensmisc_feature(1)..(98)Lambda3c light chain variable region
52Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln1
5 10 15Thr Ala Ser Ile Thr Cys Xaa Trp Tyr Gln Gln Lys Pro Gly Gln
Ser 20 25 30Pro Val Leu Val Ile Tyr Xaa Gly Ile Pro Glu Arg Phe Ser
Gly Ser 35 40 45Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr
Gln Ala Met 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
Arg Ser Leu Cys Pro Pro 85 90 95Pro Pro5398PRTHomo
sapiensmisc_feature(1)..(98)Lambda3e light chain variable region
53Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln1
5 10 15Thr Val Arg Ile Thr Cys Xaa Trp Tyr Gln Gln Lys Pro Gly Gln
Ala 20 25 30Pro Val Leu Val Ile Tyr Xaa Gly Ile Pro Asp Arg Phe Ser
Gly Ser 35 40 45Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala
Gln Ala Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe Pro Pro 85 90 95Ser Ser5494PRTHomo
sapiensmisc_feature(1)..(94)Lambda4a light chain variable region
54Gln Pro Val Leu Thr Gln Ser Ser Ser Ala Ser Ala Ser Leu Gly Ser1
5 10 15Ser Val Lys Leu Thr Cys Xaa Trp His Gln Gln Gln Pro Gly Lys
Ala 20 25 30Pro Arg Tyr Leu Met Lys Xaa Gly Val Pro Asp Arg Phe Ser
Gly Ser 35 40 45Ser Ser Gly Ala Asp Arg Tyr Leu Thr Ile Ser Asn Leu
Gln Ser Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala Ala Pro Ser
Val Thr Leu Phe 85 905595PRTHomo
sapiensmisc_feature(1)..(95)Lambda4b light chain variable region
55Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala1
5 10 15Ser Val Lys Leu Thr Cys Xaa Trp His Gln Gln Gln Pro Glu Lys
Gly 20 25 30Pro Arg Tyr Leu Met Lys Xaa Gly Ile Pro Asp Arg Phe Ser
Gly Ser 35 40 45Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser Ser Leu
Gln Ser Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Ile
Gly Gly Gly Thr65 70 75 80Lys Leu Thr Val Leu Gly Gln Pro Lys Ala
Ala Pro Ser Val Ser 85 90 955688PRTHomo
sapiensmisc_feature(1)..(75)Lambda5 light chain variable region
56Gln Ala Val Leu Thr Gln Pro Ser Ser Leu Ser Ala Ser Pro Gly Ala1
5 10 15Ser Ala Ser Leu Thr Cys Xaa Trp Tyr Gln Gln Lys Pro Gly Ser
Pro 20 25 30Pro Gln Tyr Leu Leu Arg Tyr Xaa Gly Val Pro Ser Arg Phe
Ser Gly 35 40 45Ser Lys Asp Ala Ser Ala Asn Ala Gly Ile Leu Leu Ile
Ser Gly Leu 50 55 60Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe
Gly Gly Gly Thr65 70 75 80Lys Leu Thr Val Leu Ser Gln Pro
8557101PRTHomo sapiensmisc_feature(1)..(101)Lambda6 light chain
variable region 57Asn Phe Met Leu Thr Gln Pro His Ser Val Ser Glu
Ser Pro Gly Lys1 5 10 15Thr Val Thr Ile Ser Cys Xaa Trp Tyr Gln Gln
Arg Pro Gly Ser Ala 20 25 30Pro Thr Thr Val Ile Tyr Xaa Gly Val Pro
Asp Arg Phe Ser Gly Ser 35 40 45Ile Asp Ser Ser Ser Asn Ser Ala Ser
Leu Thr Ile Ser Gly Leu Lys 50 55 60Thr Glu Asp Glu Ala Asp Tyr Tyr
Cys Xaa Phe Gly Gly Gly Thr Lys65 70 75 80Leu Thr Val Leu Gly Gln
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe 85 90 95Pro Pro Ser Ser Ser
1005889PRTHomo sapiensmisc_feature(1)..(72)Lambda7 light chain
variable region 58Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val
Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Xaa Trp Phe Gln Gln
Lys Pro Gly Gln Ala 20 25 30Pro Arg Ala Leu Ile Tyr Xaa Trp Thr Pro
Ala Arg Phe Ser Gly Ser 35 40 45Leu Leu Gly Gly Lys Ala Ala Leu Thr
Leu Ser Gly Val Gln Pro Glu 50 55 60Asp Glu Ala Glu Tyr Tyr Cys Xaa
Phe Gly Gly Gly Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys
Ala Ala Pro 855989PRTHomo sapiensmisc_feature(1)..(89)Lambda8 light
chain variable region 59Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser
Val Ser Pro Gly Gly1 5 10 15Thr Val Thr Leu Thr Cys Xaa Trp Tyr Gln
Gln Thr Pro Gly Gln Ala 20 25 30Pro Arg Thr Leu Ile Tyr Xaa Gly Val
Pro Asp Arg Phe Ser Gly Ser 35 40 45Ile Leu Gly Asn Lys Ala Ala Leu
Thr Ile Thr Gly Ala Gln Ala Asp 50 55 60Asp Glu Ser Asp Tyr Tyr Cys
Xaa Phe Gly Gly Gly Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro
Lys Ala Ala Pro 856091PRTHomo sapiensmisc_feature(1)..(91)Lambda9
light chain variable region 60Gln Pro Val Leu Thr Gln Pro Pro Ser
Ala Ser Ala Ser Leu Gly Ala1 5 10 15Ser Val Thr Leu Thr Cys Xaa Trp
Tyr Gln Gln Arg Pro Gly Lys Gly 20 25 30Pro Arg Phe Val Met Arg Xaa
Gly Ile Pro Asp Arg Phe Ser Val Leu 35 40 45Gly Ser Gly Leu Asn Arg
Tyr Leu Thr Ile Lys Asn Ile Gln Glu Glu 50 55 60Asp Glu Ser Asp Tyr
His Cys Xaa Phe Gly Gly Gly Thr Lys Leu Thr65 70 75 80Val Leu Gly
Gln Pro Lys Ala Ala Pro Ser Val 85 906187PRTHomo
sapiensmisc_feature(1)..(87)Lambda10 light chain variable region
61Gln Ala Gly Leu Thr Gln Pro Pro Ser Val Ser Lys Gly Leu Arg Gln1
5 10 15Thr Ala Thr Leu Thr Cys Xaa Trp Leu Gln Gln His Gln Gly His
Pro 20 25 30Pro Lys Leu Leu Ser Tyr Xaa Gly Ile Ser Glu Arg Phe Ser
Ala Ser 35 40 45Arg Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Leu
Gln Pro Glu 50 55 60Asp Glu Ala Asp Tyr Tyr Cys Xaa Phe Gly Gly Gly
Thr Lys Leu Thr65 70 75 80Val Leu Gly Gln Pro Lys Ala
8562354PRTHomo sapiensmisc_feature(1)..(354)IgA1 heavy chain
constant region 62Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser
Leu Cys Ser Thr1 5 10 15Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu
Val Gln Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser
Glu Ser Gly Gln Gly Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln
Asp Ala Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu
Pro Ala Thr Gln Cys Leu Ala Gly65 70 75 80Lys Ser Val Thr Cys His
Val Lys His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys
Pro Val Pro Ser Thr Pro Pro Thr Pro Ser Pro 100 105 110Ser Thr Pro
Pro Thr Pro Ser Pro Ser Cys Cys His Pro Arg Leu Ser 115 120 125Leu
His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser Glu Ala Asn 130 135
140Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly Val Thr
Phe145 150 155 160Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln
Gly Pro Pro Glu 165 170 175Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser
Ser Val Leu Pro Gly Cys 180 185 190Ala Glu Pro Trp Asn His Gly Lys
Thr Phe Thr Cys Thr Ala Ala Tyr 195 200 205Pro Glu Ser Lys Thr Pro
Leu Thr Ala Thr Leu Ser Lys Ser Gly Asn 210 215 220Thr Phe Arg Pro
Glu Val His Leu Leu Pro Pro Pro Ser Glx Glu Glu225 230 235 240Leu
Ala Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg Gly Phe 245 250
255Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln Glu Leu
260 265 270Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro
Ser Gln 275 280 285Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg
Val Ala Ala Glu 290 295 300Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys
Met Val Gly His Glu Ala305 310 315 320Leu Pro Leu Ala Phe Thr Gln
Lys Thr Ile Asp Arg Leu Ala Gly Lys 325 330 335Pro Thr His Val Asn
Val Ser Val Val Met Ala Glu Val Asp Gly Thr 340 345 350Cys Tyr
63340PRTHomo sapiensmisc_feature(1)..(340)IgA2 heavy chain constant
region 63Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu Asp
Ser Thr1 5 10 15Pro Gln Asp Gly Asn Val Val Val Ala Cys Leu Val Gln
Gly Phe Phe 20 25 30Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser
Gly Gln Asn Val 35 40 45Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp Ala
Ser Gly Asp Leu Tyr 50 55 60Thr Thr Ser Ser Gln Leu Thr Leu Pro Ala
Thr Gln Cys Pro Asp Gly65 70 75 80Lys Ser Val Thr Cys His Val Lys
His Tyr Thr Asn Pro Ser Gln Asp 85 90 95Val Thr Val Pro Cys Pro Val
Pro Pro Pro Pro Pro Cys Cys His Pro 100 105 110Arg Leu Ser Leu His
Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 115 120 125Glu Ala Asn
Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 130 135 140Ala
Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln Gly145 150
155 160Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser Ser Val
Leu 165 170 175Pro Gly Cys Ala Gln Pro Trp Asn His Gly Glu Thr Phe
Thr Cys Thr 180 185 190Ala Ala His Pro Glu Leu Lys Thr Pro Leu Thr
Ala Asn Ile Thr Lys 195 200 205Ser Gly Asn Thr Phe Arg Pro Glu Val
His Leu Leu Pro Pro Pro Ser 210 215 220Glu Glu Leu Ala Leu Asn Glu
Leu Val Thr Leu Thr Cys Leu Ala Arg225 230 235 240Gly Phe Ser Pro
Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 245 250 255Glu Leu
Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 260 265
270Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg Val Ala
275 280 285Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys Met Val
Gly His 290 295 300Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile
Asp Arg Leu Ala305 310 315 320Gly Lys Pro Thr His Val Asn Val Ser
Val Val Met Ala Glu Val Asp 325 330 335Gly Thr Cys Tyr
34064384PRTHomo sapiensmisc_feature(1)..(384)IgD heavy chain
constant region 64Ala Pro Thr Lys Ala Pro Asp Val Phe Pro Ile Ile
Ser Gly Cys Arg1 5 10 15His Pro Lys Asp Asn Ser Pro Val Val Leu Ala
Cys Leu Ile Thr Gly 20 25 30Tyr His Pro Thr Ser Val Thr Val Thr Trp
Tyr Met Gly Thr Gln Ser 35 40 45Gln Pro Gln Arg Thr Phe Pro Glu Ile
Gln Arg Arg Asp Ser Tyr Tyr 50 55 60Met Thr Ser Ser Gln Leu Ser Thr
Pro Leu Gln Gln Trp Arg Gln Gly65 70 75 80Glu Tyr Lys Cys Val Val
Gln His Thr Ala Ser Lys Ser Lys Lys Glu 85 90 95Ile Phe Arg Trp Pro
Glu Ser Pro Lys Ala Gln Ala Ser Ser Val Pro 100 105 110Thr Ala Gln
Pro Gln Ala Glu Gly Ser Leu Ala Lys Ala Thr Thr Ala 115 120 125Pro
Ala Thr Thr Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys 130 135
140Glu Lys Glu Lys Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro
Glu145 150 155 160Cys Pro Ser His Thr Gln Pro Leu Gly Val Tyr Leu
Leu Thr Pro Ala 165 170 175Val Gln Asp Leu Trp
Leu Arg Asp Lys Ala Thr Phe Thr Cys Phe Val 180 185 190Val Gly Ser
Asp Leu Lys Asp Ala His Leu Thr Trp Glu Val Ala Gly 195 200 205Lys
Val Pro Thr Gly Gly Val Glu Glu Gly Leu Leu Glu Arg His Ser 210 215
220Asn Gly Ser Gln Ser Gln His Ser Arg Leu Thr Leu Pro Arg Ser
Leu225 230 235 240Trp Asn Ala Gly Thr Ser Val Thr Cys Thr Leu Asn
His Pro Ser Leu 245 250 255Pro Pro Gln Arg Leu Met Ala Leu Arg Glu
Pro Ala Ala Gln Ala Pro 260 265 270Val Lys Leu Ser Leu Asn Leu Leu
Ala Ser Ser Asp Pro Pro Glu Ala 275 280 285Ala Ser Trp Leu Leu Cys
Glu Val Ser Gly Phe Ser Pro Pro Asn Ile 290 295 300Leu Leu Met Trp
Leu Glu Asp Gln Arg Glu Val Asn Thr Ser Gly Phe305 310 315 320Ala
Pro Ala Arg Pro Pro Pro Gln Pro Arg Ser Thr Thr Phe Trp Ala 325 330
335Trp Ser Val Leu Arg Val Pro Ala Pro Pro Ser Pro Gln Pro Ala Thr
340 345 350Tyr Thr Cys Val Val Ser His Glu Asp Ser Arg Thr Leu Leu
Asn Ala 355 360 365Ser Arg Ser Leu Glu Val Ser Tyr Val Thr Asp His
Gly Pro Met Lys 370 375 38065497PRTHomo
sapiensmisc_feature(1)..(497)IgE heavy chain constant region 65Ala
Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg Cys Cys Lys1 5 10
15Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr
20 25 30Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser
Leu 35 40 45Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu
Ser Gly 50 55 60His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala
Trp Ala Lys65 70 75 80Gln Met Phe Thr Cys Arg Val Ala His Thr Pro
Ser Ser Thr Asp Trp 85 90 95Val Asp Asn Lys Thr Phe Ser Val Cys Ser
Arg Asp Phe Thr Pro Pro 100 105 110Thr Val Lys Ile Leu Gln Ser Ser
Cys Asp Gly Gly Gly His Phe Pro 115 120 125Pro Thr Ile Gln Leu Leu
Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr 130 135 140Ile Asn Ile Thr
Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu145 150 155 160Ser
Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser 165 170
175Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr
180 185 190Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr
Lys Lys 195 200 205Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr
Leu Ser Arg Pro 210 215 220Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser
Pro Thr Ile Thr Cys Leu225 230 235 240Val Val Asp Leu Ala Pro Ser
Lys Gly Thr Val Asn Leu Thr Trp Ser 245 250 255Arg Ala Ser Gly Lys
Pro Val Asn His Ser Thr Arg Lys Glu Glu Lys 260 265 270Gln Arg Asn
Gly Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 275 280 285Arg
Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro 290 295
300His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly
Pro305 310 315 320Val Gly Pro Arg Ala Ala Pro Glu Val Tyr Ala Phe
Ala Thr Pro Glu 325 330 335Trp Pro Gly Ser Arg Asp Lys Arg Thr Leu
Ala Cys Leu Ile Gln Asn 340 345 350Phe Met Pro Glu Asp Ile Ser Val
Gln Trp Leu His Asn Glu Val Gln 355 360 365Leu Pro Asp Ala Arg His
Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly 370 375 380Ser Gly Phe Phe
Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp385 390 395 400Glu
Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser 405 410
415Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys Asp
420 425 430Val Cys Val Glu Glu Ala Glu Gly Glu Ala Pro Trp Thr Trp
Thr Gly 435 440 445Leu Cys Ile Phe Ala Ala Leu Phe Leu Leu Ser Val
Ser Tyr Ser Ala 450 455 460Ala Leu Thr Leu Leu Met Val Gln Arg Phe
Leu Ser Ala Thr Arg Gln465 470 475 480Gly Arg Pro Gln Thr Ser Leu
Asp Tyr Thr Asn Val Leu Gln Pro His 485 490 495Ala66339PRTHomo
sapiensmisc_feature(1)..(339)IgG1 heavy chain constant region 66Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170
175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asx Asn Gly Gln Pro Glu 260 265 270Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 275 280 285Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 290 295
300Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr305 310 315 320Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Thr
His Thr Cys Pro 325 330 335Pro Cys Pro67326PRTHomo
sapiensmisc_feature(1)..(326)IgG2 heavy chain constant region 67Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10
15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly
Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys
Pro Pro Cys Pro Ala Pro 100 105 110Pro Val Ala Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly145 150 155 160Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170
175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp
180 185 190Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly
Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro
Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295
300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu305 310 315 320Ser Leu Ser Pro Gly Lys 32568377PRTHomo
sapiensmisc_feature(1)..(377)IgG3 heavy chain constant region 68Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10
15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu
Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Arg Val Glu Leu Lys Thr Pro Leu Gly Asp
Thr Thr His Thr Cys Pro 100 105 110Arg Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125Cys Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140Pro Glu Pro Lys
Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro145 150 155 160Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170
175Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
180 185 190Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys
Trp Tyr 195 200 205Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu 210 215 220Gln Tyr Asn Ser Thr Phe Arg Val Val Ser
Val Leu Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255Ala Leu Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295
300Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn
Asn305 310 315 320Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu 325 330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Ile 340 345 350Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn Arg Phe Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser
Pro Gly Lys 370 37569327PRTHomo sapiensmisc_feature(1)..(327)IgG4
heavy chain constant region 69Ala Ser Thr Lys Gly Pro Ser Val Phe
Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65 70 75 80Tyr Thr Cys
Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105
110Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
Trp Tyr Val Asp145 150 155 160Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205Pro Ser Ser
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230
235 240Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser 290 295 300Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser
Leu Gly Lys 32570476PRTHomo sapiensmisc_feature(1)..(476)IgM heavy
chain constant region 70Gly Ser Ala Ser Ala Pro Thr Leu Phe Pro Leu
Val Ser Cys Glu Asn1 5 10 15Ser Pro Ser Asp Thr Ser Ser Val Ala Val
Gly Cys Leu Ala Gln Asp 20 25 30Phe Leu Pro Asp Ser Ile Thr Phe Ser
Trp Lys Tyr Lys Asn Asn Ser 35 40 45Asp Ile Ser Ser Thr Arg Gly Phe
Pro Ser Val Leu Arg Gly Gly Lys 50 55 60Tyr Ala Ala Thr Ser Gln Val
Leu Leu Pro Ser Lys Asp Val Met Gln65 70 75 80Gly Thr Asp Glu His
Val Val Cys Lys Val Gln His Pro Asn Gly Asn 85 90 95Lys Glu Lys Asn
Val Pro Leu Pro Val Ile Ala Glu Leu Pro Pro Lys 100 105 110Val Ser
Val Phe Val Pro Pro Arg Asp Gly Phe Phe Gly Asn Pro Arg 115 120
125Ser Lys Ser Lys Leu Ile Cys Gln Ala Thr Gly Phe Ser Pro Arg Gln
130 135 140Ile Gln Val Ser Trp Leu Arg Glu Gly Lys Gln Val Gly Ser
Gly Val145 150 155 160Thr Thr Asp Gln Val Gln Ala Glu Ala Lys Glu
Ser Gly Pro Thr Thr 165 170 175Tyr Lys Val Thr Ser Thr Leu Thr Ile
Lys Glu Ser Asp Trp Leu Ser 180 185 190Gln Ser Met Phe Thr Cys Arg
Val Asp His Arg Gly Leu Thr Phe Gln 195 200 205Gln Asn Ala Ser Ser
Met Cys Val Pro Asp Gln Asp Thr Ala Ile Arg 210 215 220Val Phe Ala
Ile Pro Pro Ser Phe Ala Ser Ile Phe Leu Thr Lys Ser225 230 235
240Thr Lys Leu Thr Cys Leu Val Thr Asp Leu Thr Thr Tyr Asp Ser Val
245 250 255Thr Ile Ser Trp Thr Arg Gln Asn Gly Glu Ala Val Lys Thr
His Thr 260 265 270Asn Ile Ser Glu Ser His Pro Asn Ala Thr Phe Ser
Ala Val Gly Glu 275 280 285Ala Ser Ile Cys Glu Asp Asp Trp Asn Ser
Gly Glu Arg Phe Thr Cys 290 295 300Thr Val Thr His Thr Asp Leu Pro
Ser Pro Leu Lys Gln Thr Ile Ser305 310 315 320Arg Pro Lys Gly Val
Ala Leu His Arg Pro Asp Val Tyr Leu Leu Pro 325 330 335Pro Ala Arg
Glu Gln Leu Asn Leu Arg Glu Ser Ala
Thr Ile Thr Cys 340 345 350Leu Val Thr Gly Phe Ser Pro Ala Asp Val
Phe Val Gln Trp Gln Met 355 360 365Gln Arg Gly Gln Pro Leu Ser Pro
Glu Lys Tyr Val Thr Ser Ala Pro 370 375 380Met Pro Glu Pro Gln Ala
Pro Gly Arg Tyr Phe Ala His Ser Ile Leu385 390 395 400Thr Val Ser
Glu Glu Glu Trp Asn Thr Gly Glu Thr Tyr Thr Cys Val 405 410 415Val
Ala His Glu Ala Leu Pro Asn Arg Val Thr Glu Arg Thr Val Asp 420 425
430Lys Ser Thr Gly Lys Pro Thr Ser Ala Asp Glu Glu Gly Phe Glu Asn
435 440 445Leu Trp Ala Thr Ala Ser Thr Phe Ile Val Leu Tyr Asn Val
Ser Leu 450 455 460Val Met Ser Asp Thr Ala Gly Thr Cys Tyr Val
Lys465 470 47571107PRTHomo sapiensmisc_feature(1)..(107)Light chain
kappa constant region (IgKc) 71Arg Thr Val Ala Ala Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val
Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 10572107PRTHomo
sapiensmisc_feature(1)..(107)Light chain lambda constant region
(IgLambda) 72Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
Pro Ser Ser1 5 10 15Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys
Leu Ile Ser Asp 20 25 30Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys
Ala Asp Ser Ser Pro 35 40 45Val Lys Ala Gly Val Glu Thr Thr Thr Pro
Ser Lys Gln Ser Asn Asn 50 55 60Lys Tyr Ala Ala Ser Ser Tyr Leu Ser
Leu Thr Pro Glu Gln Trp Lys65 70 75 80Ser His Arg Lys Ser Tyr Ser
Cys Gln Val Thr His Glu Gly Ser Thr 85 90 95Val Glu Lys Thr Val Ala
Pro Thr Glu Cys Ser 100 105734PRTArtificialsynthetic peptide 73Gly
Gly Gly Ser1746PRTArtificialsynthetic peptide 74Gly Ser Gly Gly Gly
Ser1 5755PRTArtificialsynthetic peptide 75Cys Pro Pro Cys Pro1
576110PRTArtificialsynthetic peptide 76Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75 80Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 85 90
95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100 105
11077110PRTArtificialsynthetic peptide 77Ala Pro Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 35 40 45Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11078107PRTArtificialsynthetic peptide 78Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp1 5 10 15Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly65 70 75
80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100
10579110PRTArtificialsynthetic peptide 79Ala Pro Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45Val Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln Phe
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11080110PRTArtificialsynthetic peptide 80Ala Pro Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys1 5 10 15Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 20 25 30Val Val Asp
Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 35 40 45Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 50 55 60Gln
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His65 70 75
80Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
85 90 95Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys 100
105 11081107PRTArtificialsynthetic peptide 81Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu1 5 10 15Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30Tyr Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly65 70 75
80Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 100
10582247PRTArtificialsynthetic peptide 82Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys
24583249PRTArtificialsynthetic peptide 83Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro 20 25 30Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr65 70 75
80Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
85 90 95Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 100 105 110Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 115 120 125Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 130 135 140Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu145 150 155 160Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200
205Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
210 215 220Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln225 230 235 240Lys Ser Leu Ser Leu Ser Pro Gly Lys
24584251PRTArtificialsynthetic peptide 84Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Ser Gly Gly Gly Ser Cys Pro Pro 20 25 30Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 50 55 60Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70 75
80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
85 90 95Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val 100 105 110Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 130 135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp145 150 155 160Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 180 185 190Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 195 200
205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
210 215 220Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr225 230 235 240Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
245 25085247PRTArtificialsynthetic peptide 85Gln Ile Gln Gly Gly
Thr Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro
Gln Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Ala Ala
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys
24586249PRTArtificialsynthetic peptide 86Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro 20 25 30Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60Val Val
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr65 70 75
80Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
85 90 95Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 100 105 110Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 115 120 125Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 130 135 140Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu145 150 155 160Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200
205Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
210 215 220Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln225 230 235 240Lys Ser Leu Ser Leu Ser Pro Gly Lys
24587247PRTArtificialsynthetic peptide 87Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Phe Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 115 120 125Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Gln Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser225 230 235 240Leu Ser Leu Ser Leu Gly Lys
24588247PRTArtificialsynthetic peptide 88Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Ala Ala Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp65 70 75
80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu 115 120 125Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser225 230 235 240Leu Ser Leu Ser Leu Gly Lys
24589249PRTArtificialsynthetic peptide 89Gln Ile Gln Gly Gly Thr
Tyr Ser Cys His Phe Gly Pro Leu Thr Trp1 5 10 15Val Cys Lys Pro Gln
Gly Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro 20 25 30Ala Pro Glu Ala
Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60Val Val
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr65 70 75
80Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
85 90 95Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His 100 105 110Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys 115 120 125Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln 130 135 140Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Gln Glu Glu Met145 150 155 160Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200
205Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
210 215 220Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln225 230 235 240Lys Ser Leu Ser Leu Ser Leu Gly Lys 245
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