U.S. patent application number 11/234482 was filed with the patent office on 2006-09-14 for polypeptide compounds for inhibiting angiogenesis and tumor growth.
This patent application is currently assigned to VasGene Therapeutics, Inc.. Invention is credited to Parkash Gill, Nathalie Kertesz, Valery Krasnoperov, Ramachandra Reddy, Sergey Zozulya.
Application Number | 20060204512 11/234482 |
Document ID | / |
Family ID | 36090699 |
Filed Date | 2006-09-14 |
United States Patent
Application |
20060204512 |
Kind Code |
A1 |
Krasnoperov; Valery ; et
al. |
September 14, 2006 |
Polypeptide compounds for inhibiting angiogenesis and tumor
growth
Abstract
In certain embodiments, this present invention provides
polypeptide compositions, including compositions containing a
modified polypeptide, and methods for inhibiting Ephrin B2 or EphB4
activity. In other embodiments, the present invention provides
methods and compositions for treating cancer or for treating
angiogenesis-associated diseases.
Inventors: |
Krasnoperov; Valery; (South
Pasadena, CA) ; Kertesz; Nathalie; (Agoura, CA)
; Reddy; Ramachandra; (Pearland, TX) ; Gill;
Parkash; (Agoura Hills, CA) ; Zozulya; Sergey;
(San Diego, CA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP;ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
VasGene Therapeutics, Inc.
Sharon Hills
PA
|
Family ID: |
36090699 |
Appl. No.: |
11/234482 |
Filed: |
September 23, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60612488 |
Sep 23, 2004 |
|
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|
Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/325; 435/69.7; 514/13.3; 514/19.3; 514/19.4;
514/19.5; 514/19.6; 514/19.8; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/385 20130101;
A61P 9/00 20180101; C07K 14/715 20130101; A61P 35/04 20180101; A61P
43/00 20180101; A61K 38/00 20130101; A61P 35/00 20180101; A61P
35/02 20180101; A61K 47/60 20170801 |
Class at
Publication: |
424/185.1 ;
435/069.7; 435/320.1; 435/325; 514/012; 530/350; 536/023.5 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07H 21/04 20060101 C07H021/04; C12P 21/04 20060101
C12P021/04; C07K 14/82 20060101 C07K014/82 |
Claims
1. An isolated soluble polypeptide comprising an amino acid
sequence of an extracellular domain of an EphB4 protein, wherein
the polypeptide is a monomer and binds specifically to an Ephrin B2
polypeptide.
2. The polypeptide of claim 1, comprising a globular domain of an
EphB4 protein or a sequence that is at least 90% identical to a
globular domain of EphB4.
3. The polypeptide of claim 1, comprising a sequence at least 90%
identical to residues 29-197 of the amino acid sequence defined by
FIG. 65 (SEQ ID NO:10).
4. The polypeptide of claim 1, further comprising a modification
that increases serum half-life.
5. The polypeptide of claim 4, wherein said modification comprises
a polyethylene glycol group.
6. The polypeptide of claim 5, wherein said modification is a
single polyethylene glycol group covalently bonded to the
polypeptide.
7. The polypeptide of claim 5, wherein said polypeptide is
covalently bonded to two polyethylene glycol groups.
8. The polypeptide of claim 5, wherein said polypeptide is
covalently bonded to multiple polyethylene glycol groups.
9. The polypeptide of claim 5, wherein said polyethylene glycol
group has a molecular weight of from about 10 to about 40 kDa.
10. The polypeptide of claim 5, wherein the polyethylene glycol
group has a molecular weight of from about 30 to about 40 kDa.
11. The polypeptide of claim 5, wherein said polyethylene glycol
group is selected from the group of linear PEG chains and branched
PEG chains.
12. The polypeptide of claim 5, wherein said polyethylene glycol
group is attached to a group selected from the lysine side chains
and the N-terminal amino group of the EphB4 polypeptide.
13. The polypeptide of claim 4, wherein said polypeptide has a
serum half-life in vivo at least 50% greater than that of an
unmodified EphB4 polypeptide.
14. The polypeptide of claim 4, wherein said polypeptide has a
serum half-life in vivo at least 100% greater than that of an
unmodified EphB4 polypeptide.
15. The polypeptide of claim 4, wherein the polypeptide is a fusion
protein.
16. The polypeptide of claim 15, wherein the polypeptide comprises
an albumin protein or fragments thereof.
17. The polypeptide of claim 16, wherein said albumin protein is
selected from the group consisting of a human serum albumin (HSA)
and bovine serum albumin (BSA).
18. The polypeptide of claim 16, wherein the albumin is a naturally
occurring variant.
19. The polypeptide of claim 1, wherein the polypeptide has one or
more activities selected from the group consisting of: (a)
inhibition of EphrinB2 activity; (b) inhibition of EphrinB2 kinase
activity; (c) inhibition of the interaction between EphB4 and
EphrinB2; (d) inhibition of EphB4 kinase activity; (e) inhibition
of clustering of Ephrin B2; and (f) inhibition of clustering of
EphB4.
20. The polypeptide of claim 4, wherein the polypeptide has
enhanced in vivo stability relative to the unmodified wildtype
polypeptide.
21. A pharmaceutical composition comprising a polypeptide of claim
1, and a pharmaceutically acceptable carrier.
22. A method of inhibiting signaling through Ephrin B2/EphB4
pathway in a cell, comprising contacting the cell with an effective
amount of a polypeptide of claim 1.
23. A method of reducing the growth rate of a tumor, comprising
administering an amount of a polypeptide of claim 1, sufficient to
reduce the growth rate of the tumor.
24. A method for treating a patient suffering from a cancer,
comprising administering to the patient a polypeptide of claim
1.
25. A method of inhibiting angiogenesis, comprising contacting a
cell with a polypeptide of claim 1.
26. A method for treating a patient suffering from an
angiogenesis-associated disease, comprising administering to the
patient a polypeptide of claim 1.
27. The polypeptide of claim 1, wherein the polypeptide comprises
one or more modified amino acid residues.
28. A cosmetic composition comprising the polypeptide of claim 1,
and a pharmaceutically acceptable carrier.
29. A method of reducing the growth rate of a tumor, comprising
administering an amount of a polypeptide agent sufficient to reduce
the growth rate of the tumor, wherein the polypeptide agent is
selected from the group consisting of: (a) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds
specifically to an Ephrin B2 polypeptide; (b) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a
monomer and binds with high affinity to an EphB4 polypeptide.
30. The method of claim 29, wherein the tumor comprises cells
expressing a higher level of EphB4 and/or EphrinB2 than
noncancerous cells of a comparable tissue.
31. A method for treating a patient suffering from a cancer,
comprising administering to the patient a polypeptide agent
selected from the group consisting of: (a) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds
specifically to an Ephrin B2 polypeptide; (b) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a
monomer and binds with high affinity to an EphB4 polypeptide.
32. The method of claim 31, wherein the cancer comprises cancer
cells expressing EphrinB2 and/or EphB4 at a higher level than
noncancerous cells of a comparable tissue.
33. The method of claim 31, wherein the cancer is metastatic
cancer.
34. The method of claim 31, wherein the tumor is selected from the
group consisting of colon carcinoma, breast tumor, mesothelioma,
prostate tumor, squamous cell carcinoma, Kaposi sarcoma, and
leukemia.
35. The method of claim 31, wherein the cancer is an
angiogenesis-dependent cancer.
36. The method of claim 31, wherein the cancer is an
angiogenesis-independent cancer.
37. The method of claim 31, wherein the polypeptide agent is a
soluble polypeptide comprising an amino acid sequence of an
extracellular domain of an Ephrin B2 protein, wherein the soluble
Ephrin B2 polypeptide is a monomer and binds with high affinity to
an EphB4 polypeptide and further comprises a modification that
increases serum half-life.
38. The method of claim 31, further including administering at
least one additional anti-cancer chemotherapeutic agent that
inhibits cancer cells in an additive or synergistic manner with the
polypeptide agent.
39. A method of inhibiting angiogenesis, comprising contacting a
cell an amount of a polypeptide agent sufficient to inhibit
angiogenesis, wherein the polypeptide agent is selected from the
group consisting of: (a) a soluble polypeptide comprising an amino
acid sequence of an extracellular domain of an EphB4 protein,
wherein the EphB4 polypeptide is a monomer and binds specifically
to an Ephrin B2 polypeptide; (b) a soluble polypeptide comprising
an amino acid sequence of an extracellular domain of an Ephrin B2
protein, wherein the soluble Ephrin B2 polypeptide is a monomer and
binds with high affinity to an EphB4 polypeptide.
40. A method for treating a patient suffering from an
angiogenesis-associated disease, comprising administering to the
patient a polypeptide agent selected from the group consisting of:
(a) a soluble polypeptide comprising an amino acid sequence of an
extracellular domain of an EphB4 protein, wherein the EphB4
polypeptide is a monomer and binds specifically to an Ephrin B2
polypeptide; (b) a soluble polypeptide comprising an amino acid
sequence of an extracellular domain of an Ephrin B2 protein,
wherein the soluble Ephrin B2 polypeptide is a monomer and binds
with high affinity to an EphB4 polypeptide.
41. An isolated soluble polypeptide comprising an amino acid
sequence of a fibronectin type 3 domain of an EphB4 protein,
wherein the polypeptide inhibits tumor growth in a mouse xenograft
model of cancer.
42. The polypeptide of claim 41, wherein the polypeptide does not
bind to EphrinB2.
43. The polypeptide of claim 41, wherein the polypeptide does not
include a substantial portion of the globular domain of an EphB4
protein.
44. The polypeptide of claim 41, wherein the polypeptide comprises
an amino acid sequence of amino acids 324-526 of the sequence of
FIG. 65 (SEQ ID NO:10).
45. The polypeptide of claim 41, wherein the polypeptide is a
monomer.
46. The polypeptide of claim 41, wherein the polypeptide further
comprises a modification that increases serum half-life.
47. A polypeptide dimer or multimers comprising two or more
polypeptides of claim 41.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 60/612,488, filed Sep. 23, 2004,
the specification of which is incorporated by reference herein in
its entirety.
BACKGROUND OF THE INVENTION
[0002] Angiogenesis, the development of new blood vessels from the
endothelium of a preexisting vasculature, is a critical process in
the growth, progression, and metastasis of solid tumors within the
host. During physiologically normal angiogenesis, the autocrine,
paracrine, and amphicrine interactions of the vascular endothelium
with its surrounding stromal components are tightly regulated both
spatially and temporally. Additionally, the levels and activities
of proangiogenic and angiostatic cytokines and growth factors are
maintained in balance. In contrast, the pathological angiogenesis
necessary for active tumor growth is sustained and persistent,
representing a dysregulation of the normal angiogenic system. Solid
and hematopoietic tumor types are particularly associated with a
high level of abnormal angiogenesis.
[0003] It is generally thought that the development of tumor
consists of sequential, and interrelated steps that lead to the
generation of an autonomous clone with aggressive growth potential.
These steps include sustained growth and unlimited self-renewal.
Cell populations in a tumor are generally characterized by growth
signal self-sufficiency, decreased sensitivity to growth
suppressive signals, and resistance to apoptosis. Genetic or
cytogenetic events that initiate aberrant growth sustain cells in a
prolonged "ready" state by preventing apoptosis.
[0004] It is a goal of the present disclosure to provide agents and
therapeutic treatments for inhibiting angiogenesis and tumor
growth.
SUMMARY OF THE INVENTION
[0005] In certain aspects, the disclosure provides polypeptide
agents that inhibit EphB4 or EphrinB2 mediated functions, including
monomeric ligand binding portions of the EphB4 and EphrinB2
proteins. As demonstrated herein, EphB4 and EphrinB2 participate in
various disease states, including cancers and diseases related to
unwanted or excessive angiogenesis. Accordingly, certain
polypeptide agents disclosed herein may be used to treat such
diseases. In further aspects, the disclosure relates to the
discovery that EphB4 and/or EphrinB2 are expressed, often at high
levels, in a variety of tumors. Therefore, polypeptide agents that
down-regulate EphB4 or EphrinB2 function may affect tumors by a
direct effect on the tumor cells as well as an indirect effect on
the angiogenic processes recruited by the tumor. In certain
embodiments, the disclosure provides the identity of tumor types
particularly suited to treatment with an agent that downregulates
EphB4 or EphrinB2 function. In preferred embodiments, polypeptides
disclosed herein are modified so as to have increased serum
half-life in vivo.
[0006] In certain aspects, the disclosure provides soluble EphB4
polypeptides comprising an amino acid sequence of an extracellular
domain of an EphB4 protein. The soluble EphB4 polypeptides bind
specifically to an EphrinB2 polypeptide. The term "soluble" is used
merely to indicate that these polypeptides do not contain a
transmembrane domain or a portion of a transmembrane domain
sufficient to compromise the solubility of the polypeptide in a
physiological salt solution. Soluble polypeptides are preferably
prepared as monomers that compete with EphB4 for binding to ligand
such as EphrinB2 and inhibit the signaling that results from EphB4
activation. Optionally, a soluble polypeptide may be prepared in a
multimeric form, by, for example, expressing as an Fc fusion
protein or fusion with another multimerization domain. Such
multimeric forms may have complex activities, having agonistic or
antagonistic effects depending on the context. In certain
embodiments the soluble EphB4 polypeptide comprises a globular
domain of an EphB4 protein. A soluble EphB4 polypeptide may
comprise a sequence at least 90% identical to residues 1-522 of the
amino acid sequence defined by FIG. 65 (SEQ ID NO:10). A soluble
EphB4 polypeptide may comprise a sequence at least 90% identical to
residues 1-412 of the amino acid sequence defined by FIG. 65 (SEQ
ID NO:10). A soluble EphB4 polypeptide may comprise a sequence at
least 90% identical to residues 1-312 of the amino acid sequence
defined by FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may
comprise a sequence encompassing the globular (G) domain (amino
acids 29-197 of FIG. 65, SEQ ID NO:10), and optionally additional
domains, such as the cysteine-rich domain (amino acids 239-321 of
FIG. 65, SEQ ID NO:10), the first fibronectin type 3 domain (amino
acids 324-429 of FIG. 65, SEQ ID NO:10) and the second fibronectin
type 3 domain (amino acids 434-526 of FIG. 65, SEQ ID NO:10).
Preferred polypeptides described herein and demonstrated as having
ligand binding activity include polypeptides corresponding to
1-537, 1-427 and 1-326, respectively, of the amino acid sequence
shown in FIG. 65 (SEQ ID NO:10). A soluble EphB4 polypeptide may
comprise a sequence as set forth in FIG. 1 or 2 (SEQ ID Nos. 1 or
2). As is well known in the art, expression of such EphB4
polypeptides in a suitable cell, such as HEK293T cell line, will
result in cleavage of a leader peptide. Although such cleavage is
not always complete or perfectly consistent at a single site, it is
known that EphB4 tends to be cleaved so as to remove the first 15
amino acids of the sequence shown in FIG. 65 (SEQ ID NO:10).
Accordingly, as specific examples, the disclosure provides
unprocessed soluble EphB4 polypeptides that bind to EphrinB2 and
comprise an amino acid sequence selected from the following group
(numbering is with respect to the sequence of FIG. 65, SEQ ID
NO:10): 1-197, 29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326,
1-412, 29-412, 1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537
and 29-537. Additionally, heterologous leader peptides may be
substituted for the endogeneous leader sequences. Polypeptides may
be used in a processed form, such forms having a predicted amino
acid sequence selected from the following group (numbering is with
respect to the sequence of FIG. 65, SEQ ID NO:10): 16-197, 16-312,
16-321, 16-326, 16-412, 16-427, 16-429, 16-526 and 16-537.
Additionally, a soluble EphB4 polypeptide may be one that comprises
an amino acid sequence at least 90%, and optionally 95% or 99%
identical to any of the preceding amino acid sequences while
retaining EphrinB2 binding activity. Preferably, any variations in
the amino acid sequence from the sequence shown in FIG. 65 (SEQ ID
NO:10) are conservative changes or deletions of no more than 1, 2,
3, 4 or 5 amino acids, particularly in a surface loop region. In
certain embodiments, the soluble EphB4 polypeptide may inhibit the
interaction between Ephrin B2 and EphB4. The soluble EphB4
polypeptide may inhibit clustering of or phosphorylation of Ephrin
B2 or EphB4. Phosphorylation of EphrinB2 or EphB4 is generally
considered to be one of the initial events in triggering
intracellular signaling pathways regulated by these proteins. As
noted above, the soluble EphB4 polypeptide may be prepared as a
monomeric or multimeric fusion protein. The soluble polypeptide may
include one or more modified amino acids. Such amino acids may
contribute to desirable properties, such as increased resistance to
protease digestion.
[0007] The present disclosure provides soluble EphB4 polypeptides
having an additional component that confers increased serum
half-life while still retaining EphrinB2 binding activity. In
certain embodiments soluble EphB4 polypeptides are monomeric and
are covalently linked to one or more polyoxyaklylene groups (e.g.,
polyethylene, polypropylene), and preferably polyethylene glycol
(PEG) groups. Accordingly, one aspect of the invention provides
modified EphB4 polypeptides, wherein the modification comprises a
single polyethylene glycol group covalently bonded to the
polypeptide. Other aspects provide modified EphB4 polypeptides
covalently bonded to one, two, three, or more polyethylene glycol
groups.
[0008] The one or more PEG may have a molecular weight ranging from
about 1 kDa to about 100 kDa, and will preferably have a molecular
weight ranging from about 10 to about 60 kDa or about 10 to about
40 kDa. The PEG group may be a linear PEG or a branched PEG. In a
preferred embodiment, the soluble, monomeric EphB4 conjugate
comprises an EphB4 polypeptide covalently linked to one PEG group
of from about 10 to about 40 kDa (monoPEGylated EphB4), or from
about 15 to 30 kDa, preferably via an .epsilon.-amino group of
EphB4 lysine or the N-terminal amino group. Most preferably, EphB4
is randomly PEGylated at one amino group out of the group
consisting of the .epsilon.-amino groups of EphB4 lysine and the
N-terminal amino group.
[0009] In one embodiment, the pegylated polypeptides provided by
the invention have a serum half-life in vivo at least 50%, 75%,
100%, 150% or 200% greater than that of an unmodified EphB4
polypeptide. In another embodiment, the pegylated EphB4
polypeptides provided by the invention inhibit EphrinB2 activity.
In a specific embodiment, they inhibit EphrinB2 receptor
clustering, EphrinB2 phosphorylation, and/or EphrinB2 kinase
activity.
[0010] Surprisingly, it has been found that monoPEGylated EphB4
according to the invention has superior properties in regard to the
therapeutic applicability of unmodified soluble EphB4 polypeptides
and poly-PEGylated EphB4. Nonetheless, the disclosure also provides
poly-PEGylated EphB4 having PEG at more than one position. Such
polyPEGylated forms provide improved serum-half life relative to
the unmodified form.
[0011] In certain embodiments, a soluble EphB4 polypeptide is
stably associated with a second stabilizing polypeptide that
confers improved half-life without substantially diminishing
EphrinB2 binding. A stabilizing polypeptide will preferably be
immunocompatible with human patients (or animal patients, where
veterinary uses are contemplated) and have little or no significant
biological activity.
[0012] In a preferred embodiment, the stabilizing polypeptide is a
human serum albumin, or a portion thereof. A human serum albumin
may be stably associated with the EphB4 polypeptide covalently or
non-covalently. Covalent attachment may be achieved by expression
of the EphB4 polypeptide as a co-translational fusion with human
serum albumin. The albumin sequence may be fused at the N-terminus,
the C-terminus or at a non-disruptive internal position in the
soluble EphB4 polypeptide. Exposed loops of the EphB4 would be
appropriate positions for insertion of an albumin sequence. Albumin
may also be post-translationally attached to the EphB4 polypeptide
by, for example, chemical cross-linking. An EphB4 polypeptide may
also be stably associated with more than one albumin polypeptide.
In some embodiments, the albumin is selected from the group
consisting of a human serum albumin (HSA) and bovine serum albumin
(BSA). In other embodiments, the albumin is a naturally occurring
variant. In one preferred embodiment, the EphB4-HSA fusion inhibits
the interaction between Ephrin B2 and EphB4, the clustering of
Ephrin B2 or EphB4, the phosphorylation of Ephrin B2 or EphB4, or
combinations thereof. In other embodiments, the EphB4-HSA fusion
has enhanced in vivo stability relative to the unmodified wildtype
polypeptide.
[0013] In certain aspects, the disclosure provides soluble EphrinB2
polypeptides comprising an amino acid sequence of an extracellular
domain of an EphrinB2 protein. The soluble EphrinB2 polypeptides
bind specifically to an EphB4 polypeptide. The term "soluble" is
used merely to indicate that these polypeptides do not contain a
transmembrane domain or a portion of a transmembrane domain
sufficient to compromise the solubility of the polypeptide in a
physiological salt solution. Soluble polypeptides are preferably
prepared as monomers that compete with EphrinB2 for binding to
ligand such as EphB4 and inhibit the signaling that results from
EphrinB2 activation. Optionally, a soluble polypeptide may be
prepared in a multimeric form, by, for example, expressing as an Fc
fusion protein or fusion with another multimerization domain. Such
multimeric forms may have complex activities, having agonistic or
antagonistic effects depending on the context. A soluble EphrinB2
polypeptide may comprise residues 1-225 of the amino acid sequence
defined by FIG. 66 (SEQ ID NO:11). A soluble EphrinB2 polypeptide
may comprise a sequence defined by FIG. 3. As is well known in the
art, expression of such EphrinB2 polypeptides in a suitable cell,
such as HEK293T cell line, will result in cleavage of a leader
peptide. Although such cleavage is not always complete or perfectly
consistent at a single site, it is known that EphrinB2 tends to be
cleaved so as to remove the first 26 amino acids of the sequence
shown in FIG. 66 (SEQ ID NO:11). Accordingly, as specific examples,
the disclosure provides unprocessed soluble EphrinB2 polypeptides
that bind to EphB4 and comprise an amino acid sequence
corresponding to amino acids 1-225 of FIG. 66 (SEQ ID NO:11). Such
polypeptides may be used in a processed form, such forms having a
predicted amino acid sequence selected from the following group
(numbering is with respect to the sequence of FIG. 66, SEQ ID
NO:11): 26-225. In certain embodiments, the soluble EphrinB2
polypeptide may inhibit the interaction between Ephrin B2 and
EphB4. The soluble EphrinB2 polypeptide may inhibit clustering of
or phosphorylation of EphrinB2 or EphB4. As noted above, the
soluble EphrinB2 polypeptide may be prepared as a monomeric or
multimeric fusion protein. The soluble polypeptide may include one
or more modified amino acids. Such amino acids may contribute to
desirable properties, such as increased resistance to protease
digestion.
[0014] In certain aspects, the disclosure provides pharmaceutical
formulations comprising a polypeptide reagent and a
pharmaceutically acceptable carrier. The polypeptide reagent may be
any disclosed herein, including, for example, soluble EphB4 or
EphrinB2 polypeptides. Additional formulations include cosmetic
compositions and diagnostic kits.
[0015] In certain aspects the disclosure provides methods of
inhibiting signaling through Ephrin B2/EphB4 pathway in a cell. A
method may comprise contacting the cell with an effective amount of
a polypeptide agent, such as (a) a soluble polypeptide comprising
an amino acid sequence of an extracellular domain of an EphB4
protein, wherein the EphB4 polypeptide is a monomer and binds
specifically to an Ephrin B2 polypeptide; (b) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a
monomer and binds with high affinity to an EphB4 polypeptide.
[0016] In certain aspects the disclosure provides methods for
reducing the growth rate of a tumor, comprising administering an
amount of a polypeptide agent sufficient to reduce the growth rate
of the tumor. The polypeptide agent may be selected from the group
consisting of: (a) a soluble polypeptide comprising an amino acid
sequence of an extracellular domain of an EphB4 protein, wherein
the EphB4 polypeptide is a monomer and binds specifically to an
Ephrin B2 polypeptide, and optionally comprises an additional
modification to increase serum half-life, such as a PEGylation or
serum albumin or both; (b) a soluble polypeptide comprising an
amino acid sequence of an extracellular domain of an Ephrin B2
protein, wherein the soluble Ephrin B2 polypeptide is a monomer and
binds with high affinity to an EphB4 polypeptide Optionally, the
tumor comprises cells expressing a higher level of EphB4 and/or
EphrinB2 than noncancerous cells of a comparable tissue.
[0017] In certain aspects, the disclosure provides methods for
treating a patient suffering from a cancer. A method may comprise
administering to the patient a polypeptide agent. The polypeptide
agent may be selected from the group consisting of: (a) a soluble
polypeptide comprising an amino acid sequence of an extracellular
domain of an EphB4 protein, wherein the EphB4 polypeptide is a
monomer and binds specifically to an Ephrin B2 polypeptide, and
optionally comprises an additional modification to increase serum
half-life, such as a PEGylation or serum albumin or both; (b) a
soluble polypeptide comprising an amino acid sequence of an
extracellular domain of an Ephrin B2 protein, wherein the soluble
Ephrin B2 polypeptide is a monomer and binds with high affinity to
an EphB4 polypeptide. Optionally, the cancer comprises cancer cells
expressing EphrinB2 and/or EphB4 at a higher level than
noncancerous cells of a comparable tissue. The cancer may be a
metastatic cancer. The cancer may be selected from the group
consisting of colon carcinoma, breast tumor, mesothelioma, prostate
tumor, squamous cell carcinoma, Kaposi sarcoma, and leukemia.
Optionally, the cancer is an angiogenesis-dependent cancer or an
angiogenesis independent cancer. The polypeptide agent employed may
inhibit clustering or phosphorylation of Ephrin B2 or EphB4. A
polypeptide agent may be co-administered with one or more
additional anti-cancer chemotherapeutic agents that inhibit cancer
cells in an additive or synergistic manner with the polypeptide
agent.
[0018] In certain aspects, the disclosure provides methods of
inhibiting angiogenesis. A method may comprise contacting a cell
with an amount of a polypeptide agent sufficient to inhibit
angiogenesis. The polypeptide agent may be selected from the group
consisting of: (a) a soluble polypeptide comprising an amino acid
sequence of an extracellular domain of an EphB4 protein, wherein
the EphB4 polypeptide is a monomer and binds specifically to an
Ephrin B2 polypeptide, and optionally comprises an additional
modification to increase serum half-life, such as a PEGylation or
serum albumin or both; (b) a soluble polypeptide comprising an
amino acid sequence of an extracellular domain of an Ephrin B2
protein, wherein the soluble Ephrin B2 polypeptide is a monomer and
binds with high affinity to an EphB4 polypeptide.
[0019] In certain aspects, the disclosure provides methods for
treating a patient suffering from an angiogenesis-associated
disease, comprising administering to the patient a polypeptide
agent. The polypeptide agent may be selected from the group
consisting of: (a) a soluble polypeptide comprising an amino acid
sequence of an extracellular domain of an EphB4 protein, wherein
the EphB4 polypeptide is a monomer and binds specifically to an
Ephrin B2 polypeptide, and optionally comprises an additional
modification to increase serum half-life, such as a PEGylation or
serum albumin or both; (b) a soluble polypeptide comprising an
amino acid sequence of an extracellular domain of an Ephrin B2
protein, wherein the soluble Ephrin B2 polypeptide is a monomer and
binds with high affinity to an EphB4 polypeptide. The soluble
polypeptide may be formulated with a pharmaceutically acceptable
carrier. An angiogenesis related disease or unwanted angiogenesis
related process may be selected from the group consisting of
angiogenesis-dependent cancer, benign tumors, inflammatory
disorders, chronic articular rheumatism and psoriasis, ocular
angiogenic diseases, Osler-Webber Syndrome, myocardial
angiogenesis, plaque neovascularization, telangiectasia,
hemophiliac joints, angiofibroma, telangiectasia psoriasis
scleroderma, pyogenic granuloma, rubeosis, arthritis, diabetic
neovascularization, vasculogenesis. A polypeptide agent may be
co-administered with at least one additional anti-angiogenesis
agent that inhibits angiogenesis in an additive or synergistic
manner with the soluble polypeptide.
[0020] In certain aspects, the disclosure provides for the use of a
polypeptide agent in the manufacture of medicament for the
treatment of cancer or an angiogenesis related disorder. The
polypeptide agent may be selected from the group consisting of: (a)
a soluble polypeptide comprising an amino acid sequence of an
extracellular domain of an EphB4 protein, wherein the EphB4
polypeptide is a monomer and binds specifically to an Ephrin B2
polypeptide, and optionally comprises an additional modification to
increase serum half-life, such as a PEGylation or serum albumin or
both; (b) a soluble polypeptide comprising an amino acid sequence
of an extracellular domain of an Ephrin B2 protein, wherein the
soluble Ephrin B2 polypeptide is a monomer and binds with high
affinity to an EphB4 polypeptide.
[0021] In certain aspects, the disclosure provides methods for
treating a patient suffering from a cancer, comprising: (a)
identifying in the patient a tumor having a plurality of cancer
cells that express EphB4 and/or EphrinB2; and (b) administering to
the patient a polypeptide agent. The polypeptide agent may be
selected from the group consisting of: (i) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
EphB4 protein, wherein the EphB4 polypeptide is a monomer and binds
specifically to an Ephrin B2 polypeptide, and optionally comprises
an additional modification to increase serum half-life, such as a
PEGylation or serum albumin or both; (ii) a soluble polypeptide
comprising an amino acid sequence of an extracellular domain of an
Ephrin B2 protein, wherein the soluble Ephrin B2 polypeptide is a
monomer and binds with high affinity to an EphB4 polypeptide.
[0022] In certain aspects, the disclosure provides methods for
identifying a tumor that is suitable for treatment with an EphrinB2
or EphB4 antagonist. A method may comprise detecting in the tumor
cell one or more of the following characteristics: (a) expression
of EphB4 protein and/or mRNA; (b) expression of EphrinB2 protein
and/or mRNA; (c) gene amplification (e.g., increased gene copy
number) of the EphB4 gene; or (d) gene amplification of the
EphrinB2 gene. A tumor cell having one or more of characteristics
(a)-(d) may be suitable for treatment with an EphrinB2 or EphB4
antagonist, such as a polypeptide agent described herein.
[0023] Surprisingly, applicants have found that an EphB4
polypeptide lacking the globular domain can in fact inhibit tumor
growth in a xenograft model, inhibit angiogenic tube formation of
vascular endothelial cells and inhibit EphrinB2-activated
autokinase activity of EphB4. While not wishing to be bound to any
mechanism of action, it is expected that the polypeptide either
prevents EphB4 aggregation or stimulates the elimination (e.g. by
endocytosis) of EphB4 from the plasma membrane. Accordingly, the
disclosure provides isolated soluble polypeptides comprising an
amino acid sequence of a fibronectin type 3 domain of an EphB4
protein. Such polypeptides will preferably have a biological
effect, such as inhibiting an activity (e.g. aggregation or kinase
activity) of an EphB4 or EphrinB2 protein, and particularly the
inhibition of tumor growth in a human or in a mouse xenograft model
of cancer. Such polypeptides may also inhibit angiogenesis in vivo
or in an cell-based assay system. Such polypeptides may not bind to
EphrinB2 and may specifically exclude all of or the functional
(e.g., EphrinB2 binding-) portions of the globular domain of an
EphB4 protein. Such a polypeptide will preferably comprise amino
acids corresponding to amino acids 324-429 and/or 434-526 of the
sequence of FIG. 65 (SEQ ID NO:10), or sequences at least 90%, 95%,
98%, 99% identical thereto. An example of such a polypeptide is
shown in SEQ ID NO:15. Such a polypeptide may be modified in any of
the ways described herein, and may be produced as a monomer or as a
dimer or multimer comprising two or more such polypeptides, such as
an Fc fusion construct. Dimers or multimers may be desirable to
enhance the effectiveness of such polypeptides. All of the methods
for producing and using such polypeptides are similar to those
described herein with respect to other EphB4 polypeptides.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1 shows amino acid sequence of the B4ECv3 protein
(predicted sequence of the precursor including uncleaved Eph B4
leader peptide is shown; SEQ ID NO:1).
[0025] FIG. 2 shows amino acid sequence of the B4ECv3NT protein
(predicted sequence of the precursor including uncleaved Eph B4
leader peptide is shown; SEQ ID NO:2).
[0026] FIG. 3 shows amino acid sequence of the B2EC protein
(predicted sequence of the precursor including uncleaved Ephrin B2
leader peptide is shown; SEQ ID NO:3).
[0027] FIG. 4 shows amino acid sequence of the B4ECv3-FC protein
(predicted sequence of the precursor including uncleaved Eph B4
leader peptide is shown; SEQ ID NO:4).
[0028] FIG. 5 shows amino acid sequence of the B2EC-FC protein
(predicted sequence of the precursor including uncleaved Ephrin B2
leader peptide is shown; SEQ ID NO:5).
[0029] FIG. 6 shows B4EC-FC binding assay (Protein A-agarose
based).
[0030] FIG. 7 shows B4EC-FC inhibition assay (Inhibition in
solution).
[0031] FIG. 8 shows B2EC-FC binding assay (Protein-A-agarose based
assay).
[0032] FIG. 9 shows chemotaxis of HUAEC in response to B4Ecv3.
[0033] FIG. 10 shows chemotaxis of HHEC in response to B2EC-FC.
[0034] FIG. 11 shows chemotaxis of HHAEC in response to B2EC.
[0035] FIG. 12 shows effect of B4Ecv3 on HUAEC tubule
formation.
[0036] FIG. 13 shows effect of B2EC-FC on HUAEC tubule
formation.
[0037] FIG. 14 is a schematic representation of human Ephrin B2
constructs.
[0038] FIG. 15 is a schematic representation of human EphB4
constructs.
[0039] FIG. 16 shows the domain structure of the recombinant
soluble EphB4EC proteins. Designation of the domains are as
follows: L--leader peptide, G--globular (ligand-binding domain),
C--Cys-rich domain, F1, F2--fibronectin type III repeats,
H--6.times.His-tag.
[0040] FIG. 17 shows purification and ligand binding properties of
the EphB4EC proteins. A. SDS-PAAG gel electrophoresis of purified
EphB4-derived recombinant soluble proteins (Coomassie-stained). B.
Binding of Ephrin B2-AP fusion to EphB4-derived recombinant
proteins immobilized on Ni-NTA-agarose beads. Results of three
independent experiments are shown for each protein. Vertical
axis--optical density at 420 nm.
[0041] FIG. 18 shows that EphB4v3 inhibits chemotaxis.
[0042] FIG. 19 shows that EphB4v3 inhibits tubule formation on
Matrigel. A displays the strong inhibition of tubule formation by
B4v3 in a representative experiment. B shows a quantitation of the
reduction of tube-length obtained with B4v3 at increasing
concentrations as well as a reduction in the number of junctions,
in comparison to cells with no protein. Results are displayed as
mean values.+-.S.D. obtained from three independent experiments
performed with duplicate wells.
[0043] FIG. 20 shows that soluble EphB4 has no detectable cytotoxic
effect as assessed by MTS assay.
[0044] FIG. 21 shows that B4v3 inhibits invasion and tubule
formation by endothelial cells in the Matrigel assay. (A) to detect
total invading cells, photographed at 20.times. magnification or
with Masson's Trichrome Top left of A B displays section of a
Matrigel plug with no GF, top right of A displays section with
B4IgG containing GF and lower left section contains GF, and lower
right shows GF in the presence of B4v3. Significant invasion of
endothelial cells is only seen in GF containing Matrigel. Top right
displays an area with a high number of invaded cells induced by
B4IgG, which signifies the dimeric form of B4v3. The left upper
parts of the pictures correspond to the cell layers formed around
the Matrigel plug from which cells invade toward the center of the
plug located in the direction of the right lower corner. Total
cells in sections of the Matrigel plugs were quantitated with Scion
Image software. Results obtained from two experiments with
duplicate plugs are displayed as mean values.+-.S.D.
[0045] FIG. 22 shows tyrosine phosphorylation of EphB4 receptor in
PC3 cells in response to stimulation with EphrinB2-Fc fusion in
presence or absence of EphB4-derived recombinant soluble
proteins.
[0046] FIG. 23 shows effects of soluble EphB4ECD on viability and
cell cycle. A) 3-day cell viability assay of two HNSCC cell lines.
B) FACS analysis of cell cycle in HNSCC-15 cells treated as in A.
Treatment of these cells resulted in accumulation in subG0/G1 and
S/G2 phases as indicated by the arrows.
[0047] FIG. 24 shows that B4v3 inhibits endovascular response in a
murine corneal hydron micropocket assay.
[0048] FIG. 25 shows that that SCC15, B16, and MCF-7 co-injected
with sB4v3 in the presence of matrigel and growth factors, inhibits
the in vivo tumor growth of these cells.
[0049] FIG. 26 shows that soluble EphB4 causes apoptosis, necrosis
and decreased angiogenesis in three tumor types, B16 (melanoma),
SCC15 (head and neck carcinoma), and MCF-7 (breast carcinoma).
Tumors were injected premixed with Matrigel plus growth factors and
soluble EphB4 subcutaneously. After 10 to 14 days, the mice were
injected intravenously with FITC-lectin (green) to assess blood
vessel perfusion. Tumors treated with control PBS displayed
abundant tumor density and a robust angiogenic response. Tumors
treated with sEphB4 displayed a decrease in tumor cell density and
a marked inhibition of tumor angiogenesis in regions with viable
tumor cells, as well as tumor necrosis and apoptosis.
[0050] FIG. 27 shows expression of EphB4 in prostate cell lines. A)
Western blot of total cell lysates of various prostate cancer cell
lines, normal prostate gland derived cell line (MLC) and acute
myeloblastic lymphoma cells (AML) probed with EphB4 monoclonal
antibody. B) Phosphorylation of EphB4 in PC-3 cells determined by
Western blot.
[0051] FIG. 28 shows expression of EphB4 in prostate cancer tissue.
Representative prostate cancer frozen section stained with EphB4
monoclonal antibody (top left) or isotype specific control (bottom
left). Adjacent BPH tissue stained with EphB4 monoclonal antibody
(top right). Positive signal is brown color in the tumor cells.
Stroma and the normal epithelia are negative. Note membrane
localization of stain in the tumor tissue, consistent with
trans-membrane localization of EphB4. Representative QRT-PCR of RNA
extracted from cancer specimens and adjacent BPH tissues (lower
right).
[0052] FIG. 29 shows downregulation of EphB4 in prostate cancer
cells by tumor suppressors and RXR expression. A) PC3 cells were
co-transfected with truncated CD4 and p53 or PTEN or vector only.
24 h later CD4-sorted cells were collected, lysed and analyzed
sequentially by Western blot for the expression of EphB4 and
.beta.-actin, as a normalizer protein. B) Western blot as in (A) of
various stable cell lines. LNCaP-FGF is a stable transfection clone
of FGF-8, while CWR22R-RXR stably expresses the RXR receptor. BPH-1
was established from benign hypertrophic prostatic epithelium.
[0053] FIG. 30 shows regulation of EphB4 in prostate cancer cells
by EGFR and IGFR-1. A) Western blot of PC3 cells treated with or
without EGFR specific inhibitor AG 1478 (1 nM) for 36 hours.
Decreased EphB4 signal is observed after AG 1478 treatment. The
membrane was stripped and reprobed with .beta.-actin, which was
unaffected. B) Western Blot of triplicate samples of PC3 cells
treated with or without IGFR-1 specific neutralizing antibody
MAB391 (2 .mu.g/ml; overnight). The membrane was sequentially
probed with EphB4, IGFR-1 and .beta.-actin antibodies. IGFR-1
signal shows the expected repression of signal with MAB391
treatment.
[0054] FIG. 31 shows effect of specific EphB4 AS-ODNs and siRNA on
expression and prostate cell functions. A) 293 cells stably
expressing full-length construct of EphB4 was used to evaluate the
ability of siRNA 472 to inhibit EphB4 expression. Cells were
transfected with 50 nM RNAi using Lipofectamine 2000. Western blot
of cell lysates 40 h post transfection with control siRNA (green
fluorescence protein; GFP siRNA) or EphB4 siRNA 472, probed with
EphB4 monoclonal antibody, stripped and reprobed with .beta.-actin
monoclonal antibody. B) Effect of EphB4 AS-10 on expression in 293
transiently expressing full-length EphB4. Cells were exposed to
AS-10 or sense ODN for 6 hours and analyzed by Western blot as in
(A). C) 48 h viability assay of PC3 cells treated with siRNA as
described in the Methods section. Shown is mean.+-.s.e.m. of
triplicate samples. D) 5-day viability assay of PC3 cells treated
with ODNs as described in the Methods. Shown is mean.+-.s.e.m. of
triplicate samples. E) Scrape assay of migration of PC3 cells in
the presence of 50 nM siRNAs transfected as in (A). Shown are
photomicrographs of representative 20.times. fields taken
immediately after the scrape was made in the monolayer (0 h) and
after 20 h continued culture. A large number of cells have filled
in the scrape after 20 h with control siRNA, but not with EphB4
siRNA 472. F) Shown is a similar assay for cells treated with AS-10
or sense ODN (both 10 .mu.M). G) Matrigel invasion assay of PC3
cells transfected with siRNA or control siRNA as described in the
methods. Cells migrating to the underside of the Matrigel coated
insert in response to 5 mg/ml fibronectin in the lower chamber were
fixed and stained with Giemsa. Shown are representative
photomicrographs of control siRNA and siRNA 472 treated cells. Cell
numbers were counted in 5 individual high-powered fields and the
average.+-.s.e.m. is shown in the graph (bottom right).
[0055] FIG. 32 shows effect of EphB4 siRNA 472 on cell cycle and
apoptosis. A) PC3 cells transfected with siRNAs as indicated were
analyzed 24 h post transfection for cell cycle status by flow
cytometry as described in the Methods. Shown are the plots of cell
number vs. propidium iodide fluorescence intensity. 7.9% of the
cell population is apoptotic (in the Sub G0 peak) when treated with
siRNA 472 compared to 1% with control siRNA. B) Apoptosis of PC3
cells detected by Cell Death Detection ELISA.sup.plus kit as
described in the Methods. Absorbance at 405 nm increases in
proportion to the amount of histone and DNA-POD in the nuclei-free
cell fraction. Shown is the mean.+-.s.e.m. of triplicate samples at
the indicated concentrations of siRNA 472 and GFP siRNA
(control).
[0056] FIG. 33 shows that EphB4 and EphrinB2 are expressed in
mesothelioma cell lines as shown by RT-PCR (A) and Western Blot
(B).
[0057] FIG. 34 shows expression of ephrin B2 and EphB4 by in situ
hybridization in mesothelioma cells. NCI H28 mesothelioma cell
lines cultured in chamber slides hybridized with antisense probe to
ephrin B2 or EphB4 (top row). Control for each hybridization was
sense (bottom row). Positive reaction is dark blue cytoplasmic
stain.
[0058] FIG. 35 shows cellular expression of EphB4 and ephrin B2 in
mesothelioma cultures. Immunofluorescence staining of primary cell
isolate derived from pleural effusion of a patient with malignant
mesothelioma and cell lines NCI H28, NCI H2373, and NCI H2052 for
ephrin B2 and EphB4. Green color is positive signal for FITC
labeled secondary antibody. Specificity of immunofluorescence
staining was demonstrated by lack of signal with no primary
antibody (first row). Cell nuclei were counterstained with DAPI
(blue color) to reveal location of all cells. Shown are merged
images of DAPI and FITC fluorescence. Original magnification
200.times..
[0059] FIG. 36 shows expression of ephrin B2 and EphB4 in
mesothelioma tumor. Immunohistochemistry of malignant mesothelioma
biopsy. H&E stained section reveals tumor architecture; bottom
left panel is background control with no primary antibody. EphB4
and ephrin B2 specific staining is brown color. Original
magnification 200.times..
[0060] FIG. 37 shows effects of EPHB4 antisense probes (A) and
EPHB4 siRNAs (B) on the growth of H28 cells.
[0061] FIG. 38 shows effects of EPHB4 antisense probes (A) and
EPHB4 siRNAs (B) on cell migration.
[0062] FIG. 39 shows that EphB4 is expressed in HNSCC primary
tissues and metastases. A) Top: Immunohistochemistry of a
representative archival section stained with EphB4 monoclonal
antibody as described in the methods and visualized with DAB (brown
color) localized to tumor cells. Bottom: Hematoxylin and Eosin
(H&E) stain of an adjacent section. Dense purple staining
indicates the presence of tumor cells. The right hand column are
frozen sections of lymph node metastasis stained with EphB4
polyclonal antibody (top right) and visualized with DAB. Control
(middle) was incubation with goat serum and H&E (bottom)
reveals the location of the metastatic foci surrounded by stroma
which does not stain. B) In situ hybridization of serial frozen
sections of a HNSCC case probed with EphB4 (left column) and ephrin
B2 (right column) DIG labeled antisense or sense probes generated
by run-off transcription. Hybridization signal (dark blue) was
detected using alkaline-phosphatase-conjugated anti-DIG antibodies
and sections were counterstained with Nuclear Fast Red. A serial
section stained with H&E is shown (bottom left) to illustrate
tumor architecture. C) Western blot of protein extract of patient
samples consisting of tumor (T), uninvolved normal tissue (N) and
lymph node biopsies (LN). Samples were fractionated by
polyacrylamide gel electrophoresis in 4-20% Tris-glycine gels and
subsequently electroblotted onto nylon membranes. Membranes were
sequentially probed with EphB4 monoclonal antibody and .beta.-actin
MoAb. Chemiluminescent signal was detected on autoradiography film.
Shown is the EphB4 specific band which migrated at 120 kD and
.beta.-actin which migrated at 40 kD. The .beta.-actin signal was
used to control for loading and transfer of each sample.
[0063] FIG. 40 shows that EphB4 is expressed in HNSCC cell lines
and is regulated by EGF: A) Survey of EphB4 expression in SCC cell
lines. Western blot of total cell lysates sequentially probed with
EphB4 monoclonal antibody, stripped and reprobed with .beta.-actin
monoclonal antibody as described for FIG. 39C. B) Effect of the
specific EGFR inhibitor AG1478 on EphB4 expression: Western blot of
crude cell lysates of SCC15 treated with 0-1000 nM AG 1478 for 24 h
in media supplemented with 10% FCS (left) or with 1 mM AG 1478 for
4, 8, 12 or 24 h (right). Shown are membranes sequentially probed
for EphB4 and .beta.-actin. C) Effect of inhibition of EGFR
signaling on EphB4 expression in SCC cell lines: Cells maintained
in growth media containing 10% FCS were treated for 24 hr with 1
.mu.M AG 1478, after which crude cell lysates were analyzed by
Western blots of cell lysates sequentially probed with for EGFR,
EphB4, ephrin B2 and .alpha.-actin antibodies. Specific signal for
EGFR was detected at 170 kD and ephrin B2 at 37 kD in addition to
EphB4 and .beta.-actin as described in FIG. 1C. .beta.-actin serves
as loading and transfer control.
[0064] FIG. 41 shows mechanism of regulation of EphB4 by EGF: A)
Schematic of the EGFR signaling pathways, showing in red the sites
of action and names of specific kinase inhibitors used. B) SCC15
cells were serum-starved for 24 h prior to an additional 24
incubation as indicated with or without EGF (10 ng/ml), 3 .mu.M
U73122, or 5 .mu.M SH-5, 5 .mu.M SP600125, 25 nM LY294002, -- .mu.M
PD098095 or 5 .mu.M SB203580. N/A indicates cultures that received
equal volume of diluent (DMSO) only. Cell lysates were subjected to
Western Blot with EphB4 monoclonal antibody. .beta.-actin signal
serves as control of protein loading and transfer.
[0065] FIG. 42 shows that specific EphB4 siRNAs inhibit EphB4
expression, cell viability and cause cell cycle arrest. A) 293
cells stably expressing full length EphB4 were transfected with 50
nM RNAi using Lipofectamine.TM.2000. 40 h post-transfection cells
were harvested, lysed and processed for Western blot. Membranes
were probed with EphB4 monoclonal antibody, stripped and reprobed
with .beta.-actin monoclonal antibody as control for protein
loading and transfer. Negative reagent control was RNAi to
scrambled green fluorescence protein (GFP) sequence and control is
transfection with Lipofectamine.TM.2000 alone. B) MTT cell
viability assays of SCC cell lines treated with siRNAs for 48 h as
described in the Methods section. Shown is mean+s.e.m. of
triplicate samples. C) SCC15 cells transfected with siRNAs as
indicated were analyzed 24 h post transfection for cell cycle
status by flow cytometry as described in the Methods. Shown are the
plots of cell number vs. propidium iodide fluorescence intensity.
Top and middle row show plots for cells 16 h after siRNA
transfection, bottom row shows plots for cells 36 h post
transfection. Specific siRNA and concentration are indicated for
each plot. Lipo=Lipofectamine.TM.200 mock transfection.
[0066] FIG. 43 shows in vitro effects of specific EphB4 AS-ODNs on
SCC cells. A) 293 cells transiently transfected with EphB4
full-length expression plasmid were treated 6 h post transfection
with antisense ODNs as indicated. Cell lysates were collected 24 h
after AS-ODN treatment and subjected to Western Blot. B) SCC25
cells were seeded on 48 well plates at equal densities and treated
with EphB4 AS-ODNs at 1, 5, and 10 .mu.M on days 2 and 4. Cell
viability was measured by MTT assay on day 5. Shown is the
mean+s.e.m. of triplicate samples. Note that AS-ODNs that were
active in inhibiting EphB4 protein levels were also effective
inhibitors of SCC15 cell viability. C) Cell cycle analysis of SCC15
cells treated for 36 h with AS-10 (bottom) compared to cells that
were not treated (top). D) Confluent cultures of SCC15 cells
scraped with a plastic Pasteur pipette to produce 3 mm wide breaks
in the monolayer. The ability of the cells to migrate and close the
wound in the presence of inhibiting EphB4 AS-ODN (AS-10) and
non-inhibiting AS-ODN (AS-1) was assessed after 48 h. Scrambled ODN
is included as a negative control ODN. Culture labeled no treatment
was not exposed to ODN. At initiation of the experiment, all
cultures showed scrapes of equal width and similar to that seen in
1 .mu.M EphB4 AS-10 after 48, h. The red brackets indicate the
width of the original scrape. E) Migration of SCC15 cells in
response to 20 mg/ml EGF in two-chamber assay as described in the
Methods. Shown are representative photomicrographs of non-treated
(NT), AS-6 and AS-10 treated cells and 10 ng/ml Taxol as positive
control of migration inhibition. F) Cell numbers were counted in 5
individual high-powered fields and the average+s.e.m. is shown in
the graph.
[0067] FIG. 44 shows that EphB4 AS-ODN inhibits tumor growth in
vivo. Growth curves for SCC15 subcutaneous tumor xenografts in
Balb/C nude mice treated with EphB4 AS-10 or scrambled ODN at 20
mg/kg/day starting the day following implantation of 5.times.106
cells. Control mice received and equal volume of diluent (PBS).
Shown are the mean+s.e.m. of 6 mice/group. * P=0.0001 by Student's
t-test compared to scrambled ODN treated group.
[0068] FIG. 45 shows that Ephrin B2, but not EphB4 is expressed in
KS biopsy tissue. (A) In situ hybridization with antisense probes
for ephrin B2 and EphB4 with corresponding H&E stained section
to show tumor architecture. Dark blue color in the ISH indicates
positive reaction for ephrin B2. No signal for EphB4 was detected
in the Kaposi's sarcoma biopsy. For contrast, ISH signal for EphB4
is strong in squamous cell carcinoma tumor cells. Ephrin B2 was
also detected in KS using EphB4-AP fusion protein (bottom left).
(B) Detection of ephrin B2 with EphB4/Fc fusion protein. Adjacent
sections were stained with H&E (left) to show tumor
architecture, black rectangle indicates the area shown in the
EphB4/Fc treated section (middle) detected with FITC-labeled
anti-human Fc antibody as described in the methods section. As a
control an adjacent section was treated with human Fc fragment
(right). Specific signal arising from EphB4/Fc binding to the
section is seen only in areas of tumor cells. (C) Co-expression of
ephrin B2 and the HHV8 latency protein LANA1. Double-label confocal
immunofluorescence microscopy with antibodies to ephrin B2 (red)
LANA1 (green), or EphB4 (red) of frozen KS biopsy material directly
demonstrates co-expression of LANA1 and ephrin B2 in KS biopsy.
Coexpression is seen as yellow color. Double label confocal image
of biopsy with antibodies to PECAM-1 (green) in cells with nuclear
propidium iodide stain (red), demonstrating the vascular nature of
the tumor.
[0069] FIG. 46 shows that HHV-8 induces arterial marker expression
in venous endothelial cells. (A) Immunofluorescence of cultures of
HUVEC and HUVEC/BC-1 for artery/vein markers and viral proteins.
Cultures were grown on chamber slides and processed for
immunofluorescence detection of ephrin B2 (a, e, i), EphB4 (m, q,
u), CD148 (j, v), and the HHV-8 proteins LANA1 (b, f, m) or ORF59
(r) as described in the Materials and Methods. Yellow color in the
merged images of the same field demonstrate co-expression of ephrin
B2 and LANA or ephrin B2 and CD148. The positions of viable cells
were revealed by nuclear staining with DAPI (blue) in the third
column (c, g, k, o, s, w). Photomicrographs are of representative
fields. (B) RT-PCR of HUVEC and two HHV-8 infected cultures
(HUVEC/BC-1 and HUVEC/BC-3) for ephrin B2 and EphB4. Ephrin B2
product (200 bp) is seen in HUVEC/BC-1, HUVEC/BC-3 and EphB4
product (400 bp) is seen in HUVEC. Shown also is .beta.-actin
RT-PCR as a control for amount and integrity of input RNA.
[0070] FIG. 47 shows that HHV-8 induces arterial marker expression
in Kaposi's sarcoma cells. (A) Western blot for ephrin B2 on
various cell lysates. SLK-vGPCR is a stable clone of SLK expressing
the HHV-8 vGPCR, and SLK-pCEFL is control stable clone transfected
with empty expression vector. SLK cells transfected with LANA or
LANA.DELTA.440 are SLK-LANA and SLK-.DELTA.440 respectively.
Quantity of protein loading and transfer was determined by
reprobing the membranes with .beta.-actin monoclonal antibody. (B)
Transient transfection of KS-SLK cells with expression vector
pvGPCR-CEFL resulted in the expression of ephrin B2 as shown by
immunofluorescence staining with FITC (green), whereas the control
vector pCEFL had no effect. KS-SLK cells (0.8.times.105/well) were
transfected with 0.8 .mu.g DNA using Lipofectamine 2000. 24 hr
later cells were fixed and stained with ephrin B2 polyclonal
antibody and FITC conjugated secondary antibody as described in the
methods. (C) Transient transfection of HUVEC with vGPCR induces
transcription from ephrin B2 luciferase constructs. 8.times.103
HUVEC in 24 well plates were transfected using Superfect with 0.8
.mu.g/well ephrin B2 promoter constructs containing sequences from
-2941 to -11 with respect to the translation start site, or two
5'-deletions as indicated, together with 80 ng/well pCEFL or
pvGPCR-CEFL. Luciferase was determined 48 h post transfection and
induction ratios are shown to the right of the graph. pGL3Basic is
promoterless luciferase control vector. Luciferase was normalized
to protein since GPCR induced expression of the cotransfected
.beta.-galactosidase. Graphed is mean+SEM of 6 replicates. Shown is
one of three similar experiments.
[0071] FIG. 48 shows that VEGF and VEGF-C regulate ephrin B2
expression. A) Inhibition of ephrin B2 by neutralizing antibodies.
Cells were cultured in full growth medium and exposed to antibody
(100 ng/ml) for 36 hr before collection and lysis for Western blot.
B) For induction of ephrin B2 expression cells were cultured in EBM
growth medium containing 5% serum lacking growth factors.
Individual growth factors were added as indicated and the cells
harvested after 36 h. Quantity of protein loading and transfer was
determined by reprobing the membranes .beta.-actin monoclonal
antibody.
[0072] FIG. 49 shows that Ephrin B2 knock-down with specific siRNA
inhibits viability in KS cells and HUVEC grown in the presence of
VEGF but not IGF, EGF or bFGF. A) KS-SLK cells were transfected
with various siRNA to ephrin B2 and controls. After 48 hr the cells
were harvested and crude cell lysates fractionated on 4-20%
SDS-PAGE. Western blot was performed with monoclonal antibody to
ephrin B2 generated in-house. The membrane was stripped and
reprobed with .beta.-actin monoclonal antibody (Sigma) to
illustrate equivalent loading and transfer. B) 3 day cell viability
assay of KS-SLK cultures in the presence of ephrin B2 and EphB4
siRNAs. 1.times.10.sup.5 cells/well in 24-well plates were treated
with 0, 10 and 100 ng/ml siRNAs as indicated on the graph.
Viability of cultures was determined by MTT assay as described in
the methods section. Shown are the mean+standard deviation of
duplicate samples. C) HUVE cells were seeded on eight wells chamber
slides coated with fibronectin. The HUVE cells were grown overnight
in EGM-2 media, which contains all growth supplements. On the
following day, the media was replaced with media containing VEGF
(10 ng/ml) or EGF, FGF and IGF as indicated. After 2 hrs of
incubation at 37.degree. C., the cells were transfected using
Lipofectamine 2000 (Invitrogen) in Opti-MEM medium containing 10 nM
of siRNA to ephrin B2, Eph B4 or green fluorescence protein (GFP)
as control. The cells were incubated for 2 hr and then the fresh
media containing growth factors or VEGF alone was added to their
respective wells. After 48 hrs, the cells were stained with crystal
violet and the pictures were taken immediately by digital camera at
10.times. magnification.
[0073] FIG. 50 shows that soluble EphB4 inhibits KS and EC cord
formation and in vivo angiogenesis. Cord formation assay of HUVEC
in Matrigel.TM. (upper row). Cells in exponential growth phase were
treated overnight with the indicated concentrations of EphB4
extracellular domain (ECD) prior to plating on Matrigel.TM.. Cells
were trypsinized and plated (1.times.10.sup.5 cells/well) in a
24-well plate containing 0.5 ml Matrigel.TM.. Shown are
representative 20.times. phase contrast fields of cord formation
after 8 hr plating on Matrigel.TM. in the continued presence of the
test compounds as shown. Original magnification 200.times.. KS-SLK
cells treated in a similar manner (middle row) in a cord formation
assay on Matrigel.TM.. Bottom row shows in vivo Matrigel.TM. assay:
Matrigel.TM. plugs containing growth factors and EphB4 ECD or PBS
were implanted subcutaneously in the mid-ventral region of mice.
After 7 days the plugs were removed, sectioned and stained with
H&E to visualize cells migrating into the matrix. Intact
vessels with large lumens are observed in the control, whereas
EphB4 ECD almost completely inhibited migration of cells into the
Matrigel.
[0074] FIG. 51 shows expression of EPHB4 in bladder cancer cell
lines (A), and regulation of EPHB4 expression by EGFR signaling
pathway (B).
[0075] FIG. 52 shows that transfection of p53 inhibit the
expression of EPHB4 in 5637 cell.
[0076] FIG. 53 shows growth inhibition of bladder cancer cell line
(5637) upon treatment with EPHB4 siRNA 472.
[0077] FIG. 54 shows results on apoptosis study of 5637 cells
transfected with EPHB4 siRNA 472.
[0078] FIG. 55 shows effects of EPHB4 antisense probes on cell
migration. 5637 cells were treated with EPHB4AS10 (10 .mu.M)
(bottom panels). Upper panels show control cells.
[0079] FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637
cells were transfected with siRNA 472 or control siRNA.
[0080] FIG. 57 shows comparison of EphB4 monoclonal antibodies by
G250 and in pull-down assay.
[0081] FIG. 58 shows that EphB4 antibodies inhibit the growth of
SCC15 xenograft tumors.
[0082] FIG. 59 shows that EphB4 antibodies cause apoptosis,
necrosis and decreased angiogenesis in SCC15, head and neck
carcinoma tumor type.
[0083] FIG. 60 shows that systemic administration of EphB4
antibodies leads to tumor regression.
[0084] FIG. 61 shows a genomic nucleotide sequence of human EphB4
(SEQ ID NO:6).
[0085] FIG. 62 shows a cDNA nucleotide sequence of human EphB4 (SEQ
ID NO:7).
[0086] FIG. 63 shows a genomic nucleotide sequence of human Ephrin
B2 (SEQ ID NO:8).
[0087] FIG. 64 shows a cDNA nucleotide sequence of human Ephrin B2
(SEQ ID NO:9).
[0088] FIG. 65 shows an amino acid sequence of human EphB4 (SEQ ID
NO:10).
[0089] FIG. 66 shows an amino acid sequence of human Ephrin B2 (SEQ
ID NO:11).
[0090] FIG. 67 shows a comparison of the EphrinB2 binding
properties of the HSA-EphB4 fusion protein and other EphB4
polypeptides.
[0091] FIG. 68 shows a comparison between the in vivo stability of
an EphB4-HSA fusion protein and an EphB4 polypeptide in mice.
[0092] FIG. 69 shows the EphrinB2 binding activity of soluble EphB4
polypeptides pegylated under specific pH conditions.
[0093] FIG. 70 shows the chromatographic separation of PEG
derivatives of EphB4 protein on SP-Sepharose columns. Purity of the
PEG-modified EphB4 protein was analyzed by PAGE. The EphrinB2
binding of the pegylation reaction products is also shown.
[0094] FIG. 71 shows the purity, as determined by SDS-PAGE, of
chromatography-separated unpegylated, monopegylated and
poly-pegylated EphB4 fractions.
[0095] FIG. 72 shows the EphrinB2-binding activity of the
chromatography fractions from the EphB4 pegylation reaction.
[0096] FIG. 73 shows the retention of EphrinB2-binding activity of
the chromatography fractions from the EphB4 pegylation reaction
after incubation in mouse serum at 37.degree. C. for three
days.
[0097] FIG. 74 shows the in vivo stability of unpegylated,
monopegylated and polypegylated EphB4 in mice over time.
DETAILED DESCRIPTION OF THE INVENTION
I. Overview
[0098] The current invention is based in part on the discovery that
signaling through the ephrin/ephrin receptor (ephrin/eph) pathway
contributes to tumorigenesis. Applicants detected expression of
ephrin B2 and EphB4 in tumor tissues and developed anti-tumor
therapeutic agents for blocking signaling through the ephrin/eph.
In addition, the disclosure provides polypeptide therapeutic agents
and methods for polypeptide-based inhibition of the function of
EphB4 and/or Ephrin B2. Accordingly, in certain aspects, the
disclosure provides numerous polypeptide compounds (agents) that
may be used to treat cancer as well as angiogenesis related
disorders and unwanted angiogenesis related processes. Applicants
have generated modified forms of EphrinB2 and EphB4 polypeptides
and have demonstrated that such modified forms have markedly
improved pharmacokinetic properties. Accordingly, in certain
aspects, the disclosure provides numerous polypeptide compounds
(agents) that may be used to treat cancer as well as angiogenesis
related disorders and unwanted angiogenesis related processes.
[0099] As used herein, the terms Ephrin and Eph are used to refer,
respectively, to ligands and receptors. They can be from any of a
variety of animals (e.g., mammals/non-mammals,
vertebrates/non-vertebrates, including humans). The nomenclature in
this area has changed rapidly and the terminology used herein is
that proposed as a result of work by the Eph Nomenclature
Committee, which can be accessed, along with previously-used names
at web site http://www.eph-nomenclature.com.
[0100] The work described herein, particularly in the examples,
refers to Ephrin B2 and EphB4. However, the present invention
contemplates any ephrin ligand and/or Eph receptor within their
respective family, which is expressed in a tumor. The ephrins
(ligands) are of two structural types, which can be further
subdivided on the basis of sequence relationships and,
functionally, on the basis of the preferential binding they exhibit
for two corresponding receptor subgroups. Structurally, there are
two types of ephrins: those which are membrane-anchored by a
glycerophosphatidylinositol (GPI) linkage and those anchored
through a transmembrane domain. Conventionally, the ligands are
divided into the Ephrin-A subclass, which are GPI-linked proteins
which bind preferentially to EphA receptors, and the Ephrin-B
subclass, which are transmembrane proteins which generally bind
preferentially to EphB receptors.
[0101] The Eph family receptors are a family of receptor
protein-tyrosine kinases which are related to Eph, a receptor named
for its expression in an erythropoietin-producing human
hepatocellular carcinoma cell line. They are divided into two
subgroups on the basis of the relatedness of their extracellular
domain sequences and their ability to bind preferentially to
Ephrin-A proteins or Ephrin-B proteins. Receptors which interact
preferentially with Ephrin-A proteins are EphA receptors and those
which interact preferentially with Ephrin-B proteins are EphB
receptors.
[0102] Eph receptors have an extracellular domain composed of the
ligand-binding globular domain, a cysteine rich region followed by
a pair of fibronectin type III repeats (e.g., see FIG. 16). The
cytoplasmic domain consists of a juxtamembrane region containing
two conserved tyrosine residues; a protein tyrosine kinase domain;
a sterile .alpha.-motif (SAM) and a PDZ-domain binding motif. EphB4
is specific for the membrane-bound ligand Ephrin B2 (Sakano, S. et
al 1996; Brambilla R. et al 1995). Ephrin B2 belongs to the class
of Eph ligands that have a transmembrane domain and cytoplasmic
region with five conserved tyrosine residues and PDZ domain. Eph
receptors are activated by binding of clustered, membrane attached
ephrins (Davis S et al, 1994), indicating that contact between
cells expressing the receptors and cells expressing the ligands is
required for Eph activation.
[0103] Upon ligand binding, an Eph receptor dimerizes and
autophosphorylate the juxtamembrane tyrosine residues to acquire
full activation (Kalo M S et al, 1999, Binns K S, 2000). In
addition to forward signaling through the Eph receptor, reverse
signaling can occur through the ephrin Bs. Eph engagement of
ephrins results in rapid phosphorylation of the conserved
intracellular tyrosines (Bruckner K, 1997) and somewhat slower
recruitment of PDZ binding proteins (Palmer A 2002). Recently,
several studies have shown that high expression of Eph/ephrins may
be associated with increased potentials for tumor growth,
tumorigenicity, and metastasis (Easty D J, 1999; Kiyokawa E, 1994;
Tang X X, 1999; Vogt T, 1998; Liu W, 2002; Stephenson S A, 2001;
Steube K G 1999; Berclaz G, 1996).
[0104] In certain embodiments, the present invention provides
polypeptide therapeutic agents that inhibit activity of Ephrin B2,
EphB4, or both. As used herein, the term "polypeptide therapeutic
agent" or "polypeptide agent" is a generic term which includes any
polypeptide that blocks signaling through the Ephrin B2/EphB4
pathway. A preferred polypeptide therapeutic agent of the invention
is a soluble polypeptide of Ephrin B2 or EphB4. Another preferred
polypeptide therapeutic agent of the invention is an antagonist
antibody that binds to Ephrin B2 or EphB4. For example, such
polypeptide therapeutic agent can inhibit function of Ephrin B2 or
EphB4, inhibit the interaction between Ephrin B2 and EphB4, inhibit
the phosphorylation of Ephrin B2 or EphB4, or inhibit any of the
downstream signaling events upon binding of Ephrin B2 to EphB4.
Such polypeptides may include EphB4 or EphrinB2 that are modified
so as to improve serum half-life, such as by PEGylation or stable
association with a serum albumin protein.
II. Soluble Polypeptides
[0105] In certain aspects, the invention relates to a soluble
polypeptide comprising an extracellular domain of an Ephrin B2
protein (referred to herein as an Ephrin B2 soluble polypeptide) or
comprising an extracellular domain of an EphB4 protein (referred to
herein as an EphB4 soluble polypeptide). Preferably, the subject
soluble polypeptide is a monomer and is capable of binding with
high affinity to Ephrin B2 or EphB4. In a specific embodiment, the
EphB4 soluble polypeptide of the invention comprises a globular
domain of an EphB4 protein. Specific examples EphB4 soluble
polypeptides are provided in FIGS. 1, 2, and 15. Specific examples
of Ephrin B2 soluble polypeptides are provided in FIGS. 3 and
14.
[0106] As used herein, the subject soluble polypeptides include
fragments, functional variants, and modified forms of EphB4 soluble
polypeptide or an Ephrin B2 soluble polypeptide. These fragments,
functional variants, and modified forms of the subject soluble
polypeptides antagonize function of EphB4, Ephrin B2 or both.
[0107] In certain embodiments, isolated fragments of the subject
soluble polypeptides can be obtained by screening polypeptides
recombinantly produced from the corresponding fragment of the
nucleic acid encoding an EphB4 or Ephrin B2 soluble polypeptides.
In addition, fragments can be chemically synthesized using
techniques known in the art such as conventional Merrifield solid
phase f-Moc or t-Boc chemistry. The fragments can be produced
(recombinantly or by chemical synthesis) and tested to identify
those peptidyl fragments that can function to inhibit function of
EphB4 or Ephrin B2, for example, by testing the ability of the
fragments to inhibit angiogenesis or tumor growth.
[0108] In certain embodiments, a functional variant of an EphB4
soluble polypeptide comprises an amino acid sequence that is at
least 90%, 95%, 97%, 99% or 100% identical to residues 1-197,
29-197, 1-312, 29-132, 1-321, 29-321, 1-326, 29-326, 1-412, 29-412,
1-427, 29-427, 1-429, 29-429, 1-526, 29-526, 1-537 and 29-537 of
the amino acid sequence defined by FIG. 65 (SEQ ID NO:10). Such
polypeptides may be used in a processed form, and accordingly, in
certain embodiments, an EphB4 soluble polypeptide comprises an
amino acid sequence that is at least 90%, 95%, 97%, 99% or 100%
identical to residues 16-197, 16-312, 16-321, 16-326, 16-412,
16-427, 16-429, 16-526 and 16-537 of the amino acid sequence
defined by FIG. 65 (SEQ ID NO:10).
[0109] In other embodiments, a functional variant of an Ephrin B2
soluble polypeptide comprises a sequence at least 90%, 95%, 97%,
99% or 100% identical to residues 1-225 of the amino acid sequence
defined by FIG. 66 (SEQ ID NO:11) or a processed form, such as one
comprising a sequence at least 90%, 95%, 97%, 99% or 100% identical
to residues 26-225 of the amino acid sequence defined by FIG. 66
(SEQ ID NO:11).
[0110] In certain embodiments, the present invention contemplates
making functional variants by modifying the structure of the
subject soluble polypeptide for such purposes as enhancing
therapeutic or prophylactic efficacy, or stability (e.g., ex vivo
shelf life and resistance to proteolytic degradation in vivo). Such
modified soluble polypeptide are considered functional equivalents
of the naturally-occurring EphB4 or Ephrin B2 soluble polypeptide.
Modified soluble polypeptides can be produced, for instance, by
amino acid substitution, deletion, or addition. For instance, it is
reasonable to expect, for example, that an isolated replacement of
a leucine with an isoleucine or valine, an aspartate with a
glutamate, a threonine with a serine, or a similar replacement of
an amino acid with a structurally related amino acid (e.g.,
conservative mutations) will not have a major effect on the
biological activity of the resulting molecule. Conservative
replacements are those that take place within a family of amino
acids that are related in their side chains.
[0111] This invention further contemplates a method of generating
sets of combinatorial mutants of the EphB4 or Ephrin B2 soluble
polypeptides, as well as truncation mutants, and is especially
useful for identifying functional variant sequences. The purpose of
screening such combinatorial libraries may be to generate, for
example, soluble polypeptide variants which can act as antagonists
of EphB4, EphB2, or both. Combinatorially-derived variants can be
generated which have a selective potency relative to a naturally
occurring soluble polypeptide. Such variant proteins, when
expressed from recombinant DNA constructs, can be used in gene
therapy protocols. Likewise, mutagenesis can give rise to variants
which have intracellular half-lives dramatically different than the
corresponding wild-type soluble polypeptide. For example, the
altered protein can be rendered either more stable or less stable
to proteolytic degradation or other cellular process which result
in destruction of, or otherwise inactivation of the protein of
interest (e.g., a soluble polypeptide). Such variants, and the
genes which encode them, can be utilized to alter the subject
soluble polypeptide levels by modulating their half-life. For
instance, a short half-life can give rise to more transient
biological effects and, when part of an inducible expression
system, can allow tighter control of recombinant soluble
polypeptide levels within the cell. As above, such proteins, and
particularly their recombinant nucleic acid constructs, can be used
in gene therapy protocols.
[0112] There are many ways by which the library of potential
homologs can be generated from a degenerate oligonucleotide
sequence. Chemical synthesis of a degenerate gene sequence can be
carried out in an automatic DNA synthesizer, and the synthetic
genes then be ligated into an appropriate gene for expression. The
purpose of a degenerate set of genes is to provide, in one mixture,
all of the sequences encoding the desired set of potential soluble
polypeptide sequences. The synthesis of degenerate oligonucleotides
is well known in the art (see for example, Narang, S A (1983)
Tetrahedron 39:3; Itakura et al., (1981) Recombinant DNA, Proc. 3rd
Cleveland Sympos. Macromolecules, ed. A G Walton, Amsterdam:
Elsevier pp 273-289; Itakura et al., (1984) Annu. Rev. Biochem.
53:323; Itakura et al., (1984) Science 198:1056; Ike et al., (1983)
Nucleic Acid Res. 11:477). Such techniques have been employed in
the directed evolution of other proteins (see, for example, Scott
et al., (1990) Science 249:386-390; Roberts et al., (1992) PNAS USA
89:2429-2433; Devlin et al., (1990) Science 249: 404-406; Cwirla et
al., (1990) PNAS USA 87: 6378-6382; as well as U.S. Pat. Nos.
5,223,409, 5,198,346, and 5,096,815).
[0113] Alternatively, other forms of mutagenesis can be utilized to
generate a combinatorial library. For example, soluble polypeptide
variants (e.g., the antagonist forms) can be generated and isolated
from a library by screening using, for example, alanine scanning
mutagenesis and the like (Ruf et al., (1994) Biochemistry
33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095-3099;
Balint et al., (1993) Gene 137:109-118; Grodberg et al., (1993)
Eur. J. Biochem. 218:597-601; Nagashima et al., (1993) J. Biol.
Chem. 268:2888-2892; Lowman et al., (1991) Biochemistry
30:10832-10838; and Cunningham et al., (1989) Science
244:1081-1085), by linker scanning mutagenesis (Gustin et al.,
(1993) Virology 193:653-660; Brown et al., (1992) Mol. Cell Biol.
12:2644-2652; McKnight et al., (1982) Science 232:316); by
saturation mutagenesis (Meyers et al., (1986) Science 232:613); by
PCR mutagenesis (Leung et al., (1989) Method Cell Mol Biol
1:11-19); or by random mutagenesis, including chemical mutagenesis,
etc. (Miller et al., (1992) A Short Course in Bacterial Genetics,
CSHL Press, Cold Spring Harbor, N.Y.; and Greener et al., (1994)
Strategies in Mol Biol 7:32-34). Linker scanning mutagenesis,
particularly in a combinatorial setting, is an attractive method
for identifying truncated (bioactive) forms of the subject soluble
polypeptide.
[0114] A wide range of techniques are known in the art for
screening gene products of combinatorial libraries made by point
mutations and truncations, and, for that matter, for screening cDNA
libraries for gene products having a certain property. Such
techniques will be generally adaptable for rapid screening of the
gene libraries generated by the combinatorial mutagenesis of the
subject soluble polypeptides. The most widely used techniques for
screening large gene libraries typically comprises cloning the gene
library into replicable expression vectors, transforming
appropriate cells with the resulting library of vectors, and
expressing the combinatorial genes under conditions in which
detection of a desired activity facilitates relatively easy
isolation of the vector encoding the gene whose product was
detected. Each of the illustrative assays described below are
amenable to high through-put analysis as necessary to screen large
numbers of degenerate sequences created by combinatorial
mutagenesis techniques.
[0115] In certain embodiments, the subject soluble polypeptides of
the invention include a small molecule such as a peptide and a
peptidomimetic. As used herein, the term "peptidomimetic" includes
chemically modified peptides and peptide-like molecules that
contain non-naturally occurring amino acids, peptoids, and the
like. Peptidomimetics provide various advantages over a peptide,
including enhanced stability when administered to a subject.
Methods for identifying a peptidomimetic are well known in the art
and include the screening of databases that contain libraries of
potential peptidomimetics. For example, the Cambridge Structural
Database contains a collection of greater than 300,000 compounds
that have known crystal structures (Allen et al., Acta Crystallogr.
Section B, 35:2331 (1979)). Where no crystal structure of a target
molecule is available, a structure can be generated using, for
example, the program CONCORD (Rusinko et al., J. Chem. Inf. Comput.
Sci. 29:251 (1989)). Another database, the Available Chemicals
Directory (Molecular Design Limited, Informations Systems; San
Leandro Calif.), contains about 100,000 compounds that are
commercially available and also can be searched to identify
potential peptidomimetics of the EphB4 or Ephrin B2 soluble
polypeptides.
[0116] In certain embodiments, the soluble polypeptides of the
invention may further comprise post-translational modifications.
Exemplary post-translational protein modification include
phosphorylation, acetylation, methylation, ADP-ribosylation,
ubiquitination, glycosylation, carbonylation, sumoylation,
biotinylation or addition of a polypeptide side chain or of a
hydrophobic group. As a result, the modified soluble polypeptides
may contain non-amino acid elements, such as lipids, poly- or
mono-saccharide, and phosphates. Effects of such non-amino acid
elements on the functionality of a soluble polypeptide may be
tested for its antagonizing role in EphB4 or Ephrin B2 function,
e.g, it inhibitory effect on angiogenesis or on tumor growth.
[0117] In one specific embodiment of the present invention,
modified forms of the subject soluble polypeptides comprise linking
the subject soluble polypeptides to nonproteinaceous polymers. In
one specific embodiment, the polymer is polyethylene glycol
("PEG"), polypropylene glycol, or polyoxyalkylenes, in the manner
as set forth in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144;
4,670,417; 4,791,192 or 4,179,337. Examples of the modified
polypeptide of the invention include PEGylated soluble Ephrin B2
and PEGylated soluble EphB4.
[0118] PEG is a well-known, water soluble polymer that is
commercially available or can be prepared by ring-opening
polymerization of ethylene glycol according to methods well known
in the art (Sandler and Karo, Polymer Synthesis, Academic Press,
New York, Vol. 3, pages 138-161). The term "PEG" is used broadly to
encompass any polyethylene glycol molecule, without regard to size
or to modification at an end of the PEG, and can be represented by
the formula:
[0119] X--O(CH.sub.2CH.sub.2O).sub.n-1CH.sub.2CH.sub.2OH (1), where
n is 20 to 2300 and X is H or a terminal modification, e.g., a
C.sub.1-4 alkyl. In one embodiment, the PEG of the invention
terminates on one end with hydroxy or methoxy, i.e., X is H or
CH.sub.3 ("methoxy PEG"). A PEG can contain further chemical groups
which are necessary for binding reactions; which results from the
chemical synthesis of the molecule; or which is a spacer for
optimal distance of parts of the molecule. In addition, such a PEG
can consist of one or more PEG side-chains which are linked
together. PEGs with more than one PEG chain are called multiarmed
or branched PEGs. Branched PEGs can be prepared, for example, by
the addition of polyethylene oxide to various polyols, including
glycerol, pentaerythriol, and sorbitol. For example, a four-armed
branched PEG can be prepared from pentaerythriol and ethylene
oxide. Branched PEG are described in, for example, EP-A 0 473 084
and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEG
side-chains (PEG2) linked via the primary amino groups of a lysine
(Monfardini, C., et al., Bioconjugate Chem. 6 (1995) 62-69).
[0120] PEG conjugation to peptides or proteins generally involves
the activation of PEG and coupling of the activated
PEG-intermediates directly to target proteins/peptides or to a
linker, which is subsequently activated and coupled to target
proteins/peptides (see Abuchowski, A. et al, J. Biol. Chem., 252,
3571 (1977) and J. Biol. Chem., 252, 3582 (1977), Zalipsky, et al.,
and Harris et. al., in: Poly(ethylene glycol) Chemistry:
Biotechnical and Biomedical Applications; (J. M. Harris ed.) Plenum
Press: New York, 1992; Chap. 21 and 22). It is noted that an
EphB4containing a PEG molecule is also known as a conjugated
protein, whereas the protein lacking an attached PEG molecule can
be referred to as unconjugated.
[0121] Any molecular mass for a PEG can be used as practically
desired, e.g., from about 1,000 Daltons (Da) to 100,000 Da (n is 20
to 2300), for conjugating to Eph4 or EphrinB2 soluble peptides. The
number of repeating units "n" in the PEG is approximated for the
molecular mass described in Daltons. It is preferred that the
combined molecular mass of PEG on an activated linker is suitable
for pharmaceutical use. Thus, in one embodiment, the molecular mass
of the PEG molecules does not exceed 100,000 Da. For example, if
three PEG molecules are attached to a linker, where each PEG
molecule has the same molecular mass of 12,000 Da (each n is about
270), then the total molecular mass of PEG on the linker is about
36,000 Da (total n is about 820). The molecular masses of the PEG
attached to the linker can also be different, e.g., of three
molecules on a linker two PEG molecules can be 5,000 Da each (each
n is about 110) and one PEG molecule can be 12,000 Da (n is about
270).
[0122] In a specific embodiment of the invention, an EphB4
polypeptide is covalently linked to one poly(ethylene glycol) group
of the formula:
--CO--(CH.sub.2).sub.x--(OCH.sub.2CH.sub.2).sub.m--OR, with the
--CO (i.e. carbonyl) of the poly(ethylene glycol) group forming an
amide bond with one of the amino groups of EphB4; R being lower
alkyl; x being 2 or 3; m being from about 450 to about 950; and n
and m being chosen so that the molecular weight of the conjugate
minus the EphB4 protein is from about 10 to 40 kDa. In one
embodiment, an EphB4 .epsilon.-amino group of a lysine is the
available (free) amino group.
[0123] The above conjugates may be more specifically presented by
formula (II): P--NHCO--(CH.sub.2).sub.x--
(OCH.sub.2CH.sub.2).sub.m--OR (II), wherein P is the group of an
EphB4 protein as described herein, (i.e. without the amino group or
amino groups which form an amide linkage with the carbonyl shown in
formula (II); and wherein R is lower alkyl; x is 2 or 3; m is from
about 450 to about 950 and is chosen so that the molecular weight
of the conjugate minus the EphB4 protein is from about 10 to about
40 kDa. As used herein, the given ranges of "m" have an
orientational meaning. The ranges of "m" are determined in any
case, and exactly, by the molecular weight of the PEG group.
[0124] One skilled in the art can select a suitable molecular mass
for PEG, e.g., based on how the pegylated EphB4 will be used
therapeutically, the desired dosage, circulation time, resistance
to proteolysis, immunogenicity, and other considerations. For a
discussion of PEG and its use to enhance the properties of
proteins, see N. V. Katre, Advanced Drug Delivery Reviews 10:
91-114 (1993).
[0125] In one embodiment of the invention, PEG molecules may be
activated to react with amino groups on EphB4, such as with lysines
(Bencham C. O. et al., Anal. Biochem., 131, 25 (1983); Veronese, F.
M. et al., Appl. Biochem., 11, 141 (1985); Zalipsky, S. et al.,
Polymeric Drugs and Drug Delivery Systems, adrs 9-110 ACS Symposium
Series 469 (1999); Zalipsky, S. et al., Europ. Polym. J., 19,
1177-1183 (1983); Delgado, C. et al., Biotechnology and Applied
Biochemistry, 12, 119-128 (1990)).
[0126] In one specific embodiment, carbonate esters of PEG are used
to form the PEG-EphB4 conjugates. N,N'-disuccinimidylcarbonate
(DSC) may be used in the reaction with PEG to form active mixed
PEG-succinimidyl carbonate that may be subsequently reacted with a
nucleophilic group of a linker or an amino group of EphB4 (see U.S.
Pat. No. 5,281,698 and U.S. Pat. No. 5,932,462). In a similar type
of reaction, 1,1'-(dibenzotriazolyl)carbonate and
di-(2-pyridyl)carbonate may be reacted with PEG to form
PEG-benzotriazolyl and PEG-pyridyl mixed carbonate (U.S. Pat. No.
5,382,657), respectively.
[0127] In one embodiment, additional sites for PEGylation are
introduced by site-directed mutagenesis by introducing one or more
lysine residues. For instance, one or more arginine residues may be
mutated to a lysine residue. In another embodiment, additional
PEGylation sites are chemically introduced by modifying amino acids
on EphB4. In one specific embodiment, carboxyl groups in EphB4 are
conjugated with diaminobutane, resulting in carboxylamidation (see
Li et al., Anal Biochem. 2004; 330(2):264-71). This reaction may be
catalyzed by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, a
water-soluble carbodiimide. The resulting amides can then
conjugated to PEG.
[0128] PEGylation of EphB4 can be performed according to the
methods of the state of the art, for example by reaction of EphB4
with electrophilically active PEGs (supplier: Shearwater Corp.,
USA, www.shearwatercorp.com). Preferred PEG reagents of the present
invention are, e.g., N-hydroxysuccinimidyl propionates (PEG-SPA),
butanoates (PEG-SBA), PEG-succinimidyl propionate or branched
N-hydroxysuccinimides such as mPEG2-NHS (Monfardini, C., et al.,
Bioconjugate Chem. 6 (1995) 62-69). Such methods may used to
PEGylated at an .epsilon.-amino group of an EphB4 lysine or the
N-terminal amino group of EphB4.
[0129] In another embodiment, PEG molecules may be coupled to
sulfhydryl groups on EphB4 (Sartore, L., et al., Appl. Biochem.
Biotechnol., 27, 45 (1991); Morpurgo et al., Biocon. Chem., 7,
363-368 (1996); Goodson et al., Bio/Technology (1990) 8, 343; U.S.
Pat. No. 5,766,897). U.S. Pat. Nos. 6,610,281 and 5,766,897
describes exemplary reactive PEG species that may be coupled to
sulfhydryl groups.
[0130] In some embodiments where PEG molecules are conjugated to
cysteine residues on EphB4, the cysteine residues are native to
Eph4, whereas in other embodiments, one or more cysteine residues
are engineered into EphB4. Mutations may be introduced into an
EphB4 coding sequence to generate cysteine residues. This might be
achieved, for example, by mutating one or more amino acid residues
to cysteine. Preferred amino acids for mutating to a cysteine
residue include serine, threonine, alanine and other hydrophilic
residues. Preferably, the residue to be mutated to cysteine is a
surface-exposed residue. Algorithms are well-known in the art for
predicting surface accessibility of residues based on primary
sequence or a protein. Alternatively, surface residues may be
predicted by comparing the amino acid sequences of EphB4 an EphB2,
given that the crystal structure of EphB2 has been solved (see
Himanen et al., Nature. (2001) 20-27; 414(6866):933-8) and thus the
surface-exposed residues identified. In one embodiment, cysteine
residues are introduced into EphB4 at or near the N- and/or
C-terminus, or within loop regions. Loop regions may be identified
by comparing the EphB4 sequence to that of EphB2.
[0131] In some embodiments, the pegylated EphB4 comprises a PEG
molecule covalently attached to the alpha amino group of the
N-terminal amino acid. Site specific N-terminal reductive amination
is described in Pepinsky et al., (2001) JPET, 297,1059, and U.S.
Pat. No. 5,824,784. The use of a PEG-aldehyde for the reductive
amination of a protein utilizing other available nucleophilic amino
groups is described in U.S. Pat. No. 4,002,531, in Wieder et al.,
(1979) J. Biol. Chem. 254, 12579, and in Chamow et al., (1994)
Bioconjugate Chem. 5, 133.
[0132] In another embodiment, pegylated EphB4 comprises one or more
PEG molecules covalently attached to a linker, which in turn is
attached to the alpha amino group of the amino acid residue at the
N-terminus of EphB4. Such an approach is disclosed in U.S. Patent
Publication No. 2002/0044921 and in WO94/01451.
[0133] In one embodiment, EphB4 is pegylated at the C-terminus. In
a specific embodiment, a protein is pegylated at the C-terminus by
the introduction of C-terminal azido-methionine and the subsequent
conjugation of a methyl-PEG-triarylphosphine compound via the
Staudinger reaction. This C-terminal conjugation method is
described in Cazalis et al., C-Terminal Site-Specific PEGylation of
a Truncated Thrombomodulin Mutant with Retention of Full
Bioactivity, Bioconjug Chem. 2004; 15(5):1005-1009.
[0134] Monopegylation of EphB4 can also be produced according to
the general methods described in WO 94/01451. WO 94/01451 describes
a method for preparing a recombinant polypeptide with a modified
terminal amino acid alpha-carbon reactive group. The steps of the
method involve forming the recombinant polypeptide and protecting
it with one or more biologically added protecting groups at the
N-terminal alpha-amine and C-terminal alpha-carboxyl. The
polypeptide can then be reacted with chemical protecting agents to
selectively protect reactive side chain groups and thereby prevent
side chain groups from being modified. The polypeptide is then
cleaved with a cleavage reagent specific for the biological
protecting group to form an unprotected terminal amino acid
alpha-carbon reactive group. The unprotected terminal amino acid
alpha-carbon reactive group is modified with a chemical modifying
agent. The side chain protected terminally modified single copy
polypeptide is then deprotected at the side chain groups to form a
terminally modified recombinant single copy polypeptide. The number
and sequence of steps in the method can be varied to achieve
selective modification at the N- and/or C-terminal amino acid of
the polypeptide.
[0135] The ratio of EphB4 (or EphrinB2) to activated PEG in the
conjugation reaction can be from about 1:0.5 to 1:50, between from
about 1:1 to 1:30, or from about 1:5 to 1:15. Various aqueous
buffers can be used in the present method to catalyze the covalent
addition of PEG to EphB4. In one embodiment, the pH of a buffer
used is from about 7.0 to 9.0. In another embodiment, the pH is in
a slightly basic range, e.g., from about 7.5 to 8.5. Buffers having
a pKa close to neutral pH range may be used, e.g., phosphate
buffer.
[0136] In one embodiment, the temperature range for preparing a
mono-PEG-EphB4 is from about 4.degree. C. to 40.degree. C., or from
about 18.degree. C. to 25.degree. C. In another embodiment, the
temperature is room temperature.
[0137] The pegylation reaction can proceed from 3 to 48 hours, or
from 10 to 24 hours. The reaction can be monitored using SE-HPLC to
distinguish EphB4, mono-PEG-EphB4 and poly-PEG-EphB4. It is noted
that mono-PEG-EphB4 forms before di-PEG-EphB4. When the
mono-PEG-EphB4 concentration reaches a plateau, the reaction can be
terminated by adding a quenching agent to react with unreacted PEG.
In some embodiments, the quenching agent is a free amino acid, such
as glycine, cysteine or lysine.
[0138] Conventional separation and purification techniques known in
the art can be used to purify pegylated EphB4 or EphrinB2 products,
such as size exclusion (e.g. gel filtration) and ion exchange
chromatography. Products may also be separated using SDS-PAGE.
Products that may be separated include mono-, di-, tri-poly- and
un-pegylated EphB4, as well as free PEG. The percentage of mono-PEG
conjugates can be controlled by pooling broader fractions around
the elution peak to increase the percentage of mono-PEG in the
composition. About ninety percent mono-PEG conjugates represents a
good balance of yield and activity. Compositions in which, for
example, at least ninety-two percent or at least ninety-six percent
of the conjugates are mono-PEG species may be desired. In an
embodiment of this invention the percentage of mono-PEG conjugates
is from ninety percent to ninety-six percent.
[0139] In one embodiment, pegylated EphB4 proteins of the invention
contain one, two or more PEG moieties. In one embodiment, the PEG
moiety(ies) are bound to an amino acid residue which is on the
surface of the protein and/or away from the surface that contacts
EphrinB2. In one embodiment, the combined or total molecular mass
of PEG in PEG-EphB4 is from about 3,000 Da to 60,000 Da, optionally
from about 10,000 Da to 36,000 Da. In a one embodiment, the PEG in
pegylated EphB4 is a substantially linear, straight-chain PEG.
[0140] In one embodiment of the invention, the PEG in pegylated
EphB4 or EphrinB2 is not hydrolyzed from the pegylated amino acid
residue using a hydroxylamine assay, e.g., 450 mM hydroxylamine (pH
6.5) over 8 to 16 hours at room temperature, and is thus stable. In
one embodiment, greater than 80% of the composition is stable
mono-PEG-EphB4, more preferably at least 90%, and most preferably
at least 95%.
[0141] In another embodiment, the pegylated EphB4 proteins of the
invention will preferably retain at least 25%, 50%, 60%, 70% least
80%, 85%, 90%, 95% or 100% of the biological activity associated
with the unmodified protein. In one embodiment, biological activity
refers to its ability to bind to EphrinB2. In one specific
embodiment, the pegylated EphB4 protein shows an increase in
binding to EphrinB2 relative to unpegylated EphB4.
[0142] In a preferred embodiment, the PEG-EphB4 has a half-life
(t.sub.1/2) which is enhanced relative to the half-life of the
unmodified protein. Preferably, the half-life of PEG-EphB4 is
enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
100%, 125%, 150%, 175%, 200%, 250%, 300%, 400% or 500%, or even by
1000% relative to the half-life of the unmodified EphB4 protein. In
some embodiments, the protein half-life is determined in vitro,
such as in a buffered saline solution or in serum. In other
embodiments, the protein half-life is an in vivo half life, such as
the half-life of the protein in the serum or other bodily fluid of
an animal.
[0143] In certain aspects, functional variants or modified forms of
the subject soluble polypeptides include fusion proteins having at
least a portion of the soluble polypeptide and one or more fusion
domains. Well known examples of such fusion domains include, but
are not limited to, polyhistidine, Glu-Glu, glutathione S
transferase (GST), thioredoxin, protein A, protein G, and an
immunoglobulin heavy chain constant region (Fc), maltose binding
protein (MBP), which are particularly useful for isolation of the
fusion proteins by affinity chromatography. For the purpose of
affinity purification, relevant matrices for affinity
chromatography, such as glutathione-, amylase-, and nickel- or
cobalt-conjugated resins are used. Another fusion domain well known
in the art is green fluorescent protein (GFP). Fusion domains also
include. "epitope tags," which are usually short peptide sequences
for which a specific antibody is available. Well known epitope tags
for which specific monoclonal antibodies are readily available
include FLAG, influenza virus haemagglutinin (HA), and c-myc tags.
In some cases, the fusion domains have a protease cleavage site,
such as for Factor Xa or Thrombin, which allows the relevant
protease to partially digest the fusion proteins and thereby
liberate the recombinant proteins therefrom. The liberated proteins
can then be isolated from the fusion domain by subsequent
chromatographic separation.
[0144] In certain embodiments, the soluble polypeptides of the
present invention contain one or more modifications that are
capable of stabilizing the soluble polypeptides. For example, such
modifications enhance the in vitro half life of the soluble
polypeptides, enhance circulatory half life of the soluble
polypeptides or reducing proteolytic degradation of the soluble
polypeptides.
[0145] In a further embodiment, a soluble polypeptide of the
present invention is fused to a cytotoxic agent. In this method,
the fusion acts to target the cytotoxic agent to a specific tissue
or cell (e.g., a tumor tissue or cell), resulting in a reduction in
the number of afflicted cells. Such an approach can thereby reduce
symptoms associated with cancer and angiogenesis-associated
disorders. Cytotoxic agents include, but are not limited to,
diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain,
curcin, crotin, phenomycin, enomycin and the like, as well as
radiochemicals.
[0146] In certain embodiments, the soluble polypeptides of the
present invention may be fused to other therapeutic proteins or to
other proteins such as Fc or serum albumin for pharmacokinetic
purposes. See for example U.S. Pat. Nos. 5,766,883 and 5,876,969,
both of which are incorporated by reference. In some embodiments,
soluble peptides of the present invention are fused to Fc variants.
In a specific embodiment, the soluble polypeptide is fused to an Fc
variant which does not homodimerize, such as one lacking the
cysteine residues which form cysteine bonds with other Fc
chains.
[0147] In some embodiments, the modified proteins of the invention
comprise fusion proteins with an Fc region of an immunoglobulin. As
is known, each immunoglobulin heavy chain constant region comprises
four or five domains. The domains are named sequentially as
follows: CH1-hinge-CH2-CH3(-CH4). The DNA sequences of the heavy
chain domains have cross-homology among the immunoglobulin classes,
e.g., the CH2 domain of IgG is homologous to the CH2 domain of IgA
and IgD, and to the CH3 domain of IgM and IgE. As used herein, the
term, "immunoglobulin Fc region" is understood to mean the
carboxyl-terminal portion of an immunoglobulin chain constant
region, preferably an immunoglobulin heavy chain constant region,
or a portion thereof. For example, an immunoglobulin Fc region may
comprise 1) a CH1 domain, a CH2 domain, and a CH3 domain, 2) a CH1
domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2
domain and a CH3 domain, or 5) a combination of two or more domains
and an immunoglobulin hinge region. In a preferred embodiment the
immunoglobulin Fc region comprises at least an immunoglobulin hinge
region a CH2 domain and a CH3 domain, and preferably lacks the CH1
domain.
[0148] In one embodiment, the class of immunoglobulin from which
the heavy chain constant region is derived is IgG (Ig.gamma.)
(.gamma. subclasses 1, 2, 3, or 4). The nucleotide and amino acid
sequences of human Fc .gamma.-1 are set forth in SEQ ID NOS: 5 and
6. The nucleotide and amino acid sequences of murine Fc.gamma.-2a
are set forth in SEQ ID NOS: 7 and 8. Other classes of
immunoglobulin, IgA (Ig.alpha.), IgD (Ig.delta.), IgE (Ig.epsilon.)
and IgM (Ig.mu.), may be used. The choice of appropriate
immunoglobulin heavy chain constant regions is discussed in detail
in U.S. Pat. Nos. 5,541,087, and 5,726,044. The choice of
particular immunoglobulin heavy chain constant region sequences
from certain immunoglobulin classes and subclasses to achieve a
particular result is considered to be within the level of skill in
the art. The portion of the DNA construct encoding the
immunoglobulin Fc region preferably comprises at least a portion of
a hinge domain, and preferably at least a portion of a CH.sub.3
domain of Fc.gamma. or the homologous domains in any of IgA, IgD,
IgE, or IgM.
[0149] Furthermore, it is contemplated that substitution or
deletion of amino acids within the immunoglobulin heavy chain
constant regions may be useful in the practice of the invention.
One example would be to introduce amino acid substitutions in the
upper CH2 region to create a Fc variant with reduced affinity for
Fc receptors (Cole et al. (1997) J. IMMUNOL. 159:3613). One of
ordinary skill in the art can prepare such constructs using well
known molecular biology techniques.
[0150] In a specific embodiment of the present invention, the
modified forms of the subject soluble polypeptides are fusion
proteins having at least a portion of the soluble polypeptide
(e.g., an ectodomain of Ephrin B2 or EphB4) and a stabilizing
domain such as albumin. As used herein, "albumin" refers
collectively to albumin protein or amino acid sequence, or an
albumin fragment or variant, having one or more functional
activities (e.g., biological activities) of albumin. In particular,
"albumin" refers to human albumin or fragments thereof (see EP 201
239, EP 322 094 WO 97/24445, WO95/23857) especially the mature form
of human albumin, or albumin from other vertebrates or fragments
thereof, or analogs or variants of these molecules or fragments
thereof.
[0151] The present invention describes that such fusion proteins
are more stable relative to the corresponding wildtype soluble
protein. For example, the subject soluble polypeptide (e.g., an
ectodomain of Ephrin B2 or EphB4) can be fused with human serum
albumin (HSA), bovine serum albumin (BSA), or any fragment of an
albumin protein which has stabilization activity. Such stabilizing
domains include human serum albumin (HSA) and bovine serum albumin
(BSA).
[0152] In particular, the albumin fusion proteins of the invention
may include naturally occurring polymorphic variants of human
albumin and fragments of human albumin (See WO95/23857), for
example those fragments disclosed in EP 322 094 (namely HA (Pn),
where n is 369 to 419). The albumin may be derived from any
vertebrate, especially any mammal, for example human, cow, sheep,
or pig. Non-mammalian albumins include, but are not limited to, hen
and salmon. The albumin portion of the albumin fusion protein may
be from a different animal than the EphB4.
[0153] In some embodiments, the albumin protein portion of an
albumin fusion protein corresponds to a fragment of serum albumin.
Fragments of serum albumin polypeptides include polypeptides having
one or more residues deleted from the amino terminus or from the
C-terminus. Generally speaking, an HA fragment or variant will be
at least 100 amino acids long, preferably at least 150 amino acids
long. The HA variant may consist of or alternatively comprise at
least one whole domain of HA. Domains, with reference to SEQ ID
NO:18 in U.S. Patent Publication No. 2004/0171123, are as follows:
domains 1 (amino acids 1-194), 2 (amino acids 195-387), 3 (amino
acids 388-585), 1+2 (1-387), 2+3 (195-585) or 1+3 (amino acids
1-194+amino acids 388-585). Each domain is itself made up of two
homologous subdomains namely 1-105, 120-194, 195-291, 316-387,
388-491 and 512-585, with flexible inter-subdomain linker regions
comprising residues Lys106 to Glu119, Glu292 to Val315 and Glu492
to Ala511.
[0154] In one embodiment, the EphB4-HSA fusion has one EphB4
soluble polypeptide linked to one HSA molecule, but other
conformations are within the invention. For example, EphB4-HSA
fusion proteins can have any of the following formula:
R.sub.1-L-R.sub.2; R.sub.2-L-R.sub.1; R.sub.1-L-R.sub.2-L-R.sub.1;
or R.sub.2-L-R1-L-R.sub.2; R.sub.1-R.sub.2; R.sub.2-R.sub.1;
R.sub.1-R.sub.2-R.sub.1; or R.sub.2-R.sub.1-R.sub.2; wherein
R.sub.1 is a soluble EphB4 sequence, R.sub.2 is HSA, and L is a
peptide linker sequence.
[0155] In a specific embodiment, the EphB4 and HSA domains are
linked to each other, preferably via a linker sequence, which
separates the EphB4 and HSA domains by a distance sufficient to
ensure that each domain properly folds into its secondary and
tertiary structures. Preferred linker sequences (1) should adopt a
flexible extended conformation, (2) should not exhibit a propensity
for developing an ordered secondary structure which could interact
with the functional EphB4 and HSA domains, and (3) should have
minimal hydrophobic or charged character, which could promote
interaction with the functional protein domains. Typical surface
amino acids in flexible protein regions include Gly, Asn and Ser.
Permutations of amino acid sequences containing Gly, Asn and Ser
would be expected to satisfy the above criteria for a linker
sequence. Other near neutral amino acids, such as Thr and Ala, can
also be used in the linker sequence.
[0156] In a specific embodiment, a linker sequence length of about
20 amino acids can be used to provide a suitable separation of
functional protein domains, although longer or shorter linker
sequences may also be used. The length of the linker sequence
separating EphB4 and HSA can be from 5 to 500 amino acids in
length, or more preferably from 5 to 100 amino acids in length.
Preferably, the linker sequence is from about 5-30 amino acids in
length. In preferred embodiments, the linker sequence is from about
5 to about 20 amino acids, and is advantageously from about 10 to
about 20 amino acids. Amino acid sequences useful as linkers of
EphB4 and HSA include, but are not limited to, (SerGly.sub.4).sub.y
wherein y is greater than or equal to 8, or
Gly.sub.4SerGly.sub.5Ser. A preferred linker sequence has the
formula (SerGly.sub.4).sub.4. Another preferred linker has the
sequence ((Ser-Ser-Ser-Ser-Gly)3-Ser-Pro).
[0157] In one embodiment, the polypeptides of the present invention
and HSA proteins are directly fused without a linker sequence. In
preferred embodiments, the C-terminus of a soluble EphB4
polypeptide can be directly fused to the N-terminus of HSA or the
C-terminus of HSA can be directly fused to the N-terminus of
soluble EphB4.
[0158] In some embodiments, the immunogenicity of the fusion
junction between HSA and EphB4 may be reduced the by identifying a
candidate T-cell epitope within a junction region spanning a fusion
protein and changing an amino acid within the junction region as
described in U.S. Patent Publication No. 2003/0166877.
[0159] In certain embodiments, soluble polypeptides (unmodified or
modified) of the invention can be produced by a variety of
art-known techniques. For example, such soluble polypeptides can be
synthesized using standard protein chemistry techniques such as
those described in Bodansky, M. Principles of Peptide Synthesis,
Springer Verlag, Berlin (1993) and Grant G. A. (ed.), Synthetic
Peptides: A User's Guide, W. H. Freeman and Company, New York
(1992). In addition, automated peptide synthesizers are
commercially available (e.g., Advanced ChemTech Model 396;
Milligen/Biosearch 9600). Alternatively, the soluble polypeptides,
fragments or variants thereof may be recombinantly produced using
various expression systems as is well known in the art (also see
below).
III. Nucleic Acids Encoding Soluble Polypeptides
[0160] In certain aspects, the invention relates to isolated and/or
recombinant nucleic acids encoding an EphB4 or Ephrin B2 soluble
polypeptide. The subject nucleic acids may be single-stranded or
double-stranded, DNA or RNA molecules. These nucleic acids are
useful as therapeutic agents. For example, these nucleic acids are
useful in making recombinant soluble polypeptides which are
administered to a cell or an individual as therapeutics.
Alternative, these nucleic acids can be directly administered to a
cell or an individual as therapeutics such as in gene therapy.
[0161] In certain embodiments, the invention provides isolated or
recombinant nucleic acid sequences that are at least 80%, 85%, 90%,
95%, 97%, 98%, 99% or 100% identical to a region of the nucleotide
sequence depicted in SEQ ID Nos. 6-9. One of ordinary skill in the
art will appreciate that nucleic acid sequences complementary to
the subject nucleic acids, and variants of the subject nucleic
acids are also within the scope of this invention. In further
embodiments, the nucleic acid sequences of the invention can be
isolated, recombinant, and/or fused with a heterologous nucleotide
sequence, or in a DNA library.
[0162] In other embodiments, nucleic acids of the invention also
include nucleotide sequences that hybridize under highly stringent
conditions to the nucleotide sequence depicted in SEQ ID Nos. 6-9,
or complement sequences thereof. As discussed above, one of
ordinary skill in the art will understand readily that appropriate
stringency conditions which promote DNA hybridization can be
varied. One of ordinary skill in the art will understand readily
that appropriate stringency conditions which promote DNA
hybridization can be varied. For example, one could perform the
hybridization at 6.0.times. sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C. For example, the salt concentration in the wash step
can be selected from a low stringency of about 2.0.times.SSC at
50.degree. C. to a high stringency of about 0.2.times.SSC at
50.degree. C. In addition, the temperature in the wash step can be
increased from low stringency conditions at room temperature, about
22.degree. C., to high stringency conditions at about 65.degree. C.
Both temperature and salt may be varied, or temperature or salt
concentration may be held constant while the other variable is
changed. In one embodiment, the invention provides nucleic acids
which hybridize under low stringency conditions of 6.times.SSC at
room temperature followed by a wash at 2.times.SSC at room
temperature.
[0163] Isolated nucleic acids which differ from the subject nucleic
acids due to degeneracy in the genetic code are also within the
scope of the invention. For example, a number of amino acids are
designated by more than one triplet. Codons that specify the same
amino acid, or synonyms (for example, CAU and CAC are synonyms for
histidine) may result in "silent" mutations which do not affect the
amino acid sequence of the protein. However, it is expected that
DNA sequence polymorphisms that do lead to changes in the amino
acid sequences of the subject proteins will exist among mammalian
cells. One skilled in the art will appreciate that these variations
in one or more nucleotides (up to about 3-5% of the nucleotides) of
the nucleic acids encoding a particular protein may exist among
individuals of a given species due to natural allelic variation.
Any and all such nucleotide variations and resulting amino acid
polymorphisms are within the scope of this invention.
[0164] In certain embodiments, the recombinant nucleic acids of the
invention may be operably linked to one or more regulatory
nucleotide sequences in an expression construct. Regulatory
nucleotide sequences will generally be appropriate for a host cell
used for expression. Numerous types of appropriate expression
vectors and suitable regulatory sequences are known in the art for
a variety of host cells. Typically, said one or more regulatory
nucleotide sequences may include, but are not limited to, promoter
sequences, leader or signal sequences, ribosomal binding sites,
transcriptional start and termination sequences, translational
start and termination sequences, and enhancer or activator
sequences. Constitutive or inducible promoters as known in the art
are contemplated by the invention. The promoters may be either
naturally occurring promoters, or hybrid promoters that combine
elements of more than one promoter. An expression construct may be
present in a cell on an episome, such as a plasmid, or the
expression construct may be inserted in a chromosome. In a
preferred embodiment, the expression vector contains a selectable
marker gene to allow the selection of transformed host cells.
Selectable marker genes are well known in the art and will vary
with the host cell used.
[0165] In certain aspect of the invention, the subject nucleic acid
is provided in an expression vector comprising a nucleotide
sequence encoding an EphB4 or Ephrin B2 soluble polypeptide and
operably linked to at least one regulatory sequence. Regulatory
sequences are art-recognized and are selected to direct expression
of the soluble polypeptide. Accordingly, the term regulatory
sequence includes promoters, enhancers, and other expression
control elements. Exemplary regulatory sequences are described in
Goeddel; Gene Expression Technology: Methods in Enzymology,
Academic Press, San Diego, Calif. (1990). For instance, any of a
wide variety of expression control sequences that control the
expression of a DNA sequence when operatively linked to it may be
used in these vectors to express DNA sequences encoding a soluble
polypeptide. Such useful expression control sequences, include, for
example, the early and late promoters of SV40, tet promoter,
adenovirus or cytomegalovirus immediate early promoter, the lac
system, the trp system, the TAC or TRC system, T7 promoter whose
expression is directed by T7 RNA polymerase, the major operator and
promoter regions of phage lambda, the control regions for fd coat
protein, the promoter for 3-phosphoglycerate kinase or other
glycolytic enzymes, the promoters of acid phosphatase, e.g., PhoS,
the promoters of the yeast .alpha.-mating factors, the polyhedron
promoter of the baculovirus system and other sequences known to
control the expression of genes of prokaryotic or eukaryotic cells
or their viruses, and various combinations thereof. It should be
understood that the design of the expression vector may depend on
such factors as the choice of the host cell to be transformed
and/or the type of protein desired to be expressed. Moreover, the
vector's copy number, the ability to control that copy number and
the expression of any other protein encoded by the vector, such as
antibiotic markers, should also be considered.
[0166] This invention also pertains to a host cell transfected with
a recombinant gene including a coding sequence for one or more of
the subject soluble polypeptide. The host cell may be any
prokaryotic or eukaryotic cell. For example, a soluble polypeptide
of the invention may be expressed in bacterial cells such as E.
coli, insect cells (e.g., using a baculovirus expression system),
yeast, or mammalian cells. Other suitable host cells are known to
those skilled in the art.
[0167] Accordingly, the present invention further pertains to
methods of producing the subject soluble polypeptides. For example,
a host cell transfected with an expression vector encoding an EphB4
soluble polypeptide can be cultured under appropriate conditions to
allow expression of the EphB4 soluble polypeptide to occur. The
EphB4 soluble polypeptide may be secreted and isolated from a
mixture of cells and medium containing the soluble polypeptides.
Alternatively, the soluble polypeptides may be retained
cytoplasmically or in a membrane fraction and the cells harvested,
lysed and the protein isolated. A cell culture includes host cells,
media and other byproducts. Suitable media for cell culture are
well known in the art. The soluble polypeptides can be isolated
from cell culture medium, host cells, or both using techniques
known in the art for purifying proteins, including ion-exchange
chromatography, gel filtration chromatography, ultrafiltration,
electrophoresis, and immunoaffinity purification with antibodies
specific for particular epitopes of the soluble polypeptides. In a
preferred embodiment, the soluble polypeptide is a fusion protein
containing a domain which facilitates its purification.
[0168] A recombinant nucleic acid of the invention can be produced
by ligating the cloned gene, or a portion thereof, into a vector
suitable for expression in either prokaryotic cells, eukaryotic
cells (yeast, avian, insect or mammalian), or both. Expression
vehicles for production of a recombinant soluble polypeptide
include plasmids and other vectors. For instance, suitable vectors
include plasmids of the types: pBR322-derived plasmids,
pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived
plasmids and pUC-derived plasmids for expression in prokaryotic
cells, such as E. coli.
[0169] The preferred mammalian expression vectors contain both
prokaryotic sequences to facilitate the propagation of the vector
in bacteria, and one or more eukaryotic transcription units that
are expressed in eukaryotic cells. The pcDNAI/amp, pcDNAI/neo,
pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7,
pko-neo and pHyg derived vectors are examples of mammalian
expression vectors suitable for transfection of eukaryotic cells.
Some of these vectors are modified with sequences from bacterial
plasmids, such as pBR322, to facilitate replication and drug
resistance selection in both prokaryotic and eukaryotic cells.
Alternatively, derivatives of viruses such as the bovine papilloma
virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205)
can be used for transient expression of proteins in eukaryotic
cells. Examples of other viral (including retroviral) expression
systems can be found below in the description of gene therapy
delivery systems. The various methods employed in the preparation
of the plasmids and transformation of host organisms are well known
in the art. For other suitable expression systems for both
prokaryotic and eukaryotic cells, as well as general recombinant
procedures, see Molecular Cloning A Laboratory Manual, 2nd Ed., ed.
by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory
Press, 1989) Chapters 16 and 17. In some instances, it may be
desirable to express the recombinant SLC5A8 polypeptide by the use
of a baculovirus expression system. Examples of such baculovirus
expression systems include pVL-derived vectors (such as pVL1392,
pVL1393 and pVL941), pAcUW-derived vectors (such as pAcUW1), and
pBlueBac-derived vectors (such as the .beta.-gal containing
pBlueBac III).
[0170] Techniques for making fusion genes are well known.
Essentially, the joining of various DNA fragments coding for
different polypeptide sequences is performed in accordance with
conventional techniques, employing blunt-ended or stagger-ended
termini for ligation, restriction enzyme digestion to provide for
appropriate termini, filling-in of cohesive ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and
enzymatic ligation. In another embodiment, the fusion gene can be
synthesized by conventional techniques including automated DNA
synthesizers. Alternatively, PCR amplification of gene fragments
can be carried out using anchor primers which give rise to
complementary overhangs between two consecutive gene fragments
which can subsequently be annealed to generate a chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology,
eds. Ausubel et al., John Wiley & Sons: 1992).
IV. Drug Screening Assays
[0171] There are numerous approaches to screening for polypeptide
therapeutic agents as antagonists of EphB4, Ephrin B2 or both. For
example, high-throughput screening of compounds or molecules can be
carried out to identify agents or drugs which inhibit angiogenesis
or inhibit tumor growth. Test agents can be any chemical (element,
molecule, compound, drug), made synthetically, made by recombinant
techniques or isolated from a natural source. For example, test
agents can be peptides, polypeptides, peptoids, sugars, hormones,
or nucleic acid molecules. In addition, test agents can be small
molecules or molecules of greater complexity made by combinatorial
chemistry, for example, and compiled into libraries. These
libraries can comprise, for example, alcohols, alkyl halides,
amines, amides, esters, aldehydes, ethers and other classes of
organic compounds. Test agents can also be natural or genetically
engineered products isolated from lysates or growth media of
cells--bacterial, animal or plant--or can be the cell lysates or
growth media themselves. Presentation of test compounds to the test
system can be in either an isolated form or as mixtures of
compounds, especially in initial screening steps.
[0172] For example, an assay can be carried out to screen for
compounds that specifically inhibit binding of Ephrin B2 (ligand)
to EphB4 (receptor), or vice-versa, e.g., by inhibition of binding
of labeled ligand- or receptor-Fc fusion proteins to immortalized
cells. Compounds identified through this screening can then be
tested in animals to assess their anti-angiogenesis or anti-tumor
activity in vivo.
[0173] In one embodiment of an assay to identify a substance that
interferes with interaction of two cell surface molecules (e.g.,
Ephrin B2 and EphB4), samples of cells expressing one type of cell
surface molecule (e.g., EphB4) are contacted with either labeled
ligand (e.g., Ephrin B2, or a soluble portion thereof, or a fusion
protein such as a fusion of the extracellular domain and the Fc
domain of IgG) or labeled ligand plus a test compound (or group of
test compounds). The amount of labeled ligand which has bound to
the cells is determined. A lesser amount of label (where the label
can be, for example, a radioactive isotope, a fluorescent or
calorimetric label) in the sample contacted with the test
compound(s) is an indication that the test compound(s) interferes
with binding. The reciprocal assay using cells expressing a ligand
(e.g., an Ephrin B2 ligand or a soluble form thereof) can be used
to test for a substance that interferes with the binding of an Eph
receptor or soluble portion thereof.
[0174] An assay to identify a substance which interferes with
interaction between an Eph receptor and an ephrin can be performed
with the component (e.g., cells, purified protein, including fusion
proteins and portions having binding activity) which is not to be
in competition with a test compound, linked to a solid support. The
solid support can be any suitable solid phase or matrix, such as a
bead, the wall of a plate or other suitable surface (e.g., a well
of a microtiter plate), column pore glass (CPG) or a pin that can
be submerged into a solution, such as in a well. Linkage of cells
or purified protein to the solid support can be either direct or
through one or more linker molecules.
[0175] In one embodiment, an isolated or purified protein (e.g., an
Eph receptor or an ephrin) can be immobilized on a suitable
affinity matrix by standard techniques, such as chemical
cross-linking, or via an antibody raised against the isolated or
purified protein, and bound to a solid support. The matrix can be
packed in a column or other suitable container and is contacted
with one or more compounds (e.g., a mixture) to be tested under
conditions suitable for binding of the compound to the protein. For
example, a solution containing compounds can be made to flow
through the matrix. The matrix can be washed with a suitable wash
buffer to remove unbound compounds and non-specifically bound
compounds. Compounds which remain bound can be released by a
suitable elution buffer. For example, a change in the ionic
strength or pH of the elution buffer can lead to a release of
compounds. Alternatively, the elution buffer can comprise a release
component or components designed to disrupt binding of compounds
(e.g., one or more ligands or receptors, as appropriate, or analogs
thereof which can disrupt binding or competitively inhibit binding
of test compound to the protein).
[0176] Fusion proteins comprising all, or a portion of, a protein
(e.g., an Eph receptor or an ephrin) linked to a second moiety not
occurring in that protein as found in nature can be prepared for
use in another embodiment of the method. Suitable fusion proteins
for this purpose include those in which the second moiety comprises
an affinity ligand (e.g., an enzyme, antigen, epitope). The fusion
proteins can be produced by inserting the protein (e.g., an Eph
receptor or an ephrin) or a portion thereof into a suitable
expression vector which encodes an affinity ligand. The expression
vector can be introduced into a suitable host cell for expression.
Host cells are disrupted and the cell material, containing fusion
protein, can be bound to a suitable affinity matrix by contacting
the cell material with an affinity matrix under conditions
sufficient for binding of the affinity ligand portion of the fusion
protein to the affinity matrix.
[0177] In one aspect of this embodiment, a fusion protein can be
immobilized on a suitable affinity matrix under conditions
sufficient to bind the affinity ligand portion of the fusion
protein to the matrix, and is contacted with one or more compounds
(e.g., a mixture) to be tested, under conditions suitable for
binding of compounds to the receptor or ligand protein portion of
the bound fusion protein. Next, the affinity matrix with bound
fusion protein can be washed with a suitable wash buffer to remove
unbound compounds and non-specifically bound compounds without
significantly disrupting binding of specifically bound compounds.
Compounds which remain bound can be released by contacting the
affinity matrix having fusion protein bound thereto with a suitable
elution buffer (a compound elution buffer). In this aspect,
compound elution buffer can be formulated to permit retention of
the fusion protein by the affinity matrix, but can be formulated to
interfere with binding of the compound(s) tested to the receptor or
ligand protein portion of the fusion protein. For example, a change
in the ionic strength or pH of the elution buffer can lead to
release of compounds, or the elution buffer can comprise a release
component or components designed to disrupt binding of compounds to
the receptor or ligand protein portion of the fusion protein (e.g.,
one or more ligands or receptors or analogs thereof which can
disrupt binding of compounds to the receptor or ligand protein
portion of the fusion protein). Immobilization can be performed
prior to, simultaneous with, or after contacting the fusion protein
with compound, as appropriate. Various permutations of the method
are possible, depending upon factors such as the compounds tested,
the affinity matrix selected, and elution buffer formulation. For
example, after the wash step, fusion protein with compound bound
thereto can be eluted from the affinity matrix with a suitable
elution buffer (a matrix elution buffer). Where the fusion protein
comprises a cleavable linker, such as a thrombin cleavage site,
cleavage from the affinity ligand can release a portion of the
fusion with compound bound thereto. Bound compound can then be
released from the fusion protein or its cleavage product by an
appropriate method, such as extraction.
V. Methods of Treatment
[0178] In certain embodiments, the present invention provides
methods of inhibiting angiogenesis and methods of treating
angiogenesis-associated diseases. In other embodiments, the present
invention provides methods of inhibiting or reducing tumor growth
and methods of treating an individual suffering from cancer. These
methods involve administering to the individual a therapeutically
effective amount of one or more polypeptide therapeutic agents as
described above. These methods are particularly aimed at
therapeutic and prophylactic treatments of animals, and more
particularly, humans.
[0179] As described herein, angiogenesis-associated diseases
include, but are not limited to, angiogenesis-dependent cancer,
including, for example, solid tumors, blood born tumors such as
leukemias, and tumor metastases; benign tumors, for example
hemangiomas, acoustic neuromas, neurofibromas, trachomas, and
pyogenic granulomas; inflammatory disorders such as immune and
non-immune inflammation; chronic articular rheumatism and
psoriasis; ocular angiogenic diseases, for example, diabetic
retinopathy, retinopathy of prematurity, macular degeneration,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasia, rubeosis; Osler-Webber Syndrome; myocardial
angiogenesis; plaque neovascularization; telangiectasia;
hemophiliac joints; angiofibroma; telangiectasia psoriasis
scleroderma, pyogenic granuloma, rubeosis, arthritis, diabetic
neovascularization, vasculogenesis, hematopoiesis.
[0180] It is understood that methods and compositions of the
invention are also useful for treating any angiogenesis-independent
cancers (tumors). As used herein, the term
"angiogenesis-independent cancer" refers to a cancer (tumor) where
there is no or little neovascularization in the tumor tissue.
[0181] In particular, polypeptide therapeutic agents of the present
invention are useful for treating or preventing a cancer (tumor),
including, but not limited to, colon carcinoma, breast cancer,
mesothelioma, prostate cancer, bladder cancer, squamous cell
carcinoma of the head and neck (HNSCC), Kaposi sarcoma, and
leukemia.
[0182] In certain embodiments of such methods, one or more
polypeptide therapeutic agents can be administered, together
(simultaneously) or at different times (sequentially). In addition,
polypeptide therapeutic agents can be administered with another
type of compounds for treating cancer or for inhibiting
angiogenesis.
[0183] In certain embodiments, the subject methods of the invention
can be used alone. Alternatively, the subject methods may be used
in combination with other conventional anti-cancer therapeutic
approaches directed to treatment or prevention of proliferative
disorders (e.g., tumor). For example, such methods can be used in
prophylactic cancer prevention, prevention of cancer recurrence and
metastases after surgery, and as an adjuvant of other conventional
cancer therapy. The present invention recognizes that the
effectiveness of conventional cancer therapies (e.g., chemotherapy,
radiation therapy, phototherapy, immunotherapy, and surgery) can be
enhanced through the use of a subject polypeptide therapeutic
agent.
[0184] A wide array of conventional compounds have been shown to
have anti-neoplastic activities. These compounds have been used as
pharmaceutical agents in chemotherapy to shrink solid tumors,
prevent metastases and further growth, or decrease the number of
malignant cells in leukemic or bone marrow malignancies. Although
chemotherapy has been effective in treating various types of
malignancies, many anti-neoplastic compounds induce undesirable
side effects. It has been shown that when two or more different
treatments are combined, the treatments may work synergistically
and allow reduction of dosage of each of the treatments, thereby
reducing the detrimental side effects exerted by each compound at
higher dosages. In other instances, malignancies that are
refractory to a treatment may respond to a combination therapy of
two or more different treatments.
[0185] When a polypeptide therapeutic agent of the present
invention is administered in combination with another conventional
anti-neoplastic agent, either concomitantly or sequentially, such
therapeutic agent is shown to enhance the therapeutic effect of the
anti-neoplastic agent or overcome cellular resistance to such
anti-neoplastic agent. This allows decrease of dosage of an
anti-neoplastic agent, thereby reducing the undesirable side
effects, or restores the effectiveness of an anti-neoplastic agent
in resistant cells.
[0186] Pharmaceutical compounds that may be used for combinatory
anti-tumor therapy include, merely to illustrate:
aminoglutethimide, amsacrine, anastrozole, asparaginase, bcg,
bicalutamide, bleomycin, buserelin, busulfan, campothecin,
capecitabine, carboplatin, carmustine, chlorambucil, cisplatin,
cladribine, clodronate, colchicine, cyclophosphamide, cyproterone,
cytarabine, dacarbazine, dactinomycin, daunorubicin, dienestrol,
diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol,
estramustine, etoposide, exemestane, filgrastim, fludarabine,
fludrocortisone, fluorouracil, fluoxymesterone, flutamide,
gemcitabine, genistein, goserelin, hydroxyurea, idarubicin,
ifosfamide, imatinib, interferon, irinotecan, ironotecan,
letrozole, leucovorin, leuprolide, levamisole, lomustine,
mechlorethamine, medroxyprogesterone, megestrol, melphalan,
mercaptopurine, mesna, methotrexate, mitomycin, mitotane,
mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin,
paclitaxel, pamidronate, pentostatin, plicamycin, porfimer,
procarbazine, raltitrexed, rituximab, streptozocin, suramin,
tamoxifen, temozolomide, teniposide, testosterone, thioguanine,
thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin,
vinblastine, vincristine, vindesine, and vinorelbine.
[0187] These chemotherapeutic anti-tumor compounds may be
categorized by their mechanism of action into, for example,
following groups: anti-metabolites/anti-cancer agents, such as
pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine,
gemcitabine and cytarabine) and purine analogs, folate antagonists
and related inhibitors (mercaptopurine, thioguanine, pentostatin
and 2-chlorodeoxyadenosine (cladribine));
antiproliferative/antimitotic agents including natural products
such as vinca alkaloids (vinblastine, vincristine, and
vinorelbine), microtubule disruptors such as taxane (paclitaxel,
docetaxel), vincristin, vinblastin, nocodazole, epothilones and
navelbine, epidipodophyllotoxins (etoposide, teniposide), DNA
damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin,
busulfan, camptothecin, carboplatin, chlorambucil, cisplatin,
cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin,
epirubicin, hexamethylmelamineoxaliplatin, iphosphamide, melphalan,
merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin,
procarbazine, taxol, taxotere, teniposide,
triethylenethiophosphoramide and etoposide (VP16)); antibiotics
such as dactinomycin (actinomycin D), daunorubicin, doxorubicin
(adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins,
plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase
which systemically metabolizes L-asparagine and deprives cells
which do not have the capacity to synthesize their own asparagine);
antiplatelet agents; antiproliferative/antimitotic alkylating
agents such as nitrogen mustards (mechlorethamine, cyclophosphamide
and analogs, melphalan, chlorambucil), ethylenimines and
methylmelamines (hexamethylmelamine and thiotepa), alkyl
sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs,
streptozocin), trazenes-dacarbazinine (DTIC);
antiproliferative/antimitotic antimetabolites such as folic acid
analogs (methotrexate); platinum coordination complexes (cisplatin,
carboplatin), procarbazine, hydroxyurea, mitotane,
aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen,
goserelin, bicalutamide, nilutamide) and aromatase inhibitors
(letrozole, anastrozole); anticoagulants (heparin, synthetic
heparin salts and other inhibitors of thrombin); fibrinolytic
agents (such as tissue plasminogen activator, streptokinase and
urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel,
abciximab; antimigratory agents; antisecretory agents (breveldin);
immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus
(rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic
compounds (TNP-470, genistein) and growth factor inhibitors
(vascular endothelial growth factor (VEGF) inhibitors, fibroblast
growth factor (FGF) inhibitors); angiotensin receptor blocker;
nitric oxide donors; anti-sense oligonucleotides; antibodies
(trastuzumab); cell cycle inhibitors and differentiation inducers
(tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin
(adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin,
eniposide, epirubicin, etoposide, idarubicin and mitoxantrone,
topotecan, irinotecan), corticosteroids (cortisone, dexamethasone,
hydrocortisone, methylpednisolone, prednisone, and prenisolone);
growth factor signal transduction kinase inhibitors; mitochondrial
dysfunction inducers and caspase activators; and chromatin
disruptors.
[0188] In certain embodiments, pharmaceutical compounds that may be
used for combinatory anti-angiogenesis therapy include: (1)
inhibitors of release of "angiogenic molecules," such as bFGF
(basic fibroblast growth factor); (2) neutralizers of angiogenic
molecules, such as an anti-.beta.bFGF antibodies; and (3)
inhibitors of endothelial cell response to angiogenic stimuli,
including collagenase inhibitor, basement membrane turnover
inhibitors, angiostatic steroids, fungal-derived angiogenesis
inhibitors, platelet factor 4, thrombospondin, arthritis drugs such
as D-penicillamine and gold thiomalate, vitamin D.sub.3 analogs,
alpha-interferon, and the like. For additional proposed inhibitors
of angiogenesis, see Blood et al., Bioch. Biophys. Acta.,
1032:89-118 (1990), Moses et al., Science, 248:1408-1410 (1990),
Ingber et al., Lab. Invest., 59:44-51 (1988), and U.S. Pat. Nos.
5,092,885, 5,112,946, 5,192,744, 5,202,352, and 6573256. In
addition, there are a wide variety of compounds that can be used to
inhibit angiogenesis, for example, peptides or agents that block
the VEGF-mediated angiogenesis pathway, endostatin protein or
derivatives, lysine binding fragments of angiostatin, melanin or
melanin-promoting compounds, plasminogen fragments (e.g., Kringles
1-3 of plasminogen), tropoin subunits, antagonists of vitronectin
.alpha..sub.v.beta..sub.3, peptides derived from Saposin B,
antibiotics or analogs (e.g., tetracycline, or neomycin),
dienogest-containing compositions, compounds comprising a MetAP-2
inhibitory core coupled to a peptide, the compound EM-138, chalcone
and its analogs, and naaladase inhibitors. See, for example, U.S.
Pat. Nos. 6,395,718, 6,462,075, 6,465,431, 6,475,784, 6,482,802,
6,482,810, 6,500,431, 6,500,924, 6,518,298, 6,521,439, 6,525,019,
6,538,103, 6,544,758, 6,544,947, 6,548,477, 6,559,126, and
6,569,845.
[0189] Depending on the nature of the combinatory therapy,
administration of the polypeptide therapeutic agents of the
invention may be continued while the other therapy is being
administered and/or thereafter. Administration of the polypeptide
therapeutic agents may be made in a single dose, or in multiple
doses. In some instances, administration of the polypeptide
therapeutic agents is commenced at least several days prior to the
conventional therapy, while in other instances, administration is
begun either immediately before or at the time of the
administration of the conventional therapy.
VI. Methods of Administration and Pharmaceutical Compositions
[0190] In certain embodiments, the subject polypeptide therapeutic
agents (e.g., soluble polypeptides or antibodies) of the present
invention are formulated with a pharmaceutically acceptable
carrier. Such therapeutic agents can be administered alone or as a
component of a pharmaceutical formulation (composition). The
compounds may be formulated for administration in any convenient
way for use in human or veterinary medicine. Wetting agents,
emulsifiers and lubricants, such as sodium lauryl sulfate and
magnesium stearate, as well as coloring agents, release agents,
coating agents, sweetening, flavoring and perfuming agents,
preservatives and antioxidants can also be present in the
compositions.
[0191] Formulations of the subject polypeptide therapeutic agents
include those suitable for oral/nasal, topical, parenteral, rectal,
and/or intravaginal administration. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any methods well known in the art of pharmacy. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will vary depending upon the host
being treated, the particular mode of administration. The amount of
active ingredient which can be combined with a carrier material to
produce a single dosage form will generally be that amount of the
compound which produces a therapeutic effect.
[0192] In certain embodiments, methods of preparing these
formulations or compositions include combining another type of
anti-tumor or anti-angiogenesis therapeutic agent and a carrier
and, optionally, one or more accessory ingredients. In general, the
formulations can be prepared with a liquid carrier, or a finely
divided solid carrier, or both, and then, if necessary, shaping the
product.
[0193] Formulations for oral administration may be in the form of
capsules, cachets, pills, tablets, lozenges (using a flavored
basis, usually sucrose and acacia or tragacanth), powders,
granules, or as a solution or a suspension in an aqueous or
non-aqueous liquid, or as an oil-in-water or water-in-oil liquid
emulsion, or as an elixir or syrup, or as pastilles (using an inert
base, such as gelatin and glycerin, or sucrose and acacia) and/or
as mouth washes and the like, each containing a predetermined
amount of a subject polypeptide therapeutic agent as an active
ingredient.
[0194] In solid dosage forms for oral administration (capsules,
tablets, pills, dragees, powders, granules, and the like), one or
more polypeptide therapeutic agents of the present invention may be
mixed with one or more pharmaceutically acceptable carriers, such
as sodium citrate or dicalcium phosphate, and/or any of the
following: (1) fillers or extenders, such as starches, lactose,
sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such
as, for example, carboxymethylcellulose, alginates, gelatin,
polyvinyl pyrrolidone, sucrose, and/or acacia; (3) humectants, such
as glycerol; (4) disintegrating agents, such as agar-agar, calcium
carbonate, potato or tapioca starch, alginic acid, certain
silicates, and sodium carbonate; (5) solution retarding agents,
such as paraffin; (6) absorption accelerators, such as quaternary
ammonium compounds; (7) wetting agents, such as, for example, cetyl
alcohol and glycerol monostearate; (8) absorbents, such as kaolin
and bentonite clay; (9) lubricants, such a talc, calcium stearate,
magnesium stearate, solid polyethylene glycols, sodium lauryl
sulfate, and mixtures thereof; and (10) coloring agents. In the
case of capsules, tablets and pills, the pharmaceutical
compositions may also comprise buffering agents. Solid compositions
of a similar type may also be employed as fillers in soft and
hard-filled gelatin capsules using such excipients as lactose or
milk sugars, as well as high molecular weight polyethylene glycols
and the like.
[0195] Liquid dosage forms for oral administration include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups, and elixirs. In addition to the active
ingredient, the liquid dosage forms may contain inert diluents
commonly used in the art, such as water or other solvents,
solubilizing agents and emulsifiers, such as ethyl alcohol,
isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,
benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in
particular, cottonseed, groundnut, corn, germ, olive, castor, and
sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene
glycols and fatty acid esters of sorbitan, and mixtures thereof.
Besides inert diluents, the oral compositions can also include
adjuvants such as wetting agents, emulsifying and suspending
agents, sweetening, flavoring, coloring, perfuming, and
preservative agents.
[0196] Suspensions, in addition to the active compounds, may
contain suspending agents such as ethoxylated isostearyl alcohols,
polyoxyethylene sorbitol, and sorbitan esters, microcrystalline
cellulose, aluminum metahydroxide, bentonite, agar-agar and
tragacanth, and mixtures thereof.
[0197] In particular, methods of the invention can be administered
topically, either to skin or to mucosal membranes such as those on
the cervix and vagina. This offers the greatest opportunity for
direct delivery to tumor with the lowest chance of inducing side
effects. The topical formulations may further include one or more
of the wide variety of agents known to be effective as skin or
stratum corneum penetration enhancers. Examples of these are
2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,
dimethylformamide, propylene glycol, methyl or isopropyl alcohol,
dimethyl sulfoxide, and azone. Additional agents may further be
included to make the formulation cosmetically acceptable. Examples
of these are fats, waxes, oils, dyes, fragrances, preservatives,
stabilizers, and surface active agents. Keratolytic agents such as
those known in the art may also be included. Examples are salicylic
acid and sulfur.
[0198] Dosage forms for the topical or transdermal administration
include powders, sprays, ointments, pastes, creams, lotions, gels,
solutions, patches, and inhalants. The subject polypeptide
therapeutic agents may be mixed under sterile conditions with a
pharmaceutically acceptable carrier, and with any preservatives,
buffers, or propellants which may be required. The ointments,
pastes, creams and gels may contain, in addition to a subject
polypeptide agent, excipients, such as animal and vegetable fats,
oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,
polyethylene glycols, silicones, bentonites, silicic acid, talc and
zinc oxide, or mixtures thereof.
[0199] Powders and sprays can contain, in addition to a subject
polypeptide therapeutic agent, excipients such as lactose, talc,
silicic acid, aluminum hydroxide, calcium silicates, and polyamide
powder, or mixtures of these substances. Sprays can additionally
contain customary propellants, such as chlorofluorohydrocarbons and
volatile unsubstituted hydrocarbons, such as butane and
propane.
[0200] Pharmaceutical compositions suitable for parenteral
administration may comprise one or more polypeptide therapeutic
agents in combination with one or more pharmaceutically acceptable
sterile isotonic aqueous or nonaqueous solutions, dispersions,
suspensions or emulsions, or sterile powders which may be
reconstituted into sterile injectable solutions or dispersions just
prior to use, which may contain antioxidants, buffers,
bacteriostats, solutes which render the formulation isotonic with
the blood of the intended recipient or suspending or thickening
agents. Examples of suitable aqueous and nonaqueous carriers which
may be employed in the pharmaceutical compositions of the invention
include water, ethanol, polyols (such as glycerol, propylene
glycol, polyethylene glycol, and the like), and suitable mixtures
thereof, vegetable oils, such as olive oil, and injectable organic
esters, such as ethyl oleate. Proper fluidity can be maintained,
for example, by the use of coating materials, such as lecithin, by
the maintenance of the required particle size in the case of
dispersions, and by the use of surfactants.
[0201] These compositions may also contain adjuvants, such as
preservatives, wetting agents, emulsifying agents and dispersing
agents. Prevention of the action of microorganisms may be ensured
by the inclusion of various antibacterial and antifungal agents,
for example, paraben, chlorobutanol, phenol sorbic acid, and the
like. It may also be desirable to include isotonic agents, such as
sugars, sodium chloride, and the like into the compositions. In
addition, prolonged absorption of the injectable pharmaceutical
form may be brought about by the inclusion of agents which delay
absorption, such as aluminum monostearate and gelatin.
[0202] Injectable depot forms are made by forming microencapsule
matrices of one or more polypeptide therapeutic agents in
biodegradable polymers such as polylactide-polyglycolide. Depending
on the ratio of drug to polymer, and the nature of the particular
polymer employed, the rate of drug release can be controlled.
Examples of other biodegradable polymers include poly(orthoesters)
and poly(anhydrides). Depot injectable formulations are also
prepared by entrapping the drug in liposomes or microemulsions
which are compatible with body tissue.
[0203] Formulations for intravaginal or rectally administration may
be presented as a suppository, which may be prepared by mixing one
or more compounds of the invention with one or more suitable
nonirritating excipients or carriers comprising, for example, cocoa
butter, polyethylene glycol, a suppository wax or a salicylate, and
which is solid at room temperature, but liquid at body temperature
and, therefore, will melt in the rectum or vaginal cavity and
release the active compound.
[0204] In other embodiments, the polypeptide therapeutic agents of
the instant invention can be expressed within cells from eukaryotic
promoters. For example, a soluble polypeptide of EphB4 or Ephrin B2
can be expressed in eukaryotic cells from an appropriate vector.
The vectors are preferably DNA plasmids or viral vectors. Viral
vectors can be constructed based on, but not limited to,
adeno-associated virus, retrovirus, adenovirus, or alphavirus.
Preferably, the vectors stably introduced in and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression. Such vectors can be repeatedly administered
as necessary. Delivery of vectors encoding the subject polypeptide
therapeutic agent can be systemic, such as by intravenous or
intramuscular administration, by administration to target cells
ex-planted from the patient followed by reintroduction into the
patient, or by any other means that would allow for introduction
into the desired target cell (for a review see Couture et al.,
1996, TIG., 12, 510).
EXEMPLIFICATION
[0205] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Example 1
Soluble Derivatives of the Extracellular Domains of Human Ephrin B2
and EphB4 Proteins
[0206] Soluble derivatives of the extracellular domains of human
Ephrin B2 and EphB4 proteins represent either truncated full-length
predicted extracellular domains of Ephrin B2 (B4ECv3, B2EC) or
translational fusions of the domains with constant region of human
immunoglobulins (IgG1 Fc fragment), such as B2EC-FC, B4ECv2-FC and
B4ECv3-FC. Representative human Ephrin B2 constructs and human
EphB4 constructs are shown FIGS. 14 and 15.
[0207] The cDNA fragments encoding these recombinant proteins were
subcloned into mammalian expression vectors, expressed in
transiently or stably transfected mammalian cell lines and purified
to homogeneity as described in detail in Materials and Methods
section (see below). Predicted amino acid sequences of the proteins
are shown in FIGS. 1-5. High purity of the isolated proteins and
their recognition by the corresponding anti-Ephrin B2 and
anti-EphB4 monoclonal or polyclonal antibodies were confirmed. The
recombinant proteins exhibit the expected high-affinity binding,
binding competition and specificity properties with their
corresponding binding partners as corroborated by the biochemical
assays (see e.g., FIGS. 6-8).
[0208] Such soluble derivative proteins human Ephrin B2 and EphB4
exhibit potent biological activity in several cell-based assays and
in vivo assays which measure angiogenesis or anti-cancer
activities, and are therefore perspective drug candidates for
anti-angiogenic and anti-cancer therapy. B4ECv3 as well as B2EC and
B2EC-FC proteins blocked chemotaxis of human endothelial cells (as
tested with umbilical cord and hepatic AECs or VECs), with a
decrease in degradation of the extracellular matrix, Matrigel, and
a decrease in migration in response to growth factor stimuli (FIGS.
9-11). B4ECv3 and B2EC-FC proteins have potent anti-angiogenic
effect as demonstrated by their inhibition of endothelial cell tube
formation (FIGS. 12-13).
[0209] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
[0210] The sequence of the Globular domain+Cys-rich domain
(B4EC-GC), precursor protein is (SEQ ID NO:12): TABLE-US-00001
MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE
EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL
PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR
PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL
TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV
TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA
VCQCRVGYFRARTDPRGAPCTTPPSAHHHHHH
[0211] For many uses, including therapeutic use, the leader
sequence (first 15 amino acids, so that the processed form begins
Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be
removed or omitted.
[0212] Sequence of the GCF precursor protein (SEQ ID NO:13):
TABLE-US-00002 MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE
EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL
PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR
PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL
TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV
TGCSCAPGFAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVC
QCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGR
EDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTY
TFEVTALNGVSSLATGPVPFEPVNVHHHHHH
[0213] For many uses, including therapeutic use, the leader
sequence (first 15 amino acids, so that the processed form begins
Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be
removed or omitted.
[0214] Amino acid sequence of encoded FL-hB4EC precursor
(His-tagged) (SEQ ID NO:14): TABLE-US-00003
MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE
EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLIWFMLECLSL
PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR
PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL
TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV
TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA
VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG
GREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDF
TYTFEVTALNGVSSLATGPVPFEPVNVITDREVPPAVSDIRVTRSSPSSL
SLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRG
ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQGSKRALLQWGKPL
PNPLLGLDSTRTGHHHHHH
[0215] For many uses, including therapeutic use, the leader
sequence (first 15 amino acids, so that the processed form begins
Leu-Glu-Glu . . . ) and the c-terminal hexahistidine tag may be
removed or omitted.
[0216] EphB4 CF2 protein, precursor (SEQ ID NO:15): TABLE-US-00004
MELRVLLCWASLAAALEETLLNTKLETQLTVNLTRFPETVPRELVVPVAG
SCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGNTKCRAC
AQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDPRGAPCT
TPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECRPGGSCA
PCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSLATGPVP
FEPVNVTTDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAWLDYEVKY
HEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYGPFGQE
HHSQTQLDESEGWREQGGRSSLEGPRFEGKPIPNPLLGLDSTRTGHHHHH H
[0217] The precursor sequence of the preferred GCF2 protein (also
referred to herein as GCF2F) is (SEQ ID NO:16): TABLE-US-00005
MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE
EQHSVRTYEVCEVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL
PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR
PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL
TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV
TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA
VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG
GREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDF
TYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSL
SLAWAVPRAPSGAWLDYEVKYHEKGAEGPSSVRPLKTSENRAELRGLKRG
ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQ
[0218] The processed sequence is (SEQ ID NO:17): TABLE-US-00006
LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCEVQR
APGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFY
YESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLR
LGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPREL
VVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGN
TKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDP
RGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECR
PGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSL
ATGPVPFEPVNVTITDREVPPAVSDIRVTRSSPSSLSLAWAVPRAPSGAW
LDYEVKYEKGAEGPSSVRFLKTSENRELRGLKRGASYLVQVRARSEAGYG
PFGQEHHSQTQLDESEGWREQ
Biochemical Assays
[0219] A. Binding Assay
[0220] 10 .mu.l of Ni-NTA-Agarose were incubated in microcentrifuge
tubes with 50 .mu.l of indicated amount of B4ECv3 diluted in
binding buffer BB (20 mM Tris-HCl, 0.15 M NaCl, 0.1% bovine serum
albumin pH 8) After incubation for 30 min on shaking platform,
Ni-NTA beads were washed twice with 1.4 ml of BB, followed by
application of 50 .mu.l of B2-AP in the final concentration of 50
nM. Binding was performed for 30 min on shaking platform, and then
tubes were centrifuged and washed one time with 1.4 ml of BB.
Amount of precipitated AP was measured colorimetrically after
application of PNPP.
[0221] B. Inhibition Assay
[0222] Inhibition in solution. Different amounts of B4ECv3 diluted
in 50 .mu.l of BB were pre-incubated with 50 .mu.l of 5 nM B2EC-AP
reagent (protein fusion of Ephrin B2 ectodomain with placental
alkaline phosphatase). After incubation for 1 h, unbound B2EC-AP
was precipitated with 5,000 HEK293 cells expressing
membrane-associated full-length EphB4 for 20 min. Binding reaction
was stopped by dilution with 1.2 ml of BB, followed by
centrifugation for 10 min. Supernatants were discarded and alkaline
phosphatase activities associated with collected cells were
measured by adding para-nitrophenyl phosphate (PNPP) substrate.
[0223] Cell based inhibition. B4ECv3 was serially diluted in 20 mM
Tris-HCl, 0.15 M NaCl, 0.1% BSA, pH 8 and mixed with 5,000 HEK293
cells expressing membrane-associated full-length Ephrin B2. After
incubation for 1 h, 50 .mu.l of 5 nM B4EC-AP reagent (protein
fusion of EphB4 ectodomain with placental alkaline phosphatase were
added into each tube for 30 min to detect unoccupied Ephrin B2
binding sites. Binding reactions were stopped by dilution with 1.2
ml of BB and centrifugation. Colorimetric reaction of
cell-precipitated AP was developed with PNPP substrate.
[0224] C. B4EC-FC binding Assay
[0225] Protein A-agarose based assay. 10 .mu.l of Protein A-agarose
were incubated in Eppendorf tubes with 50 .mu.l of indicated amount
of B4EC-FC diluted in binding buffer BB (20 mM Tris-HCl, 0.15 M
NaCl, 0.1% BSA pH 8). After incubation for 30 min on shaking
platform, Protein AAagarose beads were washed twice with 1.4 ml of
BB, followed by application of 50 .mu.l of B2ECAP reagent at the
final concentration of 50 nM. Binding was performed for 30 min on
shaking platform, and then tubes were centrifuged and washed once
with 1.4 ml of BB. Colorimetric reaction of precipitated AP was
measured after application of PNPP (FIG. 6).
[0226] Nitrocellulose based assay. B4EC-FC was serially diluted in
20 mM Tris-HCl, 0.15 M NaCl, 50 .mu.g/ml BSA, pH 8. 2 .mu.l of each
fraction were applied onto nitrocellulose strip and spots were
dried out for 3 min. Nitrocellulose strip was blocked with 5%
non-fat milk for 30 min, followed by incubation with 5 nM B2EC-AP
reagent. After 45 min incubation for binding, nitrocellulose was
washed twice with 20 mM Tris-HCl, 0.15 M NaCl, 50 .mu.g/ml BSA, pH
8 and color was developed by application of alkaline phosphatase
substrate Sigma Fast (Sigma).
[0227] D. B4EC-FC Inhibition Assay
[0228] Inhibition in solution. See above, for B4ECv3. The results
were shown in FIG. 7.
[0229] Cell based inhibition. See above, for B4ECv3.
[0230] E. B2EC-FC Binding Assay
[0231] Protein-A-agarose based assay. See above, for B4EC-FC. The
results were shown in FIG. 8.
[0232] Nitrocellulose based assay. See above, for B4EC-FC.
[0233] 6) Cell-Based Assays
[0234] A. Growth Inhibition Assay
[0235] Human umbilical cord vein endothelial cells (HUVEC)
(1.5.times.103) are plated in a 96-well plate in 100 .mu.l of EBM-2
(Clonetic # CC3162). After 24 hours (day 0), the test recombinant
protein (100 .mu.l) is added to each well at 2.times. the desired
concentration (5-7 concentration levels) in EBM-2 medium. On day 0,
one plate is stained with 0.5% crystal violet in 20% methanol for
10 minutes, rinsed with water, and air-dried. The remaining plates
are incubated for 72 h at 37.degree. C. After 72 h, plates are
stained with 0.5% crystal violet in 20% methanol, rinsed with water
and airdried. The stain is eluted with 1:1 solution of ethanol: 0.1
M sodium citrate (including day 0 plate), and absorbance is
measured at 540 nm with an ELISA reader (Dynatech Laboratories).
Day 0 absorbance is subtracted from the 72 h plates and data is
plotted as percentage of control proliferation (vehicle treated
cells). IC50 (drug concentration causing 50% inhibition) is
calculated from the plotted data.
[0236] B. Cord Formation Assay (Endothelial Cell Tube Formation
Assay)
[0237] Matrigel (60 .mu.l of 10 mg/ml; Collaborative Lab # 35423)
is placed in each well of an ice-cold 96-well plate. The plate is
allowed to sit at room temperature for 15 minutes then incubated at
37.degree. C. for 30 minutes to permit the matrigel to polymerize.
In the mean time, HUVECs are prepared in EGM-2 (Clonetic # CC3162)
at a concentration of 2.times.10.sup.5 cells/ml. The test compound
is prepared at 2.times. the desired concentration (5 concentration
levels) in the same medium. Cells (500 .mu.L) and 2.times. drug
(500 .mu.l) is mixed and 200 .mu.l of this suspension are placed in
duplicate on the polymerized matrigel. After 24 h incubation,
triplicate pictures are taken for each concentration using a
Bioquant Image Analysis system. Drug effect (IC50) is assessed
compared to untreated controls by measuring the length of cords
formed and number of junctions.
[0238] C. Cell Migration Assay
[0239] Migration is assessed using the 48-well Boyden chamber and 8
.mu.m pore size collagen-coated (10 .mu.g/ml rat tail collagen;
Collaborative Laboratories) polycarbonate filters (Osmonics, Inc.).
The bottom chamber wells receive 27-29 .mu.l of DMEM medium alone
(baseline) or medium containing chemo-attractant (bFGF, VEGF or
Swiss 3T3 cell conditioned medium). The top chambers receive 45
.mu.l of HUVEC cell suspension (1.times.10.sup.6 cells/ml) prepared
in DMEM+1% BSA with or without test compound. After 5 h incubation
at 37.degree. C., the membrane is rinsed in PBS, fixed and stained
in Diff-Quick solutions. The filter is placed on a glass slide with
the migrated cells facing down and cells on top are removed using a
Kimwipe. The testing is performed in 4-6 replicates and five fields
are counted from each well. Negative unstimulated control values
are subtracted from stimulated control and drug treated values and
data is plotted as mean migrated cell.+-.S.D. IC50 is calculated
from the plotted data.
Example 2
Extracellular Domain Fragments of EphB4 Receptor Inhibit
Angiogenesis and Tumor Growth.
A. Globular Domain of EphB4 is Required for EphrinB2 Binding and
for the Activity of EphB4-Derived Soluble Proteins in Endothelial
Tube Formation Assay.
[0240] To identify subdomain(s) of the ectopic part of EphB4
necessary and sufficient for the anti-angiogenic activity of the
soluble recombinant derivatives of the receptor, four recombinant
deletion variants of EphB4EC were produced and tested (FIG. 16).
Extracellular part of EphB4, similarly to the other members of EphB
and EphA receptor family, contains N-terminal ligand-binding
globular domain followed by cysteine-rich domain and two
fibronectin type III repeats (FNIII). In addition to the
recombinant B4-GCF2 protein containing the complete ectopic part of
EphB4, we constructed three deletion variants of EphB4EC containing
globular domain and Cys-rich domain (B4-GC); globular, Cys-rich and
the first FNIII domain (GCF1) as well as the ECD version with
deleted globular domain (CF2). Our attempts to produce several
versions of truncated EphB4EC protein containing the globular
domain alone were not successful due to the lack of secretion of
proteins expressed from all these constructs and absence of ligand
binding by the intracellularly expressed recombinant proteins. In
addition, a non-tagged version of B4-GCF2, called GCF2-F,
containing complete extracellular domain of EphB4 with no
additional fused amino acids was expressed, purified and used in
some of the experiments described here.
[0241] All four C-terminally 6.times.His tagged recombinant
proteins were preparatively expressed in transiently transfected
cultured mammalian cells and affinity purified to homogeneity from
the conditioned growth media using chromatography on
Ni.sup.2+-chelate resin (FIG. 17). Apparently due to their
glycosylation, the proteins migrate on SDS-PAAG somewhat higher
than suggested by their predicted molecular weights of 34.7 kDa
(GC), 41.5 (CF2), 45.6 kDa (GCF1) and 57.8 kDa (GCF2). Sequence of
the extracellular domain of human EphB4 contains three predicted
N-glycosylation sites (NXS/T) which are located in the Cys-rich
domain, within the first fibronectin type III repeat and between
the first and the second fibronectin repeats.
[0242] To confirm ability of the purified recombinant proteins to
bind Ephrin B2, they were tested in an in vitro binding assay. As
expected, GC, GCF1 and GCF2, but not CF2 are binding the cognate
ligand Ephrin B2 as confirmed by interaction between Ephrin
B2-alkaline phosphatase (Ephrin B2-AP) fusion protein with the B4
proteins immobilized on Ni.sup.2+ resin or on nitrocellulose
membrane (FIG. 17).
[0243] All four proteins were also tested for their ability to
block ligand-dependent dimerization and activation of Eph B4
receptor kinase in PC3 cells. The PC3 human prostate cancer cell
line is known to express elevated levels of human Eph B4.
Stimulation of PC3 cells with Ephrin B2 IgG Fc fusion protein leads
to a rapid induction of tyrosine phosphorylation of the receptor.
However, preincubation of the ligand with GCF2, GCF1 or GC, but not
CF2 proteins suppresses subsequent EphB4 autophosphorylation.
Addition of the proteins alone to the PC3 cells or preincubation of
the cells with the proteins followed by changing media and adding
the ligand does not affect EphB4 phosphorylation status.
[0244] Further, we found that globular domain of EphB4 is required
for the activity of EphB4-derived soluble proteins in endothelial
tube formation assay.
B. Effects of Soluble EphB4 on HUV/AEC In Vitro.
[0245] Initial experiments were performed to determine whether
soluble EphB4 affected the three main stages in the angiogenesis
pathway. These were carried out by establishing the effects of
soluble EphB4 on migration/invasion, proliferation and tubule
formation by HUV/AEC in vitro. Exposure to soluble EphB4
significantly inhibited both bFGF and VEGF-induced migration in the
Boyden chamber assay in a dose-dependent manner, achieving
significance at nM (FIG. 18). Tubule formation by HUV/AECS on wells
coated with Matrigel was significantly inhibited by soluble EphB4
in a dose-dependent manner in both the absence and presence of bFGF
and VEGF (FIG. 19). We also assessed in vitro, whether nM of
soluble EphB4 was cytotoxic for HUVECS. Soluble EphB4 was found to
have no detectable cytotoxic effect at these doses, as assessed by
MTS assay (FIG. 20).
C. Soluble EphB4 Receptor Inhibits Vascularization of Matrigel
Plugs, In Vivo
[0246] To demonstrate that soluble EphB4 can directly inhibit
angiogenesis in vivo, we performed a murine matrigel plug
experiment. Matrigel supplemented with bFGF and VEGF with and
without soluble EphB4 was injected s.c. into Balb/C nu/nu mice,
forming semi-solid plugs, for six days. Plugs without growth
factors had virtually no vascularization or vessel structures after
6 days (FIG. 21). In contrast, plugs supplemented with bFGF and
VEGF had extensive vascularization and vessels throughout the plug.
Plugs taken from mice treated with .mu.g of soluble EphB4 had
markedly reduced vascularization of plugs, comparable to plugs
without growth factor (FIG. 21). Furthermore, histological
examination of plugs showed decreased vessel staining (FIG. 21).
Treatment at 0 .mu.g/dose significantly inhibited the amount of
infiltration in Matrigel plugs compared to control (FIG. 21).
[0247] We examined EphB4 receptor phosphorylation in HUVECs by
performing Western blot analyses with lysates from soluble
EphB4-treated cells and antibodies against phosphor-tyrosine. We
found that soluble EphB4 treatment of serum-starved HUVECs
stimulated a rapid and transient decrease in the level of
phosphorylated EphB4, in the presence of EphrinB2Fc, EphB4 ligand
dimer. Ephrin B2Fc without the soluble EphB4 protein induced
phosphorylation of EphB4 receptor (FIG. 22).
D. Effects of Soluble EphB4 on Tumor Growth, In Vitro.
[0248] We found that soluble EphB4 inhibits the growth of SCC15
tumors grown in Balb/C Nu/Nu mice (FIG. 23).
[0249] E. Soluble EphB4 Inhibited Corneal Neovascularization
[0250] To further investigate the antiangiogenic activity of
soluble EphB4 in vivo, we studied the inhibitory effect of
administration of soluble EphB4 on neovascularization in the mouse
cornea induced by bFGF. Hydron Pellets implanted into corneal
micropocket could induce angiogenesis, in the presence of growth
factors, in a typically avascular area. The angiogenesis response
in mice cornea was moderate, the appearance of vascular buds was
delayed and the new capillaries were sparse and grew slowly.
Compared with the control group, on day 7 of implantation, the
neovascularization induced by bFGF in mice cornea was markedly
inhibited in soluble EphB4-treated group (FIG. 24).
F. Effects of Soluble EphB4 on Tumor Growth, In Vivo.
[0251] The same model was used to determine the effects of soluble
EphB4 in vivo. SCC15 tumors implanted subcutaneously, pre-incubated
with matrigel and with or w/o growth factors, as well as implanted
sc alone, and mice treated sc or ip daily with 1-5 ug of soluble
EphB4 were carried out.
[0252] Tumors in the control group continued to grow steadily over
the treatment period, reaching a final tumor volume of mm3.
However, animals injected with soluble EphB4 exhibited a
significantly (p<0.0/) reduced growth rate, reaching a final
tumor volume of only mm3 (FIG. 25). Similar results were obtained
in two further cohorts of such tumor-bearing mice. Soluble EphB4
administration appeared to be well tolerated in vivo, with no
significant effect on body weight or the general well-being of the
animals (as determined by the absence of lethargy, intermittent
hunching, tremors or disturbed breathing patterns).
G. Effects of Soluble EphB4 on Tumor Histology.
[0253] Histological analysis revealed the presence of a central
area of necrosis in all SCC15 tumors, which was usually surrounded
by a viable rim of tumor cells um in width. The central necrotic
areas were frequently large and confluent and showed loss of
cellular detail. Necrosis, assessed as a percentage of tumor
section area, was significantly (p<0.02) more extensive in the
soluble EphB4-treated group (% necrosis in treated vs. control). To
determine whether the reduced volume of soluble EphB4 treated
tumors was due to an effect of this protein on the tumor vascular
supply, endothelial cells in blood vessels were identified in tumor
sections using immunostaining with an anti-platelet cell adhesion
molecule (PECAM-1; CD31) antibody (FIG. 26) and the density of
microvessels was assessed. Microvessel density was similar in the
outer viable rim of tumor cells (the uniform layer of cells
adjacent to the tumor periphery with well defined nuclei) in
control and soluble EphB4-treated tumors. Microvessel density was
significantly in the inner, less viable region of tumor cells
abutting the necrotic central areas in soluble EphB4-treated than
control tumors. Fibrin deposition, as identified by Masson's
Trichrome staining, was increased in and around blood vessels in
the inner viable rim and the central necrotic core of soluble EphB4
treated than control tumors. In the outer viable rim of soluble
EphB4 treated tumors, although the vessel lumen remained patent and
contained red blood cells, fibrin deposition was evident around
many vessels. Soluble EphB4 was found to have no such effects on
the endothelium in the normal tissues examined (lungs, liver and
kidneys).
H. Materials and Methods
[0254] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
[0255] Cell-based EphB4 tyrosine kinase assay
[0256] The human prostate carcinoma cell line PC3 cells were
maintained in RPMI medium with 10% dialyzed fetal calf serum and 1%
penicillin/streptomycin/neomycin antibiotics mix. Cells were
maintained at 37.degree. C. in a humidified atmosphere of 5%
CO.sub.2/95% air. Typically, cells were grown in 60 mm dishes until
confluency and were either treated with mouse Ephrin B2-Fc fusion
at 1 .mu.g/ml in RPMI for 10 min to activate EphB4 receptor or
plain medium as a control. To study the effect of different
derivatives of soluble EphB4 ECD proteins on EphB4 receptor
activation, three sets of cells were used. In the first set, cells
were treated with various proteins (5 proteins; GC, GCF1, GCF2,
GCF2--F, CF2) at 5 .mu.g/ml for 20 min. In the second set of cells,
prior to application, proteins were premixed with ephrinB2-Fc at
1:5 (EphB4 protein: B2-Fc) molar ratio, incubated for 20 min and
applied on cells for 10 min. In the third set of cells, cells were
first treated with the proteins for 20 min at 5 .mu.g/ml, media was
replaced with fresh media containing 1 .mu.g/ml of EphrinB2-Fc and
incubated for another 10 min.
[0257] After the stimulation, cells were immediately harvested with
protein extraction buffer containing 20 mM Tris-HCl, pH 7.4, 150 mM
NaCl, 1% (v/v) Triton X100, 1 mM EDTA, 1 mM PMSF, 1 mM Sodium
vanadate. Protein extracts were clarified by centrifugation at
14,000 rpm for 20 min at 4.degree. C. Clarified protein samples
were incubated overnight with protein A/G coupled agarose beads
pre-coated with anti-EphB4 monoclonal antibodies. The IP complexes
were washed twice with the same extraction buffer containing 0.1%
Triton X100. The immunoprecipitated proteins were solubilized in
1.times.SDS-PAGE sample loading buffer and separated on 10%
SDS-PAGE. For EphB4 receptor activation studies, electroblotted
membrane was probed with anti-pTyr specific antibody 4G10 at 1:1000
dilution followed by Protein G-HRP conjugate at 1:5000
dilutions.
[0258] Endothelial Cell Tube Formation Assay
[0259] Matrigel (60 .mu.l of 10 mg/ml; Collaborative Lab, Cat. No.
35423) was placed in each well of an ice-cold 96-well plate. The
plate was allowed to sit at room temperature for 15 minutes then
incubated at 37.degree. C. for 30 minutes to permit Matrigel to
polymerize. In the mean time, human umbilical vein endothelial
cells were prepared in EGM-2 (Clonetic, Cat. No. CC3162) at a
concentration of 2.times.10.sup.5 cells/ml. The test protein was
prepared at 2.times. the desired concentration (5 concentration
levels) in the same medium. Cells (500 .mu.l) and 2.times. protein
(500 .mu.l) were mixed and 200 .mu.l of this suspension were placed
in duplicate on the polymerized Matrigel. After 24 h incubation,
triplicate pictures were taken for each concentration using a
Bioquant Image Analysis system. Protein addition effect (IC.sub.50)
was assessed compared to untreated controls by measuring the length
of cords formed and number of junctions.
[0260] Cell Migration Assay
[0261] Chemotaxis of HUVECs to VEGF was assessed using a modified
Boyden chamber, transwell membrane filter inserts in 24 well
plates, 6.5 mm diam, 8 .mu.m pore size, 10 .mu.m thick matrigel
coated, polycarbonate membranes (BD Biosciences). The cell
suspensions of HUVECs (2.times.10.sup.5 cells/ml) in 200 .mu.l of
EBM were seeded in the upper chamber and the soluble EphB4 protein
were added simultaneously with stimulant (VEGF or bFGF) to the
lower compartment of the chamber and their migration across a
polycarbonate filter in response to 10-20 ng/ml of VEGF with or
without 100 nM-1 .mu.M test compound was investigated. After
incubation for 4-24 h at 37.degree. C., the upper surface of the
filter was scraped with swab and filters were fixed and stained
with Diff Quick. Ten random fields at 200.times. mag were counted
and the results expressed as mean # per field. Negative
unstimulated control values were subtracted from stimulated control
and protein treated sample values and the data was plotted as mean
migrated cell.+-.S.D. IC.sub.50 was calculated from the plotted
data.
[0262] Growth Inhibition Assay
[0263] HUVEC (1.5.times.10.sup.3 cells) were plated in a 96-well
plate in 100 .mu.l of EBM-2 (Clonetic, Cat. No. CC3162). After 24
hours (day 0), the test recombinant protein (100 .mu.l) is added to
each well at 2.times. the desired concentration (5-7 concentration
levels) in EBM-2 medium. On day 0, one plate was stained with 0.5%
crystal violet in 20% methanol for 10 minutes, rinsed with water,
and air-dried. The remaining plates were incubated for 72 h at
37.degree. C. After 72 h, plates were stained with 0.5% crystal
violet in 20% methanol, rinsed with water and air-dried. The stain
was eluted with 1:1 solution of ethanol: 0.1M sodium citrate
(including day 0 plate), and absorbance measured at 540 nm with an
ELISA reader (Dynatech Laboratories). Day 0 absorbance was
subtracted from the 72 h plates and data is plotted as percentage
of control proliferation (vehicle treated cells). IC.sub.50 value
was calculated from the plotted data.
[0264] Murine Matrigel Plug Angiogenesis Assay
[0265] In vivo angiogenesis was assayed in mice as growth of blood
vessels from subcutaneous tissue into a Matrigel plug containing
the test sample. Matrigel rapidly forms a solid gel at body
temperature, trapping the factors to allow slow release and
prolonged exposure to surrounding tissues. Matrigel (8.13 mg/ml,
0.5 ml) in liquid form at 4.degree. C. was mixed with Endothelial
Cell Growth Supplement (ECGS), test proteins plus ECGS or Matrigel
plus vehicle alone (PBS containing 0.25% BSA). Matrigel (0.5 ml)
was injected into the abdominal subcutaneous tissue of female nu/nu
mice (6 wks old) along the peritoneal mid line. There were 3 mice
in each group. The animals were cared for in accordance with
institutional and NIH guidelines. At day 6, mice were sacrificed
and plugs were recovered and processed for histology. Typically the
overlying skin was removed, and gels were cut out by retaining the
peritoneal lining for support, fixed in 10% buffered formalin in
PBS and embedded in paraffin. Sections of 3 .mu.m were cut and
stained with H&E or Masson's trichrome stain and examined under
light microscope
[0266] Mouse Corneal Micropocket Assay
[0267] Mouse corneal micropocket assay was performed according to
that detailed by Kenyon et al., 1996. Briefly, hydron pellets
(polyhydroxyethylmethacrylate [polyHEMA], Interferon Sciences, New
Brunswick, N.J., U.S.A.) containing either 90 ng of bFGF (R&D)
or 180 ng of VEGF (R&D Systems, Minneapolis, Minn., U.S.A.) and
40 .mu.g of sucrose aluminium sulfate (Sigma) were prepared. Using
an operating microscope, a stromal linear keratotomy was made with
a surgical blade (Bard-Parker no. 15) parallel to the insertion of
the lateral rectus muscle in an anesthetized animal. An
intrastromal micropocket was dissected using a modified von Graefe
knife (2''30 mm). A single pellet was implanted and advanced toward
the temporal corneal limbus (within 0.+-.7.+-.1.+-.0 mm for bFGF
pellets and 0.+-.5 mm for VEGF pellets). The difference in pellet
location for each growth factor was determined to be necessary
given the relatively weaker angiogenic stimulation of VEGF in this
model. Antibiotic ointment (erythromycin) was then applied to the
operated eye to prevent infection and to decrease surface
irregularities. The subsequent vascular response was measured
extending from the limbal vasculature toward the pellet and the
contiguous circumferential zone of neovascularization Data and
clinical photos presented here were obtained on day 6 after pellet
implantation, which was found to be the day of maximal angiogenic
response.
[0268] In Vitro Invasion Assay
[0269] "Matrigel" matrix-coated 9-mm cell culture inserts (pore
size, 8 .mu.m; Becton Dickinson, Franklin Lakes, N.J.) were set in
a 24-well plate. The HUVEC cells were seeded at a density of
5.times.10.sup.3 cells per well into the upper layer of the culture
insert and cultured with serum-free EBM in the presence of EphB4
ECD for 24 h. The control group was cultured in the same media
without EphB4. Then 0.5 ml of the human SCC15 cell line,
conditioned medium was filled into the lower layer of the culture
insert as a chemo-attractant. The cells were incubated for 24 h,
then the remaining cells in the upper layer were swabbed with
cotton and penetrating cells in the lower layer were fixed with 5%
glutaraldehyde and stained with Diff Quick. The total number of
cells passing through the Matrigel matrix and each 8 .mu.m pore of
the culture insert was counted using optical microscopy and
designated as an invasion index (cell number/area).
[0270] SCC15 Tumor Growth in Mice
[0271] Subcutaneously inject logarithmically growing SCC15, head
and neck squamous cell carcinoma cell line, at 5.times.10.sup.6
cell density; with or without EphB4 ECD in the presence or absence
of human bFGF, into athymic Balb/c nude mice, along with Matrigel
(BD Bioscience) synthetic basement membrane (1:1 v/v), and examine
tumors within 2 weeks. Tumor volumes in the EphB4 ECD group, in the
presence and absence of growth factor after implantation were
three-fold smaller than those in the vehicle groups. There was no
difference in body weight between the groups. Immunohistochemical
examination of cross-sections of resected tumors and TUNEL-positive
apoptosis or necrosis, CD34 immunostaining, and BrdU proliferation
rate will be performed, after deparaffinized, rehydrated, and
quenched for endogenous peroxidase activity, and after 10 min
permeabilization with proteinase K. Quantitative assessment of
vascular densities will also be performed. Local intratumoral
delivery or IV delivery of EphB4 ECD will also be performed twice a
week.
[0272] 30 athymic nude mice, BALB/c (nu/nu), were each injected
with 1.times.10.sup.6 B16 melanoma cells with 0.1 ml PBS mixed with
0.1 ml matrigel or 1.5.times.10.sup.6 SCC15 cells resuspended in
200 .mu.l of DMEM serum-free medium and injected subcutaneously on
day 0 on the right shoulder region of mice. Proteins were injected
intravenously or subcutaneously, around the tumor beginning on day
1 at a loading dose of 4 .mu.g/mg, with weekly injections of 2
ug/mg. (10 .mu.g/g, 50 .mu.g/kg/day), and at 2 weeks
post-inoculation. Mice are sacrificed on Day 14. Control mice
received PBS 50 .mu.l each day.
[0273] Tumor Formation in Nude Mice
[0274] All animals were treated under protocols approved by the
institutional animal care committees. Cancer cells
(5.times.10.sup.6) were subcutaneously inoculated into the dorsal
skin of nude mice. When the tumor had grown to a size of about 100
mm.sup.3 (usually it took 12 days), sEphB4 was either
intraperitoneally or subcutaneously injected once/day, and
tumorigenesis was monitored for 2 weeks. Tumor volume was
calculated according to the formula a.sup.2.times.b, where a and b
are the smallest and largest diameters, respectively. A Student's t
test was used to compare tumor volumes, with P<0.05 being
considered significant.
[0275] Quantification of Microvessel Density
[0276] Tumors were fixed in 4% formaldehyde, embedded in paraffin,
sectioned by 5 .mu.m, and stained with hematoxylineosin. Vessel
density was semi-quantitated using a computer-based image analyzer
(five fields per section from three mice in each group).
Example 3
EphB4 is Upregulated and Imparts Growth Advantage in Prostate
Cancer
A. Expression of EphB4 in Prostate Cancer Cell Lines
[0277] We first examined the expression of EphB4 protein in a
variety of prostate cancer cell lines by Western blot. We found
that prostate cancer cell lines show marked variation in the
abundance of the 120 kD EphB4. The levels were relatively high in
PC3 and even higher in PC3M, a metastatic clone of PC3, while
normal prostate gland derived cell lines (MLC) showed low or no
expression of EphB4 (FIG. 27A). We next checked the activation
status of EphB4 in PC3 cells by phosphorylation study. We found
that even under normal culture conditions, EphB4 is phosphorylated
though it can be further induced by its ligand, ephrin B2 (FIG.
27B).
B. Expression of EphB4 in Clinical Prostate Cancer Samples
[0278] To determine whether EphB4 is expressed in clinical prostate
samples, tumor tissues and adjacent normal tissue from prostate
cancer surgical specimens were examined. The histological
distribution of EphB4 in the prostate specimens was determined by
immunohistochemistry. Clearly, EphB4 expression is confined to the
neoplastic epithelium (FIG. 28, top left), and is absent in stromal
and normal prostate epithelium (FIG. 28, top right). In prostate
tissue array, 24 of the 32 prostate cancers examined were positive.
We found EphB4 mRNA is expressed both in the normal and tumor
tissues of clinical samples by quantitative RT-PCR. However, tumor
EphB4 mRNA levels were at least 3 times higher than in the normal
in this case (FIG. 28, lower right).
C. p53 and PTEN Inhibited the Expression of EphB4 in PC3 Cells
[0279] PC3 cells are known to lack PTEN expression (Davis, et al.,
1994, Science. 266:816-819) and wild-type p53 function (Gale, et
al., 1997, Cell Tissue Res. 290:227-241). We investigated whether
the relatively high expression of EphB4 is related to p53 and/or
PTEN by re-introducing wild-type p53 and/or PTEN into PC3 cells. To
compensate for the transfection efficiency and the dilution effect,
transfected cells were sorted for the cotransfected truncated CD4
marker. We found that the expression of EphB4 in PC3 cells was
reduced by the re-introduction of either wild-type p53 or PTEN. The
co-transfection of p53 and PTEN did not further inhibit the
expression of EphB4 (FIG. 29A).
D. Retinoid X Receptor (RXR .alpha.) Regulates the Expression of
EphB4
[0280] We previously found that RXR.alpha. was down-regulated in
prostate cancer cell lines (Zhong, et al., 2003, Cancer Biol Ther.
2:179-184) and here we found EphB4 expression has the reverse
expression pattern when we looked at "normal" prostate (MLC),
prostate cancer (PC3), and metastatic prostate cancer (PC3M) (FIG.
27A), we considered whether RXR.alpha. regulates the expression of
EphB4. To confirm the relationship, the expression of EphB4 was
compared between CWR22R and CWR22R-RXR.alpha., which constitutively
expresses RXR.alpha.. We found a modest decrease in EphB4
expression in the RXR.alpha. overexpressing cell line, while FGF8
has no effect on EphB4 expression. Consistent with initial results,
EphB4 was not found in "normal" benign prostate hypertrophic cell
line BPH-1 (FIG. 29B).
E. Growth Factor Signaling Pathway of EGFR and IGF-R Regulates
EphB4 Expression
[0281] EGFR and IGF-1R have both been shown to have autocrine and
paracrine action on PC3 cell growth. Because we found that EphB4
expression is higher in the more aggressive cell lines, we
postulated that EphB4 expression might correlate with these
pro-survival growth factors. We tested the relationship by
independently blocking EGFR and IGF-1R signaling. EphB4 was
down-regulated after blocking the EGFR signaling using EGFR kinase
inhibitor AG 1478 (FIG. 30A) or upon blockade of the IGF-1R
signaling pathway using IGF-1R neutralizing antibody (FIG.
30B).
F. EphB4 siRNA and Antisense ODNs Inhibit PC3 Cell Viability
[0282] To define the significance of this EphB4 overexpression in
our prostate cancer model, we concentrated our study on PC3 cells,
which have a relatively high expression of EphB4. The two
approaches to decreasing EphB4 expression were siRNA and AS-ODNs. A
number of different phosphorothioate-modified AS-ODNs complementary
to different segments of the EphB4 coding region were tested for
specificity and efficacy of EphB4 inhibition. Using 293 cells
transiently transfected with full-length EphB4 expression vector
AS-10 was found to be the most effective (FIG. 31B). A Similar
approach was applied to the selection of specific siRNA. EphB4
siRNA 472 effectively knocks down EphB4 protein expression (FIG.
31A). Both siRNA 472 and antisense AS-10 ODN reduced the viability
of PC3 cells in a dose dependent manner (FIG. 31C, D). Unrelated
siRNA or sense oligonucleotide had no effect on viability.
G. EphB4 siRNA and Antisense ODNs Inhibit the Mobility of PC3
Cells
[0283] PC3 cells can grow aggressively locally and can form lymph
node metastases when injected orthotopically into mice. In an
effort to study the role of EphB4 on migration of PC3 cells in
vitro, we performed a wound-healing assay. When a wound was
introduced into a monolayer of PC3 cells, over the course of the
next 20 hours cells progressively migrated into the cleared area.
However, when cells were transfected with siRNA 472 and the wound
was introduced, this migration was significantly inhibited (FIG.
31E). Pretreatment of PC3 cells with 10 .mu.M EphB4 AS-10 for 12
hours generated the same effect (FIG. 31F). In addition, knock-down
of EphB4 expression in PC3 cells with siRNA 472 severely reduced
the ability of these cells to invade Matrigel as assessed by a
double-chamber invasion assay (FIG. 31G), compared to the control
siRNA.
H. EphB4 siRNA Induces Cell Cycle Arrest and Apoptosis in PC3
Cells
[0284] Since knock-down of EphB4 resulted in decreased cell
viability (FIG. 31C) we sought to determine whether this was due to
effects on the cell cycle. In comparison to control siRNA
transfected cells, siRNA 472 resulted in an accumulation of cells
in the sub G0 and S phase fractions compared to cells treated with
control siRNA. The sub G0 fraction increased from 1% to 7.9%, and
the S phase fraction from 14.9% to 20.8% in siRNA 472 treated cells
compared to control siRNA treated cells (FIG. 32A). Cell cycle
arrest at sub G0 and G2 is indicative of apoptosis. Apoptosis as a
result of EphB4 knock-down was confirmed by ELISA assay. A
dose-dependent increase in apoptosis was observed when PC3 cells
were transfected with siRNA 472, but not with control siRNA (FIG.
32B). At 100 nM there was 15 times more apoptosis in siRNA 472
transfected than control siRNA transfected PC3 cells.
I. Materials and Methods
[0285] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
Example 4
Expression of EPHB4 in Mesothelioma: a Candidate Target for
Therapy
[0286] Malignant mesothelioma (MM) is a rare neoplasm that most
often arises from the pleural and peritoneal cavity serous surface.
The pleural cavity is by far the most frequent site affected
(>90%), followed by the peritoneum (6-10%) (Carbone et al.,
2002, Semin Oncol. 29:2-17). There is a strong association with
asbestos exposure, about 80% of malignant mesothelioma cases occur
in individuals who have ingested or inhaled asbestos. This tumor is
particularly resistant to the current therapies and, up to now, the
prognosis of these patients is dramatically poor (Lee et al., 2000,
Curr Opin Pulm Med. 6:267-74).
[0287] Several clinical problems regarding the diagnosis and
treatment of malignant mesothelioma remain unsolved. Making a
diagnosis of mesothelioma from pleural or abdominal fluid is
notoriously difficult and often requires a thoracoscopic or
laproscopic or open biopsy and Immunohistochemical staining for
certain markers such as meosthelin expressed preferentially in this
tumor. Until now, no intervention has proven to be curative,
despite aggressive chemotherapeutic regimens and prolonged
radiotherapy. The median survival in most cases is only 12-18
months after diagnosis.
[0288] In order to identify new diagnostic markers and targets to
be used for novel diagnostic and therapeutic approaches, we
assessed the expression of EPHB4 and its ligand EphrinB2 in
mesothelioma cell lines and clinical samples.
A. EPHB4 and EphrinB2 is Expressed in Mesothelioma Cell Lines
[0289] The expression of Ephrin B2 and EphB4 in malignant
mesothelioma cell lines was determined at the RNA and protein level
by a variety of methods. RT-PCR showed that all of the four cell
lines express EphrinB2 and EPHB4 (FIG. 33A). Protein expression was
determined by Western blot in these cell lines. Specific bands for
EphB4 were seen at 120 kD. In addition, Ephrin B2 was detected in
all cell lines tested as a 37 kD band on Western blot (FIG. 33B).
No specific band for Ephrin B2 was observed in 293 human embryonic
kidney cells, which were included as a negative control.
[0290] To confirm the presence of EphB4 transcription in
mesothelioma cells, in situ hybridization was carried out on NCI
H28 cell lines cultured on chamber slides. Specific signal for
EphB4 was detected using antisense probe Ephrin B2 transcripts were
also detected in the same cell line. Sense probes for both EphB4
and Ephrin B2 served as negative controls and did not hybridize to
the cells (FIG. 34). Expression of EphB4 and Ephrin B2 proteins was
confirmed in the cell lines by immunofluorescence analysis (FIG.
35). Three cell lines showed strong expression of EphB4, whereas
expression of Ephrin B2 was present in H28 and H2052, and weakly
detectable in H2373.
B. Evidence of Expression of EPHB4 and EphrinB2 in Clinical
Samples
[0291] Tumor cells cultured from the pleural effusion of a patient
diagnosed with pleural malignant mesothelioma were isolated and
showed positive staining for both EphB4 and Ephrin B2 at passage 1
(FIG. 35, bottom row). These results confirm co-expression of EphB4
and Ephrin B2 in mesothelioma cell lines. To determine whether
these results seen in tumor cell lines were a real reflection of
expression in the disease state, tumor biopsy samples were
subjected to immunohistochemical staining for EphB4 and Ephrin B2.
Antibodies to both proteins revealed positive stain in the tumor
cells. Representative data is shown in FIG. 36.
C. EPHB4 is Involved in the Cell Growth and Migration of
Mesothelioma
[0292] The role of EphB4 in cell proliferation was tested using
EPHB4 specific antisepses oligonucleotides and siRNA. The treatment
of cultured H28 with EPHB4 antisense reduced cell viability. One of
the most active inhibitor of EphB4 expression is EPHB4AS-10 (FIG.
37A). Transfection of EPHB4 siRNA 472 generated the same effect
(FIG. 37B).
[0293] MM is a locally advancing disease with frequent extension
and growth into adjacent vital structures such as the chest wall,
heart, and esophagus. In an effort to study this process in vitro,
we perform wound healing assay using previously described
techniques (3:36). When a wound was introduced into sub confluent
H28 cells, over the course of the next 28 hours cells would
progressively migrate into the area of the wound. However, when
cells were pretreated with EPHB4AS-10 for 24 hours, and the wound
was introduced, this migration was virtually completely prevented
(FIG. 38A). The migration study with Boyden Chamber assay with
EPHB4 siRNA showed that cell migration was greatly inhibited with
the inhibition of EPHB4 expression (FIG. 38B).
[0294] D. Materials and Methods
[0295] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
Example 5
EphB4 is Expressed in Squamous Cell Carcinoma of the Head and Neck:
Regulation by Epidermal Growth Factor Signaling Pathway and Growth
Advantage
[0296] Squamous cell carcinoma of the head and neck (HNSCC) is the
sixth most frequent cancer worldwide, with estimated 900,000 cases
diagnosed each year. It comprises almost 50% of all malignancies in
some developing nations. In the United States, 50,000 new cases and
8,000 deaths are reported each year. Tobacco carcinogens are
believed to be the primary etiologic agents of the disease, with
alcohol consumption, age, gender, and ethnic background as
contributing factors.
[0297] The differences between normal epithelium of the upper
aerodigestive tract and cancer cells arising from that tissue are
the result of mutations in specific genes and alteration of their
expression. These genes control DNA repair, proliferation,
immortalization, apoptosis, invasion, and angiogenesis. For head
and neck cancer, alterations of three signaling pathways occur with
sufficient frequency and produce such dramatic phenotypic changes
as to be considered the critical transforming events of the
disease. These changes include mutation of the p53 tumor
suppressor, overexpression of epidermal growth factor receptor
(EGFR), and inactivation of the cyclin dependent kinase inhibitor
p16. Other changes such as Rb mutation, ras activation, cyclin D
amplification, and myc overexpression are less frequent in
HNSCC.
[0298] Although high expression of EphB4 has been reported in
hematologic malignancies, breast carcinoma, endometrial carcinoma,
and colon carcinoma, there is limited data on the protein levels of
EphB4, and complete lack of data on the biological significance of
this protein in tumor biology such as HNSCC.
A. HNSCC Tumors Express EphB4
[0299] We studied the expression of EphB4 in human tumor tissues by
immunohistochemistry, in situ hybridization, and Western blot.
Twenty prospectively collected tumor tissues following IRB approval
have been evaluated with specific EphB4 monoclonal antibody that
does not react with other members of the EphB and EphA family.
EphB4 expression is observed in all cases, with varying intensity
of staining. FIG. 39A (top left) illustrates a representative case,
showing that EphB4 is expressed in the tumor regions only, as
revealed by the H&E tumor architecture (FIG. 39A bottom left).
Note the absence of staining for EphB4 in the stroma. Secondly, a
metastatic tumor site in the lymph node shows positive staining
while the remainder of the lymph node is negative (FIG. 39A, top
right).
[0300] In situ hybridization was carried out to determine the
presence and location of EphB4 transcripts in the tumor tissue.
Strong signal for EphB4 specific antisense probe was detected
indicating the presence of transcripts (FIG. 39B, top left).
Comparison with the H&E stain (FIG. 39B, bottom left) to
illustrate tumor architecture reveals that the signal was localized
to the tumor cells, and was absent from the stromal areas. Ephrin
B2 transcripts were also detected in tumor sample, and as with
EphB4, the signal was localized to the tumor cells (FIG. 39B, top
right). Neither EphB4 nor ephrin B2 sense probes hybridized to the
sections, proving specificity of the signals.
B. High expression of EphB4 in Primary and Metastatic Sites of
HNSCC
[0301] Western blots of tissue from primary tumor, lymph node
metastases and uninvolved tissue were carried out to determine the
relative levels of EphB4 expression in these sites. Tumor and
normal adjacent tissues were collected on 20 cases, while lymph
nodes positive for tumor were harvested in 9 of these 20 cases.
Representative cases are shown in FIG. 39C. EphB4 expression is
observed in each of the tumor samples. Similarly, all tumor
positive lymph nodes show EphB4 expression that was equal to or
greater than the primary tumor. No or minimal expression is
observed in the normal adjacent tissue.
C. EphB4 Expression and Regulation by EGFR Activity in HNSCC Cell
Lines
[0302] Having demonstrated the expression of EphB4 limited to tumor
cells, we next sought to determine whether there was an in vitro
model of EphB4 expression in HNSCC. Six HN SCC cell lines were
surveyed for EphB4 protein expression by Western Blot (FIG. 40A). A
majority of these showed strong EphB4 expression and thus
established the basis for subsequent studies. Since EGFR is
strongly implicated in HNSCC we asked whether EphB4 expression is
associated with the activation of EGFR. Pilot experiments in
SCC-15, which is an EGFR positive cell line, established an optimal
time of 24 h and concentration of 1 mM of the specific EGFR kinase
inhibitor AG 1478 (FIG. 40B) to inhibit expression of EphB4. When
all the cell lines were studied, we noted robust EGFR expression in
all but SCC-4, where it is detectable but not strong (FIG. 40C, top
row). In response to EGFR inhibitor AG1478 marked loss in the total
amount of EphB4 was observed in certain cell lines (SCC-15, and
SCC-25) while no effect was observed in others (SCC-9, -12, -13 and
-71). Thus SCC-15 and -25 serve as models for EphB4 being regulated
by EGFR activity, while SCC-9, -12, -13 and -71 are models for
regulation of EphB4 in HNSCC independent of EGFR activity, where
there may be input from other factors such as p53, PTEN, IL-6 etc.
We also noted expression of the ligand of EphB4, namely ephrin B2,
in all of the cell lines tested. As with EphB4 in some lines ephrin
B2 expression appears regulated by EGFR activity, while it is
independent in other cell lines.
[0303] Clearly, inhibition of constitutive EGFR signaling repressed
EphB4 levels in SCC 15 cells. We next studied whether EGF could
induce EphB4. We found that EphB4 levels were induced in SCC15
cells that had been serum starved for 24 h prior to 24 h treatment
with 10 ng/ml EGF as shown in FIG. 41B (lanes 1 and 2). The
downstream signaling pathways known for EGFR activation shown in
FIG. 41A, (for review see Yarden & Slikowski 2001) were then
investigated for their input into EGF mediated induction of EphB4.
Blocking PLCg, AKT and JNK phosphorylation with the specific kinase
inhibitors U73122, SH-5 and SP600125 respectively reduced basal
levels and blocked EGF stimulated induction of EphB4 (FIG. 41B,
lanes 3-8). In contrast, inhibition of ERK1/2 with PD098095 and
P13-K with LY294002 or Wortmannin had no discernible effect on EGF
induction of EphB4 levels. However, basal levels of EphB4 were
reduced when ERK1/2 phosphorylation was inhibited. Interestingly,
inhibition of p38 MAPK activation with SB203580 increased basal,
but not EGF induced EphB4 levels. Similar results were seen in the
SCC25 cell line (data not shown).
D. Inhibition of EphB4 in High Expressing Cell Lines Results in
Reduced Viability and Causes Cell-Cycle Arrest
[0304] We next turned to the role of EphB4 expression in HNSCC by
investigating the effect of ablating expression using siRNA or
AS-ODN methods. Several siRNAs to EphB4 sequence were developed
(Table 1) which knocked-down EphB4 expression to varying degrees as
seen in FIG. 42A. Viability was reduced in SCC-15, -25 and -71 cell
lines transfected with siRNAs 50 and 472, which were most effective
in blocking EphB4 expression (FIG. 42B). Little effect on viability
was seen with EphB4 siRNA 1562 and 2302 or ephrin B2 siRNA 254.
Note that in SCC-4, which does not express EphB4 (see FIG. 40A)
there was no reduction in cell viability. The decreased cell
viability seen with siRNA 50 and 472 treatment was attributable to
accumulation of cells in sub G0, indicative of apoptosis. This
effect was both time and dose-dependant (FIG. 42C and Table 2). In
contrast, siRNA2302 that was not effective in reducing EphB4 levels
and had only minor effects on viability did not produce any changes
in the cell cycle when compared with the mock Lipofectamine.TM.2000
transfection.
[0305] A detailed description of the siRNA constructs for this
example may be found in U.S. Patent Publication No. 20050084873.
TABLE-US-00007 TABLE Effect of different EphB4 siRNA on Cell Cycle
Treatment Sub G0 G1 S G2 36 hr Lipo alone 1.9 39.7 21.3 31.8 100 nM
2302 2.0 39.3 21.2 31.2 100 nM 50 18.1 31.7 19.7 24.4 100 nM 472
80.2 10.9 5.2 2.1 16 hr Lipo alone 7.8 55.7 15.2 18.5 100 nM 2302
8.4 57.3 14.3 17.3 10 nM 50 10.4 53.2 15.7 17.7 100 nM 50 27.7 31.3
18.1 19.6 10 nM 472 13.3 50.2 15.8 17.5 100 nM 472 30.7 31.9 16.4
18.0
[0306] In addition, over 50 phosphorothioate AS-ODNs complementary
to the human EphB4 coding sequences were synthesized and tested for
their ability to inhibit EphB4 expression in 293 cells transiently
transfected with full length EphB4 expression plasmid. FIG. 43A
shows a representative sample of the effect of some of these
AS-ODNs on EphB4 expression. Note that expression is totally
abrogated with AS-10, while AS-11 has only a minor effect. The
effect on cell viability in SCC15 cells was most marked with
AS-ODNs that are most effective in inhibiting EphB4 expression as
shown in FIG. 43B. The IC.sub.50 for AS-10 was approximately 1
.mu.M, while even 10 .mu.M AS-11 was not sufficient to attain 50%
reduction of viability. When the effect that AS-10 had on the cell
cycle was investigated, it was found that the sub G0 fraction
increased from 1.9% to 10.5% compared to non-treated cells,
indicative of apoptosis (FIG. 43C).
E. EphB4 Regulates Cell Migration
[0307] We next wished to determine if EphB4 participates in the
migration of HNSCC. Involvement in migration may have implications
for growth and metastasis. Migration was assessed using the
wound-healing/scrape assay. Confluent SCC 15 and SCC25 cultures
were wounded by a single scrape with a sterile plastic Pasteur
pipette, which left a 3 mm band with clearly defined borders.
Migration of cells into the cleared area in the presence of test
compounds was evaluated and quantitated after 24, 48 and 72 hr.
Cell migration was markedly diminished in response to AS-10 that
block EphB4 expression while the inactive compounds, AS-1 and
scrambled ODN had little to no effect as shown in FIG. 43D.
Inhibition of migration with AS-10 was also shown using the Boyden
double chamber assay (FIG. 43E).
F. EphB4 AS-10 In Vivo Anti-Tumor Activity
[0308] The effect of EphB4 AS-10, which reduces cell viability and
motility, was determined in SCC15 tumor xenografts in Balb/C nude
mice. Daily treatment of mice with 20 mg/kg AS-10, sense ODN or
equal volume of PBS by I.P. injection was started the day following
tumor cell implantation. Growth of tumors in mice receiving AS-10
was significantly retarded compared to mice receiving either sense
ODN or PBS diluent alone (FIG. 44). Non-specific effects
attributable to ODN were not observed, as there was no difference
between the sense ODN treated and PBS treated groups.
G. Materials and Methods
[0309] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
Example 6
Ephrin B2 Expression in Kaposi's Sarcoma is Induced by Human
Herpesvirus Type 8: Phenotype Switch from Venous to Arterial
Endothelium
[0310] Kaposi's Sarcoma (KS) manifests as a multifocal
angioproliferative disease, most commonly of the skin and mucus
membranes, with subsequent spread to visceral organs (1) Hallmarks
of the disease are angiogenesis, edema, infiltration of
lymphomononuclear cells and growth of spindle-shaped tumor cells.
Pathologically, established lesions exhibit an extensive vascular
network of slit-like spaces. The KS vascular network is distinct
from normal vessels in the lack of basement membranes and the
abnormal spindle shaped endothelial cell (tumor cell) lining these
vessels. Defective vasculature results in an accumulation of the
blood components including albumin, red and mononuclear cells in
the lesions (1). The KS tumor is endothelial in origin; the tumor
cells express many endothelial markers, including lectin binding
sites for Ulex europeaus agglutinin-1 (UEA-1), CD34, EN-4, PAL-E
(2) and the endothelial cell specific tyrosine kinase receptors,
VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR), VEGFR-3 (Flt-4), Tie-1 and
Tie-2 (3, RM & PSG unpublished data). KS cells co-express
lymphatic endothelial cell related proteins including LYVE and
podoplanin (4).
[0311] The herpesvirus HHV-8 is considered the etiologic agent for
the disease. In 1994 sequences of this new herpes virus were
identified in KS tumor tissue (5), and subsequent
molecular-epidemiology studies have shown that nearly all KS tumors
contain viral genome. Sero-epidemiology studies show that HIV
infected patients with KS have the highest prevalence of HHV-8 and
secondly that those with HIV infection but no KS have increased
risk of developement of KS over the ensuing years if they are also
seropositive for HHV-8 (6). Direct evidence for the role of HHV-8
in KS is the transformation of bone marrow endothelial cells after
infection with HHV-8 (7). A number of HHV-8 encoded genes could
contribute to cellular transformation (reviewed in 8). However, the
most evidence has accumulated for the G-protein coupled receptor
(vGPCR) in this role (9).
[0312] We investigated whether KS tumor cells are derived from
arterial or venous endothelium. In addition, we investigated
whether HHV-8 has an effect on expression of arterial or venous
markers in a model of KS. KS tumor cells were found to express the
ephrin B2 arterial marker. Further, ephrin B2 expression was
induced by HHV-8 vGPCR in KS and endothelial cell lines. Ephrin B2
is a potential target for treatment of KS because inhibition of
ephrin B2 expression or signaling was detrimental to KS cell
viability and function.
A. KS Tumors Express Ephrin B2, but not EphB4
[0313] The highly vascular nature of KS lesions and the probable
endothelial cell origin of the tumor cells prompted investigation
of expression of EphB4 and ephrin B2 which are markers for venous
and arterial endothelial cells, respectively. Ephrin B2, but not
EphB4 transcripts were detected in tumor cells of KS biopsies by in
situ hybridization (FIG. 45A). Comparison of the positive signal
with ephrin B2 antisense probe and tumor cells as shown by H&E
staining shows that ephrin B2 expression is limited to the areas of
the biopsy that contain tumor cells. The lack of signal in KS with
EphB4 antisense probe is not due to a defect in the probe, as it
detected transcripts in squamous cell carcinoma, which we have
shown expresses this protein (18). Additional evidence for the
expression of ephrin B2 in KS tumor tissue is afforded by the
localization of EphB4/Fc signal to tumor cells, detected by FITC
conjugated anti human Fc antibody. Because ephrin B2 is the only
ligand for EphB4 this reagent is specific for the expression of
ephrin B2 (FIG. 45B, left). An adjacent section treated only with
the secondary reagent shows no specific signal. Two-color confocal
microscopy demonstrated the presence of the HHV-8 latency protein,
LANA1 in the ephrin B2 positive cells (FIG. 45C, left), indicating
that it is the tumor cells, not tumor vessels, which are expressing
this arterial marker. Staining of tumor biopsy with PECAM-1
antibody revealed the highly vascular nature of this tumor (FIG.
45C, right). A pilot study of the prevalence of this pattern of
ephrin B2 and EphB4 expression on KS biopsies was conducted by
RT-PCR analysis. All six samples were positive for ephrin B2, while
only 2 were weakly positive for EphB4 (data not shown).
B. Infection of Venous Endothelial Cells with HHV-8 Causes a
Phenotype Switch to Arterial Markers
[0314] We next asked whether HHV-8, the presumed etiologic agent
for KS, could itself induce expression of ephrin B2 and repress
EphB4 expression in endothelial cells. Co-culture of HUVEC and BC-1
lymphoma cells, which are productively infected with HHV-8, results
in effective infection of the endothelial cells (16). The attached
monolayers of endothelial cells remaining after extensive washing
were examined for ephrin B2 and EphB4 by RT-PCR and
immunofluorescence. HUVEC express EphB4 venous marker strongly at
the RNA level, but not ephrin B2 (FIG. 46B). In contrast, HHV-8
infected cultures (HUVEC/BC-1 and HUVEC/BC-3) express ephrin B2,
while EphB4 transcripts are almost absent.
[0315] Immunofluorescence analysis of cultures of HUVEC and
HUVEC/HHV-8 for artery/vein markers and viral proteins was
undertaken to determine whether changes in protein expression
mirrored that seen in the RNA. In addition, cellular localization
of the proteins could be determined. Consistent with the RT-PCR
data HUVEC are ephrin B2 negative and EphB4 positive (FIG. 46A(a
& m)). As expected they do not express any HHV-8 latency
associated nuclear antigen (LANA1) (FIG. 46A(b, n)). Co-culture of
BC-1 cells, which are productively infected with HHV-8, resulted in
infection of HUVEC as shown by presence of viral proteins LANA1 and
ORF59 (FIG. 46A(f, r)). HHV-8 infected HUVEC now express ephrin B2
but not EphB4 (FIG. 46A(e, q, u), respectively). Expression of
ephrin B2 and LANA1 co-cluster as shown by yellow signal in the
merged image (FIG. 46A(h)). HHV-8 infected HUVEC positive for
ephrin B2 and negative for Eph B4 also express the arterial marker
CD148 (19) (FIG. 46A (j, v)). Expression of ephrin B2 and CD148
co-cluster as shown by yellow signal in the merged image (FIG.
46A(l)). Uninfected HUVEC expressing Eph B4 were negative for CD148
(not shown).
C. HHV-8 vGPCR Induces Ephrin B2 Expression
[0316] To test whether individual viral proteins could induce the
expression of ephrin B2 seen with the whole virus KS-SLK cells were
stably transfected with HHV-8 LANA, or LANA.DELTA.440 or vGPCR.
Western Blot of stable clones revealed a five-fold induction of
ephrin B2 in KS-SLK transfected with vGPCR compared to SLK-LANA or
SLK-LANA.DELTA.440 (FIG. 47A). SLK transfected with vector alone
(pCEFL) was used as a control. SLK-vGPCR and SLK-pCEFL cells were
also examined for ephrin B2 and Eph B4 expression by
immunofluorescence in transiently transfected KS-SLK cells. FIG.
47B shows higher expression of ephrin B2 in the SLK-vGPCR cells
compared to SLK-pCEFL. No changes in Eph B4 were observed in
SLK-vGPCR compared to SLK-pCEFL. This clearly demonstrates that
SLK-vGPCR cells expressed high levels of ephrin B2 compared to
SLK-pCEFL cells. This suggests that vGPCR of HHV-8 is directly
involved in the induction of Ephrin B2 and the arterial phenotype
switch in KS. Since we had shown that HHV-8 induced expression of
ephrin B2 in HUVEC, we next asked if this could be mediated by a
transcriptional effect. Ephrin B2 5'-flanking DNA-luciferase
reporter plasmids were constructed as described in the Materials
and Methods and transiently transfected into HUVECs. Ephrin
B25'-flanking DNA sequences -2491/-11 have minimal activity in
HUVEC cells (FIG. 47C). This is consistent with ephrin B2 being an
arterial, not venous marker. However, we have noted that HUVEC in
culture do express some ephrin B2 at the RNA level. Cotransfection
of HHV-8 vGPCR induces ephrin B2 transcription approximately
10-fold compared to the control expression vector pCEFL. Roughly
equal induction was seen with ephrin B2 sequences -2491/-1,
-1242/-11, or -577/-11, which indicates that elements between -577
and -11 are sufficient to mediate the response to vGPCR, although
maximal activity is seen with the -1242/-11 luciferase
construct.
D. Expression of Ephrin B2 is Regulated by VEGF and VEGF-C
[0317] We next asked whether known KS growth factors could be
involved in the vGPCR-mediated induction of ephrin B2 expression.
SLK-vGPCR cells were treated with neutralizing antibodies to
oncostatin-M, IL-6, IL-8, VEGF or VEGF-C for 36 hr. FIG. 48A shows
that neutralization of VEGF completely blocked expression of ephrin
B2 in SLK-vGPCR cells. A lesser, but significant decrease in ephrin
B2 was seen neutralization of VEGF-C and IL-8. No appreciable
effect was seen with neutralization of oncostatin-M or IL-6. To
verify that VEGF and VEGF-C are integral to the induction of ephrin
B2 expression we treated HUVEC with VEGF, VEGF-C or EGF. HUVECs
were grown in EBM-2 media containing 5% FBS with two different
concentration of individual growth factor (10 ng, 100 ng/ml) for 48
h. Only VEGF-A or VEGF-C induced ephrin B2 expression in a dose
dependent manner (FIG. 48B). In contrast, EGF had no effect on
expression of ephrin B2.
E. Ephrin B2 siRNA Inhibits the Expression of Ephrin B2 in KS
[0318] Three ephrin B2 siRNA were synthesized as described in the
methods section. KS-SLK cells were transfected with siRNA and 48 h
later ephrin B2 expression was determined by Western Blot. Ephrin
B2 siRNAs 137 or 254 inhibited about 70% of ephrin B2 expression
compared to control siRNA such as siRNA Eph B4 50 or siRNA GFP.
Ephrin B2 63 siRNA was less effective than the above two siRNA
Ephrin B2 (FIG. 49A).
F. Ephrin B2 is Necessary for Full KS and EC Viability, Cord
Formation and In Vivo Angiogenesis Activities
[0319] The most effective ephrin B2 siRNA (254) was then used to
determine whether inhibiting expression of ephrin B2 has any effect
on the growth of KS-SLK or HUVEC cells. The viability of KS-SLK
cells was decreased by the same siRNAs that inhibited ephrin B2
protein levels (FIG. 49B). KS-SLK express high levels of ephrin B2
and this result shows maintenance of ephrin B2 expression is
integral to cell viability in this setting. HUVECs do not express
ephrin B2, except when stimulated by VEGF as shown in FIG. 48B.
Ephrin B2 siRNA 264 dramatically reduced growth of HUVECs cultured
with VEGF as the sole growth factor. In contrast, no significant
effect was seen when HUVECs were cultured with IGF, EGF and bFGF.
As a control, EphB4 siRNA 50 had no detrimental effect on HUVECs in
either culture condition (FIG. 49C). In addition to inhibition of
viability of KS and primary endothelial cells, EphB4-ECD inhibits
cord formation in HUVEC and KS-SLK and in vivo angiogenesis in the
Matrigel.TM. plug assay (FIG. 50).
G. Methods and Materials
[0320] A detailed description of the materials and methods for this
example may be found in U.S. Patent Publication No.
20050084873.
Example 7
Expression of EphB4 in Bladder Cancer: a Candidate Target for
Therapy
[0321] FIG. 51 shows expression of EPHB4 in bladder cancer cell
lines (A), and regulation of EPHB4 expression by EGFR signaling
pathway (B).
[0322] FIG. 52 shows that transfection of p53 inhibit the
expression of EPHB4 in 5637 cell.
[0323] FIG. 53 shows growth inhibition of bladder cancer cell line
(5637) upon treatment with EPHB4 siRNA 472.
[0324] FIG. 54 shows results on apoptosis study of 5637 cells
transfected with EPHB4 siRNA 472.
[0325] FIG. 55 shows effects of EPHB4 antisense probes on cell
migration. 5637 cells were treated with EPHB4AS10 (10 .mu.M).
[0326] FIG. 56 shows effects of EPHB4 siRNA on cell invasion. 5637
cells were transfected with siRNA 472 or control siRNA.
Example 8
Inhibition of EphB4 Gene Expression by EphB4 Antisense Probes and
RNAi Probes
[0327] Cell lines expressing EphB4 were treated with the synthetic
phosphorothioate modified oligonucleotides and harvested after 24
hr. Cell lysates were prepared and probed by western blot analysis
for relative amounts of EphB4 compared to untreated control
cells.
[0328] Studies on inhibition of cell proliferation were done in
HNSCC cell lines characterized to express EphB4. Loss of cell
viability was shown upon knock-down of EphB4 expression. Cells were
treated in vitro and cultured in 48-well plates, seeded with 10
thousand cells per well. Test compounds were added and the cell
viability was tested on day 3. The results on EphB4 antisense
probes were summarized below in Table 6. The results on EphB4 RNAi
probes were summarized below in Table 7.
[0329] A detailed description of the antisense and siRNA constructs
for this example may be found in U.S. Patent Publication No.
20050084873.
Example 9
Inhibition of Ephrin B2 Gene Expression by Ephrin B2 Antisense
Probes and RNAi Probes
[0330] KS SLK, a cell line expressing endogenous high level of
ephrin B2. Cell viability was tested using fixed dose of each
oligonuceotide (5 uM). Gene expression downregulation was done
using cell line 293 engineered to stably express full-length ephrin
B2. KS SLK expressing EphrinB2 were also used to test the viability
in response to RNAi probes tested at the fixed dose of 50 nM.
Protein expression levels were measured using 293 cells stably
expressing full-length EphrinB2, in cell lysates after 24 hr
treatment with fixed 50 nM of RNAi probes.
[0331] The results on Ephrin B2 antisense probes were summarized
below in Table 8. The results on Ephrin B2 RNAi probes were
summarized below in Table 9.
[0332] A detailed description of the antisense and siRNA constructs
for this example may be found in U.S. Patent Publication No.
20050084873.
Example 10
EphB4 Antibodies Inhibit Tumor Growth
[0333] FIG. 57 shows results on comparison of EphB4 monoclonal
antibodies by G250 and in Pull-down assay.
[0334] FIG. 58 shows that EphB4 antibodies, in the presence of
matrigel and growth factors, inhibit the in vivo tumor growth of
SCC15 cells.
[0335] BaIbC nude mice were injected subcutaneously with
2.5.times.10.sup.6 viable tumor cells SCC15 is a head and neck
squamous cell carcinoma line. Tumors were initiated in nu/nu mice
by injecting 2.5-5.times.10.sup.6 cells premixed with matrigel and
Growth factors, and Ab's subcutaneously to initiate tumor
xenografts. Mice were opened 14 days after injections. SCC15 is a
head and neck squamous cell carcinoma line, B16 is a melanoma cell
line, and MCF-7 is a breast carcinoma line. The responses of tumors
to these treatments were compared to control treated mice, which
receive PBS injections. Animals were observed daily for tumor
growth and subcutaneous tumors were measured using a caliper every
2 days. Antibodies #1 and #23 showed significant regression of
SCC15 tumor size compared to control, especially with no additional
growth factor added.
[0336] FIG. 59 shows that EphB4 antibodies cause apoptosis,
necrosis and decreased angiogenesis in SCC15, head and neck
carcinoma tumor type.
[0337] Angiogenesis was assessed by CD-31 immunohistochemistry.
Tumor tissue sections from treated and untreated mice were stained
for CD31. Apoptosis was assessed by immunohistochemical TUNNEL, and
proliferation by BrdU assay. Following surgical removal, tumors
were immediately sliced into 2 mm serial sections and embedded in
paraffin using standard procedures. Paraffin embedded tissue were
sectioned at 5 .mu.m, the wax removed and the tissue rehydrated.
The rehydrated tissues were microwave irradiated in antigen
retreival solution. Slides were rinsed in PBS, and TUNNEL reaction
mixture (Terminal deoxynucleotidyl transferase and flourescein
labeled nucleotide solution), and BrdU were added in a humidity
chamber completely shielded from light. The TUNNEL and BrdU
reaction mixture were then removed, slides were rinsed and
anti-flourescein antibody conjugated with horseradish peroxidase
was added. After incubation and rinsing, 3, 3'diaminobenzidine was
added. Masson's Trichrome and Hematoxylin and Eosin were also used
to stain the slides to visualize morphology. Masson's Trichrome
allows to visualize necrosis and fibrosis. The tumor gets blood
support from tumor/skin, muscle boundary. As tumor grows, inner
regions get depleted of nutrients. This leads to necrosis (cell
death), preferably at the tumor center. After cells die, (tumor)
tissue gets replaced with fibroblastic tissue. Slides were
visualized under 20-fold magnification with digital images
acquired. A different morphology was obtained on SCC tumors with
each antibody administered. Ab #1 showed an increase in necrosis
and fibrosis but not apoptosis. Ab #23 showed an increase in
apoptosis, necrosis and fibrosis and a decrease in vessel
infiltration. Ab #35 showed an increase in necrosis and fibrosis,
and a small increase in apoptosis and a decrease in vessel
infiltration. Ab #79 showed a large increase in apoptosis, and
necrossis and fibrosis. Ab #91 showed no change in apoptosis but an
increase in proliferation. And Ab #138 showed an increase in
apoptosis, necrosis, fibrosis and a decrease in proliferation and
vessel infiltration. Tumors treated with control PBS displayed
abundant tumor density and a robust angiogenic response. Tumors
treated with EphB4 antibodies displayed a decrease in tumor cell
density and a marked inhibition of tumor angiogenesis in regions
with viable tumor cells, as well as tumor necrosis and
apoptosis.
[0338] FIG. 60 shows that systemic administration of antibodies on
xenografts leads to tumor regression in SCC15 tumor xenografts.
[0339] Alternate day treatment with EphB4 monoclonal antibody or an
equal volume of PBS as control were initiated on day 4, after the
tumors have established, and continued for 14 days. Systemic
administration was administered either IP or SC with no significant
difference. All the experiments were carried out in a double-blind
manner to eliminate investigator bias. Mice were sacrificed at the
conclusion of the two week treatment period. Tumors were harvested
immediately postmortem and fixed and processed for
immunohistochemistry. EphB4 antibodies 40 mg per kg body weight
were administered. Treatment with EphB4 antibody significantly
inhibited human SCC tumor growth compared with control-treated mice
(p<0.05). Treatment with EphB4 antibody significantly inhibited
tumor weight compared with control-treated mice (p<0.05).
Example 11
HSA-EphB4 Ectodomain Fusion and PEG-Modified EphB4 Ectodomain
A. Generation of HSA-EphB4 Ectodomain Fusion
[0340] Human serum albumin fragment in XbaI-NotI form was
PCR-amplified out for creating a fusion with GCF2, and TA-cloned
into pEF6. In the next step, the resulting vector was cut with Xba
I (partial digestion) and the HSA fragment (1.8 kb) was cloned into
Xba I site of pEF6-GCF2-Xba to create fusion expression vector. The
resulting vector had a point mutation C to T leading to Thr to Ile
substitution in position 4 of the mature protein. It was called
pEF6-GCF2-HSAmut. In the next cloning step, the mutation was
removed by substituting wild type KpnI fragment from pEF6-GCF2-IF
(containing piece of the vector and N-terminal part of GCF2) for
the mutated one, this final vector was called pEF6-GCF2. The DNA
sequence of pEF6-GCF2 was confirmed.
[0341] The predicted amino acid of the HSA-EphB4 precursor protein
was as follows (SEQ ID NO:18): TABLE-US-00008
MELRVLLCWASLAAALEETLLNTKLETADLKWVTFPQVDGQWEELSGLDE
EQHSVRTYEVCDVQRAPGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSL
PRAGRSCKETFTVFYYESDADTATALTPAWMENPYIKVDTVAAEHLTRKR
PGAEATGKVNVKTLRLGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQL
TVNLTRFPETVPRELVVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPV
TGCSCAPGFEAAEGNTKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSA
VCQCRVGYFRARTDPRGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESG
GREDLTYALRCRECRPGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDF
TYTFEVTALNGVSSLATGPVPFEPVNVTTDREVPPAVSDIRVTRSSPSSL
SLAWAVPRAPSGAVLDYEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRG
ASYLVQVRARSEAGYGPFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRF
KDLGEENEKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAEN
CDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPN
LPRIVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRY
KAAFTECCQAADKAACLLPKIDELRDEGKASSAKQRLKCASLQKFGERAF
KAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLA
KYICENQDSISSKCCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKD
VCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAA
DPHECYAKVFDEFKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKK
VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLH
EKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADIC
TLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDK
ETCFAEEGKKLVAASQAALGL
[0342] The predicted amino acid sequence of the mature form of the
HSA-EphB4 protein was as follows (SEQ ID NO:19): TABLE-US-00009
LEETLLNTKLETADLKWVTFPQVDGQWEELSGLDEEQHSVRTYEVCDVQR
APGQAHWLRTGWVPRRGAVHVYATLRFTMLECLSLPRAGRSCKETFTVFY
YESDADTATALTPAWMENPYIKVDTVAAEHLTRKRPGAEATGKVNVKTLR
LGPLSKAGFYLAFQDQGACMALLSLHLFYKKCAQLTVNLTRFPETVPREL
VVPVAGSCVVDAVPAPGPSPSLYCREDGQWAEQPVTGCSCAPGFEAAEGN
TKCRACAQGTFKPLSGEGSCQPCPANSHSNTIGSAVCQCRVGYFRARTDP
RGAPCTTPPSAPRSVVSRLNGSSLHLEWSAPLESGGREDLTYALRCRECR
PGGSCAPCGGDLTFDPGPRDLVEPWVVVRGLRPDFTYTFEVTALNGVSSL
ATGPVPFEPVNVTTDREVPPAVSDWVTRSSPSSLSLAWAVPRAPSGAVLD
YEVKYHEKGAEGPSSVRFLKTSENRAELRGLKRGASYLVQVRARSEAGYG
PFGQEHHSQTQLDESEGWREQSRDAHKSEVAHRFKDLGEENFKALVLIAF
AQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTV
ATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAF
HDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAAC
LLPKLDELRDEGKASSAKQRLKCASLQKPGERAFKAWAVARLSQRFPKAE
FAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLEC
CEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLG
MFLYEYARRHPDYSVVLLLRLAKTYETFITLEKCCAAADPHECYAKVFDE
FKPLVEEPQNLIKQNCELFKQLGEYKFQNALLVRYTKKVPQVSTPTLVEV
SRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKC
CTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQ
TALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLV AASQAALGL
[0343] The nucleic acid sequence of the pEF6-GCF2 plasmid was as
follows (SEQ ID NO: 20): TABLE-US-00010
aatattattgaagcatttatcagggttattgtctcatgagcggatacata
tttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttcc
ccgaaaagtgccacctgacgtcgacggatcgggagatctcccgatcccct
atggtcgactctcagtacaatctgctctgatgccgcatagttaagccagt
atctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaa
atttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatct
gcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatata
cgcgttgacattgattattgactaggcttttgcaaaaagctttgcaaaga
tggataaagttttaaacagagaggaatctttgcagctaatggaccttcta
ggtcttgaaaggagtgcctcgtgaggctccggtgcccgtcagtgggcaga
gcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaatt
gaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtc
gtgtactggctccgcctttttcccgagggtgggggagaaccgtatataag
tgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaa
cacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggt
tatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgat
tcttgatcccgagcttcgggttggaagtgggtgggagagttcgaggcctt
gcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctggg
cgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgc
tgctttcgataagtctctagccatttaaaatttttgatgacctgctgcga
cgctttttttctggcaagatagtcttgtaaatgcgggccaagatctgcac
actggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtc
ccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaa
tcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctc
gcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggc
accagttgcgtgagcggaaagatggccgcttcccggccctgctgcaggga
gctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcaccc
acacaaaggaaaagggccrttccgtcctcagccgtcgcttcatgtgactc
cacggagtaccgggcgccgtccaggcacctcgattagttctcgagctttt
ggagtacgtcgtctttaggttggggggaggggttttatgcgatggagttt
ccccacactgagtgggtggagactgaagttaggccagcttggcacttgat
gtaattctccttggaatttgccctttttgagtttggatcttggttcattc
tcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcg
tgaggaattagcttggtactaatacgactcactatagggagacccaagct
ggctaggtaagcttggtaccgagctcggatccactagtccagtgtggtgg
aattgcccttCAAGCTTGCCGCCACCATGGAGCTCCGGGTGCTGCTCTGC
TGGGCTTCGTTGGCCGCAGCTTTGGAAGAGACCCTGCTGAACACAAAATT
GGAAACTGCTGATCTGAAGTGGGTGACATTCCCTCAGGTGGACGGGCAGT
GGGAGGAACTGAGCGGCCTGGATGAGGAACAGCACAGCGTGCGCACCTAC
GAAGTGTGTGACGTGCAGCGTGCCCCGGGCCAGGCCCACTGGCTTCGCAC
AGGTTGGGTCCCACGGCGGGGCGCCGTCCACGTGTACGCCACGCTGCGCT
TCACCATGCTCGAGTGCCTGTCCCTGCCTCGGGCTGGGCGCTCCTGCAAG
GAGACCTTCACCGTCTTCTACTATGAGAGCGATGCGGACACGGCCACGGC
CCTCACGCCAGCCTGGATGGAGAACCCCTACATCAAGGTGGACACGGTGG
CCGCGGAGCATCTCACCCGGAAGCGCCCTGGGGCCGAGGCCACCGGGAAG
GTGAATGTCAAGACGCTGCGCCTGGGACCGCTCAGCAAGGCTGGCTTCTA
CCTGGCCTTCCAGGACCAGGGTGCCTGCATGGCCCTGCTATCCCTGCACC
TCTTCTACAAAAAGTGCGCCCAGCTGACTGTGAACCTGACTCGATTCCCG
GAGACTGTGCCTCGGGAGCTGGTTGTGCCCGTGGCCGGTAGCTGCGTGGT
GGATGCCGTCCCCGCCCCTGGCCCCAGCCCCAGCCTCTACTGCCGTGAGG
ATGGCCAGTGGGCCGAACAGCCGGTCACGGGCTGCAGCTGTGCTCCGGGG
TTCGAGGCAGCTGAGGGGAACACCAAGTGCCGAGCCTGTGCCCAGGGCAC
CTTCAAGCCCCTGTCAGGAGAAGGGTCCTGCCAGCCATGCCCAGCCAATA
GCCACTCTAACACCATTGGATCAGCCGTCTGCCAGTGCCGCGTCGGGTAC
TTCCGGGCACGCACAGACCCCCGGGGTGCACCCTGCACCACCCCTCCTTC
GGCTCCGCGGAGCGTGGTTTCCCGCCTGAACGGCTCCTCCCTGCACCTGG
AATGGAGTGCCCCCCTGGAGTCTGGTGGCCGAGAGGACCTCACCTACGCC
CTCCGCTGCCGGGAGTGTCGACCCGGAGGCTCCTGTGCGCCCTGCGGGGG
AGACCTGACTTTTGACCCCGGCCCCCGGGACCTGGTGGAGCCCTGGGTGG
TGGTTCGAGGGCTACGTCCTGACTTCACCTATACCTTTGAGGTCACTGCA
TTGAACGGGGTATCCTCCTTAGCCACGGGGCCCGTCCCATTTGAGCCTGT
CAATGTCACCACTGACCGAGAGGTACCTCCTGCAGTGTCTGACATCCGGG
TGACGCGGTCCTCACCCAGCAGCTTGAGCCTGGCCTGGGCTGTTCCCCGG
GCACCCAGTGGGGCTGTGCTGGACTACGAGGTCAAATACCATGAGAAGGG
CGCCGAGGGTCCCAGCAGCGTGCGGTTCCTGAAGACGTCAGAAAACCGGG
CAGAGCTGCGGGGGCTGAAGCGGGGAGCCAGCTACCTGGTGCAGGTACGG
GCGCGCTCTGAGGCCGGCTACGGGCCCTTCGGCCAGGAACATCACAGCCA
GACCCAACTGGATGAGAGCGAGGGCTGGCGGGAGCAGtctagaGATGCAC
ACAAGAGTGAGGTTGCTCATCGGTTTAAAGATTTGGGAGAAGAAAATTTC
AAAGCCTTGGTGTTGATTGCCTTTGCTCAGTATCTTCAGCAGTGTCCATT
TGAAGATCATGTAAAATTAGTGAATGAAGTAACTGAATTTGCAAAAACAT
GTGTAGCTGATGAGTCAGCTGAAAATTGTGACAAATCACTTCATACCCTT
TTTGGAGACAAATTATGCACAGTTGCAACTCTTCGTGAAACCTATGGTGA
AATGGCTGACTGCTGTGCAAAACAAGAACCTGAGAGAAATGAATGCTTCT
TGCAACACAAAGATGACAACCCAAACCTCCCCCGATTGGTGAGACCAGAG
GTTGATGTGATGTGCACTGCTTTTCATGACAATGAAGAGACATTTTTGAA
AAAATACTTATATGAAATTGCCAGAAGACATCCTTACTTTTATGCCCCGG
AACTCCTTTTCTTTGCTAAAAGGTATAAAGCTGCTTTTACAGAATGTTGC
CAAGCTGCTGATAAAGCTGCCTGCCTGTTGCCAAAGCTCGATGAACTTCG
GGATGAAGGGAAGGCTTCGTCTGCCAAACAGAGACTCAAATGTGCCAGTC
TCCAAAAATTTGGAGAAAGAGCTTTCAAAGCATGGGCAGTGGCTCGCCTG
AGCCAGAGATTTCCCAAAGCTGAGTTTGCAGAAGTTTCCAAGTTAGTGAC
AGATCTTACCAAAGTCCACACGGAATGCTGCCATGGAGATCTGCTTGAAT
GTGCTGATGACAGGGCGGACCTTGCCAAGTATATCTGTGAAAATCAGGAT
TCGATCTCCAGTAAACTGAAGGAATGCTGTGAAAAACCTCTGTTGGAAAA
ATCCCACTGCATTGCCGAAGTGGAAAATGATGAGATGCCTGCTGACTTGC
CTTCATTAGCTGCTGATTTTGTTGAAAGTAAGGATGTTTGCAAAAACTAT
GCTGAGGCAAAGGATGTCTTCCTGGGCATGTTTTTGTATGAATATGCAAG
AAGGCATCCTGATTACTCTGTCGTGCTGCTGCTGAGACTTGCCAAGACAT
ATGAAACCACTCTAGAGAAGTGCTGTGCCGCTGCAGATCCTCATGAATGC
TATGCCAAAGTGTTCGATGAATTTAAACCTCTTGTGGAAGAGCCTCAGAA
TTTAATCAAACAAAACTGTGAGCTTTTTAAGCAGCTTGGAGAGTACAAAT
TCCAGAATGCGCTATTAGTTCGTTACACCAAGAAAGTACCCCAAGTGTCA
ACTCCAACTCTTGTAGAGGTCTCAAGAAACCTAGGAAAAGTGGGCAGCAA
ATGTTGTAAACATCCTGAAGCAAAAAGAATGCCCTGTGCAGAAGACTATC
TATCCGTGGTCCTGAACCAGTTATGTGTGTTGCATGAGAAAACGCCAGTA
AGTGACAGAGTCACAAAATGCTGCACAGAGTCCTTGGTGAACAGGCGACC
ATGCTTTTCAGCTCTGGAAGTCGATGAAACATACGTTCCCAAAGAGTTTA
ATGCTGAAACATTCACCTTCCATGCAGATATATGCACACTTTCTGAGAAG
GAGAGACAAATCAAGAAACAAACTGCACTTGTTGAGCTTGTGAAACACAA
GCCCAAGGCAACAAAAGAGCAACTGAAAGCTGTTATGGATGATTTCGCAG
CTTTTGTAGAGAAGTGCTGCAAGGCTGACGATAAGGAGACCTGCTTTGCC
GAGGAGGGTAAAAAACTTGTTGCTGCAAGTCAAGCTGCCTTAGGCTTATA
Atagcggccgcttaagggcaattctgcagatatccagcacagtggcggcc
gctcgagtctagagggcccgcggttcgaaggtaagcctatccctaaccct
ctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattg
agtttaaacccgctgatcagcctcgactgtgccttctagttgccagccat
ctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccact
cccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgag
taggtgtcattctattctggggggtggggtggggcaggacagcaaggggg
aggattgggaagacaatagcaggcatgctggggatgcggtgggctctatg
gcttctgaggcggaaagaaccagctggggctctagggggtatccccacgc
gccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcg
tgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttc
ccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcg
gggcatccctttagggttccgatttagtgctttacggcacctcgacccca
aaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatag
acggtttttcgccctttgacgttggagtccacgttctttaatagtggact
cttgttccaaactggaacaacactcaaccctatctcggtctattcttttg
atttataagggattttggggatttcggcctattggttaaaaaatgagctg
atttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagtta
gggtgtggaaagtccccaggctccccaggcaggcagaagtatgcaaagca
tgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctcccca
gcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagt
cccgcccctaactccgcccatcccgcccctaactccgcccagttccgccc
attctccgccccatggctgactaattttttttatttatgcagaggccgag
gccgcctctgcctctgagctattccagaagtagtgaggaggcttttttgg
aggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcg
gatctgatcagcacgtgttgacaattaatcatcggcatagtatatcggca
tagtataatacgacaaggtgaggaactaaaccatggccaagcctttgtct
caagaagaatccaccctcattgaaagagcaacggctacaatcaacagcat
ccccatctctgaagactacagcgtcgccagcgcagctctctctagcgacg
gccgcatcttcactggtgtcaatgtattcattttactgggggaccttgtg
cagaactcgtggtgctgggcactgctgctgctgcggcagctggcaacctg
acttgtatcgtcgcgatcggaaatgagaacaggggcatcttgagcccctg
cggacggtgtcgacaggtgcttctcgatctgcatcctgggatcaaagcga
tagtgaaggacagtgatggacagccgacggcagttgggattcgtgaattg
ctgccctctggttatgtgtgggagggctaagcacttcgtggccgaggagc
aggactgacacgtgctacgagatttcgattccaccgccgccttctatgaa
aggttgggcttcggaatcgttttccgggacgccggctggatgatcctcca
gcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattg
cagcttataatggttacaaataaagcaatagcatcacaaatttcacaaat
aaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaa
tgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgt
aatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaatt
ccacacaacatacgagccggaagcataaagtgtaaagcctggggtgccta
atgagtgagctaactcacattaattgcgttgcgctcactgcccgctttcc
agtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcg
gggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactga
ctcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaa
aggcggtaatacggttatccacagaatcaggggataacgcaggaaagaac
atgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt
gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc
gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccag
gcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgcc
gcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgcttt
ctcaatgctcacgctgtaggtatctcagtrcggtgtaggtcgttcgctcc
aagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgcctt
atccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc
cactggcagcagccactggtaacaggattagcagagcgaggtatgtaggc
ggtgctacagagttcttgaagtggtggcctaactacggctacactagaag
gacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaa
gagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggt
ttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaaga
agatcctttgatcttttctacggggtctgacgctcagtggaacgaaaact
cacgttaagggattttggtcatgagattatcaaaaaggatcttcacctag
atccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatga
gtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatct
cagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtg
tagataactacgatacgggagggcttaccatctggccccagtgctgcaat
gataccgcgagacccacgctcaccggctccagatttatcagcaataaacc
agccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcc
tccatccagtctattaattgttgccgggaagctagagtaagtagttcgcc
agttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgt
cacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatca
aggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctcctt
cggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactca
tggttatggcagcactgcataattctcttactgtcatgccatccgtaaga
tgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtg
tatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccg
cgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcg
gggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgta
acccactcgtgcacccaactgatcttcagcatcttttactttcaccagcg
tttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaata
agggcgacacggaaatgttgaatactcatactcttcctttttc
B. Cell Culture and Transfections: The human embryonic kidney cell
line, 293T cells, was maintained in DMEM with 10% dialyzed fetal
calf serum and 1% penicillin/streptomycin/neomycin antibiotics.
Cells were maintained at 37.degree. C. in a humidified atmosphere
of 5% CO.sub.2/95% air. Transfections of plasmids encoding EphB4
ectodomain, fragments thereof, and EphB4-HSA fusions were performed
using Lipofectamine 2000 reagent (Invitrogen) according to
suggested protocol. One day before transfections, 293T cells were
seeded at a high density to reach 80% confluence at the time of
transfection. Plasmid DNA and Lipofectamine reagent at 1:3 ratio
were diluted in Opti-MEM I reduced serum medium (Invitrogen) for 5
min and mixed together to form DNA-Lipofectamine complex. For each
10 cm culture dish, 10 .mu.g of plasmid DNA was used. After 20 min,
the above complex was added directly to cells in culture medium.
After 16 hours of transfection, medium was aspirated, washed once
with serum free DMEM and replaced with serum free DMEM. Secreted
proteins were harvested after 48 hours by collecting conditional
medium. Conditional medium was clarified by centrifugation at
10,000 g for 20 min and filtered through 0.2.mu. filter and used
for purification. C. Chromatographic Separation of EphB4 Ectodomain
and EphB4 Ectodomain-HSA Fusion Protein
[0344] The EphB4 ectodomain fused to HSA was purified as follows:
700 ml of media was harvested from transiently transfected 293
cells grown in serum free media and concentrated up to final volume
of 120 ml. Membrane: (Omega, 76 mm), 50 kDa C/O. After
concentration, pH of the sample was adjusted by adding 6 ml of 1M
NaAc, pH 5.5. Then sample was dialyzed against starting buffer
(SB): 20 mM NaAc, 20 mM NaCl, pH 5.5 for O/N. 5 ml of SP-Sepharose
was equilibrated with SB and sample was loaded. Washing: 100 ml of
SB. Elution by NaCl: 12 ml/fraction and increment of 20 mM. Most of
the EphrinB2 binding activity eluted in the 100 mM and 120 mM
fractions.
[0345] Fractions, active in EphrinB2 binding assay (See SP
chromatography, fractions # 100-120 mM) were used in second step of
purification on Q-column. Pulled fractions were dialyzed against
starting buffer#2 (SB2): 20 mM Tris-HCl, 20 mM NaCl, pH 8 for O/N
and loaded onto 2 ml of Q-Sepharose. After washing with 20 ml of
SB2, absorbed protein was eluted by NaCl: 3 ml/fraction with a
concentration increment of 25 mM. Obtained fractions were analyzed
by PAGE and in Ephrin-B2 binding assay. The 200 mM and 225 mM
fractions were found to contain the most protein and the most B2
binding activity.
[0346] Soluble EphB4 ectodomain protein was purified as follows:
300 ml of conditional medium (see: Cell culture and transfections)
were concentrated up to final volume of 100 ml, using
ultrafiltration membrane with 30 kDa C/O. After concentration, pH
of the sample was adjusted by adding 5 ml of 1 M Na-Acetate, pH
5.5. Then sample was dialyzed against starting buffer (StB): 20 mM
Na-Acetate, 20 mM NaCl, pH 5.5 for O/N. 5 ml of SP-Sepharose was
equilibrated with StB and sample was loaded. After washing the
column with 20 ml of StB, absorbed proteins were eluted by linear
gradient of concentration of NaCl (20-250 mM and total elution
volume of 20 column's volumes). Purity of the proteins was analyzed
by PAGE.
D. Biotinylation of sB4 and sB4-HSA Fusion Protein.
[0347] Both soluble EphB4 ectodomain protein (sB4) and EphB4
ectodomain fused to HSA (HSA-sB4) were biotin labeled through
carbohydrate chains using sodium meta-periodate as an oxidant and
EZ-Link Biotin Hydrazide (PIERCE, Cat. # 21339) according to
manufacture's protocol. The in vitro stability of the biotinylated
sB4 protein was tested by incubating 2.0.times.10.sup.-9 with 40
.mu.L of mouse serum at 37.degree. C. for 0, 0.5, 1, 2 and 3 days.
Two .mu.L of magnetic beads and B2-AP was added for an extra hour
at room temperature. After washing twice with buffer, pnPP was
added for 1 hour. Biotinylated sB4 protein was found to very stable
over three days, with less than 10% of the B2 binding activity
being lost over that time.
E. Ephrin-B2 Binding Properties of B4-HSA
[0348] To test whether the B4-HSA fusion property retained the
ability of the EphB4 extracellular domain to bind to EphrinB2, the
ability of the purified B4-HSA fusion was compared to that of
GCF2F, GCF2, GC, CF and B4-Fc fusion, which comprises the
extracellular domain of B4 fused to hIgG1 Fc as described in
Example 1. Biotinylated or His-tag protein samples were inoculated
with the corresponding affinity magnetic beads and B2-AP for an
hour at room temperature, before addition of PnPP. Results of
binding assays are shown on FIG. 67. B4-HSA was found to retain
most of its binding activity towards EphrinB2. Surprisingly, the
B4-HSA protein was superior to the B4-Fc fusion in binding to
EphrinB2.
[0349] An EphB4 ectodomain fusion to the C-terminus of HSA was also
generated, and found to retain the ability to bind to EphrinB2 and
was found to have enhanced stability in vivo over the EphB4
ectodomain.
F. Stability of B4-HSA vs. sB4 in Mice
[0350] The stability of the purified biotinylated sB4 and sB4-HSA
were assayed in vivo. Each of the proteins were intravenously
injected into the tail of mice in the amount of 0.5 nmoles per
mouse. Blood from the eye of each mouse was taken in time frames of
15 min (0 days), 1, 2, 3 and 6 days. 10 ml of obtained serum was
used in binding assay with Ephrin-B2-Alkaline Phosphatase fusion
protein and Streptavidin-coated magnetic beads as a solid phase.
The stability of the two proteins is shown on FIG. 68. sB4-HSA was
found to have superior stability relative to sB4. For example, one
day after injection, the levels of sB4-HSA in the blood of the mice
were 5-fold greater than those of sB4.
G. PEGylation of Biotinylated sB4
[0351] Prior to PEGylation, biotinylated sB4 protein generated as
described above was concentrated up to final concentration of 2
mg/ml using a 30 kDa MWCO ultra membrane. Sample was dialyzed O/N
against coupling buffer: 30 mM phosphate, 75 mM NaCl, pH 8.00.
Coupling to PEG was performed at 4.degree. C. for 18 hours in 10
fold molar excess of reactive linear PEG unless otherwise
indicated. The reactive PEG used was PEG-succinimidyl propionate,
having a molecular weight of about 20 kda. Coupling to PEG may be
similarly performed using branches PEGs, such as of 10 kDa, 20 kDa
or 40 kDa. Other linear PEG molecules of 10 or 40 kDa may also be
used.
[0352] After PEGylation, the protein sample containing EphB4
ectodomain was dialyzed against StB O/N. Three ml of SP-Sepharose
was equilibrated with StB and sample was loaded. Washing and
elution of absorbed proteins was performed as above (see:
Purification of soluble EphB4 ectodomain and its fusion to HSA)
with just one modification: total elution volume was 40 volumes of
column. FIG. 69 shows chromatographic separation of PEG derivatives
of EphB4 protein on SP-Sepharose columns. Purity of the
PEG-modified EphB4 protein was analyzed by SDS-PAGE.
[0353] Double modified (PEGylated Biotinylated).sub.sB4 was used on
ion-exchange chromatography to separate non-PEGylated,
mono-PEGylated and poly-PEGylated proteins from each other.
Pegylated sample was dialyzed O/N against 20 mM Na-acetate, 20 mM
NaCl, pH 5.5 and loaded onto 2 ml of SP-Sepharose. After washing
with 10 ml of buffer, absorbed proteins were separated by gradual
elution of NaCl: 3 ml/fraction and increment of 25 mM NaCl.
Obtained fractions were analyzed by PAGE and in Ephrin-B2 binding
assay.
H. Effect of PEGylation Conditions on sB4 Binding to EphrinB2
[0354] The effects of pegylating biotinylated sB4 under different
pH conditions was determined. sB4 was pegylated at pH 6, 7 or 8,
and the pegylated products were tested for binding to EphrinB2 as
shown in FIG. 69. Ephrin2B binding activity was retained when
PEGylation was performed at pH 6 and pH 7, but was partially lost
at pH 8.
[0355] Additional combinations of parameters were tested, including
temperature, pH and molar ratio of pegylation agent to sB4 protein,
and the ability of the products of the pegylation reaction to bind
to Ephrin-B2. The results of the optimization experiment are shown
in FIG. 70. These results confirm the gradual decrease in B2
binding activity at basic pH.
I. Purification of Pegylated sB4 Species
[0356] Biotinylated sB4 protein was concentrated up to final
concentration of 2 mg/ml using a 30 kDa MWCO ultra membrane. Sample
was dialyzed O/N against coupling buffer: 30 mM phosphate, 75 mM
NaCl, pH 8.00. Coupling to PEG was performed at 4.degree. C. for 18
hours in 10 fold molar excess of reactive PEG. Double modified
(PEGylated Biotinylated).sub.sB4 was used on ion-exchange
chromatography to separate non-PEGylated, mono-PEGylated and
poly-PEGylated proteins from each other. Sample was dialyzed for
O/N against 20 mM Na-Acetate, 20 mM NaCl, pH 5.5 and loaded onto 2
ml of SP-Sepharose. After washing with 10 ml of buffer, absorbed
proteins were separated by gradual elution of NaCl: 3 ml/fraction
and increment of 25 mM NaCl. Obtained fractions were analyzed by
PAGE as shown in FIG. 71. Fractions 1, 2 and 3 were found to
correspond to polypegylated, monopegylated and unpegylated
biotinylated sB4.
J. In Vitro Properties of PEGylated EphB4 Derivatives
[0357] Fractions 1, 2 and 3 of biotinylated and PEGylated sB4 from
the SP column purification, corresponding to polypegylated,
monopegylated and unpegylated biotinylated sB4, were tested for
their ability to bind EphrinB2 using the standard assay. Results of
this experiment are shown on FIG. 72. The order of binding activity
was found to be Unpegylated>monopegylated>polypegylated.
[0358] The fractions were also tested for their stability in vitro.
The fractions were tested for retention of EphrinB2 binding
activity after incubation in mouse serum at 37.degree. C. for three
days. The results of this experiment are shown in FIG. 73. The
order of in vitro stability was found to be
monopegylated>unpegylated>polypegylated.
K. In Vivo Stability Analysis of PEGylated Derivatives of EphB4
Ectodomain in Mice
[0359] Fractions 1, 2 and 3 of biotinylated. and PEGylated sB4 from
the SP column purification, corresponding to polypegylated,
monopegylated and unpegylated biotinylated sB4, were introduced by
intravenous injection into mice in the amount of 0.5 nMoles/mouse.
Blood from each mouse was taken in time frame of 10 min, 1, 2 and 3
days. 10 ml of obtained serum was used in binding assay with
Ephrin-B2-Alkaline Phosphatase fusion protein and
Streptavidin-coated magnetic beads as a solid phase. Signals,
obtained at 10 min were taken as 100%. The two mice for each
protein were of a different strain. Results are shown in FIG. 74.
Pegylation was found to increase the stability of EphB4 in vivo
relative to unpegylated EphB4.
INCORPORATION BY REFERENCE
[0360] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference.
[0361] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such variations.
Sequence CWU 1
1
22 1 570 PRT Unknown Recombinant B4ECv3 protein 1 Met Glu Leu Arg
Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15 Glu Glu
Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30
Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35
40 45 Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val
Gln 50 55 60 Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp
Val Pro Arg 65 70 75 80 Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg
Phe Thr Met Leu Glu 85 90 95 Cys Leu Ser Leu Pro Arg Ala Gly Arg
Ser Cys Lys Glu Thr Phe Thr 100 105 110 Val Phe Tyr Tyr Glu Ser Asp
Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125 Ala Trp Met Glu Asn
Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140 His Leu Thr
Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn 145 150 155 160
Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165
170 175 Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His
Leu 180 185 190 Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr
Arg Phe Pro 195 200 205 Glu Thr Val Pro Arg Glu Leu Val Val Pro Val
Ala Gly Ser Cys Val 210 215 220 Val Asp Ala Val Pro Ala Pro Gly Pro
Ser Pro Ser Leu Tyr Cys Arg 225 230 235 240 Glu Asp Gly Gln Trp Ala
Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255 Pro Gly Phe Glu
Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270 Gln Gly
Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285
Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290
295 300 Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro
Cys 305 310 315 320 Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser
Arg Leu Asn Gly 325 330 335 Ser Ser Leu His Leu Glu Trp Ser Ala Pro
Leu Glu Ser Gly Gly Arg 340 345 350 Glu Asp Leu Thr Tyr Ala Leu Arg
Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365 Ser Cys Ala Pro Cys Gly
Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380 Asp Leu Val Glu
Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe 385 390 395 400 Thr
Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410
415 Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu
420 425 430 Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser
Pro Ser 435 440 445 Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro
Ser Gly Ala Trp 450 455 460 Leu Asp Tyr Glu Val Lys Tyr His Glu Lys
Gly Ala Glu Gly Pro Ser 465 470 475 480 Ser Val Arg Phe Leu Lys Thr
Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495 Leu Lys Arg Gly Ala
Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510 Ala Gly Tyr
Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525 Asp
Glu Ser Glu Gly Trp Arg Glu Gln Gly Ser Lys Arg Ala Ile Leu 530 535
540 Gln Ile Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser
545 550 555 560 Thr Arg Thr Gly His His His His His His 565 570 2
555 PRT Unknown Recombinant B4ECv3NT protein 2 Met Glu Leu Arg Val
Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr
Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30 Val
Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40
45 Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln
50 55 60 Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val
Pro Arg 65 70 75 80 Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe
Thr Met Leu Glu 85 90 95 Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser
Cys Lys Glu Thr Phe Thr 100 105 110 Val Phe Tyr Tyr Glu Ser Asp Ala
Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125 Ala Trp Met Glu Asn Pro
Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140 His Leu Thr Arg
Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn 145 150 155 160 Val
Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170
175 Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu
180 185 190 Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg
Phe Pro 195 200 205 Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala
Gly Ser Cys Val 210 215 220 Val Asp Ala Val Pro Ala Pro Gly Pro Ser
Pro Ser Leu Tyr Cys Arg 225 230 235 240 Glu Asp Gly Gln Trp Ala Glu
Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255 Pro Gly Phe Glu Ala
Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270 Gln Gly Thr
Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285 Pro
Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295
300 Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys
305 310 315 320 Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg
Leu Asn Gly 325 330 335 Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu
Glu Ser Gly Gly Arg 340 345 350 Glu Asp Leu Thr Tyr Ala Leu Arg Cys
Arg Glu Cys Arg Pro Gly Gly 355 360 365 Ser Cys Ala Pro Cys Gly Gly
Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380 Asp Leu Val Glu Pro
Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe 385 390 395 400 Thr Tyr
Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415
Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420
425 430 Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro
Ser 435 440 445 Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser
Gly Ala Trp 450 455 460 Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly
Ala Glu Gly Pro Ser 465 470 475 480 Ser Val Arg Phe Leu Lys Thr Ser
Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495 Leu Lys Arg Gly Ala Ser
Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510 Ala Gly Tyr Gly
Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525 Asp Glu
Ser Glu Gly Trp Arg Glu Gln Gly Ser Lys Arg Ala Ile Leu 530 535 540
Gln Ile Ser Ser Thr Val Ala Ala Ala Arg Val 545 550 555 3 233 PRT
Unknown Recombinant B2EC protein 3 Met Ala Val Arg Arg Asp Ser Val
Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10 15 Met Val Leu Cys Arg Thr
Ala Ile Ser Lys Ser Ile Val Leu Glu Pro 20 25 30 Ile Tyr Trp Asn
Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 35 40 45 Val Leu
Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys Pro Lys 50 55 60
Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met 65
70 75 80 Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu
Asn Thr 85 90 95 Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile
Lys Phe Thr Ile 100 105 110 Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp
Gly Leu Glu Phe Gln Lys 115 120 125 Asn Lys Asp Tyr Tyr Ile Ile Ser
Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140 Leu Asp Asn Gln Glu Gly
Gly Val Cys Gln Thr Arg Ala Met Lys Ile 145 150 155 160 Leu Met Lys
Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr Arg Asn 165 170 175 Lys
Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg 180 185
190 Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr
195 200 205 Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly
Ser Glu 210 215 220 Val Gly Ser His His His His His His 225 230 4
771 PRT Unknown Recombinant B4ECv3-FC protein 4 Met Glu Leu Arg Val
Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr
Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys Trp 20 25 30 Val
Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40
45 Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln
50 55 60 Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val
Pro Arg 65 70 75 80 Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe
Thr Met Leu Glu 85 90 95 Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser
Cys Lys Glu Thr Phe Thr 100 105 110 Val Phe Tyr Tyr Glu Ser Asp Ala
Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125 Ala Trp Met Glu Asn Pro
Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130 135 140 His Leu Thr Arg
Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val Asn 145 150 155 160 Val
Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170
175 Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu
180 185 190 Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg
Phe Pro 195 200 205 Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala
Gly Ser Cys Val 210 215 220 Val Asp Ala Val Pro Ala Pro Gly Pro Ser
Pro Ser Leu Tyr Cys Arg 225 230 235 240 Glu Asp Gly Gln Trp Ala Glu
Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250 255 Pro Gly Phe Glu Ala
Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala 260 265 270 Gln Gly Thr
Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro Cys 275 280 285 Pro
Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295
300 Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys
305 310 315 320 Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg
Leu Asn Gly 325 330 335 Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu
Glu Ser Gly Gly Arg 340 345 350 Glu Asp Leu Thr Tyr Ala Leu Arg Cys
Arg Glu Cys Arg Pro Gly Gly 355 360 365 Ser Cys Ala Pro Cys Gly Gly
Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375 380 Asp Leu Val Glu Pro
Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe 385 390 395 400 Thr Tyr
Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu Ala 405 410 415
Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420
425 430 Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro
Ser 435 440 445 Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser
Gly Ala Trp 450 455 460 Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly
Ala Glu Gly Pro Ser 465 470 475 480 Ser Val Arg Phe Leu Lys Thr Ser
Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495 Leu Lys Arg Gly Ala Ser
Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500 505 510 Ala Gly Tyr Gly
Pro Phe Gly Gln Glu His His Ser Gln Thr Gln Leu 515 520 525 Asp Glu
Ser Glu Gly Trp Arg Glu Gln Asp Pro Glu Pro Lys Ser Cys 530 535 540
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 545
550 555 560 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met 565 570 575 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser His 580 585 590 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly Val Glu Val 595 600 605 His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn Ser Thr Tyr 610 615 620 Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly 625 630 635 640 Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 645 650 655 Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 660 665
670 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
675 680 685 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu 690 695 700 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro 705 710 715 720 Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val 725 730 735 Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met 740 745 750 His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 755 760 765 Pro Gly Lys
770 5 459 PRT Unknown Recombinant B2EC-FC protein 5 Met Ala Val Arg
Arg Asp Ser Val Trp Lys Tyr Cys Trp Gly Val Leu 1 5 10 15 Met Val
Leu Cys Arg Thr Ala Ile Ser Lys Ser Ile Val Leu Glu Pro 20 25 30
Ile Tyr Trp Asn Ser Ser Asn Ser Lys Phe Leu Pro Gly Gln Gly Leu 35
40 45 Val Leu Tyr Pro Gln Ile Gly Asp Lys Leu Asp Ile Ile Cys Pro
Lys 50 55 60 Val Asp Ser Lys Thr Val Gly Gln Tyr Glu Tyr Tyr Lys
Val Tyr Met 65 70 75 80 Val Asp Lys Asp Gln Ala Asp Arg Cys Thr Ile
Lys Lys Glu Asn Thr 85 90 95 Pro Leu Leu Asn Cys Ala Lys Pro Asp
Gln Asp Ile Lys Phe Thr Ile 100 105 110 Lys Phe Gln Glu Phe Ser Pro
Asn Leu Trp Gly Leu Glu Phe Gln Lys 115 120 125 Asn Lys Asp Tyr Tyr
Ile Ile Ser Thr Ser Asn Gly Ser Leu Glu Gly 130 135 140 Leu Asp Asn
Gln Glu Gly Gly Val Cys Gln Thr Arg Ala Met Lys Ile 145 150 155 160
Leu Met Lys Val Gly Gln Asp Ala Ser Ser Ala Gly Ser Thr Arg Asn 165
170 175 Lys Asp Pro Thr Arg Arg Pro Glu Leu Glu Ala Gly Thr Asn Gly
Arg 180 185 190 Ser Ser Thr Thr Ser Pro Phe Val Lys Pro Asn Pro Gly
Ser Ser Thr 195 200 205 Asp Gly Asn Ser Ala Gly His Ser Gly Asn Asn
Ile Leu Gly Ser Glu 210 215 220 Val Asp Pro Glu Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro 225 230 235 240 Cys Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 245 250 255 Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 260 265 270 Cys Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn 275 280
285
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 290
295 300 Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val 305 310 315 320 Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser 325 330 335 Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 340 345 350 Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp 355 360 365 Glu Leu Thr Lys Asn Gln
Val Ser Leu Thr Cys Leu Val Lys Gly Phe 370 375 380 Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 385 390 395 400 Asn
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 405 410
415 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
420 425 430 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
His Tyr 435 440 445 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 450
455 6 26000 DNA Homo sapiens 6 ggggtttcat catgttggcc aggctggtct
tgaactcctg acctcaaatg atccgcctgc 60 ctctgcctcc caaaatgctg
ggactacagg cgtgagccac cgcgcccgcc acacccacct 120 tttctttacc
gttgtttcct cgatttttct ctactcccta gcgcagctta gtgcgcgcct 180
cctctggaca tttttcaggg cttggttgcg cgcacagtag gtccccaaca ctgaatgttt
240 atggggtgac tgtgtgaacg ttcgctgcaa ggctatccaa actgggattg
ctccttgagg 300 ccccctgggc ggccgtcaat tctccaaagc ttctactccc
ttttccttcc ttttccccca 360 aaacgcagtc cctgcgccca ctagagggtg
gtgggcgcat ccaagagcgg catctagagt 420 ccgcagcaag gtcagagcgg
gctttgtgtg cgcggtgaac atttacgtgc acgcctgggc 480 ggccctccgt
gttgctgctg ggtgtgtgtt ttctctgctc cctggtgcca gccgggttcg 540
ggcctgtccc gggggtccct gggccccagc cccgacatgc tcggtcctgg acagcgcgca
600 ccgccacggc gcacatctgg gcggtcccgg ggttcctcac ccgccgcccc
tcccccttct 660 ccaaactttc tctcaacttc ccgacctgct ccactcggtg
cccctctccg cttccctcat 720 gaattattca gtagcgtgag ctccaatcag
cgcgcccggg gctcactcgc ggagcccccg 780 cgttgggaga gctgcccccg
ccccccgcgc gcccctccct cccgggcccg gcgccgcccg 840 gcccagttcc
agcgcagctc agcccctgcc cggcccggcc cgcccggctc cgcgccgcag 900
tctccctccc tcccgctccg tccccgctcg ggctcccacc atccccgccc gcgaggagag
960 cactcggccc ggcggcgcga gcagagccac tccagggagg gggggagacc
gcgagcggcc 1020 ggctcagccc ccgccacccg gggcgggacc ccgaggcccc
ggagggaccc caactccagc 1080 cacgtcttgc tgcgcgcccg cccggcgcgg
ccactgccag cacgctccgg gcccgccgcc 1140 cgcgcgcgcg gcacagacgc
ggggccacac ttggcgccgc cgcccggtgc cccgcacgct 1200 cgcatgggcc
cgcgctgagg gccccgacga ggagtcccgc gcggagtatc ggcgtccacc 1260
cgcccaggga gagtcagacc tgggggggcg agggcccccc aaactcagtt cggatcctac
1320 ccgagtgagg cggcgccatg gagctccggg tgctgctctg ctgggcttcg
ttggccgcag 1380 ctttggaagg tgagtttcct tgcggggggg ggcgcacccc
gtcactcctg ggacctcccc 1440 cccaacatct gggcctcgga gtggaggggc
cggcctctga ctacccctac ccgggcactg 1500 cagtcccaaa cacttcggac
cgatagtgct ggaacgggag gggggcgggg aagaggcgcc 1560 cgacgggtag
tggagttttc ttttgtttgg gaaagagatg gagtctggct acgacccggg 1620
acattcccct gcccgggctc cccgaactct cactgctgat tacatacgcc cctggctgcc
1680 tttcctttcc tccctacccc actattcaaa actatctgca aagtttctgt
cccagtccca 1740 cctcccgccg tacatgaggg aaggtttctg gagaagcaac
agcagacaag gcacaacttt 1800 tcgtgctagg ccctaaaacg acccccagcg
ccaattcctt agcgatcaca ccttgatcct 1860 ccagttccac actcctgcaa
caggatggcc tcctttgcat tcacacagca aacccccaaa 1920 ccgctctccc
gcccactgct cctgcccctg gtatagggtg gctccttggt ttctacaggc 1980
tgcaccccat ccctttaaat gcggtctaga ccccggcccc aggtgagtcc cgggcttccc
2040 ttgagaccta ggagcgggta gaaactgacc tacacagccc ccaggtagaa
actgacctac 2100 acagccccca catcgcccta actaacccag tctatctccc
acctcctggt ctctccaagc 2160 atttctttgg ccatggatcg ctgtccctcc
tggtccccta aagggggagc caagagccct 2220 agaaactctc ctgtgtccct
aatgtccttt cagtgagctg ccaacacccc cctttctctg 2280 tctggtatga
aagtggttat ggggcggtag gctatgaggg actcccaaag ggaaggattc 2340
agcggcgtta gaaaaaccct ctccccctgg ctgggcagga ctgccctggg ctggggatca
2400 aaggctaggt gtggggttgg gagtgagggg aggcttgccc agctcagaga
acggagaagg 2460 gggaacaaaa accatgaacg aggggaagag gaaggccaaa
ggggtggaaa aaccacgagg 2520 acgaggtgtg gtgagaagga aagacgcaaa
gaggaaatgg tgattgtgac acctattacc 2580 tgagtgtttc caagcaccag
gcctgtgctg agcgccttac aaatattaat ttcacccatc 2640 cagcaacgct
aagggtggtg ctattattgc ccccattttt cagatgagga ggctggggct 2700
tagttaaggt taagtagttt atccaaggcc ctgtgccgcg aggaacagcg agaagtggag
2760 gccgaaagcg aaggagagat agtgactgtc agaaagagaa acggaggtgg
acagagagtg 2820 gaggagagat aggtgagaga catgcgaact gacagatcaa
agcgtggctg cagctgagct 2880 gggacgcaga aagggagcct gcgcttgctc
tgggctgcgg acagcccgag gcagagacag 2940 tgtgtaaatt ggagacagga
aaacactatc ccggctggaa caatggaggg tggagacggc 3000 agcctctatc
cacccccttc ccagaacccg ggcatcctgt ccccagtgag cagggctgtc 3060
tcttgccacc catggggacc ttgcgcctct cacctcaggc tggctggctt cccatctgac
3120 ccctagctgg aggacatcat ttggtcccca ggaagaggct gcctcaccca
ccctctttct 3180 cttctctcct gcagctccca tggggtggga gccaggtgtt
ctggctcccc tctccaccct 3240 tcccagcgcc caatgccccc cacattgccg
gcccccgagg ggattcctgt accctccctc 3300 ctccactctc cactgccagg
ggctgtgcag tttttcctaa tcccccccct tcctccagtg 3360 cctgtcccct
cccccgatga tccgagccaa gccaggtgtg ttcacccctc ccattcatac 3420
cgccccccag aatctcctcc cctctgcctt cccataacca aatccagatg tgaggcctcg
3480 gcgggagcct gggaacccta gcatcccgac ctccagtgct tcctgatcag
ggcactcgtg 3540 gggagggagg tactgggatg ggggccaggg ctatgcccca
ggcacggagc gctcccttca 3600 aggagggaag gacggggtgt ttggtctgaa
agcagagagg ggtcttggac agggaatgaa 3660 attgtggggt agagaggctg
attctgggac ttaggggagg aaacgtggag gctgagacaa 3720 gaggttcccc
tcccacacca gcagcctctg ctcgtggggg tcaggaccag ggcgcagctc 3780
tcattttaac cctttctgag ctgccgcccc ttctccccgt acattttgat ctccctccct
3840 cctccaggga ggcctagatc tggggtatcc caagggagcc ccatgcctac
cagatgttgg 3900 gggtggggtt ggcacttagc agaagaggcc agaaatcagg
cgggtgcaga gggcagggct 3960 tgctcccctc ttggcccccc aactcctcta
gctcagagct aagaggatcc acctgcctcg 4020 gttcccaggg atctggtctt
cctgacctcc ctcccccacc ccaggcactg actctgtctc 4080 tctgtctgtc
tcagagaccc tgctgaacac aaaattggaa actgctgatc tgaagtgggt 4140
gacattccct caggtggacg ggcaggtgag agctgcaccc aggagctgga gctctggagg
4200 gaaactgagg gaggagaggg cgcctgtgcc gcctgctttc tgtgtgccac
tcctctcccc 4260 tgtcccccca gatgacagca gccccagcag tgtcgtctga
gcccttctca gaggcgccct 4320 cctcgcagta ccagcagccc ccctttctca
gtccctctca ctttatagga ttcaccccat 4380 gcagccctct ccctggcggc
tccccagccc ccttgctgac ctccttctct gcacagtggg 4440 aggaactgag
cggcctggat gaggaacagc acagcgtgcg cacctacgaa gtgtgtgacg 4500
tgcagcgtgc cccgggccag gcccactggc ttcgcacagg ttgggtccca cggcggggcg
4560 ccgtccacgt gtacgccacg ctgcgcttca ccatgctcga gtgcctgtcc
ctgcctcggg 4620 ctgggcgctc ctgcaaggag accttcaccg tcttctacta
tgagagcgat gcggacacgg 4680 ccacggccct cacgccagcc tggatggaga
acccctacat caaggtacct gggtgccccc 4740 agggctcagc cacagccaag
gtgggattcc agccagcagg cccgtggcct ggagggcagc 4800 cgatgtagtt
gcgaggcctc tggcccgcgc gctgggggct ggaagcagga ggcttaggtc 4860
tggggaggga agggggtgat cttctgggcg gaggagcaga atatacgggg gctgcctggc
4920 ccggccccca gggaggccca agggtcaggc ttctcctcca gtcacctcaa
ccaccctacc 4980 ccactgtgct ccagccacac tgagtttctc ccattccctg
actgcacctg gctggtttcc 5040 agctcaagac tttgcagcgg tgatgtctcc
acctgggggc ctctctgcct ctcacacccc 5100 tacttgtctt cggagttcca
gctcccgaga tcttgcctgt gccaccttgg ctgactctct 5160 cctccctaca
atcctgcata cctctgtcca cctgcctgtc tcggcactca ttttacttta 5220
tttatttttc ttttatatct atatttttaa agcggggtct tctacgttac ccaggctggt
5280 ctctaactcc tgggctcaag agatttctcc cacctcggcc tcctaaagtg
ctgggattat 5340 aggcatgagg cactacgccc ggcctcatgg tactttataa
cttccccagg attcattcat 5400 cgctgtctcc ttgactctga ggtcaaggcc
tggcatggcg tcagtgtcag taaatgtttg 5460 tagaacgagt gaataaaaag
ggggagaggt gcaggccaga ggccgggcat atcgcaggag 5520 ctttgcaagg
ctgaatggac agtgtggggg cctgcagaaa gtgtgccctg gggaaggtgg 5580
agggaagatt ctggaacggg aaccaaggag gtccgggagg gtgagctggg aagaacacaa
5640 cagtccgctg ggtcctcagg gagtggggac agcagcggtg tgcctccccc
ccgccggcag 5700 gtggacacgg tggccgcgga gcatctcacc cggaagcgcc
ctggggccga ggccaccggg 5760 aaggtgaatg tcaagacgct gcgtctggga
ccgctcagca aggctggctt ctacctggcc 5820 ttccaggacc agggtgcctg
catggccctg ctatccctgc acctcttcta caaaaagtgc 5880 gcccagctga
ctgtgaacct gactcgattc ccggagactg tgcctcggga gctggttgtg 5940
cccgtggccg gtagctgcgt ggtggatgcc gtccccgccc ctggccccag ccccagcctc
6000 tactgccgtg aggatggcca gtgggccgaa cagccggtca cgggctgcag
ctgtgctccg 6060 gggttcgagg cagctgaggg gaacaccaag tgccgaggtg
agagctggag cttcccctgc 6120 gactgctgct catccggggg agagtcctga
actccactca ggacccactt cttaagtttc 6180 cattttgtat agttagatgt
tgaaatggag gcttgctctg tcacccaggc tggagtgcag 6240 tggcacaatc
tctgctcaac tgcaaccttt gcctcccggg tccctgttca agcagttctc 6300
ctgcctcagc ctcgtgagta gctgggacta caggcacacg ccaccacgcc cggctaattt
6360 ttgtatttta gtagagacgg ggtttcgcca tgttggccag gctggtctcg
aactcctgac 6420 ctgaagtgat ttgcccgcct cggcctccca aagtgctggg
attacaggcg tgcgtcacca 6480 cacccagctg gaaaaaaaaa agactttatt
ttcacctgaa attcattaat ttccacttga 6540 aattccacct gcagttgtag
caggacctga cacttgggcc ccatggaaat cacaggtatt 6600 gcctgacaca
gtggttcatg cccatagtgc cagcactttg agatgccaag gtgggaggat 6660
cacttgagcc caggagttcg agatcagcct gggtgacaga gcaagacccc gtctctaaaa
6720 aaaatttttt tttttttttc aagacagagt cttgctctgt cgcccaggct
ggagtgcagt 6780 ggtgcgatct cggctcactg caagctccgc ctcccaagtt
aacaccattc tcctgcctca 6840 gcctcccgag tagctgggac tacaggcccc
gccaccacgc ccggctaatt tcttgtattt 6900 ttagtagaga tggagtttca
ccgtgttagc caggatggtc tcgatctcct gacctcatga 6960 tctgcccgcc
ttggcctccc aaagtgctgg gattacaggt gtgagccacc acacccggat 7020
tacaaaaact ttttagataa ttatctgggc gacctgcctg accaacatgg agaaaccctg
7080 tctctactaa aaatacaaaa ttagccggac atggtggcgc atgcctgtaa
tcccagctac 7140 ttgggaggct gaggcaggag aatcatttga acccaggaag
cagaggttgc ggtaagccga 7200 gatcatgcca ctgcactccg gtctgggagt
gcactccaac aagaaggagt ttcgctcttt 7260 ttgcccaggc tggagtgcag
tggtgggatc tcagctcacc gcaacctcca cctcccgggt 7320 tcaggcgatt
ctcctgcctc agcctcccaa ggagtagctg ggattatagg tatgcatcgt 7380
cacacccggc tacttttgta tttttagtag aggcaggttt ccaccatgtt ggccaggctg
7440 gtcttgaact caagtgatct gccctctttg gcctccttct caggaaaaaa
aaaaaatcac 7500 aggtatttac aggccattcc aagtgccaaa agattgtttt
tgctcatggt gacttcagta 7560 tcacagatgt taggagactt gctgctatat
gttaagaaag aagcacaaat gttgctgtag 7620 cccaaacttt tttcctcatg
tttcattgca tttcagctta attggtttcc ctggtattcc 7680 tatgtatttt
gtggagtgct tttaaaatca taagttggag tagaggtctt tctgtgggct 7740
tcaccagact gccgagatca gggtcgaaac aggtgaggac cccttctctg gagagagtct
7800 cctttctcct ctaagaggaa aggttttgag atcttttgtc cattttccca
ccttagcact 7860 tcatcagcct taaaagaagc tggaattttt tttttttttt
ttggagatgg gatctcgata 7920 tgttgcccag gctggtcttg aaccccttgg
ctcaagcgat cctccagcct cagcctccca 7980 aagtgctggg attcgaggca
tgagccaccg agcccaccgt gcagatggat gtttttgtgc 8040 atgcttttga
tgaatgcttt ctctctctca gcctgtgccc agggcacctt caagcccctg 8100
tcaggagaag ggtcctgcca gccatgccca gccaatagcc actctaacac cattggatca
8160 gccgtctgcc agtgccgcgt cgggtacttc cgggcacgca cagacccccg
gggtgcaccc 8220 tgcaccagta agtgaccagc acccaggtgc agttcactgg
ggaggggtca cagacctctg 8280 aggtggaccc tcacatggcc cccatcctcc
ctgggcttct tccctttgtc cctggcatgc 8340 ttgtccctag cccggaggaa
catgtggagc ccactgtctc caaggcaaga gtccagcatg 8400 gctgctggtg
cctccattgc cctctcccca ccaccgcaga gcaggtcggc ctctgcctga 8460
ctccctggtc tcctgcagcc cctccttcgg ctccgcggag cgtggtttcc cgcctgaacg
8520 gctcctccct gcacctggaa tggagtgccc ccctggagtc tggtggccga
gaggacctca 8580 cctacgccct ccgctgccgg gagtgccgac ccggaggctc
ctgtgcgccc tgcgggggag 8640 acctgacttt tgaccccggc ccccgggacc
tggtggagcc ctgggtggtg gttcgagggc 8700 tacgtcctga cttcacctat
acctttgagg tcactgcatt gaacggggta tcctccttag 8760 ccacggggcc
cgtcccattt gagcctgtca atgtcaccac tgaccgagag ggtgagactt 8820
gggggctggg gcggctggtg gtctggcggg agagatgtca ctgagggcct gaaggggaga
8880 ggcaggggct gtgaagttgg gtaccccgga agtgtgaggg gctaaggctt
tgggggcaag 8940 aggcagaaag agggcaatgg ctgggcgcag tggctcacgc
ctgtaatccc agcactttca 9000 gaggctgaga caggcggatc acttgagccc
tggagttcaa gaccagcctg ggtaacatag 9060 gaagatctct ctacaaaaaa
taaaaatatt agccaggcga ggtggtgcat gcctgtggtc 9120 ccagctactc
aagaggctga ggcaggagga ttgcttgagc ccaggagtcg gaggctgcag 9180
tgagctatga tcgcaccgct gcatgccagc ctgggtgaca gagcagtgtg agatcctctc
9240 tcaaaataaa tgaataagaa agagagggtg aggagctcgt aaagctgggc
tggagagtta 9300 agtacaggaa ggcccccagt gggactgggg ccagagagaa
tcagaaggaa ttctcgaaac 9360 agccaggggg aaattgagac aagtgtagcc
agcagaggaa gtgttggaaa agataaggga 9420 catggccagg ctgatcacaa
ggtcaggagt tcaagactag cctggccaac gtggtgaaac 9480 cccatgtcta
ctaaaaataa aaaaattagc caggcatggt ggtgggcacc tgtaatccac 9540
ttgggaagca accagaagaa ttgcttgaac ccaggaggcg gaggttgcag taagctgaga
9600 ctgcgccact gcactccagc ctgggtgata gagcacgact ccgtctcgaa
aaaaaaaatt 9660 ttttttaagt taagggacag agctaccatg cacaagggtt
ccctgtgtct ctgcctctca 9720 cagtacctcc tgcagtgtct gacatccggg
tgacgcggtc ctcacccagc agcttgagcc 9780 tggcctgggc tgttccccgg
gcacccagtg gggctgtgct ggactacgag gtcaaatacc 9840 atgagaaggt
aaggccatcc cccagccctg gggtgggtgg gcaatgggtt gtgctctcct 9900
ggctgggaca cctgggttgc aggcacctgg caggcatttg aattccagct ctgccatgga
9960 ttccctgggc agccttgggt aagccccttg gcctgtctga gcctcagact
cttcatctat 10020 aaaatagtta ctgtaatagt taccagcagc tggacacagt
ggctgaggtt gggtgcggtg 10080 gctcacgcct gtaataccaa gcactttggg
aggctgaggc gggcagaatg cttgagccta 10140 ggagtttgag accagcctgg
gcaacatggt gaaacttcat ctctataaaa aacttaaaat 10200 gggccgggcg
cggtagctta cgcctgtaat cccagcactt tgggaggccg aggtgggcgg 10260
atcacaaggt caggagtatc gagaccatcc tggctaacac ggtgaaaccc catctctact
10320 aaaaatacaa aaaattagcc aggcgcggtg gcaggcgcct gtagtcccag
ctactcggga 10380 ggctgaggca ggagaatggc gtgaacccag gaggcggagc
ttgcagtgag ccgagatagc 10440 gccactgcag tccggcctgg gcgaaagaac
aagactctgt ctccaaaaaa aaaaaaaaaa 10500 aaaaaaaacg caaaaaatac
ttaaaatgaa aaaaattaga ctgggcacag tggctcatgc 10560 ctgtaatccc
ggcactttgg gaggccgagg tgggtagaac acctggggtg aagagttcga 10620
gaccagcctg gccaacaagg tgaaatcccc gtctctacta caaatagcaa aatcagctga
10680 gtgtgttggc gggcccctgt aatcccagct actcaggagg ctgagacagg
agaatcactg 10740 gaacccaagt gattctcgac ttgaggtcga ggctgcagtg
agtcgtgttt gcaccattgc 10800 attccagcct gagaaagtga gaccttgtct
taaaaaaaag gaatgatatt atgaatacag 10860 cacatggctt gcatgcgtaa
gttctcccaa aggcctcacc agttgcaagg caggctagtg 10920 atgggagtgg
agggcgaggg aaggaggcag gaagagcaac aggaacttgg gttcccgggt 10980
gacggccacc ccactacctc tcccggacag ggcgccgagg gtcccagcag cgtgcggttc
11040 ctgaagacgt cagaaaaccg ggcagagctg cgggggctga agcggggagc
cagctacctg 11100 gtgcaggtac gggcgcgctc tgaggccggc tacgggccct
tcggccagga acatcacagc 11160 cagacccaac tggatggtga gcctggggaa
gggggtgagg gtgggggttg gaaagacccc 11220 caaagttcct gggaagaccc
caggtctcca aagtcccatc atcttttttt tttttttttt 11280 tttttgagat
ggagtcttgc tctgtccctc aggctggagt gcagtggcac catctccgct 11340
cactgcaacc tccgcctccc ggattcaagc cattctcctg cctcagcctc ccgagtagct
11400 gggattacag gcgcctgcca ccgcgcctgg ccgatttttt gtatttttag
tagagacggg 11460 gcttcaccgc gttggccagg ctggtctcga actcctgacc
ttgtgattcg cccgcctcgg 11520 cctcccgaag tgctgggatt acaggcatga
gccactgcac ccggtcaaag tcctatcttc 11580 atgtccttct tcctgtggat
cacatggcat gccctagaga ggagagaacg taagatgtcg 11640 aaaccaaaac
caacagctga gttttgtgaa gtctggcctg cttcactctg tacccccagg 11700
ctggagcgca gttgctcgat caaagctcac tgcacagcca ggcacagtgg ctcaccctgt
11760 aaccccagca ctttgggagg ctgaagcagg aggatcactt gaggtcagga
gttcgagacc 11820 agtctgacca gcatggtgaa accgcgtctc tactaaaaat
atagaagtta gctgagcgtg 11880 gtggtgcaca cctgtaatcc cagctactcg
ggaggctgag gcaggagaat cgcttgaacc 11940 tgggaggtgg aggttgcagt
gagctgagat tgtgccagtg cactccagcc tgggcaacag 12000 agcaagactc
tgtctcaaaa aaaaaaaagc tcaccgcagg cttgactttt agcaacaacc 12060
tgacccctga gctccccatt ccccatccaa caaaatggga atatcatgaa gcttcctgca
12120 gggctttgag gattggaggt aacaggttat ttttaatatg ctaggccagt
ggctttcttt 12180 tttctttcac attttttttt ttgagacgga gtctcactct
gttgcccagg ctggagtgcg 12240 gtggcgcgat ctcagctcac cgcaagctcc
acctcctggt ctcgatctgc tgacctcctg 12300 atccacccgc ctcggcttcc
cgaaatgctg ggactgctgg cgtgagccac cacgcccggc 12360 ctaacttttt
ctttttttta agagacacgg tcttttttat cacccaggct ggagtgcggt 12420
ggcaccatca tagctcattg cagcctacaa ctcccgagct caaccaatcc ttccacctta
12480 gcctcccaag tagctggggc tataggcatg tgctaccgtg ctcaactaaa
ttttttttta 12540 tgttttgttg agacagtttc cctatgttgc ccaggctggt
ctcaaattcc tgacctcgag 12600 caatcctccc gcatcggcct cccaaagtgc
tgggattaca ggcatgagcc gccacaccca 12660 gcattggacc agtggctttc
taaaccttgt aattttctgt aatagcttta ctgaaataca 12720 gttcccctgc
catacaattt gcctgttcaa agtgtacaat cgatgacttt tgatacattc 12780
acagaattgt gcagtcacca ccacaagtaa ttttgggaca ttttcagcac cctcaaaaga
12840 gaccctatag cccttagcca tcacccccca cccagatctt tctgttgcct
tagtccctgg 12900 caagcactaa cccactttct gtcttgaaat cttccagtgt
ggtcttttgt gactgttcac 12960 cgagcagaat gttttcaagg tttatgtatg
ttgtagtata tatccgtggg tttttttggt 13020 tgtggtttgt tttttgtttg
ttttggaaac agggtctcgc tctgtcaccc aggctggagt 13080 gcagtggttc
aattacagct cactgcagcc tcaacctccc aggctcaagt gatcctccca 13140
cctcagcctc ccaagcagct gggactgtag gcatgagcca ccatgcccag ctaatttttt
13200 ttggtatttt ttgtaaagac agggtttcac catgtttccc aggctggtct
cgaactcctg 13260 agctcaggca atccacccac ctcagcctcc caaagtgctg
tgattacagg catgagccac 13320 tggacctggc ctgttttttg tttttgtttt
gaacacacga ttttgctttg tcacccaggc 13380 tggaatgtaa tggtctgatc
atagtgcatt gcagcctcaa actcctgggc tcaagcgatc 13440 ctcctacctc
agcctcctga gtatctggga ccacacgtgc tcaccaccat gcttggctaa 13500
ttattattat tttttgatag agacggggtc ttgctatgtt tcccaggctg gtcttgaaca
13560 cctggcctca cacaatcctc ccacctcagt atctcagagt gctgggatta
caggcatgag 13620 ccactgctcc tggccaatat ttcatttctt tttatggaga
cgtaataatc agttgtatgg 13680 aaatagctga ttttgttttt tattgtatct
tttggtgaac atttcaattg tatcgacttt 13740 ttggataaaa acctgaaaat
gtttcacctt tagaacgttt cattgaatgg agattttttt 13800 gtggactctg
gtatttatac tagaaccaaa tcaaaaccac tctggcggct gggcatgcct 13860
aggctggttt gagactagcc tgtccaacct ggtgaaagcc catctctact aaaaatacac
13920 aaattagccg agcatggtgg tacacacctg taatcccagc tactcaggag
gctgaggcag 13980 gagaatcgca gaacccggga
ggcggagatt gcagtgagct gagattgcgc cactgcactc 14040 cagcctgggc
gacagagtga gactgcgtct caaaaaaaca aacaaaaaat tactctggca 14100
gtaagaaaag atttcgaaac ttcctccctt gccctgaggt acttcagagg agcctgctgg
14160 cccctggggg agagtttgaa acccactgtt tgttccctga ccttgcctgc
ttgtgtcctc 14220 tccctccacc tgtcccctgt actggggacc tgttctcagg
agatcacagt tcattgctca 14280 aagccggggc tggggcctcc tacaggacca
tcagtttctc ctgatcagca gcctttcctt 14340 ccgcagagag cgagggctgg
cgggagcagc tggccctgat tgcgggcacg gcagtcgtgg 14400 gtgtggtcct
ggtcctggtg gtcattgtgg tcgcagttct ctgcctcagg taagggctct 14460
gacacccaga ggcccctgga agccctcagt tgatggccac ctgcctgggt gctacaggac
14520 aagcctttct ggctgtcccc agcctctttt tacttgaaat cttctccaat
ccctgctcct 14580 tcctttggtg tgtgtgcctc ataaagatgt gtgactcagt
ttaccttttg ttcctttccc 14640 atcggctaca ggaagcagag caatgggaga
gaagcagaat attcggacaa acacggacag 14700 tatctcatcg gacatggtgg
gttgccctaa tttgatggga ataggggctt ggggccgggt 14760 gtggtggctc
ctatctataa tcccagcact ttgggaggca gaggtgggca gatcacttga 14820
ggtcaggagt tcgagaccag cctggccaac atgttgaaac tccatctcta taaaaaatac
14880 atcagtcagc caggcatggt ggtgggcacc tgtaatccca gctactcagg
aggctgaggc 14940 agaagaatca ttttaacccg ggaggcggag attgcagtga
gccaagatcg cgccactgcg 15000 ctccaggcct gggtgacaga gcgagactcc
atctcaggaa aaaaaaaaaa aaaaaaaaaa 15060 accacggaga caggggtttg
gggctaaaag ctatgagccg agcctccgag tccagtggga 15120 gttaattccc
agctgacggg gccctgcctg atttctcagg tactaaggtc tacatcgacc 15180
ccttcactta tgaagaccct aatgaggctg tgagggaatt tgcaaaagag atcgatgtct
15240 cctacgtcaa gattgaagag gtgattggtg caggtgagag ccgaaggctg
cccgggcacc 15300 tgggaacgaa gcgggggtgg gcagggccac actggagcgg
gagagctgat gacctctgcg 15360 tccttgtttg aaggtgagtt tggcgaggtg
tgccgggggc ggctcaaggc cccagggaag 15420 aaggagagct gtgtggcaat
caagaccctg aagggtggct acacggagcg gcagcggcgt 15480 gagtttctga
gcgaggcctc catcatgggc cagttcgagc accccaatat catccgcctg 15540
gagggcgtgg tcaccaacag catgcccgtc atgattctca cagagttcat ggagaacggc
15600 gccctggact ccttcctgcg ggtgagcacc ctccctggct tctgcggcca
cccggagttc 15660 ccacttacac ccagaggcca cttgggttaa gaagccagga
cagacagtgg gtcccaggtc 15720 acctcctcca gccttttcct cttgggctaa
gccctggtcc tctgcctttt ctttttttta 15780 agacagagcc tcgctctgtc
gcccaggctg gagtgcagtg gcgcgatctc ggctcattgc 15840 tgtctccacc
tccagggttc aagcgattct cctgcctcag tctcccaagt agctggtact 15900
ataggcatgc accaccatgc tgactaattt ttgtattttt agtagacaca gggtttcacc
15960 atgtaggcca ggctggtatc aaactcctga cctcaagtga tctccccacc
tcagcctccc 16020 aaagtgctgg tattacaggt gtgaggcacc acgcctggcc
agccctctgc ctttaatttt 16080 ccctctggga aaggctgggc tcctgggacc
ttcctttccc actgccccat acagctgaag 16140 gttgtcattc cttctttttt
tttttaattt tgttttaatt gaattttttt tttttgagat 16200 ggagtttcac
tcttgttgcc caggccggag tgcaatggca agatcttggc tcaccgcaac 16260
ctccgcctcc caggttcaag cgattctcct gccttagcct ccccagtagc tgggattata
16320 ggcatgtgcc accacgcttg actaattttg tatttttagt agagacgggg
gtttctctgt 16380 gttggtcagg ctggtctcga actcccgacc tcaggtgatc
cgcctgcctc ggcctcccaa 16440 agtgctggga ttacagacgt gagccaccgc
gcccggccaa tttttttttt ttttttttaa 16500 gacagagtct cactctgtcc
tctaggctgg agtgcagtgg tgcattcata gctcactgta 16560 gccttgacct
cctgggctca agtgatcctc ccgcctcagc ctcctgagta gctggaacta 16620
cactcatgta ccaccatgct cagcaaattt ttaaaatttt ttgtagagac aggatctcga
16680 taggttgccc aggctggtct gaactcctgg cctcaagcga gcctccctcc
tcagcctccc 16740 acagcactgg gattgcaggc atgagccact gtgcctggcc
tgtcattcct tcttttgaca 16800 aatatttact gagtgctttc tacgcaccgg
tcatcctccc agtccccagg aataaagcta 16860 tacacacggc aaactggatt
tctcctcttg gggagcagag ggtctaatgg ggcaggggga 16920 ctgaaaatta
gcaagtaaat agacaggctt tttaaaaaag taaacaaatc atttcaaatg 16980
tgaaaaaaag caaacggggt ccttcatgca gatgtggcta gagaggaaag agaactgctt
17040 aatttatttg gtcactttac cagattttac tgactttttt ttttttttta
actttattaa 17100 gcttttcttt tttcttgaga tggagtttcc atctgtcacc
caggctggag tgcagtggtg 17160 cgttcttggc tcaccgcaac gtccacctcc
tgggttcaag tgattctcct gcctcagcct 17220 cctgagtagc ttggaattgc
atggcatgca ccaccatacc cagctgatgt ttgtattttt 17280 agtagagaca
gggtttcatc atgttgccca ggctggtctt gaactcctgg gctcaagtga 17340
tccacccatc tcggcccctc aaagtgctgg gattacaggc atgagccacc atgcctggcc
17400 taggcatctt tttaaaaaaa tcaaaacatt tttctatgta gcaaaataac
attgcattga 17460 acagagttat agcgattccc tagcgtcatt gaatacccag
ttgattttca cgtttctcta 17520 gttgttctaa agatgtcctt cactgctgct
ttattccaac caggatccag ttcaagaccg 17580 ggctttgtac ctggttatta
tatatatttt atttatttat tttagaaaca aggtcttgcc 17640 ctttcgccca
gtttagagtg cagtggtgca atcatagctc actgcagcct ccaaactcct 17700
tggctcaggt gatcctcctg cctcagcctc ctgggtagct ggaactacag gtgcacacca
17760 ccacacctgg ctaattttta aattttttac ggagatgggg gtctcgctat
gttgcccagg 17820 ctggtctcaa actcctggac tcaagcgatc ctccctcctt
aacctctcaa agtgctggga 17880 ttacaggcgt gagccaccac gcctgctgat
tattatattt tcgagcctct ctaaatcttg 17940 agcagttcct catgatgaca
ctgacacact gaagggttag gtcccttgtc cgcctgaatg 18000 tcttgatttc
tggatttatg aaattcttct tatgggatca tttagcttgt ctctctgtat 18060
ttcctgtaag agaagctcta tctgatgtgg ggtttttttg gttttgtttg tttgtttttt
18120 gagatggagt cctgctgtcg cccaggctgg agtgcagtgg cacaatctcg
gctcactgca 18180 acctccgcct cctgggttca agagattctt ctgcctcagc
ctcctgagta gctgggacta 18240 caggcgagtg ccaccatgcc cagctaattt
ttgtattttt agtagagaca gggtttcacc 18300 atattggcca ggatggtctc
gaacttctga cctcgtgatc tgcccaccac ctcagcctcc 18360 cacagtgctg
ggattacagg catgagccac tatgcccggc taatttttgt atttttagta 18420
gagacagggc ttcgccatgt tggccaggct gatctgaaac ccctggcctc aagccatcca
18480 ccctccttgg cctcccaaag tgctgggatt aaacgcgtga gccaccgtgc
ctggtcgaag 18540 agacagaaag ggtcttaaag gttcagtgac acacacctgt
aatcccagca ctttgggaag 18600 ctgaggctgg tggatcactc gaggccagga
gttagagatc accctgggca acatggtgaa 18660 accccgtctc tacacaaaat
acaaaaatgg gcagagcatg atggtgcata tctgtagtcc 18720 cagctactcg
ggaggctgag gcgggaggat cacttaagcc tgggagatcg aggctgtagt 18780
gagccatcat tgcactactg cattccagcc tgggcgatcc catctcttaa aaagagagag
18840 agatgggaag accagcacag gtgaaactgg tgaacagagg agagatggta
gatgctgcat 18900 tgggcagtgt gacgggaacc cgctggaggg ctttggcagg
agagtagttt aagaggatcc 18960 cagctgggca cagtggctca cacttgtgat
cccagcactt ggggaggccg gggcaggtgg 19020 atcacttgag gtcaggagtt
cgagaccagc ctggccaaca tggtgaaacc ctgtctgtac 19080 taaaaataca
aaaaccagcc aggcatggtg gtgcacccct gtaatcccag ctactcagga 19140
gactaagaca ggagaatcgc ttgaactcag gaggcagagg ttgcagtgag ccaagatcac
19200 gccactttac tccagcctgg gcagtagagc gagactccat ctcaaaaaaa
taaataaata 19260 aaaagacctc tttgctgggt gctagggagc aagagcagga
gctgggagag gcctgcagca 19320 gaagcctgtt gccagcatcc aggccgtggg
gtgaagggaa gggtttggat ttgggacatg 19380 tcttggaagc atcaccagca
gaacttgctg atggattgga agtggctggt gagggagaaa 19440 agggggtcaa
aggaaactct gaggtctata ccctgaccat ctggcaagtg gtggtgttgc 19500
cacaaactga gcggggagta gggcaggtgc aggtctggag gatggattca aaattcagtt
19560 tttggagtct atgtccctgg ttctgtaggg ctgcagatgg tctgccaaat
cttagcggaa 19620 cccagaatac gggatttgtt tactgtctgt gacttgttgg
tttccctggt gagagcaaac 19680 tctttaaagg tcaaggttgg gcttcagacc
ttggtttttg caccgatcat tggtcatact 19740 gcagttcctc actcttctct
tgcaaatcca tacacagcta gtccaagaga gctgaacagc 19800 tttgtggttg
gatcagcacc aatgtatctc cacctgtaga cgggttgctc aggtgactca 19860
tgcctgtaat cccagcacct tgggaggcca aggtgggaag attgcttgag gccaggagtt
19920 ggagacaagc ctgggaaaca cagtgagacc ccatatctac caaaaaaacc
cctttgtttt 19980 aattagccag gtgcagtggt gtgcacctat agtcccagct
actaaggagg ctgaggcaga 20040 aggatcattt gagcccagga gtttaaggct
gcggtgaacc atgatcgtgc cactgcactc 20100 caacctgggg gaaagaaaga
gaccttgtct ctaaaaaaac taaaaaacag aaaagcattt 20160 gttgagtatt
tcctgggtat aaagcagtgt accaggttaa atggaaggaa aagttgaaat 20220
aatttttcaa ctcataatcc gattgggaga gactgaatgc ttaccattga agcaggaacc
20280 attgtaagca atgtgttgtg atactgtagc aagagctgag aaaacttggg
aaaagagaaa 20340 ggaggaaggc tcacctgagg gagttggggg gcttgcccta
caggtgagtt gtgaggtggg 20400 tctggaagtg acagatgcag tttaggaagt
ggacgggagg ctgggtacgg tgactcaaca 20460 tctgtaatcc cagtgctttg
ggagacccag gcggaaggat cgcttcaggc caggagttaa 20520 agaccagcct
gggcaacata gtgggaacct atctctacta aaaattaaaa aattatccag 20580
gcataatggc acatgcctat tgttccagct actcaggagg cttgcctgag cccaggaggt
20640 tgaggctgca gtgagctatg atggcaccac tgcactccag cctgggcgac
agaacaagac 20700 cctgtctcta aaaaaaaaag atgtggatgg gagggggaac
ggtgggtggg ctgtcctcac 20760 caagccccca ccctatctgc tctccagcta
aacgacggac agttcacagt catccagctc 20820 gtgggcatgc tgcggggcat
cgcctcgggc atgcggtacc ttgccgagat gagctacgtc 20880 caccgagacc
tggctgctcg caacatccta gtcaacagca acctcgtctg caaagtgtct 20940
gactttggcc tttcccgatt cctggaggag aactcttccg atcccaccta cacgagctcc
21000 ctggtaatgc tgggggtaat actgggtgtg agcttcttag ggccaggtgg
gcagggcagg 21060 ttggaaaggt gggaggctga gggtttggca gccctgctcc
agggagagga tacaggagca 21120 ggctgtgggt ggggggacag tcagctccag
gaagccgact tccagatgtc taggaaaata 21180 acagttggat aacctgggca
acatagcaag accccatctc tacaaaaaaa ttaaaagatt 21240 agccaggcgc
agtggcatgc acctgtagtc ccagctactt gggaggttga ggcaggagga 21300
ttgcttaagc ccaggagttg gaggctgcag tgagctatga atgtgccact gtactgcaga
21360 ctgggcgaca gagcaagacc ctgtctcaaa agaacagtgg ccaggtgtgg
tggctcacgc 21420 ctgtaaatcc agcactttgg gaggctgagg caggaggatc
gcctgaggtc aggagttcga 21480 gaccagcctg gccaacatgg gaaaaccctg
tcgctactaa aaatacaaaa ttagctgagg 21540 gtggtggtac acgcctgtaa
tccgagctac tcaggaggct gaggtaggag aaccagttga 21600 acccgggagg
cggagtttca gtgagccaag atcgcaccac tgcactccaa cctgggcaaa 21660
cagagttgga gagtaggagg cttggggcct gagctagggg gaaaaagcag aggcaggtgg
21720 gggactgggg ggcagtgtgc tgggtctggt gagtccctca gtgagtcccc
cagctcacct 21780 tttctccttt ttctgcaggg aggaaagatt cccatccgat
ggactgcccc ggaggccatt 21840 gccttccgga agttcacttc cgccagtgat
gcctggagtt acgggattgt gatgtgggag 21900 gtgatgtcat ttggggagag
gccgtactgg gacatgagca atcaggacgt aagtgtcccg 21960 tggtcctacc
aagctttcct cgagtgttct ctcacctggg atttggggtg aagggtgggt 22020
tcccagagag tcatcactgc tgggttcttg agaccatgga gatgacaaaa aggagaattg
22080 atctttgtat caaagagttg agatacaggg ccaggcctag tggctcaagc
ctgtaatccc 22140 agcactttgg gaggccaagg tgggcagatc acctaaggtt
aggagttcaa gaccagcctg 22200 gccaacatgg tgaaaccccg tctctaaaaa
aatacaaaaa attagcccag catgatgggc 22260 gggtgcctgt aatcccagct
actcaggagg ctgagacagg ataatcgctt gaacccagga 22320 acagaggttg
cagtgagctg agatcacgcc attgctttcc agcctgggca actgagcgag 22380
actctgtctt aataaataaa taaaagagtt gggtacagca tatttgggtc gcagaaggat
22440 gcagagatgg agggcagggt tgagaggtaa catgtctgta tcatagccca
agagctgctg 22500 gggccttcag ccacagagag cttcaactcc ggctaggagg
attcctggat ctgttatttt 22560 ttggggggct gtggctccta tcctaccatc
ttccaagtca ccatttcctg ggcctgttag 22620 catctttgct tttcctggac
agcctcaccc agagcttctt cccctctttc caggtgatca 22680 atgccattga
acaggactac cggctgcccc cgcccccaga ctgtcccacc tccctccacc 22740
agctcatgct ggactgttgg cagaaagacc ggaatgcccg gccccgcttc ccccaggtgg
22800 tcagcgccct ggacaagatg atccggaacc ccgccagcct caaaatcgtg
gcccgggaga 22860 atggcgggtg aggactgcag agaatgggcc ctccttcccg
ctctctgccc ccactccttg 22920 cccagaagtg tccgttcatt ggtgttgggt
gggagggcct ctgtccgcct ctgcaaggct 22980 gggttccacc tcctcccccg
gacctgggcc tggtactcag cattcctccc catccttgcc 23040 ccctagggcc
tcacaccctc tcctggacca gcggcagcct cactactcag cttttggctc 23100
tgtgggcgag tggcttcggg ccatcaaaat gggaagatac gaagaaagtt tcgcagccgc
23160 tggctttggc tccttcgagc tggtcagcca gatctctgct gagtaagcag
tggcaggagc 23220 tggagtgggg ctgggagagc ggggcagctg gagtcaggcc
cacggggtct ccaggggctt 23280 ttggggtcag cttcgggtgc caatgctgtc
ttcttgcact gcgctcatgc catgcctaga 23340 agggccccag aggagcagtc
acagccccat ggagctgagg acccaaggac tctttggggc 23400 cagcctgccc
gcctcacctc ctcctgccat cacagccctg ggccatcgcg cttccgcctc 23460
tcacttctag ctatctttgt gcatctatct gcattccagg cccggctctc acggtaacaa
23520 tgtgtcaact cgggttctct ttttccaacc ataaaaggag aagattgggc
taggttttgg 23580 agatcctctt cagcttttat gtgaaatggt tttatgattc
cttgcctccc aaaggctgcg 23640 tatccccact tggcctttgt ctgctactcc
ccctttctgc cttcccgttc ctctcccaag 23700 atctcctctc accccaggtt
gaataacaga aatagaagga atagaaatct gaaggccggg 23760 catggtggct
catgcctgta atgccagcac tttgggaggc cgaggtgggc agatcacttg 23820
aggttaggag ttcgagacca ttgtggacaa cttggtgaaa ccttatgtct actaaaaata
23880 caaaaattag ctgggcatgg tggtgcgtgc ctgtaatacc agctactgag
gaggctgagg 23940 caggagaatc gcttgaaccc gggaggtgga ggttgcagtg
agccgagatc gcaccactgc 24000 actccagcct ggatgacaga gtgaaattcc
atctcaaaaa aaaaaaaaaa aaaaaaaaag 24060 aaatgtgaag gccaggtggt
ggctcacgcc tgtaatctca gcactttggg aggctcaggt 24120 ggaccgattg
cttgagccca ggagtttgag agcagcctgg ccaaaatagc aaaaccccat 24180
ctctacaaaa caaaaacaaa aaaattagct gggcatggtg gtgcgtgcct gtggtcccag
24240 ctactcagga ggctagagcc agagggtctc aggccagtct gcccctgccc
cacggggcct 24300 gggcacatcc ctccctaatt cttcccagcc tctctctgac
ccagggggcc tcctctccct 24360 tttttcccct tatctcagcc tccagccatc
agcaacctcc tcttcctctc cacccagctc 24420 ttcctctccc acttcggcct
tttctttctc acactccatt tccctctacg gcaatctgtg 24480 cagcctcttc
ccccagtctc attttgcggg cttttctctc ttttctttcc ttccctggca 24540
cccaagccaa aggccctgcc tctggcctcc agccctaccc ccttctgcgg ttgcacagaa
24600 ggatggctgc ccagctctta aaaaaactgc ccgggaactg ttgacatctg
ttctccctcc 24660 cccgctggct tttctgattg gcttacaatc ctgaggctag
gaccgtctca ggagccaaga 24720 gaggagagcg gccacaggga acctagggtc
tcaccaagct ctcctttcct tctgcaggga 24780 cctgctccga atcggagtca
ctctggcggg acaccagaag aaaatcttgg ccagtgtcca 24840 gcacatgaag
tcccaggcca agccgggaac cccgggtggg acaggaggac cggccccgca 24900
gtactgacct gcaggaactc cccaccccag ggacaccgcc tccccatttt ccggggcaga
24960 gtggggactc acagaggccc ccagccctgt gccccgctgg attgcacttt
gagcccgtgg 25020 ggtgaggagt tggcaatttg gagagacagg atttgggggt
tctgccataa taggagggga 25080 aaatcacccc ccagccacct cggggaactc
cagaccaagg gtgagggcgc ctttccctca 25140 ggactgggtg tgaccagagg
aaaaggaagt gcccaacatc tcccagcctc cccaggtgcc 25200 cccctcacct
tgatgggtgc gttcccgcag accaaagaga gtgtgactcc cttgccagct 25260
ccagagtggg ggggctgtcc cagggggcaa gaaggggtgt cagggcccag tgacaaaatc
25320 attggggttt gtagtcccaa cttgctgctg tcaccaccaa actcaatcat
ttttttccct 25380 tgtaaatgcc cctcccccag ctgctgcctt catattgaag
gtttttgagt tttgtttttg 25440 gtcttaattt ttctccccgt tccctttttg
tttcttcgtt ttgtttttct accgtccttg 25500 tcataacttt gtgttggagg
gaacctgttt cactatggcc tcctttgccc aagttgaaac 25560 aggggcccat
catcatgtct gtttccagaa cagtgccttg gtcatcccac atccccggac 25620
cccgcctggg acccccaagc tgtgtcctat gaaggggtgt ggggtgaggt agtgaaaagg
25680 gcggtagttg gtggtggaac ccagaaacgg acgccggtgc ttggaggggt
tcttaaatta 25740 tatttaaaaa agtaactttt tgtataaata aaagaaaatg
ggacgtgtcc cagctccagg 25800 ggtgatgggg gtgatggact agatttctaa
ggagagtggg gctgggtagg gagggctttg 25860 tggctgaccg agaggtgtca
gaggtctgga ggctgcaggg ctgtaggggc tggaacttgg 25920 ttatcagccc
cagggtatgt ttgaggtggt ggggtggggg ccgagcgaga tgaatcattc 25980
gcagctgctt ctaacgtctc 26000 7 4235 DNA Homo sapiens 7 ctcggcccgg
cggcgcgagc agagccactc cagggagggg gggagaccgc gagcggccgg 60
ctcagccccc gccacccggg gcgggacccc gaggccccgg agggacccca actccagcca
120 cgtcttgctg cgcgcccgcc cggcgcggcc actgccagca cgctccgggc
ccgccgcccg 180 cgcgcgcggc acagacgcgg ggccacactt ggcgccgccg
cccggtgccc cgcacgctcg 240 catgggcccg cgctgagggc cccgacgagg
agtcccgcgc ggagtatcgg cgtccacccg 300 cccagggaga gtcagacctg
ggggggcgag ggccccccaa actcagttcg gatcctaccc 360 gagtgaggcg
gcgccatgga gctccgggtg ctgctctgct gggcttcgtt ggccgcagct 420
ttggaagaga ccctgctgaa cacaaaattg gaaactgctg atctgaagtg ggtgacattc
480 cctcaggtgg acgggcagtg ggaggaactg agcggcctgg atgaggaaca
gcacagcgtg 540 cgcacctacg aagtgtgtga cgtgcagcgt gccccgggcc
aggcccactg gcttcgcaca 600 ggttgggtcc cacggcgggg cgccgtccac
gtgtacgcca cgctgcgctt caccatgctc 660 gagtgcctgt ccctgcctcg
ggctgggcgc tcctgcaagg agaccttcac cgtcttctac 720 tatgagagcg
atgcggacac ggccacggcc ctcacgccag cctggatgga gaacccctac 780
atcaaggtgg acacggtggc cgcggagcat ctcacccgga agcgccctgg ggccgaggcc
840 accgggaagg tgaatgtcaa gacgctgcgt ctgggaccgc tcagcaaggc
tggcttctac 900 ctggccttcc aggaccaggg tgcctgcatg gccctgctat
ccctgcacct cttctacaaa 960 aagtgcgccc agctgactgt gaacctgact
cgattcccgg agactgtgcc tcgggagctg 1020 gttgtgcccg tggccggtag
ctgcgtggtg gatgccgtcc ccgcccctgg ccccagcccc 1080 agcctctact
gccgtgagga tggccagtgg gccgaacagc cggtcacggg ctgcagctgt 1140
gctccggggt tcgaggcagc tgaggggaac accaagtgcc gagcctgtgc ccagggcacc
1200 ttcaagcccc tgtcaggaga agggtcctgc cagccatgcc cagccaatag
ccactctaac 1260 accattggat cagccgtctg ccagtgccgc gtcgggtact
tccgggcacg cacagacccc 1320 cggggtgcac cctgcaccac ccctccttcg
gctccgcgga gcgtggtttc ccgcctgaac 1380 ggctcctccc tgcacctgga
atggagtgcc cccctggagt ctggtggccg agaggacctc 1440 acctacgccc
tccgctgccg ggagtgccga cccggaggct cctgtgcgcc ctgcggggga 1500
gacctgactt ttgaccccgg cccccgggac ctggtggagc cctgggtggt ggttcgaggg
1560 ctacgtcctg acttcaccta tacctttgag gtcactgcat tgaacggggt
atcctcctta 1620 gccacggggc ccgtcccatt tgagcctgtc aatgtcacca
ctgaccgaga ggtacctcct 1680 gcagtgtctg acatccgggt gacgcggtcc
tcacccagca gcttgagcct ggcctgggct 1740 gttccccggg cacccagtgg
ggctgtgctg gactacgagg tcaaatacca tgagaagggc 1800 gccgagggtc
ccagcagcgt gcggttcctg aagacgtcag aaaaccgggc agagctgcgg 1860
gggctgaagc ggggagccag ctacctggtg caggtacggg cgcgctctga ggccggctac
1920 gggcccttcg gccaggaaca tcacagccag acccaactgg atgagagcga
gggctggcgg 1980 gagcagctgg ccctgattgc gggcacggca gtcgtgggtg
tggtcctggt cctggtggtc 2040 attgtggtcg cagttctctg cctcaggaag
cagagcaatg ggagagaagc agaatattcg 2100 gacaaacacg gacagtatct
catcggacat ggtactaagg tctacatcga ccccttcact 2160 tatgaagacc
ctaatgaggc tgtgagggaa tttgcaaaag agatcgatgt ctcctacgtc 2220
aagattgaag aggtgattgg tgcaggtgag tttggcgagg tgtgccgggg gcggctcaag
2280 gccccaggga agaaggagag ctgtgtggca atcaagaccc tgaagggtgg
ctacacggag 2340 cggcagcggc gtgagtttct gagcgaggcc tccatcatgg
gccagttcga gcaccccaat 2400 atcatccgcc tggagggcgt ggtcaccaac
agcatgcccg tcatgattct cacagagttc 2460 atggagaacg gcgccctgga
ctccttcctg cggctaaacg acggacagtt cacagtcatc 2520 cagctcgtgg
gcatgctgcg gggcatcgcc tcgggcatgc ggtaccttgc cgagatgagc 2580
tacgtccacc gagacctggc tgctcgcaac atcctagtca acagcaacct cgtctgcaaa
2640 gtgtctgact ttggcctttc ccgattcctg gaggagaact cttccgatcc
cacctacacg 2700 agctccctgg gaggaaagat tcccatccga tggactgccc
cggaggccat tgccttccgg 2760 aagttcactt ccgccagtga tgcctggagt
tacgggattg tgatgtggga ggtgatgtca 2820 tttggggaga ggccgtactg
ggacatgagc aatcaggacg tgatcaatgc cattgaacag 2880 gactaccggc
tgcccccgcc cccagactgt cccacctccc tccaccagct catgctggac 2940
tgttggcaga aagaccggaa tgcccggccc cgcttccccc aggtggtcag
cgccctggac
3000 aagatgatcc ggaaccccgc cagcctcaaa atcgtggccc gggagaatgg
cggggcctca 3060 caccctctcc tggaccagcg gcagcctcac tactcagctt
ttggctctgt gggcgagtgg 3120 cttcgggcca tcaaaatggg aagatacgaa
gaaagtttcg cagccgctgg ctttggctcc 3180 ttcgagctgg tcagccagat
ctctgctgag gacctgctcc gaatcggagt cactctggcg 3240 ggacaccaga
agaaaatctt ggccagtgtc cagcacatga agtcccaggc caagccggga 3300
accccgggtg ggacaggagg accggccccg cagtactgac ctgcaggaac tccccacccc
3360 agggacaccg cctccccatt ttccggggca gagtggggac tcacagaggc
ccccagccct 3420 gtgccccgct ggattgcact ttgagcccgt ggggtgagga
gttggcaatt tggagagaca 3480 ggatttgggg gttctgccat aataggaggg
gaaaatcacc ccccagccac ctcggggaac 3540 tccagaccaa gggtgagggc
gcctttccct caggactggg tgtgaccaga ggaaaaggaa 3600 gtgcccaaca
tctcccagcc tccccaggtg cccccctcac cttgatgggt gcgttcccgc 3660
agaccaaaga gagtgtgact cccttgccag ctccagagtg ggggggctgt cccagggggc
3720 aagaaggggt gtcagggccc agtgacaaaa tcattggggt ttgtagtccc
aacttgctgc 3780 tgtcaccacc aaactcaatc atttttttcc cttgtaaatg
cccctccccc agctgctgcc 3840 ttcatattga aggtttttga gttttgtttt
tggtcttaat ttttctcccc gttccctttt 3900 tgtttcttcg ttttgttttt
ctaccgtcct tgtcataact ttgtgttgga gggaacctgt 3960 ttcactatgg
cctcctttgc ccaagttgaa acaggggccc atcatcatgt ctgtttccag 4020
aacagtgcct tggtcatccc acatccccgg accccgcctg ggacccccaa gctgtgtcct
4080 atgaaggggt gtggggtgag gtagtgaaaa gggcggtagt tggtggtgga
acccagaaac 4140 ggacgccggt gcttggaggg gttcttaaat tatatttaaa
aaagtaactt tttgtataaa 4200 taaaagaaaa tgggacgtgt cccagctcca ggggt
4235 8 43948 DNA Homo sapiens 8 gcgcctcgga gctgcctgcg ggcgcacgcc
gtcttccccg ccagtctgcc ccggaggatt 60 gggggtccca gcctgcgtcc
cgtcagtccc ttcttggccc ggagtgcgcg gagctgggag 120 tggcttcgcc
atggctgtga gaagggactc cgtgtggaag tactgctggg gtgttttgat 180
ggttttatgc agaactgcga tttccaaatc gatagtttta gagcctatct attggaattc
240 ctcgaactcc aagtaagtgg cgtccgcgat ccccctatgt ccccgccccg
gggtccgccg 300 cgccgtccgg gcgggaggag gggtcagtcc gcggggcctc
ggagcctgtt tctggaacct 360 cggttccccg tcccccaccc ccaacccccg
ccccatttca ctaggtggag actcctcgct 420 cggctttcca acccgagccc
cgctggaacg gacggtctct ccgcctttcc tcccccgaac 480 gctcccaggc
gctaaaagct actatcggct cgggtgtcaa gtccgggaag gtgtccgatg 540
gcgatacctg accctctcct gttttcgagg acgaaggaca tggccacaat ctaggctggc
600 cggcacgcgg ggactggtgg gctctggaga gaggcggaga tgctgcattc
gcggggagcg 660 cgggcggcgt ggtccggggc ccgcgggcgg gcgaccgggg
tggcaggacg ctggcagcga 720 agcgcgttct ggagagggga gcctggagtc
gctacgctgc ccgcagagcc ctggagccgg 780 ggcgccttgg caccgcgccg
ccagcccgag ggtgcgcggg gagctcgcct gcttcgcagg 840 agaactcggg
cgtcgagccc tttcctccgc gccggggaga cgggccttag gcttctccct 900
gagggcccgc cgcacctcgg cctcccgctt cgttcataag ccggtagccc cggagtatgc
960 ggtctcgatg gccgacctga ttgtaatgca cttcctataa aagcttaggg
ccctgcccag 1020 tcgacactgc tcctgaagcc ttctccctcg ggaccctggt
aggaatggga tccttaggat 1080 cagatttgct cttaccggac tctacagccg
ggagcgagcc aggccttgtg gagagtaact 1140 ttcagtttgg gccaccagag
tgcattcaga atttagaaaa tcccatccat ccctaaatct 1200 gtgtggtcat
aactcgtagt catctgggta ttcagtactg tgtatcccct tatttcgaat 1260
cacagccaaa acatatttta cagaatcttg gaattgtagt ctcgggaaac ttggagaaga
1320 agtatgcaga cattagctgg tttctggaga aaacgtttga gatcagaagc
aaaatcaatg 1380 gcctaattga agttgagcaa gttgggcctg gttttaggag
aaaagaaatg ggggattgat 1440 ttagaaatca cgtcttaaag gagtgtgtcc
attctcttaa aagtgtcaaa tttcaaattc 1500 actaacatgt taaccaagaa
tcccttcatg aaaagggcga aaacgtcggt tacaaatcgg 1560 tttaaacaaa
tgtttgtatg atgctagaag gcactttcaa caccgctcat acggagaagt 1620
tacttagctc tgcctccttc catgtagtct gctcttgcat ggattatatt tttaatgtaa
1680 attgttgtat ttgctgatga agtactggcg gcggcatctt tgcatcgatg
ccggctcggg 1740 aggcgccagg tggtgccgga aggagccggg ctaggacctc
gcgcagcagc gggtcccgga 1800 gtccgggaga ggcgggcggg cgggcgaggc
ggtcgcgggg agcccgcggc gccgctgccc 1860 gcccggtgcc tccagaggtc
actcttccat gcggaatcgc gcagcgccag gcctcgcccc 1920 tcccccaggc
cgcctgctcc agccactctg cactttcact gaccggttct ctttgaggct 1980
gttttttttt ttcttatgag gatttaatat ttctgtttaa atctagttga aagcaattcc
2040 gttagcctct tcagcgttta gttcggtgtg tgtatcttta tctttgcgct
atattaacta 2100 ttagtttgtg tgtatccggt aggagaatta gaaataccta
gttgggagaa aaagaaaagt 2160 agaacaatag ttatttcaac ctaaggttta
gacgttaata acttcttttt gtaatgtgtc 2220 gagatggggg gtcctggggg
gaggtgacag gtactcacca ctcccccccc ccattctgat 2280 gatgaagatg
agtctgtctt tccagctatg tccagacctg cgagggccct gcgtttctgg 2340
aagcctgccg tttgcgcggt tgaggttgct gctgctgtct tgtcctccac agcagcattt
2400 cttttaaaat tctcctgata acggcctgcc tggatgactg gataatgtgt
gcctggaaaa 2460 ggtctccctt gcagctgaat gctagctcca gagatcagaa
agatttcttc ctgtaggagc 2520 cataggaaag agtcctctct aagtttttga
gaatgcatac aaccccctga tgacaggggg 2580 tcgctttcct tggggaagtt
ttatatttat ttccagagga aagtttgaat cggtaaatat 2640 gatgtggcag
gaaggtaatc aaatgcattg aagtttcaca tcagttccta tgaactgtgg 2700
aacaattcat ttgtaatgaa gccgccatca gtaattagat ttgtttcatt cagaggtcag
2760 cttttttagc aggtggtcga cacagggagc atgcagcagc tgtttggata
cagggtccag 2820 aaaacccttt gtaaattcag cgtctccgta actactttaa
tcacattgtc ggctctcccg 2880 tccctgactg tatgtaataa tggaaagatg
tcctgcgtgc tgaaacagta gctgccctgt 2940 taggttattc acattgcttt
gatacgttct ggtagagttg ggtccgttgt agccattttg 3000 gttgtttaaa
gttttggttt tttttttgtt ttttttttaa ttcagcagag aacagtaatg 3060
cctagcttcc gtttttaact taacacttca gtagaacatt ttcttccaag agggagattt
3120 tggcctaagt aaagtagtgg gctctttttt aaaaaaaaat taattttact
ttaatgtgag 3180 caaatctgta ttggtatggt gttctgcaat gcattacact
gactttgaaa atttcgagta 3240 ctaatgcctt atgtctgggg ttaccattcc
ctgtgcatca catactagtt agttaacata 3300 gcattttgct tttcccatgt
aattttttcc ctatataata ctggattcct gatactaatt 3360 gacttgatac
aaaagaatgg ctggatgata tccagataac gtataataca tgggcttcac 3420
cacaatcagg ctctgaataa atacagacct gtcagagatt gataaaataa actacaatgg
3480 atagtgctgt ttaaacagtc cattcaataa catatataag ccagcctgcc
ttccattgtg 3540 tctgaaattc ttatttttgt aggtaaacaa atgcacattc
agcactgatt gaatagcccc 3600 ttgaactatg ctccacagtt tgcgtttggg
ttaatcttgt cggttttaat atagagagaa 3660 aaaagctcaa agcaccaggg
gtggaattgt tagtgctttc acatccacat tcctcacatt 3720 ttgtcaggat
gataaactgt aggtaatgga ctgtcgttgt tctgcaggac aactgagcca 3780
ggcagagcac aaagactaag ctaaagcgat acctcacaac atgcttggta gccttctttt
3840 cagatgagaa tttatttgag aatcatgtgt ctagggactg cacatcttaa
cctcaacagt 3900 tacagcttca agccccagaa acaggagctg gaggttaaga
tgatttgcta agcacctggt 3960 tctaaatctt ttacaaagca taagctgttg
acgctggttc tgccgacgca aagacatgca 4020 gatgactcca acatttccag
aggcttctga cttaagctaa agtgtgtgga caggtgaatt 4080 cgccatgggc
ctggagacca gcttgctaaa aactatgtgt ttgaatggtt cctccagaca 4140
gagtcagctg aagaacaatt ggtggattta tattaaaacc tcttgtctgt aaacttactg
4200 aggtgcatcc ttcggttggt ggatcagtga gataattgcc ttcagatgga
cattgcaact 4260 ggagcaacta aatccttgct gtctttcctt cctctgaaat
cttccaggta gctcccgaga 4320 gcttcagtat gacaccaaac ttcgggcgac
gttttagagt gcgttcacct aatgggaaac 4380 tattcgagat cccagcgtga
ctgcagtaat gcgtcatagg aatgggagtg gcaggggaaa 4440 aggaaataca
gattgtagac cctaataaaa aaatttttag gaaagatatt tctttaacgt 4500
tttatgagaa cttcattctt aaaatactta attgcaaatt agacaaatag aagtgctctt
4560 ctaaggaagg tgattaaact ggtcctccta tcagcctaat ctctgcctgc
ctttgctgct 4620 gacataaaga acctgttttt caggtcactt aatatacatc
tacatagatt tgcttatgag 4680 ctcacccttt gtgtagcgga gtagagcctt
aaagaggagt gctcaactgt ttaaaatatt 4740 ttgattaaaa tatgcagaac
ccatagaact ataagcttct agtcaggaat tagctctttc 4800 agggaacagc
tccccccttc tttttaaggg gggaattaga aggaggctgg gggaggaata 4860
taagaacagc aaagaaggaa ggatagcaaa tgggacatgt tccgaacagc ttggaaaaac
4920 tcctgtggct tcattgtctc tataaagcca aagaatacaa agacataagc
aattcagccc 4980 ttctcccatg atggaagatg taaaccgttg acatgcctcc
cctgtttaac ttgtttaatt 5040 ctcattttaa attcagcacg atactagccg
tgtgaactct gaagatttct ttagtaatcc 5100 attttgtagt tccgaatcaa
aaacaaagtg aaagggtctg acacaatttg cttttatttt 5160 taggcaaatc
aaccctggtc atagttaata aggggattac aactcagact aggtctttac 5220
agatgtgatg taaatcaagg gcagagtata aagaaactga tcccttttga ttgaagtata
5280 gtaaaaaggc atagagaaac tagcagcagt aatctgattg tatggcaata
aaaccaccat 5340 tttctgtctt tcagataaaa ataatgtggt aaatccatgc
agttcataag atgtaaaggc 5400 agataaaggg tgaagccatg gcaacatata
gattagcttg atgttagaaa tgacacgtct 5460 ctgaaaaggg cgcgggacga
aggcccttgc ctccaggctg ttgggcatta tgtgagaacc 5520 acacagactt
ggaaactggg attaggaagt atgaaagctc tacttgtggt ctgggatggc 5580
tgaggcagta aagaaaagct gctcagttct tgctcattgg tggtggataa tatggcaaag
5640 gtagatttca ttgactgcct tttttataga ttgagattgg ggctgattaa
aacttcagat 5700 cactgcagtt gttagggcct gggagatttt cctttttaac
tcctggccta acagcagcag 5760 ccgttctgta ggattaactg cacttcgcgg
tcgttgcctt aatctatttg ggcttcaggc 5820 agggacatgc tgggaaggaa
cagagaccag aggggatagg tagggctggg gttatctgaa 5880 aagaaaacag
agaccttttg atttcagcca tcttttcaga cccagctccc tctcccgctg 5940
catgggagaa gcaaaggtaa acaggacaca ttgtccctct ccctcagcca cagagctctt
6000 ctgtgagttt tgtctttccc accctggaaa aaaagataaa atacaatttt
taaaagggga 6060 gggaggaatt tagttttaat tcaaatgagt agtaatccaa
tatgccaaaa gcagtgggct 6120 ctacctagat gtaattttac tcgtaaatgt
gagtcttaaa ctttgagttg aatggggcag 6180 gctgttagag gtggtgtaaa
ttacaggatt ataaaaatgt tagtgctgcc cagccttaaa 6240 gtcaaaaaca
gaaaaatctc tgtgctgttg agtcttcccg ccctctctcc tgaacaacct 6300
tgtaagtaag ctagactttt gtttttgcct tccatacttt ccatttcagc cattaaacaa
6360 aataagccat tgaaaccacg attgggttcc atgcagagtg acatccgcaa
tcgggtcaag 6420 ccagaaggaa atacttgctc gattgccccc tatttggcat
tacaggaaag tctccacact 6480 ttggaagagt ctgaactctc aagacattga
aaatgccaaa ggctgcaaac accctgtgtc 6540 tttcttgatg gagtgcatct
tggtgtgttt tacaaagggg aattcagtgc tgtttttttg 6600 ttgttgttgt
tgtttttttt ttttaaagag cagcataggg cccttctaga ctcttggatt 6660
ctgtgtctga caaaaatggt cattaaatga gcaatattat aatttagacc catttcactg
6720 attttgttcc aaattctcaa ctgacttgag catctgtttg gggctgtaga
tacattgccc 6780 ttgttgactg tttttctcgt ttctatggga attactgtag
ccattactat gtagctttca 6840 tagactcaaa acatttttaa agtattgcat
ataggctggc catatccagt gcctgttact 6900 ttaccttctt tttctaactt
aatgcagcag tctgtattaa cagatccatt tcatttgtct 6960 agcttcatca
gagagaggct accccctgat ttacaggctg ctcacatcca agcaccttgc 7020
attctacact tgacagtgat tgctaatggc ccattcaact aaagtatttg cttgttaaca
7080 gggaacagaa catgataaat gtccagcaag cttgctgcct ccttcagctt
ttcaaacgca 7140 gactggtgca tatttatggc aggcaaatga caaaagaaaa
agctgaattg ccctggcctc 7200 cagctttcta tcagaaacag ggttaaagtg
attaaagcaa tcattcaaga aagccctgcc 7260 gtttgtttac taaccttcat
ccaacattta gctttgtagt ctacctgtga gaagatattt 7320 cagaagtatt
agagataagg aaggaggatc tagcaaacca gtgaaaagag taggtgacca 7380
gttataaaat gctttccatg cacattgaat gccaggcgaa cctatttctg ttattccagc
7440 agacaatcag cagtggctct agattattaa catattttcc tttcatgtat
aaattcaaat 7500 atgtaattct agtccaaagc attctgtggc tggtaagcac
atacttgctg atttcaaata 7560 agaaaacata gcaagggaaa gctccattaa
acaagttgtt tctgccctta gtaattctct 7620 aaacaagata ggaagaaaaa
gtggacagta gtggagtatt aatagtgtgc tcttttcatt 7680 ctctaaagca
cgagtaagta agcgttcaaa ctactctgtg gtgggcatac atttagagcg 7740
ctgtgaatga accactgctg ttctgccata cttaatttat ttatattatt atttttattt
7800 tattgttgtt tttatgtatt attataatta tttatttata ttactaattt
attttctcaa 7860 tttaaatcct gttgcatcca attttaatta cagtttttgt
atctgccttc ccatacttgc 7920 tacccacgtc cccattgcca ctgcggcctt
atccatgttt tctgtgtaca ccactctcgt 7980 atcaccccag aataattatg
agtgctaccc agacttttga aaccactaga gtcaacatgt 8040 ttgtctttga
ggaaagccaa tgatgcttta gcatttttgg caggggtgga tgtgtgttta 8100
agtggggtgg gtgcagctcc ttattgtctg cctattctac tgttgttccc aatccacatt
8160 ccctgcgggg cacctaacct gtgtgcatag caaagaattt ccgaccttca
gagccagaag 8220 tgtttctcaa ttgatctctt ccagcctagg gttatagctg
atgaattata atccttgctc 8280 tttccacacc tttacctggg cttaccatgg
ccctaaaaca tttgcccaga atcagaattg 8340 tctcatgagt gagtggggca
aggcaaatcc tgttccagac cagctgagaa tgtacctagc 8400 tgcagaagaa
gttagaaagt gtcatctttt acttatctac cagaactata ttcgaggtac 8460
attttagatt taaaaaaaaa gcaagttctc gtaggccttg aatccccccc ttgctatggg
8520 aaaatggatc attattataa tggactgtcc agtaaagttc atgatttctc
ctagacatgt 8580 tctctctctt tatgacctag atcaagagtg atctctttaa
gtcttttctt cataatccca 8640 cagcactttg tacttagatg tacttagaaa
gaaccatata cacggtacgt catgattgat 8700 atgcaagcct tcaccactct
acctgtccta aaagtcaggg acacaccttc ttcatttcat 8760 cagtccctac
ttctatccag cattggcatc cagtaagtat tagtggaatg gacagacaac 8820
ccgaatttgt gctgatggca gtttaccctg ttttaactgt catccttctg ctactagaca
8880 tggatgagac ctgagacgat gggactgctc agaggtccct ggctcttgaa
ctttagggca 8940 ccagaatccc ctgcagggct tgagaaaaca ggggtttctg
ggccccaccc ccagagttcc 9000 tgattcctga ggtctggggt ggggcttgaa
gatggacatg tttaacaagc tcccaggtga 9060 cgctggcaac tgctgcctca
gggccatgct gagaaccctc gccctacaca aacctttctg 9120 ggaaaacaac
tcaacattaa agctgtttgg ggatctctga agaaatctgt agtccttgcc 9180
ttgttggggg agcatcaggg atctaaccat tgatggtgga gtatttgttg ttaattcagc
9240 aagcaactat taagtgttag gcctgttact cggctctaac aatacaaggc
agagtgacct 9300 gtaccctcga gatttaaagt ctaagtcctg tagagagaag
cccaggtggg agcaagcaca 9360 tttagagtta ggtgcttggt gcaaggtggg
gacacagaag aagggaatgg catttgcctc 9420 tggaggggtc cggaaacagc
ctagggagga ggagcttgag tcttgaaata ctgtgggcat 9480 ctctaagcaa
agtcacagta gacagctgaa ataaagaaaa tagtaagcaa gccaaagaaa 9540
cagtatttca gccaagggca gcgtgtgtct atcacgtcca cctgtgaaca cgtcccagga
9600 ttctctgcat ccggccattg ctcaagacag atccctcaca ggaacagcta
agccactgat 9660 ttcagctacc tgttcacgtg agaattatca gtacctactg
cttttcaaaa tgagtatgat 9720 catggatagg tgaggcaatt cagtttcgca
gagacagtag ggcaagtgcc actgtagttt 9780 agttaagggc acatgcttta
gagtttggct atgtgagtcc aatcccagtt tagccattta 9840 ttagctgggt
agctttagga gcagtagcct tagtgtctct cagttgtccc atctctataa 9900
tagggacaat aacataatag tgctgaataa aagagtaaca aaattttggt caacatttaa
9960 tgtatttaaa gagctaagct ccgtgattgg cacaatgaac caatcaatca
aacaccagtt 10020 gttattaata aaagtcagtt gaatatgtac tgtgtgcctg
gccgtggttc aatttgcctt 10080 tgcatacaag gaaaaaatta aaatactctg
ttaataaaga ctatagcata atactttcac 10140 cttaaacttc ttgatgttaa
tttattttgt ttacctgcca aacttctact cattccttat 10200 gactttctgc
tacatgaaac accctttgta attcttttgt cctattaaat taagttctct 10260
ctcctctgct ttcctgcttt tggtgctttc taataacact tttaaccctg gactttctca
10320 ttcagctgtg caactgtgga ctgagaggag gctctttgaa ttcattttgt
atattctagt 10380 agagagtact gtgagcagtt gggttgttga atgaatacat
taattcaacc tggagggatg 10440 ggcagtattg cattttttac attgatatta
catgatattt agaaaactgc ttaactggtg 10500 gacgttgttt tattaacagc
attttgtgta tagcactcac tatgtgccag ctgctattct 10560 aactgcctga
caaatactcc tgaaaccttc atggtaacca tatgagggaa gcacttttaa 10620
tatatccata ataccaacgg ggagactgtg gccaaattgg ttaattaact tagccaaagt
10680 catattgaac taataagtgg atttaaaccc agctagtctg gggccagggt
ccctctttta 10740 atcttctgcc tcctgcttat gctgttgcat ggagtagtct
ttatcatata actaaattaa 10800 gcatgcattt gcttaaagca gtgcatacat
gatggatcaa aaagtttgtg gtataattgg 10860 tttaattctg tcattatcca
ttttgattta tagtcacttt cttatgatgg tcgtgtagtt 10920 ttaaatggaa
cctttgaatc tttgatataa taaggttatg tcaaatcttg ggtataataa 10980
ggttataccc aatggaaaca gaataatgat cagcccattt aaaggatgac tggagagtta
11040 ttacaataca taatagtcat gcatatattg agtagtattc ctttggtaac
attttccttt 11100 taaaaattgt aacatttgat tgttccttgt tgggagaaaa
ggaggtcaga tttttgaggg 11160 gagatccatt tggtgagatg ctgagtgtgt
gtcaagctaa ggagatagta tgacatcttt 11220 tttagagtct agtcacaatt
aaatgccatt ttattttgga ttttgggatc cgtgccagct 11280 tccagcttgt
cagagctgag aagactcaaa tcaagtccag gcttatttct acagcaaact 11340
gggattctgg cttcttgccg gtggattcat tcagtacagc ccatctggct tttgatgttc
11400 tgcaagtttg gagccatttg ttgaaggaag ccaggcggtg aatattggtg
gtcctggggt 11460 tctcttgact ccaagtggtg ccccttggtt tgcattttca
ccatgcttag catctgctta 11520 cctggagacc atgcagccgc cggccagagg
tctccaacaa ccaaatcttc atgcctttta 11580 gaactcagag tccccagcac
atcctccttc ctcctccttg tccaattact ttcatgcagt 11640 tctcagtagc
tgcttgtttg aatcacttat agtatttaac ttctagggtg tttttgggtt 11700
ttggtcaagg taattccagg ctgaatgtgg tgactaagca ggaaataaat gggtcgtcct
11760 caaagttaca gtggagcgct gtttctattt tcctaaggta cacagttgtg
ggggcgatcc 11820 gtatggaagt caggaaccca gtctgatttt gcttcctttt
gatggtagca gtacagacct 11880 ggctgttttg tagcctgctt tgtttttctt
ccttttcttc cctaacttca cgggctgtgg 11940 caaagccctg agacgtgcag
gaaaatgtct cctgtcatac gcccacagca gacctagccc 12000 tgaccctcct
ctgaagccca ggaaggaggt atctgtgaag cagcctgctt gtaaagcaat 12060
tgcacacagc cttgtaaact gtgttactgg gctgattata cttgattggc aaggtgaatc
12120 tcttatagca aaagagaact tggagagttt tatctcatct tatgccttat
taatttgttc 12180 attctttaat tacacagcca cctattgagc accctattta
tgcaaggtac ctggtcgggg 12240 gtcagaggga gggtcccatg gtaaacgaga
cagactcaat cctggaggag caggaatggc 12300 agcccctcgc tgggctgttg
gccccaccaa aagggaaagg tttcatttta ataatacatg 12360 ggtgaatcat
ttttgtcaat aggcaaaatt ctttgtagtt aaaaaaaaat atgatggtag 12420
gaaggaaagg gatgggcaga gggttaaaac aaaagatatg ctctccctaa ctctagattg
12480 tagtattgtt atgcttgtca ctgtagctga attccatttc tttgagtttt
ttcaatgcca 12540 aggcattccc tgtatgactt acgtgagcct ttcatctccg
cgatttttcc cattcaggta 12600 aatgagcaaa tggatttgaa cactcatatc
taaaacaaga gagaaccagc tggaaatgcc 12660 ctttgaattt ctttctctat
gtaaaccatt tttctttctg gtgcctcacc tataaataac 12720 aggagttcca
ccttccttta tagactcttg ctgaaagcat ggtttggaac aagaccgtac 12780
aggtgcacac aaattacagt tgggaaagaa gcctgcagtg catcttgtct ctgaaggtta
12840 tgaaatcctc cttttagtaa tggagctggc gtgatcaagc cagcaggatg
aaatttggca 12900 tttgtgagat cacccccctt ctcacttgcc cactgtacat
agcatcccag ccttactctt 12960 caaatctcca cattttttct tatctagcta
caaaattcat aggctgattt ttttggggtg 13020 cgtgtgtggt tttttttttg
tttttttggt aaataaagac ctgcattttt attttgatat 13080 aggtggttga
gttttgtctt taatttcatg acagagattt aactagtctc aacttttgaa 13140
aagacaacaa tgatatttgg ggatcacaca cttaaagtta gatttctaga tgattaatac
13200 caaagtagat gattttttag cctcagccat ttataggtat gcccttctgt
gaatttttta 13260 tgacagtgaa aatcatggca cagataaaaa ttaaataaat
acttctgtta ttttcctgaa 13320 gaaaaaaaaa aaaagcttaa actatgagaa
tactgtcttt gagcacttta aaataaaatt 13380 gacttcagcc agcaggattt
tgagcattac atcacaaata aaaaacaaga ttaacatcaa 13440 aaggagtcag
ttttcattca attgtgcagc actgtgggct gtgaaattta atattatttt 13500
gactcatatg ctaattgtag actgacagag gaaaatggat tgtgtttaaa taaaaggata
13560 cacagcatca cacgcagctg tatcaaatac aagttgaggt ctttgggcca
ggaactgggg 13620 gccctctagc tctgttattg cagattcaag tttgacaaat
aaaactttcc tttagactgt 13680 agtttaatta ctttttttca aaggtatgcg
tgatgaagag gcacaaatac acctcacctt 13740 gaagagttgc taaactggtt
tgtgtgccga
tcagttcacc gtgtgtttga atttctgtgc 13800 ttctcatctt tccttttctt
gaaaagattt tgcttgtcat tggtgtgaat tgtacccccc 13860 acccccaccc
atctagtctt tgctctcaga tttataacac tttaatggtt ccaaattgta 13920
tagcctgctc ttagacccct tttcttttcc ttgaataaat caggttcatg ttgcagacga
13980 tatttgtttt aggaaagtgt gaaagaaggg gcacctgtga aaacacgcaa
ttgttccaac 14040 acacatatac atccaaatta aagcagaaaa tgtcaaagcc
tccaatcact accttatttc 14100 ttggaggttt aaagccgctg agaagatagt
ggtgccctcg ctggaagttt taaggtaatt 14160 actttttact ctaagcagta
gtatctggta acctaattcc gtataaacct gacaccctat 14220 cgctacaccc
cagtatttct ctgatttcag aataagtctg cgtagaaact tgttctgatg 14280
ttaaagtgca aaagggggca gtaaagtgct atccacaaaa aaggaaaaac attttccaag
14340 tatttcttat tactgcctgt gtctttcgta ggccctgcct ttatttattc
attttataac 14400 aaaactctta tgtttggggc attcagagaa taccttatta
agctgttgca gcaatctagc 14460 attaaatgga agacatgcaa gactgaagat
cctgcctgtt tatgaagtgt gccatcaaat 14520 tcacatgctc atgatgcaga
gtccttcttt gggagtattc gtattcccaa gtgcacagag 14580 cacttcggaa
aggagccttg gtctttggtg ttaatgctct cctagctccg tatagatgtg 14640
gcaggcccaa agtacatggt ggggtgaagg gtcaagggtt tgggcttatc cagagcagcg
14700 tgcatccttt gtcaggaggt gactggaaac accagccaat tacagcagaa
ctgcagactg 14760 ctcatctgca ttcggaattg cagatgaacc agtttgtact
cgacttctct tcttcactgt 14820 aggctttgac atttaattaa aaattaaagc
cttttatgga aaaagtacat gttttccaaa 14880 atggggtaaa ttcgaagtat
acttgataca gaacactggc ttgggaataa acctgtgata 14940 ttacatgact
tttggtttgc aactgctagg ctgagcctct ttgtaaagct gggatttaga 15000
atctttgaaa tgtttgtaca gttcaatgat taagcataaa ttgtatatat tccctttttt
15060 tcacttattt gagtaaacaa gtttgttact acagcttctg tggactcaga
gatttatgta 15120 ttaaataggc cacaacttca actaggataa ttttatttat
ctgcttgtta gggaattgca 15180 tcaaaagttt aagtctgtag gcattaaata
ttttaaatgc ttatttttaa agtcaattat 15240 gaaagatagc acaaagtttt
tctgaaacta cattaaaaaa ataatgtttt aatcttatca 15300 caaaagcatt
gactatttat tgcaaagaaa acacagaaag ctaaaaatca ttctaagtcc 15360
accattcagt agcccaaagt ggtctcaggt aaaggcggtg tgtgtgacca tttgtttatg
15420 gttgtctccg tgcagtcagc aaaataaaca gaacaacatg ccatatatta
ttgatgtgta 15480 tattttcaac tgaaattagc catctgctta caatgatcat
atacactaat ggtataattt 15540 tgaaatgaaa agaaaaataa aataattctt
tgtggagagt aatgcgaatt gacttatgaa 15600 tctcgccctg cttggcagtt
tgctctagag gtagaagagc tttatgtgtg ggcctcctcc 15660 ccccccacac
atttattctg ctcacacttg caccagcatc catgtcagga ctcaccttgt 15720
cctgttacat gagtaacatg gccctgattc tcaagtgcat gataactgcc ataattacac
15780 ataaatatta aatatttaaa tagatcttta cgtgtgtaat attaggtaga
agtggctctg 15840 gatcgaatct gatgcttttt aaatagaagc tttcccacaa
catttccaag cactgtcatc 15900 gtgtctgtct cgatttgggg tttacctggc
ctagttatct gtctgggtgt agaaactggt 15960 agttcctgtt tgtatctttt
ttgttctgat ctctttattc tgtgtcagct aaatattctt 16020 gcagtcagtt
actaacatat taactcatcc ttgtttggaa actttggcat atccttccat 16080
ggtttccttc cgtggacctg tcgcgtctct caggagagcc accaggtata ttgtcacaca
16140 tttcgcatgt attttcagag actacagcag catcaagtgg ccccccagcg
atttgggttt 16200 tcttctcggt taatctacac tctttggcca accgtgagaa
aacttgtaag aaggcatcag 16260 atgtttgtgc taaggtgcgt gtagtatggt
cagaggaaga aagaagcagg gaaaatggag 16320 tggccgtggg tgggagggga
agcagggagt gcaatttcgg gttcactaca cagctctcca 16380 taaacttctc
cactgctggc ttcccacgga tcctcctatt acactgggca aagtgcagaa 16440
atagatcagg cgaccactgc ctccgtccat ttcccaggca ccctgtgaga cccgataatg
16500 caatacaggt cagcagaaaa gtccagactt gacatcccaa cgtgccatgg
tctggtctgt 16560 gaatgaaaat cacatgaggt gacctctgaa ctctaagtgg
ctggtttatg ttttcagtgt 16620 attaggcccg tgttttaaac aagcatgtgc
tcgtagtgta ggttaaaact ttctgttgtc 16680 ttcattaatt atgctgtgtt
ctagtctatt aatattaaag aatattgtgt tgcataatga 16740 ctaatttttt
tattttttgg agacggagtc ttgctctgtc acccaggctg gagtgcagta 16800
gtgcgatctc ggctcactgc aacctccgcc tctcggattc aagcaattct ctgtctcagc
16860 ctccgagtaa ctaggactac aggcgcccgc caccatgccc agctaagtgt
tgtattttta 16920 atagagacgg ggttttacca tcttggccag gctggtcttg
aactcctgac ctcgtgatcc 16980 acccgcctca gcctcccaaa gtgctgggat
tataggcgtg agccaccacg cctggcaaca 17040 taaggactat tttttaaagt
ttttacaatt atgactgtga agttgaaatg tctaaattat 17100 tagagatcca
gtttagatta ctaaatattt atgtctaatt gagatgatta gacttagcca 17160
aagtatccat gtagaagtat tagagtctag attggtgaaa aacttgaaaa agcttggctt
17220 aagttcaata ggtaatccaa gagtaaaaac agattccaat atcagatctt
ttcaccatag 17280 tcatgttaag tttggaagcc ctacttgagt gtttccagtt
ttttccacat tatattgtgt 17340 ctatatttga ttcaaaggca gggcatctat
tgtcttgctt aggactgatt cactgggaaa 17400 agccactgga gttgcctatt
tccactcagt atgcctcact cttagagtag cttcccatgg 17460 ttcccaggca
ggccctccag tgagaatgca ccaagccaca cgccatggcc tgggaagcag 17520
tcctgaacct ggagattgtc ttgatggaaa ggaagaggca gccttcccct cccaggaaga
17580 tagtagagag cctgctctga cttcgctcag ggatggaact ggtctggctc
agttctctct 17640 cctgtgtggg acatgaatca ctcttggtgg tctttgcttt
ttatttgggc ttaaaatcag 17700 cagactttat taaatgacac ctctctctaa
ccactctctg tctgggcgaa gtttaacaag 17760 aacagcctcc ccccatgtgg
tatgggttgt aactgtggcg gtttccctct gctgtttttg 17820 gttacaagat
gaacattatc tgaacacaca gaaagaaatc tgtatttggc atccataatg 17880
gaaagtcagt ttagtaattt aaacttagcc agttatcatc atcataattc tttttaacac
17940 tttcaaagtc agcataggag aagtgtattg ttgaatatta caaaatattt
agggcataga 18000 tagatgtgct gtgtagtttg atttgttaat gtgtctaagc
aatcaaagca acagaattca 18060 aatataaacc ccatcacttc caaaatagga
actctgttta ctgacttgat tataacatat 18120 ggaactcaat tgttttccat
taaaaaatga tactattagg aaactcaccc cattttcttt 18180 tcatatatat
tctgctattt gcataattgt ctggagtcca tatgtaatat taaatgtaaa 18240
acacaaatgc catgtagctg gtctgtttct tcctcacctt ttggttcctg gcctcctggg
18300 gaagggttgc acatctgagc cgtggtctca gatgactgcc tcggaagaag
cctcttccct 18360 tcaggcacca ctgatgtgtg cttggtgtgg agctagactt
tccctggctc tccatgtgac 18420 gctcacatgt gcgtgtcttg atttccctta
acttcatggc ttatctatga acagcttgat 18480 ttgggggaaa aaaatgtgtt
tcccaatgct ggagttataa ttgaatgtgc tgcagtcaaa 18540 actgaaatgt
gtgcagagaa agggggcttt tcctgtcatg ctcattgggc accagtgtgt 18600
cttcacctgt tttgtgtgtt aggtccatgc gtcatgctga aatgaagaac atgggatgta
18660 tggggctttg gacagtgctg agccaaaagc aagtgctcaa aagcagctgt
gtttgtatta 18720 ttagtggttc tggaggtggc tgattgcctt gcattttaag
tagagaggga ttgtagaaga 18780 ctgccaatac ttagaacttt ttccagagag
gaagggtcag aaactgcatc tgcagggctc 18840 cttgctctcc agaaatgcca
gtgtgcctgg gagggcatct tcagaaatcc agtctctcct 18900 cctcagtgtg
tcctgtaccg actcagtggt tctgtcttca gaattcctat catgtctgtg 18960
atctgcaaat agtggtattt aatttgactt caatttgtat aaatgttagc ttctatttgt
19020 tcattcctat tttttgttca attaatacat tatttattga gcatctactc
tgtgtcagcc 19080 ccttgggtgt ttaatactga attagtcaca tgtgggactt
gcctgccctc agggagctag 19140 actataaatt cctaatgatc agtggtctcc
acttttctgt cactcataat gtctggcaca 19200 acataggtta cttgagttgt
tacactcaca gtactgttgt ttgctgccat ggtgctttag 19260 gaagtgtgag
agttcccggg aggcagagtc aataatgcag actacacgta gtgaaaacat 19320
ggccaggaga gctgtagttc aggctctcag ctcaactgca ctctgtccac tgagaagcca
19380 taatttcttc acttaaagtg actgtgcgct atggctgttt atatatacgc
ttaaaaagta 19440 aaagctgcta aaccactcaa ggattggggc cttttgtatt
gatttaatta aaggaacaat 19500 cattgtttta atgagctcta gaaacaatta
cttttgaaga gccgaggatc aaattcttgc 19560 ctcacgtttt gccacagtgt
gttctgaaag gtgaattaat gcttttggaa tcatcaggaa 19620 tagtgagctt
tgtcacgatt tactttttac aagcgtatct aatatgcata ttgaaatgtg 19680
agcctcccca ccacacttcc gctttgataa gcatcccccg gattgccgtc actgaccatt
19740 atagattttt aacaaagttg gacagtacac actgaatgaa aactttacat
caaggaaggc 19800 ctggcgtgtt tgtaaaatga attaaaaggc tcattaaatg
atttatatga cttacgcctt 19860 ctgaaaatat ggcctcaaac acagagatcc
ccaaagccac accgacccct gcgtcccatg 19920 ttctcgacct caccgcatca
gcaccagcaa gacctgtcgc tgagacggtg agtgatgaga 19980 gtcaagagga
gtgacttgca tggcctggga ggaaacctcc tgtgaatctt tagttaagca 20040
ggaaaaaaaa aatcctcatg aaggaaacag gatcttggga gcattttgaa tgaagaagga
20100 gcttagtgag ccaaacttga gacatagggt gtaatgtggg agagttttaa
gatttgcaga 20160 gatgtacagc ttgggagggg gtgtaatgca ttttcttaaa
agagctgaat gaatggttga 20220 ggaaatgggt acatctggtt tggttaagga
tcctaatctc tgaagcctgg gatgccccca 20280 gggcttgtaa tttaggaata
cttcccctaa tagtagctaa cccttatata gtgctgtctg 20340 tgcaggctac
aaaaggagca gattaaggat agaaaaggtt tggagtgtat gagaaaccct 20400
aggcaggaat tgactcctgg tgtttgtaaa ccttaaagat gtcctaaaaa ggtcaaggaa
20460 taagacagga gaaaaaggaa atgtcaggaa gatgatcaat ttaatgttta
tggaatttag 20520 tttgtactta ctgcccggca tcttgcctga ggtttttaac
ctcagcagca catcagaatt 20580 actgtgtgtg tgttggaggg gctgggggag
ataaagaaat tagcctcatc ccaaacattc 20640 tgattcagtc tgttacttga
gaaactgaat tgtgttttgt ccataaagaa gatgaaattg 20700 tctacagaga
acacattgcc attcacaagg ttgaggggat accacagaga ggctcccact 20760
gtgatttgca tttgtcaaaa gttctagaga attcttcaac agtacacaca tggttgtttt
20820 aaatatatca ttgttataaa aattcgtttt gagttctgtt tcacagaaag
tttttttgaa 20880 tgaatgaatg tcatatatcc ttgctaaagg agctcagtta
aaaaaaaagg gaccatcctt 20940 ctcttttggg ggttgtacag taacacattc
ccaagaaaga ggtaacagcc acatacattt 21000 ttcttcccaa taaagagtgt
gggtttttaa tatgaatcca tagtatgatt tctgttatgt 21060 tttgtgctgc
ttcataacca cactcatgca cttttcagaa aattaatacc attcattagc 21120
ataaatcata aactattccc ttggtatggg tttgaaattg ggggtgccct atcatccttg
21180 ctttatctct tagtgaatta tgaccctgta gtcatcatgg ctggtgggcg
tctctggtta 21240 aagaaagggt tggattggaa ggattcagag gcgattcttt
gttcttaggc tttaatattt 21300 taatgagcct gcaggcttgg ctgcttacga
acgagctgag atttctaagt gtgttgttag 21360 tgttagcact tgtagaagga
tgttcattag gaagttcttg tttcagtttt tcagagaaac 21420 tccccattaa
gaaagatcat tcaggaacat ggctaccaag aaagaggaaa gggaggaggg 21480
aggctttcag ctataagcat taaggggata ttgtatcagt agtcttagtt ctaaagattt
21540 gcttctgaga attaattgga gcaaatacat ctcaagggaa gaaaaaaaaa
gatttatagg 21600 gcagggacag tagttgtcct tgcaagtaga ggacacttca
ttttgcagct gaatcaatac 21660 cacaactaat tatttctggt tatcttttac
gcatttgtaa gacattgctt ttgttcagtg 21720 taataaaaaa cccattgttt
gatcagtgac tgactaatta tgataagtaa tttgaaacat 21780 tcttgatgaa
acttgtctgt taattaacat caacagcaca gggaaactaa caggacaaca 21840
aagtattagt ggatccactg ttccctccaa ttgacgagct ttctctgtgg catgcccaat
21900 aaactaaagc tgccaatggt taaaaaataa caaacatgtg ggagatctga
ctcaccacgg 21960 aggaagagtt atggtaaagt tacacaaagg agtactgaaa
tattacaagc gagggggtgg 22020 taaagaaatg tcagcaggta gcctgatcct
acagcttaga gtaaggaaag tggtttcttt 22080 ctgtctttcc tttttctttt
aaagcttaat tccaaaatac attcatccca tattgatctg 22140 aagtaagaga
cttttgataa attaaagtgt gaatctgaaa atgtgtagtt tgggattatg 22200
ggcattgcct ggctatcttg taactgtcat taatactgtt aatttttatc aactcaatgg
22260 cttttttttc ttatgctttt agatttctac ctggacaagg actggtacta
tacccacaga 22320 taggagacaa attggatatt atttgcccca aagtggactc
taaaactgtt ggccagtatg 22380 aatattataa agtttatatg gttgataaag
accaagcaga cagatgcact attaagaagg 22440 aaaatacccc tctcctcaac
tgtgccaaac cagaccaaga tatcaaattc accatcaagt 22500 ttcaagaatt
cagccctaac ctctggggtc tagaatttca gaagaacaaa gattattaca 22560
ttatatgtaa gtataatttt attcatttat tttatagaaa ttaagataag ctatataggt
22620 ttgtatcaat tttttgtttc cttaaaatta ttgtgacaaa taatttgatg
aaaatctatg 22680 tggaaaaatt gtcccccccc cctttttttt tttcaaagaa
aacttcattg aatttgggac 22740 cctgtgctac cagtattcat taagtataca
tacccaaaga gaaaaaaaaa cactagaatt 22800 cttaatagta ttgaaataaa
tgtattatat gaatatattc agcatctcta ctgacaaaac 22860 catttttaag
gaccattggt ggattttgat aggtaaatct tgtgcattgc cttttctctt 22920
cacccatcca tccattcatt cactcattca tttcgtattt attctgtgcc agagactgtg
22980 cttaagggct agggattcag cagtgaaagg tggtaaaata gcatgttttc
ctcaagaagt 23040 taacagtcta gagaagatgg agctcataaa ttcgaaagat
ggggatgaca ggtcacatta 23100 aaaccagatt cagaagaaaa agacgaaact
tggtttgctt agtacattac tcttttttgc 23160 atacatatat ataatttgac
acgctgtttc aagaagagat ggtacgtatc ccttgggtca 23220 tatctgaggc
tgacttgtga ggatgtgaag tcagctgatg agcacatttg gagcccacgc 23280
ctactatgtg cagatctctc gtcagcgtca ttcccagggc cccaggtggt gttaaagtct
23340 aggtgactca gacagctgtt cgcgtcattc aagcaatgaa gtcttttttc
ttaatttctt 23400 tggtttaaaa ttatactcat aattaattgg gttgaatttt
ccagtggctt ggttaccata 23460 gacttcagtt tattagggaa ctgctatctg
ccactggttt attatttgcc ccaaggtgga 23520 ctctaaaact ttaggtagga
gactcttggt gatcaaactg aaactcttgc atctcaacct 23580 atgagccgca
ctttattgtt attttatttt tttagagaca gggtctagct ttgttgccga 23640
ggctggcgtg cagtggcatg atcacagctc actgtagcct tgaactccag ggctcaagtg
23700 atcctcccac ctcagcctcc aagtagctcg gactacaggc atgtgccact
gcacccagct 23760 caagagctac acttcaaagc acagaatgaa aacctatttt
taaagccaac ttgatacata 23820 gagtagctta ccaagaatta gtaacaacaa
caacaagaaa aaaaagagag aatgtggtag 23880 agtatatact tagtaaggag
taattattat aaaataaaag cattctgaaa tgaaacaggt 23940 agatggggtg
gccaagtatg cagcatagta gggaaatctt tgaaaatgta aaatagttac 24000
caggtaaaat aaatggaaac tttaagcttt tggaagccta acaatgtatt tatattagta
24060 aagactttat ttttttattt tattttattt tatttttgag acggagtctc
tctctttcgt 24120 caggctggag tgcagtggcg tgatctcggc tcactgcaac
ctccacctcc tgggttcaag 24180 tgattctcct gcctcagcct cccaagtagc
tgggactaca ggtgtgcgct aatttttgta 24240 tttttagtca agacggggtt
tcaccatgtt ggccaggatc atctggatct cttgaccttg 24300 tgatccttcc
gccttggcct cccaaagtac tgggattcca ggcgtgagcc accgcgcctg 24360
gccttagtaa agacttttaa agtaagactt tttcagtgaa agctactgtt aggcatgaca
24420 tttacaggca actgaaactg atcagatgca tttattaaga aggttaatgc
ccctaggtgg 24480 ggtgggagaa agaaggtcgt ggtacgggaa gaggggacac
actagagatg agatgcccta 24540 gggcagtgaa cgcatgtccc taatgcgtgg
atgcagccca cgtccaccga taatgccgac 24600 acacccagag tctctcttct
tactttagct tatgacttca cgaagaatgc tttgcaaatt 24660 ctaagttcgc
actgggcgca agtggaattt tagtaaacat taagagttta acctttagtg 24720
tgaaataata tgcaagatat gcaaataatt gtttaccaac atctctttgc ttaatgtggt
24780 gagcatttaa taattgcttt ttattaatac atgagagatt tgtatttaga
agcagtttaa 24840 tttataatta taatattaat ctacacaata acgacatcta
ttattttctt tttttggaaa 24900 ctcttcatac cacactaaca ggttcattgc
agttactgaa ctactctggc catcagagct 24960 ctccttagag ttacgattta
ccatgcaaaa gcatatggta gcctgggata aatgaatctt 25020 tcttaataca
gaattgaggg tctcaagttt gaaactacga gaggctattt gaatgttgct 25080
ttgggggact gtcataaggg ctgggtggag gactcagggc taagaagttt gccaggaagt
25140 ccagttgaga ctttcagcag agttgaaaga cttccacgat ggcgtaggca
gaggaaggcg 25200 tttcagatac ttgggaaaat atagaagcca atttctcacc
caccctacag caaagctcat 25260 tgatctacaa gtttccctag aaaggaaatg
ggaaatgcag agaacaaatg ttaaaatagt 25320 tttagaaatt aatattgact
ttgtattgct tctgcataag ttccaagaca ccaaaacaat 25380 gaatggattt
taaaaagtca ctactttgca tatcagacaa atgcacacac acacacacac 25440
acacacacac acacacacac acacacagtc aagctctgta ctggcttttt tgagaaggaa
25500 agtgtttgaa gttagtaatt tttatatcag tacatttata aatagtgcta
ggtagcatga 25560 cggaaagtat taaaatttac atgtatattt ttaacacttc
aaatcgttgg ttcactttga 25620 gacagtaaat aatattagca tttgagttca
gctttaataa attctacatg ggtttaaccc 25680 caaatctgag tgtctagttg
gtaagcgcct tcagaacgag cagtgttata ataaatatgt 25740 tattgtgtgc
tggtttcttt ccatggagag gaaaaagaga cctgatgctt tggaggagtg 25800
cttgactttt ccccagtgag gagtagtcca gagggactga cttgcattgg ggagtaccct
25860 acatgaacag catttcagaa gaattaaacc aggaacctag agtcctactt
gctagtcctg 25920 cttcctaagc ttaatgagaa agtcaatttt atttctttga
actttaattt atttccctaa 25980 aaaacgcttt tagtattgtc attgttctgg
ctaatgatgg cggtctcctc cagtttcaag 26040 ccaccttagg gctgggcata
caaatgcaat ataggatcac ttgttagtgt ggtttcaaat 26100 ggacatgatc
ctctgtaaat tctttaaaaa catttaattt gatttgtggt gttacctgct 26160
ttaaaatata gtcatcacac ttgtgagttt cagacgtgaa tatgaatttt taatttgaac
26220 tgtattttta aacacactaa gtattaacta agtcccctta ggagatatgt
ggcaaactga 26280 tatgcatcct cattcattct tctcatagat ggttatttgt
tttttaactt gtggcaaaat 26340 tatatatgaa tggtcaccga cttaaaatag
ttccacttaa atttttcaac tttctgatgg 26400 gtttattgga gtattaaatg
tattttcaat ttaatgatat tttcagctta ccttgtgctt 26460 atcaagtatc
aagacatagc cccacctaag tcatggagca tctgtatatg ggtttttatt 26520
cttgtttaga attgactttt tcaagtgacc tatttcagta attagccctg ggcctgattt
26580 gcataatgag atctcctaat cttcaagtaa tgcaaagatg gagatattat
ggccatgtgg 26640 tctgaagaga ccttttcttt attatgttca gatctttaat
tgccttaaaa atagagtagc 26700 taatttacct aacctctagt tattttatta
ttgtctttaa agtttttttt aatgttcatg 26760 aaataactgt tctgaaattg
cctattttca agggaagctg tgtcttagac ttactaaatg 26820 ctccagttga
tactgggaaa gccttcttgt gttcgtagcc tttatccgta gagttttctt 26880
tgcagcattt tctgtgcctg gtttagtttc ttttcagagg cgacacccag agctgaatga
26940 gtcagcaggt ttggtgtgtc gaccctttgc aacagctgtc cttacgaagg
ttctgtgggc 27000 tggttattct accttcgcat aaaaccttgc aaaataaccc
acaaagaggt tttcgtcaca 27060 ctaccaaaat catgtgagtc agagatggat
gaaaaatgaa tgccattgtg ttcatacttt 27120 tccagtgaac agtagctaca
gcagagctgt tagacaaaga aaaccgtatt aatgaagcgc 27180 ctcccaattt
agcttcatat ggcttttgca ttattttgct gcaaatccat agctaagaca 27240
catcttgtgg catagtccgt aagtcatctt tccgaaggac tgtttgatta aaggttgttc
27300 tgtgagatcc accctgtgtt gttcatggca tcctcttgga ggcctccctc
actctccatg 27360 ccttggcaaa gtcttcctta aggaacactg aacaagtctg
gagaagctgc catttcttag 27420 ggccctcatt ggttcagttg tctatagctt
tttatttttt attttttttt taataaagag 27480 tatgtaaaat tggaaagctt
cacaaacagc tttgctattt tttagacatg tactccactt 27540 ctaagcaaaa
tcacaaaata aagtaaaatg cttccacaaa tataatgaaa caatattctt 27600
aaagaatcaa agcagaagaa cttcagagtc tgttgcttat gttaagcata tatttgtttt
27660 cttctctgct tttgatttac ttatttctgg ggtgtaggtt tggcaagtag
tactgaaacg 27720 tactgaatgc actgttcttt agcaagatag ttacaggagc
tttcaaatgt cctcttaaca 27780 tatagatttc ttttagaata tagaataatg
tgtgggctgt ataaagcgat tatgtgcttt 27840 atttgatgaa ttatttatgt
acgataaatg tagcaaaagc cacatttcca tcattaaatg 27900 taatcccatt
tggtgataca gcaacatcag cctgtcattt gggtcctctg attgaggggt 27960
gaggatttct gtttgatacc ttgtgcataa tggctgcgtt caagcattta aactcatttt
28020 tatttctaac ctacagctgt catctttgta ataggatatt catcagaatc
ttgccagaga 28080 ctgtgcattt gggatcttgg gggatacagc accaccacca
ccctccccct gtccaagaga 28140 aacagatcaa catcttaggt tgagagtctg
gggtctggaa gacccgagtt cctgagtgcc 28200 ctttgacaag taacttaacc
cctgtctgcc tcagtctctt catctgtaaa gtggggataa 28260 tgacagcacc
tgcttcacag ggttgatggg aatccagatg tggtgggata tagaaaatgc 28320
ttattacttc cacctttgac accaaataca tataactaag agttaacttt ggagcagggg
28380 aggaagtgtg aggctccagg ctggaggcag acctgtgttc ggctgcaagc
tggagaggat 28440 ggaccccaaa agcttggctg atttgaagtc catccataaa
atggaactcc agagagttta 28500 cacgtttcag taatgctgca taacttaatt
ataagatctt ctctctttgt cttctttcag 28560 tgttataaaa gctcttttgt
ccttgagctt cctttaccaa gaaacatgca tttatgtatc 28620 tttttgttca
tggaattgcc caagcttgtt agcagatcct ttgtaagacc caaaagagac 28680
agacagggga ggagtcttca gatacatata atcatttttc ccaatttcca tgttaccagc
28740 cttgccagga ctttttctca gttccctgtt acacaatgaa aatagtgtct
ctttattgat 28800 aattttagta gcatcctaat gtggtataaa
tcgtcttcca gagaagaaaa tgtgtcaggg 28860 ttgcgttatc actgaggcta
gctgggaaag tagatcagcc cattagtctg ataattcgaa 28920 gcgttgtttc
tgttatttct gaacatcatg tgaactcctt ttctgggtgt attaaaggtt 28980
ttcccagtgt gtgtcagtga gactcctgat tgaatttaat atgaataaag ataaattctt
29040 tacatttaag gattaaagtc tcagcttctg cttaacttga gattgcactg
agaaactcct 29100 ggctctcggg tatagcggag tcacgacctg gggatgtctg
tcccatatgg ctctgtgtgt 29160 aagaagaaaa agctgctgtg gacggagact
ctgttcacat taaatgacat cacctaagcc 29220 atcatgacag caagaattat
ttaggaattg ctcagaataa aactgccttc attatttcat 29280 aaaatgtatc
ttggtatctt tagcacctta tttatggctt tttaaaggtt cactgggatt 29340
tataaataat tggacaatgc tagagaccta gtacaagaat gaaagaggac aggcttcttt
29400 cttaataacc tttaaacatt catcaggaag ataaaacttt aaagcaaaat
aaaacacatg 29460 aaaatagcca agatgcacag accagacaag caaatactac
tttaacttat ttgtatagtt 29520 cttaagagtc acatttgttc ctgaagtttc
aaaatctcgg gctgagtgtt tgatcactta 29580 gggaagtgtt gtggccttca
catactcttg tctcactttg aagtctagaa acacaggtct 29640 tagagcaatt
tttatcactg tgagaaagct gaaacttagt gtgagtagct tagtacaatt 29700
cagttggcca tcaaatgtca gaaacaaaac tcagtccagg gccgctggac ccttaggccg
29760 gcgttgttag tttacaacag tgcctcctgg gtccaaacat ctaagtgcac
atgtagcaat 29820 agtaaagata gtatgtatgc atacataaca catatgtaga
gacagcagag tatacgtaca 29880 cacatgttgc atacatagca acagcagaga
agctcatgaa ctataaagga tggactgtat 29940 gcttgtatca gacattttgg
tactgacgct ttgtcatata ttgtgtaaca tataaccagc 30000 ttgcaatcat
ctgcccccaa agttgaacta agaaaatcct acagggtact aggaaaggaa 30060
ggccattggg aaaaggtggt tatagtggca atttgttagc tcttatgaat tttctttttc
30120 tttttagaca tactcttaat tccatttttt caataaatct atactatttt
gtgtttttat 30180 gttagcaagt actttaagcc cctcaataga aagttgctac
atcatatagt gattaaaaat 30240 aaaaatctct caaacataca agtagaggtg
gtatgagact tcaaattccc ttagccaagt 30300 acaagtgcag cagttttgtt
ggctggctgg ctgcatagaa ggactgatgg attggcagac 30360 cctcaagctg
gagtgtaatt gatctcatta cagaggagcc aggctgggtg acagttgtgc 30420
tttgcaagtg gttttttgca ttggtgaagt agcccatttt gttgttcctg atgttaaaca
30480 ggggatgaag gtattctttt attggcacaa acgcgggaaa ttgctctgga
ttcttagagg 30540 atagaacatg tcccctggac ggaataaggt tcatgtgtag
ggcaaattta gataggggca 30600 ccttattggg gttactactg gtctctagat
ggtcaaagca aacaacatgt ccatctaagc 30660 tgtgatgtcc atctaagctg
tgtgtgtcca tgagagtgac gcattttctc ctctgcagtg 30720 ttgttatatt
ctaaactgtc agcagacatt aattcggtcg ctggtgaagt cccaccgcct 30780
agagatgaac tctgcctccg atggatgttt tccacttcag tgccactcgt ctcgcaatta
30840 ctgggtcatt aatatcattg catgcaatta gtgacagtag aaagagctag
agggttgtgg 30900 gatgtgcacc ctccccacca tgaacttttt actctgaccc
tttcccagct agaccttttc 30960 gtatcttggc aaggatattt taatgattga
gactgtcaga atcttcagag caggcactgg 31020 attatgtgct ggaaataatt
cactcaaaca cctgcttctc catggttcag aatattttca 31080 ttagatatta
tcactatccc ttccctggga agtttcattt ttaaaaatct gatgcttaag 31140
tacagctaat atagacaata gggaattatg ttttatcttt agaactctta cattattctt
31200 ttctttaaaa atgtgagctg agtcattgct attgcagtgg tcatctggcc
gcctattttt 31260 aaaacacaat tcctctatct tagtagattt tggcccatat
taagcatatc aagaatgact 31320 tttttttttt caagacatgg ggttttattg
ggggcttata tacaaggaaa gagagagtcc 31380 agtggcagtg ggctggacaa
gatatccaca tggccctgtg gcagtgagct gggcaggaaa 31440 actgcaactg
cttgcaaaca gcatgtagtt catctatagc attttcactt aacaccaccc 31500
agctaatgac ttccacctgg caaccttcat ttaatccaga acttaggacc tcgagtccct
31560 gtacggccca tgttccacag gatgggccga gggctcagct gttcctcata
gacaaggaat 31620 gactctccac attggccact cccggattcc ctagctcagg
acacatattc aggtgtgtct 31680 aaggctggct cttctatgtg aagttactta
ttcttttacc attgactctc atgttcccac 31740 tatattaagt ttttctgaat
tactgtggca ataagaaacg gtcccttaaa ttatactaga 31800 agaaaagctt
tttttttgtt ttgtttttta ttttgaaatt atgttaaatt ttttttctta 31860
actgagagat tccacctgca taaatcgtca taacttttaa cagtaagatc ttagacttag
31920 aaagtgatgt ttttcctcaa cagaatttat taaaaatcaa gacaccaagc
tgttccaaac 31980 aatagtttga ggggaaataa aataaacaac tccataaata
atcttatgtt gttaaacatg 32040 tctctagcaa aacaaacaaa caaaaaagtc
gggggttggg ggaggtgcag tttattgcca 32100 gtactgtctg gtctttctca
gaaaagcgtc agtgtacatc actgagcctg gacggtatgt 32160 tttcttgatc
tataccccct atgtgtacat gtgcttgcac gcacacacat gtagacacgc 32220
acacatgtgc acctgccatc actttctgct cttccgtctt ttcactcttg agtgtctgta
32280 gccagtagct ttccaggtct gtatagtcaa agatacctat ggccctgaat
gtcttcactg 32340 attgctattt gacattcata cggtttttaa tggttaaaag
gctttatgcg aaagctgtga 32400 tagaatttct cctgttctag atgtggtgtt
tattgcttta ttttgtgact tttctctcag 32460 tagattgacc ttctccctca
gtgtccaagc ctcgcatagc atgatggcac ctgtaaactc 32520 agttctgtat
cctggtatcc tttctcttcc caagtagaag caattaagta atatatgtca 32580
tcaaaacctt ttaagtgcac atacaaacaa aatcaactta ccaaactgct tcaaagttgt
32640 tccatgttta acactcttct ttctgagctc tgggtagaat gtcctattat
tgttcatcat 32700 gaatatttga aattaaagaa ataaaactgt accattttct
ttaagagcat ccatttgtac 32760 ttgataacat cttcagtcat atttcaatgc
tggcaaagag gaggggagtt ctaaactgtg 32820 actcaatttt agaatctact
ttttccaaat tattctgttt agtgcagaaa actaattaat 32880 agtgttgcat
agaaaagtca ctgaagctaa gccagttatt acttcttaat gcatgattta 32940
ctgctttaag ttttcaaaac acaaccatag caatgtggta ttaattcaag tgattcttcc
33000 tatcatattg aacgatattt tcacgggtga aaaactcaca catcctacat
cactgatagt 33060 ttatacagtg ttttagctgt ggctccctgc atgcaaaata
agagttaatc aaatgtcagt 33120 gagaaccatc tcatcaagta gagggcttgt
tttgtttaaa ttaactttgc taagtataaa 33180 tttcttcttg aaaataaatt
ctgggccggg cgcggtggct cacgcctgta atcctagcac 33240 tttgggaggc
cgaggcgggc ggatcacgag gtcaggagat cgagaccaaa ctggctaaca 33300
ctgtgaaacc ccgtctctac taaaaataca aaaaatgagc cgggtgtggt ggcgggctcc
33360 tgtagtccca gctactcggg aggctgaggc aggagaatgg cgtgaacctg
ggaggcagag 33420 cttgtggtga gccaagatca caccactgca ctccagcctg
ggtgacagag cgagactccg 33480 tctcaaaaaa aaaaaaaagg aaaataaatt
cttctgtatt tttctttctt caagtgaggc 33540 catttagggg aaagtatacc
ataaaacttg ctctaagata aggcaaattt ggtattatag 33600 gatgaagtgc
tatgtgattt gaagtaatgc tgaatttttt aaatatatta aactaaacaa 33660
gaataatgag gccctcggaa agtcatgatt atatttctca tttttctcat tttaaagcca
33720 cagtgaaaaa cacataaaag gaagaagtta gaaaaaaaaa tgaatgaaat
tctttttttc 33780 cttttggcaa attaaataga tgtttctgtt tcagaagatt
ttattaatta actttaaaga 33840 aacagtcatt tatttttggc attcagtgaa
cactatcatt tccatgttta gaacttttct 33900 tctaagttag catcttaaaa
gataactgtg aaactcaagg cattcaacta cattaatttg 33960 agtttcagaa
attgaattct tgtttctaga gtacatagtt tgaattgatg tcagggtgtt 34020
aaatagataa atcttagctt cctaggttgt atattcacac taattatttt tttatcagcc
34080 ttcttatttt tcaacttacc ttattctttt tgtttttttg acactcagat
ttgatagccc 34140 tgtggtagaa gaaaacagta atacagtttg gtttgttgtt
gtgtttgtgt ttattttaaa 34200 gtcacggctt tgctttccat gttgttactg
gattatgctt tttttaattc ttcagtttgc 34260 caagataaca gtcttccgat
cttcagaagt ctgtatcaag cttaaggaaa ctgatgtgta 34320 ggaagactcg
cctaagaagt ccaaattagc aaggctagca tgtgaggaca tgctggaaaa 34380
gaatagttcc catagatatt gacagagaat gttcataaaa tgctacttgt tttgtggtta
34440 catgagagta acttgtgtcc agtgcagctg tatgtaaggg caacgttttt
attctgacga 34500 ctctgtggtt ttcatgaccc tggatgctta tcatgtctct
ctgttggact tcttcaacgg 34560 agttgataca aatacttgct tccaagtgtc
catctgccct ctcctccatc ctggccccat 34620 acaaatacgc tacattttta
aataatttga aataccctca atagtattta tatttcctgg 34680 tgcttcattc
tttccataag aactgtgata ccattattct gtaggatttt tttgtgcttc 34740
cccgtttcac atctctgtgc cagtgagacc catatatcgg tgcaaatcca gaagtttgat
34800 tgtccatctg attagcacac tgttagcaat gtggtggact aaacacagcc
aagatgtggg 34860 gctggagctt agcctcctgg gagcagagcg gtgaacatca
gatgaagaca tgtgaaaatg 34920 gagtactact tcctcttcct ggggatgggc
taaaaagcac agccagaaat attcttgccc 34980 ttccagtctg ctttacagtt
actcactggt tctctttttt ttcctactca gataaccagt 35040 atactcttcc
cagtgactaa gaactgcaga taagtatagg tgcaaataga tggcaaaccg 35100
cagatggcag ctgtgtggtt tcagatgtgc tgcagaactt ttagacgatg tgaacgcaag
35160 gaactttttt gctgagcagt aatctctacc cactggaaat taggccctgg
ggggaacaat 35220 gtagtgactt ctatatactt actacatgca gttagacccc
tgaagcaaaa gcttttaaaa 35280 acaggctgta aaatgcccat gtatctttat
taagcctatt ttccaactgg atagagaaat 35340 tttctggtaa tttttaaatt
tgtaaagtct atttttttcc tgagccaagg gaaaaaaaat 35400 atctgggccc
taaaagctta gttataacaa tgttattttt tctatctctg aatgattaaa 35460
tgtgatttca tttatgtagc aatactatga ttgtggctgc attagatcac gctgatagaa
35520 agatacaaag aaaaactaag tataatgaac taacaattta ttttcactct
ttctctaagt 35580 taaaaattcc cagtacattc aaatgaacaa tgaaaataat
tgcagaattg tctcctgaaa 35640 tggaaataga ttttttttcc caagcattag
caatttcttg ttatttttca aaatcagcca 35700 ctaagccttt cagagcttct
tggtgactat tgcaggagaa atcagaatat taatcttgtg 35760 gttttatttc
agagttcgct gccaggaagg aggtataatt gggataggag actttttttt 35820
tttagctgtg tcactgttca aggagggggg tttggaacct cagcataaga attacactct
35880 gtgatgagga tgtagcaggg gagaagaaag gtgattttca ctatgggaag
ctatacttac 35940 atcaagtata aaatagactg aagtcatttt gaattacgtt
atacttgtaa agtttacctc 36000 ctggagtttc agttagtacc agtgtactaa
ctgggttaaa acagttcatg gcaccttaga 36060 tcatttctaa ctcatggcaa
aaatctttcc tggtggaacg tgtaactgta ttttaaatgc 36120 ccctttataa
gcaaccaagt atttgggatg ttattttgat attagtagtg aatttttcag 36180
tatcttccag taccctttgc aagtcacagg ttgacttaaa aggaaaagaa gcaaaatgct
36240 gaatatagca gaaaaactgt ctgcattcag actgttcagc ccacttttgc
tccccacgtg 36300 gcaagcacac tcccccaaac aagcaatagc ctgtggcttc
agaggaacct acaaaggcag 36360 catctgtaga tttttccttc ttcaactcta
agacttgaat gtttccctct tccccacaca 36420 cttttttttt aaaccaagaa
ataaaaaagt tttcactctt aaaggtgcaa agcagtttca 36480 ttcttatgca
acacagcctt cctcctactg tcttatagtc tgtggatgtt aaattataga 36540
ttccaattga attttaatac tctagagatt ttacatttgt ggttgtcaag accccgtttt
36600 ggtaaaccta gggagctccg cacaaaagca ttgatattca gaaaaggcac
tgacctacaa 36660 attaaaagaa aaaaaaatca aataatgtgc acctcttgtg
cttccagttt gacaaagcag 36720 aagtcatcag cagtttctcc ctctgcagac
gcagttctca attctattta caagtaactg 36780 ctctactgtg cctgtttttc
tcttgctgat actcatttaa ttgtttttct tttggatctg 36840 aatctttgac
tgtcttttcc ccctcaagat taaaataaat acatctgtat tcctcccctt 36900
tctttctgtg cactgccctt cagatctcat tttgtcattt ttcagcttag tgttgaaact
36960 tttagcaaca aaaagtcagt tacttacttt gagtaagtaa ctcaaagtaa
gttaactttg 37020 agtttgagtg cacttttgcg tgtaggttca tttatgtgct
tgtgaattta aaaacattgg 37080 gattccacct gaatgaagta aaccaaacat
tttaaactat cagccagata gagacatcag 37140 cctttcactt ctttctatat
gcagacatat cctaattttt tagaaaaatc aaataggaaa 37200 attctcaaca
attaattgaa gattatagct ctgctctgaa atggtccaga aataggatct 37260
gctcatagaa actcatagtt tgaagcctct gggaggaaag gatactttaa aatttagtca
37320 catatttgga ggagggaaaa gggaaagagc agaatgaaga actgaaaaaa
atcacacacc 37380 ggggcctgtc gtgaggtggg ggactggggg agggatagca
ttaggagata tacctaatgt 37440 aaatgacgag ttaacaggcg cagcccacca
acatggcaca cgtatacata tgtaacaaac 37500 ctgcacgttg tgcacatgta
ccctagaact taaagtataa taaaaaaaaa ttttaatagc 37560 cccattaaat
aattaaaaag atttttttta gattcacaga agtgtacaaa atttttaggt 37620
tttttttttt ttaagctgtc tgctgaatag tttcttaatg gtctacaatg tttgtatcta
37680 caaacagata ctgtctgctt cttactaccc ttccaagaca agtattatta
tggcaattat 37740 tgcccagttt cccgggaaaa atttatccac agttacagaa
gaatgagatg caattgtgag 37800 actgtaaagt ttaagcaagc actcagagaa
gcacagtgat atgtatgcac agaagaggca 37860 gtctttgttt tgaggaaaac
agtgaaagta aagttaattc aagaccacaa agacaagtaa 37920 ataagtgcct
tatttttgta gttaatataa tttcagtgga atgcatattt ctaccataaa 37980
tgcatataga acttgtttgc tgacctactg tttggaaaac aaacaatccc attagaagaa
38040 tgtctttggg atttattttt accagaaaat caatcctttt ttcagtccct
tgcaaagtac 38100 agtgttacaa gccaagactt tgataatcag gtagaaaatg
gatttaaatt gcagaaatgt 38160 atatgaaaca cttttgttcc ttgccccttg
aactttaggg gaatgaaaat gtctagcact 38220 ctccaccttc ttttctctcc
tggaacttga actgtaattc aaagcctgtt tctcattaaa 38280 gtacctggca
gcctatctct ttacagcttg agttacaaag ctattcagag acctcgctgg 38340
tctaaagaga cagaacaagg atgtgtttaa atagagcata ggctgttgaa aaaaaaaatg
38400 ctgaaaatgg taaaatgatt ctgtccttcc ttccactcct cactgctgag
gtggagaggg 38460 aattcagttg gtgaacacca gcaagtggct ggtaaaagtc
cccactttct ctccagggct 38520 gccacaggac ccagaatgag tggtgggcat
gtgtgtgaac cctctattca gccagagttt 38580 tcccgcaaca ggtagtttgg
ttgaagaggt tgactaaggt tgacattggc agtaataaca 38640 cgtatgttct
tctgatttac aaaacgatgg aggaaaaagg ggagattttg aagacctgat 38700
ttctggtata cttcttaagc atgcataagg ctgaaaaaag aagacaaggg ttgtgggagg
38760 ctcctggtct agtgtttaca gaacttggat gcttgacaaa cagagcgtca
agctaattgt 38820 tcttgaagca ggaaatctgc agtggaggaa gcaggtgtgg
ggggatgatt accacgtttg 38880 gaaatggctg cattaactat tttgctcttc
tgagtttggc cccaaaagag tccatagact 38940 ttttgaagga tgccatccct
tttatttata gactaacatt aaatcagtca tttgtgaagg 39000 aaggagaaag
tgcctaaata aatttggagt cagatagcat acgtgcggca gtgtttccga 39060
tatccatttc tctttatttc tttttctttt tctttttggc tttcagcatc cccatacttt
39120 cagaaaactt gtgactaaga gtgaattctt atttttcaaa ttgttttcag
acatttcatg 39180 ttcatgtaaa cttggcttat tgatttcctg atttttcttt
atttttttgt tttgtccatt 39240 ttatttttaa tcagctacat caaatgggtc
tttggagggc ctggataacc aggagggagg 39300 ggtgtgccag acaagagcca
tgaagatcct catgaaagtt ggacaaggta aagaccatct 39360 gctgcttcat
gacgccactg tgacctggtg tagcccccag ctagtatggt gctaatgttg 39420
ccgatgccca ccttcattcg ctcttctttt tagttttcaa agcaaaccct tctgcacttt
39480 gagccactga cagatttcct caagtcaatg tactaagctt ttattggaga
tctaagagtt 39540 aagatcagca aggtagaatg tctattgcca tagatagata
gatagataga tagataatag 39600 atagatagat agatagatag atatttcttt
ttaaaaagca aaacactttg gttcaaaatc 39660 aaaatatcca gaatgaaaac
taaaagcttg tgcagttttg ctcatttctg aatcttgact 39720 acagaagagt
tttgttcatt gtgacttttc caatatagat aacctattgt gcagaaagaa 39780
ataattattc ttctaattaa aaattggtat agtagtcaat caacttgctc agttaaattg
39840 aaatgtcatc tgcaatgctt tgcctgccaa atgcaagaat ccctatagtt
tccacagatg 39900 gcctcacgtt ctaaacctct gaaataacta gtataaccat
tttgttttaa aagaaaaatt 39960 atattcttgt atttcacagt actttgcata
aagactctta tgttcattgc tattcatgcc 40020 tgttgaaata tatatgcagc
tcctaaagct agatattgtc agatgtctgt gccgtaatta 40080 atcatttgtt
tttcatatag atgcaagttc tgctggatca accaggaata aagatccaac 40140
aagacgtcca gaactagaag ctggtacaaa tggaagaagt tcgacaacaa gtccctttgt
40200 aaaaccaaat ccaggtataa cagcatgatc tgtgtgtatg gaggtctgtg
ggtaccacat 40260 tcttagtagt atcttaaaag gtagggcaga gtctaaagac
ttctaaccag ttaggattag 40320 ctggaagtta cagtgatcag gaatctttgc
tgtcagtgag tcattattaa ttacactcaa 40380 taagaacaaa ataactcatt
ccaatgaaag tcatatattc aaaggagtag agttcatgag 40440 ctgtaagtgc
cagttattag aactactctg tcaggccaaa ggtttcattg gctgacattt 40500
tatcaagctg gttgtcaact ccagcttaaa gctgatgtta atgtatatgt aattaatgtg
40560 ctaatccctc atctaattat atctaagcca cagagggttt aattgatcct
cttctaaatt 40620 ttaaatggta acatttttaa atattgcata atagtatttt
ttcaggtggt tatcgttatt 40680 ttgtttcaca ttttccatgt aaaagaaaat
attaaacagg tccctgacaa aagtgtagaa 40740 taccagataa aattgtccgt
cgttgacctt cgttttctta acagtcttgg aacaaatagt 40800 tctgtatttg
ttaccatgct aatgaaggtt ttatagagta gctgttgagc agacatcagc 40860
agttttgtat taggattgtt gtgtgcttgc ttggtcgttg tgcaaattta tcgtctgcag
40920 caatattcca tccctttcca agagtcaagg agggaagttg ttatttctaa
ctttcaatga 40980 caagatgtgt caaattcttg tgacaaactg ataaatggat
aatataatga tgccaggcag 41040 ttttttagtg cttaacattt gggctggcag
tctgttcggt gtgagagttt ctgctgcctt 41100 ccaaatatat tttaagtgta
aatcaaataa tacagacgag ttacgagctg aacattttcc 41160 caggccccct
cactccttcc gcgttcccga gctgttctgt tctgccagga ggcagggctc 41220
ttctttagaa ggcaggccct ttgaaggttt gcatgaaact ccctttctca aaggaggcgg
41280 aagagcaata ccacataaac gctcaccgct gacctggaga attggccact
tccctttttc 41340 ttccctgccg ctgccccagg ctggctgaca cgggttagaa
gatgaagcaa gatcaagggc 41400 tggctgtcac cgacagtctg tgctcttgct
ggataatgat acaaaggaaa ccctgtggct 41460 tgggagggta gggaagtccc
tcctagagat acctctcatt tccttttgcg ttgagctctt 41520 agacgaggta
ttggcgaggc aaagtccagc ttctagttag taataagcct ggcttatttt 41580
tcacattttt aagggtcata aaagcagtcc gtctgcactg ggacagcagt aactatctct
41640 gaccttttct gtctccgcgt ctgcaggttc tagcacagac ggcaacagcg
ccggacattc 41700 ggggaacaac atcctcggtt ccgaagtggc cttatttgca
gggattgctt caggatgcat 41760 catcttcatc gtcatcatca tcacgctggt
ggtcctcttg ctgaagtacc ggaggagaca 41820 caggaagcac tcgccgcagc
acacgaccac gctgtcgctc agcacactgg ccacacccaa 41880 gcgcagcggc
aacaacaacg gctcagagcc cagtgacatt atcatcccgc taaggactgc 41940
ggacagcgtc ttctgccctc actacgagaa ggtcagcggc gactacgggc acccggtgta
42000 catcgtccag gagatgcccc cgcagagccc ggcgaacatt tactacaagg
tctgagaggg 42060 accctggtgg tacctgtgct ttcccagagg acacctaatg
tcccgatgcc tcccttgagg 42120 gtttgagagc ccgcgtgctg gagaattgac
tgaagcacag caccggggga gagggacact 42180 cctcctcgga agagcccgtc
gcgctggaca gcttacctag tcttgtagca ttcggccttg 42240 gtgaacacac
acgctccctg gaagctggaa gactgtgcag aagacgccca ttcggactgc 42300
tgtgccgcgt cccacgtctc ctcctcgaag ccatgtgctg cggtcactca ggcctctgca
42360 gaagccaagg gaagacagtg gtttgtggac gagagggctg tgagcatcct
ggcaggtgcc 42420 ccaggatgcc acgcctggaa gggccggctt ctgcctgggg
tgcatttccc ccgcagtgca 42480 taccggactt gtcacacgga cctcgggcta
gttaaggtgt gcaaagatct ctagagttta 42540 gtccttactg tctcactcgt
tctgttaccc agggctctgc agcacctcac ctgagacctc 42600 cactccacat
ctgcatcact catggaacac tcatgtctgg agtcccctcc tccagccgct 42660
ggcaacaaca gcttcagtcc atgggtaatc cgttcataga aattgtgttt gctaacaagg
42720 tgccctttag ccagatgcta ggctgtctgc gaagaaggct aggagttcat
agaagggagt 42780 ggggctgggg aaagggctgg ctgcaattgc agctcactgc
tgctgcctct gaaacagaaa 42840 gttggaaagg aaaaaagaaa aaagcaatta
ggtagcacag cactttggtt ttgctgagat 42900 cgaagaggcc agtaggagac
acgacagcac acacagtgga ttccagtgca tggggaggca 42960 ctcgctgtta
tcaaatagcg atgtgcagga agaaaagccc ctcttcattc cggggaacaa 43020
agacgggtat tgttgggaaa ggaacaggct tggagggaag ggagaaagta ggccgctgat
43080 gatatattcg ggcaggactg ttgtggtact ggcaataaga tacacagctc
cgagctgtag 43140 gagagtcggt ctgctttgga tgatttttta agcagactca
gctgctatac ttatcacatt 43200 ttattaaaca cagggaaagc atttaggaga
atagcagaga gccaaatctg acctaaaagt 43260 tgaaaagcca aaggtcaaac
aggctgtaat tccatcatca tcgttgttat taaagaatcc 43320 ttatctataa
aaggtaggtc agatccccct ccccccaggt tcctccttcc cctcccgatt 43380
gagccttacg acactttggt ttatgcggtg ctgtccgggt gccagggctg cagggtcggt
43440 actgatggag gctgcagcgc ccggtgctct gtgtcaaggt gaagcacata
cggcagacct 43500 cttagagtcc ttaagacgga agtaaattat gatgtccagg
gggagaagga agataggacg 43560 tatttataat aggtatatag aacacaaggg
atataaaatg aaagattttt actaatatat 43620 attttaaggt tgcacacagt
acacaccaga agatgtgaaa ttcatttgtg gcaattaagt 43680 ggtcccaatg
ctcagcgctt aaaaaaacaa attggacagc tacttctggg aaaaacaaca 43740
tcattccaaa aagaacaata atgagagcaa atgcaaaaat aaccaagtcc tccgaaggca
43800 tctcacggaa ccgtagacta ggaagtacga gccccacaga gcaggaagcc
gatgtgactg 43860 catcatatat ttaacaatga caagatgttc
cggcgtttat ttctgcgttg ggttttccct 43920 tgccttatgg gctgaagtgt
tctctaga 43948 9 4335 DNA Homo sapiens 9 gcgcggagct gggagtggct
tcgccatggc tgtgagaagg gactccgtgt ggaagtactg 60 ctggggtgtt
ttgatggttt tatgcagaac tgcgatttcc aaatcgatag ttttagagcc 120
tatctattgg aattcctcga actccaaatt tctacctgga caaggactgg tactataccc
180 acagatagga gacaaattgg atattatttg ccccaaagtg gactctaaaa
ctgttggcca 240 gtatgaatat tataaagttt atatggttga taaagaccaa
gcagacagat gcactattaa 300 gaaggaaaat acccctctcc tcaactgtgc
caaaccagac caagatatca aattcaccat 360 caagtttcaa gaattcagcc
ctaacctctg gggtctagaa tttcagaaga acaaagatta 420 ttacattata
tctacatcaa atgggtcttt ggagggcctg gataaccagg agggaggggt 480
gtgccagaca agagccatga agatcctcat gaaagttgga caagatgcaa gttctgctgg
540 atcaaccagg aataaagatc caacaagacg tccagaacta gaagctggta
caaatggaag 600 aagttcgaca acaagtccct ttgtaaaacc aaatccaggt
tctagcacag acggcaacag 660 cgccggacat tcggggaaca acatcctcgg
ttccgaagtg gccttatttg cagggattgc 720 ttcaggatgc atcatcttca
tcgtcatcat catcacgctg gtggtcctct tgctgaagta 780 ccggaggaga
cacaggaagc actcgccgca gcacacgacc acgctgtcgc tcagcacact 840
ggccacaccc aagcgcagcg gcaacaacaa cggctcagag cccagtgaca ttatcatccc
900 gctaaggact gcggacagcg tcttctgccc tcactacgag aaggtcagcg
gggactacgg 960 gcacccggtg tacatcgtcc aggagatgcc cccgcagagc
ccggcgaaca tttactacaa 1020 ggtctgagag ggaccctggt ggtacctgtg
ctttcccaga ggacacctaa tgtcccgatg 1080 cctcccttga gggtttgaga
gcccgcgtgc tggagaattg actgaagcac agcaccgggg 1140 gagagggaca
ctcctcctcg gaagagcccg tcgcgctgga cagcttacct agtcttgtag 1200
cattcggcct tggtgaacac acacgctccc tggaagctgg aagactgtgc agaagacgcc
1260 cattcggact gctgtgccgc gtcccacgtc tcctcctcga agccatgtgc
tgcggtcact 1320 caggcctctg cagaagccaa gggaagacag tggtttgtgg
acgagagggc tgtgagcatc 1380 ctggcaggtg ccccaggatg ccacgcctgg
aagggccggc ttctgcctgg ggtgcatttc 1440 ccccgcagtg cataccggac
ttgtcacacg gacctcgggc tagttaaggt gtgcaaagat 1500 ctctagagtt
tagtccttac tgtctcactc gttctgttac ccagggctct gcagcacctc 1560
acctgagacc tccactccac atctgcatca ctcatggaac actcatgtct ggagtcccct
1620 cctccagccg ctggcaacaa cagcttcagt ccatgggtaa tccgttcata
gaaattgtgt 1680 ttgctaacaa ggtgcccttt agccagatgc taggctgtct
gcgaagaagg ctaggagttc 1740 atagaaggga gtggggctgg ggaaagggct
ggctgcaatt gcagctcact gctgctgcct 1800 ctgaaacaga aagttggaaa
ggaaaaaaga aaaaagcaat taggtagcac agcactttgg 1860 ttttgctgag
atcgaagagg ccagtaggag acacgacagc acacacagtg gattccagtg 1920
catggggagg cactcgctgt tatcaaatag cgatgtgcag gaagaaaagc ccctcttcat
1980 tccggggaac aaagacgggt attgttggga aaggaacagg cttggaggga
agggagaaag 2040 taggccgctg atgatatatt cgggcaggac tgttgtggta
ctggcaataa gatacacagc 2100 tccgagctgt aggagagtcg gtctgctttg
gatgattttt taagcagact cagctgctat 2160 acttatcaca ttttattaaa
cacagggaaa gcatttagga gaatagcaga gagccaaatc 2220 tgacctaaaa
gttgaaaagc caaaggtcaa acaggctgta attccatcat catcgttgtt 2280
attaaagaat ccttatctat aaaaggtagg tcagatcccc ctccccccag gttcctcctt
2340 cccctcccga ttgagcctta cgacactttg gtttatgcgg tgctgtccgg
gtgccagggc 2400 tgcagggtcg gtactgatgg aggctgcagc gcccggtgct
ctgtgtcaag gtgaagcaca 2460 tacggcagac ctcttagagt ccttaagacg
gaagtaaatt atgatgtcca gggggagaag 2520 gaagatagga cgtatttata
ataggtatat agaacacaag ggatataaaa tgaaagattt 2580 ttactaatat
atattttaag gttgcacaca gtacacacca gaagatgtga aattcatttg 2640
tggcaattaa gtggtcccaa tgctcagcgc ttaaaaaaac aaattggaca gctacttctg
2700 ggaaaaacaa catcattcca aaaagaacaa taatgagagc aaatgcaaaa
ataaccaagt 2760 cctccgaagg catctcacgg aaccgtagac taggaagtac
gagccccaca gagcaggaag 2820 ccgatgtgac tgcatcatat atttaacaat
gacaagatgt tccggcgttt atttctgcgt 2880 tgggttttcc cttgccttat
gggctgaagt gttctctaga atccagcagg tcacactggg 2940 ggcttcaggt
gacgatttag ctgtggctcc ctcctcctgt cctcccccgc accccctccc 3000
ttctgggaaa caagaagagt aaacaggaaa cctacttttt atgtgctatg caaaatagac
3060 atctttaaca tagtcctgtt actatggtaa cactttgctt tctgaattgg
aagggaaaaa 3120 aaatgtagcg acagcatttt aaggttctca gacctccagt
gagtacctgc aaaaatgagt 3180 tgtcacagaa attatgatcc tctatttcct
gaacctggaa atgatgttgg tccaaagtgc 3240 gtgtgtgtat gtgtgagtgg
gtgcgtggta tacatgtgta catatatgta taatatatat 3300 ctacaatata
tattatatat atctatatca tatttctgtg gagggttgcc atggtaacca 3360
gccacagtac atatgtaatt ctttccatca ccccaacctc tcctttctgt gcattcatgc
3420 aagagtttct tgtaagccat cagaagttac ttttaggatg ggggagaggg
gcgagaaggg 3480 gaaaaatggg aaatagtctg attttaatga aatcaaatgt
atgtatcatc agttggctac 3540 gttttggttc tatgctaaac tgtgaaaaat
cagatgaatt gataaaagag ttccctgcaa 3600 ccaattgaaa agtgttctgt
gcgtctgttt tgtgtctggt gcagaatatg acaatctacc 3660 aactgtccct
ttgtttgaag ttggtttagc tttggaaagt tactgtaaat gccttgcttg 3720
tatgatcgtc cctggtcacc cgactttgga atttgcacca tcatgtttca gtgaagatgc
3780 tgtaaatagg ttcagatttt actgtctatg gatttggggt gttacagtag
ccttattcac 3840 ctttttaata aaaatacaca tgaaaacaag aaagaaatgg
cttttcttac ccagattgtg 3900 tacatagagc aatgttggtt ttttataaag
tctaagcaag atgttttgta taaaatctga 3960 attttgcaat gtatttagct
acagcttgtt taacggcagt gtcattcccc tttgcactgt 4020 aatgaggaaa
aaatggtata aaaggttgcc aaattgctgc atatttgtgc cgtaattatg 4080
taccatgaat atttatttaa aatttcgttg tccaatttgt aagtaacaca gtattatgcc
4140 tgagttataa atattttttt ctttctttgt tttattttaa tagcctgtca
taggttttaa 4200 atctgcttta gtttcacatt gcagttagcc ccagaaaatg
aaatccgtga agtcacattc 4260 cacatctgtt tcaaactgaa tttgttctta
aaaaaataaa atattttttt cctatggaaa 4320 aaaaaaaaaa aaaaa 4335 10 987
PRT Homo sapiens 10 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu
Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu
Thr Ala Asp Leu Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp Gly
Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His Ser
Val Arg Thr Tyr Glu Val Cys Asp Val Gln 50 55 60 Arg Ala Pro Gly
Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg Gly
Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95
Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100
105 110 Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr
Pro 115 120 125 Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val
Ala Ala Glu 130 135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala
Thr Gly Lys Val Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly Pro
Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln Gly
Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys Lys
Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu Thr
Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220
Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg 225
230 235 240 Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser
Cys Ala 245 250 255 Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys
Arg Ala Cys Ala 260 265 270 Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu
Gly Ser Cys Gln Pro Cys 275 280 285 Pro Ala Asn Ser His Ser Asn Thr
Ile Gly Ser Ala Val Cys Gln Cys 290 295 300 Arg Val Gly Tyr Phe Arg
Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys 305 310 315 320 Thr Thr Pro
Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335 Ser
Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345
350 Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly
355 360 365 Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly
Pro Arg 370 375 380 Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu
Arg Pro Asp Phe 385 390 395 400 Thr Tyr Thr Phe Glu Val Thr Ala Leu
Asn Gly Val Ser Ser Leu Ala 405 410 415 Thr Gly Pro Val Pro Phe Glu
Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430 Val Pro Pro Ala Val
Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445 Ser Leu Ser
Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Val 450 455 460 Leu
Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser 465 470
475 480 Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg
Gly 485 490 495 Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala
Arg Ser Glu 500 505 510 Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His
Ser Gln Thr Gln Leu 515 520 525 Asp Glu Ser Glu Gly Trp Arg Glu Gln
Leu Ala Leu Ile Ala Gly Thr 530 535 540 Ala Val Val Gly Val Val Leu
Val Leu Val Val Ile Val Val Ala Val 545 550 555 560 Leu Cys Leu Arg
Lys Gln Ser Asn Gly Arg Glu Ala Glu Tyr Ser Asp 565 570 575 Lys His
Gly Gln Tyr Leu Ile Gly His Gly Thr Lys Val Tyr Ile Asp 580 585 590
Pro Phe Thr Tyr Glu Asp Pro Asn Glu Ala Val Arg Glu Phe Ala Lys 595
600 605 Glu Ile Asp Val Ser Tyr Val Lys Ile Glu Glu Val Ile Gly Ala
Gly 610 615 620 Glu Phe Gly Glu Val Cys Arg Gly Arg Leu Lys Ala Pro
Gly Lys Lys 625 630 635 640 Glu Ser Cys Val Ala Ile Lys Thr Leu Lys
Gly Gly Tyr Thr Glu Arg 645 650 655 Gln Arg Arg Glu Phe Leu Ser Glu
Ala Ser Ile Met Gly Gln Phe Glu 660 665 670 His Pro Asn Ile Ile Arg
Leu Glu Gly Val Val Thr Asn Ser Met Pro 675 680 685 Val Met Ile Leu
Thr Glu Phe Met Glu Asn Gly Ala Leu Asp Ser Phe 690 695 700 Leu Arg
Leu Asn Asp Gly Gln Phe Thr Val Ile Gln Leu Val Gly Met 705 710 715
720 Leu Arg Gly Ile Ala Ser Gly Met Arg Tyr Leu Ala Glu Met Ser Tyr
725 730 735 Val His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Asn Ser
Asn Leu 740 745 750 Val Cys Lys Val Ser Asp Phe Gly Leu Ser Arg Phe
Leu Glu Glu Asn 755 760 765 Ser Ser Asp Pro Thr Tyr Thr Ser Ser Leu
Gly Gly Lys Ile Pro Ile 770 775 780 Arg Trp Thr Ala Pro Glu Ala Ile
Ala Phe Arg Lys Phe Thr Ser Ala 785 790 795 800 Ser Asp Ala Trp Ser
Tyr Gly Ile Val Met Trp Glu Val Met Ser Phe 805 810 815 Gly Glu Arg
Pro Tyr Trp Asp Met Ser Asn Gln Asp Val Ile Asn Ala 820 825 830 Ile
Glu Gln Asp Tyr Arg Leu Pro Pro Pro Pro Asp Cys Pro Thr Ser 835 840
845 Leu His Gln Leu Met Leu Asp Cys Trp Gln Lys Asp Arg Asn Ala Arg
850 855 860 Pro Arg Phe Pro Gln Val Val Ser Ala Leu Asp Lys Met Ile
Arg Asn 865 870 875 880 Pro Ala Ser Leu Lys Ile Val Ala Arg Glu Asn
Gly Gly Ala Ser His 885 890 895 Pro Leu Leu Asp Gln Arg Gln Pro His
Tyr Ser Ala Phe Gly Ser Val 900 905 910 Gly Glu Trp Leu Arg Ala Ile
Lys Met Gly Arg Tyr Glu Glu Ser Phe 915 920 925 Ala Ala Ala Gly Phe
Gly Ser Phe Glu Leu Val Ser Gln Ile Ser Ala 930 935 940 Glu Asp Leu
Leu Arg Ile Gly Val Thr Leu Ala Gly His Gln Lys Lys 945 950 955 960
Ile Leu Ala Ser Val Gln His Met Lys Ser Gln Ala Lys Pro Gly Thr 965
970 975 Pro Gly Gly Thr Gly Gly Pro Ala Pro Gln Tyr 980 985 11 333
PRT Homo sapiens 11 Met Ala Val Arg Arg Asp Ser Val Trp Lys Tyr Cys
Trp Gly Val Leu 1 5 10 15 Met Val Leu Cys Arg Thr Ala Ile Ser Lys
Ser Ile Val Leu Glu Pro 20 25 30 Ile Tyr Trp Asn Ser Ser Asn Ser
Lys Phe Leu Pro Gly Gln Gly Leu 35 40 45 Val Leu Tyr Pro Gln Ile
Gly Asp Lys Leu Asp Ile Ile Cys Pro Lys 50 55 60 Val Asp Ser Lys
Thr Val Gly Gln Tyr Glu Tyr Tyr Lys Val Tyr Met 65 70 75 80 Val Asp
Lys Asp Gln Ala Asp Arg Cys Thr Ile Lys Lys Glu Asn Thr 85 90 95
Pro Leu Leu Asn Cys Ala Lys Pro Asp Gln Asp Ile Lys Phe Thr Ile 100
105 110 Lys Phe Gln Glu Phe Ser Pro Asn Leu Trp Gly Leu Glu Phe Gln
Lys 115 120 125 Asn Lys Asp Tyr Tyr Ile Ile Ser Thr Ser Asn Gly Ser
Leu Glu Gly 130 135 140 Leu Asp Asn Gln Glu Gly Gly Val Cys Gln Thr
Arg Ala Met Lys Ile 145 150 155 160 Leu Met Lys Val Gly Gln Asp Ala
Ser Ser Ala Gly Ser Thr Arg Asn 165 170 175 Lys Asp Pro Thr Arg Arg
Pro Glu Leu Glu Ala Gly Thr Asn Gly Arg 180 185 190 Ser Ser Thr Thr
Ser Pro Phe Val Lys Pro Asn Pro Gly Ser Ser Thr 195 200 205 Asp Gly
Asn Ser Ala Gly His Ser Gly Asn Asn Ile Leu Gly Ser Glu 210 215 220
Val Ala Leu Phe Ala Gly Ile Ala Ser Gly Cys Ile Ile Phe Ile Val 225
230 235 240 Ile Ile Ile Thr Leu Val Val Leu Leu Leu Lys Tyr Arg Arg
Arg His 245 250 255 Arg Lys His Ser Pro Gln His Thr Thr Thr Leu Ser
Leu Ser Thr Leu 260 265 270 Ala Thr Pro Lys Arg Ser Gly Asn Asn Asn
Gly Ser Glu Pro Ser Asp 275 280 285 Ile Ile Ile Pro Leu Arg Thr Ala
Asp Ser Val Phe Cys Pro His Tyr 290 295 300 Glu Lys Val Ser Gly Asp
Tyr Gly His Pro Val Tyr Ile Val Gln Glu 305 310 315 320 Met Pro Pro
Gln Ser Pro Ala Asn Ile Tyr Tyr Lys Val 325 330 12 332 PRT Unknown
Recombinant B4EC-GC 12 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser
Leu Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu
Glu Thr Ala Asp Leu Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp
Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His
Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60 Arg Ala Pro
Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg
Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90
95 Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr
100 105 110 Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu
Thr Pro 115 120 125 Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr
Val Ala Ala Glu 130 135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu
Ala Thr Gly Lys Val Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly
Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln
Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys
Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu
Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215
220 Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg
225 230 235 240 Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys
Ser Cys Ala 245 250 255 Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys
Cys Arg Ala Cys Ala 260 265 270 Gln Gly Thr Phe Lys Pro Leu Ser Gly
Glu Gly Ser Cys Gln Pro Cys 275 280 285 Pro Ala Asn Ser His Ser Asn
Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300 Arg Val Gly Tyr Phe
Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys 305 310 315 320 Thr Thr
Pro Pro Ser Ala His His His His His His 325 330 13 431 PRT Unknown
Recombinant GCF 13 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu
Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu
Thr Ala Asp Leu Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp Gly
Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His
Ser
Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60 Arg Ala Pro Gly
Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg Gly
Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95
Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100
105 110 Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr
Pro 115 120 125 Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val
Ala Ala Glu 130 135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala
Thr Gly Lys Val Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly Pro
Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln Gly
Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys Lys
Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu Thr
Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220
Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg 225
230 235 240 Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser
Cys Ala 245 250 255 Pro Gly Phe Ala Glu Gly Asn Thr Lys Cys Arg Ala
Cys Ala Gln Gly 260 265 270 Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser
Cys Gln Pro Cys Pro Ala 275 280 285 Asn Ser His Ser Asn Thr Ile Gly
Ser Ala Val Cys Gln Cys Arg Val 290 295 300 Gly Tyr Phe Arg Ala Arg
Thr Asp Pro Arg Gly Ala Pro Cys Thr Thr 305 310 315 320 Pro Pro Ser
Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly Ser Ser 325 330 335 Leu
His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg Glu Asp 340 345
350 Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly Ser Cys
355 360 365 Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg
Asp Leu 370 375 380 Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro
Asp Phe Thr Tyr 385 390 395 400 Thr Phe Glu Val Thr Ala Leu Asn Gly
Val Ser Ser Leu Ala Thr Gly 405 410 415 Pro Val Pro Phe Glu Pro Val
Asn Val His His His His His His 420 425 430 14 570 PRT Unknown
Recombinant FL-hB4EC 14 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser
Leu Ala Ala Ala Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu
Glu Thr Ala Asp Leu Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp
Gly Gln Trp Glu Glu Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His
Ser Val Arg Thr Tyr Glu Val Cys Glu Val Gln 50 55 60 Arg Ala Pro
Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg
Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90
95 Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr
100 105 110 Val Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu
Thr Pro 115 120 125 Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr
Val Ala Ala Glu 130 135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu
Ala Thr Gly Lys Val Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly
Pro Leu Ser Lys Ala Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln
Gly Ala Cys Met Ala Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys
Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu
Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215
220 Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg
225 230 235 240 Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys
Ser Cys Ala 245 250 255 Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys
Cys Arg Ala Cys Ala 260 265 270 Gln Gly Thr Phe Lys Pro Leu Ser Gly
Glu Gly Ser Cys Gln Pro Cys 275 280 285 Pro Ala Asn Ser His Ser Asn
Thr Ile Gly Ser Ala Val Cys Gln Cys 290 295 300 Arg Val Gly Tyr Phe
Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro Cys 305 310 315 320 Thr Thr
Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335
Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340
345 350 Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly
Gly 355 360 365 Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro
Gly Pro Arg 370 375 380 Asp Leu Val Glu Pro Trp Val Val Val Arg Gly
Leu Arg Pro Asp Phe 385 390 395 400 Thr Tyr Thr Phe Glu Val Thr Ala
Leu Asn Gly Val Ser Ser Leu Ala 405 410 415 Thr Gly Pro Val Pro Phe
Glu Pro Val Asn Val Thr Thr Asp Arg Glu 420 425 430 Val Pro Pro Ala
Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro Ser 435 440 445 Ser Leu
Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460
Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser 465
470 475 480 Ser Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu
Arg Gly 485 490 495 Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg
Ala Arg Ser Glu 500 505 510 Ala Gly Tyr Gly Pro Phe Gly Gln Glu His
His Ser Gln Thr Gln Leu 515 520 525 Asp Glu Ser Glu Gly Trp Arg Glu
Gln Gly Ser Lys Arg Ala Ile Leu 530 535 540 Gln Ile Glu Gly Lys Pro
Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser 545 550 555 560 Thr Arg Thr
Gly His His His His His His 565 570 15 401 PRT Unknown Recombinant
B4-CF2 15 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala
Ala Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Gln
Leu Thr Val Asn 20 25 30 Leu Thr Arg Phe Pro Glu Thr Val Pro Arg
Glu Leu Val Val Pro Val 35 40 45 Ala Gly Ser Cys Val Val Asp Ala
Val Pro Ala Pro Gly Pro Ser Pro 50 55 60 Ser Leu Tyr Cys Arg Glu
Asp Gly Gln Trp Ala Glu Gln Pro Val Thr 65 70 75 80 Gly Cys Ser Cys
Ala Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys 85 90 95 Cys Arg
Ala Cys Ala Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly 100 105 110
Ser Cys Gln Pro Cys Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser 115
120 125 Ala Val Cys Gln Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp
Pro 130 135 140 Arg Gly Ala Pro Cys Thr Thr Pro Pro Ser Ala Pro Arg
Ser Val Val 145 150 155 160 Ser Arg Leu Asn Gly Ser Ser Leu His Leu
Glu Trp Ser Ala Pro Leu 165 170 175 Glu Ser Gly Gly Arg Glu Asp Leu
Thr Tyr Ala Leu Arg Cys Arg Glu 180 185 190 Cys Arg Pro Gly Gly Ser
Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe 195 200 205 Asp Pro Gly Pro
Arg Asp Leu Val Glu Pro Trp Val Val Val Arg Gly 210 215 220 Leu Arg
Pro Asp Phe Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly 225 230 235
240 Val Ser Ser Leu Ala Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val
245 250 255 Thr Thr Asp Arg Glu Val Pro Pro Ala Val Ser Asp Ile Arg
Val Thr 260 265 270 Arg Ser Ser Pro Ser Ser Leu Ser Leu Ala Trp Ala
Val Pro Arg Ala 275 280 285 Pro Ser Gly Ala Trp Leu Asp Tyr Glu Val
Lys Tyr His Glu Lys Gly 290 295 300 Ala Glu Gly Pro Ser Ser Val Arg
Phe Leu Lys Thr Ser Glu Asn Arg 305 310 315 320 Ala Glu Leu Arg Gly
Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val 325 330 335 Arg Ala Arg
Ser Glu Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His 340 345 350 Ser
Gln Thr Gln Leu Asp Glu Ser Glu Gly Trp Arg Glu Gln Gly Gly 355 360
365 Arg Ser Ser Leu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn
370 375 380 Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His
His His 385 390 395 400 His 16 537 PRT Unknown Recombinant B4-GCF2F
16 Met Glu Leu Arg Val Leu Leu Cys Trp Ala Ser Leu Ala Ala Ala Leu
1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu
Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu
Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His Ser Val Arg Thr Tyr
Glu Val Cys Glu Val Gln 50 55 60 Arg Ala Pro Gly Gln Ala His Trp
Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg Gly Ala Val His Val
Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95 Cys Leu Ser Leu
Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110 Val Phe
Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120 125
Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu 130
135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val
Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala
Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln Gly Ala Cys Met Ala
Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys Lys Cys Ala Gln Leu
Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu Thr Val Pro Arg Glu
Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220 Val Asp Ala Val
Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg 225 230 235 240 Glu
Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245 250
255 Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys Ala
260 265 270 Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln
Pro Cys 275 280 285 Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala
Val Cys Gln Cys 290 295 300 Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp
Pro Arg Gly Ala Pro Cys 305 310 315 320 Thr Thr Pro Pro Ser Ala Pro
Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335 Ser Ser Leu His Leu
Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350 Glu Asp Leu
Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365 Ser
Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370 375
380 Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp Phe
385 390 395 400 Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val Ser
Ser Leu Ala 405 410 415 Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val
Thr Thr Asp Arg Glu 420 425 430 Val Pro Pro Ala Val Ser Asp Ile Arg
Val Thr Arg Ser Ser Pro Ser 435 440 445 Ser Leu Ser Leu Ala Trp Ala
Val Pro Arg Ala Pro Ser Gly Ala Trp 450 455 460 Leu Asp Tyr Glu Val
Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser 465 470 475 480 Ser Val
Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490 495
Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu 500
505 510 Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr Gln
Leu 515 520 525 Asp Glu Ser Glu Gly Trp Arg Glu Gln 530 535 17 522
PRT Unknown Recombinant processed B4-GCF2F 17 Leu Glu Glu Thr Leu
Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys 1 5 10 15 Trp Val Thr
Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly 20 25 30 Leu
Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Glu Val 35 40
45 Gln Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val Pro
50 55 60 Arg Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe Thr
Met Leu 65 70 75 80 Glu Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser Cys
Lys Glu Thr Phe 85 90 95 Thr Val Phe Tyr Tyr Glu Ser Asp Ala Asp
Thr Ala Thr Ala Leu Thr 100 105 110 Pro Ala Trp Met Glu Asn Pro Tyr
Ile Lys Val Asp Thr Val Ala Ala 115 120 125 Glu His Leu Thr Arg Lys
Arg Pro Gly Ala Glu Ala Thr Gly Lys Val 130 135 140 Asn Val Lys Thr
Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr 145 150 155 160 Leu
Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His 165 170
175 Leu Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg Phe
180 185 190 Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala Gly
Ser Cys 195 200 205 Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser Pro
Ser Leu Tyr Cys 210 215 220 Arg Glu Asp Gly Gln Trp Ala Glu Gln Pro
Val Thr Gly Cys Ser Cys 225 230 235 240 Ala Pro Gly Phe Glu Ala Ala
Glu Gly Asn Thr Lys Cys Arg Ala Cys 245 250 255 Ala Gln Gly Thr Phe
Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro 260 265 270 Cys Pro Ala
Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln 275 280 285 Cys
Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro 290 295
300 Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu Asn
305 310 315 320 Gly Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu Glu
Ser Gly Gly 325 330 335 Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys Arg
Glu Cys Arg Pro Gly 340 345 350 Gly Ser Cys Ala Pro Cys Gly Gly Asp
Leu Thr Phe Asp Pro Gly Pro 355 360 365 Arg Asp Leu Val Glu Pro Trp
Val Val Val Arg Gly Leu Arg Pro Asp 370 375 380 Phe Thr Tyr Thr Phe
Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu 385 390 395 400 Ala Thr
Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg 405 410 415
Glu Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro 420
425 430 Ser Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser Gly
Ala 435 440 445 Trp Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly Ala
Glu Gly Pro 450 455 460 Ser Ser Val Arg Phe Leu Lys Thr Ser Glu Asn
Arg Ala Glu Leu Arg 465 470 475 480 Gly Leu Lys Arg Gly Ala Ser Tyr
Leu Val Gln Val Arg Ala Arg Ser 485 490 495 Glu Ala Gly Tyr Gly Pro
Phe Gly Gln Glu His His Ser Gln Thr Gln 500 505 510 Leu Asp Glu Ser
Glu Gly Trp Arg Glu Gln 515 520 18 1124 PRT Unknown Recombinant
HSA-EphB4 precursor protein 18 Met Glu Leu Arg Val Leu Leu Cys Trp
Ala Ser Leu Ala Ala Ala
Leu 1 5 10 15 Glu Glu Thr Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp
Leu Lys Trp 20 25 30 Val Thr Phe Pro Gln Val Asp Gly Gln Trp Glu
Glu Leu Ser Gly Leu 35 40 45 Asp Glu Glu Gln His Ser Val Arg Thr
Tyr Glu Val Cys Asp Val Gln 50 55 60 Arg Ala Pro Gly Gln Ala His
Trp Leu Arg Thr Gly Trp Val Pro Arg 65 70 75 80 Arg Gly Ala Val His
Val Tyr Ala Thr Leu Arg Phe Thr Met Leu Glu 85 90 95 Cys Leu Ser
Leu Pro Arg Ala Gly Arg Ser Cys Lys Glu Thr Phe Thr 100 105 110 Val
Phe Tyr Tyr Glu Ser Asp Ala Asp Thr Ala Thr Ala Leu Thr Pro 115 120
125 Ala Trp Met Glu Asn Pro Tyr Ile Lys Val Asp Thr Val Ala Ala Glu
130 135 140 His Leu Thr Arg Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys
Val Asn 145 150 155 160 Val Lys Thr Leu Arg Leu Gly Pro Leu Ser Lys
Ala Gly Phe Tyr Leu 165 170 175 Ala Phe Gln Asp Gln Gly Ala Cys Met
Ala Leu Leu Ser Leu His Leu 180 185 190 Phe Tyr Lys Lys Cys Ala Gln
Leu Thr Val Asn Leu Thr Arg Phe Pro 195 200 205 Glu Thr Val Pro Arg
Glu Leu Val Val Pro Val Ala Gly Ser Cys Val 210 215 220 Val Asp Ala
Val Pro Ala Pro Gly Pro Ser Pro Ser Leu Tyr Cys Arg 225 230 235 240
Glu Asp Gly Gln Trp Ala Glu Gln Pro Val Thr Gly Cys Ser Cys Ala 245
250 255 Pro Gly Phe Glu Ala Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys
Ala 260 265 270 Gln Gly Thr Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys
Gln Pro Cys 275 280 285 Pro Ala Asn Ser His Ser Asn Thr Ile Gly Ser
Ala Val Cys Gln Cys 290 295 300 Arg Val Gly Tyr Phe Arg Ala Arg Thr
Asp Pro Arg Gly Ala Pro Cys 305 310 315 320 Thr Thr Pro Pro Ser Ala
Pro Arg Ser Val Val Ser Arg Leu Asn Gly 325 330 335 Ser Ser Leu His
Leu Glu Trp Ser Ala Pro Leu Glu Ser Gly Gly Arg 340 345 350 Glu Asp
Leu Thr Tyr Ala Leu Arg Cys Arg Glu Cys Arg Pro Gly Gly 355 360 365
Ser Cys Ala Pro Cys Gly Gly Asp Leu Thr Phe Asp Pro Gly Pro Arg 370
375 380 Asp Leu Val Glu Pro Trp Val Val Val Arg Gly Leu Arg Pro Asp
Phe 385 390 395 400 Thr Tyr Thr Phe Glu Val Thr Ala Leu Asn Gly Val
Ser Ser Leu Ala 405 410 415 Thr Gly Pro Val Pro Phe Glu Pro Val Asn
Val Thr Thr Asp Arg Glu 420 425 430 Val Pro Pro Ala Val Ser Asp Ile
Arg Val Thr Arg Ser Ser Pro Ser 435 440 445 Ser Leu Ser Leu Ala Trp
Ala Val Pro Arg Ala Pro Ser Gly Ala Val 450 455 460 Leu Asp Tyr Glu
Val Lys Tyr His Glu Lys Gly Ala Glu Gly Pro Ser 465 470 475 480 Ser
Val Arg Phe Leu Lys Thr Ser Glu Asn Arg Ala Glu Leu Arg Gly 485 490
495 Leu Lys Arg Gly Ala Ser Tyr Leu Val Gln Val Arg Ala Arg Ser Glu
500 505 510 Ala Gly Tyr Gly Pro Phe Gly Gln Glu His His Ser Gln Thr
Gln Leu 515 520 525 Asp Glu Ser Glu Gly Trp Arg Glu Gln Ser Arg Asp
Ala His Lys Ser 530 535 540 Glu Val Ala His Arg Phe Lys Asp Leu Gly
Glu Glu Asn Phe Lys Ala 545 550 555 560 Leu Val Leu Ile Ala Phe Ala
Gln Tyr Leu Gln Gln Cys Pro Phe Glu 565 570 575 Asp His Val Lys Leu
Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys 580 585 590 Val Ala Asp
Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu 595 600 605 Phe
Gly Asp Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly 610 615
620 Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys
625 630 635 640 Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu Pro Arg
Leu Val Arg 645 650 655 Pro Glu Val Asp Val Met Cys Thr Ala Phe His
Asp Asn Glu Glu Thr 660 665 670 Phe Leu Lys Lys Tyr Leu Tyr Glu Ile
Ala Arg Arg His Pro Tyr Phe 675 680 685 Tyr Ala Pro Glu Leu Leu Phe
Phe Ala Lys Arg Tyr Lys Ala Ala Phe 690 695 700 Thr Glu Cys Cys Gln
Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys 705 710 715 720 Leu Asp
Glu Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg 725 730 735
Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala 740
745 750 Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe
Ala 755 760 765 Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys Val His
Thr Glu Cys 770 775 780 Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp
Arg Ala Asp Leu Ala 785 790 795 800 Lys Tyr Ile Cys Glu Asn Gln Asp
Ser Ile Ser Ser Lys Leu Lys Glu 805 810 815 Cys Cys Glu Lys Pro Leu
Leu Glu Lys Ser His Cys Ile Ala Glu Val 820 825 830 Glu Asn Asp Glu
Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe 835 840 845 Val Glu
Ser Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val 850 855 860
Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr 865
870 875 880 Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr
Thr Leu 885 890 895 Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu Cys
Tyr Ala Lys Val 900 905 910 Phe Asp Glu Phe Lys Pro Leu Val Glu Glu
Pro Gln Asn Leu Ile Lys 915 920 925 Gln Asn Cys Glu Leu Phe Lys Gln
Leu Gly Glu Tyr Lys Phe Gln Asn 930 935 940 Ala Leu Leu Val Arg Tyr
Thr Lys Lys Val Pro Gln Val Ser Thr Pro 945 950 955 960 Thr Leu Val
Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys 965 970 975 Cys
Lys His Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu 980 985
990 Ser Val Val Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val
995 1000 1005 Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser Leu Val
Asn Arg Arg 1010 1015 1020 Pro Cys Phe Ser Ala Leu Glu Val Asp Glu
Thr Tyr Val Pro Lys Glu 1025 1030 1035 1040 Phe Asn Ala Glu Thr Phe
Thr Phe His Ala Asp Ile Cys Thr Leu Ser 1045 1050 1055 Glu Lys Glu
Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val 1060 1065 1070
Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp
1075 1080 1085 Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys Ala Asp
Asp Lys Glu 1090 1095 1100 Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu
Val Ala Ala Ser Gln Ala 1105 1110 1115 1120 Ala Leu Gly Leu 19 1109
PRT Unknown Recombinant HSA-EphB4 mature protein 19 Leu Glu Glu Thr
Leu Leu Asn Thr Lys Leu Glu Thr Ala Asp Leu Lys 1 5 10 15 Trp Val
Thr Phe Pro Gln Val Asp Gly Gln Trp Glu Glu Leu Ser Gly 20 25 30
Leu Asp Glu Glu Gln His Ser Val Arg Thr Tyr Glu Val Cys Asp Val 35
40 45 Gln Arg Ala Pro Gly Gln Ala His Trp Leu Arg Thr Gly Trp Val
Pro 50 55 60 Arg Arg Gly Ala Val His Val Tyr Ala Thr Leu Arg Phe
Thr Met Leu 65 70 75 80 Glu Cys Leu Ser Leu Pro Arg Ala Gly Arg Ser
Cys Lys Glu Thr Phe 85 90 95 Thr Val Phe Tyr Tyr Glu Ser Asp Ala
Asp Thr Ala Thr Ala Leu Thr 100 105 110 Pro Ala Trp Met Glu Asn Pro
Tyr Ile Lys Val Asp Thr Val Ala Ala 115 120 125 Glu His Leu Thr Arg
Lys Arg Pro Gly Ala Glu Ala Thr Gly Lys Val 130 135 140 Asn Val Lys
Thr Leu Arg Leu Gly Pro Leu Ser Lys Ala Gly Phe Tyr 145 150 155 160
Leu Ala Phe Gln Asp Gln Gly Ala Cys Met Ala Leu Leu Ser Leu His 165
170 175 Leu Phe Tyr Lys Lys Cys Ala Gln Leu Thr Val Asn Leu Thr Arg
Phe 180 185 190 Pro Glu Thr Val Pro Arg Glu Leu Val Val Pro Val Ala
Gly Ser Cys 195 200 205 Val Val Asp Ala Val Pro Ala Pro Gly Pro Ser
Pro Ser Leu Tyr Cys 210 215 220 Arg Glu Asp Gly Gln Trp Ala Glu Gln
Pro Val Thr Gly Cys Ser Cys 225 230 235 240 Ala Pro Gly Phe Glu Ala
Ala Glu Gly Asn Thr Lys Cys Arg Ala Cys 245 250 255 Ala Gln Gly Thr
Phe Lys Pro Leu Ser Gly Glu Gly Ser Cys Gln Pro 260 265 270 Cys Pro
Ala Asn Ser His Ser Asn Thr Ile Gly Ser Ala Val Cys Gln 275 280 285
Cys Arg Val Gly Tyr Phe Arg Ala Arg Thr Asp Pro Arg Gly Ala Pro 290
295 300 Cys Thr Thr Pro Pro Ser Ala Pro Arg Ser Val Val Ser Arg Leu
Asn 305 310 315 320 Gly Ser Ser Leu His Leu Glu Trp Ser Ala Pro Leu
Glu Ser Gly Gly 325 330 335 Arg Glu Asp Leu Thr Tyr Ala Leu Arg Cys
Arg Glu Cys Arg Pro Gly 340 345 350 Gly Ser Cys Ala Pro Cys Gly Gly
Asp Leu Thr Phe Asp Pro Gly Pro 355 360 365 Arg Asp Leu Val Glu Pro
Trp Val Val Val Arg Gly Leu Arg Pro Asp 370 375 380 Phe Thr Tyr Thr
Phe Glu Val Thr Ala Leu Asn Gly Val Ser Ser Leu 385 390 395 400 Ala
Thr Gly Pro Val Pro Phe Glu Pro Val Asn Val Thr Thr Asp Arg 405 410
415 Glu Val Pro Pro Ala Val Ser Asp Ile Arg Val Thr Arg Ser Ser Pro
420 425 430 Ser Ser Leu Ser Leu Ala Trp Ala Val Pro Arg Ala Pro Ser
Gly Ala 435 440 445 Val Leu Asp Tyr Glu Val Lys Tyr His Glu Lys Gly
Ala Glu Gly Pro 450 455 460 Ser Ser Val Arg Phe Leu Lys Thr Ser Glu
Asn Arg Ala Glu Leu Arg 465 470 475 480 Gly Leu Lys Arg Gly Ala Ser
Tyr Leu Val Gln Val Arg Ala Arg Ser 485 490 495 Glu Ala Gly Tyr Gly
Pro Phe Gly Gln Glu His His Ser Gln Thr Gln 500 505 510 Leu Asp Glu
Ser Glu Gly Trp Arg Glu Gln Ser Arg Asp Ala His Lys 515 520 525 Ser
Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys 530 535
540 Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe
545 550 555 560 Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu Phe
Ala Lys Thr 565 570 575 Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp
Lys Ser Leu His Thr 580 585 590 Leu Phe Gly Asp Lys Leu Cys Thr Val
Ala Thr Leu Arg Glu Thr Tyr 595 600 605 Gly Glu Met Ala Asp Cys Cys
Ala Lys Gln Glu Pro Glu Arg Asn Glu 610 615 620 Cys Phe Leu Gln His
Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val 625 630 635 640 Arg Pro
Glu Val Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu 645 650 655
Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr 660
665 670 Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala
Ala 675 680 685 Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala Cys
Leu Leu Pro 690 695 700 Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala
Ser Ser Ala Lys Gln 705 710 715 720 Arg Leu Lys Cys Ala Ser Leu Gln
Lys Phe Gly Glu Arg Ala Phe Lys 725 730 735 Ala Trp Ala Val Ala Arg
Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe 740 745 750 Ala Glu Val Ser
Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu 755 760 765 Cys Cys
His Gly Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu 770 775 780
Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys 785
790 795 800 Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His Cys Ile
Ala Glu 805 810 815 Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser
Leu Ala Ala Asp 820 825 830 Phe Val Glu Ser Lys Asp Val Cys Lys Asn
Tyr Ala Glu Ala Lys Asp 835 840 845 Val Phe Leu Gly Met Phe Leu Tyr
Glu Tyr Ala Arg Arg His Pro Asp 850 855 860 Tyr Ser Val Val Leu Leu
Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr 865 870 875 880 Leu Glu Lys
Cys Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys 885 890 895 Val
Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile 900 905
910 Lys Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu Tyr Lys Phe Gln
915 920 925 Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro Gln Val
Ser Thr 930 935 940 Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys
Val Gly Ser Lys 945 950 955 960 Cys Cys Lys His Pro Glu Ala Lys Arg
Met Pro Cys Ala Glu Asp Tyr 965 970 975 Leu Ser Val Val Leu Asn Gln
Leu Cys Val Leu His Glu Lys Thr Pro 980 985 990 Val Ser Asp Arg Val
Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg 995 1000 1005 Arg Pro
Cys Phe Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys 1010 1015
1020 Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr
Leu 1025 1030 1035 1040 Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr
Ala Leu Val Glu Leu 1045 1050 1055 Val Lys His Lys Pro Lys Ala Thr
Lys Glu Gln Leu Lys Ala Val Met 1060 1065 1070 Asp Asp Phe Ala Ala
Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys 1075 1080 1085 Glu Thr
Cys Phe Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln 1090 1095
1100 Ala Ala Leu Gly Leu 1105 20 9244 DNA Artificial Sequence
PEF6-GCF2 plasmid sequence 20 aatattattg aagcatttat cagggttatt
gtctcatgag cggatacata tttgaatgta 60 tttagaaaaa taaacaaata
ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg 120 tcgacggatc
gggagatctc ccgatcccct atggtcgact ctcagtacaa tctgctctga 180
tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
240 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
tgaagaatct 300 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg
gccagatata cgcgttgaca 360 ttgattattg actaggcttt tgcaaaaagc
tttgcaaaga tggataaagt tttaaacaga 420 gaggaatctt tgcagctaat
ggaccttcta ggtcttgaaa ggagtgcctc gtgaggctcc 480 ggtgcccgtc
agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg 540
gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc
600 gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag
tgcagtagtc 660 gccgtgaacg ttctttttcg caacgggttt gccgccagaa
cacaggtaag tgccgtgtgt 720 ggttcccgcg ggcctggcct ctttacgggt
tatggccctt gcgtgccttg aattacttcc 780 acctggctgc agtacgtgat
tcttgatccc gagcttcggg ttggaagtgg gtgggagagt 840 tcgaggcctt
gcgcttaagg agccccttcg cctcgtgctt gagttgaggc ctggcctggg 900
cgctggggcc gccgcgtgcg aatctggtgg caccttcgcg cctgtctcgc tgctttcgat
960 aagtctctag ccatttaaaa tttttgatga cctgctgcga cgcttttttt
ctggcaagat 1020 agtcttgtaa atgcgggcca agatctgcac actggtattt
cggtttttgg ggccgcgggc 1080 ggcgacgggg cccgtgcgtc ccagcgcaca
tgttcggcga ggcggggcct gcgagcgcgg 1140 ccaccgagaa tcggacgggg
gtagtctcaa gctggccggc ctgctctggt gcctggcctc 1200 gcgccgccgt
gtatcgcccc gccctgggcg gcaaggctgg cccggtcggc accagttgcg 1260
tgagcggaaa gatggccgct tcccggccct gctgcaggga
gctcaaaatg gaggacgcgg 1320 cgctcgggag agcgggcggg tgagtcaccc
acacaaagga aaagggcctt tccgtcctca 1380 gccgtcgctt catgtgactc
cacggagtac cgggcgccgt ccaggcacct cgattagttc 1440 tcgagctttt
ggagtacgtc gtctttaggt tggggggagg ggttttatgc gatggagttt 1500
ccccacactg agtgggtgga gactgaagtt aggccagctt ggcacttgat gtaattctcc
1560 ttggaatttg ccctttttga gtttggatct tggttcattc tcaagcctca
gacagtggtt 1620 caaagttttt ttcttccatt tcaggtgtcg tgaggaatta
gcttggtact aatacgactc 1680 actataggga gacccaagct ggctaggtaa
gcttggtacc gagctcggat ccactagtcc 1740 agtgtggtgg aattgccctt
caagcttgcc gccaccatgg agctccgggt gctgctctgc 1800 tgggcttcgt
tggccgcagc tttggaagag accctgctga acacaaaatt ggaaactgct 1860
gatctgaagt gggtgacatt ccctcaggtg gacgggcagt gggaggaact gagcggcctg
1920 gatgaggaac agcacagcgt gcgcacctac gaagtgtgtg acgtgcagcg
tgccccgggc 1980 caggcccact ggcttcgcac aggttgggtc ccacggcggg
gcgccgtcca cgtgtacgcc 2040 acgctgcgct tcaccatgct cgagtgcctg
tccctgcctc gggctgggcg ctcctgcaag 2100 gagaccttca ccgtcttcta
ctatgagagc gatgcggaca cggccacggc cctcacgcca 2160 gcctggatgg
agaaccccta catcaaggtg gacacggtgg ccgcggagca tctcacccgg 2220
aagcgccctg gggccgaggc caccgggaag gtgaatgtca agacgctgcg cctgggaccg
2280 ctcagcaagg ctggcttcta cctggccttc caggaccagg gtgcctgcat
ggccctgcta 2340 tccctgcacc tcttctacaa aaagtgcgcc cagctgactg
tgaacctgac tcgattcccg 2400 gagactgtgc ctcgggagct ggttgtgccc
gtggccggta gctgcgtggt ggatgccgtc 2460 cccgcccctg gccccagccc
cagcctctac tgccgtgagg atggccagtg ggccgaacag 2520 ccggtcacgg
gctgcagctg tgctccgggg ttcgaggcag ctgaggggaa caccaagtgc 2580
cgagcctgtg cccagggcac cttcaagccc ctgtcaggag aagggtcctg ccagccatgc
2640 ccagccaata gccactctaa caccattgga tcagccgtct gccagtgccg
cgtcgggtac 2700 ttccgggcac gcacagaccc ccggggtgca ccctgcacca
cccctccttc ggctccgcgg 2760 agcgtggttt cccgcctgaa cggctcctcc
ctgcacctgg aatggagtgc ccccctggag 2820 tctggtggcc gagaggacct
cacctacgcc ctccgctgcc gggagtgtcg acccggaggc 2880 tcctgtgcgc
cctgcggggg agacctgact tttgaccccg gcccccggga cctggtggag 2940
ccctgggtgg tggttcgagg gctacgtcct gacttcacct atacctttga ggtcactgca
3000 ttgaacgggg tatcctcctt agccacgggg cccgtcccat ttgagcctgt
caatgtcacc 3060 actgaccgag aggtacctcc tgcagtgtct gacatccggg
tgacgcggtc ctcacccagc 3120 agcttgagcc tggcctgggc tgttccccgg
gcacccagtg gggctgtgct ggactacgag 3180 gtcaaatacc atgagaaggg
cgccgagggt cccagcagcg tgcggttcct gaagacgtca 3240 gaaaaccggg
cagagctgcg ggggctgaag cggggagcca gctacctggt gcaggtacgg 3300
gcgcgctctg aggccggcta cgggcccttc ggccaggaac atcacagcca gacccaactg
3360 gatgagagcg agggctggcg ggagcagtct agagatgcac acaagagtga
ggttgctcat 3420 cggtttaaag atttgggaga agaaaatttc aaagccttgg
tgttgattgc ctttgctcag 3480 tatcttcagc agtgtccatt tgaagatcat
gtaaaattag tgaatgaagt aactgaattt 3540 gcaaaaacat gtgtagctga
tgagtcagct gaaaattgtg acaaatcact tcataccctt 3600 tttggagaca
aattatgcac agttgcaact cttcgtgaaa cctatggtga aatggctgac 3660
tgctgtgcaa aacaagaacc tgagagaaat gaatgcttct tgcaacacaa agatgacaac
3720 ccaaacctcc cccgattggt gagaccagag gttgatgtga tgtgcactgc
ttttcatgac 3780 aatgaagaga catttttgaa aaaatactta tatgaaattg
ccagaagaca tccttacttt 3840 tatgccccgg aactcctttt ctttgctaaa
aggtataaag ctgcttttac agaatgttgc 3900 caagctgctg ataaagctgc
ctgcctgttg ccaaagctcg atgaacttcg ggatgaaggg 3960 aaggcttcgt
ctgccaaaca gagactcaaa tgtgccagtc tccaaaaatt tggagaaaga 4020
gctttcaaag catgggcagt ggctcgcctg agccagagat ttcccaaagc tgagtttgca
4080 gaagtttcca agttagtgac agatcttacc aaagtccaca cggaatgctg
ccatggagat 4140 ctgcttgaat gtgctgatga cagggcggac cttgccaagt
atatctgtga aaatcaggat 4200 tcgatctcca gtaaactgaa ggaatgctgt
gaaaaacctc tgttggaaaa atcccactgc 4260 attgccgaag tggaaaatga
tgagatgcct gctgacttgc cttcattagc tgctgatttt 4320 gttgaaagta
aggatgtttg caaaaactat gctgaggcaa aggatgtctt cctgggcatg 4380
tttttgtatg aatatgcaag aaggcatcct gattactctg tcgtgctgct gctgagactt
4440 gccaagacat atgaaaccac tctagagaag tgctgtgccg ctgcagatcc
tcatgaatgc 4500 tatgccaaag tgttcgatga atttaaacct cttgtggaag
agcctcagaa tttaatcaaa 4560 caaaactgtg agctttttaa gcagcttgga
gagtacaaat tccagaatgc gctattagtt 4620 cgttacacca agaaagtacc
ccaagtgtca actccaactc ttgtagaggt ctcaagaaac 4680 ctaggaaaag
tgggcagcaa atgttgtaaa catcctgaag caaaaagaat gccctgtgca 4740
gaagactatc tatccgtggt cctgaaccag ttatgtgtgt tgcatgagaa aacgccagta
4800 agtgacagag tcacaaaatg ctgcacagag tccttggtga acaggcgacc
atgcttttca 4860 gctctggaag tcgatgaaac atacgttccc aaagagttta
atgctgaaac attcaccttc 4920 catgcagata tatgcacact ttctgagaag
gagagacaaa tcaagaaaca aactgcactt 4980 gttgagcttg tgaaacacaa
gcccaaggca acaaaagagc aactgaaagc tgttatggat 5040 gatttcgcag
cttttgtaga gaagtgctgc aaggctgacg ataaggagac ctgctttgcc 5100
gaggagggta aaaaacttgt tgctgcaagt caagctgcct taggcttata atagcggccg
5160 cttaagggca attctgcaga tatccagcac agtggcggcc gctcgagtct
agagggcccg 5220 cggttcgaag gtaagcctat ccctaaccct ctcctcggtc
tcgattctac gcgtaccggt 5280 catcatcacc atcaccattg agtttaaacc
cgctgatcag cctcgactgt gccttctagt 5340 tgccagccat ctgttgtttg
cccctccccc gtgccttcct tgaccctgga aggtgccact 5400 cccactgtcc
tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 5460
tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
5520 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
cagctggggc 5580 tctagggggt atccccacgc gccctgtagc ggcgcattaa
gcgcggcggg tgtggtggtt 5640 acgcgcagcg tgaccgctac acttgccagc
gccctagcgc ccgctccttt cgctttcttc 5700 ccttcctttc tcgccacgtt
cgccggcttt ccccgtcaag ctctaaatcg gggcatccct 5760 ttagggttcc
gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat 5820
ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
5880 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
tatctcggtc 5940 tattcttttg atttataagg gattttgggg atttcggcct
attggttaaa aaatgagctg 6000 atttaacaaa aatttaacgc gaattaattc
tgtggaatgt gtgtcagtta gggtgtggaa 6060 agtccccagg ctccccaggc
aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 6120 accaggtgtg
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 6180
aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
6240 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc
agaggccgag 6300 gccgcctctg cctctgagct attccagaag tagtgaggag
gcttttttgg aggcctaggc 6360 ttttgcaaaa agctcccggg agcttgtata
tccattttcg gatctgatca gcacgtgttg 6420 acaattaatc atcggcatag
tatatcggca tagtataata cgacaaggtg aggaactaaa 6480 ccatggccaa
gcctttgtct caagaagaat ccaccctcat tgaaagagca acggctacaa 6540
tcaacagcat ccccatctct gaagactaca gcgtcgccag cgcagctctc tctagcgacg
6600 gccgcatctt cactggtgtc aatgtatatc attttactgg gggaccttgt
gcagaactcg 6660 tggtgctggg cactgctgct gctgcggcag ctggcaacct
gacttgtatc gtcgcgatcg 6720 gaaatgagaa caggggcatc ttgagcccct
gcggacggtg tcgacaggtg cttctcgatc 6780 tgcatcctgg gatcaaagcg
atagtgaagg acagtgatgg acagccgacg gcagttggga 6840 ttcgtgaatt
gctgccctct ggttatgtgt gggagggcta agcacttcgt ggccgaggag 6900
caggactgac acgtgctacg agatttcgat tccaccgccg ccttctatga aaggttgggc
6960 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga
tctcatgctg 7020 gagttcttcg cccaccccaa cttgtttatt gcagcttata
atggttacaa ataaagcaat 7080 agcatcacaa atttcacaaa taaagcattt
ttttcactgc attctagttg tggtttgtcc 7140 aaactcatca atgtatctta
tcatgtctgt ataccgtcga cctctagcta gagcttggcg 7200 taatcatggt
catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 7260
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
7320 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
ccagctgcat 7380 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
ttgggcgctc ttccgcttcc 7440 tcgctcactg actcgctgcg ctcggtcgtt
cggctgcggc gagcggtatc agctcactca 7500 aaggcggtaa tacggttatc
cacagaatca ggggataacg caggaaagaa catgtgagca 7560 aaaggccagc
aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 7620
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
7680 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
ctctcctgtt 7740 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
cttcgggaag cgtggcgctt 7800 tctcaatgct cacgctgtag gtatctcagt
tcggtgtagg tcgttcgctc caagctgggc 7860 tgtgtgcacg aaccccccgt
tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 7920 gagtccaacc
cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 7980
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
8040 tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
cttcggaaaa 8100 agagttggta gctcttgatc cggcaaacaa accaccgctg
gtagcggtgg tttttttgtt 8160 tgcaagcagc agattacgcg cagaaaaaaa
ggatctcaag aagatccttt gatcttttct 8220 acggggtctg acgctcagtg
gaacgaaaac tcacgttaag ggattttggt catgagatta 8280 tcaaaaagga
tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 8340
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
8400 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
gtagataact 8460 acgatacggg agggcttacc atctggcccc agtgctgcaa
tgataccgcg agacccacgc 8520 tcaccggctc cagatttatc agcaataaac
cagccagccg gaagggccga gcgcagaagt 8580 ggtcctgcaa ctttatccgc
ctccatccag tctattaatt gttgccggga agctagagta 8640 agtagttcgc
cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 8700
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
8760 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
gatcgttgtc 8820 agaagtaagt tggccgcagt gttatcactc atggttatgg
cagcactgca taattctctt 8880 actgtcatgc catccgtaag atgcttttct
gtgactggtg agtactcaac caagtcattc 8940 tgagaatagt gtatgcggcg
accgagttgc tcttgcccgg cgtcaatacg ggataatacc 9000 gcgccacata
gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 9060
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
9120 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
aggaaggcaa 9180 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
gaatactcat actcttcctt 9240 tttc 9244 21 11 PRT Unknown linker
peptide sequence 21 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser 1 5
10 22 17 PRT Unknown linker peptide sequence 22 Ser Ser Ser Ser Gly
Ser Ser Ser Ser Gly Ser Ser Ser Ser Gly Ser 1 5 10 15 Pro
* * * * *
References