U.S. patent application number 13/652203 was filed with the patent office on 2013-04-18 for methods for treating vascular leak syndrome and cancer.
This patent application is currently assigned to AERPIO THERAPEUTICS, INC.. The applicant listed for this patent is AERPIO THERAPEUTICS, INC.. Invention is credited to Kevin Peters, Robert Shalwitz.
Application Number | 20130095065 13/652203 |
Document ID | / |
Family ID | 48082581 |
Filed Date | 2013-04-18 |
United States Patent
Application |
20130095065 |
Kind Code |
A1 |
Peters; Kevin ; et
al. |
April 18, 2013 |
Methods for Treating Vascular Leak Syndrome and Cancer
Abstract
Disclosed are methods for treating Vascular Leak Syndrome and
preventing cancer metastasis. Further disclosed are methods for
treating vascular leakage due to inflammatory diseases, sepsis,
cancer or the presence of pathogens.
Inventors: |
Peters; Kevin; (Cincinnati,
OH) ; Shalwitz; Robert; (Bexley, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AERPIO THERAPEUTICS, INC.; |
Cincinnati |
OH |
US |
|
|
Assignee: |
AERPIO THERAPEUTICS, INC.
Cincinnati
OH
|
Family ID: |
48082581 |
Appl. No.: |
13/652203 |
Filed: |
October 15, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61546748 |
Oct 13, 2011 |
|
|
|
61546697 |
Oct 13, 2011 |
|
|
|
Current U.S.
Class: |
424/85.2 ;
424/133.1; 424/146.1; 424/178.1; 436/501 |
Current CPC
Class: |
A61P 9/00 20180101; A61P
31/10 20180101; A61P 27/02 20180101; A61K 38/2013 20130101; C07K
14/55 20130101; A61P 33/02 20180101; A61P 35/04 20180101; C07K
16/40 20130101; A61P 13/12 20180101; A61P 31/04 20180101; A61P
17/02 20180101; A61P 11/00 20180101; A61P 29/00 20180101; A61P
43/00 20180101; A61P 31/00 20180101; A61K 2039/505 20130101; C07K
16/28 20130101; A61P 35/00 20180101; A61P 25/00 20180101; A61P 9/14
20180101; A61P 17/00 20180101; A61P 31/12 20180101; A61P 7/00
20180101 |
Class at
Publication: |
424/85.2 ;
424/146.1; 424/133.1; 424/178.1; 436/501 |
International
Class: |
A61K 39/395 20060101
A61K039/395; A61P 29/00 20060101 A61P029/00; A61P 35/00 20060101
A61P035/00; A61P 31/00 20060101 A61P031/00; G01N 33/566 20060101
G01N033/566; A61P 31/04 20060101 A61P031/04; A61P 31/12 20060101
A61P031/12; A61P 31/10 20060101 A61P031/10; A61P 35/04 20060101
A61P035/04; A61P 9/14 20060101 A61P009/14; A61P 11/00 20060101
A61P011/00 |
Claims
1. A method for treating or preventing vascular leak syndrome in a
patient comprising administering to the patient a composition
comprising an effective amount of an HPTPO-ECD binding agent or a
pharmaceutically acceptable salt thereof.
2. The method according to claim 1, wherein the HPTP.beta.-ECD
binding agent, or a pharmaceutically acceptable salt thereof, is
administered in combination with an additional therapeutic agent,
wherein the HPTP.beta.-ECD binding agent or pharmaceutically
acceptable salt are administered together or in any order.
3. The method according to claim 1, wherein the patient is
suffering from an inflammatory disease or condition, cancer,
trauma, shock, sepsis, adult respiratory distress syndrome, acute
lung injury, or infection with a pathogen.
4. The method according to claim 3, wherein the HPTP.beta.-ECD
binding agent, or a pharmaceutically acceptable salt thereof, is
administered in combination with an additional therapeutic agent,
wherein the HPTP.beta.-ECD binding agent or pharmaceutically
acceptable salt are administered together or in any order.
5. The method according to claim 4, wherein the additional
therapeutic agent is IL-2.
6. The method according to claim 4, wherein patient is suffering
from cancer and the additional therapeutic agent is a
chemotherapeutic agent.
7. The method according to claim 4, wherein the patient suffering
from an infection with a pathogen and the additional therapeutic
agent is an anti-pathogenic agent.
8. The method according to claim 7, wherein the pathogen is a
bacterium, a virus, or a fungus.
9. The method according to claim 8, wherein the anti-pathogenic
agent is an antibacterial agent, an antiviral agent, an anti-fungal
agent, or any combination thereof.
10. The method according to claim 1, wherein the dose of the
HPTP.beta.-ECD binding agent, or pharmaceutically acceptable salt
thereof, is from about 0.01 mg/kg to about 10 mg/kg by weight of
the patient.
11. The method according to claim 1, wherein the dose is
administered once daily, three-times weekly, twice weekly, once
weekly, three times monthly, twice monthly, once monthly, and once
every other month.
12. The method according to claim 1, wherein the HPTP.beta.-ECD
binding agent is conjugated to a vehicle.
13. The method according to claim 1, wherein the HPTP.beta.-ECD
binding agent is administered by intravenous injection or
subcutaneous injection
14. A method for determining the course of treatment for a patient
suffering from vascular leak syndrome, comprising: a) administering
to a patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent; b) monitoring the level of
angiopoietin-2 present in the patient during the course of
treatment; and c) discontinuing treatment when the angiopoietin-2
level returns to within a normal range.
15. A method for treating or preventing metastasis in a patient
with cancer, by administering to a patient having cancer, a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof.
16. The method according to claim 15, wherein the HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof, is
administered in combination with an additional therapeutic agent
wherein the HPTP.beta.-ECD binding agent or pharmaceutically
acceptable salt are administered together or in any order.
17. The method according to claim 16, wherein the additional
therapeutic agent is a chemotherapeutic agent.
18. The method according to claim 17, wherein the HPTP.beta.-ECD
binding agent is administered before the chemotherapeutic
agent.
19. The method according to claim 15, wherein the dose of the
HPTP.beta.-ECD binding agent, or pharmaceutically acceptable salt
thereof, is from about 0.01 mg/kg to about 10 mg/kg by weight of
the patient.
20. The method according to claim 15, wherein the dose is
administered once daily, three-times weekly, twice weekly, once
weekly, three times monthly, twice monthly, once monthly, and once
every other month.
21. The method according to any one of claims 15, wherein the
HPTP.beta.-ECD binding agent is conjugated to a vehicle.
22. The method according to claim 15, wherein the HPTP.beta.-ECD
binding agent is administered by intravenous injection or
subcutaneous injection.
23. A method for stabilizing the vasculature of a patient in need
thereof, comprising administering to the patient a composition
comprising an effective amount of an HPTP.beta.-ECD binding agent
or a pharmaceutically acceptable salt thereof.
24. The method of claim 23, wherein the HPTP.beta.-ECD binding
agent is administered prior to the patient undergoing a surgical
treatment.
25. The method of claim 23, wherein the HPTP.beta.-ECD binding
agent is administered prior to the patient undergoing
chemotherapeutic treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Provisional
Application Ser. No. 61/546,748 filed Oct. 13, 2011 and to U.S.
Provisional Application Ser. No. 61/546,697 filed Oct. 13, 2011.
The entire content of U.S. Provisional Application Ser. No.
61/546,748 and U.S. Provisional Application Ser. No. 61/546,697 is
incorporated herein by reference.
INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety is a
computer-readable sequence listing submitted concurrently herewith
and identified as follows: One 92 KB ASCII (Text) file named
"233106-331561_Seq_Listing_ST25," created on Oct. 15, 2012, at
11:51 am.
FIELD
[0003] Methods for treating cancer, preventing metastasis and
vascular leak syndrome by administration of a HPTP.beta.
inhibitor.
BACKGROUND
[0004] Vascular leak syndrome (VLS) is characterized by
hypotension, peripheral edema and hypoalbuminemia. VLS can occur as
a side effect of illness especially illnesses due to pathogens,
inter alia, viruses and bacteria. Vascular leak complicates the
healing process and can itself be a direct result of certain
therapies. For example, patients suffering from malignant renal
carcinoma are given Interleukin-2 (IL-2) to help boost their immune
system. However, this treatment must be withdrawn in many patients
due to the onset of severe VLS well before the full course of
treatment can be administered. VLS restricts the doses of IL-2
which can be administered to humans and, in some cases,
necessitates the cessation of therapy before the therapy is
maximally effective.
[0005] VLS is characterized by an increase in vascular permeability
accompanied by extravasation of fluids and proteins resulting in
interstitial edema and organ failure. Manifestations of VLS include
fluid retention, increase in body weight, peripheral edema, pleural
and pericardial effusions, ascites, anasarca and, in severe form,
signs of pulmonary and cardiovascular failure. Symptoms are highly
variable among patients and the causes are poorly understood.
Endothelial cell modifications or damage are thought to be
important is vascular leak. The pathogenesis of endothelial cell
(EC) damage is complex and can involve activation or damage to ECs
and leukocytes, release of cytokines and of inflammatory mediators,
alteration in cell-cell and cell-matrix adhesion and in
cytoskeleton function.
[0006] One of the most frightening aspects of cancer is its ability
to spread, or metastasize. Initially, cancer cells are found
grouped together thereby forming one or more tumors. After
formation of the primary tumor, cancer cells can gain the ability
to separate from the original tumor and travel to other areas of
the body. Lung cancer cells that take up in the liver and form
tumors are still lung cancer cells. Thus, the propensity for one
particular form of cancer to metastasize is dependent on many
factors, including type of cancer; however, the overall process of
how cells begin the process of metastasis is still not completely
understood.
[0007] If a single localized tumor is discovered before it has had
a chance to metastasize, then the prognosis of patient survival is
higher. This is because the tumor can be effectively excised or
destroyed by radiation or chemotherapy. There is, therefore, a
difference between tumor growth and metastasis of the tumor cells;
the first does not always lead to the other. Cancers that have
metastasized, however, are difficult to cure because of extent to
which they have spread throughout the body.
[0008] In order to metastasize, a cancer cell must break away from
its tumor and invade either the circulatory or lymph system. The
free cells are then carried to a new location where they establish
themselves. Although the body has natural safeguards that prevent
cell from surviving after being detached from their natural
location, some cancer cells have the ability to overcome these
safeguards. Therefore, if metastasis is stopped or significantly
reduced, the extent of cancer can be determined and subsequently
treated. As such, a follow up treatment to cancer therapy wherein a
tumor has been excised or radiation/chemotherapy has been used,
would be the treatment of the patient to an anti-metastasizing
agent. There is a long felt need for methods of preventing cancer
cell metastasis.
[0009] The growth of primary tumors also presents a challenge to
treatment. If the growth of a primary tumor goes unchecked, the
initial tumor can grow to a size that adversely effects organ
function at the primary site and in nearby tissues. Metastasis of
the primary tumor are also more likely if the primary tumor's
growth is uncontrolled. There is a need for methods of slowing or
preventing tumor growth.
[0010] During the course of antiviral and antibacterial infections,
patients can develop vascular leak that is induced as result of the
initial infection. There is now a long felt need for a method of
preventing vascular leak due to viral or bacterial infection, and
for providing methods of increasing the survival of humans or other
mammals infected with one or more pathogens. In addition, there is
a long felt need for a method of preventing vascular leakage due to
certain anticancer drugs or other anticancer therapies such that
the administration of anticancer drugs or anticancer therapies can
be given to humans or other mammals for a longer course of
treatment or therapy.
SUMMARY
[0011] The present disclosure provides methods for treating a
patient having vascular leak syndrome comprising administering to
the patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt
thereof.
[0012] Also provided are methods for treating a patient having
vascular leak syndrome comprising administering to the patient, a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof, and
one or more pharmaceutically acceptable excipient.
[0013] The present disclosure provides for methods of treating
vascular leak wherein the patient being treated is suffering from
an inflammatory disease or condition, trauma, shock, adult
respiratory distress syndrome, acute lung injury, or sepsis
comprising administering to the patient, a composition comprising
an effective amount of an HPTP.beta.-ECD binding agent or a
pharmaceutically acceptable salt thereof.
[0014] The present disclosure also provides for methods of treating
vascular leak wherein the patient being treated is suffering from
an inflammatory disease or condition, trauma, shock, adult
respiratory distress syndrome, acute lung injury, or sepsis
comprising administering to the patient, a composition comprising
an effective amount of an HPTP.beta.-ECD binding agent or a
pharmaceutically acceptable salt thereof, and one or more
pharmaceutically acceptable excipient.
[0015] Another method provided by the present disclosure, is a
method for determining the course of treatment for a patient
suffering from vascular leak syndrome, comprising: a) administering
to a patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent; b) monitoring the level of
angiopoietin-2 present in the patient during the course of
treatment; and c) discontinuing treatment when the angiopoietin-2
level returns to within a normal range.
[0016] A further method provided by the present disclosure is a
method for treating cancer in a patient, comprising administering
to a patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt
thereof.
[0017] A further method provided by the present is a method for
treating cancer in a patient, comprising administering to a patient
a composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable excipient.
[0018] Still another method provided by the present disclosure is a
method for preventing metastasis in a patient with cancer, by
administering to a patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent or a pharmaceutically
acceptable salt thereof.
[0019] Still another method provided by the present disclosure is a
method for preventing metastasis in a patient with cancer, by
administering to a patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable
excipient.
[0020] In the methods of the present disclosure, HPTP.beta.-ECD
binding agents include, but are not limited to antibodies,
proteins, peptides, aptamers, peptibodies, adnectins, or nucleic
acids, that binds to the extracellular portion of HPTP.beta..
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIG. 1. The monoclonal antibody R15E6 recognizes endogenous
HPTP.beta. on endothelial Cells. (Panel A) Endothelial cell lysates
are immunoprecipitated with a control antibody (Lane 1), with R15E6
(Lane 2), or with a mixture of anti-Tie2 and anti-VEGFR2 antibodies
(Lane 3). Immunoprecipitates are resolved by SDS-PAGE, transferred
to a PVDF membrane and probed by western blot with a mixture of
R15E6, anti-Tie2 and anti-VEGFR2 antibodies. A single major high
molecular weight band consistent with HPTP.beta. is seen with R15E6
(Lane 2), and not with the control antibody (Lane 1), or the
mixture of anti-Tie2 and anti-VEGFR2 (Lane 3). (Panel B)
Endothelial cells are subjected to FACS analysis with R15E6 (white
peak) or a no primary antibody control (black peak). The robust
shift in fluorescence indicates that R15E6 binds to HPTP.beta. on
the surface of intact endothelial cells.
[0022] FIG. 2 The monoclonal antibody R15E6 enhances Tie2 Receptor
Activation in HUVECs. Tie2 activation is measured in human
endothelial cells as described in Example 4. R15E6 dose dependently
enhances both basal and Ang1-induced Tie2 activation.
[0023] FIG. 3. Is a graphical representation of the mean area of
choroidal neovascularization in C57BL/6 mice 14 days post laser
treatment in eyes treated with intravitreal injection of 1 .mu.g or
2 .mu.g of an anti-VE-PTP extracellular domain antibody in one eye
versus similar treatment of the fellow eye with vehicle.
[0024] FIG. 4. Shows the mean area (mm.sup.2) of retinal
neovascularization in C57BL/6 mice on day P17 after containment in
a 75% oxygen atmosphere from P5 to P12 and intravitreal injection
of an anti-VE-PTP extracellular domain antibody at P12 when the
mice were returned to room air.
[0025] FIG. 5. Show representative fluorescent micrographs of mouse
retinas in the oxygen-induced retinopathy model after intravitreal
injection of vehicle or 2 .mu.g of an anti-VE-PTP extracellular
domain antibody.
[0026] FIG. 6. Shows the mean area (mm.sup.2) of retinal
neovascularization in C57BL/6 mice on day P17 after containment in
a 75% oxygen atmosphere from P5 to P12 followed by return to room
air on P12 with subcutaneous administration of 1 mg/kg of an
anti-VE-PTP extracellular domain antibody on days P12, 14 and
16.
[0027] FIG. 7. Shows the mean area (mm.sup.2) of retinal
neovascularization in C57BL/6 mice on day P17 after containment in
a 75% oxygen atmosphere from P5 to P12 follow by return to room air
on P12 with subcutaneous administration of 2 mg/kg of an
anti-VE-PTP extracellular domain antibody on days P12, 14 and
16.
DETAILED DESCRIPTION
General Definitions
[0028] In this specification and in the claims that follow,
reference will be made to a number of terms, which shall be defined
to have the following meanings:
[0029] The term "HPTP.beta.-ECD binding agent" and "specific
binding agent" are used interchangeably herein and refer to a
molecule that specifically binds to the extracellular portion of
HPTP.beta., and variants and derivatives thereof, as defined
herein, that inhibits the Tie2 dephosphorylase activity of
HPTP.beta..
[0030] "Agent" as used herein refers to an "HPTP.beta. binding
agent" or unless otherwise noted.
[0031] "Specifically binds HPTP.beta.-ECD" refers to the ability of
a specific binding agent of the present invention to recognize and
bind to an epitope of the extracellular domain of HPTP.beta. with
higher affinity than to other related and/or unrelated molecules.
Specific binding agents preferentially bind to HPTP.beta. in a
complex mixture of proteins and/or macromolecules. The specific
binding agent is preferably selective for HPTP.beta.. "Selective"
means that the agent has significantly greater activity toward
HPTP.beta. compared with other related and/or unrelated molecules,
not that it is completely inactive with regard to other molecules.
For example, a selective agent may show 10-fold, 100-fold, or
1000-fold selectivity toward HPTP.beta. than to other related or
unrelated molecules.
[0032] The term "anti-HPTP.beta.-ECD antibodies" refers to
antibodies or antibody fragments that bind to the extracellular
domain of HPTP.beta.. Anti-HPTP.beta.-ECD antibodies are a type of
HPTP.beta.-ECD binding agent as defined herein.
[0033] The term "VE-PTP" refers to the mouse ortholog of
HPTP.beta..
[0034] All percentages, ratios and proportions herein are by
weight, unless otherwise specified. All temperatures are in degrees
Celsius (.degree. C.) unless otherwise specified.
[0035] Ranges may be expressed herein as from one particular value
to another particular value, the endpoints are included in the
range. For example for the range from "1 mg to 50 mg" includes the
specific values 1 mg and 50 mg. The antecedent "about" indicates
that the values are approximate. For example for the range from
"about 1 mg to about 50 mg" indicates that the values are
approximate values. Additionally, when such a range is expressed,
the range includes the range "from 1 mg to 50 mg". It will be
further understood that the endpoints of each of the ranges are
significant both in relation to the other endpoint, and
independently of the other endpoint. For example the range "from 1
mg to 50 mg", includes the range "from 30 mg to 40 mg."
[0036] As used herein, the term "in combination" refers to the use
of more than one prophylactic and/or therapeutic agent. The use of
the term "in combination" does not restrict the order in which
prophylactic and/or therapeutic agents are administered to a
patient. A first prophylactic or therapeutic agent can be
administered prior to (e.g., 1 minute, 5 minutes, 15 minutes, 30
minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours,
24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4
weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before),
concomitantly with, or subsequent to (e.g., 1 minute, 5 minutes, 15
minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours,
12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks,
3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the
administration of a second prophylactic or therapeutic agent to a
patient which had, has, or is susceptible to a disorder. The
prophylactic or therapeutic agents are administered to a patient in
a sequence and within a time interval such that the agent of the
present disclosure can act together with the other agent to provide
an increased benefit than if they were administered otherwise. Any
additional prophylactic or therapeutic agent can be administered in
any order with the other additional prophylactic or therapeutic
agents
[0037] "Effective amount" means an amount of an active agent or
combination of agents effective to ameliorate or prevent the
symptoms, or prolong the survival of the patient being treated. An
effective amount may vary according to factors known in the art,
such as the disease state, age, sex, and weight of the human or
animal being treated. Although particular dosage regimes may be
described in examples herein, a person skilled in the art would
appreciated that the dosage regime may be altered to provide
optimum therapeutic response. For example, several divided doses
may be administered daily or the dose may be proportionally reduced
as indicated by the exigencies of the therapeutic situation. In
addition, the compositions of this disclosure can be administered
as frequently as necessary to achieve a therapeutic amount.
Determination of a therapeutically effective amount is well within
the capabilities of those skilled in the art, especially in light
of the detailed disclosure provided herein.
[0038] As used herein the term "inhibit" or "inhibiting" refers to
a statistically significant and measurable reduction in activity,
preferably a reduction of at least about 10% versus control, more
preferably a reduction of about 50% or more, still more preferably
a reduction of about 80% or more.
[0039] As used herein the term "increase" or "increasing" refers to
a statistically significant and measurable increase in activity,
preferably an increase of at least about 10% versus control, more
preferably an increase of about 50% or more, still more preferably
an increase of about 80% or more.
[0040] "HPTP beta" or "HPTP.beta." are used interchangeably herein
and are abbreviations for human protein tyrosine phosphatase
beta.
[0041] As used herein, "subject" means an individual. Thus, the
"subject" can include domesticated animals (e.g., cats, dogs,
etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.),
laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.),
and birds. "Patient" can also include a mammal, such as a primate
or a human. "Subject" and "patient" are used interchangeably
herein. Preferably the subject is a human.
[0042] By "reduce" or other forms of the word, such as "reducing"
or "reduction," is meant lowering of an event or characteristic
(e.g., vascular leakage). It is understood that this is typically
in relation to some standard or expected value, in other words it
is relative, but that it is not always necessary for the standard
or relative value to be referred to.
[0043] The terms "treatment", "treating", "treat" and the like,
refer to obtaining a desired pharmacologic and/or physiologic
effect such as mitigating a disease or a disorder in a host and/or
reducing, inhibiting, or eliminating a particular characteristic or
event associated with a disorder (e.g., vascular leak). Thus, the
term "treatment" includes, preventing a disorder from occurring in
a host, particularly when the host is predisposed to acquiring the
disease, but has not yet been diagnosed with the disease;
inhibiting the disorder; and/or alleviating or reversing the
disorder. Insofar as the methods of the present invention are
directed to preventing disorders, it is understood that the term
"prevent" does not require that the disease state be completely
thwarted. Rather, as used herein, the term preventing refers to the
ability of the skilled artisan to identify a population that is
susceptible to disorders, such that administration of the
HPTP.beta.-ECD binding agents of the disclosure may occur prior to
onset of a disease. The term does not imply that the disease state
is completely avoided.
[0044] As used herein, the term "cancer treatment" means any
treatment for cancer known in the art including, but not limited
to, chemotherapy and radiation therapy.
[0045] As used herein, the term "cancer treatment" means any
treatment for cancer known in the art including, but not limited
to, chemotherapy and radiation therapy.
[0046] Throughout the description and claims of this specification
the word "comprise" and other forms of the word, such as
"comprising" and "comprises," means including but not limited to,
and is not intended to exclude, for example, other additives,
components, integers, or steps.
[0047] As used in the description and the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a composition" includes mixtures of two or more such
compositions.
[0048] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0049] "Specifically binds HPTP.beta." refers to the ability of an
agent of the present invention to recognize and bind to an epitope
of the extracellular domain of HPTP.beta. with higher affinity than
to the other related and/or unrelated molecules. The agent is
preferably selective for HPTP.beta..
[0050] "Specific" means that the agent has significantly greater
activity toward HPTP.beta. compared with other related and/or
unrelated molecules, not that it is completely inactive with regard
to other molecules. For example, a selective agent may show
10-fold, 100-fold, or 1000-fold selectivity toward HPTP.beta. than
to other related or unrelated molecules.
[0051] The term "epitope" refers to any portion of any molecule
capable of being recognized by and bound by an agent at one or more
of the agent's antigen binding regions. Epitopes usually consist of
distinct surface groupings such as amino acids, sugars, lipids,
phosphoryl, or sulfonyl, and, in certain embodiments, may have
specific three dimensional structural characteristics, and/or
specific charge characteristics. Epitopes as used herein may be
conformational or linear.
[0052] "Peptibody" is a molecule comprising an antibody Fc domain
attached to at least one peptide. The production of peptibodies is
generally described in WO2002/24782.
[0053] "Fragment" refers to a portion of an agent. A fragment may
retain the desired biological activity of the agent and may be
considered to be an agent itself. For example a truncated protein
in which the amino terminus and/or carboxy terminus and/or an
internal amino acid residue is deleted is a fragment of the protein
and an Fab of an immunoglobulin molecule is a fragment of the
immunoglobulin. Such fragments may also be connected to another
molecule by way of a direct connection (e.g. a peptide or disulfide
bond) or by way of a linker.
[0054] "Protein" is used herein interchangeably with peptide and
polypeptide.
[0055] Peptides of the present invention include, but are not
limited to amino acid sequences having from about 3 to about 75
amino acids, or from about 5 to about 50 amino acids, or from about
10 to about 25 amino acids. Peptides may be naturally occurring or
artificial amino acid sequences.
[0056] A protein of the invention may be obtained by methods well
known in the art, for example, using standard direct peptide
synthesizing techniques such as via solid-phase synthesis. If the
gene sequence is known or can be deduced then the protein may be
produced by standard recombinant methods. The proteins may be
isolated or purified in a variety of ways known to one skilled in
the art. Standard purification methods include precipitation with
salts, electrophoretic, chromatographic techniques and the
like.
[0057] "Derivatives" include those binding agents that have been
chemically modified in some manner distinct from insertion,
deletion, or substitution variants. For example, wherein the
binding agent is a protein, the carboxyl terminus may be capped
with an amino group, such as NH.sub.2.
[0058] In some embodiments one or more molecules are linked
together to form the agent. For example antibody fragments may be
connected by a linker. In general the chemical structure of the
linker is not critical as it serves primarily as a space. In one
embodiment the linker is made of amino acids linked together by way
of peptide bonds. In another embodiment the linker is a non-peptide
linker such as a non-sterically hindering C.sub.1-C.sub.6 alkyl
group. In another embodiment the linker is a PEG linker. It will
further be appreciated that the linker can be inserted in a number
of locations on the molecule.
[0059] Variants of an agent are included within the scope of the
present invention. "Variant" or "Variants" as used herein means an
agent having a protein or nucleotide sequence which is
substantially similar to the protein or nucleotide sequence of the
non-variant agent and which shares a similar activity of the
non-variant agent. A protein or nucleotide sequence may be altered
in various ways to yield a variant encompassed by the present
invention, including substitutions, deletions, truncations,
insertions and other modifications. Methods for such manipulations
are well known in the art. See, for example, Current Protocols in
Molecular Biology (and updates) Ausubel et al., Eds (1996), John
Wiley and Sons, New York: Methods in Molecular Biology, Vol. 182,
In vitro Mutagenesis Protocols, 2nd Edition, Barman Ed. (2002),
Humana Press), and the references cited therein. For example,
variants include peptides and polypeptides wherein amino acid
residues are inserted into, deleted from and/or substituted into
the known amino acid sequence for the binding agent. In one
embodiment, the substitution of the amino acid is conservative in
that it minimally alters the biochemical properties of the variant.
In other embodiments, the variant may be an active fragment of a
full-length protein, a chemically modified protein, a protein
modified by addition of affinity or epitope tags, or fluorescent or
other labeling moieties, whether accomplished by in vivo or in
vitro enzymatic treatment of the protein, by chemical modification,
or by the synthesis of the protein using modified amino acids.
[0060] Fusions proteins are also contemplated herein. Using known
methods, one of skill in the art would be able to make fusion
proteins of the proteins of the invention; that, while different
from native form, may be useful. For example, the fusion partner
may be a signal (or leader) polypeptide sequence that
co-translationally or post-translationally directs transfer of the
protein from its site of synthesis to another site (e.g., the yeast
alpha-factor leader). Alternatively, it may be added to facilitate
purification or identification of the protein of the invention
(e.g., poly-His, Flag peptide, or fluorescent proteins).
[0061] Standard techniques may be used for recombinant DNA,
oligonucleotide synthesis, and tissue culture and transformation
(e.g., electroporation, lipofection). Enzymatic reactions and
purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein. The techniques and procedures are
generally performed according to conventional methods known in the
art and as described in various general and more specific
references that are cited and discussed throughout the present
specification. Unless specific definitions are provided, the
nomenclature utilized in connection with, and the laboratory
procedures and techniques of, analytical chemistry, synthetic
organic chemistry, and medicinal and pharmaceutical chemistry
described herein are those known and commonly used in the art.
Standard techniques may be used for chemical syntheses, chemical
analyses, pharmaceutical preparation, formulation, delivery, and
treatment of patients.
Sequence Listing
TABLE-US-00001 [0062] TABLE 1 SEQ ID NO: 1 Full length Human
HPTP.beta. nucleotide sequence (X54131) SEQ ID NO: 2 Full length
Human HPTP.beta. amino acid sequence (P23467) SEQ ID NO: 3
Extracellular Portion of Human HPTP.beta. with (His).sub.6Gly Tag
SEQ ID NO: 4 Extracellular Portion of Human HPTP.beta. SEQ ID NO: 5
Full length mouse VE-PTP nucleotide sequence (AY077755) SEQ ID NO:
6 Full length mouse VE-PTP amino acid sequence (AAL75813) SEQ ID
NO: 7 Extracellular portion of mouse VE-PTP amino acid sequence
HPTP.beta.-ECD binding agents
[0063] Agents useful in the present invention include, but are not
limited to, antibodies, proteins, darpins, peptides, aptamers,
adnectins, peptibodies, or nucleic acids that bind specifically to
the extracellular portion of HPTP.beta. and inhibit at least one
phosphatase activity of HPTP.beta.. As used herein, "phosphatase
activity" includes enzymatic activity and biologic activity where
biological activity is measured by assessing Tie2
phosphorylation.
[0064] Agents useful in the present invention further include:
antibodies, or antigen binding fragments thereof which bind to the
extracellular portion of HPTP.beta. wherein the antibody or
antigen-binding fragment inhibits at least one phosphatase activity
of HPTP.beta.. These agents include monoclonal and polyclonal
antibodies. An agent may be a fragment of an antibody, wherein the
fragment comprises the heavy and light chain variable regions, or
the fragment is an F(ab').sub.2, or the fragment is a dimer or
trimer of an Fab, Fv, scFv, or a dia-, tria-, or tetrabody derived
from the antibody.
[0065] For example, the agent may be, without limitation, an
antibody or antibody fragment that binds the extracellular portion
of HPTP.beta.; or in particular an antibody that binds an FN3
repeat of HPTP.beta., or more specifically an antibody that binds
the first FN3 repeat of HPTP.beta..
[0066] Agents further include: the monoclonal antibody R15E6 which
is described in U.S. Pat. No. 7,973,142, which is hereby
incorporated in its entirety. (The mouse hybridoma, Balbc spleen
cells (B cells) which may be used to produce the antibody are
deposited with American Type Culture Collection (ATCC), P.O. Box
1549, Manassas, Va. 20108 USA on 4 May 2006, assigned ATCC No.
PTA-7580) (Referred to herein as R15E6)), antibodies having the
same or substantially the same biological characteristics of R15E6;
antibody fragments of R15E6, wherein the fragment comprises the
heavy and light chain variable regions; an F(ab').sub.2 of R15E6;
dimers or trimers of an Fab, Fv, scFv; and dia-, tria-, or
tetrabodies derived from R15E6.
[0067] In particular, an agent suitable for use in the present
invention is an antibody, antibody fragment, variant or derivatives
thereof, either alone or in combination with other amino acid
sequences, provided by known techniques. Such techniques include,
but are not limited to enzymatic cleavage, chemical cleavage,
peptide synthesis or recombinant techniques. The invention further
embraces derivative agents, e.g. peptibodies.
[0068] Thus, one embodiment of an HPTP.beta.-ECD binding agent is
an antibody, another embodiment is a protein, yet another
embodiment is a peptide, and another embodiment is a darpin,
another embodiment is an aptamer, another embodiment is a
peptibody, still another embodiment is an adnectin, another
embodiment is a nucleic acid. In some embodiments the
HPTP.beta.-ECD binding agent is an monoclonal antibody, or is a
polyclonal antibody. In particular embodiments, the HPTP.beta.-ECD
binding agent is an antibody fragment that is capable of binding to
HPTP.beta.-ECD. Preferably the HPTP.beta.-ECD binding agent is an
antibody, or an antibody fragment, including but not limited to, an
F(ab').sub.2, an Fab, a dimer of an Fab, an Fv, a dimer of an Fv, a
scFv, a dimer of a scFv, a dimer an Fab, an Fv, a dimer of an Fv, a
scFv, a dimer of a scFv, a trimer of an Fab, a trimer of an Fv, a
trimer of a scFv, minibodies, a diabody, a triabody, a tetrabody, a
linear antibody, a protein, a peptide, an aptamer, a peptibody, an
adnectin, or a nucleic acid, that binds to the extracellular
portion of HPTP.beta.. In certain embodiments the HPTP.beta.-ECD
binding agent is and F(ab').sub.2 of a monoclonal antibody. In some
embodiments the HPTP.beta.-ECD binding agent comprises a plurality
of HPTP.beta.-ECD binding sites, for example where the
HPTP.beta.-ECD binding agent is an intact antibody or an
F(ab').sub.2, or a dimer of an Fab, or a trimer of an Fab. For
example, in some embodiments an HPTP.beta.-ECD binding agent is
able to bind to two HPTP.beta. molecules simultaneously at the same
or different epitope, thereby bringing the two HPTP.beta. molecules
into close proximity with one and other. In other embodiments the
HPTP.beta.-ECD binding agent is able to bind to three HPTP.beta.
molecules simultaneously at the same or different epitope, thereby
bringing the three HPTP.beta. molecules into close proximity with
one and other. In another embodiment, the HPTP.beta.-ECD binding
agent is the monoclonal antibody produced by hybridoma cell line
ATCC No. PTA-7680. In yet another embodiment, the HPTP.beta.-ECD
binding agent is an antigen binding fragment of the monoclonal
antibody produced by hybridoma cell line ATCC No. PTA-7680. In
still another embodiment, the HPTP.beta.-ECD binding agent is an
antibody having the same or substantially the same biological
characteristics the monoclonal antibody produced by hybridoma cell
line ATCC No. PTA-7680 or an antigen binding fragment thereof.
[0069] Any of the embodiments of HPTP.beta.-ECD binding agents
disclosed in the present application, may be covalently or
non-covalently conjugated to a vehicle. The term "vehicle" refers
to a molecule that affects a biological property of an agent. For
example, a vehicle may prevent degradation, and/or increase
half-life, absorption, reduce toxicity, reduce immunogenicity, or
increase biological activity of the agent. Exemplary vehicles
include, but are not limited to, Fc domains of immunoglobulins;
polymers, for example: polyethylene glycol (PEG), polylysine,
dextran; lipids; cholesterol groups (such as a steroid);
carbohydrates, dendrimers, oligosaccharides, or peptides that binds
to a salvage receptor. In some embodiments the vehicle is
polyethylene glycol (PEG), in other embodiments the vehicle is
polylysine, in yet other embodiments the vehicle is dextran, in
still other embodiments the vehicle is a lipid
[0070] Water soluble polymer attachments, such as polyethylene
glycol, polyoxyethylene glycol, or polypropylene glycol, as
described U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144,
4,670,417, 4,791,192, and 4,179,337, which are incorporated herein
in their entirety. Still other useful polymers known in the art
include monomethoxy-polyethylene glycol, dextran, cellulose, or
other carbohydrate based polymers, poly-(N-vinyl
pyrrolidone)-polyethylene glycol, propylene glycol homopolymers, a
polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated
polyols (e.g., glycerol) and polyvinyl alcohol, as well as mixtures
of these polymers. Particularly preferred are peptibodies
covalently modified with polyethylene glycol (PEG) subunits. Water
soluble polymers may be bonded at specific positions, for example
at the amino terminus of the peptibodies, or randomly attached to
one or more side chains of the polypeptide. The use of PEG for
improving the therapeutic capacity for agents, e.g. peptibodies,
and for humanized antibodies in particular, is described in U.S.
Pat. No. 6,133,426. The invention also contemplates derivatizing
the peptide and/or vehicle portion of the agents. Such derivatives
may improve the solubility, absorption, biological half-life, and
the like of the agents. The moieties may alternatively eliminate or
attenuate any undesirable side-effect of the agents and the
like.
[0071] The term "antibody" (Ab) as used herein includes monoclonal
antibodies, polyclonal antibodies, multi-specific antibodies (e.g.
bispecific antibodies), single chain antibodies, e.g., antibodies
from llama and camel, antibody fragments, e.g., variable regions
and/or constant region fragments, so long as they exhibit a desired
biological activity, e.g., antigen-binding activity. The term
"immunoglobulin" (Ig) is used interchangeably with "antibody"
herein.
[0072] An "antigen binding fragment" as used herein is a fragment
of an agent that binds to a portion of HPTP.beta. and inhibits at
least one phosphatase activity of HPTP.beta..
[0073] The basic four-chain antibody unit is a heterotetrameric
glycoprotein composed of two identical light (L) chains and two
identical heavy (H) chains (an IgM antibody consists of 5 of the
basic heterotetramer units along with an additional polypeptide
called J chain, and therefore contain 10 antigen binding sites,
while secreted IgA antibodies may polymerize to form polyvalent
assemblages comprising 2-5 of the basic 4-chain units along with J
chain). In the case of IgGs, the four-chain unit is generally about
150 kilo Daltons (kDa). Each L chain is linked to an H chain by one
covalent disulfide bond, while the two H chains are linked to each
other by one or more disulfide bonds depending on the H chain
isotype. Each H and L chain also has regularly spaced intrachain
disulfide bridges. Each H chain has at the N-terminus, a variable
domain (V.sub.H) followed by three constant domains (C.sub.H) for
each of the alpha and gamma chains and four C.sub.H domains for mu
and epsilon isotypes. Each L chain has at the N-terminus, a
variable domain (V.sub.L) followed by a constant domain (C.sub.L)
at its other end. The V.sub.L is aligned with the V.sub.H and the
C.sub.L is aligned with the first constant domain of the heavy
chain (C.sub.H1). Particular amino acid residues are believed to
form an interface between the light chain and heavy chain variable
domains. The pairing of a V.sub.H and V.sub.L together forms a
single antigen-binding site. For the structure and properties of
the different classes of antibodies, see, e.g., Basic and Clinical
Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and
Tristram G. Parslow (eds.), Appleton & Lange, 1994, page 71 and
Chapter 6.
[0074] The L chain from any vertebrate species may be assigned to
one of two clearly distinct types, called kappa and lambda, based
on the amino acid sequences of their constant domains. Depending on
the amino acid sequence of the constant domain of their heavy
chains (C.sub.H), immunoglobulins may be assigned to different
classes or isotypes. There are five classes of immunoglobulins:
IgA, IgD, IgE, IgG and IgM, having heavy chains designated alpha,
delta, epsilon, gamma and mu, respectively. The gamma and alpha
classes are further divided into subclasses on the basis of
relatively minor differences in C.sub.H sequence and function,
e.g., humans express the following subclasses: IgG1, IgG2, IgG3,
IgG4, IgA1 and IgA2.
[0075] Members of the Camelidae family, e.g., llama, camel, and
dromedaries, contain a unique type of antibody, that are devoid of
light chains, and further lack the C.sub.H1 domain (Muyldermans,
S., Rev. Mol. Biotechnol., 74, 277-302 (2001)). The variable region
of these heavy chain antibodies are termed V.sub.HH or VHH, and
constitute the smallest available intact antigen binding fragment
(15 kDa) derived from a functional immunoglobulin.
[0076] The term "variable" refers to the fact that certain segments
of the variable domains differ extensively in sequence among
antibodies. The V domain mediates antigen binding and defines
specificity of a particular antibody for its antigen. However, the
variability is not evenly distributed across the 110-amino acid
span of the variable domains. Instead, the V regions consist of
relatively invariant stretches called framework regions (FR) of
15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-12 amino
acids long. The variable domains of native heavy and light chains
each comprise four FRs, largely adopting a .beta.-sheet
configuration, connected by three hypervariable regions, which form
loops connecting, and in some cases forming part of, the
.beta.-sheet structure. The hypervariable regions in each chain are
held together in close proximity by the FRs and, with the
hypervariable regions from the other chain, contribute to the
formation of the antigen-binding site of antibodies. The constant
domains are not involved directly in binding an antibody to an
antigen, but exhibit various effector functions, such as
participation of the antibody in antibody dependent cellular
cytotoxicity (ADCC).
[0077] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody which are responsible for
antigen-binding. The hypervariable region generally comprises amino
acid residues from a "complementarity determining region" or "CDR"
(e.g. around about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3)
in the V.sub.L, and around about 1-35 (H1), 50-65 (H2) and 95-102
(H3) in the V.sub.H; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop".
[0078] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. In contrast to
polyclonal antibody preparations which include different antibodies
directed against different epitopes, each monoclonal antibody is
directed against a single epitope, i.e., a single antigenic
determinant. In addition to their specificity, the monoclonal
antibodies are advantageous in that they may be synthesized
uncontaminated by other antibodies. The modifier "monoclonal" is
not to be construed as requiring production of the antibody by any
particular method. For example, the monoclonal antibodies useful in
the present invention may be prepared by the hybridoma methodology
or may be made using recombinant DNA methods in bacterial,
eukaryotic animal or plant cells (see, e.g., U.S. Pat. No.
4,816,567). The "monoclonal antibodies" may also be isolated from
phage antibody libraries, using the available techniques, e.g.,
Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).
[0079] The monoclonal antibodies herein include "chimeric"
antibodies in which a portion of the heavy and/or light chain is
identical with or homologous to corresponding sequences in
antibodies derived from a particular species or belonging to a
particular antibody class or subclass, while the remainder of the
chain(s) is identical with or homologous to corresponding sequences
in antibodies derived from another species or belonging to another
antibody class or subclass, as well as fragments of such
antibodies, so long as they exhibit the desired biological activity
(see U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.
Acad. Sci. USA, 81, 6851-6855 (1984)).
[0080] An "antibody fragment" comprises a portion of a multimeric
antibody, preferably the antigen binding or variable region of the
intact antibody. Examples of antibody fragments include Fab, Fab',
F(ab').sub.2, dimers and trimers of Fabs, Fv, scFv, minibodies;
dia-, tria-, and tetrabodies; linear antibodies (See Hudson et al.,
Nature Med. 9, 129-134 (2003)).
[0081] "Fv" is the minimum antibody fragment which contains a
complete antigen binding site. This fragment consists of a dimer of
one heavy- and one light-chain variable region domain in tight,
non-covalent association. From the folding of these two domains
emanate six hypervariable loops (3 loops each from the H and L
chain) that contribute the amino acid residues for antigen binding
and confer antigen binding specificity to the antibody. However,
even a single variable domain (or half of an Fv comprising only
three CDRs specific for an antigen) has the ability to recognize
and bind antigen, and are therefore included in the definition of
Fv.
[0082] A single-chain variable fragment (scFv) is a fusion protein
of the variable regions of the heavy (V.sub.H) and light chains
(V.sub.L) of immunoglobulins, connected with a short linker peptide
of ten to about 25 amino acids. The linker is usually rich in
glycine for flexibility, as well as serine or threonine for
solubility, and can either connect the N-terminus of the V.sub.H
with the C-terminus of the V.sub.L, or vice versa. This protein
retains the specificity of the original immunoglobulin, despite
removal of the constant regions and the introduction of the
linker.
[0083] Divalent (or bivalent) single-chain variable fragments
(di-scFvs, bi-scFvs) can be engineered by linking two scFvs. This
can be done by producing a single peptide chain with two V.sub.H
and two V.sub.L regions, yielding tandem scFvs. Another possibility
is the creation of scFvs with linker peptides that are too short
for the two variable regions to fold together (about five amino
acids), forcing scFvs to dimerize. This type is known as diabodies.
Diabodies have been shown to have dissociation constants up to
40-fold lower than corresponding scFvs, meaning that they have a
much higher affinity to their target. Consequently, diabody drugs
could be dosed much lower than other therapeutic antibodies and are
capable of highly specific targeting of tumors in vivo. Still
shorter linkers (one or two amino acids) lead to the formation of
trimers, so-called triabodies or tribodies. Tetrabodies are known
and have been shown to exhibit an even higher affinity to their
targets than diabodies.
[0084] The term "humanized antibody" or "human antibody" refers to
antibodies which comprise heavy and light chain variable region
sequences from a non-human species (e.g., a mouse) but in which at
least a portion of the V.sub.H and/or V.sub.L sequence has been
altered to be more "human-like", i.e., more similar to human
germline variable sequences. One type of humanized antibody is a
CDR-grafted antibody, in which human CDR sequences are introduced
into non-human V.sub.H and V.sub.L sequences to replace the
corresponding nonhuman CDR sequences. Means for making chimeric,
CDR-grafted and humanized antibodies are known to those of ordinary
skill in the art (see, e.g., U.S. Pat. Nos. 4,816,567 and
5,225,539). One method for making human antibodies employs the use
of transgenic animals, such as a transgenic mouse. These transgenic
animals contain a substantial portion of the human antibody
producing genome inserted into their own genome and the animal's
own endogenous antibody production is rendered deficient in the
production of antibodies. Methods for making such transgenic
animals are known in the art. Such transgenic animals may be made
using XenoMouse.TM. technology or by using a "minilocus" approach.
Methods for making XenoMice.TM. are described in U.S. Pat. Nos.
6,162,963, 6,150,584, 6,114,598 and 6,075,181. Methods for making
transgenic animals using the "minilocus" approach are described in
U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, and WO
93/12227.
[0085] Humanization of a non-human antibody has become routine in
recent years, and is now within the knowledge of one skilled in the
art. Several companies provide services to make a humanized
antibody, e.g., Xoma, Aries, Medarex, PDL, and Cambridge Antibody
Technologies. Humanization protocols are extensively described in
technical literature, e.g., Kipriyanov and Le Gall, Molecular
Biotechnol., Vol. 26, pp. 39-60 (2004), Humana Press, Totowa, N.J.;
Lo, Methods Mol. Biol., Vol. 248, pp. 135-159 (2004), Humana Press,
Totowa, N.J.; Wu et al., J. Mol. Biol., 294, pp. 151-162
(1999).
[0086] In certain embodiments, antibodies useful in the present
invention may be expressed in cell lines other than hybridoma cell
lines. Sequences encoding particular antibodies may be used for
transformation of a suitable mammalian host cell by known methods
for introducing polynucleotides into a host cell, including, for
example packaging the polynucleotide in a virus (or into a viral
vector) and transducing a host cell with the virus (or vector), or
by transfection procedures known in the art, as exemplified by U.S.
Pat. Nos. 4,399,216, 4,912,040, 4,740,461 and 4,959,455. The
transformation procedure used may depend upon the host to be
transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are known in the art and
include; but are not limited to, dextran-mediated transfection,
calcium phosphate precipitation, polybrene mediated transfection,
protoplast fusion, electroporation, encapsulation of the
polynucleotide(s) in liposomes, mixing nucleic acid with
positively-charged lipids, and direct microinjection of the DNA
into nuclei.
[0087] A nucleic acid molecule encoding the amino acid sequence of
a heavy chain constant region, a heavy chain variable region, a
light chain constant region, or a light chain variable region of an
antibody, or a fragment thereof in a suitable combination if
desired, is/are inserted into an appropriate expression vector
using standard ligation techniques. The antibody heavy chain or
light chain constant region may be appended to the C-terminus of
the appropriate variable region and is ligated into an expression
vector. The vector is typically selected to be functional in the
particular host cell employed (i.e., the vector is compatible with
the host cell machinery such that amplification of the gene and/or
expression of the gene may occur). For a review of expression
vectors, see Methods Enzymol. Vol. 185 (Goeddel, ed.), 1990,
Academic Press.
Identification of Specific Binding Agents
[0088] Suitable selective binding agents may be identified using a
variety of techniques known in the art. For example candidate
agents can be screened for binding to HPTP.beta., and screened for
activity. Generally the candidate agents will first be screened for
binding and those that show selective binding will then be screened
to determine ability to inhibit the HPTP.beta.-mediated
dephosphorylation of Tie2. In some cases however the candidate
agents may be first screened in vitro for activity.
Determination of Binding Activity
[0089] The selection of a suitable assay for use in identification
of a specific binding agent depends on the nature of the candidate
agent to be screened. One of skill in the art would be able to
choose the appropriate assays for the particular candidate
agent.
[0090] For example, where the candidates are antibodies or
peptibodies which comprises an Fc moiety, FACS analysis as
described in Example 3B allows the candidate agent to be selected
based on its ability to bind to cells which express HPTP.beta.. The
cell may endogenously express HPTP.beta. or may be genetically
engineered to express HPTP.beta..
[0091] For other candidate agents such as aptamers, other
techniques are known in the art. For example, aptamers which
specifically bind to HPTP.beta. can be selected using a technique
known as SELEX (systematic evolution of ligands by exponential
enrichment) which selects specific aptamers through repeated rounds
of in vitro selection.
Determination of Inhibitor Activity by Western Blot
[0092] As exemplified in Example 4, in one suitable assay HUVECs
are cultured in serum-free media in the presence or absence of
various concentrations of candidate agent and lysates of the cells
are prepared, immunoprecipitated with a Tie2 antibody, resolved by
polyacrylamide gel electrophoresis and transferred to a PVDF
membrane. Membrane-bound immunoprecipitated proteins are then
serially western blotted with an antiphosphotyrosine antibody to
quantify Tie2 phosphorylation followed by a Tie2 antibody to
quantify total Tie2. Tie2 phosphorylation is expressed as the ratio
of the anti-phosphotyrosine signal over the total Tie2 signal.
Greater levels of the anti-phosphotyrosine signal indicate greater
HPTP.beta. inhibition by the candidate agent.
[0093] Candidate agents that can be screened include, but are not
limited to, libraries of known agents, including natural products,
such as plant or animal extracts, biologically active molecules
including proteins, peptides including but not limited to members
of random peptide libraries and combinatorial chemistry derived
molecular library made of D- or L-configuration amino acids,
antibodies including, but not limited to, polyclonal, monoclonal,
chimeric, human, single chain antibodies, Fab, F(ab).sub.2 and Fab
expression library fragments and eptiope-binding fragments
thereof).
[0094] As used herein "antibody fragments" include, but are not
limited, to an F(ab').sub.2, a dimer or trimer of an Fab, Fv, scFv,
or a dia-, tria-, or tetrabody derived from an antibody.
[0095] Throughout this application, various publications are
referenced. The disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the
art.
[0096] The vascular endothelium lines the inside of all blood
vessels, forming a non-thrombogenic surface that controls the entry
and exit of plasma and white blood cells to and from the
bloodstream. The quiescent endothelium has turnover rates of months
to years, and proliferates only following angiogenic activation.
The loss of endothelial quiescence is a common feature of
conditions such as inflammation, atherosclerosis, restenosis,
angiogenesis and various types of vasculopathies.
[0097] Vasculogenesis and angiogenesis are down-regulated in the
healthy adult and are, except for the organs of the female
reproductive system, almost exclusively associated with pathology
when angiogenesis is induced by microenvironmental factors such as
hypoxia or inflammation. These pathological processes associated
with, or induced by, angiogenesis include diseases as diverse as
cancer, psoriasis, macular degeneration, diabetic retinopathy,
thrombosis, and inflammatory disorders including arthritis and
atherosclerosis. However, in certain instances insufficient
angiogenesis can lead to diseases such as ischemic heart disease
and pre-eclampsia.
[0098] The quiescent vascular endothelium forms a tight barrier
that controls the passage of plasma and cells from the bloodstream
to the underlying tissues. Endothellial cells adhere to each other
through junctional transmembrane proteins that are linked to
specific intracellar structural and signaling complexes. The
endothelial layer can undergo a transition from the resting state
to the active state wherein activation of the endothelium results
in the expression of adhesion molecules. This endothelium
activation is a prerequisite for initiating angiogensesis,
inflammation and inflammation associated diseases.
[0099] Tie-2, a receptor-like tyrosine kinase exclusively expressed
in endothelial cells that controls endothelial differentiation.
Tie-2 binds and is activated by the stimulatory ligand
angiopoeitin-1 (Ang-1) which promotes autophosphorylation of the
Tie-2 receptor leading to a cascade of events that results in
stabilization of vascular structures by promoting endothelial cell
viability and preventing basement membrane dissolution. As such,
Tie-2 activation is a method for attenuating leaking vasculature by
maintaining a quiescent, intact vascular endothelium. Tie-2
activation is inhibited by Ang-2, which exhibits Ang-1 antagonism
by competitively binding to Tie-2 and thus blocking phosphorylation
of Tie-2. Elevated levels of Ang-2 have been found to be associated
with inflammatory diseases, inter alia, sepsis, lupus, inflammatory
bowel disease and metastatic diseases such as cancer.
[0100] During periods of high Ang-2 levels, fissures or breaks in
the endothelium form which results in vascular leak syndrome.
Vascular leak syndrome results in life-threatening effects such as
tissue and pulmonary edema. For many disease states elevated Ang-2
levels are clear markers that a disease state or condition exists.
Once a disease state has been resolved, the Ang-1/Ang-2 balance
returns and the vascular endothelium is stabilized. In conditions
wherein the normal balance between Ang-1 and Ang-2 has been
disrupted, the disclosed agents have been found to amplify Tie-2
signaling by inhibiting dephosphorylation of phosphorylated Tie-2
via inhibition of Human Protein Tyrosine Phosphatase-.beta.
(HPTP-.beta.). In addition, the disclosed agents can be used in
varying amounts to increase the Tie-2 signaling in a very
controlled manner, and to therefore titrate the level of Tie-2
amplification.
[0101] The present disclosure provides methods for treating a
patient having vascular leak syndrome comprising administering to
the patient a composition comprising effective amount of an
HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt
thereof. The compositions of the present disclosure may also
comprise one or more pharmaceutically acceptable excipients.
[0102] Disclosed herein, are compositions comprising an
HPTP.beta.-ECD binding agent wherein the compositions are useful
for treatment of the disclosed conditions, illness, injuries,
courses of treatment, cellular treatments and the like.
[0103] In one embodiment, the method comprises treating vascular
leak in a patient wherein the patient suffers from an inflammatory
disease or condition which comprises administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent. Another embodiment is a method of treating a patient
suffering from a physical trauma comprising administering to the
patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent. In a particular embodiment the trauma
is surgical trauma. In one embodiment the method is a method of
treating a patient suffering from shock comprising administering to
the patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent.
[0104] Particular embodiments include post-hemorrhagic shock, or
post-traumatic shock or septic shock. The present disclosure also
provides for a method of treating a patient suffering from adult
respiratory distress syndrome by administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent. Another embodiment is a method of treating a patient
with an acute lung injury comprising administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent. Cancer metastasis and bacterial and viral infections
are covered below.
[0105] In some embodiments an HPTP.beta.-ECD binding agent is
administered prophylactically to stabilize the patient's
vasculature prior to an event that places the patient at risk for
vascular leak. In one embodiment the HPTP.beta.-ECD binding agent
is administered prophylactically to stabilize the patient's
vasculature prior to surgery. Still another embodiment is a method
of preventing vascular leak syndrome in a patient wherein an
effective amount of an HPTP.beta.-ECD binding agent is administered
to the patient prior to undergoing chemotherapy. Another embodiment
is a method of treating a patient at risk of shock comprising
administering to the patient an effective amount of an
HPTP.beta.-ECD binding agent.
[0106] The disclosed HPTP.beta.-ECD binding agents can be used to
prevent, abate, minimize, control, and/or lessen tumor metastasis
in humans and animals. The disclosed HPTP.beta.-ECD binding agents
can also be used to slow the rate of primary tumor growth. As such,
the agents disclosed herein can be administered as part of a
combination therapy with one or more drugs or other pharmaceutical
agents. When used as part of the combination therapy, the decrease
in metastasis and reduction in primary tumor growth afforded by the
disclosed agents allows for a more effective and efficient use of
any pharmaceutical or drug therapy being used to treat the patient.
In addition, control of metastasis by the disclosed agent affords
the subject a greater ability to concentrate the disease in one
location.
[0107] Thus, one embodiment of the present disclosure is a method
of treating cancer in a patient comprising administering a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof.
Another embodiment is a method of preventing metastasis in a
patient suffering from cancer comprising administering a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof. Yet
another embodiment is a method of minimizing tumor metastasis in a
patient suffering from cancer comprising administering a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof.
Disclosed herein are methods for preventing metastasis of malignant
tumors or to reduce the rate of tumor growth. Thus, another
embodiment is a method of treating a patient diagnosed with a
malignant tumor comprising administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent or a pharmaceutically acceptable salt thereof.
Another embodiment is a method of preventing metastasis in a
patient diagnosed with a malignant tumor comprising administering
to the patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent or a pharmaceutically acceptable salt
thereof. Yet another embodiment is a method of reducing the rate of
tumor growth in a patient diagnosed with a tumor comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent or a pharmaceutically
acceptable salt thereof.
[0108] The following are non-limiting examples of cancers that can
be treated by the disclosed methods and compositions: leukemia, for
example, chronic myelogenous leukemia; acute lymphoblastic
leukemia, acute childhood myeloid leukemia; adult acute myeloid
leukemia, hairy cell leukemia; lymphoma, for example, Burkitt's
lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, cutaneous
t-cell lymphoma, central nervous system lymphoma; astrocytomas, for
example, cerebellar astrocytoma, childhood astrocytoma, pilocytic
astrocytoma, diffuse astrocytoma, anaplastic astrocytoma, gliomas,
oligodendroglioma, cerebral astrocytoma visual pathway glioma and
hypothalamic glioma, brain stem glioma, visual pathway,
hypothalamic glioma, cerebral astrocytoma/malignant glioma;
carcinoma, for example, thymoma carcinoma, thymic carcinoma,
squamous cell carcinoma, skin carcinoma, Merkel cell carcinoma,
adrenocortical carcinoma, adrenocortical carcinoma, basal cell
carcinoma; sarcoma, for example, rhabdomyosarcoma sarcoma, Ewing
sarcoma, Kaposi sarcoma, soft tissue sarcoma, uterine sarcoma,
osteosarcoma, malignant fibrous histiocytoma of the bone; appendix
cancer; extrahepatic bile duct cancer; bladder cancer; bone cancer;
salivary gland cancer; brain tumor; childhood central nervous
system atypical teratoid/rhabdoid tumor; central nervous system
embryonal tumors; craniopharyngioma; ependymoblastoma; ependymoma;
medulloblastoma; medulloepithelioma; pineal parenchymal tumors of
intermediate differentiation; supratentorial primitive
neuroectodermal tumors and pineoblastoma; brain and spinal cord
tumors; breast cancer; bronchial tumors; carcinoid tumor;
gastrointestinal carcinoid tumor; central nervous system embryonal
tumors; cervical cancer; chordoma, childhood; chronic
myeloproliferative disorders; colon cancer; colorectal cancer;
craniopharyngioma; extragonadal germ cell tumor; testicular germ
cell tumor; retinoblastoma; gallbladder cancer; gastric (stomach)
cancer; gastrointestinal carcinoid tumor; gastrointestinal stromal
tumor (gist); extracranial germ cell tumor; gestational
trophoblastic tumor; glioblastoma; head and neck cancer;
hepatocellular (liver) cancer; Langerhans cell histiocytosis;
hypopharyngeal cancer; islet cell tumors; kidney (renal cell)
cancer; laryngeal cancer; lip and oral cavity cancer; liver cancer;
non-small cell lung cancer; small-cell lung cancer; mesothelioma;
metastatic squamous neck cancer with occult primary; mouth cancer;
childhood multiple endocrine neoplasia syndrome; multiple
myeloma/plasma cell neoplasm; mycosis fungoides; myelodysplastic
syndromes; multiple myeloma; myeloproliferative disorders, chronic;
nasal cavity and paranasal sinus cancer; neuroblastoma; oral
cancer; oropharyngeal cancer; ovarian cancer, for example, ovarian
epithelial cancer, ovarian germ cell tumor, ovarian low malignant
potential tumor; pancreatic cancer; islet cell tumors;
papillomatosis; thyroid cancer; parathyroid cancer; penile cancer;
esophageal cancer; pharyngeal cancer; nasopharyngeal cancer;
pheochromocytoma; pineal parenchymal tumors; pituitary tumor;
plasma cell neoplasm/multiple myeloma; pleuropulmonary blastoma;
prostate cancer; rectal cancer; renal cell (kidney) cancer; renal
pelvis and ureter cancer; Sezary syndrome; skin cancer
(nonmelanoma); skin cancer (melanoma); intraocular melanoma;
malignant melanoma, small intestine cancer; metastatic squamous
neck cancer; stomach (gastric) cancer; testicular cancer; throat
cancer; transitional cell cancer of the renal pelvis and ureter;
gestational trophoblastic tumor; urethral cancer; uterine cancer,
endometrial; vaginal cancer; vulvar cancer; Waldenstrom
macroglobulinemia; and Wilms tumor.
[0109] The HPTP.beta.-ECD binding agents can be administered in
combination with one or more chemotherapeutic agent.
[0110] A "chemotherapeutic agent" or "chemotherapeutic compound" is
a chemical compound useful in the treatment of cancer.
Chemotherapeutic cancer agents that can be used in combination with
an HPTP.beta.-ECD binding agent disclosed herein, include but are
not limited to, mitotic inhibitors (vinca alkaloids). These include
vincristine, vinblastine, vindesine and Navelbine.TM.
(vinorelbine-5'-noranhydroblastine). In yet other embodiments,
chemotherapeutic cancer agents include topoisomerase I inhibitors,
such as camptothecin compounds. As used herein, "camptothecin
compounds" include Camptosar.TM. (irinotecan HCL), Hycamtin.TM.
(topotecan HCL) and other compounds derived from camptothecin and
its analogues. Another category of chemotherapeutic cancer agents
that may be used in the methods and compositions of the present
disclosure are podophyllotoxin derivatives, such as etoposide,
teniposide and mitopodozide. The present disclosure further
encompasses other chemotherapeutic cancer agents known as
alkylating agents, which alkylate the genetic material in tumor
cells. These include without limitation cisplatin,
cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide,
carmustine, busulfan, chlorambucil, belustine, uracil mustard,
chlomaphazin and dacarbazine. The present disclosure encompasses
antimetabolites as chemotherapeutic agents. Examples of these types
of agents include cytosine arabinoside, fluorouracil, methotrexate,
mercaptopurine, azathioprime and procarbazine. An additional
category of chemotherapeutic cancer agents that may be used in the
methods and compositions of the present disclosure include
antibiotics. Examples include without limitation doxorubicin,
bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin,
mytomycin C and daunomycin. There are numerous liposomal
formulations commercially available for these compounds. The
present disclosure further encompasses other chemotherapeutic
cancer agents including without limitation anti-tumor antibodies,
dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide,
taxol and its derivatives, L-asparaginase, mitoxantrone, IF-2,
gemcitabine, erlotinib, doxil, irinortecan and bevacizumab.
[0111] Other anti-cancer agents that can be used in combination
with the disclosed HPTP.beta.-ECD binding agent include, but are
not limited to: acivicin, aclarubicin, acodazole hydrochloride,
acronine, adozelesin, aldesleukin, altretamine, ambomycin,
ametantrone acetate, aminoglutethimide, anastrozole, anthramycin,
asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa,
bicalutamide, bisantrene hydrochloride, bisnafide dimesylate,
bizelesin, bleomycin sulfate, brequinar sodium, bropirimine,
cactinomycin, calusterone, caracemide, carbetimer, carboplatin,
carubicin hydrochloride, carzelesin, cedefingol, cirolemycin,
cladribine, crisnatol mesylate, cytarabine, daunorubicin
hydrochloride, decitabine, dexormaplatin, dezaguanine, dezaguanine
mesylate, diaziquone, docetaxel, doxorubicin hydrochloride,
droloxifene, droloxifene citrate, dromostanolone propionate,
duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin,
enloplatin, enpromate, epipropidine, epirubicin hydrochloride,
erbulozole, esorubicin hydrochloride, estramustine, estramustine
phosphate sodium, etanidazole, etoposide phosphate, etoprine,
fadrozole hydrochloride, fazarabine, fenretinide, floxuridine,
fludarabine phosphate, fluorocitabine, fosquidone, fostriecin
sodium, gemcitabine hydrochloride, hydroxyurea, idarubicin
hydrochloride, ilmofosine, interleukin 2 (including recombinant
interleukin 2, or rIL2), interferon alfa-2a, interferon alfa-2b,
interferon alfa-n1, interferon alfa-n3, interferon beta-1a,
interferon gamma-1b, iproplatin, irinotecan hydrochloride,
lanreotide acetate, letrozole, leuprolide acetate, liarozole
hydrochloride, lometrexol sodium, lomustine, losoxantrone
hydrochloride, masoprocol, maytansine, mechlorethamine
hydrochloride, megestrol acetate, melengestrol acetate, menogaril,
methotrexate sodium, metoprine, meturedepa, mitindomide,
mitocarcin, mitocromin, mitogillin, mitomalcin, mitosper, mitotane,
mitoxantrone hydrochloride, mycophenolic acid, nocodazole,
nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase,
peliomycin, pentamustine, peplomycin sulfate, perfosfamide,
pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin,
plomestane, porfimer sodium, porfiromycin, prednimustine,
procarbazine hydrochloride, puromycin, puromycin hydrochloride,
pyrazofurin, riboprine, rogletimide, safingol, safingol
hydrochloride, semustine, simtrazene, sparfosate sodium,
sparsomycin, spirogermanium hydrochloride, spiromustine,
spiroplatin, streptonigrin, streptozocin, sulofenur, talisomycin,
tecogalan sodium, tegafur, teloxantrone hydrochloride, temoporfin,
teroxirone, testolactone, thiamiprine, thioguanine, thiotepa,
tiazofurin, tirapazamine, toremifene citrate, trestolone acetate,
triciribine phosphate, trimetrexate, trimetrexate glucuronate,
triptorelin, tubulozole hydrochloride, uredepa, vapreotide,
verteporfin, vinblastine sulfate, vincristine sulfate, vindesine
sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine
sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine
sulfate, vorozole, zeniplatin, zinostatin, zorubicin hydrochloride.
Other anti-cancer drugs include, but are not limited to:
20-epi-1,25 dihydroxyvitamin D3, 5-ethynyluracil, abiraterone,
aclarubicin, acylfulvene, adecypenol, adozelesin, aldesleukin,
ALL-TK antagonists, altretamine, ambamustine, amidox, amifostine,
aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,
andrographolide, angiogenesis inhibitors, antagonist D, antagonist
G, antarelix, anti-dorsalizing morphogenetic protein-1,
antiandrogen, prostatic carcinoma, antiestrogen, antineoplaston,
aphidicolin glycinate, apoptosis gene modulators, apoptosis
regulators, apurinic acid, ara-CDP-DL-PTBA, arginine deaminase,
asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,
axinastatin 3, azasetron, azatoxin, azatyrosine, baccatin III
derivatives, balanol, batimastat, BCR/ABL antagonists,
benzochlorins, benzoylstaurosporine, beta lactam derivatives,
beta-alethine, betaclamycin B, betulinic acid, bFGF inhibitor,
bicalutamide, bisantrene, bisaziridinylspermine, bisnafide,
bistratene A, bizelesin, breflate, bropirimine, budotitane,
buthionine sulfoximine, calcipotriol, calphostin C, canarypox IL-2,
capecitabine, carboxamide-amino-triazole, carboxyamidotriazole,
CaRest M3, CARN 700, cartilage derived inhibitor, carzelesin,
casein kinase inhibitors (ICOS), castanospermine, cecropin B,
cetrorelix, chlorins, chloroquinoxaline sulfonamide, cicaprost,
cis-porphyrin, cladribine, clomifene analogues, clotrimazole,
collismycin A, collismycin B, combretastatin A4, combretastatin
analogue, conagenin, crambescidin 816, crisnatol, cryptophycin 8,
cryptophycin A derivatives, curacin A, cyclopentanthraquinones,
cycloplatam, cypemycin, cytarabine ocfosfate, cytolytic factor,
cytostatin, dacliximab, decitabine, dehydrodidemnin B, deslorelin,
dexamethasone, dexifosfamide, dexrazoxane, dexverapamil,
diaziquone, didemnin B, didox, diethylnorspermine,
dihydro-5-azacytidine, dihydrotaxol, 9-, dioxamycin, diphenyl
spiromustine, docetaxel, docosanol, dolasetron, doxifluridine,
droloxifene, dronabinol, duocarmycin SA, ebselen, ecomustine,
edelfosine, edrecolomab, eflornithine, elemene, emitefur,
epirubicin, epristeride, estramustine analogue, estrogen agonists,
estrogen antagonists, etanidazole, etoposide phosphate, exemestane,
fadrozole, fazarabine, fenretinide, filgrastim, finasteride,
flavopiridol, flezelastine, fluasterone, fludarabine,
fluorodaunorunicin hydrochloride, forfenimex, formestane,
fostriecin, fotemustine, gadolinium texaphyrin, gallium nitrate,
galocitabine, ganirelix, gelatinase inhibitors, gemcitabine,
glutathione inhibitors, hepsulfam, heregulin, hexamethylene
bisacetamide, hypericin, ibandronic acid, idarubicin, idoxifene,
idramantone, ilmofosine, ilomastat, imidazoacridones, imiquimod,
immunostimulant peptides, insulin-like growth factor-1 receptor
inhibitor, interferon agonists, iobenguane, iododoxorubicin,
ipomeanol, 4-, iroplact, irsogladine, isobengazole,
isohomohalicondrin B, itasetron, jasplakinolide, kahalalide F,
lamellarin-N triacetate, lanreotide, leinamycin, lenograstim,
lentinan sulfate, leptolstatin, letrozole, leukemia inhibiting
factor, leukocyte alpha interferon,
leuprolide+estrogen+progesterone, leuprorelin, levamisole,
liarozole, linear polyamine analogue, lipophilic disaccharide
peptide, lipophilic platinum compounds, lissoclinamide 7,
lobaplatin, lombricine, lometrexol, lonidamine, losoxantrone,
lovastatin, loxoribine, lurtotecan, lutetium texaphyrin,
lysofylline, lytic peptides, maitansine, mannostatin A, marimastat,
masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinase
inhibitors, menogaril, merbarone, meterelin, methioninase,
metoclopramide, MIF inhibitor, mifepristone, miltefosine,
mirimostim, mismatched double stranded RNA, mitoguazone,
mitolactol, mitomycin analogues, mitonafide, mitotoxin fibroblast
growth factor-saporin, mofarotene, molgramostim, monoclonal
antibody, human chorionic gonadotrophin, monophosphoryl lipid
A+myobacterium cell wall sk, mopidamol, multiple drug resistance
gene inhibitor, multiple tumor suppressor 1-based therapy, mustard
anticancer agent, mycaperoxide B, mycobacterial cell wall extract,
myriaporone, N-acetyldinaline, N-substituted benzamides, nafarelin,
nagrestip, naloxone+pentazocine, napavin, naphterpin, nartograstim,
nedaplatin, nemorubicin, neridronic acid, neutral endopeptidase,
nilutamide, nisamycin, nitric oxide modulators, nitroxide
antioxidant, nitrullyn, O6-benzylguanine, octreotide, okicenone,
oligonucleotides, onapristone, ondansetron, ondansetron, oracin,
oral cytokine inducer, ormaplatin, osaterone, oxaliplatin,
oxaunomycin, paclitaxel, paclitaxel analogues, paclitaxel
derivatives, palauamine, palmitoylrhizoxin, pamidronic acid,
panaxytriol, panomifene, parabactin, pazelliptine, pegaspargase,
peldesine, pentosan polysulfate sodium, pentostatin, pentrozole,
perflubron, perfosfamide, perillyl alcohol, phenazinomycin,
phenylacetate, phosphatase inhibitors, picibanil, pilocarpine
hydrochloride, pirarubicin, piritrexim, placetin A, placetin B,
plasminogen activator inhibitor, platinum complex, platinum
compounds, platinum-triamine complex, porfimer sodium,
porfiromycin, prednisone, propyl bis-acridone, prostaglandin J2,
proteasome inhibitors, protein A-based immune modulator, protein
kinase C inhibitor, protein kinase C inhibitors, microalgal,
protein tyrosine phosphatase inhibitors, purine nucleoside
phosphorylase inhibitors, purpurins, pyrazoloacridine,
pyridoxylated hemoglobin polyoxyethylene conjugate, raf
antagonists, raltitrexed, ramosetron, ras farnesyl protein
transferase inhibitors, ras inhibitors, ras-GAP inhibitor,
retelliptine demethylated, rhenium Re 186 etidronate, rhizoxin,
ribozymes, RII retinamide, rogletimide, rohitukine, romurtide,
roquinimex, rubiginone B1, ruboxyl, safingol, saintopin, SarCNU,
sarcophytol A, sargramostim, Sdi 1 mimetics, semustine, senescence
derived inhibitor 1, sense oligonucleotides, signal transduction
inhibitors, signal transduction modulators, single chain antigen
binding protein, sizofuran, sobuzoxane, sodium borocaptate, sodium
phenylacetate, solverol, somatomedin binding protein, sonermin,
sparfosic acid, spicamycin D, spiromustine, splenopentin,
spongistatin 1, squalamine, stem cell inhibitor, stem-cell division
inhibitors, stipiamide, stromelysin inhibitors, sulfinosine,
superactive vasoactive intestinal peptide antagonist, suradista,
suramin, swainsonine, synthetic glycosaminoglycans, tallimustine,
tamoxifen methiodide, tauromustine, tazarotene, tecogalan sodium,
tegafur, tellurapyrylium, telomerase inhibitors, temoporfin,
temozolomide, tetrachlorodecaoxide, tetrazomine, thaliblastine,
thiocoraline, thrombopoietin, thrombopoietin mimetic, thymalfasin,
thymopoietin receptor agonist, thymotrinan, thyroid stimulating
hormone, tin ethyl etiopurpurin, tirapazamine, titanocene
bichloride, topsentin, toremifene, totipotent stem cell factor,
translation inhibitors, tretinoin, triacetyluridine, triciribine,
trimetrexate, triptorelin, tropisetron, turosteride, tyrosine
kinase inhibitors, tyrphostins, UBC inhibitors, ubenimex,
urogenital sinus-derived growth inhibitory factor, urokinase
receptor antagonists, vapreotide, variolin B, vector system,
erythrocyte gene therapy, velaresol, veramine, verdins,
verteporfin, vinxaltine, vitaxin, vorozole, zanoterone, zeniplatin,
zilascorb, and zinostatin stimalamer. In one embodiment, the
anti-cancer drug is 5-fluorouracil or leucovorin.
[0112] Anti-angiogenic agents are also useful in the treatment of
cancer. Anti-angiogenic agents are well known to those of skill in
the art. Suitable anti-angiogenic agents for use in the methods and
compositions of the present disclosure include anti-VEGF
antibodies, including humanized and chimeric antibodies, anti-VEGF
aptamers and antisense oligonucleotides. Other known inhibitors of
angiogenesis include angiostatin, endostatin, interferons,
interleukin 1 (including .alpha. and .beta.) interleukin 12,
retinoic acid, and tissue inhibitors of metalloproteinase-1 and
metalloproteinase-2. (TIMP-1 and -2). Small molecules, including
topoisomerases such as razoxane, a topoisomerase II inhibitor with
anti-angiogenic activity, can also be used.
[0113] One embodiment of the disclosure is a method for treating a
patient diagnosed with a carcinoma, comprising administering to the
patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent. Yet another embodiment is a method
for treating a patient diagnosed with a carcinoma, comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent in combination with an
effective amount of a chemotherapeutic agent, wherein the
HPTP.beta.-ECD binding agent and chemotherapeutic agent are
administered together or in any order.
[0114] One embodiment of the disclosure is a method for preventing
or reducing metastasis in a patient diagnosed with a carcinoma,
comprising administering to the patient a composition comprising an
effective amount of an HPTP.beta.-ECD binding agent. Yet another
embodiment is a method for preventing or reducing metastasis in a
patient diagnosed with a carcinoma, comprising administering to the
patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent in combination with an effective
amount of a chemotherapeutic agent, wherein the HPTP.beta.-ECD
binding agent and chemotherapeutic agent are administered together
or in any order.
[0115] In yet another embodiment of the disclosure is a method for
treating a patient diagnosed with a sarcoma, comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. Yet another embodiment
is a method for treating a patient diagnosed with a sarcoma,
comprising administering to the patient a composition comprising an
effective amount of an HPTP.beta.-ECD binding agent in combination
with an effective amount of a chemotherapeutic agent, wherein the
HPTP.beta.-ECD binding agent and the chemotherapeutic agent are
administered together or in any order.
[0116] Yet another embodiment of the disclosure is a method for
preventing or reducing metastasis in a patient diagnosed with a
sarcoma, comprising administering to the patient a composition
comprising an effective amount of an HPTP.beta.-ECD binding agent.
Yet another embodiment is a method for preventing or reducing
metastasis in a patient diagnosed with a sarcoma, comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent in combination with an
effective amount of one or more chemotherapeutic agent, wherein the
HPTP.beta.-ECD binding agent and one or more chemotherapeutic agent
are administered together or in any order.
[0117] Yet another embodiment of the disclosure is a method for
treating a patient diagnosed with pancreatic cancer, comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. Still another embodiment
is a method for treating a patient diagnosed with pancreatic
cancer, comprising administering to the patient a composition
comprising an effective amount of an HPTP.beta.-ECD binding agent
in combination with an effective amount of one or more
chemotherapeutic agents, wherein the HPTP.beta.-ECD binding agent
and one or more chemotherapeutic agents are administered together
or in any order.
[0118] Yet another embodiment of the disclosure is a method for
preventing or reducing metastasis in a patient diagnosed with
pancreatic cancer, comprising administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent. Still another embodiment is a method for preventing
or reducing metastasis in a patient diagnosed with pancreatic
cancer, comprising administering to the patient a composition
comprising an effective amount of an HPTP.beta.-ECD binding agent
in combination with an effective amount of one or more
chemotherapeutic agent, wherein the HPTP.beta.-ECD binding agent
and one or more chemotherapeutic agents are administered together
or in any order.
[0119] In some embodiments the chemotherapeutic agent used in the
treatment of pancreatic cancer is gemcitabine, or 5-fluorouracil,
or cisplatin or capecitabine, or oxaliplatin, or mitomycin, or any
combination thereof.
[0120] Still another embodiment is a method for treating a patient
diagnosed with malignant melanoma, comprising administering to the
patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent. Yet another embodiment is a method
for treating a patient diagnosed with metastatic melanoma,
comprising administering to the patient a composition comprising an
effective amount of an HPTP.beta.-ECD binding agent in combination
with an effective amount of one or more chemotherapeutic agent,
wherein the HPTP.beta.-ECD binding agent and one or more
chemotherapeutic agents are administered together or in any
order.
[0121] Still another embodiment is a method for preventing or
reducing metastasis in a patient diagnosed with malignant melanoma,
comprising administering to the patient a composition comprising an
effective amount of an HPTP.beta.-ECD binding agent. Yet another
embodiment is a method for preventing or reducing metastasis in a
patient diagnosed with metastatic melanoma, comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent in combination with an
effective amount of one or more chemotherapeutic agent, wherein the
HPTP.beta.-ECD binding agent and one or more chemotherapeutic
agents are administered together or in any order.
[0122] In some embodiments the chemotherapeutic agent is used to
treat melanoma is cisplatin, or vinblastine, or dacarbazine, or any
combination thereof.
[0123] Still another embodiment is a method for treating a patient
diagnosed with breast cancer comprising administering to the
patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent. Yet another embodiment is a method
for treating a patient diagnosed with breast cancer comprising
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent in combination with an
effective amount of one or more chemotherapeutic agent, wherein the
HPTP.beta.-ECD binding agent and one or more chemotherapeutic
agents are administered together or in any order. Yet another
embodiment is a method for preventing or reducing metastasis in a
patient diagnosed with breast cancer comprising administering to
the patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent. Yet another embodiment is a method
for preventing or reducing metastasis in a patient diagnosed with
breast cancer comprising administering to the patient a composition
comprising an effective amount of an HPTP.beta.-ECD binding agent
in combination with an effective amount of one or more
chemotherapeutic agent, wherein the HPTP.beta.-ECD binding agent
and one or more chemotherapeutic agent are administered together or
in any order. In some embodiments the chemotherapeutic agent is
used in the treatment of breast cancer is taxol or an analog of
taxol.
[0124] In particular embodiments the HPTP.beta.-ECD binding agent
is administered in combination with IL-2.
IL-2 Induced Vascular Leak: Treatment of Metastatic Cancers
[0125] Immunotherapy is one method of treating cancer.
Up-regulation of the body's own immune system is one aspect of
immunotherapy. Among the many immune system signaling molecules is
interleukin-2 (IL-2) which is instrumental in the body's natural
response to microbial infection and in discriminating between
foreign (non-self) and self. High-dose interleukin-2 is an FDA
approved treatment for patients with metastatic renal cell
carcinoma and metastatic melanoma. Although it has been reported
that only 23% of those subjects given this therapy show a tumor
response, the duration of this response can exceed 10 years (Elias
L. et al., "A literature analysis of prognostic factors for
response and quality of response of patients with renal cell
carcinoma to interleukin-2-based therapy." Oncology, (2001), Vol.
61, pp. 91-101). As such, IL-2 therapy is the only available
treatment that offers the potential for cure.
[0126] Gallagher (Gallagher, D. C. et al., "Angiopoietin 2 Is a
Potential Mediator of High-Dose Interleukin 2-Induced Vascular
Leak" Clin. Cancer Res., (2007), Vol. 13, No. 7, pp. 2115-2120)
reports that elevated levels of angiopoietin-2 are found in
patients treated with high doses of IL-2 and suggests that
overcoming Ang-2 blockade of Tie-2 signaling might be curative for
vascular leak syndrome which is a side effect of this therapy.
[0127] IL-2 is known to cause endothelial cell activation, however,
with loss of proper barrier function. Amplification of Tie-2
signaling during high dose IL-2 immunotherapy would lead to
attenuation of vascular leakage since Tie-2 stimulation promotes
endothelial cell stability. As such, by administering an agent that
can amplify Tie-2 signaling, vascular stability can be increased
and, hence, the side effects of high IL-2 dosing mitigated. The
disclosed HPTP.beta.-ECD binding agents can amplify Tie-2 signaling
under the conditions of low angiopoietin-1 concentrations or when
high concentrations of angiopoietin-2 are present as in IL-2
treated patients.
[0128] By amplifying Tie-2 signaling without affecting Ang-2
levels, the use of elevated levels of Ang-2 as a potential
pathology marker is retained. For example, a patient suffering from
an inflammatory disease such as sepsis will normally have an
elevated Ang-2 level that acts to suppress Ang-1 stimulation of
Tie-2. This elevated Ang-2 results in edema which is a symptom of
vascular leakage. The present methods, by amplifying Tie-2
signaling without affecting the Ang-2 level, provide a method for
alleviating the symptoms that are associated with vascular leak
while retaining the ability to use Ang-2 levels as a measure of
disease progress and resolution.
[0129] As many as 65% of patients receiving this IL-2 therapy will
necessarily interrupt or discontinue treatment due to VLS. The
major dose-limiting toxicity of interleukin-2 (IL-2) and of
immunotoxin (IT) therapies is vascular leak syndrome (VLS). VLS is
characterized by an increase in vascular permeability accompanied
by extravasation of fluids and proteins resulting in interstitial
edema and organ failure. Manifestations of VLS include fluid
retention, hypotension, increase in body weight, peripheral edema,
pulmonary edema, pleural and pericardial effusions, ascites,
anasarca and, in severe form, signs of pulmonary and cardiovascular
failure.
[0130] The disclosed HPTP.beta.-ECD binding agents can be used as
an effective therapy to reduce vascular leak caused by treatment
with IL-2. Therefore an embodiment of the present invention is a
method of treating, reducing or preventing vascular leak in a
patient being administered IL-2 wherein the method comprises
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. The HPTP.beta.-ECD
binding agent can be co-administered with IL-2 or administered
separately. The IL-2 and the HPTP.beta.-ECD binding agent may be
administered in any order and by any method, for example,
intravenously, orally, by patch, subcutaneous injection and the
like.
[0131] One embodiment of the present disclosure is a method for
treating renal cell carcinoma comprising administering to a patient
a composition comprising: a) an effective amount of interleukin-2
such that an immune response is provided; and b) an effective
amount of an HPTP.beta.-ECD binding agent; wherein the
interleukin-2 and the HPTP.beta.-ECD binding agent can be
administered together or in any order. Another embodiment disclosed
herein is a method for treating renal cell carcinoma comprising
administering to a patient a composition comprising: a) a high dose
of interleukin-2; and b) an effective amount of an HPTP.beta.-ECD
binding agent.
[0132] Further disclosed is a method for treating metastatic
melanoma comprising administering to a patient a series of
compositions, wherein the compositions can be administered in any
order and at any effective amount, a first composition comprising,
a high dose of interleukin-2 and the second composition comprising
an effective amount of an HPTP.beta.-ECD binding agent.
[0133] Still further disclosed is a method for treating renal cell
carcinoma comprising administering to a patient a series of
compositions, wherein the compositions can be administered in any
order and at any effective amount, a first composition comprising a
high dose of interleukin-2 and the second composition comprising an
effective amount of an HPTP.beta.-ECD binding agent.
[0134] Disclosed herein is a method for treating metastatic
melanoma by administering to a patient in need of treatment a
therapy that comprises: a) an effective amount of interleukin-2
such that an immune response is provided; and b) an effective
amount of an HPTP.beta.-ECD binding agent; wherein the
interleukin-2 and the HPTP.beta.-ECD binding agent can be
administered together or in any order.
[0135] Also disclosed herein is a method for treating metastatic
melanoma by administering to a patient in need of treatment a
therapy that comprises: a) an effective amount of interleukin-2
such that an immune response is provided; and b) an effective
amount of an HPTP.beta.-ECD binding agent; wherein the
interleukin-2 and the HPTP.beta.-ECD binding agent can be
administered together or in any order.
[0136] Disclosed herein are compositions which can be used to treat
patients with cancer, wherein the patient having cancer is treated
with one or more cancer agents that induce vascular leak syndrome
in the patient. As such, disclosed herein are compositions
effective in reducing vascular leak resulting from a cancer
treatment, the compositions comprising an effective amount of an
HPTP.beta.-ECD binding agent.
[0137] Another aspect disclosed herein are compositions effective
for treating humans or other mammals having a medical condition or
disease state wherein the treatment for the medical condition or
disease state induces vascular leak syndrome, the composition
comprising: a) an effective amount of an HPTP.beta.-ECD binding
agent; and b) one or more pharmaceutical drugs; wherein at least
one of the pharmaceutical drugs induces vascular leak syndrome.
[0138] In a further aspect, disclosed herein are compositions
comprising: a) an effective amount of an HPTP.beta.-ECD binding
agent; and b) one or more chemotherapeutic agent.
[0139] Also disclosed herein are compositions which can be used to
control vascular leakage, the compositions comprising an effective
amount of one or more of the agents disclosed herein. Still further
disclosed herein are compositions which can be used to treat
patients with an inflammatory disease, non-limiting examples of
which include sepsis, lupus, and inflammatory bowel disease, the
compositions comprising an effective amount of an HPTP.beta.-ECD
binding agents disclosed herein. The HPTP.beta.-ECD binding agents
inhibit the Tie2 dephosphorylase activity of HPTP.beta. acting as
Tie-2 signaling amplifiers.
[0140] Tumor growth is often a multi-step process that starts with
the loss of control of cell proliferation. The cancerous cell then
begins to divide rapidly, resulting in a microscopically small,
spheroid tumor: an in situ carcinoma. As the tumor mass grows, the
cells will find themselves further and further away from the
nearest capillary. Finally the tumor stops growing and reaches a
steady state, in which the number of proliferating cells
counterbalances the number of dying cells. The restriction in size
is caused by the lack of nutrients and oxygen. In tissues, the
oxygen diffusion limit corresponds to a distance of 100 .mu.m
between the capillary and the cells, which is in the range of 3-5
lines of cells around a single vessel. In situ carcinomas may
remain dormant and undetected for many years and metastasis are
rarely associated with these small (2 to 3 mm.sup.2), avascular
tumors.
[0141] When a tumor's growth is stopped due to a lack of nutrients
and/or oxygen, this reduction in tumor vasculature also limits the
ability of anti-tumor drugs to be delivered to the malignant cells.
Moreover, if there is a slight increase in tumor vasculature, this
will allow delivery of anti-tumor therapies to the malignant cells
without initiating metastasis. As such, the disclosed agents when
used to slightly amplify Tie-2 signaling can be used to increase
blood flow to the tumor cells without setting off metastasis or
uncontrolled tumor cell proliferation while providing a method for
delivering anti-cancer drugs to malignant cells.
[0142] Disclosed herein, is a method for treating cancer comprising
administering to a patient in need an effective amount of an
HPTP.beta.-ECD binding agent in conjunction with one or more
chemotherapeutic compound or immunotherapeutic compound. To
"slightly amplify Tie-2 signaling" means that a sufficient amount
of a disclosed compound is administered to a patient such that the
amount of tumor cell vasculature is increased such that the
increased circulation allows for delivery of the anti-tumor
compound or therapy without instigating tumor growth wherein the
rate of tumor cell growth is less than the rate of tumor cell
death. It is recognized that amplifying Tie2 signaling would
stabilize the tumor vasculature making it resistant to angiogenic
signals reducing tumor angiogenesis and tumor growth while
improving tumor blood flow and the delivery of chemotherapeutic
agents.
[0143] Angiopoietin-2 is significantly correlated to Gleason Score,
metastasis and to cancer specific survival (Lind A. J. et al.,
"Angiopoietin-2 expression is related to histological grade,
vascular density, metastasis and outcome in prostate cancer"
Prostate, (2005), Vol. 62, pp. 394-299). Angiopoietin-2 was found
to be expressed in prostate cancer bone, liver and lymph node
metastasis, but with little to no angiopoietin-1 expression in
prostate cancer tumor cells in bone, liver and lymph nodes
(Morrissey C. et al., "Differential expression of angiogenesis
associated genes in prostate cancer bone, live and lymph node
metastasis" Clin. Exp. Metastasis, (2008), Vol. 25, pp. 377-388).
As such, monitoring the level of Ang-2 provides a method for
evaluating the presence of prostate cancer and the spread of
prostate cancer cells throughout the body due to vascular
leakage.
[0144] Thus, another embodiment of the disclosure is a method of
evaluating efficacy of treatment comprising monitoring the Ang-2
level of the patient while the patient is undergoing treatment.
Vasculature Stabilization in Diseases Caused by Pathogens
[0145] Disclosed herein is a method for preventing or treating
vascular leak syndrome caused by one or more pathogens, comprising
administering to a human or other mammal in need of treatment an
effective amount of one or more HPTP.beta.-ECD binding agent.
[0146] One embodiment is a method for treating vascular leak
syndrome caused by one or more pathogens, comprising administering
to a human or other mammal in need of treatment a composition
comprising: a) an effective amount of one or more compounds
effective against a pathogen present in the human or mammal; and b)
an effective amount of an HPTP.beta.-ECD binding agent; wherein the
one or more compounds effective against a pathogen and the
HPTP.beta.-ECD binding agent can be administered together or in any
order.
[0147] Further disclosed is a method for preventing vascular leak
syndrome in a human or other mammal diagnosed with a pathogen
infection that can produce vascular leak syndrome in a human or
mammal, comprising administering to a human or mammal a composition
comprising: a) an effective amount of one or more compounds
effective against a pathogen present in the human or mammal; and b)
an effective amount of one or more HPTP.beta.-ECD binding agent;
wherein the one or more compounds effective against a pathogen and
the one or more HPTP.beta.-ECD binding agent can be administered
together or in any order.
[0148] The following are non-limiting examples of viruses, bacteria
and other pathogens where virulence can be controlled by mitigating
the degree of vascular leak that is induced by the organism.
Staphylococcus aureus, Bacillus anthracis, Pseudomonas,
Streptococcus pyogenes, and dengue virus.
[0149] One embodiment is a method for treating vascular leak
syndrome in a patient suffering from a bacterial infection by
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. Further disclosed is a
method for preventing vascular leak syndrome in a human or other
mammal diagnosed with a bacterial infection.
[0150] Thus one embodiment of the present disclosure is a method of
treating a patient suffering from a bacterial infection by
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. In particular
embodiments the bacterial infection is a Bacillus anthracis
infection. In other embodiments the bacterial infection is a
Pseudomonas infection. In yet other embodiments the bacterial
infection is a Streptococcus pyogenes infection.
[0151] One embodiment is a method for treating vascular leak
syndrome in a patient suffering from a viral infection by
administering to the patient a composition comprising an effective
amount of an HPTP.beta.-ECD binding agent. Further disclosed is a
method for preventing vascular leak syndrome in a human or other
mammal diagnosed with a viral infection.
[0152] Another embodiment is a method of treating a patient
suffering from a viral infection by administering to the patient a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent. In particular embodiments the viral infection is a
dengue virus infection.
[0153] The HPTP.beta.-ECD binding agent may be administered in
combination with one or more antibacterial or antiviral agent
wherein the HPTP.beta.-ECD binding agent and the antiviral or
antibacterial agents can be administered together or in any order.
Thus, an embodiment of the present disclosure is a method of
treating a patient suffering from a bacterial infection comprising
administering: a) an HPTP.beta.-ECD binding agent; and b) one or
more antibacterial agents, wherein the HPTP.beta.-ECD binding agent
and antibacterial agent can be administered together or in any
order. Another embodiment of the present disclosure is a method of
treating a patient suffering from a viral infection comprising
administering: a) an HPTP.beta.-ECD binding agent; and b) one or
more antiviral agents wherein the HPTP.beta.-ECD binding agent and
antiviral agent can be administered together or in any order.
[0154] Another method provided by the present disclosure, is a
method for determining the course of treatment for a patient
suffering from vascular leak syndrome, comprising: a) administering
to a patient a composition comprising an effective amount of an
HPTP.beta.-ECD binding agent; b) monitoring the level of
angiopoietin-2 present in the patient during the course of
treatment; and c) discontinuing treatment when the angiopoietin-2
level returns to within a normal range.
[0155] A further aspect relates to methods of treating vascular
leak in a patient infected with anthrax comprising administering a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent in combination with an effective amount of one or
more antibacterial agents effective against anthrax, wherein the
HPTP.beta.-ECD binding agent and the antibacterial agents effective
against anthrax are administered together or in any order.
[0156] Yet another aspect relates to methods of treating vascular
leak in a patient infected with a virus comprising administering a
composition comprising an effective amount of an HPTP.beta.-ECD
binding agent in combination with an effective amount of one or
more antiviral agents, wherein the HPTP.beta.-ECD binding agent and
the antiviral agent are administered together or in any order.
[0157] Increased amplification of Tie-2 signaling using the
disclosed agents provides a method for stabilizing vasculature
without the need to affect Ang-1 and/or Ang-2 levels. Disclosed
herein are methods for stabilizing vasculature, comprising
administering to a patient an effective amount of an HPTP.beta.-ECD
binding agents.
[0158] Because the disclosed agents can amplify Tie-2 signaling
without increasing the amount of Ang-2, monitoring the amount of
Ang-2 in blood serum of a patient while administering to a patient
an HPTP.beta.-ECD binding agent, serves as a method for determining
the course of various illnesses or disease states associated with
vascular leak syndrome, for example, sepsis as a result of
infection. As such, disclosed is a method for stabilizing
vasculature in a patient suffering from an inflammatory disease
wherein the level of angiopoietin-2 is elevated, comprising: a)
administering to a patient an effective amount of an HPTP.beta.-ECD
binding agent; b) monitoring the level of angiopoietin-2 present in
the patient; and c) discontinuing treatment when the angiopoietin-2
level returns to a normal range.
[0159] What is meant herein by "normal angiopoietin-2 level" is an
amount of Ang-2 in blood serum of from about 1 ng/mL to about 2
ng/mL. Alternatively, the level of Ang-2 can be determined for an
individual suffering from a disease state, for example, severe
sepsis and the level of Ang-2 can be monitored until the amount of
Ang-2 in the patient's serum drop to a level that is nearer the
normal range. In this case, the co-administration of a drug can be
continued or discontinued.
[0160] Therefore, disclosed herein is a method for stabilizing the
vasculature of a patient during a course of treatment, comprising:
a) co-administering to a patient an effective amount of an
HPTP.beta.-ECD binding agent and one or more drugs as a treatment;
b) monitoring the level of angiopoietin-2 present in the patient;
and c) discontinuing the administration of the one or more drugs,
and selecting one or more other drugs for use as a treatment if the
level of serum angiopoetin-2 does not decrease.
[0161] The HPTP.beta.-ECD binding agent, while stabilizing the
vasculature of a patient such that a course of treatment against a
pathogen can be sustained, can also be used to stabilize a patient
during a period wherein an effective treatment against a pathogen
is being determined. That is, the HPTP.beta.-ECD binding agents by
themselves can have a beneficial effect on the outcome of diseases
caused by pathogens by reducing vascular leak and its
complications.
[0162] Any of the foregoing compositions comprising an
HPTP.beta.-ECD binding agent are suitable for use in the
manufacture of a medicament for treatment of any of the diseases or
disorder described above. In addition, any of the foregoing
compositions comprising an HPTP.beta.-ECD binding agent are
suitable for use in treating any of the diseases or disorder
described above.
In Vivo Vascular Leak
[0163] The Miles assay (Miles, A. A. and E. M. Miles (1952)
Vascular reactions to histamine, histamine-liberator and
leukotaxine in the skin of guinea-pigs. J. Physiol., Vol. 118, pp.
228-257 incorporated herein by reference in its entirety) can be
used to directly investigate and quantify lethal toxin, as well as
edema toxin (ET [PA plus EF])-mediated vascular leakage in the
mouse model.
[0164] The following is a modified Miles assay as described by
Gozes Y. et al., Anthrax Lethal Toxin Induces Ketotifen-Sensitive
Intradermal Vascular Leakage in Certain Inbred Mice Infect. Immun.,
2006 February, Vol. 74, No. 2, pp. 1266-1272 incorporated herein by
reference in its entirety, that can be used to evaluate the
disclosed HPTP.beta.-ECD binding agents for their ability to
prevent vascular leakage in humans and animals exposed to
anthrax.
[0165] Highly pure PA, LF, and mutant LF E687C are purified as
previously described (Varughese M. et al., (1998) Internalization
of a Bacillus anthracis protective antigen-c-Myc fusion protein
mediated by cell surface anti-c-Myc antibodies. Mol. Med. 4:87-95
included herein by reference in its entirety). Doses of ET or LT
refer to the amount of each component (i.e., 100 .mu.g LT is 100
.mu.g PA plus 100 .mu.g of LF). All drugs except for azelastine can
be purchased from Sigma Aldrich (St. Louis, Mo.); azelastine can be
purchased from LKT Laboratories (St. Paul, Minn.). LT is an
abbreviation for lethal toxin; PA is an abbreviation for protective
antigen; LF is an abbreviation for lethal factor; and EP is an
abbreviation for edema factor.
Animals.
[0166] BALB/cJ, DBA/2J, C3H/HeJ, C3H/HeOuJ,
WBB6F1/J-Kit.sup.W/Kit.sup.W-v, and colony-matched wild-type
homozygous control mice can be purchased from The Jackson
Laboratory (Bar Harbor, Me.). BALB/c nude, C57BL/6J nude, and C3H
hairless (C3.Cg/TifBomTac-hr) mice can be purchased from Taconic
Farms (Germantown, N.Y.). C3H nude mice can be purchased from The
National Cancer Institute Animal Production Area (Frederick, Md.).
Mice are used when they are 8 to 12 weeks old. Except for C3H
hairless and nude animals, all mice are shaved 24 hours prior to
intradermal (i.d.) injections. In order to assess the
susceptibility to systemic LT, mice are injected intraperitoneally
(i.p.) with 100 .mu.g LT and observed over 5 days for signs of
malaise or death. Fischer 344 rats can be purchased from Taconic
Farms (Germantown, N.Y.) and used at weights of 150 to 180 g. Rats
are injected intravenously (i.v.) in the tail vein with 12 .mu.g
LT, with or without 250 .mu.g of the mast cell stabilizer drug
ketotifen and monitored for the exact time to death.
Miles Assay.
[0167] The Miles assay uses i.v. injection of Evans blue dye (which
binds to endogenous serum albumin) as a tracer to assay
macromolecular leakage from peripheral vessels after i.d. injection
of test substances. Nude mice and normal shaved mice are injected
i.v. with 200 .mu.l of 0.1% Evans blue dye (Sigma Chemical Co., St.
Louis, Mo.). After 10 min, 30 .mu.l of test toxin or control
samples (PA only, LF only, EF only, or phosphate-buffered saline)
are injected i.d. in both left and right flanks, as well as at
single or dual dorsal sites. To quantify the extents of leakage,
equally sized (1.0- to 1.5-cm diameter) skin regions surrounding
i.d. injection sites are removed 60 min after injection and placed
in formamide (1 ml) at 41.degree. C. for 48 h, allowing for dye
extraction. The A.sub.620 of samples is read, and the extent of
leakage is calculated by comparison with phosphate-buffered
saline-, PA-, or LF-treated controls.
[0168] In experiments wherein the effectiveness of the
HPTP.beta.-ECD binding agents are tested for LT-mediated leakage,
mice are injected i.v. with Evans blue as described above, and the
test agent introduced systemically through i.p. injection 10 min
after dye injection. LT was introduced by i.d. injection 30 min
after the injection of Evans blue. In another embodiment, the agent
to be tested can be introduced locally by i.d. injection and LT
injected in the same site after 10 min.
Cytotoxicity Experiments.
[0169] MC/9 mast cells can be obtained from ATCC (Manassas, Va.)
and grown in Dulbecco's modified Eagle's medium supplemented with
1-glutamine (2 mM), 2-mercaptoethanol (0.05 mM), Rat T-STIM (BD
Biosciences-Discovery Labware, Bedford, Mass.) (10%), and fetal
bovine serum (FBS, 10% final concentration; Invitrogen-GIBCO BRL,
Gaithersburg, Md.). Cells are then seeded at a density of
10.sup.4/well in 96-well plates prior to treatment with various LT
concentrations or PA-only controls. After 6, 12, and 24 hours,
viability is assessed using Promega's CellTiter 96 Aq.sub.ueous One
Solution cell proliferation assay (Promega, Madison, Wis.) per the
manufacturer's protocol. Alternatively, toxicity assays can be
performed in medium provided with all supplements except FBS
(serum-free medium). In other embodiments, pooled human umbilical
vein endothelial cells (HUVECs) at third to fifth passage can be
obtained from Cambrex Corp. (Cambrex, Walkersville, Md.) and grown
in an EGM-MV Bulletkit (Cambrex, Walkersville, Md.) in flasks
pretreated with endothelial cell attachment factor (Sigma, St.
Louis, Mo.). For cytotoxicity experiments, cells are typically
seeded in 96-well plates in an EGM-MV Bulletkit. On the day of
assays, this medium is then replaced with M199 medium (Sigma, St.
Louis, Mo.) supplemented with 10% FBS or human serum (Sigma, St.
Louis, Mo.), and cells are reseeded in 96-well plates at a density
of 2.times.10.sup.3/0.1 ml/well and treated with various
concentrations of LT in triplicate. Cell viability is typically
assessed as for MC/9 cells at 24, 48 and 72 hour time points.
HUVEC Permeability Assay
[0170] HUVEC monolayers can be effectively cultured on
Transwell-Clear cell culture inserts (6.5-mm diameter, 0.4-1 .mu.m
pore size; Corning-Costar, Acton, MA) in 24-well plates, creating a
two-chamber culturing system consisting of a luminal compartment
(inside the insert) and a subluminal compartment (the tissue
culture plate well). Prior to seeding cells, the inserts are coated
with endothelial cell attachment factor (Sigma, St. Louis, Mo.).
Pre-warmed CS-C medium (Sigma, St. Louis, Mo.) containing 10%
iron-supplemented calf serum and 1% endothelial cell growth factor
(Sigma, St. Louis, Mo.) is added to wells prior to insert
placement. A HUVEC cell suspension (200 .mu.L of 5.times.10.sup.5
cells/ml) is then added to each insert. Cells are cultured at
37.degree. C. in 5% CO.sub.2 for up to 21 days to ensure proper
formation of a monolayer. For testing barrier function, medium can
be changed to RPMI supplemented with 10% FBS or to RPMI without
serum. To assess barrier function, horseradish peroxidase enzyme
(Sigma, St. Louis, Mo.) is added to the inserts (10 .mu.g/well). LT
(1 .mu.g/mL) or control treatments of PA alone (1 .mu.g/mL) or LF
alone (1 .mu.g/mL) are added to duplicate wells, and every hour
(for 12 hours), a sample of 10 .mu.L was taken from the subluminal
compartment and tested for the enzymatic activity of horseradish
peroxidase by adding 100 .mu.L substrate
[2',2'-azino-bis(3-ethylbenzthizolin 6-sulfonic acid)] (A-3219;
Sigma, St. Louis, Mo.) and reading at 405 nm.
Anthrax Combination Therapy
[0171] Increased stabilization of vascular tissue can increase the
effectiveness of known antimicrobials against anthrax infection. As
such, HPTP.beta.-ECD binding agents can be evaluated as a
combination therapy for the treatment of anthrax. The following
describes a series of assays that can be used to determine the
effectiveness of an HPTP.beta.-ECD binding agent as one part of a
combination therapy useful for treating anthrax infections.
[0172] LF has been found to cleave mitogen-activated protein kinase
kinases (MAPKK), disrupts signal transduction, and leads to
macrophage lysis. As such, in addition to the Miles Assay, the
following cell-based and peptide cleavage assay can be used to
confirm the potency of the HPTP.beta.-ECD binding agents to inhibit
the effect of LT activity. For the following assay, MAPKKide can be
purchased from List Biological Laboratories (Campbell, Calif.
Fluorinated peptide substrate is available from Anaspec (San Jose,
Calif.).
In Vivo Assays
[0173] One week before beginning an evaluation of a combination
course of treatment for anthrax, test agents (200 mg each) are
dissolved in 800 .mu.L of DMSO and stored at -20.degree. C.
Immediately before injection, each test agent is diluted in PBS,
resulting in a final concentration of 0.5 mg/mL in 2% DMSO. Test
animal are challenged on day 0 with 2.times.10.sup.7 spores per
mouse in PBS through i.p. injection. Treatment was started 24 hours
after challenge. One example of a suitable treatment regimen is the
combination of ciprofloxacin (50 mg/kg) and an HPTP.beta.-ECD
binding agent (5 mg/kg). A control sample of untreated animals,
ciprofloxacin alone, a disclosed agent alone and ciprofloxacin in
combination with a disclosed agent are given to the animals and
they are monitored twice per day until day 14 after injection.
[0174] Ciprofloxacin and the agent to be tested can be conveniently
administered through parenteral injection with a volume of 200
.mu.L for each once per day for 10 days. All surviving animals are
sacrificed on day 14. Sick animals that appear moribund (i.e.,
exhibiting a severely reduced or absent activity or locomotion
level, an unresponsiveness to external stimuli, or an inability to
obtain readily available food or water, along with any of the
following accompanying signs: ruffled haircoat, hunched posture,
inability to maintain normal body temperature, signs of
hypothermia, respiratory distress, or other severely debilitating
condition) should be sacrificed on the same day these symptoms are
manifested.
Modulation of Bacterium-Induced Vascular Leak
[0175] Pathogenic bacteria are known to cause vascular leak. This
induced vascular leakage inhibits the ability of antimicrobials and
other pharmaceuticals from targeting the invading microorganism. As
such, HPTP.beta.-ECD binding agents can be used alone or in
combination with other pharmaceutical ingredients to boost the host
immune system by preventing excess vascular leakage that occurs as
a result of a bacterial infection.
[0176] The following describe tests and assays that can be used to
determine the effectiveness of an HPTP.beta.-ECD binding agent,
either alone, or a combination therapy.
[0177] Staphylococcus aureus (S. aureus) is a major pathogen of
gram-positive septic shock and is associated with consumption of
plasma kininogen. The effect of an HPTP.beta.-ECD binding agent on
S. aureus induced vascular leakage activity can be determined by
measuring the activity of these agents with respect to two cysteine
proteinases that are secreted by S. aureus. Proteolytically active
staphopain A (ScpA) induces vascular leakage in a bradykinin (BK)
B.sub.2-receptor-dependent manner in guinea pig skin. This effect
is augmented by staphopain B (SspB), which, by itself, had no
vascular leakage activity. ScpA also produces vascular leakage
activity from human plasma.
[0178] An important pathophysiologic mechanism of septic shock is
hypovolemic hypotension that is caused by plasma leakage into the
extravascular space. It has been found that ScpA induced vascular
leakage at a concentration as low as 20 nM within 5 minute after
injection into the guinea pig skin--with the reaction being
augmented by coexisting SspB indicating that vascular leakage
induction by these proteinases occurs efficiently in vivo (Imamura
T. et al., Induction of vascular leakage through release of
bradykinin and a novel kinin by cysteine proteinases from
Staphylococcus aureus (2005) J. Experimental Medicine, Vol. 201,
No. 10, pp. 1669-1676).
Vascular Leakage Assay.
[0179] Animals can be evaluated for vascular leakage using the
following procedure. 100 .mu.L of a 1% solution of Evans blue dye
(Sigma Aldrich) in saline is injected into the tail vein. Thirty
minutes later, mice are sacrificed and perfused with saline via the
right ventricle to remove intravascular Evans blue. Lungs are
excised and extracted in 1 mL of formamide at 55.degree. C.
overnight. Evans blue content is determined as OD.sub.620 minus
OD.sub.500 of the formamide extract.
[0180] The agents disclosed herein can be used as a single
pharmaceutical therapy to reduce the severity of influenza by
mediating the effects of vascular leak caused by viruses, and,
hence, allowing the body's own immune system to affect greater
resistance to these pathogens. The following assays can be used to
determine the effect of an HPTP.beta.-ECD binding agent to inhibit
viral severity because of improved vascular integrity.
[0181] The disclosed assays can use inhibition of viral plaques,
viral cytopathic effect (CPE), and viral hemagglutitin.
Proteolytic Sensitivity Assay
[0182] An HPTP.beta.-ECD binding agent can be determined to bind to
hemagglutinin and thereby destabilize the protein assembly. The
following procedure can be used to determine the increase in
destabilization and therefore the increased sensitivity of
hemagglutinin to proteolytic attack caused by an HPTP.beta.-ECD
binding agent. At the fusion conformation, HA becomes more
sensitive to protease digestion. This property can be used to
verify if a fusion inhibitor interacts with HA (Luo G. et al.,
"Molecular mechanism underlying the action of a novel fusion
inhibitor of influenza A virus." J. Virol., (1997), Vol. 71, No. 5,
pp. 4062-4070). Thus, an HPTP.beta.-ECD binding agent, due to the
control of vascular leakage, can be evaluated for its ability to
indirectly effect HA digestion by enhancing the body's immune
response.
[0183] The purified trimer of hemagglutinin ectodomain is incubated
with the agent to be tested at a concentration of 5 .mu.M. The
trimers are subjected to trypsin digestion at pH 7.0 and pH 5.0
with controls of untreated HA and HA treated with DMSO which is the
solvent used to dissolve the test agent. For the pH 5.0 sample, the
HA trimers are treated with a pH 5.0 buffer for 15 minutes and
neutralized to pH 7.0. Trypsin (20 ng) is added to the sample in 10
.mu.L and the digestion allowed to proceed for 1 hour at 37.degree.
C., The amount of HA present is assessed by a western blot gel
electrophoresis using anti-HA (H3) antisera. Samples containing
effective inhibitors will provide an increase in digestion of HA by
trypsin.
[0184] In addition, combination therapies can provide a method for
treating influenza by providing an antiviral medication together
with an agent that prevents the severity of vascular leakage due to
influenza viruses.
[0185] An antiviral compound, for example, oseltamivir, can be used
for an in vivo evaluation of the disclosed combination therapy and
to evaluate the effectiveness of an HPTP.beta.-ECD binding agent.
The drug combination is administered in a single dose to mice
infected with the influenza A/NWS/(H1N1) virus. In some instances,
infection of the animals will include multiple passage of the virus
through their lungs. One convenient protocol involves administering
20 mg/kg per day twice daily for 5 days beginning 4 hours prior to
virus exposure. The animals are then challenged with different
concentrations of virus, ranging 10-fold from 10.sup.-2
(10.sup.5.75 cell culture 50% infectious doses (CCID.sub.50) per
mL). Four mice in each group are sacrificed on day 6 and their
lungs removed, assigned a consolidation score ranging from 0
(normal) to 4 (maximal plum coloration), weighted, homogenized, the
homogenates centrifuged at 2000.times.g for 10 minutes, and varying
10-fold dilutions of the supernatant assayed for virus titer in
MDCK cells using CPE produced after a 96-hour incubation at
37.degree. C. as endpoint.
[0186] The serum taken from mice on day 6 is assayed for a.sub.1-AG
using single radial immunodiffusion kites. Eight additional mice in
each group are continually observed daily for death for 21 days,
and their arterial oxygen saturation (SaO.sub.2) values determined
by pulse oximetery (Sidwell R. et al., (1992) Utilization of pulse
oximetry for the study of the inhibitory effects of antiviral
agents on influenza virus in mice. Antimicrob. Agents Chemother.
36, 473-476) on day 3, when SaO.sub.2 decline usually begins to
occur, through day 11, when the values are seen to decline to the
maximum degree of the animals otherwise die.
Inhibition of Protein Tyrosine Phosphatase Beta in a Cell
[0187] Disclosed herein are methods for inhibiting protein tyrosine
phosphatase beta (HPTP-.beta.) activity in a cell, comprising
contacting a cell with an effective amount of an HPTP.beta.-ECD
binding agents. The cell can be contacted in vivo, ex vivo, or in
vitro.
Administration
[0188] Depending on the nature of the particular agent, agents of
the present disclosure can be administered to humans and other
animal, parenterally, (e.g., by intravenous or intraperitoneal
injection), subcutaneously, orally, topically, rectally, buccally,
as an oral or nasal spray.
[0189] The HPTP.beta.-ECD binding agents of the disclosure are
preferably formulated in dosage unit form for ease of
administration and uniformity of dosage. The expression "dose" or
"dosage unit form" as used herein refers to a physically discrete
unit of agent appropriate for the patient to be treated. It will be
understood, however, that the total daily usage of the
HPTP.beta.-ECD binding agents and compositions of the present
invention will be decided by the attending physician within the
scope of sound medical judgment.
Dosing
[0190] Effective dosages and schedules for administering the
HPTP.beta.-ECD binding agent may be determined empirically, and
making such determinations is within the skill in the art. Those
skilled in the art will understand that the dosage of the agent
that must be administered will vary depending on, for example, the
subject which will receive the agent, the route of administration,
the particular type of agent used and other drugs being
administered to the subject. For example, guidance in selecting
appropriate doses for antibodies is found in the literature on
therapeutic uses of antibodies, e.g., Handbook of Monoclonal
Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge,
N.J., (1985) ch. 22 and pp. 303-357; Smith et al., Antibodies in
Human Diagnosis and Therapy, Haber et al., eds., Raven Press, New
York (1977) pp. 365-389. A typical dose of the agent used alone
might range from about 0.01 mg/kg to up to 500 mg/kg of body weight
or more per day, or from about 0.01 mg/kg to about 50 mg/kg, or
from 0.1 mg/kg to about 50 mg/kg, or from about 0.1 mg/kg to up to
about 10 mg/kg, or from about 0.2 mg/kg to about 1 mg/kg, or from
about 1 mg/kg to about depending on the factors mentioned
above.
[0191] The dosing schedules for administration of an HPTP.beta.-ECD
binding agent include, but are not limited to, once daily,
three-times weekly, twice weekly, once weekly, three times, twice
monthly, once monthly and once every other month.
Formulations
[0192] In one aspect of the present invention, pharmaceutically
acceptable compositions are provided, wherein these compositions
comprise any of the agents as described herein, and a
pharmaceutically acceptable carrier and, in addition, can include
other pharmaceutical agents, adjuvants or diluents. For example,
pharmaceutical compositions can also include one or more additional
active ingredients such as antimicrobial agents, anti-inflammatory
agents, anesthetics and the like.
[0193] The formulation may vary depending on the mode of
administration. The pharmaceutical compositions can be in the form
of solid, semi-solid or liquid dosage forms, such as, for example,
tablets, suppositories, pills, capsules, powders, liquids,
suspensions, lotions, creams, gels, or the like, preferably in unit
dosage form suitable for single administration of a precise
dosage.
[0194] For the purposes of the present disclosure the term
"excipient" and "carrier" are used interchangeably throughout the
description of the present disclosure and said terms are defined
herein as, "ingredients which are used in the practice of
formulating a safe and effective pharmaceutical composition." The
formulator will understand that excipients are used primarily to
serve in delivering a safe, stable and functional pharmaceutical,
serving not only as part of the overall vehicle for delivery but
also as a means for achieving effective absorption by the recipient
of the active ingredient. An excipient may fill a role as simple
and direct as being an inert filler, or an excipient as used herein
may be part of a pH stabilizing system or coating to insure
delivery of the ingredients.
[0195] "Pharmaceutically acceptable" means a material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to a patient without causing any undesirable
biological effects or interacting in a deleterious manner with any
of the other components of the pharmaceutical formulation in which
it is contained. The carrier would naturally be selected to
minimize any degradation of the active ingredient and to minimize
any adverse side effects in the patient, as would be well known to
one of skill in the art. See Remington's Pharmaceutical Sciences,
18th ed., Gennaro, AR. Ed., Mack Publishing, Easton Pa. (1990),
which discloses typical carriers and conventional methods of
preparing pharmaceutical compositions that can be used in
conjunction with the preparation of formulations of the agents
described herein. It will be apparent to those persons skilled in
the art that certain carriers can be more preferable depending
upon, for instance, the route of administration and concentration
of composition being administered.
[0196] For solid compositions, conventional nontoxic solid carriers
include, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharin, talc, cellulose,
glucose, sucrose, magnesium carbonate and the like. Liquid
pharmaceutically administrable compositions can, for example, be
prepared by dissolving, dispersing, etc., an active agent as
described herein and optional pharmaceutical adjuvants in an
excipient, such as, for example, water, saline aqueous dextrose,
glycerol, ethanol and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered can also contain minor amounts of nontoxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like, for example, sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, etc. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this art.
[0197] The disclosed agents can also be present in liquids,
emulsions, or suspensions for delivery of active therapeutic
agents. Liquid pharmaceutically administrable compositions can, for
example, be prepared by dissolving, dispersing, etc., an active
agent as described herein and optional pharmaceutical adjuvants in
an excipient, such as, for example, water, saline aqueous dextrose,
glycerol, ethanol and the like, to thereby form a solution or
suspension. If desired, the pharmaceutical composition to be
administered can also contain minor amounts of nontoxic auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like, for example, sodium acetate, sorbitan
monolaurate, triethanolamine sodium acetate, triethanolamine
oleate, etc. Actual methods of preparing such dosage forms are
known, or will be apparent, to those skilled in this art; for
example see Remington's Pharmaceutical Sciences, 18th ed., Gennaro,
AR. Ed., Mack Publishing, Easton Pa. (1990).
[0198] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0199] The injectable formulations can be sterilized, for example,
by filtration through a bacterial-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions which can be dissolved or dispersed in sterile water
or other sterile injectable medium prior to use.
[0200] The disclosed agents can also be present in liquids,
emulsions, or suspensions for delivery of active therapeutic agents
in aerosol form to cavities of the body such as the nose, throat,
or bronchial passages. The ratio of agents to the other compounding
agents in these preparations will vary as the dosage form
requires.
[0201] Depending on the intended mode of administration, the
pharmaceutical compositions can be in the form of solid, semi-solid
or liquid dosage forms, such as, for example, tablets,
suppositories, pills, capsules, powders, liquids, suspensions,
lotions, creams, gels, or the like, preferably in unit dosage form
suitable for single administration of a precise dosage. The
compositions will include, as noted above, an effective amount of
the agents in combination with a pharmaceutically acceptable
carrier and, in addition, can include other medicinal agents,
pharmaceutical agents, carriers, adjuvants, diluents, etc.
[0202] When the agents are to be delivered into a mammal other than
a human, the mammal can be a non-human primate, horse, pig, rabbit,
dog, sheep, goat, cow, cat, guinea pig, or rodent. The terms human
and mammal do not denote a particular age or sex. Thus, adult and
newborn subjects, as well as fetuses, whether male or female, are
intended to be covered. A patient, subject, human or mammal refers
to a subject afflicted with a disease or disorder. The term
"patient" includes human and veterinary subjects.
KITS
[0203] Also disclosed are kits comprising the agents be delivered
into a human, mammal, or cell. The kits can comprise one or more
packaged unit doses of a composition comprising an HPTP.beta.-ECD
binding agent to be delivered into a human, mammal, or cell. The
kit optionally includes directions for using the components of the
kit. The agents can be packaged as a sterile formulation, and the
hermetically sealed container is designed to preserve sterility of
the formulation until use.
EXAMPLES
Example 1
Production of the HPTP.beta. Extracellular Domain Protein
[0204] Full length HPTP.beta. cDNA (SEQ ID NO:1) is cloned from a
human placental library according to the manufacturer's (Origene)
instructions. A cDNA encoding the entire soluble extracellular
domain (ECD) of HPTP.beta. is cloned by PCR from the full length
cDNA coding for amino acids 1-1621 with an added c-terminal
His-His-His-His-His-His-Gly (6His-Gly) (SEQ ID NO:3). The resulting
cDNA is cloned into mammalian expression vectors for transient
(pShuttle-CMV) or stable (pcDNA3.1(-)) expression in HEK293 cells.
To obtain purified HPTP.beta. ECD (.beta.ED), HEK293 cells
transfected with a 13ECD expression vector are incubated in
OptiMEM-serum free (Gibco) for 24 hours under normal growth
conditions. The conditioned media is then recovered, centrifuged to
remove debris, and 1 mL of washed Ni-NTA agarose (Qiagen) (500
.mu.L packed material) is added to each 10 .mu.L of cleared media
and allowed to rock overnight at 4.degree. C. On the following day,
the mixture is loaded into a column and washed with 20 bed volumes
of 50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8. The
purified HPTP.beta. extracellular domain protein (SEQ ID NO:4) is
then eluted with 200 .mu.L/elution in 50 mM NaH.sub.2PO.sub.4, 300
mM NaCl, 250 mM Imidazole, pH 8. Fractions are analyzed for protein
content using reducing-denaturing SDS-polyacrylimide gel
electrophoresis and detected by silver stain (Invitrogen) and
confirmed by mass spectrometry.
Example 2
[0205] Purified HPTP.beta. extracellular domain protein is
produced, for example by the procedure described in Example 1. For
production of the HPTP.beta. extracellular domain immunogen, the
purified HPTP.beta.extracellular domain-6-His protein is conjugated
to porcine thyroglobulin (Sigma) using EDC coupling chemistry
(Hockfield, S. et al., (1993) Cold Spring Habor Laboratory Press.,
Vol. 1 pp. 111-201, Immunocytochemistry). The resulting HPTP.beta.
extracellular domain-thyroglobulin conjugate is dialyzed against
PBS, pH 7.4. Adult Balb/c mice are then immunized subcutaneously
with the conjugate (100-200 .mu.g) and complete Freund's adjuvant
in a 1:1 mixture. After 2-3 weeks, the mice are injected
intraperitoneally or subcutaneously with incomplete Freund's
adjuvant and the conjugate in a 1:1 mixture. The injection is
repeated at 4-6 weeks. Sera are collected from mice 7 days
post-third-injection and assayed for immunoreactivity to HPTP.beta.
extracellular domain antigen by ELISA and western blotting. Mice
that display a good response to the antigen are boosted by a single
intra-spleen injection with 50 .mu.l of purified HPTP.beta.
extracellular domain protein mixed 1:1 with Alum hydroxide using a
31 gauge extra long needle (Goding, J. W., (1996) Monoclonal
Antibodies: Principles and Practices. Third Edition, Academic Press
Limited. p. 145). Briefly, mice are anesthetized with 2.5% avertin,
and a 1 centimeter incision is created on the skin and left oblique
body wall. The antigen mixture is administered by inserting the
needle from the posterior portion to the anterior portion of the
spleen in a longitudinal injection. The body wall is sutured and
the skin is sealed with two small metal clips. Mice are monitored
for safe recovery. Four days after surgery the mouse spleen is
removed and single cell suspensions are made for fusion with mouse
myeloma cells for the creation of hybridoma cell lines (Spitz, M.,
(1986) Methods In Enzymology, Vol. 121. Eds. John J, Lagone and
Helen Van Vunakis. pp. 33-41 (Academic Press, New York, N.Y.)).
Resulting hybridomas are cultured in Dulbeccos modified media
(Gibco) supplemented with 15% fetal calf serum (Hyclone) and
hypoxathine, aminopterin and thymidine.
[0206] Screening for positive hybridomas begins 8 days after the
fusion and continues for 15 days. Hybridomas producing
anti-HPTP.beta. extracellular domain antibodies are identified by
ELISA on two sets of 96-well plates: one coated with the histidine
tagged-HPTP.beta. extracellular domain and another one coated with
a histidine-tagged bacterial MurA protein as a negative control.
The secondary antibody is a donkey anti-mouse IgG labeled with
horseradish peroxidase (HRP) (Jackson Immunoresearch).
Immunoreactivity is monitored in wells using color development
initiated by ABTS tablets dissolved in TBS buffer, pH 7.5. The
individual HRP reaction mixtures are terminated by adding 100
microliters of 1% SDS and reading absorbance at 405 nm with a
spectrophotometer. Hybridomas producing antibodies that interact
with HPTP.beta. extracellular domain-6His, and not with the
murA-6His protein are used for further analysis. Limiting dilutions
(0.8 cells per well) are performed twice on positive clones in 96
well plates, with clonality defined as having greater than 99% of
the wells with positive reactivity. Isotypes of antibodies are
determined using the iso-strip technology (Roche). To obtain
purified antibody for further evaluation, tissue culture
supernatants are affinity purified using a protein A or a protein G
column.
[0207] Five monoclonal antibodies immunoreactive to HPTP.beta.-ECD
protein were isolated and given the following nomenclature, R15E6,
R12A7, R3A2, R11C3, R15G2 and R5A8. Based on its reaction with the
HPTP.beta.-ECD protein in ELISA and in western blots, R15E6 was
selected for further study.
Example 3
The Monoclonal Antibody R15E6
[0208] The monoclonal antibody R15E6 was identified and
characterized as described in Example 2 of the present application
and in U.S. Pat. No. 7,973,142; the procedure and results are
summarized below.
A. R15E6 Binds Endogenous HPTP.beta. as Demonstrated by
Demonstrated by Immunoprecipitation.
[0209] Materials: Human umbilical vein endothelial cells (HUVECs),
EGM media, and trypsin neutralizing solution from Cambrex; OPTIMEM
I (Gibco), bovine serum albumin (BSA; Santa Cruz), phosphate
buffered saline (PBS; Gibco), Growth Factors including Angiopoietin
1 (Ang1), vascular endothelial growth factor (VEGF) and fibroblast
growth factor (FGF) (R&D Systems), Tie2 monoclonal antibody
(Duke University/P&GP), VEGF receptor 2 (VEGFR2) polyclonal
antibody (Whitaker et. al), protein A/G agarose (Santa Cruz),
Tris-Glycine pre-cast gel electrophoresis/transfer system (6-8%)
(Invitrogen), PVDF membranes (Invitrogen), lysis buffer (20 mm
Tris-HCl, 137 mm NaCl, 10% glycerol, 1% triton-X-100, 2 mM EDTA, 1
mM NaOH, 1 mM NaF, 1 mM PMSF, 1 .mu.g/ml leupeptin, 1 .mu.g/ml
pepstatin).
[0210] Method: HUVECs are pre-treated for 30 min with antibody (in
OPTIMEM) or OPTIMEM I alone. After removal of pre-treatment, cells
are treated with Ang1 (100 ng/ml) for 6 minutes in PBS+0.2% BSA and
lysed in lysis buffer. Lysates are run directly on a Tris-Glycine
gel or immunoprecipitated with 2-5 .mu.g/ml Tie-2 antibody or 10
.mu.g/ml R15E6 antibody and protein A/G agarose. Immunoprecipitated
samples are rinsed once with lysis buffer and boiled for 5 min in
1.times. times sample buffer. Samples are resolved on a
Tris-Glycine gel, transferred to a PVDF membrane, and detected by
western blot using the indicated antibodies (pTYR Ab (PY99, Santa
Cruz), Tie-2, VEGFR2 and/or R15E6).
[0211] Results: By IP/western blotting, R15E6 recognizes a major,
high molecular weight band consistent with the size of HPTP.beta.
(FIG. 1, Panel A, Lane 2). The less intense, lower molecular weight
bands likely represent less glycosylated precursor forms of
HPTP.beta.. An immunoprecipitation (IP) with control, non-immune
IgG shows no bands in the molecular weight range of HPTP.beta.
(FIG. 1, Panel A, Lane 1), and a combined Tie2/VEGFR2 IP shows
bands of the expected molecular weight (FIG. 1, Panel A, Lane 3).
This result demonstrates that R15E6 recognizes and is specific for
HPTP.beta..
B. R15E6 Binds Endogenous HPTP.beta. as Demonstrated by FACS
Analysis
[0212] Materials: HUVECs, EGM media, and trypsin neutralizing
solution from Cambrex; Secondary Alexfluor 488-tagged antibody from
Molecular Probes; Hanks balanced salt solution (Gibco); FACSCAN
flow cytometer and CellQuest software from Becton Dickenson.
[0213] Method: HUVECs are trypsinized, treated with trypsin
neutralizing solution and rinsed with HBSS. R15E6 antibody (0.6
.mu.g) is added to 250,000 cells in 500 of HBSS and incubated on
ice for 20 minutes. Cells are rinsed with 1 ml HBSS followed by
adding 2 .mu.g of fluorescent-conjugated secondary antibody for 20
minutes on ice. Cells are rinsed and resuspended in 1 ml HBSS then
analyzed on the FACSCAN flow cytometer with CellQuest software.
Control cells are treated with fluorescent-conjugated secondary
antibody only.
[0214] Results: By FACS analysis, intact HUVECs, R15E6 causes a
robust shift (>90% of cells) in the fluorescence signal compared
to the secondary antibody alone (FIG. 1, Panel B). This result
indicates that R15E6 binds to endogenous HPTP.beta. presented on
the surface of intact endothelial cells.
Example 4
R15E6 Enhances Tie2 Activation
[0215] R15E6 enhances Tie2 phosphorylation in the absence and
presence of the angiopoietin 1 (Ang1), the Tie2 ligand.
[0216] Methods: HUVECs are cultured in serum free media as
described above in the presence or absence of various
concentrations of R15E6 and with or without added Ang1. Lysates are
prepared, immunoprecipitated with a Tie2 antibody, resolved by
polyacrylamide gel electrophoresis and transferred to a PVDF
membrane. Membrane-bound immunoprecipitated proteins are then
serially western blotted with an antiphosphotyrosine antibody to
quantify Tie2 phosphorylation followed by a Tie2 antibody to
quantify total Tie2. Tie2 phosphorylation is expressed as the ratio
of the antiphosphotyrosine signal over the total Tie2 signal.
[0217] Results: R15E6 enhances Tie2 phosphorylation both in the
absence and presence of Ang1 (FIG. 2). This result indicates that
binding of R15E6 to HPTP.beta. on the surface of endothelial cells
modulates its biological function resulting in enhanced activation
of Tie2 in the absence or presence of ligand.
Example 5
Generation of Anti-VE-PTP Extracellular Domain Antibodies
A. Production of Mouse VE-PTP Extracellular Domain Protein
(VE-PTP-ECD)
[0218] VE-PTP-ECD may be produced by any suitable method. Such
methods are well known in the art. For example, VE-PTP-ECD can be
produced using a method similar to Example 1 of the present
disclosure where VE-PTP-ECD cDNA is used in place of cDNA encoding
HPTP.beta.-ECD. SEQ ID NO:7 provides a nucleotide sequence that
encodes VE-PTP-ECD. SEQ ID NO:8 provides the amino acid sequence of
VE-PTP-ECD.
B. Generation of Antibodies to VE-PTP ECD
[0219] Anti-VE-PTP antibodies are readily generated by methods that
are well known in the art. For example, anti VE-PTP antibodies can
be generated using the method of Example 2 of the present
disclosure by substituting VE-PTP-ECD for the HPTP.beta.
extracellular domain and immunizing rats with the resulting
protein. The rat anti-mouse VE-PTP antibody used in the present
studies was kindly provided by Dr. D. Vestweber (mAb 109). The
antibody was generated as described in Baumer S. et al., Blood,
2006, Vol. 107, pp. 4754-4762. Briefly, the antibody was generated
by immunizing rats with a VE-PTP-Fc fusion protein. Immunization,
hybridoma-fusion, and screening were conducted as described in
Gotsch U., et al., J Cell Sci., 1997, Vol. 110, pp. 583-588 and
Bosse R. and Vestweber D., Eur J. Immunol., 1994, Vol. 24, pp.
3019-3024.
[0220] The fusion protein was constructed such that the first 8
fibronectin type III-like repeats ending with the amino acid
proline at position 732 of VE-PTP were fused in frame with the Fc
part of human IgG1 (starting with amino acid proline at position
239). This construct cloned into pcDNA3 (Invitrogen) was stably
transfected into CHO cells, and the fusion protein was purified by
protein A Sepharose affinity purification.
Example 6
Intravitreal Injections of an Anti-VE-PTP ECD Antibody
[0221] Laser-induced Choroidal Neovascularization Model: The
choroidal neovascularization model is considered to represent a
model of neovascular age-related macular degeneration. Choroidal NV
was generated as previously described. See To be T, et al., Am. J.
Pathol., 1998, Vol. 153, pp. 1641-1646. Adult C57BL/6 mice had
laser-induced rupture of Bruch's membrane in three locations in
each eye and were then given 1 .mu.L intravitreal injections of 1
or 2 .mu.g of a rat anti-mouse VE-PTP-ECD antibody (IgG2a), in one
eye and vehicle (5% dextrose) in the fellow eye. These treatments
were repeated on day 7. Fourteen days after laser, the mice were
perfused with fluorescein-labeled dextran (2.times.10.sup.6 average
MW, Sigma, St. Louis, Mo.) and the extent of neovascularization was
assessed in choroidal flat mounts by fluorescence microscopy. The
area of CNV at each Bruch's membrane rupture site was measured by
image analysis by an observer masked with respect to treatment
group. The area of CNV is the average of the three rupture sites in
one eye. As shown in FIG. 3, treatment with the VE-PTP-ECD antibody
significantly reduced choroidal neovascularization at both 1 and 2
.mu.g doses versus treatment with vehicle control.
Example 7
Oxygen-Induced Ischemic Retinopathy
[0222] The oxygen-induced ischemic retinopathy model is considered
to represent a model of proliferative diabetic retinopathy.
Ischemic retinopathy was produced in C57BL/6 mice by a method
described by Smith, L. E. H., et al. Oxygen-induced retinopathy in
the mouse. Invest. Ophthalmol. Vis. Sci. 35, 101-111 (1994).
[0223] C57BL/6 mice at postnatal day 7 (P7) and their mothers were
placed in an airtight chamber and exposed to hyperoxia (75.+-.3%
oxygen) for five days. Oxygen was continuously monitored with a
PROOX model 110 oxygen controller (Reming Bioinstruments Co.,
Redfield, N.Y.). On P12, mice were returned to room air and under a
dissecting microscope, a Harvard Pump Microinjection System and
pulled glass pipettes were used to deliver a 1 .mu.l intravitreal
injection of 1 or 2 .mu.g of a VE-PTP-ECD antibody was made in one
eye and vehicle was injected in the fellow eye. At P17, the area of
NV on the surface of the retina was measured at P17 as previously
described. See Shen J, et al., Invest. Ophthalmol. Vis. Sci., 2007,
Vol. 48, pp. 4335-4341. Briefly, mice were given an intraocular
injection of 1 .mu.l containing 0.5 .mu.g rat anti-mouse PECAM
antibody (Pharmingen, San Jose, Calif.). Twelve hours later, the
mice were euthanized, the eyes fixed in 10% formalin. The retinas
were dissected, incubated for 40 minutes in 1:500 goat anti-rat IgG
conjugated with Alexa488 (Invitrogen, Carlsbad, Calif.), washed,
and whole mounted. An observer masked with respect to treatment
group examined the slides with a Nikon Fluorescence microscope and
measured the area of NV per retina by computerized image analysis
using ImagePro Plus software (Media Cybernetics, Silver Spring,
Md.). FIG. 4 shows that treatment with the VE-PTP-ECD antibody
significantly reduced retinal neovascularization at both 1 and 2
.mu.g doses versus treatment with vehicle control. FIG. 5 shows
representative retinal whole mounts from a mouse treated with
vehicle versus a mouse treated with 2 .mu.g of the VE-PTP-ECD
antibody.
Example 8
Subcutaneous Injection of a VE-PTP-ECD Antibody
[0224] The oxygen-induced ischemic retinopathy model was conducted
as described in Example 7 (containment in a 75% oxygen atmosphere
from P5 to P12) for intravitreal dosing except that the VE-PTP-ECD
antibody (1 mg/kg) was dosed subcutaneously at P12 when the mice
were returned to room air and again on days P14 and P16 (three
total doses). Neovascularization was assessed as described above on
day (P17). FIG. 6 shows that subcutaneous dosing of the VE-PTP-ECD
antibody reduces the area of retinal neovascularization.
Example 9
[0225] The experiment described in Example 8 was repeated at a
subcutaneous dose of 2 mg/kg. (FIG. 7)
[0226] While a number of embodiments of this disclosure are
described, it is apparent that the basic examples may be altered to
provide other embodiments that utilize or encompass the
HPTP.beta.-ECD binding agent, methods and processes of this
invention. The embodiments and examples are for illustrative
purposes and are not to be interpreted as limiting the disclosure,
but rather, the appended claims define the scope of this invention.
Sequence CWU 1
1
716045DNAHomo sapiens 1atgctgagcc atggagccgg gttggccttg tggatcacac
tgagcctgct gcagactgga 60ctggcggagc cagagagatg taacttcacc ctggcggagt
ccaaggcctc cagccattct 120gtgtctatcc agtggagaat tttgggctca
ccctgtaact ttagcctcat ctatagcagt 180gacaccctgg gggccgcgtt
gtgccctacc tttcggatag acaacaccac atacggatgt 240aaccttcaag
atttacaagc aggaaccatc tataacttca agattatttc tctggatgaa
300gagagaactg tggtcttgca aacagatcct ttacctcctg ctaggtttgg
agtcagtaaa 360gagaagacga cttcaaccgg cttgcatgtt tggtggactc
cttcttccgg aaaagtcacc 420tcatatgagg tgcaattatt tgatgaaaat
aaccaaaaga tacagggggt tcaaattcaa 480gaaagtactt catggaatga
atacactttt ttcaatctca ctgctggtag taaatacaat 540attgccatca
cagctgtttc tggaggaaaa cgttcttttt cagtttatac caatggatca
600acagtgccat ctccagtgaa agatattggt atttccacaa aagccaattc
tctcctgatt 660tcctggtccc atggttctgg gaatgtggaa cgataccggc
tgatgctaat ggataaaggg 720atcctagttc atggcggtgt tgtggacaaa
catgctactt cctatgcttt tcacgggctg 780tcccctggct acctctacaa
cctcactgtt atgactgagg ctgcagggct gcaaaactac 840aggtggaaac
tagtcaggac agcccccatg gaagtctcaa atctgaaggt gacaaatgat
900ggcagtttga cctctctaaa agtcaaatgg caaagacctc ctggaaatgt
ggattcttac 960aatatcaccc tgtctcacaa agggaccatc aaggaatcca
gagtattagc accttggatt 1020actgaaactc actttaaaga gttagtcccc
ggtcgacttt atcaagttac tgtcagctgt 1080gtctctggtg aactgtctgc
tcagaagatg gcagtgggca gaacatttcc agacaaagtt 1140gcaaacctgg
aggcaaacaa taatggcagg atgaggtctc ttgtagtgag ctggtcgccc
1200cctgctggag actgggagca gtatcggatc ctactcttca atgattctgt
ggtgctgctc 1260aacatcactg tgggaaagga agaaacacag tatgtcatgg
atgacacggg gctcgtaccg 1320ggaagacagt atgaggtgga agtcattgtt
gagagtggaa atttgaagaa ttctgagcgt 1380tgccaaggca ggacagtccc
cctggctgtc ctccagcttc gtgtcaaaca tgccaatgaa 1440acctcactga
gtatcatgtg gcagacccct gtagcagaat gggagaaata catcatttcc
1500ctagctgaca gagacctctt actgatccac aagtcactct ccaaagatgc
caaagaattc 1560acttttactg acctggtgcc tggacgaaaa tacatggcta
cagtcaccag tattagtgga 1620gacttaaaaa attcctcttc agtaaaagga
agaacagtgc ctgcccaagt gactgacttg 1680catgtggcca accaaggaat
gaccagtagt ctgtttacta actggaccca ggcacaagga 1740gacgtagaat
tttaccaagt cttactgatc catgaaaatg tggtcattaa aaatgaaagc
1800atctccagtg agaccagcag atacagcttc cactctctca agtccggcag
cctgtactcc 1860gtggtggtaa caacagtgag tggagggatc tcttcccgac
aagtggttgt ggagggaaga 1920acagtccctt ccagtgtgag tggagtaacg
gtgaacaatt ccggtcgtaa tgactacctc 1980agcgtttcct ggctcgtggc
gcccggagat gtggataact atgaggtaac attgtctcat 2040gacggcaagg
tggttcagtc ccttgtcatt gccaagtctg tcagagaatg ttccttcagc
2100tccctcaccc caggccgcct ctacaccgtg accataacta caaggagtgg
caagtatgaa 2160aatcactcct tcagccaaga gcggacagtg cctgacaaag
tccagggagt cagtgttagc 2220aactcagcca ggagtgacta tttaagggta
tcctgggtgc atgccactgg agactttgat 2280cactatgaag tcaccattaa
aaacaaaaac aacttcattc aaactaaaag cattcccaag 2340tcagaaaacg
aatgtgtatt tgttcagcta gtccctggac ggttgtacag tgtcactgtt
2400actacaaaaa gtggacaata tgaagccaat gaacaaggga atgggagaac
aattccagag 2460cctgttaagg atctaacatt gcgcaacagg agcactgagg
acttgcatgt gacttggtca 2520ggagctaatg gggatgtcga ccaatatgag
atccagctgc tcttcaatga catgaaagta 2580tttcctcctt ttcaccttgt
aaataccgca accgagtatc gatttacttc cctaacacca 2640ggccgccaat
acaaaattct tgtcttgacg attagcgggg atgtacagca gtcagccttc
2700attgagggct tcacagttcc tagtgctgtc aaaaatattc acatttctcc
caatggagca 2760acagatagcc tgacggtgaa ctggactcct ggtgggggag
acgttgattc ctacacggtg 2820tcggcattca ggcacagtca aaaggttgac
tctcagacta ttcccaagca cgtctttgag 2880cacacgttcc acagactgga
ggccggggag cagtaccaga tcatgattgc ctcagtcagc 2940gggtccctga
agaatcagat aaatgtggtt gggcggacag ttccagcatc tgtccaagga
3000gtaattgcag acaatgcata cagcagttat tccttaatag taagttggca
aaaagctgct 3060ggtgtggcag aaagatatga tatcctgctt ctaactgaaa
atggaatcct tctgcgcaac 3120acatcagagc cagccaccac taagcaacac
aaatttgaag atctaacacc aggcaagaaa 3180tacaagatac agatcctaac
tgtcagtgga ggcctcttta gcaaggaagc ccagactgaa 3240ggccgaacag
tcccagcagc tgtcaccgac ctgaggatca cagagaactc caccaggcac
3300ctgtccttcc gctggaccgc ctcagagggg gagctcagct ggtacaacat
ctttttgtac 3360aacccagatg ggaatctcca ggagagagct caagttgacc
cactagtcca gagcttctct 3420ttccagaact tgctacaagg cagaatgtac
aagatggtga ttgtaactca cagtggggag 3480ctgtctaatg agtctttcat
atttggtaga acagtcccag cctctgtgag tcatctcagg 3540gggtccaatc
ggaacacgac agacagcctt tggttcaact ggagtccagc ctctggggac
3600tttgactttt atgagctgat tctctataat cccaatggca caaagaagga
aaactggaaa 3660gacaaggacc tgacggagtg gcggtttcaa ggccttgttc
ctggaaggaa gtacgtgctg 3720tgggtggtaa ctcacagtgg agatctcagc
aataaagtca cagcggagag cagaacagct 3780ccaagtcctc ccagtcttat
gtcatttgct gacattgcaa acacatcctt ggccatcacg 3840tggaaagggc
ccccagactg gacagactac aacgactttg agctgcagtg gttgcccaga
3900gatgcactta ctgtcttcaa cccctacaac aacagaaaat cagaaggacg
cattgtgtat 3960ggtcttcgtc cagggagatc ctatcaattc aacgtcaaga
ctgtcagtgg tgattcctgg 4020aaaacttaca gcaaaccaat ttttggatct
gtgaggacaa agcctgacaa gatacaaaac 4080ctgcattgcc ggcctcagaa
ctccacggcc attgcctgtt cttggatccc tcctgattct 4140gactttgatg
gttatagtat tgaatgccgg aaaatggaca cccaagaagt tgagttttcc
4200agaaagctgg agaaagaaaa atctctgctc aacatcatga tgctagtgcc
ccataagagg 4260tacctggtgt ccatcaaagt gcagtcggcc ggcatgacca
gcgaggtggt tgaagacagc 4320actatcacaa tgatagaccg cccccctcct
ccacccccac acattcgtgt gaatgaaaag 4380gatgtgctaa ttagcaagtc
ttccatcaac tttactgtca actgcagctg gttcagcgac 4440accaatggag
ctgtgaaata cttcacagtg gtggtgagag aggctgatgg cagtgatgag
4500ctgaagccag aacagcagca ccctctccct tcctacctgg agtacaggca
caatgcctcc 4560attcgggtgt atcagactaa ttattttgcc agcaaatgtg
ccgaaaatcc taacagcaac 4620tccaagagtt ttaacattaa gcttggagca
gagatggaga gcttaggtgg aaaacgcgat 4680cccactcagc aaaaattctg
tgatggacca ctgaagccac acactgccta cagaatcagc 4740attcgagctt
ttacacagct ctttgatgag gacctgaagg aattcacaaa gccactctat
4800tcagacacat ttttttcttt acccatcact actgaatcag agcccttgtt
tggagctatt 4860gaaggtgtga gtgctggtct gtttttaatt ggcatgctag
tggctgttgt tgccttattg 4920atctgcagac agaaagtgag ccatggtcga
gaaagaccct ctgcccgtct gagcattcgt 4980agggatcgac cattatctgt
ccacttaaac ctgggccaga aaggtaaccg gaaaacttct 5040tgtccaataa
aaataaatca gtttgaaggg catttcatga agctacaggc tgactccaac
5100taccttctat ccaaggaata cgaggagtta aaagacgtgg gccgaaacca
gtcatgtgac 5160attgcactct tgccggagaa tagagggaaa aatcgataca
acaatatatt gccctatgat 5220gccacgcgag tgaagctctc caatgtagat
gatgatcctt gctctgacta catcaatgcc 5280agctacatcc ctggcaacaa
cttcagaaga gaatacattg tcactcaggg accgcttcct 5340ggcaccaagg
atgacttctg gaaaatggtg tgggaacaaa acgttcacaa catcgtcatg
5400gtgacccagt gtgttgagaa gggccgagta aagtgtgacc attactggcc
agcggaccag 5460gattccctct actatgggga cctcatcctg cagatgctct
cagagtccgt cctgcctgag 5520tggaccatcc gggagtttaa gatatgcggt
gaggaacagc ttgatgcaca cagactcatc 5580cgccactttc actatacggt
gtggccagac catggagtcc cagaaaccac ccagtctctg 5640atccagtttg
tgagaactgt cagggactac atcaacagaa gcccgggtgc tgggcccact
5700gtggtgcact gcagtgctgg tgtgggtagg actggaacct ttattgcatt
ggaccgaatc 5760ctccagcagt tagactccaa agactctgtg gacatttatg
gagcagtgca cgacctaaga 5820cttcacaggg ttcacatggt ccagactgag
tgtcagtatg tctacctaca tcagtgtgta 5880agagatgtcc tcagagcaag
aaagctacgg agtgaacaag aaaacccctt gtttccaatc 5940tatgaaaatg
tgaatccaga gtatcacaga gatccagtct attcaaggca ttgagaatgt
6000acctgaagag ctcctggata aaaattattc actgtgtgat ttgtt
604521997PRTHomo sapiens 2Met Leu Ser His Gly Ala Gly Leu Ala Leu
Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu Ala Glu Pro
Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys Ala Ser Ser
His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly Ser Pro Cys
Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55 60 Ala Ala
Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys 65 70 75 80
Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg Ile Ile 85
90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr Asp Pro Leu
Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys Thr Thr Ser
Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser Gly Lys Val
Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn Asn Gln Lys
Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr Ser Trp Asn
Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser Lys Tyr Asn
Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185 190 Phe Ser
Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp 195 200 205
Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp Ser His 210
215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu Met Asp Lys
Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp Lys His Ala
Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly Tyr Leu Tyr
Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu Gln Asn Tyr
Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu Val Ser Asn
Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser Leu Lys Val
Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310 315 320 Asn
Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val Leu 325 330
335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val Pro Gly Arg
340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly Glu Leu Ser
Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro Asp Lys Val
Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met Arg Ser Leu
Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp Trp Glu Gln
Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val Leu Leu Asn
Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430 Met Asp Asp
Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435 440 445 Ile
Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly Arg 450 455
460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn Glu
465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro Val Ala Glu
Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg Asp Leu Leu
Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys Glu Phe Thr
Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met Ala Thr Val
Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser Ser Val Lys
Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555 560 His Val
Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr 565 570 575
Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His Glu 580
585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu Thr Ser Arg
Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu Tyr Ser Val
Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val
Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser Val Ser Gly
Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr Leu Ser Val
Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn Tyr Glu Val
Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680 685 Val Ile
Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro 690 695 700
Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys Tyr Glu 705
710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro Asp Lys Val
Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu
Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe Asp His Tyr
Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile Gln Thr Lys
Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe Val Gln Leu
Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800 Thr Thr Lys
Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805 810 815 Thr
Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser Thr 820 825
830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp Val Asp Gln
835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro
Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr Arg Phe Thr
Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile Leu Val Leu
Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe Ile Glu Gly
Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His Ile Ser Pro
Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925 Thr Pro Gly
Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930 935 940 His
Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe Glu 945 950
955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr Gln Ile Met
Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln Ile Asn Val
Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly Val Ile Ala
Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile Val Ser Trp
Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr Asp Ile Leu
Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg Asn Thr Ser
Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045 1050 Asp Leu
Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val 1055 1060 1065
Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly Arg Thr 1070
1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr Glu Asn Ser
Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala Ser Glu Gly
Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp
Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp Pro Leu Val
Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln Gly Arg Met
Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly Glu Leu Ser
Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165 1170 Ala Ser
Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp 1175 1180 1185
Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe Asp Phe 1190
1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu
Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp Arg Phe Gln
Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu Trp Val Val
Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val Thr Ala Glu
Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu Met Ser Phe
Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile Thr Trp Lys
Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285 1290 Glu Leu
Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro 1295 1300 1305
Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu Arg 1310
1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr Val Ser Gly
Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile Phe Gly Ser
Val Arg Thr 1340 1345 1350 Lys Pro
Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln Asn Ser 1355 1360 1365
Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370
1375 1380 Gly Tyr Ser Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Val
Glu 1385 1390 1395 Phe Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu
Asn Ile Met 1400 1405 1410 Met Leu Val Pro His Lys Arg Tyr Leu Val
Ser Ile Lys Val Gln 1415 1420 1425 Ser Ala Gly Met Thr Ser Glu Val
Val Glu Asp Ser Thr Ile Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro
Pro Pro Pro Pro His Ile Arg Val Asn 1445 1450 1455 Glu Lys Asp Val
Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys
Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485
Thr Val Val Val Arg Glu Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490
1495 1500 Glu Gln Gln His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His
Asn 1505 1510 1515 Ala Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala
Ser Lys Cys 1520 1525 1530 Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser
Phe Asn Ile Lys Leu 1535 1540 1545 Gly Ala Glu Met Glu Ser Leu Gly
Gly Lys Cys Asp Pro Thr Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly
Pro Leu Lys Pro His Thr Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg
Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe
Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser Leu Pro 1595 1600 1605
Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Ala Ile Glu Gly Val 1610
1615 1620 Ser Ala Gly Leu Phe Leu Ile Gly Met Leu Val Ala Val Val
Ala 1625 1630 1635 Leu Leu Ile Cys Arg Gln Lys Val Ser His Gly Arg
Glu Arg Pro 1640 1645 1650 Ser Ala Arg Leu Ser Ile Arg Arg Asp Arg
Pro Leu Ser Val His 1655 1660 1665 Leu Asn Leu Gly Gln Lys Gly Asn
Arg Lys Thr Ser Cys Pro Ile 1670 1675 1680 Lys Ile Asn Gln Phe Glu
Gly His Phe Met Lys Leu Gln Ala Asp 1685 1690 1695 Ser Asn Tyr Leu
Leu Ser Lys Glu Tyr Glu Glu Leu Lys Asp Val 1700 1705 1710 Gly Arg
Asn Gln Ser Cys Asp Ile Ala Leu Leu Pro Glu Asn Arg 1715 1720 1725
Gly Lys Asn Arg Tyr Asn Asn Ile Leu Pro Tyr Asp Ala Thr Arg 1730
1735 1740 Val Lys Leu Ser Asn Val Asp Asp Asp Pro Cys Ser Asp Tyr
Ile 1745 1750 1755 Asn Ala Ser Tyr Ile Pro Gly Asn Asn Phe Arg Arg
Glu Tyr Ile 1760 1765 1770 Val Thr Gln Gly Pro Leu Pro Gly Thr Lys
Asp Asp Phe Trp Lys 1775 1780 1785 Met Val Trp Glu Gln Asn Val His
Asn Ile Val Met Val Thr Gln 1790 1795 1800 Cys Val Glu Lys Gly Arg
Val Lys Cys Asp His Tyr Trp Pro Ala 1805 1810 1815 Asp Gln Asp Ser
Leu Tyr Tyr Gly Asp Leu Ile Leu Gln Met Leu 1820 1825 1830 Ser Glu
Ser Val Leu Pro Glu Trp Thr Ile Arg Glu Phe Lys Ile 1835 1840 1845
Cys Gly Glu Glu Gln Leu Asp Ala His Arg Leu Ile Arg His Phe 1850
1855 1860 His Tyr Thr Val Trp Pro Asp His Gly Val Pro Glu Thr Thr
Gln 1865 1870 1875 Ser Leu Ile Gln Phe Val Arg Thr Val Arg Asp Tyr
Ile Asn Arg 1880 1885 1890 Ser Pro Gly Ala Gly Pro Thr Val Val His
Cys Ser Ala Gly Val 1895 1900 1905 Gly Arg Thr Gly Thr Phe Ile Ala
Leu Asp Arg Ile Leu Gln Gln 1910 1915 1920 Leu Asp Ser Lys Asp Ser
Val Asp Ile Tyr Gly Ala Val His Asp 1925 1930 1935 Leu Arg Leu His
Arg Val His Met Val Gln Thr Glu Cys Gln Tyr 1940 1945 1950 Val Tyr
Leu His Gln Cys Val Arg Asp Val Leu Arg Ala Arg Lys 1955 1960 1965
Leu Arg Ser Glu Gln Glu Asn Pro Leu Phe Pro Ile Tyr Glu Asn 1970
1975 1980 Val Asn Pro Glu Tyr His Arg Asp Pro Val Tyr Ser Arg His
1985 1990 1995 31631PRTHomo sapiens 3Met Leu Ser His Gly Ala Gly
Leu Ala Leu Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu
Ala Glu Pro Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys
Ala Ser Ser His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly
Ser Pro Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55
60 Ala Ala Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys
65 70 75 80 Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg
Ile Ile 85 90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr
Asp Pro Leu Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys
Thr Thr Ser Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser
Gly Lys Val Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn
Asn Gln Lys Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr
Ser Trp Asn Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser
Lys Tyr Asn Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185
190 Phe Ser Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp
195 200 205 Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp
Ser His 210 215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu
Met Asp Lys Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp
Lys His Ala Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly
Tyr Leu Tyr Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu
Gln Asn Tyr Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu
Val Ser Asn Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser
Leu Lys Val Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310
315 320 Asn Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val
Leu 325 330 335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val
Pro Gly Arg 340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly
Glu Leu Ser Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro
Asp Lys Val Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met
Arg Ser Leu Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp
Trp Glu Gln Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val
Leu Leu Asn Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430
Met Asp Asp Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435
440 445 Ile Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly
Arg 450 455 460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His
Ala Asn Glu 465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro
Val Ala Glu Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg
Asp Leu Leu Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys
Glu Phe Thr Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met
Ala Thr Val Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser
Ser Val Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555
560 His Val Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr
565 570 575 Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile
His Glu 580 585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu
Thr Ser Arg Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu
Tyr Ser Val Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser
Arg Gln Val Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser
Val Ser Gly Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr
Leu Ser Val Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn
Tyr Glu Val Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680
685 Val Ile Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro
690 695 700 Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys
Tyr Glu 705 710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro
Asp Lys Val Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser
Asp Tyr Leu Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe
Asp His Tyr Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile
Gln Thr Lys Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe
Val Gln Leu Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800
Thr Thr Lys Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805
810 815 Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser
Thr 820 825 830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp
Val Asp Gln 835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys
Val Phe Pro Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr
Arg Phe Thr Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile
Leu Val Leu Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe
Ile Glu Gly Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His
Ile Ser Pro Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925
Thr Pro Gly Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930
935 940 His Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe
Glu 945 950 955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr
Gln Ile Met Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln
Ile Asn Val Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly
Val Ile Ala Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile
Val Ser Trp Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr
Asp Ile Leu Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg
Asn Thr Ser Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045
1050 Asp Leu Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val
1055 1060 1065 Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly
Arg Thr 1070 1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr
Glu Asn Ser Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala
Ser Glu Gly Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr
Asn Pro Asp Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp
Pro Leu Val Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln
Gly Arg Met Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly
Glu Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165
1170 Ala Ser Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp
1175 1180 1185 Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe
Asp Phe 1190 1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr
Lys Lys Glu Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp
Arg Phe Gln Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu
Trp Val Val Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val
Thr Ala Glu Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu
Met Ser Phe Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile
Thr Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285
1290 Glu Leu Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro
1295 1300 1305 Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly
Leu Arg 1310 1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr
Val Ser Gly Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile
Phe Gly Ser Val Arg Thr 1340 1345 1350 Lys Pro Asp Lys Ile Gln Asn
Leu His Cys Arg Pro Gln Asn Ser 1355 1360 1365 Thr Ala Ile Ala Cys
Ser Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370 1375 1380 Gly Tyr Ser
Ile Glu Cys Arg Lys Met Asp Thr Gln Glu Val Glu 1385 1390 1395 Phe
Ser Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile Met 1400 1405
1410 Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val Gln
1415 1420 1425 Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr
Ile Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro Pro Pro Pro Pro His
Ile Arg Val Asn 1445 1450 1455 Glu Lys Asp Val Leu Ile Ser Lys Ser
Ser Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys Ser Trp Phe Ser Asp
Thr Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485 Thr Val Val Val Arg
Glu Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490 1495 1500 Glu Gln Gln
His Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His Asn 1505 1510 1515 Ala
Ser Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys Cys 1520 1525
1530 Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser Phe Asn Ile Lys Leu
1535 1540 1545 Gly Ala Glu Met Glu Ser Leu Gly Gly Lys Cys Asp Pro
Thr Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His
Thr Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg Ala Phe Thr Gln Leu
Phe Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe Thr Lys Pro Leu Tyr
Ser Asp Thr Phe Phe
Ser Leu Pro 1595 1600 1605 Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly
Ala Ile Glu Arg Gly 1610 1615 1620 Arg His His His His His His Gly
1625 1630 41621PRTHomo sapiens 4Met Leu Ser His Gly Ala Gly Leu Ala
Leu Trp Ile Thr Leu Ser Leu 1 5 10 15 Leu Gln Thr Gly Leu Ala Glu
Pro Glu Arg Cys Asn Phe Thr Leu Ala 20 25 30 Glu Ser Lys Ala Ser
Ser His Ser Val Ser Ile Gln Trp Arg Ile Leu 35 40 45 Gly Ser Pro
Cys Asn Phe Ser Leu Ile Tyr Ser Ser Asp Thr Leu Gly 50 55 60 Ala
Ala Leu Cys Pro Thr Phe Arg Ile Asp Asn Thr Thr Tyr Gly Cys 65 70
75 80 Asn Leu Gln Asp Leu Gln Ala Gly Thr Ile Tyr Asn Phe Arg Ile
Ile 85 90 95 Ser Leu Asp Glu Glu Arg Thr Val Val Leu Gln Thr Asp
Pro Leu Pro 100 105 110 Pro Ala Arg Phe Gly Val Ser Lys Glu Lys Thr
Thr Ser Thr Ser Leu 115 120 125 His Val Trp Trp Thr Pro Ser Ser Gly
Lys Val Thr Ser Tyr Glu Val 130 135 140 Gln Leu Phe Asp Glu Asn Asn
Gln Lys Ile Gln Gly Val Gln Ile Gln 145 150 155 160 Glu Ser Thr Ser
Trp Asn Glu Tyr Thr Phe Phe Asn Leu Thr Ala Gly 165 170 175 Ser Lys
Tyr Asn Ile Ala Ile Thr Ala Val Ser Gly Gly Lys Arg Ser 180 185 190
Phe Ser Val Tyr Thr Asn Gly Ser Thr Val Pro Ser Pro Val Lys Asp 195
200 205 Ile Gly Ile Ser Thr Lys Ala Asn Ser Leu Leu Ile Ser Trp Ser
His 210 215 220 Gly Ser Gly Asn Val Glu Arg Tyr Arg Leu Met Leu Met
Asp Lys Gly 225 230 235 240 Ile Leu Val His Gly Gly Val Val Asp Lys
His Ala Thr Ser Tyr Ala 245 250 255 Phe His Gly Leu Thr Pro Gly Tyr
Leu Tyr Asn Leu Thr Val Met Thr 260 265 270 Glu Ala Ala Gly Leu Gln
Asn Tyr Arg Trp Lys Leu Val Arg Thr Ala 275 280 285 Pro Met Glu Val
Ser Asn Leu Lys Val Thr Asn Asp Gly Ser Leu Thr 290 295 300 Ser Leu
Lys Val Lys Trp Gln Arg Pro Pro Gly Asn Val Asp Ser Tyr 305 310 315
320 Asn Ile Thr Leu Ser His Lys Gly Thr Ile Lys Glu Ser Arg Val Leu
325 330 335 Ala Pro Trp Ile Thr Glu Thr His Phe Lys Glu Leu Val Pro
Gly Arg 340 345 350 Leu Tyr Gln Val Thr Val Ser Cys Val Ser Gly Glu
Leu Ser Ala Gln 355 360 365 Lys Met Ala Val Gly Arg Thr Phe Pro Asp
Lys Val Ala Asn Leu Glu 370 375 380 Ala Asn Asn Asn Gly Arg Met Arg
Ser Leu Val Val Ser Trp Ser Pro 385 390 395 400 Pro Ala Gly Asp Trp
Glu Gln Tyr Arg Ile Leu Leu Phe Asn Asp Ser 405 410 415 Val Val Leu
Leu Asn Ile Thr Val Gly Lys Glu Glu Thr Gln Tyr Val 420 425 430 Met
Asp Asp Thr Gly Leu Val Pro Gly Arg Gln Tyr Glu Val Glu Val 435 440
445 Ile Val Glu Ser Gly Asn Leu Lys Asn Ser Glu Arg Cys Gln Gly Arg
450 455 460 Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala
Asn Glu 465 470 475 480 Thr Ser Leu Ser Ile Met Trp Gln Thr Pro Val
Ala Glu Trp Glu Lys 485 490 495 Tyr Ile Ile Ser Leu Ala Asp Arg Asp
Leu Leu Leu Ile His Lys Ser 500 505 510 Leu Ser Lys Asp Ala Lys Glu
Phe Thr Phe Thr Asp Leu Val Pro Gly 515 520 525 Arg Lys Tyr Met Ala
Thr Val Thr Ser Ile Ser Gly Asp Leu Lys Asn 530 535 540 Ser Ser Ser
Val Lys Gly Arg Thr Val Pro Ala Gln Val Thr Asp Leu 545 550 555 560
His Val Ala Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp Thr 565
570 575 Gln Ala Gln Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His
Glu 580 585 590 Asn Val Val Ile Lys Asn Glu Ser Ile Ser Ser Glu Thr
Ser Arg Tyr 595 600 605 Ser Phe His Ser Leu Lys Ser Gly Ser Leu Tyr
Ser Val Val Val Thr 610 615 620 Thr Val Ser Gly Gly Ile Ser Ser Arg
Gln Val Val Val Glu Gly Arg 625 630 635 640 Thr Val Pro Ser Ser Val
Ser Gly Val Thr Val Asn Asn Ser Gly Arg 645 650 655 Asn Asp Tyr Leu
Ser Val Ser Trp Leu Leu Ala Pro Gly Asp Val Asp 660 665 670 Asn Tyr
Glu Val Thr Leu Ser His Asp Gly Lys Val Val Gln Ser Leu 675 680 685
Val Ile Ala Lys Ser Val Arg Glu Cys Ser Phe Ser Ser Leu Thr Pro 690
695 700 Gly Arg Leu Tyr Thr Val Thr Ile Thr Thr Arg Ser Gly Lys Tyr
Glu 705 710 715 720 Asn His Ser Phe Ser Gln Glu Arg Thr Val Pro Asp
Lys Val Gln Gly 725 730 735 Val Ser Val Ser Asn Ser Ala Arg Ser Asp
Tyr Leu Arg Val Ser Trp 740 745 750 Val His Ala Thr Gly Asp Phe Asp
His Tyr Glu Val Thr Ile Lys Asn 755 760 765 Lys Asn Asn Phe Ile Gln
Thr Lys Ser Ile Pro Lys Ser Glu Asn Glu 770 775 780 Cys Val Phe Val
Gln Leu Val Pro Gly Arg Leu Tyr Ser Val Thr Val 785 790 795 800 Thr
Thr Lys Ser Gly Gln Tyr Glu Ala Asn Glu Gln Gly Asn Gly Arg 805 810
815 Thr Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Arg Asn Arg Ser Thr
820 825 830 Glu Asp Leu His Val Thr Trp Ser Gly Ala Asn Gly Asp Val
Asp Gln 835 840 845 Tyr Glu Ile Gln Leu Leu Phe Asn Asp Met Lys Val
Phe Pro Pro Phe 850 855 860 His Leu Val Asn Thr Ala Thr Glu Tyr Arg
Phe Thr Ser Leu Thr Pro 865 870 875 880 Gly Arg Gln Tyr Lys Ile Leu
Val Leu Thr Ile Ser Gly Asp Val Gln 885 890 895 Gln Ser Ala Phe Ile
Glu Gly Phe Thr Val Pro Ser Ala Val Lys Asn 900 905 910 Ile His Ile
Ser Pro Asn Gly Ala Thr Asp Ser Leu Thr Val Asn Trp 915 920 925 Thr
Pro Gly Gly Gly Asp Val Asp Ser Tyr Thr Val Ser Ala Phe Arg 930 935
940 His Ser Gln Lys Val Asp Ser Gln Thr Ile Pro Lys His Val Phe Glu
945 950 955 960 His Thr Phe His Arg Leu Glu Ala Gly Glu Gln Tyr Gln
Ile Met Ile 965 970 975 Ala Ser Val Ser Gly Ser Leu Lys Asn Gln Ile
Asn Val Val Gly Arg 980 985 990 Thr Val Pro Ala Ser Val Gln Gly Val
Ile Ala Asp Asn Ala Tyr Ser 995 1000 1005 Ser Tyr Ser Leu Ile Val
Ser Trp Gln Lys Ala Ala Gly Val Ala 1010 1015 1020 Glu Arg Tyr Asp
Ile Leu Leu Leu Thr Glu Asn Gly Ile Leu Leu 1025 1030 1035 Arg Asn
Thr Ser Glu Pro Ala Thr Thr Lys Gln His Lys Phe Glu 1040 1045 1050
Asp Leu Thr Pro Gly Lys Lys Tyr Lys Ile Gln Ile Leu Thr Val 1055
1060 1065 Ser Gly Gly Leu Phe Ser Lys Glu Ala Gln Thr Glu Gly Arg
Thr 1070 1075 1080 Val Pro Ala Ala Val Thr Asp Leu Arg Ile Thr Glu
Asn Ser Thr 1085 1090 1095 Arg His Leu Ser Phe Arg Trp Thr Ala Ser
Glu Gly Glu Leu Ser 1100 1105 1110 Trp Tyr Asn Ile Phe Leu Tyr Asn
Pro Asp Gly Asn Leu Gln Glu 1115 1120 1125 Arg Ala Gln Val Asp Pro
Leu Val Gln Ser Phe Ser Phe Gln Asn 1130 1135 1140 Leu Leu Gln Gly
Arg Met Tyr Lys Met Val Ile Val Thr His Ser 1145 1150 1155 Gly Glu
Leu Ser Asn Glu Ser Phe Ile Phe Gly Arg Thr Val Pro 1160 1165 1170
Ala Ser Val Ser His Leu Arg Gly Ser Asn Arg Asn Thr Thr Asp 1175
1180 1185 Ser Leu Trp Phe Asn Trp Ser Pro Ala Ser Gly Asp Phe Asp
Phe 1190 1195 1200 Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys
Lys Glu Asn 1205 1210 1215 Trp Lys Asp Lys Asp Leu Thr Glu Trp Arg
Phe Gln Gly Leu Val 1220 1225 1230 Pro Gly Arg Lys Tyr Val Leu Trp
Val Val Thr His Ser Gly Asp 1235 1240 1245 Leu Ser Asn Lys Val Thr
Ala Glu Ser Arg Thr Ala Pro Ser Pro 1250 1255 1260 Pro Ser Leu Met
Ser Phe Ala Asp Ile Ala Asn Thr Ser Leu Ala 1265 1270 1275 Ile Thr
Trp Lys Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp Phe 1280 1285 1290
Glu Leu Gln Trp Leu Pro Arg Asp Ala Leu Thr Val Phe Asn Pro 1295
1300 1305 Tyr Asn Asn Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu
Arg 1310 1315 1320 Pro Gly Arg Ser Tyr Gln Phe Asn Val Lys Thr Val
Ser Gly Asp 1325 1330 1335 Ser Trp Lys Thr Tyr Ser Lys Pro Ile Phe
Gly Ser Val Arg Thr 1340 1345 1350 Lys Pro Asp Lys Ile Gln Asn Leu
His Cys Arg Pro Gln Asn Ser 1355 1360 1365 Thr Ala Ile Ala Cys Ser
Trp Ile Pro Pro Asp Ser Asp Phe Asp 1370 1375 1380 Gly Tyr Ser Ile
Glu Cys Arg Lys Met Asp Thr Gln Glu Val Glu 1385 1390 1395 Phe Ser
Arg Lys Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile Met 1400 1405 1410
Met Leu Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val Gln 1415
1420 1425 Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile
Thr 1430 1435 1440 Met Ile Asp Arg Pro Pro Pro Pro Pro Pro His Ile
Arg Val Asn 1445 1450 1455 Glu Lys Asp Val Leu Ile Ser Lys Ser Ser
Ile Asn Phe Thr Val 1460 1465 1470 Asn Cys Ser Trp Phe Ser Asp Thr
Asn Gly Ala Val Lys Tyr Phe 1475 1480 1485 Thr Val Val Val Arg Glu
Ala Asp Gly Asn Asp Glu Leu Lys Pro 1490 1495 1500 Glu Gln Gln His
Pro Leu Pro Ser Tyr Leu Glu Tyr Arg His Asn 1505 1510 1515 Ala Ser
Ile Arg Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys Cys 1520 1525 1530
Ala Glu Asn Pro Asn Ser Asn Ser Lys Ser Phe Asn Ile Lys Leu 1535
1540 1545 Gly Ala Glu Met Glu Ser Leu Gly Gly Lys Cys Asp Pro Thr
Gln 1550 1555 1560 Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr
Ala Tyr Arg 1565 1570 1575 Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe
Asp Glu Asp Leu Lys 1580 1585 1590 Glu Phe Thr Lys Pro Leu Tyr Ser
Asp Thr Phe Phe Ser Leu Pro 1595 1600 1605 Ile Thr Thr Glu Ser Glu
Pro Leu Phe Gly Ala Ile Glu 1610 1615 1620 56199DNAMus musculus
5ggcacgaggg cggctgccac ggcccttgag catcgccagc cccgagggta gcgcgctgcg
60cccgcccgca gggcgcgctg agcgctcaac aagtggtacc cagaagtgcc ctggctggcc
120tcccagcgag atgctgaggc atggagccct aacggccttg tggataacac
tgagcgtcgt 180gcagactgga gtggcagagc aagtgaaatg taacttcaca
ctgttggagt ccagggtctc 240tagcttgtca gcgtctatcc agtggaggac
tttcgcgtca ccctgtaact ttagcctcat 300ctacagcagt gatacctcgg
ggcccatgtg gtgccatcct attcggatag acaactttac 360ctacggatgt
aaccccaagg atttacaagc agggaccgtc tataacttca ggattgtttc
420tctggatgga gaagagagca ctctggtctt acagacagat ccgttgcctc
ctgccaggtt 480tgaagtcaat cgggagaaaa cagcatcaac caccctgcag
gtccggtgga ctccctcttc 540tggaaaagtc tcctggtatg aggtgcaatt
atttgatcat aacaatcaaa agatacaaga 600agtccaagtt caagaaagta
ccacctggag ccaatatact tttctgaacc tcactgaggg 660taacagttac
aaagttgcca tcacagctgt ttcgggagaa aagcgctcct ttccggtgta
720tatcaatggc tctacagtac catctccagt gaaagatctt ggcatttccc
ccaatcctaa 780ttctctccta atttcctggt ctcgtggttc tgggaatgtg
gaacaataca ggctggtgct 840aatggataaa ggggccatcg ttcaagacac
aaacgtggac aggcgtgata cttcttatgc 900ttttcacgag ctgacccctg
gccacctcta caacctcact attgtcacca tggcctcggg 960actgcaaaac
tccaggtgga aactggtgag gaccgctccc atggaagtct caaatctgaa
1020ggtgacaaat gacgggaggt tgacctctct aaatgtgaag tggcagaaac
cccctgggga 1080tgtagattcc tacagcatta ccctgtctca ccaagggacc
atcaaagaat ccaaaacatt 1140agcacctcct gttactgaaa ctcaatttaa
ggacttagtc cctggacggc tttaccaagt 1200gaccatcagc tgcatctctg
gtgagctctc tgctgagaag tcagcagcgg ggagaacagt 1260tccagaaaaa
gtgaggaatc tggtttccta caacgagatt tggatgaagt cctttacagt
1320gaactggacg ccccctgctg gagattggga gcattatcgt atcgtgctct
tcaatgaatc 1380cttggtcttg ctcaacacca cagtgggaaa ggaagaaacg
cactatgcct tggatggctt 1440ggagctcata ccaggaagac agtatgagat
agaagtcatt gttgagagcg gaaatctgcg 1500gaattccgag cgctgtcaag
gcaggacagt acccctggct gtcctccagc ttcgcgtcaa 1560acacgctaac
gaaacttcac tgggcatcac gtggcgggcc cctctaggcg aatgggagaa
1620atacatcatt tcgttgatgg acagagagct cttggtcatc cacaagtcac
tctccaaaga 1680tgccaaagaa ttcactttta cagacctgat gcctggacgg
aattacaagg ctactgtcac 1740tagcatgagt ggagatttaa aacagtcatc
ttcaatcaaa ggaagaacag tgcctgccca 1800ggtgactgac ctgcacgtca
acaaccaagg gatgaccagt agtctgttca ctaactggac 1860aaaggcactg
ggagatgtag agttctacca agttttactg atccatgaaa atgtggttgt
1920caagaacgag agtgtttcca gtgataccag cagatacagc ttccgcgccc
tgaaacccgg 1980cagcctctac tccgtggtgg tgaccacggt gagtggaggg
atctcctccc ggcaggtggt 2040ggcggaagga agaacagtcc cgtccagcgt
gagtggggtg acagtcaaca attctggccg 2100gaatgactac ctcagcgttt
cctggctgcc ggcgcctgga gaagtggatc actacgtggt 2160gagcctctcc
cacgagggca aggtggatca gttcctcatc atcgccaaat ctgtcagcga
2220gtgttccttc agctccctca ccccgggccg cctctacaac gtcactgtaa
ccaccaagag 2280cggcaattat gcaagccact ccttcaccga ggaacggaca
gtgccagaca aggtccaggg 2340aatcagtgtt agcaactctg ccagaagcga
ctacttaaag gtgtcctggg tgcatgccac 2400tggagacttt gaccactatg
aagtcaccat caaaaacaga gaaagcttca ttcaaaccaa 2460aaccatcccc
aagtcagaaa atgagtgtga atttattgag ctggttcctg gacgcctgta
2520cagcgtcact gtcagtacaa agagtggaca atatgaagcc agtgaacagg
ggacagggag 2580aacgatccca gagcctgtga aggatctcac ccttctcaac
aggagtacgg aggatctcca 2640tgtgacttgg tcaagagcca atggggatgt
tgatcagtac gaggtccagc tgctcttcaa 2700cgacatgaaa gtcttccctc
atattcacct tgtgaacaca gcaactgagt ataagttcac 2760ggcgctcacg
ccggggcgcc attacaaaat cctcgtcctg accatcagtg gcgatgtcca
2820gcagtcagcc ttcattgaag gcctcccagt tcccagcact gtcaaaaaca
ttcacatttc 2880tgccaatgga gccacggata ggctgatggt aacctggagc
cctggtggcg gggatgtgga 2940ctcctatgtg gtgtctgcat tcagacagga
cgagaaggtt gactctcaga ccattcccaa 3000gcatgcctcg gagcacacgt
tccacaggct ggaggccgga gccaagtaca ggatcgccat 3060tgtttctgtc
agtgggtccc tgagaaacca gatagatgcg ctcggacaga cagtcccagc
3120gtctgtccag ggagtcgtcg cagccaatgc atacagcagt aattccttaa
cagtaagttg 3180gcagaaagcc cttggtgtgg cagaaagata cgatatcctg
cttctaaacg agaatgggct 3240tcttttgagc aacgtgtcag agccagctac
ggcaagacag cacaaatttg aagatctaac 3300gccaggcaag aaatacaaga
tgcagatcct gactgtcagc ggaggcctct tcagtaaaga 3360atctcaggct
gaaggccgaa cagtcccagc agctgtcacc aatctgagga tcacagagaa
3420ctccagtaga tacctgtcct tcggctggac cgcctcggag ggtgaactca
gctggtacaa 3480catcttcctc tacaacccag acaggactct tcaggagcga
gctcaagttg acccgctagt 3540ccagagcttc tctttccaga acttgctaca
aggcagaatg tacaagatgg tgattgtcac 3600tcacagtggg gagctgtcca
atgagtcatt tatattcggc agaacagttc ctgctgccgt 3660gaaccatctc
aaaggctccc atcggaacac gacagacagc ctgtggttca gctggagccc
3720agcctccggg gactttgact tctatgagct gattctttac aatcccaacg
gcacgaagaa 3780ggagaactgg aaagaaaagg acgtgacaga gtggcgtttc
caaggtcttg ttcctggaag 3840gaaatacacc ctgtatgtgg tgactcacag
tggggacctc agcaataaag tcacagggga 3900gggcagaaca gccccaagtc
ctccgagtct tttgtcattc gctgatgttg caaacacctc 3960cttggctatc
acctggaagg gacccccaga ctggacagat tacaatgact
ttgagctgca 4020gtggttccct ggagatgcac ttaccatctt caacccctac
agcagcagaa agtcagaagg 4080acgcattgtg tacgggcttc acccagggag
gtcctatcaa ttcagtgtca agactgtgag 4140cggggactcc tggaaaacct
acagcaaacc aatttctggg tctgtgagga caaagccaga 4200caagatacaa
aacctgcatt gccgccccca gaactccacg gccattgcct gctcttggat
4260acctcctgac tccgactttg atggctacag cattgagtgc cgaaaaatgg
atacccaaga 4320aatcgagttt tccagaaagc tggagaaaga aaaatcactg
ctcaacatca tgatgttagt 4380acctcataag aggtacctgg tgtccatcaa
ggtgcagtcg gccggcatga ccagtgaggt 4440ggttgaagat agcaccatca
ccatgataga ccgcccgcct caaccgcctc cacacatccg 4500tgtgaatgaa
aaggatgtgc taatcagcaa atcttccatc aactttactg tcaactgcag
4560ctggttcagc gacaccaacg gagcggttaa atactttgct gtggtggtga
gagaggccga 4620cagcatggat gagttgaagc cagaacagca gcaccctctc
ccttcctacc tggagtacag 4680acacaacgcc tccatccgag tctaccagac
caattatttt gccagcaaat gtgctgaaag 4740tcccgacagc agttctaaaa
gtttcaacat taagcttgga gcagagatgg acagcctcgg 4800tggcaaatgt
gatcccagtc agcagaaatt ctgtgatgga ccgctgaagc cacacaccgc
4860ctacagaatc agcatccggg cttttacaca gctatttgac gaggacttga
aagagttcac 4920caaacctctc tactcggata cgttcttctc tatgcccatc
accacagagt cagagccctt 4980gtttggagtt attgaaggtg tgagtgctgg
cctgtttcta attggcatgc tggtggccct 5040tgttgccttc ttcatctgca
gacagaaagc tagccacagc agggaaaggc catctgcccg 5100gctcagcatt
cgtagggacc ggcctttgtc tgtccatctg aatctgggcc agaaaggcaa
5160ccggaaaact tcttgcccca taaagatcaa tcagtttgaa gggcatttca
tgaagctgca 5220ggcagactcc aactaccttc tatccaagga atatgaggac
ttaaaagacg tgggtagaag 5280ccagtcatgt gacattgccc tcttgcctga
gaatcgaggg aaaaatcgat acaacaacat 5340attgccttat gatgcctcaa
gagtgaagct ctcgaatgtc gatgacgacc cttgctctga 5400ctacatcaac
gccagctaca tccccggtaa caacttcaga cgagaataca tcgccactca
5460gggaccgctt ccaggcacca aggatgactt ctggaagatg gcgtgggagc
agaacgttca 5520caacatcgtc atggtgaccc agtgtgttga aaagggccga
gtgaagtgtg accattactg 5580gccagcagac caggaccccc tctactacgg
tgatctcatc ctacagatgg tctcggagtc 5640cgtgctcccc gagtggacca
tcagggagtt taagatatgc agtgaagaac agttggatgc 5700acacagactc
atccgtcact ttcactacac ggtgtggcca gaccatgggg tcccagagac
5760cacccagtcc ctgatccaat ttgtgaggac agtcagggac tacatcaaca
gaagccccgg 5820ggctgggccc accgtagtgc actgcagcgc tggtgtgggc
agaacaggga cgttcgttgc 5880cctggaccgg atcctccagc agttggactc
taaggactcc gtggacattt atggggcagt 5940gcatgaccta agactccaca
gggttcacat ggtccagacc gagtgtcaat atgtgtatct 6000gcatcagtgt
gtaagagacg tcctcagagc aaagaaactg cggaacgagc aagagaaccc
6060cctgtttccg atttatgaga atgtgaatcc agagtatcac agagatgcaa
tctactcgag 6120acattaagaa ttcacctgaa gatcccctgg ataaaagcgt
ttcactgtgt gactttaaaa 6180aaaaaaaaaa aaaaaaaaa 619961998PRTMus
musculus 6Met Leu Arg His Gly Ala Leu Thr Ala Leu Trp Ile Thr Leu
Ser Val 1 5 10 15 Val Gln Thr Gly Val Ala Glu Gln Val Lys Cys Asn
Phe Thr Leu Leu 20 25 30 Glu Ser Arg Val Ser Ser Leu Ser Ala Ser
Ile Gln Trp Arg Thr Phe 35 40 45 Ala Ser Pro Cys Asn Phe Ser Leu
Ile Tyr Ser Ser Asp Thr Ser Gly 50 55 60 Pro Met Trp Cys His Pro
Ile Arg Ile Asp Asn Phe Thr Tyr Gly Cys 65 70 75 80 Asn Pro Lys Asp
Leu Gln Ala Gly Thr Val Tyr Asn Phe Arg Ile Val 85 90 95 Ser Leu
Asp Gly Glu Glu Ser Thr Leu Val Leu Gln Thr Asp Pro Leu 100 105 110
Pro Pro Ala Arg Phe Glu Val Asn Arg Glu Lys Thr Ala Ser Thr Thr 115
120 125 Leu Gln Val Arg Trp Thr Pro Ser Ser Gly Lys Val Ser Trp Tyr
Glu 130 135 140 Val Gln Leu Phe Asp His Asn Asn Gln Lys Ile Gln Glu
Val Gln Val 145 150 155 160 Gln Glu Ser Thr Thr Trp Ser Gln Tyr Thr
Phe Leu Asn Leu Thr Glu 165 170 175 Gly Asn Ser Tyr Lys Val Ala Ile
Thr Ala Val Ser Gly Glu Lys Arg 180 185 190 Ser Phe Pro Val Tyr Ile
Asn Gly Ser Thr Val Pro Ser Pro Val Lys 195 200 205 Asp Leu Gly Ile
Ser Pro Asn Pro Asn Ser Leu Leu Ile Ser Trp Ser 210 215 220 Arg Gly
Ser Gly Asn Val Glu Gln Tyr Arg Leu Val Leu Met Asp Lys 225 230 235
240 Gly Ala Ile Val Gln Asp Thr Asn Val Asp Arg Arg Asp Thr Ser Tyr
245 250 255 Ala Phe His Glu Leu Thr Pro Gly His Leu Tyr Asn Leu Thr
Ile Val 260 265 270 Thr Met Ala Ser Gly Leu Gln Asn Ser Arg Trp Lys
Leu Val Arg Thr 275 280 285 Ala Pro Met Glu Val Ser Asn Leu Lys Val
Thr Asn Asp Gly Arg Leu 290 295 300 Thr Ser Leu Asn Val Lys Trp Gln
Lys Pro Pro Gly Asp Val Asp Ser 305 310 315 320 Tyr Ser Ile Thr Leu
Ser His Gln Gly Thr Ile Lys Glu Ser Lys Thr 325 330 335 Leu Ala Pro
Pro Val Thr Glu Thr Gln Phe Lys Asp Leu Val Pro Gly 340 345 350 Arg
Leu Tyr Gln Val Thr Ile Ser Cys Ile Ser Gly Glu Leu Ser Ala 355 360
365 Glu Lys Ser Ala Ala Gly Arg Thr Val Pro Glu Lys Val Arg Asn Leu
370 375 380 Val Ser Tyr Asn Glu Ile Trp Met Lys Ser Phe Thr Val Asn
Trp Thr 385 390 395 400 Pro Pro Ala Gly Asp Trp Glu His Tyr Arg Ile
Val Leu Phe Asn Glu 405 410 415 Ser Leu Val Leu Leu Asn Thr Thr Val
Gly Lys Glu Glu Thr His Tyr 420 425 430 Ala Leu Asp Gly Leu Glu Leu
Ile Pro Gly Arg Gln Tyr Glu Ile Glu 435 440 445 Val Ile Val Glu Ser
Gly Asn Leu Arg Asn Ser Glu Arg Cys Gln Gly 450 455 460 Arg Thr Val
Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn 465 470 475 480
Glu Thr Ser Leu Gly Ile Thr Trp Arg Ala Pro Leu Gly Glu Trp Glu 485
490 495 Lys Tyr Ile Ile Ser Leu Met Asp Arg Glu Leu Leu Val Ile His
Lys 500 505 510 Ser Leu Ser Lys Asp Ala Lys Glu Phe Thr Phe Thr Asp
Leu Met Pro 515 520 525 Gly Arg Asn Tyr Lys Ala Thr Val Thr Ser Met
Ser Gly Asp Leu Lys 530 535 540 Gln Ser Ser Ser Ile Lys Gly Arg Thr
Val Pro Ala Gln Val Thr Asp 545 550 555 560 Leu His Val Asn Asn Gln
Gly Met Thr Ser Ser Leu Phe Thr Asn Trp 565 570 575 Thr Lys Ala Leu
Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His 580 585 590 Glu Asn
Val Val Val Lys Asn Glu Ser Val Ser Ser Asp Thr Ser Arg 595 600 605
Tyr Ser Phe Arg Ala Leu Lys Pro Gly Ser Leu Tyr Ser Val Val Val 610
615 620 Thr Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val Val Ala Glu
Gly 625 630 635 640 Arg Thr Val Pro Ser Ser Val Ser Gly Val Thr Val
Asn Asn Ser Gly 645 650 655 Arg Asn Asp Tyr Leu Ser Val Ser Trp Leu
Pro Ala Pro Gly Glu Val 660 665 670 Asp His Tyr Val Val Ser Leu Ser
His Glu Gly Lys Val Asp Gln Phe 675 680 685 Leu Ile Ile Ala Lys Ser
Val Ser Glu Cys Ser Phe Ser Ser Leu Thr 690 695 700 Pro Gly Arg Leu
Tyr Asn Val Thr Val Thr Thr Lys Ser Gly Asn Tyr 705 710 715 720 Ala
Ser His Ser Phe Thr Glu Glu Arg Thr Val Pro Asp Lys Val Gln 725 730
735 Gly Ile Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu Lys Val Ser
740 745 750 Trp Val His Ala Thr Gly Asp Phe Asp His Tyr Glu Val Thr
Ile Lys 755 760 765 Asn Arg Glu Ser Phe Ile Gln Thr Lys Thr Ile Pro
Lys Ser Glu Asn 770 775 780 Glu Cys Glu Phe Ile Glu Leu Val Pro Gly
Arg Leu Tyr Ser Val Thr 785 790 795 800 Val Ser Thr Lys Ser Gly Gln
Tyr Glu Ala Ser Glu Gln Gly Thr Gly 805 810 815 Arg Thr Ile Pro Glu
Pro Val Lys Asp Leu Thr Leu Leu Asn Arg Ser 820 825 830 Thr Glu Asp
Leu His Val Thr Trp Ser Arg Ala Asn Gly Asp Val Asp 835 840 845 Gln
Tyr Glu Val Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro His 850 855
860 Ile His Leu Val Asn Thr Ala Thr Glu Tyr Lys Phe Thr Ala Leu Thr
865 870 875 880 Pro Gly Arg His Tyr Lys Ile Leu Val Leu Thr Ile Ser
Gly Asp Val 885 890 895 Gln Gln Ser Ala Phe Ile Glu Gly Leu Pro Val
Pro Ser Thr Val Lys 900 905 910 Asn Ile His Ile Ser Ala Asn Gly Ala
Thr Asp Arg Leu Met Val Thr 915 920 925 Trp Ser Pro Gly Gly Gly Asp
Val Asp Ser Tyr Val Val Ser Ala Phe 930 935 940 Arg Gln Asp Glu Lys
Val Asp Ser Gln Thr Ile Pro Lys His Ala Ser 945 950 955 960 Glu His
Thr Phe His Arg Leu Glu Ala Gly Ala Lys Tyr Arg Ile Ala 965 970 975
Ile Val Ser Val Ser Gly Ser Leu Arg Asn Gln Ile Asp Ala Leu Gly 980
985 990 Gln Thr Val Pro Ala Ser Val Gln Gly Val Val Ala Ala Asn Ala
Tyr 995 1000 1005 Ser Ser Asn Ser Leu Thr Val Ser Trp Gln Lys Ala
Leu Gly Val 1010 1015 1020 Ala Glu Arg Tyr Asp Ile Leu Leu Leu Asn
Glu Asn Gly Leu Leu 1025 1030 1035 Leu Ser Asn Val Ser Glu Pro Ala
Thr Ala Arg Gln His Lys Phe 1040 1045 1050 Glu Asp Leu Thr Pro Gly
Lys Lys Tyr Lys Met Gln Ile Leu Thr 1055 1060 1065 Val Ser Gly Gly
Leu Phe Ser Lys Glu Ser Gln Ala Glu Gly Arg 1070 1075 1080 Thr Val
Pro Ala Ala Val Thr Asn Leu Arg Ile Thr Glu Asn Ser 1085 1090 1095
Ser Arg Tyr Leu Ser Phe Gly Trp Thr Ala Ser Glu Gly Glu Leu 1100
1105 1110 Ser Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp Arg Thr Leu
Gln 1115 1120 1125 Glu Arg Ala Gln Val Asp Pro Leu Val Gln Ser Phe
Ser Phe Gln 1130 1135 1140 Asn Leu Leu Gln Gly Arg Met Tyr Lys Met
Val Ile Val Thr His 1145 1150 1155 Ser Gly Glu Leu Ser Asn Glu Ser
Phe Ile Phe Gly Arg Thr Val 1160 1165 1170 Pro Ala Ala Val Asn His
Leu Lys Gly Ser His Arg Asn Thr Thr 1175 1180 1185 Asp Ser Leu Trp
Phe Ser Trp Ser Pro Ala Ser Gly Asp Phe Asp 1190 1195 1200 Phe Tyr
Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu 1205 1210 1215
Asn Trp Lys Glu Lys Asp Val Thr Glu Trp Arg Phe Gln Gly Leu 1220
1225 1230 Val Pro Gly Arg Lys Tyr Thr Leu Tyr Val Val Thr His Ser
Gly 1235 1240 1245 Asp Leu Ser Asn Lys Val Thr Gly Glu Gly Arg Thr
Ala Pro Ser 1250 1255 1260 Pro Pro Ser Leu Leu Ser Phe Ala Asp Val
Ala Asn Thr Ser Leu 1265 1270 1275 Ala Ile Thr Trp Lys Gly Pro Pro
Asp Trp Thr Asp Tyr Asn Asp 1280 1285 1290 Phe Glu Leu Gln Trp Phe
Pro Gly Asp Ala Leu Thr Ile Phe Asn 1295 1300 1305 Pro Tyr Ser Ser
Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu 1310 1315 1320 His Pro
Gly Arg Ser Tyr Gln Phe Ser Val Lys Thr Val Ser Gly 1325 1330 1335
Asp Ser Trp Lys Thr Tyr Ser Lys Pro Ile Ser Gly Ser Val Arg 1340
1345 1350 Thr Lys Pro Asp Lys Ile Gln Asn Leu His Cys Arg Pro Gln
Asn 1355 1360 1365 Ser Thr Ala Ile Ala Cys Ser Trp Ile Pro Pro Asp
Ser Asp Phe 1370 1375 1380 Asp Gly Tyr Ser Ile Glu Cys Arg Lys Met
Asp Thr Gln Glu Ile 1385 1390 1395 Glu Phe Ser Arg Lys Leu Glu Lys
Glu Lys Ser Leu Leu Asn Ile 1400 1405 1410 Met Met Leu Val Pro His
Lys Arg Tyr Leu Val Ser Ile Lys Val 1415 1420 1425 Gln Ser Ala Gly
Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile 1430 1435 1440 Thr Met
Ile Asp Arg Pro Pro Gln Pro Pro Pro His Ile Arg Val 1445 1450 1455
Asn Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn Phe Thr 1460
1465 1470 Val Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly Ala Val Lys
Tyr 1475 1480 1485 Phe Ala Val Val Val Arg Glu Ala Asp Ser Met Asp
Glu Leu Lys 1490 1495 1500 Pro Glu Gln Gln His Pro Leu Pro Ser Tyr
Leu Glu Tyr Arg His 1505 1510 1515 Asn Ala Ser Ile Arg Val Tyr Gln
Thr Asn Tyr Phe Ala Ser Lys 1520 1525 1530 Cys Ala Glu Ser Pro Asp
Ser Ser Ser Lys Ser Phe Asn Ile Lys 1535 1540 1545 Leu Gly Ala Glu
Met Asp Ser Leu Gly Gly Lys Cys Asp Pro Ser 1550 1555 1560 Gln Gln
Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr 1565 1570 1575
Arg Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu Asp Leu 1580
1585 1590 Lys Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr Phe Phe Ser
Met 1595 1600 1605 Pro Ile Thr Thr Glu Ser Glu Pro Leu Phe Gly Val
Ile Glu Gly 1610 1615 1620 Val Ser Ala Gly Leu Phe Leu Ile Gly Met
Leu Val Ala Leu Val 1625 1630 1635 Ala Phe Phe Ile Cys Arg Gln Lys
Ala Ser His Ser Arg Glu Arg 1640 1645 1650 Pro Ser Ala Arg Leu Ser
Ile Arg Arg Asp Arg Pro Leu Ser Val 1655 1660 1665 His Leu Asn Leu
Gly Gln Lys Gly Asn Arg Lys Thr Ser Cys Pro 1670 1675 1680 Ile Lys
Ile Asn Gln Phe Glu Gly His Phe Met Lys Leu Gln Ala 1685 1690 1695
Asp Ser Asn Tyr Leu Leu Ser Lys Glu Tyr Glu Asp Leu Lys Asp 1700
1705 1710 Val Gly Arg Ser Gln Ser Cys Asp Ile Ala Leu Leu Pro Glu
Asn 1715 1720 1725 Arg Gly Lys Asn Arg Tyr Asn Asn Ile Leu Pro Tyr
Asp Ala Ser 1730 1735 1740 Arg Val Lys Leu Ser Asn Val Asp Asp Asp
Pro Cys Ser Asp Tyr 1745 1750 1755 Ile Asn Ala Ser Tyr Ile Pro Gly
Asn Asn Phe Arg Arg Glu Tyr 1760 1765 1770 Ile Ala Thr Gln Gly Pro
Leu Pro Gly Thr Lys Asp Asp Phe Trp 1775 1780 1785 Lys Met Ala Trp
Glu Gln Asn Val His Asn Ile Val Met Val Thr 1790 1795 1800 Gln Cys
Val Glu Lys Gly Arg Val Lys Cys Asp His Tyr Trp Pro 1805 1810 1815
Ala Asp Gln Asp Pro Leu Tyr Tyr Gly Asp Leu Ile Leu Gln Met 1820
1825 1830 Val Ser Glu Ser Val Leu Pro Glu Trp Thr Ile Arg Glu Phe
Lys 1835 1840 1845 Ile Cys Ser Glu Glu Gln Leu Asp Ala His Arg Leu
Ile Arg His 1850 1855 1860 Phe His Tyr Thr Val Trp Pro Asp His Gly
Val Pro Glu Thr Thr 1865 1870 1875 Gln Ser Leu Ile Gln Phe Val Arg
Thr Val Arg Asp Tyr Ile Asn 1880 1885 1890 Arg Ser Pro Gly Ala Gly
Pro Thr Val Val His Cys Ser Ala Gly 1895 1900 1905 Val Gly Arg Thr
Gly Thr Phe Val Ala Leu Asp Arg
Ile Leu Gln 1910 1915 1920 Gln Leu Asp Ser Lys Asp Ser Val Asp Ile
Tyr Gly Ala Val His 1925 1930 1935 Asp Leu Arg Leu His Arg Val His
Met Val Gln Thr Glu Cys Gln 1940 1945 1950 Tyr Val Tyr Leu His Gln
Cys Val Arg Asp Val Leu Arg Ala Lys 1955 1960 1965 Lys Leu Arg Asn
Glu Gln Glu Asn Pro Leu Phe Pro Ile Tyr Glu 1970 1975 1980 Asn Val
Asn Pro Glu Tyr His Arg Asp Ala Ile Tyr Ser Arg His 1985 1990 1995
71619PRTMus musculus 7Met Leu Arg His Gly Ala Leu Thr Ala Leu Trp
Ile Thr Leu Ser Val 1 5 10 15 Val Gln Thr Gly Val Ala Glu Gln Val
Lys Cys Asn Phe Thr Leu Leu 20 25 30 Glu Ser Arg Val Ser Ser Leu
Ser Ala Ser Ile Gln Trp Arg Thr Phe 35 40 45 Ala Ser Pro Cys Asn
Phe Ser Leu Ile Tyr Ser Ser Asp Thr Ser Gly 50 55 60 Pro Met Trp
Cys His Pro Ile Arg Ile Asp Asn Phe Thr Tyr Gly Cys 65 70 75 80 Asn
Pro Lys Asp Leu Gln Ala Gly Thr Val Tyr Asn Phe Arg Ile Val 85 90
95 Ser Leu Asp Gly Glu Glu Ser Thr Leu Val Leu Gln Thr Asp Pro Leu
100 105 110 Pro Pro Ala Arg Phe Glu Val Asn Arg Glu Lys Thr Ala Ser
Thr Thr 115 120 125 Leu Gln Val Arg Trp Thr Pro Ser Ser Gly Lys Val
Ser Trp Tyr Glu 130 135 140 Val Gln Leu Phe Asp His Asn Asn Gln Lys
Ile Gln Glu Val Gln Val 145 150 155 160 Gln Glu Ser Thr Thr Trp Ser
Gln Tyr Thr Phe Leu Asn Leu Thr Glu 165 170 175 Gly Asn Ser Tyr Lys
Val Ala Ile Thr Ala Val Ser Gly Glu Lys Arg 180 185 190 Ser Phe Pro
Val Tyr Ile Asn Gly Ser Thr Val Pro Ser Pro Val Lys 195 200 205 Asp
Leu Gly Ile Ser Pro Asn Pro Asn Ser Leu Leu Ile Ser Trp Ser 210 215
220 Arg Gly Ser Gly Asn Val Glu Gln Tyr Arg Leu Val Leu Met Asp Lys
225 230 235 240 Gly Ala Ile Val Gln Asp Thr Asn Val Asp Arg Arg Asp
Thr Ser Tyr 245 250 255 Ala Phe His Glu Leu Thr Pro Gly His Leu Tyr
Asn Leu Thr Ile Val 260 265 270 Thr Met Ala Ser Gly Leu Gln Asn Ser
Arg Trp Lys Leu Val Arg Thr 275 280 285 Ala Pro Met Glu Val Ser Asn
Leu Lys Val Thr Asn Asp Gly Arg Leu 290 295 300 Thr Ser Leu Asn Val
Lys Trp Gln Lys Pro Pro Gly Asp Val Asp Ser 305 310 315 320 Tyr Ser
Ile Thr Leu Ser His Gln Gly Thr Ile Lys Glu Ser Lys Thr 325 330 335
Leu Ala Pro Pro Val Thr Glu Thr Gln Phe Lys Asp Leu Val Pro Gly 340
345 350 Arg Leu Tyr Gln Val Thr Ile Ser Cys Ile Ser Gly Glu Leu Ser
Ala 355 360 365 Glu Lys Ser Ala Ala Gly Arg Thr Val Pro Glu Lys Val
Arg Asn Leu 370 375 380 Val Ser Tyr Asn Glu Ile Trp Met Lys Ser Phe
Thr Val Asn Trp Thr 385 390 395 400 Pro Pro Ala Gly Asp Trp Glu His
Tyr Arg Ile Val Leu Phe Asn Glu 405 410 415 Ser Leu Val Leu Leu Asn
Thr Thr Val Gly Lys Glu Glu Thr His Tyr 420 425 430 Ala Leu Asp Gly
Leu Glu Leu Ile Pro Gly Arg Gln Tyr Glu Ile Glu 435 440 445 Val Ile
Val Glu Ser Gly Asn Leu Arg Asn Ser Glu Arg Cys Gln Gly 450 455 460
Arg Thr Val Pro Leu Ala Val Leu Gln Leu Arg Val Lys His Ala Asn 465
470 475 480 Glu Thr Ser Leu Gly Ile Thr Trp Arg Ala Pro Leu Gly Glu
Trp Glu 485 490 495 Lys Tyr Ile Ile Ser Leu Met Asp Arg Glu Leu Leu
Val Ile His Lys 500 505 510 Ser Leu Ser Lys Asp Ala Lys Glu Phe Thr
Phe Thr Asp Leu Met Pro 515 520 525 Gly Arg Asn Tyr Lys Ala Thr Val
Thr Ser Met Ser Gly Asp Leu Lys 530 535 540 Gln Ser Ser Ser Ile Lys
Gly Arg Thr Val Pro Ala Gln Val Thr Asp 545 550 555 560 Leu His Val
Asn Asn Gln Gly Met Thr Ser Ser Leu Phe Thr Asn Trp 565 570 575 Thr
Lys Ala Leu Gly Asp Val Glu Phe Tyr Gln Val Leu Leu Ile His 580 585
590 Glu Asn Val Val Val Lys Asn Glu Ser Val Ser Ser Asp Thr Ser Arg
595 600 605 Tyr Ser Phe Arg Ala Leu Lys Pro Gly Ser Leu Tyr Ser Val
Val Val 610 615 620 Thr Thr Val Ser Gly Gly Ile Ser Ser Arg Gln Val
Val Ala Glu Gly 625 630 635 640 Arg Thr Val Pro Ser Ser Val Ser Gly
Val Thr Val Asn Asn Ser Gly 645 650 655 Arg Asn Asp Tyr Leu Ser Val
Ser Trp Leu Pro Ala Pro Gly Glu Val 660 665 670 Asp His Tyr Val Val
Ser Leu Ser His Glu Gly Lys Val Asp Gln Phe 675 680 685 Leu Ile Ile
Ala Lys Ser Val Ser Glu Cys Ser Phe Ser Ser Leu Thr 690 695 700 Pro
Gly Arg Leu Tyr Asn Val Thr Val Thr Thr Lys Ser Gly Asn Tyr 705 710
715 720 Ala Ser His Ser Phe Thr Glu Glu Arg Thr Val Pro Asp Lys Val
Gln 725 730 735 Gly Ile Ser Val Ser Asn Ser Ala Arg Ser Asp Tyr Leu
Lys Val Ser 740 745 750 Trp Val His Ala Thr Gly Asp Phe Asp His Tyr
Glu Val Thr Ile Lys 755 760 765 Asn Arg Glu Ser Phe Ile Gln Thr Lys
Thr Ile Pro Lys Ser Glu Asn 770 775 780 Glu Cys Glu Phe Ile Glu Leu
Val Pro Gly Arg Leu Tyr Ser Val Thr 785 790 795 800 Val Ser Thr Lys
Ser Gly Gln Tyr Glu Ala Ser Glu Gln Gly Thr Gly 805 810 815 Arg Thr
Ile Pro Glu Pro Val Lys Asp Leu Thr Leu Leu Asn Arg Ser 820 825 830
Thr Glu Asp Leu His Val Thr Trp Ser Arg Ala Asn Gly Asp Val Asp 835
840 845 Gln Tyr Glu Val Gln Leu Leu Phe Asn Asp Met Lys Val Phe Pro
His 850 855 860 Ile His Leu Val Asn Thr Ala Thr Glu Tyr Lys Phe Thr
Ala Leu Thr 865 870 875 880 Pro Gly Arg His Tyr Lys Ile Leu Val Leu
Thr Ile Ser Gly Asp Val 885 890 895 Gln Gln Ser Ala Phe Ile Glu Gly
Leu Pro Val Pro Ser Thr Val Lys 900 905 910 Asn Ile His Ile Ser Ala
Asn Gly Ala Thr Asp Arg Leu Met Val Thr 915 920 925 Trp Ser Pro Gly
Gly Gly Asp Val Asp Ser Tyr Val Val Ser Ala Phe 930 935 940 Arg Gln
Asp Glu Lys Val Asp Ser Gln Thr Ile Pro Lys His Ala Ser 945 950 955
960 Glu His Thr Phe His Arg Leu Glu Ala Gly Ala Lys Tyr Arg Ile Ala
965 970 975 Ile Val Ser Val Ser Gly Ser Leu Arg Asn Gln Ile Asp Ala
Leu Gly 980 985 990 Gln Thr Val Pro Ala Ser Val Gln Gly Val Val Ala
Ala Asn Ala Tyr 995 1000 1005 Ser Ser Asn Ser Leu Thr Val Ser Trp
Gln Lys Ala Leu Gly Val 1010 1015 1020 Ala Glu Arg Tyr Asp Ile Leu
Leu Leu Asn Glu Asn Gly Leu Leu 1025 1030 1035 Leu Ser Asn Val Ser
Glu Pro Ala Thr Ala Arg Gln His Lys Phe 1040 1045 1050 Glu Asp Leu
Thr Pro Gly Lys Lys Tyr Lys Met Gln Ile Leu Thr 1055 1060 1065 Val
Ser Gly Gly Leu Phe Ser Lys Glu Ser Gln Ala Glu Gly Arg 1070 1075
1080 Thr Val Pro Ala Ala Val Thr Asn Leu Arg Ile Thr Glu Asn Ser
1085 1090 1095 Ser Arg Tyr Leu Ser Phe Gly Trp Thr Ala Ser Glu Gly
Glu Leu 1100 1105 1110 Ser Trp Tyr Asn Ile Phe Leu Tyr Asn Pro Asp
Arg Thr Leu Gln 1115 1120 1125 Glu Arg Ala Gln Val Asp Pro Leu Val
Gln Ser Phe Ser Phe Gln 1130 1135 1140 Asn Leu Leu Gln Gly Arg Met
Tyr Lys Met Val Ile Val Thr His 1145 1150 1155 Ser Gly Glu Leu Ser
Asn Glu Ser Phe Ile Phe Gly Arg Thr Val 1160 1165 1170 Pro Ala Ala
Val Asn His Leu Lys Gly Ser His Arg Asn Thr Thr 1175 1180 1185 Asp
Ser Leu Trp Phe Ser Trp Ser Pro Ala Ser Gly Asp Phe Asp 1190 1195
1200 Phe Tyr Glu Leu Ile Leu Tyr Asn Pro Asn Gly Thr Lys Lys Glu
1205 1210 1215 Asn Trp Lys Glu Lys Asp Val Thr Glu Trp Arg Phe Gln
Gly Leu 1220 1225 1230 Val Pro Gly Arg Lys Tyr Thr Leu Tyr Val Val
Thr His Ser Gly 1235 1240 1245 Asp Leu Ser Asn Lys Val Thr Gly Glu
Gly Arg Thr Ala Pro Ser 1250 1255 1260 Pro Pro Ser Leu Leu Ser Phe
Ala Asp Val Ala Asn Thr Ser Leu 1265 1270 1275 Ala Ile Thr Trp Lys
Gly Pro Pro Asp Trp Thr Asp Tyr Asn Asp 1280 1285 1290 Phe Glu Leu
Gln Trp Phe Pro Gly Asp Ala Leu Thr Ile Phe Asn 1295 1300 1305 Pro
Tyr Ser Ser Arg Lys Ser Glu Gly Arg Ile Val Tyr Gly Leu 1310 1315
1320 His Pro Gly Arg Ser Tyr Gln Phe Ser Val Lys Thr Val Ser Gly
1325 1330 1335 Asp Ser Trp Lys Thr Tyr Ser Lys Pro Ile Ser Gly Ser
Val Arg 1340 1345 1350 Thr Lys Pro Asp Lys Ile Gln Asn Leu His Cys
Arg Pro Gln Asn 1355 1360 1365 Ser Thr Ala Ile Ala Cys Ser Trp Ile
Pro Pro Asp Ser Asp Phe 1370 1375 1380 Asp Gly Tyr Ser Ile Glu Cys
Arg Lys Met Asp Thr Gln Glu Ile 1385 1390 1395 Glu Phe Ser Arg Lys
Leu Glu Lys Glu Lys Ser Leu Leu Asn Ile 1400 1405 1410 Met Met Leu
Val Pro His Lys Arg Tyr Leu Val Ser Ile Lys Val 1415 1420 1425 Gln
Ser Ala Gly Met Thr Ser Glu Val Val Glu Asp Ser Thr Ile 1430 1435
1440 Thr Met Ile Asp Arg Pro Pro Gln Pro Pro Pro His Ile Arg Val
1445 1450 1455 Asn Glu Lys Asp Val Leu Ile Ser Lys Ser Ser Ile Asn
Phe Thr 1460 1465 1470 Val Asn Cys Ser Trp Phe Ser Asp Thr Asn Gly
Ala Val Lys Tyr 1475 1480 1485 Phe Ala Val Val Val Arg Glu Ala Asp
Ser Met Asp Glu Leu Lys 1490 1495 1500 Pro Glu Gln Gln His Pro Leu
Pro Ser Tyr Leu Glu Tyr Arg His 1505 1510 1515 Asn Ala Ser Ile Arg
Val Tyr Gln Thr Asn Tyr Phe Ala Ser Lys 1520 1525 1530 Cys Ala Glu
Ser Pro Asp Ser Ser Ser Lys Ser Phe Asn Ile Lys 1535 1540 1545 Leu
Gly Ala Glu Met Asp Ser Leu Gly Gly Lys Cys Asp Pro Ser 1550 1555
1560 Gln Gln Lys Phe Cys Asp Gly Pro Leu Lys Pro His Thr Ala Tyr
1565 1570 1575 Arg Ile Ser Ile Arg Ala Phe Thr Gln Leu Phe Asp Glu
Asp Leu 1580 1585 1590 Lys Glu Phe Thr Lys Pro Leu Tyr Ser Asp Thr
Phe Phe Ser Met 1595 1600 1605 Pro Ile Thr Thr Glu Ser Glu Pro Leu
Phe Gly 1610 1615
* * * * *