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THIS IS VOLUME 1 OF 2
NOTE: For additional vohxmes please contact the Canadian Patent Oi~ice.
CA 02559167 2006-09-11
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MODIFIED ANNEXIN PROTEINS AND METHODS FOR PREVENTING
THROMBOSIS
FIELD OF THE INVENTION
[0001 ] The present invention relates generally to methods and compositions
for
treating thrombosis. More particularly, it relates to modified annexin
proteins and
methods for their use.
BACKGROUND OF THE INVENTION
[0002] Thrombosis-the formation, development, or presence of a blood clot
(thronlbuS) in a blood vessel-is the most cO1n111011 Severe 111ed1Ca1
dlSOrder. The most
frequent example of arterial thrombosis is coronary thrombosis, which leads to
occlusion
of the coronary arteries and often to myocardial infarction (heart attack).
More than 1.3
million patients are admitted to the hospital for myocardial infarction each
year in North
America. The standard therapy is administration of a thrombolytic protein by
infusion.
Thrombolytic treatment of acute myocardial infarction is estimated to save 30
lives per
1000 patients treated; nevertheless the 30-day mortality for this disorder
remains
substantial (Mehta et al., Lancet 356:449-454 (2000)) The disclsosure of
Mehta, et al.,
and the disclsosure of all other patents, patent applications and publications
refewed to
herein, are incorporated herein by reference in their entirety). It would be
convenient to
administer antithrombotic and thrombolytic agents by bolus injection, since
they might be
used before admission to hospital with additional benefit (Rawles, J. Am.
Goll. Cardiol.
30:1181-1186 (1997), incorporated herein by reference). However, bolus
injection (as
opposed to a more gradual intravenous infusion) significantly increases the
risk of cerebral
hemorrhage (Mehta et al., 2000). The development of an agent able to prevent
thrombosis
and/or increase thrombolysis, without augmenting the risk of bleeding, would
be desirable.
[0003] Unstable angina, caused by inadequate oxygen delivery to the heart due
to
coronary occlusion, is the most common cause of admission to hospital, with
1.5 I111111011
cases a year in the United States alone. When patients with occlusion of
coronary arteries
are treated with angioplasty and stenting, the use of an antibody against
platelet
gp IIb / IIIa decreases the likelihood of restenosis. However, the same
antibody has shown
no benefit in unstable angina without angioplasty, and a better method for
preventing
coronary occlusion in these patients is needed.
1
CA 02559167 2006-09-11
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[0004] Another important example of arterial thrombosis is cerebral
thrombosis.
Intravenous recombinant tissue plasminogen activator (z-tPA) is the only
treatment for
acute ischemic stroke that is approved by the Food and Drug Administration.
The earlier
it is administered the better (Ernst et al., Stroke 31:2552-2557 (2000),
incorporated herein
by reference). However, intravenous z-tPA administration is associated with
increased risk
of intracerebral hemorrhage. Full-blown strokes are often preceded by
transient ischemic
attacks (TIA), and it is estimated that about 300,000 persons suffer TIA every
year in the
United States. It would be desirable to have a safe and effective agent that
could be
administered as a bolus and would for several days prevent recurrence of
cerebral
thrombosis without increasing the risk of cerebral hemorrhage. Thrombosis also
contributes to peripheral arterial occlusion in diabetics and other patients,
and an
efficacious and safe antithrombotic agent for use in such patients is needed.
[0005] Venous thrombosis is a frequent complication of surgical procedures
such
as hip and knee arthroplasties. It would be desirable to prevent thrombosis
without
increasing hemorrhage into the field of operation. Similar considerations
apply to venous
thrombosis associated with pregnancy and parturition. Some persons are prone
to repeated
venous thrombotic events and are currently treated by antithrombotic agents
such as
coumarin-type drugs. The dose of such drugs must be titrated in each patient,
and the
margin between effective antithrombotic doses and those increasing hemorrhage
is small.
Having a treatment with better separation of antithrombotic activity from
increased risk of
bleeding is desirable. All of the recently introduced antithrombotic
therapies, including
ligands of platelet gp IIb / IIIa, Iow molecular weight heparins, and a
pentasaccharide
inhibitor of factor Xa, carry an increased risk of bleeding (Levine et al.,
Chest 119:1085-
1215 (2001), incorporated herein by reference). Hence there is a need to
explore
altezmative strategies for preventing arterial and venous thrombosis without
augmenting
the risk of hemorrhage.
[0006] To inhibit the extension of arterial or venous thrombi without
increasing
hemorrhage, it is necessary to exploit potential differences between
mechanisms involved
in hemostasis and those involved in thrombosis in large blood vessels. Primary
hemostatic
mechanisms include the formation of platelet microaggregates, which plug
capillaries and
accumulate over damaged or activated endothelial cells in small blood vessels.
Inhibitors
of platelet aggregation, including agents suppressing the formation or action
of
thromboxane A2, ligands of gp IIa / IIIb, and drugs acting on ADP receptors
such as
2
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WO 2005/086955 PCT/US2005/008193
clopidogrel (Hallopeter, Nature 409:202-207 (2001 ), incorporated herein by
reference),
interfere with this process and therefore increase the risk of bleeding
(Levine et al., 2001).
In contrast to microaggregate formation, occlusion by an arterial or venous
thrombus
requires the continued recruitment and incorporation of platelets into the
thrombus. To
overcome detachment by shear forces in large blood vessels, platelets must be
bound
tightly to one another and to the fibrin network deposited around them.
[0007] Evidence has accumulated that the formation of tight macroaggregates of
platelets is facilitated by a cellular and a humoral amplification mechanism,
which
reinforce each other. In the cellular mechanism, the formation of relatively
loose
microaggregates of platelets, induced by moderate concentrations of agonists
such as
ADP, thromboxane A?, or collagen, is accompanied by the release from platelet
a.-granules of the 85-1cD protein Gash (Angelillo-Schemer et al., Nature
Medicine
7:215-221 (2001), incorporated herein by reference). Binding of released Gash
to receptor
tyrosine kinases (Axl, Sky, Mer) expressed on the surface of platelets induces
complete
degranulation and the formation of tight macroaggregates of these cells. In
the humoral
amplification mechanism, a prothrombinase complex is formed on the surface of
activated
platelets and microvesicles. This generates thrombin and fibrin. Thrombin is
itself a
potent platelet activator and inducer of the release of Gash (Ishimoto and
Nakano, FEBS
Lett. 446:197-199 (2000), incorporated herein by reference). Fully activated
platelets bind
tightly to the fibrin network deposited around them. Histological observations
show that
both platelets and fibrin are necessary for the formation of a stable coronary
thrombus in
humans (Falls et al. Interrelationship between atherosclerosis and thrombosis.
In
Vanstraete et al. (editors), Cardiovascular Thrombosis: Thrombocardiolog
Thromboneurolo~y. Philadelphia: Lipincott-Raven Publishers (1998), pp. 45-58,
incorporated herein by reference). Another platelet adhesion molecule,
amphoterin, is
translocated to the platelet surface during activation, and binds anionic
phospholipid
(Rouhainen et al., Thromb. Hemost. 84:1087-1094 (2000), incorporated herein by
reference). Like Gas6, amphoterin could form a bridge during platelet
aggregation.
[0008] The question arises whether it is possible to inhibit these
amplification
meCha111S111S but not the initial platelet aggregation step, thereby
preventing thrombosis
without increasing hemorrhage. The importance of cellular amplification has
recently
been established by studies of mice with targeted inactivation of Gash
(Angelillo-Scherrer
et al., 2001). The Gas6-/- mice were found to be protected against thrombosis
and
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
embolism induced by collagen and epinephrine. However, the Gas6-/- mice did
not suffer
from spontaneous hemorrhage and had normal bleeding after tail clipping. Ful-
thermore,
antibodies against Gash inhibited platelet aggregation ifz vita°o as
well as thrombosis
induced ifz vivo by collagen and epinephrine. In principle, such antibodies,
or ligands
competing for Gash binding to receptor tyrosine lcinases, might be used to
inhibit
thrombosis. However, in view of the potency of humoral amplification, it nnght
be
preferable to inhibit that step. Ideally such an inhibitor would also have
additional
suppressive activity on the Gas6-mediated cellular amplification mechanism.
[0009] A strategy for preventing both cellular and humoral amplification of
platelet aggregation is provided by the annexins, a family of highly
homologous
antithrombotic proteins of which ten are expressed in several human tissues
(Benz and
Hofinann, Biol. Chem. 378:177-183 (1997), incorporated herein be reference).
Annexins
share the property of binding calcium and negatively charged phospholipids,
both of
which are required for blood coagulation. Under physiological conditions,
negatively
charged phospholipid is mainly supplied by phosphatidylserine (PS) in
activated or
damaged cell membranes. In intact cells, PS is confined to the inner leaflet
of the plasma
membrane bilayer and is not accessible on the surface. When platelets are
activated, the
amounts of PS accessible on their surface, and therefore the extent of annexin
binding, are
greatly increased (Sun et al., Thrombosis Res. 69:289-296 (1993), incorporated
herein by
reference). During activation of platelets, microvesicles are released from
their surfaces,
greatly increasing the surface area expressing anionic phospholipids with
procoagulant
activity (Merten et al., Circulation 99:2577-2582 (1999); Chow et al., J. Lab.
Clin. Med.
135:66-72 (2000), both incorporated herein by reference). These may play an
impol-tant
role in the propagation of platelet-mediated arterial thrombi.
[0010] Proteins involved in the blood coagulation cascade (factors X, Xa, and
Va)
bind to membranes bearing PS on their surfaces, and to one another, forming a
stable,
tightly bound proth rombinase complex. Several annexins, including II, V, and
VIII, bind
PS with high affinity, thereby preventing the formation of a prothrombinase
complex and
exerting antithrombotic activity. Annexin V binds PS with very high affinity
(Kd = 1.7
nmol/L), greater than the affinity of factors X, Xa, and Va for negatively
charged
phospholipids (Thiagarajan and Tait, J. Biol. Chem. 265:17420-17423 (1990),
incorporated herein by reference). Tissue factor-dependent blood coagulation
on the
surface of activated or damaged endothelial cells also requires surface
expression of PS,
4
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
and annexin V can inhibit this process (van Heerde et al., Arterioscl. Thromb.
14:824-830
(1994), incorporated herein by reference), although annexin is less effective
in this activity
than in inhibition of prothrombinase generation (Rao et al., Thromb. Res.
62:517-531
( 1992), incorporated herein by reference).
[0011] The binding of annexin V to activated platelets and to damaged cells
probably explains the selective retention ofthe protein in thrombi. This has
been shown in
experimental animal models of venous and arterial thrombosis (Stratton et al.,
Circulation
92:3113-3121 (1995); Thiagarajan and Benedict, Circulation 96:2339-2347
(1997), both
incorporated herein by reference), and labeled annexin has been proposed for
medical
imaging of vascular thrombi in humans, with reduced noise and increased safety
(Reno
and Kasina, International Patent Application PCT/US95/07599 (WO 95/34315)
(published
December 21, 1995), incorporated herein by reference). The binding to thrombi
of a
potent antithrombotic agent such as annexin V provides a strategy for
preventing the
extension or recurrence of thrombosis. Transient myocardial ischemia also
increases
annexin V binding (Dumont et al., Circulation 102:1564-1568 (2000),
incorporated herein
by reference). Annexin V imaging in humans has shown increased binding of the
protein
in transplanted heal-ts when endomyocardial biopsy has demonstrated vascular
rejection
(Acio et al., J. Nuclear Med. 41 (5 Suppl.):127P (2000), incorporated herein
by reference).
This binding is presumably due to PS exteriorized on the surface of damaged
endothelial
cells, as well as of apoptotic myocytes in hearts that are being rejected. It
follows that
administration of annexin after myocardial infarction should prevent the
formation of pro-
thrombotic complexes on both platelets and endothelial cells, thereby
preventing the
extension or recurrence of thrombosis. Annexin V binding is also augmented
following
cerebral hypoxia in humans (D'Arceuil et al., Stroke 2000: 2692-2700 (2000),
incorporated herein by reference), which supports the hypothesis that
administration of
annexin following TIA may decrease the likelihood of developing a full-blown
stroke.
[0012] Allllex111s have shown anticoagulant activity in several ire oilro
thrombln-
depelldellt aSSayS, as well as in experimental animal models of VeIlOLIS
thI'OnlbOSlS
(Romisch et al., Thrombosis Res. 61:93-104 (1991); Van Ryn-McI~enna et al.,
Thrombosis Hemostasis 69:227-230 (1993), both incorporated herein by
reference) and
arterial thrombosis (Thiagarajan and Benedict, 1997). Remarkably, annexin in
antithrombotic doses had no demonstrable effect on traditional G.~ 1~11?O
C10tt1llg tests in
created rabbits (Thiagarajan and Benedict, 1997) and did not significantly
prolong
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
bleeding times of treated rats (Van Ryn-McKenna et al., 1993). In treated
rabbits annexin
did not increase bleeding into a surgical incision (Thiagarajan and Benedict,
1997). Thus,
uniquely among all the agents so far investigated, annexins exert
antithrombotic activity
without increasing hemorrhage. Annexins do not inhibit platelet aggregation
triggered by
agonists other than thrombin (van Heerde et al., 1994), and platelet
aggregation is the
primary hemostatic mechanism. In the walls of damaged blood vessels and in
extravascular tissues, the tissue factor / VIIa complex also exerts hemostatic
effects, and
this system is less susceptible to inhibition by annexin V than is the
prothrombinase
complex (Rao et al., 199?). This is one argument for confining administered
annexin V to
the vascular compartment as far as possible; the risk of hemorrhage is likely
to be reduced.
[0013] Despite such promising results for preventing thrombosis, a major
problem
associated with the therapeutic use of annexins is their short half life in
the circulation,
estimated in experimental animals to be 5 to IS minutes (Romisch et al., 1991;
Stratton
et al., 1995; Thiagarajan. and Benedict, 1997); annexin V also has a short
half life in the
circulation of humans (Strauss et al., J. Nuclear Med. 41 (5 Suppl.):149P
(2000),
incorporated herein by reference). Most of the annexin is lost into the urine,
as expected
of a 36 kDa protein (Thiagarajan and Benedict, 1997). There is a need,
therefore, for a
method of preventing annexin loss from the vascular compartment into the
extravascular
compartment and urine, thereby prolonging antithrombotic activity following a
single
inj ection.
SUMMARY OF THE INVENTION
[0014] The present invention provides compounds and methods for preventing
arterial or venous thrombosis. Recombinant human annexins are modified in such
a way
that its half life in the vascular compartment is prolonged. This can be
achieved in a
variety of ways; three embodiments are an annexin coupled to polyethylene
glycol, a
homopolymer or heteropolymer of annexin, and a fusion protein of annexin with
another
protein (e.g., the Fc portion of immunoglobulin). The modified annexin binds
with high
affinity to phosphatidylserine on the surface of activated platelets or
injured cells, thereby
preventing the binding of Gash as well as procoagulant proteins and the
formation of a
prothrombinase complex. Modified annexin therefore inhibits both the cellular
and
humoral mechanisms by which platelet aggregation is amplified, thereby
preventing
thrombosis.
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[0015] In one embodiment, the present invention provides an isolated modified
annexin protein containing an annexin protein, preferably annexin V, coupled
to
polyethylene glycol (PEG). Preferably, at least two PEG chains are coupled to
a single
annexin molecule, with each PEG having a molecular weight of at least 5 kDa,
more
preferably at least 10 kDa, and most preferably at least 15 kDa. In an
alternative
embodiment, an isolated modified annexin protein contains an annexin protein
coupled to
at least one additional protein, such as an additional annexin protein
(forming a
homodimer) or the Fc poution of innnunoglobulin. The additional protein
preferably has a
molecular weight of at least 30 lcDa. Also provided by the present invention
are
pharmaceutical compositions containing an antithrombotically effective amount
of any of
the modified annexin proteins of the invention.
[0016] In methods of the invention, the modified annexin is administered to a
subject at risk of thrombosis in a pharmaceutical composition having an
antithrombotically
effective amount of any one of the modified annexin proteins of the present
invention. For
example, the pharmaceutical composition can be administered after an arterial
thrombosis
such as coronary thrombosis, cerebral thrombosis, or a transient cerebral
ischemic attack.
It can also be administered after a surgical operation associated with venous
thrombosis.
Additionally, it can be administered to subjects having conditions subject to
arterial or
venous thrombosis, such as diabetes, pregnancy, or parturition.
[0017] Also provided by the present invention are an isolated nucleic acid
molecule encoding a homodimer of annexin, a recombinant molecule containing at
least a
portion of the nucleic acid molecule, and a recombinant cell containing at
least a portion
of the nucleic acid molecule. The recombinant cell is cultured under suitable
conditions in
a method of the invention to produce a homodimer of annexin.
[0018] The present invention also provides a method for screening for a
modified
annexin protein that modulates thrombosis using a thrombosis test system. The
test
system is contacted with a test modified annexin protein, after which the
thrombolytic
activity is assessed and compared with the activity of the system in the
absence of the test
modified annexin protein. Preferably, the activated partial thromboplastin
time is
measured. Also provided is a method for identifying a modified annexin protein
by
contacting activated platelets with a test modified annexin protein and
assessing the
platelet-binding and protein S-binding activity.
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[0019] Also provided by the present invention is a method for in vivo
screening
for a modified annexin protein. In this method, a thrombosis animal model is
contacted
with a test modified annexin protein, after which the in vivo anticoagulation
activity and
increase in hemorrhage of the test modified annexin protein is assessed. The
anticoagulation activity and time are compared with the anticoagulation
activity and time
of annexin, and the amount of hemorrhage is compared with hemorrhage in the
animal
model in the absence of the test modified annexin protein.
[0020] Thus the invention provides a method of treating a subject at risk of
thrombosis comprising administering to said subject an antithrombotically
effective
amount of an isolated modified annexin protein comprising an annexin dimer.
The
isolated modified annexin protein is administered after coronary thrombosislc,
after a overt
cerebral thrombosis, after, transient cerebral ischemic attack, after a
surgical operation
associated with venous thrombosis, wherein said subject is diabetic and said
thrombosis is
arterial thrombosis, or during a condition selected from the group consisting
of pregnancy
and parturition. The isolated modified annexin protein is administered in a
range from 0.2
mg/kg to 1.0 mg/lcg.
[0021] The present invention also provides a method of inhibiting the
attachment
of leukocytes to endothelial cells comprising administering an effective
amount of an
isolated modified annexin protein comprising an annexin dimer to a patient in
need
thereof. In some elnbodilnents, the method further comprises reducing
endothelial cell
damage.
[0022] The present invention also provides a method of treating a subject at
risk
of thl'OnlboSlS COInpI'lslIlg administering to said subject an
antithrombotically effective
amount of a protein having an affinity for phosphatidylserine that is at least
90% of the
affinity of annexin V for phosphatidylserine, including wherein said protein
is a
1110110C1011a1 or polyclonal antibody.
BRIEF DESCRIPTION OF THE FIGURES
[0023] FIGS. lA-C show the structural scheme of two modified annexin
e111bOd1111e11tS. FIG. lA shows the structural scheme of human annexin V
hOlllod1111er Wlth
a His-tag; FIG. 1 B shows the structural scheme of the human annexin V
homodimer
without a His-tag. FIG. 1 C shows a DNA construct for making a homodimer of
annexin
V.
8
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[0024] FIGS. 2A-D show the results of flowcytometric analysis of a mixture of
normal (1 x 107/ml) and PS exposing (1 x 107/ml) RBCs incubated with 0.2 qg/ml
biotinylated AV (FIG. 2A); 0.2 ~g/ml biotinylated DAV (FIG. 2B); 0.2 ~g/ml
biotinylated
AV and 0.2 qg/ml nonbiotinylated DAV (FIG. 2C); and 0.2 qg/ml biotinylated DAV
and
0.2 ~g/ml nonbiotinylated AV (FIG. 2D), in each case, followed by R-
phycoerythrein-
conjugated streptavidin.
[0025] FIGS. 3A-E illustrate the levels of AV or DAV in mouse circulation at
various times after injection. FIGS. 3A-B show serum samples recovered 5
minutes and
20 minutes after injection of AV into mice, respectively. FIGS. 3C-E show
serum samples
recovered 5 minutes, 25 minutes and 120 minutes after injection of annexin V
homodimer
(DAV) into mice, respectively.
[0026] FIG. 4 shows PLA2-induced hemolysis of PS-exposing RBC. A mixture
of normal (1 x 107/ml) and PS exposing (1 x 107/ml) RBCs was incubated with
100 ng/ml
pancreatic PLA2 (pPLA2) or secretory PLA2 (sPLA2). Hemolysis was measured as a
function of time and expressed relative to 100% hemolysis induced by osmotic
shock.
The percentage of PS-exposing cells was determined by flow cytometry of the
cell
suspension after labeling with biotinylated DAV and R-phycoerythrein-
conjugated
streptavidin. FIG. 4A shows hemolysis induced by 100 ng/ml pPLA2 in absence
(triangles) or presence of 2 pg/ml DAV (circles) or AV (squares). FIG. 4B
shows
hemolysis induced by 100 ng/ml pPLA2 in the presence of various amounts of DAV
(circles) or AV (squares). FIG. 4C shows PS-exposing cells in the cell
suspension after 60
minutes incubation with 100 ng/ml pPLA2 in the presence of 2 ~.glml DAV.
[0027] FIG. 5 shows serum alanine aminotransferase (ALT) levels in mice sham
operated (Sham), mice given saline, mice given HEPES buffer 6 hrs. before
clamping the
hepatic artery, mice given pegylated amiexin (PEG Anex) or annexin diner 6
hrs. before
clamping the artery, and mice given monomeric annexin (Anex). The asterisk
above PEG
ANNEX and ANNEX DIMER indicates p<0.001.
[0028] FIG. 6 is a plot of clotting time of an irT vitro clotting assay
comparing the
anticoagulant potency of recombinant human annexin V and pegylated recombinant
human annexin V.
[0029] FIG. 7 shows thrombus weight in the five treatment groups of the 10-
minute thrombosis study (mean ~ sd; n=8).
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[0030] FIG. 8 shows APTT in the five treatment groups of the thrombosis study
(mean ~ sd; n=8).
[0031] FIG. 9 shows bleeding time in the three groups of the tail bleeding
study
(mean ~ sd; n=8).
[0032] FIG. 10 shows blood loss in the three groups of the tail bleeding study
(mean ~ sem; n=8).
[0033] FIG. 11 SNOWS APTT in the three groups of the tail bleeding study (mean
~
sd; n=8).
[0034] FIG. 12A shows attachment of leukocytes to endothelial cells during
ischemia-reperfusion injury with and without diannexin for peripol-tal
sinusoids. FIG. 12B
shows attachment of leukocytes to endothelial cells during ischemia-
reperfusion injury
with and without diannexin for centrilobular sinusoids.
[0035] FIG. 13A shows attachment of platelets to endothelial cells during
ischemia-reperfusion injury with and without diannexin for periportal
sinusoids. FIG. 13B
shows attachment of platelets to endothelial cells during ischemia-reperfusion
injury with
and without diannexin for centrilobular sinusoids.
[0036] FIG. 14A shows swelling of endothelial cells during ischemia-
reperfusion
injury with and without diannexin for periportal sinusoids. FIG. 14B shows
swelling of
endothelial cells during ischemia-reperfusion injury with and without
diannexin for
centrilobular sinusoids.
[0037] FIG. 15A shows phagocytic activity of Kupffer cells during ischemia-
reperfusion injury with and without diannexin for periportal sinusoids. FIG.
15B shows
phagocytic activity of Kupffer cells during ischemia-reperfusion injury with
and without
diannexin for centrilobular sinusoids.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The present invention provides compounds and methods for preventing
thrombosis in mammals without increasing hemorrhage. The invention relies in
part on
the recognition that the primary mechanisms of platelet aggregation are
different from the
mechanisms of amplifying platelet aggregation, which are required for the
formation of an
arterial or venous thrombus. By inhibiting thrombus formation but not primary
platelet
aggregation, thrombosis can be prevented without increasing hemorrhage.
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
[0039] Compounds of the invention include any product containing annexin
amino acid sequences that have been modified to increase the half life of the
product in
humans or other mammals. Where ''amino acid sequence'' is recited herein to
refer to an
amino acid sequence of a naturally-occurring protein molecule, "amino acid
sequence''
and like terms, such as "polypeptidi:"' or "protein,'' are not meant to limit
the amino acid
sequence to the complete, native amino acid sequence associated with the
recited proteins.
The annexins are a family of homologous phospholipid-binding membrane
proteins, of
which ten represent distinct gene products expressed in mammals (Benz and
Hofinann,
1997). Crystallographic analysis has revealed a common tertiary structure for
all the
family members so far studied, exemplified by annexin V (Huber et al., EMBO
Journal
9:3867 (1990), incorporated herein by reference). The core domain is a concave
discoid
structure that can be closely apposed to phospholipid membranes. It contains
four
subdomains, each consisting of a 70-amino-acid annexin repeat made up of five
a-helices.
The annexins also have a more hydrophilic tail domain that varies in length
and amino
acid sequence among the different annexins. The sequences of genes encoding
annexins
are well known (e.g., Funakoshi et al., Biochemistry 26:8087-8092 (1987)
(annexin V),
incorporated herein by reference).
[0040] Annexin proteins include proteins of the annexin family, , such as
Annexin
II (lipocortin 2, calpactin 1, protein I, p36, chromobindin 8), Annexin III
(lipocortin 3,
PAP-III), Annexin IV (lipocortin 4, endonexin I, protein II, chromobindin 4 ),
Annexin V
(Lipocortin 5, Endonexin 2, VAC-alpha, Anchorin CII, PAP-I), Annexin VI
(Lipocortin 6,
Protein III, Chromobindin 20, p68, p70), Annexin VII (Synexin), Annexin VIII
(VAC-
beta), Annexin XI (CAP-50), and Annexin XIII (ISA),
[0041 ] Annexin IV shares many of the same properties of Annexin V. Like
annexin V, Annexin IV binds to acidic phospholipids membranes in the presence
of
calcium. Annexin IV is a close structural homologue of Annexin V. The sequence
of
i
Annexin IV is known. Hamman et al., Biochem. Biophys. Res. Comm., 156:660-667
(1988). Annexin IV belonds to the annexin family of calcium-dependent
phospholipids
binding proteins. Its functions are still not clearly defined.
[0042] Annexin IV (endonexin) is a 321cDa, calcium-dependent membrane-
binding protein. The translated amino acid sequence of Annexin IV shows the
four
domain structure characteristic of proteins in this class. Annexin IV has 45 -
59% identity
with other members of its family and shares a similar size and exon-intron
organization.
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Isolated from human placenta, Annexin IV encodes a protein that has in vitro
anticoagulant activity and inhibits _ phospholipase A2 activity. Annexin IV is
almost
exclusively expressed in epithelial cells.
[0043] Annexin VIII belonds to the family of CA (2+) dependent phospholipids
binding proteins (annexins) and has high identity to Annexin V (56%).
Hauptmann, et al.,
Eur J Biochem. 1989 Oct 20;185(1):63-71. It was initially isolated as a 2.2 kb
vascular
anticoagulant-beta. Annexin VIII is neither an extracellular protein nor
associated with
the cell surface. It may not play a role in blood coagulation in vivo. Its
physiological, rule
remains unknown. It is expressed at low levels in human placenta and shows
restricted
expression in lung, endothelia and skin, liver and kidney.
[0044] In the present invention, annexin proteins are modified to increase
their
half life in humans or other mammals. In some embodiments, the annexin protein
is
annexin V, annexin IV or annexin VIII. One suitable modification of annexin is
an
increase in its effective size, which prevents loss from the vascular
compartment into the
extravascular compartment and urine, thereby prolonging antithrombotic
activity
following a single injection. Any increase in effective size that maintains a
sufficient
binding affinity with phosphatidylserine is within the scope of the present
invention.
[0045] In one embodiment of the invention, a modiFed annexin contains a
recombinant human annexin protein coupled to polyethylene glycol (PEG) in such
a way
that the modified annexin is capable of performing the function of annexin in
a
phosphatidylserine (PS)-binding assay. The antithrombotic action of the
intravenously
administered annexin-PEG conjugate is prolonged as compared with that of the
free
annexin. The recombinant annexin protein coupled to PEG can be annexin V
protein or
another annexin protein. In one embodiment, the annexin protein is annexin V,
annexin
IV or annexin VIII.
[0046] PEG consists of repeating units of ethylene oxide that terminate in '
hydroxyl groups on either end of a linear or, in some cases, branched chain.
The size and
molecular weight of the coupled PEG chain depend upon the number of ethylene
oxide
units it contains, which can be selected. Por the present invention, any size
of PEG and
number of PEG chains per annexin molecule can be used such that the half life
of the
modified annexin is increased, relative to annexin, while preserving the
function of
binding of the modified molecule to PS. As stated above, sufficient binding
includes
binding that is diminished from that of the unmodified annexin, but still
competitive with
12
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the binding of Gash and factors of the prothrombinase complex and therefore
able to
prevent thrombosis. The optimal molecular weight of the conjugated PEG varies
with the
number of PEG chains. In one embodiment, two PEG molecules of molecular weight
of at
least about 15 kDa each are coupled to each annexin molecule. The PEG
molecules can
be linear or branched. The calcium-dependent binding of annexins to PS is
affected not
only by the size of the coupled PEG molecules, belt also the sites on the
proiein to which
PEG is bound. Optimal selection ensures that desirable properties are
retained. Selection
of PEG attachment sites is facilitated by knowledge of the three-dimensional
structure of
the molecule and by mutational and crystallographic analyses of the
interaction of the
molecule with phospholipid membranes (Campos et al., Biochemistry 37:8004-8008
(1998), incorporated herein by reference).
[0047] In the area of drug delivery, PEG derivatives have been widely used in
covalent attachment (referred to as pegylation) to proteins to enhance
solubility, as well as
to reduce immunogenicity, proteolysis, and kidney clearance. The superior
clinical
efficacy of recombinant products coupled to PEG is well established. For
example, PEG-
interferon alpha-2a administered once weekly is significantly more effective
against
hepatitis C virus than three weekly doses of the free interferon (Heathcote et
al., N. En~l.
J. Med. 343:1673-1680 (2000), incorporated herein by reference). Coupling to
PEG has
been used to prolong the half life of recombinant proteins in vivo (Knauf et
al., J. Biol.
Chem. 266:2796-2804 (1988), incorporated herein by reference), as well as to
prevent the
enzymatic degradation of recombinant proteins and to decrease the
immunogenicity
sometimes observed with homologous products (references in Hermanson,
Bioconjugate
techniques. New York, Academic Press (1996), pp. 173-176, incorporated herein
by
reference).
[0048] In another embodiment of the invention, the modified annexin protein is
a
polymer of annexin proteins that has an increased effective size. It is
believed that the
increase in effective size results in prolonged half life in the vascular
compartment and
prolonged antithrombotic activity. One such modified annexin is a diner of
annexin
proteins. In one embodiment, the diner of annexin is a homodimer of annexin V,
annexin
IV or annexin VIII. In another embodiment, the diner of annexin is a
heterodimer of
annexin V and other annexin protein (e.g., annexin IV or annexin VIII),
annexin IV and
another annexin protein (e.g., annexin V or annexin VIII) or annexin VIII and
another
annexin protein (e.g., annexin V or annexin IV). Another such polymer is the
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heterotetramer of annexin II with p 11, a member of the S 100 family of
calcium-binding
proteins. The binding of an 5100 protein to an annexin increases the affinity
of the
annexin for Ca2+. The annexin homopolymer or heterotetramer can be produced by
bioconjugate methods or recombinant methods, and be administered by itself or
in a PEG-
conjugated form.
[0049] In some embodiments, the modified annexins have increased affinity for
PS. As described in Example 4, a homodimer of human annexin V (DAV) was
prepared
in using well-established methods of recombinant DNA technology. The annexin
molecules of the homodimer are joined through peptide bonds to a flexible
linker (FIG. 1 ).
In some embodiments, the flexible linker contains a sequence of amino acids
flanked by a
glycine and a serine residue at either end to serve as swivels. The linker
preferably
comprises one or more such ''swivels." Preferably, the linker comprises 2
swivels which
may be separated by at least 2 amino acids, more particularly by at least 4
amino acids,
more particularly by at least 6 amino acids, more particularly by at least 8
amino acids,
more particularly by at least 10 amino acids. Preferably, the overall length
of the linker is
5-30 amino acids, 5-20 amino acids, 5-10 amino acids, 10-15 amino acids, or 10-
20 amino
acids. The diner can fold in such a way that the convex surfaces of the
monomer, which
bind Ca2+ and PS, can both gain access to externalized PS. Flexible linkers
are known in
the art, for example, (GGGGS)(n) (n = 3-4), and helical linkers, (EAAAK)(n) (n
= 2-5),
described in Arai, et al., Proteins. 2004 Dec 1;57(4):829-38. As described in
Example II,
the annexin V homodimer out-competes annexin monomer in binding to PS on cell
surfaces (FIG. 2).
[0050] In another embodiment of the invention, recombinant annexin is
expressed
with, or chemically coupled to, another protein such as the Fc portion of
immunoglobulin.
Such expression or coupling increases the effective size of the molecule,
preventing the
loss of annexin from the vascular compartment and prolonging its anticoagulant
action.
[0051] Preferably, a modified annexin protein of the invention is an isolated
modified annexin protein. The modified annexin protein can contain amlexin II,
annexin
IV, annexin V, or annexin VIII. IIl SOllle embOd1I11e11tS, the protein is
modified human
annexin. In some embodiments, the modified annexin contains recombinant human
annexin. According to the present invention, an isolated or biologically pure
protein is a
protein that has been removed from its natural environment. As such,
"isolated" and
"biologically pure" do not necessarily reflect the extent to which the protein
has been
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purified. An isolated modified annexin protein of the present invention can be
obtained
from its natural source, can be produced using recombinant DNA technology, or
can be
produced by chemical synthesis. As used herein, an isolated modified annexin
protein can
be a full-length modified protein or any homologue of such a protein. It can
also be (e.g.,
for a pegylated protein) a modified full-length protein or a modified
homologue of such a
protein.
[0052] The minimal size of a protein homologue of the present invention is a
size
sufficient to be encoded by a nucleic acid molecule capable of forming a
stable hybrid
with the complementary sequence of a nucleic acid molecule encoding the
corresponding
natural protein. As such, the size of the nucleic acid molecule encoding such
a protein
homologue is dependent on nucleic acid composition and percent homology
between the
nucleic acid molecule and complementary sequence as well as upon hybridization
conditions per se (e.g., temperature, salt concentration, and formamide
concentration).
The minimal size of such nucleic acid molecules is typically at least about 12
to about 15
nucleotides in length if the nucleic acid molecules are GC-rich and at least
about 15 to
about 17 bases in length if they are AT-rich. As such, the minimal size of a
nucleic acid
molecule used to encode a protein homologue of the present invention is from
about 12 to
about 18 nucleotides in length. There is no limit on the maximal size of such
a nucleic
acid molecule in that the nucleic acid molecule can include a portion of a
gene, an entire
gene, or multiple genes or portions thereof. Similarly, the minimal size of an
annexin
protein homologue or a modified annexin protein homologue of the present
invention is
from about 4 to about 6 amino acids in length, with sizes depending on whether
a full-
length, multivalent (i.e., fusion protein having more than one domain, each of
which has a
function) protein, or functional portions of such proteins are desired.
Annexin and
modified annexin homologues of the present invention preferably have activity
col~l~esponding to the natural subunit, such as being able to perform the
activity of the
annexin protein in preventing thrombus formation.
[0053] Annexin protein and modified aI111ex1I1 h0111010gLleS Call be the
result of
natural allelic variation or natural 111L1tat1011. The protein h0111010gLleS
Of the present
invention can also be produced using techniques known in the al-t, including,
but not
limited to, direct modifications to the protein or modifications to the gene
encoding the
protein using, for example, classic or recombinant DNA techniques to effect
random or
targeted mutagenesis.
CA 02559167 2006-09-11
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[0054] Also included is a modified annexin protein containing an amino acid
sequence that is at least about 75%, more preferably at least about 80%, more
preferably at
least about 85%, more preferably at least about 90%, more preferably at least
about 95%,
and most preferably at least about 98% identical to amino acid sequence SEQ ID
N0:3,
SEQ .ID N0:6, SEQ ID NO:12, SEQ ID NO:15, or a protein encoded by an allelic
variant
of a nucleic acid molecule encoding a protein containing any of these
sequences. Also
included is a modified annexin protein comprising more than one of SEQ ID
NO:3, SEQ
ID N0:6, SEQ ID N0:12, SEQ ID NO:15; for example, a protein comprising SEQ ID
N0:3 and SEQ ID N0:12 and separated by a linker. Methods to determine percent
identities between amino acid sequences and between nucleic acid sequences are
known to
those skilled in the art. Methods to determine percent identities between
sequences
include computer programs such as the GCG~'' Wisconsin paclcageTM (available
from
Accelrys Corporation), the DNAsisl~M program (available from Hitachi Software,
San
Bruno, CA), the Vector NTI Suite (available from Informax, Inc., North
Bethesda, MD),
or the BLAST software available on the NCBI website.
[0055] In one embodiment, a modified annexin protein includes an amino acid
sequence of at least about 5 amino acids, preferably at least about 50 amino
acids, more
preferably at least about 100 amino acids, more preferably at least about 200
amino acids,
more preferably at least about 250 amino acids, more preferably at least about
275 amino
acids, more preferably at least about 300 amino acids, and most preferably at
least about
319 amino acids or the full-length annexin protein, whichever is shorter. In
another
embodiment, annexin proteins contain full-length proteins, i.e., proteins
encoded by full-
length coding regions, or post-translationally modified proteins thereof, such
as mature
proteins fr0111 Whlch lllltlatlng methionine and/or signal sequences or "pro"
sequences
have been removed.
[0056] A fragment of a modified annexin protein of the present invention
preferably contains at least about 5 amino acids, more preferably at least
about 10 amino
acids, more preferably at least about 15 amino acids, more preferably at least
about 20
amino acids, more preferably at least about 25 amino acids, more preferably at
least about
30 amino acids, more preferably at least about 35 a111111o acids, more
preferably at least
about 40 amino acids, more preferably at least about 45 amino acids, more
preferably at
least about 50 amino acids, more preferably at least about 55 amino acids,
more preferably
at least about 60 amino acids, more preferably at least about 65 amino acids,
more
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preferably at least about 70 amino acids, more preferably at least about 75
amino acids,
more preferably at least about 80 amino acids, more preferably at least about
85 amino
acids, more preferably at least about 90 amino acids, more preferably at least
about 95
amino acids, and even more preferably at least about 100 amino acids in
length.
[0057] In one embodiment, an isolated modified annexin protein of the present
invention contains a protein encoded by a nucleic acid molecule having the
nucleic acid
sequence SEQ ID N0:4. Alternatively, the modified annexin protein contains a
protein
encoded by a nucleic acid molecule having the nucleic acid sequence SEQ ID
NO:l or by
an allelic variant of a nucleic acid molecule having this sequence.
Alternatively, the
modified annexin protein contains more than one protein sequence encoded by a
nucleic
acid molecule having the nucleic acid sequence SEQ ID NO:l or by an allelic
variant of a
nucleic acid molecule having this sequence.
[0058] In one embodiment, an isolated modified annexin protein of the present
invention contains a protein encoded by a nucleic acid molecule having the
nucleic acid
sequence SEQ ID N0:10 or by an allelic variant of a nucleic acid molecule
having this
sequence. Alternatively, the modified annexin protein contains more than one
protein
sequence encoded by a nucleic acid molecule having the nucleic acid sequence
SEQ ID
NO:10 or by an allelic variant of a nucleic acid molecule having this sequence
(e.g., SEQ
ID NO: 12-linker-SEQ ID NO: 12).
[0059] In another embodiment, an isolated modified annexin protein of the
present invention is a modified protein encoded by a nucleic acid molecule
having the
nucleic acid sequence SEQ ID N0:13 or by an allelic variant of a nucleic acid
molecule
having this sequence. Alternatively, the modified annexin protein contains
more than one
protein sequence encoded by a nucleic acid molecule having the nucleic acid
sequence
SEQ ID N0:13 or by an allelic variant of a nucleic acid molecule having this
sequence
(e.g., SEQ ID NO: 15-linker-SEQ ID NO: 15).
[0060] In another embodiment, an isolated modiFed annexin protein of the
present invention is a modified protein which contains a protein encoded by a
nucleic acid
molecule having the nucleic acid sequence SEQ ID NO:1 and a protein encoded by
a
nucleic acid molecule having the nucleic acid sequence SEQ ID NO:10, or by
allelic
variants of these nucleic acid molecules (e.g., SEQ ID NO: 3-linker-SEQ ID
N0:12 or
SEQ ID N0:12-linker-SEQ ID N0:3).
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[0061] In another embodiment, an isolated modified annexin protein of the
present invention is a modified protein which contains a protein encoded by a
nucleic acid
molecule having the nucleic acid sequence SEQ ID NO:1 and a protein encoded by
a
nucleic acid molecule having the nucleic acid sequence SEQ ID N0:13, or by
allelic
variants of these nucleic acid molecules (e.g., SEQ ID N0:3-linker-SEQ ID
NO:15 or
SEQ ID NO:15-linker-SEQ ID N0:3).
[0062] In another embodiment, an isolated modified annexin protein of the
present invention is a modified protein which contains a protein encoded by a
nucleic acid
molecule having the nucleic acid sequence SEQ ID NO:10 and a protein encoded
by a
nucleic acid molecule having the nucleic acid sequence SEQ ID N0:13, or by
allelic
variants of these nucleic acid molecules (e.g., SEQ ID NO:12-linker-SEQ ID
NO:15 or
SEQ ID NO:15-linker-SEQ ID N0:12).
[0063] One embodiment of the present invention includes a non-native modified
annexin protein encoded by a nucleic acid molecule that hybridizes under
stringent
hybridization conditions with an annexin gene. As used herein, stringent
hybridization
conditions refer to standard hybridization conditions under which nucleic acid
molecules,
including oligonucleotides, are used to identify molecules having similar
nucleic acid
sequences. Such standard conditions are disclosed, for example, in Sambroolc
et al.,
Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989),
incorporated herein by reference. Stringent hybridization conditions typically
permit
isolation of nucleic acid molecules having at least about 70% nucleic acid
sequence
identity with the nucleic acid molecule being used to probe in the
hybridization reaction.
Formulae to calculate the appropriate hybridization and wash conditions to
achieve
hybridization permitting 30% or less mismatch of nucleotides are disclosed,
for example,
in Meinkoth et al., Anal. Biochem. 138:267-284 (1984), incorporated herein by
reference.
In some embodiments, hybridization conditions will permit isolation of nucleic
acid
molecules having at least about 80% nucleic acid sequence identity with the
nucleic acid
molecule being used to probe. In other embodiments, hybridization conditions
will permit
isolation of nucleic acid molecules having at least about 90% nucleic acid
sequence
identity with the nucleic acid molecule being used to probe. In still other
embodiments,
hybridization conditions will permit isolation of nucleic acid molecules
having at least
about 95% nucleic acid sequence identity with the nucleic acid molecule being
used to
probe.
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[0064] A modified annexin protein includes a protein encoded by a nucleic acid
molecule that is at least about 50 nucleotides and that hybridizes under
conditions that
preferably allow about 20% base pair mismatch, more preferably under
conditions that
allow about 15% base pair mismatch, more preferably under conditions that
allow about
10% base pair mismatch, more preferably under conditions that allow about 5%
base pair
mismatch, and even more preferably under conditions that allow about 2% base
pair
mismatch with a nucleic acid molecule selected from the group consisting of
SEQ ID
NO:1, SEQ ID N0:4, SEQ ID NO:10, SEQ ID N0:13, or a complement of any of these
nucleic acid molecules.
[0065] As used herein, an annexin gene includes all nucleic acid sequences
related
to a natural annexin gene such as regulatory regions that control production
of the annexin
protein encoded by that gene (such as, but not limited to, transcription,
translation or post-
translation control regions) as well as the coding region itself. In one
embodiment, an
annexin gene includes the nucleic acid sequence SEQ ID NO:1. In another
embodiment,
an annexin gene includes the nucleic acid sequence SEQ ID NO:10. In another
embodiment, an annexin gene includes the nucleic acid sequence SEQ ID N0:13.
It
should be noted that since nucleic acid sequencing technology is not entirely
ewor-free,
SEQ ID NO:l (as well as other sequences presented herein), at best, represents
an
apparent nucleic acid sequence of the nucleic acid molecule encoding an
annexin protein
of the present invention.
[0066] In another embodiment, an annexin gene can be an allelic variant that
includes a similar but not identical sequence to SEQ ID NO:l . In another
embodiment, an
annexin gene can be an allelic variant that includes a similar but IlOt
identical sequence to
SEQ ID NO:10. In another embodiment, an annexin gene can be an allelic variant
that
includes a similar but not identical sequence to SEQ ID N0:13. An allelic
variant of an
annexin gene including SEQ ID NO:1 is a gene that occurs at essentially the
same locus
(or loci) in the genome as the gene including SEQ ID NO:1, but which, due to
natural
variations caused by, for example, mutation or recombination, has a similar
but not
identical sequence. Allelic variants typically encode proteins having similar
activity to
that of the protein encoded by the gene to which they are being compared.
Allelic variants
can also comprise alterations in the 5' or 3' untranslated regions of the gene
(e.g., in
regulatory control regions). Allelic variants are well known to those skilled
in the art and
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WO 2005/086955 PCT/US2005/008193
would be expected to be found within a given human since the genome is diploid
and/or
among a population comprising two or more humans.
[0067] An isolated modified annexin protein of the present invention can be
obtained from its natural source, can be produced using recombinant DNA
technology, or
can be produced by chemical synthesis. As used herein, an isolated modified
annexin
protein can contain a full-length protein or any homologue of such a protein.
Examples of
annexin and modified annexin homologues include annexin and modified annexin
proteins
in which amino acids have been deleted (e.g., a truncated version of the
protein, such as a
peptide or by a protein splicing reaction when an intron has been removed or
two exons
are joined), inserted, inverted, substituted and/or derivatized (e.g., by
glycosylation,
phosphorylation, acetylation, methylation, myristylation, prenylation,
palmitoylation,
amidation and/or addition of glycerophosphatidyl inositol) such that the
homologue
includes at least one epitope capable of eliciting an immune response against
an annexin
protein. That is, when the homologue is administered to an animal as an
immunogen,
using techniques known to those skilled in the art, the animal will produce a
humoral
and/or cellular immune response against at least one epitope of an annexin
protein.
AnneXin and modified annexin homologues can also be selected by their ability
to
selectively bind to immune serum. Methods to measure such activities are
disclosed
herein. Annexin and modified annexin homologues also include those proteins
that are
capable of performing the function of native annexin in a functional assay;
that is, are
capable of binding to phosphatidylserine or to activated platelets or
exhibiting
antithrombotic activity. Methods for such assays are described in the Examples
section
and elsewhere herein.
[0068] A modified annexin protein of the present invention may be identified
by
its ability to perform the function of an annexin protein in a functional
assay. The phrase
"capable of performing the function of that in a functional assay" means that
the protein or
modified protein has at least about 10% of the activity of the Ilatllral
pl'Ote111 Ill the
functional assay. In other embodiments, it has at least about 20% of the
activity of the
natural protein in the functional assay. In other embodiments, it has at least
about 30% of
the activity of the natural protein in the functional assay. In other
embodiments, it has at
least about 40% of the activity of the natural protein in the f1111CtlOllal
assay. In other
embodiments, it has at least aboLlt 50% of the activity of the natural protein
111 the
functional assay. In other embodiments, the protein or modified protein has at
least about
CA 02559167 2006-09-11
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60% of the activity of the natural protein in the functional assay. In still
other
embodiments, the protein or modified protein has at least about 70% of the
activity of the
natural protein in the functional assay. In yet other embodiments, the protein
or modified
protein has at least about 80% of the activity of the natural protein in the
functional assay.
In other embodiments, the protein or modified protein has at least about 90%
of the
activity bf the natural protein in the functional assay. Examples of
functional assays are
described herein. e'
[0069] An isolated protein of the present invention can be produced in a
variety of
ways, including recovering such a protein from a bacterium and producing such
a protein
recombinantly. One embodiment of the present invention is a method to produce
an
isolated modified annexin protein of the present invention using recombinant
DNA
technology. Such a method includes the steps of (a) culturing a recombinant
cell
containing a nucleic acid molecule encoding a modified annexin protein of the
present
invention to produce the protein and (b) recovering the protein therefrom.
Details on
producing recombinant cells and culturing thereof are presented below. The
phrase
"recovering the protein" refers simply to collecting the whole fermentation
medium
containing the protein and need not nnply additional steps of separation or
purification.
Proteins of the present invention can be purified using a variety of standard
protein
purification techniques.
[0070] Isolated proteins of the present invention are preferably retrieved in
"substantially pure" f01'111. As used herein, ''substantially pure'' refers to
a purity that
allows for the effective use of the protein in a functional assay.
Modified Annexin Nucleic Acid Molecules or Genes
[0071] Another embodiment of the present invention is an isolated nucleic acid
molecule capable of hybridizing under stringent conditions with a gene
ellCOdlllg a
modified annexin protein such as a homodimer of annexin V, a hOlllOdllllel' of
annexin IV,
a homodimer of annexin VIII, a heterodimer of annexin V and amiexin VIII, a
heterodimer
of annexin V and annexin IV or a heterodimer of annexin IV and annexin VIII.
Such a
nucleic acid molecule is also referred to herein as a modified annexin nucleic
acid
molecule. Included is an isolated nucleic acid molecule that hybridizes under
stringent
conditions with a modified annexin gene. The characteristics of such genes are
disclosed
herein. In accordance with the present invention, an isolated nucleic acid
molecule is a
nucleic acid molecule that has been removed iiom its natural milieu (i.e.,
that has been
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subject to human manipulation). As such, "isolated" does not reflect the
extent to which
the nucleic acid molecule has been purified. An isolated nucleic acid molecule
can
include DNA, RNA, or derivatives of either DNA or RNA.
[0072] As stated above, a modified annexin gene includes all nucleic acid
sequences related to a natural annexin gene, such as regulatory regions that
control
production of an annexin protein encoded by that gene (such as, but not
limited to,
transcriptional, translational, or post-translational control regions) as well
as the coding
region itself. A nucleic acid molecule of the present invention can be an
isolated modified
annexin nucleic acid molecule or a homologue thereof. A nucleic acid molecule
of the
present invention can include one or more regulatory regions, full-length or
partial coding
regions, or combinations thereof. The minimal size of a modified annexin
nucleic acid
molecule of the present invention is the minimal size capable of forming a
stable hybrid
under stringent hybridization conditions with a corresponding natural gene.
Annexin
nucleic acid molecules can also include a nucleic acid molecule encoding a
hybrid protein,
a fusion protein, a multivalent protein or a truncation fragment.
[0073] An isolated nucleic acid molecule of the present invention can be
obtained
from its natural source either as an entire (i.e., complete) gene or a portion
thereof capable
of forming a stable hybrid with that gene. As used herein, the phrase "at
least a portion
of an entity refers to an amount of the entity that is at least sufficient to
have the
functional aspects of that entity. For example, at least a portion of a
nucleic acid
sequence, as used herein, is an amount of a nucleic acid sequence capable of
forming a
stable hybrid with the corresponding gene under stringent hybridization
conditions.
[0074] An isolated nucleic acid molecule of the present invention can also be
produced using recombinant DNA technology (e.g., polymerase chain reaction
(PCR)
amplification, cloning. etc.) or chemical synthesis. Isolated modified annexin
nucleic acid
molecules include natural nucleic acid molecules and homologues thereof,
including, but
IlOt limited to, natural allelic variants and modified nucleic acid
111o1ecL11eS 111 Whlch
nucleotides have been inserted, deleted, substituted, and/or inverted in such
a manner that
such modifications do not substantially interfere with the ability of the
nucleic acid
molecule to encode an annexin protein of the present 111Ve11t1011 oI' to form
stable hybrids
under stringent conditions with natural nucleic acid molecule isolates.
[0075] A modified annexin nucleic acid molecule homologue can be produced
using a number of methods known to those skilled in the alt (see, e.g.,
Sambroole et al.,
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I 989). For example, nucleic acid molecules can be modified using a variety of
techniques
including, but not limited to, classic mutagenesis techniques and recombinant
DNA
techniques, such as site-directed mutagenesis, chemical treatment of a nucleic
acid
molecule to induce mutations, restriction enzyme cleavage of a nucleic acid
fragment,
ligation of nucleic acid fragments, polymerase chain reaction (PCR)
amplification and/or
mutagenesis of selected regions of a nucleic acid sequence, synthesis of
oligonucleotide
mixtures, and ligation of mixture groups to "build" a mixture of nucleic acid
molecules
and combinations thereof. Nucleic acid molecule homologues can be selected
from a
mixture of modified nucleic acids by screening for the function of the protein
encoded by
the nucleic acid (e.g., the ability of a homologue to elicit an immune
response against an
annexin protein and/or to function in a clotting assay, or other functional
assay), and/or by
hybridization with isolated annexin-encoding nucleic acids under stringent
conditions.
[0076] An isolated modified annexin nucleic acid molecule of the present
invention can include a nucleic acid sequence that encodes at least one
modified annexin
protein of the present invention, examples of such proteins being disclosed
herein.
Although the phrase "nucleic acid molecule" primarily refers to the physical
nucleic acid
molecule and the phrase "nucleic acid sequence" primarily refers to the
sequence of
nucleotides on the nucleic acid molecule, the two phrases can be used
interchangeably,
especially with respect to a nucleic acid molecule, or a nucleic acid
sequence, being
capable of encoding a modified annexin protein.
[0077] One embodiment of the present invention is a modified annexin nucleic
acid molecule that is capable of hybridizing under stringent conditions to a
nucleic acid
strand that encodes at least a portion of a modified annexin protein or a
homologue thereof
or to the complement of such a nucleic acid strand. A nucleic acid sequence
complement
of any nucleic acid sequence of the present invention refers to the nucleic
acid sequence of
the nucleic acid strand that is complementary to (i.e., can form a complete
double helix
with) the strand for which the sequence is cited. It is to be noted that a
double-stranded
nucleic acid molecule of the present invention for which a nucleic acid
sequence has been
determined for one strand, that is represented by a SEQ ID NO, also comprises
a
complementary strand having a sequence that is a complement of that SEQ ID NO.
As
such, nucleic acid molecules of the present invention, which can be either
double-stranded
or single-stranded, include those nucleic acid molecules that form stable
hybrids under
stringent hybridization conditions with either a given SEQ ID NO denoted
herein and/or
23
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with the complement of that SEQ ID NO, which may or may not be denoted herein.
Methods to deduce a complementary sequence are known to those skilled in the
art.
Included is a modified annexin nucleic acid molecule that includes a nucleic
acid sequence
having at least about 65 percent, preferably at least about 70 percent, more
preferably at
least about 75 percent, more preferably at least about 80 percent, more
preferably at least
about 85 percent, more preferably at least about 90 percent and even more
preferably at
least about 95 percent homology with the corresponding regions) of the nucleic
acid
sequence encoding at least a portion of a modified annexin protein. Included
is a modified
annexin nucleic acid molecule capable of encoding a homodimer of an annexin
protein or
homologue thereof.
[0078] Annexin nucleic acid molecules include SEQ ID N0:4 and an allelic
variants of SEQ ID N0:4, SEQ ID NO:1 and an allelic variants of SEQ ID NO:1,
SEQ ID
NO:10 and an allelic variants of SEQ ID NO:10; and SEQ ID N0:13 and an allelic
variants of SEQ ID NO:13.
[0079] Knowing a nucleic acid molecule of a modified annexin protein of the
present invention allows one skilled in the art to make copies of that nucleic
acid molecule
as well as to obtain a nucleic acid molecule including additional portions of
annexin
protein-encoding genes (e.g., nucleic acid molecules that include the
translation stal-t site
and/or transcription and/or translation control regions), and/or annexin
nucleic acid
molecule homologues. KIlOWlllg a portion of an amino acid sequence of an
annexin
protein of the present invention allows one skilled in the al-t to clone
nucleic 'acid
sequences encoding such an annexin protein. In addition, a desired modified
annexin
nucleic acid molecule can be obtained in a variety of ways including screening
appropriate
expression libraries with antibodies that bind to annexin proteins of the
present invention;
traditional cloning techniques using oligonucleotide probes of the present
invention to
screen appropriate libraries or DNA; and PCR amplification of appropriate
libraries, or
RNA or DNA using oligonucleotide primers of the present invention (genomic
and/or
cDNA libraries can be used).
[0080] The present invention also includes nucleic acid molecules that are
oligonucleotides capable of hybridizing, under stringent conditions, with
complementary
regions of other, preferably longer, nucleic acid molecules of the present
invention that
encode at least a portion of a modified annexin protein. Oligonucleotides of
the present
invention can be RNA, DNA, or derivatives of either. The llllllllllal size of
such
24
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oligonucleotides is the size required to form a stable hybrid between a given
oligonucleotide and the complementary sequence on another nucleic acid
molecule of the
present invention. Minimal size characteristics are disclosed herein. The size
of the
oligonucleotide must also be sufficient for the use of the oligonucleotide in
accordance
with the present invention. Oligonucleotides of the present invention can be
used in a
variety of applications including, but not limited to, as probes to identify
additional nucleic
acid molecules, as primers to amplify or extend nucleic acid molecules or in
therapeutic
applications to modulate modified annexin production. Such therapeutic
applications
include the use of such oligonucleotides in, for example, antisense-, triplex
formation-,
ribozyme- and/or RNA drug-based technologies. The present invention,
therefore,
includes such oligonucleotides and methods to modulate the production of
modified
annexin proteins by use~of one or more of such technologies.
Natural, Wild-Type Bacterial Cells and Recombinant Molecules and Cells
[0081] The present invention also includes a recombinant vector, which
includes a
modified annexin nucleic acid molecule of the present invention inserted into
any vector
capable of delivering the nucleic acid molecule into a host cell. Such a
vector contains
heterologous nucleic acid sequences, that is, nucleic acid sequences that are
not naturally
found adjacent to modified annexin nucleic acid molecules of the present
invention. The
vector can be either RNA or DNA, either prokaryotic or eukaryotic, and
typically is a
virus or a plasmid. Recombinant vectors can be used in the cloning,
sequencing, and/or
otherwise manipulating of modified annexin nucleic acid molecules of the
present
invention. One type of recombinant vector, herein refel'red to as a
recombinant molecule
and described in more detail below, can be used in the expression of nucleic
acid
molecules of the present invention. Some recombinant vectors are capable of
replicating
in the transformed cell. Nucleic acid molecules to include in recombinant
vectors of the
present invention are disclosed herein.
[0082] As heretofore disclosed, one embodiment of the present lllVellt1011 15
a
method t0 produce a modified a1111eX111 prOtelll Of the present lnVentlOll by
CLlltLlrlllg a cell
capable of expressing the protein under conditions effective to produce the
protein, and
recovering the protein. In an alternative embodiment, the method includes
producing an
annexin protein by culturing a cell capable of expressing the protein under
conditions
effective to produce the annexin protein, recovering the protein, and
modifying the protein
by coupling it to an agent that increases its effective size.
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[0083] In one embodiment, the cell to culture is a natural bacterial cell, and
modified annexin is isolated from these cells. In another embodiment, a cell
to culture is a
recombinant cell that is capable of expressing the modified annexin protein,
the
recombinant cell being produced by transforming a host cell with one or more
nucleic acid
molecules of the present lI1Ve11t10I1. Transformation of a nucleic acid
molecule into a cell
can be accomplished by any method by which a nucleic acid molecule can be
inserted into
the cell. Transformation techniques include, but are not limited-to,
transfection,
electroporation, microinjection, lipofection, adsorption, and protoplast
fusion. A
recombinant cell may remain unicellular or may grow into a tissue, organ or a
multicellular organism. Transformed nucleic acid molecules of the present
invention can
remain extrachromosomal or can integrate into one or more sites within a
chromosome of
the transformed (i.e., recombinant) cell in such a manner that their ability
to be expressed
is retained. Nucleic acid molecules with which to transform a host cell are
disclosed
herein.
[0084] Suitable host cells to transform include any cell that can be
transformed
and that can express the introduced modified annexin protein. Such cells are,
therefore,
capable of producing modified annexin proteins of the present invention after
being
transformed with at least one nucleic acid molecule of the present invention.
Host cells
can be either untransformed cells or cells that are already transformed with
at least one
nucleic acid molecule. Suitable host cells of the present invention can
include bacterial,
fungal (including yeast), insect, animal, and plant cells. Host cells include
bacterial cells,
with E. colt cells being particularly preferred. Alternative host cells are
untransformed
(wild-type) bacterial cells producing cognate modified annexin proteins,
including
attenuated strains with reduced pathogenicity, as appropriate.
[0085] A recombinant cell is preferably produced by transforming a host cell
with
one or more recombinant molecules, each comprising one or more nucleic acid
molecules
of the present invention operatively linked to an expression vector containing
one or more
transcription control sequences. The phrase ''operatively linked" refers to
insertion of a
nucleic acid molecule into an expression vector in a manner such that the
molecule is able
to be expressed when transformed into a host cell. As used herein, an
expression vector is
a DNA or RNA vector that is capable of transforming a host cell and of
effecting
expression of a specified nucleic acid molecule. Preferably, the expression
vector is also
capable of replicating within the host cell. Expression vectors can be either
prokaryotic or
26
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eulcaryotic, and are typically, viruses or plasmids. Expression vectors of the
present
invention include any vectors that function (i.e., direct gene expression) in
recombinant
cells of the present invention, including in bacterial, fungal, insect,
animal, and/or plant
cells. As such, nucleic acid molecules of the present invention can be
operatively linked
to expression vectors containing regulatory sequences such as promoters,
operators,
repressors, enhancers, termination sequences, origins of replication, and
other regulatory
sequences that are compatible with the recombinant cell and that control the
expression of
nucleic acid molecules of the present invention. As used herein, a
transcription control
sequence includes a sequence that is capable of controlling the initiation,
elongation, and
termination of transcription. Particularly important transcription control
sequences are
those that control transcription initiation, such as promoter, enhances,
operator and
repressor sequences. Suitable transcription control sequences include any
transcription
control sequence that can function in at least one of the recombinant cells of
the present
invention. A variety of such transcription control sequences are known to the
art.
Transcription control sequences include those which function in bacterial,
yeast, insect and
mammalian cells, such as, but not limited to, tac, lac, tzp, trc, oxy-pro,
omp/lpp, rrnB,
bacteriophage lambda (~,) (such as 7~p~ and 7~p~z and fusions that include
such promoters),
bacteriophage T7, T7lac, bacteriophage T3, bacteriophage SP6, bacteriophage
SPOT,
metallothionein, alpha mating factor, Pichia alcohol oxidase, alphavirus
subgenomic
promoters (such as Sindbis virus subgenomic promoters), baculovirus, Heliothis
zea insect
virus, vaccinia virus, herpesvirus, poxvirus, adenovirus, simian virus 40,
retrovirus actin,
retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and
nitrate
transcription control sequences as well as other sequences capable of
controlling gene
expression in prokaryotic or eukaryotic cells. Additional suitable
transcription control
sequences include tissue-specific promoters and enhancers as well as
lymphokine-
inducible promoters (e.g., promoters inducible by interferons or
interleulcins).
Transcription control sequences of the present invention can also include
naturally
occurring transcription control sequences naturally associated with a DNA
sequence
encoding an annexin protein. One transcription control sequence is the I~ozak
strong
promotor and initiation sequence.
[0086] Expression vectors of the present invention may also contain secretory
signals (i.e., signal segment nucleic acid sequences) to enable an expressed
annexin
protein to be secreted from the cell that produces the protein. Suitable
signal segments
27
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include an annexin protein signal segment or any heterologous signal segment
capable of
directing the secretion of an annexin protein, including fusion proteins, of
the present
invention. Signal segments include, but are not limited to, tissue plasminogen
activator (t-
PA), interferon, interleukin, growth hormone, histocompatibility and viral
envelope
glycoprotein signal segments.
[0087] Expression vectors of the present invention may also contain fusion
sequences which lead to the expression of inserted nucleic acid molecules of
the present
invention as fusion proteins. Inclusion of a fusion sequence as part of a
modified annexin
nucleic acid molecule of the present invention can enhance the stability
during production,
storage and/or use of the protein encoded by the nucleic acid molecule.
Furthermore, a
fusion segment can function as a tool to simplify purification of a modified
annexin
protein, such as to enable purification of tile resultant fusion protein using
affinity
chromatography. One fusion segment that can be used for protein purification
is the 8-
amino acid peptide sequence asp-tyr-lys-asp-asp-asp-asp-lys (SEQ ID NO:9).
[0088] A suitable fusion segment can be a domain of any size that has the
desired
function (e.g., increased stability and/or purification tool). It is within
the scope of the
present invention to use one or more fusion segments. Fusion segments can be
joined to
amino and/or carboxyl termini of an annexin protein. Another type of fusion
protein is a
fusion protein wherein the fusion segment connects two or more annexin
proteins or
modified annexin proteins. Linkages between fusion segments and annexin
proteins can
be constructed to be susceptible to cleavage to enable straightforward
recovery of the
annexin ol: modified annexin proteins. Fusion proteins are preferably produced
by
culturing a recombinant cell transformed with a fusion nucleic acid sequence
that encodes
a protein including the fusion segment attached to either the carboxyl and/or
amino
terminal end of an annexin protein.
[0089] A recombinant molecule of the present invention is a molecule that can
include at least one of any nucleic acid molecule heretofore described
operatively linked to
at least one of any transcription control sequence capable of effectively
regulating
expression of the nucleic acid molecules in the cell to be transformed. A
recombinant
molecule includes one or more nucleic acid molecules of the present invention,
including
those that encode one or more modified annexin proteins. Recombinant molecules
of the
present 111Ve11t1011 alld their prOdL1Ct1011 al'e described in the Examples
section. Similarly, a
recombinant cell includes one or more nucleic acid molecules of the present
invention,
28
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WO 2005/086955 PCT/US2005/008193
with those that encode one or more annexin proteins. Recombinant cells of the
present
invention include those disclosed in the Examples section.
[0090] It may be appreciated by one skilled in the art that use of recombinant
DNA technologies can improve expression of transformed nucleic acid molecules
by
manipulating, for example, the number of copies of the nucleic acid molecules
within a
host cell, the efficiency with which those nucleic acid molecules are
transcribed, the
efficiency with which the resultant transcripts are translated, and the
efficiency of post-
translational modifications. Recombinant techniques useful for increasing the
expression
of nucleic acid molecules of the present invention include, but are not
limited to,
operatively linking nucleic acid molecules to high-copy number plasmids,
integration of
the nucleic acid molecules into one or more host cell chromosomes, addition of
vector
stability sequences to plasmids, substitutions or modifications of
transcription control
signals (e.g., promoters, operators, enhancers), substitutions or
modifications of
translational control signals (e.g., ribosome binding sites, Shine-Dalgarno
sequences),
modification of nucleic acid molecules of the present invention to colTespond
to the codon
usage of the host cell, deletion of sequences that destabilize transcripts,
and use of control
signals that temporally separate recombinant cell growth from recombinant
protein
production during fermentation. The activity of an expressed recombinant
protein of the
present invention may be improved by fragmenting, modifying, or derivatizing
the
resultant protein.
[0091] In accordance with the present invention, recombinant cells can be used
to
produce annexin or modified annexin proteins of the present invention by
culturing such
cells under conditions effective to produce such a protein, and recovering the
protein.
Effective conditions to produce a protein include, but are not limited to,
appropriate
media, bioreactor, temperature, pH and oxygen conditions that permit protein
prOdL1Ct1011.
An appropriate, or effective, medium refers t0 ally medl11111 111 WhlCh a Cell
Of the present
invention, when cultured, is capable of producing an annexin or modified
annexin protein.
Such a medium is typically an aqueous medium comprising assimilable
carbohydrate,
nitrogen and phosphate sources, as well as appropriate salts, minerals, metals
and other
nutrients, such as vitamins. The medium may COI11pr1Se Co111pleX, nutrients or
may be a
defined minimal medIL1I11.
[0092] Cells of the present lIlVeI1t10I1 Call be cultured 111 COI1Ve11t1011a1
fe1'Illelltatloll
bioreactors, which include, belt are not limited to, batch, fed-batch, cell
recycle, and
29
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continuous fermentors. Culturing can also be conducted in shake flash, test
tubes,
microtiter dishes, and petri plates. Culturing is carried out at a
temperature, pH and
oxygen content appropriate for the recombinant cell. Such culturing conditions
are well
within the expertise of one of ordinary skill in the aut.
[0093] Depending on the vector and host system used for production, resultant
annexin proteins may either remain within the recombinant cell; be secreted
into the
fermentation medium; be secreted into a space between two cellular membranes,
such as
the periplasmic space in E. c~li; or be retained on the outer surface of a
cell or viral
membrane. Methods to purify such proteins are disclosed in the Examples
section.
Antibodies
[0094] The present invention also includes isolated anti-modified annexin
antibodies and their use. An anti-modified annexin antibody is an antibody
capable of
selectively binding to a modified annexin protein. Isolated antibodies are
antibodies that
have been removed from their natural milieu. The term "isolated" does not
refer to the
state of purity of such antibodies. As such, isolated antibodies can include
anti-sera
containing such antibodies, or antibodies that have been purified to varying
degrees. As
used herein, the term "selectively binds to" refers to the ability of such
antibodies to
preferentially bind to the protein against which the antibody was raised
(i.e., to be able to
distinguish that protein from unrelated components in a mixture). Binding
affinities,
commonly expressed as equilibrium association constants, typically range from
about 103
M-1 to about 1012 M-~. Binding can be measured using a variety of methods
known to
those skilled in the art including innnunoblot assays, immunoprecipitation
assays,
radioinnnunoassays, enzyme immunoassays (e.g., ELISA), immunofluorescent
antibody
assays and immunoelectron microscopy; see, e.g., Sambrook et al., 1989.
[0095] Antibodies of the present invention can be either polyclonal or
monoclonal
antibodies. Antibodies of the present invention include functional equivalents
such as
antibody fragments and genetically-engineered antibodies, including single
chain
antibodies, that are capable of selectively binding to at least one of the
epitopes of the
protein used to obtain the antibodies. Antibodies of the present invention
also include
chimeric antibodies that can bind to more than one epitope. Antibodies are
raised in
response to proteins that are encoded, at least in part, by a modified annexin
nucleic acid
molecule of the present invention.
CA 02559167 2006-09-11
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[0096] Anti-modified annexin antibodies of the present invention include
antibodies raised in an animal administered a modified annexin. Anti-modified
annexin
antibodies of the present invention also include antibodies raised in an
animal against one
or more modified annexin proteins of the present invention that are then
recovered from
the cell using techniques known to those skilled in the art. Yet additional
antibodies of the
present invention are produced recombinantly using techniques as heretofore
disclosed for
modified annexin proteins of the present invention. Antibodies produced
against defined
proteins can be advantageous because such antibodies are not substantially
contaminated
with antibodies against other substances that might otherwise cause
interference in a
diagnostic assay or side effects if used in a therapeutic composition.
[009'x.] Anti-modified annexin antibodies of the present 111Ve11t1011 have a
variety
of uses that are within the scope of the present invention. Anti-modified
annexin
antibodies can be used as tools to screen expression libraries and/or to
recover desired
proteins of the present 111VeI1t1011 from a mixture of proteins and other
contaminants.
[0098] An anti-modified annexin antibody of the present invention can
selectively
bind to a modified annexin protein.
Therapeutic Methods
[0099] Any of the above-described modified annexin proteins is used in methods
of the invention to treat arterial or venous thrombosis caused by any medical
procedure or
condition. Generally, the therapeutic agents used in the invention are
administered to an
animal in an effective amount. Generally, an effective amount is an amount
effective
either (1) to reduce the symptoms of the disease sought to be treated or (2)
to induce a
pharmacological change relevant to treating the disease sought to be treated.
[00100] For thrombosis, an effective amount includes an amount effective to
exert
prolonged antithrombotic activity without substantially increasing the risk of
hemorrhage
or to increase the life expectancy of the affected animal. As used herein,
prolonged
antithrombotic activity refers to the time of activity of the modified annexin
protein with
respect to the time of activity of the same amount (molar) of an unmodified
annexin
protein. Preferably, antith rombotic activity is prolonged by at least about a
factor of two,
more preferably by at least about a factor of five, and most preferably by at
least about a
factor of ten. Preferably, the effective amount does not substantially
increase the risk of
hemorrhage compared with the hemorrhage risk of the same subject to whom the
modified
annexin has not been administered. Preferably, the hemorrhage risk is very
small and, at
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most, below that provided by alternative antithrombotic treatments available
in the prior
art. Therapeutically effective amounts of the therapeutic agents can be any
amount or
dose sufficient to bring about the desired antitlwombotic effect and depends,
in part, on the
condition, type, and location of the thrombus, the size and condition of the
patient, as well
as other factors known to those skilled in the art. The dosages can be given
as a single
dose, or as several doses, for example, divided over the course of several
weeks.
[00101] Administration preferably occurs by bolus injection or by intravenous
infusion, either after thrO111bOS1S to prevent further thrombosis or under
conditions in
which the subject is susceptible to or at risk of thrombosis.
[00102] The therapeutic agents of the present invention can be administered by
any
suitable means, including, for example, parenteral or local administration,
such as
intravenous or subcutaneous injection, or by aerosol. A therapeutic
composition can be
administered in a variety of unit dosage forms depending upon the method of
administration. Delivery methods for a therapeutic composition of the present
invention
include intravenous administration and local administration by, for example.
injection.
For pal-ticular modes of delivery, a therapeutic composition of the present
invention can be
formulated in an excipient of the present invention. A therapeutic agent of
the present
invention can be administered to any animal, preferably to mammals, and more
preferably
to humans.
[00103] One suitable administration time occurs following coronary thrombosis,
thereby preventing the recurrence of thrombosis without substantially
increasing the risk
of hemorrhage. Bolus injection of the modified annexin is preferably performed
soon
after thrombosis, e.g., before admission to hospital. The modified annexin can
be
administered in conjunction with a thrombolytic therapeutic such as tissue
plasminogen
activator, urolcinase, or a bacterial enzyme.
[00104] Methods of use of modified annexin proteins of the present invention
include methods to treat cerebral thrombosis, including overt cerebral
thrombosis or
transient cerebral ischemic attacks, by administering an effective amount of
modified
annexin protein to a patient in need thereof. Transient cerebral ischemic
attaclcs frequently
precede full-blown strokes. The modified annexin can also be administered to
diabetic
and other patients who are at increased risk for thrombosis in peripheral
arteries.
Accordingly, the present invention provides a method for reducing the risk of
thrombosis
in a patient having an increased risk for thl'O111boS1S lnCludlllg
administering an effective
32
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amount of a modified annexin protein to a patient in need thereof. For an
adult patient, the
modified annexin can be administered intravenously or as a bolus in the dosage
range of
about 1 to about 100 mg.
[00105] The present invention also provides a method for decreasing the risk
of
venous thrombosis associated with some surgical procedures, such as hip and
knee
arthroplasties, by administering an effective amount of a modified annexin
protein of the
present invention to a patient in need thereof. The modified annexin treatment
can prevent
thrombosis without increasing hemorrhage into the operating field. In another
embodiment, the present invention provides a method for preventing thrombosis
associated with pregnancy and parturition without increasing hemorrhage, by
administering an effective amount of a modified annexin protein of the present
invention
to a patient in need thereof. In a further embodiment, the present invention
provides a
method for the treatment of recurrent venous thrombosis, by administering an
effective
amount of a modified annexin protein of the present invention to a patient in
need thereof.
For an adult patient, the modified annexin can be administered intravenously
as a bolus in
the dosage range of about 1 to about 100 mg.
[00106] The present invention also provides a method of screening for a
modified
annexin protein that modulates thrombosis, by contacting a thrombosis test
system with at
least one test modified annexin protein under conditions permissive for
thrombosis, and
comparing the antithrombotic activity in the presence of the test modified
annexin protein
with the antithrombotic activity in the absence of the test modified annexin
protein,
wherein a change in the antithrombotic activity in the presence of the test
modified
annexin protein is indicative of a modified annexin protein that modulates
thrOIllbOtlc
activity. In one embodiment, the thrombosis test system is a system for
measuring
activated partial thromboplastin time. Also included within the scope of the
present
invention are modified annexin proteins that modulate thrombosis as identified
by this
method.
[00107] The present invention also provides a method for identifying a
modified
annexin protein for annexin activity, including contacting activated platelets
with at least
one test modified annexin protein under conditions permissive for binding, and
comparing
the test modified annexin-binding activity and protein S-binding activity of
the platelets in
the presence of the test modified annexin protein with the annexin-binding
activity and
protein S-binding activity in the presence of unmodified annexin protein,
whereby a
33
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modified annexin protein with annexin activity may be identified. Also
included within
the scope of the invention are modified annexin proteins identified by the
method.
[00108] In an additional embodiment, the present invention provides a method
of
screening for a modified annexin protein that modulates thrombosis, by
contacting an in
vivo thrombosis test system with at least one test modified annexin protein
under
conditions permissive for thrombosis, and comparing the antithrombotic
activity in the
presence of the test modified annexin protein with the antithrombotic activity
in the
absence of the test modified annexin protein. A change in the antithrombotic
activity in
the presence of the test modified annexin protein is indicative of a modified
annexin
protein that modulates thrombotic activity. Additionally, the time over which
antithrombotic activity is sustained in the presence of the test modified
annexin protein is
compared with a time of antithroW botic activity in the presence of unmodified
annexin to
determine the prolongation of antithrombotic activity associated with the test
modified
annexin protein. The extent of hemorrhage in the presence of the test modified
annexin
protein is assessed, e.g., by measuring tail bleeding time, and compared with
the extent of
hemorrhage in the absence of the test modified annexin protein. In one
embodiment, the
in vivo thrombosis test system is a mouse model of photochemically-induced
thrombus in
cremaster muscles. Also included within the scope of the present invention are
modified
annexin proteins that modulate thrombosis as identified by this method.
[00109] In a further embodiment, the therapeutic agents of the present
invention
are useful for gene therapy. As used herein, the phrase ''gene therapy" refers
to the
transfer of genetic material (e.g., DNA or RNA) of interest into a host to
treat or prevent a
genetic or acquired disease or condition. The genetic material of interest
encodes a
product (e.g., a protein polypeptide, peptide or functional RNA) whose
production in vivo
is desired. For example, the genetic material of interest can encode a
hormone, receptor,
enzyme or (poly)peptide of therapeutic value. In a specific embodiment, the
subject
invention utilizes a class of lipid molecules for use in non-viral gene
therapy which can
complex with nucleic acids as described in Hughes et al., U.S. Patent No.
6,169,078,
incorporated herein by reference, in which a disulfide linker is provided
between a polar
head group and a lipophilic tail group of a lipid.
[00110] These therapeutic compounds of the present invention effectively
complex
with DNA and facilitate the transfer of DNA through a cell membrane into the
intracellular space of a cell to be transformed with heterologous DNA.
Furthermore, these
34
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
lipid molecules facilitate the release of heterologous DNA in the cell
cytoplasm thereby
increasing gene transfection during gene therapy in a human or animal.
[OO11I] Cationic lipid-polyanionic macromolecule aggregates may be formed by a
variety of methods known in the art. Representative methods are disclosed by
Felgner et
al., Proc. Natl. Acad. Sci. USA 86: 7413-7417 (1987); Eppstein et al., U.S.
Patent No.
4,897,355; Behr et al., Proc. Natl. Acad. Sci. USA 86:6982-6986 (1989) ;
Bangham et al.,
J. Mol. Biol. 23:238-252 (1965); Olson et al., Biochim. Bio~ys. Acta SS7:9
(1979);
Szoka, et al., Proc. Natl. Acad. Sci. 75:4194 (1978); Mayhew et al., Biochim.
Biophys.
Acta 775:169 (1984); Kim et al., Biochim. Biophys. Acta 728:339 (1983); and
Fukunaga
et al., Endocrinol. 115:757 (1984), all incorporated herein by reference. In
general,
aggregates may be formed by preparing lipid particles consisting of either (1)
a cationic
lipid or (2) a cationic lipid mixed with a colipid, followed by adding a
polyanionic
macromolecule to the lipid particles at about 'room temperature (about 18 to
26 °C.). In
general, conditions are chosen that are not conducive to deprotection of
protected groups.
In one embodiment, the mixture is then allowed to form an aggregate over a
period of
about 10 minutes to about 20 hours, with about 1 S to 60 minutes most
conveniently used.
Other time periods may be appropriate for specific lipid types. The complexes
may be
formed over a longer period, but additional enhancement of transfection
efficiency will not
usually be gained by a longer period of complexing.
[00112] The compounds and methods of the subject invention can be used to
intracellularly deliver a desired molecule, such as, for example, a
polynucleotide, to a
target cell. The desired polynucleotide can be composed of DNA or RNA or
analogs
thereof. The desired polynucleotides delivered using the present invention can
be
composed of nucleotide sequences that provide different functions or
activities, such as
nucleotides that have a regulatory function, e.g., promoter sequences, or that
encode a
polypeptide. The desired polynucleotide can also provide nucleotide sequences
that are
antisense to other nucleotide sequences in the cell. For example, the desired
polynucleotide when transcribed in the cell can provide a polynucleotide that
has a
sequence that is antisense to other nucleotide sequences in the cell. The
antisense
sequences can hybridize to the sense strand sequences in the cell.
Polynucleotides that
provide antisense sequences can be readily prepared by the ordinarily skilled
artisan. The
desired polynucleotide delivered into the cell can also comprise a nucleotide
sequence that
is capable of forming a triplex complex with double-stranded DNA in the cell.
3S
CA 02559167 2006-09-11
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[00113] The present invention provides compounds and methods for preventing or
attenuating reperfusion injury in mammals. Reperfusion injury (RI) occurs when
the
blood supply to an organ or tissue is cut off and after an interval restored.
The loss of
phospholipids asymmetry in endothelial cells and other cells is considered a
significant
event in the pathogenesis of RI. The PS exposed on the surfaces of these cells
allows the
binding of activated monocytes. This bmdlng triggers a sequence of events
leading to
irreversible apoptosis of endothelial and other cells, another significant
event in RI. In
addition, PS on the surfaces of cells, and vesicles derived therefrom, is
accessible to
phospholipases that generate lipid mediators. These lipid mediators amplify
the damage
occurring by mechanisms described above and produce serious complications such
as
ventricular arrhythmia following acute myocardial infarction.
[00114] A recombinant human amlexin, preferably annexin V, is modified in such
a way that its half life in the vascular compartment is prolonged. This can be
achieved in
a variety of ways; three embodiments are an annexin coupled to polyethylene
glycol, a
homopolymer or heteropolymer of annexin, and a fusion protein of annexin with
another
protein (e.g., the Fc portion of innnunoglobulin). See Allison, ''Modified
Annexin
Proteins and Methods for Preventing Thrombosis," U.S. patent application
Serial No.
10/080,370 (filed February 21, 2002) and Allison, "Modified Annexin Proteins
and
Methods for Treating Vaso-Occlusive Sickle-Cell Disease,'' U.S. patent
application Serial
No. 10/632,694 (filed August 1, 2003), both incorporated by reference herein
in their
entirety.
[00115] The modified annexin binds with high affinity to phosphatidylserine on
the
surface of epithelial and other cells, thereby preventing the binding of
phagocytes and the
operation of phospholipases, which release lipid mediators. The modified
annexin
therefore inhibits both cellular and humoral mechanisms of reperfusion injury.
[00116] In one embodiment, the present invention provides an isolated modified
annexin protein containing an annexin protein coupled to at least one
additional protein,
such as an additional annexin protein (forming a hO1110d1111e1'), polyethylene
glycol, or the
Fc portion of immunoglobulin. The additional protein preferably has a
molecular weight
of at least 30 kDa. Also provided by the present invention are pharmaceutical
compositions containing an amount of any of the modified annexin proteins of
the
invention that is effective for preventing or reducing reperfusion injury.
36
CA 02559167 2006-09-11
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[00117] In some methods of the invention, the modified annexin is administered
to
a subject at risk of reperfusion injury in a pharmaceutical composition having
an amount
of any one of the modified annexin proteins of the present invention effective
for
preventing or attenuating reperfusion injury. For example, the pharmaceutical
composition may be administered before and after organ transplantation,
arthroplasty or
other surgical procedure in which the blood supply to organ or tissue is cut
off and after an
interval restored. It can also be administered after a coronary or cerebral
thrombosis.
[00118] The modified annexin binds PS accessible on cell surfaces (shielding
the
cells), thereby preventing the attachment of monocytes and the irreversible
stage of
apoptosis. In addition, the modified annexin inhibits the activity of
phospholipases that
generate lipid mediators that also contribute to RI. The modified annexin will
be useful to
prevent or attenuate RI in organs transplanted from cadaver donors, in
patients with
coronary and cerebral thrombosis, in patients undergoing arthroplasties, and
in other
situations. In addition the modified annexin will exel-t prolonged
antithrombotic activity
without increasing hemolThage. This combination of antithrombotic potency with
capacity to attenuate RI presents a unique profile of desirable activities not
displayed by
any therapeutic agent currently used or known to be in development.
[00119] As described in Example 6, the annexin homodimer is a potent inhibitor
of
sPLA2 (FIG. 4). Because annexin V binds to PS on cell surfaces with high
affinity, it
shields PS from degradation by sPLA2 and other phospholipases.
[00120] Producing a homodimer of human annexin V both increased its affinity
for
PS, thereby improving its efficacy as a therapeutic agent; and augmented its
size, thereby
prolonging its survival in the circulation and duration of action. The 36 lcDa
monomer is
lost rapidly from the blood stream into the kidneys. In the rabbit more than
80% of
labeled annexin V injected into the circulation disappears in 7 minutes
(Thiagarajan and
Benedict, Circulation 96: 2339, 1997). In cynomolgus monkeys the half life of
injected
annexin V was found to be 11 to 15 minutes (Romisch et al., Thrombosis Res.,
61: 93,
1991). In humans injected Wlth a1111eX111 V labeled with 99MTc, the half life
with respect
to the major (a) compartment was 24 minutes (Kemerinle et al., J. Nucl. Med.
44: 947,
2003).
[00121] The annexin holllodlnler may be produced by any convenient method. In
some embodiments, the annexin homodimer is produced by recombinant DNA
technology
as this avoids the necessity for post-translation procedures such as linkage
to the one
37
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WO 2005/086955 PCT/US2005/008193
available sulflzydryl group in the monomer or coupling with polyethylene
glycol.
Recombinant homodimerization was achieved by the use of a flexible peptide
linker
attached to the amino terminus of one annexin monomer and the carboxy terminus
of the
other (FIG. 1). The three-dimensional structure of annexin V, and the residues
binding
Ga2+ and PS, are known from X-ray crystallography and site-specific
mutagenesis (Huber
et al., J. Mol. Biol. 223: 683, 1992; Campos et al., 37: 8004, 1998). The Ca2+-
and PS-
binding sites are on the convex surface of the molecule while the amino
terminus forms a
loose tail on the concave surface. The annexin V homodimer shown in FIG. 1 is
designed
so that the convex surfaces could fold 111 SLICK a way that both could gain
access to PS on
cell surfaces. Thus, for this reason, the diner would have a higher affinity
for PS than that
of the monomer. As repouted in Example 4, this was verified experimentally.
Another
advantage of the homodimer of annexin V is that while a molecule of 36kDa (the
monomer) would be lost rapidly from circulation into the kidney, one of 73kDa
(the
diner), exceeding the renal filtration threshold, would not. Hence, the
therapeutically
useful activity would be prolonged in the diner. This prediction was confimned
in
experiments.
[00122] To prevent or attenuate reinfarction and RI, it is desirable, in some
instances, to have a longer duration of activity. Increasing the molecular
weight of
annexin V by homodimerization to 76 kDa prevents renal loss and extends
survival in the
circulation. Accordingly, such modified annexins may effectively attenuate RI,
even
when administered several hours before the blood supply to an organ is cut
off.
[00123] The teachings of the present invention are contrary to reports in the
literature suggesting that annexin V does not inhibit RI. For example, d'Amico
et al.
repout that annexin V did not inhibit RI in the rat heart whereas lipocortin I
(annexin I) did
(d'Amico et al., FASEB J. 14: 1867, 2000). A fragment of lipocortin I,
injected into the
cerebral ventricle of rats, was reported to decrease infarct size and cerebral
edema after
cerebral ischemia (Pelton et al., J. Exp. Med. 174: 305, 1991); these authors
did not study
reperfusion. In a comprehensive review of strategies to prevent ischemic
injury of the
liver (Selzner et al., Gastroenterology 15:917, 2003), annexin is not
mentioned.
[00124] As described in Example 7, the ability of the annexin V homodimer to
attenuate RI was tested in a mouse liver model (Teoh et al., Hepatology 36:94,
2002). In
this model, the blood supply to the left lateral and median lobes of the liver
is cut off for
90 minutes and then restored. After 24 hours, the severity of liver injury is
assessed by
38
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
serum levels of alanine aminotransferase (ALT) and hepatic histology. Both the
annexin
V homodimer (DAV), molecular weight 73kDa, and annexin V coupled to
polyethylene
glycol (PEG-AV), molecular weight 57 leDa, injected 6 hours before clamping
the hepatic
arteries, were highly effective in attenuating RI as shown by serum ALT levels
(FIG. 5)
and hepatic histology. The annexin V monomer (AV) was less protective in this
model.
[00125] The experimental evidence therefore confirms that the modified
annexins
of the present invention will be useful to attenuate RI in subjects. As
discussed above,
similar pathogenetic mechanisms are involved in the forms of RI occurring in
different
organs, thus, the annexin V homodimer may be used to attenuate RI in all of
them.
[00126] Because of its high affinity for PS and reduced loss from the
circulation,
the annexin V homodimer will exert prolonged antithrombotic activity. This is
clinically
useful to prevent reinfarction, which is known to be an important event
following coronary
thrombosis (Andersen et al., N. Engl. J. Med. 349: 733, 2003), and is likely
to be
important in stroke. Prevention of thrombosis in patients undergoing al-
throplasty is also a
major clinical need. The additional activity of a modified annexin as an
anticoagulant is
therefore valuable. In several experimental animal models, annexin V inhibits
arterial and
venous thrombosis without increasing hemorrhage (Romisch et al., Thromb. Res.
61: 93,
1991; Van Ryn-McKenna et al., Thromb. Hemost. 69: 227, 1993; Thiagarajan and
Benedict, Circulation 96: 2339, 1997). A modified annexin has the capacity to
exert
anticoagulant activity without lllCreaSlllg he111orrh age alld to attenuate
reperfusion injury.
This combination of actions could be useful in several clinical situations. No
other
therapeutic agent currently used, or known to be in development, shares this
desirable
profile of activities.
[00127] Several annexins, other than annexin V, bind Ca2+ and PS. Any of these
llllght be used to prevent or diminish reperfusion injury. The molecular
weight of annexin
V, or another annexin, may be increased by procedures other than
homodimerization.
Such procedures include the preparation of other homopolymers or
heteropolymers.
Alternatively, an annexin might be conjugated to another protein by
recombinant DNA
technology or chemical manipulation. Conjugation of an annexin to polyethylene
glycol
or another nonpeptide compound are also envisaged.
[00128] It is expected that the annexin V homodimer will be well-tolerated.
Another annexin, annexin VI, is a naturally existing homodimer of the
conserved annexin
sequence. However, annexin VI does not bind PS with high affinity A PS-binding
protein
39
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
other than an annexin may also be used in the methods of the invention. For
example, a
monoclonal or polyclonal antibody with a high affinity for PS (Diaz et al.,
Bioconjugate
Chem. 9:250, 1998; Thorpe et al., U. S. Patent No. 6,312,694) may be used
according to
the present invention (e.g., for decreasing or preventing reperfusion injury).
[00129] Diannexin (PLEASE DEFINE-IS THIS SEQ ID NO: 6) has dose-related
antithrombotic activity in the rat (fig. 7). Even when Diannexin is
administered at 5.0
mg/kg (approximately 7x the antithrombotic dose) it does not significantly
increase blood
loss after transacting rat tails. In contrast, Fragmin (low molecular weight
heparin)
administered at 140 aXa units/lcg (approx. 7x therapeutic dose) significantly
increased
blood loss in experiments conducted simultaneously (table 4 and fig. 10).
Regarding the
APTT (activated prothrombin time), none of the doses of Diannexin used
increased the
APTT, whereas both 20 aXa units/kg (table 2) of Fragmin, and 140 aXa units/kg
(table 5
and Figure 11 ) significantly increased the APTT. Clearance of iodine-labeled
Diannexin
could be described by a two-compartment model, an a-phase of 9-14 min and a (3-
phase of
6-7 hrs. The latter is significantly longer than previously reported for
annexin IV
monomer in several species. The 6.5 hour half life is convenient
therapeutically because a
single bolus injection should suffice for many clinical applications of
Diannexin. In the
unlikely event that Diannexin induces hemorrhage its effects will disappear
fairly soon.
Both Diannexin and Fragmin significantly increase the bleeding time in the rat
following
tail transaction (fig. 9 and table 4). In the case of Diannexin this may be
due to inhibition
of phospholipase A2 action and thromboxane generation. In humans bleeding
times are
increased when cyclooxygenase is irhbited by a drug or as a result of a
genetic deficiency.
Because Diannexin does not significantly increase blood loss, despite
increasing the
bleeding time, it is clear that Diannexin has ~ no major effect on early
hemostatic
mechanisms. Diannexin administration has no effect on body weight.
[00130] The present invention is also directed Toward therapeutic compositions
COlllpl'lSlllg the 1110dlfled annexin proteins of the present 111Ve11t1011.
Compositions of the
present invention can also include other components such as a pharmaceutically
acceptable excipient, an adjuvant, and/or a carrier. For example, compositions
of the
present invention can be formulated in an excipient that the animal to be
treated can
tolerate. Examples of such excipients include water, saline, Ringer's
solution, dextrose
solution, mannitol, Hanlc's solution, and other aqueous physiologically
balanced salt
solutions. Nonaqueous vehicles, such as triglycerides may also be used.
Excipients can
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
also contain minor amounts of additives, such as substances that enhance
isotonicity and
chemical stability. Examples of buffers include phosphate buffer, bicarbonate
buffer, Tris
buffer. histidine, citrate, and glycine, or mixtures thereof, while examples
of preservatives
include thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard
formulations
can either be liquid injectables or solids which can be taken up in a suitable
liquid as a
suspension or solution for injection. Thus, in a non-liquid formulation, the
excipient can
comprise dextrose, human serum albumin, preservatives, etc., to which sterile
water or
saline can be added prior to administration.
[00131 ] One embodiment of the present invention is a controlled release
formulation that is capable of slowly releasing a composition of the present
invention into
an animal. As used herein, a controlled release formulation comprises a
composition of
the present invention in a controlled release vehicle. Suitable controlled
release vehicles
include, but are not limited to, biocompatible polymers, other polymeric
matrices,
capsules, microcapsules, microparticles, bolus preparations, osmotic pumps,
diffusion
devices, liposomes, lipospheres, and transdermal delivery systems. Other
controlled
release formulations of the present invention include liquids that, upon
administration to
an animal, form a solid or a gel in ,situ. Preferred controlled release
formulations are
biodegradable (i.e., bioerodible).
[00132] Generally, the therapeutic agents used in the invention are
administered to
an animal in an effective amount. Generally, an effective amount is an amount
effective to
either (1) reduce the symptoms of the disease sought to be treated or (2)
induce a
pharmacological change relevant to treating the disease sought to be treated.
[00133] Therapeutically effective amounts of the therapeutic agents can be any
amount or doses sufficient to bring about the desired effect and depend, in
part, on the
condition, type and location of the cancer, the size and condition of the
patient, as well as
other factors readily known to those skilled in the art. The dosages can be
given as a
single dose, or as several doses, for example, divided over the course of
several weela.
[00134] The present invention is also directed toward methods of treatment
utilizing the therapeutic compositions of the present invention. The method
comprises
administering the therapeutic agent to a subject in need of such
administration.
[00135] The therapeutic agents of the instant invention can be administered by
any
suitable means, including, for example. parenteral, topical, oral or local
administration,
such as intradermally, by injection, or by aerosol. hl One e111bod1111eI1t of
the invention, the
41
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WO 2005/086955 PCT/US2005/008193
agent is administered by injection. Such injection can be locally administered
to any
affected area. A therapeutic composition can be administered in a variety of
unit dosage
forms depending upon the method of administration. Suitable delivery methods
fot~ a
therapeutic composition of the present invention include intravenous
administration and
local administration by, for example, injection. For particular modes of
delivery, a
therapeutic composition of the present invention can be formulated in an
excipient. A
therapeutic reagent of the present invention can be administered to any
animal, preferably
to mammals, and more preferably to humans.
[00136] The particular mode of administration will depend on the condition to
be
treated. It is contemplated that administration of the agents of the present
invention may
be via any bodily fluid, or any target or any tissue accessible through a body
fluid.
[00137] The following examples illustrate the preparation of modified annexin
proteins of the invention and in vitro and in vivo assays for anticoagulant
activity of
modified annexin proteins. It is to be understood that the invention is not
limited to the
exemplary work described or to the specific details set forth in the examples.
EXAMPLES
Example 1: Modified Annexin Preparation
[00138] Annexins can be purified from human tissues or produced by recombinant
technology. For instance, annexin V can be purified from human placentas as
described
by Funakoshi et al. (1987). Examples of recombinant products are the
expression of
annexin II and annexin V in Escher~ichia colt (Kang, H.-M., Trends Cardiovasc.
Med.
9:92-102 (1999); Thiagarajan and Benedict, 1997, 2000). A rapid and efficient
purification method for recombinant annexin V, based on Ca2+-enhanced binding
to
phosphatidylserine-containing liposomes and subsequent elution by EDTA, has
been
described by Berger, FEBS Lett. 329:25-28 (1993). This procedure can be
improved by
the use of phosphatidylserine coupled to a solid phase support.
[00139] Annexins can be coupled to polyethylene glycol (PEG) by any of several
well-established procedures (reviewed by Hermanson, 1996) in a process
referred to as
pegylation. The present invention includes chemically-derivatized annexin
molecules
having mono- or poly-(e.g., 2-4) PEG moieties. Methods for preparing a
pegylated
annexin generally include the steps of (a) reacting the annexin with
polyethylene glycol
(such as a reactive ester or aldehyde derivative of PEG) under conditions
whereby the
42
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
annexin becomes attached to one or more PEG groups and (b) obtaining the
reaction
product or products. In general, the optimal reaction conditions for the
reactions must be
determined case by case based on known parameters and the desired result.
Furthermore,
the reaction may produce different products having a different number of PEG
chains, and
further purification may be needed to obtain the desired product.
[00140] Conjugation of PEG to annexin V can be performed using the EDC plus
sulfo-NHS procedure. EDC (1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide
hydrochloride) is used to form active ester groups with carboxylate groups
using sulfo-
NHS (N-hydroxysulfosuccinamide). This increases the stability of the active
intermediate,
which reacts with an amine to give a stable amide linkage. The conjugation can
be carried
out as described in Hennanson, 1996.
[00141] Bioconjugate methods can be used to produce homopolymers or
heteropolymers of annexin; methods are reviewed by Hennanson, 1996.
Recombinant
methods can also be used to produce fusion proteins, e.g., annexin expressed
with the Fc
portion of immunoglobulin or another protein. The heterotetramer of annexin II
with Pl 1
has also been produced in E. coli (Kang et al., 1999). All of these procedures
increase the
molecular weight of annexin and have the potential to increase the half life
of annexin in
the circulation and prolong its anticoagulant effect.
[00142] A homodimer of annexin V can be produced using a DNA construct
shown schematically in FIG. 1 C (5'-3' sense strand) (SEQ ID N0:4) and coding
for an
amino acid sequence represented by SEQ ID N0:6. In this example, the annexin V
gene
is cloned into the expression vector pCMV FLAG 2 (available from Sigma-
Aldrich) at
EcoRI and Bgl II sites. The exact sequences prior to and after the annexin V
sequence are
unknown and denoted as "x''. It is therefore necessary to sequence the
construct prior to
modification to assure proper colon alignment. The pCMV FLAG 2 vector comes
with a
strong promotor and initiation sequence (Kozalc) and star site (ATG) built in.
The start
colon before each annexin V gene must therefore be removed, and a strong stop
for tight
expression should be added at the terminus of the second annexin V gene. The
vector also
comes with an ~-amino acid peptide sequence that can be used for protein
purification
(asp-tyr-lys-asp-asp-asp-asp-lys) (SEQ ID N0:9). A 14-amino acid spacer with
glycine-
serine swivel ends allows optimal rotation between between tandem gene-encoded
proteins. Addition of restriction sites PvuII and ScaI allow removal of the
linker if
necessary. Addition of a protease site allows cleavage of tandem proteins
following
43
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
expression. PreScissionTM protease is available from Amersham Pharmacia
Biotech and
can be used to cleave tandem proteins.Two annexin V homodimers were generated.
In the
first, a ''His tag'' was placed at~ the amino terminal end of the diner to
facilitate
purification (FIG. 1 A). The linker sequence of 12 amino acids was flanked by
a glycine
and a serine residue at either end to serve as swivels. The structural scheme
is shown in
FIG. lA. The amino acid sequence of the His-tagged annexin V homodimer is
provided
below:
MHHHHHHQAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEI
SAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEI
IASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVE
QDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNL
EQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFA
TSLYSMIKGDTSGDYKKALLLLCGEDDGSLEVLFQGPSGKLAQVLRGTVTDFPGFDERAD
AETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKL
IVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDD
VVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGT
RSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMK
GAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD
[00143] The "swivel'' amino acids of the linker are bolded and underlined.
This
His-tagged annexin V homodimer was expressed at a high level in Eschericlzaa
coli and
purified using a nickel column. The DNA in the construct was shown to have the
correct
sequence and the diner had the predicted molecular weight (74kDa). MALDI-TOF
mass
spectrometry was accomplished using a PerSeptive Biosystems Voyager-DE Pro
workstation operating in linear, positive ion mode with a static accelerating
voltage of
?SkV and a delay time of 40 nsec.
[00144] A SeCOlld hlllllall alllleX111 V homodimer was synthesized without the
H1S
tag. The structural scheme is shown in FIG. 1 B. The amino acid sequence of
the (non-
IIis-tagged) annexin V homodimer is provided below:
MAQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISA.AFKTL
FGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVLTEIIASRTPE
ELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDAGIDEAQVEQDAQALF
QAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQIEETIDRETSGNLEQLLLAV
44
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
VKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMI
KGDTSGDYKKALLLLCGEDDGSLEVLFQGPSGKLAQVLRGTVTDFPGFDERADAETLRKA
MKGLGTDEESILTLLTSRSNAQRQEISAAFKTLFGRDLLDDLKSELTGKFEKLIVALMKP
SRLYDAYELKHALKGAGTNEKVLTEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSG
YYQRMLVVLLQANRDPDAGIDEAQVEQDAQALFQAGELKV~IGTDEEKFITIFGTRSVSHLR
KVFDKYMTISGFQIEETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDH
TLIRVMVSRSEIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD
[00145] Again, the "swivel" amino acids of the linker are bolded and
underlined.
This diner was expressed at a high level in E.coli and purified by ion-
exchange
chromatography followed by heparin affinity chromatography. The ion-exchange
column
was from Bio-Rad (Econo-pak HighQ Support) and the heparin affinity column was
from
Amersham Biosciences (HiTrap Heparin HP). Both were used according to
manufacturers' instructions. Again, the DNA sequence of the annexin V
homodimer was
found to be correct. Mass spectrometry showed a protein of 73kDa, as expected.
The
amino acid sequence of armexin and other proteins is routinely determined in
this
laboratory by mass spectrometry of peptide fragments. Expected sequences were
obtained.
[00146] Human Annexin V has the following amino acid sequence:
AQVLRGTVTDFPGFDERADAETLRKAMKGLGTDEESILTLLTSRSNAQRQEISAA
FKTLFGRDLLDDLKSELTGKFEKLIVALMKPSRLYDAYELKHALKGAGTNEKVL
TEIIASRTPEELRAIKQVYEEEYGSSLEDDVVGDTSGYYQRMLVVLLQANRDPDA
GIDEAQVEQDAQALFQAGELKWGTDEEKFITIFGTRSVSHLRKVFDKYMTISGFQI
EETIDRETSGNLEQLLLAVVKSIRSIPAYLAETLYYAMKGAGTDDHTLIRVMVSRS
EIDLFNIRKEFRKNFATSLYSMIKGDTSGDYKKALLLLCGEDD (SEQ ID N0:3)
[00147] The nucleotide sequence of human annexin V, inserted as indicated in
the
DNA construct illustrated in FIG. 1 C, is as follows:
GCACAGGTTCTCAGAGGCACTGTGACTGACTTCCCTGGATTTGATGAGCGGGC
TGATGCAGAAACTCTTCGGAAGGCTATGAAAGGCTTGGGCACAGATGAGGAG
AGCATCCTGACTCTGTTGACATCCCGAAGTAATGCTCAGCGCCAGGAAATCTC
TGCAGCTTTTAAGACTCTGTTTGGCAGGGATCTTCTGGATGACCTGAAATCAG
AACTAACTGGAAAATTTGAAAAATTAATTGTGGCTGTGATGAAACCCTCTCGG
CTTTATGATGCTTATGAACTGAAACATGCCTTGAAGGGAGCTGGAAGAAATG
AAAAAGTACTGACAGAAATTATTGCTTCAAGGACACCTGAAGAACTGAGAGC
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GATCAAACAAGTTTATGAAGAAGAATATGGCTCAAGCCTGGAAGATGACGTG
GTGGGGGACACTTGAGGGTACTACCAGCGGATGTTGGTGGTTCTCCTTCAGGC
TAACAGAGACCCTGATGCTGGAATTGATGAAGCTCAAGTTGAACAAGATGCT
CAGGCTTTATTTCAGGCTGGAGAACTTAAATGGGGGACAGATGAAGAAAAGT
TTATCACCATCTTTGGAACACGAAGTGTGTCTCATTTGAGAAAGGTGTTTGAC
AAGTACATGACTATATCAGGATTTCAAATTGAGGAAACCATTGACCGCGAGA
GTTCTGGCAATTTAGAGCAACTACTCCTTGCTGTTGTGAAATCTATTCGAAGT
ATACCTGCCTACCTTGCAGAGACCCTCTATTATGCTATGAAGGGAGCTGGGAC
AGATGATCATACCCTCATCAGAGTCATGGTTTCCAGGAGTGAGATTGATCTGT
TTAACATCAGGAAGGAGTTTAGGAAGAATTTTGCCACCTCTCTTTATTCCATG
ATTAAGGGAGATACATCTGGGGACTATAAGAAAGCTCTTCTGCTGCTCTGTGG
AGAAGATGAC (SEQ ID NO:1)
Example 2: Ira Lritr°o and In Yivo Assay
[00148] In vitro assays determine the ability of modified annexin proteins to
bind
to activated platelets. Annexin V binds to platelets, and this binding is
markedly increased
in vitro by activation of the platelets with thrombin (Thiagarajan and Tait,
1990; Sun et al.,
1993). Preferably, the modified annexin proteins of the present invention are
prepared in
such a way that perform the function of annexin in that they bind to platelets
and prevent
protein S from binding to platelets (Sun et al., 1993). The modified annexin
proteins also
perform the function of exhibiting the same anticoagulant activity in
vitr°o that unmodified
annexin proteins exhibit. A method for measuring the clotting time is the
activated partial
thromboplastin time (Fritsma, ire Hemostasis and thrombosis in the clinical
laboratory
(Corriveau, D.M. and Fritsma, G.A., eds) J.P. Lipincott Co., Philadelphia
(1989), pp. 92-
124, incorporated herein by reference).
[00149] IrZ vivo assays determine the antitln-ombotic activity of annexin
proteins.
Annexin V has been shown to decrease venous thrombosis induced by a laser or
photochemically in rats (Romisch et al., 1991). The maximal anticoagulant
effect was
observed between 15 and 30 minutes after intravenous administration of annexin
V, as
determined functionally by thromboelastography. The modified annexin proteins
of the
present invention preferably show more prolonged activity in such a model than
unmodified annexin. Annexin V was also found to decrease f brie accretion in a
rabbit
model of jugular vein thI'0111bOS15 (Van Ryn-McKenna et al., 1993). Air
injection was
used to remove the endothelium, and annexin V was shown to bind to the treated
vein belt
46
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not to the control contralateral vein. Decreased fibrin accumulation in the
injured vein
was not associated with systemic anticoagulation. Heparin did not inhibit
fibrlll
accumulation in the injured vein. The modified annexin proteins of the present
invention
preferably perform the function of annexin in this model of venous thrombosis.
A rabbit
model of arterial thrombosis was used by Thiagarajan and Benedict, 1997. A pal-
tially
occlusive thrombus was formed in the left carotid al-tery by application of an
electric
current. Annexin V infusion strongly inhibited thrombosis as manifested by
measurements of blood flow, thrombll5 Welght, labeled fibrin deposition and
labeled
platelet accumulation. Recently, a mouse model of photochemically-induced
thrombus in
cremaster muscles was introduced (Volhnar et al. Thromb. Haemost. 85:160-164
(2001),
incorporated herein by reference). Using this technique, thrombosis can be
induced in any
desired artery or vein. The modified annexin proteins of the present invention
preferably.
perform the function of annexin in such models, even when administered by
bolus
injection.
Example 3
[00150] The anticoagulant ability of human recombinant annexin V and pegylated
human recombinant annexin V were compared in vita°o.
[00151] Annexin V production. The polymerase chain reaction was used to
amplify the cDNA from the initiator methionine to the stop codon with specific
oligonucleotide primers from a human placental cDNA library. The forward
primer was
5'-ACCTGAGTAGTCGCCATGGCACAGGTTCTC-3' (SEQ ID N0:7) and the reverse
primer was 5'-CCCGAATTCACGTTAGTCATCTTCTCCACAGAGCAG-3' (SEQ ID
N0:8). The amplified 1.1-kb fragment was digested with Nco I and Eco RI and
ligated
into the prokaryotic expression vector pTRC 99A. The ligation product was used
to
transform competent Esche~ichia coli strain JM 105 and sequenced.
[00152] Recombinant annexin V was isolated from the bacterial lysates as
described by Berger et al., 1993, Wlth 50111e I11od1f1Cat1011. An overnight
culture of E. coli
JM 105 transformed with pTRC 99A-annexin V was expanded 50-fold in fresh Luria-
Bertrani medium containing 100 mg/L ampicillin. After 2 hours, isopropyl (3-D-
thiogalactopyranoside was added to a final concentration of 1 11111101/L.
Atter 16 hours of
induction, the bacteria were pelleted at 35008 for 15 minutes at 4°C.
The bacterial pellet
was suspended in TBS, pH 7.5, containing 1 mmol/L PMSF, 5 mmol/L EDTA, and 6
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mol/L urea. The bacterial suspension was sonicated with an ultrasonic probe at
a setting
of 6 on ice for 3 minutes. The lysate was centrifuged at 10,000g for 15
minutes, and the
supernatant was dialyzed twice against 50 vol TBS containing 1 mmol/L EDTA and
once
against 50 vol TBS.
[00153] Multilamcllar liposomes were prepared by dissolving
phosphatidylserine,
lyophilized bovine brain extract, cholesterol, and dicetylphosphate in
chloroform in a
molar ration of 10:15:1 and dried in a stream of nitrogen in a conical flask.
TBS (5 mL)
was added to the flask and agitated vigorously in a vortex mixer for 1 minute.
The
liposomes were washed by centrifugation at 3500g for 15 minutes, then
incubated with the
bacterial extract, and calcium chloride was added to a final concentration of
5 mn~ol/L.
After 15 minutes of incubation at 37°C, the liposomes were sedimented
by centrifugation
at 10,000g for 10 minutes, and the bound annexin V was eluted with 10 mmol/L
EDTA.
The eluted annexin V was concentrated by Amicon ultrafiltration and loaded
onto a
Sephacryl S 200 column. The annexin V was recovered in the included volume,
whereas
most of the liposomes were in the void volume. Fractions containing annexin V
were
pooled and dialyzed in 10 mmol/L Tris and 2 mmol/L EDTA, pH 8.1, loaded onto
an
anion exchange column, and eluted with a linear gradient of 0 to 200 nnnol/L
NaCI in the
same buffer. The purified preparation showed a single band in SDS-PAGE under
reducing conditions.
[00154] The annexin V produced as above was pegylated using the method of
Hermanson, 1996, as described above.
[00155] Anti-coagulation assays. Prolongation of the clotting time (activated
partial th romboplastin time) induced by annexin V and pegylated annexin V
were
compared. Activated partial thromboplastin times were assayed with citrated
normal
pooled plasma as described in Fritsma, 1989. Using different concentrations of
annexin V
and pegylated annexin V, produced as described above, dose-response curves for
prolongation of clotting times were obtained. Results are shown in FIG. 6, a
plot of
clotting time versus annexin V and pegylated annexin V dose. As shown in the
figure, the
anticoagulant potency of the recombinant human annexin V and the pegylated
recombinant human annexin V are substantially equivalent. The small difference
observed is attributable to the change in molecular weight after pegylation.
This
experiment validates the assertion made herein that pegylation of annexin V
can be
achieved without significantly reducing its antithrombotic effects.
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Example 4
[00156] The affinities of recombinant annexin V (AV) and recombinant annexin V
ho1110d1111er (DAV) for PS on the surface of cells were compared. To produce
cells with
PS exposed on their surfaces, human peripheral red blood cells (RBCs) were
treated with a
Ca'+ ionophore (A23187). The phospholipid translocase (flipase), which moves
PS to the
inner leaflet of the plasma membrane bilayer, was inactivated by treatment
with N-ethyl
maleimide (NEM), which binds covalently to free sulfhydryl groups. Raising
intracellular
Ca'+ activates the scramblase enzyme, thus increasing the amount of PS in the
outer leaflet
of the plasma membrane bilayer.
[00157] Washed human RBCs were resuspended at 30% hematocrit in K-buffer
(80mM KCI, 7mM NACI, lOmM HEPES, pH 7.4). They were incubated for 30 minutes
at 37 °C in the presence of l OmM NEM to inhibit the flipase. The NEM-
treated cells were
washed and suspended at 16% hematocrit in the salve buffer with added 2mM
CaCl2. The
scramblase enzyme was activated by incubation for 30 minutes at 37 °C
with A23187
(final concentration 4pM). As a result of this procedure, more than 95% of the
RBCs had
PS demonstrable on their surface by flow cytometry.
[00158] Recombinant AV and DAV were biotinylated using the FluReporter
protein-labeling lcit (Molecular Probes, Eugene OR). Biotin-AV and biotin-DAV
conjugates were visualized with R-phycoerythrin-conjugated streptavidin (PE-
SA) at a
final concentration of 2~g/ml. Flow cytometry was performed on a Becton
Dickinson
FACScaliber and data were analyzed with Cell Quest software (Becton Dickinson,
San
Jose CA).
[00159] No binding of AV or DAV was detectable when normal RBCs were used.
However, both AV and DAV were bound to at least 95% of RBCs exposing PS. RBCs
exposing PS were incubated with various amounts of AV and DAV, either (a)
separately
or (b) mixed in a 1:1 molar ratio, before addition of PE-SA and flow
cytometry. In such
mixtures, either AV or DAV was biotinylated and the amount of each protein
bOlllld was
assayed as described above. The experiments were controlled for higher biotin
labeling in
DAV than AV.
[00160] Representative results are shown in FIG. 2. In this set of
experiments,
RBCs exposing PS were incubated with (a) 0.2 yg of biotinylated DAV (FIG. 2A);
(b)
0.2 pg of biotinylated DAV (FIG. 2B); (c) 0.2 pg of biotinylated AV and 0.2 yg
49
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nonbiotinylated DAV; and (d) 0.2 pg of biotinylated DAV and 0.2 pg
nonbiotinylated AV
(FIG. 2D). Comparing FIG. 2B and FIG. 2D shows that the presence of 0.2 ~g of
nonbiotinylated AV had no effect on the binding of biotinylated DAV. However,
comparing FIG. 2A and FIG. 2C shows that the presence of 0.2 ~g of
nonbiotinylated
DAV strongly reduced the amount of biotinylated AV bound to PS-exposing cells.
These
results indicate that DAV and AV compete for the same PS-binding sites on
RBCs, but
with different affinities; DAV binds to PS that is exposed on the surface of
cells with a
higher affinity than does AV.
Example 5
[00161) A cell-binding assay was established using known amounts of annexin V
monomer (AV) and dimes (DAV) added to mouse serum. RBCs with externalized PS,
as
described above, were incubated with serum containing dilutions of AV and DAV.
After
washing, addition of labeled streptavidin and washing again, AV and DAV bound
to the
RBCs were assayed by flow cytometry. No binding was detectable when RBCs
without
externalized PS were used. Concentrations.of AV and DAV in mouse serum,
assayed by
cell binding, were highly correlated with those determined by independent
ELISA assays.
Hence, AV and DAV in mouse plasma are not bound to other plasma proteins in a
way
that impairs their capacity to interact with externalized PS on cell surfaces.
These
observations validated the application of the cell-binding assay to compare
the survival of
AV and DAV in the circulation.
[00162] Mice were injected intravenously with AV and DAV, and peripheral blood
samples were recovered at several times thereafter. Different mice were used
for each
time point. Representative results are shown in FIG. 3. Observations in the
rabbit
(Thiagarajan and Benedict, Circulation 96: 2339, 1977), cynomolgus monkey,
(Rornisch
et al., Thrombosis Res. 61: 93, 1991) alld h11ma11S (Kemerink et al., J. Nucl.
Med. 44: 947,
2003) show that AV has a short half life in the circulation (7 to 24 minutes,
respectively),
with a major loss into the kidney. Consistent with these reports, 20 minutes
after injection
of AV into the mouse, virtually none was detectable in the peripheral blood
(FIG. 3B).
However, even 120 minutes after intravenous injection of DAV into mice,
substantial
amounts of the protein were detectable in the circulation (FIG. 3E). Thus
dimerization of
annexin V increases its survival in the circulation and hence the duration of
its therapeutic
efficacy.
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Example 6
[00163] The inhibitory effects of annexin V (AV) and the annexin V homodimer
(DAV) on the activity of human sPLA2 (Cayman, A1111 Arbor MI) were compared.
PS
externalized on RBCs Treated with NEM and A23187, as described above, was used
as the
substrate. In control cells, AV and DAV were found to bind to PS-exposing RBCs
as
demonstrable by flow cytometry. Incubation of the PS-exposing cells with sPLA?
removes PS, so that the cells no longer bind annexin. If the PS-exposing cells
are treated
with AV or DAV before incubation with PLAN, the PS is not removed. The cells
can be
exposed to a Ca'~- chelating agent, which dissociates AV or DAV from PS, and
subsequent binding of labeled AV reveals the residual PS on cell surfaces.
Titration of
AV and DAV in such assays shows that both are potent inhibitors of the
activity of sPLA~
on cell-surface PS.
[00164] The inhibition of phospholipase is also demonstrable by another
method.
Activity of sPLA2 releases lysophosphatidylcholine (LPS), which is hemolytic.
It is
therefore possible to compare the inhibitory effects of AV and DAV on' PLA2 in
a
hemolytic assay. As shown in FIG. 4, both AV and DAV inhibit the action of
PLAN, with
DAV being somewhat more efficacious. Hemolysis induced after 60 minutes
incubation
with pPLA2 was strongly reduced in the presence of DAV or AV compared to their
absence. From these results it can be concluded that the homodimer of annexin
V is a
potent inhibitor of secretory PLAN. It should therefore decrease the formation
of
mediators such as thromboxane A~ as well as lysophophatidylcholine and
lysophosphatidic acid, which are believed to contribute to the pathogenesis of
reperfusion
injury (Hashizume et al. Jpn. Heart J., 38: 1 l, 1997; Okuza et al., J.
Physiol., 285: F565,
2003).
Example 7
[00165] A mouse liver model of warm ischemia-reperfusion injury was used to
ascertain whether modified annexins protect against reperfusion injury (RI),
compare the
activity of annexin V with modified annexins, and determine the duration of
activity of
modified annexins. The model has been described by Teoh et al. (Hepatology
36:94,
2002). Female C57BL6 mice weighing 18 to 25 g were used. Under
ketamine/xylazine
anesthesia, the blood supply to the left lateral and median lobes of the liver
was occluded
with an atraumatic microvascular clamp for 90 minutes. Reperfusion was then
established
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by removal of the vascular clamp. The animals were allowed to recover, and 24
hours
later they were killed by exsanguination. Liver damage was assessed by
measurement of
serum alanine aminotransferase (ALT) activity and histological examination. A
control
group was subjected to anesthesia and sham laparotomy. To assay the activity
of annexin
V and modified annexins, groups of 4 mice were used. Each of the mice in the
first group
was injected intravenously with 25 micrograms of annexin V (AV), each of the
second
group received 25 micrograms of annexin homodimer (DAV), and each of the third
group
received 2.5 micrograms of annexin V coupled to polyethylene glycol (PEG-AV,
57 lcDa).
Controls received saline or the HEPES buffer in which the annexins were
stored. In the
first set of experiments, the annexins were administered minutes before
clamping branches
of the hepatic artery. In the second set of experiments, annexins and HEPES
were
administered 6 hours before initiating ischemia. Representative experimental
results are
summarized in FIG. 5.
[00166] In animals receiving annexin V (AV) just before ischemia, slight
protection was observed. By contrast, animals receiving the annexin dimes
(DAV) or
PEG-AV, either just before or 6 hours before ischemia, showed dramatic
protection
against RI. Histological studies confirmed that there was little or no
hepatocellular
necrosis in these groups. The results show that the modified annexins (DAV and
PEG-
AV) are significantly more protective against ischemia reperfusion injury in
the liver than
is AV. Furthermore, the modified annexins (DAV and PEG-AV) retain their
capacity to
attenuate RI for at least 6 hours.
[00167] In sham-operated animals, levels of ALT in the circulation were very
low.
In animals receiving saline just before ischemia, or HEPES 6 hours before
ischemia, levels
of ALT were very high, and histology confirmed that there was severe
hepatocellular
necrosis. HEPES administered just before ischemia was found to have protective
activity
against RI.
Example 8
[00168] Thrombosis study
[00169] Six groups of eight rats each were used. The rats for this study were
male
Wistar rats" weighing about 300 grams (Charles River Nederland, Maastricht,
the
Netherlands). Animals were housed in macrolon cages, and given standard rodent
food
pellets and acidified tap water ad lib. Experiments conformed to the rules and
regulations
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set forward by the Netherlands Law on Animal Experiments. Rats were
anaesthetized
with FFM (Fentanyl / Fluanison / Midazolam), and placed on a heating pad. A
cannula
wras inserted into the femoral vein and filled with saline. The vena cava
inferior was
isolated, and side branches were closed by ligation or cauterization. A loose
ligature was
applied around the caval vein below the left renal vein. A second loose
ligature was
applied 1.5 cm upstream from the first one, above the bifurcation. The test
(or control)
compound was given intravenously via the femoral vein cannula, and the cannula
was then
flushed with saline.
[00170] Test or control compounds include phosphate-buffered saline 1.0 ml/Icg
bodyweight (10 min); Phosphate-buffered saline 1.0 ml/kg bodyweight (12 hrs);
Diannexin 0.04 mg/kg body weight; Diannexin 0.2 mg/lcg body weight; Diannexin
1.0
mg/kg body weight (10 min); Diannexin 1.0 mg/kg body weight (12 hrs); Fragmin
20 aXa
U/kg body weight. Ten minutes later (or in two groups: 12 hrs later),
recombinant human
thromboplastin (O.lSmL/lcg) was rapidly injected into the venous cannula, the
cannula was
flushed with saline, and exactly ten seconds later the downstream ligature
near the renal
vein was closed. After nine minutes, a citrated venous blood sample was
obtained and put
on ice.
[00171] One minute later (at ten minutes) the upstream ligature near the
bifurcation
was closed and the thrombus that had formed in the segment was recovered. The
thrombus
was briefly washed in saline, blotted, and its wet weight was determined.
Citrated plasma
was prepared by centrifugation for 15 min at 2000g at 4°C, and stored
at -60°C for
analysis. In the two groups in which thrombus induction tools place at 12 hrs
after
compound injection, a different i.v. injection procedure was used. Rats were
anaesthetized
with s.c. DDF (Domitor/Dormicum/Fentanyl) and injected via the vein of the
penis. Rats
were then s.c. given an antidote (Anexate / Antisedan / Naloxon) and kept
overnight in
their cage.
[00172] After insertion of a femoral vein cannula, rats were intravenously
injected
with At 10 minutes after the intravenous injection of compound (in two groups:
at 12 hrs
after injection), diluted thromboplastin was injected i.v., and ten seconds
later the vena
cava inferior ligated. At nine minutes after ligation, blood was collected and
citrated
plasma was prepared. At ten minutes after ligation, the thrombosed segment was
ligated,
and the thrombus was recovered alld weighed. aPTT (sec) was also measured. At
0.04
mg/kg , Diannexin reduced thrombus weight by about 40%. At 12 hrs after
injection of
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Diannexin (1 mg/kg), thrombus formation was not different from controls. Body
weight
did not differ between groups by parametric ANOVA. Thrombus wet weights (Table
1 )
ranged from 0 to 44.5 mg.
[00173] Table 1: Effect of Treatment on Thrombus Wet Weight (mg) in the 10-
min Thrombosis study.
Saline Diannexin DiannexinDiannexin Fragmin
1 m /k 0.2 m 0.04 mglkg 20
/kg aXa U/k
21.0 1.8 0.0 15.5 0.5
43.8 0.0 4.3 19.6 1.5
26.6 3.2 2.1 220 4.6
44.5 0.5 6.0 7.5 00
17.6 3.5 3.1 10.5 4.3
24.0 2.7 2.8 15.6 30
10.6 4.3 5.2 16.6 0.0
17.8 0.5 4.7 15.3 0.0
mean 25.7 2.1 3.5 15.3 1.7
sd 12.3 1.6 1.9 4.6 20
By parametric ANOVA; F=24.48; p < 0.00001
All groups < saline controls (p<0.01)
By parametric ANOVA of the three Diannexin groups:
F = 4600, p < 0.0001
1 mg=0.2 mg<0.04mg;p<0.001
[00174] Treatment had a significant effect on thrombus weight. Both Fragmin
(20
aXa U/kg) and Diannexin (0.04, 0.2 and 1.0 mg/kg) significantly reduced
thrombus weight
(p<0.0001), see Figure 7 and the text table. For Diannexin, the effect was
dose-dependent.
The APTT values are shown in Table 2 and in Figure 8.
[00175] Table 2: Effect of Treatment on the APTT (seconds) in the 10-Minute
Thrombosis Study
Saline Diannexin DiannexinDiannexin ~ Fragmin~20
1 m~/k~ 0.2 m 0.04 mg/kg aXa U/k
/k
20.7 26.1 17.6 20.7 n.a.
20.0 22.0 20.8 23.5 27.1
17.6 19.0 20.7 22.0 37.9
21.6 16.5 20.2 21.7 19.5
17.5 21.5 21.3 24.9 24.2
14.7 23.0 23.0 21.5 24.4
20.2 22.5 19.0 19.9 29.7
18.7 19.3 20.4 19.4 25.0
mean 18.9 21.2 20.4 21.7 26.8
sd 2.2 2.9 1.6 1.8 5.8
By parametric ANOVA; F=G.GG; p = 0.0005
Fragmin group > all other groups (p < 0.05)
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Saline and Diannexin groups not significantly different
[00176] Fragmin increased the APPT significantly, compared to all other
groups.
The APTT was slightly, though significantly increased only in the Fragmin
group. The
Diannexin groups did not differ from the saline control group.
[00177] In the second thrombosis study, in which rats were treated at 12 hrs
before
the induction of thrombus formation, no significant difference between the
saline-injected
control group and the Diannexin-treated group was found (Table 3).
[00178] Table 3: Effect of Treatment on ThrolIlbLlS Wet Weight (mg) in the 12-
hr
Thrombosis study.
Saline Diannexin
1 m /k
16.1 22
21.2 9.5
17.1 13.5
23.2 29.0
15.3 22.1
19.2 18.3
15.6 22.3
20.8 37.9
mean 18.6 21.8
3 - l 8.8
~~mean time to thrombus induction: 13.6 hrs
no significant difference by t-test
[00179] Thrombus weights in the saline group were also not significantly
different
from thr0111buS WelghtS 111 the saline control group of the 1 O-111111
thrombosis study (25.7 ~
12.3 111g, see Table 1 ). APTT values were not different (not shown).
[00180] Intravenous injection of Diannexin (at 0.2 mg/lcg and at 1.0 mg/lcg)
at 10
min before thrombus induction strongly inhibited thrombus formation in the
Wessler rat
venous thr0111bOS1S 1110de1.
Example 9
[00181] Bleeding study Three groups were studied. Groups of eight rats, as
described in Example 8, were used. Rats were anaesthetized with isoflurane,
intubated
and ventilated, and placed on a heating pad. A cannula was inserted into the
femoral vein,
and filled with saline. Test or control compounds were i.v. injected via the
cannula, and
the cannula was then flushed with saline. Test or control compound were
phosphate-
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buffered saline 1.0 ml/lcg bodyweight; Diannexin 5.0 mg/lcg body weight;
Fragmin 140
aXa Ulcg body weight. At 10 min after injection of test compound, the rat tail
was put in a
horizontal position, and then transected at a defined fixed distance from the
tail by
scissors. Subsequently, bleeding from the tail was determined by gently
blotting-off all
blood protruding from the tail by filter paper. The time when bleeding stopped
was
determined. Any was noted. The experiment was terminated at 30 min after tail
transection. Just prior to the end of the experiment, a citrated blood sample
was obtained
from the cannula. Citrated plasma was prepared by centrifugation for 15 min at
2000g at
4°C, and stored at -60°C for analysis. The filter papers were
extracted in 20 ml of 10111M
phosphate buffer (pH = 7.8), containing 0.05% Triton X-100 ~z . The amount of
blood lost
was determined by measuring the hemoglobin content of the phosphate buffer
(potassium
cyanide 1 pOta5Sl11111 ferricyanide procedure of Drablcin). Body weight (Table
3) did not
differ between groups by ,parametric ANOVA. Treatment by either Diannexin (5
mgllcg)
or by Fragmin (140 Ulcg) approximately doubled bleeding time (Figure 9, Table
3),
although these effects were only borderline significant (nonparametric ANOVA;
KW =
5.72, p = 0.057). Blood loss (Figure 10, Table 4) was slightly increased in
the Diannexin
group, and approximately doubled in the Fragmin group, compared to the control
group.
[00182] Table 4. Bleeding times and Blood Loss in the Tail Bleeding Study
SALINE
GROUP
rat # primary bleedingSecondary bleedingblood loss (mL)
time (min) (min)
1 2.5 # 0.049
2 30.0 # 0.400
3 17.67 # 0.58
4 110 5.5 0.035
30.0 # 0.384
6 10 # 0.001
7 7.5 2.0 0.009
8 8.67 # 0.034
mean 13.5 0.19
sd 11.4 0.23
median 9.8 0.042
DIANNEXIN
GROUP
rat # primary bleedingSecondary bleedingblood loss (mL)
time (min) (min)
1 30.0 # 0.257
2 16.16 # 0.016
56
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
3 3 00 # 0.02
2
4 180 10.0 _
0.098
30.0 # 0.263
6 17.0 10.0 1,868
7 30.0 # 0.107
8 30.0 # 0.037
mean 25.1 0.33
sd 6.7 0.63
median 30 0.104
FRAGMIN
GROUP
rat # primary bleeding Secondary bleedingblood loss (mL)
time (min (min)
1 12.0 12.0 0.034
2 9.0 8.67 0.069
3 30.0 # 0.263
4 30.0 # 0.093
5 15.0 # nd
6 30.0 # 1.846
7 30.0 # 1.520
8 30.0 # 0.213
mean 23.3 0.58
sd 9.5 0.77
median 30 0.213
[00183] These differences were, however, not significant (non-parametric
ANOVA, p = 0.490). The APTT values are shown in Table 5 and in Figure 11.
[00184] Table 5. Effect of Treatment on the APTT (seconds) in the Tail
Bleeding
Study.
Saline Diannexin Fragmin
5 m lk~ 20
aXa U/k
24.3 26.3 46.6
17.8 27.0 32.1
17.3 24.1 62.9
16.5 25.5 69.8
19.9 27.7 69.1
20.3 25.1 52.4
21.4 21.0 45.7
21.9 23.2 56.5
mean 19.9 25.2 54.4
sd 2.6 2.2 12.9
57
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
[00185] Fragmin approximately doubled the APTT, while the APTT in the
Diannexin group did not differ from the saline control.group.
[00186] Blood loss and the aPTT were approximately twice as large in the
Fragmin
group as in the Diannexin group in the tail bleeding study. At 5.0 mg/lcg i.v.
Diannexin
induced bleeding from a transected rat tail, though less blood was lost than
after injection
of 140 aXa U/lcg of Fragmin.
Example 10
[00187] Clearance study Rats were injected with radiolabeled Diannexin, blood
samples were obtained at 5, 10, 15, 20, 30, 46, and 60 min and 2, 3, 4, 8, 16
and 24 hrs,
and blood radioactivity was determined to construct a blood disappearance
curve.
Disappearance of Diannexin from blood could be described by a two-compartment
model,
with about 75-80% disappearing in the a-phase (t/2 about 10 min), and 15-20%
in the (3-
phase (tl2 about 400 min). Clearance could be described by a two-compartment
model,
with half lives of 9-14 min and 6-7 hrs, respectively. Two experiments were
performed,
each with three male Wistar rats (300 gram). Diannexin was labelled with '''I
by the
method of Macfarlane, and the labeled protein was separated from free Sephadex
G-50.
After injection of NaI (5 mg/lcg) to prevent thyroid uptake of label, about 8
x 106 cpm (50
~L of protein solution diluted to 0.5 mL with saline) were injected via a
femoral vein
catheter (rats 1 and 2) or via the vein of the penis (rat 3). At specified
times thereafter (see
Table below), blood samples (150 pL) were obtained from a tail vein and 100 ~L
was
counted. After the last blood sample, rats were sacrificed by Nembutal i.v.,
and (pieces
of) liver, lung, heart, spleen and kidneys were collected for counting.
[00188] Sampling scheme for clearance study:
Rat 1 Rat 2 Rat 3
111111 5 111111 5 111111
min 2 hrs 16 hrs
min 3 hrs 24 hrs
111111 4 hrs
min 8 hrs
45 min
60 min
90 min
- 120 min
L
58
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
[00189] The (3-phase parameters were calculated from the data collected
between
45 min and 24 hrs. The a-phase parameters were then calculated from the data
between 5
and 45 min by the subtraction method. The blood radioactivity curves were
analysed by a
two-compartment model, using the subtraction method. The linear correlation
coefficients
for the a- and the (3-phase were -0.99 and -0.99 in experiment l, and -0.95
and -0.96 in
experiment 2. The clearance parameters are shown in Table 6.
[00190] Table 6. Diannexin clearance parameters.
Experiment Experiment
1 2
t/2 a1 ha phase 9.2 min 14.1 min
t/2 beta phase 385 min 433 min
in alpha phase 85% 79%
% in beta phase 15% 21
Isotype recovery in 89% 52%
blood (%)
[00191 ] Figures 15 and 16 show the clearance curves with the alpha- and beta-
phases superimposed. In Table 7 are shown the cpm recovered in lung, heart,
liver spleen
and kidneys (after digestion of the tissues). Of note is the high number of
COLllltS 111 the
lung at 2 hrs after Diannexin injection.
[00192] 'table 7. Radioactivity Recovered in Selected Tissues at 2, 8 and 24
hours
after injection of ~ZSI-Diannexin.
Exp 1 cpmJtissue % of
total
counts
at 2 at 8 at 24 at 2 at 8 at 24
hrs hrs hrs hrs hrs hrs
lung 166740 41622 4228 28 16 5
spleen 82425 15211 4074 14 6 5
heart 22582 11144 1610 4 4 2
liver 181832 85359 19730 30 33 24
kidneys 151858 108241 53046 25 41 64
sum 605437 261577 82688 100 100 100
% of 2 100 43 14
hrs
>;xp 2 cpm/tissue % of
total
counts
at 2 at 8 at 24 at 2 at 8 at 24
hrs hrs hrs hrs hrs hrs
lung 242130 12495 4025. 47 8 6
spleen 55377 11466 5019 11 7 7
heart 14966 8127 1645 3 5 2
~
liver 37628 7152 1642 7 5 2
kidneys 168560 114030 60774 32 74 83
sum 518661 153270 73105 100 100 100
% of 2 100 30 14
hrs
59
CA 02559167 2006-09-11
WO 2005/086955 PCT/US2005/008193
Example 11
[00193] Studies were undertaken to confirm the pathogenesis of ischemia-
reperfusion injury (IRI) and mode of action of Diannexin. According to the
hypothesis of
the pathogenesis of ischemia-reperfusion injury, during ischemia,
phosphatidylserine (PD)
becomes accessible on the luminal surface of endothetial cells (EC) in the
hepatic
microvasculature. During the reperfusion phase leukocytes and platelets become
attached
to PS on the surface of EC and trigger the terminal stages of apoptosis in EC.
Diannexin
binds to PS on the surface of EC and decreases the attachment of leukocytes
and platelets
to them. By this mechanism Diannexin prevents irreversible damage to EC and
thereby
attenLiates ischemia-reperfusion injury.
[00194] This hypothesis was tested by observing the microcirculation in the
mouse
liver in vivo using published methods (McCuskey et al., Hepatology 40:
386,2004). As
described in example 7, 90 minutes of ischemia was followed by various times
of
reperfusion. Figures 12A and 12B show that during reperfusion many leukocytes
become
attached to EC in both the periportal and centrilobular areas (IR). Diannexin
(1 mg/kg)
IV) reduces such attachment in a statistically significant manner (IR+D). Figs
13A and
13B show that this is also true of the adherence of platelets to EC during
reperfusion. As
predicted, EC damage (reflected by swelling) is prominent during reperfusion
and is
significantly decreased by Diannexin (Fig 14A and 14B). ~ur hypothesis of the
mode of
action of Diannexin in attenuating ischemia-reperfusion injury is therefore
confirmed.
As shown in figs. 15A and 15B, Diannexin does not influence the phagocytic
activity of
Kupffer cells in either location. Hence, Diannexin has no effect on this
defense
IllechalllSln aga111St pathogenic organisms. This finding supports other
evidence that
Diannexin does not have adverse effects.
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