U.S. patent application number 11/988196 was filed with the patent office on 2009-12-24 for anticoagulation agent and uses thereof.
This patent application is currently assigned to Baker IDI Heart & Diabetes Institute Holdings Limited. Invention is credited to Karlheinz Peter.
Application Number | 20090317329 11/988196 |
Document ID | / |
Family ID | 37604044 |
Filed Date | 2009-12-24 |
United States Patent
Application |
20090317329 |
Kind Code |
A2 |
Peter; Karlheinz |
December 24, 2009 |
ANTICOAGULATION AGENT AND USES THEREOF
Abstract
The present invention provides an anticoagulant agent including
a first element capable of inhibiting coagulation and a second
element capable of targeting an activated platelet wherein upon
administration of the agent to a subject the second element directs
the first element to the activated platelet. Also provided is a
probe for detecting a blood vessel abnormality including (a) a
binding element capable of targeting an activated platelet and (b)
a label. Applicant has shown that agents and probes directed to
activated platelets are useful in the diagnosis and therapy of
coagulation disorders.
Inventors: |
Peter; Karlheinz; (Hawthorne
East, Victoria, AU) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
UNITED STATES
617-428-0200
617-428-7045
patentadministrator@clarkelbing.com
|
Assignee: |
Baker IDI Heart & Diabetes
Institute Holdings Limited
75 Commercial Road
Melbourne, Victoria
AU
3004
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20090148383 A1 |
June 11, 2009 |
|
|
Family ID: |
37604044 |
Appl. No.: |
11/988196 |
Filed: |
December 18, 2008 |
Current U.S.
Class: |
424/9.1;
424/178.1; 435/7.21; 530/387.3; 530/391.3; 536/23.53 |
Current CPC
Class: |
C07K 16/2848 20130101;
A61K 49/16 20130101; A61P 11/00 20180101; C07K 2317/34 20130101;
A61K 51/1027 20130101; C07K 2319/00 20130101; C07K 2317/622
20130101; A61K 49/1818 20130101; A61K 47/6849 20170801; A61K
2039/505 20130101; A61K 49/04 20130101; C07K 14/811 20130101; C07K
2317/76 20130101 |
Class at
Publication: |
424/009.1;
530/387.3; 530/391.3; 435/007.21; 424/178.1; 536/023.53 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07K 16/18 20060101 C07K016/18; A61K 39/395 20060101
A61K039/395; C12N 15/11 20060101 C12N015/11; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 5, 2005 |
AU |
2005903570 |
Oct 6, 2005 |
AU |
2005905522 |
Claims
1-50. (canceled)
51. A composition comprising: a) a single chain antibody or
derivative thereof, wherein, i) the single chain antibody or
derivative thereof specifically binds the activated state of the
platelet integrin receptor GP IIb/IIIa; and ii) the single chain
antibody or derivative thereof has substantially no effect upon
thrombosis when bound to the activated state of the platelet
integrin receptor GP IIb/IIIa.
52. The composition of claim 51, further comprising a label bound
to the single chain antibody or derivative thereof.
53. The composition of claim 52, wherein the label comprises a
contrast agent for magnetic resonance imaging.
54. The composition of claim 52, wherein the label comprises a
paramagnetic bead.
55. The composition of claim 52, wherein a histidine-tag is bound
to the antibody or derivative thereof.
56. The composition of claim 55, wherein a label is conjugated to
the histidine-tag.
57. The composition of claim 56, wherein the label comprises a
paramagnetic bead.
58. The composition of claim 56, wherein the label comprises a
superparamagnetic iron oxide particle (SPIO).
59. The composition of claim 56, wherein the label comprises a
micron-sized paramagnetic iron oxide (MPIO).
60. The composition of claim 59, wherein the label comprises an
autofluorescent cobalt-functionalized MPIO.
61. The composition of claim 51, wherein the single chain antibody
or derivative thereof comprises a segment of an immunoglobulin
comprising a heavy variable chain, the heavy variable chain
comprising an amino acid sequence selected from the group
consisting of GFTFSSYIMS (SEQ ID NO: 7), TIRSGGDNTYYPDSVKG (SEQ ID
NO: 8), and YYGNYGGLAY (SEQ ID NO: 9).
62. The composition of claim 51, wherein the heavy variable chain
comprises the amino acid sequence beginning at position 36 and
ending at position 158 of SEQ ID NO: 2.
63. The composition of claim 51, wherein the single chain antibody
or derivative thereof comprises a segment of an immunoglobulin
comprising a light variable chain, the light variable chain
comprising an amino acid sequence selected from the group
consisting of RASGNIHNYLA (SEQ ID NO: 10), NAKTLAD (SEQ ID NO: 11),
and QHFWSTPYT (SEQ ID NO: 12).
64. The composition of claim 51, wherein the light variable chain
comprises the amino acid sequence beginning at position 168 and
ending at position 284 of SEQ ID NO: 2.
65. A method of assaying for an activated blood platelet
comprising: a) contacting blood or tissue with a single chain
antibody or derivative thereof, wherein, i) the single chain
antibody or derivative thereof specifically binds the activated
state of the platelet integrin receptor GP IIb/IIIa; and ii) the
single chain antibody or derivative thereof has substantially no
effect upon thrombosis when bound to the activated state of the
platelet integrin receptor GP IIb/IIIa under conditions that
provide for specific binding of the antibody or derivative thereof
with the activated state of the platelet integrin receptor GP
IIb/IIIa to form a bound complex; and b) imaging the bound complex,
wherein an image of the bound complex signals the detection of an
activated blood platelet.
66. The method of claim 65, wherein the bound complex is imaged by
a second antibody specific to the single chain antibody or
derivative thereof.
67. The method of claim 65, wherein the single chain antibody or
derivative thereof is coupled to a label.
68. The method of claim 67, wherein the label comprises a contrast
agent for magnetic resonance imaging.
69. The method of claim 67, wherein the label comprises a
paramagnetic bead.
70. The method of claim 67, wherein a histidine-tag is bound to the
antibody or derivative thereof.
71. The method of claim 70, wherein the bound complex is imaged by
detection of the histidine-tag.
72. The method of claim 70, wherein a label is conjugated to the
histidine-tag.
73. The method of claim 72, wherein the label comprises a
paramagnetic bead.
74. The method of claim 73, wherein the label comprises a
superparamagnetic iron oxide particle (SPIO).
75. The method of claim 73, wherein the label comprises a
micron-sized paramagnetic iron oxide (MPIO).
76. The method of claim 75, wherein the label comprises an
autofluorescent cobalt-functionalized MPIO.
77. The method of claim 65, wherein the imaging comprises magnetic
resonance imaging (MRI).
78. The method of claim 65, wherein the platelet integrin receptor
GP IIb/IIIa is activated by the binding of fibrinogen or its
mimetics.
79. The method of claim 65, wherein the single chain antibody or
derivative thereof comprises a segment of an immunoglobulin
comprising a heavy variable chain, the heavy variable chain
comprising an amino acid sequence selected from the group
consisting of GFTFSSYIMS (SEQ ID NO: 7), TIRSGGDNTYYPDSVKG (SEQ ID
NO: 8), and YYGNYGGLAY (SEQ ID NO: 9).
80. The method of claim 65, wherein the heavy variable chain
comprises the amino acid sequence beginning at position 36 and
ending at position 158 of SEQ ID NO: 2.
81. The method of claim 65, wherein the single chain antibody or
derivative thereof comprises a segment of an immunoglobulin
comprising a light variable chain, the light variable chain
comprising an amino acid sequence selected from the group
consisting of RASGNIHNYLA (SEQ ID NO: 10), NAKTLAD (SEQ ID NO: 11),
and QHFWSTPYT (SEQ ID NO: 12).
82. The method of claim 65, wherein the light variable chain
comprises the amino acid sequence beginning at position 168 and
ending at position 284 of SEQ ID NO: 2.
83. The method of claim 65, wherein the activated platelet is in
vivo.
84. The method of claim 65, wherein the activated platelet is ex
vivo.
85. A therapeutic composition comprising: a) a single chain
antibody or derivative thereof, wherein, i) the single chain
antibody or derivative thereof specifically binds the activated
state of the platelet integrin receptor GP IIb/IIIa; and ii) the
single chain antibody or derivative thereof has substantially no
effect upon thrombosis when bound to the activated state of the
platelet integrin receptor GP IIb/IIIa; and b) a pharmaceutically
active element bound to the single chain antibody or derivative
thereof.
86. The composition of claim 85, wherein the pharmaceutically
active element comprises an anticoagulant.
87. The composition of claim 86, wherein the pharmaceutically
active element is tick anticoagulant protein (TAP).
88. The composition of claim 86, wherein the pharmaceutically
active element is hirudin or a hirudin derivative.
89. The composition of claim 85, wherein a histidine-tag is bound
to the antibody or derivative thereof.
90. The composition of claim 89, wherein the pharmaceutically
active element is bound to the histidine-tag.
91. The composition of claim 88, wherein the pharmaceutically
active element is tick anticoagulant protein (TAP).
92. The composition of claim 88, wherein the pharmaceutically
active element is hirudin or a hirudin derivative.
93. The composition of claim 85, wherein the single chain antibody
or derivative thereof comprises a segment of an immunoglobulin
comprising a heavy variable chain, the heavy variable chain
comprising an amino acid sequence selected from the group
consisting of GFTFSSYIMS (SEQ ID NO: 7), TIRSGGDNTYYPDSVKG (SEQ ID
NO: 8), and YYGNYGGLAY (SEQ ID NO: 9).
94. The composition of claim 85, wherein the heavy variable chain
comprises the amino acid sequence beginning at position 36 and
ending at position 158 of SEQ ID NO: 2.
95. The composition of claim 85, wherein the single chain antibody
or derivative thereof comprises a segment of an immunoglobulin
comprising a light variable chain, the light variable chain
comprising an amino acid sequence selected from the group
consisting of RASGNIHNYLA (SEQ ID NO: 10), NAKTLAD (SEQ ID NO: 11),
and QHFWSTPYT (SEQ ID NO: 12).
96. The composition of claim 85, wherein the light variable chain
comprises the amino acid sequence beginning at position 168 and
ending at position 284 of SEQ ID NO: 2.
97. A method of treating a coagulation disorder comprising
administering a single chain antibody or derivative thereof,
wherein, i) the single chain antibody or derivative thereof
specifically binds the activated state of the platelet integrin
receptor GP IIb/IIIa; and ii) the single chain antibody or
derivative thereof has substantially no effect upon thrombosis when
bound to the activated state of the platelet integrin receptor GP
IIb/IIIa; and b) a pharmaceutically active element bound to the
single chain antibody or derivative thereof.
98. The method of claim 97, wherein the pharmaceutically active
element comprises an anticoagulant.
99. The method of claim 98, wherein the pharmaceutically active
element is tick anticoagulant protein (TAP).
100. The method of claim 99, wherein the pharmaceutically active
element is hirudin or a hirudin derivative.
101. The method of claim 97, wherein a histidine-tag is bound to
the antibody or derivative thereof.
102. The method of claim 101, wherein the pharmaceutically active
element is bound to the histidine-tag.
103. The method of claim 102, wherein the pharmaceutically active
element is tick anticoagulant protein (TAP).
104. The method of claim 103, wherein the pharmaceutically active
element is hirudin or a hirudin derivative.
105. The method of claim 97, wherein the single chain antibody or
derivative thereof comprises a segment of an immunoglobulin
comprising a heavy variable chain, the heavy variable chain
comprising an amino acid sequence selected from the group
consisting of GFTFSSYIMS (SEQ ID NO: 7), TIRSGGDNTYYPDSVKG (SEQ ID
NO: 8), and YYGNYGGLAY (SEQ ID NO: 9).
106. The method of claim 97, wherein the heavy variable chain
comprises the amino acid sequence beginning at position 36 and
ending at position 158 of SEQ ID NO: 2.
107. The method of claim 97, wherein the single chain antibody or
derivative thereof comprises a segment of an immunoglobulin
comprising a light variable chain, the light variable chain
comprising an amino acid sequence selected from the group
consisting of RASGNIHNYLA (SEQ ID NO: 10), NAKTLAD (SEQ ID NO: 11),
and QHFWSTPYT (SEQ ID NO: 12).
108. The method of claim 97, wherein the light variable chain
comprises the amino acid sequence beginning at position 168 and
ending at position 284 of SEQ ID NO: 2.
109. An isolated nucleic acid sequence encoding a single chain
antibody or derivative thereof, wherein, the single chain antibody
or derivative thereof specifically binds the activated state of the
platelet integrin receptor GP IIb/IIa; and the single chain
antibody or derivative thereof has substantially no effect upon
thrombosis when bound to the activated state of the platelet
integrin receptor GP IIb/IIIa.
110. An isolated nucleic acid sequence encoding the amino acid
sequence of SEQ ID NO: 2.
111. An isolated and purified polypeptide comprising the sequence
of SEQ ID NO: 2.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to the field of haematology
and particularly the sub-speciality of haemostasis. More
specifically, the present invention relates to 1) agents that
inhibit coagulation in mammalian blood, and uses for these agents
in the treatment and prevention of diseases such as stroke,
myocardial infarction, and deep vein thrombosis and 2) probes that
allow diagnosis and identification of activated platelets in
clinical settings such as thrombosis, thrombotic emboli as well as
unstable plaques.
BACKGROUND TO THE INVENTION
[0002] The ability of the body to control the flow of blood
following vascular injury is paramount to continued survival. The
process of blood clotting and then the subsequent dissolution of
the clot, following repair of the injured tissue, is termed
hemostasis. Hemostasis is composed of a number of events that occur
in a set order following the loss of vascular integrity:
[0003] The initial phase of the process is vascular constriction.
This limits the flow of blood to the area of injury. Next,
platelets become activated by thrombin and aggregate at the site of
injury, forming a temporary, loose platelet plug. The protein
fibrinogen is primarily responsible for stimulating platelet
clumping. Platelets clump by binding to collagen that becomes
exposed following rupture of the endothelial lining of vessels.
Upon activation, platelets release adenosine-5'-diphosphate, ADP
and TXA2 (which activate additional platelets), serotonin,
phospholipids, lipoproteins, and other proteins important for the
coagulation cascade. In addition to induced secretion, activated
platelets change their shape to accommodate the formation of the
plug.
[0004] To insure stability of the initially loose platelet plug, a
fibrin mesh (also called the clot) forms and entraps the plug.
Finally, the clot must be dissolved in order for normal blood flow
to resume following tissue repair. The dissolution of the clot
occurs through the action of plasmin.
[0005] Two pathways lead to the formation of a fibrin clot: the
intrinsic and extrinsic pathway. Although they are initiated by
distinct mechanisms, the two converge on a common pathway that
leads to clot formation. The formation of a red thrombus or a clot
in response to an abnormal vessel wall in the absence of tissue
injury is the result of the intrinsic pathway. Fibrin clot
formation in response to tissue injury is the result of the
extrinsic pathway. Both pathways are complex and involve numerous
different proteins termed clotting factors
Platelet Activation and von Willebrand Factor (vWF).
[0006] In order for hemostasis to occur, platelets must adhere to
exposed collagen, release the contents of their granules, and
aggregate. The adhesion of platelets to the collagen exposed on
endothelial cell surfaces is mediated by von Willebrand factor
(vWF). The function of vWF is to act as a bridge between a specific
glycoprotein on the surface of platelets (GPIb/IX) and collagen
fibrils. In addition to its role as a bridge between platelets and
exposed collagen on endothelial surfaces, vWF binds to and
stabilizes coagulation factor VIII. Binding of factor VIII by vWF
is required for normal survival of factor VIII in the
circulation.
[0007] Von Willebrand factor is a complex multimeric glycoprotein
that is produced by and stored in the platelets. It is also
synthesized by megakaryocytes and found associated with
subendothelial connective tissue. The initial activation of
platelets is induced by thrombin binding to specific receptors on
the surface of platelets, thereby initiating a signal transduction
cascade. The thrombin receptor is coupled to a G-protein that, in
turn, activates phospholipase C-.gamma. (PLC-.gamma.). PLC-.gamma.
hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP2) leading to
the formation of inositol trisphosphate (IP3) and diacylglycerol
(DAG). IP3 induces the release of intracellular Ca2+ stores, and
DAG activates protein kinase C (PKC).
[0008] The collagen to which platelets adhere as well as the
release of intracellular Ca2+ leads to the activation of
phospholipase A2 (PLA2), which then hydrolyzes membrane
phospholipids, leading to liberation of arachidonic acid. The
arachidonic acid release leads to an increase in the production and
subsequent release of thromboxane A2 (TXA2). This is another
platelet activator that functions through the PLC-.gamma. pathway.
Another enzyme activated by the released intracellular Ca2+ stores
is myosin light chain kinase (MLCK). Activated MLCK phosphorylates
the light chain of myosin which then interacts with actin,
resulting in altered platelet morphology and motility.
[0009] One of the many effects of PKC is the phosphorylation and
activation of a specific 47,000-Dalton platelet protein. This
activated protein induces the release of platelet granule contents;
one of which is ADP. ADP further stimulates platelets increasing
the overall activation cascade; it also modifies the platelet
membrane in such a way as to allow fibrinogen to adhere to the
platelet surface, resulting in fibrinogen-induced platelet
aggregation.
[0010] Activation of platelets is required for their consequent
aggregation to a platelet plug. However, equally significant is the
role of activated platelet surface phospholipids in the activation
of the coagulation cascade.
[0011] The intrinsic clotting cascade is initiated when contact is
made between blood and exposed endothelial cell surfaces. The
extrinsic and intrinsic pathways converge at the point where factor
X is activated to factor Xa. Factor Xa has a role in the further
activation of factor VII to VIIa. Active factor Xa also hydrolyzes
and activates prothrombin to thrombin. Thrombin can then activate
factors XI, VIII and V furthering the cascade. Ultimately the role
of thrombin is to convert fribrinogen to fibrin and to activate
factor XIII to XIIIa. Factor XIIIa (also termed transglutaminase)
cross-links fibrin polymers solidifying the clot.
[0012] The intrinsic pathway requires the clotting factors VIII,
IX, X, XI, and XII. Also required are the proteins prekallikrein
and high-molecular-weight kininogen, as well as calcium ions and
phospholipids secreted from platelets. Each of these pathway
constituents leads to the conversion of factor X (inactive) to
factor Xa (active). Initiation of the intrinsic pathway occurs when
prekallikrein, high-molecular-weight kininogen, factor XI and
factor XII are exposed to a negatively charged surface. This is
termed the contact phase. Exposure of collagen to a vessel surface
is the primary stimulus for the contact phase.
[0013] The assemblage of contact phase components results in
conversion of prekallikrein to kallikrein, which in turn activates
factor XII to factor XIIa. Factor XIIa can then hydrolyze more
prekallikrein to kallikrein, establishing a reciprocal activation
cascade. Factor XIIa also activates factor XI to factor XIa and
leads to the release of bradykinin, a potent vasodilator, from
high-molecular-weight kininogen.
[0014] In the presence of Ca2+, factor XIa activates factor IX to
factor IXa. Factor IX is a proenzyme that contains vitamin
K-dependent .gamma.-carboxyglutamate (gla) residues, whose serine
protease activity is activated following Ca2+ binding to these gla
residues. Several of the serine proteases of the cascade (II, VII,
IX, and X) are gla-containing proenzymes. Active factor IXa cleaves
factor X at an internal arg-ile bond leading to its activation to
factor Xa.
[0015] The activation of factor Xa requires assemblage of the
tenase complex (Ca2+ and factors VIIIa, IXa and X) on the surface
of activated platelets. One of the responses of platelets to
activation is the presentation of phosphatidylserine and
phosphatidylinositol on their surfaces. The exposure of these
phospholipids allows the tenase complex to form. The role of factor
VIII in this process is to act as a receptor, in the form of factor
VIIIa, for factors IXa and X. Factor VIIIa is termed a cofactor in
the clotting cascade. The activation of factor VIII to factor VIIIa
(the actual receptor) occurs in the presence of minute quantities
of thrombin. As the concentration of thrombin increases, factor
VIIIa is ultimately cleaved by thrombin and inactivated. This dual
action of thrombin, upon factor VIII, acts to limit the extent of
tenase complex formation and thus the extent of the coagulation
cascade.
[0016] As discussed supra activated factor Xa is the site at which
the intrinsic and extrinsic coagulation cascades converge. The
extrinsic pathway is initiated at the site of injury in response to
the release of tissue factor (factor III). Tissue factor is a
cofactor in the factor VIIa-catalyzed activation of factor X.
Factor VIIa, a gla residue containing serine protease, cleaves
factor X to factor Xa in a manner identical to that of factor IXa
of the intrinsic pathway. The activation of factor VII occurs
through the action of thrombin or factor Xa. The ability of factor
Xa to activate factor VII creates a link between the intrinsic and
extrinsic pathways. An additional link between the two pathways
exists through the ability of tissue factor and factor VIIa to
activate factor IX. While there is some uncertainty it appears the
formation of complex between factor VIIa and tissue factor is
believed to be a principal step in the overall clotting cascade. A
major mechanism for the inhibition of the extrinsic pathway occurs
at the tissue factor-factor VIIa-Ca2+-Xa complex. The protein,
lipoprotein-associated coagulation inhibitor, LACI specifically
binds to this complex. LACI is also referred to as extrinsic
pathway inhibitor, EPI or tissue factor pathway inhibitor, TFPI and
was formerly named anticonvertin. LACI is composed of 3 tandem
protease inhibitor domains. Domain 1 binds to factor Xa and domain
2 binds to factor VIIa only in the presence of factor Xa
Activation of Prothrombin to Thrombin
[0017] The common point in both extrinsic and intrinsic pathways is
the activation of factor X to factor Xa. Factor Xa activates
prothrombin (factor II) to thrombin (factor IIa). Thrombin, in
turn, converts fibrinogen to fibrin. The activation of thrombin
occurs on the surface of activated platelets and requires formation
of a prothrombinase complex. This complex is composed of the
platelet phospholipids, phosphatidylinositol and
phosphatidylserine, Ca2+, factors Va and Xa, and prothrombin.
Factor V is a cofactor in the formation of the prothrombinase
complex, similar to the role of factor VIII in tenase complex
formation. Like factor VIII activation, factor V is activated to
factor Va by means of minute amounts and is inactivated by
increased levels of thrombin. Factor Va binds to specific receptors
on the surfaces of activated platelets and forms a complex with
prothrombin and factor Xa.
[0018] Prothrombin is a 72,000-Dalton, single-chain protein
containing ten gla residues in its N-terminal region. Within the
prothrombinase complex, prothrombin is cleaved at 2 sites by factor
Xa. This cleavage generates a 2-chain active thrombin molecule
containing an A and a B chain which are held together by a single
disulfide bond.
[0019] In addition to its role in activation of fibrin clot
formation, thrombin plays an important regulatory role in
coagulation. Thrombin combines with thrombomodulin present on
endothelial cell surfaces forming a complex that converts protein C
to protein Ca. The cofactor protein S and protein Ca degrade
factors Va and VIIIa, thereby limiting the activity of these two
factors in the coagulation cascade.
[0020] Thrombin also binds to and leads to the release of
G-protein-coupled protease activated receptors (PARs), specifically
PAR-1, -3 and 4. The release of these proteins leads to the
activation of numerous signaling cascades that in turn increase
release of the interleukins, ILs, IL-1 and IL-6, increases
secretion of intercellular adhesion molecule-1 (ICAM-1) and
vascular cell adhesion molecule-1 (VCAM-1). The thrombin-induced
signaling also leads to increased platelet activation and leukocyte
adhesion. Thrombin also activates thrombin-activatable fibrinolysis
inhibitor (TAFI) thus modulating fibrinolysis (degradation of
fibrin clots). TAFI is also known as carboxypeptidase U (CPU) whose
activity leads to removal of C-terminal lysines from partially
degraded fibrin. This leads to an impairment of plasminogen
activation, thereby reducing the rate of fibrin clot dissolution
(i.e. fibrinolysis).
Control of Thrombin Levels
[0021] The inability of the body to control the circulating level
of active thrombin would lead to dire consequences. There are two
principal mechanisms by which thrombin activity is regulated. The
predominant form of thrombin in the circulation is the inactive
prothrombin, whose activation requires the pathways of proenzyme
activation described above for the coagulation cascade. At each
step in the cascade, feedback mechanisms regulate the balance
between active and inactive enzymes.
[0022] The activation of thrombin is also regulated by four
specific thrombin inhibitors. Antithrombin III is the most
important since it can also inhibit the activities of factors IXa,
Xa, XIa and XIIa. The activity of antithrombin III is potentiated
in the presence of heparin by the following means: heparin binds to
a specific site on antithrombin III, producing an altered
conformation of the protein, and the new conformation has a higher
affinity for thrombin as well as its other substrates. This effect
of heparin is the basis for its clinical use as an anticoagulant.
The naturally occurring heparin activator of antithrombin III is
present as heparin and heparin sulfate on the surface of vessel
endothelial cells. It is this feature that controls the activation
of the intrinsic coagulation cascade.
[0023] However, thrombin activity is also inhibited by
.alpha.2-macroglobulin, heparin cofactor II and
.alpha.1-antitrypsin. Although a minor player in thrombin
regulation .alpha.1-antitrypsin is the primary serine protease
inhibitor of human plasma. Its physiological significance is
demonstrated by the fact that lack of this protein plays a
causative role in the development of emphysema.
Activation of Fibrinogen to Fibrin
[0024] Fibrinogen (factor I) consists of 3 pairs of polypeptides
([A-.alpha.][B-.beta.][.gamma.]).sub.2. The 6 chains are covalently
linked near their N-terminals through disulfide bonds. The A and B
portions of the A-.alpha. and B-.beta. chains comprise the
fibrinopeptides, A and B, respectively. The fibrinopeptide regions
of fibrinogen contain several glutamate and aspatate residues
imparting a high negative charge to this region and aid in the
solubility of fibrinogen in plasma. Active thrombin is a serine
protease that hydrolyses fibrinogen at four arg-gly bonds between
the fibrinopeptide and the a and b portions of the protein.
[0025] Thrombin-mediated release of the fibrinopeptides generates
fibrin monomers with a subunit structure
(.alpha.-.beta.-.gamma.).sub.2. These monomers spontaneously
aggregate in a regular array, forming a somewhat weak fibrin clot.
In addition to fibrin activation, thrombin converts factor XIII to
factor XIIIa, a highly specific transglutaminase that introduces
cross-links composed of covalent bonds between the amide nitrogen
of glutamines and e-amino group of lysines in the fibrin
monomers.
Dissolution of Fibrin Clots
[0026] Degradation of fibrin clots is the function of plasmin, a
serine protease that circulates as the inactive proenzyme,
plasminogen. Any free circulating plasmin is rapidly inhibited by
.alpha.2-antiplasmin. Plasminogen binds to both fibrinogen and
fibrin, thereby being incorporated into a clot as it is formed.
Tissue plasminogen activator (tPA) and, to a lesser degree,
urokinase are serine proteases which convert plasminogen to
plasmin. Inactive tPA is released from vascular endothelial cells
following injury; it binds to fibrin and is consequently activated.
Urokinase is produced as the precursor, prourokinase by epithelial
cells lining excretory ducts. The role of urokinase is to activate
the dissolution of fibrin clots that may be deposited in these
ducts.
[0027] Active tPA cleaves plasminogen to plasmin which then digests
the fibrin; the result is soluble degradation product to which
neither plasmin nor plasminogen can bind. Following the release of
plasminogen and plasmin they are rapidly inactivated by their
respective inhibitors. The inhibition of tPA activity results from
binding to specific inhibitory proteins. At least four distinct
inhibitors have been identified, of which 2-plasminogen
activator-inhibitors type I (PAI-1) and type 2 (PAI-2) are of
greatest physiological significance.
[0028] Thus, from the above it can be seen that the physiological
mechanisms involved in coagulation are exceedingly complex, and it
will be appreciated that great difficulty exists in designing or
identifying agents that are capable of safely modulating the many
inter-related pathways in coagulation. The multilevel cascade of
blood clotting system permits enormous amplification of its
triggering signals. Moving down the extrinsic pathway, for example,
proconvertin (VII), Stuart factor (X), prothrombin, and fibrinogen
are present in plasma in concentrations of <1, 8, 150, and
.about.4000 mg.mL-.sup.1, respectively. Thus a small signal is very
quickly amplified to bring about effective hemostatic control.
[0029] Clotting must be very strictly regulated because even one
inappropriate clot can have fatal consequences. Indeed, blood clots
are the leading cause of strokes and heart attack, the two major
causes of human death. Thus, the control of clotting is a major
medical concern. Several inhibitors have been developed with
different mechanisms of anticoagulant action. These include the
heparins, the coumarins, and the 1,3-indanediones.
[0030] Heparin is a mucopolysaccharide with a molecular weight
ranging from 6,000 to 40,000 Da. The average molecular of most
commercial heparin preparations is in the range of 12,000-15,000.
The polymeric chain is composed of repeating disaccharide unit of
D-glucosamine and uronic acid linked by interglycosidic bonds. The
uronic acid residue could be either D-glucuronic acid or L-iduronic
acid. Few hydroxyl groups on each of these monosaccharide residues
may be sulfated giving rise to a polymer with that is highly
negatively charged. The average negative charge of individual
saccharide residues is about 2.3.
[0031] The key structural unit of heparin is a unique
pentasaccharide sequence. This sequence consists of three
D-glucosamine and two uronic acid residues. The central
D-glucosamine residue contains a unique 3-O-sulfate moiety that is
rare outside of this sequence.
[0032] Heparin forms a high-affinity complex with antithrombin. The
formation of antithrombin--heparin complex greatly increases the
rate of inhibition of two principle procoagulant proteases, factor
Xa and thrombin. The normally slow rate of inhibition of both these
enzymes (.about.10.sup.3-10.sup.4 M-1 s-1) by antithrombin alone is
increased about a 1,000-fold by heparin. Accelerated inactivation
of both the active forms of proteases prevents the subsequent
conversion of fibrinogen to fibrin that is crucial for clot
formation.
[0033] Heparin is relatively non-toxic, however heparin overdose or
hypersensitivity may result in excessive bleeding. Protamines, are
used as anti-dote for excessive bleeding complications.
[0034] Coumarin and its derivatives are principal oral
anticoagulants. Warfarin is a coumarin derivative marketed as a
racemic mixture of R and S isomers.
[0035] Coumarins are slow to act, exerting their effect in vivo
only after a latent period of 12 to 4 hours and their effect lasts
for 1.5 to 5 days. The observed slow onset may be due to the time
required to decrease predrug prothrombin blood levels, whereas the
long duration of action observed with warfarin may be due to the
lag time required for the liver to resynthesize prothrombin to
predrug blood levels.
[0036] Coumarins and 1,3-indandiones (see infra) have a further
disadvantage in that they interact with certain drugs. For example,
the action of oral anticoagulants can be enhanced by drugs such as
phyenylbutazone and salicylates while antagonized by barbiturates
and vitamin K. Coumarins are competitive inhibitors of vitamin K in
the biosynthesis of prothrombin.
[0037] The coagulation cascade relies on the conversion of
prothrombin to thrombin in a very important step. However, this
conversion depends on the presence of 10 g-carboxyglutamic acid
(GLA) residues in the N-terminus of prothrombin. The multiple Gla
residues form a binding site for Ca.sup.2+. Under normal
circumstances 10 glutamic acid (Glu) residues of prothrombin are
converted to Gla residues in a post-translational modification.
[0038] This post-translation modification is catalyzed by an
enzymes vitamin K reductase and vitamin K epoxide reductase.
Vitamin K is a co-factor in this conversion reaction. Thus it
cycles between a reduced form and an epoxide form. Because of their
structural similarity with vitamin K coumarins are thought to bind
the enzymes, vitamin K reductase and vitamin K epoxide reductase,
without facilitating the conversion of Glu residues of prothrombin
to Gla. Thus prothrombin cannot be acted upon by factor Xa.
[0039] The 1,3-indanediones have been known in the art to be
anticoagulant since the 1940s. The onset and duration of action of
anisindione are similar to those for coumarins. The chief
disadvantage of indandiones is their side effects. Some patients
are hypersensitive to it and develop a rash, pyrexia, and
leukopenia.
[0040] Despite the overall benefits achieved, the currently used
therapeutic anticoagulants are also a major source of mortality and
morbidity, caused by limitations in efficacy and even more so by
bleeding complications. In an effort to overcome these problems, a
number of new agents have been developed. However, it appears that
therapeutic anticoagulation inevitably comes with the inherent
problem that increased efficiency is only achieved by an increase
in bleeding complications. Targeting of anticoagulants to the clot
may represent a means to break this fatal linkage. The fusion of
anticoagulants to antibodies that are directed against
clot-specific epitopes allows enrichment of the anticoagulants at
the clot whereas the concentration of the anticoagulants in the
circulating blood can be kept at a low level.
[0041] The success of clot targeting is dependent on the abundance
and specificity of the epitope chosen as target. It has been
previously demonstrated that fibrin, may be used for clot
targeting. However fibrin or fibrin degradation products may
circulate in the blood leading to mis-targeting of anticoagulants
in the circulation.
[0042] A further problem in the art relates to the diagnosis of
clotting disorders. It is accepted that many clotting disorders may
be prevented or at least prevented from advancing to a more serious
problem. It is therefore desirable for the clinician to have an
indicator of early clotting disorders.
[0043] It is an aspect of the present invention to overcome or
alleviate a problem of the prior art by providing an anticoagulant
agent that is efficacious, yet does not result in extended clotting
time. The present invention further provides methods and reagents
for diagnosing a clotting-related disorder.
[0044] The discussion of documents, acts, materials, devices,
articles and the like is included in this specification solely for
the purpose of providing a context for the present invention. It is
not suggested or represented that any or all of these matters
formed part of the prior art base or were common general knowledge
in the field relevant to the present invention as it existed before
the priority date of each claim of this application.
SUMMARY OF THE INVENTION
[0045] In one aspect, the present invention provides an
anticoagulant agent including a first element capable of inhibiting
coagulation and a second element capable of targeting an activated
platelet wherein upon administration of the agent to a subject the
second element directs the first element to the activated platelet.
Applicants have demonstrated that targeting of anticoagulants to
activated platelets provides a means to inhibit coagulation without
the danger of excessive bleeding. In one form of the invention the
second element is targeted against ligand-induced binding sites
(LIBS) on the activated, fibrinogen/fibrin-binding GP IIb/IIIa
represent a clot-specific target, which is abundantly and
specifically expressed on clots. For clot-targeting, an anti-LIBS
single-chain antibody (scFv.sub.anti-LIBS) was produced. As the
first element, a highly potent, direct factor Xa (fXa) inhibitor,
the tick anticoagulant peptide (TAP) was used. Specific antibody
binding of the fusion molecule scFv.sub.anti-LIBS-TAP was proven in
flow cytometry, and anti-fXa activity was demonstrated in
chromogenic assays. In vivo anticoagulative efficiency was
determined as occlusion time (OT) by doppler flow measurements in a
ferric-chloride induced thrombosis model of the carotid artery in
mice. scFv.sub.anti-LIBS-TAP prolonged OT comparable to enoxaparin,
and equimolar doses of recombinant TAP, and a non-targeted
mutant-scFv-TAP, even at low doses where the latter control did not
reveal antithrombotic effects. In contrast to the other
anticoagulants tested, bleeding time as measured by tail
transection was not prolonged by scFv.sub.anti-LIBS-TAP.
[0046] The present invention also provides pharmaceutical
compositions, and methods for treating or preventing a coagulation
disorder, said method including the steps of administering to a
mammal in need thereof an effective amount of a composition as
described herein. Also included are diagnostic methods for
screening compounds useful as anticoagulants, and methods for
identifying the presence of thrombosis, thrombotic emboli, unstable
plaques, and the like using probes directed to activated
platelets.
BRIEF DESCRIPTION OF THE FIGURES
[0047] FIG. 1 shows the ScFv-anti-LIBS-TAP: The Signal Peptide
Sequence of Bacterial Pectate Lyase (pelB) includes the nucleotides
from position 40 to position 105, the variable region of the heavy
chain includes nucleotides from position 106 to position 483, the
Linker (YOL epitope) includes nucleotides from position 484 to
position 510; the variable region of the light chain includes
nucleotides from position 511 to position 861; the TAP region
includes nucleotides from position 862 to position 1041, and the
His6-tag commences at position 1069 and terminates at position
1086.
[0048] FIG. 2 shows a map of pHOG21-scFvanti-LIBS-TAP. RAMP:
ampicillin resistance gene; ColE1ORI: origin of replication of E.
coli; f1 IG, filamentous intergenic region; pelB: leader peptide
sequence of pectate lyases pelB; VH/VL: heavy/light chain; TAP:
tick anticoagulant peptide; His6: repeat of 6 histidines.
[0049] FIG. 3 shows flow cytometry histograms of specific binding
of IgG anti-LIBS, scFv anti-LIBS, and scFv anti-LIBS-TAP to
activated but not to non-activated human platelets. Binding of
ADP-activated platelets is given by open histograms; binding to
non-activated platelets is given by shaded histograms. Binding of
the IgG antibody is detected by a DTAF-conjugated goat anti mouse
antibody, binding of the scFvs is detected by an Alexa Fluor 488
conjugated anti-His tag antibody.
[0050] FIG. 4 shows Western blot analysis of Ni 2+-purified scFv
anti-LIBS, scFv anti-LIBS-TAP, and non-targeted mut-scFv-TAP. MW:
molecular weight marker (6.times.His protein ladder), 1: scFv
anti-LIBS, 2: scFv anti-LIBS-TAP, 3: non-targeted scFv-TAP.
[0051] FIG. 5 shows inhibition of factor Xa activity by rTAP, scFv
anti-LIBS-TAP, and non-targeted mut-scFv-TAP, but not by scFv
anti-LIBS. The cleavage of chromogenic substrate (spectrozyme FXa
#222) by factor Xa (500 pM) was determined at 405 nm. Bars show
optical density (OD) as mean and standard deviation of triplicate
measurements of a representative experiment.
[0052] FIG. 6 shows flow cytometry histograms of specific binding
of IgG anti-LIBS, scFv anti-LIBS, and scFv anti-LIBS-TAP to
activated but not to non-activated mouse platelets. Binding of
thrombin-activated platelets is given by open histograms; binding
to non-activated platelets is given by shaded histograms. Binding
of the IgG antibody is detected by a DTAF-conjugated goat
anti-mouse antibody, binding of the scFvs is detected by an Alexa
Fluor 488 conjugated anti-His tag antibody.
[0053] FIG. 7 shows antithrombotic effects of scFv anti-LIBS-TAP at
high and low doses in a mouse model with ferric chloride-induced
thrombosis in the carotid artery. Thrombus development was
evaluated by occlusion time measurements as determined by flow
measurement with a nano doppler flow probe at the carotid artery.
Saline (0.9% NaCl) and the single-chain antibody scFv anti-LIBS are
used as negative control. Enoxaparin as a clinically used agent is
used as a positive control. rTAP, scFv anti-LIBS-TAP, and
non-targeted mut-scFv-TAP were used at a high equimolar dose and
scFv anti-LIBS-TAP and non-targeted scFv-TAP were used at a low
equimolar dose. Mean and standard deviation (SD) of 4 mice per
group are given.
[0054] FIG. 8 shows the clot-targeted anticoagulant scFv
anti-LIBS-TAP does not cause bleeding time prolongation in contrast
to enoxaparin, rTAP, and non-targeted mut-scFv-TAP. Bleeding time
in mice was determined by tail transection. Saline (0.9% NaCl) and
the single-chain antibody scFv anti-LIBS are used as negative
control. rTAP and non-targeted mut-scFv-TAP demonstrated
considerable prolongation of bleeding time in contrast to scFv
anti-LIBS-TAP. Mean and standard deviation (SD) of 4 mice per group
are given.
[0055] FIG. 9 shows an overview of the thrombus adhesion assay.
Platelets immobilized on fibrinogen were targeted with the
anti-LIBS bead contrast agent via the activated GP IIb/IIIa
receptor. Co-staining of platelets was performed using P-selectin
antibodies and fluorescein-avidin in order to demonstrate selective
binding of the contrast agent to platelets only.
[0056] FIG. 10 Left panel: Confocal microscopy of the adhesion
assay. P-Selectin and fluorescein avidin-stained platelets appear
as green conglomerates, surrounded by the red fluorescent beads of
the anti-LIBS bead contrast agent. Right panel: 3D-reconstruction
of a Z-stack from 60 images of confocal microscopy.
[0057] FIG. 11 shows an MRI of human thrombi, 3D FLASH-images
reconstructed perpendicular to the longitudinal axes. Thrombi
exposed to different concentrations of anti-LIBS bead contrast
agent show negative contrast as caused by SPIO beads in T2*(black
ring around the thrombus). Thrombi exposed to irrelevant antibodies
on beads do not show this negative contrast.
[0058] FIG. 12 shows immunohistochemistry of platelets using mouse
anti-human P-selectin and Nova Red (brown). The anti-LIBS bead
contrast agent appears yellow and is only present in the areas of
platelet aggregates on the thrombus surface.
[0059] FIG. 13 shows immunofluorescence of fibrinogen-fixed human
platelets stained for avidin-fluorescein using a CD62P antibody.
(A) Platelets incubated with the red autofluorescing LIBS-MPIO
contrast agent show specific binding to platelets represented by
the green avidin-fluorescein induced signal, whereas incubation of
platelets with Control-MPIO contrast agent shows no binding (B).
(C) represents a 3D reconstruction of a z-stack from platelets
incubated with LIBS-MPIO and stained for P-selectin, demonstrating
the principle of targeting LIBS on activated GP IIb/IIIa
receptors.
[0060] FIG. 14 shows 11.7 T ex vivo MRI, 3D gradient echo sequence
(TE=4 Ms/TR=90 ms, field of view 13.times.13.times.19.5 mm, matrix
size 256.times.256.times.384), isotropic resolution of 25
.mu.m.sup.3. (A) shows the injured femoral artery in a LIBS-MPIO
perfused mouse. Black intrinsic vessel-wall signal can be observed
in the LIBS-MPIO mouse as well as in the Control-MPIO mouse (B),
but attached to the luminal side of the femoral artery signal void
can be observed as an indicator for MPIO-binding in the LIBS-MPIO
mouse (A, arrows). Quantification of MPIO-induced MRI signal-void
reveals a significant difference between LIBS-MPIO and Control-MPIO
perfused mice (p<0.05).
[0061] FIG. 15 shows histology of representative injured femoral
artery segments. (A) shows multiple bead-binding to the injured
wall after perfusion with LIBS-MPIO (arrows), whereas no binding
can be observed after Control-MPIO perfusion (B). Tissue of (A) and
(B) was iron-stained, although polystyrene-coating of the beads
allow typical blue staining of the iron core. However, blue
intrinsic signal of the iron core could be observed depending upon
the focus of the microscope. (C) Binding of the LIBS-MPIO contrast
agent to platelets was confirmed using immunohistochemistry for
CD61 and NovaRed stain. (a) represents a wall-adherent platelet and
(b) a MPIO bound directly to the platelet. (D) Quantification of
MPIOs bound per representative histology-section in LIBS-MPIO and
Control-MPIO perfused mice, showing highly specific binding of the
LIBS-targeted contrast agent (p<0.01).
[0062] FIG. 16 shows a correlation analysis between MPIO-signal per
injured leg in MRI and MPIOs bound per section in histology,
showing a significant correlation (R.sup.2=0.72)
[0063] FIG. 17 shows a three-dimensional reconstruction of the
femoral artery of a LIBS-MPIO animal using the 3D Constructor
plug-in for ImagePro Plus. (A) shows the femoral artery lumen
appearing as a green tube. (B) shows red MPIO-signal alone, and in
(C) merged information with the anatomical picture is
demonstrated.
DETAILED DESCRIPTION OF THE INVENTION
[0064] In a first aspect, the present invention provides an
anticoagulant agent including a first element capable of inhibiting
coagulation and a second element capable of targeting an activated
platelet wherein upon administration of the agent to a subject the
second element directs the first element to the activated platelet.
Applicants have found that by targeting anticoagulants to activated
platelets the nexus between increased anticoagulant efficacy and
bleeding complications is broken, thereby overcoming a significant
problem in the art. Applicants have found that activated platelets
can be used as an effective target for efficient clot-targeting.
Platelets are highly abundant in particular in thrombi within the
arterial system, as with atherosclerosis-induced thrombi e.g. in
myocardial infarction. Activated platelets are highly specific for
clots and are typically not found in the circulation. Thus, both
requirements for efficient clot-targeting, abundance and
specificity, are satisfied. Besides these favorable properties, the
use of activated platelets as epitopes for clot-targeting may be
further advantageous compared to fibrin, since platelet activation
may precede fibrin formation.
[0065] The anticoagulant agent may take any form, so long as the
two elements are capable of performing their required functions as
described supra.
[0066] The anticoagulant element may be any size so long as the
agent is able to effectively localize at the site of a clot.
However, the anticoagulant element is preferably small, since this
may improve thrombus accessibility and penetration. In a preferred
form the invention the first element has a molecular weight of
about 7,000 Da or less. While the prior art discloses a number of
small anticoagulants, It is preferred that it potently inhibit a
central and important coagulation factor. In one form of the
invention the first element is a peptide anticoagulant derived from
the soft tick Ornithodoros moubata that uses an anti-factor Xa
inhibitor to facilitate extraction of blood from its host. This
anticoagulant was initially described in 1990 (Waxman et al.
Science 1990; 248: 2473) and named TAP (tick anticoagulant
peptide). Recombinant TAP has been described as a selective factor
Xa inhibitor allowing effective anticoagulation because of the
central, up-stream, and rate-determining position of factor Xa in
the coagulation cascade (Neeper et al. J Biol Chem 1990; 265:
17746). TAP is one of the most potent anticoagulants found in
nature and it is a small molecule with only 60 amino acids. The
sequence of a suitable TAP is available from the Genbank database
under accession number M60480.
[0067] The first element may act upon any component of either the
extrinsic or intrinsic coagulation pathways. In a preferred form of
the invention the first element acts on the enzyme factor Xa.
Direct inhibition of fXa has been proposed to be advantageous
compared to the indirect, antithrombin-III-mediated inhibition,
e.g. as mediated by heparins, since clot-bound fXa and
prothrombinase-associated fXa seem to be significantly better
inhibited by direct fXa inhibitors. In comparative studies
investigating anti-thrombotic potency and the prevention of
re-occlusion, TAP has been shown to be advantageous to indirect fXa
inhibitors as well as to thrombin inhibitors. Despite these
advantages, high bleeding rates were expected for a therapeutic use
in humans, similar to hirudin, and the development for a human drug
has not been preceded. Without wishing to be limited by theory
targeting of TAP to developing clots could decrease systemic
anticoagulation and thus bleeding complications and a long lasting
local anticoagulative effect may be achievable due to a stable
fixation of anti-fXa activity at the clot. Thus, TAP being a small
molecule, demonstrating a direct inhibition with no need for a
cofactor, and targeting the early centre of the coagulation pathway
is a preferred candidate for targeting.
[0068] While TAP is used in one embodiment of the invention, many
other anticoagulants will be suitable, including other inhibitors
of coagulation factors such as hirudin. Also the targeting of
fibrinolytics promises highly efficient thrombolysis with less
bleeding complications.
[0069] As used herein, the term "inhibiting coagulation" is
intended to mean not only a complete inhibition, but also a partial
inhibition of clot formation. Without wishing to be limited by
theory, it is contemplated that a partial inhibition is preferable
since complete inhibition may lead to uncontrolled bleeding.
[0070] The function of the second element is to bring the first
element into physiological proximity to the activated platelet.
This may be accomplished by the second element having the ability
to bind to the activated platelet, or to bind to a molecule
associated with the activated platelet. Typically, this will be
achieved by the second element being capable of binding to a marker
on the surface of an activated platelet. To afford the agent the
highest possible specificity, the marker should be one that is
expressed only on the surface of activated platelets. However it
will be understood that such an absolute requirement is not
strictly necessary, and so long as the second element is capable of
targeting predominantly activated platelets, then the invention
will provide the advantages disclosed herein.
[0071] There are a number of markers that are predominant on
activated platelets including activated GPIIb/IIIa. The marker may
be one that takes an inactive and an active form such that one form
is found to predominate over the other in activated platelets, as
compared with other components of the coagulation system. One of
the most abundantly expressed molecules on the platelet surface is
the glycoprotein receptor (GP) IIb/IIIa (CD41/CD61). This receptor
belongs to the adhesion molecule family of integrins and is also
termed .alpha..sub.IIb.beta..sub.3. Integrins consist of two
non-covalently linked subunits that undergo a conformational change
from a low affinity to a high affinity receptor in respect to the
binding of the GPIIb/IIIa ligand fibrinogen. Besides the exposure
of the ligand binding pocket, this conformational change also
induces the exposure of so-called ligand-induced binding sites
(LIBS) on GPIIb/IIIa. These binding sites are specific for the
activated and/or ligand bound GP IIb/IIIa receptor. GPIIb/IIIa is
highly abundant with around 60 000 to 80 000 molecules on the
surface of each platelet.
[0072] This receptor is transformed from an inactive state to an
active state upon platelet activation, the mechanism of which is a
conformational change of the receptor such that new epitopes are
exposed. Thus, in one form of the invention the second element is
capable of binding to a new epitope formed upon activation of the
GP IIb/IIIa receptor.
[0073] The skilled person will appreciate that it is not strictly
necessary for the first and second elements to be physically
linked. For example the first and second elements may be physically
separate, with the first element including means for binding to the
second element. Under this proposal, the first element may be
administered first to the subject, and travel to a new dot to bind
to the activated platelet. The second element may then be
administered, and bind to the first element. Thus, the two elements
are physically separate until a functional anticoagulation agent is
achieved at the site of the activated platelet.
[0074] In a preferred form of the invention the anticoagulation
agent is in the form of a single molecule, and typically a single
protein molecule. A convenient means for achieving the two elements
in a single molecule is by including both elements in the framework
of a single-chain antibody molecule. These molecules are
particularly suitable for specifically targeting epitopes given
their inclusion of a variable region. The variable region is
designed such that it has an affinity for the targeted epitope.
Single-chain antibodies are a promising format for the design of
recombinant therapeutic agents. They consist of only the variable
regions of the antibodies' heavy and light chains fused together
via a short linker molecule on a single peptide chain. Thus,
single-chain antibodies (scFvs) comprise the smallest fragments
containing a complete antibody binding site. Since size is a
determinant of immunogenicity, it is expected that scFvs are only
minimal, if at all, immunogenic.
[0075] Another advantage of single chained antibodies is that
coupling of the first and second elements leads to little loss of
biological function of the elements. It will be appreciated however
that chemical coupling typically results in a significant loss of
both the antibody binding function as well as of the activity of
the coupled effector molecules, scFv can be coupled without
functional loss using molecular biology techniques. Finally, scFvs
can be produced in bacteria in large quantities, in a short period
of time, at low cost, and they can be highly purified by affinity
chromatography. Means for producing single chained antibodies are
well known to the skilled person with a review on the topic being
found in Recombinant Antibodies (Breitling & Duebel, 1999,
Publisher Wiley & sons, ISBN 0471178470), the contents of which
is herein incorporated by reference.
[0076] Preferably, the cloning of an anti-LIBS single-chain
antibody (scFv) based on a hybridoma cell line expressing IgG
anti-LIBS 145. An antibody against a LIBS (ligand-induced binding
sites) epitope was chosen for the targeting of anticoagulants to
clots. As previously demonstrated, the mAb anti-LIBS 145
(IgG.sub.anti-LIBS) demonstrates ligand-induced binding to
GPIIb/IIIa after incubation of platelets with RGD-peptides,
abciximab, tirofiban and eptifibatide (Schwarz et al. JPET 2004,
308: 1002). Furthermore, IgG.sub.anti-LIBS demonstrate a strong
binding to ADP-activated platelets in the presence of fibrinogen
(FIG. 2). Thus, this antibody provides a targeting propensity that
is highly abundant and specific.
[0077] The mAb anti-LIBS 145 expressing hybridoma cell line was
used as the basis for the cloning of an anti-LIBS single-chain
antibody (scFv). mRNA of this hybridoma cell line was prepared and
reverse transcribed using an oligo-dT primer. The variable regions
of the antibody's heavy and light chain were amplified by PCR using
primers that anneal to conserved regions (see methods described
elsewhere herein for detail) at the 5' and 3' ends of the variable
regions. The PCR products were cloned into the pHOG21 expression
vector that allows high-level expression in bacteria. After
transformation of TG1 E. coli individual clones were assessed for
LIBS-typical binding to GP IIb/IIIa. One clone that revealed a
stronger binding compared to the original IgG anti-LIBS 145 mAb in
flow cytometry (FIG. 2) was chosen for further use. This clone was
sequenced and it revealed all the typical features of a
single-chain antibody (FIG. 1). Furthermore, Western blot analysis
revealed the correct size with around 32 kDa (FIG. 3).
[0078] Preferably, the single chain antibody is expressed as a scFv
fusion protein. Based on previous results showing that TAP can be
fused without functional loss (TH), this highly potent direct
factor Xa inhibitor was chosen to couple with the cloned
single-chain antibody. TAP was originally synthesized according to
published sequence information (Genbank database under accession
number M60480) and was cloned into the pHOG21 expression vector
directly at the C-terminus of the variable region of the light
chain (see FIG. 1A, B). pHOG21 contains a pelB-leader-sequence
facilitating purification via inclusion bodies and a His(6)-tag for
Ni.sup.2+-purification as well as detection (FIG. 1A, B). The yield
of purified scFv.sub.anti-LIBS-TAP was around 0.4 to 0.8 mg from 1
L bacterial culture. After expression and purification, the size of
the single-chain antibody constructs was assessed by Western blot
analysis (FIG. 3). The molecular weight of the scFv.sub.anti-LIBS
alone was .about.32 kDa, of the intact fusion protein
scFv.sub.anti-LIBS-TAP was .about.39 kDa., and of the non-targeted
mut-scFv-TAP was .about.42 kDa (FIG. 3).
[0079] In a highly preferred form of the invention, the
single-chain antibody is essentially as shown in FIG. 1. The
skilled person will understand that the degeneracy of the genetic
code and the ability to substitute amino acids for other similar
amino acids means that derivatives and equivalents of the molecule
specified by FIG. 1 can be easily generated. These derivatives and
equivalents are included in the scope of the present
application.
[0080] In another aspect, the present invention provides a
pharmaceutical composition including an anticoagulant agent as
described herein. The skilled person will be enabled to devise
compositions suitable for delivering the anticoagulant agents
described herein by routine methods. Where the anticoagulation
agent is a protein the composition may simply contain NaCl at an
isotonic concentration. It may be necessary to add carrier
proteins, stabilizer , buffers, non-aqueous solvents, salts,
preservatives and the like.
[0081] In another aspect the present invention provides a method
for treating or preventing a coagulation disorder, said method
including the steps of administering to a mammal in need thereof an
effective amount of a composition as described herein. Typically,
the composition will be administered systemically by intravenous or
intra-arterial boli or infusion. In terms of dosage, where the
anticoagulant agent is a protein the dosage is between about 30
mg/kg to about 300 mg/kg. It is well within the ability of a
clinician to titrate the dosage upwards or downwards to achieve the
desired clinical effect for any given subject, or for any given
disorder of coagulation.
[0082] The coagulation disorder may be any disorder that requires
an inhibition of coagulation. Such disorders include all clinical
settings that are associated with thrombosis such as coronary
artery disease, acute coronary syndrome including myocardial
infarction, stroke, atherosclerosis of the carotid artery or aorta,
deep vein thrombosis, pulmonary embolism, and atherosclerosis or
thrombosis of an organ.
[0083] The preferred bi-functional fusion molecule
scFv.sub.anti-LIBS-TAP has been evaluated by the Applicant. The
function of the single-chain antibody part of the fusion molecule
scFv.sub.anti-LIBS-TAP was evaluated by flow cytometry.
ScFv.sub.anti-LIBS-TAP and scFv.sub.anti-LIBS demonstrated similar
binding properties to fibrinogen-bound, activated platelets (FIG.
2). Thus, the genetic fusion did not significantly alter the
single-chain antibody's binding property. The factor Xa inhibitory
activity of the fusion constructs was evaluated by a chromogenic
assay. Factor Xa was incubated with a specific chromogenic
substrate in the presence of scFv.sub.anti-LIBS-TAP, non-targeted
mut-scFv-TAP, scFv.sub.anti-LIBS and recombinant TAP (FIG. 4).
Compared to rTAP, TAP activity was slightly reduced in the fusion
constructs, but was clearly present (FIG. 4). Thus, both functions,
antibody binding and factor Xa inhibition were retained.
[0084] To show superiority of the targeting of anticoagulants to
the LIBS epitopes of GP IIb/IIIa on activated platelets compared to
the conventional, non-targeted use of anticoagulant a
well-established mouse thrombosis model was chosen (Farrehi et al.
1998; 97:1002). However, it was first demonstrated that the
anti-LIBS antibodies could be used for targeting to
fibrinogen-bound, activated platelets of mice. Applicants obtained
mouse blood and evaluated the binding of the original
IgG.sub.anti-LIBS, of the scFv.sub.anti-LIBS, and of the fusion
construct scFv.sub.anti-LIBS-TAP to mouse platelets by flow
cytometry. Similar to the results in human platelets, a specific
binding of the IgG.sub.anti-LIBS was seen, but an even stronger
specific binding was noted with the scFv.sub.anti-LIBS antibody
alone as well as binding of its fusion protein
scFv.sub.anti-LIBS-TAP to fibrinogen-bound, activated mouse
platelets (FIG. 5). Thus, it is proposed that targeting of mouse
platelets will work with the generated anti-LIBS fusion
constructs.
[0085] Thrombi were induced in the carotid artery of mice using
ferric chloride. The termination of blood flow measured by a nano
flow probe was used as an indicator of an occlusive thrombus in the
vessel. Sodium chloride solution and the single-chain antibody
anti-LIBS were used as a negative controls and enoxaparin was used
as a positive control representing the current clinical standard.
Enoxaparin nearly doubled the occlusion time (FIG. 8). Equimolar
amounts of recombinant TAP, non-targeted mut-scFv-TAP and
scFv.sub.anti-LIBS-TAP caused significant prolongation of the
occlusion time close to the effects of enoxaparin. A reduction to
1/10 (0.03 .mu.g/g body weight) of the original dose delivered
still caused a significant prolongation of the occlusion time
(p=0.002) with the scFv.sub.anti-LIBS-TAP, whereas the non-targeted
mut-scFv-TAP at the same dose did not cause a significant
prolongation of the occlusion time. Thus, the
scFv.sub.anti-LIBS-TAP delivers a strong anticoagulant effect, even
at a dose at which the direct control, the non-targeted
mut-scFv-TAP does not cause significant anticoagulation.
[0086] A major advantage of activated platelet-targeted
anticoagulation is a reduction of bleeding complications. Bleeding
time was determined by a standardized surgical tail transection in
mice. As expected, saline and scFv.sub.anti-LIBS did not cause
bleeding time prolongations, whereas enoxaparin, and in particular
recombinant TAP caused a considerable prolongation. At the dose of
0.3 .mu.g/g BW at which both, non-targeted mut-scFv-TAP and
scFv.sub.ant-LIBS-TAP demonstrated a strong anticoagulant effect
(FIG. 6), only the non-targeted mut scFv-TAP caused a highly
significant prolongation in bleeding time (p<0.001, FIG. 7). The
clot-targeted scFv.sub.anti-LIBS-TAP did not cause a prolongation
in the bleeding time at all. Also the lower dose of
scFv.sub.anti-LIBS-TAP, which still demonstrated a clear
anticoagulant effect at the carotid artery of the mouse, did not
cause bleeding time prolongation. Thus, highly effective
anticoagulative effects could be achieved by the newly generated
fusion of TAP to the anti-LIBS single-chain antibody without
prolongation of bleeding time.
[0087] It will be appreciated that while avoidance of prolongation
of bleeding time is an advantage of the present invention, the
anticoagulant agents described herein may increase bleeding time in
some embodiments.
[0088] In another aspect the present invention provides the use of
an anticoagulant agent as described herein in the preparation of a
medicament for the prevention or treatment of a coagulation
disorder.
[0089] In a further aspect the present invention provides a method
of screening for a compound useful for targeting an anticoagulant
to a clot, said method including the steps of providing a candidate
compound, exposing the compound to an activated platelet and at
least one other component of a clot, assessing whether the compound
binds to the activated platelet, and assessing whether the compound
binds to the at least one other component of a clot wherein the
compound is useful if it is capable of higher affinity binding to
the activated platelet as compared the at least on other component
of a clot.
[0090] Thus, it will be possible to identify compounds useful as a
second element in the context of the present invention based on the
Applicant's finding that activated platelets are an advantageous
target for anticoagulation therapy. The skilled artisan will be
familiar with a number of methods useful in determining the binding
of one molecule to another, including immunoadsorbent methods,
chromatographic methods, surface plasmon resonance methods and the
like.
[0091] In a further aspect the present invention provides a
compound identified by a screening method described herein.
[0092] Another aspect of the present invention provides a method of
diagnosis or prognosis of a coagulation disorder in a subject, the
method including the detection of an activated platelet in a blood
vessel of the subject.
[0093] To the best of the Applicant's knowledge, the prior art
fails to disclose the use of activated platelets for diagnosis or
prognosis. The detection of activated platelets will provide the
clinician with a relevant marker useful in a number of medical
applications. One application is to image activated platelets found
on ruptured coronary plaques or those plaques that are prone to
rupture. This will allow for an early non-invasive diagnosis of
such as myocardial infarction syndromes with following prophylactic
implantation of stents into relevant lesions possible. This is of
special clinical interest as coronary angiography (as described in
the prior art) only provides information about the vessel lumen,
but not about the morphology of the vessel wall itself. Thus,
possibly ruptured or rupture-prone plaques are not detected with
coronary angiography.
[0094] In consideration of the usefulness of activated platelets as
a diagnostic marker, the present invention also provides a probe
for detecting a blood vessel abnormality including (a) a binding
element capable of targeting an activated platelet and (b) a label.
The skilled person will understand that probes useful in the
context of the present invention are typically provided as an
aqueous composition and injected into an artery or a vein of the
subject prior to or during the diagnostic method. The probe is then
transported to the site of interest in the body by the blood and
binds to an activated platelet if present. The bound probe is then
detected by an appropriate means such as MRI.
[0095] The binding element may be capable of binding to a marker on
the surface of an activated platelet. A non-limiting example of a
suitable marker is the activated GPIIb/IIIa receptor molecule.
Other markers such as PAC-1 and CD62-P are also contemplated. In
one form of the invention the binding element and label are in the
framework of a single-chain antibody molecule.
[0096] The probes and methods using the probes described herein may
be used to detect any accumulation of activated platelets, for
example in pulmonary or peripheral embolism, or on ruptured
atherosclerotic plaques in peripheral or cerebral arteries. These
lesions could be detected early in the disease process and
selectively treated.
[0097] The skilled person will understand that the probes and
methods described herein may be useful in identifying individuals
having a predisposition to a coagulation disorder, without
necessarily demonstrating as a clinically recognizable sign or
symptom of a coagulation disorder.
[0098] In a preferred form of the method, the probe used for the
step of detecting an activated platelet is a single-chained
antibody as described herein. Preferably the single-chained
antibody is the same or similar to the anti-LIBS antibody as
described herein. It will be appreciated that for diagnostic
purposes, the single-chained antibody does not need to include the
anticoagulant component. Indeed, the skilled person will understand
that it may be possible to use a fragment of the single-chained
antibody, so long as that fragment includes the site responsible
for binding to activated platelets. Without wishing to be limited
by theory it is thought that the compact dimensions of a
single-chain antibody is of particular advantage in this
application. It is proposed that the antibody is capable of
penetrating beyond the surface of a thrombus into areas where a
greater number of activated platelets are present. This allows for
more effective detection of the bound antibody, and therefore
higher sensitivity imaging. The antibody probe may also adhere to
the surface of a blood vessel where an activated platelet has
deposited.
[0099] The method may be used to diagnose and identify thrombi
(e.g. deep vein thrombosis), thrombotic emboli (e.g. pulmonary
embolism) and deposition of activated platelets (e.g. at the site
of unstable atherosclerotic plaques). Early detection will be
highly advantageous allowing the administration of clot dissolving
agents and/or anticoagulant therapy and/or interventional
procedures.
[0100] The skilled artisan will understand that the probe used for
diagnostic and prognostic methods may be labelled by any method
known in the art. Depending on the functionalization of the
particles, different strategies can be used for this purpose. One
way is to build peptide bonds between carboxy-functionalized SPIOs
and free amino groups of the single-chain antibody. The skilled
person is familiar with a range of commercially available coupling
agents and kits that may be used for this chemical crosslinking
approach. Another way would be to use the histidine-tag of the
antibody for conjugation with commercially available
cobalt-functionalized 1 .mu.m SPIO-beads, whereby the single-chain
antibody/bead complex is maintained by the binding of histidine to
cobalt. Briefly, with this approach single-chain antibodies and
SPIO-beads are incubated at room temperature for 10 minutes,
thereafter the suspension is separated by a magnet and washed
several times. Appropriate controls are generated by conjugating an
irrelevant single-chain antibody to SPIOs using the same
protocol.
[0101] The skilled person will understand that any label useful in
an X-Ray imaging method could be incorporated in the probe. As a
non-limiting example of the method, a paramagnetic label could be
coupled to a probe targeted to activated platelets. Upon
administration of the probe, the paramagnetic label would localize
at the site of a clot, embolus or unstable atherosclerotic plaque
that could then be visualised by a magnetic resonance imaging
technique.
[0102] Alternatively, the probe could be radiolabelled (for example
with technetium-99m, rubidium-82, thallium 201, F-18, gallium-67,
or indium-111), with the activated platelets being visualized using
a gamma camera. Also the labelling of activated platelets using
computer tomography and ultrasonic methods (e.g. targeting of micro
bubbles) is contemplated to be useful with the described
antibody.
[0103] Applicant discloses herein the use of a probe that targets
activated platelets and allows quantification of contrast binding
using MRI in an in vivo setting. In one form of the invention a
single chain antibody that recognizes only the active conformation
of GpIIb/IIIa is used, the antibody being coupled to micro-meter
sized paramagnetic iron oxide particles. Intravascular structures
are accessible to micron-sized particles that are several orders of
magnitude larger than the iron oxide nanoparticles typically used
in the art. To the best of the Applicant's knowledge, functional
imaging of activated platelets using single-chain antibodies is
described herein for the first time.
[0104] To demonstrate the use of labeled probes targeted to
activate platelets in an in vivo setting, the femoral wire injury
model in mice was used (Roque, M., et al., Mouse model of femoral
artery denudation injury associated with the rapid accumulation of
adhesion molecules on the luminal surface and recruitment of
neutrophils. Arterioscler Thromb Vasc Biol, 2000. 20(2): p.
335-42), which leads to a monolayer of platelets 24 hours after
injury. The time course of cellular events following femoral wire
injury in the mouse is well described and, as demonstrated in FIG.
15C, consistently shows confluent platelet deposition on the
denuded endothelium after 24 hours. This was used as the basis for
targeting activated platelets with single-chain antibodies against
ligand-induced binding sites on the activated GP IIb/IIIa receptor.
This antibody confers functional specificity since its binding is
dependent upon the presence of fibrinogen or its analogues. These
properties make this antibody attractive as a ligand for MPIOs to
mediate imaging of platelet thrombus. Using 11.7 T MRI and
T2*-weighted MRI, signal void at the areas of strong bead binding
was detected. The histologically confirmed quantity of MPIOs bound
to the vessel wall correlated significantly to the extent of signal
extinction caused by the MPIOs in T.sub.2* weighted MRI. This MRI
method possesses sufficient resolution for imaging small vessels of
only 200 .mu.m diameter. The MPIO-induced signal void was
sufficiently sensitive to detect signal void even extending the
intrinsic negative contrast caused by the arterial vessel wall in
T2*-weighted MRI.
[0105] Previous targeted contrast agent approaches have included
integrin-conjugated gadolinium rich perfluorocarbon nanoparticles,
peptide conjugated nanoparticles of iron oxide, and fibrin specific
cyclic peptide labelled with gadolinium. However, the quantity of
contrast agent that can be delivered, and therefore the intensity
of contrast effect achieved is relatively limited, particularly for
low-abundance targets.
[0106] Applicants have found that MPIOs carry a high payload of
contrast that is not readily dispersed and that is conspicuous on
MRI and propose the use of MPIOs for molecular imaging. The
versatility of this approach allows generic endovascular imaging of
vascular receptors even for different receptor conformations.
Sparse epitopes can be efficiently targeted by MPIOs despite their
size compared to the ligand itself as demonstrated herein,
simultaneously the high iron payload would allow detection of even
individual beads and therefore individual receptors depending upon
the MPIO-size. Furthermore, the phage display methods described
herein offer the possibility of constructing selective ligands to
sparse or functional epitopes, therefore allowing deep insights
into pathophysiological processes in various diseases. Other
important issues are the minimal immunogenicity of single chain
antibodies as they only consist of the variable regions, and the
size which facilitates the access to clandestine epitopes.
[0107] It will be understood that the probes described herein may
be used in methods for imaging a blood vessel abnormality in a
subject by detection of an activated platelet in a blood vessel. In
one form of the method, the detection includes the use of a probe
as described herein. It is contemplated that probes the probes are
useful with X-ray and CT apparatus found in a standard cardiac
catheterization laboratory. The 64-slice CT or the 128-slice CT is
proposed to be suitable for the imaging methods described herein.
The probes are also proposed to be useful in the context of
near-infrared spectrometric imaging (thermography) by using a
catheter to introduce the probe.
[0108] Abnormalities that may be detected include a ruptured
atherosclerotic plaque or an atherosclerotic plaque that is prone
to rupture, a thrombus, an embolus, and an accumulation of
activated platelets.
[0109] The invention will now be further described by reference to
the following non-limiting examples.
EXAMPLES
Example 1
Generation of the Single-Chain Antibody scFv.sub.anti-LIBS and the
Fusion Construct scFv.sub.anti-LIBS-TAP
[0110] The generation of the hybridoma cell line expressing a
monoclonal antibody against a LIBS epitope on GPIIb/IIIa and its
functional characterisation has been described earlier (Schwarz et
al. JPET 2004; 308: 1002). Briefly, GPIIb/IIIa purified and eluted
with RGD peptides was used as immunogen for hybridoma production.
Clones were screened with activated platelets as well as with
immobilized GPIIb/IIIa saturated with RGD peptides. One of these
clones, monoclonal antibody (mAb) clone 145 demonstrated increased
binding to ADP-activated platelets and to platelets pre-incubated
with RGD peptides (GRGDSP, BIOMOL Research Laboratories, Plymouth
Meeting, Pa.), eptifibatide (Integrilin.RTM., Essex Pharma,
Muenchen, Germany), tirofiban (Aggrastat.RTM., MSD, Whitehouse
Station, N.J.), and abciximab (ReoPro.RTM., Eli Lilly & Co,
Indianapolis, Ind.). The hybridoma was maintained in RPMI, 10%
fetal calf serum, 1 mM sodium pyruvat, 10 .mu.M mercaptoethanol,
100 units/ml penicillin, 100 g/ml streptomycin (all from Gibco),
and 1.times.HAT supplement (H0262, Sigma). The IgG.sub.anti-LIBS
mAb was prepared by affinity purification of hybridoma supernatant
using ImmunoPure.RTM. IgG Protein G purification (Pierce, Rockford,
Ill., USA).
[0111] For single-chain antibody cloning, cDNA of the hybridoma was
prepared using mRNA purification columns (oligo-dT) and M-MuLV
(both Amersham-Pharmacia, Freiburg, Germany). Amplification of the
antibody variable regions was achieved by polymerase chain reaction
(PCR) using Pfu.RTM. Polymerase (Strategene, La Jolla, Calif.,
USA). The following primers based on sequences from conserved
regions of the variable regions of the heavy (V.sub.H) and light
chain (V.sub.L) (Welschof et al. J Immunol Methods 1995; 179: 203)
were used: V.sub.H sense: 5'-CCG GCC ATG GCG CAG GTG CAG CTG CAG
CAG-3', V.sub.H antisense: 5'-CC AGG GGC CAG TGG ATA GAC AAG CTT
GGG TGT CGT TTT-3', V.sub.L sense: 5'-M TTT TCA GAA GCA CGC GTA GAT
ATC .sup.G/.sub.TTG .sup.A/.sub.CT.sup.G/.sub.C ACC CAA
.sup.T/.sub.ACT CC, V.sub.L antisense: 5'-GM GAT GGA TCC AGC GGC
CGC AGC ATC AGC-3'. The PCR constructs were cloned into the pHOG21
vector system (Kipriyanov et al. J Immunol Methods. 1997; 200: 69,
Schwarz et al. FASEB J 2004: 18: 1704) using the restriction sites
Nco I and Hind III for V.sub.H and the restriction sites Mlu I and
Not I for V.sub.L. The resulting single-chain antibody was termed
scFv.sub.anti-LIBS. TAP has been cloned previously (Hagemeyer et
al. Thromb Haemost. 2004; 92: 47) and was transferred to pHOG21
that already included scFv.sub.anti-LIBS using the restriction
sites Not I and Xba I, thereby creating scFv.sub.anti-LIBS-TAP
(FIG. 1A). As a control without binding function of the scFv part,
a non-targeted mut-scFv-TAP was generated that contains a
single-chain antibody, which originally bound to GP IIb/IIIa, but
its heavy-chain CDR3 (complexity determining region) was mutated
(RND to AND) and thereby its binding property was lost. All
construct were sequenced (FIG. 1B).
Example 2
Expression and Purification of scFv Constructs in E. coli
[0112] E. coli (TG1) cells were transformed with the pHOG21
plasmids described above and individual colonies from a freshly
streaked agar plate were grown in LB media containing 100 .mu.g/mL
ampicillin and 100 mM glucose at 37.degree. C. in 500 mL flasks.
Cultures were shaken at 200 rpm for approximate 4-6 hours until an
OD (600 nm) of .about.0.8 was reached. Bacteria were pelleted by
centrifugation at 5000 rpm for 10 min at 4.degree. C. and
resuspended with LB media containing 100 .mu.g/ml ampicillin and
0.4 M sucrose. IPTG was added to a final concentration of 0.25 mM
for induction of scFv production and incubated at room temperature
(22-24.degree. C.) with 200 rpm for 16-20 hours. For purification
of soluble protein from whole cell extract, bacteria were harvested
by centrifugation at 5000 rpm for 10 min at 4.degree. C. Pelleted
bacteria were resuspended in 5 mL 1.times. BugBuster.RTM. (Novagen,
Madison, USA) solution/g pellet and incubated for 15 min at room
temperature with gentle shaking. After an additional centrifugation
step at 15 000 rpm for 20 min at 4.degree. C., the supernatant
containing soluble protein was kept on ice and a protease inhibitor
(Complete.RTM. Roche, Basel, Switzerland) diluted 1:50 was added.
The supernatant containing soluble protein extract was mixed with
500 .mu.L Ni.sup.2+-Agarose (QIAGEN, Hilden, Germany) and incubated
for 1 hour at 4.degree. C. with constant shaking at 150 rpm.
Ni.sup.2+-Agarose, now binding His(6)-tagged proteins, was allowed
to settle for 30 min before washed with buffer (50 mM
NaH.sub.2PO.sub.4, 300 mM NaCl, 20 mM imidazole, pH 8). This batch
washing procedure was repeated twice. Finally, scFv fusion proteins
were eluted at high imidazole concentrations (250 mM) and
subsequently analyzed by gradient SDS-PAGE and Western blotting
under reducing conditions. Proteins were transferred onto an
Immobilon P membrane (Millipore Corporation, Bedford, USA) for
immunoblotting. After blocking the membrane overnight with
phosphate buffered saline containing 0.2% Tween20 (PBS-Tween) and
1% BSA, a HRP-labeled anti-His(6)-antibody (Roche, Mannheim,
Germany) was added (dilution 1:500) and incubated for 2 hours at
room temperature. The membrane was washed several times with
PBS-Tween buffer before visualization of peroxidase activity by
addition of SuperSignal.RTM. Chemiluminescent Substrate (Pierce,
Rockford, USA) on a ChemiDoc XRS.RTM. (BioRad, Regents Park, NSW,
Australia). As a size marker and His(6)-tag positive control a
6.times.His protein Ladder.RTM. (QIAGEN) was used.
Example 3
In Vitro Functional Characterization of the scFv Anti-LIBS-TAP
Blood Preparation
[0113] Human blood was collected by venipuncture with a 21-gauge
butterfly needle from healthy volunteers and anticoagulated with
citric acid. Platelet-rich plasma was obtained by centrifugation
(GS-6R centrifuge, Beckmann Coulter, Gladesville, NSW, Australia)
at 100.times.g in plastic tubes at room temperature for 10 min in a
centrifuge.
[0114] Mouse Blood was collected by intracardial puncture with a
27-gauge needle from C57BL/6 mice and anticoagulated with
unfractionated heparin (20 U/mL). A volume of 50 .mu.l was
resuspended with 1 mL modified Tyrode's buffer (150 mM NaCl, 2.5 mM
KCl, 1.2 mM NaHCO.sub.3, 2 mM MgCl.sub.2, 2 mM CaCl.sub.2, 0.1%
BSA, 0.1% Glucose) and centrifuged at 1300.times.g for 5 min. The
supernatant was discarded and the pellet was resuspended with 1 mL
modified Tyrode's buffer.
Flow Cytometry
[0115] Human citrated whole blood was diluted 1/50 in modified
Tyrode's buffer, either activated by addition of 20 .mu.M ADP or
non-activated and then preincubated for 10 min with 10 .mu.g/mL
IgG.sub.anti-LIBS, scFv.sub.anti-LIBS, and scFv.sub.anti-LIBS-TAP.
ScFvs were detected by a secondary antibody (Penta His Alexa Fluor
488 Conjugat.RTM., QIAGEN) directed against the Histidin(6)-tag of
the scFv. The IgG.sub.anti-LIBS was detected by a DTAF-conjugated
goat anti-mouse IgG+IgM (H+L) (Jackson Immuno Research, West Grove,
Pa., USA)
[0116] Mouse platelets were either activated by addition of 0.1
U/mL Thrombin (Enzyme Research Laboratories, South Bend, Ind., USA)
or not activated, and then incubated for 10 min with 10 .mu.g/mL
IgG.sub.anti-LIBS, scFv.sub.anti-LIBS, and scFv.sub.anti-LIBS-TAP.
Fluorescence detection was performed as described above. Samples
were measured in a FACSCalibur.RTM. flow cytometer (Becton
Dickinson, San Jose, Calif., USA), after fixation with CellFIX.RTM.
(Becton Dickinson).
Example 4
Anti-Factor Xa Activity Assay
[0117] Inhibition of fXa was determined by the degradation of the
chromogenic substrate spectrozyme fXa #222 (American Diagnostica
Inc., Greenwich, Conn., USA). Probes were dialyzed against modified
Tyrode's buffer (150 mM NaCl, 2.5 mM KCl, 12 mM NaHCO.sub.3, 2 mM
MgCl.sub.2, 2 mM CaCl.sub.2, pH 7.4) and adjusted to get 100 nM of
scFv.sub.anti-LIBS, scFv.sub.anti-LIBS-TAP and non-targeted
mut-scFv-TAP, or free recombinant TAP in a final volume of 165
.mu.l. After adding 10 .mu.l 0.1% human albumin, probes were mixed
with 10 .mu.L of 500 .mu.M fXa (Haemochrom, Enzyme Research
Laboratories) and compared to fXa alone as positive control. After
incubation at room temperature for 10 min, 15 .mu.L chromogenic
substrate solution (5 mM) was added and plates were incubated for
15 minutes at room temperature. Finally, reaction was terminated by
adding 50 .mu.l stop-solution and absorption was measured at 405 nm
in an ELISA reader (Victor.sup.3.RTM., Perkin Elmer, Melbourne,
Australia).
Example 5
In Vivo Functional Evaluation of Antithrombotic Efficacy and
Protection from Bleeding in a Mouse Model
[0118] C57BL/6 mice with weights 22-38 g (Charles River
Laboratories, Wilmington, Mass., USA) were used for the present
study. Care and use of laboratory animals followed the national
guidelines and was approved by the institutional animal care and
ethics committee at the University of Freiburg and at the Baker
Heart Research Institute. Mice were anesthetized with Isoflurane
using a exicator for a few seconds and i.p. injection with Ketamin
(Ketanest.RTM. 100 mg/kg BW) and Xylazin (Rompun.RTM. 5 mg/kg BW)
and placed under a dissecting microscope. After absence of any
reflexes, an incision of the skin was made directly on the top of
the right common carotid artery region. The fascia was bluntly
dissected and a segment of the right common carotid artery was
exposed. Then a nano doppler flow probe (Model 0.5 VB, Transonic
Systems, Ithaca, N.Y., USA) was positioned over the artery and the
carotid blood flow was measured by a flow meter (model T106,
Transonic Systems, Ithaca, N.Y., USA). Thrombosis was induced by
applying a piece of filter paper (1.times.2 mm) (Gel Blot Paper,
GB003, Schleicher and Schuell, Keene, N.H., USA) saturated with
ferric chloride (10% solution) (Sigma, St. Louis, Mo., USA) under
the right carotid artery and removed after 3 min. Thrombotic
occlusion was considered to occur when flow decreased to 0.0.+-.0.2
mL/min, a range corresponding to the accuracy of the system as
specified by the manufacturer.
[0119] One minute prior the ferric chloride treatment, mice were
infused through the tail vein with saline (negative control) (0.9%
sodium chloride) 100 .mu.l, enoxaparin (positive control)
(Clexane.RTM. Sanofi Aventis, Paris, France) with 1 mg/kg BW
diluted with saline to volume of 100 .mu.l, and with various doses
of purified recombinant scFv.sub.anti-LIBS-TAP, scFv.sub.anti-LIBS,
non-targeted mut-scFv-TAP, and rTAP. All doses of used scFvs and
rTAP were dissolved to a volume of 100 .mu.l. To ensure that all
drugs were infused in mice the aditus was flushed with 100 .mu.l
saline.
[0120] Mouse bleeding time was measured as described previously by
Xinkang. The anesthetized mice were placed under the dissecting
microscope. About 1-2 mm from the tip of the mouse tail (in about 1
mm diameter) a cut was made with a disposable surgical blade. The
time at which the tail first stopped bleeding for more than 30 sec
was recorded in seconds.
Statistical Analysis
[0121] Data are presented as mean.+-.standard deviations for the
indicated number (N) of mice. The statistical comparisons were made
by analysis of variance (ANOVA following a Newmann-Keuls-test) and
differences were considered to be significant at p<0.05.
Example 6
Use of Labelled Single-Chain Antibodies for Diagnostic Imaging
[0122] The single-chain antibody anti-LIBS as described in Example
1 were mixed with Superparamagnetic Iron Oxide particles (SPIOs)
that were functionalized to interact with proteins that include a
His-tag (Dynabeads.RTM. TALON.TM.; Dynal Biotech). Other methods
(e.g. chemical coupling) to couple antibodies to paramagnetic beads
can also be used. Binding of the contrast agent to activated
platelets (which are a major and essential constituent of thrombi
and emboli) was demonstrated by an adhesion assay. The activation
of platelets used in the assay was monitored by fluorescence
microscopy demonstrating the upregulation of P-selectin expression
on the platelet surface as well as with fluorescence detection of
an increase in intracellular Ca.sup.2+ level. The assay involved
the immobilization of activated platelets on a fibrinogen-covered
cover slip by incubating the platelets for 30 minutes at 37.degree.
C. After washing of the cover slips, the fibrinogen-platelet matrix
was exposed to the contrast agent for 30 minutes. In order to
exclude unspecific binding and to demonstrate binding of the
contrast agent to platelets only, a co-staining of platelets was
performed using P-selectin antibodies and secondary staining with
fluorescein-avidin (FIG. 9). Using confocal microscopy, binding of
the red-appearing auto-fluorescent contrast agent to platelets was
demonstrated by simultaneous green fluorescence of the
P-selectin-stained platelets. A 3D-reconstruction using the z-stack
from confocal microscopy is shown in FIG. 10.
[0123] Furthermore, to assess the suitability of the imaging method
in vitro, magnetic resonance experiments were performed to show
binding of single-chain antibody to activated platelets on thrombus
surfaces. For these experiments, human thrombi were generated
artificially by adding actin, adenosine diphosphate and calcium
chloride to human platelet rich plasma and incubating the mixture
for 15-30 minutes at 37.degree. C. Thrombi were exposed to
different concentrations of the contrast agent and incubated for
another 30 minutes at 37.degree. C. Finally, the thrombi were
washed twice in PBS buffer and fixed with 4% paraformaldehyde.
After 4 hours of fixation, the thrombi were embedded into wells of
a 24-well cell-culture plate, surrounded by gadolinium-spiked 2%
agarose. Magnetic Resonance Imaging (MRI) was performed on a 3
Tesla clinical scanner, employing the standard wrist coil. A 3D
FLASH sequence with TE/TR 9.3 ms/700 ms was run with a resolution
of 130.times.130.times.200 .mu.m and images were reconstructed
perpendicular to the longitudinal axes of the clots in an overnight
run. Negative contrast, as caused by SPIO in T2*weighted MRI, was
observed as a black ring around the thrombi incubated with the
LIBS-targeted antibody in a dose-dependent manner (FIG. 11).
Furthermore, bead binding was confirmed using immunohistochemistry
with an anti-P-selectin antibody and staining with NovaRed in
paraffin-embedded sections: FIG. 4 shows conglomerates of platelets
(brown) with binding of beads (yellow) to areas with platelets.
These results indicate successful binding of the designed contrast
agent to activated platelets in vitro, which can be detected with
clinically relevant field strengths by MRI.
Example 7
Use of Labelled Single-Chain Antibodies for Diagnostic Imaging in a
Mouse Model
Single-Chain Antibody Generation and Conjugation to 1 .mu.m Iron
Oxide Microparticles
[0124] The LIBS epitope on GPIIb/IIIa represents an abundant and
highly specific target for activated platelets. The mAb anti-LIBS
145 binds to GPIIb/IIIa only in its active conformation and it
demonstrates strong binding to ADP-activated platelets in the
presence of fibrinogen (Schwarz JPET 2004). The mAb anti-LIBS 145
expressing hybridoma cell line was used as the basis for the
cloning of an anti-LIBS single-chain antibody (scFv). mRNA of this
hybridoma cell line was prepared and reverse transcribed using an
oligo-dT primer. The variable regions of the antibody's heavy and
light chain were amplified by PCR using primers that anneal to
conserved regions at the 5' and 3' ends of the variable regions.
The PCR products were cloned into the pHOG21 vector, TG1 E. coli
were transformed, and individual clones were assessed for
LIBS-typical binding to GPIIb/IIIa was tested in flow cytometry
with activated platelets. Finally the best binding scFv.sub.LIBS
was produced in LB media containing 100 .mu.g/mL ampicillin and 100
mM glucose at 37.degree. C. in 500 mL flasks. Cultures were shaken
at 200 rpm for approximate 4-6 hours until an OD (600 nm) of
.about.0.8 was reached. Bacteria were pelleted by centrifugation at
5000 rpm for 10 min at 4.degree. C. and resuspended with LB media
containing 100 .mu.g/ml ampicillin and 0.4 M sucrose. IPTG was
added to a final concentration of 0.25 mM for induction of scFv
production and incubated at room temperature (22-24.degree. C.)
with 200 rpm for 16-20 hours. Bacteria were harvested by
centrifugation at 5000 rpm for 10 min at 4.degree. C., the pelleted
bacteria were resuspended in 5 mL 1.times. BugBuster.RTM. (Novagen)
solution/g pellet and incubated for 15 min at room temperature with
gentle shaking. After an additional centrifugation step at 15 000
rpm for 20 min at 4.degree. C., the supernatant containing soluble
protein was kept on ice and a protease inhibitor (Complete.RTM.
Roche) diluted 1:50 was added. The supernatant was mixed with 500
.mu.L Ni.sup.2+-Agarose (Qiagen) and incubated for 1 hour at
4.degree. C. with constant shaking at 150 rpm. Ni.sup.2+-Agarose,
now binding His(6)-tagged proteins, was allowed to settle for 30
min before washed with buffer (50 mM NaH.sub.2PO.sub.4, 300 mM
NaCl, 20 mM imidazole, pH 8). This batch washing procedure was
repeated twice. Finally, the scFv was eluted at high imidazole
concentrations (250 mM) and dialyzed. Functionality of the scFv
preparations were evaluated in flow cytometry.
[0125] Autofluorescent cobalt-functionalized MPIOs (1 .mu.m) were
conjugated to the histidine-tag of the LIBS single-chain antibody
referring to the protocol of the manufacturer (Dynal Biotech, Oslo,
Norway). In brief, after washing 1 mg of beads was incubated with
the LIBS antibody for 10 min at room temperature (RT) to bind
approximately 10 .mu.g of histidine-tagged antibody. The tube
containing the suspension was then placed on a magnet until the
beads had migrated to the side of the tube and the supernatant
discarded. This washing was repeated four times using a binding and
washing buffer containing 50 mM NaP (pH 8), 300 mM NaCl and 0.01%
Tween-20.
LIBS-MPIO Binding to Activated Platelets
[0126] An adhesion assay was performed to demonstrate binding of
the LIBS-MPIO to activated platelets. Blood from a healthy
volunteer taking no medication was anticoagulated with citric acid
and centrifuged at 1000 rpm for 10 min. The resulting platelet rich
plasma was diluted with PBS (1:10) and 100 .mu.l added onto
fibrinogen-covered cover slips, which had been preincubated with 20
.mu.g/ml fibrinogen for 1 hour at 38.degree. C. and blocked with 1%
BSA for 1 hour at room temperature. After 30 min incubation at
38.degree. C., cover-slips were washed with PBS and under
continuous rotation either incubated with 0.5 .mu.g of LIBS-MPIO
(LIBS-MPIO) or an equivalent conjugated irrelevant single-chain
antibody control (Control-MPIO) for another 30 min at 38.degree. C.
Cover-slips were then washed twice for 5 min with PBS and blocked
with 10% goat-serum (Vector, Burlingame, Calif./USA) for 1 hour at
RT. To demonstrate specific binding of the contrast agent,
platelets were co-stained for P-selectin using a monoclonal mouse
anti-human CD62 antibody (1:100, R&D Systems, Abingdon, UK)
with a biotinylated goat anti-mouse IgG (Vector, Burlingame,
Calif./USA) serving as secondary antibody. Finally, 1:200 diluted
Fluorescin Avidin D (Vector, Burlingame, Calif./USA) was added and
incubated for 1 hour and RT. Cover-slips were fixed using CellFix
(BD Biosciences, Heidelberg, Germany) and evaluated by confocal
microscopy.
Mice
[0127] Wire injury was performed in male C57BL/6 mice weeks
(Jackson Laboratories, UK), mean age of 10.+-.0.8 weeks. Mice
received water and standard chow diet ad libitum. All procedures
were performed in accordance with the UK Home Office Animals
(Scientific Procedures) Act 1986.
Femoral Wire Injury, Bead Perfusion and Sample Preparation
[0128] Single-sided femoral wire injury was performed under general
anesthesia, using a combination of Hypnorm (25 mg/kg, Bayer,
Germany) and Hypnoval (25 mg/kg, Bayer, Germany) administered
subcutaneously, as described previously (Roque, M., et al., Mouse
model of femoral artery denudation injury associated with the rapid
accumulation of adhesion molecules on the luminal surface and
recruitment of neutrophils. Arterioscler Thromb Vasc Biol, 2000.
20(2): p. 335-42). Under a surgical microscope, a groin incision
was made. The femoral artery was exposed, and an arteriotomy was
made distal to the epigastric branch using 30G injection cannula
(BD, Erembodegem, Belgium). A 0.010'' guidewire (Boston Scientific,
Natick, USA) was inserted, advanced to the aortic bifurcation and
pulled back. After removal of the wire, the arteriotomy site was
ligated and the skin closed using silk sutures. After 24 hours,
mice were terminally anesthetized by inhalation of isoflurane. The
chest was opened by thoracotomy, the heart exposed and the right
atrium cut. A 30G needle was inserted through the apex of the left
ventricle and the animal perfused with 10 ml of PBS to eliminate
the blood. Perfusion was continued with 5 mL PBS containing either
LIBS-MPIO or control-MPIO (1.5.times.10.sup.8 beads/ml for each).
After 30 minutes, mice were again perfused under physiological
pressure with 10 mL PBS followed by 5 mL 4% Paraformaldehyde (PFA)
containing 2 mM gadoteridiol (Prohance, Bracco, UK). The skin was
removed, the leg with the area of injury cut, kept in 4% PFA/2 mM
gadoteridiol for 24 hours and then embedded in a glass MR tube
containing 2% high-grade, with low melting point agarose. (Cambrex,
Rockland, Me./USA).
Ex Vivo MRI
[0129] Ex vivo MRI was performed at 11.7 T using a 13 mm .sup.1H
birdcage radiofrequency coil (RAPID Biomedical, Wurzburg, Germany).
A 3D gradient echo sequence (TE=4 ms/TR=90 ms, field of view
13.times.13.times.19.5 mm, matrix size 256.times.256.times.384, two
averages, imaging time .about.7 h per sequence) was used in an
unattended overnight run. Data reconstruction was performed
off-line with a final isotropic resolution of 25 .mu.m.sup.3.
Histology and Quantification of MPIO Binding in the Injured Femoral
Artery
[0130] After MRI, specimens were decalcified in 10% Formic Acid
overnight, dehydrated through graded ethanol solutions and Neoclear
(VMR, UK), paraffin embedded and serially sectioned (8 .mu.m
thick). Specimens were stained for iron (Accustain, Sigma, Germany)
referring to the manufacturers protocol. The number of conjugated
MPIOs bound to the injured luminal vessel wall was quantified and
averaged in 20-25 sections per animal from the injured vessel site
using light microscopy.
[0131] For platelet visualization with immunohistochemistry,
deparaffinized and rehydrated sections were saturated in 1%
H.sub.2O.sub.2 for 20 min, added to simmering citrate buffer and
boiled for 4 min in a pressure cooker for antigen retrieval.
Specimens were washed in PBS Tween, incubated with protein block
solution (DakoCytomation, Hamburg, Germany) for 4 hours, and
incubated overnight at 4.degree. C. with rat anti-mouse CD61
antibody (1:8000, InterCell Technologies, Fl/USA). After washing
with PBS, biotinylated goat anti-hamster IgG (1:200, Vector,
Burlingame, Calif./USA) secondary antibody. Slides were washed with
PBS, and peroxidase reaction was performed using Vectastain RTU
Elite ABC-reagent and Vector NovaRed (both Vector, Burlingame,
Calif./USA). Finally, sections were deyhdrated, mounted with
Permount (Biomeda, Foster City, Calif./USA) and bead binding to
platelets was evaluated on a light microscope.
MPIO Binding in the Femoral Artery by Ex Vivo MRI
[0132] Quantification of the MPIO binding was performed blinded.
Antibody-conjugated MPIO binding was defined as a clear circular
signal void on the luminal surface of the femoral artery in >2
consecutive slices. MPIOs appearing in multiple sections were
counted only once. Segmented images were reconstructed in three
dimensions using the 3D Constructor plug-in for ImagePro Plus to
visualize the distribution of MPIO binding throughout the femoral
artery.
Statistical Methods
[0133] Data are expressed as mean.+-.standard deviation. Parametric
data were compared using t-tests. Statistical significance was
assigned to P<0.05.
Results
LIB-MPIO Detects Activated Glycoprotein IIb/IIIa Receptors on
Platelets
[0134] In FIG. 13, human platelet thrombi labeled with
anti-P-selectin antibody fluorescence bright green. Superimposed on
the platelet thrombi are red areas corresponding to autofluorescent
LIBS-MPIO (Panel A). The MPIOs are confined to the platelet thrombi
without non-specific background retention. By contrast, in Panel B,
there is complete absence of binding of control-MPIO conjugated to
an irrelevant single chain antibody. A 3D z-stack reconstruction in
confocal microscopy shows LIBS-MPIO binding (red) to
P-selectin-stained platelets (green), emphasizing their relative
size and spatial relations.
LIBS-MPIO Bound to Wall-Adherent Platelets Detected by Ex Vivo
MRI
[0135] Unilateral femoral artery wire-injury was performed in 13
mice without complication. Seven mice were perfused with LIBS-MPIO
and 6 with control-MPIO via the left ventricle. One control animal
was excluded from the quantification analysis, of marked variation
in the quantification of MPIOs between two observers.
[0136] Ex vivo T2*-weighted MRI of injured arterial segment often
demonstrated intrinsic low signal areas within the arterial wall
(FIG. 14B). Distinct from this was the appearance of circular
signal voids within the vessel lumen but adjacent to the vessel
wall. This feature was observed in the wire-injured arteries of all
mice injected with LIBS-MPIO (FIG. 14A). In quantitative analysis
luminal areas of low signal suggesting MPIO-accumulation were
significantly higher in LIBS-MPIO injected animals than in
control-MPIO perfused animals (23.72 vs. 6.2; P<0.01, FIG.
14C).
[0137] MPIO binding was confirmed in histology (FIG. 15), with
significantly higher MPIO-binding in LIBS-MPIO injected animals
(9.98 vs. 0.5 beads per section, P<0.01; FIG. 15D).
Colocalization of MPIOs and platelet adhenerce to the arterial wall
was confirmed by immunohistochemistry. In FIG. 15C, MPIOs are
demonstrated to be present in association with positive
immunostaining for the platelet marker CD61.
[0138] An analysis of bead quantification in histology compared to
quantification by ex vivo MRI revealed a strong correlation
(R.sup.2=0.7219, P<0.001; FIG. 17). Therefore, MPIO signal
quantity determined by MRI directly reflected the quantity of MPIOs
bound to the injured vessel wall.
[0139] Finally, it is to be understood that various other
modifications and/or alterations may be made without departing from
the spirit of the present invention as outlined herein.
* * * * *