U.S. patent application number 10/310113 was filed with the patent office on 2006-10-19 for use of anti-tissue factor antibodies for treating thromboses.
Invention is credited to Jin-An Jiao, Luis A. Mosquera, Esperanza Liliana Nieves, Hing C. Wong.
Application Number | 20060235209 10/310113 |
Document ID | / |
Family ID | 46281656 |
Filed Date | 2006-10-19 |
United States Patent
Application |
20060235209 |
Kind Code |
A9 |
Jiao; Jin-An ; et
al. |
October 19, 2006 |
Use of anti-tissue factor antibodies for treating thromboses
Abstract
Disclosed is a method for preventing or treating thrombosis in a
mammal such as a primate and particularly a human patient. A
preferred method includes administering to the mammal a
therapeutically effective amount of at least one humanized
antibody, chimeric antibody, or fragment thereof that binds
specifically to human tissue factor (TF). Additional methods and
kits are provided.
Inventors: |
Jiao; Jin-An; (Fort
Lauderdale, FL) ; Wong; Hing C.; (Fort Lauderdale,
FL) ; Nieves; Esperanza Liliana; (Newark, DE)
; Mosquera; Luis A.; (Miami, FL) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Prior
Publication: |
|
Document Identifier |
Publication Date |
|
US 20030176664 A1 |
September 18, 2003 |
|
|
Family ID: |
46281656 |
Appl. No.: |
10/310113 |
Filed: |
December 4, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09990586 |
Nov 21, 2001 |
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10310113 |
Dec 4, 2002 |
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09293854 |
Apr 16, 1999 |
6555319 |
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10310113 |
Dec 4, 2002 |
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08814806 |
Mar 10, 1997 |
5986065 |
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09293854 |
Apr 16, 1999 |
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60343306 |
Oct 29, 2001 |
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Current U.S.
Class: |
530/389.2 ;
424/131.1 |
Current CPC
Class: |
A61K 2039/505 20130101;
C07K 16/467 20130101; A61P 7/02 20180101; C07K 2319/00 20130101;
C07K 2317/24 20130101; C07K 16/36 20130101 |
Class at
Publication: |
530/389.2 ;
424/131.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 16/42 20060101 C07K016/42 |
Claims
1. A method for preventing or treating a thrombosis in a mammal,
the method comprising administering to the mammal a therapeutically
effective amount of at least one antibody, or fragment thereof that
binds specifically to human tissue factor (TF) to form a complex,
the administration being sufficient to prevent or treat the
thrombosis in the mammal, wherein the antibody or the fragment has
a binding specificity for the TF about equal or greater than the
antibody obtained from cell line H36.D2.B7 deposited under ATCC
Accession No. HB-12255
2. The method of claim 1, wherein after administration of the
antibody or fragment, the mammal exhibits a blood clotting time of
between from about 50 to about 350 seconds as determined by a
standard prothrombin (PT) time assay.
3. The method of claim 1, wherein the amount of the administered
antibody, or fragment is sufficient to inhibit platelet deposition
time by at least about 50% as determined by a standard platelet
deposition assay.
4. The method of claim 1, wherein the amount of the administered
antibody, or fragment reduces platelet deposition as determined by
vessel injury to blood ratio by at least about 50% as determined by
a standard vessel injury to blood ratio assay.
5. The method of claim 1, wherein the amount of the administered
antibody, or fragment increases vessel patency by at least about
100%.
6. The method of claim 1, wherein the mammal is a primate.
7. The method of claim 6, wherein the primate is a human
patient.
8. The method of claim 1-5, wherein the antibody is a humanized or
chimeric antibody and the fragment is a Fab, Fab', F(ab').sub.2
where the fragment can be derived from a humanized or chimeric
antibody or single chain Fv engineered using the variable domains
of an antibody.
9. The method of claim 1, wherein the antibody or the fragment
exhibits at least one of: 1) a dissociation constant (K.sub.d) for
the TF of less than about 0.5 nM ; and 2) an affinity constant
(K.sub.a) for the TF of less than about 10.times.10.sup.10
M.sup.-1.
9. The method of claim 1, wherein the humanized antibody used in
the method comprises at least one fully murine complimentarity
determining region (CDR).
10. The method of claim 9, wherein the humanized antibody further
comprises at least one fully human framework (FR) region.
11. The method of claim 10, wherein the humanized antibody has at
least about 90% amino acid sequence identity to a human
antibody.
12. The method of claim 10, wherein the variable region of the
humanized antibody has at least about 70% amino acid sequence
identity to a human antibody variable region.
13. The method of claim 10, wherein each of frameworks (FRs) 1, 2,
3 and 4 of the humanized antibody has at least about 95% amino acid
sequence identity to the light chain FR sequences shown in FIG. 6A
(SEQ ID NO. ___).
14. The method of claim 10, wherein the antibody comprises a light
chain constant region having at least about 95% amino acid sequence
identity to the sequence shown in FIG. 8A or 9A (SEQ ID NO.
___).
15. The method of claim 10, wherein each of frameworks (FRs) 1, 2,
3 and 4 of the humanized antibody has at least about 95% amino acid
sequence identity to the heavy chain sequences shown in FIG. 7A
(SEQ ID NO. ___).
16. The method of claim 10, wherein the antibody further comprises
a heavy chain constant region having at least about 95% amino acid
sequence identity to sequence shown in FIG. 8B or 9B (SEQ ID NO.
___).
17. The method of claim 1, wherein the humanized antibody has an
IgG1 (hOAT) or IgG4 (hFAT) isotype.
18. The method of claim 1, wherein the human TF binding fragment is
Fab, Fab', or F(ab).sub.2.
19. The method of claim 1, wherein the humanized antibody is a
monoclonal antibody.
20. The method of claim 1, wherein the humanized antibody is a
single-chain.
21. A method for performing plastic, reconstructive, or transplant
surgery in a mammal, the method comprising introducing a graft into
the mammal and contacting the graft with a therapeutically
effective amount of at least one humanized antibody, chimeric
antibody, or fragment thereof that binds specifically to human
tissue factor (TF) to form a complex, the contact being sufficient
to maintain or increase patency of the graft, wherein the humanized
antibody, chimeric antibody or the fragment has a binding
specificity for the TF about equal or greater than the antibody
obtained from cell line H36.D2.B7 deposited under ATCC Accession
No. HB-12255
22. The method of claim 21 wherein the amount of the humanized
antibody, chimeric antibody or fragment administered to the mammal
is sufficient to prevent or reduce thrombosis by at least about 50%
as determined by a standard skin flap assay.
23. The method of claim 21, wherein the method further comprises
preventing or reducing devascularization of the graft as determined
by a standard skin flap assay.
24. The method of claim 21, wherein the method further comprises
preventing or reducing at( least one of edema, erythema, and
necrosis in the graft.
25. The method of claim 1 or 21, wherein the amount of the
humanized antibody, chimeric antibody or fragment administered to
the mammal is between about 0.01 to about 25 mg/kg.
26. The method of claim 1, wherein the thrombosis is associated
with cardiovascular disease.
27. The method of claim 26, wherein the cardiovascular disease is
at least one of coronary artery disease, acute coronary syndrome,
and atherosclerosis.
28. The method of claim 1, wherein the thrombosis is associated
with angioplasty or restenosis.
29. A kit for performing the method of claim 1 or 21, wherein the
kit includes at least one of the humanized antibody, chimeric
antibody, or fragment thereof and optionally, directions for using
the kit.
30. The kit of claim 29, wherein the humanized antibody, chimeric
antibody, or fragment thereof is provided in a pharmaceutically
acceptable vehicle.
31. The kit of claim 29, wherein the humanized antibody, chimeric
antibody, or fragment thereof is lyophilized and the kit further
includes a pharmaceutically acceptable vehicle for dissolving the
humanized antibody, chimeric antibody or fragment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 09/990,586 entitled Antibodies For Inhibiting
Blood Coagulation and Methods of Use Thereof by Jiao, J. et al. as
filed on Nov. 21, 2001, which application claims priority to U.S
Provisional Application U.S. Ser. No. 60/343,306 as filed on Oct.
29, 2001. The present application claims further priority to U.S.
Ser. No. 09/293,854 as filed on Apr. 16, 1999 which application is
a continuation of U.S. Ser. No. 08/814,806 (now U.S. Pat.
No.5,986,065) as filed on Mar. 10, 1997 and published as PCT
Application No. PCT/US98/04644 (WO 98/40408). The disclosures of
said U.S. application Ser. Nos. 09/990,586; 60/343,306; 09/293,854;
08/814,806; and WO 98/40408 are each incorporated herein by
reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to novel human tissue factor
antibodies and methods of using the antibodies to inhibit tissue
factor-related functions such as blood coagulation, angiogenesis,
tumor growth and metastasis, and inflammation. In particular, the
invention relates to novel antibodies that can specifically bind
native human tissue factor with high affinity and prevent factor X
or factor IX binding and activation. The antibodies of the
invention are useful for a variety of applications, particularly
for reducing thrombosis in vivo.
[0004] 2. Background
[0005] Blood clotting assists homeostasis by minimizing blood loss.
Generally, blood clotting requires vessel damage, platelet
aggregation, activation of coagulation factors, and inhibition of
fibrinolysis. The coagulation factors act through a cascade that
relates the vessel damage to formation of a blood clot (see
generally L. Stryer, Biochemistry, 3rd Ed, W. H. Freeman Co., New
York; and A. G. Gilman et al., The Pharmacological Basis of
Therapeutics, 8th Edition, McGraw Hill Inc., New York, pp.
1311-1331).
[0006] There is general agreement that factor X (FX) activation to
factor Xa (FXa) (or factor IX activation to factor IXa) is a
critical step in the blood coagulation process. Generally, FX (or
FIX) is converted to FXa (or FIXa) by binding a catalytically
active complex that includes "tissue factor" (TF). TF is a
controllably-expressed cell membrane protein that binds factor
VII/VIIa (FVII/FVIIa) to produce the catalytically active complex
(TF:FVIIa). A blood clot follows FXa-mediated activation of
prothrombin to thrombin, which then converts fibrinogen to fibrin
and activates platelets. Blood clotting can be minimized by
inactivation of TF to non-native forms which cannot optimally
produce the TF:FVIIa complex. Excessive activation of the
coagulation cascade through formation of FXa (or FIXa) by TF:FVIIa
complex is believed to contribute to various thromboses including
restenosis.
[0007] Thrombosis may be associated with invasive medical
procedures including but not limited to cardiac surgery (e.g.
angioplasty), abdominothoracic surgery, arterial surgery,
peripheral vascular bypass grafts or coronary artery bypass grafts,
deployment of an implementation (e.g., a stent or catheter),
arterio-venous shunts or fistulas, reconstructive or plastic
surgery or endarterectomy. Further, thrombosis may accompany
various thromboembolic disorders and coagulopathies such as stroke,
pulmonary embolism (e.g., atrial fibrillation with embolization),
coronary artery disease or acute coronary syndromes (e.g., unstable
angina or myocardial infarction), atherosclerosis or other
thrombo-occlusive disorders, deep vein thrombosis and disseminated
intravascular coagulation, etc. Manipulation of body fluids can
also result in an undesirable thrombus, particularly in blood
transfusions or fluid sampling, as well as procedures involving
extracorporeal circulation (e.g., cardiopulmonary bypass surgery)
and renal dialysis.
[0008] More generally, thromboses particularly amenable to
prevention or treatment include those associated with
cardiovascular disease, for instance, coronary artery disease,
acute coronary syndrome, and atherosclerosis. Other particular
thromboses include those associated with angioplasty or
restenosis.
[0009] Anti-coagulants are frequently used to alleviate or avoid
blood clots associated with thrombosis. Blood clotting often can be
minimized or eliminated by administering a suitable anti-coagulant
or mixture thereof, including one or more of a coumarin derivative
(e.g., warfarin, Coumadin or dicumarol) or a charged polymer (e.g.,
heparin, low molecular weight heparin, pentosan, hirudin or
hirulog) or anti-platelet agents (e.g., ReoPro, Integrilin,
Aggrestat, Plavix, Ticlid or aspirin). See e.g., Gilman et al.,
supra, R. J. Beigering et al., Ann. Hematol., 72:177 (1996); J. D.
Willerson, Circulation, 94:866 (1996).
[0010] However, use of anti-coagulants is often associated with
side effects such as hemorrhaging, re-occlusion, "white-clot"
syndrome, irritation, birth defects, thrombocytopenia and hepatic
dysfunction. Long-term administration of anti-coagulants can
particularly increase risk of life-threatening illness (see e.g.,
Gilman et al., supra).
[0011] Certain antibodies with anti-platelet activity have also
been used to alleviate various thromboses. For example, ReoPro.RTM.
is a therapeutic antibody fragment that is routinely administered
to alleviate various thromboembolic disorders such as those arising
from angioplasty, myocardial infarction, unstable angina and
coronary artery stenoses. Additionally, ReoPro.RTM. can be used as
a prophylactic to reduce the risk of myocardial infarction and
angina (J. T. Willerson, Circulation, 94:866 (1996); M. L. Simmons
et al., Circulation, 89:596 (1994)).
[0012] Certain anti-coagulant antibodies are also known.
Particularly, certain TF-binding antibodies have been reported to
inhibit blood coagulation, presumably by interfering with assembly
of a catalytically active TF:FVIIa complex (see e.g., Jeske et al.,
SEM in THROM. and HEMO, 22:213 (1996); Ragni et al., Circulation,
93:1913 (1996); European Patent No. 0 420 937 B1; W. Ruf et al.,
Throm. Haemost., 66:529 (1991); M. M. Fiorie et al., Blood, 8:3127
(1992)).
[0013] However, current TF-binding antibodies exhibit significant
disadvantages which can minimize their suitably as anti-coagulants.
For example, current TF-binding antibodies do not exhibit
sufficient binding affinity for optimal anti-coagulant activity.
Accordingly, for many thrombotic conditions, to compensate for such
ineffective binding affinities, unacceptably high antibody levels
must be administered to minimize blood coagulation.
[0014] It would thus be desirable to have an anti-coagulant
antibody that binds native human TF with high affinity and
selectivity to thereby inhibit undesired blood coagulation and the
formation of blood clots. It would be further desirable to have
such an anti-coagulant antibody that prevents the binding of factor
X (or factor IX) to TF:FVIIa complex.
SUMMARY OF THE INVENTION
[0015] The present invention features methods for preventing or
treating thrombosis in a mammal. In general, the invention methods
feature use of humanized immunoglobins including partially
humanized antibodies such as chimeric antibodies. Also envisioned
are use of fragments of such immunoglobins, preferably those that
bind tissue factor (TF) specifically. Practice of the invention has
a variety of important uses including reducing potentially
life-threatening vascular thromboses in primates and especially
human subjects.
[0016] Particular methods of the invention help prevent or treat
one or a combination of vascular thromboses, particularly those
impacting arteries and associated vasculature. Additionally
preferred methods help to maintain vessel patency often without
significantly impairing homeostatic function. Preferred use of the
invention provides an especially effective means to block
thrombosis at the initial stage of the blood coagulation cascade.
Internal bleeding complications often associated with standard
anti-thrombotic therapies is reduced or avoided by the
invention.
[0017] Accordingly, and in one aspect, the invention provides a
method for preventing or treating a thrombosis in the mammal. In
one embodiment, the method includes administering to the mammal a
therapeutically effective amount of at least one humanized
antibody, chimeric antibody; or fragment of such antibodies that
binds specifically to human TF. Preferred binding forms a specific
complex in which factor X or factor IX binding thereto and the FX
or FIX activation by TF:VIIa are inhibited. Preferred
administration of the humanized antibodies, chimeric antibodies and
fragments are sufficient to prevent or treat one or more thromboses
in the mammal.
[0018] In another aspect, the invention provides a method for
performing plastic, reconstructive, or transplant surgery in the
mammal. In one embodiment, the method includes introducing a graft
into the mammal and contacting the graft with a therapeutically
effective amount of at least one humanized antibody, chimeric
antibody, or fragment of the humanized antibody or chimeric
antibody that binds specifically to human TF to form a specific
binding complex. Typical contact with the graft is generally
sufficient to maintain or increase patency of the graft as
determined by conventional methods such as inspection.
[0019] Particular humanized antibodies for use with the invention
can be monoclonal. Most specifically bind a conformational epitope
predominant to native human TF, usually with strong binding
affinity. Indeed, preferred humanized antibodies of the invention
bind to native human TF at least about 5 times greater, more
typically at least about ten times greater than the binding
affinity exhibited by certain current anti-TF antibodies (see e.g.,
Jeske et al., SEM in THROM. and HEMO, 22:213 (1996); Ragni et al.,
Circulation, 93:1913 (1996); European Patent No. 0 420 937 B1; W.
Ruf et al., Throm. Haemost., 66:529 (1991); M. M. Fiorie et al.,
Blood, 8:3127 (1992)). Additionally, preferred humanized antibodies
of the invention are specific for native human TF, and do not
substantially bind non-native or denatured TF.
[0020] Additionally preferred humanized antibodies, chimeric
antibodies, and fragments thereof include at least one
hypervariable region from a murine monoclonal antibody, preferably
the H36.D2.B7 monoclonal (secreted by hybridoma ATCC HB-12255 and
often referred to as H36). More preferred humanized immunoglobins
and fragments for use with the invention include multiple
hypervariable regions of the H36.D2.B7 antibody including all of
same. Specifically preferred antibodies for use with the invention
have a binding specificity for the TF about equal or greater than
the antibody obtained from the ATCC deposited cell line H36.D2.B7.
Nucleic acid and amino acid sequences (SEQ ID:NOS ) of the
hypervariable regions of the murine H36.D2.B7 antibody are set
forth in FIGS. 1A and 1B.
[0021] Practice of the invention is compatible with use of a wide
variety of immunoglobins, specifically humanized antibodies,
humanized chimeric antibodies and fragments of such antibodies that
bind human TF specifically. Preferably, substantially of the
immunoglobin outside of the hypervariable region is humanized
although less humanization (or more) may be desirable for some
applications. For instance, FIGS. 6A-D and 13A-D show sequences of
partially and fully humanized light (LC) and heavy chain (HC)
variable regions of antibodies well suite for use with the present
invention. FIGS. 8A-B and 9A-B show drawings of preferred humanized
antibody constant regions.
[0022] In preferred aspects, the invention methods prevent or treat
thrombosis in the mammal and may also desirably inhibit blood
coagulation and blood clot formation, as well as reduce levels of
unwanted TF in the mammal.
[0023] The invention has a wide variety of uses and finds
particular use in the prevention or treatment of various
thromboses, particularly to prevent or inhibit restenosis, or other
thromboses. Such indications can follow an invasive medical
procedure such as arterial or cardiac surgery (e.g., angioplasty).
However, the invention is additionally employed to reduce or even
effectively eliminate thrombotic occlusion arising from activation
of blood coagulation in such non-surgical cardiovascular conditions
including but not limited to coronary artery disease, acute
coronary syndromes (e.g., unstable angina and myocardial
infarction) and atherosclerosis. The invention also can be used to
reduce or even effectively eliminate blood coagulation arising from
use of a medical implementation (e.g., a catheter, stent or other
medical device). Preferred invention methods are generally
compatible with many anti-coagulant, anti-platelet and thrombolytic
therapies, thereby allowing administration in a cocktail format,
for instance, to boost or prolong inhibition of blood coagulation
and thrombus formation.
[0024] The invention methods provided herein can also be employed
to assist anti-thrombosis efforts by helping to decrease
coagulation potential in the extracorporeal circulation of a mammal
such as a primate and particularly a human subject. In such
methods, one or more antibodies disclosed herein can be
administered to the mammal in an amount sufficient to inhibit blood
coagulation prior to or during extracorporeal circulation such as
may occur with cardiopulmonary bypass surgery, organ transplant
surgery or other prolonged surgeries.
[0025] In another aspect, the invention provides a kit for
performing the methods described herein. A particular kit includes
at least one humanized antibody, chimeric antibody, or fragment
thereof that binds specifically to human tissue factor (TF) to form
a complex.
[0026] Other aspects of the invention are discussed infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIGS. 1A and 1B shows the nucleic acid (SEQ ID NOS: 1 and 3)
and amino acid (SEQ ID NOS: 2 and 4) sequences of light chain and
heavy chain variable regions of H36.D2.B7 with hypervariable
regions (CDRs or Complementarity Determining Regions) underlined
(single underline for nucleic acid sequences and double underline
for amino acid sequences).
[0028] FIGS. 2A-B are drawings showing human IgG1-cH36 HC variable
region cloning and expression vectors. HC cloning vector (2A) and
HC expression vector (2B).
[0029] FIGS. 3A-B are drawings showing human IgG4-cH36 HC variable
region cloning and expression vectors. HC cloning vector (3C) and
HC expression vector (3D).
[0030] FIGS. 4A-B are drawings showing cH36 LC variable region
cloning and expression vectors. LC cloning vector (4A) and LC
expression vector (4B).
[0031] FIG. 5 is a drawing showing a plasmid map of humanized
anti-TF IgG1 antibody expression vector (pSUN 34).
[0032] FIGS. 6A-D are drawings showing sequences of partially and
fully humanized light chain (LC) variable regions. Light chain CDR
sequences of cH36 are shown in FIGS. 6B-D. Sequence named "LC-09"0
is representative of a fully humanized LC framework region.
Immediately following the last residue of the framework 4 (FR4)
variable domain is the first residue of the constant domain for
hOAT or HFAT.
[0033] FIGS. 7A-D are sequences of partially and fully humanized
heavy chain (LC) variable regions. Heavy chain CDR sequences for
cH36 and HC-08 are shown in FIGS. 7B-D. Sequence named "HC-08" is
fully humanized HC framework region. Except for HC-08, all other HC
mutants and cH36 contain F at position 64 in CDR2. HC-08 has the
mutation F64 to L (change from F to L at position 64). Immediately
following the last residue of the framework 4 (FR4) variable domain
is the first residue of the constant domain for OAT or FAT. LC
constant are the same for hOAT and hFAT.
[0034] FIGS. 8A-B are drawings showing humanized IgG1 anti-tissue
factor antibody (hOAT (IgG1) constant regions.
[0035] FIGS. 9A-B are drawings showing humanized IgG four
anti-tissue factor antibody (hFAT) (IgG4) constant regions.
Immediately following the last residue of the framework 4 (FR4)
variable domain is the first residue of the constant domain for
hOAT or hFAT. LC constant domain is the same for hOAT and hFAT.
[0036] FIG. 10 is a graph showing platelet deposition in
endarterectomized chimpanzees
[0037] FIG. 11 is a graph showing vessel injury/blood ratios in
endarterectomized chimpanzees
[0038] FIG. 12 is a graph showing mean bleeding times in an
angioplasty study performed in cynomolgus monkeys. Post cH36
represents the mean (with standard deviation) of all bleeding time
determinations taken at 1.5 or 4 hours after administration of
cH36.
[0039] FIG. 13 is a graph showing mean target/background ratio for
.sup.111In-Platelet Deposition.
DETAILED DESCRIPTION OF THE INVENTION
[0040] As discussed above, the invention provides a method for
preventing or treating thrombosis in a mammal that includes
administering to the mammal a therapeutically effective amount of
at least one humanized immunoglobin, typically a humanized
antibody; chimeric antibody, or fragment of the humanized antibody
or chimeric antibody that binds specifically to human TF (free or
in complex with FVIIa or FVII) to form a complex. Preferably,
factor X or factor IX binding to the TF-antibody complex and the FX
or FIX activation by TF:VIIa are inhibited.
[0041] Thus in one embodiment, the invention provides a method for
preventing or treating a thrombosis in a mammal and particularly a
primate such as a human subject. A preferred method involves
administering to the mammal a therapeutically effective amount of
at least one humanized antibody, chimeric antibody, or fragment
thereof that binds specifically to human TF (free or in complex
with FVIIa or FVII) to form a complex in which factor X or factor
IX binding to the complex and the FX or FIX activation by TF:VIIa
are inhibited. Preferred administration routes are sufficient to
prevent or treat the thrombosis in the mammal.
[0042] Preferably, the humanized antibody, humanized chimeric
antibody or the fragment of the antibody or chimera for use with
the invention exhibits at least one of: 1) a dissociation constant
(K.sub.d) for the TF of less than about 0.5 nM; and 2) an affinity
constant (K.sub.a) for the TF of less than about 2.times.10.sup.9
M.sup.-1. An example of a preferred CDR for inclusion in the
humanized immunoglobins is one derived from the murine monoclonal
antibody designated H36.D2.B7 (secreted by hybridoma ATCC HB-12255
and often referred to as H36). See FIGS. 1A and 1B, for instance as
well U.S. Pat. No. 5,986,065 and the PCT Application No.
PCT/US98/04644 (WO 98/40408).
[0043] More particular antibody or chimeric fragments for use with
the invention will have a molecular weight of at least about 40,000
daltons for Fab and about 80,000 daltons for Fab(2)', preferably
between from about 45,000 daltons to about 52,000 daltons for Fab,
and between from about 90,000 daltons to about 104,000 daltons for
Fab(2)', more preferably about 50,000 for Fab and 100,000 for
Fab(2)' as determined by routine protein sizing assays such as gel
electrophoresis.
[0044] By the phrase "humanized" is meant an immunoglobin
(typically an antibody) that includes a human framework region and
one or more CDRs from a non-human source, usually rodent such as a
rat or mouse immunoglobin. The non-human antibody providing the
CDRs is called a "donor" and the human immunoglobin called the
"acceptor". Constant regions need not be present, as in, for
example, certain TF binding fragments of such immunoglobins.
Preferred constant regions, if present, are substantially identical
to human immunoglobin constant regions i.e., at least about 90%
identical with regard to the amino acid sequence, preferably at
least about 95% identical or greater. Accordingly, nearly all parts
of the humanized immunoglobin, with the possible exception of the
CDRs, are substantially identical to corresponding parts of
naturally-occurring human immunoglobin sequences.
[0045] By the phrase "humanized antibody" is meant an antibody that
includes a humanized light chain and a humanized heavy chain
immunoglobin. Also included are partially humanized (chimeric)
antibodies and fragments of chimeric and non-chimeric antibodies
that bind TF specifically. Methods for making and using such
antibodies have been disclosed, for example, in the U.S.
application Ser. No. 09/990,586 entitled Antibodies For Inhibiting
Blood Coagulation and Methods of Use Thereof by Jiao, J. et al.;
U.S. Provisional Application No. 60/343,306 and U.S. Ser. No.
10/230,880 entitled Method of Humanizing Immune System Molecules as
filed on Aug. 29, 2002.
[0046] As discussed throughout the U.S Pat. No. 5,986,065 and the
PCT Application No. PCT/US98/04644 (WO 98/40408), antibodies that
include the preferred CDRs of the murine H36.D2.B7 antibody
typically exhibit substantial affinity for native human TF. In
particular, such antibodies exhibit an association constant
(K.sub.a, M.sup.-1) for native human TF of at least about
1.times.10.sup.8 M.sup.-1 as determined by surface plasmon analysis
(particularly, BIACore analysis in accordance with the procedures
of the U.S Pat. No. 5,986,065; and WO 98/40408 which has been
disclosed), more preferably at least about 5.times.10.sup.8
M.sup.-1 as determined by surface plasmon analysis, still more
preferably a K.sub.a (K.sub.a, M.sup.-1) for native human TF of at
least about 1.times.10.sup.10 M.sup.-1 as determined by surface
plasmon resonance analysis.
[0047] As also discussed throughout the U.S Pat. No. 5,986,065 and
the PCT Application No. PCT/US98/04644 (WO 98/40408), antibodies
that include such preferred CDRs particularly bind with TF so that
FX (or FIX) does not effectively bind to the TF:FVIIa complex
whereby FX (or FIX) is not effectively converted to its activated
form (FXa or FIXa). Particularly preferred humanized antibodies
will strongly inhibit FX activation by a TF:FVIIa complex, e.g. an
inhibition of at least about 50%, more preferably at least about
80%, and even more preferably at least about 90% or 95%, even at
low lipidated TF concentrations such as less than about 1.0 nM TF,
or even less than about 0.20 nM or 0.10 nM TF, as determined by a
standard in vitro binding assay (described fully in the WO 98/40408
and the U.S Pat. No. 5,986,065).
[0048] By the phrase "chimeric antibody" or related phrase
including plural forms is meant antibodies whose light and heavy
chain genes have been constructed, typically by genetic
engineering, from immunoglobulin gene segments belonging to
different species. For example, the variable (V) segments of the
genes from a mouse monoclonal antibody may be joined to human
constant (C) segments, such as .gamma. (.gamma..sub.1,
.gamma..sub.2, .gamma..sub.3, or .gamma..sub.4). A typical
therapeutic chimeric antibody is thus a hybrid protein consisting
of the V or antigen-binding domain from a mouse antibody and the C
or effector domain from a human antibody, although other mammalian
species may be used. A specifically preferred chimeric antibody is
the cH36 mouse-human chimera disclosed herein.
[0049] The humanized antibodies of the present invention can be
polyclonal or monoclonal, as needed, and may have an IgG1 or IgG4
isotype.
[0050] More particular murine immunoglobin hypervariable regions
that are preferred antigen binding regions for many invention
applications are shown in FIGS. 1A and 1B and SEQ ID NOS. 2 and 4).
See also the WO 98/40408 application, for example.
[0051] Also preferred for use with the present invention methods
are humanized antibodies, humanized chimeric antibodies and
fragments of same that have at least one hypervariable region of a
light chain variable region that has high sequence identity (at
least 90% or 95% amino acid sequence identity) to or be the same as
one, two or three of the corresponding hypervariable regions of the
light chain variable region of H36.D2.B7 as shown in the FIGS. 1A
and 1B as well as the PCT/US98/04644 (WO 98/40408). See in
particular those hypervariable regions shown with underlining in
FIG. 1A and are the following: 1) LASQTID (SEQ ID NO: 5); 2)
AATNLAD (SEQ ID NO: 6); and 3) QQVYSSPFT (SEQ ID NO: 7).
[0052] Especially preferred antibodies for use with the present
invention will have one, two or three hypervariable regions of a
heavy chain variable region that have high sequence identity (at
least 90% or 95% amino acid sequence identity) to or be the same as
one, two or three of the corresponding hypervariable regions of the
heavy chain variable region of H36.D2.B7 (those hypervariable
regions shown with underlining in FIG. 1B and are the following: 1)
TDYNVY (SEQ ID NO: 8); 2) YIDPYNGITIYDQNFKG (SEQ ID NO: 9); and 3)
DVTTALDF (SEQ ID NO: 10). See also the PCT/US98/04644 (WO 98/40408)
application.
[0053] Thus in an especially preferred invention embodiment,
humanized antibodies, humanized chimeric antibodies, and fragments
of same will be substantially the same as and preferably identical
to the hypervariable regions of the murine H36.D2.B7 antibody.
[0054] Certain humanized chimeric antibodies are also suitable for
use with the present invention, for example, antibody molecules
that combine a non-human animal variable region and a human
constant region, thereby rendering the antibodies less immunogenic
in a human subject than the corresponding non-chimeric antibody. A
variety of types of such chimeric antibodies can be prepared,
including e.g. by producing human variable region chimeras, in
which parts of the variable regions, especially conserved regions
of the antigen-binding domain, are of human origin and only the
hypervariable regions are of non-human origin. See the U.S. Pat.
No. 5,986,065 and the PCT/US98/04644 (WO 98/40408) application. See
also discussions of humanized chimeric antibodies and methods of
producing same in S. L. Morrison, Science, 229:1202-1207 (1985); Oi
et al., BioTechniques, 4:214 (1986); Teng et al., Proc. Natl. Acad.
Sci. U.S.A., 80:7308-7312 (1983); Kozbor et al., Immunology Today,
4:7279 (9183); Olsson et al., Meth. Enzymol., 9:3-16 (1982).
Additionally, transgenic mice can be employed. For example,
transgenic mice carrying human antibody repertoires have been
created which can be immunized with native human TF. Splenocytes
from such immunized transgenic mice can then be used to create
hybridomas that secrete human monoclonal antibodies that
specifically react with native human TF as described above. See N.
Lonberg et al., Nature, 368:856-859 (1994); L. L. Green et al.,
Nature Genet., 7:13-21 (1994); S. L. Morrison, Proc. Natl. Acad.
Sci. U.S.A., 81:6851-6855 (1994).
[0055] See the U.S. Pat. No. 5,986,065 and the PCT/US98/04644 (WO
98/40408) application for more information relating to making and
using the murine H36.D2.B7 immunoglobin as well as related
antibodies.
[0056] "Humanized antibody of the invention" or other similar term
refers to humanized whole immunoglobulin as well as immunologically
active fragments which bind native human TF. The humanized
immunoglobulins and immunologically active fragments thereof
include an epitope-binding site (i.e., a site or epitope capable of
being specifically bound by an antibody recognizing native human
TF). Exemplary antibody fragments include, for example, Fab, F(v),
Fab', F(ab').sub.2 fragments, "half molecules" derived by reducing
the disulfide bonds of immunoglobulins, single chain
immunoglobulins, or other suitable antigen binding fragments (see
e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al.,
PNAS, (USA), 85:5879 (1988); Webber et al., Mol. Immunol., 32:249
(1995)). The antibody or immunologically active fragment thereof
may be of animal (e.g., a rodent such as a mouse or a rat), or
chimeric form (see Morrison et al., PNAS, 81:6851 (1984); Jones et
al., Nature, 321: 522 (1986)). Single chain antibodies can be
preferred for some invention applications as well as TF binding
fragments thereof.
[0057] By the phrase "specific binding" is meant that the humanized
antibodies form a detectable binding complex with the TF and no
other antigen as determined by standard immunological techniques
such as RIA, Western blot or ELISA.
[0058] Typically preferred humanized antibodies for use with the
invention have a binding specificity for tissue factor, preferably
human TF, that is about equal or greater than the antibody obtained
from H36.D2.B7 deposited under ATCC Accession No. HB-12255. Methods
for determining binding specificity and affinity are known in the
field and include specific assays described in the U.S. Pat. No.
5,986,065 and the PCT/US98/04644 (WO 98/40408) application, for
instance.
[0059] As mentioned, it is an object of the present invention to
provide effective methods for preventing, treating, delaying onset
of or relieving symptoms associated with one or a combination of
thromboses that can inflict a mammal.
[0060] In one embodiment, following about 5 minutes after
administration of the humanized antibody, chimeric antibody or
fragment, the mammal exhibits a blood clotting time of between from
about 50 to about 350 seconds as determined by a standard
prothrombin (PT) time assay. The standard PT assay is discussed
below. See also Example 3 and the U.S. Pat. No. 5,986,065 and the
PCT/US98/04644 (WO 98/40408) for related disclosure.
[0061] In another embodiment of the method, the amount of the
administered humanized antibody, chimeric antibody, or fragment of
the humanized antibody or chimera is sufficient to inhibit platelet
deposition time by at least about 50% as determined by a standard
platelet deposition assay, preferably at least about 70% or about
95%.
[0062] By the phrase "standard platelet deposition assay" or
related phrase is meant performing at least one and preferably all
of the following steps. [0063] a) exposing tissue factor in a
vessel of a subject e.g., by performing surgery such as an
endarterectomy on a major vessel, preferably an artery, [0064] b)
detectably-labeling platelets deposited at the site of injury e.g.,
by labeling the platelets with radioactive indium according to
conventional methods; and [0065] c) detecting and preferably
quantifying the amount of deposited (labeled) platelets such as by
camera imaging.
[0066] The standard platelet deposition assay is preferably
performed by administering at least one of the humanized antibodies
(including chimerics) described herein including fragments to the
subject prior to step c), for instance, after step a) or b).
Performance of a particular antibody or fragment can be compared
against a suitable control such as buffer or physiological saline.
A preferred assay is described below in Example 4.
[0067] Other embodiments of the present invention methods are
contemplated. For example, according to one embodiment, the
administered humanized antibody, humanized chimeric antibody, or
fragment reduces vessel injury to blood ratio by at least about 50%
as determined by a standard vessel injury to blood ratio assay as
described below in Example 4, preferably at least about 70% or
about 95%.
[0068] Reference herein to "standard vessel injury to blood ratio
assay" or related phrase means performing at least one and
preferably all of the following steps. [0069] a) exposing tissue
factor in a vessel of a subject e.g., by performing surgery such as
an endarterectomy on a major vessel, preferably an artery, [0070]
b) detectably-labeling platelets deposited at the site of injury
e.g., by use of radioactive indium, [0071] c) detecting and
preferably quantifying the amount of detectably-labeled platelets
at the site of vessel injury, [0072] d) detecting and preferably
quantifying the amount of detectably-labeled platelets in the blood
of the subject; and [0073] e) expressing the amounts of c) and d)
as the ratio of platelet deposition at vessel injury site to
circulating platelets in the blood.
[0074] The standard vessel injury to blood ratio assay is
preferably performed by administering at least one of the humanized
antibodies (including chimerics) described herein including
fragments to the subject prior to step e), for instance, after step
a) or b). Performance of a particular antibody or fragment can be
compared against a suitable control as already described. A
preferred assay is described below in Example 4.
[0075] In another embodiment, the amount of the administered
humanized antibody, humanized chimeric antibody, or fragment
administered to the mammal increases vessel patency by at least
about 50%, preferably at least about 80%, more preferably at least
about 100% as determined by a standard vessel patency assay.
Patency was determined at 30 days following surgery by harvesting
the vessels and placing them in fixative.
[0076] Reference herein to a "standard vessel patency assay" or
related phrase means detecting and preferably quantifying an
increase in vessel patency usually by inspection. Those of skill in
the field that changes in vessel patency can be readily ascertained
by visualization which may or may not be assisted by magnification
of subject tissue such as with the aid of a microscopic lens or
related device. Typically, the increase is compared to a suitable
control which in many instances can be administration of buffer,
physiological saline, etc. to the mammal in lieu of the antibody or
fragment.
[0077] As will be appreciated, immunoglobin light and heavy chain
share certain structural similarities e.g., each includes a
framework of four regions (FR1-4) whose sequences are relatively
conserved. Each of FR1-4 (FR1, FR2, FR3, FR4) are covalently
connected by three CDRs i.e., CDR1, CDR2, CDR3. There is general
recognition that the four FRs largely adopt a beta-sheet
configuration and the interconnected CDRs form loops connecting,
and in some instances, forming part of the beta-sheet structure.
Most CDRs are held close to adjoining FRs, and with a corresponding
CDR from the opposite light or heavy chain, help form the antigen
binding site. A wide range of CDRs and FRs have been disclosed. See
e.g., Kabat et al. in Sequences of Proteins of Immunological
Interest US Dept. of Health and Human Services, US Government
Printing Office (1987).
[0078] See also EP-A-0239400 and U.S. Pat. No. 5,985,279
(describing methods of making altered antibodies in which CDRs are
derived from different species than the FR).
[0079] For example, an illustrative humanized antibody for use with
the present invention methods includes: 1) light and heavy chain
frameworks (FRs) that are each at least about 90% identical in
amino acid sequence, preferably at least 95% identical to
corresponding human FRs, 2) at least one CDR derived from the
murine H36.D2.B7 antibody, preferably all the CDRs being derived
from same, 3) and an immunoglobin constant region that is at least
about 90% identical, preferably at least 95% identical to a
corresponding human immunoglobin constant region. It will be
appreciated that the donor antibody has been "humanized" by the
process of "humanization" because the resultant humanized antibody
is expected to bind to the same antigen as the donor antibody that
provides the CDRs.
[0080] It will be further appreciated that the humanized antibodies
provided herein may have one or more additional conservative amino
acid substitutions which can be contiguous or non-contiguous as
needed. For example, such substitutions will typically have
substantially little or no effect on antigen binding or other
immunoglobin functions. By the phrase "conservative substitution"
including plural forms is meant combinations of:
gly.revreaction.ala; val.revreaction.ile.revreaction.leu;
asp.revreaction.glu; asn.revreaction.gln; ser.revreaction.thr,
lys.revreaction.arg; and phe.revreaction.tyr.
[0081] Additional humanized antibodies feature a variable region
that is at least 70% identical in amino acid sequence (e.g., about
73% to 75% identical), to the corresponding variable region of one
or more native human immunoglobin sequences. Further humanized
antibodies in accord with the invention have at least 90% identity
over the entire antibody to one or more human antibodies.
[0082] The U.S. Provisional Application 60/343,306; and U.S patent
application Ser. Nos. 09/990,586 and 10/230,880, for example,
disclose additionally suitable humanized molecules suitable for use
with the present invention.
[0083] Thus as described in the U.S patent application Ser. No.
10/230,880, for instance, more specific humanized antibodies for
use with the invention are those in each of frameworks (FRs) 1, 2,
3 and 4 has at least about 90% amino acid sequence identity,
preferably at least about 95% or greater identity to the light
chain FR sequences shown in FIG. 6A (SEQ ID NO. ___), preferably,
the sequence shown as "LC-09" in FIG. 6A. Further preferred are
those humanized antibodies that include a light chain constant
region having at least about 90% amino acid sequence identity,
preferably at least about 95% sequence identity or greater to the
sequence shown in FIG. 8A or 9A (SEQ ID NO. ___).
[0084] Further specific humanized antibodies are those in which
each of frameworks (FRs) 1, 2, 3 and 4 has at least about 90% amino
acid sequence identity, preferably about 95% identity or greater to
the heavy chain sequences shown in FIG. 7A (SEQ ID NO. ___),
preferably, the sequence shown as "HC-08" in FIG. 7A. Additional
humanized antibodies have a heavy chain constant region with at
least about 90% amino acid sequence identity, preferably at least
about 95% identity or greater, to sequence shown in FIG. 8B or 9B
(SEQ ID NO. ___).
[0085] In certain embodiments, the humanized antibody will have an
IgG1 (hOAT) or IgG4 (hFAT) isotype. See Example 7.
[0086] Also provided by the present invention are functional
fragments of the humanized immunoglobins disclosed herein
including, but not limited to, fragments of humanized antibodies
and chimeric antibodies. Preferred fragments specifically bind TF
with an affinity constant (Kd) of less than about 1 nM, preferably
less than about 0.5 nM, more preferably between from about 0.01 nM
to about 0.4 nM. Specifically preferred are antigen binding Fab,
Fab', and F(ab).sub.2 fragments.
[0087] As disclosed in the U.S patent application Ser. No.
10/230,880, for instance, additional humanized antibodies can be
used with the present invention methods such as those featuring at
least one murine complementarity determining region (CDR) from
H36.D2.B7, e.g., CDR1, CDR2, CDR3 of that antibody. In a preferred
embodiment, the antibodies bind specifically to human tissue factor
(TF) to form a complex. Typically, the factor X or factor IX
binding to TF or TF:VIIa and activation by TF:FVIIa thereto is
inhibited. As mentioned above, preferred CDRs (light and heavy
chain) are from a rodent source, typically the mouse, and
preferably the murine H36.D2.B7 antibody.
[0088] In one embodiment, the antibodies further include at least
one human framework (FR) region. Preferably, all the FR regions
(light and heavy chain) are human.
[0089] In a more particular embodiment of the present invention,
the first CDR (CDR1) of the heavy chain hypervariable region is at
least 90% identical to the CDR1 amino acid sequence shown in FIG.
7B (SEQ ID NO. ___), preferably at least about 95% identical or
greater to that sequence. Typically, the second CDR (CDR2) of the
heavy chain hypervariable region is at least 90% identical to the
CDR2 amino acid sequence shown in FIG. 7C (SEQ ID NO. ___),
preferably at least about 95% identical or greater. Preferably
also, the third CDR (CDR3) of the heavy chain hypervariable region
is at least 90% identical to the CDR3 sequence shown in FIG. 7D
(SEQ ID NO. ___), more preferably about 95% identical or greater to
that sequence. See the U.S. patent application Ser. No. 10/230,880
for additional information relating to FIGS. 7A-D.
[0090] Identity between two nucleic acid sequences can be
determined by inspection and/or use of conventional computer
software such as BLAST and FASTA. Identity between related amino
acid sequences can be determined by related programs such as
TFASTA.
[0091] In another invention embodiment, the first CDR (CDR1) of the
light chain hypervariable region is at least 90% identical to the
CDR1 amino acid sequence shown in FIG. 6B (SEQ ID NO. ___),
preferably at least about 95% identical or greater. Typically, the
second CDR (CDR2) of the light chain hypervariable region is at
least 90% identical to the CDR2 amino acid sequence shown in FIG.
6C (SEQ ID NO. ___), preferably about 95% identical or greater.
Preferably, the third CDR (CDR3) of the light chain hypervariable
region is at least 90% identical to the CDR3 amino acid sequence
shown in FIG. 6D (SEQ ID NO. ___), more preferably about 95%
identical or greater to that sequence. See the U.S patent
application Ser. No. 10/230,880 for additional information relating
to FIGS. 6A-C.
[0092] Additional humanized antibodies for use with the invention
include a first framework (FR1) of the heavy chain hypervariable
region which FR1 is at least 90% identical to the amino acid
sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR1 HC-08",
preferably about 95% identical or greater to that sequence. In one
embodiment, the FR1 comprises at least one of the following amino
acid changes: E1 to Q; Q5 to V; P9 to G; L11 to V; V12 to K; Q19 to
R; and T24 to A. Preferably, the FR1 includes two, three, four,
five, or six of those changes with all of those amino acid changes
being preferred for many applications.
[0093] Further humanized antibodies suitable for use with the
invention methods include a second framework (FR2) of the heavy
chain hypervariable region which FR2 is at least 90% identical to
the sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR2 HC-08",
preferably about 95% identical or greater to that sequence. In one
embodiment, the FR2 at least one of the following amino acid
changes: 41H to P; and 44S to G. A preferred FR2 includes both of
those amino acid changes.
[0094] The invention also features humanized antibodies in which a
third framework (FR3) of the heavy chain hypervariable region is at
least 90% identical to the sequence shown in FIG. 7A (SEQ ID NO.
___) as "FR3 HC-08", preferably about 95% identical or greater to
that sequence. In one embodiment, the FR3 includes at least one of
the following amino acid changes: 76S to T; 77T to S; 80F to Y; 82H
to E; 84N to S; 87T to R; 89D to E; and 91S to T. A preferred FR3
includes two, three, four, five or six of those amino acid changes
with all seven of those amino acid changes being generally
preferred.
[0095] Also featured for use with the present methods are humanized
antibodies in which the fourth framework (FR4) of the heavy chain
hypervariable region is at least 90% identical to the amino acid
sequence shown in FIG. 7A (SEQ ID No. ___) as "FR4 HC-08",
preferably at least about 95% identical or greater to that
sequence. Preferably, the FR4 includes the following amino acid
change: 113L to V.
[0096] Additional humanized antibodies for use in accord with the
invention feature a first framework (FR1) of the light chain
hypervariable region which is at least about 90% identical to the
amino acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR1
LC-09", preferably at least about 95% identical or greater to that
sequence. In one embodiment, the FR1 comprises at least one of the
following amino acid changes: 11Q to L; 15L to V; 17E to D; and 18
to R. A preferred FR1 includes two or three of such amino acid
changes with all four amino acid changes being generally
preferred.
[0097] The present invention also features use of humanized
antibodies in which a second framework (FR2) of the light chain
hypervariable region is at least about 90% identical to the amino
acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR2 LC-09",
preferably at least about 95% identical or greater to that
sequence. A preferred FR2 has the following amino acid change: 37Q
to L.
[0098] Also encompassed by the invention is the use of humanized
antibodies in which a third framework (FR3) of the light chain
hypervariable region is at least about 90% identical to the amino
acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR3 LC-09",
preferably at least about 95% identical or greater to that
sequence. In one embodiment, the FR3 has at least one of the
following amino acid changes: 70K to D, 74K to T, 80A to P, 84A to
V, and 85N to T. Preferably, the FR3 has two, three, or four of
such amino acid changes with all five of the changes being
generally preferred.
[0099] Additional humanized antibodies for use with the present
invention include a fourth framework (FR4) of the light chain
hypervariable region which FR4 is at least about 90% identical to
the sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR4 LC-09",
preferably at least about 95% identical or greater to that
sequence. In one embodiment, the FR4 includes at least one and
preferably all of the following amino acid changes: 100A to Q; and
106L to I.
[0100] The invention also features a human TF binding fragment of
the foregoing humanized antibodies as provided in the U.S. patent
application Ser. Nos. 60/343,306; 09/990,586; and 10/230,880.
Examples of such fragments include Fab, Fab',and F(ab).sub.2.
[0101] In a particular embodiment, the invention features use of a
humanized antibody that includes at least one murine
complementarity determining region (CDR) from the H36.D2.B7
antibody. Preferably, that antibody binds specifically to human
tissue factor (TF) to form a complex in which factor X or factor IX
binding to TF or TF/VIIa and activation by TF/VIIa thereto is
inhibited. Also preferably, the humanized antibody includes, on the
heavy chain, at least one of and more preferably all of the
following components: [0102] a) a first CDR (CDR1) which is at
least 95% identical to CDR1 amino acid sequence shown in FIG. 7B
(SEQ ID NO. ___), [0103] b) a second CDR (CDR2) which is at least
95% identical to the CDR2 amino acid sequence shown in FIG. 7C (SEQ
ID NO. ___), [0104] c) a third CDR (CDR3) which is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 7D (SEQ ID
NO. ___), [0105] d) a first framework (FR1) which is at least 95%
identical to the amino acid sequence shown in FIG. 7A (SEQ ID NO.
___) as "FR1 HC-08", [0106] e) a second framework (FR2) which is at
least 95% identical to the amino acid sequence shown in FIG. 7A
(SEQ ID NO. ___) as "FR2 HC-08", [0107] f) a third framework (FR3)
which is at least 95% identical to the amino acid sequence shown in
FIG. 7A (SEQ ID NO. ___) as "FR3 HC-08", and [0108] g) a fourth
framework (FR4) which is at least 95% identical to the amino acid
sequence shown in FIG. 7A (SEQ ID No. ___) as "FR4 HC-08".
[0109] In a particular embodiment, the humanized antibody also
includes, on the light chain, at least one of and preferably all of
the following components: [0110] h) a first CDR (CDR1) which is at
least 95% identical to CDR1 amino acid sequence shown in FIG. 6B
(SEQ ID NO. ___), [0111] i) a second CDR (CDR2) which is at least
95% identical to the CDR2 amino acid sequence shown in FIG. 6C (SEQ
ID NO. ___), [0112] j) a third CDR (CDR3) which is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 6C (SEQ ID
NO. ___), [0113] k) a first framework (FR1) which is at least 95%
identical to the amino acid sequence shown in FIG. 6A (SEQ ID NO.
___) as "FR1 LC-09", [0114] l) a second framework (FR2) which is at
least 95% identical to the amino acid sequence shown in FIG. 6A
(SEQ ID ___) as "FR2 LC-09", [0115] m) a third framework (FR3)
which is at least 95% identical to the amino acid sequence shown in
FIG. 6A (SEQ ID NO. ___) as "FR3 LC-09", and [0116] n) a fourth
framework (FR4) which is at least 95% identical to the amino acid
sequence shown in FIG. 6A (SEQ ID No. ___) as "FR4 LC-09".
Preferably, the humanized antibody further includes the light chain
constant sequence of FIG. 8A (SEQ ID No. ___) or FIG. 9A (SEQ ID
No. ___). Also preferably, the antibody includes the heavy chain
constant region of FIG. 8B (SEQ ID No. ___) or FIG. 9B (SEQ ID No.
___).
[0117] The invention also features use of a humanized antibody that
includes, on the heavy chain, at least one of and preferably all of
the following components: [0118] a) a first CDR (CDR1) identical to
the CDR1 amino acid sequence shown in FIG. 7B (SEQ ID NO. ___),
[0119] b) a second CDR (CDR2) identical to the CDR2 amino acid
sequence shown in FIG. 7C (SEQ ID NO. ___), [0120] c) a third CDR
(CDR3) identical to the CDR3 amino acid sequence shown in FIG. 7D
(SEQ ID NO. ___), [0121] d) a first framework (FR1) identical to
the amino acid sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR1
HC-08", [0122] e) a second framework (FR2) identical to the amino
acid sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR2 HC-08",
[0123] f) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR3 HC-08"; and
[0124] g) a fourth framework (FR4) identical to the amino acid
sequence shown in FIG. 7A (SEQ ID No. ___) as "FR4 HC-08".
[0125] In one embodiment, the humanized antibody used in the
present methods further includes, on the light chain, at least one
of and preferably all of the following components: [0126] h) a
first CDR (CDR1) identical to CDR1 amino acid sequence shown in
FIG. 6B (SEQ ID NO. ___), [0127] i) a second CDR (CDR2) identical
to the CDR2 amino acid sequence shown in FIG. 6C (SEQ ID NO. ___),
[0128] j) a third CDR (CDR3) identical to the CDR3 amino acid
sequence shown in FIG. 6D (SEQ ID NO. ___), [0129] k) a first
framework (FR1) identical to the amino acid sequence shown in FIG.
6A (SEQ ID NO. ___) as "FR1 LC-09", [0130] l) a second framework
(FR2) identical to the amino acid sequence shown in FIG. 6A (SEQ ID
NO. ___) as "FR2 LC-09", [0131] m) a third framework (FR3)
identical to the amino acid sequence shown in FIG. 6A (SEQ ID NO.
___) as "FR3 LC-09", and [0132] n) a fourth framework (FR4)
identical to the amino acid sequence shown in FIG. 6A (SEQ ID No.
___) as "FR4 LC-09". Preferably, the humanized antibody further
includes the light chain constant sequence of FIG. 8A (SEQ ID No.
___) or FIG. 9A (SEQ ID No. ___). Also preferably, the antibody
includes the heavy chain constant region of FIG. 8B (SEQ ID No.
___) or FIG. 9B (SEQ ID No. ___).
[0133] In another example, the invention provides methods of
inhibiting one or a combination of thromboses in a mammal such as a
primate and particularly a human subject in need of such treatment
that includes administering to the mammal, a therapeutically
effective amount of a humanized immunoglobin, more particularly a
humanized antibody, humanized chimeric antibody or suitable
fragment thereof. Typically, the humanized antibody binds
specifically to human tissue factor (TF) to form the binding
complex. Preferably, the humanized antibody or fragment includes,
on the heavy chain, at least one of and preferably all of the
following components: [0134] a) a first CDR (CDR1) which is at
least 95% identical to CDR1 amino acid sequence shown in FIG. 7B
(SEQ ID NO. ___), [0135] b) a second CDR (CDR2) which is at least
95% identical to the CDR2 amino acid sequence shown in FIG. 7C (SEQ
ID NO. ___), [0136] c) a third CDR (CDR3) which is at least 95%
identical to the CDR3 amino acid sequence shown in FIG. 7D (SEQ ID
NO. ___), [0137] d) a first framework (FR1) which is at least 95%
identical to the amino acid sequence shown in FIG. 7A (SEQ ID NO.
___) as "FR1 HC-08", [0138] e) a second framework (FR2) which is at
least 95% identical to the amino acid sequence shown in FIG. 7A
(SEQ ID NO. ___) as "FR2 HC-08", [0139] f) a third framework (FR3)
which is at least 95% identical to the amino acid sequence shown in
FIG. 7A (SEQ ID NO. ___) as "FR3 HC-08", [0140] g) a fourth
framework (FR4) which is at least 95% identical to the amino acid
sequence shown in FIG. 7A (SEQ ID No. ___) as "FR4 HC-08".
[0141] In a more specific invention embodiment, the humanized
antibody includes, on the light chain, at least one of, and
preferably all of the following components: [0142] h) a first CDR
(CDR1) which is at least 95% identical to CDR1 amino acid sequence
shown in FIG. 6B (SEQ ID NO. ___), [0143] i) a second CDR (CDR2)
which is at least 95% identical to the CDR2 amino acid sequence
shown in FIG. 6C (SEQ ID NO. ___), [0144] j) a third CDR (CDR3)
which is at least 95% identical to the CDR3 amino acid sequence
shown in FIG. 6D (SEQ ID NO. ___), [0145] k) a first framework
(FR1) which is at least 95% identical to the amino acid sequence
shown in FIG. 6A (SEQ ID NO. ___) as "FR1 LC-09", [0146] l) a
second framework (FR2) which is at least 95% identical to the amino
acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR2 LC-09",
[0147] m) a third framework (FR3) which is at least 95% identical
to the amino acid sequence shown in FIG. 6A (SEQ ID NO. ___) as
"FR3 LC-09", [0148] n) a fourth framework (FR4) which is at least
95% identical to the amino acid sequence shown in FIG. 6A (SEQ ID
No. ___) as "FR4 LC-09", [0149] o) a light chain constant region
which is at least 95% identical to the amino acid sequence shown in
FIG. 8A (SEQ ID No. ___) or FIG. 9A (SEQ ID No. ___); and [0150] p)
a heavy chain constant region which is at least 95% identical to
the amino acid sequence shown in FIG. 8B (SEQ ID No. ___) or FIG.
9B (SEQ ID No. ___).
[0151] In a more specific embodiment, the humanized antibody or
fragment thereof includes, on the heavy chain, at least one of and
preferably all of the following components: [0152] a) a first CDR
(CDR1) identical to CDR1 amino acid sequence shown in FIG. 7B (SEQ
ID NO. ___), [0153] b) a second CDR (CDR2) identical to the CDR2
amino acid sequence shown in FIG. 7C (SEQ ID NO. ___), [0154] c) a
third CDR (CDR3) identical to the CDR3 amino acid sequence shown in
FIG. 7D (SEQ ID NO. ___), [0155] d) a first framework (FR1)
identical to the amino acid sequence shown in FIG. 7A (SEQ ID NO.
___) as "FR1 HC-08", [0156] e) a second framework (FR2) identical
to the amino acid sequence shown in FIG. 7A (SEQ ID NO. ___) as
"FR2 HC-08", [0157] f) a third framework (FR3) identical to the
amino acid sequence shown in FIG. 7A (SEQ ID NO. ___) as "FR3
HC-08", [0158] g) a fourth framework (FR4) identical to the amino
acid sequence shown in FIG. 7A (SEQ ID No. ___) as "FR HC-08";
[0159] and on the light chain: [0160] h) a first CDR (CDR1)
identical to CDR1 amino acid sequence shown in FIG. 6B (SEQ ID NO.
___), [0161] i) a second CDR (CDR2) identical to the CDR2 amino
acid sequence shown in FIG. 6C (SEQ ID NO. ___), [0162] j) a third
CDR (CDR3) identical to the CDR3 amino acid sequence shown in FIG.
6D (SEQ ID NO. ___), [0163] k) a first framework (FR1) identical to
the amino acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR1
LC-09", [0164] l) a second framework (FR2) identical to the amino
acid sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR2 LC-09",
[0165] m) a third framework (FR3) identical to the amino acid
sequence shown in FIG. 6A (SEQ ID NO. ___) as "FR3 LC-09", [0166]
n) a fourth framework (FR4) identical to the amino acid sequence
shown in FIG. 6A (SEQ ID No. ___) as "FR4 LC-09", [0167] o) a light
chain constant region which is identical to the amino acid sequence
shown in FIG. 8A (SEQ ID No. ___) or FIG. 9A (SEQ ID No. ___), and
[0168] p) a heavy chain constant region which is identical to the
amino acid sequence shown in FIG. 8B (SEQ ID No. ___) or FIG. 9B
(SEQ ID No. ___).
[0169] Thus in one embodiment of the present methods for preventing
or treating thromboses, a suitable humanized antibody, humanized
chimeric antibody or fragment thereof includes at least one fully
human framework (FR) region. Additionally, such an antibody can
have at least 90%, preferably at least 95%, more preferably at
least 98% sequence identity to a human antibody. Further humanized
antibodies suitable for use with the invention have at least 70%
amino acid sequence identity to a human antibody variable region.
Additionally preferred humanized antibodies are preferably
monoclonal and may be in a single-chain format as desired.
[0170] The humanized antibodies of the present invention may exist
in a variety of suitable forms in addition to whole antibodies;
including, for example, Fv, Fab, and F(ab').sub.2 as well as
bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J
Immunol. 17: 105 (1987)) and in single chains (e.g., Huston et al.,
Proc. Natl. Acad. Sci. U.S.A., 85: 5879-5883 (1988) and Bird et
al., Science 242: 423-426 (1988), which are incorporated herein by
reference). (See, Hood et al., Immunology, Benjamin, N.Y., 2.sup.nd
ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold
Spring Harbor Laboratory (1988) and Hunkapiller and Hood, Nature,
323: 15-16 (1986), which are incorporated herein by
reference.).
[0171] The humanized antibodies disclosed herein can be produced by
one or a combination of strategies including those already
referenced above. See e.g., S. L. Morrison, supra; Oi et al.,
supra; Teng et al., supra; Kozbor et al., supra; Olsson et
al.,supra; and other references cited previously.
[0172] See also the U.S. Pat. No. 5,693,762 by Queen et al. and
references disclosed therein.
[0173] Particularly useful methods have been disclosed the U.S.
patent application Ser. Nos. 60/343,306; 09/990,586; and
10/230,880, for instance. Briefly, four general steps can be
employed to humanize an antibody. First, the amino acid sequences
of the mouse antibody light and heavy chains were obtained from the
cH36 mouse-human chimeric antibody. Second, the cH36 antibody was
humanized by determining which human antibody framework region gave
the "best fit" i.e., most closely resembled the mouse framework
region amino acid sequence. Third, relevant light and heavy chain
FR sequences were humanized, and fourth, transfection and
expression of isolated nucleic acid(s) that encode the humanized
light or heavy chain (or humanized light and heavy chain e.g., see
the mega vectors described below).
[0174] The following three nucleic acid vectors pSUN36 (humanized
anti-TF antibody Ig G1-HC expression vector), pSUN37 (humanized
anti-TF antibody Ig G4-HC expression vector), and pSUN38 (humanized
anti-TF antibody LC expression vector) may be useful in some
approaches to make the humanized immunoglobins disclosed herein.
Such vectors have been deposited pursuant to the Budapest Treaty
with the American Type Culture Collection (ATCC) at 10801
University Boulevard, Manassas Va. 20110-2209. The vectors were
assigned the following Accession Numbers: PTA-3727 (pSUN36);
PTA-3728 (pSUN37); and PTA-3729 (pSUN38).
[0175] As provided in the U.S. patent application Ser. Nos.
60/343,306; 09/990,586; and 10/230,880, humanized antibodies and
fragments thereof according to the invention can be tested for
function by one or a combination of standard methods. A preferred
method is what is sometimes referred to herein as a "standard
prothrombin time" assay or related phrase. The standard prothrombin
time (PT) assay typically involves at least one and preferably all
of the following steps: [0176] a) combining a solution containing
lipidated TF and calcium with human plasma; and [0177] b) measuring
and recording the clotting times using a coagulation analyzer.
[0178] See also the U.S. Pat. No. 5,986,065 and the PCT/US98/04644
(WO 98/40408) application for additional disclosure relating to the
standard PT assay including preferred reagent sources.
[0179] The humanized antibodies and fragments thereof provided
herein can be readily tested in standard PT assay. An aliquot of
the purified antibody or fragment, preferably about 200 nM to about
2000 nM, is added to the human plasma (Ci-Trol Coagulation
Control), and a solution containing lipidated recombinant TF and
calcium (such as Innovin from Dade Behring) is added to the human
plasma to start the PT reaction.
[0180] Highly preferred humanized antibodies and fragments thereof
including whole IgG, Fab, Fab', F(ab).sub.2, and single chain
antibodies (comprising the antigen binding variable regions of the
humanized antibodies) will increase blood clotting time by at least
about 5 seconds when tested in the standard assay at a final
concentration of at least about 1 nM to about 20 nM, preferably
about 5 nM to about 15 nM, more preferably about 10 nM in the
assay. A typical control is a standard PT assay performed without
adding any antibody of fragment. Additionally preferred antibodies
and fragments of the invention achieve at least about 90%
inhibition of TF-dependent coagulation, preferably at least about
95% inhibition or greater when compared to the control.
[0181] Humanized antibodies, humanized chimerics and fragments
suitable for use with the invention are preferably substantially
pure. References to an antibody being "substantially pure" mean an
antibody or protein which has been separated from components which
naturally accompany it. For example, by using standard
immunoaffinity or Protein A affinity purification techniques, an
antibody of the invention can be purified from a hybridoma culture
by using recombinant human TF coupled resin or Protein A resin.
Similarly, recombinant human TF or native human TF can be obtained
in substantially pure form by using an antibody of the invention
with standard immunoaffinity purification techniques. Particularly,
an antibody or protein is substantially pure when at least 50% of
the total protein (weight % of total protein in a given sample) is
an antibody or protein of the invention. Preferably the antibody or
protein is at least 60 weight % of the total protein, more
preferably at least 75 weight %, even more preferably at least 90
weight %, and most preferably at least 98 weight % of the total
material. Purity can be readily assayed by known methods such as
SDS (PAGE) gel electrophoresis, column chromatography (e.g.,
affinity chromatography) or HPLC analysis.
[0182] As discussed above, humanized immunoglobins for use with the
invention can be administered to a mammal, preferably a primate
such as a human, to prevent or reduce thrombotic occlusive
disorders such as those attributable to TF-mediated activation of
coagulation. Typical administration routes include use of a
composition including one or more pharmaceutically acceptable
non-toxic carriers such as sterile water or saline, glycols such as
polyethylene glycol, oils of vegetable origin, and the like. In
particular, biocompatible, biodegradable lactide polymer, lactide
glycolide copolymer or polyoxyethylene, polyoxypropylene copolymers
may be useful excipients to control the release of the
antibody-containing compositions described herein. Other
potentially useful administration systems include ethylene vinyl
acetate copolymer particles, osmotic pumps, and implantable
infusion systems and liposomes. Generally, an anti-coagulant
composition of the invention will be in the form of a solution or
suspension (or a lyophilized form that can be reconstituted to a
solution or suspension), and will preferably include approximately
0.01% to 10% (w/w) of the antibody of the present invention,
preferably approximately 0.01% to 5% (w/w) of the antibody. The
antibody can be administered as a sole active ingredient in the
composition, or as a cocktail including one or more other
anti-coagulant (e.g., heparin, hirudin or hirulog, coumadin,
warfarin), anti-platelet (e.g., aspirin, Plavix, Ticlid, ReoPro,
Integrilin or Aggrestat), or thrombolytic agents (e.g., tissue
plasminogen activator, strepokinase and urokinase).
[0183] Methods of the invention can be used before, during or after
administration of one or more suitable anti-coagulant,
anti-platelet or thrombolytic agents to boost or prolong desired
anti-thrombosis effect.
[0184] Therapeutic anti-thrombotic compositions according to the
invention (including cocktails) are suitable for use in parenteral
or intravenous administration, particularly in the form of liquid
solutions. Such compositions may be conveniently administered in
unit dose and may be prepared in accordance with methods known in
the pharmaceutical art. See Remington's Pharmaceutical Sciences,
(Mack Publishing Co., Easton Pa., (1980)). By the term "unit dose"
is meant a therapeutic composition of the present invention
employed in a physically discrete unit suitable as unitary dosages
for a primate such as a human, each unit containing a
pre-determined quantity of active material calculated to produce
the desired therapeutic effect in association with the required
diluent or carrier. The unit dose will depend on a variety of
factors including the type and severity of thrombosis to be
treated, capacity of the subject's blood coagulation system to
utilize the administered composition, etc. Precise amounts of the
antibody to be administered typically will be guided by judgment of
the practitioner, however, the unit dose will generally depend on
the route of administration and be in the range of 10 ng/kg body
weight to 50 mg/kg body weight per day, more typically in the range
of 100 ng/kg body weight to about 10 mg/kg body weight per day.
Suitable regimens for initial administration in booster shots are
also variable but are typified by an initial administration
followed by repeated doses at one or more hour intervals by a
subsequent injection or other administration. Alternatively,
continuous or intermittent intravenous infusions may be made
sufficient to maintain concentrations of at least from about 10
nanomolar to 10 micromolar of the antibody in the blood.
[0185] In practice, the invention methods can be used as separately
administered compositions given in conjunction with other
anti-clotting agents including aspirin, coumadin, heparin, hirudin,
or hirulog. Also envisioned is co-administration with anti-platelet
(e.g., ReoPro, Integrilin, Aggrestat, Plavix, and/or Ticlid) and/or
thrombolytic agents (e.g., tissue plasminogen activator,
strepokinase and urokinase).
[0186] In some instances, it may be desirable to modify the
antibody of the present invention to impart a desirable biological,
chemical or physical property thereto. More particularly, it may be
useful to conjugate (i.e. covalently link) the antibody to a
pharmaceutical agent, e.g. a fibrinolytic drug such as t-PA,
streptokinase, or urokinase to provide fibrinolytic activity or to
a targeting agent such as a fibrin-binding domain. Such linkage can
be accomplished by several methods including use of a linking
molecule such as a heterobifunctional protein cross-linking agent,
e.g. SPDP, carbodimide, or the like, or by recombinant methods.
[0187] As discussed, the invention is useful for preventing or
treating thrombosis. Particularly, the invention can be employed to
prevent or treat restenosis associated with, e.g., an invasive
medical procedure such as percutanous transluminal coronary
intervention, cardiopulmonary bypass surgery, endarterectomy,
peripheral vascular bypass grafts, reconstructive or plastic
surgery, joint replacement; a heart ailment such as myocardial
infarction, cardiomyopathy, valvular heart disease, stable angina,
unstable angina, or artrial fibrillation associated with
embolization; a coagulopathy including disseminated intravascular
coagulation, deep vein thrombosis, deployment of an implementation
such as a stent or catheter; shock (e.g., septic shock syndrome),
vascular trauma, liver disease, hemorrhagic stroke, heat stroke,
malignancies (e.g., pancreatic, ovarian, or small lung cell
carcinoma), lupus, eclampsia, perivascular occlusive disease, and
renal disease.
[0188] Although a range of therapeutic anti-coagulant compositions
of the invention have been described above, other compositions that
include the humanized antibodies, humanized chimeric antibodies and
fragments of same are contemplated. For example, such antibodies
and fragments may be used as the sole therapeutic agent or in
combination with one or more other humanized antibodies or
fragments to achieve a desired outcome. Such antibodies and
fragments may also be used in combination with other antibodies,
particularly human monoclonal antibodies reactive with other
markers on cells responsible for the disease.
[0189] In embodiments in which the therapeutic anti-coagulant
compositions described herein include one or more humanized
antibodies or fragments, that composition may include a solution of
the antibody or a cocktail thereof dissolved in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers have already been referenced such as water, buffered
water, 0.4% saline, 0.3% glycine and the like. These solutions are
preferably sterile and generally free of particulate matter. These
compositions may be sterilized by conventional, well-known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjustment agents and the like, for
example sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate, etc. The concentration of
antibody in these formulations can vary widely, for example from
less than about 0.5%, usually at or at least about 1% to as much as
15 or 20% by weight and will be selected primarily based on fluid
volumes, viscosities, etc., in accordance with the particular mode
of administration selected. See generally, Remington's
Pharmaceutical Sciences, supra.
[0190] If desired, the therapeutic anti-coagulant compositions
described herein can be lyophilized for storage and reconstituted
in a suitable carrier prior to use. This technique has been shown
to be effective with conventional immune globulins. Any suitable
lyophilization and reconstitution techniques can be employed. It
will be appreciated by those skilled in the art that lyophilization
and reconstitution can lead to varying degrees of antibody activity
loss (e.g., with conventional immune globulins, IgM antibodies tend
to have greater activity loss than IgG antibodies) and that use
levels may have to be adjusted to compensate.
[0191] For some prophylactic applications, it will be helpful to
use the present invention methods with a patient not already in a
detectable disease state to enhance the patient's resistance to the
disease. Such an amount is defined to be a "prophylactically
effective dose". In this use, the precise amounts again depend upon
the patient's state of health and general level of immunity, but
generally range from 0.03 to 25 mg/kg, especially 0.25 to 2.5
mg/kg. Such administration amounts are generally suitable for use
in the methods described herein however they may be adjusted
depending on recognized parameters such as the humanized
immunoglobin selected, the indication to be treated, health of the
individual, etc.
[0192] As discussed, the invention also features kits that include
subject antibodies or fragments thereof. In one embodiment, the
humanized antibodies or fragments thereof can be supplied for use
against or in the detection of TF antigen. Thus, for instance, one
or more humanized antibodies, fragments thereof, or single chain
antibodies may be provided, usually in a lyophilized form in a
container. Such antibodies, fragments, or single chain antibodies,
which may be conjugated to a previously mentioned label or toxin,
or unconjugated, are included in the kits with buffers, such as
Tris, phosphate, carbonate, etc., stabilizers, biocides, inert
proteins, e.g., serum albumin, or the like. Generally, these
materials will be present in less than about 5% by weight based on
the amount of active antibody, and usually present in total amount
of at least about 0.001% wt. based again on the antibody
concentration. Frequently, it will be desirable to include an inert
extender or excipient to dilute the active ingredients, where the
excipient may be present in from about 1 to 99% wt. of the total
composition. Where a second antibody capable of binding to the
chimeric antibody is employed in an assay, this will usually be
present in a separate vial. The second antibody is typically
conjugated to a label and formulated in an analogous manner with
the antibody formulations described above. The kit will generally
also include a set of instructions for use.
[0193] As discussed, the invention also provides a variety of
methods of inhibiting blood coagulation in a mammal, preferably a
primate such as a human patient.
[0194] For example, in one embodiment, the methods include
administering to the mammal a therapeutically effective amount of
at least one of, preferably one, two or three of the humanized
antibodies, chimeric or fragment thereof as provided herein. In
most embodiments, the methods further include forming a specific
complex between the antibody and the TF to inhibit the blood
coagulation.
[0195] Also provided are methods of inhibiting blood coagulation in
a mammal that include administering to the mammal, a
therapeutically effective amount of the humanized antibodies
disclosed herein or a fragment thereof. Typical antibodies and
fragments bind specifically to human tissue factor (TF) to form a
complex, and further wherein factor X or factor IX binding to TF or
TF:FVIIa and activation by TF:FVIIa thereto is inhibited. In most
embodiments, the methods further include forming a specific complex
between the antibody and the TF to inhibit the blood
coagulation.
[0196] As discussed, the invention also provides methods for
performing plastic, reconstructive, or transplant surgery in a
mammal that includes, in one embodiment, introducing a graft into
the mammal and contacting the graft with a therapeutically
effective amount of at least one any of the humanized antibodies,
chimeric antibodies, or fragments disclosed herein. An animal model
designed to examine thrombosis during surgery is the skin flap
surgery model described in detail in Example 5.
[0197] By the phrase "standard skin flap assay" or related phrase
is meant conducting at least one and preferably all of the
following steps: [0198] a) making a skin flap in a subject mammal
and reducing blood supply therein, [0199] b) ligating the skin flap
to the mammal, [0200] c) administering to the ligated skin flap a
detectable label capable of reporting blood perfusion e.g, a
fluorscent dye such as fluorescein; and [0201] d) detecting and
optionally quantifying sites of poor blood perfusion (ie. dark
regions).
[0202] The standard skin flap assay is preferably performed by
administering at least one of the humanized antibodies (including
chimerics) or fragments thereof to the mammal prior to step c), for
instance, after step a) or b). A preferred skin flap assay is
described below in Example 5.
[0203] In one embodiment of the method, amount of the humanized
antibody, humanized chimeric antibody or fragment administered to
the mammal is sufficient to prevent or reduce thrombosis by at
least about 50% as determined by a standard skin flap model,
preferably at least about 70% or 80% according to that model as
described in Example 5. Typical methods further include preventing
or reducing devascularization of the graft as determined by a
standard skin flap model e.g., by preventing or reducing at least
one of edema, erythema, and necrosis in the graft.
[0204] The following conditions, among others disclosed herein, can
be prevented or treated by using the present invention. [0205] 1.
Angioplasty and Thrombosis: Percutaneous coronary interventional
(PCI) techniques are now widely used to treat patients with acute
and chronic coronary artery disease. During PCI, blood vessel is
injured and TF and other procoagulant components are exposed to
blood coagulation factors and platelets, triggering coagulation and
platelet activation, and leading to thrombosis. Therefore, abrupt
vessel closure in the short term and restenosis in the long term
are the limitations to the event free survival following balloon
angioplasty. cH36 was shown in Example 6 to inhibit
angioplasty-induced thrombosis. [0206] 2. Acute Coronary Syndrome
and Thrombosis: Atherosclerothesis is a systemic disease
responsible for acute coronary syndromes (ACS) such as unstable
angina, acute myocardial infarction, and sudden cardiac death. ACS
is the major cause of morbidity and mortality in developed
countries. Most ACS patients share a common pathophysiological
phenomenon: coronary thrombosis. Coronary thrombosis is caused by
plaque disruption or plaque erosion. The consequence of plaque
disruption and erosion is the exposure of TF within the plaques to
the flowing blood, leading to the activation of coagulation cascade
and platelets. An electrolytic model in cynomolgus monkeys was
employed to mimic the events of plague disruption in Example 7.
[0207] 3. In-Stent Thrombosis: Coronary stents are a major
advancement in percutaneous coronary intervention. However,
in-stent thrombosis (restenosis) is a problem, occurring in 20% to
30% of cases. In a perfusion system, a thrombus forms on stent
surfaces. The stent thrombus contains fibrin, platelets, and
leucocytes. Very interestingly, the thrombus also contains TF,
which is carried over to stent surface by leucocytes from the blood
during perfusion (see Peter L. A. G. et al., PNAS96:2311-2315
(1999); Tullio P et al., J. Am. Coll. Cardiol. 40:360-366 (2002)).
cH36 can significantly reduce the thrombus formation on stent
surfaces. Once the thrombus is formed, it can trigger further
thrombin generation due to the presence of TF in the thrombus.
Thus, cH36 can strongly inhibit thrombin generation induced by
thrombus.
[0208] The invention also provides a kit for performing or
assisting in the performance of the present invention methods. A
typical kit includes at least one of the humanized immunoglobins
referred to herein including particular humanized antibodies,
chimeric antibodies, or fragments thereof that binds specifically
to human TF to a complex. Typically, such humanized immunoglobins
will be provided in at least one pharmaceutically acceptable
vehicle such as those mentioned herein including those useful in
dissolving, stabilizing or otherwise providing such immunoglobins
in a form useful to practice the invention.
[0209] All documents mentioned herein are fully incorporated by
reference in their entirety.
[0210] The following non-limiting examples are illustrative of the
invention.
[0211] As discussed, the U.S. Pat. No. 5,986,065 and the
PCT/US98/04644 (WO 98/40408) disclose how to make and use the
murine H36.D2 antibody as well as related molecules. In those
documents, the antibodies H36 and H36.D2 are referred to. Those
antibodies are the same antibody as H36.D2.B7, but H36 is derived
from the mother clone, and H36.D2 is obtained from the primary
clone, whereas H36.D2.B7 is obtained from the secondary clone. As
pointed out in the U.S. Pat. No.5,986,065 and the PCT/JS98/04644
(WO 98/40408), no differences have been observed between those
three clones with respect to ability to inhibit TF or other
physical properties. In general usage, H36 is often used to
indicate anti-TF antibody produced by any of these clones or
related cell lines producing the antibody. In addition the
derivatives of H36, including the chimeric antibody cH36 and the
humanized versions hOAT (containg human IgG1 Fc domain) and hFAT
(containg human IgG4 Fc domain), bind to tissue factor
similarly.
[0212] Certain of the following Examples have been disclosed in the
U.S. patent application Ser. No. 10/230,880 entitled Method of
Humanizing Immune System Molecules as filed on Aug. 29, 2002.
EXAMPLE 1
Humanization of Anti-Tissue Factor Antibody
[0213] The previous examples describe how to make and use a
particular murine antibody called H36.D2 (sometimes also called H36
as discussed above). The present example shows how to make and use
a humanized version of that antibody. A humanized H36 antibody has
a variety of uses including helping to minimize potential for human
anti-mouse antibody (HAMA) immunological responses. These and other
undesired responses pose problems for use of the H36 antibody in
human therapeutic applications.
A. Preparation of Chimeric Anti-Tissue Factor Antibody (cH36)
[0214] The H36 antibody described previously is an IgG2a murine
antibody. H36 was first converted to a mouse-human chimeric
antibody for clinical development. To do this, the heavy and light
chain genes for H36 were cloned (see U.S. Pat. No.5,986,065). The
heavy chain variable region was fused to a human IgG4 constant (Fc)
domain and the light chain variable region was fused to a human
kappa light chain constant domain. The resulting IgG4.kappa.
chimeric antibody was designated cH36. For multiple uses of H36 or
cH36 in patients with chronic diseases, a fully humanized cH36 is
preferred so that it will decease or eliminate any human anti-mouse
antibody immunological response. The humanization of cH36 is
described below.
B. Humanization of cH36 Antibody
[0215] Humanization of the chimeric anti-tissue factor antibody
cH36 was achieved by using a "best-fit" method. This method takes
full advantage of the fact that a great number of human IgGs with
known amino acid sequences are available in the public database.
The individual frameworks of the mouse heavy and light variable
regions in cH36 are compared with their corresponding human
frameworks in the Kabat database (see http://immuno.bme.nwu.edu).
The following criteria were used to select the desired human IgG
frameworks for humanization: (1) The number of mismatched amino
acids was kept as low as possible. (2) Amino acids inside the
"vernier" zone (amino acids in this zone may adjust CDR structure
and fine-tune the fit to antigen, see Foote, J. and Winter, G., J.
Mol. Bio. 224(2): 487-499 [1992]) were left unchanged. (3)
Conservative amino acid substitutions were favored when evaluating
similar candidates. The matching program used for this comparison
can be found in Kabat's home page. See also Johnson G, Wu T. "Kabat
database and its application: Future directions." Nucleic Acids
Res. 29:205-206(2001). The program finds and aligns regions of
homologies between the mouse sequences and human sequences in the
Kabat's database. By using this unique best-fit method, it is
anticipated that the humanized LC or HC variable region of the
target IgG may have all the four FRs derived from as few as one
human IgG molecule or to as many as four different human IgG
molecules.
(i). Selection of Human Kappa Light Chain Variable Region
Frameworks
[0216] The amino acid sequence in each of the frameworks of cH36 LC
was compared with the amino acid sequence in the corresponding FR
in human kappa light chain variable region in Kabat Database. The
best-fit FR was selected based on the three criteria described
above.
[0217] The amino acid sequence of human kappa light chain variable
region with a Kabat Database ID No. 005191 was selected for
humanization of cH36 LC FR1. The amino acid sequence of human kappa
light chain variable region with a Kabat Database ID No. 019308 was
selected for humanization of cH36 LC FR2. The following mutations
were made in cH36 LC FR1 to match the amino acid sequence of a
human kappa light chain variable region with a Kabat Database ID
No. 005191: Q11 L, L15 V, E17 D, S18 R. One mutation Q37 L was made
in cH36 LC FR2 to match the amino acid sequence of a human kappa
light chain variable region with a Kabat Database ID No. 019308
(see Table 1A for sequence information).
[0218] The amino acid sequence of a human kappa light chain
variable region with a Kabat Database ID No. 038233 was selected
for humanization of cH36 LC FR3. The amino acid sequence of a human
kappa light chain variable region with a Kabat Database ID No.
004733 was selected for humanization of cH36 LC FR4. The following
mutations were made in cH36 LC FR3 to match the amino acid sequence
of a human kappa light chain variable region with a Kabat Database
ID No. 038233: K70 D, K74 T, A80 P, V84 A, N85 T. Two mutations
A100 Q and L106 I were made cH36 LC FR4 to match the amino acid
sequence of a human kappa light chain variable region with a Kabat
Database ID No. 004733 (see Table 1B for sequence information).
(ii). Selection of Human IgG Heavy Chain Variable Region
Frameworks
[0219] The amino acid sequence in each of the frameworks of cH36 HC
was compared with the amino acid sequence in the corresponding FR
in human IgG heavy chain variable region in Kabat Database. The
best-fit FR was selected based on the three criteria described
above.
[0220] The amino acid sequence of a human IgG heavy chain variable
region with a Kabat Database ID No. 000042 was selected for
humanization of cH36 HC FR1. The amino acid sequence of a human IgG
heavy chain variable region with a Kabat Database ID No. 023960 was
selected for humanization of cH36 HC FR2. The following mutations
were made in cH36 HC FR1 to match the amino acid sequence of a
human IgG heavy chain variable region with a Kabat Database ID No.
000042: E1Q, Q5 V, P9 G, L 11 V, V12 K, Q19 R, T24 A. Two mutations
H41 P and S44 G were made in cH36 HC FR2 to match the amino acid
sequence of a human IgG heavy chain variable region with a Kabat
Database ID No. 023960 (see Table 2A for sequence information).
[0221] The amino acid sequence of a human IgG heavy chain variable
region with a Kabat Database ID No. 037010 was selected for
humanization of cH36 HC FR3. The amino acid sequence of a human IgG
heavy chain variable region with a Kabat Database ID No. 000049 was
selected for humanization of cH36 HC FR4. The following mutations
were made in cH36 HC FR3 to match the amino acid sequence of a
human IgG heavy chain variable region with a Kabat Database ID No.
037010: S76 T, T77 S, F80 Y, H82 E, N84 S, T87 R, D89 E, S91 T. One
mutation L113 V was made in cH36 HC FR2 to match the amino acid 5
sequence of a human IgG heavy chain variable region with a Kabat
Database ID No. 000049 (see Table 2B for sequence information).
[0222] Table 1A and 1B: Comparison of cH36 and Human Light Chain
(LC) FR Sequences TABLE-US-00001 TABLE 1A FR1 (23 AA) FR2 (15 AA)
Names 1 10 20 35 48 DIQMTQSPASQSASLGESVTITC WYQQKPGKSPQLLIY cH36
-LC DIQMTQSPASLSASVGDRVTITC WYLQKPGKSPQLLTY Human-LC
[0223] TABLE-US-00002 TABLE 1B FR3 (32 AA) FR4 (10 AA) Names 57 60
70 80 86 98 107 GVPSRFSGSGSGTK FSFKTSSLQAEDFVNYYC FGAGTKLELK
cH36-LC GVPSRFSGSGSGTDFSFTISSLQPEDFATYYC FGQGTKLEIK Human-LC
[0224] Table 2A and 2B: Comparison of cH36 and Human Heavy Chain
(HV) FR Sequences TABLE-US-00003 TABLE 2A FR1 (30 AA) FR2 (14 AA)
Names 1 10 20 29 36 44 EIQLQQSGPELVKPGASVQVSCKTSGYSFT
WVRQSHGKSLEWIG cH36-HC QIQLVQSGGEVKKPGASVRVSCKASGYSFT
WVRQSPGKGLEWTG Human-HC
[0225] TABLE-US-00004 TABLE 2B FR3 (32 AA) FR4 (11 AA) Names 67 75
85 95 107 117 KATLTVDKSSTTAFMHLNSLTSDDSAVYFCAR WGQGTTLTVSS cH36-HC
KATLTVDKSTSTAYMELSSLRSEDTAVYFCAR WGQGTTVTVSS Human-HC
[0226] Once the decisions on the desired human frameworks were
made, the following three techniques were used to achieve the
desired amino acid substitutions in both the light and heavy
chains: (1) Regular PCR was used for cloning, to introduce cloning
or diagnostic endonuclease sites, and to change amino acid residues
located at the ends of the variable regions. (2) PCR-based
mutagenesis was used to change multiple amino acid residues at a
time, especially when these residues were in the middle of the
variable regions. (3) Site directed mutagenesis was used to
introduce one or two amino acid substitutions at a time. Site
directed mutagenesis was done following the protocol described in
Stratagene's "QuickChange Site-Directed Mutagenesis Kit" (Catalog
#200518).
[0227] After each step, the partially humanized clones were
sequenced and some of these variable regions were later cloned into
expression vectors. The plasmid tKMC180 was used to express LC
mutants, and pJRS 355 or pLAM 356 vector was used to express HC
mutants as IgG1 or IgG4, respectively. Some of these clones were
then combined and expressed transiently in COS cells to determine
the expression levels by ELISA.
[0228] The final fully humanized forms of the anti-TF heavy and
light variable regions were cloned into what is sometimes referred
to herein as a "mega vector" and transfected into CHO and NSO cells
for IgG expression. Stable cell lines were then used to produce
amounts of humanized anti-TF sufficient for analysis. The resulting
humanized versions are 100% human in origin (when the CDR sequences
are not considered). The humanized IgG4 kappa version is designated
hFAT (humanized IgG Four Anti-Tissue Factor antibody) and the IgG1
kappa version is designated hOAT (humanized IgG One Anti-Tissue
Factor antibody). These fully humanized versions of cH36 are
intended for treating chronic indications, such as thrombosis,
cancer and inflammatory diseases.
C. Humanization of Anti-TF Antibody Heavy Chain
[0229] 1. PCR amplification and cloning into pGem T-easy of anti-TF
mAb cH36 heavy chain (HC) variable region were performed using
plasmid pJAIgG4TF.A8 (an expression vector for chimeric H36) as
template and primers TFHC1s2 and TFHC1as2. Primer TFHC1s2
introduced a BsiW1 site upstream of the initiation codon and also
an amino acid change E1 to Q in framework (FR) 1. Primer TFHC1 as
introduced an amino acid change L113 to V in FR4. This step
resulted in the construct HC01.
[0230] 2. PCR-based mutagenesis using the previous construct (HC01)
and the following four primers generated construct HC02. Upstream
PCR used primers TFHC1s2 and TFHC7as. Downstream PCR used primers
TFHC7s and TFHC1as2. Overlap PCR using upstream and downstream PCR
products as templates and with primers TFHC1s2 and TFHC1as2 yielded
HC02. The use of primers TFHC7s and TFHC7as introduced two amino
acid changes in FR2: H41 to P and S44 to G.
[0231] 3. PCR-based mutagenesis using HC02 as template and the
following four primers generated construct HC03. Upstream PCR used
primers TFHC1s2 and TFHC5as2. Downstream PCR used primers TFHC5s
and TFHC1as2. PCR using upstream and downstream PCR products as
templates and with primers TFHC1s2 and TFHC1as2 yielded HC03. The
use of primers TFHC5s and TFHC5as2 introduced three amino acid
changes in FR3: T87 to R, D89 to E, and S91 to T. A Bgl II site was
also introduced at position. 87.
[0232] 4. PCR amplification was performed using primers TFHC2s and
TFHC3as and HC03 in pGem as template. TFHC2s sits upstream of the
cloning site in pGem. TFHC3as sits in framework 3 and introduces
two amino acid changes in FR3: H82 to E and N84 to S. The resulting
PCR band was cloned into pGem and then the proper size insert was
digested with BsiW1 and Bgl II. Cloning of this fragment into HC03
yields HC04.
[0233] 5. PCR-based mutagenesis using HC04 as template and the
following primers resulted in HC05. Upstream PCR used primers
TFHC1s2 and TFHC6as. Downstream PCR used primers TFHC6s and
TFHC1as2. Mutagenic PCR using upstream and downstream PCR products
as templates and with primers TFHC1s2 and TFHC1as2 yielded HC05.
This step introduced the following amino acid changes in FR3: S76
to T, T77 to S, and F80 to Y.
[0234] 6. PCR-based mutagenesis using HC05 as template and the
following four primers generated HC06. Upstream PCR used primers
TFHC2s and TFHC2as2. Downstream PCR used primers TFHC3s2 and
TFHC1as2. Amplification using TFHC2as2 introduced an amino acid
change in FR1: P9 to G. Primer TFHC3s2 changes Q19 to R and T24 to
A. PCR using upstream and downstream PCR products as template and
with primers TFHC1s2 and TFHC1as2 yielded HC0 6.
[0235] 7. A point mutation from I to M in position 2 of FR1 was
spontaneously introduced during construction of HC06. PCR
amplification using HC06 as template and TFHC1s3 and TFHC1as2 as
primers, corrected this erroneous substitution and also introduced
an amino acid. change in FR1: Q5 to V. The resulting construct was
HC07.
[0236] 8. Construct HC08 was made by PCR-based mutagenesis using
HC07 as template and the following primers. TFHC2s and TFHC2as3
were used for the upstream product. The downstream product was
previously amplified using TFHC1s3 and TFHC1as2 (see step 7). The
use of primer TFHC2as3 introduced two amino acid changes in FR1:
L11 to V and V12 to K. A spontaneous point mutation resulted in a F
to L change at position 64 in CDR2. Further screening and
sequencing yielded construct HC08R1, which has the correct sequence
of F at position 64 in CDR2.
[0237] 9. Two constructs, HC11 and HC22, were generated by
site-directed mutagenesis from HC07. Two complementary primers
TFHC8sP and TFHC8asP were used along with HC07 as template to
produce HC11 which contains three amino acid changes in FR1: G9 P,
L11 to V, and V12 to K. Then, HC11 was methylated and column
purified for the next round of site directed mutagenesis. PCR using
HC11 as a template and the complementary primers TFHC9sL and
TFHC0asL generated HC12 which has a mutation from V11 to L in
FR1.
[0238] 10. Construct HC09 was derived from HC12 by performing PCR
using HC12 as a template and the complementary primers TFHC10sK and
TFHC10asK. HC09 contains an amino acid change: K12 to V in FR1.
[0239] 11. Construct HC10 was made from HC09. PCR using HC09 as a
template and the complementary primers LV-1 and LV-2 resulted in
the generation of HC10, which contains a mutation from L11 to V in
FR1.
[0240] After each mutation step, the partially humanized or fully
humanized clones were sequenced and some of these variable regions
were later cloned into expression vectors. pJRS 355 or pLAM 356
vector was used to express HC mutants fused to Fe of human IgG1 or
IgG4.
[0241] FIG. 7A summarizes steps 1-11 and shows incremental amino
acid changes introduced into FR1-4. Except HC08, all other heavy
chain mutants and cH36 contain F at position 64 in CDR2. HC08 has a
mutation from F to L at position 64. FIGS. 7B-D show the heavy
chain CDR sequences. TABLE-US-00005 Primers Used for Heavy Chain
Humanization TFHC1s2 5' TTTCGTACGTCTTGTCCCAGATCCAGCTGCAGCAGTC 3'
TFHC1as2 5' AGCGAATTCTGAGGAGACTGTGACAGTGGTGCCTTGGCCCCAG 3' TFHC7s
5' GTGAGGCAGAGCCCTGGAAAGGGCCTTGAGTGGATTGG 3' TFHC7as 5'
CCAATCCACTCAAGGCCCTTTCCAGGGCTCTGCCTCAC 3' TFHC5s 5'
GCATCTCAACAGCCTGAGATCTGAAGACACTGCAGTTTATTTCTGTG 3' TFHC5as2 5'
CTGCAGTGTCTTCAGATCTCAGGCTGTTGAGATGCATGAAGGC 3' TFHC3as 5'
GTCTTCAGATCTCAGGCTGCTGAGCTCCATGAAGGCTGTGGTG 3' TFHC2s 5'
TACGACTCACTATAGGGCGAATTGG 3' TFHC6s 5'
CTGTTGACAAGTCTACCAGCACAGCCTACATGGAGCTCAGCAG 3' TFHC6as 5'
CTGCTGAGCTCCATGTAGGCTGTGCTGGTAGACTTGTCAACAG 3' TFHC2as2 5'
GCACTGAAGCCCCAGGCTTCACCAGCTCACCTCCAGACTGCTGCAGC 3' TFHC3s2 5'
CTGGGGCTTCAGTGCGGGTATCCTGCAAGGCTTCTGGTTACTCATTCAC 3' TFHC1s3 5'
TCGTACGTCTTGTCCCAGATCCAGCTGGTGCAGTCTGGAGGTGAGC 3' TFHC2as3 5'
GCACTGAAGCCCCAGGCTTCTTCACCTCACCTCCAGACTGCACC 3' TFHC9sL 5'
GCAGTCTGGACCTGAGCTGAAGAAGCCTGGGG 3' TFHC9asL 5'
CCCCAGGCTTCTTCAGCTCAGGTCCAGACTGC 3' TFHC8sP 5'
GCTGGTGCAGTCTGGACCTGAGGTGAAGAAGCC 3' TFHC8asP 5'
GGCTTCTTCACCTCAGGTCCAGACTGCACCAGC 3' TFHC10sK 5'
GCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTC 3' TFHC10asK 5'
GAAGCCCCAGGCTTCACCAGCTCAGGTCCAGACTGC 3' LV-1 5'
CAGTCTGGACCTGAGGTGGTGAAGCCTGGG 3' LV-2 5'
CCCAGGCTTCACCACCTCAGGTCCAGACTG 3'
D. Humanization of Anti-TF Antibody Light Chain
[0242] 1. PCR amplification was performed using plasmid
pJAIgG4TF.A8 (an expression vector for chimeric H36) as template
and primers TFLC1s2.1 and TFLC1as2. This step introduced a cloning
site, AgeI, upstream of the coding region. It also introduced the
L106I mutation in FR4. This step yielded the construct LC03.
[0243] 2. Site-directed mutagenesis was performed using
complementary primers TFLC5s and TFLC5as and LC03 as template. This
step introduced the mutation Q37L in FR2 and added a PstI site for
diagnostic purposes. This new construct is named LC04.
[0244] 3. PCR amplification was performed using LC04 as template
and primers TFHC2s and TFLC2as1. This step generated Fragment A
that will be used in step 6. This step introduced Q11L and L15V
mutations in FR1.
[0245] 4. PCR amplification was performed using LC04 as template
and primers TFLC1s2.1 and TFLC1asR. This introduced the KpnI site
at the end of LC variable region. Cloning of this PCR fragment into
pGEM yields pGEM04K that will be used in step 6.
[0246] 5. PCR amplification was performed using LC04 as template
and primers TFLC2s and TFLC4as. This step generated Fragment C that
will be used in step 6. Three mutations E17D, S18R in FR1 and A100Q
in FR4 were introduced in this step.
[0247] 6. PCR-based mutagenesis using Fragment A and Fragment C as
templates and primers TFHC2s and TFLC4as yielded Fragment D.
Cloning of Fragment D into pGEM04K yielded the construct LC05.
[0248] 7. PCR amplification was performed using pGEM04K as template
and primers TFLC1s2.1 and TFLC4as. This step generated Fragment H,
which is then cloned into pGEM04K. This introduced the A100Q
mutation in FR4 and the construct is named LC06.
[0249] 8. PCR amplification was performed using LC06 as template
and primers TFLC1s2.1 and TFLC3as. This step generated Fragment I
that will be used in step 10. This introduced the K70D and the K74T
mutations in FR3.
[0250] 9. PCR amplification was performed using LC06 as template
and primers TFLC3s2 and TFLC4as. This step generated Fragment F
that will be used in step 10. This introduced the A80P mutation in
FR3.
[0251] 10. PCR using Fragment I and Fragment F as templates and
primers TFLC1s2.1 and TFLC4as yielded Fragment J. Cloning of
Fragment J into pGEM yielded the construct LC07.
[0252] 11. Site-directed mutagenesis was conduced using
complementary primers TFLC08sds and TFLC08sdsa and LC07 as
template. This step introduced the mutations V84A and N85T in FR3.
This construct is named LC08.
[0253] 12. The AgeI to EcoO109I fragment from LC05 containing the
mutations Q11L, L15V, E17D, S18R and Q37L is cloned into LC08. This
yielded the construct LC09.
[0254] 13. Site-directed mutagenesis was conduced using LC09 as
template and the complementary primers LC105 and LC103. This step
introduced the T85N mutation in FR3 and yielded the construct
LC10.
[0255] 14. Site-directed mutagenesis was conducted using LC10 as
template and the complementary primers LC115 and LC113. This step
introduced the D70K mutation in FR3. This yielded the construct
LC11.
[0256] 15. Site-directed mutagenesis was conducted using LC11 as
template and the complementary primers LC125a and LC123a. This step
introduced the K42Q mutation in FR2. This yielded the construct
LC12.
[0257] After each mutation step, the partially humanized or fully
humanized LC clones were sequenced and some of these variable
regions were later cloned into expression vector tKMC180.
[0258] FIG. 6 A summarizes steps 1-15 and shows incremental amino
acid changes introduced into FR1-4 of the light chain. FIGS. 6B-D
show the light chain CDR sequences. TABLE-US-00006 Oligonucleotide
Primers Used for Light Chain Humanization TFLC1as2: 5'
TTCGAAAAGTGTACTTACGTTTGATCTCCAGCTTGGTCCCAG 3' TFLC1s2.1: 5'
ACCGGTGATATCCAGATGACCCAGTCTCC 3' TFLC5s: 5'
GGTTAGCATGGTATCTGCAGAAACCAGGG 3' TFLC5as: 5'
CCCTGGTTTCTGCAGATACCATGCTAACC 3' TFHC2s: 5'
TACGACTCACTATAGGGCGAATTGG 3' TFLC2as1: 5'
CCACAGATGCAGACAGGGAGGCAGGAGACTG 3' TFLC1asR: 5'
TTCGAAAAGTGTACTTACGTTTGATCTCCAGCTTGGTACCAGCACCGAACG 3' TFLC2s: 5'
CCTGTCTGCATCTGTGGGAGATAGGGTCACCATCACATGC 3' TFLC4as: 5'
GATCTCCAGCTTGGTACCCTGACCGAACGTGAATGG 3' TFLC3as: 5'
GTAGGCTGCTGATCGTGAAAGAAAAGTCTGTGCCAGATCC 3' TFLC3s2: 5'
CACGATCAGCAGCCTACAGCCTGAAGATTTTGTAAATTATTACTGTC 3' TFLC08sds: 5'
GCAGCCTACAGCCTGAAGATTTTGCAACTTATTACTGTCAACAAG 3' TFLC08sdsa: 5'
CTTGTTGACAGTAATAAGTTGCAAAATCTTCAGGCTGTAGGCTGC 3' LC105: 5'
CAGCAGCCTACAGCCTGAAGATTTTGCAAATTATTACTGTCAAC 3' LC103: 5'
GTTGACAGTAATAATTTGCAAAATCTTCAGGCTGTAGGCTGCTG 3' LC115: 5'
CAGTGGATCTGGCACAAAGTTTTCTTTCACGATCAGCAGC 3' LC113: 5'
GCTGCTGATCGTGAAAGAAAACTTTGTGCCAGATCCACTG 3' LC125a: 5'
CTGCAGAAACCAGGGCAATCTCCTCAGCTCCTG 3' LC123a: 5'
CAGGAGCTGAGGAGATTGCCCTGGTTTCTGCAG 3'
[0259] FIG. 8 shows hOAT (humanized cH36-IgG1) constant region
sequences of the light (FIG. 8A) and heavy chain (FIG. 8B). FIG. 9
shows hFAT (humanized cH36-IgG4) constant region sequences of the
light (FIG. 9A) and heavy chain (FIG. 9B). In each figure, the last
amino acid residue of the framework 4 (FR4) variable region is
connected to the first amino acid residue of the constant region
for hOAT and hFAT.
EXAMPLE 2
Expression and Purification of Humanized anti-TF Antibodies
[0260] The partially humanized or fully humanized LC and HC clones
were cloned into expression vectors. The plasmid tKMC180 (see FIGS.
4A-B) was used to express LC mutants fused to human kappa chain,
and pJRS 355 (see FIGS. 2A-B) or pLAM 356 (see FIGS. 3A-B) vector
was used to express HC mutants fused to Fc of human IgG1 or IgG4.
Some combinations of the HC and LC clones were then co-transfected
into COS cells. The transiently expressed IgGs in COS cells were
assayed for the whole IgG production and binding to TF by
ELISA.
[0261] The final fully-humanized forms of the anti-TF heavy and
light variable regions (combination of HC08 and LC09) were cloned
into Sunol's Mega expression vector (pSUN34, see FIG. 5) and
transfected into CHO and NSO cells for IgG expression. Stably
transfected cell lines producing the IgG4.kappa. or IgG1.kappa.
humanized anti-TF antibody were cloned. The selected stable cell
lines were then used to produce amounts of humanized anti-TF
sufficient for analysis. The resulting humanized versions are
approximately 95% human in origin (the CDR sequences are not
considered). The humanized IgG4 kappa version is designated hFAT
(humanized IgG Four Anti-Tissue Factor antibody) and the IgG1 kappa
version is designated hOAT (humanized IgG One Anti-Tissue Factor
antibody). These fully humanized versions of cH36 are intended for
treating chronic indications, such as cancer and inflammatory
diseases.
[0262] One of the NSO cell lines (OAT-NSO-P10A7) that expresses
hOAT (combination of HC08 and LC09) was thawed and extended in 10
mL of IMDM medium supplemented with 10% FBS in a 15 mL tube and
centrifuged. The cell pellet was resuspended in 10 mL of fresh
media and passed to a T25 flask and incubated at 37.degree. C. in
5% CO.sub.2. In order to prepare a sufficient number of cells to
inoculate a hollow fiber bioreactor, the cells were expanded to
obtain a total of 6.times.10.sup.8 cells. A bioreactor was set up
as per manufacturer's instruction manual. The harvested cells were
pelleted and resuspended in 60 mL of IMDM containing 35% FBS and
injected into the extracapillary space of the bioreactor.
Concentrations of glucose and lactate were monitored daily and the
harvest material was centrifuged and pooled. The harvested material
was tested for anti-TF antibody concentrations by ELISA assay. The
pooled sample containing anti-TF antibody (hOAT) were then purified
and analyzed as described below.
A. rProtein A Sepharose Fast Flow Chromatography
[0263] Recombinant humanized anti-TF monoclonal antibody consists
of two light and two heavy chains. Heavy chain is a fusion of mouse
variable region (unaltered or humanized as described above) and
human IgG1 or IgG4 Fe domain, while light chain contains mouse
variable region (unaltered or humanized as described above) and
human K domain. It is well established that human IgG Fe region has
high affinity for Protein A or recombinant Protein A (rprotein
A).
[0264] Harvest pools containing humanized anti-TF antibody (hOAT)
were adjusted to pH 8.0+0.1 by adding 0.08 ml of 1 M Tris-HCl, pH
8.0 per ml of sample. Then the sample is filtered through low
protein-binding 0.22 micron filters (e.g., Nalgene sterile
disposable tissue culture filter units with polyethersulfone
membrane from Nalge Nunc International, Cat. No. 167-0020).
Following sample application, rProtein A column (from Pharmacia) is
washed with 5 bed volumes of 20 mM Tris-HCl, pH 8.0 to remove
unbound materials such as media proteins. Since the harvest medium
contains high content of bovine serum, a stepwise pH gradient wash
was used to remove bovine IgG from the column. The stepwise pH
gradient was achieved by increasing the relative percentage of
Buffer B (100 mM acetic acid) in Buffer A (100 mM sodium acetate).
A typical pH stepwise wash employed 20%, 40%, and 60% Buffer B.
Elute the column with 100% Buffer B and collect fractions based on
A.sub.280. The pooled fractions were adjusted to pH 8.5 with
addition of 1 M Tris base.
B. Q Sepharose Fast Flow Chromatography
[0265] Anion ion exchange chromatography is very effective in
separating proteins according to their charges. The eluted and
pH-adjusted sample from rProtein A column was diluted with two
volumes of water, and the pH is checked and adjusted to 8.5. The
sample was then loaded to a 5 ml (1.6.times.2.5 cm) Q Sepharose
Fast Flow equilibrated with 20 mM Tris-HCl, pH 8.5 and the column
washed with (1) 5 bed volumes of 20 mM Tris-HCl, pH 8.5; and (2) 4
bed volumes of 20 mM Tris-HCl, pH 8.5 containing 100 mM NaCl. The
IgG protein was then eluted with bed volumes of 20 mM Tris-HCl, pH
8.5 containing 500 mM NaCl. The protein peaks were pooled and
buffer-exchanged into PBS using ultrafiltration device.
[0266] Using the same transfection, cell culture, and purification
methods, hFAT was also produced and purified.
EXAMPLE 3
Properties of Humanized Anti-TF Antibodies
A. Inhibition of TF Function by Humanized Anti-TF Antibody
[0267] One of the key properties of anti-TF antibodies is its
ability to inhibit tissue factor-initiated blood coagulation. The
purified hOAT and hFAT were measured for their ability to inhibit
TF activity in a standard PT assay. PT assay is widely used to
measure tissue factor-dependent blood clotting times. The principal
of this assay is that tissue factor (TF) forms complex with factor
VIIa in plasma. This complex then activates factor X to FXa; FXa
then converts prothrombin to thrombin in the presence of factor Va
and phospholipids. Thrombin eventually leads to formation of a
blood clot. In standard PT assays, lipidated TF is added to plasma
to initiate blood coagulation and the clotting is recorded by an
Organon Teknika Coag-A-Mate Coagulation Analyzer or equivalent.
[0268] The anti-TF antibody, H36, inhibits human TF activity by a
unique mechanism. It binds to TF (free or in complex with factor
VIIa) in such a way that factor X and IX binding to TF:VIIa complex
is prohibited, thus FX and FIX activation by TF:VIIa is blocked
(see U.S. Pat. No. 5,986,065). In PT tests, the prolongation of
clotting times by anti-TF antibody added into human plasma is a
clear indication that this TF-dependent coagulation is inhibited.
The clotting time is related to the amount of TF activity. A TF
standard curve is generated by measuring PT clotting times of
serially diluted TF. From the data of TF standard curve, the
inhibition of TF activity by anti-TF antibody is determined.
[0269] Reagents for standard PT assay: Innovin (Cat No 68100-392)
as a recombinant human TF source and Ci-Trol Coagulation Control,
Level I (Cat No 68100-336) as a human plasma source are obtained
from VWR. PT Assay Method: PT test is performed at 37 C. using a
Coagulation Analyzer. PT reaction is initiated by adding 0.2 ml of
lipidated recombinant human tissue factor (e.g., Innovin) into 0.1
ml of human plasma (Ci-Trol Control Level I) containing 0.01 ml
buffer (50 mM Tris-HCl, pH 7.5, 0.1% BSA) or anti-TF antibody.
[0270] 1. Add purified water to a vial of Innovin according to
manufacturer's instruction. Warm the reagent to 37.degree. C. The
reagent is stable for a few days if stored at 4-8.degree. C. [0271]
2. Add 1 ml purified water to each vial of Ci-Trol. Mix to
solubilize. If more one vials are used, combine them into one
container (e.g., a 10 ml test tube). 1 ml Ci-Trol can run 5 assays
(each assay uses 2.times.0.1 ml=0.2 ml). Ci-Trol can be stored on
ice and last for a few hours. [0272] 3. From anti-TF antibody
stock, make a series of anti-TF antibody solutions (200 nM to 1600
nM) with 50 mM Tris-HCl, pH 7.5, 0.1% BSA [0273] 4. Add 10 .mu.l of
50 mM Tris-HCl, pH 7.5, 0.1% BSA or 10 .mu.l of diluted anti-TF
antibody to each well of the twin-well cuvette that contains 0.1 ml
of Ci-Trol. Use a pipette with 0.1 ml tip to mix each well. Make
sure no air bubbles are in the well. Following mixing anti-TF (or
buffer) with plasma (Ci-Trol), measure clotting times within 10 min
by adding 0.2 ml of Innovin to the plasma. [0274] 5. For TF
standard curve, first dilute Innovin (100% TF) to 20%, 10%, 5%and
2.5% with 50 mM Tris-HCl, pH 7.5, 0.1% BSA. Then PT assays were
performed as in Step 4 but using diluted Innovin samples.
[0275] Table 3 is the summary of the effect of cH36, hOAT, and HFAT
on PT clotting times. Compared to the data in Table 4, cH36, hFAT,
and hOAT showed very potent inhibition of TF function. At a protein
concentration of above 12.9 nM, all antibodies achieved about 95%
inhibition. The results in Table 3 also indicate that humanization
of anti-TF, cH36, by the method described above did not have any
significant effect on cH36 inhibitory activity since both HFAT and
hOAT showed very similar ability to inhibit TF-dependent blood
coagulation as seen for cH36. TABLE-US-00007 TABLE 3 Effect on
Prothrombin Times by Chimeric (cH36) and Humanized) Anti-TF
Antibodies (hFAT and hOAT).sup.# Anti-TF Antibody Concentrations
(nm) PT Time (in seconds) in PT Assays scH36 hOAT hFAT 0 12.2 12.2
12.2 6.45 14.9 nd nd 9.7 17.8 16.5 nd 12.9 19.8 18.9 20.5 25.8 40
33.7 41.7 51.6 101.3 82.1 94.8
[0276] #All assays used the same 100% TF activity (concentration)
sample as in Table 4. TABLE-US-00008 TABLE 4 Clotting Times and
Relative Tissue Factor Activities (Concentrations) Relative TF
Activities (Concentrations) PT Clotting Times (Seconds) 100% (neat)
11.90 20% 13.225 10% 14.675 5% 16.700 2.5% 20.000
B. Determination of Affinity Constants
[0277] The affinity of humanized anti-TF antibody for TF was
determined by surface plasmon resonance (BIAcore from Pharmacia
Biosensor) with recombinant human tissue factor covalently
immobilized on a CM5 sensor chip. The affinity constants were the
average data calculated from four anti-TF monoclonal antibody
concentrations (0.125 nM, 0.25 nM, 0.5 nM, and 1 nM) by the BlAcore
computer software. The results in Table 5 indicate that
humanization of anti-TF, cH36, by the method described above did
not have any significant effect on cH36 affinity for TF since both
cH36 and hFAT have similar affinity for human TF. TABLE-US-00009
TABLE 5 Apparent Affinity and Dissociation Constants of Anti-TF
Antibodies Anti-IF Antibody Apparent K.sub.a (M.sup.-1) Apparent
K.sub.d (M) H36 1.56 .times. 10.sup.10 6.4 .times. 10.sup.-11 cH36
7.94 .times. 10.sup.9 1.26 .times. 10.sup.-10 hFAT 2.99 .times.
10.sup.9 3.35 .times. 10.sup.-10
EXAMPLE 4
Effects of Chimeric Anti-Tissue Factor Monoclonal Antibody on the
Formation of Vascular Thrombosis in Chimpanzees
[0278] The following example shows results of using a particular
chimeric antibody of the invention (cH36) to prevent or treat
vascular thrombosis and vascular lesion formation. As described
above, cH36 is a chimeric antibody comprising mouse antibody light
and heavy chains variable domains fused to human antibody light and
heavy chain constant domains. In the examples that follows, the
cH36 antibody will sometimes be referred to as "Sunol-cH36" or
related phrase.
[0279] Briefly, TF was exposed by performing an endarterectomy on
the femoral artery in chimpanzees. .sup.111Indium-oxine labeled
platelets were deposited at the endarterectomy site in the controls
and deposition was significantly reduced with cH36 treatment. As
shown below, the vessels from antibody-treated animals were 80%
patent whereas the controls were 20% patent 30 days following the
endarterectomy. Cutaneous bleeding time and other hematology
parameters were not significantly different between the
antibody-treated and the controls with the exception of PT times
(which are expected to increase due to the presence of the antibody
in plasma which is used to perform PT assay with Innovin).
[0280] Results of the example show that the cH36 antibody and other
antibodies of the invention, particularly humanized TF binding
antibodies, should be efficacious in treating patients with
cardiovascular diseases due to thrombotic occlusion (such as stable
or unstable angina, myocardial infarction (MI), peripheral vascular
bypass grafts (PVBG), deep vein thrombosis (DVT) or restenosis
following percutaneous transluminal coronary intervention, etc.)
and that cH36 is safe with respect to bleeding.
[0281] The cH36 antibody was shown to inhibit chimpanzee
TF-dependent FX activation, indicating that chimpanzees could be
used for in vivo studies.
[0282] In the in vivo study, five chimpanzees underwent sequential
surgical endarterectomies on the right and left superficial femoral
arteries (30 days apart). Autologous platelets were obtained prior
to surgery and labeled using .sup.111Indium-oxine. Each chimpanzee
was administered .sup.111In-labeled platelets intravenously,
followed by administration of a placebo or a 1 mg/kg dose of cH36
immediately prior to restoration of flow in the endarterectomized
femoral artery. Post-surgical gamma camera imaging of
.sup.111In-platelet deposition at the endarterectomy sites was
performed, and the manipulated segments were harvested after 30
days. Each chimpanzee was subjected to the procedure (using placebo
or cH36) on the right or left side, and after 30 days the procedure
was repeated on the opposite side with the other treatment, so that
each animal served as its own control for comparison of platelet
deposition and neointimal lesion formation at the sites of
endarterectomy with and without the cH36 treatment.
[0283] Deposition of autologous .sup.111In-platelets was measured
in five-minute intervals for the first 30 minutes by gamma camera
imaging to assess acute vascular thrombosis at the endarterectomy
sites. Platelet deposition was significantly decreased with
Sunol-cH36 when evaluated at 5 and 30 minutes post-operatively
compared to placebo. Since platelets play a pivotal role in
thrombus formation, the results indicate that Sunol-cH36 inhibit
acute thrombus formation.
[0284] Autologous platelets were labeled with 1 mCi .sup.111Indium
(.sup.111In) oxine as described (Lumsden A B, Kelly A B, Schneider
P A, Krupski W C, Dodson T, Hanson S R, Harker L A. "Lasting safe
interruption of endarterectomy thrombosis by transiently infused
antithrombin peptide D-Phe-Pro-ArgCH2Cl in baboons." Blood 1993;
81:1762-1770; Kelly A S, Marzec U M, Krupski W, Bass A, Cadroy Y,
Hanson S R, Harker L A. "Hirudin interruption of heparin-resistant
arterial thrombus formation in baboons." Blood 1991; 77:1006-1012)
and re-injected. Gamma camera images were acquired continuously for
30 min in 5 min intervals with a G E 400T scintillation camera
(General Electric, Milwaukee, Wis.) or equvalent. The data were
stored and analyzed on a Medical Data System A Computer (Medasys
Inc., Ann Arbor, Mich.) or equivalent. A medium-energy collimator
was placed close to the animal. Measurements of .sup.111In-activity
were corrected for background activity. Activity of a 5-mL whole
blood standard was also determined. The activity of the blood
standard was also corrected for the small fraction of circulating
non-platelet radioactivity to give platelet-associated
.sup.111In-activity per mL of whole blood. The total platelet
deposition, including both labeled and unlabeled platelets, was
calculated by dividing the deposited CPM by circulating platelet
CPM (blood standard) and multiplying by the circulating platelet
count (platelets per mL of whole blood) as measured in the blood
standard sample.
[0285] Vessel injury site to blood ratios were also determined and
found to be decreased between control and Sunol-cH36 treated
chimpanzees at 5 and 30 minutes post-operatively. At 24 hours, the
platelet deposition effects of Sunol-cH36 treatment on vessel
injury to blood ratio were less statistically significant. Table 6
and FIGS. 10 and 11 summarize the results of platelet deposition
and vessel injury/blood ratio determinations.
[0286] Vessel Injury Site/Blood Ratio: Because circulating
.sup.111In platelet activity is cleared continuously through normal
physiological mechanisms, platelet accumulation after the acute
postoperative period was expressed as the ratio of the .sup.111In
platelet activity at the endarterectomy site to the .sup.111In
platelet activity in the blood. This measurement was independent of
the size of the animal, the amount of isotope injected, or the
extent to which the isotope may have decayed. Radioactivity values
in these calculations refer to platelet activity only, with blood
and standard values corrected for the small fraction of nonplatelet
activity (see Schneider P A, Hanson S R, Price T M, Harker L A.
"Confluent durable endothelialization of endarterectomized baboon
aorta by early attachment of cultured endothelial cells," J. Vasc.
Surg. 11:3 65-3 72 [1990]). This vessel injury site/blood ratio was
a reproducible measure of thrombus, allowing comparisons at 24
hours post surgery. TABLE-US-00010 TABLE 6 Platelet Deposition and
Vessel Injury/Blood Ratio in Chimpanzees Controls Sunol-cH36 p
Platelet Deposition (.times.10.sup.9) At 5 minutes 1.29 .+-. 0.42
0.36 .+-. 0.19 0.001 At 30 minutes 1.53 .+-. 0.93 0.37 .+-. 0.03
0.02 Vessel Injury/Blood Ratio At 5 minutes 5.22 .+-. 1.98 1.62
.+-. 0.92 0.002 At 30 minutes 6.81 .+-. 4.29 1.29 .+-. 0.82 0.02 At
24 hours 3.72 .+-. 1.61 2.26 .+-. 1.47 0.17 30 Day Vessel Patency
(%) 20 80 -- Values = mean .+-. standard deviation p < 0.05 was
considered statistically significant
[0287] The endarterectomized vessel segments were harvested 30 days
following surgery to assess longer-term effects of Sunol-cH36. The
patency of these vessels was examined and 20% (1/5) of the vessels
from the controls were patent, whereas 80% (4/5) of those treated
with Sunol-cH36 were patent (Table 6). It was thus concluded that a
single dose of Sunol-cH36 at 1 mg/kg contributed significantly to
maintaining patency.
[0288] The data presented in Table 7 show that the mean bleeding
time was 2 minutes in control chimpanzees and 2.5 minutes in the
treated chimpanzees, and this difference is not significant. The
surgical blood loss was not significantly different for the five
control procedures compared to the procedures in which the
chimpanzees received the test article. All other hematological
markers similarly did not show a difference in values considered
significant for controls versus the Sunol-cH36 treated animals (see
Table 7). TABLE-US-00011 TABLE 7 Hematology & Other Assessments
in Endarterectomized Chimpanzees Control Sunol-cH36 p Bleeding Time
(min) 2.0 .+-. 0.2 2.5 .+-. 0.6 ns Surgical Blood Loss (mL) 5.91
.+-. 2.2 4.7 .+-. 3.0 ns Prothrombin Time (sec) Baseline
(pre-surgery) -- 11.2 .+-. 0.7 -- 5 min (post RF) -- 180 .+-. 40
0.0001 15 min (post RF) -- 157 .+-. 52 0.0001 30 min (post RF) --
161 .+-. 60 0.0001 60 min (post RF) -- 177 .+-. 27 0.0001 90 min
(post RF) -- 202 .+-. 7.7 0.0001 24 hours (post RF) -- 18.1 .+-.
5.1 0.036 Platelet Count (.times.10.sup.3/.mu.L) Baseline 222 .+-.
73 233 .+-. 52 ns Post Surgery 236 .+-. 72 249 .+-. 54 ns 24 hr
Post Surgery 273 .+-. 61 247 .+-. 90 ns WBC (.times.10.sup.3/.mu.L)
Baseline 17.8 .+-. 1.5 9.8 .+-. 1.8 ns Post Surgery 16.9 .+-. 3.2
13.9 .+-. 42 ns 24 hr Post Surgery 18.1 .+-. 4.5 19.7 .+-. 6.5 ns
Antibody Levels by Prothrombin Time Assay (.mu.g/mL) 5 min 0 26.5
.+-. 5.3 -- 90 min -- 27.0 .+-. 2.1 -- 24 hour -- 8.9 .+-. 3.7 --
Antibody Levels by Factor X Activation Assay (.mu.g/mL) 5 min --
18.8 .+-. 2.8 -- 90 min -- 16.5 .+-. 1.4 -- 24 hour -- 6.3 .+-. 0.5
-- Monocyte expression of CD64 (number) Baseline 7160 .+-. 1930
8600 .+-. 1800 ns 90 min 6900 .+-. 1700 8250 .+-. 1200 ns 24 hr
10600 .+-. 4900 10400 .+-. 3300 ns Monocyte expression of tissue
factor (ratio of clotting time of unstimulated/stimulated) Baseline
4.8 .+-. 0.8 4.0 .+-. 1.2 0.3 90 min 4.7 .+-. 0.6 1.4 .+-. 0.1
0.005 24 hr -- 3.0 .+-. 0.8 -- Values are mean .+-. standard
deviation; n = 5 ns = not significant; p < 0.05 considered
statistically significant --means value not determined post RF
means post restoration of flow following conclusion of
endarterectomy
[0289] See also Smyth et al, (1995) British Journal of Surgery
82:588-595 (disclosing other methods of measuring platelet
deposition).
EXAMPLE 5
Effect of Anti-Tissue Factor Antibody (Sunol-cH36) When
Administered During the Performance of Skin Flap Surgery on
Cynomolgus Monkeys
[0290] In this example, a large skin flap was surgically elevated
and its blood supply limited to a single perforating vessel. In
control animals and those treated with lower doses of cH36, the
skin flap does not receive adequate perfusion and is slow to heal.
At high cH36 doses, the flap showed better perfusion and faster
healing. This suggests that cH36 will also improve the outcome of
plastic and reconstructive surgery.
[0291] A surgical study was also conducted to evaluate treatments
with Sunol-cH36 in preventing potential thrombosis of the vascular
supply to an abdominal skin flap on cynomolgus monkeys and to
determine whether or not excessive bleeding occurs during the
procedure according to the dose administered. In this surgical
study, five cynomolgus monkeys (1 male and 4 females) underwent a
surgical skin flap procedure to induce surgical trauma. The study
consisted of 5 groups of 1 animal each. The procedure consisted of
raising a rectangular skin paddle, measuring approximately 10 cm by
9 cm, which was elevated from the abdominal wall, limiting blood
supply and then reattaching the skin flap. Following ligation of
the inferior epigastric artery, the blood supply to the skin flap
is limited to vessels on the left side. Group 1 served as a control
and received PBS. The remaining groups were administered different
doses of Sunol-cH36. Table 8 below, shows the experimental
design.
[0292] Skin flap viability was evaluated over a 4-week observation
period to determine if treatments with the test article have an
effect on preventing thrombosis of the vascular supply to the
abdominal skin flap on cynomolgus monkeys. Fluorescein was injected
and the skin flap examined under ultraviolet light. When the tissue
was adequately perfused, the area would fluoresce while areas with
poor perfusion appeared dark. For all but one animal (Group 5
animal 5101; 5 mg/kg), the left side (predictably) showed good
perfusion following surgery, and the right side typically did not
indicate acceptable perfusion, also an expected observation. For
some reason, this was reversed in the animal 5101, in that the
right side showed acceptable perfusion, rather than the left. This
was apparently due to an anatomical variation in this one animal.
With time, both sides demonstrated adequate perfusion in all
animals; however there seemed to be a trend toward earlier and more
complete perfusion as the dose of the test article was increased.
TABLE-US-00012 TABLE 8 Study Design for the Skin Flap Study in
Cynomolgus Monkeys Treatment Dose Dose Dose Group Animal Test Level
Concentration Volume Number Number Article (mg/kg) (mg/mL) (mL/kg)
1 1001 PBS 0 0 0.5 2 2101 Sunol- 0.04 0.515 0.08 3 3101 cH36 0.2
0.4 4 4101 1.0 10.3 0.1 5 5101 5.0 0.5
[0293] Macroscopic and microscopic pathology evaluations were also
performed. Evidence of possible continuing compromise to the skin
flap was observed for groups 2 and 3 animals (0.04 and 0.2 mg/kg
respectively) at the scheduled time of euthanasia, Day 29. The
group 2 animal 2101 had a dark area of discoloration in the skin to
the left and right skin flap regions. Animal 3101 had a single
6.times.40 mm area of ulceration in the skin of the cranial portion
of the right skin flap region.
[0294] Microscopic analysis consisted of examination of four
sections from within the skin flap site for all animals: one each
from the left and right cranial regions of the flap site, and one
each from the left and right caudal regions of the flap site. Table
9 summarizes notable microscopic findings.
[0295] Although this study involved only one animal per dose group,
there appeared to be a few notable differences in the severity of
changes between the skin flap sites of the control animal (left and
right), and the skin flap sites of the test article-treated
animals. Macroscopic study indicated the degree of edema, erythema,
and even necrosis of the right skin flap tended to decrease in
severity as the dose of the test article increased. The degree of
fluorescein staining of the skin flap tended to increase with
increasing dose of Sunol-cH36.
[0296] Microscopically, there appeared to be a small but notable,
dose-dependent decrease in granulation tissue formation and
subintimal vascular smooth muscle proliferation in the panniculus
on the left side of the skin flap site in the test article-treated
animals compared to the left side of the skin flap site in the
control animal. These results suggest that the test article could
prevent microthrombosis following procedures of this nature, thus
preventing devascularization of a graft. TABLE-US-00013 TABLE 9
Severity* of Selected Microscopic Findings in the Skin Flap Study
in Cynomolgus Monkeys Animal Numbers & Dose Level mg/kg) 1001
2101 3101 4101 5101 Tissue/Lesion 0 0.04 0.2 1.0 5.0 Left Skin Flap
Site Chronic Inflammation 2.0 2.0 1.5 0.5 1.0 Granulation Tissue
2.0 3.0 1.5 0.5 0.5 Subintimal Vascular Proliferation 2.0 2.5 1.0
-- -- Hemosiderin Accumulation 1.0 1.0 0.5 -- -- Ulceration -- --
-- -- -- Right Skin Flap Site{circumflex over ( )} Chronic
Inflammation 1.0 2.0 2.5 1.0 1.0 Granulation Tissue 2.0 3.0 2.5 2.5
1.0 Subimtimal Vascular Proliferation -- 3.0 2.5 -- 1.5 Hemosiderin
Accumulation -- 0.5 2.0 -- -- Ulceration -- -- 2.0 -- -- *The
severity of the change is the average severity for the 2 sites
examined (cranial and caudal) where 1 is least severe and 3 is most
severe. (Average severity equals the summation of severity scores
for each section/number of sections affected) -- means microscopic
findings not present {circumflex over ( Right skin flap site had
perforating vessel ligated
[0297] All surgical procedures proceeded without incident. There
were no observed increases in bleeding. The sponge count procedure
described in the study protocol was not performed due to the
negligible amounts of blood loss. Except for a slight increase in
the white blood cell count during the surgical procedure, there
were no hematological changes. The slight increase in white blood
cell count is expected due to the traumatic nature of the surgical
procedure. No significant change in hematocrit and hemoglobin
levels was detected. Hematology results showed several red blood
cell parameters to be outside of the expected ranges in groups 2
and 3. These variations were not considered to be test
article-related. Table 10 summarizes the hematology parameters and
study results.
[0298] In all groups, ACT, APTT and fibrinogen levels remained
within normal ranges and did not deviate from baseline during the
procedure. No change in PT values was observed in groups 1, 2, 3
and 4. However, in the group 5 animal, PT dramatically increased
immediately following administration of 5 mg/kg of Sunol-cH36.
Prothrombin Times increased from 10.1 seconds to >212 seconds
(approximately 5 minutes after dosing). Prothrombin Times slowly
decreased from >212 seconds to 198.1 seconds at 15 minutes post
administration and to 135.2 seconds at 45 minutes post
administration. This elevation was considered an expected
Sunol-cH36-related effect. TABLE-US-00014 TABLE 10 Hematology
Parameters and Individual Study Results for the Skin Flap Study in
Cynomolgus Monkeys Animal Number/cH36 Dose (mg/kg) 1001 2101 3101
4101 5101 Parameter Time Points 0 (PBS) 0.04 0.2 1.0 5.0 Total Post
Catheter 7.6 4.5 5.5 5.1 5.1 Leukocyte Pre-Treatment 7.7 4.4 5.3
5.1 4.8 Count 15 minutes 7.8 4.58 5.8 5.8 4.6 (WBC) 30 minutes 9.7
4.8 7.8 5.4 5.2 (10.sup.3/.mu.L) 45 minutes N/A 5.4 8.9 6.9 5.2
Erythiocyte Post Catheter 6.00 3.99 4.32 6.00 6.33 Count
Pre-Treatment 5.87 4.02 4.13 6.05 6.03 (RBC) 15 minutes 5.70 3.96
4.10 6.09 5.94 (10.sup.6/.mu.L) 30 minutes 5.41 3.87 4.24 5.69 5.95
45 minutes N/A 3.83 3.87 5.77 5.55 Hemoglobin Post Catheter 11.4
10.2 11.2 10.8 11.6 Concentration Pre-Treatment 11.2 10.2 10.9 10.6
11.3 (HGB) 15 minutes 10.5 10.0 10.7 10.9 11.2 (g/dL) 30 minutes
10.9 9.9 11.0 9.9 11.0 45 minutes N/A 9.9 10.7 10.6 10.5 Hematocrit
Post Catheter 37.9 32.9 34.6 35.8 39.6 Value Pre-Treatment 37.3
33.2 33.3 36.3 37.4 (HCT).sup.a 15 minutes 36.5 32.7 32.9 36.4 36.9
% 30 minutes 34.5 31.9 34.0 34.1 37.0 45 minutes N/A 31.5 30.8 34.5
34.6 Mean Post Catheter 63.2 82.5 80.1 59.7 62.6 Corpuscular
Pre-Treatment 63.5 82.6 80.6 60.0 62.0 Volume 15 minutes 64.0 82.6
80.2 59.8 62.1 (MCV).sup.a 30 minutes 63.8 82.4 80.2 59.9 62.2 (fL)
45 minutes N/A 82.2 79.6 59.8 62.3 Mean Post Catheter 19.0 25.6
25.9 18.0 18.3 Corpuscular Pre-Treatment 19.1 25.4 26.4 17.5 18.7
Hemoglobin 15 minutes 18.4 25.3 26.1 17.9 18.9 (MCH).sup.a 30
minutes 20.1 25.6 25.9 17.4 18.5 (pg) 45 minutes N/A 25.8 27.6 18.4
18.9 Mean Post Catheter 30.1 31.0 32.4 30.2 29.3 Corpuscular
Pre-Treatment 30.0 30.7 32.7 29.2 30.2 Hemoglobin 15 minutes 28.8
30.6 32.5 29.9 30.4 Concentration 30 minutes 31.6 31.0 32.4 29.0
29.7 (MCHC).sup.a 45 minutes N/A 31.4 34.7 30.7 30.3 (g/dL)
Platelet Post Catheter 377 333 324 252 253 Count Pre-Treatment 315
318 292 279 220 (PLT) 15 minutes 365 332 300 274 233
(10.sup.3/.mu.L) 30 minutes 327 325 320 262 227 45 minutes N/A 315
285 266 175 .sup.aCalculated value
[0299] TABLE-US-00015 TABLE 11 Coagulation Parameters &
Individual Study Results (Day 1) for the Skin Flap Study in
Cynomolgus Monkeys Animal Activated Prothrombin Activated Partial
Fibrinogen Number/ Clotting Time Time (PT).sup.b Thromboplastin
(FIB) Dose (mg/kg) Time Points (ACT) (sec) (sec) Time (APTT) (sec)
(mg/dL) 1001 Post Catheter 114 10.9 20.8 95 0 (PBS) Pre-Treatment
-- 10.7 20.3 213 15 min post mAb.sup.a 37 -- -- -- 30 min post mAb
53 11.3 20.5 170 2101 Post Catheter 124 9.8 19.8 162 0.04
Pre-Treatment 94 9.8 19.8 174 15 min post mAb 68 10.0 19.9 159 30
min post mAb 146 9.7 19.6 162 45 min post mAb 107 10.0 19.5 153
3101 Post Catheter 131 9.3 21.7 170 0.2 Pre-Treatment 150 9.4 21.7
162 15 min post mAb 97 10.4 21.2 159 30 min post mAb 107 10.6 21.2
174 45 min post mAb 108 11.2 22.1 125 4101 Post Catheter 166 9.6
24.0 219 1.0 Pre-Treatment 114 9.3 23.3 209 15 min post mAb 105 9.3
23.5 198 30 min post mAb 103 9.7 24.5 338 45 min post mAb 115 10.3
26.5 117 5101 Post Catheter 155 10.1 20.7 181 5.0
Pre-Treatment.sup.b 108 >212 22.5 184 15 min post mAb 113 198.1
20.9 198 30 min.sup.apost mAb 128 -- -- -- 45 min post mAb not
taken 135.2 21.8 150 .sup.aSample clotted .sup.bSample collected 2
minutes post treatment
EXAMPLE 6
Safety and Effects of Chimeric Anti-Tissue Factor Antibody
(Sunol-cH36) on the Formation of Vascular Thrombosis in Cynomolgus
Monkeys
[0300] In this example, an angioplasty induced injury to the
arterial vessel was used to expose TF and induce thrombosis using
cynomolgus monkeys. The results indicate that cH36 may be reducing
thrombotic occlusion. The results also show the safety of the
antibody.
[0301] The objectives of this study were to determine the safety
(and efficacy) of Sunol-cH36 in a model of thrombosis in cynomolgus
monkeys. The objectives included a pharmacokinetic aspect, a
toxicological component and a pharmacodynamic model in monkeys
undergoing a thrombosis-inducing surgical injury.
[0302] A pilot study using 2 male cynomolgus monkeys was conducted
prior to the start of the main study to establish feasibility of
using angioplasty injury as a model for initiation of thrombosis in
this species. One animal was dosed intravenously (IV) with cH36 (5
mg/kg) approximately 30 to 45 minutes prior to angioplasty. The
second animal was scheduled for dosing with the vehicle; however,
this animal did not receive the vehicle. All other procedures were
performed in the same manner for both animals.
.sup.111Indium-labeled platelets were injected IV after
administration of test article. Animals were then allowed to
recover from anesthesia and were observed for approximately 24
hours. Pilot animals were imaged at 1-2 hours post-surgery and at
approximately 24 hours, at which time they were euthanized and
their injured vessels were perfused and harvested. The segment of
the injured vessel was counted in a gamma counter and evaluated
grossly for thrombosis. Sections were then fixed for future
possible analysis. Based on this pilot study, the main study was
conducted.
[0303] In the main study, 24 cynomolgus monkeys (12 male and 12
female) were anesthetized and angioplasty was performed. Surgeries
were conducted over a four-week period and staggered so that Day 1
was not the same day for all animals. On Day 1, a blood sample was
obtained from each animal prior to the surgical procedure for
labeling of autologous platelets with .sup.111Indium. Animals were
dosed with the vehicle or test article via a single intravenous
slow (<3 min) bolus injection followed by intravenous
administration of the autologous platelets (approximately 20
minutes prior to angioplasty). Endothelial denudation via balloon
angioplasty in a common iliac artery was then performed. Acute
platelet deposition and thrombosis were monitored within the vessel
by gamma scintigraphy. Toxicity was assessed by clinical
observations, body weight measurements, and cutaneous bleeding
times. In addition, blood was collected for hematology, coagulation
and serum chemistry analyses, and urine was collected for
urinalysis. Blood was also collected to provide plasma for analysis
of Sunol-cH36 by ELISA. A comprehensive necropsy was performed on
animals that were euthanized as scheduled on Day 15.
Protocol-specified organs were weighed and comprehensive tissue
collection was performed. Microscopic evaluation was performed on
selected tissues from Group 1 and 5 animals. In addition, the
angioplasty sites from all animals were evaluated. Table 15
summarizes the design of this study.
[0304] This study to determine the safety and efficacy of
Sunol-cH36 in monkeys undergoing a thrombosis-inducing surgical
injury induced by angioplasty included pharmacokinetic and
pharmacodynamic components, which were presented above. The
following summarizes the toxicological aspects of Sunol-cH36 in
this model of thrombosis in cynomolgus monkeys.
[0305] Dose administration for this study was generally performed
according to the study design in Table 15. The order of treatment
was maintained with test article or vehicle administration first
(approximately 30 to 45 minutes prior to angioplasty) and
radiolabeled platelets administered second (approximately 20
minutes prior to angioplasty). The intended time frames of
administration did not always fall into the ranges specified due to
the variable difficulty in accomplishment of the angioplasty
procedure.
[0306] One control animal (Group 1 female No. 1101) was removed
from the study and euthanized on Day 1 without further evaluation.
This animal showed unusual anatomy, making the angioplasty
difficult, and did not recover from anesthesia. Three other study
animals died or were euthanized on Day 1 (Group 2 male No. 2001,
0.04 mg/kg; Group 3 female No. 3102, 0.2 mg/kg; and Group 4 male
No. 4001, 1.0 mg/kg). The deaths were all considered
anesthesia/procedure related, and all animals were replaced.
Complete histopathology was performed for these animals in an
attempt to further identify a cause of death.
[0307] There were no clinical observations that were considered
cH36-related. The most common observations were bruising of the
inguinal areas due to blood collection, and unequal pupil diameter
that was most likely due to the mechanical trauma of angioplasty.
These observations were present in control and cH36-treated groups.
Other observations including soft feces, scant feces, and apparent
blood in the feces were noted at low incidences only. Some animals
showed signs of difficulty breathing and wheezing which was most
likely related to intubation trauma. None of these observations
appeared to be test article related. One animal (Group 4 female
No.4102) was treated with aminophylline and cefotaxime due to
respiratory difficulties after treatment/angioplasty. Macroscopic
and microscopic assessment of urine samples collected prior to
treatment and prior to necropsy revealed no apparent test
article-related effects.
[0308] Individual body weights were recorded prior to surgery, on
Day 8, prior to necropsy (Day 15) for all animals, and on Day 14
for some animals. Body weights were unaffected by test article
treatment and remained constant or fluctuated from week to
week.
[0309] There were no cH36-related differences in cutaneous bleeding
times between groups. Of the 60 bleeding times that were scheduled
in the study protocol, 37 times were obtained. Of these, most
bleeding times were between one and three minutes; there were 6
bleeding times that were exceptions where bleeding times ranged
from 3.5 (n=2), 4.5 (n=2), 6 (n=1), and 7 minutes (n=1). Although
only two of the twenty 4-hour cutaneous bleeding times were taken
due to technical difficulties and error, there were no bleeding
difficulties noted for any animals during attempted blood
collection at the 4-hour time point or at any other time during the
study. Individual cutaneous bleeding times for this study are
provided in Table 13 below along with a chart of the mean bleeding
times (with standard deviation) for each dose level in FIG. 12.
[0310] There were no test article-related alterations in hematology
parameters. There were decreases in red blood cell parameters (red
blood cell count [RBC], hemoglobin [HGB] and hematocrit [HCT]), and
increases in white blood cells that were present in animals from
all groups after treatment with placebo or Sunol-cH36. These
alterations showed no TABLE-US-00016 TABLE 12 Cutaneous Bleeding
Times in the Angioplasty Study in Cynomolgus Monkeys Group Animal
Bleeding Time Duration (min:sec) No./Dose Number Date Pre-Treatment
@ 1.5 hours @ 4 hours 1 1001 Dec. 19, 2000 01:30 a 03:30 0 (PBS)
1002 Jan. 4, 2001 07:00 a a 1102 Jan. 10-11, 2001 02:00 01:00 a
1103 Jan. 12, 2001 02:00 03:30 a 2 2002 Jan, 18, 2001 02:30 01:30 a
0.04 2003 Feb. 8, 2001 01:00 02:30 a mg/kg 2101 Jan. 16, 2001 03:00
01:30 a 2102 Feb. 6, 2001 01:00 04:30 a 3 3001 Jan. 16, 2001 01:30
02:20 a 0.2 3002 Feb. 6, 2001 01:00 04:30 a mg/kg 3101 Jan. 16,
2001 01:30 02:00 a 3103 Feb. 13, 2001 02:00 02:00 a 4 4002 Feb. 7,
2001 01:00 02:30 a 1.0 4003 Feb. 13, 2001 02:30 06:00 a mg/kg 4101
Jan. 18, 2001 02:20 02:30 a 4102 Feb. 8, 2001 02:00 01:30 a 5 5001
Dec. 19, 2000 03:30 01:00 a 5.0 5002 Jan. 10-11, 2001 03:00 b a
mg/kg 5101 Jan. 4, 2001 02:30 a 03:00 5102 Jan. 11, 2001 03:00 b a
a Data not collected. b No bleeding.
[0311] TABLE-US-00017 TABLE 13 Hematology Parameters and Group Mean
Study Results in the Angioplasty Study in Cynomolgus Monkeys Group
Number/Dose (mg/kg) 1 2 3 4 5 Time 0 (PBS) 0.04 0.2 2.0 5.0
Parameter Points M F M F M F M F M F Total Leukocyte Pre-Treat.
20.95 7.75 5.75 9.30 8.80 7.20 6.65 9.80 15.35 3.95 Count (WBC) 1.5
hours 23.30 22.75 11.50 16.55 9.50 12.90 8.35 21.85 16.45 13.80
(10.sup.3/.mu.L) 24 hours 21.65 26.80 16.40 23.55 19.25 16.30 21.35
19.90 30.95 16.90 Day 3 21.65 15.45 10.05 19.95 15.10 13.30 15.60
18.30 18.15 13.60 Day 7 25.55 20.75 10.95 19.10 17.60 14.35 16.15
21.30 12.10 14.10 Day 15 14.55 13.45 11.00 15.45 17.30 10.00 11.90
13.30 15.55 8.45 Erythrocyte Pre-Treat. 7.225 5.910 5.445 5.320
6.325 5.000 5.025 5.205 6.990 4.890 Count (RBC) 1.5 hours 5.910
5.310 4.950 4.715 5.335 4.110 4.920 4.865 5.270 4.700
(10.sup.6/.mu.L) 24 hours 5.605 5.375 4.440 4.755 5.675 4.350 4.130
4.390 6.140 4.650 Day 3 5.330 5.285 5.005 4.680 5.855 4.250 3.930
4.660 5.820 4.200 Day 7 5.625 5.120 5.040 5.135 5.865 4.120 4.115
4.185 5.550 4.270 Day 15 5.865 5.355 5.530 5.505 5.370 4.755 4.730
4.955 6.020 5.035 Hemoglobin Pre-Treat. 14.20 11.70 11.05 10.45
12.20 11.50 11.20 11.20 14.90 10.15 Concentration 1.5 hours 12.00
10.40 10.05 9.20 10.25 9.50 10.95 10.50 11.15 9.45 (HGB) (g/dL) 24
hours 10.90 10.65 9.40 9.15 10.95 10.15 9.15 9.45 13.05 9.25 Day 3
10.40 10.45 10.10 9.10 11.00 10.05 8.75 9.90 12.20 8.35 Day 7 10.85
10.25 10.45 10.10 11.10 9.80 9.35 9.30 11.90 8.55 Day 15 11.60
11.00 11.55 11.20 10.90 11.30 10.80 10.95 13.05 10.45 Hematocrit
Pre-Treat. 44.05 37.65 35.70 32.65 39.25 36.80 35.45 35.10 47.30
32.55 Value (HCT).sup.a 1.5 hours 37.70 33.80 32.05 29.05 33.20
30.30 34.35 32.85 35.60 30.90 (%) 24 hours 35.05 34.15 30.60 29.40
35.45 32.05 28.75 29.95 41.75 30.65 Day 3 33.60 34.10 33.10 29.00
36.70 31.40 27.45 31.30 40.00 27.80 Day 7 36.05 33.60 33.65 32.30
36.80 30.95 29.95 28.75 39.50 29.40 Day 15 38.85 36.70 37.50 35.80
35.90 36.40 35.30 34.65 42.30 35.80 Platelet Count Pre-Treat. 294.0
219.5 289.5 374.5 365.5 471.0 385.5 340.0 320.5 312.5 (PLT) 1.5
hours 425.0 226.5 185.5 321.0 279.5 378.5 393.0 311.5 262.0 271.5
(10.sup.3/.mu.L) 24 hours 276.0 306.0 233.0 388.0 378.0 430.0 308.5
316.0 323.5 314.5 Day 3 209.5 320.5 259.5 399.0 383.5 430.5 370.5
369.0 345.5 312.5 Day 7 525.5 513.5 484.0 550.0 668.5 605.5 487.5
617.0 477.0 414.0 Day 15 434.0 375.0 434.5 598.0 546.0 776.5 576.0
453.5 472.0 393.0 .sup.aCalculated value; M = male; F = female
5 cH36-related patterns and were most likely due to repeated blood
sampling and the stress of the surgical procedure. Group mean
hematology values are summarized in Table 13.
[0312] Group mean coagulation values are summarized in Table 14.
There were expected cH36-related prolongation of Prothrombin Times
present for Group 4 (1.0 mg/kg) and Group 5 (5 mg/kg) animals.
There were 2 to 6 fold increases in PT times for Group 4 animals at
approximately 1 hour post-treatment (one Group 4 animal showed no
notable increase). The values generally returned to baseline by Day
2. The PT times for Group 5 animals were increased 4 to 17 fold at
1-hour post treatment and 3 to 6 fold at Day 2. Two of the Group 5
animals still had a notable increase in PT time at Day 3 (animal
5002-5 fold). The Group 5 PT times otherwise generally returned to
baseline by Day 3 or 7. There were no test article-related effects
noted for activated partial thromboplastin time (APTT) or
fibrinogen (FIB) levels.
[0313] There were no alterations in serum chemistry parameters that
were considered test article related. There were elevations in
alanine aminotransferase (ALT) and aspartate aminotransferase (AST)
present for some animals from all groups, generally at Day 2 or 3.
These changes may have been due to the surgical and sample
collection procedures concentrated during the first few days of
study. The values mainly returned to baseline by Day 7 or 15, and
there were no corresponding histopathologic findings noted in Group
1 or 5 animals. Macroscopic and microscopic assessment of urine
samples collected prior to treatment and prior to necropsy revealed
no apparent test article-related effects.
[0314] A comprehensive necropsy was performed on all animals,
including those that died early (the exception is one control
animal, Group 1 female No. 1101, removed from the study due to
unusual anatomy which would have made the angioplasty procedure
more difficult to perform and also because the animal did not
recover from the anesthesia). There were three animals that died or
were euthanized on Day 1 (Group 2 male No. 2001, Group 3 female No.
3102 and Group 4 male No. 4001). Since the early death animals came
from three different groups, and did not include any deaths in the
high dose group, and there were no toxicologically relevant
findings in any of the animals that survived until termination, the
deaths were considered related to the surgical procedure and
unrelated to test article. Comparison of microscopic evaluation of
tissues from Group 5 (5 mg/kg) and Group 1 (vehicle control)
animals revealed no findings that were considered test article
related.
[0315] Numerous organs and tissues were collected at necropsy. Of
these, seven organs were weighed. Organ weights were determined
prior to fixation and paired organs were weighed together. No
effects on organ weights were observed.
[0316] To summarize, there were no toxicologically relevant
findings noted in the clinical observations, body weight
measurements, cutaneous bleeding times, clinical pathology results,
organ weight measurements and the pathologic evaluation. A single
intravenous administration of Sunol-cH36 at doses of 0.04, 0.2, 1.0
and 5.0 mg/kg, in a model of thrombosis, was well tolerated by
cynomolgus monkeys. All findings other than normal, including early
deaths, were attributed to the surgical procedure for endothelial
denudation by angioplasty. TABLE-US-00018 TABLE 14 Group Mean
Coagulation Parameters in the Angioplasty Study in Cynomolgus
Monkeys Group Number/Dose (mg/kg) Time 1/0 2/0.04 3/0.2 4/1.0 5/5.0
Parameter Points M F M F M F M F M F PT (sec) Pre-Treat 8.85 9.75
10.10 10.00 10.25 9.85 10.40 10.25 9.70 9.85 1.5 hours 11.00 10.80
12.60 10.45 13.30 12.00 48.05 16.75 102.85 73.40 24 hours 8.20 8.55
8.50 7.90 8.95 8.80 13.20 9.55 37.80 35.85 Day 3 7.95 8.25 7.95
7.80 8.20 8.45 9.50 8.40 59.15 13.20 Day 7 7.80 8.25 8.85 8.30 8.30
8.35 8.70 9.20 10.05 8.40 Day 15 11.00 9.60 9.05 9.10 9.80 9.45
9.45 9.45 9.15 9.30 APTT Pre-Treat 20.90 23.20 21.00 23.80 24.95
21.25 26.25 24.20 21.65 22.60 (sec) 1.5 hours 23.50 25.55 26.25
23.80 26.90 22.25 29.10 23.80 22.95 23.40 24 hours 20.00 21.35
20.55 20.00 20.55 19.50 22.50 20.75 21.80 20.25 Day 3 20.40 20.65
19.15 19.75 20.00 18.00 21.85 19.80 19.50 19.00 Day 7 17.20 20.35
20.10 20.90 21.20 18.40 22.35 23.20 19.35 17.40 Day 15 25.80 22.70
18.80 20.85 22.00 20.30 23.70 21.05 20.90 20.90 FIB Pre-Treat 165.5
174.5 153.0 164.0 145.0 239.5 176.0 141.5 180.5 117.0 (mg/dL) 1.5
hours 144.0 131.0 90.5 135.5 87.0 168.5 163.5 126.0 175.0 93.5 24
hours 446.5 412.5 360.0 470.0 313.0 496.5 237.5 514.0 533.0 319.5
Day 3 638.5 663.0 624.5 433.0 578.0 500.5 383.0 421.5 623.5 418.5
Day 7 329.0 317.0 344.0 244.0 384.0 367.5 332.5 507.0 315.0 371.0
Day 15 185.5 181.5 219.5 235.0 97.0 254.0 313.5 201.5 244.5 200.0 M
= male; F = female
[0317] TABLE-US-00019 TABLE 15 Study Design for the Angioplasty
Study in Cynomolgus Monkeys Treatment Administration Number of
Surgical Dose Dose Dose Group Animals Procedure Test Level
concentration Volume Number Male Female on Day 1 Article (mg/kg)
(mg/mL) (mL/kg) 1 2 3* Angioplasty PBS 0 0 0.5 2 3* 2 Sunol- 0.04
0.515 0.08 3 2 3* cH36 0.2 0.4 4 3* 2 1.0 10.3 0.1 5 2 2 5.0 0.5
One animal from each of the indicated groups died on Day 1 after
most of the Day 1 procedures were completed. One animal was added
to each of these groups to provide a total of 2 males and 2 females
per group.
[0318] For the pharmacodynamic component of this study, acute
vascular thrombosis and platelet deposition at the sites of
vascular injury were measured by gamma scintigraphy of radiolabeled
platelets. Radiolabeled platelets were injected into each animal
after treatment with the test article. Gamma camera images were
acquired for 2 hours following the angioplasty procedure. Not all
animals had all images acquired at the same time due to logistical
and technical limitations, so the time of acquisition is only
approximate. Region-of-interest analyses were performed at the site
of injury and compared to that of background (i.e. soft tissue
adjacent to the target injury site). A target injury site to
background (T/B) ratio was calculated and the mean T/B ratio is
presented in FIG. 13. The mean T/B ratio in Group 1 increased from
3 at 30 minutes to 3.55 at 2 hours suggesting that continued
deposition of platelets is occurring at the injury site. Animals
treated with Sunol-cH36 at the higher doses (0.2-5 mg/kg) have
reduced T/B ratios indicating that Sunol-cH36 had an effect of
inhibiting platelet deposition at the site of vessel injury.
TABLE-US-00020 TABLE 16 Gamma Scintigraphy Analysis of
.sup.111Indium-labeled Platelet Deposition Sunol-cH36/ Animal
Activity at 30 minutes Activity at 2 hours Dose Number arget
Background T/B arget Background T/B Group 1 1001 30 15 2.0 37 16
2.31 0 (PBS) 1002 14 5 2.8 14 5 2.8 1101* 57 33 1.73 49 17 2.89
1102 103 17 6.06 92 12 7.67 1103 32 13 2.46 42 20 2.1 Mean .+-.
S.D. 3.01 .+-. 1.75 3.55 .+-. 2.32 Group 2 2001* 129 10 12.9 119 9
13.22 0.04 2002 36 18 2.0 35 22 1.59 mg/kg 2101 37 22 1.68 34 23
1.48 2102 53 33 1.61 45 24 1.88 Mean .+-. S.D. 4.55 .+-. 5.57 4.54
.+-. 5.79 Group 3 3001 62 29 2.14 51 36 1.42 0.2 3002 70 26 2.69 76
23 3.3 mg/kg 3101 37 19 1.95 31 20 1.55 3103 48 21 2.29 15 14 1.07
Mean .+-. S.D. 2.27 .+-. 0.31 1.84 .+-. 0.99 Group 4 4001* 14 6
2.33 12 5 2.4 1.0 4002 47 30 1.57 42 27 1.56 mg/kg 4003 22 16 1.37
28 13 2.15 4101 11 8 1.4 10 9 1.11 Mean .+-. S.D. 1.67 .+-. 0.45
1.81 .+-. 0.58 Group 5 5001 42 26 1.62 44 31 1.42 5.0 5002 43 26
1.65 41 23 1.78 mg/kg 5101 36 20 1.8 31 19 1.63 5102 18 10 1.8 31
19 1.63 Mean .+-. S.D. 1.72 .+-. 0.1 1.62 .+-. 0.15 *These animals
were imaged but later died during the initial 24 hours of the
study.
EXAMPLE 7
Electrolytic Injury Model in Cynomolgus Monkeys
[0319] The electrolytic injury model used in this study is similar
to that described for carotid artery thrombosis in cynomolgus
monkeys (see Rote W E, Nedelman M A, Xu D -X, Manley P J, Weisman
H, Cunnihgharm M R, Lucchesi B R, Stroke 25:1223-1233 (1994)). In
the electrolytic injury model, continuous anodal current is applied
to the intimal surface of the cartoid or femoral arteries. This
treatment will lead to the denudation of the endothelium and a deep
injury of the subendothelial layers. The resulting vessel wall
injury and endothelial lesion provide stimuli (TF and collagen
exposure) for coagulation and platelet activation, which then leads
to the formation of a platelet-rich intravascular thrombus at the
injury site. In this study, the efficacy of Sunol-cH36 was tested
in cynomolgus monkeys using the arterial thrombosis model induced
by electrolytic injury. This thrombosis model mimics the clinical
settings of unstable angina and non-ST-segment elevation myocardial
infarction where atherosclerotic plaque rupture leads to exposure
of thrombogenic surfaces resulting in coagulation and platelet
activation.
[0320] Table 17 shows the result from a study with two animals. In
two control procedures the vessels occluded earlier than in the
procedures when cH36 was used. All procedures were conducted with
heparin and aspirin present. This indicates that cH36 can retard or
prevent occlusion in a thrombosis model where heparin and aspirin
are not effective. TABLE-US-00021 TABLE 17 Effect of Sunol-cH36 on
Blocking Thrombotic Occlusion Induced by Arterial Injury Time to
Occlusion Animal No. #1 #2 Placebo 54 min 25 min Sunol-cH36, 5
mg/kg >2 hr 82 min* *Vessel re-perfused 8 mm after occlusion for
9 min before reocciuding. Animals pre-treated with aspirin and
bolus heparin followed by heparin infusion during procedure.
[0321] Although Examples 1-7 have been described with particular
reference to the cH36 chimeric antibody, the work described could
have been practiced with other suitable antibodies of the invention
e.g., hFAT or Fab (where the Fab can be derived from cH36, hFAT or
hOAT).
[0322] The invention has been described in detail with reference to
preferred embodiments thereof. However, it will be appreciated that
those skilled in the art, upon consideration of the disclosure, may
make modification and improvements within the spirit and scope of
the invention.
* * * * *
References