U.S. patent application number 14/197672 was filed with the patent office on 2014-07-24 for cancer treatment comprising therapeutics that bind to phosphatidylserine.
This patent application is currently assigned to Board of Regents, The University of Texas System. The applicant listed for this patent is Board of Regents, The University of Texas System. Invention is credited to Rolf A. Brekken, Sophia Ran, Philip E. Thorpe.
Application Number | 20140205544 14/197672 |
Document ID | / |
Family ID | 26785826 |
Filed Date | 2014-07-24 |
United States Patent
Application |
20140205544 |
Kind Code |
A1 |
Thorpe; Philip E. ; et
al. |
July 24, 2014 |
Cancer Treatment Comprising Therapeutics That Bind To
Phosphatidylserine
Abstract
Disclosed is the surprising discovery that aminophospholipids,
such as phosphatidylserine and phosphatidylethanolamine, are
specific, accessible and stable markers of the luminal surface of
tumor blood vessels. The present invention thus provides
aminophospholipid-targeted diagnostic and therapeutic constructs
for use in tumor intervention. Antibody-therapeutic agent
conjugates and constructs that bind to aminophospholipids are
particularly provided, as are methods of specifically delivering
therapeutic agents, including toxins and coagulants, to the
stably-expressed aminophospholipids of tumor blood vessels, thereby
inducing thrombosis, necrosis and tumor regression.
Inventors: |
Thorpe; Philip E.; (Dallas,
TX) ; Ran; Sophia; (Riverton, IL) ; Brekken;
Rolf A.; (Dallas, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System |
Austin |
TX |
US |
|
|
Assignee: |
Board of Regents, The University of
Texas System
Austin
TX
|
Family ID: |
26785826 |
Appl. No.: |
14/197672 |
Filed: |
March 5, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11254137 |
Oct 19, 2005 |
8709430 |
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14197672 |
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09351149 |
Jul 12, 1999 |
7067109 |
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11254137 |
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60092589 |
Jul 13, 1998 |
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60110600 |
Dec 2, 1998 |
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Current U.S.
Class: |
424/9.42 ;
424/1.53; 424/181.1; 424/450; 424/451; 530/391.7; 600/1 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
49/04 20130101; A01K 2217/05 20130101; A61K 39/395 20130101; C07K
2317/31 20130101; A61K 47/6835 20170801; C07K 2317/24 20130101;
A61K 51/1027 20130101; C07K 16/2836 20130101; C07K 2319/00
20130101; A61K 47/6849 20170801; A61N 5/10 20130101; A61P 35/00
20180101; A61K 39/395 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/9.42 ;
424/450; 424/451; 424/181.1; 424/1.53; 530/391.7; 600/1 |
International
Class: |
A61K 47/48 20060101
A61K047/48; A61K 49/04 20060101 A61K049/04; A61N 5/10 20060101
A61N005/10; A61K 51/10 20060101 A61K051/10 |
Goverment Interests
[0002] The U.S. Government owns rights in the present invention
pursuant to grant numbers 1RO1CA74951-01 and 5RO1CA54168-05 from
the National Institutes of Health.
Claims
1. A phosphatidylserine-targeted therapeutic agent comprising, in
operative association, a selected therapeutic agent and a binding
protein, or active fragment thereof, that binds to
phosphatidylserine on tumor blood vessels; wherein said selected
therapeutic agent is a phospholipid liposome or nanocapsule.
2. A composition comprising a phosphatidylserine-targeted
therapeutic agent, comprising a phosphatidylserine binding protein,
or active fragment thereof, operatively associated with a
phospholipid liposome or nanocapsule; wherein said phospholipid
liposome or nanocapsule entraps said phosphatidylserine binding
protein, or active fragment thereof, to form a construct that binds
to phosphatidylserine and has an anti-tumor effect.
3. A pharmaceutical composition comprising an amount of at least a
first anti-cancer agent effective to treat cancer and a
pharmaceutically acceptable carrier; wherein said at least a first
anti-cancer agent is a phosphatidylserine-targeted therapeutic
agent that comprises a phosphatidylserine binding protein, or
active fragment thereof, in operative association with the lipid
bilayer of a phospholipid liposome or nanocapsule.
4. The composition of claim 3, further comprising at least a second
anti-cancer agent.
5. The composition of claim 4, wherein said at least a second
anti-cancer agent is contained in the core of said phospholipid
liposome or nanocapsule.
6. A kit comprising, in at least one container, therapeutically
effective amounts of: (a) at least a first anti-cancer agent;
wherein said at least a first anti-cancer agent is a
phosphatidylserine-targeted therapeutic agent that comprises a
phosphatidylserine binding protein, or active fragment thereof, in
operative association with the lipid bilayer of a phospholipid
liposome or nanocapsule; and (b) at least a second, distinct
anti-cancer agent.
7. The kit of claim 6, wherein said at least a first anti-cancer
agent and said at least a second, distinct anti-cancer agent are
comprised within distinct containers.
8. The kit of claim 6, wherein said at least a second, distinct
anti-cancer agent is a chemotherapeutic agent, radiotherapeutic
agent, anti-angiogenic agent or apoptosis-inducing agent.
9. The kit of claim 6, wherein said kit further comprises, in
another container, a diagnostically effective amount of a
detectably-labeled phosphatidylserine binding construct.
10. A composition comprising: (a) a liposome or nanocapsule formed
from phospholipids that comprises a lipid bilayer and a core; (b) a
phosphatidylserine binding protein, or active fragment thereof,
that binds to phosphatidylserine on a target cell plasma membrane;
and (c) a pharmaceutically acceptable carrier; wherein said
liposome or nanocapsule entraps said phosphatidylserine binding
protein, or active fragment thereof, in said lipid bilayer; and
wherein said composition binds to phosphatidylserine and has an
anti-tumor effect.
11. The composition of claim 10, wherein said liposome or
nanocapsule has a diameter of from 25 nm to 4 .mu.m.
12. The composition of claim 10, wherein the liposome or
nanocapsule has a size of about 0.1 .mu.m.
13. The composition of claim 10, wherein said composition further
comprises a supplementary active ingredient.
14. The composition of claim 13, wherein said supplementary active
ingredient is contained in said core of said phospholipid liposome
or nanocapsule.
15. A method for treating an animal having a vascularized tumor,
comprising administering to said animal a therapeutically effective
amount of a phosphatidylserine-targeted therapeutic agent that
comprises a phosphatidylserine binding protein, or active fragment
thereof, in operative association with a phospholipid liposome or
nanocapsule; wherein said phospholipid liposome or nanocapsule
entraps said phosphatidylserine binding protein, or active fragment
thereof, to form a construct that binds to phosphatidylserine in
said vascularized tumor and exerts an anti-tumor effect.
16. The method of claim 15, wherein said method induces apoptosis,
causes tumor cell death or induces necrosis in said vascularized
tumor.
17. The method of claim 15, wherein said
phosphatidylserine-targeted therapeutic agent is administered to
said animal intravenously.
18. The method of claim 15, further comprising: (a) subjecting said
animal to surgery or radiotherapy; or (b) simultaneously or
sequentially administering to said animal a therapeutically
effective amount of another anti-cancer agent.
19. The method of claim 18, wherein said another anti-cancer agent
is a chemotherapeutic agent, radiotherapeutic agent,
anti-angiogenic agent or apoptosis-inducing agent.
20. The method of claim 15, wherein said animal is a human
patient.
21. A method for delivering a diagnostic or therapeutic agent to a
vascularized tumor, comprising: (a) preparing a
phosphatidylserine-targeted liposome or nanocapsule construct that
contains said diagnostic or therapeutic agent; wherein said
construct comprises a phosphatidylserine binding protein, or active
fragment thereof, operatively associated with a phospholipid
liposome or nanocapsule; and (b) administering a biologically
effective amount of said construct to an animal having a
vascularized tumor, wherein said construct binds to
phosphatidylserine in said vascularized tumor and thereby delivers
said diagnostic or therapeutic agent thereto.
22. The method of claim 21, wherein said phospholipid liposome or
nanocapsule comprises a lipid bilayer and a core; wherein said
phosphatidylserine binding protein, or active fragment thereof, is
entrapped in said lipid bilayer; and wherein said diagnostic or
therapeutic agent is contained within said core.
23. A method for imaging a vascularized tumor, comprising: (a)
preparing a phosphatidylserine-targeted liposome or nanocapsule
construct that contains a detectable or imaging agent; wherein said
construct comprises a phosphatidylserine binding protein, or active
fragment thereof, operatively associated with a phospholipid
liposome or nanocapsule; (b) administering a diagnostically
effective amount of said construct to an animal having a
vascularized tumor, wherein said construct binds to
phosphatidylserine in said vascularized tumor; and (c) detecting
the image of the tumor so formed.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 11/254,137 filed Oct. 19, 2005, which is a
continuation of U.S. application Ser. No. 09/351,149, filed Jul.
12, 1999, now granted as U.S. Pat. No. 7,067,109 on Jun. 27, 2006,
which claims priority to first provisional application Ser. No.
60/092,589, filed Jul. 13, 1998, and second provisional application
Ser. No. 60/110,600, filed Dec. 2, 1998, the entire text and
figures of which applications are incorporated herein by reference
without disclaimer.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the fields of
blood vessels and tumor biology. More particularly, it embodies the
surprising findings that aminophospholipids, such as
phosphatidylserine and phosphatidylethanolamine, are accessible,
stable and specific markers of tumor vasculature. The invention
thus provides therapeutic constructs and conjugates that bind to
aminophospholipids for use in delivering toxins and coagulants to
tumor blood vessels and for inducing thrombosis and tumor
regression.
[0005] 2. Description of the Related Art
[0006] Tumor cell resistance to chemotherapeutic agents represents
a significant problem in clinical oncology. In fact, this is one of
the main reasons why many of the most prevalent forms of human
cancer still resist effective chemotherapeutic intervention,
despite certain advances in the field of chemotherapy.
[0007] A significant problem to address in tumor treatment regimens
is the desire for a "total cell kill". This means that the more
effective treatment regimens come closer to a total cell kill of
all so-called "clonogenic" malignant cells, i.e., cells that have
the ability to grow uncontrolled and replace any tumor mass that
might be removed by the therapy. Due to the goal of developing
treatments that approach a total cell kill, certain types of tumors
have been more amenable to therapy than others. For example, the
soft tissue tumors, e.g., lymphomas, and tumors of the blood and
blood-forming organs, e.g., leukemias, have generally been more
responsive to chemotherapeutic therapy than have solid tumors, such
as carcinomas.
[0008] One reason for the susceptibility of soft and blood-based
tumors to chemotherapy is the greater accessibility of lymphoma and
leukemic cells to chemotherapeutic intervention. Simply put, it is
much more difficult for most chemotherapeutic agents to reach all
of the cells of a solid tumor mass than it is the soft tumors and
blood-based tumors, and therefore much more difficult to achieve a
total cell kill. Increasing the dose of chemotherapeutic agents
most often results in toxic side effects, which generally limits
the effectiveness of conventional anti-tumor agents.
[0009] Another tumor treatment strategy is the use of an
"immunotoxin", in which an anti-tumor cell antibody is used to
deliver a toxin to the tumor cells. However, in common with the
chemotherapeutic approaches described above, immunotoxin therapy
also suffers from significant drawbacks. For example,
antigen-negative or antigen-deficient cells can survive and
repopulate the tumor or lead to further metastases. Also, in the
treatment of solid tumors, the tumor mass is generally impermeable
to molecules of the size of antibodies and immunotoxins. Both the
physical diffusion distances and the interstitial pressure within
the tumor are significant limitations to this type of therapy.
[0010] A more recent strategy has been to target the vasculature of
solid tumors. Targeting the blood vessels of the tumors, rather
than the tumor cells themselves, has certain advantages in that it
is not likely to lead to the development of resistant tumor cells,
and that the targeted cells are readily accessible. Moreover,
destruction of the blood vessels leads to an amplification of the
anti-tumor effect, as many tumor cells rely on a single vessel for
their oxygen and nutrients (Denekamp, 1990). Exemplary vascular
targeting strategies are described in U.S. Pat. Nos. 5,855,866 and
5,965,132, which particularly describe the targeted delivery of
anti-cellular agents and toxins to protein markers of tumor
vasculature.
[0011] Another effective version of the vascular targeting approach
is to target a coagulation factor to a protein marker expressed or
adsorbed within the tumor vasculature (Huang et al., 1997; U.S.
Pat. Nos. 5,877,289, 6,004,555 and 6,093,399). The delivery of
coagulants, rather than toxins, to tumor vasculature has the
further advantages of reduced immunogenicity and even lower risk of
toxic side effects. As disclosed in U.S. Pat. No. 5,877,289, a
preferred coagulation factor for use in such tumor-specific
thrombogens, or "coaguligands", is a truncated version of the human
coagulation-inducing protein, Tissue Factor (TF). TF is the major
initiator of blood coagulation (Ruf et al., 1991; Edgington et al.,
1991; Ruf and Edgington, 1994). Treatment of tumor-bearing mice
with such coaguligands results in significant tumor necrosis and
even complete tumor regression in many animals (Huang et al., 1997;
U.S. Pat. Nos. 5,877,289, 6,004,555 and 6,093,399).
[0012] Although the specific delivery of therapeutic agents, such
as anti-cellular agents, toxins and coagulation factors, to protein
markers of tumor vessels represents a significant advance in tumor
treatment protocols, there is still room for additional vascular
targeting therapies. The identification of additional stable
targets to allow specific tumor vessel destruction in vivo would
naturally be of benefit in expanding the number of targeting
options. More particularly, the development of targeting agents for
delivering therapeutics even closer to the tumor vascular
endothelial cell membrane would represent an important advance.
SUMMARY OF THE INVENTION
[0013] The present invention addresses the needs of the prior art
by providing new compositions and methods for tumor vasculature
imaging and destruction. The invention is based, in part, on the
finding that aminophospholipid membrane components, such as
phosphatidylserine and phosphatidylethanolamine, are accessible,
stable markers of tumor vasculature. The invention thus provides
binding ligands and antibodies against aminophospholipids that are
operatively attached to therapeutic agents, and methods of using
constructs in the specific delivery of diagnostics and therapeutics
to the actual surface of tumor vascular endothelial cell
membranes.
[0014] Important aspects of the invention are that therapeutic
agents can be delivered in intimate contact with the tumor vascular
endothelial cell membrane, allowing either rapid entry into the
target cell or rapid association with effector cells, components of
the coagulation cascade, and such like. Certain surprising features
of the invention include the discovery that translocation of
aminophospholipids, such as phosphatidylserine (PS), to the surface
of tumor vascular endothelial cells occurs, at least in a
significant part, independently of cell damage and apoptopic or
other cell-death mechanisms. Thus, PS surface expression in this
environment is not a consequence of cell death, nor does it trigger
immediate cell destruction.
[0015] The discovery of sufficiently stable PS expression on
morphologically intact tumor-associated vascular endothelial cells
is important to the targeting nature of the present invention.
Should PS translocation to the outer surface of tumor vascular
endothelium occur only in dying cells, or should it inevitably
trigger cell death, then PS expression would be expected to be
transient and PS would not likely be a good candidate target for
therapeutic intervention. Surprisingly, the present invention shows
that significant stable PS expression occurs in viable endothelial
cells in a tumor environment, thus providing ample targeting
opportunities.
[0016] The present invention therefore basically provides methods
for delivering selected diagnostic and therapeutic agents to tumor
or intratumoral vasculature, comprising administering to an animal
having a vascularized tumor a biologically effective amount of a
binding ligand that comprises a selected diagnostic or therapeutic
agent operatively attached to a targeting agent that binds to an
aminophospholipid, preferably one that binds to phosphatidylserine
or phosphatidylethanolamine, on the luminal surface of blood
vessels or intratumoral blood vessels of the vascularized
tumor.
[0017] The methods of the invention provide for killing, or
specifically killing, tumor or intratumoral vascular endothelial
cells, and comprise administering to an animal or patient having a
vascularized tumor a biologically effective amount of at least a
first pharmaceutical composition comprising a binding ligand that
comprises a selected therapeutic agent operatively attached to a
targeting agent that binds to an aminophospholipid, preferably one
that binds to phosphatidylserine or phosphatidylethanolamine, on
the luminal surface of tumor or intratumoral vascular endothelial
cells.
[0018] The "binding ligands" of the present invention are thus
"aminophospholipid binding ligands", "therapeutic aminophospholipid
binding ligand constructs", "aminophospholipid-targeted therapeutic
agents", "aminophospholipid-targeted therapeutics",
"aminophospholipid-targeted therapeutic agent constructs", or
"therapeutic agent-aminophospholipid targeting agent constructs".
For simplicity, these agents are referred to herein as "binding
ligands" or "therapeutic agent-targeting agent constructs", with
the understanding that such terms are used as a succinct way of
referring to a conjugate or other operative association of a
selected therapeutic agent and a targeting agent, antibody, binding
protein or active fragment thereof, that binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, expressed on the luminal surface of tumor
or intratumoral vascular endothelial cells.
[0019] "Biologically effective amounts" are amounts of the
therapeutic agent-targeting agent construct effective to
specifically kill at least a portion, and preferably a significant
portion, of the tumor or intratumoral vascular endothelial cells,
as opposed to endothelial cells in normal vessels, upon binding to
an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, expressed on the luminal surface of the
tumor or intratumoral vascular endothelial cells. As such, it is an
"endothelial cell killing amount" or a "tumor vascular endothelial
cell killing amount" of a therapeutic agent-targeting agent
construct.
[0020] As used throughout the entire application, the terms "a" and
"an" are used in the sense that they mean "at least one", "at least
a first", "one or more" or "a plurality" of the referenced
components or steps, except in instances wherein an upper limit is
thereafter specifically stated. Therefore a "therapeutic
agent-targeting agent construct" means "at least a first
therapeutic agent-targeting agent construct". The operable limits
and parameters of combinations, as with the amounts of any single
agent, will be known to those of ordinary skill in the art in light
of the present disclosure.
[0021] The "a" and "an" terms are also used to mean "at least one",
"at least a first", "one or more" or "a plurality" of steps in the
recited methods, except where specifically stated. This is
particularly relevant to the administration steps in the treatment
methods. Thus, not only may different doses be employed with the
present invention, but different numbers of doses, e.g.,
injections, may be used, up to and including multiple
injections.
[0022] An "aminophospholipid", as used herein, means a phospholipid
that includes within its structure at least a first primary amino
group. Preferably, the term "aminophospholipid" is used to refer to
a primary amino group-containing phospholipid that occurs naturally
in mammalian cell membranes. However, this is not a limitation on
the meaning of the term "aminophospholipid", as this term also
extends to non-naturally occurring or synthetic aminophospholipids
that nonetheless have uses in the invention, e.g., as an immunogen
in the generation of anti-aminophospholipid antibodies
("cross-reactive antibodies") that do bind to aminophospholipids of
mammalian plasma membranes. The aminophospholipids of U.S. Pat. No.
5,767,298, incorporated herein by reference, are appropriate
examples.
[0023] The prominent aminophospholipids found in mammalian
biological systems are the negatively-charged phosphatidylserine
("PS") and the neutral or zwitterionic phosphatidylethanolamine
("PE"), which are therefore preferred aminophospholipids for
targeting by the present invention. However, the invention is by no
means limited to the targeting of phosphatidylserines and
phosphatidylethanolamines, and any other aminophospholipid target
may be employed (White et al., 1978; incorporated herein by
reference) so long as it is expressed, accessible or complexed on
the luminal surface of tumor vascular endothelial cells.
[0024] All aminophospholipid-, phosphatidylserine- and
phosphatidylethanolamine-based components are encompassed as
targets of the invention irrespective of the type of fatty acid
chains involved, including those with short, intermediate or long
chain fatty acids, and those with saturated, unsaturated and
polyunsaturated fatty acids. Preferred compositions for raising
antibodies for use in the present invention may be
aminophospholipids with fatty acids of C18, with C18:1 being more
preferred (Levy et al., 1990; incorporated herein by reference). To
the extent that they are accessible on tumor vascular endothelial
cells, aminophospholipid degradation products having only one fatty
acid (lyso derivatives), rather than two, may also be targeted
(Qamar et al., 1990; incorporated herein by reference).
[0025] Another group of potential aminophospholipid targets
include, for example, phosphatidal derivatives (plasmalogens), such
as phosphatidalserine and phosphatidalethanolamine (having an ether
linkage giving an alkenyl group, rather than an ester linkage
giving an acyl group). Indeed, the targets for therapeutic
intervention by the present invention include any substantially
lipid-based component that comprises a nitrogenous base and that is
present, expressed, translocated, presented or otherwise complexed
in a targetable form on the luminal surface of tumor vascular
endothelial cells, not excluding phosphatidylcholine ("PC"). Lipids
not containing glycerol may also form appropriate targets, such as
the sphingolipids based upon sphingosine and derivatives.
[0026] The biological basis for including a range of lipids in the
group of targetable components lies, in part, with the observed
biological phenomena of lipids and proteins combining in membranous
environments to form unique lipid-protein complexes. Such
lipid-protein complexes extend to antigenic and immunogenic forms
of lipids such as phosphatidylserine, phosphatidylethanolamine and
phosphatidylcholine with, e.g., proteins such as
.beta..sub.2-glycoprotein 1, prothrombin, kininogens and
prekallikrein. Therefore, as proteins and polypeptides can have one
or more free primary amino groups, it is contemplated that a range
of effective "aminophospholipid targets" may be formed in vivo from
lipid components that are not aminophospholipids in the strictest
sense. Nonetheless, all such targetable complexes that comprise
lipids and primary amino groups constitute an "aminophospholipid"
within the scope of the present invention.
[0027] The inventive methods also act to arrest blood flow, or
specifically arrest blood flow, in tumor vasculature. This is
achieved by administering to an animal or patient having a
vascularized tumor at least one dose of at least a first
pharmaceutical composition comprising a coagulation-inducing
amount, or a vessel-occluding amount, of at least a first cytotoxic
or coagulative agent operatively attached to a targeting agent that
binds to an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, translocated to the luminal surface of
tumor vasculature.
[0028] The "coagulation-inducing amount" or "vessel-occluding
amount" is an amount of the therapeutic agent-targeting agent
construct effective to specifically promote coagulation in, and
hence occlude, at least a portion, and preferably a significant
portion, of tumor or intratumoral blood vessels, as opposed to
normal blood vessels, upon binding to an aminophospholipid,
preferably phosphatidylserine or phosphatidylethanolamine,
translocated to the luminal surface of tumor or intratumoral blood
vessels. The "vessel-occluding amount" is therefore a functionally
effective amount, and is not a physical mass of therapeutic
agent-targeting agent construct sufficient to span the breadth of a
vessel.
[0029] Methods for destroying, or specifically destroying, tumor
vasculature are provided that comprise administering to an animal
or patient having a vascularized tumor one or more doses of at
least a first pharmaceutical composition comprising a
tumor-destructive amount of at least a first occluding or
destructive agent operatively attached to a targeting agent that
binds to an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, presented on the luminal surface of tumor
or intratumoral vasculature. The "tumor-destructive amount" is an
amount of the therapeutic agent-targeting agent construct effective
to specifically destroy or occlude at least a portion, and
preferably a significant portion, of tumor or intratumoral blood
vessels, as opposed to normal blood vessels, upon binding to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, presented on the luminal surface of the
vascular endothelial cells of the tumor or intratumoral blood
vessels.
[0030] The invention further encompasses methods for treating
cancer and solid tumors, comprising administering to an animal or
patient having a vascularized tumor a tumor necrosis-inducing
amount or amounts of at least a first pharmaceutical composition
comprising at least a first therapeutic or necrotic agent
operatively attached to a targeting agent that binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, on the luminal surface of blood vessels
or intratumoral blood vessels of the vascularized tumor. The "tumor
necrosis-inducing amount" is an amount of the therapeutic
agent-targeting agent construct effective to specifically induce
hemorrhagic necrosis in at least a portion, and preferably a
significant portion, of the tumor upon binding to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, complexed at the luminal surface of the
vascular endothelial cells of the tumor or intratumoral blood
vessels, while exerting little adverse side effects on normal,
healthy tissues.
[0031] The methods of the invention may thus be summarized as
methods for treating an animal or patient having a vascularized
tumor, comprising administering to the animal or patient a
therapeutically effective amount of at least a first pharmaceutical
composition comprising at least a first therapeutic agent-targeting
agent construct that binds to an aminophospholipid, preferably
phosphatidylserine or phosphatidylethanolamine, present, expressed,
translocated, presented or complexed at the luminal surface of
blood transporting vessels of the vascularized tumor.
[0032] The essence of the invention may also be defined as a
composition comprising at least a first diagnostic agent-targeting
agent construct, or preferably a therapeutic agent-targeting agent
construct, preferably that binds to phosphatidylserine or
phosphatidylethanolamine, for use in the preparation of a
medicament for use in tumor vasculature imaging and/or destruction
and for human tumor diagnosis and/or treatment. This can also be
defined as a composition comprising at least a first diagnostic
agent-targeting agent construct, or preferably a therapeutic
agent-targeting agent construct, for use in the preparation of a
medicament for use in binding to an aminophospholipid, preferably
phosphatidylserine or phosphatidylethanolamine, present, expressed,
translocated, presented or complexed at the luminal surface of
blood transporting vessels of a vascularized tumor and for use in
forming an image of tumor vasculature and/or for use in inducing
tumor vasculature destruction and for human tumor diagnosis and/or
treatment.
[0033] In the methods, medicaments and uses of the present
invention, one of the advantages lies in the fact that the
provision of the diagnostic or therapeutic agent-targeting agent
construct, preferably one that binds to phosphatidylserine or
phosphatidylethanolamine, into the systemic circulation of an
animal or patient results in the preferential or specific
localization to the tumor vascular surface membranes themselves,
and not to some protein complex more distant from the membrane. The
invention thus provides for more intimate cell contact than the
methods and anti-vascular agents of the prior art.
[0034] In the context of the present invention, the term "a
vascularized tumor" most preferably means a vascularized, malignant
tumor, solid tumor or "cancer". The invention is particularly
advantageous in treating vascularized tumors of at least about
intermediate size, and in treating large vascularized
tumors--although this is by no means a limitation on the invention.
The invention may therefore be used in the treatment of any tumor
that exhibits aminophospholipid-positive blood vessels, preferably
phosphatidylserine- and/or phosphatidylethanolamine-positive blood
vessels.
[0035] In preferred embodiments, the tumors to be treated by the
present invention will exhibit a killing effective number of
aminophospholipid-positive blood vessels. "A killing effective
number of aminophospholipid-positive blood vessels", as used
herein, means that at least about 3% of the total number of blood
vessels within the tumor will be positive for aminophospholipid
expression, preferably phosphatidylserine and/or
phosphatidylethanolamine expression. Preferably, at least about 5%,
at least about 8%, or at least about 10% or so, of the total number
of blood vessels within the tumor will be positive for
aminophospholipid expression. Given the aminophospholipid-negative,
particularly PS-negative, nature of the blood vessels within normal
tissues, the tumor vessels will act as sink for the administered
antibodies. Furthermore, as destruction of only a minimum number of
tumor vessels can cause widespread thrombosis, necrosis and an
avalanche of tumor cell death, antibody localization to all, or
even a majority, of the tumor vessels is not necessary for
effective therapeutic intervention.
[0036] Nonetheless, in more preferred embodiments, tumors to be
treated by this invention will exhibit a significant number of
aminophospholipid-positive blood vessels. "A significant number of
aminophospholipid-positive blood vessels", as used herein, means
that at least about 10-12% of the total number of blood vessels
within the tumor will be positive for aminophospholipid expression,
preferably phosphatidylserine and/or phosphatidylethanolamine
expression. Even more preferably, the % of
aminophospholipid-expressing tumor vessels will be at least about
15%, at least about 20%, at least about 30%, at least about 40%, at
least about 50%, at least about 60%, at least about 70%, or at
least about 80% or so of the total number of blood vessels within
the tumor, up to and including even at least about 90% or 95% of
the vessels.
[0037] The "therapeutically effective amounts" for use in the
invention are amounts of therapeutic agent-targeting agent
constructs, preferably PS- or PE-binding constructs, effective to
specifically kill at least a portion of tumor or intratumoral
vascular endothelial cells; to specifically promote coagulation in
at least a portion of tumor or intratumoral blood vessels; to
specifically occlude or destroy at least a portion of blood
transporting vessels of the tumor; to specifically induce necrosis
in at least a portion of a tumor; and/or to induce tumor regression
or remission upon administration to selected animals or patients.
Such effects are achieved while exhibiting little or no binding to,
or little or no killing of, vascular endothelial cells in normal,
healthy tissues; little or no coagulation in, occlusion or
destruction of blood vessels in healthy, normal tissues; and
exerting negligible or manageable adverse side effects on normal,
healthy tissues of the animal or patient.
[0038] The terms "preferentially" and "specifically", as used
herein in the context of promoting coagulation in, or destroying,
tumor vasculature, and/or in the context of causing tumor necrosis,
thus mean that the therapeutic agent-targeting agent constructs
function to achieve coagulation, destruction and/or tumor necrosis
that is substantially confined to the tumor vasculature and tumor
site, and does not substantially extend to causing coagulation,
destruction and/or tissue necrosis in normal, healthy tissues of
the animal or subject. The structure and function of healthy cells
and tissues is therefore maintained substantially unimpaired by the
practice of the invention.
[0039] Although understanding the mechanism of action is not
necessary to the practice of the present invention, the methods
will generally operate on the basis of the mode of action of the
particular therapeutic agent or agents chosen for attachment to the
targeting agent. As such, the aminophospholipid binding agents that
are conjugated to, or operatively associated with, cytotoxic or
anticellular agents ("anti-aminophospholipid immunotoxins") will
act initially via cellular destruction. Likewise, aminophospholipid
binding agents that are conjugated to, or operatively associated
with, coagulation factors ("anti-aminophospholipid coaguligands")
will act initially via coagulation. However, these mechanisms will
have some cross-over, as cell destruction exposes basement
membranes and results in coagulation, and as coagulation deprives
the cells of oxygen and nutrients and results in cell
destruction.
[0040] Naked or unconjugated antibodies against aminophospholipid
components are also capable of specifically inducing tumor blood
vessel destruction and tumor necrosis in vivo. Such methods of
tumor treatment are also contemplated by the present inventors, and
are disclosed and claimed in first and second provisional
application Ser. Nos. 60/092,672 (filed Jul. 13, 1998) and
60/110,608 (filed Dec. 2, 1998) and in co-filed U.S. and PCT patent
applications (Attorney Docket Nos. 4001.002200, 4001.002282 and
4001.002210), each specifically incorporated herein by reference.
In light of the beneficial effects of naked anti-aminophospholipid
antibodies, the mechanism of action of the present conjugates may
extend beyond the mode of action of the particular therapeutic
agent or agents employed.
[0041] Therefore, the following mechanisms may contribute to the
success of the invention: cell-mediated cytotoxicity,
complement-mediated lysis, apoptosis, antibody-induced cell
signaling (direct signaling), or mimicking or altering signal
transduction pathways (indirect signaling).
[0042] The treatment methods thus include administering to an
animal or patient having a vascularized tumor at least a first
pharmaceutical composition comprising an amount of at least a first
therapeutic agent-targeting agent construct effective to induce, or
specifically induce, cell-mediated cytotoxicity of at least a
portion of the tumor or intratumoral vascular endothelial cells.
Herein, the first therapeutic agent-targeting agent construct binds
to an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of tumor or
intratumoral vascular endothelial cells and induces cell-mediated
cytotoxicity of at least a portion of the tumor or intratumoral
vascular endothelial cells, as opposed to endothelial cells in
normal vessels. As used herein, "cell-mediated cytotoxicity or
destruction" includes ADCC (antibody-dependent, cell-mediated
cytotoxicity) and NK (natural killer) cell killing.
[0043] The methods further include administering to an animal or
patient having a vascularized tumor at least a first pharmaceutical
composition comprising an amount of at least a first therapeutic
agent-targeting agent construct effective to induce, or
specifically induce, complement-mediated lysis of at least a
portion of the tumor or intratumoral vascular endothelial cells.
Herein, the first therapeutic agent-targeting agent construct binds
to an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of tumor or
intratumoral vascular endothelial cells and induces
complement-mediated lysis of at least a portion of the tumor or
intratumoral vascular endothelial cells, as opposed to endothelial
cells in normal vessels.
[0044] As used herein, "complement-mediated or complement-dependent
lysis or cytotoxicity" means the process by which the
complement-dependent coagulation cascade is activated,
multi-component complexes are assembled, ultimately generating a
lytic complex that has direct lytic action, causing cell
permeabilization. Therapeutic agent-targeting agents for use in
inducing complement-mediated lysis will generally include an
antibody Fc portion.
[0045] The complement-based mechanisms by which the present
invention may operate further include "complement-activated ADCC".
In such aspects, the administered therapeutic agent-targeting agent
contains an antibody, or fragment thereof, that binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of tumor or
intratumoral vascular endothelial cells and induces
complement-activated ADCC of at least a portion of the tumor or
intratumoral vascular endothelial cells, as opposed to endothelial
cells in normal vessels. "Complement-activated ADCC" is used to
refer to the process by which complement, not an antibody Fe
portion per se, holds a multi-component complex together and in
which cells such as neutrophils lyse the target cell.
[0046] In other embodiments, the methods include administering to
an animal or patient having a vascularized tumor at least a first
pharmaceutical composition comprising an amount of at least a first
therapeutic agent-targeting agent construct effective to induce, or
specifically induce, apoptosis in at least a portion of the tumor
or intratumoral vascular endothelial cells. Herein, the first
therapeutic agent-targeting agent construct binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of tumor or
intratumoral vascular endothelial cells and induces apoptosis in
least a portion of the tumor or intratumoral vascular endothelial
cells, as opposed to endothelial cells in normal vessels.
[0047] As used herein, "induces apoptosis" means induces the
process of programmed cell death that, during the initial stages,
maintains the integrity of the cell membrane, yet transmits the
death-inducing signals into the cell. This is opposed to the
mechanisms of cell necrosis, during which the cell membrane loses
its integrity and becomes leaky at the onset of the process.
[0048] Therapeutic benefits may be realized by the administration
of at least two, three or more therapeutic agent-targeting agent
constructs. The therapeutic agent-targeting agent constructs may
also be combined with other therapies to provide combined
therapeutically effective amounts, as disclosed herein.
[0049] The treatment methods of the present invention will
generally involve the administration of the pharmaceutically
effective composition to the animal systemically, such as via
intravenous injection. However, any route of administration that
allows the therapeutic agent-targeting agent construct to localize
to the tumor or intratumoral vascular endothelial cells will be
acceptable.
[0050] "Administration", as used herein, therefore means provision
or delivery of therapeutic agent-targeting agent constructs in an
amount(s) and for a period of time(s) effective to allow binding to
an aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of blood transporting
vessels of the vascularized tumor, and to exert a tumor vasculature
destructive and tumor-regressive effect. The passive administration
of proteinaceous therapeutic agent-targeting agent constructs is
generally preferred, in part, for its simplicity and
reproducibility.
[0051] However, the term "administration" is herein used to refer
to any and all means by which therapeutic agent-targeting agent
constructs are delivered or otherwise provided to the tumor
vasculature. "Administration" therefore includes the provision of
cells that produce the therapeutic agent-targeting agent constructs
in a manner effective to result in the delivery of the therapeutic
agent-targeting agent constructs to the tumor vasculature, and/or
their localization to such vasculature. In such embodiments, it may
be desirable to formulate or package the cells in a selectively
permeable membrane, structure or implantable device, generally one
that can be removed to cease therapy. Exogenous therapeutic
agent-targeting agent administration will still generally be
preferred, as this represents a non-invasive method that allows the
dose to be closely monitored and controlled.
[0052] The "therapeutic agent-targeting agent administration
methods" of the invention also extend to the provision of nucleic
acids that encode therapeutic agent-targeting agent constructs in a
manner effective to result in the expression of the therapeutic
agent-targeting agent constructs in the vicinity of the tumor
vasculature, and/or in the expression of therapeutic
agent-targeting agent constructs that can localize to the tumor
vasculature. Any gene therapy technique may be employed, such as
naked DNA delivery, recombinant genes and vectors, cell-based
delivery, including ex vivo manipulation of patients' cells, and
the like.
[0053] One of the benefits of the present invention is that
aminophospholipids, particularly phosphatidylserine and
phosphatidylethanolamine, are generally expressed or available
throughout the tumor vessels. Aminophospholipid expression on
established, intratumoral blood vessels is advantageous as
targeting and destroying such vessels will rapidly lead to
anti-tumor effects. However, so long as the administered
therapeutic agent-targeting agent constructs bind to at least a
portion of the blood transporting vessels, significant anti-tumor
effects will ensue. This will not be problematical as
aminophospholipids, such as phosphatidylserine and
phosphatidylethanolamine, are expressed on the large, central
vessels, and also on veins, venules, arteries, arterioles and blood
transporting capillaries in all regions of the tumor.
[0054] In any event, the ability of the therapeutic agent-targeting
agent constructs to destroy the tumor vasculature means that tumor
regression can be achieved, rather than only tumor stasis. Tumor
stasis is most often the result of anti-angiogenic therapies that
target only the budding vessels at the periphery of a solid tumor
and stop the vessels proliferating. Even if the present invention
targeted more of the peripheral regions of the tumor in certain
tumor types, which is not currently believed to be the case,
destruction of the blood transporting vessels in such areas would
still lead to widespread thrombosis and tumor necrosis.
[0055] The targeting portions of the diagnostic and/or therapeutic
agent-targeting agent constructs of the present invention, whether
binding to phosphatidylethanolamine or phosphatidylserine, may be
either antibody-based or binding ligand or binding protein based.
Any aminophospholipid binding ligand or protein known in the art
may thus now be advantageously used in the delivery of therapeutic
agents to tumor vasculature.
[0056] By way of example only, suitable aminophospholipid binding
ligands and proteins include low and high molecular weight
kininogens and other rat, bovine, monkey or human
phosphatidylethanolamine binding proteins; and any one or more of a
number of phosphatidylserine-serine binding annexins. The protein
and DNA sequences for such binding ligands are known in the art and
incorporated herein by reference, facilitating the production of
recombinant fusion proteins for use in the present invention.
[0057] Aminophospholipid binding reagents encompassed by the term
"aminophospholipid binding ligands or binding proteins" extend to
all aminophospholipid binding ligands and proteins from all
species, and aminophospholipid binding fragments thereof, including
dimeric, trimeric and multimeric ligands and proteins; bispecific
ligands and proteins; chimeric ligands and proteins; human ligands
and proteins; recombinant and engineered ligands and proteins, and
fragments thereof.
[0058] Where antibody-based targeting portions are employed,
whether binding to phosphatidylethanolamine or phosphatidylserine,
the term "anti-aminophospholipid antibody", as used herein, refers
broadly to any immunologic binding agent, such as polyclonal and
monoclonal IgG, IgM, IgA, IgD and IgE antibodies. Generally, IgG
and/or IgM are preferred because they are the most common
antibodies in the physiological situation and because they are most
easily made in a laboratory setting.
[0059] Polyclonal anti-aminophospholipid antibodies, obtained from
antisera, may be employed in the invention. However, the use of
monoclonal anti-aminophospholipid antibodies (MAbs) will generally
be preferred. MAbs are recognized to have certain advantages, e.g.,
reproducibility and large-scale production, that makes them
suitable for clinical treatment. The invention thus provides
monoclonal antibodies of the murine, human, monkey, rat, hamster,
rabbit and even frog or chicken origin. Due to the ease of
preparation and ready availability of reagents, murine monoclonal
antibodies will be used in certain embodiments.
[0060] As will be understood by those in the art, the immunologic
binding reagents encompassed by the term "anti-aminophospholipid
antibody" extend to all anti-aminophospholipid antibodies from all
species, and antigen binding fragments thereof, including dimeric,
trimeric and multimeric antibodies; bispecific antibodies; chimeric
antibodies; human and humanized antibodies; recombinant and
engineered antibodies, and fragments thereof.
[0061] The term "anti-aminophospholipid antibody" is thus used to
refer to any anti-aminophospholipid antibody-like molecule that has
an antigen binding region, and includes antibody fragments such as
Fab', Fab, F(ab').sub.2, single domain antibodies (DABs), Fv, scFv
(single chain Fv), and the like. The techniques for preparing and
using various antibody-based constructs and fragments are well
known in the art.
[0062] In certain embodiments, the antibodies employed in the
therapeutic agent-targeting agent constructs will be "humanized" or
human antibodies. "Humanized" antibodies are generally chimeric
monoclonal antibodies from mouse, rat, or other non-human species,
bearing human constant and/or variable region domains ("part-human
chimeric antibodies"). Mostly, humanized monoclonal antibodies for
use in the present invention will be chimeric antibodies wherein at
least a first antigen binding region, or complementarity
determining region (CDR), of a mouse, rat or other non-human
anti-aminophospholipid monoclonal antibody is operatively attached
to, or "grafted" onto, a human antibody constant region or
"framework".
[0063] "Humanized" monoclonal antibodies for use herein may also be
anti-aminophospholipid monoclonal antibodies from non-human species
wherein one or more selected amino acids have been exchanged for
amino acids more commonly observed in human antibodies. This can be
readily achieved through the use of routine recombinant technology,
particularly site-specific mutagenesis.
[0064] Entirely human, rather than "humanized",
anti-aminophospholipid antibodies may also be prepared and used in
the therapeutic agent-targeting agent constructs of the present
invention. Such human antibodies may be polyclonal antibodies, as
obtained from human patients that have any one or more of a variety
of diseases, disorders or clinical conditions associated with the
production of anti-aminophospholipid antibodies. Such antibodies
may be concentrated, partially purified or substantially purified
for use herein.
[0065] A range of techniques are also available for preparing human
monoclonal antibodies. As human patients with
anti-aminophospholipid antibody-producing diseases exist, the
anti-aminophospholipid antibody-producing cells from such patients
may be obtained and manipulated in vitro to provide a human
monoclonal antibody for use in a therapeutic agent-targeting agent
construct. The in vitro manipulations or techniques include fusing
to prepare a monoclonal antibody-producing hybridoma, and/or
cloning the gene(s) encoding the anti-aminophospholipid antibody
from the cells ("recombinant human antibodies").
[0066] Human anti-aminophospholipid antibody-producing cells may
also be obtained from human subjects without an
anti-aminophospholipid antibody-associated disease, i.e. "healthy
subjects" in the context of the present invention. To achieve this,
one would simply obtain a population of mixed peripheral blood
lymphocytes from a human subject, including antigen-presenting and
antibody-producing cells, and stimulate the cell population in
vitro by admixing with an immunogenically effective amount of an
aminophospholipid sample. Again, the human anti-aminophospholipid
antibody-producing cells, once obtained, could be used in hybridoma
and/or recombinant antibody production prior to therapeutic
agent-targeting agent construct preparation.
[0067] Further techniques for human monoclonal antibody production
include immunizing a transgenic animal, preferably a transgenic
mouse, that comprises a human antibody library with an
immunogenically effective amount of an aminophospholipid sample.
This also generates human anti-aminophospholipid antibody-producing
cells for further manipulation in hybridoma and/or recombinant
antibody production, with the advantage that spleen cells, rather
than peripheral blood cells, can be readily obtained from the
transgenic animal or mouse.
[0068] Preferred anti-aminophospholipid antibodies for use in the
therapeutic agent-targeting agent constructs of the present
invention are anti-phosphatidylserine (anti-PS) and
antiphosphatidylethanolamine (anti-PE) antibodies. Anti-PS
antibodies will generally recognize, bind to or have
immunospecificity for the PS molecule present, expressed,
translocated, presented or complexed at the luminal surface of
tumor vascular endothelial cells. Suitable antibodies will thus
bind to phosphatidyl-L-serine (Umeda et al., 1989; incorporated
herein by reference). Anti-PE antibodies will generally recognize,
bind to or have immunospecificity for the PE molecule present,
expressed, translocated, presented or complexed at the luminal
surface of tumor vascular endothelial cells.
[0069] Administering diagnostic and/or therapeutic agent-targeting
agent constructs to an animal with a tumor will result in specific
binding to the aminophospholipid molecules present, expressed or
translocated to the luminal surface of the tumor blood vessels,
i.e., the therapeutic agent-targeting agent constructs will bind to
the aminophospholipid molecules in a natural, biological
environment. Therefore, no particular manipulation will be
necessary to ensure binding.
[0070] However, in terms of antibody binding, it is of scientific
interest to note that aminophospholipids may be most frequently
recognized, or bound, by anti-aminophospholipid antibodies when the
aminophospholipid molecules are associated with one or more
proteins or other non-lipid biological components. For example,
anti-PS antibodies that occur as a sub-set of anti-phospholipid
(anti-PL) antibodies in patients with certain diseases and
disorders are now believed to bind to PS in combination with
proteins such as .beta..sub.2-glycoprotein I (.beta..sub.2-GPI or
apolipoprotein H, apoH) and prothrombin (U.S. Pat. No. 5,344,758;
Rote, 1996; each incorporated herein by reference). Similarly,
anti-PE antibodies that occur in disease states are now believed to
bind to PE in combination with proteins such as low and high
molecular weight kininogen (HK), prekallikrein and even factor XI
(Sugi and McIntyre, 1995; 1996a; 1996b; each incorporated herein by
reference).
[0071] This is the meaning of the terms "presented" and "complexed
at" the luminal surface of tumor blood vessels, as used herein,
which mean that the aminophospholipid molecules are present at the
surface of tumor blood vessels in a binding competent state, or
antibody-binding competent state, irrespective of the molecular
definition of that particular state. PS may even be targeted as a
complex with factor II/IIa, VII/VIIa, IX/IXa and X/Xa. Moreover,
the nature of the aminophospholipid target may change during
practice of the invention, as the initial aminophospholipid
antibody binding, anti-endothelial cell and anti-tumor effects may
result in biological changes that alter the number, conformation
and/or type of the aminophospholipid target epitope(s).
[0072] The term "anti-aminophospholipid antibody", as used in the
context of the present invention, therefore means any antibody,
immunological binding agent or antisera; monoclonal, human,
humanized, dimeric, trimeric, multimeric, chimeric, bispecific,
recombinant or engineered antibody; or Fab', Fab, F(ab').sub.2,
DABs, Fv or scFv antigen binding fragment thereof; that at least
binds to a lipid and amino group-containing complex or
aminophospholipid target, preferably a phosphatidylserine- or
phosphatidylethanolamine-based target.
[0073] The requirement that the antibody "at least bind to an
aminophospholipid target" is met by the antibody binding to any
and/or all physiologically relevant forms of aminophospholipids,
including so-called "hexagonal" and "hexagonal phase II" PS and PE
(HexII PS and HexII PE) (Rauch et al., 1986; Rauch and Janoff,
1990; Berard et al., 1993; each incorporated herein by reference)
and PS and PE in combination with any other protein, lipid,
membrane component, plasma or serum component, or any combination
thereof. Thus, an "anti-aminophospholipid antibody" is an antibody
that binds to an aminophospholipid in the tumor blood vessels,
notwithstanding the fact that bilayer or micelle aminophospholipids
may be considered to be immunogenically neutral.
[0074] The anti-aminophospholipid antibodies may recognize, bind to
or have immunospecificity for aminophospholipid molecules, or an
immunogenic complex thereof (including hexagonal aminophospholipids
and protein combinations), to the exclusion of other phospholipids
or lipids. Such antibodies may be termed
"aminophospholipid-specific or aminophospholipid-restricted
antibodies", and their use in the therapeutic agent-targeting agent
constructs of the invention will often be preferred.
"Aminophospholipid-specific or aminophospholipid-restricted
antibodies" will generally exhibit significant binding to
aminophospholipids, while exhibiting little or no significant
binding to other lipid components, such as phosphatidylinositol
(PI), phosphatidylglycerol (PG) and even phosphatidylcholine (PC)
in certain embodiments.
[0075] "PS-specific or PS-restricted antibodies" will generally
exhibit significant binding to PS, while exhibiting little or no
significant binding to lipid components such as
phosphatidylethanolamine and cardiolipin (CL), as well as PC, PI
and PG. "PE-specific or PE-restricted antibodies" will generally
exhibit significant binding to PE, while exhibiting little or no
significant binding to lipid components such as phosphatidylserine
and cardiolipin, as well as PC, PI and PG. The preparation of
specific anti-aminophospholipid antibodies is readily achieved,
e.g., as disclosed by Rauch et al. (1986); Umeda et al. (1989);
Rauch and Janoff (1990); and Rote et al. (1993); each incorporated
herein by reference.
[0076] "Cross-reactive anti-aminophospholipid antibodies" that
recognize, bind to or have immunospecificity for an
aminophospholipid molecule, or an immunogenic complex thereof
(including hexagonal aminophospholipids and protein combinations),
in addition to exhibiting lesser but detectable binding to other
phospholipid or lipid components are by no means excluded from use
in the invention. Such "cross-reactive anti-aminophospholipid
antibodies" may be employed so long as they bind to an
aminophospholipid present, expressed, translocated, presented or
complexed at the luminal surface of tumor vascular endothelial
cells in vivo.
[0077] Further suitable aminophospholipid-specific or
aminophospholipid-restricted antibodies are those
anti-aminophospholipid antibodies that bind to both PS and PE.
While clearly being specific or restricted to aminophospholipids,
as opposed to other lipid components, antibodies exist that bind to
each of the preferred targets of the present invention. Examples of
such antibodies for use in the therapeutic agent-targeting agent
constructs of the invention include, but are not limited to, PS3A,
PSF6, PSF7, PSB4, PS3H1 and PS3E10 (Igarashi et al., 1991;
incorporated herein by reference)
[0078] Further exemplary anti-PS antibodies for use in the
therapeutic agent-targeting agent constructs include, but are not
limited to BA3B5C4, PS4A7, PS1G3 and 3SB9b; with PS4A7, PS1G3 and
3SB9b generally being preferred. Monoclonal antibodies, humanized
antibodies and/or antigen-binding fragments based upon the 3SB9b
antibody (Rote et al., 1993; incorporated herein by reference) are
currently most preferred.
[0079] Although aminophospholipids, such as PS and PE, in bilayer
or micelle form have been reported to be non- or weakly antigenic,
or non- or weakly-immunogenic, the scientific literature has
reported no difficulties in generating anti-aminophospholipid
antibodies, such as anti-PS and anti-PE antibodies.
Anti-aminophospholipid antibodies or monoclonal antibodies may
therefore be readily prepared by preparative processes and methods
that comprise: [0080] (a) preparing an anti-aminophospholipid
antibody-producing cell; and [0081] (b) obtaining an
anti-aminophospholipid antibody or monoclonal antibody from the
antibody-producing cell.
[0082] The processes of preparing anti-aminophospholipid
antibody-producing cells and obtaining anti-aminophospholipid
antibodies therefrom may be conduced in situ in a given patient.
That is, simply providing an immunogenically effective amount of an
immunogenic aminophospholipid sample to a patient will result in
anti-aminophospholipid antibody generation. Thus, the
anti-aminophospholipid antibody is still "obtained" from the
antibody-producing cell, but it does not have to be isolated away
from a host and subsequently provided to a patient, being able to
spontaneously localize to the tumor vasculature and exert its
biological anti-tumor effects.
[0083] As disclosed herein, anti-aminophospholipid
antibody-producing cells may be obtained, and antibodies
subsequently isolated and/or purified, from human patients with
anti-aminophospholipid antibody-producing diseases, from
stimulating peripheral blood lymphocytes with aminophospholipids in
vitro, and also by immunization processes and methods. The latter
of which generally comprise: [0084] (a) immunizing an animal by
administering to the animal at least one dose, and optionally more
than one dose, of an immunogenically effective amount of an
immunogenic aminophospholipid sample (such as a hexagonal, or
hexagonal phase II form of an aminophospholipid), preferably an
immunogenic PS or PE sample; and [0085] (b) obtaining an
anti-aminophospholipid antibody-producing cell from the immunized
animal.
[0086] The immunogenically effective amount of the
aminophospholipid sample or samples may be a Salmonella-coated
aminophospholipid sample (Umeda et al., 1989; incorporated herein
by reference); an aminophospholipid micelle, liposome, lipid
complex or lipid formulation sample; or an aminophospholipid sample
fabricated with SDS. Any such aminophospholipid sample may be
administered in combination with any suitable adjuvant, such as
Freund's complete adjuvant (Rote et al., 1993; incorporated herein
by reference). Any empirical technique or variation may be employed
to increase immunogenicity, and/or hexagonal or hexagonal phase II
forms of the aminophospholipids may be administered.
[0087] The immunization may be based upon one or more intrasplenic
injections of an immunogenically effective amount of an
aminophospholipid sample (Umeda et al., 1989; incorporated herein
by reference).
[0088] Irrespective of the nature of the immunization process, or
the type of immunized animal, anti-aminophospholipid
antibody-producing cells are obtained from the immunized animal
and, preferably, further manipulated by the hand of man. "An
immunized animal", as used herein, is a non-human animal, unless
otherwise expressly stated. Although any antibody-producing cell
may be used, most preferably, spleen cells are obtained as the
source of the antibody-producing cells. The anti-aminophospholipid
antibody-producing cells may be used in a preparative process that
comprises: [0089] (a) fusing an anti-aminophospholipid
antibody-producing cell with an immortal cell to prepare a
hybridoma that produces an anti-aminophospholipid monoclonal
antibody and [0090] (b) obtaining an anti-aminophospholipid
monoclonal antibody from the hybridoma.
[0091] Hybridoma-based monoclonal antibody preparative methods thus
include those that comprise: [0092] (a) immunizing an animal by
administering to the animal at least one dose, and optionally more
than one dose, of an immunogenically effective amount of an
immunogenic aminophospholipid sample (such as a hexagonal, or
hexagonal phase II form of an aminophospholipid), preferably an
immunogenic PS or PE sample; [0093] (b) preparing a collection of
monoclonal antibody-producing hybridomas from the immunized animal;
[0094] (c) selecting from the collection at least a first hybridoma
that produces at least a first anti-aminophospholipid monoclonal
antibody, and preferably, at least a first
aminophospholipid-specific monoclonal antibody; and [0095] (d)
culturing the at least a first anti-aminophospholipid-producing or
aminophospholipid-specific hybridoma to provide the at least a
first anti-aminophospholipid monoclonal antibody or
aminophospholipid-specific monoclonal antibody; and preferably
[0096] (e) obtaining the at least a first anti-aminophospholipid
monoclonal antibody or aminophospholipid-specific monoclonal
antibody from the cultured at least a first hybridoma.
[0097] As non-human animals are used for immunization, the
anti-aminophospholipid monoclonal antibodies obtained from such a
hybridoma will often have a non-human make up. Such antibodies may
be optionally subjected to a humanization process, grafting or
mutation, as known to those of skill in the art and further
disclosed herein. Alternatively, transgenic animals, such as mice,
may be used that comprise a human antibody gene library.
Immunization of such animals will therefore directly result in the
generation of human anti-aminophospholipid antibodies.
[0098] After the production of a suitable antibody-producing cell,
most preferably a hybridoma, whether producing human or non-human
antibodies, the monoclonal antibody-encoding nucleic acids may be
cloned to prepare a "recombinant" monoclonal antibody. Any
recombinant cloning technique may be utilized, including the use of
PCR to prime the synthesis of the antibody-encoding nucleic acid
sequences. Therefore, yet further appropriate monoclonal antibody
preparative methods include those that comprise using the
anti-aminophospholipid antibody-producing cells as follows: [0099]
(a) obtaining at least a first anti-aminophospholipid
antibody-encoding nucleic acid molecule or segment from an
anti-aminophospholipid antibody-producing cell, preferably a
hybridoma; and [0100] (b) expressing the nucleic acid molecule or
segment in a recombinant host cell to obtain a recombinant
anti-aminophospholipid monoclonal antibody.
[0101] However, other powerful recombinant techniques are available
that are ideally suited to the preparation of recombinant
monoclonal antibodies. Such recombinant techniques include the
phagemid library-based monoclonal antibody preparative methods
comprising: [0102] (a) immunizing an animal by administering to the
animal at least one dose, and optionally more than one dose, of an
immunogenically effective amount of an immunogenic
aminophospholipid sample (such as a hexagonal, or hexagonal phase
II form of an aminophospholipid), preferably an immunogenic PS or
PE sample; [0103] (b) preparing a combinatorial immunoglobulin
phagemid library expressing RNA isolated from the
antibody-producing cells, preferably from the spleen, of the
immunized animal; [0104] (c) selecting from the phagemid library at
least a first clone that expresses at least a first
anti-aminophospholipid antibody, and preferably, at least a first
aminophospholipid-specific antibody; [0105] (d) obtaining
anti-aminophospholipid antibody-encoding nucleic acids from the at
least a first selected clone and expressing the nucleic acids in a
recombinant host cell to provide the at least a first
anti-aminophospholipid antibody or aminophospholipid-specific
antibody; and preferably [0106] (e) obtaining the at least a first
anti-aminophospholipid antibody or aminophospholipid-specific
antibody expressed by the nucleic acids obtained from the at least
a first selected clone.
[0107] Again, in such phagemid library-based techniques, transgenic
animals bearing human antibody gene libraries may be employed, thus
yielding recombinant human monoclonal antibodies.
[0108] Irrespective of the manner of preparation of a first
anti-aminophospholipid antibody nucleic acid segment, further
suitable anti-aminophospholipid antibody nucleic acid segments may
be readily prepared by standard molecular biological techniques. In
order to confirm that any variant, mutant or second generation
anti-aminophospholipid antibody nucleic acid segment is suitable
for use in the present invention, the nucleic acid segment will be
tested to confirm expression of an antibody that binds to an
aminophospholipid. Preferably, the variant, mutant or second
generation anti-aminophospholipid antibody nucleic acid segment
will also be tested to confirm hybridization to an
anti-aminophospholipid antibody nucleic acid segment under
standard, more preferably, standard stringent hybridization
conditions. Exemplary suitable hybridization conditions include
hybridization in about 7% sodium dodecyl sulfate (SDS), about 0.5 M
NaPO.sub.4, about 1 mM EDTA at about 50.degree. C.; and washing
with about 1% SDS at about 42.degree. C.
[0109] As a variety of recombinant monoclonal antibodies, whether
human or non-human in origin, may be readily prepared, the
treatment methods of the invention may be executed by providing to
the animal or patient at least a first nucleic acid segment that
expresses a biologically effective amount of at least a first
therapeutic agent-targeting agent construct in the patient. The
"nucleic acid segment that expresses a therapeutic agent-targeting
agent construct" will generally be in the form of at least an
expression construct, and may be in the form of an expression
construct comprised within a virus or within a recombinant host
cell. Preferred gene therapy vectors of the present invention will
generally be viral vectors, such as comprised within a recombinant
retrovirus, herpes simplex virus (HSV), adenovirus,
adeno-associated virus (AAV), cytomegalovirus (CMV), and the
like.
[0110] Once a targeting agent has been selected, whether
antibody-based or binding ligand-based, and whether binding to
phosphatidylethanolamine and/or phosphatidylserine, the targeting
agent is operatively attached to one or more diagnostic and/or
therapeutic agents or "effector" portions. The therapeutic agents
of the present constructs will generally be either anti-cellular,
cytotoxic or anti-angiogenic agents, or coagulation factors
(coagulants).
[0111] The use of anti-cellular, cytotoxic and/or anti-angiogenic
agents results in "aminophospholipid immunotoxins" (or
anti-aminophospholipid immunotoxins), whereas the use of
coagulation factors results in "aminophospholipid coaguligands" (or
anti-aminophospholipid coaguligands). These terms are again used
for simplicity and succinctly refer to aminophospholipid binding
ligands or therapeutic agent-aminophospholipid targeting agent
constructs in terms of their attached therapeutic moiety.
[0112] The present invention further provides binding ligands, and
methods of use, comprising at least two therapeutic agents
operatively attached to a targeting agent comprising a single
aminophospholipid binding site. The binding ligands may comprise at
least two therapeutic agents operatively attached to a targeting
agent that comprises at least two aminophospholipid binding sites;
or a plurality of therapeutic agents operatively attached to a
targeting agent that comprises a plurality of aminophospholipid
binding sites, generally at regions distinct from the
aminophospholipid binding sites.
[0113] Combinations of anti-cellular and cytotoxic agents with
coagulation factors are also contemplated, irrespective of the
number of aminophospholipid binding sites. The combined use of
therapeutic agents of different classes, such as cytotoxins and
coagulants, is also contemplated in embodiments where two or more
binding ligands are administered to the animal, each containing a
single type of therapeutic agent. Different cytotoxins may also be
employed in one or more binding ligands or methods, such as DNA
synthesis inhibitors combined with classic cytotoxins, such as
ricin.
[0114] In certain applications, the aminophospholipid-targeted
constructs will be operatively attached to cytotoxic, cytostatic or
otherwise anti-cellular agents that have the ability to kill or
suppress the growth or cell division of endothelial cells. Suitable
anti-cellular agents include chemotherapeutic agents, as well as
cytotoxins and cytostatic agents. Cytostatic agents are generally
those that disturb the natural cell cycle of a target cell,
preferably so that the cell is taken out of the cell cycle.
[0115] Exemplary chemotherapeutic agents include: steroids;
cytokines; anti-metabolites, such as cytosine arabinoside,
fluorouracil, methotrexate or aminopterin; anthracyclines;
mitomycin C; vinca alkaloids; antibiotics; demecolcine; etoposide;
mithramycin; and anti-tumor alkylating agents, such as chlorambucil
or melphalan. Indeed, any of the agents disclosed herein in Table C
could be used. Certain preferred anti-cellular agents are DNA
synthesis inhibitors, such as daunorubicin, doxorubicin,
adriamycin, and the like.
[0116] In other embodiments, aminophospholipid-targeted constructs
of the invention may be operatively attached to anti-angiogenic
agents that, acting either alone or in concert with other host
factors, or administered therapeutic agents, have the ability to
prevent or inhibit vascularization and/or to induce regression of
blood vessels. Suitable anti-angiogenic agents include those listed
in Table D, as well as other anti-angiogenic agents known to those
of skill in the art. By way of example only, one may mention the
angiopoietins, preferably, angiopoietin-2 (Ang-2; SEQ ID NO:3 and
SEQ ID NO:4), but also angiopoietin-1 (Ang-1; SEQ ID NO:1 and SEQ
ID NO:2), angiopoietin fusion proteins (for example, as in SEQ ID
NO:5), and even angiopoietin-3 and angiopoietin-4.
[0117] In certain therapeutic applications, toxin moieties will be
preferred, due to the much greater ability of most toxins to
deliver a cell killing effect, as compared to other potential
agents. Therefore, certain preferred anti-cellular agents for
aminophospholipid-targeted constructs are plant-, fungus- or
bacteria-derived toxins. Exemplary toxins include
epipodophyllotoxins; bacterial endotoxin or the lipid A moiety of
bacterial endotoxin; ribosome inactivating proteins, such as
saporin or gelonin; .alpha.-sarcin; aspergillin; restrictocin;
ribonucleases, such as placental ribonuclease; diphtheria toxin and
pseudomonas exotoxin.
[0118] Preferred toxins for certain embodiments are gelonin and/or
the A chain toxins, such as ricin A chain. The most preferred toxin
moiety is often ricin A chain that has been treated to modify or
remove carbohydrate residues, so called "deglycosylated A chain"
(dgA). Deglycosylated ricin A chain is preferred because of its
extreme potency, longer half-life, and because it is economically
feasible to manufacture it a clinical grade and scale. Recombinant
and/or truncated ricin A chain may also be used.
[0119] For tumor targeting and treatment with immunotoxins, the
following patents and patent applications are specifically
incorporated herein by reference for the purposes of even further
supplementing the present teachings regarding anticellular and
cytotoxic agents: U.S. Pat. Nos. 5,855,866; 5,776,427; 5,863,538;
6,004,554; 5,965,132; 6,051,230 and 5,660,827; and U.S. application
Ser. No. 07/846,349.
[0120] The aminophospholipid-targeted constructs of the invention
may comprise a component that is capable of promoting coagulation,
i.e., a coagulant. Here, the targeting antibody or ligand may be
directly or indirectly, e.g., via another antibody, linked to a
factor that directly or indirectly stimulates coagulation.
[0121] Preferred coagulation factors for such uses are Tissue
Factor (TF) and TF derivatives, such as truncated TF (tTF),
dimeric, trimeric, polymeric/multimeric TF, and mutant TF deficient
in the ability to activate Factor VII. Other suitable coagulation
factors include vitamin K-dependent coagulants, such as Factor
II/IIa, Factor VII/VIIa, Factor IX/IXa and Factor X/Xa; vitamin
K-dependent coagulation factors that lack the Gla modification;
Russell's viper venom Factor X activator; platelet-activating
compounds, such as thromboxane A.sub.2 and thromboxane A.sub.2
synthase; and inhibitors of fibrinolysis, such as
.alpha.2-antiplasmin.
[0122] Tumor targeting and treatment with coaguligands is described
in the following patents and patent applications, each of which are
specifically incorporated herein by reference for the purposes of
even further supplementing the present teachings regarding
coaguligands and coagulation factors: U.S. Pat. Nos. 5,855,866;
5,965,132; 6,036,955; 6,093,399 and 5,877,289; U.S. application
Ser. Nos. 07/846,349.
[0123] As somewhat wider distribution of a coagulating agent will
not be associated with severe side effects, there is a less
stringent requirement imposed on the targeting element of
coaguligands than with immunotoxins. Therefore, to achieve specific
targeting means that coagulation is promoted in the tumor
vasculature relative to the vasculature in non-tumor sites. Thus,
specific targeting of a coaguligand is a functional term, rather
than a purely physical term relating to the biodistribution
properties of the targeting agent.
[0124] The preparation of immunotoxins is generally well known in
the art (see, e.g., U.S. Pat. No. 4,340,535, incorporated herein by
reference). Each of the following patents and patent applications
are further incorporated herein by reference for the purposes of
even further supplementing the present teachings regarding
immunotoxin generation, purification and use: U.S. Pat. Nos.
5,855,866; 5,776,427; 5,863,538; 6,004,554; 5,965,132; 6,051,230;
and 5,660,827; and U.S. application Ser. No. 07/846,349.
[0125] In the preparation of immunotoxins, advantages may be
achieved through the use of certain linkers. For example, linkers
that contain a disulfide bond that is sterically "hindered" are
often preferred, due to their greater stability in vivo, thus
preventing release of the toxin moiety prior to binding at the site
of action. It is generally desired to have a conjugate that will
remain intact under conditions found everywhere in the body except
the intended site of action, at which point it is desirable that
the conjugate have good "release" characteristics.
[0126] Depending on the specific toxin compound used, it may be
necessary to provide a peptide spacer operatively attaching the
targeting agent and the toxin compound, wherein the peptide spacer
is capable of folding into a disulfide-bonded loop structure.
Proteolytic cleavage within the loop would then yield a
heterodimeric polypeptide wherein the targeting agent and the toxin
compound are linked by only a single disulfide bond.
[0127] When certain other toxin compounds are utilized, a
non-cleavable peptide spacer may be provided to operatively attach
the targeting agent and the toxin compound. Toxins that may be used
in conjunction with non-cleavable peptide spacers are those that
may, themselves, be converted by proteolytic cleavage, into a
cytotoxic disulfide-bonded form. An example of such a toxin
compound is a Pseudonomas exotoxin compound.
[0128] A variety of chemotherapeutic and other pharmacological
agents can also be successfully conjugated to aminophospholipid
antibodies or targeting ligands. Exemplary antineoplastic agents
that have been conjugated to antibodies include doxorubicin,
daunomycin, methotrexate and vinblastine. Moreover, the attachment
of other agents such as neocarzinostatin, macromycin, trenimon and
.alpha.-amanitin has been described (see U.S. Pat. No. 5,855,866;
and U.S. Pat. No. 5,965,132 and references incorporated
therein).
[0129] In light of one of the present inventors earlier work, the
preparation of coaguligands is now also easily practiced. The
operable association of one or more coagulation factors with an
aminophospholipid targeting agent may be a direct linkage, such as
those described above for the immunotoxins. Alternatively, the
operative association may be an indirect attachment, such as where
the targeting agent is operatively attached to a second binding
region, preferably and antibody or antigen binding region of an
antibody, that binds to the coagulation factor. The coagulation
factor should be attached to the targeting agent at a site distinct
from its functional coagulating site, particularly where a covalent
linkage is used to join the molecules.
[0130] Indirectly linked coaguligands are often based upon
bispecific antibodies. The preparation of bispecific antibodies is
also well known in the art. One preparative method involves the
separate preparation of antibodies having specificity for the
targeted tumor component, on the one hand, and the coagulating
agent on the other. Peptic F(ab'.gamma.).sub.2 fragments from the
two chosen antibodies are then generated, followed by reduction of
each to provide separate Fab'.gamma..sub.SH fragments. The SH
groups on one of the two partners to be coupled are then alkylated
with a cross-linking reagent, such as o-phenylenedimaleimide, to
provide free maleimide groups on one partner. This partner may then
be conjugated to the other by means of a thioether linkage, to give
the desired F(ab'.gamma.).sub.2 heteroconjugate (Glennie et al.,
1987; incorporated herein by reference). Other approaches, such as
cross-linking with SPDP or protein A may also be carried out.
[0131] Another method for producing bispecific antibodies is by the
fusion of two hybridomas to form a quadroma. As used herein, the
term "quadroma" is used to describe the productive fusion of two B
cell hybridomas. Using now standard techniques, two antibody
producing hybridomas are fused to give daughter cells, and those
cells that have maintained the expression of both sets of clonotype
immunoglobulin genes are then selected.
[0132] A preferred method of generating a quadroma involves the
selection of an enzyme deficient mutant of at least one of the
parental hybridomas. This first mutant hybridoma cell line is then
fused to cells of a second hybridoma that had been lethally
exposed, e.g., to iodoacetamide, precluding its continued survival.
Cell fusion allows for the rescue of the first hybridoma by
acquiring the gene for its enzyme deficiency from the lethally
treated hybridoma, and the rescue of the second hybridoma through
fusion to the first hybridoma. Preferred, but not required, is the
fusion of immunoglobulins of the same isotype, but of a different
subclass. A mixed subclass antibody permits the use if an
alternative assay for the isolation of a preferred quadroma.
[0133] Microtiter identification embodiments, FACS,
immunofluorescence staining, idiotype specific antibodies, antigen
binding competition assays, and other methods common in the art of
antibody characterization may be used to identify preferred
quadromas. Following the isolation of the quadroma, the bispecific
antibodies are purified away from other cell products. This may be
accomplished by a variety of antibody isolation procedures, known
to those skilled in the art of immunoglobulin purification (see,
e.g., Antibodies: A Laboratory Manual, 1988; incorporated herein by
reference). Protein A or protein G sepharose columns are
preferred.
[0134] In the preparation of both immunotoxins and coaguligands,
recombinant expression may be employed. The nucleic acid sequences
encoding the chosen targeting agent, and toxin or coagulant, are
attached in-frame in an expression vector. Recombinant expression
thus results in translation of the nucleic acid to yield the
desired targeting agent-toxin/coagulant compound. Chemical
cross-linkers and avidin:biotin bridges may also join the
therapeutic agent(s) to the targeting agent(s).
[0135] The following patents and patent applications are each
incorporated herein by reference for the purposes of even further
supplementing the present teachings regarding coaguligand
preparation, purification and use, including bispecific antibody
coaguligands: U.S. Pat. Nos. 5,855,866; 5,965,132; 6,004,555;
6,036,955; 6,093,399 and 5,877,289; U.S. application Ser. No.
07/846,349.
[0136] In certain embodiments, the vasculature of the vascularized
tumor of the animal or patient to be treated may be first imaged.
Generally this is achieved by first administering to the animal or
patient a diagnostically effective amount of at least a first
pharmaceutical composition comprising at least a first
detectably-labeled aminophospholipid binding construct that binds
to and identifies an aminophospholipid, preferably
phosphatidylserine or phosphatidylethanolamine, present, expressed,
translocated, presented or complexed at the luminal surface of
blood vessels or intratumoral blood vessels of the vascularized
tumor. The invention thus further provides compositions for use in,
and methods of, distinguishing between tumor and/or intratumoral
blood vessels and normal blood vessels. The "distinguishing" is
achieved by administering one or more of the detectably-labeled
aminophospholipid binding constructs described.
[0137] The detectably-labeled aminophospholipid binding construct
may comprise an X-ray detectable compound, such as bismuth (III),
gold (III), lanthanum (III) or lead (II); a radioactive ion, such
as copper.sup.67, gallium.sup.67, gallium.sup.68, indium.sup.111,
indium.sup.113, iodine.sup.123, iodine.sup.125, iodine.sup.131,
mercury.sup.197, mercury.sup.203, rhenium.sup.186, rhenium.sup.188,
rubidium.sup.97, rubidium.sup.103, technetium.sup.99m or
yttrium.sup.90; a nuclear magnetic spin-resonance isotope, such as
cobalt (II), copper (II), chromium (III), dysprosium (III), erbium
(III), gadolinium (III), holmium (III), iron (II), iron (III),
manganese (II), neodymium (III), nickel (II), samarium (III),
terbium (III), vanadium (II) or ytterbium (III); or rhodamine or
fluorescein.
[0138] Pre-imaging before tumor treatment may thus be carried out
by: [0139] (a) administering to the animal or patient a
diagnostically effective amount of a pharmaceutical composition
comprising at least a first detectably-labeled aminophospholipid
binding construct that comprises a diagnostic agent operatively
attached to an antibody, binding protein or ligand, or
aminophospholipid binding fragment thereof, that binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, present, expressed, translocated,
presented or complexed at the luminal surface of blood vessels or
intratumoral blood vessels of the vascularized tumor; and [0140]
(b) subsequently detecting the detectably-labeled aminophospholipid
binding construct bound to an aminophospholipid, preferably
phosphatidylserine or phosphatidylethanolamine, on the luminal
surface of tumor or intratumoral blood vessels, thereby obtaining
an image of the tumor vasculature.
[0141] Cancer treatment may also be carried out by: [0142] (a)
forming an image of a vascularized tumor by administering to an
animal or patient having a vascularized tumor a diagnostically
minimal amount of at least a first detectably-labeled
aminophospholipid binding construct comprising a diagnostic agent
operatively attached to an antibody, binding protein or ligand, or
aminophospholipid binding fragment thereof, that binds to an
aminophospholipid, preferably phosphatidylserine or
phosphatidylethanolamine, on the luminal surface of tumor or
intratumoral blood vessels of the vascularized tumor, thereby
forming a detectable image of the tumor vasculature; and [0143] (b)
subsequently administering to the same animal or patient a
therapeutically optimized amount of at least a first therapeutic
agent-targeting agent construct that binds to an aminophospholipid,
preferably phosphatidylserine or phosphatidylethanolamine, on the
tumor or intratumoral blood vessel luminal surface and thereby
destroys the tumor vasculature.
[0144] Imaging and treatment formulations or medicaments are thus
provided, which generally comprise: [0145] (a) a first
pharmaceutical composition comprising a diagnostically effective
amount of a detectably-labeled aminophospholipid binding construct
that comprises a detectable agent operatively attached to an
antibody, binding protein or ligand, or aminophospholipid binding
fragment thereof, that binds to an aminophospholipid, preferably
phosphatidylserine or phosphatidylethanolamine, on the luminal
surface of tumor or intratumoral blood vessels of the vascularized
tumor; and [0146] (b) a second pharmaceutical composition
comprising a therapeutically effective amount of at least one
therapeutic agent-targeting agent construct, preferably one that
binds to phosphatidylserine or phosphatidylethanolamine.
[0147] In such methods and medicaments, advantages will be realized
wherein the first and second pharmaceutical compositions comprise
the same targeting agents, e.g., anti-aminophospholipid antibodies,
or fragments thereof, from the same antibody preparation, or
preferably, from the same antibody-producing hybridoma. The
foregoing medicaments may also further comprise one or more
anti-cancer agents.
[0148] In the vasculature imaging aspects of the invention, it is
recognized that the administered detectably-labeled
aminophospholipid binding construct, or anti-aminophospholipid
antibody-detectable agent, may itself have a therapeutic effect.
Whilst this would not be excluded from the invention, the amounts
of the detectably-labeled constructs to be administered would
generally be chosen as "diagnostically effective amounts", which
are typically lower than the amounts required for therapeutic
benefit.
[0149] In the imaging embodiments, as with the therapeutics, the
targeting agent may be either antibody-based or binding ligand- or
binding protein-based. Although not previously connected with
tumors or tumor vasculature, detectably labeled aminophospholipid
binding ligand compositions are known in the art and can now, in
light of this motivation and the present disclosure, be used in the
present invention. The detectably-labeled annexins of U.S. Pat. No.
5,627,036; WO 95/19791; WO 95/27903; WO 95/34315; WO 96/17618; and
WO 98/04294; each incorporated herein by reference; may thus be
employed.
[0150] In still further embodiments, the animals or patients to be
treated by the present invention are further subjected to surgery
or radiotherapy, or are provided with a therapeutically effective
amount of at least a first anti-cancer agent. The "at least a first
anti-cancer agent" in this context means "at least a first
anti-cancer agent in addition to the therapeutic agent-targeting
agent construct of the invention. The "at least a first anti-cancer
agent" may thus be considered to be "at least a second anti-cancer
agent", where the therapeutic agent-targeting agent construct is a
first anti-cancer agent. However, this is purely a matter of
semantics, and the practical meaning will be clear to those of
ordinary skill in the art.
[0151] The at least a first anti-cancer agent may be administered
to the animal or patient substantially simultaneously with the
therapeutic agent-targeting agent construct; such as from a single
pharmaceutical composition or from two pharmaceutical compositions
administered closely together.
[0152] Alternatively, the at least a first anti-cancer agent may be
administered to the animal or patient at a time sequential to the
administration of the at least a first therapeutic agent-targeting
agent construct. "At a time sequential", as used herein, means
"staggered", such that the at least a first anti-cancer agent is
administered to the animal or patient at a time distinct to the
administration of the at least a first therapeutic agent-targeting
agent construct. Generally, the two agents are administered at
times effectively spaced apart to allow the two agents to exert
their respective therapeutic effects, i.e., they are administered
at "biologically effective time intervals".
[0153] The at least a first anti-cancer agent may be administered
to the animal or patient at a biologically effective time prior to
the therapeutic agent-targeting agent construct, or at a
biologically effective time subsequent to the therapeutic
agent-targeting agent construct. Administration of a
non-aminophospholipid targeted anti-cancer agent at a
therapeutically effective time subsequent to the therapeutic
agent-targeting agent construct may be particularly desired wherein
the anti-cancer agent is an anti-tumor cell immunotoxin designed to
kill tumor cells at the outermost rim of the tumor, and/or wherein
the anti-cancer agent is an anti-angiogenic agent designed to
prevent micrometastasis of any remaining tumor cells. Such
considerations will be known to those of skill in the art.
[0154] Administration of one or more non-aminophospholipid targeted
anti-cancer agents at a therapeutically effective time prior to a
therapeutic agent-targeting agent construct may be particularly
employed where the anti-cancer agent is designed to increase
aminophospholipid expression. This may be achieved by using
anti-cancer agents that injure, or induce apoptosis the tumor
endothelium. Exemplary anti-cancer agent include, e.g., taxol,
vincristine, vinblastine, neomycin, combretastatin(s),
podophyllotoxin(s), TNF-.alpha., angiostatin, endostatin,
vasculostatin, .alpha..sub.v.beta..sub.3 antagonists, calcium
ionophores, calcium-flux inducing agents, any derivative or prodrug
thereof.
[0155] The one or more additional anti-cancer agents may be
chemotherapeutic agents, radiotherapeutic agents, cytokines,
anti-angiogenic agents, apoptosis-inducing agents or anti-cancer
immunotoxins or coaguligands. "Chemotherapeutic agents", as used
herein, refer to classical chemotherapeutic agents or drugs used in
the treatment of malignancies. This term is used for simplicity
notwithstanding the fact that other compounds may be technically
described as chemotherapeutic agents in that they exert an
anti-cancer effect. However, "chemotherapeutic" has come to have a
distinct meaning in the art and is being used according to this
standard meaning.
[0156] A number of exemplary chemotherapeutic agents are described
herein. Those of ordinary skill in the art will readily understand
the uses and appropriate doses of chemotherapeutic agents, although
the doses may well be reduced when used in combination with the
present invention. A new class of drugs that may also be termed
"chemotherapeutic agents" are agents that induce apoptosis. Any one
or more of such drugs, including genes, vectors and antisense
constructs, as appropriate, may also be used in conjunction with
the present invention.
[0157] Anti-cancer immunotoxins or coaguligands are further
appropriate anti-cancer agents. "Anti-cancer immunotoxins or
coaguligands", or targeting-agent/therapeutic agent constructs, are
based upon targeting agents, including antibodies or antigen
binding fragments thereof, that bind to a targetable component of a
tumor cell, tumor vasculature or tumor stroma, and that are
operatively attached to a therapeutic agent, generally a cytotoxic
agent (immunotoxin) or coagulation factor (coaguligand). A
"targetable component" of a tumor cell, tumor vasculature or tumor
stroma, is preferably a surface-expressed, surface-accessible or
surface-localized component, although components released from
necrotic or otherwise damaged tumor cells or vascular endothelial
cells may also be targeted, including cytosolic and/or nuclear
tumor cell antigens.
[0158] Both antibody and non-antibody targeting agents may be used,
including growth factors, such as VEGF and FGF; peptides containing
the tripeptide R-G-D, that bind specifically to the tumor
vasculature; and other targeting components such as annexins and
related ligands.
[0159] Anti-tumor cell immunotoxins or coaguligands may comprise
antibodies exemplified by the group consisting of B3 (ATCC HB
10573), 260F9 (ATCC HB 8488), D612 (ATCC HB 9796) and KS1/4, said
KS1/4 antibody obtained from a cell comprising the vector pGKC2310
(NRRL B-18356) or the vector pG2A52 (NRRL B-18357).
[0160] Anti-tumor stroma immunotoxins or coaguligands will
generally comprise antibodies that bind to a connective tissue
component, a basement membrane component or an activated platelet
component; as exemplified by binding to fibrin, RIBS or LIBS.
[0161] Anti-tumor vasculature immunotoxins or coaguligands may
comprise ligands, antibodies, or fragments thereof, that bind to a
surface-expressed, surface-accessible or surface-localized
component of the blood transporting vessels, preferably the
intratumoral blood vessels, of a vascularized tumor. Such
antibodies include those that bind to surface-expressed components
of intratumoral blood vessels of a vascularized tumor, including
aminophospholipids themselves, and intratumoral vasculature cell
surface receptors, such as endoglin (TEC-4 and TEC-11 antibodies),
a TGF.beta. receptor, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA,
a VEGF/VPF receptor, an FGF receptor, a TIE,
.alpha..sub.v.beta..sub.3 integrin, pleiotropia, endosialin and MHC
Class II proteins. The antibodies may also bind to
cytokine-inducible or coagulant-inducible components of
intratumoral blood vessels.
[0162] Other anti-tumor vasculature immunotoxins or coaguligands
may comprise antibodies, or fragments thereof, that bind to a
ligand or growth factor that binds to an intratumoral vasculature
cell surface receptor. Such antibodies include those that bind to
VEGF/VPF (GV39 and GV97 antibodies), FGF, TGF.beta., a ligand that
binds to a TIE, a tumor-associated fibronectin isoform, scatter
factor/hepatocyte growth factor (HGF), platelet factor 4 (PF4),
PDGF and TIMP. The antibodies, or fragments thereof, may also bind
to a ligand:receptor complex or a growth factor:receptor complex,
but not to the ligand or growth factor, or to the receptor, when
the ligand or growth factor or the receptor is not in the
ligand:receptor or growth factor:receptor complex.
[0163] Anti-tumor cell, anti-tumor stroma or anti-tumor vasculature
antibody-therapeutic agent constructs may comprise cytotoxic agents
such as plant-, fungus- or bacteria-derived toxins (immunotoxins).
Ricin A chain and deglycosylated ricin A chain will often be
preferred, and gelonin and angiopoietins are also contemplated.
Anti-tumor cell, anti-tumor stroma or anti-tumor vasculature
antibody-therapeutic agent constructs may comprise coagulation
factors or second antibody binding regions that bind to coagulation
factors (coaguligands). The operative association with Tissue
Factor or Tissue Factor derivatives, such as truncated Tissue
Factor, will often be preferred.
[0164] The invention still further provides a series of novel
therapeutic binding ligands, binding ligand compositions and
pharmaceutical compositions, each of which comprise at least a
first targeting agent that binds to an aminophospholipid,
operatively attached to at least a first therapeutic agent, such as
a cytotoxin, anti-angiogenic agent or coagulant. Radiolabels are
generally excluded from the binding ligands and binding ligand
compositions; although not from the diagnostic methods, or even
from the therapeutic methods described above.
[0165] The targeting agents of the binding ligands preferably bind
to phosphatidylethanolamine and/or phosphatidylserine. The entire
range of binding ligands described above in the context of the
therapeutic and combined methods may be employed in the present
compositions. Annexin conjugates and constructs; anti-PS, anti-PE,
human, humanized and monoclonal antibody conjugates and constructs;
ricin conjugates; and Tissue Factor conjugates and constructs are
currently preferred. Compositions comprising one or more anti-PS
antibodies operatively attached to one or more Tissue Factor
derivatives, preferably, truncated Tissue Factor, are currently
particularly preferred.
[0166] Direct or indirect attachment and linkages may be employed
in the binding ligand compositions, including all variations of
bispecific antibodies. Operative combinations of a first
antigen-binding region of an antibody that binds to an
aminophospholipid, with a second antigen-binding region of an
antibody that binds Tissue Factor or a Tissue Factor derivative are
also preferred. In the aminophospholipid binding protein constructs
or conjugates, annexins are preferred, with Annexin V being more
preferred, and Annexin V operatively attached to truncated Tissue
Factor currently being most preferred.
[0167] Components of the invention therefore include an antibody
construct, comprising at least a first anti-aminophospholipid
antibody, or antigen-binding fragment thereof, operatively attached
to at least a first therapeutic agent; and a bispecific antibody,
comprising a first antigen-binding region that binds to an
aminophospholipid operatively attached to a second antigen-binding
region that binds to a therapeutic agent.
[0168] The compositions and pharmaceutical compositions may
comprise at least a first and second binding ligand that each
comprise at least a first targeting agent operatively attached to
at least a first therapeutic agent; wherein each targeting agent
binds to an aminophospholipid. Compositions and pharmaceutical
compositions that comprise at least a first binding ligand that
binds to phosphatidylethanolamine and at least a second binding
ligand that binds to phosphatidylserine are exemplary combined
compositions.
[0169] The present invention yet further provides a series of novel
therapeutic kits, medicaments and/or cocktails for use in
conjunction with the methods of the invention. The kits,
medicaments and/or cocktails generally comprise a combined
effective amount of an anti-cancer agent and a therapeutic
agent-targeting agent construct, preferably one that binds to
phosphatidylserine or phosphatidylethanolamine. Imaging components
may also be included.
[0170] The kits and medicaments will comprise, preferably in
suitable container means, a biologically effective amount of at
least a first therapeutic agent-targeting agent construct,
preferably binding to phosphatidylserine or
phosphatidylethanolamine; in combination with a biologically
effective amount of at least a first anti-cancer agent. The
components of the kits and medicaments may be comprised within a
single container or container means, or comprised within distinct
containers or container means. The cocktails will generally be
admixed together for combined use.
[0171] The entire range of therapeutic agent-targeting agent
construct, as described above, may be employed in the kits,
medicaments and/or cocktails, with annexin conjugates and
constructs; anti-PS, anti-PE, human, humanized and monoclonal
antibody conjugates and constructs; ricin conjugates; and Tissue
Factor conjugates and constructs being preferred. The anti-cancer
agents are also those as described above, including
chemotherapeutic agents, radiotherapeutic agents, anti-angiogenic
agents, apoptopic agents, immunotoxins and coaguligands. Agents
formulated for intravenous administration will often be
preferred.
BRIEF DESCRIPTION OF THE DRAWINGS
[0172] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0173] FIG. 1A and FIG. 1B. Activity of cell-bound
anti-VCAM-1.cndot.tTF in vitro. FIG. 1A. Binding of
anti-VCAM-1.cndot.tTF coaguligand to unstimulated (control) and
IL-1.alpha.-activated bEnd.3 cells. FIG. 1B. Generation of factor
Xa by cell-bound anti-VCAM-1.cndot.tTF coaguligand.
[0174] FIG. 2. Retardation of growth of L540 tumors in mice treated
with anti-VCAM-1.cndot.tTF. L540 tumor bearing mice were injected
i.v. with either saline, 20 .mu.g of anti-VCAM-1.cndot.tTF, 4 .mu.g
of unconjugated tTF or 20 .mu.g of control IgG.cndot.tTF.
Injections were repeated on day 4 and 8 after the first treatment.
Tumors were measured daily. Mean tumor volume and SD of 8 mice per
group is shown.
[0175] FIG. 3. Annexin V blocks coaguligand activation of Factor X
in vitro. IL-1.alpha.-stimulated bEnd.3 cells were incubated with
anti-VCAM-.cndot.tTF coaguligand in 96-well microtiter plates, as
described in Example V. Annexin V was added at concentrations
ranging from 0.1 to 10 .mu.g/ml (as shown) and cells were incubated
for 30 min. before addition of diluted Proplex T. The amount of
Factor Xa generated in the presence or absence of Annexin V was
determined using a chromogenic substrate, as described in Example
V.
[0176] FIG. 4A and FIG. 4B. Anti-tumor effects of naked anti-PS
antibodies in animals with syngeneic and xenogeneic tumors.
1.times.10.sup.7 cells of murine colorectal carcinoma Colo 26 (FIG.
4A) or human Hodgkin's lymphoma L540 (FIG. 48) were injected
subcutaneously into the right flank of Balb/c mice (FIG. 4A) or
male CB17 SCID mice (FIG. 4B), respectively. Tumors were allowed to
grow to a size of about 0.6-0.9 cm.sup.3 and then the mice (4
animals per group) were injected i.p. with 20 .mu.g of naked
anti-PS antibody (open squares) or saline (open circles) (control
mouse IgM gave similar results to saline.). Treatment was repeated
3 times with a 48 hour interval. Animals were monitored daily for
tumor measurements and body weight. Tumor volume was calculated as
described in Example VII. Mice were sacrificed when tumors had
reached 2 cm.sup.3, or earlier if tumors showed signs of necrosis
or ulceration.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
A. Tumor Destruction Using VCAM-1 Coaguligand
[0177] Solid tumors and carcinomas account for more than 90% of all
cancers in man. Although the use of monoclonal antibodies and
immunotoxins has been investigated in the therapy of lymphomas and
leukemias (Vitetta et al., 1991), these agents have been
disappointingly ineffective in clinical trials against carcinomas
and other solid tumors (Abrams and Oldham, 1985). A principal
reason for the ineffectiveness of antibody-based treatments is that
macromolecules are not readily transported into solid tumors. Even
once within a tumor mass, these molecules fail to distribute evenly
due to the presence of tight junctions between tumor cells, fibrous
stroma, interstitial pressure gradients and binding site barriers
(Dvorak et al., 1991).
[0178] In developing new strategies for treating solid tumors, the
methods that involve targeting the vasculature of the tumor, rather
than the tumor cells, offer distinct advantages. An effective
destruction or blockade of the tumor vessels arrests blood flow
through the tumor and results in an avalanche of tumor cell death.
Antibody-toxin and antibody-coagulant constructs have already been
effectively used in the specific targeting and destruction of tumor
vessels, resulting in tumor necrosis (Burrows at al., 1992; Burrows
and Thorpe, 1993; WO 93/17715; WO 96/01653; U.S. Pat. Nos.
5,855,866; 5,877,289; 5,965,132; 6,004,555; and 6,093,399; each
incorporated herein by reference).
[0179] Where antibodies, growth factors or other binding ligands
are used to specifically deliver a coagulant to the tumor
vasculature, such agents are termed "coaguligands". A currently
preferred coagulant for use in coaguligands is truncated Tissue
Factor (tTF) (Huang et al., 1997; WO 96/01653; U.S. Pat. No.
5,877,289). TF is the major initiator of blood coagulation (Ruf et
al., 1991). At sites of injury, Factor VII/VIIa in the blood comes
into contact with, and binds to, TF on cells in the perivascular
tissues. The TF:VIIa complex, in the presence of the phospholipid
surface, activates factors IX and X. This, in turn, leads to the
formation of thrombin and fibrin and, ultimately, a blood clot (Ruf
and Edgington, 1994).
[0180] The recombinant, truncated form of tissue factor (tTF),
lacking the cytosolic and transmembrane domains, is a soluble
protein that has about five orders of magnitude lower coagulation
inducing ability than native TF (Stone et al., 1995; Huang et al.,
1997). This is because TF needs to be associated with phospholipids
for the complex with VIIa to activate IXa or Xa efficiently.
However, when tTF is delivered to tumor vascular endothelium by
means of a targeting antibody or agent, it is brought back into
proximity to a lipid surface and regains thrombogenic activity
(Huang et al., 1997; U.S. Pat. Nos. 5,877,289; 6,004,555; and
6,093,399). A coaguligand is thus created that selectively
thromboses tumor vasculature.
[0181] Truncated IF has several advantages that commend its use in
vascular targeted coaguligands: human tTF is readily available, and
the human protein will have negligible or low immunogenicity in
man; human tTF is fully functional in experimental animals,
including mice; and targeted tTF is highly potent because it
triggers the activation of a cascade of coagulation proteins,
giving a greatly amplified effect (U.S. Pat. Nos. 5,877,289;
6,004,555; and 6,093,399).
[0182] A range of suitable target molecules that are available on
tumor endothelium, but largely absent from normal endothelium, have
been described. For example, expressed targets may be utilized,
such as endoglin, E-selectin, P-selectin, VCAM-1, ICAM-1, PSMA, a
TIE, a ligand reactive with LAM-1, a VEGF/VPF receptor, an FGF
receptor, .alpha..sub.v.beta..sub.3 integrin, pleiotropia or
endosialin (U.S. Pat. Nos. 5,855,866; 5,5,877,289 and 6,004,555;
Burrows et al., 1992; Burrows and Thorpe, 1993; Huang et al., 1997;
Liu et al., 1997; Ohizumi et al., 1997; each incorporated herein by
reference).
[0183] Adsorbed targets are another suitable group, such as VEGF,
FGF, TGF.beta., HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE
or a tumor-associated fibronectin isoform (U.S. Pat. Nos.
5,877,289; 5,965,132 and 6,004,555; each incorporated herein by
reference). Fibronectin isoforms are ligands that hind to the
integrin family of receptors. Tumor-associated fibronectin isoforms
are targetable components of both tumor vasculature and tumor
stroma. The monoclonal antibody BC-1 (Carnemolla et al., 1989)
specifically binds to tumor-associated fibronectin isoforms.
[0184] Other targets inducible by the natural tumor environment or
following intervention by man are also targetable entities, as
described in U.S. Pat. Nos. 5,776,427, 5,863,538 and 6,036,955.
When used in conjunction with prior suppression in normal tissues
and tumor vascular induction, MHC Class II antigens may also be
employed as targets (U.S. Pat. Nos. 5,776,427; 5,863,538; 6,004,554
and 6,036,955; each incorporated herein by reference).
[0185] One currently preferred target for clinical applications is
vascular endothelial adhesion molecule-1 (VCAM-1) (U.S. Pat. Nos.
5,855,866, 5,877,289, 6,004,555 and 6,093,399; each incorporated
herein by reference). VCAM-1 is a cell adhesion molecule that is
induced by inflammatory cytokines IL-1.alpha., IL-4 (Thornhill et
al., 1990) and TNF.alpha. (Munro, 1993) and whose role in vivo is
to recruit leukocytes to sites of acute inflammation (Bevilacqua,
1993).
[0186] VCAM-1 is present on vascular endothelial cells in a number
of human malignant tumors including neuroblastoma (Patey et al.,
1996), renal carcinoma (Droz et al., 1994), non-small lung
carcinoma (Staal-van den Brekel et al, 1996), Hodgkin's disease
(Patey et al., 1996), and angiosarcoma (Kuzu et al., 1993), as well
as in benign tumors, such as angioma (Patey et al., 1996) and
hemangioma (Kuzu et al., 1993). Constitutive expression of VCAM-1
in man is confined to a few vessels in the thyroid, thymus and
kidney (Kuzu et al., 1993; Bruijn and Dinklo, 1993), and in the
mouse to vessels in the heart and lung (Fries et al., 1993).
[0187] Certain of the data presented herein even further supplement
those provided in U.S. Pat. Nos. 5,855,866, 5,877,289 and
6,004,555; each incorporated herein by reference) and show the
selective induction of thrombosis and tumor infarction resulting
from administration of an anti-VCAM-1.cndot.tTF coaguligand. The
results presented were generated using mice bearing L540 human
Hodgkin lymphoma. When grown as a xenograft in SCID mice, this
tumor shows close similarity to the human disease with respect to
expression of inflammatory cytokines (Diehl et al., 1985) and the
presence of VCAM-1 and other endothelial cell activation molecules
on its vasculature.
[0188] Using a covalently-linked anti-VCAM-1.cndot.tTF coaguligand,
in which tTF was directly linked to the anti-VCAM-1 antibody, it is
shown herein that the coaguligand localizes selectively to tumor
vessels, induces thrombosis of those vessels, causes necrosis to
develop throughout the tumor and retards tumor growth in mice
bearing solid L540 Hodgkin tumors. Tumors generally needed to be at
least about 0.3 cm in diameter to respond to the coaguligand,
because VCAM-1 was absent from smaller tumors. Presumably, in small
tumors, the levels of cytokines secreted by tumor cells or host
cells that infiltrate the tumor are too low for VCAM-1 induction.
This is in accordance with the studies in U.S. Pat. Nos. 5,855,866,
5,877,289, 5,776,427, 6,004,555 and 6,036,955, where the inventions
were shown to be most useful in larger solid tumors.
[0189] Although VCAM-1 staining was initially observed more in the
periphery of the tumor, the coaguligand evidently bound to and
occluded blood transporting vessels--as it was capable of
curtailing blood flow in all tumor regions. Furthermore, one of the
inventors contemplates that the thrombin generation caused by the
initial administration of the coaguligand likely leads to further
VCAM-1 induction on central vessels (Sluiter et al., 1993),
resulting in an amplified signal and evident destruction of the
intratumoral region. This type of coagulant-induced expression of
further targetable markers, and hence signal amplification, is also
disclosed in U.S. Pat. No. 6,036,955.
B. Mechanism of VCAM-1-Targeted Tumor Destruction
[0190] As shown herein, although localization to VCAM-1-expressing
vessels in the heart and lungs of mice was observed upon
administration of an anti-VCAM-1 coaguligand, this construct did
not induce thrombosis in such non-tumor sites. Furthermore, the
anti-VCAM-1 coaguligand was no more toxic to mice than was a
control coaguligand of irrelevant specificity, again indicating
that the constitutive expression of VCAM-1 on heart and lung
vessels did not lead to toxicity. This data is important to the
immediate clinical progress of coaguligand therapy, given that
VCAM-1 is a naturally occurring marker of tumor vascular
endothelium in humans. However, this phenomenon also provided the
inventors with a unique insight, leading to other approaches for
tumor vasculature destruction.
[0191] The inventors sought to understand the mechanism behind the
ability of the anti-VCAM-1 coaguligand to bind to the VCAM-1
constitutively expressed on blood vessels in the heart and lungs,
and yet not to cause thrombosis in those vessels. There are
numerous scientific possibilities for this empirical observation,
generally connected with the prothrombotic nature of the tumor
environment and any fibrinolytic predisposition in the heart and
lungs.
[0192] Generally, there is a biological equilibrium between the
coagulation system (fibrin deposition) and the fibrinolytic system
(degradation of fibrin by enzymes). However, in malignant disease,
particularly carcinomas, this equilibrium is disrupted, resulting
in the abnormal activation of coagulation (hypercoagulability or
the "prothrombotic state"). Evidence also indicates that various
components of these pathways may contribute to the disorderly
characteristics of malignancy, such as proliferation, invasion, and
metastasis (Zacharski et al., 1993).
[0193] Donati (1995) reviewed the complex interplay between the
original clinical observations of thrombotic complications of
malignant diseases, and the subsequent progress in the cell biology
and biochemistry of tumor cell activities. However, despite
extensive research, a clear molecular explanation for the
prothrombotic nature of the tumor environment could not be provided
(Donati, 1995). Donati did emphasize, though, the role of tumor
cells in this process. It was explained that tumor cells express
procoagulant activities, such as tissue thromboplastin and cancer
procoagulant (CP) (Donati, 1995). WO 91/07187 also reported a
procoagulant activity of tumor cells.
[0194] Numerous other studies have also identified the tumor cells
themselves as being responsible for the prothrombotic state within
a tumor. For example, Nawroth et al. (1988) reported that factor(s)
elaborated by sarcoma cells enhance the procoagulant response of
nearby endothelium to TNF. These authors reported that fibrin
formation occurred throughout the tumor vascular bed 30 minutes
after TNF infusion, but that fibrin deposition and platelet
aggregates were not observed in normal vasculature (Nawroth et al.,
1988). TNF was later shown to enhance the expression of tissue
factor on the surface of endothelial cells (Murray et al., 1991).
This was proposed to explain earlier studies showing that cultured
endothelial cells incubated with recombinant TNF have enhanced
procoagulant activity, tissue factor, and concomitant suppression
of the protein C pathway, an anti-thrombotic mechanism that
functions on the surface of quiescent endothelial cells (Nawroth et
al., 1985; Nawroth and Stern, 1986).
[0195] Data from Sugimura et al. (1994) also implicated tumor cells
as the key components of the procoagulant activity of the tumor. It
was reported that four tumor cell lines were able to support
different stages of the extrinsic pathway of coagulation (Sugimura
et al., 1994). Another study reported that a human ovarian
carcinoma cell line, OC-2008, constitutively expressed surface
membrane Tissue Factor activity and exhibited cell
surface-dependent prothrombinase complex activity (Rao et al.,
1992). Connor et al. (1989) further suggested that it is the
pathologic cells that control coagulation. Their results indicated
that tumorigenic, undifferentiated murine erythroleukemic cells
exhibit a 7- to 8-fold increase in the potency of their
procoagulant activity (Connor et al., 1989).
[0196] Zacharski et al. (1993) also focused on tumor cells and
sought to define the mode of interaction of ovarian carcinoma cells
with the coagulation (procoagulant-initiated) and fibrinolysis
(urokinase-type plasminogen activator-initiated, u-PA) pathways.
They reported that tumor cells expressed Tissue Factor and
coagulation pathway intermediates that resulted in local thrombin
generation--as evidenced by the conversion of fibrinogen, present
in tumor connective tissue, to fibrin that was found to hug the
surfaces of tumor nodules and individual tumor cells. Detected
fibrin could not be accounted for on the basis of necrosis or a
local inflammatory cell infiltrate (Zacharski et al., 1993). These
authors concluded that there exists a dominant tumor
cell-associated procoagulant pathway that leads to thrombin
generation and hypercoagulability.
[0197] Other hypotheses have proposed that it is changes in the
tumor blood vessels that render these vessels better able to
support the formation of thrombi and/or less able to dissolve
fibrin. For example, tumor vessels have been reported to exhibit
upregulation of Tissue Factor, down-regulation of plasminogen
activators and/or upregulation of the inhibitor of plasminogen
activators, PAI-1 (Nawroth and Stern, 1986; Nawroth et al., 1988).
Such effects are believed to be magnified by tumor derived factors
(Murray et al., 1991; Ogawa et al., 1990), possibly VEGF.
[0198] For example, Ogawa et al. (1990) reported that hypoxia
caused endothelial cell surface coagulant properties to be shifted
to promote activation of coagulation. This was accompanied by
suppression of the anticoagulant cofactor, thrombomodulin, and
induction of an activator of factor X, distinct from the classical
extrinsic and intrinsic systems (Ogawa et al., 1990). Also, there
could be an increase in the local concentration of Factors VIIa,
IXa, Xa, or other molecules that interact with TF, within the tumor
vessels, thus encouraging thrombosis.
[0199] Additionally, platelets are a major component of any
procoagulant state. Recently, the procoagulant potential of
platelets has been linked to their ability to shed procoagulant
microparticles from the plasma membrane (Zwaal et al., 1989; 1992;
Dachary-Prigent et al., 1996). It has been proposed that an
increased proportion of circulating microparticles, vesicles or
membrane fragments from platelets contributes to `prethrombotic`
(prothrombotic) states in various pathological conditions (Zwaal et
al., 1989; 1992; Dachary-Prigent et al., 1996, pp. 159 and
references cited therein). McNeil et al. (1990) also reported that
.beta..sub.2-GPI exerts multiple inhibitory effects on coagulation
and platelet aggregation. Tumor platelet biology could thus explain
the effectiveness of the anti-VCAM-1 coaguligand.
[0200] Further tenable explanations include the simple possibility
that VCAM-1 is expressed at higher levels in tumor vessels than on
blood vessels in the heart and lungs, probably due to induction by
tumor-derived cytokines, and that binding to the healthy vessels
cannot tip the balance into sustained thrombosis. Also the
fibrinolytic mechanisms could be upregulated in the heart, as
exemplified by increased Tissue Factor pathway inhibitor (TFPI),
increased plasminogen activators, and/or decreased plasminogen
activator inhibitors. Should the fibrinolytic physiology of the
heart and lung vessels prove to be the major reason underlying the
tumor-specific effects of the anti-VCAM-1 coaguligand, this would
generally preclude the development of additional anti-tumor
therapies targeted to unique aspects of tumor biology.
[0201] Despite all the possible options, the inventors reasoned
that the failure of the anti-VCAM-1 coaguligand to cause thrombosis
in vessels of normal tissues was due to the absence of the
aminophospholipid, phosphatidylserine (PS), from the luminal
surface of such vessels. To complete the theory, therefore, not
only would phosphatidylserine have to be shown to be absent from
these normal vessels, but its presence on the luminal side of
tumor-associated vessels would have to be conclusively
demonstrated.
[0202] The inventors therefore used immunohistochemical staining to
evaluate the distribution of a monoclonal anti-phosphatidylserine
(anti-PS) antibody injected intravenously into tumor-bearing mice.
These studies revealed that the VCAM-1 expressing vessels in the
heart and lungs lacked PS, whereas the VCAM-1 expressing vessels in
the tumor expressed PS. The need for surface PS expression in
coaguligand action is further indicated by the inventors' finding
that annexin V, which binds to PS, blocks anti-VCAM-1.cndot.tTF
coaguligand action, both in vitro and in vivo.
[0203] The lack of thrombotic effect of the anti-VCAM-1 coaguligand
on normal heart and lung vessels can thus be explained, at least in
part: the absence of the aminophospholipid, phosphatidylserine,
means that the normal vessels lack a procoagulant surface upon
which coagulation complexes can assemble. In the absence of surface
PS, anti-VCAM-1.cndot.tTF binds to VCAM-1 expressing heart and lung
vessels, but cannot induce thrombosis. In contrast, VCAM-1
expressing vessels in the tumor show coincident expression of
surface PS. The coaguligand thus binds to tumor vessels and
activates coagulation factors locally to form an occlusive
thrombus.
[0204] In addition to delineating the tumor-specific thrombotic
effects of anti-VCAM-1 coaguligands, the specific expression of the
aminophospholipid, phosphatidylserine, on the luminal surface of
tumor blood vessels also allowed the inventors to explain the
prothrombotic phenotype observed, but not understood, in earlier
studies (Zacharski et al., 1993; Donati, 1995). Rather than being
predominantly due to tumor cells or elaborated factors; platelets,
procoagulant microparticles or membrane fragments; or due to
imbalances in thromboplastin, thrombomodulin, cancer procoagulant,
Tissue Factor, protein C pathway, plasminogen activators or
plasminogen activator inhibitors (e.g., PAI-1), the inventors'
studies indicate that it is PS expression that plays a significant
role in the prothrombotic state of tumor vasculature.
C. Aminophospholipids as Markers of Tumor Vasculature
[0205] Following their discovery that the representative
aminophospholipid, phosphatidylserine, was specifically expressed
on the luminal surface of tumor blood vessels, but not in normal
blood vessels, the inventors reasoned that aminophospholipids had
potential as targets for therapeutic intervention. The present
invention therefore provides compositions and methods for the
targeted delivery of therapeutic agents to aminophospholipid
membrane constituents, particularly phosphatidylserine (PS) and
phosphatidylethanolamine (PE). Although anti-tumor effects from
aminophospholipid-targeted delivery are demonstrated herein, using
art-accepted animal models, the ability of aminophospholipids to
act as safe and effective targetable markers of tumor vasculature
could not have been predicted from previous studies.
[0206] For example, although tumor vessels are generally
prothrombotic in nature, as opposed to other blood vessels, it is
an inherent property of the tumor to maintain a network of blood
vessels in order to deliver oxygen and nutrients to the tumor
cells. Evidently, tumor-associated blood vessels cannot be so
predisposed towards thrombosis that they spontaneously and readily
support coagulation, as such coagulation would necessarily cause
the tumor to self-destruct. It is thus unexpected that any
thrombosis-associated tumor vessel marker, such as the presently
identified phosphatidylserine, could be discovered that is
expressed in quantities sufficient to allow effective therapeutic
intervention by targeting, and yet is expressed at levels low
enough to ordinarily maintain blood flow through the tumor.
[0207] The present identification of aminophospholipids as safe and
effective tumor vasculature targets is even more surprising given
(1) the previous speculations regarding the role of other cell
types and/or various factors, activators and inhibitors underlying
the complex, prothrombotic state of the tumor (as discussed above);
and (2) the confusing and contradictory state of the art concerning
aminophospholipid biology, in terms of both expression and function
in various cell types.
[0208] Phosphatidylserine and phosphatidylethanolamine are normally
segregated to the inner surface of the plasma membrane bilayer in
different cells (Gaffet et al., 1995; Julien et al., 1995). In
contrast, the outer leaflet of the bilayer membrane is rich in
phosphatidylcholine analogs (Zwaal et al., 1989; Gaffet et al.,
1995). This lipid segregation creates an asymmetric transbilayer.
Although the existence of membrane asymmetry has been discussed for
some time, the reason for its existence and the mechanisms for its
generation and control are poorly understood (Williamson and
Schlegel, 1994), particularly in cells other than platelets.
[0209] There are even numerous conflicting reports regarding the
presence or absence of PS and PE in different cells and tissues,
let alone concerning the likely role that these aminophospholipids
may play. For example, the many PS studies conducted with
platelets, key components in blood coagulation (Dachary-Prigent et
al., 1996), have yielded highly variable results. Bevers et al.
(1982) measured the platelet prothrombin-converting activity of
non-activated platelets after treatment with various phospholipases
or proteolytic enzymes. They concluded that negatively charged
phosphatidylserine, and possibly phosphatidylinositol, were
involved in the prothrombin-converting activity of non-activated
platelets (Bevers et al., 1982).
[0210] Bevers et al. (1983) then reported an increased exposure of
phosphatidylserine, and a decreased exposure of sphingomyelinase,
in activated platelets. However, these alterations were much less
apparent in platelets activated either by thrombin or by collagen
alone, in contrast to collagen plus thrombin, diamide, or a calcium
ionophore (Bevers et al., 1983). The surface expression of PS in
response to diamide was contradicted by studies in erythrocytes,
which showed no diamide-stimulated PS exposure (de Jong et al.,
1997). While echoing their earlier results, Bevers and colleagues
then later reported that changes in the plasma
membrane-cytoskeleton interaction, particularly increased
degradation of cytoskeletal actin-binding protein, was important to
platelet surface changes (Bevers et al., 1985; pages 368-369).
[0211] Maneta-Peyret et al. (1989) also reported the detection of
PS on human platelets. These authors noted that the platelet
procoagulant surface could be formed by negatively charged
phospholipids, such as phosphatidylserine and
phosphatidylethanolamine (generally neutral or zwitterionic), or
both. The role of phosphatidylserine in the process of coagulation
has been questioned in favor of phosphatidylethanolamine
(Maneta-Peyret et al., 1989; Schick et al., 1976; 1978). For
example, studies have reported that 18% of phosphatidylethanolamine
becomes surface-accessible after 2 hours, in contrast to zero
phosphatidylserine (Schick et al., 1976).
[0212] Ongoing studies with platelets were also reported as showing
a further 16% increase in phosphatidylethanolamine exposure after
thrombin treatment, with no increase in the phosphatidylserine
levels (Schick et al., 1976). Therefore, PS was said not to be a
component of the functional surface of the platelet plasma membrane
(Schick et al., 1976; 1978). Nonetheless, current evidence does
seem to indicate that both PS and PE are involved in the
phospholipid asymmetry observed in the outer membrane of platelets
and erythrocytes, and that PS is involved in the procoagulant
activity of platelets (Gaffet et al., 1995; de Jong et al., 1997;
U.S. Pat. No. 5,627,036).
[0213] The mechanisms for achieving and maintaining differential
aminophospholipid distribution, let alone the functional
significance of doing so, have long been the subject of
controversial speculations. In reviewing the regulation of
transbilayer phospholipid movement, Williamson and Schlegel (1994)
indicated that elevating intracellular Ca.sup.2+ allows the major
classes of phospholipids to move freely across the bilayer,
scrambling lipids and dissipating asymmetry. de Jong et al. (1997)
also reported that an increase of intracellular calcium leads to a
rapid scrambling of the lipid bilayer and the exposure of PS, which
could be partially inhibited by cellular oxidation. The interaction
of aminophospholipids with cytoskeletal proteins has also been
proposed as a mechanism for regulating membrane phospholipid
asymmetry (Zwaal et al., 1989).
[0214] Gaffet et al. (1995) stated that the transverse
redistribution of phospholipids during human platelet activation is
achieved by a vectorial outfox of aminophospholipids, not
counterbalanced by a rapid reciprocal influx of choline head
phospholipids, i.e. not scrambling. They suggested that the
specific vectorial outflux of aminophospholipids could be catalyzed
by a "reverse aminophospholipid translocase" activity (Gaffet et
al., 1995). An alternative hypothesis would be that the activity of
an inward translocase was inhibited. Zwaal et al. (1989) proposed
the involvement of a phospholipid-translocase that catalyzed both
the outward and inward movement of aminophospholipids.
[0215] The presence of an energy- and protein-dependent
aminophospholipid translocase activity that transports
phosphatidylethanolamine from the outer to the inner leaflet of the
lipid bilayer was reported by Julien et al. (1993). They then
showed that the aminophospholipid translocase activity could also
transfer phosphatidylserine, and that the activity could be
maintained, suppressed and restored depending on the conditions of
cell incubation (Julien et al., 1993), and inhibited by the tumor
promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Julien et
al., 1997).
[0216] A 35 kD phospholipid scramblase that promotes the
Ca.sup.2+-dependent bidirectional movement of phosphatidylserine
and other phospholipids was recently cloned from a cDNA library
(Zhou et al., 1997). This "PL scramblase" protein is a
proline-rich, type II plasma membrane protein with a single
transmembrane segment near the C terminus. Subsequent studies
confirmed that this protein was responsible for the rapid movement
of phospholipids from the inner to the outer plasma membrane
leaflets in cells exposed to elevated cytosolic calcium
concentrations (Zhao et al., 1998).
[0217] The aminophospholipid translocase activity reported by
Julien et al. (1993; 1997), which transports PS and PE from the
outer to the inner leaflet, is different to the bidirectional
Ca.sup.2+-dependent scramblase (Zhou et al., 1997; Zhao et al.,
1998). The scramblase is activated by Ca.sup.2+, and mostly
functions to move PS from the inner to the outer leaflet in
response to increased Ca.sup.2+ levels. It is now generally
believed that the aminophospholipid translocase maintains membrane
asymmetry during normal conditions, but that the scramblase is
activated by Ca.sup.2+ influx, over-riding the translocase and
randomizing aminophospholipid distribution.
[0218] The normal segregation of PS and PE to the inner surface of
the plasma membrane is thus now generally accepted, and certain
membrane components involved in the asymmetric processes have even
been identified. However, doubts remain about the conditions,
mechanisms and cell types that are capable of re-locating
aminophospholipids to the outer leaflet of the membrane, and the
biological implications of such events.
[0219] Contradictory reports concerning aminophospholipid
expression are not limited to studies of platelets.
Phosphatidylserine and phosphatidylethanolamine are generally about
7% and about 10%, respectively, of the phospholipid composition of
cultured human endothelial cells from human artery, saphenous and
umbilical vein (7.1% and 10.2%, respectively; Murphy et al., 1992).
However, an important example of the contradictions in the
literature concerns the ability of anti-PS antibodies to bind to
endothelial cells (Lin et al., 1995).
[0220] The anti-PS antibodies present in recurrent pregnancy loss
(Rote et al., 1995; Rote, 1996; Vogt et al., 1996; Vogt et al.,
1997) were believed to modulate endothelial cell function, without
evidence of binding to endothelial cells. In an attempt to explain
this discrepancy, Lin et al. (1995) tried but failed to demonstrate
anti-PS antibody binding to resting endothelial cells. They
concluded that PS antigenic determinants are not expressed on the
surface of resting endothelial cells, although a PS-dependent
antigenic determinant was associated with cytoskeletal-like
components in acetone-fixed cells (Lin et al., 1995).
[0221] Van Heerde et al. (1994) reported that vascular endothelial
cells in vitro can catalyze the formation of thrombin by the
expression of binding sites at which procoagulant complexes can
assemble. In contrast to other studies with activated platelets
(Bevers et al., 1982; 1983; 1985; Maneta-Peyret et al., 1989;
Schick et al., 1976; 1978), stimulated HUVEC endothelial cells did
not exhibit an increase in PS binding sites as compared to
quiescent cells (Van Heerde et al., 1994). Phosphatidylserine was
reported to be necessary for Factor Xa formation via the extrinsic
as well as the intrinsic route (Van Heerde et al., 1994).
Nonetheless, Brinkman et al. (1994) published contradictory
results, indicating that other membrane constituents besides
negatively charged phospholipids are involved in endothelial cell
mediated, intrinsic activation of factor X.
[0222] Ravanat et al. (1992) also studied the catalytic potential
of phospholipids in pro- and anti-coagulant reactions in purified
systems and at the surface of endothelial cells in culture after
stimulation. Their seemingly contradictory results were proposed to
confirm a role for phospholipid-dependent mechanisms in both
procoagulant Tissue-Factor activity and anticoagulant activities
(activation of protein C by the thrombin-thrombomodulin complex and
by Factor Xa) (Ravanat et al., 1992). The Ravanat et al. (1992)
results were also said to provide evidence of phospholipid exposure
during activation of human endothelial cells, which was not
observed by Van Heerde et al. (1994) or Brinkman et al. (1994).
However, they did note that anionic phospholipids are of restricted
accessibility in the vicinity of cellular Tissue Factor. The
situation is further complicated as, even after Tissue Factor
induction, other events are likely necessary for coagulation, as
the Tissue Factor remains inaccessible, being under the cell.
[0223] Ravanat et al. (1992) went on to suggest that the different
extent of inhibition of Tissue Factor and thrombomodulin activities
on stimulated endothelial cells means that the cofactor
environments differ for the optimal expression of these opposite
cellular activities. However, the acknowledged difficulties in
trying to reproduce exact cellular phospholipid environments
(Ravanat et al., 1992), raise the possibility of artifactual data
from these in vitro studies. Indeed, irrespective of the Ravanat et
al. (1992) data, it is generally acknowledged that meaningful
information regarding tumor biology, and particularly therapeutic
intervention, can only be gleaned from in vivo studies in
tumor-bearing animals, such as those conducted by the present
inventors.
[0224] In addition to the disagreements regarding aminophospholipid
expression, as discussed above, there are also conflicting reports
concerning the function of aminophospholipids in various cell
types. Although it is now generally accepted that PS expression on
activated platelets is connected with the procoagulant surface, in
discussing the physiological significance of membrane phospholipid
asymmetry in platelets and red blood cells, Zwaal et al. (1989)
highlighted other important functions. Moreover, Toti et al. (1996)
stated that the physiological implications of a loss of asymmetric
phospholipid distribution remain poorly understood in cell types
other than blood cells.
[0225] Zwaal et al. (1989) stated that the membrane phospholipid
asymmetry of platelets and red cells is undone when the cells are
activated in various ways, presumably mediated by the increased
transbilayer movement of phospholipids. These changes, coupled with
the release of shed microparticles, were explained to play a role
in local blood clotting reactions. A similar phenomenon was
described to occur in sickled red cells: phospholipid vesicles
breaking off from reversibly sickled cells contribute to
intravascular clotting in the crisis phase of sickle cell disease
(Zwaal et al., 1989).
[0226] Both Zwaal et al. (1989) and Williamson and Schlegel (1994)
have indicated that the physiological significance of surface
phospholipid changes is not restricted to hemostasis. In fact, the
surface exposure of PS by blood cells was said to significantly
alter their recognition by the reticuloendothelial system, and was
to likely represent at least part of the homeostatic mechanism for
the clearance of blood cells from the circulation (Zwaal et al.,
1989). Thus, PS acts as a signal for the elimination of activated
platelets after bleeding has stopped. Recognition of PS exposed on
sickle cells and malarially infected cells by phagocytes and
macrophages explains their counter-pathophysiological effects
(Zwaal et al., 1989). Furthermore, PS-dependent phagocytosis marks
virally infected cells for phagocytic uptake (WO 97/17084). The
surface expression of aminophospholipids could also confer "fusion
competence" to a cell (Williamson and Schlegel, 1994).
[0227] Williamson and Schlegel (1994) also speculated that there is
a more general raison d' tre for lipid asymmetry. For example,
although the different head groups have received most attention, it
could well be that fatty acid asymmetry is the important factor
(Williamson and Schlegel, 1994). A further hypothesis is that the
asymmetric distribution of transbilayer phospholipids has no
function in itself, but that it is the dynamic process of lipid
movement that is important to biological systems (Williamson and
Schlegel, 1994).
[0228] Many groups have reported that tumor cells are responsible
for the prothrombinase activity of the tumor (Connor et al., 1989;
Rao et al., 1992; Zacharski et al., 1993; Sugimura et al., 1994;
Donati, 1995). This could have been reasoned to be due to PS (WO
91/07187). However, the results of Sugimura et al. (1994) argue
against this: they reported that although both the prothrombinase
activity and total procoagulant activity of the tumorigenic cells,
HepG2 and MKN-28, fell on reaching confluency, the PS levels
remained constant.
[0229] Rather than supporting a role for tumor cell PS in
prothrombinase activity, Connor et al. (1989) suggested that the
increased expression of PS in tumorigenic cells is relevant to
their ability to be recognized and bound by macrophages. Utsugi et
al. (1991) similarly proposed that the presence of PS in the outer
membrane of human tumor cells explains their recognition by
monocytes.
[0230] Jamasbi et al. (1994) suggested a totally different role for
lipid components in tumorigenic cells, proposing that the lipids
interfere with tumor antigen accessibility. Thus, tumor cell lipids
would act to modify the tumor cell surface antigen(s), thus
protecting the tumor cells from host immune destruction (Jamasbi et
al., 1994). This hypothesis is not unlike that proposed by Qu et
al. (1996), in terms of endothelial cells. These authors showed
that T cells adhered to thrombin-treated human umbilical
endothelial cells by virtue of binding to PS (Qu et al., 1996).
[0231] It has thus been proposed that PS-mediated T cell adhesion
to endothelial cells in vivo is important to both immune
surveillance, and also to the disease processes of atherosclerosis
(Qu et al., 1996; Moldovan et al., 1994). Bombeli et al. (1997) and
Flynn et al. (1997) also suggested that cells within
atherosclerotic plaques may contribute to disease progression by
exposing PS, although this was based solely on in vitro studies. Qu
et al (1996) and Moldovan et al. (1994) even hinted at an approach
opposite to that of the present invention, i.e., the manipulation
of phosphatidylserine interactions as an anticoagulant approach.
U.S. Pat. No. 5,658,877 and U.S. Pat. No. 5,296,467 have proposed
annexin (or "annexine") for use as anti-endotoxins and
anti-coagulants. U.S. Pat. No. 5,632,986 (incorporated herein by
reference) suggests the use of the phosphatidylserine-binding
ligand, annexin V, as a conjugate with a component, such as
urokinase, that lyses thrombi.
[0232] Toti et al. (1996) suggested that Scott syndrome, an
inherited bleeding disorder, may reflect the deletion or mutation
of a putative outward phosphatidylserine translocase or
"scramblase". Although an interesting notion, Stout et al. (1997)
later isolated a membrane protein from Scott erythrocytes that
exhibited normal PL scramblase activity when reconstituted in
vesicles with exogenous PLs. It was suggested that the defect in
Scott syndrome is related to an altered interaction of Ca.sup.2+
with PL scramblase on the endofacial surface of the cell membrane,
due either to an intrinsic constraint upon the protein, preventing
interaction with Ca.sup.2+ in situ, or due to an unidentified
inhibitor or cofactor in the Scott cell that is dissociated by
detergent (Stout et al., 1997).
[0233] More variable results have been reported in connection with
the possible role of PS in apoptosis. Williamson and Schlegel
(1994) discussed the theme of PS as a marker of programmed cell
death (PCD or apoptosis). It is generally accepted that programmed
cell death, at least in the hematopoietic system, requires the
phagocytic sequestration of the apoptopic cells before the loss of
membrane integrity or "rupture". The loss of membrane asymmetry in
apoptopic cells, and particularly the appearance of PS in the
external leaflet, was proposed to be the trigger for their
recognition by phagocytic macrophages (Williamson and Schlegel,
1994).
[0234] Martin et al. (1995) further reported PS externalization to
be an early and widespread event during apoptosis of a variety of
murine and human cell types, regardless of the initiating stimulus.
They also indicated that, under conditions in which the
morphological features of apoptosis were prevented (macromolecular
synthesis inhibition, overexpression of Bcl-2 or Abl), the
appearance of PS on the external leaflet of the plasma membrane was
similarly prevented (Martin et al., 1995).
[0235] However, other analyses argue against the Williamson and
Schlegel (1994) and Martin et al. (1995) proposals to some extent
(Vermes et al., 1995). Although these authors indicate that the
translocation of PS to the outer membrane surface is a marker of
apoptosis, they reason that this is not unique to apoptosis, but
also occurs during cell necrosis. The difference between these two
forms of cell death is that during the initial stages of apoptosis
the cell membrane remains intact, while at the very moment that
necrosis occurs the cell membrane loses its integrity and becomes
leaky. Therefore, according to this reasoning, PS expression at the
cell surface does not indicate apoptosis unless a dye exclusion
assay has been conducted to establish cell membrane integrity
(Vermes et al., 1995).
[0236] Nonetheless, the body of literature prior to the present
invention does seem to indicate that the appearance of PS on the
outer surface of a cell identifies an apoptotic cell and signals
that cell's ingestion (Hampton et al., 1996; WO 95/27903). Hampton
et al. (1996) concluded that while an elevation of intracellular
Ca.sup.2+ was an ineffective trigger of apoptosis in the cells
investigated, extracellular Ca.sup.2+ was required for efficient PS
exposure during apoptosis. In contrast, the proposal of Martin et
al. (1995) that activation of an inside-outside PS translocase is
an early widespread event during apoptosis would seem to require at
least some intracellular Ca.sup.2+ (Zhou et al., 1997; Zhao et al.,
1998).
[0237] Blankenberg et al. (1998) very recently reported that
annexin V, an endogenous human protein with a high affinity for PS,
can be used to concentrate at sites of apoptotic cell death in
vivo. Radiolabeled annexin V localized to sites of apoptosis in
three models, including acute cardiac allograft rejection
(Blankenberg et al., 1998). Staining of cardiac allografts for
exogenously administered annexin V revealed myocytes at the
periphery of mononuclear infiltrates, of which only a few
demonstrated positive apoptotic nuclei.
[0238] Finally, the transbilayer movement of phospholipids in the
plasma membrane has even been analyzed in ram sperm cells, where
the existence of a transverse segregation of phospholipids has been
implicated in the fertilization process (Muller et al., 1994).
Phospholipid asymmetry has thus been receiving increasing
attention, although a clear understanding of this phenomenon, or
its relationship to health or disease, has not been realized.
[0239] Irrespective of the confusing state of the art regarding
aminophospholipid biology, the present inventors discovered, in
controlled in vivo studies, that aminophospholipids, such as PS and
PE, were specific markers of tumor blood vessels. This is
surprising in light of the earlier studies of aminophospholipid
function, particularly those indicating that the cell surface
expression of PS is accompanied by binding of circulating cells,
such as T cells (Qu et al., 1996), macrophages (Connor et al.,
1989), monocytes (Utsugi et al., 1991) or phagocytes (Zwaal et al.,
1989; Williamson and Schlegel, 1994) and is a marker of apoptopic
cells (Hampton et al., 1996; Martin et al., 1995; Zhou et al.,
1997; Zhao et al., 1998).
[0240] Thus, prior to this invention, the possibility of using
aminophospholipids as targetable markers of any disease, let alone
of tumor vasculature, would be unlikely to be contemplated, due to
the perceived masking of these molecules by the binding of one or
more cell types. In fact, speculative suggestions have concerned
the disruption of PS-cellular interactions, such as in preventing
leukocyte binding, an initial event in atherosclerosis (Qu et al.,
1996).
[0241] Other surprising aspects of this discovery are evident in a
comparison to earlier work concerning the shedding of procoagulant
microparticles from plasma membranes and the demarcation of cells
for phagocytosis (WO 97/17084). Zwaal et al. (1989; 1992) and
Dachary-Prigent et al. (1996) explained that PS translocation to
the plasma membrane is followed by release of microparticles,
microvesicles or microspheres from the cells. Zwaal et al. (1989)
and Williamson and Schlegel (1994) indicated that PS surface
expression prompts clearance by the reticuloendothelial system. In
light of these fates of PS-expressing cells, and the various
documented bilayer translocase activities (Julien et al., 1995;
Zhou et al., 1997; Zhao et al, 1998), it is surprising that cell
surface aminophospholipids such as PS and PE can form static and
stable enough markers to allow antibody localization and
binding.
[0242] Prior to the present invention, there was mounting evidence
that surface PS appears as part of the apoptopic process, marking
cells for rapid destruction (Hampton et al., 1996; Martin et al.,
1995). Therefore, although reasonable for use as a diagnostic
marker for certain disease states, such as graft rejection
(Blankenberg et al., 1998), the apparently limited life time of
surface PS would also advise against its use as a viable marker for
targeting in therapeutic intervention.
[0243] Nonetheless, the present study did indeed discover
aminophospholipids to be markers of tumor vascular endothelial
cells suitable for targeting. After postulating that PS expression
was necessary for VCAM coaguligand action, the presence of PS on
tumor blood vessels, but normal vessels, was demonstrated in vivo.
The in vivo observations allowed the inventors to explain the
safety and effectiveness of the anti-VCAM coaguligands. This is due
to the requirement for coincident expression of a targeted marker
(e.g., VCAM) and PS on tumor endothelium. Even if the target
molecule is present on endothelium in normal or pathological
conditions, thrombosis will not result if surface PS expression is
lacking.
[0244] The value of the present invention is not limited to
explaining coaguligand action, nor to the surprising development of
naked antibody therapies (provisional application Ser. Nos.
60/092,672 and 60/110,608, each incorporated herein by reference).
In fact, the present discoveries have allowed the inventors to
show, for the first time, that PS translocation in endothelial
cells can occur without significant cell damage or cell death
(Example XIV). In the inventors' new model of tumor biology, the
translocation of PS to the surface of tumor blood vessel
endothelial cells occurs, at least in a significant part,
independently of apoptopic or other cell-death mechanisms. Thus, PS
surface expression in the tumor environment is not a consequence of
cell death, nor does it trigger immediate cell destruction. This is
of fundamental importance and represents a breakthrough in the
scientific understanding of PS biology, membrane translocation,
cell signaling and apoptosis pathways.
[0245] The separation of endothelial cell PS translocation from
apoptosis (Example XIV) is also integral to methods of therapeutic
intervention based upon PS surface expression. Should PS
translocation to the outer membrane in tumor vascular endothelial
cells occur only in dying cells, or should it inevitably trigger
cell death, then the PS marker would not likely be sufficiently
available to serve as a target for the delivery of therapeutic
agents. That is not to say that PS expression on certain tumor
vascular endothelial cells is not transient, and that turnover and
cell death do not occur in this endothelial cell population, but
the finding that significant stable PS expression can be achieved
without cell death is a landmark discovery important to various
fields of biology and to the new targeted therapeutics described
below.
D. Aminophospholipid-Targeted Therapeutics
[0246] The in vivo aminophospholipid tumor vasculature expression
studies further support the use of coaguligands directed against
previously identified tumor vasculature markers, e.g., VCAM-1 and
E-selectin, as selective thrombotic agents for the treatment of
solid tumors. However, these observations also led the inventors to
develop additional tumor treatment methods. For example, naked or
unconjugated antibodies against aminophospholipid components were
surprisingly found to be capable of specifically inducing tumor
blood vessel destruction and tumor necrosis in vivo in the absence
of additional effector moieties. Such uses are disclosed and
claimed in first and second provisional application Ser. Nos.
60/092,672 (filed Jul. 13, 1998) and 60/110,608 (filed Dec. 2,
1998) and in co-filed U.S. and PCT patent applications (Attorney
Docket Nos. 4001.002200, 4001.002282 and 4001.002210), each
specifically incorporated herein by reference.
[0247] The studies of first and second provisional application Ser.
Nos. 60/092,672 (filed Jul. 13, 1998) and 60/110,608 (filed Dec. 2,
1998) are in contrast to those recently reported by Nakamura et al.
(1998). These authors analyzed antibody fractions from patients
with lupus anticoagulant (LAC), a disorder associated with arterial
and venous thrombosis, thrombocytopenia, and recurrent fetal loss.
Plasma with LAC activity was initially reported to induce apoptosis
in endothelial cells (Nakamura et al., 1994). The apoptotic
activities of LAC antisera were then reported to be localized in an
annexin V-binding antibody fraction in 10/10 patients studied
(Nakamura et al., 1998). As annexin binds to PS, the apparent
ability of anti-annexin antibodies to induce apoptosis would be the
opposite of the ability of an anti-PS antibody to induce
apoptosis.
[0248] The ability of LAC antibody fractions to induce apoptosis
was further reported to be inhibited by preincubation with annexin
V (Nakamura et al., 1998). In contrast, removal of
anti-phospholipid antibodies from the patients' IgG fractions with
phospholipid liposomes did not abolish the apoptosis-inducing
activities or annexin V binding (Nakamura et al., 1998). These
results reasonably implied that patients with LAC often have
antibodies that do not bind phospholipids and yet are responsible
for the induction of apoptosis in endothelial cells (Nakamura et
al., 1998).
[0249] Without needing to equate the Nakamura et al. (1998) LAC
data with the inventors' observations from in vivo studies of
tumors and tumor vasculature, due to the evidently disparate nature
of these clinical conditions, the inventors nonetheless have
certain unifying theories. Nakamura et al. (1998) attempted to
remove anti-phospholipid antibodies from patients' antisera using
phospholipid Liposomes, and observed that this did not abolish the
apoptosis-inducing activity. These results led Nakamura et al.
(1998) to conclude that the anti-phospholipids antibodies cannot be
responsible for apoptopic activity. However, the present inventors
now have the insight to suggest that the incubation with
phospholipid liposomes may not have removed the anti-phospholipids
antibodies from the antisera, as phospholipids are antigenically
neutral in bilayer and liposomal form, and largely only bind
antibodies in hexagonal form (Rauch et al., 1986; Rauch and Janoff,
1990; Berard et al., 1993; each incorporated herein by reference)
or in association with membrane proteins. Thus, anti-phospholipids
antibodies may remain in the LAC antisera and may cause, or
contribute to, the observed apoptopic activity.
[0250] The invention disclosed herein is directed to the use of
aminophospholipids as targets for anti-tumor vasculature
immunotoxin and/or coaguligand therapy. Although the identification
of any additional target to allow specific tumor vessel
localization in vascular targeting therapies is valuable, the
present discovery of aminophospholipids as suitable targets is
particularly important as it brings another entire group of targets
into the picture: lipids rather than the proteins previously
preferred. The aminophospholipid discovery is also functionally
significant as it allows therapeutic agents to be delivered into
even more intimate contact with the target cell membrane, rather
than binding to a protein complex more distant from the
membrane.
[0251] One of the most surprising aspects of the present discovery
is that PS expression on intact tumor-associated endothelial cells
is sufficiently stable to allow targeting. The present in vivo and
in vitro data definitively show that PS is expressed on viable
tumor-associated endothelial cells with normal morphology and
intact cytoskeletons. As PS expression is not limited to cells
undergoing cell death or about to enter an apoptopic pathway,
targeting with diagnostic and therapeutic agents is both
practicable and surprising (given that PS expression was thought to
be associated only with cell destruction).
[0252] A precise molecular understanding of exactly how and why
aminophospholipid-targeted therapeutic agents are suitable for use
in tumor treatment is not necessary in order to practice the
present invention. Given that the administration of
aminophospholipid-directed therapeutic agents is herein shown to
advantageously result in specific anti-tumor effects in vivo, the
invention can be utilized irrespective of the molecular mechanisms
that underlie the aminophospholipid expression in tumor
vasculature.
[0253] However, it is interesting to note that a review of the
scientific literature to date reveals features that argue against
the present surprisingly effective uses, and even proposes directly
opposite uses for distinct aminophospholipid binding
agent-conjugates. For example, annexin, a phosphatidylserine
binding protein, has itself been proposed for use as an
anticoagulant (WO 91/07187; U.S. Pat. No. 5,296,467; each
incorporated herein by reference). This use of annexin was said to
be based upon the inhibition of the procoagulant activity of tumor
cells (WO 91/07187).
[0254] Even more telling is the disclosure of U.S. Pat. No.
5,632,986 which, in complete contrast to the present invention,
proposes the use of annexin as a conjugate with compounds that lyse
thrombi, or precursors of such thrombolytic compounds. The
referenced combination of an aminophospholipid binding protein,
annexin, with a lytic agent is, evidently, the opposite of the
present invention, which concerns the combination of annexin and
other aminophospholipid binding proteins with agents that induce
thrombosis, either directly or indirectly.
[0255] In the preparation of both immunotoxins and coaguligands
based upon aminophospholipid binding agents and antibodies,
recombinant expression may be employed to create a fusion protein,
as is known to those of skill in the art and further disclosed
herein. Equally, immunotoxins and coaguligands may be generated
using avidin:biotin bridges or any of the chemical conjugation and
cross-linker technologies, mostly developed in reference to
antibody conjugates. Therefore, any of the following
aminophospholipid binding proteins and ligands may be conjugated to
a toxin or coagulant in the same manner as used for antibody
conjugates, described herein.
[0256] D1. Aminophospholipid Binding Proteins
[0257] In addition to antibodies (see below), aminophospholipid
binding ligands or binding proteins may be used in the therapeutic
agent-targeting agent constructs of the present invention.
Naturally occurring proteins are known that bind to both
phosphatidylethanolamine and phosphatidylserine with
specificity.
[0258] A series of studies by Sugi and McIntyre revealed that
kininogens can bind to membrane-exposed PE, at least in platelets
(Sugi and McIntyre 1995; 1996a; 1996b; each incorporated herein by
reference). Kininogens are naturally occurring proteins that
normally have anti-thrombotic effects. The present inventors
propose that low or high molecular weight kininogens may therefore
be attached to therapeutic agents and used in the delivery of
therapeutics to phosphatidylethanolamine, newly discovered to be a
marker of tumor vasculature.
[0259] Various mammalian and human kininogen genes have now been
cloned, and such genes and proteins can be used in the various
recombinant and/or chemical embodiments of the present invention.
For example, the complete nucleotide and amino acid sequences of
the genes and proteins described in Nakanishi et al., 1983, are
incorporated herein by reference for such purposes.
[0260] Nawa et al. (1983; incorporated herein by reference)
reported cDNA and protein sequences for bovine low molecular weight
kininogens. FIG. 2 of Nawa et al. (1983) is specifically
incorporated herein by reference for purposes of providing these
complete nucleotide and amino acid sequences. Kitamura et al.
(1983; incorporated herein by reference) then reported that a
single gene encodes the bovine high molecular weight and low
molecular weight kininogens. FIG. 2 of Kitamura et al. (1983) is
again incorporated herein by reference to provide the referenced
gene and protein sequences. Kitamura et al. (1987) is also
specifically incorporated herein by reference for purposes of
providing further information concerning the bovine, rat and human
kininogens, including low molecular weight, high molecular weight
and T-kininogens.
[0261] Preferred high and low molecular weight kininogens for use
in these aspects of the invention will be the human genes and
proteins, as described by Takagaki et al. (1985), Kitamura et al.
(1985) and Kellemiann et al. (1986), each incorporated herein by
reference. Each of FIG. 2 and FIG. 3 of Takagaki et al. (1985) are
specifically incorporated herein by reference to provide the
complete nucleotide and amino acid sequences of human low and high
molecular weight prekininogens, respectively. FIGS. 1 and 8 of the
protein analysis paper of Kellermann et al. (1986) are similarly
incorporated herein.
[0262] Kitamura et al. (1985) is also specifically incorporated
herein by reference for purposes of providing further information
regarding the structural organization of the human kininogen gene,
as may be used, e.g., to design particular expression constructs
for use herewith. Kitamura et al. (1988) is further incorporated by
reference for purposes of providing detailed information regarding
the cloning of cDNAs and genomic kininogens, such that any desired
kininogen may be cloned.
[0263] In addition to the T-kininogens described by Kitamura et al.
(1987; incorporated herein by reference), Anderson et al. (1989) is
also specifically incorporated herein by reference for purposes of
providing the gene and protein sequences of T-kininogen. FIG. 3 of
Anderson et al. (1989) is specifically incorporated.
[0264] Other phosphatidylethanolamine binding proteins are known
that can be used in such embodiments. A number of studies,
particularly by Jones and Hall, and Bernier and Jolles, have
concerned the purification, characterization and cloning of
phosphatidylethanolamine binding proteins. For example, Bernier and
Jolles (1984; incorporated herein by reference) first reported the
purification and characterization of a basic .about.23 kDa
cytosolic protein from bovine brain that was later characterized as
a phosphatidylethanolamine-binding protein (Bernier et al., 1986;
incorporated herein by reference). Schoentgen et al. (1987;
incorporated herein by reference) reported the complete amino acid
sequence of this bovine protein, then shown to be 21 kDa. FIG. 2 of
Schoentgen et al, (1987) is specifically incorporated herein by
reference for purposes of providing the complete amino acid
sequence of this bovine phosphatidylethanolamine binding
protein.
[0265] Jones and Hall (1991; incorporated herein by reference)
later purified and partially sequenced a .about.23 kDa protein from
rat sperm plasma membranes that showed sequence similarity and
phospholipid binding properties similar to the bovine brain
cytosolic protein of Bernier and Jolles (Bernier and Jolles, 1984;
Bernier et al., 1986; Schoentgen et al., 1987). The rat 23 kDa
protein of Jones and Hall (1991; incorporated herein by reference)
also showed selective affinity for phosphatidylethanolamine
(Kd=1.6.times.10.sup.-5 M).
[0266] Perry et al. (1994; incorporated herein by reference) then
cloned and sequenced rat and monkey versions of the
phosphatidylethanolamine binding protein of Jones and Hall (1991).
FIGS., 4, 5 and 6 of Perry et al. (1994) are specifically
incorporated herein by reference for purposes of providing the
complete DNA and amino acid sequences of the rat and monkey
phosphatidylethanolamine binding proteins, and comparison to the
bovine protein sequence. Any of the foregoing mammalian
phosphatidylethanolamine binding proteins, or their human
counterparts, may be attached to therapeutic agents and used in the
present invention. These mammalian sequences have EMBL Nucleotide
Sequence Database Accession Numbers X71873 (rat) and X73137
(monkey), and are each incorporated herein by reference.
[0267] To counterpart human phosphatidylethanolamine binding
protein has also been cloned (Hori et al., 1994; incorporated
herein by reference). Both FIG. 1 of Hori et al. (1994) and
GenBank, EMBL and DDBJ Accession Number D16111 are incorporated
herein by reference for purposes of providing the complete DNA and
amino acid sequences of the human phosphatidylethanolamine binding
proteins. The mammalian and human sequences, as incorporated
herein, may be employed in well-known expression techniques, either
to express the proteins themselves or therapeutic agent-fusions
thereof. Phosphatidylethanolamine binding proteins and genes from
other sources, such as yeast, Drosophila, simian, T. canis and O.
volvulus may also be employed in these embodiments (Gems et al.,
1995; incorporated herein by reference).
[0268] Variant, mutant or second generation
phosphatidylethanolamine binding protein nucleic acids may also be
readily prepared by standard molecular biological techniques, and
may optionally be characterized as hybridizing to any of the
phosphatidylethanolamine binding protein nucleotide sequences set
forth in any one or more of Nakanishi et al. (1983); Nawa et al.
(1983); Kitamura et al. (1983; 1985; 1987; 1988); Takagaki et al.
(1985); Kellerman et al. (1986); Anderson et al. (1989); Bernier
and Jolles (1984); Bernier et al. (1986); Schoentgen et al. (1987);
Jones and Hall (1991); Perry et al. (1994); and Hori et al. (1994);
each incorporated herein by reference. Exemplary suitable
hybridization conditions include hybridization in about 7% sodium
dodecyl sulfate (SDS), about 0.5 M NaPO.sub.4, about 1 mM EDTA at
about 50.degree. C.; and washing with about 1% SDS at about
42.degree. C.
[0269] In addition to the foregoing phosphatidylethanolamine
binding proteins or "ligands", naturally occurring proteins exist
that specifically bind phosphatidylserine. Preferred amongst these
are annexins (sometimes spelt "annexins"), a group of
calcium-dependent phospholipid binding proteins. At least nine
members of the annexin family have been identified in mammalian
tissues (Annexin I through Annexin IX). Most preferred amongst
these is annexin V (also known as PAP-I).
[0270] U.S. Pat. No. 5,658,877, incorporated herein by reference,
describes Annexin I, effective amounts of Annexin I and
pharmaceutical compositions thereof. Also described are methods of
treating an animal to prevent or alleviate the adverse effects of
endotoxin in the lung that comprise administering into the airway
of an animal a safe amount of 33 kDa Annexin 1 fragment.
[0271] Annexin V contains one free sulfhydryl group and does not
have any attached carbohydrate chains. The primary structure of
annexin V deduced from the cDNA sequence shows that annexin V
comprises four internal repeating units (U.S. Pat. No. 4,937,324;
incorporated herein by reference).
[0272] U.S. Pat. No. 5,296,467 and WO 91/07187 are also each
incorporated herein by reference as they provide pharmaceutical
compositions comprising `annexine` (annexin). Although proposed for
use as anticoagulants, the annexins of U.S. Pat. No. 5,296,467 and
WO 91/07187 may now be used as part of the conjugates of the
present invention.
[0273] WO 91/07187 provides natural, synthetic or genetically
prepared derivatives and analogues of `annexine` (annexin), which
may now be used in the conjugates of the present invention.
Particular annexins are provided of 320 amino acids, containing
variant amino acids and, optionally, a disulphide bridge between
the 316-Cys and the 2-Ala.
[0274] U.S. Pat. No. 5,296,467 is incorporated herein by reference
in its entirety, including all figures and sequences, for purposes
of even further describing annexins and pharmaceutical compositions
thereof. U.S. Pat. No. 5,296,467 describes annexin cloning,
recombinant expression and preparation. Aggregates of two or more
annexines, e.g., linked by disulfide bonds between one or more
cysteine groups on the respective annexine, are also disclosed. Yet
a further example of suitable annexin starting materials is
provided by WO 95/27903 (incorporated herein by reference), which
provides annexins for use in detecting apoptotic cells.
[0275] WO 97/17084 is also incorporated herein by reference for
purposes of describing annexin starting materials for preparing
constructs of the present invention. WO 97/17084 particularly
concerns the use of Annexin V to alter phosphatidylserine-dependent
phagocytosis. It is said that blocking PS-dependent phagocytosis
means that PS-carrying cells undergo phagocytosis by other
pathways, leading to greater immune responses, such that Annexin V
may be used as an adjuvant to increase immunogenicity of vaccines.
The treatment of sickle cell anemia and malaria is also described.
WO 97/17084 also provides certain expression vector systems that
may be adapted for use herein.
[0276] To the extent that they clearly describe appropriate annexin
starting materials for preparing therapeutic constructs of the
present invention, each of the diagnostic approaches of U.S. Pat.
No. 5,627,036; WO 95/19791; WO 95/27903; WO 95/34315; WO 96/17618;
and WO 98/04294; are also specifically incorporated herein by
reference. Various of these documents also concern recombinant
expression vectors useful for adaptation into the present
invention.
[0277] Although totally counter-intuitive prior to the present
invention, the annexin conjugation technology of U.S. Pat. No.
5,632,986 may now be adapted for use in the present tumor treatment
methods. U.S. Pat. No. 5,632,986 (incorporated herein by reference)
provides annexin conjugates using compounds that lyse thrombi, or
precursors of such compounds. Annexin-plasminogen activator
conjugates and annexin-urokinase conjugates were particularly
provided for thrombolysis and for treating disorders resulting from
thrombosis. By switching the thrombolytic compounds of U.S. Pat.
No. 5,632,986 for the toxic and coagulative compounds disclosed
herein, the basic conjugate technology of U.S. Pat. No. 5,632,986
can be easily adapted for use in the present invention.
[0278] U.S. Pat. No. 5,632,986 is thus provided for purposes of
further describing annexin isolation from tissue extracts (U.S.
Pat. No. 4,937,324; also incorporated herein by reference) and
annexin production by recombinant methods. Each of the cDNA clones
and expression vectors of U.S. Pat. No. 5,632,986 are thus
specifically incorporated herein by reference.
[0279] U.S. Pat. No. 5,632,986 is also specifically incorporated
herein by reference for purposes of further describing mutants and
variants of the annexin molecule that are subdivided or altered at
one or more amino acid residues so long as the phospholipid binding
capability is not reduced substantially. Appropriate annexins for
use in the present invention can thus be truncated, for example, to
include one or more domains or contain fewer amino acid residues
than the native protein, or can contain substituted amino acids.
Any changes are acceptable within the scope of the invention so
long as the mutein or second generation annexin molecule does not
contain substantially lower affinity for aminophospholipid. Such
guidance can also be applied to phosphatidylethanolamine binding
proteins.
[0280] Second generation, variant and mutant annexin-encoding
nucleic acids may also be readily prepared by standard molecular
biological techniques, and may optionally be characterized as
hybridizing to any of the foregoing annexin-encoding nucleic acid
sequences under hybridization conditions such as those including
hybridization in about 7% sodium dodecyl sulfate (SDS), about 0.5 M
NaPO.sub.4, about 1 mM EDTA at about 50.degree. C.; and washing
with about 1% SDS at about 42.degree. C.
[0281] The chemical cross-linking of annexins and other agents is
also described in U.S. Pat. No. 5,632,986, incorporated herein by
reference. All such techniques can be adapted for use herewith
simply by substituting the thrombolytic agents for those described
herein. Aliphatic diamines; succinimide esters; hetero-bifunctional
coupling reagents, such as SPDP; maleimide compounds; linkers with
spacers; and the like, may thus be used.
[0282] U.S. Pat. No. 5,632,986 is yet further specifically
incorporated herein by reference for purposes of describing the
recombinant production of annexin-containing conjugates.
Appropriate nucleic acid sequences are thus joined to produce
chimeric coding sequences that, in turn, produce chimeric proteins.
Exemplary expression vectors are said to be pKK233-2 (E. coli),
DPOT (yeast) and pDSP1.1BGH (mammalian). Such teaching is
supplemented by further information provided herein.
[0283] D2. Biologically Functional Equivalents
[0284] Equivalents, or even improvements, of aminophospholipid
binding proteins can now be made, generally using the materials
provided above as a starting point. Modifications and changes may
be made in the structure of an aminophospholipid binding protein
and still obtain a molecule having like or otherwise desirable
characteristics. For example, certain amino acids may substituted
for other amino acids in a protein structure without appreciable
loss of interactive binding capacity, such as, binding to the
aminophospholipids, PS and PE. These considerations also apply to
toxins and coagulants.
[0285] Since it is the interactive capacity and nature of a protein
that defines that protein's biological functional activity, certain
amino acid sequence substitutions can be made in a protein sequence
(or of course, the underlying DNA sequence) and nevertheless obtain
a protein with like (agonistic) properties. It is thus contemplated
that various changes may be made in the sequence of known
aminophospholipid binding proteins or peptides (or underlying DNA
sequences) without appreciable loss of their biological utility or
activity. Biological functional equivalents made from mutating an
underlying DNA sequence can be made using the codon information
provided herein in Table A, and the supporting technical details on
site-specific mutagenesis.
[0286] It also is well understood by the skilled artisan that,
inherent in the definition of a "biologically functional
equivalent" protein or peptide, is the concept that there is a
limit to the number of changes that may be made within a defined
portion of the molecule and still result in a molecule with an
acceptable level of equivalent biological activity. Biologically
functional equivalent proteins and peptides are thus defined herein
as those proteins and peptides in which certain, not most or all,
of the amino acids may be substituted. Of course, a plurality of
distinct proteins/peptides with different substitutions may easily
be made and used in accordance with the invention.
[0287] Amino acid substitutions are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like.
An analysis of the size, shape and type of the amino acid
side-chain substituents reveals that arginine, lysine and histidine
are all positively charged residues; that alanine, glycine and
serine are all a similar size; and that phenylalanine, tryptophan
and tyrosine all have a generally similar shape. Therefore, based
upon these considerations, arginine, lysine and histidine; alanine,
glycine and serine; and phenylalanine, tryptophan and tyrosine; are
defined herein as biologically functional equivalents.
[0288] In making more quantitative changes, the hydropathic index
of amino acids may be considered. Each amino acid has been assigned
a hydropathic index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0289] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). It is known that certain amino
acids may be substituted for other amino acids having a similar
hydropathic index or score and still retain a similar biological
activity. In making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indices are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred.
[0290] It is thus understood that an amino acid can be substituted
for another having a similar hydrophilicity value and still obtain
a biologically equivalent protein. As detailed in U.S. Pat. No.
4,554,101 (incorporated herein by reference), the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate
(+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (-0.5);
histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine
(-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0291] In making changes based upon hydrophilicity values, the
substitution of amino acids whose hydrophilicity values are within
.+-.2 is preferred, those which are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred.
[0292] D3. Toxic and Anti-Cellular Agents
[0293] For certain applications, the therapeutic agents will be
cytotoxic or pharmacological agents, particularly cytotoxic,
cytostatic, anti-cellular or anti-angiogenic agents having the
ability to kill or suppress the growth or cell division of
endothelial cells. In general, these aspects of the invention
contemplate the use of any pharmacological agent that can be
conjugated to a targeting agent, and delivered in active form to
the targeted endothelium.
[0294] Exemplary anti-cellular agents include chemotherapeutic
agents, as well as cytotoxins. Chemotherapeutic agents that may be
used include: hormones, such as steroids; anti-metabolites, such as
cytosine arabinoside, fluorouracil, methotrexate or aminopterin;
anthracyclines; mitomycin C; vinca alkaloids; demecolcine;
etoposide; mithramycin; anti-tumor alkylating agents, such as
chlorambucil or melphalan. Other embodiments may include agents
such as cytokines. Basically, any anti-cellular agent may be used,
so long as it can be successfully conjugated to, or associated
with, a targeting agent or antibody in a manner that will allow its
targeting, internalization, release and/or presentation to blood
components at the site of the targeted endothelial cells.
[0295] There may be circumstances, such as when the target antigen
does not internalize by a route consistent with efficient
intoxication by the toxic compound, where one will desire to target
chemotherapeutic agents, such as anti-tumor drugs, cytokines,
antimetabolites, alkylating agents, hormones, and the like. A
variety of chemotherapeutic and other pharmacological agents have
now been successfully conjugated to antibodies and shown to
function pharmacologically, including doxorubicin, daunomycin,
methotrexate, vinblastine, neocarzinostatin, macromycin, trenimon
and .alpha.-amanitin.
[0296] In other circumstances, any potential side-effects from
cytotoxin-based therapy may be eliminated by the use of DNA
synthesis inhibitors, such as daunorubicin, doxorubicin,
adriamycin, and the like. These agents are therefore preferred
examples of anti-cellular agents for use in the present invention.
In terms of cytostatic agents, such compounds generally disturb the
natural cell cycle of a target cell, preferably so that the cell is
taken out of the cell cycle. Exemplary cytostatic agents
include.
[0297] A wide variety of cytotoxic agents are known that may be
conjugated to anti-aminophospholipid antibodies or binding ligands.
Examples include numerous useful plant-, fungus- or
bacteria-derived toxins, which, by way of example, include various
A chain toxins, particularly ricin A chain; ribosome inactivating
proteins, such as saporin or gelonin; .alpha.-sarcin; aspergillin;
restrictocin; ribonucleases, such as placental ribonuclease;
diphtheria toxin; and pseudomonas exotoxin, to name just a few.
[0298] Of the toxins, ricin A chains are preferred. The most
preferred toxin moiety for use herewith is toxin A chain that has
been treated to modify or remove carbohydrate residues, so-called
deglycosylated A chain (dgA). Deglycosylated ricin A chain is
preferred because of its extreme potency, longer half-life, and
because it is economically feasible to manufacture it in a clinical
grade and scale.
[0299] It may be desirable from a pharmacological standpoint to
employ the smallest molecule possible that nevertheless provides an
appropriate biological response. One may thus desire to employ
smaller A chain peptides that will provide an adequate
anti-cellular response. To this end, it has been discovered that
ricin A chain may be "truncated" by the removal of 30 N-terminal
amino acids by Nagarase (Sigma), and still retain an adequate toxin
activity. It is proposed that where desired, this truncated A chain
may be employed in conjugates in accordance with the invention.
[0300] Alternatively, one may find that the application of
recombinant DNA technology to the toxin A chain moiety will provide
additional benefits in accordance the invention. In that the
cloning and expression of biologically active ricin A chain has
been achieved, it is now possible to identify and prepare smaller
or otherwise variant peptides which nevertheless exhibit an
appropriate toxin activity. Moreover, the fact that ricin A chain
has now been cloned allows the application of site-directed
mutagenesis, through which one can readily prepare and screen for A
chain-derived peptides and obtain additional useful moieties for
use in connection with the present invention.
[0301] Other agents for use in immunoconjugate targeting of PS
expressed on tumor vasculature are the angiopoietins. The
angiopoietins, like the members of the VEGF family, are growth
factors largely specific for vascular endothelium (Davis and
Yancopoulos, 1999; Holash et al., 1999; incorporated herein by
reference). The angiopoietins first described were a naturally
occurring agonist, angiopoietin-1 (Ang-1; SEQ ID NO:1 and SEQ ID
NO:2), and a naturally occurring antagonist, angiopoietin-2 (Ang-2;
SEQ ID NO:3 and SEQ ID NO:4), both of which act by means of the
endothelial cell tyrosine kinase receptor, Tie2.
[0302] Two new angiopoietins, angiopoietin-3 (mouse) and
angiopoietin-4 (human) have also been identified (Valenzuela et
al., 1999). Angiopoietin-3 appears to act as an antagonist, whereas
angiopoietin-4 appears to function as an agonist (Valenzuela et
al., 1999). A protein termed angiopoietin-3 was also cloned from
human heart and reported not to have mitogenic effects on
endothelial cells (Kim et al., 1999).
[0303] Whereas VEGF is necessary for the early stages of vascular
development, angiopoietin-1 is generally required for the later
stages of vascular remodeling. Angiopoietin-1 is thus a maturation
or stabilization factor, which converts immature vessels to mature
vessels.
[0304] Angiopoietin-1 has been shown to augment revascularization
in ischemic tissue (Shyu et al., 1998) and to increase the survival
of vascular networks exposed to either VEGF or a form of aFGF
(Papapetropoulos et al., 1999). These authors also showed that
angiopoietin-1 prevents apoptotic death in HUVEC triggered by
withdrawal of the same form of aFGF (Papapetropoulos at al., 1999).
Such data are consistent with the direct role of angiopoietin-1 on
human endothelial cells and its interaction with other angiogenic
molecules to stabilize vascular structures by promoting the
survival of differentiated endothelial cells.
[0305] Of the angiopoietins, angiopoietin-2 is a preferred agent
for use in PS-targeted therapy, particularly in tumors with low
VEGF levels and/or in combination with VEGF Angiopoietin-2 is also
a ligand for Tie2, but generally counteracts blood vessel
maturation/stability mediated by angiopoietin-1. It is thus an
antagonist of angiopoietin-1, and acts to disturb capillary
structure. However, as angiopoietin-2 renders endothelial cells
responsive to angiogenic stimuli, it can initiate
neovascularization in combination with other appropriate signals,
particularly VEGF (Asahara et al., 1998; Holash et al., 1999;
incorporated herein by reference).
[0306] In the absence of another angiogenic signal, angiopoietin-2
causes vessels to destabilize and become immature. In the presence
of a stimulus, such as VEGF, angiopoietin-2 promotes angiogenesis.
Indeed, the angiogenic effects of a number of regulators are
believed to be achieved, at least in part, through the regulation
of an autocrine loop of angiopoietin-2 activity in microvascular
endothelial cells (Mandriota and Pepper, 1998).
[0307] Angiopoietin-2 expression in tumor tissue has been reported
(Tanaka et al., 1999), where it presumably acts in combination with
VEGF to promote angiogenesis (Stratmann et al., 1998). However, as
angiopoietin-2 provides a negative signal when VEGF is low or
absent, provision of angiopoietin-2 can be a useful therapeutic
approach. In addition to tumor-targeted forms, angiopoietin-2 can
also be administered as a protein or gene therapy therapeutic (see
combination therapies described herein). Fusion proteins of
angiopoietins are also envisioned for use in this invention, such
as the stable Ang-1-Ang-2 fusion protein included herein as SEQ ID
NO:5.
[0308] D4. Coagulation Factors
[0309] The antibody and ligand targeting agents of the invention
may be linked to a component that is capable of directly or
indirectly stimulating coagulation, to form a coaguligand. Here,
the targeting agents may be directly linked to the coagulant or
coagulation factor, or may be linked to a second binding region
that binds and then releases the coagulant or coagulation factor.
As used herein, the terms "coagulant" and "coagulation factor" are
each used to refer to a component that is capable of directly or
indirectly stimulating coagulation under appropriate conditions,
preferably when provided to a specific in vivo environment, such as
the tumor vasculature.
[0310] Preferred coagulation factors are Tissue Factor
compositions, such as truncated TF (tTF), dimeric, multimeric and
mutant TF molecules. "Truncated TF" (tTF) refers to TF constructs
that are rendered membrane-binding deficient by removal of
sufficient amino acid sequences to effect this change in property.
A "sufficient amount" in this context is an amount of transmembrane
amino acid sequence originally sufficient to enter the TF molecule
in the membrane, or otherwise mediate functional membrane binding
of the TF protein. The removal of such a "sufficient amount of
transmembrane spanning sequence" therefore creates a truncated
Tissue Factor protein or polypeptide deficient in phospholipid
membrane binding capacity, such that the protein is substantially a
soluble protein that does not significantly bind to phospholipid
membranes. Truncated TF thus substantially fails to convert Factor
VII to Factor VIIa in a standard TF assay, and yet retains
so-called catalytic activity including activating Factor X in the
presence of Factor VIIa.
[0311] U.S. Pat. No. 5,504,067 is specifically incorporated herein
by reference for the purposes of further describing such truncated
Tissue Factor proteins. Preferably, the Tissue Factors for use in
these aspects of the present invention will generally lack the
transmembrane and cytosolic regions (amino acids 220-263) of the
protein. However, there is no need for the truncated TF molecules
to be limited to molecules of the exact length of 219 amino
acids.
[0312] Tissue Factor compositions may also be useful as dimers. Any
of the truncated, mutated or other Tissue Factor constructs may be
prepared in a dimeric form for use in the present invention. As
will be known to those of ordinary skill in the art, such TF dimers
may be prepared by employing the standard techniques of molecular
biology and recombinant expression, in which two coding regions are
prepared in-frame and expressed from an expression vector. Equally,
various chemical conjugation technologies may be employed in
connection with the preparation of TF dimers. The individual TF
monomers may be derivatized prior to conjugation. All such
techniques would be readily known to those of skill in the art.
[0313] If desired, the Tissue Factor dimers or multimers may be
joined via a biologically-releasable bond, such as a
selectively-cleavable linker or amino acid sequence. For example,
peptide linkers that include a cleavage site for an enzyme
preferentially located or active within a tumor environment are
contemplated. Exemplary forms of such peptide linkers are those
that are cleaved by urokinase, plasmin, thrombin, Factor IXa,
Factor Xa, or a metalloproteinase, such as collagenase, gelatinase
or stromelysin.
[0314] In certain embodiments, the Tissue Factor dimers may further
comprise a hindered hydrophobic membrane insertion moiety, to later
encourage the functional association of the Tissue Factor with the
phospholipid membrane, but only under certain defined conditions.
As described in the context of the truncated Tissue Factors,
hydrophobic membrane-association sequences are generally stretches
of amino acids that promote association with the phospholipid
environment due to their hydrophobic nature. Equally, fatty acids
may be used to provide the potential membrane insertion moiety.
[0315] Such membrane insertion sequences may be located either at
the N-terminus or the C-terminus of the IF molecule, or generally
appended at any other point of the molecule so long as their
attachment thereto does not hinder the functional properties of the
IF construct. The intent of the hindered insertion moiety is that
it remains non-functional until the TF construct localizes within
the tumor environment, and allows the hydrophobic appendage to
become accessible and even further promote physical association
with the membrane. Again, it is contemplated that
biologically-releasable bonds and selectively-cleavable sequences
will be particularly useful in this regard, with the bond or
sequence only being cleaved or otherwise modified upon localization
within the tumor environment and exposure to particular enzymes or
other bioactive molecules.
[0316] In other embodiments, the tTF constructs may be multimeric
or polymeric. In this context a "polymeric construct" contains 3 or
more Tissue Factor constructs. A "multimeric or polymeric TF
construct" is a construct that comprises a first TF molecule or
derivative operatively attached to at least a second and a third TF
molecule or derivative. The multimers may comprise between about 3
and about 20 such TF molecules. The individual TF units within the
multimers or polymers may also be linked by selectively-cleavable
peptide linkers or other biological-releasable bonds as desired.
Again, as with the TF dimers discussed above, the constructs may be
readily made using either recombinant manipulation and expression
or using standard synthetic chemistry.
[0317] Even further TF constructs useful in context of the present
invention are those mutants deficient in the ability to activate
Factor VII. Such "Factor VII activation mutants" are generally
defined herein as TF mutants that bind functional Factor VII/VIIa,
proteolytically activate Factor X, but are substantially free from
the ability to proteolytically activate Factor VII. Accordingly,
such constructs are TF mutants that lack Factor VII activation
activity.
[0318] The ability of such Factor VII activation mutants to
function in promoting tumor-specific coagulation is based upon
their specific delivery to the tumor vasculature, and the presence
of Factor Vila at low levels in plasma. Upon administration of such
a Factor VII activation mutant-targeting agent conjugate, the
mutant will be localized within the vasculature of a vascularized
tumor. Prior to localization, the TF mutant would be generally
unable to promote coagulation in any other body sites, on the basis
of its inability to convert Factor VII to Factor VIIa. However,
upon localization and accumulation within the tumor region, the
mutant will then encounter sufficient Factor VIIa from the plasma
in order to initiate the extrinsic coagulation pathway, leading to
tumor-specific thrombosis. Exogenous Factor VIIa could also be
administered to the patient.
[0319] Any one or more of a variety of Factor VII activation
mutants may be prepared and used in connection with the present
invention. There is a significant amount of scientific knowledge
concerning the recognition sites on the TF molecule for Factor
VII/VIIa. It will thus be understood that the Factor VII activation
region generally lies between about amino acid 157 and about amino
acid 167 of the TF molecule. However, it is contemplated that
residues outside this region may also prove to be relevant to the
Factor VII activating activity, and one may therefore consider
introducing mutations into any one or more of the residues
generally located between about amino acid 106 and about amino acid
209 of the TF sequence (WO 94/07515; WO 94/28017; each incorporated
herein by reference).
[0320] A variety of other coagulation factors may be used in
connection with the present invention, as exemplified by the agents
set forth below. Thrombin, Factor V/Va and derivatives, Factor
VIII/VIIIa and derivatives, Factor IX/IXa and derivatives, Factor
X/Xa and derivatives, Factor XI/XIa and derivatives, Factor
XII/XIIa and derivatives, Factor XIII/XIIIa and derivatives, Factor
X activator and Factor V activator may be used in the present
invention.
[0321] Russell's viper venom Factor X activator is contemplated for
use in this invention. Monoclonal antibodies specific for the
Factor X activator present in Russell's viper venom have also been
produced, and could be used to specifically deliver the agent as
part of a bispecific binding ligand.
[0322] Thromboxane A.sub.2 is formed from endoperoxides by the
sequential actions of the enzymes cyclooxygenase and thromboxane
synthetase in platelet microsomes. Thromboxane A.sub.2 is readily
generated by platelets and is a potent vasoconstrictor, by virtue
of its capacity to produce platelet aggregation. Both thromboxane
A.sub.2 and active analogues thereof are contemplated for use in
the present invention.
[0323] Thromboxane synthase, and other enzymes that synthesize
platelet-activating prostaglandins, may also be used as
"coagulants" in the present context. Monoclonal antibodies to, and
immunoaffinity purification of, thromboxane synthase are known; as
is the cDNA for human thromboxane synthase.
[0324] .alpha.2-antiplasmin, or .alpha.2-plasmin inhibitor, is a
proteinase inhibitor naturally present in human plasma that
functions to efficiently inhibit the lysis of fibrin clots induced
by plasminogen activator. .alpha.2-antiplasmin is a particularly
potent inhibitor, and is contemplated for use in the present
invention.
[0325] As the cDNA sequence for .alpha.2-antiplasmin is available,
recombinant expression and/or fusion proteins are preferred.
Monoclonal antibodies against .alpha.2-antiplasmin are also
available that may be used in the bispecific binding ligand
embodiments of the invention. These antibodies could both be used
to deliver exogenous .alpha.2-antiplasmin to the target site or to
garner endogenous .alpha.2-antiplasmin and concentrate it within
the targeted region.
[0326] D5. Fusion Proteins and Recombinant Expression
[0327] The therapeutic agent-targeting agent compositions of the
present invention may be readily prepared as fusion proteins using
molecular biological techniques. The use of recombinant DNA
techniques to achieve such ends is now standard practice to those
of skill in the art. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques and in vivo
recombination/genetic recombination. DNA and RNA synthesis may,
additionally, be performed using an automated synthesizers (see,
for example, the techniques described in Sambrook et al.,
1989).
[0328] The preparation of such a fusion protein generally entails
the preparation of a first and second DNA coding region and the
functional ligation or joining of such regions, in frame, to
prepare a single coding region that encodes the desired fusion
protein. In the present context, the targeting agent DNA sequence
will be joined in frame with a DNA sequence encoding a therapeutic
agent. It is not generally believed to be particularly relevant
which portion of the therapeutic agent-targeting agent is prepared
as the N-terminal region or as the C-terminal region.
[0329] Once the desired coding region has been produced, an
expression vector is created. Expression vectors contain one or
more promoters upstream of the inserted DNA regions that act to
promote transcription of the DNA and to thus promote expression of
the encoded recombinant protein. This is the meaning of
"recombinant expression".
[0330] To obtain a so-called "recombinant" version of the
therapeutic agent-targeting agent protein, it is expressed in a
recombinant cell. The engineering of DNA segment(s) for expression
in a prokaryotic or eukaryotic system may be performed by
techniques generally known to those of skill in recombinant
expression. It is believed that virtually any expression system may
be employed in the expression of the therapeutic agent-targeting
agent constructs.
[0331] Such proteins may be successfully expressed in eukaryotic
expression systems, e.g., CHO cells, however, it is envisioned that
bacterial expression systems, such as E. coli pQE-60 will be
particularly useful for the large-scale preparation and subsequent
purification of the therapeutic agent-targeting agent constructs.
cDNAs may also be expressed in bacterial systems, with the encoded
proteins being expressed as fusions with .beta.-galactosidase,
ubiquitin, Schistosoma japonicum glutathione S-transferase, and the
like. It is believed that bacterial expression will have advantages
over eukaryotic expression in terms of ease of use and quantity of
materials obtained thereby.
[0332] In terms of microbial expression, U.S. Pat. Nos. 5,583,013;
5,221,619; 4,785,420; 4,704,362; and 4,366,246 are incorporated
herein by reference for the purposes of even further supplementing
the present disclosure in connection with the expression of genes
in recombinant host cells.
[0333] Recombinantly produced therapeutic agent-targeting agent
constructs may be purified and formulated for human administration.
Alternatively, nucleic acids encoding the therapeutic
agent-targeting agent constructs may be delivered via gene therapy.
Although naked recombinant DNA or plasmids may be employed, the use
of liposomes or vectors is preferred. The ability of certain
viruses to enter cells via receptor-mediated endocytosis and to
integrate into the host cell genome and express viral genes stably
and efficiently have made them attractive candidates for the
transfer of foreign genes into mammalian cells. Preferred gene
therapy vectors for use in the present invention will generally be
viral vectors.
[0334] Retroviruses have promise as gene delivery vectors due to
their ability to integrate their genes into the host genome,
transferring a large amount of foreign genetic material, infecting
a broad spectrum of species and cell types and of being packaged in
special cell-lines. Other viruses, such as adenovirus, herpes
simplex viruses (HSV), cytomegalovirus (CMV), and adeno-associated
virus (AAV), such as those described by U.S. Pat. No. 5,139,941
(incorporated herein by reference), may also be engineered to serve
as vectors for gene transfer.
[0335] Although some viruses that can accept foreign genetic
material are limited in the number of nucleotides they can
accommodate and in the range of cells they infect, these viruses
have been demonstrated to successfully effect gene expression.
However, adenoviruses do not integrate their genetic material into
the host genome and therefore do not require host replication for
gene expression, making them ideally suited for rapid, efficient,
heterologous gene expression. Techniques for preparing
replication-defective infective viruses are well known in the
art.
[0336] In certain further embodiments, the gene therapy vector will
be HSV. A factor that makes HSV an attractive vector is the size
and organization of the genome. Because HSV is large, incorporation
of multiple genes or expression cassettes is less problematic than
in other smaller viral systems. In addition, the availability of
different viral control sequences with varying performance (e.g.,
temporal, strength) makes it possible to control expression to a
greater extent than in other systems. It also is an advantage that
the virus has relatively few spliced messages, further easing
genetic manipulations. HSV also is relatively easy to manipulate
and can be grown to high titers.
[0337] Of course, in using viral delivery systems, one will desire
to purify the virion sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering viral
particles or endotoxins and other pyrogens such that it will not
cause any untoward reactions in the cell, animal or individual
receiving the vector construct. A preferred means of purifying the
vector involves the use of buoyant density gradients, such as
cesium chloride gradient centrifugation.
E. Anti-Aminophospholipid Antibodies and Conjugates
[0338] E1. Polyclonal Anti-Aminophospholipid Antibodies
[0339] Means for preparing and characterizing antibodies are well
known in the art (see, e.g., Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, 1988; incorporated herein by reference).
To prepare polyclonal antisera an animal is immunized with an
immunogenic aminophospholipid composition, and antisera collected
from that immunized animal. A wide range of animal species can be
used for the production of antisera. Typically the animal used for
production of anti-antisera is a rabbit, mouse, rat, hamster,
guinea pig or goat. Because of the relatively large blood volume of
rabbits, a rabbit is a preferred choice for production of
polyclonal antibodies.
[0340] The amount of immunogen composition used in the production
of polyclonal antibodies varies upon the nature of the immunogen as
well as the animal used for immunization. A variety of routes can
be used to administer the present aminophospholipid immunogen;
subcutaneous, intramuscular, intradermal, intravenous,
intraperitoneal and intrasplenic. The production of polyclonal
antibodies may be monitored by sampling blood of the immunized
animal at various points following immunization. A second, booster
injection, may also be given. The process of boosting and titering
is repeated until a suitable titer is achieved. When a desired
titer level is obtained, the immunized animal can be bled and the
serum isolated and stored. The animal can also be used to generate
monoclonal antibodies.
[0341] As is well known in the art, the immunogenicity of a
particular composition can be enhanced by the use of non-specific
stimulators of the immune response, known as adjuvants. Exemplary
adjuvants include complete Freund's adjuvant, a non-specific
stimulator of the immune response containing killed Mycobacterium
tuberculosis; incomplete Freund's adjuvant; and aluminum hydroxide
adjuvant.
[0342] It may also be desired to boost the host immune system, as
may be achieved by associating aminophospholipids with, or coupling
aminophospholipids to, a carrier. Exemplary carriers are keyhole
limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other
albumins such as ovalbumin, mouse serum albumin or rabbit serum
albumin can also be used as carriers.
[0343] As is also known in the art, a given composition may vary in
its immunogenicity. However, the generation of antibodies against
aminophospholipids is not particularly difficult. For example,
highly specific anti-phosphatidylserine antibodies were raised in
rabbits immunized by intramuscular injections of
phosphatidylserine-containing polyacrylamide gels and with
phosphatidylserine-cytochrome c vesicles (Maneta-Peyret et al.,
1988; 1989; each incorporated herein by reference). The use of
acrylamide implants enhanced the production of antibodies
(Maneta-Peyret et al., 1988; 1989). The anti-phosphatidylserine
antibodies raised in this manner are able to detect
phosphatidylserine in situ on human platelets (Maneta-Peyret et
al., 1988). The groups of Inoue, Rote and Rauch have also developed
anti-PS and anti-PE antibodies (see below).
[0344] E2. Monoclonal Anti-Aminophospholipid Antibodies
[0345] Various methods for generating monoclonal antibodies (MAbs)
are also now very well known in the art. The most standard
monoclonal antibody generation techniques generally begin along the
same lines as those for preparing polyclonal antibodies
(Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
1988; incorporated herein by reference). A polyclonal antibody
response is initiated by immunizing an animal with an immunogenic
aminophospholipid composition and, when a desired titer level is
obtained, the immunized animal can be used to generate MAbs.
[0346] MAbs may be readily prepared through use of well-known
techniques, such as those exemplified in U.S. Pat. No. 4,196,265,
incorporated herein by reference. Typically, this technique
involves immunizing a suitable animal with the selected
aminophospholipid immunogen composition. The immunizing composition
is administered in a manner effective to stimulate antibody
producing cells. Rodents such as mice and rats are preferred
animals, however, the use of rabbit, sheep and frog cells is also
possible. The use of rats may provide certain advantages (Goding,
1986, pp. 60-61; incorporated herein by reference), but mice are
preferred, with the BALB/c mouse being most preferred as this is
most routinely used and generally gives a higher percentage of
stable fusions.
[0347] Following immunization, somatic cells with the potential for
producing aminophospholipid antibodies, specifically B lymphocytes
(B cells), are selected for use in the MAb generating protocol.
These cells may be obtained from biopsied spleens, tonsils or lymph
nodes, or from a peripheral blood sample. Spleen cells and
peripheral blood cells are preferred, the former because they are a
rich source of antibody-producing cells that are in the dividing
plasmablast stage, and the latter because peripheral blood is
easily accessible. Often, a panel of animals will have been
immunized and the spleen of animal with the highest antibody titer
will be removed and the spleen lymphocytes obtained by homogenizing
the spleen with a syringe. Typically, a spleen from an immunized
mouse contains approximately 5.times.10.sup.7 to 2.times.10.sup.8
lymphocytes.
[0348] The anti-aminophospholipid antibody-producing B lymphocytes
from the immunized animal are then fused with cells of an immortal
myeloma cell, generally one of the same species as the animal that
was immunized. Myeloma cell lines suited for use in
hybridoma-producing fusion procedures preferably are
non-antibody-producing, have high fusion efficiency, and enzyme
deficiencies that render then incapable of growing in certain
selective media which support the growth of only the desired fused
cells (hybridomas).
[0349] Any one of a number of myeloma cells may be used, as are
known to those of skill in the art (Goding, pp. 65-66, 1986;
Campbell, pp. 75-83, 1984; each incorporated herein by reference).
For example, where the immunized animal is a mouse, one may use
P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO, NSO/U,
MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, one may use
R210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or one of the above listed
mouse cell lines; and U-266, GM1500-GRG2, LICR-LON-HMy2 and
UC729-6, are all useful in connection with human cell fusions.
[0350] Methods for generating hybrids of antibody-producing spleen
or lymph node cells and myeloma cells usually comprise mixing
somatic cells with myeloma cells in a 4:1 proportion, though the
proportion may vary from about 20:1 to about 1:1, respectively, in
the presence of an agent or agents (chemical or electrical) that
promote the fusion of cell membranes. Fusion methods using Sendai
virus have been described by Kohler and Milstein (1975; 1976; each
incorporated herein by reference), and those using polyethylene
glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. (1977;
incorporated herein by reference). The use of electrically induced
fusion methods is also appropriate (Goding pp. 71-74, 1986;
incorporated herein by reference).
[0351] Fusion procedures usually produce viable hybrids at low
frequencies, about 1.times.10.sup.-6 to 1.times.10.sup.-8. However,
this does not pose a problem, as the viable, fused hybrids are
differentiated from the parental, unfused cells (particularly the
unfused myeloma cells that would normally continue to divide
indefinitely) by culturing in a selective medium. The selective
medium is generally one that contains an agent that blocks the de
novo synthesis of nucleotides in the tissue culture media.
Exemplary and preferred agents are aminopterin, methotrexate, and
azaserine. Aminopterin and methotrexate block de novo synthesis of
both purines and pyrimidines, whereas azaserine blocks only purine
synthesis. Where aminopterin or methotrexate is used, the media is
supplemented with hypoxanthine and thymidine as a source of
nucleotides (HAT medium). Where azaserine is used, the media is
supplemented with hypoxanthine.
[0352] The preferred selection medium is HAT. Only cells capable of
operating nucleotide salvage pathways are able to survive in HAT
medium. The myeloma cells are defective in key enzymes of the
salvage pathway, e.g., hypoxanthine phosphoribosyl transferase
(HPRT), and they cannot survive. The B cells can operate this
pathway, but they have a limited life span in culture and generally
die within about two weeks. Therefore, the only cells that can
survive in the selective media are those hybrids formed from
myeloma and B cells.
[0353] This culturing provides a population of hybridomas from
which specific hybridomas are selected. Typically, selection of
hybridomas is performed by culturing the cells by single-clone
dilution in microtiter plates, followed by testing the individual
clonal supernatants (after about two to three weeks) for the
desired anti-aminophospholipid reactivity. The assay should be
sensitive, simple and rapid, such as radioimmunoassays, enzyme
immunoassays, cytotoxicity assays, plaque assays, dot immunobinding
assays, and the like.
[0354] The selected hybridomas would then be serially diluted and
cloned into individual anti-aminophospholipid antibody-producing
cell lines, which clones can then be propagated indefinitely to
provide MAbs. The cell lines may be exploited for MAb production in
two basic ways. A sample of the hybridoma can be injected (often
into the peritoneal cavity) into a histocompatible animal of the
type that was used to provide the somatic and myeloma cells for the
original fusion. The injected animal develops tumors secreting the
specific monoclonal antibody produced by the fused cell hybrid. The
body fluids of the animal, such as serum or ascites fluid, can then
be tapped to provide MAbs in high concentration. The individual
cell lines could also be cultured in vitro, where the MAbs are
naturally secreted into the culture medium from which they can be
readily obtained in high concentrations.
[0355] MAbs produced by either means will generally be further
purified, e.g., using filtration, centrifugation and various
chromatographic methods, such as HPLC or affinity chromatography,
all of which purification techniques are well known to those of
skill in the art. These purification techniques each involve
fractionation to separate the desired antibody from other
components of a mixture. Analytical methods particularly suited to
the preparation of antibodies include, for example, protein
A-Sepharose and/or protein G-Sepharose chromatography.
[0356] Umeda et al. (1989; incorporated herein by reference)
reported the effective production of monoclonal antibodies
recognizing stereo-specific epitopes of phosphatidylserine. The
Umeda system is based on the direct immunization of
phosphatidylserine into mouse spleen using a Salmonella-coated
aminophospholipid sample (Umeda et al., 1989; incorporated herein
by reference). The Umeda protocol gives a high frequency of anti-PS
MAbs, which exhibit three distinct reactivity profiles ranging from
highly specific to broadly cross-reactive. Umeda is therefore also
incorporated herein by reference for purposes of further describing
screening assays to identify MAbs that bind specifically to PS,
e.g., and do not bind to phosphatidylcholine.
[0357] Any of the 61 hybridomas generated by Umeda could
potentially be employed in the therapeutic agent-targeting agent
constructs of the present invention. Examples are PSC8, PSF11,
PSG3, PSD11, PSF10, PS1B, PS3D12, PS2C11; PS3A, PSF6, PSF7, PSB4,
PS3H1; PS4A7 and PS1G3. More preferred are PS3A, PSF6, PSF7, PSB4
and PS3H1 as they bind only to phosphatidylserine and
phosphatidylethanolamine. Preferred anti-PS antibodies are PS4A7
(IgM) and PS (IgG.sub.3), as they are highly specific for PS and
exhibit no cross-reaction with other phospholipids. PS4A7
recognizes the stereo-specific configuration of the serine residue
in (FIG. 1 Umeda et al., 1989; incorporated herein by
reference).
[0358] Igarashi et al. (1991; incorporated herein by reference)
also reported the effective induction of anti-PS antibodies of the
IgG isotype by intrasplenic immunization. Only a slight increase of
the titer was observed when the antigen was again injected
intravenously. A high frequency of anti-PS MAbs of the IgG isotype
was also observed even when MAbs were produced 10 days after the
intrasplenic injection of the antigen. These antibodies were also
employed by Schuurmans Stekhoven et al. (1994).
[0359] The other significant anti-PS antibody production has been
by Rote and colleagues. Rote et al. (1993; incorporated herein by
reference) particularly employed PS micelles in combination with
Freund's complete adjuvant to generate specific anti-PS antibodies.
Rote et al. (1993) also generated monoclonal antibodies that
differentiate between cardiolipin (CL) and PS. Rote et al. (1993)
is therefore also incorporated herein by reference for purposes of
further describing screening assays to identify MAbs that bind
specifically to PS by testing against resting and
thrombin-activated platelets using flow cytometry.
[0360] The 3SB9b antibody produced by Rote et al. (1993) reacted
with only with PS, and is a preferred antibody for use in the
therapeutic agent-targeting agent constructs of the present
invention. BA3B5C4 may also be used as it reacts with both PS and
CL. These antibodies are also described in Lin et al. (1995),
Obringer et al. (1995) and Katsuragawa et al. (1997).
[0361] E3. Anti-Aminophospholipid Antibodies from Phagemid
Libraries
[0362] Recombinant technology now allows the preparation of
antibodies having the desired specificity from recombinant genes
encoding a range of antibodies (Van Dijk et al., 1989; incorporated
herein by reference). Certain recombinant techniques involve the
isolation of the antibody genes by immunological screening of
combinatorial immunoglobulin phage expression libraries prepared
from RNA isolated from the spleen of an immunized animal (Morrison
et al., 1986; Winter and Milstein, 1991; each incorporated herein
by reference).
[0363] For such methods, combinatorial immunoglobulin phagemid
libraries are prepared from RNA isolated from the spleen of the
immunized animal, and phagemids expressing appropriate antibodies
are selected by panning using cells expressing the antigen and
control cells. The advantages of this approach over conventional
hybridoma techniques are that approximately 10.sup.4 times as many
antibodies can be produced and screened in a single round, and that
new specificities are generated by H and L chain combination, which
further increases the percentage of appropriate antibodies
generated.
[0364] One method for the generation of a large repertoire of
diverse antibody molecules in bacteria utilizes the bacteriophage
lambda as the vector (Huse et al., 1989; incorporated herein by
reference). Production of antibodies using the lambda vector
involves the cloning of heavy and light chain populations of DNA
sequences into separate starting vectors. The vectors are
subsequently combined randomly to form a single vector that directs
the co-expression of heavy and light chains to form antibody
fragments. The heavy and light chain DNA sequences are obtained by
amplification, preferably by PCR.TM. or a related amplification
technique, of mRNA isolated from spleen cells (or hybridomas
thereof) from an animal that has been immunized with a selected
antigen. The heavy and light chain sequences are typically
amplified using primers that incorporate restriction sites into the
ends of the amplified DNA segment to facilitate cloning of the
heavy and light chain segments into the starting vectors.
[0365] Another method for the generation and screening of large
libraries of wholly or partially synthetic antibody combining
sites, or paratopes, utilizes display vectors derived from
filamentous phage such as M13, fl or fd. These filamentous phage
display vectors, referred to as "phagemids", yield large libraries
of monoclonal antibodies having diverse and novel
immunospecificities. The technology uses a filamentous phage coat
protein membrane anchor domain as a means for linking gene-product
and gene during the assembly stage of filamentous phage
replication, and has been used for the cloning and expression of
antibodies from combinatorial libraries (Kang et al., 1991; Barbas
et al., 1991; each incorporated herein by reference).
[0366] This general technique for filamentous phage display is
described in U.S. Pat. No. 5,658,727, incorporated herein by
reference. In a most general sense, the method provides a system
for the simultaneous cloning and screening of pre-selected
ligand-binding specificities from antibody gene repertoires using a
single vector system. Screening of isolated members of the library
for a pre-selected ligand-binding capacity allows the correlation
of the binding capacity of an expressed antibody molecule with a
convenient means to isolate the gene that encodes the member from
the library.
[0367] Linkage of expression and screening is accomplished by the
combination of targeting of a fusion polypeptide into the periplasm
of a bacterial cell to allow assembly of a functional antibody, and
the targeting of a fusion polypeptide onto the coat of a
filamentous phage particle during phage assembly to allow for
convenient screening of the library member of interest. Periplasmic
targeting is provided by the presence of a secretion signal domain
in a fusion polypeptide. Targeting to a phage particle is provided
by the presence of a filamentous phage coat protein membrane anchor
domain (i.e., a cpIII- or cpVIII-derived membrane anchor domain) in
a fusion polypeptide.
[0368] The diversity of a filamentous phage-based combinatorial
antibody library can be increased by shuffling of the heavy and
light chain genes, by altering one or more of the complementarity
determining regions of the cloned heavy chain genes of the library,
or by introducing random mutations into the library by error-prone
polymerase chain reactions. Additional methods for screening
phagemid libraries are described in U.S. Pat. Nos. 5,580,717;
5,427,908; 5,403,484; and 5,223,409, each incorporated herein by
reference.
[0369] Another method for the screening of large combinatorial
antibody libraries has been developed, utilizing expression of
populations of diverse heavy and light chain sequences on the
surface of a filamentous bacteriophage, such as M13, fl or fd (U.S.
Pat. No. 5,698,426; incorporated herein by reference). Two
populations of diverse heavy (Hc) and light (Lc) chain sequences
are synthesized by polymerase chain reaction (PCR.TM.). These
populations are cloned into separate M13-based vector containing
elements necessary for expression. The heavy chain vector contains
a gene VIII (gVIII) coat protein sequence so that translation of
the heavy chain sequences produces gVIII-Hc fusion proteins. The
populations of two vectors are randomly combined such that only the
vector portions containing the Hc and Lc sequences are joined into
a single circular vector.
[0370] The combined vector directs the co-expression of both Hc and
Lc sequences for assembly of the two polypeptides and surface
expression on M13 (U.S. Pat. No. 5,698,426; incorporated herein by
reference). The combining step randomly brings together different
Hc and Lc encoding sequences within two diverse populations into a
single vector. The vector sequences donated from each independent
vector are necessary for production of viable phage. Also, since
the pseudo gVIII sequences are contained in only one of the two
starting vectors, co-expression of functional antibody fragments as
Lc associated gVIII-Hc fusion proteins cannot be accomplished on
the phage surface until the vector sequences are linked in the
single vector.
[0371] Surface expression of the antibody library is performed in
an amber suppressor strain. An amber stop codon between the Hc
sequence and the gVIII sequence unlinks the two components in a
non-suppressor strain. Isolating the phage produced from the
non-suppressor strain and infecting a suppressor strain will link
the Hc sequences to the gVIII sequence during expression. Culturing
the suppressor strain after infection allows the coexpression on
the surface of M13 of all antibody species within the library as
gVIII fusion proteins (gVIII-Fab fusion proteins). Alternatively,
the DNA can be isolated from the non-suppressor strain and then
introduced into a suppressor strain to accomplish the same
effect.
[0372] The surface expression library is screened for specific Fab
fragments that bind preselected molecules by standard affinity
isolation procedures. Such methods include, for example, panning
(Parmley and Smith, 1988; incorporated herein by reference),
affinity chromatography and solid phase blotting procedures.
Panning is preferred, because high titers of phage can be screened
easily, quickly and in small volumes. Furthermore, this procedure
can select minor Fab fragments species within the population, which
otherwise would have been undetectable, and amplified to
substantially homogenous populations. The selected Fab fragments
can be characterized by sequencing the nucleic acids encoding the
polypeptides after amplification of the phage population.
[0373] Another method for producing diverse libraries of antibodies
and screening for desirable binding specificities is described in
U.S. Pat. No. 5,667,988 and U.S. Pat. No. 5,759,817, each
incorporated herein by reference. The method involves the
preparation of libraries of heterodimeric immunoglobulin molecules
in the form of phagemid libraries using degenerate oligonucleotides
and primer extension reactions to incorporate the degeneracies into
the CDR regions of the immunoglobulin variable heavy and light
chain variable domains, and display of the mutagenized polypeptides
on the surface of the phagemid. Thereafter, the display protein is
screened for the ability to bind to a preselected antigen.
[0374] The method for producing a heterodimeric immunoglobulin
molecule generally involves (1) introducing a heavy or light chain
V region-coding gene of interest into the phagemid display vector;
(2) introducing a randomized binding site into the phagemid display
protein vector by primer extension with an oligonucleotide
containing regions of homology to a CDR of the antibody V region
gene and containing regions of degeneracy for producing randomized
coding sequences to form a large population of display vectors each
capable of expressing different putative binding sites displayed on
a phagemid surface display protein; (3) expressing the display
protein and binding site on the surface of a filamentous phage
particle; and (4) isolating (screening) the surface-expressed phage
particle using affinity techniques such as panning of phage
particles against a preselected antigen, thereby isolating one or
more species of phagemid containing a display protein containing a
binding site that binds a preselected antigen.
[0375] A further variation of this method for producing diverse
libraries of antibodies and screening for desirable binding
specificities is described in U.S. Pat. No. 5,702,892, incorporated
herein by reference. In this method, only heavy chain sequences are
employed, the heavy chain sequences are randomized at all
nucleotide positions which encode either the CDRI or CDRIII
hypervariable region, and the genetic variability in the CDRs is
generated independent of any biological process.
[0376] In the method, two libraries are engineered to genetically
shuffle oligonucleotide motifs within the framework of the heavy
chain gene structure. Through random mutation of either CDRI or
CDRIII, the hypervariable regions of the heavy chain gene were
reconstructed to result in a collection of highly diverse
sequences. The heavy chain proteins encoded by the collection of
mutated gene sequences possessed the potential to have all of the
binding characteristics of an immunoglobulin while requiring only
one of the two immunoglobulin chains.
[0377] Specifically, the method is practiced in the absence of the
immunoglobulin light chain protein. A library of phage displaying
modified heavy chain proteins is incubated with an immobilized
ligand to select clones encoding recombinant proteins that
specifically bind the immobilized ligand. The bound phage are then
dissociated from the immobilized ligand and amplified by growth in
bacterial host cells. Individual viral plaques, each expressing a
different recombinant protein, are expanded, and individual clones
can then be assayed for binding activity.
[0378] E4. Anti-Aminophospholipid Antibodies from Human
Patients
[0379] Antibodies against aminophospholipids, particularly
phosphatidylserine and phosphatidylethanolamine, occur in the human
population, where they are correlated with certain disease states.
Anti-aminophospholipid antibodies are part of the heterogeneous
anti-phospholipid antibodies (aPL), observed to have families of
different specificities and classes. Primary anti-phospholipid
syndrome (APS) has even been separated from other forms of
autoimmune disease associated with anti-phospholipid antibody
production.
[0380] Anti-PS antibodies are particularly associated with
recurrent pregnancy loss (Rote et al., 1995; Rote, 1996; Vogt et
al., 1996; Vogt et al., 1997; incorporated herein by reference) and
with the autoimmune disease, systemic lupus erythematosus (SLE or
"lupus") (Branch et al., 1987; incorporated herein by reference).
Anti-PE antibodies have also been reported in human patients,
particularly those with autoimmune diseases (Staub et al., 1989).
Branch et al. (1987) reported that 80% of patients with lupus
anticoagulant (LA or LAC) had autoantibodies that recognized PE;
with Drouvalakis and Buchanan (1998) increasing this number to 95%
PE-positives from autoimmune LAC sera.
[0381] Anti-phospholipid antibodies are not to be confused with
anti-endothelial cell antibodies (AECA), although they can be found
in the same patient. The existence of AECA has been documented in a
variety of clinical settings associated with vasculitis, such as
systemic sclerosis (SS). To study AECA, antibodies are obtained
from patients that do not have anti-phospholipid antibodies
(aPL-negative sera).
[0382] The pathogenic role of AECA remains unclear, although
Bordron et al. (1998) very recently suggested that AECA may
initiate apoptosis in endothelial cells, which would be followed by
PS transfer to the outer face of the membrane. They proposed that
this would account for the subsequent generation of the
anti-phospholipid antibodies that are sometimes seen in conjunction
with AECA in patients with skin lesions or connective tissue
disease (Bordron et al., 1998). However, although AECA binding to
an apoptosis-inducing antigen was postulated, these studies did not
lead to the further characterization of AECA, still said to
represent an extremely heterogeneous family of antibodies reacting
with different (non-lipid) structures on endothelial cells (Bordron
et al., 1998).
[0383] Anti-phosphatidylserine antibodies are closely associated
with pregnancy loss, pregnancy-induced hypertension and
intrauterine growth retardation. A phosphatidylserine-dependent
antigen has been shown to be expressed on the surface of a
choriocarcinoma model (BeWo) of differentiating cytotrophoblastic
cells, indicating that it should be accessible in vivo to
circulating anti-phosphatidylserine antibodies (Rote et al., 1995).
Indeed, Vogt et al. (1996) showed that the monoclonal antibody
3SB9b, which reacts with phosphatidylserine but not cardiolipin,
induced a significant reduction in both fetal and placental weights
in a mouse model for the anti-phospholipid antibody syndrome
[0384] These authors developed a model for explaining miscarriages
associated with anti-phospholipid antibodies:
anti-phosphatidylserine antibody reveals sites for prothrombin
binding on the surface of the trophoblast, most likely by removing
Annexin V (Vogt et al., 1997). Trophoblast differentiation is
associated with externalization of phosphatidylserine from the
inner to the outer surface of the plasma membrane. Normally,
externalization of phosphatidylserine is concurrent with binding of
Annexin V, which prevents the phosphatidylserine-rich surface from
acting as a site for activation of coagulation. Thus, when
anti-phospholipid antibodies are present, they prevent Annexin V
binding and lead to a procoagulant state (Vogt et al., 1997).
[0385] Anti-PE antibodies are frequently associated with lupus
anticoagulants (LAC sera). The role of PE and anti-PE in LAC is
extremely complex, see, e.g., Smirnov et al. (1995; incorporated
herein by reference), where various hypotheses are set forth.
Smirnov et al. (1995) report that, in the presence of activated
protein C and PE, LAC plasma clots faster than normal plasma. Rauch
et al. (1986) characterize LAC anti-phospholipid antibodies as
prolonging the clotting time in in vitro coagulation assays.
[0386] Vlachoyiannopoulos et al. (1993; incorporated herein by
reference) tested SLE and APS sera by ELISA for antibodies to
phosphatidylethanolamine and cardiolipin, as compared to healthy
blood donors. Both SLE and APS patients were reported to present a
higher titer of IgM anti-PE antibodies than normal subjects, while
the IgG and IgA anti-PE reactivity reportedly did not differ. It
was suggested that IgA and IgG anti-PE antibodies may occur in low
titers as natural autoantibodies in normal subjects
(Vlachoyiannopoulos et al., 1993; incorporated herein by
reference).
[0387] Rauch et al. (1986; incorporated herein by reference)
produced hybridomas by fusing lymphocytes from 13 systemic lupus
erythematosus patients with a lymphoblastoid line. They
demonstrated that the autoantibodies that prolonged clotting time
bound to hexagonal phase phospholipids, including natural and
synthetic forms of phosphatidylethanolamine (Rauch et al., 1986;
incorporated herein by reference). In contrast, lamellar
phospholipids, such as phosphatidylcholine and synthetic lamellar
forms of phosphatidylethanolamine, had no effect on the
anticoagulant activity (Rauch et al., 1986).
[0388] Rauch and Janoff (1990; incorporated herein by reference)
went on to show that immunization of mice with
phosphatidylethanolamine in the hexagonal II phase, but not in the
bilayer phase, resulted in the induction of anti-phospholipid
antibodies. These antibodies were strongly reactive with
phosphatidylethanolamine and had functional lupus anticoagulant
activity characteristic of autoantibodies from patients with
autoimmune disease (Rauch and Janoff, 1990).
[0389] The hexagonal II phase form of aminophospholipids should
thus be advantageously used to generate antibodies for use in the
present invention. Indeed, Trudell reported that antibodies raised
against TFA-(trifluoroacetyl-) protein adducts bind to
TFA-phosphatidylethanolamine in hexagonal phase phospholipid
micelles, but not in lamellar liposomes (Trudell et al., 1991a;
incorporated herein by reference). The authors suggested that
TFA-phosphatidylethanolamine adducts that reside in non-lamellar
domains on the hepatocyte surface could be recognition sites for
anti-TFA-adduct antibodies and potentially participate in
immune-mediated halothane hepatotoxicity (Trudell et al., 1991a).
It was later shown that these same antibodies cross-react with
TFA-dioleoylphosphatidylethanolamine when this adduct is
incorporated into the surface of hepatocytes (Trudell et al.,
1991b; incorporated herein by reference), thus supporting this
hypothesis.
[0390] Berard further explained the hexagonal II phase form of
aminophospholipids, such as PE (Berard et al., 1993; incorporated
herein by reference). In bilayers, phospholipids generally adopt a
gel structure, crystalline lattice or lamellar phase (Berard et
al., 1993). However, depending on the cholesterol content, protein
and ionic environments, phospholipids can easily change phases,
adopting a hexagonal II phase (Berard et al., 1993; incorporated
herein by reference). It is this hexagonal II phase of
aminophospholipids that is believed to be immunogenic, as initial
proposed for autoantibody generation in disease situations (Berard
et al., 1993; incorporated herein by reference).
[0391] Qamar et al. (1990; incorporated herein by reference) have
developed a variation on the hexagonal aminophospholipid
recognition theme. Using phosphatidylethanolamine as a model, these
authors reported that anti-PE antibodies from aPL-positive SLE sera
do not bind to PE, but in fact are directed to
lysophosphatidylethanolamine (IPE), a natural PE degradation
product and a likely contaminant of most PE preparations (Qamar et
al., 1990; incorporated herein by reference).
[0392] Other recent data indicate that most anti-phospholipid
antibodies recognize phospholipid in the context of nearby proteins
(Rote, 1996; Chamley et al., 1991). In plasma membranes, the
majority of the phospholipid appears to be naturally in
non-antigenic bilaminar form (Rote, 1996). Accessory molecules may
help facilitate the transition to hexagonal antigenic forms and
stabilize their expression (Galli et al., 1993). For example,
naturally occurring anti-phospholipid antibodies were first
reported to recognize complexes of cardiolipin or
phosphatidylserine with .beta..sub.2-glycoprotein I
(.beta..sub.2-GPI or apolipoprotein H, apoH) (Galli et al., 1990;
1993). .beta..sub.2-GPI is believed to stabilize phospholipids in
antigenic conformations that do not exist in pure phospholipids
(McNeil et al., 1990; U.S. Pat. No. 5,344,758; Chamley et al.,
1991; Matsuura et al., 1994). Prothrombin has also been implicated
in the phospholipid stabilization process (Bevers et al.,
1991).
[0393] Phospholipid-binding plasma proteins are also generally
necessary for antibody recognition of the electrically neutral or
zwitterionic phospholipid, phosphatidylethanolamine. Sugi and
McIntyre (1995; incorporated herein by reference) identified two
prominent PE-binding plasma proteins as high molecular weight
kininogen (HMWK or HK) and low molecular weight kininogen (LMWK or
LK). Anti-PE antibodies from patients with SLE and/or recurrent
spontaneous abortions were shown not to recognize PE, HMWK or LMWK
when they were presented independently as sole antigens on ELISA
plates (Sugi and McIntyre, 1995). Other anti-PE-positive sera that
did not react with PE-HMWK or PE-LMWK were suggested to recognize
factor XI or prekallikrein, which normally bind to HMWK (Sugi and
McIntyre, 1995; incorporated herein by reference).
[0394] The validity of these results was confirmed by showing that
intact HMWK binds to various phospholipids, such as cardiolipin,
phosphatidylserine, phosphatidylcholine and
phosphatidylethanolamine; but that anti-PE antibodies recognize
only a kininogen-PE complex, and do not recognize kininogens
presented with other phospholipid substrates (Sugi and McIntyre,
1996a; incorporated herein by reference). This indicates that PE
induces unique antigenic conformational changes in the kininogens
that are not induced when the kininogens bind to other
phospholipids (Sugi and McIntyre, 1996a).
[0395] It has further been suggested that kininogens can bind to
platelets by virtue of exposed PE in the platelet membrane (Sugi
and McIntyre, 1996b; incorporated herein by reference). Exogenously
added kininogen-dependent anti-PE was shown to increase
thrombin-induced platelet aggregation in vitro, but not to alter
ADP-induced aggregation (Sugi and McIntyre, 1996b; incorporated
herein by reference). In contrast, kininogen independent anti-PE,
which recognized PE per se, was reported not augment
thrombin-induced platelet aggregation. It was thus proposed that
kininogen dependent anti-PE may disrupt the normal anti-thrombotic
effects of kininogen (Sugi and McIntyre, 1996b; incorporated herein
by reference).
[0396] Anti-aminophospholipid antibodies from human patients are
therefore a mixture of antibodies that generally recognize
aminophospholipids stabilized by protein interactions (Rote, 1996).
The antibodies may bind to stabilized phospholipid epitopes, or may
bind to an epitope formed from the interaction of the phospholipid
and amino acids on the stabilizing protein (Rote, 1996). Either
way, such antibodies clearly recognize aminophospholipids in
natural membranes in the human body, probably associated with
plasma proteins (McNeil et al., 1990; Bevers et al., 1991). These
antibodies would thus be appropriate as starting materials for
generating an antibody for use in the therapeutic agent-targeting
agent constructs of the present invention.
[0397] To prepare an anti-aminophospholipid antibody from a human
patient, one would simply obtain human lymphocytes from an
individual having anti-aminophospholipid antibodies, for example
from human peripheral blood, spleen, lymph nodes, tonsils or the
like, utilizing techniques that are well known to those of skill in
the art. The use of peripheral blood lymphocytes will often be
preferred.
[0398] Human monoclonal antibodies may be obtained from the human
lymphocytes producing the desired anti-aminophospholipid antibodies
by immortalizing the human lymphocytes, generally in the same
manner as described above for generating any monoclonal antibody.
The reactivities of the antibodies in the culture supernatants are
generally first checked, employing one or more selected
aminophospholipid antigen(s), and the lymphocytes that exhibit high
reactivity are grown. The resulting lymphocytes are then fused with
a parent line of human or mouse origin, and further selection gives
the optimal clones.
[0399] The recovery of monoclonal antibodies from the immortalized
cells may be achieved by any method generally employed in the
production of monoclonal antibodies. For instance, the desired
monoclonal antibody may be obtained by cloning the immortalized
lymphocyte by the limiting dilution method or the like, selecting
the cell producing the desired antibody, growing the selected cells
in a medium or the abdominal cavity of an animal, and recovering
the desired monoclonal antibody from the culture supernatant or
ascites.
[0400] Such techniques have been used, for example, to isolate
human monoclonal antibodies to Pseudomonas aeruginosa epitopes
(U.S. Pat. Nos. 5,196,337 and 5,252,480, each incorporated herein
by reference); polyribosylribitol phosphate capsular
polysaccharides (U.S. Pat. No. 4,954,449, incorporated herein by
reference); the Rh(D) antigen (U.S. Pat. No. 5,665,356,
incorporated herein by reference); and viruses, such as human
immunodeficiency virus, respiratory syncytial virus, herpes simplex
virus, varicella zoster virus and cytomegalovirus (U.S. Pat. Nos.
5,652,138; 5,762,905; and 4,950,595, each incorporated herein by
reference).
[0401] The applicability of the foregoing techniques to the
generation of human anti-aminophospholipid antibodies is clear.
Rauch et al. (1986; incorporated herein by reference) generally
used such methods to produce hybridomas by fusing lymphocytes from
SLE patients with a lymphoblastoid line. This produced human
antibodies that bound to hexagonal phase phospholipids, including
natural and synthetic forms of phosphatidylethanolamine (Rauch et
al., 1986; incorporated herein by reference).
[0402] Additionally, the methods described in U.S. Pat. No.
5,648,077 (incorporated herein by reference) can be used to form a
trioma or a quadroma that produces a human antibody against a
selected aminophospholipid. In a general sense, a hybridoma cell
line comprising a parent rodent immortalizing cell, such as a
murine myeloma cell, e.g. SP-2, is fused to a human partner cell,
resulting in an immortalizing xenogeneic hybridoma cell. This
xenogeneic hybridoma cell is fused to a cell capable of producing
an anti-aminophospholipid human antibody, resulting in a trioma
cell line capable of generating human antibody effective against
such antigen in a human. Alternately, when greater stability is
desired, a trioma cell line which preferably no longer has the
capability of producing its own antibody is made, and this trioma
is then fused with a further cell capable of producing an antibody
useful against the aminophospholipid antigen to obtain a still more
stable hybridoma (quadroma) that produces antibody against the
antigen.
[0403] E5. Anti-Aminophospholipid Antibodies from Human
Lymphocytes
[0404] In vitro immunization, or antigen stimulation, may also be
used to generate a human anti-aminophospholipid antibody. Such
techniques can be used to stimulate peripheral blood lymphocytes
from both anti-aminophospholipid antibody-producing human patients,
and also from normal, healthy subjects. Indeed, Vlachoyiannopoulos
et al. (1993; incorporated herein by reference) reported that low
titer anti-aminophospholipid antibodies occur in normal subjects.
Even if this were not the case, anti-aminophospholipid antibodies
can be prepared from healthy human subjects, simply by stimulating
antibody-producing cells with aminophospholipids in vitro.
[0405] Such "in vitro immunization" involves antigen-specific
activation of non-immunized B lymphocytes, generally within a mixed
population of lymphocytes (mixed lymphocyte cultures, MLC). In
vitro immunizations may also be supported by B cell growth and
differentiation factors and lymphokines. The antibodies produced by
these methods are often IgM antibodies (Borrebaeck et al., 1986;
incorporated herein by reference).
[0406] Another method has been described (U.S. Pat. No. 5,681,729,
incorporated herein by reference) wherein human lymphocytes that
mainly produce IgG (or IgA) antibodies can be obtained. The method
involves, in a general sense, transplanting human lymphocytes to an
immunodeficient animal so that the human lymphocytes "take" in the
animal body; immunizing the animal with a desired antigen, so as to
generate human lymphocytes producing an antibody specific to the
antigen; and recovering the human lymphocytes producing the
antibody from the animal. The human lymphocytes thus produced can
be used to produce a monoclonal antibody by immortalizing the human
lymphocytes producing the antibody, cloning the obtained
immortalized human-originated lymphocytes producing the antibody,
and recovering a monoclonal antibody specific to the desired
antigen from the cloned immortalized human-originated
lymphocytes.
[0407] The immunodeficient animals that may be employed in this
technique are those that do not exhibit rejection when human
lymphocytes are transplanted to the animals. Such animals may be
artificially prepared by physical, chemical or biological
treatments. Any immunodeficient animal may be employed. The human
lymphocytes may be obtained from human peripheral blood, spleen,
lymph nodes, tonsils or the like.
[0408] The "taking" of the transplanted human lymphocytes in the
animals can be attained by merely administering the human
lymphocytes to the animals. The administration route is not
restricted and may be, for example, subcutaneous, intravenous or
intraperitoneal. The dose of the human lymphocytes is not
restricted, and can usually be 10.sup.6 to 10.sup.8 lymphocytes per
animal. The immunodeficient animal is then immunized with the
desired aminophospholipid antigen.
[0409] After the immunization, human lymphocytes are recovered from
the blood, spleen, lymph nodes or other lymphatic tissues by any
conventional method. For example, mononuclear cells can be
separated by the Ficoll-Hypaque (specific gravity: 1.077)
centrifugation method, and the monocytes removed by the plastic
dish adsorption method. The contaminating cells originating from
the immunodeficient animal may be removed by using an antiserum
specific to the animal cells. The antiserum may be obtained by, for
example, immunizing a second, distinct animal with the spleen cells
of the immunodeficient animal, and recovering serum from the
distinct immunized animal. The treatment with the antiserum may be
carried out at any stage. The human lymphocytes may also be
recovered by an immunological method employing a human
immunoglobulin expressed on the cell surface as a marker.
[0410] By these methods, human lymphocytes mainly producing IgG and
IgA antibodies specific to one or more selected
aminophospholipid(s) can be obtained. Monoclonal antibodies are
then obtained from the human lymphocytes by immortalization,
selection, cell growth and antibody production.
[0411] E6. Transgenic Mice Containing Human Antibody Libraries
[0412] Recombinant technology is now available for the preparation
of antibodies. In addition to the combinatorial immunoglobulin
phage expression libraries disclosed above, another molecular
cloning approach is to prepare antibodies from transgenic mice
containing human antibody libraries. Such techniques are described
in U.S. Pat. No. 5,545,807, incorporated herein by reference.
[0413] In a most general sense, these methods involve the
production of a transgenic animal that has inserted into its
germline genetic material that encodes for at least part of an
immunoglobulin of human origin or that can rearrange to encode a
repertoire of immunoglobulins. The inserted genetic material may be
produced from a human source, or may be produced synthetically. The
material may code for at least part of a known immunoglobulin or
may be modified to code for at least part of an altered
immunoglobulin.
[0414] The inserted genetic material is expressed in the transgenic
animal, resulting in production of an immunoglobulin derived at
least in part from the inserted human immunoglobulin genetic
material. It is found the genetic material is rearranged in the
transgenic animal, so that a repertoire of immunoglobulins with
part or parts derived from inserted genetic material may be
produced, even if the inserted genetic material is incorporated in
the germline in the wrong position or with the wrong geometry.
[0415] The inserted genetic material may be in the form of DNA
cloned into prokaryotic vectors such as plasmids and/or cosmids.
Larger DNA fragments are inserted using yeast artificial chromosome
vectors (Burke et al., 1987; incorporated herein by reference), or
by introduction of chromosome fragments (Richer and Lo, 1989;
incorporated herein by reference). The inserted genetic material
may be introduced to the host in conventional manner, for example
by injection or other procedures into fertilized eggs or embryonic
stem cells.
[0416] In preferred aspects, a host animal that initially does not
carry genetic material encoding immunoglobulin constant regions is
utilized, so that the resulting transgenic animal will use only the
inserted human genetic material when producing immunoglobulins.
This can be achieved either by using a naturally occurring mutant
host lacking the relevant genetic material, or by artificially
making mutants e.g., in cell lines ultimately to create a host from
which the relevant genetic material has been removed.
[0417] Where the host animal carries genetic material encoding
immunoglobulin constant regions, the transgenic animal will carry
the naturally occurring genetic material and the inserted genetic
material and will produce immunoglobulins derived from the
naturally occurring genetic material, the inserted genetic
material, and mixtures of both types of genetic material. In this
case the desired immunoglobulin can be obtained by screening
hybridomas derived from the transgenic animal, e.g., by exploiting
the phenomenon of allelic exclusion of antibody gene expression or
differential chromosome loss.
[0418] Once a suitable transgenic animal has been prepared, the
animal is simply immunized with the desired immunogen. Depending on
the nature of the inserted material, the animal may produce a
chimeric immunoglobulin, e.g. of mixed mouse/human origin, where
the genetic material of foreign origin encodes only part of the
immunoglobulin; or the animal may produce an entirely foreign
immunoglobulin, e.g. of wholly human origin, where the genetic
material of foreign origin encodes an entire immunoglobulin.
[0419] Polyclonal antisera may be produced from the transgenic
animal following immunization. Immunoglobulin-producing cells may
be removed from the animal to produce the immunoglobulin of
interest. Preferably, monoclonal antibodies are produced from the
transgenic animal, e.g., by fusing spleen cells from the animal
with myeloma cells and screening the resulting hybridomas to select
those producing the desired antibody. Suitable techniques for such
processes are described herein.
[0420] In an alternative approach, the genetic material may be
incorporated in the animal in such a way that the desired antibody
is produced in body fluids such as serum or external secretions of
the animal, such as milk, colostrum or saliva. For example, by
inserting in vitro genetic material encoding for at least part of a
human immunoglobulin into a gene of a mammal coding for a milk
protein and then introducing the gene to a fertilized egg of the
mammal, e.g., by injection, the egg may develop into an adult
female mammal producing milk containing immunoglobulin derived at
least in part from the inserted human immunoglobulin genetic
material. The desired antibody can then be harvested from the milk.
Suitable techniques for carrying out such processes are known to
those skilled in the art.
[0421] The foregoing transgenic animals are usually employed to
produce human antibodies of a single isotype, more specifically an
isotype that is essential for B cell maturation, such as IgM and
possibly IgD. Another preferred method for producing human
anti-aminophospholipid antibodies is to use the technology
described in U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126;
5,633,425; 5,661,016; and 5,770,429; each incorporated by
reference, wherein transgenic animals are described that are
capable of switching from an isotype needed for B cell development
to other isotypes.
[0422] In the development of a B lymphocyte, the cell initially
produces IgM with a binding specificity determined by the
productively rearranged V.sub.H and V.sub.L regions. Subsequently,
each B cell and its progeny cells synthesize antibodies with the
same L and H chain V regions, but they may switch the isotype of
the H chain. The use of mu or delta constant regions is largely
determined by alternate splicing, permitting IgM and IgD to be
coexpressed in a single cell. The other heavy chain isotypes
(gamma, alpha, and epsilon) are only expressed natively after a
gene rearrangement event deletes the C mu and C delta exons. This
gene rearrangement process, termed isotype switching, typically
occurs by recombination between so called switch segments located
immediately upstream of each heavy chain gene (except delta). The
individual switch segments are between 2 and 10 kb in length, and
consist primarily of short repeated sequences.
[0423] For these reasons, it is preferable that transgenes
incorporate transcriptional regulatory sequences within about 1-2
kb upstream of each switch region that is to be utilized for
isotype switching. These transcriptional regulatory sequences
preferably include a promoter and an enhancer element, and more
preferably include the 5' flanking (i.e., upstream) region that is
naturally associated (i.e., occurs in germline configuration) with
a switch region. Although a 5' flanking sequence from one switch
region can be operably linked to a different switch region for
transgene construction, in some embodiments it is preferred that
each switch region incorporated in the transgene construct have the
5' flanking region that occurs immediately upstream in the
naturally occurring germline configuration. Sequence information
relating to immunoglobulin switch region sequences is known (Mills
et al., 1990; Sideras et al., 1989; each incorporated herein by
reference).
[0424] In the method described in U.S. Pat. Nos. 5,545,806;
5,569,825; 5,625,126; 5,633,425; 5,661,016; and 5,770,429, the
human immunoglobulin transgenes contained within the transgenic
animal function correctly throughout the pathway of B-cell
development, leading to isotype switching. Accordingly, in this
method, these transgenes are constructed so as to produce isotype
switching and one or more of the following: (1) high level and
cell-type specific expression, (2) functional gene rearrangement,
(3) activation of and response to allelic exclusion, (4) expression
of a sufficient primary repertoire, (5) signal transduction, (6)
somatic hypermutation, and (7) domination of the transgene antibody
locus during the immune response.
[0425] An important requirement for transgene function is the
generation of a primary antibody repertoire that is diverse enough
to trigger a secondary immune response for a wide range of
antigens. The rearranged heavy chain gene consists of a signal
peptide exon, a variable region exon and a tandem array of
multi-domain constant region regions, each of which is encoded by
several exons. Each of the constant region genes encode the
constant portion of a different class of immunoglobulins. During
B-cell development, V region proximal constant regions are deleted
leading to the expression of new heavy chain classes. For each
heavy chain class, alternative patterns of RNA splicing give rise
to both transmembrane and secreted immunoglobulins.
[0426] The human heavy chain locus consists of approximately 200 V
gene segments spanning 2 Mb, approximately 30 D gene segments
spanning about 40 kb, six J segments clustered within a 3 kb span,
and nine constant region gene segments spread out over
approximately 300 kb. The entire locus spans approximately 2.5 Mb
of the distal portion of the long arm of chromosome 14. Heavy chain
transgene fragments containing members of all six of the known
V.sub.H families, the D and J gene segments, as well as the mu,
delta, gamma 3, gamma 1 and alpha 1 constant regions are known
(Berman et al., 1988; incorporated herein by reference). Genomic
fragments containing all of the necessary gene segments and
regulatory sequences from a human light chain locus is similarly
constructed.
[0427] The expression of successfully rearranged immunoglobulin
heavy and light transgenes usually has a dominant effect by
suppressing the rearrangement of the endogenous immunoglobulin
genes in the transgenic nonhuman animal. However, in certain
embodiments, it is desirable to effect complete inactivation of the
endogenous Ig loci so that hybrid immunoglobulin chains comprising
a human variable region and a non-human (e.g., murine) constant
region cannot be formed, for example by trans-switching between the
transgene and endogenous Ig sequences. Using embryonic stem cell
technology and homologous recombination, the endogenous
immunoglobulin repertoire can be readily eliminated. In addition,
suppression of endogenous Ig genes may be accomplished using a
variety of techniques, such as antisense technology.
[0428] In other aspects of the invention, it may be desirable to
produce a trans-switched immunoglobulin. Antibodies comprising such
chimeric trans-switched immunoglobulins can be used for a variety
of applications where it is desirable to have a non-human (e.g.,
murine) constant region, e.g., for retention of effector functions
in the host. The presence of a murine constant region can afford
advantages over a human constant region, for example, to provide
murine effector functions (e.g., ADCC, murine complement fixation)
so that such a chimeric antibody may be tested in a mouse disease
model. Subsequent to the animal testing, the human variable region
encoding sequence may be isolated, e.g., by PCR amplification or
cDNA cloning from the source (hybridoma clone), and spliced to a
sequence encoding a desired human constant region to encode a human
sequence antibody more suitable for human therapeutic use.
[0429] E7. Humanized Anti-Aminophospholipid Antibodies
[0430] Human antibodies generally have at least three potential
advantages for use in human therapy. First, because the effector
portion is human, it may interact better with the other parts of
the human immune system, e.g., to destroy target cells more
efficiently by complement-dependent cytotoxicity (CDC) or
antibody-dependent cellular cytotoxicity (ADCC). Second, the human
immune system should not recognize the antibody as foreign. Third,
the half-life in the human circulation will be similar to naturally
occurring human antibodies, allowing smaller and less frequent
doses to be given.
[0431] Various methods for preparing human anti-aminophospholipids
are provided herein. In addition to human antibodies, "humanized"
antibodies have many advantages. "Humanized" antibodies are
generally chimeric or mutant monoclonal antibodies from mouse, rat,
hamster, rabbit or other species, bearing human constant and/or
variable region domains or specific changes. Techniques for
generating a so-called "humanized" anti-aminophospholipid antibody
are well known to those of skill in the art.
[0432] Humanized antibodies also share the foregoing advantages.
First, the effector portion is still human. Second, the human
immune system should not recognize the framework or constant region
as foreign, and therefore the antibody response against such an
injected antibody should be less than against a totally foreign
mouse antibody. Third, injected humanized antibodies, as opposed to
injected mouse antibodies, will presumably have a half-life more
similar to naturally occurring human antibodies, also allowing
smaller and less frequent doses.
[0433] A number of methods have been described to produce humanized
antibodies. Controlled rearrangement of antibody domains joined
through protein disulfide bonds to form new, artificial protein
molecules or "chimeric" antibodies can be utilized (Konieczny et
al., 1981; incorporated herein by reference). Recombinant DNA
technology can also be used to construct gene fusions between DNA
sequences encoding mouse antibody variable light and heavy chain
domains and human antibody light and heavy chain constant domains
(Morrison et al., 1984; incorporated herein by reference).
[0434] DNA sequences encoding the antigen binding portions or
complementarity determining regions (CDR's) of murine monoclonal
antibodies can be grafted by molecular means into the DNA sequences
encoding the frameworks of human antibody heavy and light chains
(Jones et al., 1986; Riechmann et al., 1988; each incorporated
herein by reference). The expressed recombinant products are called
"reshaped" or humanized antibodies, and comprise the framework of a
human antibody light or heavy chain and the antigen recognition
portions, CDR's, of a murine monoclonal antibody.
[0435] Another method for producing humanized antibodies is
described in U.S. Pat. No. 5,639,641, incorporated herein by
reference. The method provides, via resurfacing, humanized rodent
antibodies that have improved therapeutic efficacy due to the
presentation of a human surface in the variable region. In the
method: (1) position alignments of a pool of antibody heavy and
light chain variable regions is generated to give a set of heavy
and light chain variable region framework surface exposed
positions, wherein the alignment positions for all variable regions
are at least about 98% identical; (2) a set of heavy and light
chain variable region framework surface exposed amino acid residues
is defined for a rodent antibody (or fragment thereof); (3) a set
of heavy and light chain variable region framework surface exposed
amino acid residues that is most closely identical to the set of
rodent surface exposed amino acid residues is identified; (4) the
set of heavy and light chain variable region framework surface
exposed amino acid residues defined in step (2) is substituted with
the set of heavy and light chain variable region framework surface
exposed amino acid residues identified in step (3), except for
those amino acid residues that are within 5 .ANG. of any atom of
any residue of the complementarity determining regions of the
rodent antibody; and (5) the humanized rodent antibody having
binding specificity is produced.
[0436] A similar method for the production of humanized antibodies
is described in U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and
5,530,101, each incorporated herein by reference. These methods
involve producing humanized immunoglobulins having one or more
complementarity determining regions (CDR's) and possible additional
amino acids from a donor immunoglobulin and a framework region from
an accepting human immunoglobulin. Each humanized immunoglobulin
chain usually comprises, in addition to the CDR's, amino acids from
the donor immunoglobulin framework that are capable of interacting
with the CDR's to effect binding affinity, such as one or more
amino acids that are immediately adjacent to a CDR in the donor
immunoglobulin or those within about 3 .ANG. as predicted by
molecular modeling. The heavy and light chains may each be designed
by using any one, any combination, or all of the various position
criteria described in U.S. Pat. Nos. 5,693,762; 5,693,761;
5,585,089; and 5,530,101, each incorporated herein by reference.
When combined into an intact antibody, the humanized
immunoglobulins are substantially non-immunogenic in humans and
retain substantially the same affinity as the donor immunoglobulin
to the original antigen.
[0437] An additional method for producing humanized antibodies is
described in U.S. Pat. Nos. 5,565,332 and 5,733,743, each
incorporated herein by reference. This method combines the concept
of humanizing antibodies with the phagemid libraries also described
in detail herein. In a general sense, the method utilizes sequences
from the antigen binding site of an antibody or population of
antibodies directed against an antigen of interest. Thus for a
single rodent antibody, sequences comprising part of the antigen
binding site of the antibody may be combined with diverse
repertoires of sequences of human antibodies that can, in
combination, create a complete antigen binding site.
[0438] The antigen binding sites created by this process differ
from those created by CDR grafting, in that only the portion of
sequence of the original rodent antibody is likely to make contacts
with antigen in a similar manner. The selected human sequences are
likely to differ in sequence and make alternative contacts with the
antigen from those of the original binding site. However, the
constraints imposed by binding of the portion of original sequence
to antigen and the shapes of the antigen and its antigen binding
sites, are likely to drive the new contacts of the human sequences
to the same region or epitope of the antigen. This process has
therefore been termed "epitope imprinted selection" (EIS).
[0439] Starting with an animal antibody, one process results in the
selection of antibodies that are partly human antibodies. Such
antibodies may be sufficiently similar in sequence to human
antibodies to be used directly in therapy or after alteration of a
few key residues. Sequence differences between the rodent component
of the selected antibody with human sequences could be minimized by
replacing those residues that differ with the residues of human
sequences, for example, by site directed mutagenesis of individual
residues, or by CDR grafting of entire loops. However, antibodies
with entirely human sequences can also be created. EIS therefore
offers a method for making partly human or entirely human
antibodies that bind to the same epitope as animal or partly human
antibodies respectively. In EIS, repertoires of antibody fragments
can be displayed on the surface of filamentous phase and the genes
encoding fragments with antigen binding activities selected by
binding of the phage to antigen.
[0440] Additional methods for humanizing antibodies contemplated
for use in the present invention are described in U.S. Pat. Nos.
5,750,078; 5,502,167; 5,705,154; 5,770,403; 5,698,417; 5,693,493;
5,558,864; 4,935,496; and 4,816,567, each incorporated herein by
reference.
[0441] E8. Mutagenesis by PCR
[0442] Site-specific mutagenesis is a technique useful in the
preparation of individual antibodies through specific mutagenesis
of the underlying DNA. The technique further provides a ready
ability to prepare and test sequence variants, incorporating one or
more of the foregoing considerations, whether humanizing or not, by
introducing one or more nucleotide sequence changes into the
DNA.
[0443] Although many methods are suitable for use in mutagenesis,
the use of the polymerase chain reaction (PCR.TM.) is generally now
preferred. This technology offers a quick and efficient method for
introducing desired mutations into a given DNA sequence. The
following text particularly describes the use of PCR.TM. to
introduce point mutations into a sequence, as may be used to change
the amino acid encoded by the given sequence. Adaptations of this
method are also suitable for introducing restriction enzyme sites
into a DNA molecule.
[0444] In this method, synthetic oligonucleotides are designed to
incorporate a point mutation at one end of an amplified segment.
Following PCR.TM., the amplified fragments are blunt-ended by
treating with Klenow fragments, and the blunt-ended fragments are
then ligated and subcloned into a vector to facilitate sequence
analysis.
[0445] To prepare the template DNA that one desires to mutagenize,
the DNA is subcloned into a high copy number vector, such as pUC19,
using restriction sites flanking the area to be mutated. Template
DNA is then prepared using a plasmid miniprep. Appropriate
oligonucleotide primers that are based upon the parent sequence,
but which contain the desired point mutation and which are flanked
at the 5' end by a restriction enzyme site, are synthesized using
an automated synthesizer. It is generally required that the primer
be homologous to the template DNA for about 15 bases or so. Primers
may be purified by denaturing polyacrylamide gel electrophoresis,
although this is not absolutely necessary for use in PCR.TM.. The
5' end of the oligonucleotides should then be phosphorylated.
[0446] The template DNA should be amplified by PCR.TM., using the
oligonucleotide primers that contain the desired point mutations.
The concentration of MgCl.sub.2 in the amplification buffer will
generally be about 15 mM. Generally about 20-25 cycles of PCR.TM.
should be carried out as follows: denaturation, 35 sec. at
95.degree. C.; hybridization, 2 min. at 50.degree. C.; and
extension, 2 min. at 72.degree. C. The PCR.TM. will generally
include a last cycle extension of about 10 min. at 72.degree. C.
After the final extension step, about 5 units of Klenow fragments
should be added to the reaction mixture and incubated for a further
15 min. at about 30.degree. C. The exonuclease activity of the
Klenow fragments is required to make the ends flush and suitable
for blunt-end cloning.
[0447] The resultant reaction mixture should generally be analyzed
by nondenaturing agarose or acrylamide gel electrophoresis to
verify that the amplification has yielded the predicted product.
One would then process the reaction mixture by removing most of the
mineral oils, extracting with chloroform to remove the remaining
oil, extracting with buffered phenol and then concentrating by
precipitation with 100% ethanol. Next, one should digest about half
of the amplified fragments with a restriction enzyme that cuts at
the flanking sequences used in the oligonucleotides. The digested
fragments are purified on a low gelling/melting agarose gel.
[0448] To subclone the fragments and to check the point mutation,
one would subclone the two amplified fragments into an
appropriately digested vector by blunt-end ligation. This would be
used to transform E. coli, from which plasmid DNA could
subsequently be prepared using a miniprep. The amplified portion of
the plasmid DNA would then be analyzed by DNA sequencing to confirm
that the correct point mutation was generated. This is important as
Taq DNA polymerase can introduce additional mutations into DNA
fragments.
[0449] The introduction of a point mutation can also be effected
using sequential PCR.TM. steps. In this procedure, the two
fragments encompassing the mutation are annealed with each other
and extended by mutually primed synthesis. This fragment is then
amplified by a second PCR.TM. step, thereby avoiding the blunt-end
ligation required in the above protocol. In this method, the
preparation of the template DNA, the generation of the
oligonucleotide primers and the first PCR.TM. amplification are
performed as described above. In this process, however, the chosen
oligonucleotides should be homologous to the template DNA for a
stretch of between about 15 and about 20 bases and must also
overlap with each other by about 10 bases or more.
[0450] In the second PCR.TM. amplification, one would use each
amplified fragment and each flanking sequence primer and carry
PCR.TM. for between about 20 and about 25 cycles, using the
conditions as described above. One would again subclone the
fragments and check that the point mutation was correct by using
the steps outlined above.
[0451] In using either of the foregoing methods, it is generally
preferred to introduce the mutation by amplifying as small a
fragment as possible. Of course, parameters such as the melting
temperature of the oligonucleotide, as will generally be influenced
by the GC content and the length of the oligo, should also be
carefully considered. The execution of these methods, and their
optimization if necessary, will be known to those of skill in the
art, and are further described in various publications, such as
Current Protocols in Molecular Biology, 1995, incorporated herein
by reference.
[0452] When performing site-specific mutagenesis, Table A can be
employed as a reference.
TABLE-US-00001 TABLE A Amino Acids Codons Alanine Ala A GCA GCC GCG
GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic
acid Glu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA
GGC GGG GGU Histidine His H CAC CAU Isoleucine Ile I AUA AUC AUU
Lysine Lys K AAA AAG Leucine Leu L UUA UUG CUA CUC CUG CUU
Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC
CCG CCU Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG
CGU Serine Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC
ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine
Tyr Y UAC UAU
[0453] E9. Antibody Fragments
[0454] Irrespective of the source of the original
anti-aminophospholipid antibody, either the intact antibody,
antibody multimers, or any one of a variety of functional,
antigen-binding regions of the antibody may be used in the present
invention. Exemplary functional regions include scFv, Fv, Fab', Fab
and F(ab').sub.2 fragments of the anti-aminophospholipid
antibodies. Techniques for preparing such constructs are well known
to those in the art and are further exemplified herein.
[0455] The choice of antibody construct may be influenced by
various factors. For example, prolonged half-life can result from
the active readsorption of intact antibodies within the kidney, a
property of the Fc piece of immunoglobulin. IgG based antibodies,
therefore, are expected to exhibit slower blood clearance than
their Fab' counterparts. However, Fab' fragment-based compositions
will generally exhibit better tissue penetrating capability.
[0456] Fab fragments can be obtained by proteolysis of the whole
immunoglobulin by the non-specific thiol protease, papain. Papain
must first be activated by reducing the sulphydryl group in the
active site with cysteine, 2-mercaptoethanol or dithiothreitol.
Heavy metals in the stock enzyme should be removed by chelation
with EDTA (2 mM) to ensure maximum enzyme activity. Enzyme and
substrate are normally mixed together in the ratio of 1:100 by
weight. After incubation, the reaction can be stopped by
irreversible alkylation of the thiol group with iodoacetamide or
simply by dialysis. The completeness of the digestion should be
monitored by SDS-PAGE and the various fractions separated by
protein A-Sepharose or ion exchange chromatography.
[0457] The usual procedure for preparation of F(ab').sub.2,
fragments from IgG of rabbit and human origin is limited
proteolysis by the enzyme pepsin. The conditions, 100.times.
antibody excess w/w in acetate buffer at pH 4.5, 37.degree. C.,
suggest that antibody is cleaved at the C-terminal side of the
inter-heavy-chain disulfide bond. Rates of digestion of mouse IgG
may vary with subclass and it may be difficult to obtain high
yields of active F(ab').sub.2, fragments without some undigested or
completely degraded IgG. In particular, IgG.sub.2b is highly
susceptible to complete degradation. The other subclasses require
different incubation conditions to produce optimal results, all of
which is known in the art.
[0458] Digestion of rat IgG by pepsin requires conditions including
dialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for
four hours with 1% w/w pepsin; IgG.sub.1 and IgG.sub.2a digestion
is improved if first dialyzed against 0.1 M formate buffer, pH 2.8,
at 4.degree. C., for 16 hours followed by acetate buffer.
IgG.sub.2b gives more consistent results with incubation in
staphylococcal V8 protease (3% w/w) in 0.1 M sodium phosphate
buffer, pH 7.8, for four hours at 37.degree. C.
[0459] The following patents and patent applications are
specifically incorporated herein by reference for the purposes of
even further supplementing the present teachings regarding the
preparation and use of functional, antigen-binding regions of
antibodies, including scFv, Fv, Fab', Fab and F(ab').sub.2
fragments of the anti-aminophospholipid antibodies: U.S. Pat. Nos.
5,855,866; 5,965,132; 6,004,555; and 6,093,399 and 5,877,289.
[0460] E10. Antibody Conjugates
[0461] Anti-aminophospholipid antibodies may be conjugated to
anti-cellular or cytotoxic agents, to prepare "immunotoxins"; or
operatively associated with components that are capable of directly
or indirectly stimulating coagulation, thus forming a
"coaguligand". In coaguligands, the targeting agents may be
directly linked to a direct or indirect coagulation factor, or may
be linked to a second binding region that binds and then releases a
direct or indirect coagulation factor. The `second binding region`
approach generally uses a coagulant-binding antibody as a second
binding region, thus resulting in a bispecific antibody construct.
The preparation and use of bispecific antibodies in general is well
known in the art, and is further disclosed herein.
[0462] In the preparation of immunotoxins, coaguligands and
bispecific antibodies, recombinant expression may be employed. The
nucleic acid sequences encoding the chosen antibody-based targeting
agent are attached, in-frame, to nucleic acid sequences encoding
the chosen toxin, coagulant, or second binding region to create an
expression unit or vector. Recombinant expression results in
translation of the new nucleic acid, to yield the desired protein
product. Although antibody-encoding nucleic acids are employed,
rather than protein binding ligands, the recombinant approach is
essentially the same as those described hereinabove.
[0463] Returning to conjugate technology, the preparation of
immunotoxins is generally well known in the art. However, certain
advantages may be achieved through the application of certain
preferred technology, both in the preparation of the immunotoxins
and in their purification for subsequent clinical administration.
For example, while IgG based immunotoxins will typically exhibit
better binding capability and slower blood clearance than their
Fab' counterparts, Fab' fragment-based immunotoxins will generally
exhibit better tissue penetrating capability as compared to IgG
based immunotoxins.
[0464] Additionally, while numerous types of disulfide-bond
containing linkers are known that can be successfully employed to
conjugate the toxin moiety to the targeting agent, certain linkers
will generally be preferred over other linkers, based on differing
pharmacological characteristics and capabilities. For example,
linkers that contain a disulfide bond that is sterically "hindered"
are to be preferred, due to their greater stability in vivo, thus
preventing release of the toxin moiety prior to binding at the site
of action.
[0465] A wide variety of cytotoxic agents are known that may be
conjugated to anti-aminophospholipid antibodies, including plant-,
fungus- and bacteria-derived toxins, such as ricin A chain or
deglycosylated A chain. The cross-linking of a toxin A chain to a
targeting agent, in certain cases, requires a cross-linker that
presents disulfide functions. The reason for this is unclear, but
is likely due to a need for certain toxin moieties to be readily
releasable from the targeting agent once the agent has "delivered"
the toxin to the targeted cells.
[0466] Each type of cross-linker, as well as how the cross-linking
is performed, will tend to vary the pharmacodynamics of the
resultant conjugate. Ultimately, in cases where a releasable toxin
is contemplated, one desires to have a conjugate that will remain
intact under conditions found everywhere in the body except the
intended site of action, at which point it is desirable that the
conjugate have good "release" characteristics. Therefore, the
particular cross-linking scheme, including in particular the
particular cross-linking reagent used and the structures that are
cross-linked, will be of some significance.
[0467] Depending on the specific toxin compound used as part of the
fusion protein, it may be necessary to provide a peptide spacer
operatively attaching the targeting agent and the toxin compound
which is capable of folding into a disulfide-bonded loop structure.
Proteolytic cleavage within the loop would then yield a
heterodimeric polypeptide wherein the targeting agent and the toxin
compound are linked by only a single disulfide bond. An example of
such a toxin is a Ricin A-chain toxin.
[0468] When certain other toxin compounds are utilized, a
non-cleavable peptide spacer may be provided to operatively attach
the targeting agent and the toxin compound of the fusion protein.
Toxins which may be used in conjunction with non-cleavable peptide
spacers are those which may, themselves, be converted by
proteolytic cleavage, into a cytotoxic disulfide-bonded form. An
example of such a toxin compound is a Pseudonomas exotoxin
compound.
[0469] There may be circumstances, such as when the target antigen
does not internalize by a route consistent with efficient
intoxication by targeting agent/toxin compounds, such as
immunotoxins, where one will desire to target chemotherapeutic
agents such as anti-tumor drugs, other cytokines, antimetabolites,
alkylating agents, hormones, and the like. A variety of
chemotherapeutic and other pharmacological agents have now been
successfully conjugated to antibodies and shown to function
pharmacologically. Exemplary antineoplastic agents that have been
investigated include doxorubicin, daunomycin, methotrexate,
vinblastine, and various others. Moreover, the attachment of other
agents such as neocarzinostatin, macromycin, trenimon and
.alpha.-amanitin has been described.
[0470] Where coagulation factors are used in connection with the
present invention, any covalent linkage to the antibody or
targeting agent should be made at a site distinct from its
functional coagulating site. The compositions are thus "linked" in
any operative manner that allows each region to perform its
intended function without significant impairment. Thus, the
targeting agents hind to aminophospholipids, and the coagulation
factor promotes blood clotting.
[0471] E11. Biochemical Cross-Linkers
[0472] In additional to the general information provided above,
anti-aminophospholipid antibodies may be conjugated to
anti-cellular or cytotoxic agents using certain preferred
biochemical cross-linkers. Cross-linking reagents are used to form
molecular bridges that tie together functional groups of two
different molecules. To link two different proteins in a step-wise
manner, hetero-bifunctional cross-linkers can be used that
eliminate unwanted homopolymer formation. Exemplary
hetero-bifunctional cross-linkers are referenced in Table B.
TABLE-US-00002 TABLE B HETERO-BIFUNCTIONAL CROSS-LINKERS Spacer Arm
Length linker Reactive Toward Advantages and Applications after
cross-linking SMPT Primary amines Greater stability 11.2 A
Sulfhydryls SPDP Primary amines Thiolation 6.8 A Sulfhydryls
Cleavable cross-linking LC-SPDP Primary amines Extended spacer arm
15.6 A Sulfhydryls Sulfo-LC-SPDP Primary amines Extended spacer arm
15.6 A Sulfhydryls Water-soluble SMCC Primary amines Stable
maleimide reactive group 11.6 A Sulfhydryls Enzyme-antibody
conjugation Hapten-carrier protein conjugation Sulfo-SMCC Primary
amines Stable maleimide reactive group 11.6 A Sulfhydryls
Water-soluble Enzyme-antibody conjugation MBS Primary amines
Enzyme-antibody conjugation 9.9 A Sulfhydryls Hapten-carrier
protein conjugation Sulfo-MBS Primary amines Water-soluble 9.9 A
Sulfhydryls SIAB Primary amines Enzyme-antibody conjugation 10.6 A
Sulfhydryls Sulfo-SIAB Primary amines Water-soluble 10.6 A
Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 A
Sulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary amines
Extended spacer arm 14.5 A Sulfhydryls Water-soluble EDC/Sulfo-NHS
Primary amines Hapten-Carrier conjugation 0 Carboxyl groups ABH
Carbohydrates Reacts with sugar groups 11.9 A Nonselective
[0473] Hetero-bifunctional cross-linkers contain two reactive
groups: one generally reacting with primary amine group (e.g.,
N-hydroxy succinimide) and the other generally reacting with a
thiol group (e.g., pyridyl disulfide, maleimides, halogens, etc.).
Through the primary amine reactive group, the cross-linker may
react with the lysine residue(s) of one protein (e.g., the selected
antibody or fragment) and through the thiol reactive group, the
cross-linker, already tied up to the first protein, reacts with the
cysteine residue (free sulfhydryl group) of the other protein
(e.g., the coagulant).
[0474] Compositions therefore generally have, or are derivatized to
have, a functional group available for cross-linking purposes. This
requirement is not considered to be limiting in that a wide variety
of groups can be used in this manner. For example, primary or
secondary amine groups, hydrazide or hydrazine groups, carboxyl
alcohol, phosphate, or alkylating groups may be used for binding or
cross-linking.
[0475] The spacer arm between the two reactive groups of a
cross-linkers may have various length and chemical compositions. A
longer spacer arm allows a better flexibility of the conjugate
components while some particular components in the bridge (e.g.,
benzene group) may lend extra stability to the reactive group or an
increased resistance of the chemical link to the action of various
aspects (e.g., disulfide bond resistant to reducing agents). The
use of peptide spacers, such as L-Leu-L-Ala-L-Leu-L-Ala, is also
contemplated.
[0476] It is preferred that a cross-linker having reasonable
stability in blood will be employed. Numerous types of
disulfide-bond containing linkers are known that can be
successfully employed to conjugate targeting and toxic or
coagulating agents. Linkers that contain a disulfide bond that is
sterically hindered may prove to give greater stability in vivo,
preventing release of the agent prior to binding at the site of
action. These linkers are thus one preferred group of linking
agents.
[0477] One of the most preferred cross-linking reagents for use in
immunotoxins is SMPT, which is a bifunctional cross-linker
containing a disulfide bond that is "sterically hindered" by an
adjacent benzene ring and methyl groups. It is believed that steric
hindrance of the disulfide bond serves a function of protecting the
bond from attack by thiolate anions such as glutathione which can
be present in tissues and blood, and thereby help in preventing
decoupling of the conjugate prior to the delivery of the attached
agent to the tumor site. It is contemplated that the SMPT agent may
also be used in connection with the bispecific ligands of this
invention.
[0478] The SMPT cross-linking reagent, as with many other known
cross-linking reagents, lends the ability to cross-link functional
groups such as the SH of cysteine or primary amines (e.g., the
epsilon amino group of lysine). Another possible type of
cross-linker includes the hetero-bifunctional photoreactive
phenylazides containing a cleavable disulfide bond such as
sulfosuccinimidyl-2-(p-azido salicylamido)
ethyl-1,3'-dithiopropionate. The N-hydroxy-succinimidyl group
reacts with primary amino groups and the phenylazide (upon
photolysis) reacts non-selectively with any amino acid residue.
[0479] In addition to hindered cross-linkers, non-hindered linkers
can also be employed in accordance herewith. Other useful
cross-linkers, not considered to contain or generate a protected
disulfide, include SATA, SPDP and 2-iminothiolane. The use of such
cross-linkers is well understood in the art.
[0480] Once conjugated, the conjugate is separated from
unconjugated targeting and therapeutic agents and from other
contaminants. A large a number of purification techniques are
available for use in providing conjugates of a sufficient degree of
purity to render them clinically useful. Purification methods based
upon size separation, such as gel filtration, gel permeation or
high performance liquid chromatography, will generally be of most
use. Other chromatographic techniques, such as Blue-Sepharose
separation, may also be used.
[0481] E12. Bispecific Antibodies
[0482] Bispecific antibodies are particularly useful in the
coaguligand aspects of the present invention. However, bispecific
antibodies in general may be employed, so long as one arm binds to
an aminophospholipid and the bispecific antibody is attached to a
therapeutic agent, generally at a site distinct from the antigen
binding sites. Bispecific antibodies that bind to both PS and PE
may also be used.
[0483] In general, the preparation of bispecific antibodies is also
well known in the art. One method involves the separate preparation
of antibodies having specificity for the targeted antigen, on the
one hand, and (as herein) a coagulating agent on the other. Peptic
F(ab'.gamma.).sub.2 fragments are prepared from the two chosen
antibodies, followed by reduction of each to provide separate
Fab'.gamma..sub.SH fragments. The SH groups on one of the two
partners to be coupled are then alkylated with a cross-linking
reagent such as o-phenylenedimaleimide to provide free maleimide
groups on one partner. This partner may then be conjugated to the
other by means of a thioether linkage, to give the desired
F(ab'.gamma.).sub.2 heteroconjugate. Other techniques are known
wherein cross-linking with SPDP or protein A is carried out, or a
trispecific construct is prepared.
[0484] Another method for producing bispecific antibodies is by the
fusion of two hybridomas to form a quadroma. As used herein, the
term "quadroma" is used to describe the productive fusion of two B
cell hybridomas. Using now standard techniques, two antibody
producing hybridomas are fused to give daughter cells, and those
cells that have maintained the expression of both sets of clonotype
immunoglobulin genes are then selected.
[0485] A preferred method of generating a quadroma involves the
selection of an enzyme deficient mutant of at least one of the
parental hybridomas. This first mutant hybridoma cell line is then
fused to cells of a second hybridoma that had been lethally
exposed, e.g., to iodoacetamide, precluding its continued survival.
Cell fusion allows for the rescue of the first hybridoma by
acquiring the gene for its enzyme deficiency from the lethally
treated hybridoma, and the rescue of the second hybridoma through
fusion to the first hybridoma. Preferred, but not required, is the
fusion of immunoglobulins of the same isotype, but of a different
subclass. A mixed subclass antibody permits the use if an
alternative assay for the isolation of a preferred quadroma.
[0486] In more detail, one method of quadroma development and
screening involves obtaining a hybridoma line that secretes the
first chosen MAb and making this deficient for the essential
metabolic enzyme, hypoxanthine-guanine phosphoribosyltransferase
(HGPRT). To obtain deficient mutants of the hybridoma, cells are
grown in the presence of increasing concentrations of 8-azaguanine
(1.times.10.sup.-7M to 1.times.10.sup.-5M). The mutants are
subcloned by limiting dilution and tested for their
hypoxanthine/aminopterin/thymidine (HAT) sensitivity. The culture
medium may consist of, for example, DMEM supplemented with 10% FCS,
2 mM L-Glutamine and 1 mM penicillin-streptomycin.
[0487] A complementary hybridoma cell line that produces the second
desired MAb is used to generate the quadromas by standard cell
fusion techniques. Briefly, 4.5.times.10.sup.7 HAT-sensitive first
cells are mixed with 2.8.times.10.sup.7 HAT-resistant second cells
that have been pre-treated with a lethal dose of the irreversible
biochemical inhibitor iodoacetamide (5 mM in phosphate buffered
saline) for 30 minutes on ice before fusion. Cell fusion is induced
using polyethylene glycol (PEG) and the cells are plated out in 96
well microculture plates. Quadromas are selected using
HAT-containing medium. Bispecific antibody-containing cultures are
identified using, for example, a solid phase isotype-specific ELISA
and isotype-specific immunofluorescence staining.
[0488] In one identification embodiment to identify the bispecific
antibody, the wells of microtiter plates (Falcon, Becton Dickinson
Labware) are coated with a reagent that specifically interacts with
one of the parent hybridoma antibodies and that lacks
cross-reactivity with both antibodies. The plates are washed,
blocked, and the supernatants (SNs) to be tested are added to each
well. Plates are incubated at room temperature for 2 hours, the
supernatants discarded, the plates washed, and diluted alkaline
phosphatase-anti-antibody conjugate added for 2 hours at room
temperature. The plates are washed and a phosphatase substrate,
e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added to each
well. Plates are incubated, 3N NaOH is added to each well to stop
the reaction, and the OD.sub.410 values determined using an ELISA
reader.
[0489] In another identification embodiment, microtiter plates
pre-treated with poly-L-lysine are used to bind one of the target
cells to each well, the cells are then fixed, e.g. using 1%
glutaraldehyde, and the bispecific antibodies are tested for their
ability to bind to the intact cell. In addition, FACS,
immunofluorescence staining, idiotype specific antibodies, antigen
binding competition assays, and other methods common in the art of
antibody characterization may be used in conjunction with the
present invention to identify preferred quadromas.
[0490] Following the isolation of the quadroma, the bispecific
antibodies are purified away from other cell products. This may be
accomplished by a variety of protein isolation procedures, known to
those skilled in the art of immunoglobulin purification. Means for
preparing and characterizing antibodies are well known in the art
(See, e.g., Antibodies: A Laboratory Manual, 1988).
[0491] For example, supernatants from selected quadromas are passed
over protein A or protein G sepharose columns to bind IgG
(depending on the isotype). The bound antibodies are then eluted
with, e.g. a pH 5.0 citrate buffer. The elute fractions containing
the BsAbs, are dialyzed against an isotonic buffer. Alternatively,
the eluate is also passed over an anti-immunoglobulin-sepharose
column. The BsAb is then eluted with 3.5 M magnesium chloride.
BsAbs purified in this way are then tested for binding activity by,
e.g., an isotype-specific ELISA and immunofluorescence staining
assay of the target cells, as described above.
[0492] Purified BsAbs and parental antibodies may also be
characterized and isolated by SDS-PAGE electrophoresis, followed by
staining with silver or Coomassie. This is possible when one of the
parental antibodies has a higher molecular weight than the other,
wherein the band of the BsAbs migrates midway between that of the
two parental antibodies. Reduction of the samples verifies the
presence of heavy chains with two different apparent molecular
weights.
F. Pharmaceutical Compositions
[0493] The most basic pharmaceutical compositions of the present
invention will generally comprise an effective amount of at least a
first therapeutic agent-targeting agent construct, dissolved or
dispersed in a pharmaceutically acceptable carrier or aqueous
medium. Combined therapeutics are also contemplated, and the same
type of underlying pharmaceutical compositions may be employed for
both single and combined medicaments.
[0494] The phrases "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce an adverse, allergic or other untoward reaction when
administered to an animal, or a human, as appropriate. As used
herein, "pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutical active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the active ingredient, its use in the
therapeutic compositions is contemplated. For human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards. Supplementary active ingredients can also be
incorporated into the compositions.
[0495] F1. Parenteral Formulations
[0496] The therapeutic agent-targeting agent constructs of the
present invention will most often be formulated for parenteral
administration, e.g., formulated for injection via the intravenous,
intramuscular, sub-cutaneous, transdermal, or other such routes,
including peristaltic administration and direct instillation into a
tumor or disease site (intracavity administration). The preparation
of an aqueous composition that contains a therapeutic
agent-targeting agent construct as an active ingredient will be
known to those of skill in the art in light of the present
disclosure. Typically, such compositions can be prepared as
injectables, either as liquid solutions or suspensions; solid forms
suitable for using to prepare solutions or suspensions upon the
addition of a liquid prior to injection can also be prepared; and
the preparations can also be emulsified.
[0497] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions; formulations including
sesame oil, peanut oil or aqueous propylene glycol; and sterile
powders for the extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the form should be sterile
and fluid to the extent that syringability exists. It should be
stable under the conditions of manufacture and storage and should
be preserved against the contaminating action of microorganisms,
such as bacteria and fungi.
[0498] The therapeutic agent-targeting agent compositions can be
formulated into a sterile aqueous composition in a neutral or salt
form. Solutions of the therapeutic agent-targeting agents as free
base or pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Pharmaceutically acceptable salts, include the acid addition salts
(formed with the free amino groups of the protein), and those that
are formed with inorganic acids such as, for example, hydrochloric
or phosphoric acids, or such organic acids as acetic,
trifluoroacetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0499] Suitable carriers include solvents and dispersion media
containing, for example, water, ethanol, polyol (for example,
glycerol, propylene glycol, and liquid polyethylene glycol, and the
like), suitable mixtures thereof, and vegetable oils. In many
cases, it will be preferable to include isotonic agents, for
example, sugars or sodium chloride. The proper fluidity can be
maintained, for example, by the use of a coating, such as lecithin,
by the maintenance of the required particle size in the case of
dispersion and/or by the use of surfactants.
[0500] Under ordinary conditions of storage and use, all such
preparations should contain a preservative to prevent the growth of
microorganisms. The prevention of the action of microorganisms can
be brought about by various antibacterial and antifungal agents,
for example, parabens, chlorobutanol, phenol, sorbic acid,
thimerosal, and the like. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0501] Prior to or upon formulation, the therapeutic
agent-targeting agent constructs should be extensively dialyzed to
remove undesired small molecular weight molecules, and/or
lyophilized for more ready formulation into a desired vehicle,
where appropriate. Sterile injectable solutions are prepared by
incorporating the active therapeutic agent-targeting agents in the
required amount in the appropriate solvent with various of the
other ingredients enumerated above, as desired, followed by
filtered sterilization. Generally, dispersions are prepared by
incorporating the various sterilized active ingredients into a
sterile vehicle that contains the basic dispersion medium and the
required other ingredients from those enumerated above.
[0502] In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum-drying and freeze-drying techniques that yield a powder
of the active therapeutic agent-targeting agent ingredient, plus
any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0503] Suitable pharmaceutical compositions in accordance with the
invention will generally include an amount of the therapeutic
agent-targeting agent construct admixed with an acceptable
pharmaceutical diluent or excipient, such as a sterile aqueous
solution, to give a range of final concentrations, depending on the
intended use. The techniques of preparation are generally well
known in the art as exemplified by Remington's Pharmaceutical
Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated
herein by reference. It should be appreciated that endotoxin
contamination should be kept minimally at a safe level, for
example, less that 0.5 ng/mg protein. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards.
[0504] Upon formulation, therapeutic agent-targeting agent
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. Formulations are easily administered in a variety of
dosage forms, such as the type of injectable solutions described
above, but other pharmaceutically acceptable forms are also
contemplated, e.g., tablets, pills, capsules or other solids for
oral administration, suppositories, pessaries, nasal solutions or
sprays, aerosols, inhalants, liposomal forms and the like.
Pharmaceutical "slow release" capsules or compositions may also be
used. Slow release formulations are generally designed to give a
constant drug level over an extended period and may be used to
deliver therapeutic agent-targeting agent constructs in accordance
with the present invention.
[0505] F2. Liposomes and Nanocapsules
[0506] In certain embodiments, liposomes and/or nanoparticles may
also be employed with the therapeutic agent-targeting agent
constructs. The formation and use of liposomes is generally known
to those of skill in the art, as summarized below.
[0507] Liposomes are formed from phospholipids that are dispersed
in an aqueous medium and spontaneously form multilamellar
concentric bilayer vesicles (also termed multilamellar vesicles
(MLVs). MLVs generally have diameters of from 25 nm to 4 .mu.m.
Sonication of MLVs results in the formation of small unilamellar
vesicles (SUVs) with diameters in the range of 200 to 500 .ANG.,
containing an aqueous solution in the core.
[0508] Phospholipids can form a variety of structures other than
liposomes when dispersed in water, depending on the molar ratio of
lipid to water. At low ratios the liposome is the preferred
structure. The physical characteristics of liposomes depend on pH,
ionic strength and the presence of divalent cations. Liposomes can
show low permeability to ionic and polar substances, but at
elevated temperatures undergo a phase transition which markedly
alters their permeability. The phase transition involves a change
from a closely packed, ordered structure, known as the gel state,
to a loosely packed, less-ordered structure, known as the fluid
state. This occurs at a characteristic phase-transition temperature
and results in an increase in permeability to ions, sugars and
drugs.
[0509] Liposomes interact with cells via four different mechanisms:
Endocytosis by phagocytic cells of the reticuloendothelial system
such as macrophages and neutrophils; adsorption to the cell
surface, either by nonspecific weak hydrophobic or electrostatic
forces, or by specific interactions with cell-surface components;
fusion with the plasma cell membrane by insertion of the lipid
bilayer of the liposome into the plasma membrane, with simultaneous
release of liposomal contents into the cytoplasm; and by transfer
of liposomal lipids to cellular or subcellular membranes, or vice
versa, without any association of the liposome contents. Varying
the liposome formulation can alter which mechanism is operative,
although more than one may operate at the same time.
[0510] Nanocapsules can generally entrap compounds in a stable and
reproducible way. To avoid side effects due to intracellular
polymeric overloading, such ultrafine particles (sized around 0.1
.mu.m) should be designed using polymers able to be degraded in
vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet
these requirements are contemplated for use in the present
invention, and such particles may be are easily made.
G. Therapeutic Kits
[0511] This invention also provides therapeutic kits comprising
therapeutic agent-targeting agent constructs for use in the present
treatment methods. Such kits will generally contain, in suitable
container means, a pharmaceutically acceptable formulation of at
least one therapeutic agent-targeting agent construct. The kits may
also contain other pharmaceutically acceptable formulations, either
for diagnosis/imaging or combined therapy. For example, such kits
may contain any one or more of a range of chemotherapeutic or
radiotherapeutic drugs; anti-angiogenic agents; anti-tumor cell
antibodies; and/or anti-tumor vasculature or anti-tumor stroma
immunotoxins or coaguligands.
[0512] The kits may have a single container (container means) that
contains the therapeutic agent-targeting agent construct, with or
without any additional components, or they may have distinct
containers for each desired agent. Where combined therapeutics are
provided, a single solution may be pre-mixed, either in a molar
equivalent combination, or with one component in excess of the
other. Alternatively, each of the therapeutic agent-targeting agent
construct and other anti-cancer agent components of the kit may be
maintained separately within distinct containers prior to
administration to a patient.
[0513] When the components of the kit are provided in one or more
liquid solutions, the liquid solution is preferably an aqueous
solution, with a sterile aqueous solution being particularly
preferred. However, the components of the kit may be provided as
dried powder(s). When reagents or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container.
[0514] The containers of the kit will generally include at least
one vial, test tube, flask, bottle, syringe or other container
means, into which the therapeutic agent-targeting agent construct,
and any other desired agent, may be placed and, preferably,
suitably aliquoted. Where separate components are included, the kit
will also generally contain a second vial or other container into
which these are placed, enabling the administration of separated
designed doses. The kits may also comprise a second/third container
means for containing a sterile, pharmaceutically acceptable buffer
or other diluent.
[0515] The kits may also contain a means by which to administer the
therapeutic agent-targeting agent construct to an animal or
patient, e.g., one or more needles or syringes, or even an eye
dropper, pipette, or other such like apparatus, from which the
formulation may be injected into the animal or applied to a
diseased area of the body. The kits of the present invention will
also typically include a means for containing the vials, or such
like, and other component, in close confinement for commercial
sale, such as, e.g., injection or blow-molded plastic containers
into which the desired vials and other apparatus are placed and
retained.
H. Tumor Treatment
[0516] The most important use of the present invention is in the
treatment of vascularized, malignant tumors; with the treatment of
benign tumors, such as BPH, also being contemplated. The invention
may also be used in the therapy of other diseases and disorders
having, as a component of the disease, prothrombotic blood vessels.
Such vasculature-associated diseases include diabetic retinopathy,
macular degeneration, vascular restenosis, including restenosis
following angioplasty, arteriovenous malformations (AVM),
meningioma, hemangioma, neovascular glaucoma and psoriasis; and
also angiofibroma, arthritis, rheumatoid arthritis, atherosclerotic
plaques, corneal graft neovascularization, hemophilic joints,
hypertrophic scars, osier-weber syndrome, pyogenic granuloma
retrolental fibroplasia, scleroderma, trachoma, vascular adhesions,
synovitis, dermatitis, various other inflammatory diseases and
disorders, and even endometriosis.
[0517] The therapeutic agent-targeting agent construct treatment of
the invention is most preferably exploited for the treatment of
solid tumors. Such uses may employ therapeutic agent-targeting
agent constructs alone or in combination with chemotherapeutic,
radiotherapeutic, apoptopic, anti-angiogenic agents and/or
immunotoxins or coaguligands. The therapeutic agent-targeting agent
construct methods provided by this invention are broadly applicable
to the treatment of any malignant tumor having a vascular
component. Typical vascularized tumors are the solid tumors,
particularly carcinomas, which require a vascular component for the
provision of oxygen and nutrients. Exemplary solid tumors that may
be treated using the invention include, but are not limited to,
carcinomas of the lung, breast, ovary, stomach, pancreas, larynx,
esophagus, testes, liver, parotid, biliary tract, colon, rectum,
cervix, uterus, endometrium, kidney, bladder, prostate, thyroid,
squamous cell carcinomas, adenocarcinomas, small cell carcinomas,
melanomas, gliomas, neuroblastomas, and the like.
[0518] The present invention is contemplated for use in the
treatment of any patient that presents with a solid tumor. However,
in that this invention is particularly successful in the treatment
of solid tumors of moderate or large sizes, patients in these
categories are likely to receive more significant benefits from
treatment in accordance with the methods and compositions provided
herein.
[0519] Therefore, in general, the invention can be used to treat
tumors of about 0.3-0.5 cm and upwards, although it is a better use
of the invention to treat tumors of greater than 0.5 cm in size.
From the studies already conducted in acceptable animal models, it
is believed that patients presenting with tumors of between about
1.0 and about 2.0 cm in size will be in the preferred treatment
group of patients for therapeutic agent-targeting agent therapy,
although tumors up to and including the largest tumors found in
humans may also be treated.
[0520] Although the present invention is not generally intended as
a preventative or prophylactic treatment, use of the invention is
certainly not confined to the treatment of patients having tumors
of only moderate or large sizes. There are many reasons underlying
this aspect of the breadth of the invention. For example, a patient
presenting with a primary tumor of moderate size or above may also
have various other metastatic tumors that are considered to be
small-sized or even in the earlier stages of metastatic tumor
seeding. Given that the therapeutic agent-targeting agent
constructs, or combinations, of the invention are generally
administered into the systemic circulation of a patient, they will
naturally have effects on the secondary, smaller and metastatic
tumors, although this may not be the primary intent of the
treatment. Furthermore, even in situations where the tumor mass as
a whole is a single small tumor, certain beneficial anti-tumor
effects will result from the use of the present therapeutic
agent-targeting agent treatment.
[0521] The guidance provided herein regarding the most suitable
patients for use in connection with the present invention is
intended as teaching that certain patient's profiles may assist
with the selection of patients for treatment by the present
invention. The pre-selection of certain patients, or categories of
patients, does not in any way negate the basic usefulness of the
present invention in connection with the treatment of all patients
having a vascularized tumor. A further consideration is the fact
that the assault on the tumor provided by the therapeutic
agent-targeting agent construct of the invention may predispose the
tumor to further therapeutic treatment, such that the subsequent
treatment results in an overall synergistic effect or even leads to
total remission or cure.
[0522] It is not believed that any particular type of tumor should
be excluded from treatment using the present invention. However,
the type of tumor cells may be relevant to the use of the invention
in combination with secondary therapeutic agents, particularly
chemotherapeutics and anti-tumor cell immunotoxins. As the effect
of the present therapy is to destroy the tumor vasculature, and as
the vasculature is substantially or entirely the same in all solid
tumors, it will be understood that the present therapeutic
agent-targeting agent methodology is widely or entirely applicable
to the treatment of all solid tumors, irrespective of the
particular phenotype or genotype of the tumor cells themselves.
[0523] Therapeutically effective closes of therapeutic
agent-targeting agent constructs are readily determinable using
data from an animal model, as shown in the studies detailed herein.
Experimental animals bearing solid tumors are frequently used to
optimize appropriate therapeutic doses prior to translating to a
clinical environment. Such models are known to be very reliable in
predicting effective anti-cancer strategies. For example, mice
bearing solid tumors, such as used in the Examples, are widely used
in pre-clinical testing. The inventors have used such art-accepted
mouse models to determine working ranges of therapeutic
agent-targeting agent constructs that give beneficial anti-tumor
effects with minimal toxicity.
[0524] As is known in the art, there are realistic objectives that
may be used as a guideline in connection with pre-clinical testing
before proceeding to clinical treatment. However, due to the safety
already demonstrated in accepted models, pre-clinical testing of
the present invention will be more a matter of optimization, rather
than to confirm effectiveness. Thus, pre-clinical testing may be
employed to select the most advantageous therapeutic
agent-targeting agent constructs, doses or combinations.
[0525] Any therapeutic agent-targeting agent dose, or combined
medicament, that results in any consistent detectable tumor
vasculature destruction, thrombosis and anti-tumor effects will
still define a useful invention. Destructive, thrombotic and
necrotic effects should be observed in between about 10% and about
40-50% of the tumor blood vessels and tumor tissues, upwards to
between about 50% and about 99% of such effects being observed. The
present invention may also be effective against vessels downstream
of the tumor, i.e., target at least a sub-set of the draining
vessels, particularly as cytokines released from the tumor will be
acting on these vessels, changing their antigenic profile.
[0526] It will also be understood that even in such circumstances
where the anti-tumor effects of the therapeutic agent-targeting
agent dose, or combined therapy, are towards the low end of this
range, it may be that this therapy is still equally or even more
effective than all other known therapies in the context of the
particular tumor targets. It is unfortunately evident to a
clinician that certain tumors cannot be effectively treated in the
intermediate or long term, but that does not negate the usefulness
of the present therapy, particularly where it is at least about as
effective as the other strategies generally proposed.
[0527] In designing appropriate doses of therapeutic
agent-targeting agent constructs, or combined therapeutics, for the
treatment of vascularized tumors, one may readily extrapolate from
the animal studies described herein in order to arrive at
appropriate doses for clinical administration. To achieve this
conversion, one would account for the mass of the agents
administered per unit mass of the experimental animal and,
preferably, account for the differences in the body surface area
between the experimental animal and the human patient. All such
calculations are well known and routine to those of ordinary skill
in the art.
[0528] For example, in taking the successful doses of annexin-TF
constructs in the mouse studies, and applying standard calculations
based upon mass and surface area, effective doses for use in human
patients would be between about 1 mg and about 500 mgs antibody per
patient, and preferably, between about 10 mgs and about 100 mgs
antibody per patient.
[0529] Accordingly, using this information, the inventors
contemplate that useful low doses of therapeutic agent-targeting
agent constructs for human administration will be about 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 15, 20, 25 or about 30 mgs or so per patient;
and useful high doses of therapeutic agent-targeting agent
constructs for human administration will be about 250, 275, 300,
325, 350, 375, 400, 425, 450, 475 or about 500 mgs or so per
patient. Useful intermediate doses of therapeutic agent-targeting
agent constructs for human administration are contemplated to be
about 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200 or about
225 mgs or so per patient.
[0530] Any particular range using any of the foregoing recited
exemplary doses or any value intermediate between the particular
stated ranges is contemplated. It will also be understood that
therapeutic agent-targeting agent constructs with coagulants can
generally be used at higher doses than those with toxins.
[0531] In general, dosage ranges of between about 5-100 mgs, about
10-80 mgs, about 20-70 mgs, about 25-60 mgs, or about 30-50 mgs or
so of therapeutic agent-targeting agent construct per patient will
be preferred. Notwithstanding these stated ranges, it will be
understood that, given the parameters and detailed guidance
presented herein, further variations in the active or optimal
ranges will be encompassed within the present invention. Although
doses in and around about 5 or 10 to about 70, 80, 90 or 100 rugs
per patient are currently preferred, it will be understood that
lower doses may be more appropriate in combination with other
agents, and that high doses can still be tolerated, particularly
given the enhanced safety of the coagulant constructs. The use of
human or humanized antibodies or binding proteins renders the
present invention even safer for clinical use, further reducing the
chances of significant toxicity or side effects in healthy
tissues.
[0532] The intention of the therapeutic regimens of the present
invention is generally to produce significant anti-tumor effects
whilst still keeping the dose below the levels associated with
unacceptable toxicity. In addition to varying the dose itself, the
administration regimen can also be adapted to optimize the
treatment strategy. A currently preferred treatment strategy is to
administer between about 1-500 mgs, and preferably, between about
10-100 mgs of the therapeutic agent-targeting agent construct, or
therapeutic cocktail containing such, about 3 times within about a
7 day period. For example, doses would be given on about day 1, day
3 or 4 and day 6 or 7.
[0533] In administering the particular doses themselves, one would
preferably provide a pharmaceutically acceptable composition
(according to FDA standards of sterility, pyrogenicity, purity and
general safety) to the patient systemically. Intravenous injection
is generally preferred, and the most preferred method is to employ
a continuous infusion over a time period of about 1 or 2 hours or
so. Although it is not required to determine such parameters prior
to treatment using the present invention, it should be noted that
the studies detailed herein result in at least some thrombosis
being observed specifically in the blood vessels of a solid tumor
within about 12-24 hours of injection, and that widespread tumor
necrosis is also observed in this period.
[0534] Naturally, before wide-spread use, clinical trials will be
conducted. The various elements of conducting a clinical trial,
including patient treatment and monitoring, will be known to those
of skill in the art in light of the present disclosure. The
following information is being presented as a general guideline for
use in establishing such trials.
[0535] Patients chosen for the first therapeutic agent-targeting
agent construct treatment studies will have failed to respond to at
least one course of conventional therapy, and will have objectively
measurable disease as determined by physical examination,
laboratory techniques, and/or radiographic procedures. Any
chemotherapy should be stopped at least 2 weeks before entry into
the study. Where murine monoclonal antibodies or antibody portions
are employed, the patients should have no history of allergy to
mouse immunoglobulin.
[0536] Certain advantages will be found in the use of an indwelling
central venous catheter with a triple lumen port. The therapeutic
agent-targeting agent constructs should be filtered, for example,
using a 0.22.mu. filter, and diluted appropriately, such as with
saline, to a final volume of 100 ml. Before use, the test sample
should also be filtered in a similar manner, and its concentration
assessed before and after filtration by determining the A.sub.280.
The expected recovery should be within the range of 87% to 99%, and
adjustments for protein loss can then be accounted for.
[0537] The therapeutic agent-targeting agent constructs may be
administered over a period of approximately 4-24 hours, with each
patient receiving 2-4 infusions at 2-7 day intervals.
Administration can also be performed by a steady rate of infusion
over a 7 day period. The infusion given at any dose level should be
dependent upon any toxicity observed. Hence, if Grade II toxicity
was reached after any single infusion, or at a particular period of
time for a steady rate infusion, further doses should be withheld
or the steady rate infusion stopped unless toxicity improved.
Increasing doses of therapeutic agent-targeting agent constructs
should be administered to groups of patients until approximately
60% of patients showed unacceptable Grade III or IV toxicity in any
category. Doses that are 2/3 of this value are defined as the safe
dose.
[0538] Physical examination, tumor measurements, and laboratory
tests should, of course, be performed before treatment and at
intervals up to 1 month later. Laboratory tests should include
complete blood counts, serum creatinine, creatine kinase,
electrolytes, urea, nitrogen, SGOT, bilirubin, albumin, and total
serum protein. Serum samples taken up to 60 days after treatment
should be evaluated by radioimmunoassay for the presence of the
administered therapeutic agent-targeting agent constructs, and
antibodies against any portions thereof. Immunological analyses of
sera, using any standard assay such as, for example, an ELISA or
RIA, will allow the pharmacokinetics and clearance of the
anti-aminophospholipid therapeutic agent to be evaluated.
[0539] To evaluate the anti-tumor responses, the patients should be
examined at 48 hours to 1 week and again at 30 days after the last
infusion. When palpable disease was present, two perpendicular
diameters of all masses should be measured daily during treatment,
within 1 week after completion of therapy, and at 30 days. To
measure nonpalpable disease, serial CT scans could be performed at
1-cm intervals throughout the chest, abdomen, and pelvis at 48
hours to 1 week and again at 30 days. Tissue samples should also be
evaluated histologically, and/or by flow cytometry, using biopsies
from the disease sites or even blood or fluid samples if
appropriate.
[0540] Clinical responses may be defined by acceptable measure. For
example, a complete response may be defined by the disappearance of
all measurable tumor 1 month after treatment. Whereas a partial
response may be defined by a 50% or greater reduction of the sum of
the products of perpendicular diameters of all evaluable tumor
nodules 1 month after treatment, with no tumor sites showing
enlargement. Similarly, a mixed response may be defined by a
reduction of the product of perpendicular diameters of all
measurable lesions by 50% or greater 1 month after treatment, with
progression in one or more sites.
[0541] In light of results from clinical trials, such as those
described above, an even more precise treatment regimen may be
formulated. Even so, some variation in dosage may later be
necessary depending on the condition of the subject being treated.
The physician responsible for administration will, in light of the
present disclosure, be able to determine the appropriate dose for
the individual subject. Such optimization and adjustment is
routinely carried out in the art and by no means reflects an undue
amount of experimentation.
I. Tumor Imaging
[0542] The present invention further provides combined tumor
treatment and imaging methods, based upon anti-aminophospholipid
binding ligands. Anti-aminophospholipid binding proteins or
antibodies that are linked to one or more detectable agents are
envisioned for use in pre-imaging the tumor, forming a reliable
image prior to the treatment, which itself targets the
aminophospholipid markers.
[0543] The anti-aminophospholipid imaging ligands or antibodies, or
conjugates thereof, will generally comprise an
anti-aminophospholipid antibody or binding ligand operatively
attached, or conjugated to, a detectable label. "Detectable labels"
are compounds or elements that can be detected due to their
specific functional properties, or chemical characteristics, the
use of which allows the component to which they are attached to be
detected, and further quantified if desired. Preferably, the
detectable labels are those detectable in vivo using non-invasive
methods.
[0544] Antibody and binding protein conjugates for use as
diagnostic agents generally fall into two classes, those for use in
in vitro diagnostics, such as in a variety of immunoassays, and
those for use in vivo diagnostic protocols. It is the in vivo
imaging methods that are particularly intended for use with this
invention.
[0545] Many appropriate imaging agents are known in the art, as are
methods for their attachment to antibodies and binding ligands
(see, e.g., U.S. Pat. Nos. 5,021,236 and 4,472,509, both
incorporated herein by reference). Certain attachment methods
involve the use of a metal chelate complex employing, for example,
an organic chelating agent such a DTPA attached to the antibody
(U.S. Pat. No. 4,472,509). Monoclonal antibodies may also be
reacted with an enzyme in the presence of a coupling agent such as
glutaraldehyde or periodate. Conjugates with fluorescein markers
are prepared in the presence of these coupling agents or by
reaction with an isothiocyanate.
[0546] An example of detectable labels are the paramagnetic ions.
In this case, suitable ions include chromium (III), manganese (II),
iron (III), iron (II), cobalt (II), nickel (II), copper (II),
neodymium (III), samarium (III), ytterbium (III), gadolinium (III),
vanadium (II), terbium (III), dysprosium (III), holmium (III) and
erbium (III), with gadolinium being particularly preferred.
[0547] Ions useful in other contexts, such as X-ray imaging,
include but are not limited to lanthanum (III), gold (III), lead
(II), and especially bismuth (III). Fluorescent labels include
rhodamine, fluorescein and renographin. Rhodamine and fluorescein
are often linked via an isothiocyanate intermediate.
[0548] In the case of radioactive isotopes for diagnostic
applications, suitable examples include .sup.14carbon,
.sup.51chromium, .sup.36chlorine, .sup.57cobalt, .sup.58cobalt,
copper.sup.67, .sup.152Eu, gallium.sup.67, .sup.3hydrogen,
iodine.sup.123, iodine.sup.125, iodine.sup.131, indium.sup.111,
.sup.59iron, .sup.32phosphorus, rhenium.sup.186, rhenium.sup.188,
.sup.75selenium, .sup.35sulphur, technetium.sup.99m and
yttrium.sup.90. .sup.125I is often being preferred for use in
certain embodiments, and technicium.sup.99m and indium.sup.111 are
also often preferred due to their low energy and suitability for
long range detection.
[0549] Radioactively labeled anti-aminophospholipid antibodies and
binding ligands for use in the present invention may be produced
according to well-known methods in the art. For instance,
intermediary functional groups that are often used to bind
radioisotopic metallic ions to antibodies are
diethylenetriaminepentaacetic acid (DTPA) and ethylene
diaminetetracetic acid (EDTA).
[0550] Monoclonal antibodies can also be iodinated by contact with
sodium or potassium iodide and a chemical oxidizing agent such as
sodium hypochlorite, or an enzymatic oxidizing agent, such as
lactoperoxidase. Anti-aminophospholipid antibodies according to the
invention may be labeled with technetium-.sup.99m by ligand
exchange process, for example, by reducing pertechnate with
stannous solution, chelating the reduced technetium onto a Sephadex
column and applying the antibody to this column; or by direct
labeling techniques, e.g., by incubating pertechnate, a reducing
agent such as SNCl.sub.2, a buffer solution such as
sodium-potassium phthalate solution, and the antibody.
[0551] Any of the foregoing type of detectably labeled
anti-aminophospholipid antibodies and aminophospholipid binding
ligands may be used in the imaging aspects of the present
invention. Although not previously proposed for use in combined
tumor imaging and treatment, the detectably-labeled annexins of
U.S. Pat. No. 5,627,036; WO 95/19791; WO 95/27903; WO 95/34315; WO
96/17618; and WO 98/04294; each incorporated herein by reference;
may also be employed.
[0552] WO 95/27903 (incorporated herein by reference) provides
annexins for use in detecting apoptotic cells. Any of the
annexin-detectable agent markers of WO 95/27903 may be used herein,
although it will be known that certain of these are more suitable
for in vitro uses. WO 95/27903 is also specifically incorporated
herein by reference for purposes of providing detectable kits that
may be adapted for combined use with the therapeutics of the
present invention.
[0553] Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO
98/04294; are also incorporated herein by reference for purposes of
further describing radiolabelled annexin conjugates for diagnostic
imaging. The intent of each of the foregoing documents is to
provide radiolabelled annexins for use in imaging vascular
thromboses, particularly in or near the heart, such as in deep vein
thrombosis, pulmonary embolism, myocardial infarction, atrial
fibrillation, problems with prosthetic cardiovascular materials,
stroke, and the like. These radiolabelled annexins were also
proposed for use in imaging activated platelets, e.g., in
conditions such as abscesses, restenosis, inflammation of joints,
clots in cerebral arteries, etc.
[0554] U.S. Pat. No. 5,627,036 (incorporated herein by reference)
also generally concerns `annexine` (annexin) binding ligands for
use in analyzing platelet phosphatidylserine. It is explained in
U.S. Pat. No. 5,627,036 that hemostatic disorders, such as
arterial, coronary and venous thrombosis, are usually idiopathic,
which makes prediction and prevention difficult. To recognize such
hemostatic disorders earlier, the detection of activated platelets
is proposed. The detectably labeled annexins compositions are thus
disclosed in order to detect activated platelets in hemostatic
disorders (U.S. Pat. No. 5,627,036).
[0555] Although proposing a wide range of diagnostic uses, none of
WO 95/19791; WO 95/34315; WO 96/17618; or WO 98/04294 make
reference to imaging the vasculature of solid tumors. Neither does
U.S. Pat. No. 5,627,036 make any such suggestions. Nonetheless, the
disclosed detectable and radiolabelled annexin compositions per se
may now be used to advantage in this regard, in light of the
surprising discoveries disclosed herein.
[0556] In particular, U.S. Pat. No. 5,627,036 (incorporated herein
by reference) discloses annexins detectably labeled with
fluorescein isothiocyanate; radioisotopes of halogens, technetium,
lead, mercury, thallium or indium; and paramagnetic contrast
agents.
[0557] WO 95/19791 (incorporated herein by reference) provides
conjugates of annexin bonded to an N.sub.2S.sub.2 chelate that can
be radiolabelled by complexing a radionuclide to the chelate. WO
95/34315 (incorporated herein by reference) provides annexin
conjugates comprising one or more galactose residues with the
N.sub.2S.sub.2 chelate. The galactose moiety is said to facilitate
the rapid elimination of the radiolabelled conjugate from the
circulation, reducing radiation damage to non-target tissues and
background `noise.`
[0558] WO 96/17618 (incorporated herein by reference) in turn
provides annexin conjugates suitable for radiolabeling with
diagnostic imaging agents that comprise an annexin with a cluster
of galactose residues and an N.sub.2S.sub.2 chelate. These are
reported to have a shorter circulating half-life and a higher
binding affinity for target sites than the foregoing radiolabeled
annexin-galactose conjugates.
[0559] Still further radiolabeled annexin conjugates are provided
by WO 98/04294 (incorporated herein by reference). These conjugates
comprise an annexin that is modified to provide an accessible
sulphydryl group conjugated to a hexose moiety that is recognized
by a mammalian liver receptor. Annexin multimer conjugates and
chelating compounds conjugated via esterase-sensitive bonds are
also provided.
[0560] Each of WO 95/19791; WO 95/34315; WO 96/17618; and WO
98/04294; are also specifically incorporated herein by reference
for purposes of providing annexin conjugate components for
radiolabelling that are amenable to packaging in "cold kits", i.e.,
wherein the components are provided in separate vials. U.S. Pat.
No. 5,627,036 similarly provides kits comprising a carrier being
compartmentalized to receive detectably labeled annexins that may
be adapted for use herewith.
[0561] Although suitable for use in in vitro diagnostics, the
present aminophospholipid detection methods are more intended for
forming an image of the tumor vasculature of a patient prior to
treatment with therapeutic agent-targeting agent constructs. The in
vivo diagnostic or imaging methods generally comprise administering
to a patient a diagnostically effective amount of an
anti-aminophospholipid antibody or binding ligand that is
conjugated to a marker that is detectable by non-invasive methods.
The antibody- or binding ligand-marker conjugate is allowed
sufficient time to localize and bind to the aminophospholipid
expressed on the luminal surface of the tumor vasculature. The
patient is then exposed to a detection device to identify the
detectable marker, thus forming an image of the tumor
vasculature.
[0562] The nuclear magnetic spin-resonance isotopes, such as
gadolinium, are detected using a nuclear magnetic imaging device;
and radioactive substances, such as technicium.sup.99m or
indium.sup.111, are detected using a gamma scintillation camera or
detector. U.S. Pat. No. 5,627,036 is also specifically incorporated
herein by reference for purposes of providing even further guidance
regarding the safe and effective introduction of such detectably
labeled constructs into the blood of an individual, and means for
determining the distribution of the detectably labeled annexin
extracorporally, e.g., using a gamma scintillation camera or by
magnetic resonance measurement.
[0563] Dosages for imaging embodiments are generally less than for
therapy, but are also dependent upon the age and weight of a
patient. A one time dose of between about 0.1, 0.5 or about 1 mg
and about 9 or 10 mgs, and more preferably, of between about 1 mg
and about 5-10 mgs of anti-aminophospholipid antibody- or
aminophospholipid binding ligand-conjugate per patient is
contemplated to be useful. U.S. Pat. No. 5,627,036; and WO
95/19791, each incorporated herein by reference, are also
instructive regarding doses of detectably-labeled annexins.
J. Combination Therapies
[0564] The therapeutic agent-targeting agent treatment methods of
the present invention may be combined with any other methods
generally employed in the treatment of the particular tumor,
disease or disorder that the patient exhibits. So long as a
particular therapeutic approach is not known to be detrimental to
the patient's condition in itself, and does not significantly
counteract the therapeutic agent-targeting agent treatment, its
combination with the present invention is contemplated.
[0565] In connection solid tumor treatment, the present invention
may be used in combination with classical approaches, such as
surgery, radiotherapy, chemotherapy, and the like. The invention
therefore provides combined therapies in which therapeutic
agent-targeting agent constructs are used simultaneously with,
before, or after surgery or radiation treatment; or are
administered to patients with, before, or after conventional
chemotherapeutic, radiotherapeutic or anti-angiogenic agents, or
targeted immunotoxins or coaguligands.
[0566] Combination therapy for other vascular diseases is also
contemplated. A particular example of such is benign prostatic
hyperplasia (BPH), which may be treated with therapeutic
agent-targeting agent constructs in combination other treatments
currently practiced in the art. For example, targeting of
immunotoxins to markers localized within BPH, such as PSA.
[0567] When one or more agents are used in combination with the
therapeutic agent-targeting agent therapy, there is no requirement
for the combined results to be additive of the effects observed
when each treatment is conducted separately. Although at least
additive effects are generally desirable, any increased anti-tumor
effect above one of the single therapies would be of benefit. Also,
there is no particular requirement for the combined treatment to
exhibit synergistic effects, although this is certainly possible
and advantageous.
[0568] To practice combined anti-tumor therapy, one would simply
administer to an animal a therapeutic agent-targeting agent
construct in combination with another anti-cancer agent in a manner
effective to result in their combined anti-tumor actions within the
animal. The agents would therefore be provided in amounts effective
and for periods of time effective to result in their combined
presence within the tumor vasculature and their combined actions in
the tumor environment. To achieve this goal, the therapeutic
agent-targeting agent constructs and anti-cancer agents may be
administered to the animal simultaneously, either in a single
composition, or as two distinct compositions using different
administration routes.
[0569] Alternatively, the therapeutic agent-targeting agent
treatment may precede, or follow, the anti-cancer agent treatment
by, e.g., intervals ranging from minutes to weeks. In certain
embodiments where the anti-cancer agent and therapeutic
agent-targeting agent construct are applied separately to the
animal, one would ensure that a significant period of time did not
expire between the time of each delivery, such that the anti-cancer
agent and therapeutic agent-targeting agent composition would still
be able to exert an advantageously combined effect on the tumor. In
such instances, it is contemplated that one would contact the tumor
with both agents within about 5 minutes to about one week of each
other and, more preferably, within about 12-72 hours of each other,
with a delay time of only about 12-48 hours being most
preferred.
[0570] Exemplary anti-cancer agents that would be given prior to
the therapeutic agent-targeting agent construct are agents that
induce the expression of aminophospholipids within the tumor
vasculature. For example, agents that stimulate localized calcium
production and/or that induce apoptosis will generally result in
increased PS expression, which can then be targeted using a
subsequent anti-PS therapeutic agent-targeting agent construct.
Therapeutic agent-targeting agent constructs would be first
administered in other situations to cause tumor destruction,
followed by, e.g., anti-angiogenic therapies or therapies directed
to targeting necrotic tumor cells.
[0571] The general use of combinations of substances in cancer
treatment is well know. For example, U.S. Pat. No. 5,710,134
(incorporated herein by reference) discloses components that induce
necrosis in tumors in combination with non-toxic substances or
"prodrugs". The enzymes set free by necrotic processes cleave the
non-toxic "prodrug" into the toxic "drug", which leads to tumor
cell death. Also, U.S. Pat. No. 5,747,469 (incorporated herein by
reference) discloses the combined use of viral vectors encoding p53
and DNA damaging agents. Any such similar approaches can be used
with the present invention.
[0572] In some situations, it may even be desirable to extend the
time period for treatment significantly, where several days (2, 3,
4, 5, 6 or 7), several weeks (1, 2, 3, 4, 5, 6, 7 or 8) or even
several months (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the
respective administrations. This would be advantageous in
circumstances where one treatment was intended to substantially
destroy the tumor, such as the therapeutic agent-targeting agent
treatment, and another treatment was intended to prevent
micrometastasis or tumor re-growth, such as the administration of
an anti-angiogenic agent. The EN 7/44 antibody of Hagemeier et al.
(1986) is not believed to be an effective anti-angiogenic agent,
lacking binding to a surface accessible antigen, amongst other
deficiencies.
[0573] It also is envisioned that more than one administration of
either the therapeutic agent-targeting agent construct or the
anti-cancer agent will be utilized. The therapeutic agent-targeting
agent constructs and anti-cancer agents may be administered
interchangeably, on alternate days or weeks; or a sequence of
therapeutic agent-targeting agent treatment may be given, followed
by a sequence of anti-cancer agent therapy. In any event, to
achieve tumor regression using a combined therapy, all that is
required is to deliver both agents in a combined amount effective
to exert an anti-tumor effect, irrespective of the times for
administration.
[0574] In terms of surgery, any surgical intervention may be
practiced in combination with the present invention. In connection
with radiotherapy, any mechanism for inducing DNA damage locally
within tumor cells is contemplated, such as .gamma.-irradiation,
X-rays, UV-irradiation, microwaves and even electronic emissions
and the like. The directed delivery of radioisotopes to tumor cells
is also contemplated, and this may be used in connection with a
targeting antibody or other targeting means.
[0575] Cytokine therapy also has proven to be an effective partner
for combined therapeutic regimens. Various cytokines may be
employed in such combined approaches. Examples of cytokines include
IL-1.alpha. IL-1.beta., IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,
IL-9, IL-10, IL-11, IL-12, IL-13, TGF-.beta., GM-CSF, M-CSF, G-CSF,
TNF.alpha., TNF.beta., LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF,
OSM, TMF, PDGF, IFN-.alpha., IFN-.beta., IFN-.gamma.. Cytokines are
administered according to standard regimens, consistent with
clinical indications such as the condition of the patient and
relative toxicity of the cytokine. Uteroglobins may also be used to
prevent or inhibit metastases (U.S. Pat. No. 5,696,092;
incorporated herein by reference).
[0576] J1. Chemotherapeutics
[0577] In certain embodiments, the therapeutic agent-targeting
agent constructs of the present invention may be administered in
combination with a chemotherapeutic agent. Chemotherapeutic drugs
can kill proliferating tumor cells, enhancing the necrotic areas
created by the overall treatment. The drugs can thus enhance the
thrombotic action of the therapeutic agent-targeting agent
constructs.
[0578] By inducing the formation of thrombi in tumor vessels, the
therapeutic agent-targeting agent constructs can enhance the action
of the chemotherapeutics by retaining or trapping the drugs within
the tumor. The chemotherapeutics are thus retained within the
tumor, while the rest of the drug is cleared from the body. Tumor
cells are thus exposed to a higher concentration of drug for a
longer period of time. This entrapment of drug within the tumor
makes it possible to reduce the dose of drug, making the treatment
safer as well as more effective.
[0579] Irrespective of the underlying mechanism(s), a variety of
chemotherapeutic agents may be used in the combined treatment
methods disclosed herein. Chemotherapeutic agents contemplated as
exemplary include, e.g., tamoxifen, taxol, vincristine,
vinblastine, etoposide (VP-16), adriamycin, 5-fluorouracil (5FU),
camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP),
combretastatin(s) and derivatives and prodrugs thereof.
[0580] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents will be generally
around those already employed in clinical therapies wherein the
chemotherapeutics are administered alone or in combination with
other chemotherapeutics. By way of example only, agents such as
cisplatin, and other DNA alkylating may be used. Cisplatin has been
widely used to treat cancer, with efficacious doses used in
clinical applications of 20 mg/m.sup.2 for 5 days every three weeks
for a total of three courses. Cisplatin is not absorbed orally and
must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
[0581] Further useful agents include compounds that interfere with
DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for adriamycin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally.
[0582] Agents that disrupt the synthesis and fidelity of
polynucleotide precursors may also be used. Particularly useful are
agents that have undergone extensive testing and are readily
available. As such, agents such as 5-fluorouracil (5-FU) are
preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
[0583] Exemplary chemotherapeutic agents that are useful in
connection with combined therapy are listed in Table C. Each of the
agents listed therein are exemplary and by no means limiting. The
skilled artisan is directed to "Remington's Pharmaceutical
Sciences" 15th Edition, chapter 33, in particular pages 624-652.
Some variation in dosage will necessarily occur depending on the
condition of the subject being treated. The physician responsible
for administration will be able to determine the appropriate dose
for the individual subject.
TABLE-US-00003 TABLE C CHEMOTHERAPEUTIC AGENTS USEFUL IN NEOPLASTIC
DISEASE NONPROPRIETARY TYPE OF NAMES CLASS AGENT (OTHER NAMES)
DISEASE Alkylating Nitrogen Mechlorethamine (HN.sub.2) Hodgkin's
disease, non-Hodgkin's lymphomas Agents Mustards Cyclophosphamide
Acute and chronic lymphocytic leukemias, Ifosfamide Hodgkin's
disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma,
breast, ovary, lung, Wilms' tumor, cervix, testis, soft-tissue
sarcomas Melphalan Multiple myeloma, breast, ovary (L-sarcolysin)
Chlorambucil Chronic lymphocytic leukemia, primary
macroglobulinemia, Hodgkin's disease, non-Hodgkin's lymphomas
Ethylenimenes Hexamethylmelamine Ovary and Methylmelamines Thiotepa
Bladder, breast, ovary Alkyl Sulfonates Busulfan Chronic
granulocytic leukemia Nitrosoureas Carmustine (BCNU) Hodgkin's
disease, non-Hodgkin's lymphomas, primary brain tumors, multiple
myeloma, malignant melanoma Lomustine (CCNU) Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung
Semustine (methyl-CCNU) Primary brain tumors, stomach, colon
Streptozocin Malignant pancreatic insulinoma, (streptozotocin)
malignant carcinoid Triazines Dacarbazine (DTIC; Malignant
melanoma, Hodgkin's disease, dimethyltriazeno- soft-tissue sarcomas
imidazolecarboxamide) Antimetabolites Folic Acid Methotrexate Acute
lymphocytic leukemia, choriocarcinoma, Analogs (amethopterin)
mycosis fungoides, breast, head and neck, lung, osteogenic sarcoma
Pyrimidine Fluouracil Breast, colon, stomach, pancreas, ovary,
Analogs (5-fluorouracil; 5-FU) head and neck, urinary bladder,
Floxuridine premalignant skin lesions (topical)
(fluorodeoxyuridine; FUdR) Cytarabine Acute granulocytic and acute
(cytosine arabinoside) lymphocytic leukemias Mercaptopurine Acute
lymphocytic, acute granulocytic and (6-mercaptopurine; 6-MP)
chronic granulocytic leukemias Purine Analogs and Thioguanine Acute
granulocytic, acute lymphocytic and Related Inhibitors
(6-thioguanine; TG) chronic granulocytic leukemias Pentostatin
Hairy cell leukemia, mycosis fungoides, (2-deoxycoformycin) chronic
lymphocytic leukemia Natural Vinca Alkaloids Vinblastine (VLB)
Hodgkin's disease, non-Hodgkin's lymphomas, Products breast, testis
Vincristine Acute lymphocytic leukemia, neuroblastoma, Wilms'
tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's
lymphomas, small-cell lung Epipodophyllotoxins Etoposide Testis,
small-cell lung and other lung, Tertiposide breast, Hodgkin's
disease, non-Hodgkin's lymphomas, acute granulocytic leukemia,
Kaposi's sarcoma Antibiotics Dactinomycin Choriocarcinoma, Wilms'
tumor, (actinomycin D) rhabdomyosarcoma, testis, Kaposi's sarcoma
Daunorubicin Acute granulocytic and acute (daunomycin; lymphocytic
leukemias rubidomycin) Doxorubicin Soft-tissue, osteogenic and
other sarcomas; Hodgkin's disease, non-Hodgkin's lymphomas, acute
leukemias, breast, genitourinary, thyroid, lung, stomach,
neuroblastoma Bleomycin Testis, head and neck, skin, esophagus,
lung and genitourinary tract; Hodgkin's disease, non-Hodgkin's
lymphomas Plicamycin Testis, malignant hypercalcemia (mithramycin)
Mitomycin Stomach, cervix, colon, breast, pancreas, (mitomycin C)
bladder, head and neck Enzymes L-Asparaginase Acute lymphocytic
leukemia Biological Interferon alfa Hairy cell leukemia., Kaposi's
sarcoma, Response melanoma, carcinoid, renal cell, ovary, Modifiers
bladder, non-Hodgkin's lymphomas, mycosis fungoides, multiple
myeloma, chronic granulocytic leukemia Miscellaneous Platinum
Cisplatin (cis-DDP) Testis, ovary, bladder, head and neck, Agents
Coordination Carboplatin lung, thyroid, cervix, endometrium,
Complexes neuroblastoma, osteogenic sarcoma Anthracenedione
Mitoxantrone Acute granulocytic leukemia, breast Substituted Urea
Hydroxyurea Chronic granulocytic leukemia, polycythemia vera,
essental thrombocytosis, malignant melanoma Methyl Hydrazine
Procarbazine Hodgkin's disease Derivative (N-methylhydrazine, MlH)
Adrenocortical Mitotane (o,p'-DDD) Adrenal cortex Suppressant
Aminoglutethimide Breast Hormones and Adrenocortico- Prednisone
(several other Acute and chronic lymphocytic leukemias, Antagonists
steroids equivalent preparations non-Hodgkin's lymphomas, Hodgkin's
available) disease, breast Progestins Hydroxyprogesterone
Endometrium, breast caproate Medroxyprogesterone acetate Megestrol
acetate Estrogens Diethylstilbestrol Breast, prostate Ethinyl
estradiol (other preparations available) Antiestrogen Tamoxifen
Breast Androgens Testosterone propionate Breast Fluoxymesterone
(other preparations available) Antiandrogen Flutamide Prostate
Gonadotropin-releasing Leuprolide Prostate hormone analog
[0584] J2. Anti-Angiogenics
[0585] The term "angiogenesis" refers to the generation of new
blood vessels, generally into a tissue or organ. Under normal
physiological conditions, humans or animals undergo angiogenesis
only in very specific restricted situations. For example,
angiogenesis is normally observed in wound healing, fetal and
embryonic development and formation of the corpus luteum,
endometrium and placenta. Uncontrolled (persistent and/or
unregulated) angiogenesis is related to various disease states, and
occurs during tumor growth and metastasis.
[0586] Both controlled and uncontrolled angiogenesis are thought to
proceed in a similar manner. Endothelial cells and pericytes,
surrounded by a basement membrane, form capillary blood vessels.
Angiogenesis begins with the erosion of the basement membrane by
enzymes released by endothelial cells and leukocytes. The
endothelial cells, which line the lumen of blood vessels, then
protrude through the basement membrane. Angiogenic stimulants
induce the endothelial cells to migrate through the eroded basement
membrane. The migrating cells form a "sprout" off the parent blood
vessel, where the endothelial cells undergo mitosis and
proliferate. The endothelial sprouts merge with each other to form
capillary loops, creating the new blood vessel.
[0587] As persistent, unregulated angiogenesis occurs during tumor
development and metastasis, the treatment methods of this invention
may be used in combination with any one or more "anti-angiogenic"
therapies. Exemplary anti-angiogenic agents that are useful in
connection with combined therapy are listed in Table D. Each of the
agents listed therein is exemplary and by no means limiting.
TABLE-US-00004 TABLE D Inhibitors and Negative Regulators of
Angiogenesis Substances References Angiostatin O'Reilly et al.,
1994 Endostatin O'Reilly et al., 1997 16 kDa prolactin fragment
Ferrara et al., 1991; Clapp et al., 1993; D'Angelo et al., 1995;
Lee et al., 1998 Laminin peptides Kleinman et al., 1993; Yamamura
et al., 1993; Iwamoto et al., 1996; Tryggvason, 1993 Fibronectin
peptides Grant et al., 1998; Sheu et al., 1997 Tissue
metalloproteinase Sang, 1998 inhibitors (TIMP 1, 2, 3, 4)
Plasminogen activator Soff et al., 1995 inhibitors (PAI-1, -2)
Tumor necrosis factor .alpha. Frater-Schroder et al., 1987 (high
dose, in vitro) TGF-.beta.1 RayChadhury and D'Amore, 1991; Tada et
al., 1994 Interferons (IFN-.alpha., -.beta., .gamma.) Moore et al.,
1998; Lingen et al., 1998 ELR- CXC Chemokines: IL-12; Moore et al.,
1998; Hiscox and Jiang, 1997; SDF-1; MIG; Platelet factor Coughlin
et al., 1998; Tanaka et al., 1997 4 (PF-4); IP-10 Thrombospondin
(TSP) Good et al., 1990; Frazier, 1991; Bornstein, 1992; Tolsma et
al., 1993; Sheibani and Frazier, 1995; Volpert et al., 1998 SPARC
Hasselaar and Sage, 1992; Lane et al., 1992; Jendraschak and Sage,
1996 2-Methoxyoestradiol Fotsis et al., 1994 Proliferin-related
protein Jackson et al., 1994 Suramin Gagliardi et al., 1992; Takano
et al., 1994; Waltenberger et al., 1996; Gagliardi et al., 1998;
Manetti et al., 1998 Thalidomide D'Amato et al., 1994; Kenyon et
al., 1997 Wells, 1998 Cortisone Thorpe et al., 1993 Folkman et al.,
1983 Sakamoto et al., 1986 Linomide Vukanovic et al., 1993; Ziche
et al., 1998; Nagler et al., 1998 Fumagillin (AGM-1470; TNP-470)
Sipos et al., 1994; Yoshida et al., 1998 Tamoxifen Gagliardi and
Collins, 1993; Linder and Borden, 1997; Haran et al., 1994 Korean
mistletoe extract Yoon et al., 1995 (Viscum album coloratum)
Retinoids Oikawa et al., 1989; Lingen et al., 1996; Majewski et al.
1996 CM101 Hellerqvist et al., 1993; Quinn et al., 1995; Wamil et
al., 1997; DeVore et al., 1997 Dexamethasone Hori et al., 1996;
Wolff et al., 1997 Leukemia inhibitory factor (LIF) Pepper et al.,
1995
[0588] A certain preferred component for use in inhibiting
angiogenesis is a protein named "angiostatin". This component is
disclosed in U.S. Pat. Nos. 5,776,704; 5,639,725 and 5,733,876,
each incorporated herein by reference. Angiostatin is a protein
having a molecular weight of between about 38 kD and about 45 kD,
as determined by reducing polyacrylamide gel electrophoresis, which
contains approximately Kringle regions 1 through 4 of a plasminogen
molecule. Angiostatin generally has an amino acid sequence
substantially similar to that of a fragment of murine plasminogen
beginning at amino acid number 98 of an intact murine plasminogen
molecule.
[0589] The amino acid sequence of angiostatin varies slightly
between species. For example, in human angiostatin, the amino acid
sequence is substantially similar to the sequence of the above
described murine plasminogen fragment, although an active human
angiostatin sequence may start at either amino acid number 97 or 99
of an intact human plasminogen amino acid sequence. Further, human
plasminogen may be used, as it has similar anti-angiogenic
activity, as shown in a mouse tumor model.
[0590] Certain anti-angiogenic therapies have already been shown to
cause tumor regressions, and angiostatin is one such agent.
Endostatin, a 20 kDa COOH-terminal fragment of collagen XVIII, the
bacterial polysaccharide CM101, and the antibody LM609 also have
angiostatic activity. However, in light of their other properties,
they are referred to as anti-vascular therapies or tumor vessel
toxins, as they not only inhibit angiogenesis but also initiate the
destruction of tumor vessels through mostly undefined mechanisms.
Their combination with the present invention is clearly
envisioned.
[0591] Angiostatin and endostatin have become the focus of intense
study, as they are the first angiogenesis inhibitors that have
demonstrated the ability to not only inhibit tumor growth but also
cause tumor regressions in mice. There are multiple proteases that
have been shown to produce angiostatin from plasminogen including
elastase, macrophage metalloelastase (MME), matrilysin (MMP-7), and
92 kDa gelatinase B/type IV collagenase (MMP-9).
[0592] MME can produce angiostatin from plasminogen in tumors and
granulocyte-macrophage colony-stimulating factor (GMCSF)
upregulates the expression of MME by macrophages inducing the
production of angiostatin. The role of MME in angiostatin
generation is supported by the finding that MME is in fact
expressed in clinical samples of hepatocellular carcinomas from
patients. Another protease thought to be capable of producing
angiostatin is stromelysin-1 (MMP-3). MMP-3 has been shown to
produce angiostatin-like fragments from plasminogen in vitro.
[0593] The mechanism of action for angiostatin is currently
unclear, it is hypothesized that it binds to an unidentified cell
surface receptor on endothelial cells inducing endothelial cell to
undergo programmed cell death or mitotic arrest. Endostatin appears
to be an even more powerful anti-angiogenesis and anti-tumor agent
although its biology is much less clear. Endostatin is effective at
causing regressions in a number of tumor models in mice. Tumors do
not develop resistance to endostatin and, after multiple cycles of
treatment, tumors enter a dormant state during which they do not
increase in volume. In this dormant state, the percentage of tumor
cells undergoing apoptosis was increased, yielding a population
that essentially stays the same size. Endostatin is also thought to
bind an unidentified endothelial cell surface receptor that
mediates its effect.
[0594] CM101 is a bacterial polysaccharide that has been well
characterized in its ability to induce neovascular inflammation in
tumors. CM101 binds to and cross-links receptors expressed on
dedifferentiated endothelium that stimulates the activation of the
complement system. It also initiates a cytokine-driven inflammatory
response that selectively targets the tumor. It is a uniquely
antipathoangiogenic agent that downregulates the expression VEGF
and its receptors. CM101 is currently in clinical trials as an
anti-cancer drug, and can be used in combination herewith.
[0595] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also
be used in combination with the present invention. These are both
angiogenesis inhibitors that associate with heparin and are found
in platelet .alpha.-granules. TSP-1 is a large 450 kDa multi-domain
glycoprotein that is constituent of the extracellular matrix. TSP-1
binds to many of the proteoglycan molecules found in the
extracellular matrix including, HSPGs, fibronectin, laminin, and
different types of collagen. TSP-1 inhibits endothelial cell
migration and proliferation in vitro and angiogenesis in vivo.
TSP-1 can also suppress the malignant phenotype and tumorigenesis
of transformed endothelial cells. The tumor suppressor gene p53 has
been shown to directly regulate the expression of TSP-1 such that,
loss of p53 activity causes a dramatic reduction in TSP-1
production and a concomitant increase in tumor initiated
angiogenesis.
[0596] PF4 is a 70aa protein that is member of the CXC ELR-family
of chemokines that is able to potently inhibit endothelial cell
proliferation in vitro and angiogenesis in vivo. PF4 administered
intratumorally or delivered by an adenoviral vector is able to
cause an inhibition of tumor growth.
[0597] Interferons and metalloproteinase inhibitors are two other
classes of naturally occurring angiogenic inhibitors that can be
combined with the present invention. The anti-endothelial activity
of the interferons has been known since the early 1980s, however,
the mechanism of inhibition is still unclear. It is known that they
can inhibit endothelial cell migration and that they do have some
anti-angiogenic activity in vivo that is possibly mediated by an
ability to inhibit the production of angiogenic promoters by tumor
cells. Vascular tumors in particular are sensitive to interferon,
for example, proliferating hemangiomas can be successfully treated
with IFN.alpha..
[0598] Tissue inhibitors of metalloproteinases (TIMPs) are a family
of naturally occurring inhibitors of matrix metalloproteases (MMPs)
that can also inhibit angiogenesis and can be used in combined
treatment protocols with the present invention. MMPs play a key
role in the angiogenic process as they degrade the matrix through
which endothelial cells and fibroblasts migrate when extending or
remodeling the vascular network. In fact, one member of the MMPs,
MMP-2, has been shown to associate with activated endothelium
through the integrin .alpha.v.beta.3 presumably for this purpose.
If this interaction is disrupted by a fragment of MMP-2, then
angiogenesis is downregulated and in tumors growth is
inhibited.
[0599] There are a number of pharmacological agents that inhibit
angiogenesis, any one or more of which may be used in combination
with the present invention. These include AGM-1470/TNP-470,
thalidomide, and carboxyamidotriazole (CAI). Fumagillin was found
to be a potent inhibitor of angiogenesis in 1990, and since then
the synthetic analogues of fumagillin, AGM-1470 and TNP-470 have
been developed. Both of these drugs inhibit endothelial cell
proliferation in vitro and angiogenesis in vivo. TNP-470 has been
studied extensively in human clinical trials with data suggesting
that long-term administration is optimal.
[0600] Thalidomide was originally used as a sedative but was found
to be a potent teratogen and was discontinued. In 1994 it was found
that thalidomide is an angiogenesis inhibitor. Thalidomide is
currently in clinical trials as an anti-cancer agent as well as a
treatment of vascular eye diseases.
[0601] CAI is a small molecular weight synthetic inhibitor of
angiogenesis that acts as a calcium channel blocker that prevents
actin reorganization, endothelial cell migration and spreading on
collagen IV. CAI inhibits neovascularization at physiological
attainable concentrations and is well tolerated orally by cancer
patients. Clinical trials with CAI have yielded disease
stabilization in 49% of cancer patients having progressive disease
before treatment.
[0602] Cortisone in the presence of heparin or heparin fragments
was shown to inhibit tumor growth in mice by blocking endothelial
cell proliferation. The mechanism involved in the additive
inhibitory effect of the steroid and heparin is unclear although it
is thought that the heparin may increase the uptake of the steroid
by endothelial cells. The mixture has been shown to increase the
dissolution of the basement membrane underneath newly formed
capillaries and this is also a possible explanation for the
additive angiostatic effect. Heparin-cortisol conjugates also have
potent angiostatic and anti-tumor effects activity in vivo.
[0603] Further specific angiogenesis inhibitors, including, but not
limited to, Anti-Invasive Factor, retinoic acids and paclitaxel
(U.S. Pat. No. 5,716,981; incorporated herein by reference);
AGM-1470 (Ingber et al., 1990; incorporated herein by reference);
shark cartilage extract (U.S. Pat. No. 5,618,925; incorporated
herein by reference); anionic polyimide or polyurea oligomers (U.S.
Pat. No. 5,593,664; incorporated herein by reference); oxindole
derivatives (U.S. Pat. No. 5,576,330; incorporated herein by
reference); estradiol derivatives (U.S. Pat. No. 5,504,074;
incorporated herein by reference); and thiazolopyrimidine
derivatives (U.S. Pat. No. 5,599,813; incorporated herein by
reference) are also contemplated for use as anti-angiogenic
compositions for the combined uses of the present invention.
[0604] Compositions comprising an antagonist of an
.alpha..sub.v.beta..sub.3 integrin may also be used to inhibit
angiogenesis in combination with the present invention. As
disclosed in U.S. Pat. No. 5,766,591 (incorporated herein by
reference), RGD-containing polypeptides and salts thereof,
including cyclic polypeptides, are suitable examples of
.alpha..sub.v.beta..sub.3 integrin antagonists.
[0605] The antibody LM609 against the .alpha..sub.v.beta..sub.3
integrin also induces tumor regressions. Integrin
.alpha..sub.v.beta..sub.3 antagonists, such as LM609, induce
apoptosis of angiogenic endothelial cells leaving the quiescent
blood vessels unaffected. LM609 or other .alpha..sub.v.beta..sub.3
antagonists may also work by inhibiting the interaction of
.alpha..sub.v.beta..sub.3 and MMP-2, a proteolytic enzyme thought
to play an important role in migration of endothelial cells and
fibroblasts.
[0606] Apoptosis of the angiogenic endothelium in this case may
have a cascade effect on the rest of the vascular network.
Inhibiting the tumor vascular network from completely responding to
the tumor's signal to expand may, in fact, initiate the partial or
full collapse of the network resulting in tumor cell death and loss
of tumor volume. It is possible that endostatin and angiostatin
function in a similar fashion. The fact that LM609 does not affect
quiescent vessels but is able to cause tumor regressions suggests
strongly that not all blood vessels in a tumor need to be targeted
for treatment in order to obtain an anti-tumor effect.
[0607] Non-targeted angiopoietins, such as angiopoietin-2, may also
be used in combination with the present invention. As described
above in the context of targeted delivery, the angiogenic effects
of various regulators involve an autocrine loop connected with
angiopoietin-2. The use of angiopoietin-2, angiopoietin-1,
angiopoietin-3 and angiopoietin-4, is thus contemplated in
conjunction with the present invention. Other methods of
therapeutic intervention based upon altering signaling through the
Tie2 receptor can also be used in combination herewith, such as
using a soluble Tie2 receptor capable of blocking Tie2 activation
(Lin et al., 1998). Delivery of such a construct using recombinant
adenoviral gene therapy has been shown to be effective in treating
cancer and reducing metastases (Lin et al., 1998).
[0608] J3. Apoptosis-Inducing Agents
[0609] Therapeutic agent-targeting agent treatment may also be
combined with treatment methods that induce apoptosis in any cells
within the tumor, including tumor cells and tumor vascular
endothelial cells. Although many anti-cancer agents may have, as
part of their mechanism of action, an apoptosis-inducing effect,
certain agents have been discovered, designed or selected with this
as a primary mechanism, as described below.
[0610] A number of oncogenes have been described that inhibit
apoptosis, or programmed cell death. Exemplary oncogenes in this
category include, but are not limited to, bcr-abl, bcl-2 (distinct
from bcl-1, cyclin D1; GenBank accession numbers M14745, X06487;
U.S. Pat. Nos. 5,650,491; and 5,539,094; each incorporated herein
by reference) and family members including Bcl-x1, MeI-1, Bak, A1,
A20. Overexpression of bcl-2 was first discovered in T cell
lymphomas. bcl-2 functions as an oncogene by binding and
inactivating Bax, a protein in the apoptotic pathway. Inhibition of
bcl-2 function prevents inactivation of Bax, and allows the
apoptotic pathway to proceed. Thus, inhibition of this class of
oncogenes, e.g., using antisense nucleotide sequences, is
contemplated for use in the present invention in aspects wherein
enhancement of apoptosis is desired (U.S. Pat. Nos. 5,650,491;
5,539,094; and 5,583,034; each incorporated herein by
reference).
[0611] Many forms of cancer have reports of mutations in tumor
suppressor genes, such as p53. Inactivation of p53 results in a
failure to promote apoptosis. With this failure, cancer cells
progress in tumorigenesis, rather than become destined for cell
death. Thus, provision of tumor suppressors is also contemplated
for use in the present invention to stimulate cell death. Exemplary
tumor suppressors include, but are not limited to, p53,
Retinoblastoma gene (Rb), Wilm's tumor (WT1), bax alpha,
interleukin-1b-converting enzyme and family, MEN-1 gene,
neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal
cancer gene (DCC), familial adenomatosis polyposis gene (FAP),
multiple tumor suppressor gene (MTS-1), BRCA1 and BRCA2.
[0612] Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469;
5,677,178; and 5,756,455; each incorporated herein by reference),
Retinoblastoma, BRCA1 (U.S. Pat. Nos. 5,750,400; 5,654,155;
5,710,001; 5,756,294; 5,709,999; 5,693,473; 5,753,441; 5,622,829;
and 5,747,282; each incorporated herein by reference), MEN-1
(GenBank accession number U93236) and adenovirus E1A (U.S. Pat. No.
5,776,743; incorporated herein by reference) genes.
[0613] Other compositions that may be used include genes encoding
the tumor necrosis factor related apoptosis inducing ligand termed
TRAIL, and the TRAIL polypeptide (U.S. Pat. No. 5,763,223;
incorporated herein by reference); the 24 kD apoptosis-associated
protease of U.S. Pat. No. 5,605,826 (incorporated herein by
reference); Fas-associated factor 1, FAF1 (U.S. Pat. No. 5,750,653;
incorporated herein by reference). Also contemplated for use in
these aspects of the present invention is the provision of
interleukin-1.beta.-converting enzyme and family members, which are
also reported to stimulate apoptosis.
[0614] Compounds such as carbostyril derivatives (U.S. Pat. Nos.
5,672,603; and 5,464,833; each incorporated herein by reference);
branched apogenic peptides (U.S. Pat. No. 5,591,717; incorporated
herein by reference); phosphotyrosine inhibitors and
non-hydrolyzable phosphotyrosine analogs (U.S. Pat. Nos. 5,565,491;
and 5,693,627; each incorporated herein by reference); agonists of
RXR retinoid receptors (U.S. Pat. No. 5,399,586; incorporated
herein by reference); and even antioxidants (U.S. Pat. No.
5,571,523; incorporated herein by reference) may also be used.
Tyrosine kinase inhibitors, such as genistein, may also be linked
to ligands that target a cell surface receptor (U.S. Pat. No.
5,587,459; incorporated herein by reference).
[0615] J4. Immunotoxins and Coaguligands
[0616] The anti-aminophospholipid-conjugate based treatment methods
of the invention may be used in combination with other immunotoxins
and/or coaguligands in which the targeting portion thereof, e.g.,
antibody or ligand, is directed to a relatively specific marker of
the tumor cells, tumor vasculature or tumor stroma. In common with
the chemotherapeutic and anti-angiogenic agents discussed above,
the combined use of other targeted toxins or coagulants will
generally result in additive, markedly greater than additive or
even synergistic anti-tumor results.
[0617] Generally speaking, antibodies or ligands for use in these
additional aspects of the invention will preferably recognize
accessible tumor antigens that are preferentially, or specifically,
expressed in the tumor site. The antibodies or ligands will also
preferably exhibit properties of high affinity; and the antibodies,
ligands or conjugates thereof, will not exert significant in vivo
side effects against life-sustaining normal tissues, such as one or
more tissues selected from heart, kidney, brain, liver, bone
marrow, colon, breast, prostate, thyroid, gall bladder, lung,
adrenals, muscle, nerve fibers, pancreas, skin, or other
life-sustaining organ or tissue in the human body. The term
"significant side effects", as used herein, refers to an antibody,
ligand or antibody conjugate, that, when administered in vivo, will
produce only negligible or clinically manageable side effects, such
as those normally encountered during chemotherapy.
[0618] At least one binding region of these second anti-cancer
agents employed in combination with the invention will be a
component that is capable of delivering a toxin or coagulation
factor to the tumor region, i.e., capable of localizing within a
tumor site. Such targeting agents may be directed against a
component of a tumor cell, tumor vasculature or tumor stroma. The
targeting agents will generally bind to a surface-expressed,
surface-accessible or surface-localized component of a tumor cell,
tumor vasculature or tumor stroma. However, once tumor vasculature
and tumor cell destruction begins, internal components will be
released, allowing additional targeting of virtually any tumor
component.
[0619] Many tumor cell antigens have been described, any one which
could be employed as a target in connection with the combined
aspects of the present invention. Appropriate tumor cell antigens
for additional immunotoxin and coaguligand targeting include those
recognized by the antibodies B3 (U.S. Pat. No. 5,242,813;
incorporated herein by reference; ATCC HB 10573); KSI/4 (U.S. Pat.
No. 4,975,369; incorporated herein by reference; obtained from a
cell comprising the vectors NRRL B-18356 and/or NRRL B-18357);
260F9 (ATCC HB 8488); and D612 (U.S. Pat. No. 5,183,756;
incorporated herein by reference; ATCC HB 9796). One may also
consult the ATCC Catalogue of any subsequent year to identify other
appropriate cell lines producing anti-tumor cell antibodies.
[0620] For tumor vasculature targeting, the targeting antibody or
ligand will often bind to a marker expressed by, adsorbed to,
induced on or otherwise localized to the intratumoral blood vessels
of a vascularized tumor. Appropriate expressed target molecules
include, for example, endoglin, E-selectin, P-selectin, VCAM-1,
ICAM-1, PSMA (Liu et al., 1997), a TIE, a ligand reactive with
LAM-1, a VEGF/VPF receptor, an FGF receptor,
.alpha..sub.v.beta..sub.3 integrin, pleiotropin and endosialin.
Suitable adsorbed targets are those such as VEGF, FGF, TGF.beta.,
HGF, PF4, PDGF, TIMP, a ligand that binds to a TIE and
tumor-associated fibronectin isoforms. Antigens naturally and
artificially inducible by cytokines and coagulants may also be
targeted, such as ELAM-1, VCAM-1, ICAM-1, a ligand reactive with
LAM-1, endoglin, and even MHC Class II (cytokine-inducible, e.g.,
by IL-1, TNF-.alpha., IFN-.gamma., IL-4 and/or TNF-.beta.); and
E-selectin, P-selectin, PDGF and ICAM-1 (coagulant-inducible e.g.,
by thrombin, Factor IX/IXa, Factor X/Xa and/or plasmin).
[0621] The following patents and patent applications are
specifically incorporated herein by reference for the purposes of
even further supplementing the present teachings regarding the
preparation and use of immunotoxins directed against expressed,
adsorbed, induced or localized markers of tumor vasculature: U.S.
Pat. Nos. 5,855,866; 5,776,427; 5,863,538; 5,660,827; 5,855,866;
6,004,554; 5,965,132; 6,051,230; 6,093,399 and 5,877,289; and U.S.
application Ser. No. 07/846,349.
[0622] Suitable tumor stromal targets include components of the
tumor extracellular matrix or stroma, or components those bound
therein; including basement membrane markers, type IV collagen,
laminin, heparan sulfate, proteoglycan, fibronectins, activated
platelets, LIBS and tenascin. A preferred target for such uses is
RIBS.
[0623] The following patents and patent applications are
specifically incorporated herein by reference for the purposes of
even further supplementing the present teachings regarding the
preparation and use of tumor stromal targeting agents: U.S. Pat.
Nos. 5,877,289; 6,004,555; 6,036,955; and 6,093,399.
[0624] The second anti-cancer therapeutics may be operatively
attached to any of the cytotoxic or otherwise anti-cellular agents
described herein for use in the anti-aminophospholipid
immunotoxins. However, suitable anti-cellular agents also include
radioisotopes. Toxin moieties will be preferred, such as ricin A
chain and deglycosylated A chain (dgA) or even gelonin. Any one or
more of the angiopoietins, or fusions thereof, may also be used as
part of a second immunoconjugate for combined therapy.
[0625] The second, targeted agent for optional use with the
invention may comprise a targeted component that is capable of
promoting coagulation, i.e., a coaguligand. Here, the targeting
antibody or ligand may be directly or indirectly, e.g., via another
antibody, linked to any factor that directly or indirectly
stimulates coagulation, including any of those described herein for
use in the anti-aminophospholipid coaguligands. Preferred
coagulation factors for such uses are Tissue Factor (TF) and TF
derivatives, such as truncated TF (tTF), dimeric and multimeric TF,
and mutant TF deficient in the ability to activate Factor VII.
[0626] Effective doses of immunotoxins and coaguligands for
combined use in the treatment of cancer will be between about 0.1
mg/kg and about 2 mg/kg, and preferably, of between about 0.8 mg/kg
and about 1.2 mg/kg, when administered via the IV route at a
frequency of about 1 time per week. Some variation in dosage will
necessarily occur depending on the condition of the subject being
treated. The physician responsible for administration will
determine the appropriate dose for the individual subject.
[0627] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example I
VCAM-1 Expression on Tumor and Normal Blood Vessels
A. Materials and Methods
1. Materials
[0628] Na.sup.125I was obtained from Amersham (Arlington Heights,
Ill.). Dulbecco's modified Eagle's tissue culture medium (DMEM) and
Dulbecco PBS containing Ca.sup.2+ and Mg.sup.2+ were obtained from
Gibco (Grand Island, N.Y.). Fetal calf serum was obtained from
Hyclone (Logan, Utah). O-phenylenediamine, hydrogen peroxide,
3-aminopropyltriethoxy-silane and sterile, endotoxin-free saline
(0.9% NaCl in 100 ml of water) were from Sigma (St. Louis, Mo.).
SMPT was from Pierce (Rockford, Ill.). Proplex T containing factor
VII (74 IU/ml), factor X and factor IX (17 IU/ml) was purchased
from Baxter Diagnostics Inc. (McGraw Park, Ill.). Chromogenic
substrate, S-2765, for measuring factor Xa proteolytic activity was
obtained from Chromogenix (Franklin, Ohio). Purified factor Xa was
purchased from American Diagnostica (Greenwich, Conn.). 96 and 48
flat bottom microtiter plates were obtained from Falcon (Becton
Dickinson and Co., Lincoln Park, N.J.). Sepharose-Protein G beads
and 5200 Superdex were purchased from Pharmacia (Piscataway, N.J.).
Recombinant murine IL-1.alpha. was purchased from R&D Systems
(Minneapolis, Minn.).
2. Antibodies
[0629] The MK2.7 hybridoma, secreting a rat IgG1 antibody against
murine VCAM-1, was obtained from the American Type Culture
Collection (ATCC, Rockville, Md.; ATCC CRL 1909). The
characterization of this anti-VCAM-1 antibody has been reported by
Miyake et al. (1991, incorporated herein by reference). The R187
hybridoma, secreting a rat IgG1 antibody against murine viral
protein p30 gag, was also obtained from the ATCC, and was used as
an isotype matched control for the anti-VCAM-1 antibody.
[0630] Mouse monoclonal antibody, 10H10, against human tissue
factor was prepared as described in Morrissey et al. (1988), and in
U.S. application Ser. No. 08/482,369, each incorporated herein by
reference.
[0631] MECA 32, a pan anti-mouse vascular endothelial cell
antibody, was prepared as described by Leppink et al. (1989,
incorporated herein by reference). MJ 7/18 rat IgG, reactive with
murine endoglin, was prepared as described by Ge and Butcher (1994,
incorporated herein by reference). The MECA 32 and MJ 7/18
antibodies served as positive controls for immunohistochemical
studies.
[0632] Rabbit anti-rat and rat anti-mouse secondary antibodies
conjugated with horseradish peroxidase (HRP) were purchased from
Dako (Carpinteria, Calif.).
3. Antibody Purification
[0633] Anti-VCAM-1 hybridoma, MK 2.7, and the irrelevant control
hybridoma, R187, were grown in bioreactors (Heraeus, Inc., Germany)
for 12 days. Supernatants were centrifuged, filtered through 0.22
.mu.m filters and loaded onto Sepharose-Protein G columns. IgG was
eluted with citric acid buffer, pH 3.5, dialyzed into PBS and
stored thereafter at 4.degree. C. in the same buffer. Purity was
estimated by SDS-PAGE and was routinely >90%. Binding capacity
of the purified anti-VCAM-1 antibody was assessed
immunohistochemically on frozen sections of L540 tumor and by
cell-based ELISA using IL-1.alpha. stimulated bEnd.3 cells, as
described herein below.
4. Tumor-Bearing Mice and Immunohistochemistry
[0634] Male CB17 SCID mice (Charles River, Wilmington, Mass.)
weighing approximately 25 g were injected with 1.times.10.sup.7
L540 Hodgkin's lymphoma cells subcutaneously into the right flank.
Tumors were allowed to grow to a size of 0.4-0.7 cm.sup.3. Animals
were anesthetized with metafane and their blood circulation was
perfused with heparinized saline as described by Burrows et al.
(1992, incorporated herein by reference). The tumor and major
organs were removed and snap-frozen in liquid nitrogen.
[0635] Cryostat sections of the tissues were cut, incubated with
the anti-VCAM-1 antibody and stained immunohistochemically to
detect VCAM-1. Rat IgG was detected using rabbit anti-rat IgG
conjugated to HRP followed by development with carbazole (Fries et
al., 1993).
B. Results
[0636] The blood vessels of the major organs and a tumor from mice
bearing subcutaneous L540 human Hodgkin's tumors were examined
immunohistochemically for VCAM-1 expression using an anti-VCAM-1
antibody. VCAM-1 expression on tumor blood vessels was more
peripheral than central. However, as demonstrated in Example VI and
Example VII, the anti-VCAM-1 antibody and coaguligand were
evidently binding to blood transporting vessels, as clearly shown
by the ability of the coaguligand to arrest blood flow in all tumor
regions and to cause destruction of the intratumoral region.
[0637] Overall, VCAM-1 expression was observed on 20-30% of total
tumor blood vessels stained by the anti-endoglin antibody, MJ 7/18.
VCAM-1 staining of the tumor vessels was largely observed on
venules. VCAM-1 expression was similar in tumors up to 1500
mm.sup.3, but larger tumors appeared to have reduced staining, with
5-10% of MJ 7/18 positive vessels being positive for VCAM-1.
[0638] Constitutive vascular expression of VCAM-1 was found in
heart and lungs in both tumor-bearing and normal animals (Table 1).
In the heart, strong staining was observed on venules and veins.
Approximately 10% of MECA 32 positive vessels were positive for
VCAM-1. Staining in lung endothelium was weak in comparison to
heart and tumor, and was confined to a few large blood vessels.
Strong stromal staining was observed in testis where VCAM-1
expression was strictly extravascular. Similar findings regarding
constitutive VCAM-1 expression in rodent lung and testis were
previously reported (Fries et al., 1993).
TABLE-US-00005 TABLE 1 Expression of VCAM-1 on Endothelium in
Tissues of L540 Tumor Bearing Mice and Localization of Anti-VCAM-1
Antibody anti-VCAM-1 antibody Tissue VCAM-1 expression.sup.a
localization.sup.b Adrenal -.sup.c - Brain Cerebellum - - Brain
Cortex - - Heart ++ ++ Kidney - - Large Intestine - - Liver - -
Lung + + Pancreas - - Small Intestine - - Spleen - - Testis -.sup.d
- L540 Hodgkin's tumor +++ +++ .sup.aVCAM-1 was detected by
anti-VCAM-1 antibody followed by anti-rat IgG-HRP.
.sup.bLocalization of anti-VCAM-1 antibody in vivo was determined
by injecting the antibody, exsanguinating the mice and staining
tissues staining with anti-rat IgG-HRP. .sup.cIntensity of staining
was compared to pan-endothelial markers MJ 7/18 and MECA 32; - no
staining; + weak; ++ moderate; +++ strong. .sup.dNo vascular
expression was observed; however, extravascular stroma of testis
was stained by anti-VCAM-1 antibody.
Example 11
Localization of Anti-VCAM-1 Antibody In Vivo
A. Methods
[0639] Male CB17 SCID mice (Charles River, Wilmington, Mass.)
weighing approximately 25 g were injected with 1.times.10.sup.7
L540 Hodgkin's lymphoma cells subcutaneously into the right flank.
Tumors were allowed to grow to a size of 0.4-0.7 cm.sup.3.
[0640] Mice were injected intravenously with 30 .mu.g/25 g body
weight of anti-VCAM-1 antibody, R187 antibody or corresponding
coaguligands in 200 .mu.l of saline. Two hours later, animals were
anesthetized with metafane and their blood circulation was perfused
with heparinized saline as described (Burrows et al., 1992;
incorporated herein by reference). The tumor and major organs were
removed and snap-frozen in liquid nitrogen.
[0641] Cryostat sections of the tissues were cut and were stained
immunohistochemically for the presence of rat IgG or TF. Rat IgG
was detected using rabbit anti-rat IgG conjugated to HRP followed
by development with carbazole (Fries et al., 1993). Coaguligand was
detected using the 10H10 antibody that recognizes human tissue
factor, followed by HRP-labeled anti-mouse IgG. 10H10 antibody does
not cross-react detectably with murine tissue factor (Morrissey et
al., 1988, incorporated herein by reference) or other murine
proteins.
B. Results
[0642] Mice bearing subcutaneous L540 tumors were injected
intravenously with anti-VCAM-1 antibody and, two hours later, the
mice were exsanguinated. The tumor and normal organs were removed
and frozen sections were prepared and examined
immunohistochemically to determine the location of the antibody.
Serial sections of the tissues were examined. Localized rat IgG was
detected by HRP-labeled anti-rat Ig; and murine blood vessels were
identified by pan-endothelial antibody, MECA 32.
[0643] Anti-VCAM-1 antibody was detected on endothelium of tumor,
heart and lung (Table 1). The intensity and number of stained
vessels was identical to that on serial sections of the same
tissues stained directly with anti-VCAM-1 antibody (Table 1).
Staining was specific as no staining of endothelium was observed in
the tumor and organs of mice injected with a species isotype
matched antibody of irrelevant specificity, R187. No localization
of anti-VCAM-1 antibody was found in testis or any normal organ
except heart and lung.
Example III
Preparation of Anti-VCAM-1.cndot.tTF Coaguligand
[0644] An anti-VCAM-1.cndot.tTF conjugate or "coaguligand" was
prepared as follows. Truncated tissue factor (tTF), with an
additional added cysteine introduced at N-terminus (U.S.
application Ser. No. 08/482,369, incorporated herein by reference),
was expressed in E. coli and purified as described by Stone et al.
(1995, incorporated herein by reference). After purification, the
sulfhydryl group of N' cysteine-tTF was protected by reaction with
Ellman's reagent. The tTF derivative was stored in small volumes at
-70.degree. C.
[0645] To prepare the anti-VCAM-1 coaguligand, 5 ml of anti-VCAM-1
antibody IgG (2 mg/ml) in PBS were mixed with 36 .mu.l of SMPT (10
mM) dissolved in dry DMF and incubated at room temperature for 1 h.
The mixture was filtered through a column of Sephadex G25
equilibrated in PBS containing 1 mM EDTA. The fractions containing
the SMPT-derivatized antibody were concentrated to 4 ml by
ultrafiltration in an Amicon cell equipped with a 10,000 Da cut-off
filter. Freshly thawed tTF derivative was incubated with 30 .mu.l
of DTT (10 mM) in H.sub.20 for 10 min. at room temperature and was
filtered through a column of Sephadex G25 equilibrated in PBS
containing 1 mM EDTA. The eluted fractions containing reduced tTF
were concentrated by ultrafiltration under nitrogen to a final
volume of 3 ml.
[0646] The reduced tTF was mixed with the SMPT-derivatized antibody
and the mixture was allowed to react for 24 h at room temperature.
At the end of the incubation, the reaction mixture was resolved by
gel filtration on a column of Superdex S200 equilibrated in PBS.
Fractions containing anti-VCAM-1.cndot.tTF having a M.sub.r of
180,000 and corresponding to one molecule of antibody linked to one
molecule of tTF were collected.
Example IV
Binding of Anti-VCAM-1 Coaguligand to Activated Endothelial
Cells
A. Methods
1. Iodination of 10H10 Antibody
[0647] Anti-human tissue factor antibody, 10H10, was radiolabeled
with .sup.125I using Chloramine T as described by Bocci (1964,
incorporated herein by reference). The specific activity was
approximately 10,000 cpm/.mu.g, as calculated from protein
determinations measured by a Bradford assay (Bradford, 1976).
2. Cells
[0648] L540 Hodgkin cells (L540 Cy), derived from a patient with
end-stage disease, were prepared as described in Diehl et al.
(1985, incorporated herein by reference), and were obtained from
Prof. Volker Diehl (Klinik fur Innere Medizin der Universitaet,
Koeln, Germany). bEnd.3 cells (murine brain endothelioma) were
prepared as described in Bussolino et al. (1991) and Montesano et
al. (1990), each incorporated herein by reference, and were
obtained from Prof. Werner Risau (Max Planck Institute, Bad
Nauheim, Germany).
3. Tissue Culture
[0649] bEnd.3 cells and hybridomas were maintained in DMEM
supplemented with 10% fetal calf serum, 2 mM L-glutamine, 2
units/ml penicillin G and 2 .mu.g/ml streptomycin. L540 cells were
maintained in RPMI 1640 containing the same additives. All cells
were subcultured once a week. bEnd.3 trypsinization was performed
using 0.125% trypsin in PBS solution containing 0.2% EDTA. For
binding studies, cells were seeded at a density of 5.times.10.sup.4
cells/ml in 0.5 ml of medium in 48 well plates and incubated for
48-96 h. Medium was refreshed 24 h before each study.
4. Binding of Coaguligand to Activated Endothelial Cells
[0650] Binding of the anti-VCAM-1 antibody and coaguligand to
VCAM-1 on activated bEnd.3 cells was determined using a cell based
ELISA, as described by Hahne (1993, incorporated herein by
reference). bEnd.3 cells were incubated with 10 units/ml of
IL-1.alpha. for 4 h at 37.degree. C. in 96-well microtiter plates.
At the end of this incubation, medium was replaced by DPBS
containing 2 mM Ca.sup.2+ and Mg.sup.2+ and 0.2% (w/v) gelatin as a
carrier protein. The same buffer was used for dilution of
antibodies and for washing of cell monolayers between steps.
[0651] Cells were incubated with 4 .mu.g/ml of
anti-VCAM-1.cndot.tTF conjugate, anti-VCAM.cndot.1 antibody or
control reagents for 2 h, and were then washed and incubated for 1
h with rabbit anti-rat IgG-HRP conjugate (1:500 dilution). All
steps were performed at room temperature. HRP activity was measured
by adding O-phenylenediamine (0.5 mg/ml) and hydrogen peroxide
(0.03% w/v) in citrate-phosphate buffer, pH 5.5. After 30 min., 100
.mu.l of supernatant were transferred to 96 well plates, 100 .mu.l
of 0.18 M H.sub.2SO.sub.4 were added and the absorbance was
measured at 492 nm. Each study was performed in duplicate and
repeated at least twice.
5. Detection of Coaguligand Bound to Endothelial Cells
[0652] Anti-VCAM-1 coaguligand and appropriate controls were
incubated with IL-1.alpha. stimulated bEnd.3 cells in 96-well
microtiter plates, as described above. Bound coaguligands were
detected by identifying both the tissue factor component and the
rat IgG component bound to bEnd.3 cells.
[0653] After removing the excess of unbound antibody, cells were
incubated with 100 .mu.l/well of .sup.125I-labeled 10H10 antibody
(0.2 .mu.g/ml) or .sup.125I-labeled rabbit anti-rat Ig (0.2
.mu.g/ml) in binding buffer. After 2 h incubation at room
temperature, cells were washed extensively and dissolved in 0.5 M
of NaOH. The entire volume of 0.5 ml was transferred to plastic
tubes and counted in a .gamma. counter. Each study was performed in
duplicate and repeated at least twice.
B. Results
[0654] The ability of an anti-VCAM-1.cndot.tTF coaguligand to bind
to IL-1.alpha. activated murine bEnd.3 cells was determined by
measuring the binding of radioiodinated anti-TF antibody to
coaguligand-treated cells in vitro. VCAM-1 expression by bEnd.3
cells is transiently inducible by IL-1.alpha. with a peak of VCAM-1
expression being obtained 4-6 h after addition of the cytokine
(Hahne et al., 1993). Strong binding of the coaguligand to
activated bEnd.3 cells was observed (FIG. 1A).
[0655] At saturation, 8.7 fmoles of anti-TF antibody was bound to
the cells, which is equivalent to 540,000 molecules of anti-TF
antibody per cell. Binding of the coaguligand was specific; no
detectable binding over background was observed with an isotype
matched control coaguligand of irrelevant specificity. Binding of
coaguligand to unstimulated cells was about half of that to
activated cells and is probably attributable to constitutive VCAM-1
expression by cultured endothelioma cells.
[0656] In further studies, the anti-VCAM-1.cndot.tTF coaguligand
was found to bind as strongly as unconjugated anti-VCAM-1 antibody
to activated bEnd.3 cells, using detection by peroxidase-labeled
anti rat IgG in the assay. This was done at both saturating and
subsaturating concentrations. Thus, the conjugation procedure
(Example III) did not diminish antibody's capacity to bind to
VCAM-1 on intact endothelial monolayers.
Example V
Factor X Activation by Endothelial Cell-Bound Coaguligand
A. Methods
[0657] The activity of the anti-VCAM-1.cndot.tTF coaguligand bound
to activated bEnd.3 cells was determined indirectly by using a
chromogenic assay to detect factor Xa (Schorer et al., 1985;
Nawroth et al., 1985; each incorporated herein by reference).
IL-1.alpha.-stimulated and unstimulated bEnd.3 cells were incubated
with specific and control coaguligands in 96-well microtiter plates
as described above. The cells were washed with PBS containing 2
mg/ml of BSA and were incubated with 150 .mu.l/well of freshly
prepared Proplex T solution diluted 1:20 in 50 mM Tris-HCl (pH
8.1), 150 mM NaCl, 2 mg/ml BSA (tissue culture grade,
endotoxin-free) and 2.5 in mM CaCl.sub.2. After incubation for 60
min. at 37.degree. C., 100 .mu.l were withdrawn from each well,
transferred to 96-well plates and mixed with 100 .mu.l of the same
buffer containing 12.5 mM EDTA (pH 8.1).
[0658] Chromogenic substrate S2765 for measuring factor Xa
proteolytic activity was added in 50 .mu.l, giving a final
concentration of 300 .mu.M. The breakdown of the substrate was
determined by reading the absorbance at 405 nm over a 2 h period in
a microplate reader (Molecular Devices, Palo Alto, Calif.).
[0659] Production of the chromogenic product was completely
dependent on the presence of Proplex T and bEnd.3 cells
preincubated with the specific coaguligand. Background hydrolysis
of the substrate by Proplex T in the absence of cells was
approximately 10% of the maximal value and was subtracted from each
determination. Free coaguligands diluted in Proplex T solution were
unable to generate factor Xa. The amount of Xa generated was
calculated by reference to a standard curve constructed with known
concentrations of purified factor Xa.
[0660] At the end of the study, cells were detached with
trypsin-EDTA and counted. The results are expressed as the amount
of factor Xa generated per 10.sup.4 cells. Each study was performed
in duplicate and was repeated at least 3 times.
B. Results
1. Factor X Activation
[0661] Anti-VCAM-1.cndot.tTF coaguligand bound to
IL-1.alpha.-activated bEnd.3 cells was capable of specifically
activating factor X. The rate of generation of factor Xa by
anti-VCAM-1.cndot.tTF coated cells was 3.2 ng per 10.sup.4 cells
per hour, which is 7-10 fold higher than was observed with
activated cells treated with a control coaguligand of irrelevant
specificity or with tTF alone (FIG. 1B). Anti-VCAM-1.cndot.tTF in
the absence of cells had undetectable factor X activating activity,
confirming that cell binding is essential for coaguligand
activity.
[0662] Anti-VCAM-1.cndot.tTF bound to unstimulated bEnd.3 cells
activated factor X at a rate of 1.6 ng per 10.sup.4 cells per hour.
This rate is about half that observed with the
IL-1.alpha.-stimulated cells, in accordance with the 50% lower
amount of coaguligand that binds to unstimulated as compared with
stimulated cells. Similar results to those shown in FIG. 1B were
obtained in three separate studies.
2. Effect of Endothelial Cell Permeabilization
[0663] Permeabilization of bEnd.3 monolayers with saponin after
treating them with anti-VCAM-1.cndot.tTF coaguligand increased the
ability of the bound coaguligand to activate factor X by about
30-fold (Table 2). The rate of factor Xa generation by unstimulated
cells treated with anti-VCAM-1.cndot.tTF increased from 1.6 to 49.2
ng per 10.sup.4 cells per hour after permeabilization, while that
of IL-1.alpha. stimulated cells increased from 3.2 to 98.8 ng per
10.sup.4 cells per hour. The factor Xa generating activity of the
permeabilized cells was due to the bound coaguligand rather than to
endogenous TF since permeabilized untreated cells or cells treated
with control coaguligand of irrelevant specificity had low factor
Xa generating activity (2 ng per 10.sup.4 cells per hour).
[0664] These results indicate that the coaguligand is able to
function more efficiently in the environment of a permeabilized
cell. Possibly, permeabilization exposes negatively-charged
phospholipids from within the cell that accelerate the formation of
the coagulation-initiation complexes, or else prevents the
inactivation of such complexes by TFPI.
TABLE-US-00006 TABLE 2 Generation of Factor Xa by
Anti-VCAM-1.cndot.tTF Bound to Intact or Permeabilized bEnd.3 cells
(ng per 10.sup.4 cells per 60 min.) Intact cells Permeabilized
cells.sup.b Treatment.sup.a Control IL-1.alpha. Control IL-1.alpha.
Buffer 0.25.sup.c 0.43 0.45 2.0 tTF 0.26 0.42 0.39 2.1 Control
IgG.cndot.tTF 0.26 0.43 0.41 2.1 Anti-VCAM-1.cndot.tTF 1.64 3.17
49.2 98.8 .sup.aIL-1.alpha. stimulated and unstimulated bEnd.3
cells were incubated with buffer alone or with 4 .mu.g/ml of tTF,
control IgG.cndot.tTF or anti-VCAM.cndot.tTF followed by 60 min.
incubation with Proplex T solution at 37.degree. C. .sup.bCells
were treated with 0.2% saponin 5 min. before addition of Proplex T.
.sup.cAmount of factor Xa was determined as described above.
Results are expressed as ng of factor Xa generated per 10.sup.4
cells per 60 min. The arithmetic mean values from triplicate wells
are shown. SE were less than 5 percent of the mean values.
Example VI
Tumor Blood Vessel Thrombosis by Anti-VCAM-1 Coaguligand
A. Methods
[0665] SCID mice bearing L540 tumors (0.4-0.7 cm.sup.3) were
injected intravenously with 40 .mu.g (total protein) of
anti-VCAM-1.cndot.tTF or R187.cndot.tTF. This dose corresponds to
32 .mu.g of antibody and 8 .mu.g of tTF. Other animals received
equivalent quantities of free antibody, free tTF or a mixture of
both. Animals were anesthetized 4 or 24 h later and their blood
circulations were perfused with heparinized saline. The tumor and
major organs were removed and were fixed in formalin and
paraffin-embedded or snap-frozen for cryosectioning. Sections were
cut through the center of the tissue or tumor. The number of
thrombosed and non-thrombosed blood vessels in 5 cross-sections
were counted. The percentage of thrombosed vessels was
calculated.
B. Results
1. Thrombosis of Tumor Blood Vessels
[0666] This study shows that intravenous administration of the
anti-VCAM-1.cndot.tTF coaguligand induces selective thrombosis of
tumor blood vessels, as opposed to vessels in normal tissues, in
tumor-bearing mice.
[0667] The anti-VCAM-1.cndot.tTF coaguligand was administered to
mice bearing subcutaneous L540 tumors of 0.4 to 0.6 cm in diameter.
Before coaguligand injection, tumors were healthy, having a uniform
morphology lacking regions of necrosis. The tumors were well
vascularized and had a complete absence of spontaneously thrombosed
vessels or hemorrhages. Within four hours of coaguligand injection,
40-70% of blood vessels were thrombosed, despite the initial
staining of only 20-30% of tumor blood vessels shown in Example I.
The thrombosed vessels contained occlusive platelet aggregates,
packed erythrocytes and fibrin. In several regions, the blood
vessels had ruptured, spilling erythrocytes into the tumor
interstitium.
[0668] By 24 h after coaguligand injection, the blood vessels were
still occluded and extensive hemorrhage had spread throughout the
tumor. Tumor cells had separated from one another, had pyknotic
nuclei and were undergoing cytolysis. By 72 h, advanced necrosis
was evident throughout the tumor. Necrosis was clearly present in
the intratumoral region of the tumor, where VCAM-1 expression on
the vessels was not originally prominent. The coaguligand binding
was evidently effective to curtail blood flow in all tumor regions,
resulting in widespread tumor destruction. Furthermore, it is
likely that the initial coaguligand-induced thrombin deposition
results in increased induction of the VCAM-1 target antigen on
central vessels, thus amplifying targeting and tumor
destruction.
[0669] The thrombotic action of anti-VCAM-1.cndot.tTF on tumor
vessels was antigen specific. None of the control reagents
administered at equivalent quantities (tTF alone, anti-VCAM-1
antibody alone, tTF plus anti-VCAM-1 antibody or the control
coaguligand of irrelevant specificity) caused thrombosis (Table
3).
TABLE-US-00007 TABLE 3 Anti-VCAM-1.cndot.tTF-Mediated Thrombosis in
L540 Tumor Bearing Mice Thrombosed Vessels (%).sup.b L540 Heart and
Other Treatment.sup.a Tumor Lung Organs Saline 0-2 0 0 tTF 0-2 0 0
Anti-VCAM-1 Antibody 0-2 0 0 Anti-VCAM-1 Antibody + tTF 0-2 0 0
Control IgG.cndot.tTF 0-2 0 0 Anti-VCAM-1.cndot.tTF (<0.3
cm3).sup.c 0-10 0 0 Anti-VCAM-1.cndot.tTF (>0.3 cm.sup.3) 40-70
0 0 .sup.aL540 tumor-bearing mice were injected i.v. with one of
the following reagents: saline; 8 .mu.g of unconjugated tTF; 32
.mu.g of unconjugated anti-VCAM-1 antibody; mixture of 8 .mu.g of
tTF and 32 .mu.g of anti-VCAM-1 antibody; 40 .mu.g of control
IgG.cndot.tTF coaguligand; or 40 .mu.g of anti- VCAM-1.cndot.tTF
coaguligand. Animals were sacrificed 4 h after injection. Tissues
were removed and fixed in formalin. .sup.bHistological
quantification was performed by counting numbers of thrombosed
blood vessels in 5 cross sections of tissue. The number of
thrombosed vessels is expressed as a percentage of total vessels.
The range of results from three mice is given. .sup.cL540 tumor
bearing mice were divided into two groups (5-8 animals per group)
having tumors smaller or larger than 0.3 cm.sup.3.
2. Lack of Thrombosis of Normal Blood Vessels
[0670] In addition to the thrombosis of tumor blood vessels, this
study also shows that intravenous administration of the
anti-VCAM-1.cndot.tTF coaguligand does not induce thrombosis of
blood vessels in normal organs.
[0671] Despite expression of VCAM-1 on vessels in the heart and
lung of normal or L540 tumor-bearing mice (Table 1), thrombosis did
not occur after anti-VCAM-1.cndot.tTF coaguligand administration.
No signs of thrombosis, tissue damage or altered morphology were
seen in 25 mice injected with 5 to 45 .mu.g of coaguligand 4 or 24
h earlier. There was a normal histological appearance of the heart
and lung from the same mouse that had major tumor thrombosis. All
other major organs (brain, liver, kidney, spleen, pancreas,
intestine, testis) also had unaltered morphology.
[0672] Frozen sections of organs and tumors from
coaguligand-treated mice gave coincident staining patterns when
developed with either the anti-TF antibody, 10H10, or an anti-rat
IgG antibody and confirmed that the coaguligand had localized to
vessels in the heart, lung and tumor. The intensity of staining was
equal to that seen when coaguligand was applied directly to the
sections at high concentrations followed by development with
anti-TF or anti-rat IgG, indicating that saturation of binding had
been attained in vivo.
[0673] These studies show that binding of coaguligand to VCAM-1 on
normal vasculature in heart and lung is not sufficient to induce
thrombosis, and that tumor vasculature provides additional factors
to support coagulation.
Example VII
In Vivo Tumor Destruction by Anti-VCAM-1 Coaguligand
A. Methods
[0674] Male CB17 SCID mice were injected subcutaneously with
1.times.10.sup.7 L540 cells as described above. When the tumors had
reached a volume of 0.4-0.6 cm.sup.3, the mice were injected
intravenously with either 20 .mu.g of anti-VCAM-1.cndot.tTF, 16
.mu.g anti-VCAM-1 antibody, 4 .mu.g tTF, a mixture of 16 .mu.g of
anti-VCAM-1 antibody and 4 .mu.g of tTF, 20 .mu.g control
IgG.cndot.tTF or saline. In some studies, the treatment was given 3
times, on days 0, 4 and 8. A minimum of 8 animals were treated in
each group.
[0675] Animals were monitored daily for tumor measurements and body
weight. Mice were sacrificed when tumors had reached a diameter of
2 cm.sup.3, or earlier if tumors showed signs of necrosis or
ulceration. Tumor volume was calculated according to the formula:
.pi./6.times.D.times.d.sup.2, where D is the larger tumor diameter
and d is the smaller diameter. Differences in tumor growth rates
were tested for statistical significance using a non-parametric
test (Mann-Whitney rank sum test) that makes no assumptions about
tumor size being normally distributed (Gibbons, 1976).
B. Results
[0676] The anti-tumor activity of anti-VCAM-1.cndot.tTF coaguligand
was determined in SCID mice bearing 0.3-0.4 cm.sup.3 L540 tumors.
The drug was administered i.v. 3 times at intervals of 4 days. The
pooled results from 3 separate studies are presented in FIG. 2 and
Table 4. Mean tumor volume of anti-VCAM-1.cndot.tTF treated mice
was significantly reduced at 21 days of treatment (P<0.001) in
comparison to all other groups. Nine of a total of 15 mice treated
with the specific coaguligand showed more than 50% reduction in
tumor volume. This effect was specific since unconjugated tTF,
control IgG coaguligand and mixture of free anti-VCAM-1 antibody
and tTF did not affect tumor growth.
TABLE-US-00008 TABLE 4 Inhibition of Tumor Growth by
Anti-VCAM-1.cndot.tTF Coaguligand Mean tumor Tumor volume
(mm.sup.3).sup.b Growth P versus Treatment.sup.a N Day 0 Day 21
Index.sup.c saline.sup.d Saline 14 331 .+-. 61 2190 .+-. 210 6.91
-- TTF 13 341 .+-. 22 2015 .+-. 205 5.90 NS Anti-VCAM-1 16 363 .+-.
24 1920 .+-. 272 5.28 NS Anti-VCAM-1 + tTF 13 349 .+-. 42 2069 .+-.
362 5.92 NS Control IgG.cndot.tTF 8 324 .+-. 30 2324 .+-. 304 7.17
NS Anti-VCAM-1.cndot.tTF 15 365 .+-. 28 1280 .+-. 130 3.50
<0.001 .sup.aL540 tumor bearing mice were injected i.v. with one
of the following reagents: saline; 8 .mu.g of unconjugated tTF; 32
.mu.g of unconjugated anti-VCAM-1 antibody; mixture of 8 .mu.g of
tTF and 32 .mu.g of anti-VCAM-1 antibody; 40 .mu.g of control
IgG.cndot.tTF (R187) coaguligand; or 40 .mu.g of
anti-VCAM-1.cndot.tTF coaguligand. The treatment was repeated on
day 4 and 7 after first injection. .sup.bMean tumor volume .+-. SD.
.sup.cThe tumor growth index is the ratio of mean tumor volume on
day 21 to mean tumor volume on day 0. .sup.dTwo tailed P values are
for differences in tumor volume (day 21) for the treated groups
versus the saline group as determined by the Mann-Whitney rank sum
test.
Example VIII
Phosphatidylserine Expression on Tumor Blood Vessels
A. Methods
1. Antibodies
[0677] Anti-phosphatidylserine (anti-PS) and anti-cardiolipin
antibodies, both mouse monoclonal IgM antibodies, were produced as
described by Rote (Rote et al., 1993). Details of the
characterization of the anti-PS and anti-cardiolipin antibodies
were also reported by Rote et al, (1993, incorporated herein by
reference).
2. Detection of PS Expression on Vascular Endothelium
[0678] L540 tumor-bearing mice were injected i.v. with 20 .mu.g of
either anti-PS or anti-cardiolipin mouse IgM antibodies. After 10
min., mice were anesthetized and their blood circulations were
perfused with heparinized saline. Tumors and normal tissues were
removed and snap-frozen. Serial sections of organs and tumors were
stained with either HRP-labeled anti-mouse IgM for detection of
anti-PS antibody or with anti-VCAM-1 antibody followed by
HRP-labeled anti-rat Ig.
[0679] To preserve membrane phospholipids on frozen sections, the
following protocol was developed. Animals were perfused with DPBS
containing 2.5 mM Ca.sup.2+. Tissues were mounted on
3-aminopropyltriethoxysilane-coated slides and were stained within
24 h. No organic solvents, formaldehyde or detergents were used for
fixation or washing of the slides. Slides were re-hydrated by DPBS
containing 2.5 mM Ca.sup.2+ and 0.2% gelatin. The same solution was
also used to wash sections to remove the excess of reagents.
Sections were incubated with HRP-labeled anti-mouse IgM for 3.5 h
at room temperature to detect anti-PS IgM.
B. Results
[0680] To explain the lack of thrombotic effect of
anti-VCAM-1.cndot.tTF on VCAM-1 positive vasculature in heart and
lungs, the inventors developed a concept of differential PS
localization between normal and tumor blood vessels. Specifically,
they hypothesized that endothelial cells in normal tissues
segregate PS to the inner surface of the plasma membrane
phospholipid bilayer, where it is unable to participate in
thrombotic reactions; whereas endothelial cells in tumors
translocate PS to the external surface of the plasma membrane,
where it can support the coagulation action of the coaguligand. PS
expression on the cell surface allows coagulation because it
enables the attachment of coagulation factors to the membrane and
coordinates the assembly of coagulation initiation complexes (Ortel
et al., 1992).
[0681] The inventors' model of PS translocation to the surface of
tumor blood vessel endothelial cells, as developed herein, is
surprising in that PS expression does not occur after, and does not
inevitably trigger, cell death. PS expression at the tumor
endothelial cell surface is thus sufficiently stable to allow PS to
serve as a targetable entity for therapeutic intervention.
[0682] To confirm the hypothesis that tumor blood vessel
endothelium expresses PS on the luminal surface of the plasma
membrane, the inventors used immunohistochemistry to determine the
distribution of anti-PS antibody after intravenous injection into
L540 tumor bearing mice. Anti-PS antibody localized within 10 min.
to the majority of tumor blood vessels, including vessels in the
central region of the tumor that can lack VCAM-1. Vessels that were
positive for VCAM-1 were also positive for PS. Thus, there is
coincident expression of PS on VCAM-1-expressing vessels in
tumors.
[0683] In the in vivo localization studies, none of the vessels in
normal organs, including VCAM-1-positive vasculature of heart and
lung, were stained, indicating that PS is absent from the external
surface of the endothelial cells. In contrast, when sections of
normal tissues and tumors were directly stained with anti-PS
antibody in vitro, no differences were visible between normal and
tumor, endothelial or other cell types, showing that PS is present
within these cells but only becomes expressed on the surface of
endothelial cells in tumors.
[0684] The specificity of PS detection was confirmed by two
independent studies. First, a mouse IgM monoclonal antibody
directed against a different negatively charged lipid, cardiolipin,
did not home to tumor or any organs in vivo. Second, pretreatment
of frozen sections with acetone abolished staining with anti-PS
antibody, presumably because it extracted the lipids together with
the bound anti-PS antibody.
Example IX
Annexin V Blocks Coaguligand Activation of Factor X In Vitro
A. Methods
[0685] The ability of Annexin V to affect Factor Xa formation
induced by coaguligand was determined by a chromogenic assay
described above in Example V. IL-1.alpha.-stimulated bEnd.3 cells
were incubated with anti-VCAM-.cndot.tTF and permeabilized by
saponin. Annexin V was added at concentrations ranging from 0.1 to
10 .mu.g/ml and cells were incubated for 30 min. before addition of
diluted Proplex T. The amount of Factor Xa generated in the
presence or absence of Annexin V was determined as described in
Example V. Each treatment was performed in duplicate and repeated
at least twice.
B. Results
[0686] The need for surface PS expression in coaguligand action is
further indicated by the inventors' finding that annexin V, which
binds to PS with high affinity, blocks the ability of
anti-VCAM-1.cndot.tTF bound to bEnd.3 cells to generate factor Xa
in vitro.
[0687] Annexin V added to permeabilized cells preincubated with
anti-VCAM-1.cndot.tTF inhibited the formation of factor Xa in a
dose-dependent manner (FIG. 3). In the absence of Annexin V,
cell-bound coaguligand produced 95 ng of factor Xa per 10,000 cells
per 60 min. The addition of increasing amounts of Annexin V (in the
.mu.g per ml range) inhibited factor Xa production. At 10 .mu.g per
ml, Annexin V inhibited factor Xa production by 58% (FIG. 3). No
further inhibition was observed by increasing the concentration of
Annexin V during the assay, indicating that annexin V saturated all
available binding sites at 10 .mu.g per ml.
Example X
Annexin V Blocks Coaguligand Activity In Vivo
A. Methods
[0688] The ability of Annexin V to inhibit coaguligand-induced
thrombosis in vivo was examined in L540 Hodgkin-bearing SCID mice.
Tumors were grown in mice as described above in Example II. Two
mice per group (tumor size 0.5 cm in diameter) were injected
intravenously via the tail vein with one of the following reagents:
a) saline; b) 100 .mu.g of Annexin V; c) 40 .mu.g of
anti-VCAM-1.cndot.tTF; d) 100 .mu.g of Annexin V followed 2 hours
later by 40 .mu.g of anti-VCAM-1.cndot.tTF.
[0689] Four hours after the last injection mice were anesthetized
and perfused with heparinized saline. Tumors were removed, fixed
with 4% formalin, paraffin-embedded and stained with
hematoxylene-eosin. The number of thrombosed and non-thrombosed
blood vessels were counted and the percentage of thrombosis was
calculated.
B. Results
[0690] Annexin V also blocks the activity of the
anti-VCAM-1.cndot.tTF coaguligand in vivo. Groups of tumor-bearing
mice were treated with one of the control or test reagents, as
described in the methods. Mice were given (a) saline; (b) 100 .mu.g
of Annexin V; (c) 40 .mu.g of anti-VCAM-1.cndot.tTF coaguligand; or
(d) 100 .mu.g of Annexin V followed 2 hours later by 40 .mu.g of
anti-VCAM-1.cndot.tTF coaguligand. Identical results were obtained
in both mice per group.
[0691] No spontaneous thrombosis, hemorrhages or necrosis were
observed in tumors derived from saline-injected mice. Treatment
with Annexin V alone did not alter tumor morphology.
[0692] In accordance with other data presented herein, 40 .mu.g of
anti-VCAM-1.cndot.tTF coaguligand caused thrombosis in 70% of total
tumor blood vessels. The majority of blood vessels were occluded
with packed erythrocytes and clots, and tumor cells were separated
from one another. Both coaguligand-induced anti-tumor effects,
i.e., intravascular thrombosis and changes in tumor cell
morphology, were completely abolished by pre-treating the mice with
Annexin V.
[0693] These findings confirm that the anti-tumor effects of
coaguligands are mediated through the blockage of tumor
vasculature. These data also demonstrate that PS is essential for
coaguligand-induced thrombosis in vivo.
Example XI
Externalized Phosphatidylserine is a Global Marker of Tumor Blood
Vessels
A. Methods
[0694] PS exposure on tumor and normal vascular endothelium was
examined in three animal tumor models: L540 Hodgkin lymphoma,
NCI-H358 non-small cell lung carcinoma, and HT 29 colon
adenocarcinoma (ATCC). To grow the tumors in vivo, 2.times.10.sup.6
cells were injected into the right flank of SCID mice and allowed
to reach 0.8-1.2 cm in diameter. Mice bearing large tumors (volume
above 800 mm.sup.3) were injected intravenously via the tail vein
with 20 .mu.g of either anti-PS or anti-cardiolipin antibodies. The
anti-cardiolipin antibody served as a control for all studies since
both antibodies are directed against negatively charged lipids and
belong to the same class of immunoglobulins (mouse IgM).
[0695] One hour after injection, mice were anesthetized and their
blood circulation was perfused with heparinized saline. Tumors and
normal organs were removed and snap-frozen. Frozen sections were
stained with anti-mouse IgM-peroxidase conjugate (Jackson
Immunoresearch Labs) followed by development with carbazole.
B. Results
[0696] The anti-PS antibodies specifically homed to the vasculature
of all three tumors (HT 29, L540 and NCI-H358) in vivo, as
indicated by detection of the mouse IgM. The average percentages of
vessels stained in the tumors were 80% for HT 29, 30% for L540 and
50% for NCI-H358. Vessels in all regions of the tumors were stained
and there was staining both of small capillaries and larger
vessels.
[0697] No vessel staining was observed with anti-PS antibodies in
any normal tissues. In the kidney, tubules were stained both with
anti-PS and anti-CL, and this likely relates to the secretion of
IgMs by this organ (Table 5). Anti-cardiolipin antibodies were not
detected in any tumors or normal tissues, except kidney.
[0698] These findings indicate that only tumor endothelium exposes
PS to the outer site of the plasma membrane.
TABLE-US-00009 TABLE 5 Vessel Localization of Anti-PS and Anti-
Cardiolipin Abs in Tumor-Bearing Mice* Tissue Anti-PS.dagger.
Anti-Cardiolipin.dagger. L540 Cy tumor ++ - H358 tumor ++ - HT29
tumor +++ - Adrenal - - Brain Cerebellum - - Brain Cortex - - Heart
- - Kidney -.dagger-dbl. -.dagger-dbl. Large Intestine - - Liver -
- Lung - - Pancreas - - Small Intestine - - Spleen - - Testes - -
*Biodistribution in normal organs of both anti-PS and
anti-cardiolipin Abs was identical in all three tumor animal
models. .dagger.Anti-PS and anti-cardiolipin antibodies were
detected on frozen sections using anti-mouse IgM-peroxidase
conjugate. - no staining; + weak; ++ moderate; +++ strong,
equivalent to pan endothelial marker Meca 32. .dagger-dbl.Tubular
staining was observed in the kidneys of both and-PS and anti-CL
recipients.
[0699] To estimate the time at which tumor vasculature loses the
ability to segregate PS to the inner side of the membrane, the
inventors examined anti-PS localization in L540 tumors ranging in
volume from 140 to 1,600 mm.sup.3. Mice were divided into 3 groups
according to their tumor size: 140-300, 350-800 and 800-1,600
mm.sup.3. Anti-PS Ab was not detected in three mice bearing small
L540 tumors (up to 300 mm.sup.3). Anti-PS Ab localized in 3 animals
of 5 in the group of intermediate size L540 tumors and in all mice
(4 out of 4) bearing large L540 tumors (Table 6). Percent of
PS-positive blood vessels from total (identified by pan endothelial
marker Meca 32) was 10-20% in the L540 intermediate group and
20-40% in the group of large L540 tumors (Table 6).
TABLE-US-00010 TABLE 6 PS Externalization Detected in Mid and Large
Sized Tumors No. Positive % PS-Positive Tumor Size (mm.sup.3)
Tumors/Total* Vessels/Total.dagger. 350-800 3/5 10-20 850-1,600 4/4
20-40 *Mice bearing L540 Cy tumors were divided into three groups
according to tumor size. 20 .mu.g of anti-PS antibodies were
injected i.v. and allowed to circulate for 1 hour. Mouse antibodies
were detected on frozen sections using anti-mouse IgM-peroxidase
conjugate. .dagger.Total number of blood vessels was determined
using pan-endothelial Ab Meca 32. PS-positive and Meca-positive
vessels were counted in 4 fields per cross section of tumor. Range
of % PS-positive vessels within the same group is shown.
Example XII
Anti-Tumor Effects of Unconjugated Anti-Phosphatidylserine
Antibodies
A. Methods
[0700] The effects of anti-PS antibodies were examined in syngeneic
and xenogeneic tumor models. For the syngeneic model,
1.times.10.sup.7 cells of murine colorectal carcinoma Colo 26
(obtained from Dr. Ian Hart, ICRF, London) were injected
subcutaneously into the right flank of Balb/c mice. In the
xenogeneic model, a human Hodgkin's lymphoma L540 xenograft was
established by injecting 1.times.10.sup.7 cells subcutaneously into
the right flank of male CB17 SCID mice. Tumors were allowed to grow
to a size of about 0.6-0.9 cm.sup.3 before treatment.
[0701] Tumor-bearing mice (4 animals per group) were injected i.p.
with 20 .mu.g of naked anti-PS antibody (IgM), control mouse IgM or
saline. Treatment was repeated 3 times with a 48 hour interval.
Animals were monitored daily for tumor measurements and body
weight. Tumor volume was calculated as described in Example VII.
Mice were sacrificed when tumors had reached 2 cm.sup.3, or earlier
if tumors showed signs of necrosis or ulceration.
B. Results
[0702] The growth of both syngeneic and xenogeneic tumors was
effectively inhibited by treatment with naked anti-PS antibodies
(FIG. 4A and FIG. 4B). Anti-PS antibodies caused tumor vascular
injury, accompanied by thrombosis, and tumor necrosis. The presence
of clots and disintegration of tumor mass surrounding blocked blood
vessels was evident.
[0703] Quantitatively, the naked anti-PS antibody treatment
inhibited tumor growth by up to 60% of control tumor volume in mice
bearing large Colo 26 (FIG. 4A) and L540 (FIG. 4B) tumors. No
retardation of tumor growth was found in mice treated with saline
or control IgM. No toxicity was observed in mice treated with
anti-PS antibodies, with normal organs preserving unaltered
morphology, indistinguishable from untreated or saline-treated
mice.
[0704] Tumor regression started 24 hours after the first treatment
and tumors continue to decline in size for the next 6 days. This
was observed in both syngeneic and immunocompromised tumor models,
indicating that the effect was mediated by immune
status-independent mechanism(s). Moreover, the decline in tumor
burden was associated with the increase of alertness and generally
healthy appearance of the animals, compared to control mice bearing
tumors larger than 1500 mm.sup.3. Tumor re-growth occurred 7-8 days
after the first treatment.
[0705] The results obtained with anti-PS treatment of L540 tumors
are further compelling for the following reasons. Notably, the
tumor necrosis observed in L540 tumor treatment occurred despite
the fact that the percentage of vessels that stained positive for
PS in L540 tumors was less than in HT 29 and NCI-H358 tumors. This
implies that even more rapid necrosis would likely result when
treating other tumor types. Furthermore, L540 tumors are generally
chosen as an experimental model because they provide clean
histological sections and they are, in fact, known to be resistant
to necrosis.
Example XIII
Anti-Tumor Effects of Annexin Conjugates
[0706] The surprising finding that aminophospholipids are stable
markers of tumor vasculature also means that antibody-therapeutic
agent constructs can be used in cancer treatment. In addition to
using antibodies as targeting agents, the inventors reasoned that
annexins, and other aminophospholipid-binding proteins, could also
be used to specifically deliver therapeutic agents to tumor
vasculature. The following data shows the anti-tumor effects that
result from the in vivo administration of annexin-TF
constructs.
A. Methods
[0707] An annexin V-tTF conjugate was prepared and administered to
nu/nu mice with solid tumors. The tumors were formed from human
HT29 colorectal carcinoma cells that formed tumors of at least
about 1.2 cm.sup.3. The annexin V-tTF coaguligand (10 .mu.g) was
administered intravenously and allowed to circulate for 24 hours.
Saline-treated mice were separately maintained as control animals.
After the one day treatment period, the mice were sacrificed and
exsanguinated and the tumors and major organs were harvested for
analysis.
B. Results
[0708] The annexin V-tTF conjugate was found to induce specific
tumor blood vessel coagulation in HT29 tumor bearing mice.
Approximately 55% of the tumor blood vessels in the annexin V-tTF
conjugate treated animals were thrombosed following a single
injection. In contrast, there was minimal evidence of thrombosis in
the tumor vasculature of the control animals.
Example XIV
Phosphatidylserine Translocation in the Tumor Environment
[0709] The discovery of PS as an in vivo surface marker unique to
tumor vascular endothelial cells prompted the inventors to further
investigate the effect of a tumor environment on PS translocation
and outer membrane expression. The present example shows that
exposing endothelial cells in vitro to certain conditions that
mimic those in a tumor duplicates the observed PS surface
expression in intact, viable cells.
A. Methods
[0710] Mouse bEnd.3 endothelial cells were seeded at an initial
density of 50,000 cells/well. Twenty-fours later cells were
incubated with increasing concentrations of H.sub.2O.sub.2 (from 10
.mu.M to 500 .mu.M) for 1 hour at 37.degree. C. or left untreated.
At the end of the incubation, cells were washed 3 times with PBS
containing 0.2% gelatin and fixed with 0.25% glutaraldehyde.
Identical wells were either stained with anti-PS IgM or trypsinized
and evaluated for viability by the Trypan Blue exclusion test. For
the anti-PS staining, after blocking with 2% gelatin for 10 min.,
cells were incubated with 2 .mu.g/ml of anti-PS antibody, followed
by detection with anti-mouse IgM-HRP conjugate.
[0711] Wells seeded with mouse bEnd.3 endothelial cells were also
incubated with different effectors and compared to control,
untreated wells after the same period of incubation at 37.degree.
C. The panel of effectors tested included TNF, LPS, bFGF,
IL-1.alpha., IL-1.beta. and thrombin. After incubation, cells were
washed and fixed and were again either stained with anti-PS IgM or
evaluated for viability using the Trypan Blue exclusion test, as
described above.
B. Results
1. PS Induction by H.sub.2O.sub.2
[0712] Exposing endothelial cells to H.sub.2O.sub.2 at
concentrations higher than 100 .mu.M caused PS translocation in
.about.90% cells. However, this was accompanied by detachment of
the cells from the substrate and cell viability decreasing to about
50-60%. The association of surface PS expression with decreasing
cell viability is understandable, although it is still interesting
to note that .about.90% PS translocation is observed with only a
50-60% decrease in cell viability.
[0713] Using concentrations of H.sub.2O.sub.2 lower than 100 .mu.M
resulted in significant PS expression without any appreciable
reduction in cell viability. For example, PS was detected at the
cell surface of about 50% of cells in all H.sub.2O.sub.2 treated
wells using H.sub.2O.sub.2 at concentrations as low as 20 .mu.M. It
is important to note that, under these low H.sub.2O.sub.2
concentrations, the cells remained firmly attached to the plastic
and to each other, showed no morphological changes and had no signs
of cytotoxicity. Detailed analyses revealed essentially 100%
cell-cell contact, retention of proper cell shape and an intact
cytoskeleton.
[0714] The 50% PS surface expression induced by low levels of
H.sub.2O.sub.2 was thus observed in cell populations in which cell
viability was identical to the control, untreated cells (i.e.,
95%). The PS expression associated with high H.sub.2O.sub.2
concentrations was accompanied by cell damage, and the PS-positive
cells exposed to over 100 .mu.M H.sub.2O.sub.2 were detached,
floating and had disrupted cytoskeletons.
[0715] The maintenance of cell viability in the presence of low
concentrations H.sub.2O.sub.2 is consistent with data from other
laboratories. For example, Schorer et al. (1985) showed that human
umbilical vein endothelial cells (HUVEC) treated with 15 .mu.M
H.sub.2O.sub.2 averaged 90 to 95% viability (reported as 5% to 10%
injury), whilst those exposed to 1500 .mu.M H.sub.2O.sub.2 were
only 0%-50% viable (50% to 100% injured).
[0716] The use of H.sub.2O.sub.2 to mimic the tumor environment in
vitro is also appropriate in that the tumor environment is rich in
inflammatory cells, such as macrophages, PMNs and granulocytes,
which produce H.sub.2O.sub.2 and other reactive oxygen species.
Although never before connected with stable tumor vascular markers,
inflammatory cells are known to mediate endothelial cell injury by
mechanisms involving reactive oxygen species that require the
presence of H.sub.2O.sub.2 (Weiss et al., 1981; Yamada et al.,
1981; Schorer et al., 1985). In fact, studies have shown that
stimulation of PMNs in vitro produces concentrations of
H.sub.2O.sub.2 sufficient to cause sublethal endothelial cell
injury without causing cell death (measured by chromium release
assays) or cellular detachment; and that these H.sub.2O.sub.2
concentrations are attainable locally in vivo (Schorer et al.,
1985).
[0717] The present in vitro translocation data correlates with the
earlier results showing that anti-PS antibodies localize
specifically to tumor vascular endothelial cells in vivo, and do
not bind to cells in normal tissues. The finding that in vivo-like
concentrations of H.sub.2O.sub.2 induce PS translocation to the
endothelial cell surface without disrupting cell integrity has
important implications in addition to validating the original in
vivo data and the inventors' therapeutic approaches.
[0718] Human, bovine and murine endothelial cells are all known to
be PS-negative under normal conditions. Any previously documented
PS expression has always been associated with cell damage and/or
cell death. This is simply not the case in the present studies,
where normal viability is maintained. This shows that PS
translocation in tumor vascular endothelium is mediated by
biochemical mechanisms unconnected to cell damage. This is believed
to be the first demonstration of PS surface expression in
morphologically intact endothelial cells and the first indication
that PS expression can be disconnected from the apoptosis
pathway(s). Returning to the operability of the present invention,
these observations again confirm that PS is a sustainable, rather
than transient, marker of tumor blood vessels and a suitable
candidate for therapeutic intervention.
2. PS Expression does not Correlate with Cell Activation
[0719] The relevance of this in vitro data to the tumor environment
is also strengthened by the fact that other, general cell
activators are without effect on PS translocation in endothelial
cells. For example, the inventors tested TNF in similarly
controlled studies and found it unable to induce PS surface
expression, despite the expected increases in E-selectin and VCAM
expression. Likewise, LPS, bFGF, IL-1.alpha. and IL-1.beta. were
all without effect on PS expression in appropriately controlled
studies.
3. PS Induction by Thrombin
[0720] In contrast to the lack of effects of other cell activators,
thrombin was observed to increase PS expression, although not to
the same extent as H.sub.2O.sub.2. This data is also an integral
part of the tumor-induction model of PS expression developed by the
present inventors (thrombin-induced PS surface expression in normal
tissues would also further coagulation as PS expression coordinates
the assembly of coagulation initiation complexes (Ortel et al.,
1992)).
[0721] The tumor environment is known to be prothrombotic, such
that tumor vasculature is predisposed to coagulation (U.S. Pat. No.
5,877,289). As thrombin is a product of the coagulation cascade, it
is present in tumor vasculature. In fact, the presence of thrombin
induces VCAM expression, contributing to the inventors' ability to
exploit VCAM as a targetable marker of tumor vasculature (U.S. Pat.
Nos. 5,855,866; 5,877,289). The present data showing that thrombin
also induces PS expression is thus both relevant to targeting
aminophospholipids with naked antibodies and therapeutic
conjugates, and further explains the beneficial effects of the
anti-VCAM coaguligand containing Tissue Factor (Example VII).
[0722] All of the compositions and methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
512149DNAHomo sapiens 1cagctgactc aggcaggctc catgctgaac ggtcacacag
agaggaaaca ataaatctca 60gctactatgc aataaatatc tcaagtttta acgaagaaaa
acatcattgc agtgaaataa 120aaaattttaa aattttagaa caaagctaac
aaatggctag ttttctatga ttcttcttca 180aacgctttct ttgaggggga
aagagtcaaa caaacaagca gttttacctg aaataaagaa 240ctagttttag
aggtcagaag aaaggagcaa gttttgcgag aggcacggaa ggagtgtgct
300ggcagtacaa tgacagtttt cctttccttt gctttcctcg ctgccattct
gactcacata 360gggtgcagca atcagcgccg aagtccagaa aacagtggga
gaagatataa ccggattcaa 420catgggcaat gtgcctacac tttcattctt
ccagaacacg atggcaactg tcgtgagagt 480acgacagacc agtacaacac
aaacgctctg cagagagatg ctccacacgt ggaaccggat 540ttctcttccc
agaaacttca acatctggaa catgtgatgg aaaattatac tcagtggctg
600caaaaacttg agaattacat tgtggaaaac atgaagtcgg agatggccca
gatacagcag 660aatgcagttc agaaccacac ggctaccatg ctggagatag
gaaccagcct cctctctcag 720actgcagagc agaccagaaa gctgacagat
gttgagaccc aggtactaaa tcaaacttct 780cgacttgaga tacagctgct
ggagaattca ttatccacct acaagctaga gaagcaactt 840cttcaacaga
caaatgaaat cttgaagatc catgaaaaaa acagtttatt agaacataaa
900atcttagaaa tggaaggaaa acacaaggaa gagttggaca ccttaaagga
agagaaagag 960aaccttcaag gcttggttac tcgtcaaaca tatataatcc
aggagctgga aaagcaatta 1020aacagagcta ccaccaacaa cagtgtcctt
cagaagcagc aactggagct gatggacaca 1080gtccacaacc ttgtcaatct
ttgcactaaa gaaggtgttt tactaaaggg aggaaaaaga 1140gaggaagaga
aaccatttag agactgtgca gatgtatatc aagctggttt taataaaagt
1200ggaatctaca ctatttatat taataatatg ccagaaccca aaaaggtgtt
ttgcaatatg 1260gatgtcaatg ggggaggttg gactgtaata caacatcgtg
aagatggaag tctagatttc 1320caaagaggct ggaaggaata taaaatgggt
tttggaaatc cctccggtga atattggctg 1380gggaatgagt ttatttttgc
cattaccagt cagaggcagt acatgctaag aattgagtta 1440atggactggg
aagggaaccg agcctattca cagtatgaca gattccacat aggaaatgaa
1500aagcaaaact ataggttgta tttaaaaggt cacactggga cagcaggaaa
acagagcagc 1560ctgatcttac acggtgctga tttcagcact aaagatgctg
ataatgacaa ctgtatgtgc 1620aaatgtgccc tcatgttaac aggaggatgg
tggtttgatg cttgtggccc ctccaatcta 1680aatggaatgt tctatactgc
gggacaaaac catggaaaac tgaatgggat aaagtggcac 1740tacttcaaag
ggcccagtta ctccttacgt tccacaacta tgatgattcg acctttagat
1800ttttgaaagc gcaatgtcag aagcgattat gaaagcaaca aagaaatccg
gagaagctgc 1860caggtgagaa actgtttgaa aacttcagaa gcaaacaata
ttgtctccct tccagcaata 1920agtggtagtt atgtgaagtc accaaggttc
ttgaccgtga atctggagcc gtttgagttc 1980acaagagtct ctacttgggg
tgacagtgct cacgtggctc gactatagaa aactccactg 2040actgtcgggc
tttaaaaagg gaagaaactg ctgagcttgc tgtgcttcaa actactactg
2100gaccttattt tggaactatg gtagccagat gataaatatg gttaatttc
21492498PRTHomo sapiens 2Met Thr Val Phe Leu Ser Phe Ala Phe Leu
Ala Ala Ile Leu Thr His 1 5 10 15Ile Gly Cys Ser Asn Gln Arg Arg
Ser Pro Glu Asn Ser Gly Arg Arg 20 25 30Tyr Asn Arg Ile Gln His Gly
Gln Cys Ala Tyr Thr Phe Ile Leu Pro 35 40 45Glu His Asp Gly Asn Cys
Arg Glu Ser Thr Thr Asp Gln Tyr Asn Thr 50 55 60Asn Ala Leu Gln Arg
Asp Ala Pro His Val Glu Pro Asp Phe Ser Ser 65 70 75 80Gln Lys Leu
Gln His Leu Glu His Val Met Glu Asn Tyr Thr Gln Trp 85 90 95Leu Gln
Lys Leu Glu Asn Tyr Ile Val Glu Asn Met Lys Ser Glu Met 100 105
110Ala Gln Ile Gln Gln Asn Ala Val Gln Asn His Thr Ala Thr Met Leu
115 120 125Glu Ile Gly Thr Ser Leu Leu Ser Gln Thr Ala Glu Gln Thr
Arg Lys 130 135 140Leu Thr Asp Val Glu Thr Gln Val Leu Asn Gln Thr
Ser Arg Leu Glu145 150 155 160Ile Gln Leu Leu Glu Asn Ser Leu Ser
Thr Tyr Lys Leu Glu Lys Gln 165 170 175Leu Leu Gln Gln Thr Asn Glu
Ile Leu Lys Ile His Glu Lys Asn Ser 180 185 190Leu Leu Glu His Lys
Ile Leu Glu Met Glu Gly Lys His Lys Glu Glu 195 200 205Leu Asp Thr
Leu Lys Glu Glu Lys Glu Asn Leu Gln Gly Leu Val Thr 210 215 220Arg
Gln Thr Tyr Ile Ile Gln Glu Leu Glu Lys Gln Leu Asn Arg Ala225 230
235 240Thr Thr Asn Asn Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met
Asp 245 250 255Thr Val His Asn Leu Val Asn Leu Cys Thr Lys Glu Gly
Val Leu Leu 260 265 270Lys Gly Gly Lys Arg Glu Glu Glu Lys Pro Phe
Arg Asp Cys Ala Asp 275 280 285Val Tyr Gln Ala Gly Phe Asn Lys Ser
Gly Ile Tyr Thr Ile Tyr Ile 290 295 300Asn Asn Met Pro Glu Pro Lys
Lys Val Phe Cys Asn Met Asp Val Asn305 310 315 320Gly Gly Gly Trp
Thr Val Ile Gln His Arg Glu Asp Gly Ser Leu Asp 325 330 335Phe Gln
Arg Gly Trp Lys Glu Tyr Lys Met Gly Phe Gly Asn Pro Ser 340 345
350Gly Glu Tyr Trp Leu Gly Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln
355 360 365Arg Gln Tyr Met Leu Arg Ile Glu Leu Met Asp Trp Glu Gly
Asn Arg 370 375 380Ala Tyr Ser Gln Tyr Asp Arg Phe His Ile Gly Asn
Glu Lys Gln Asn385 390 395 400Tyr Arg Leu Tyr Leu Lys Gly His Thr
Gly Thr Ala Gly Lys Gln Ser 405 410 415Ser Leu Ile Leu His Gly Ala
Asp Phe Ser Thr Lys Asp Ala Asp Asn 420 425 430Asp Asn Cys Met Cys
Lys Cys Ala Leu Met Leu Thr Gly Gly Trp Trp 435 440 445Phe Asp Ala
Cys Gly Pro Ser Asn Leu Asn Gly Met Phe Tyr Thr Ala 450 455 460Gly
Gln Asn His Gly Lys Leu Asn Gly Ile Lys Trp His Tyr Phe Lys465 470
475 480Gly Pro Ser Tyr Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro
Leu 485 490 495Asp Phe32269DNAHomo sapiens 3tgggttggtg tttatctcct
cccagccttg agggagggaa caacactgta ggatctgggg 60agagaggaac aaaggaccgt
gaaagctgct ctgtaaaagc tgacacagcc ctcccaagtg 120agcaggactg
ttcttcccac tgcaatctga cagtttactg catgcctgga gagaacacag
180cagtaaaaac caggtttgct actggaaaaa gaggaaagag aagactttca
ttgacggacc 240cagccatggc agcgtagcag ccctgcgttt cagacggcag
cagctcggga ctctggacgt 300gtgtttgccc tcaagtttgc taagctgctg
gtttattact gaagaaagaa tgtggcagat 360tgttttcttt actctgagct
gtgatcttgt cttggccgca gcctataaca actttcggaa 420gagcatggac
agcataggaa agaagcaata tcaggtccag catgggtcct gcagctacac
480tttcctcctg ccagagatgg acaactgccg ctcttcctcc agcccctacg
tgtccaatgc 540tgtgcagagg gacgcgccgc tcgaatacga tgactcggtg
cagaggctgc aagtgctgga 600gaacatcatg gaaaacaaca ctcagtggct
aatgaagctt gagaattata tccaggacaa 660catgaagaaa gaaatggtag
agatacagca gaatgcagta cagaaccaga cggctgtgat 720gatagaaata
gggacaaacc tgttgaacca aacagctgag caaacgcgga agttaactga
780tgtggaagcc caagtattaa atcagaccac gagacttgaa cttcagctct
tggaacactc 840cctctcgaca aacaaattgg aaaaacagat tttggaccag
accagtgaaa taaacaaatt 900gcaagataag aacagtttcc tagaaaagaa
ggtgctagct atggaagaca agcacatcat 960ccaactacag tcaataaaag
aagagaaaga tcagctacag gtgttagtat ccaagcaaaa 1020ttccatcatt
gaagaactag aaaaaaaaat agtgactgcc acggtgaata attcagttct
1080tcaaaagcag caacatgatc tcatggagac agttaataac ttactgacta
tgatgtccac 1140atcaaactca gctaaggacc ccactgttgc taaagaagaa
caaatcagct tcagagactg 1200tgctgaagta ttcaaatcag gacacaccac
aaatggcatc tacacgttaa cattccctaa 1260ttctacagaa gagatcaagg
cctactgtga catggaagct ggaggaggcg ggtggacaat 1320tattcagcga
cgtgaggatg gcagcgttga ttttcagagg acttggaaag aatataaagt
1380gggatttggt aacccttcag gagaatattg gctgggaaat gagtttgttt
cgcaactgac 1440taatcagcaa cgctatgtgc ttaaaataca ccttaaagac
tgggaaggga atgaggctta 1500ctcattgtat gaacatttct atctctcaag
tgaagaactc aattatagga ttcaccttaa 1560aggacttaca gggacagccg
gcaaaataag cagcatcagc caaccaggaa atgattttag 1620cacaaaggat
ggagacaacg acaaatgtat ttgcaaatgt tcacaaatgc taacaggagg
1680ctggtggttt gatgcatgtg gtccttccaa cttgaacgga atgtactatc
cacagaggca 1740gaacacaaat aagttcaacg gcattaaatg gtactactgg
aaaggctcag gctattcgct 1800caaggccaca accatgatga tccgaccagc
agatttctaa acatcccagt ccacctgagg 1860aactgtctcg aactattttc
aaagacttaa gcccagtgca ctgaaagtca cggctgcgca 1920ctgtgtcctc
ttccaccaca gagggcgtgt gctcggtgct gacgggaccc acatgctcca
1980gattagagcc tgtaaacttt atcacttaaa cttgcatcac ttaacggacc
aaagcaagac 2040cctaaacatc cataattgtg attagacaga acacctatgc
aaagatgaac ccgaggctga 2100gaatcagact gacagtttac agacgctgct
gtcacaacca agaatgttat gtgcaagttt 2160atcagtaaat aactggaaaa
cagaacactt atgttataca atacagatca tcttggaact 2220gcattcttct
gagcactgtt tatacactgt gtaaataccc atatgtcct 22694496PRTHomo sapiens
4Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala 1
5 10 15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys
Lys 20 25 30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu
Leu Pro 35 40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val
Ser Asn Ala 50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser
Val Gln Arg Leu 65 70 75 80Gln Val Leu Glu Asn Ile Met Glu Asn Asn
Thr Gln Trp Leu Met Lys 85 90 95Leu Glu Asn Tyr Ile Gln Asp Asn Met
Lys Lys Glu Met Val Glu Ile 100 105 110Gln Gln Asn Ala Val Gln Asn
Gln Thr Ala Val Met Ile Glu Ile Gly 115 120 125Thr Asn Leu Leu Asn
Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135 140Val Glu Ala
Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu145 150 155
160Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp
165 170 175Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe
Leu Glu 180 185 190Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile
Gln Leu Gln Ser 195 200 205Ile Lys Glu Glu Lys Asp Gln Leu Gln Val
Leu Val Ser Lys Gln Asn 210 215 220Ser Ile Ile Glu Glu Leu Glu Lys
Lys Ile Val Thr Ala Thr Val Asn225 230 235 240Asn Ser Val Leu Gln
Lys Gln Gln His Asp Leu Met Glu Thr Val Asn 245 250 255Asn Leu Leu
Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr 260 265 270Val
Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe 275 280
285Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn
290 295 300Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly
Gly Gly305 310 315 320Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly
Ser Val Asp Phe Gln 325 330 335Arg Thr Trp Lys Glu Tyr Lys Val Gly
Phe Gly Asn Pro Ser Gly Glu 340 345 350Tyr Trp Leu Gly Asn Glu Phe
Val Ser Gln Leu Thr Asn Gln Gln Arg 355 360 365Tyr Val Leu Lys Ile
His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr 370 375 380Ser Leu Tyr
Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg385 390 395
400Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile
405 410 415Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn
Asp Lys 420 425 430Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly
Trp Trp Phe Asp 435 440 445Ala Cys Gly Pro Ser Asn Leu Asn Gly Met
Tyr Tyr Pro Gln Arg Gln 450 455 460Asn Thr Asn Lys Phe Asn Gly Ile
Lys Trp Tyr Tyr Trp Lys Gly Ser465 470 475 480Gly Tyr Ser Leu Lys
Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe 485 490 4955495PRTHomo
sapiens 5Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val
Leu Ala 1 5 10 15Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser
Ile Gly Lys Lys 20 25 30Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr
Thr Phe Leu Leu Pro 35 40 45Glu Met Asp Asn Cys Arg Ser Ser Ser Ser
Pro Tyr Val Ser Asn Ala 50 55 60Val Gln Arg Asp Ala Pro Leu Glu Tyr
Asp Phe Ser Ser Gln Lys Leu 65 70 75 80Gln His Leu Glu His Val Met
Glu Asn Tyr Thr Gln Trp Leu Gln Lys 85 90 95Leu Glu Asn Tyr Ile Val
Glu Asn Met Lys Ser Glu Met Ala Gln Ile 100 105 110Gln Gln Asn Ala
Val Gln Asn His Thr Ala Thr Met Leu Glu Ile Gly 115 120 125Thr Ser
Leu Leu Ser Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp 130 135
140Val Glu Thr Gln Val Leu Asn Gln Thr Ser Arg Leu Glu Ile Gln
Leu145 150 155 160Leu Glu Asn Ser Leu Ser Thr Tyr Lys Leu Glu Lys
Gln Leu Leu Gln 165 170 175Gln Thr Asn Glu Ile Leu Lys Ile His Glu
Lys Asn Ser Leu Leu Glu 180 185 190His Lys Ile Leu Glu Met Glu Gly
Lys His Lys Glu Glu Leu Asp Thr 195 200 205Leu Lys Glu Glu Lys Glu
Asn Leu Gln Gly Leu Val Thr Arg Gln Thr 210 215 220Tyr Ile Ile Gln
Glu Leu Glu Lys Gln Leu Asn Arg Ala Thr Thr Asn225 230 235 240Asn
Ser Val Leu Gln Lys Gln Gln Leu Glu Leu Met Asp Thr Val His 245 250
255Asn Leu Val Asn Leu Ser Thr Lys Glu Gly Val Leu Leu Lys Gly Gly
260 265 270Lys Arg Glu Glu Glu Lys Pro Phe Arg Asp Cys Ala Asp Val
Tyr Gln 275 280 285Ala Gly Phe Asn Lys Ser Gly Ile Tyr Thr Ile Tyr
Ile Asn Asn Met 290 295 300Pro Glu Pro Lys Lys Val Phe Cys Asn Met
Asp Val Asn Gly Gly Gly305 310 315 320Trp Thr Val Ile Gln His Arg
Glu Asp Gly Ser Leu Asp Phe Gln Arg 325 330 335Gly Trp Lys Glu Tyr
Lys Met Gly Phe Gly Asn Pro Ser Gly Glu Tyr 340 345 350Trp Leu Gly
Asn Glu Phe Ile Phe Ala Ile Thr Ser Gln Arg Gln Tyr 355 360 365Met
Leu Arg Ile Glu Leu Met Asp Trp Glu Gly Asn Arg Ala Tyr Ser 370 375
380Gln Tyr Asp Arg Phe His Ile Gly Asn Glu Lys Gln Asn Tyr Arg
Leu385 390 395 400Tyr Leu Lys Gly His Thr Gly Thr Ala Gly Lys Gln
Ser Ser Leu Ile 405 410 415Leu His Gly Ala Asp Phe Ser Thr Lys Asp
Ala Asp Asn Asp Asn Cys 420 425 430Met Cys Lys Cys Ala Leu Met Leu
Thr Gly Gly Trp Trp Phe Asp Ala 435 440 445Cys Gly Pro Ser Asn Leu
Asn Gly Met Phe Tyr Thr Ala Gly Gln Asn 450 455 460His Gly Lys Leu
Asn Gly Ile Lys Trp His Tyr Phe Lys Gly Pro Ser465 470 475 480Tyr
Ser Leu Arg Ser Thr Thr Met Met Ile Arg Pro Leu Asp Phe 485 490
495
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