U.S. patent application number 15/207955 was filed with the patent office on 2016-10-27 for constructs binding to phosphatidylserine and their use in disease treatment and imaging.
The applicant listed for this patent is Board of Regents, The University of Texas System, Peregrine Pharmaceuticals, Inc.. Invention is credited to Steven W. King, Troy A. Luster, Phillip E. Thorpe.
Application Number | 20160311886 15/207955 |
Document ID | / |
Family ID | 36693034 |
Filed Date | 2016-10-27 |
United States Patent
Application |
20160311886 |
Kind Code |
A1 |
Thorpe; Phillip E. ; et
al. |
October 27, 2016 |
Constructs Binding to Phosphatidylserine and Their Use in Disease
Treatment and Imaging
Abstract
Disclosed are new phosphatidylserine binding constructs with
surprising combinations of properties, and a range of diagnostic
and therapeutic conjugates thereof. The new constructs effectively
bind phosphatidylserine targets in disease and enhance their
destruction, and can also specifically deliver attached imaging or
therapeutic agents to the disease site. Also disclosed are methods
of using the new construct compositions, therapeutic conjugates and
combinations thereof in tumor vasculature targeting, cancer
diagnosis and treatment, and for treating viral infections and
other diseases.
Inventors: |
Thorpe; Phillip E.; (Dallas,
TX) ; Luster; Troy A.; (Norfolk, MA) ; King;
Steven W.; (Tustin, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Board of Regents, The University of Texas System
Peregrine Pharmaceuticals, Inc. |
Austin
Tustin |
TX
CA |
US
US |
|
|
Family ID: |
36693034 |
Appl. No.: |
15/207955 |
Filed: |
July 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14611634 |
Feb 2, 2015 |
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15207955 |
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11339392 |
Jan 24, 2006 |
8956616 |
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14611634 |
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60646333 |
Jan 24, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 5/14 20180101; A61K
49/0002 20130101; A61K 47/6831 20170801; A61K 38/1709 20130101;
A61P 29/00 20180101; C07K 16/3076 20130101; C07K 2319/30 20130101;
A61P 31/16 20180101; A61P 27/02 20180101; A61K 45/06 20130101; A61P
9/10 20180101; A61P 31/06 20180101; C07K 2319/00 20130101; A61P
35/00 20180101; A61P 27/06 20180101; C07K 16/44 20130101; A61P
31/18 20180101; C07K 2317/622 20130101; A61K 38/00 20130101; A61K
47/6835 20170801; A61K 47/6803 20170801; B82Y 5/00 20130101; A61K
47/6811 20170801; A61P 17/06 20180101; A61K 38/17 20130101; A61K
2039/505 20130101; A61P 31/12 20180101; C07K 14/775 20130101; A61P
31/22 20180101; A61K 47/6851 20170801; A61K 47/6898 20170801; C07K
2317/77 20130101; C07K 2317/73 20130101; A61P 19/02 20180101; A61P
9/00 20180101 |
International
Class: |
C07K 14/775 20060101
C07K014/775; A61K 38/17 20060101 A61K038/17; A61K 49/00 20060101
A61K049/00 |
Claims
1. A construct comprising an antibody Fc region operatively
attached to two .beta.2-glycoprotein I (.beta.2GPI) polypeptides,
wherein said .beta.2GP1 polypeptides each comprise at least an
intact domain V of .beta.2GPI, wherein said intact domain V binds
to phosphatidylserine, wherein said .beta.2GPI polypeptides form a
dimer when attached to said antibody Fc region and wherein said
construct retains the property of binding to
phosphatidylserine.
2. A method of inhibiting virus replication, infection or spread,
comprising contacting a population of cells with a construct in
accordance with claim 1 in an amount effective to inhibit virus
replication, infection or spread in said population of cells.
3. The method of claim 2, wherein said population of cells is
located within an animal and said construct is administered to said
animal.
4. A method for treating an animal with a viral infection,
comprising administering to said animal a construct in accordance
with claim 1 in an amount effective to inhibit viral replication,
infection or spread in said animal, thereby treating said viral
infection.
5. The method of claim 4, wherein said animal is a human
patient.
6. A method for treating an animal with cancer, comprising
administering to said animal a construct in accordance with claim 1
in an amount effective to treat said cancer in said animal.
7. The method of claim 6, further comprising administering a second
anti-cancer agent to said animal.
8. The method of claim 6, wherein said animal is a human
patient.
9. A method for forming an image of a tumor in an animal having a
tumor, comprising administering to said animal a diagnostically
effective amount of a construct operatively attached to an in vivo
detectable agent, thereby forming a detectable image of said tumor;
wherein said construct comprises an antibody Fc region operatively
attached to two .beta.2-glycoprotein I (.beta.2GPI) polypeptides,
wherein said .beta.2GPI polypeptides each comprise at least an
intact domain V of .beta.2GPI, wherein said intact domain V binds
to phosphatidylserine, wherein said .beta.2GPI polypeptides form a
dimer when attached to said antibody Fc region and wherein said
construct retains the property of binding to
phosphatidylserine.
10. The method of claim 9, wherein said .beta.2GPI polypeptides
each comprise at least an intact domain V of .beta.2GPI and domain
I of .beta.2GPI.
11. The method of claim 9, wherein said .beta.2GPI polypeptides
each comprise domain 1, domain II, domain III, domain IV and an
intact domain V of .beta.2GPI.
12. The method of claim 9, wherein said .beta.2GPI polypeptides
each comprise at least an intact domain V of human .beta.2GPI.
13. The method of claim 9, wherein said antibody Fc region is a
human antibody Fc region.
14. The method of claim 9, wherein said antibody Fc region is
operatively attached to said .beta.2GPI polypeptides via a peptide
cross-linker.
15. The method of claim 9, wherein said construct is prepared by
recombinant expression as a fusion protein.
16. The method of claim 9, wherein said method further comprises
subsequently administering to said animal a therapeutically
effective amount of said construct, thereby causing an anti-tumor
effect.
17. The method of claim 9, wherein said animal is a human
patient.
18. A method for forming an image of a tumor in an animal having a
tumor, comprising administering to said animal a diagnostically
effective amount of a construct operatively attached to an in vivo
detectable agent, thereby forming a detectable image of said tumor;
wherein said construct comprises an antibody Fc region operatively
attached to two .beta.2-glycoprotein I (.beta.2GPI) polypeptides,
wherein said .beta.2GPI polypeptides each comprise at least domain
I of .beta.2GPI and an intact domain V of .beta.2GPI, wherein said
intact domain V binds to phosphatidylserine, wherein said
.beta.2GPI polypeptides form a dimer when attached to said antibody
Fc region and wherein said construct retains the property of
binding to phosphatidylserin.
19. A method for forming an image of a tumor in an animal having a
tumor, comprising administering to said animal a diagnostically
effective amount of a construct operatively attached to an in vivo
detectable agent, thereby forming a detectable image of said tumor,
wherein said construct comprises an antibody Fc region operatively
attached to two .beta.2-glycoprotein I (.beta.2GPI) polypeptides,
wherein said .beta.2GPI polypeptides each comprise at least an
intact domain V of .beta.2GPI and one or more of the other four
domains of .beta.2GPI, wherein said intact domain V binds to
phosphatidylserine, wherein said .beta.2GPI polypeptides form a
dimer when attached to said antibody Fc region and wherein said
construct retains the property of binding to phosphatidylserine.
Description
[0001] The present application is a continuation of U.S.
application Ser. No. 14/611,634 filed on Feb. 2, 2015, which is a
divisional of U.S. application Ser. No. 11/339,392, filed Jan. 24,
2006, which issued as U.S. Pat. No. 8,956,616 on Feb. 17, 2015,
which claims priority to U.S. provisional application Ser. No.
60/646,333, filed Jan. 24, 2005, the disclosure of which
applications, including the specification, claims, drawings and
sequences, are specifically incorporated herein by reference
without disclaimer.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to the fields of
phosphatidylserine biology, and to treating tumors and viral
infections. It provides surprising new constructs, compositions,
methods and combinations for tumor vasculature targeting and cancer
treatment, and for treating viral infections and other diseases.
The invention particularly provides new phosphatidylserine binding
constructs with surprising combinations of properties and
diagnostic and therapeutic conjugates thereof. The new constructs
effectively bind phosphatidylserine disease targets and enhancing
their destruction, and can also deliver therapeutic agents to
specific sites, and thus provide a range of methods for treating
cancer, viral infections and other diseases.
[0004] 2. Description of the Related Art
[0005] Tumor cell resistance to chemotherapeutic agents represents
a significant problem in clinical oncology. Another major problem
to address in tumor treatment is the desire for a "total cell
kill", i.e., killing all so-called "clonogenic" malignant cells
that have the ability to grow uncontrolled and replace any tumor
mass that might be removed by the therapy. Despite certain advances
in the field, these are two of the main reasons why many prevalent
forms of human cancer still resist effective chemotherapeutic
intervention.
[0006] 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.
[0007] 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.
[0008] 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
chemotherapeutic approaches, immunotoxin therapy also suffers from
significant drawbacks when applied to solid tumors. For example,
antigen-negative or antigen-deficient cells can survive and
repopulate the tumor or lead to further metastases. A further
reason for solid tumor resistance to antibody-based therapies is
that the tumor mass is generally impermeable to macromolecular
agents such as antibodies and immunotoxins. Both the physical
diffusion distances and the interstitial pressure within the tumor
are significant limitations to this type of therapy.
[0009] An improved treatment strategy is 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. Exemplary vascular targeting agents
(VTAs) are described in U.S. Pat. Nos. 5,855,866, 5,965,132,
6,261,535, 6,051,230 and 6,451,312, which describe the targeted
delivery of anti-cellular agents and toxins to markers of tumor
vasculature.
[0010] Another effective version of the vascular targeting approach
is to target a coagulation factor to a marker expressed or adsorbed
within the tumor vasculature or stroma (Huang et al., 1997; U.S.
Pat. Nos. 6,093,399, 6,004,555, 5,877,289, and 6,036,955). 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 "coaguligands" is a truncated version of the human
coagulation-inducing protein, Tissue Factor (TF), the major
initiator of blood coagulation.
[0011] More recently, phosphatidylserine (PS) was identified as a
specific marker of tumor vasculature (Ran et al., 1998). This led
to the development of new anti-PS immunoconjugates for delivering
anti-cellular agents, toxins and coagulation factors to tumor blood
vessels (U.S. Pat. Nos. 6,312,694, 6,783,760 and 6,818,213). In
addition, it was discovered that unconjugated antibodies to PS
exerted an anti-cancer effect without attachment to a therapeutic
agent, which became known as the phosphatidylserine "naked
antibody" approach to tumor vascular targeting and treatment (U.S.
Pat. No. 6,406,693).
[0012] Although the foregoing methods have furthered the art of
tumor treatment, the development of additional therapeutic and
vascular targeting agents is needed to expand the number and
effectiveness of therapeutic options. An important advance would be
the identification of a group of therapeutic agents with
anti-cancer properties and therapeutic effects in other systems,
such as in treating viral infections. The generation of new
targeted constructs that can be made from two human components,
particularly those that do not rely on the use of antibodies for
targeting, would be a significant development, providing improved
safety. Designing and developing new agents that enhance a
patients' own response against disease, i.e., that increase host
effector functions, would be of great value in maximizing
therapeutic responses, particularly where the same mechanisms could
be leveraged against cancer and other diseases, such as viral
infections and diseases.
SUMMARY OF THE INVENTION
[0013] The present invention addresses the foregoing and other
needs of the prior art by providing new constructs, compositions,
methods and combinations for tumor vasculature targeting and cancer
treatment, and for treating viral infections and other diseases.
The invention particularly provides new phosphatidylserine binding
constructs with surprising combinations of properties, which
effectively bind phosphatidylserine targets in disease and enhance
their destruction, such as by increasing host effector functions. A
range of conjugate compositions are also provided, in which the new
constructs are attached to further biological, diagnostic and
therapeutic agents, which can be specifically delivered to disease
sites. The invention further provides effective methods for using
the new constructs and conjugates and combinations thereof in tumor
vasculature targeting, cancer treatment and for treating viral
infections and other diseases.
[0014] 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 "construct", as used
herein, means "at least a first 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.
[0015] ReceptorBodies and BetaBodies:
[0016] The invention first provides a range of phosphatidylserine
binding construct compositions, in which the constructs comprise at
least a first phosphatidylserine binding protein, polypeptide or
receptor operatively attached to at least a first antibody Fc
region. Joining a phosphatidylserine binding protein, polypeptide
or "receptor" to an "antibody" Fc region gives rise to the terms
"receptorbody" and "receptorbodies", which are used herein to refer
to the phosphatidylserine-binding Fc constructs of the
invention.
[0017] The constructs or receptorbodies of the invention typically
comprise at least a first antibody Fc region operatively attached
to at least a first phosphatidylserine binding protein or
polypeptide, receptor, ligand or peptide. The term
"phosphatidylserine binding protein" is succinctly used herein to
refer to all phosphatidylserine binding proteins, polypeptides,
receptors, ligands and peptides.
[0018] In many embodiments, the "phosphatidylserine binding
protein" will retain the phosphatidylserine binding property when
attached to an antibody Fc region to form a construct of the
invention. Naturally, retention of phosphatidylserine binding
function is important in those constructs intended for use in
targeting phosphatidylserine exposed in disease sites. However, not
all embodiments of the invention are limited to phosphatidylserine
binding proteins that retain phosphatidylserine binding properties
when attached to an antibody Fc region.
[0019] Notably, the invention encompasses an antibody Fc region
linked to "nicked .beta.2-glycoprotein I", wherein nicked
.beta.2-glycoprotein I no longer binds phosphatidylserine (see
below). As nicked .beta.2-glycoprotein I is known to be an
inhibitor of angiogenesis (U.S. patent application publication No.
US 2003/0219406, specifically incorporated herein by reference),
the "Fc-nicked .beta.2" of the present invention will be useful as
an inhibitor of angiogenesis and has the advantage of having a
longer half life and additional effector functions, if needed.
[0020] Accordingly, the term "phosphatidylserine binding protein"
refers to the origin of the protein, polypeptide, receptor, ligand
or peptide for use in the constructs of the invention,
notwithstanding that some constructs of the invention will not bind
phosphatidylserine and yet will have important biological and
therapeutic uses, as set forth above. That is, .beta.2-glycoprotein
I is known as a phosphatidylserine binding protein and yet nicked
.beta.2 no longer binds phosphatidylserine.
[0021] In terms of binding phosphatidylserine, the original
phosphatidylserine binding proteins and the phosphatidylserine
binding proteins of the resultant constructs will bind to
phosphatidylserine under biologically appropriate conditions,
preferably under physiological conditions. Such phosphatidylserine
binding proteins may optionally bind to other anionic
phospholipids, under biologically appropriate conditions,
preferably under physiological conditions.
[0022] In certain preferred embodiments, the phosphatidylserine
binding proteins of the constructs do not substantially bind to the
aminophospholipid, phosphatidylethanolamine (PE). In other
preferred embodiments, the phosphatidylserine binding proteins of
the constructs show no detectable binding to
phosphatidylethanolamine.
[0023] A range of phosphatidylserine binding proteins may be used
in the constructs of the invention. Certain exemplary
phosphatidylserine binding proteins that may be used include
Protein C, Protein S, Factor II (prothrombin), Factor V, Factor
VII, Factor IX or Factor X.
[0024] Other exemplary phosphatidylserine binding proteins that may
be used include Mer, a PS-binding scavenger receptor,
.alpha..sub.5.beta..sub.3 integrin, the CR3 complement receptor,
the CR4 complement receptor and the phosphatidylserine receptor,
PSr (Balasubramanian and Schroit, 2003, specifically incorporated
herein by reference, see Table 2 in particular).
[0025] Other exemplary phosphatidylserine binding proteins that may
be used in the constructs of the invention are annexins, preferably
annexin V, which are particularly contemplated for use in certain
embodiments, such as in further conjugates, liposomes and the like.
However, in certain embodiments, the present invention provides
constructs comprising an antibody Fc region operatively attached to
at least a first phosphatidylserine binding protein, wherein said
phosphatidylserine binding protein is not an annexin or a
phosphatidylserine binding fragment thereof, i.e., is not annexin V
or a phosphatidylserine binding fragment thereof.
[0026] Preferred examples of phosphatidylserine binding proteins,
polypeptides and peptides for use in the constructs of the
invention are Beta2-glycoprotein I (.beta.2-glycoprotein I or
.beta.2GP1) proteins, polypeptides and peptides. Joining a
"Beta"2-glycoprotein I binding protein, polypeptide or peptide to
an "antibody" Fc region gives rise to the terms "betabody" and
"betabodies", which are used herein to refer to the preferred
Fc-.beta.2GP1 constructs of the invention.
[0027] .beta.2GP1, previously known as apolipoprotein H, is a 50
kDa plasma glycoprotein that binds phosphatidylserine. The DNA and
amino acid sequences of .beta.2GPI from various mammalian species
are known, including mouse, rat, dog, cow, chimp and human
.beta.2GPI. .beta.2GP1 has five domains, I, II, III, IV and V, and
the domain structure is conserved across mammals, as represented by
domains of mouse and human .beta.2GPI shown in FIG. 18A and FIG.
18B, respectively.
[0028] .beta.2GP1 binds phosphatidylserine through its C terminal
domain, domain V. As the lipid and phosphatidylserine binding
region(s) from .beta.2GPI domain V are known, the
phosphatidylserine binding part of the constructs of the invention
need only contain "a lipid binding region from domain V of
.beta.2GPI".
[0029] With exemplary reference to the human .beta.2GPI amino acid
sequence provided herein as SEQ ID NO:22 (Accession number 1C1ZA),
as shown in FIG. 18B, and the counterpart mouse .beta.2GPI sequence
shown in FIG. 18A, the lipid binding regions from domain V of
.beta.2GPI include a cluster of positively charged amino acids
(282-287) and a conserved hydrophobic region (311-317) responsible
for binding of .beta.2GPI to anionic phospholipids (underlined
italics and double underlined italics in FIG. 18B and FIG.
18A).
[0030] Accordingly, in certain embodiments, the phosphatidylserine
binding part of the constructs of the invention will comprise a
peptide having an amino acid sequence as set forth by amino acids
282-287 of SEQ ID NO:22, or a peptide having an amino acid sequence
as set forth by amino acids 311-317 of SEQ ID NO:22. In other
embodiments, the phosphatidylserine binding part of the constructs
of the invention will comprise a peptide spanning these regions,
i.e., a peptide having the amino acid sequence of SEQ ID NO:24
(human) or a peptide having the amino acid sequence of SEQ ID NO:20
(mouse).
[0031] As the foregoing smaller peptides and polypeptides may not
be optimal, other preferred constructs of the invention are those
in which the phosphatidylserine binding portion is a .beta.2GPI
polypeptide that contains the full or intact domain V of
.beta.2GPI, including .beta.2GPI polypeptides that comprise at
least domain V and those that contain only domain V.
[0032] Although there are no concerns regarding the safety of the
methods of the present invention, antibodies from patients with
Anti-Phospholipid Syndrome(s) or APS commonly recognize domain I of
.beta.2GPI (de Laat et al., 2005a). Antibodies that recognize
.beta.2GPI domain II are not pathogenic. Therefore, in certain
embodiments, the phosphatidylserine binding part of the constructs
of the invention may comprise a .beta.2GPI polypeptide that
comprises at least a lipid binding region from .beta.2GPI domain V
and that substantially lacks domain I of .beta.2GPI. In preferred
embodiments, these constructs will comprise a .beta.2GPI
polypeptide that comprises at least domain V of .beta.2GPI and that
substantially lacks domain I of .beta.2GPI.
[0033] Exemplary .beta.2GPI DNA and amino acid sequences are
provided herein, including the human .beta.2GPI amino acid sequence
of SEQ ID NO:22 (Accession number 1C1ZA), as shown in FIG. 18B, and
the counterpart mouse .beta.2GPI amino acid sequence shown in FIG.
18A (the sequence of an expression plasmid encoding an exemplary
Fc-.beta.2GPI construct of the invention is also provided as SEQ ID
NO:25). With reference to these figures, sequences and the
Accession numbers, optionally in light of the working examples
herein, those of ordinary skill in the art will be able to make and
use a range of polypeptides that comprise at least domain V of
.beta.2GPI.
[0034] As set forth above, certain preferred .beta.2GPI
polypeptides will comprise at least .beta.2GPI domain V and
substantially lack .beta.2GPI domain I. .beta.2GPI polypeptides in
accordance with this description may comprise at least domain IV
and domain V of .beta.2GPI; comprise at least domain III, domain IV
and domain V of .beta.2GPI; or comprise domain II, domain III,
domain IV and domain V of .beta.2GPI.
[0035] Nonetheless, the presence of .beta.2GPI domain I will not be
a significant drawback in the use of the invention, and it may be
convenient to prepare constructs in which the .beta.2GPI
polypeptide comprises domain I, e.g., for ease of preparation and
use. Accordingly, the invention further comprises constructs in
which the phosphatidylserine binding portion is a full length
.beta.2GPI polypeptide, i.e., a .beta.2GPI polypeptide comprising
domain I, domain II, domain III, domain IV and domain V. A full
length .beta.2GPI polypeptide may be a human .beta.2GPI polypeptide
that comprises the amino acid sequence of SEQ ID NO:22.
[0036] In certain embodiments of the invention, it is important
that the construct retains phosphatidylserine binding functions
when the at least a first phosphatidylserine binding protein is
attached to the Fc region, particularly where the construct is
intended for use in targeting phosphatidylserine exposed in disease
sites. An example where phosphatidylserine binding is not necessary
is where at least a first nicked .beta.2GPI polypeptide is attached
to an antibody Fc region, which will have use as an angiogenesis
inhibitor.
[0037] A "nicked" .beta.2GPI polypeptide is where the .beta.2GPI
sequence has been nicked, such as by cleavage with the enzyme
plasmin, at the Lys317/Thr318 cleavage site. A construct comprising
a "nicked .beta.2GPI polypeptide", as used herein, may contain only
a nicked domain V. Alternatively, a nicked .beta.2GPI polypeptide
may comprise "at least a nicked domain V", such that the
polypeptide may comprise a nicked domain V in conjunction with any
one or more of domains IV, III, II and I.
[0038] Where retention of phosphatidylserine binding is required in
a construct of the invention, the present invention provides
further surprising and advantageous features in connection with the
use of .beta.2GPI polypeptides attached to an Fc region. It was
surprisingly found that when an antibody Fc region was operatively
attached to two .beta.2GPI polypeptides, the .beta.2GPI
polypeptides formed a dimer in the resultant Fc-.beta.2GPI
construct and the dimeric construct effectively bound
phosphatidylserine.
[0039] Thus, additional embodiments of the invention are wherein an
antibody Fc region is operatively attached to two .beta.2GPI
polypeptides, preferably where each .beta.2GPI polypeptide
comprises at least domain V of .beta.2GPI. More preferably, wherein
the antibody Fc region is operatively attached to two .beta.2GPI
polypeptides, which each comprise at least domain V of .beta.2GPI,
and wherein the .beta.2GPI polypeptides form a dimer when attached
to the Fc region. Preferably, wherein the antibody Fc region is
operatively attached to two .beta.2GPI polypeptides, which each
comprise at least domain V of .beta.2GPI, and wherein the
.beta.2GPI polypeptides form a dimer when attached to the Fc region
and wherein the resultant construct binds phosphatidylserine, i.e.,
the .beta.2GPI polypeptides form a dimer and bind to
phosphatidylserine when attached to the Fc region.
[0040] Also, preferably wherein an antibody Fc region is
operatively attached to two .beta.2GPI polypeptides; wherein the
.beta.2GPI polypeptides each comprise at least domain V of
.beta.2GPI and substantially lack domain I of .beta.2GPI; and
wherein the .beta.2GPI polypeptides form a dimer and bind to
phosphatidylserine when attached to the Fc region. Further,
preferably wherein first and second .beta.2GPI polypeptides, which
each comprise at least domain V of .beta.2GPI, are operatively
attached to the antibody Fc region, thereby forming an
Fc-.beta.2GPI dimer.
[0041] However, even where phosphatidylserine binding is required
in a .beta.2GPI-containing construct of the invention, it is not
necessary that the construct comprise two .beta.2GPI polypeptides,
nor that any two or more .beta.2GPI polypeptides form a dimer when
attached to the Fc region. For example, an Fc region may be
attached to a single .beta.2GPI polypeptide, or attached to two or
more .beta.2GPI polypeptides that do not form a dimer when
attached, and the resultant constructs can still be used to bind
phosphatidylserine, e.g., by formulation in a scaffold that
provides the required valency. Examples of such formulations or
scaffolds are nanoparticles, liposomes and such like, containing
the constructs.
[0042] For use in humans, any phosphatidylserine binding protein,
such as one or more .beta.2GPI polypeptides, should be a human
phosphatidylserine binding protein, such as a human .beta.2GPI
polypeptide(s). As human Fc regions can also be used, this has the
advantage of providing a totally human therapeutic agent, which
should reduce side-effects, such as those associated with
immunogenicity.
[0043] The Fc regions for use in the invention are derived from an
antibody or "immunoglobulin", including polyclonal and monoclonal
antibodies. The Fc region provides the resultant construct with the
ability to stimulate host effector functions, in order to enhance
disease treatment.
[0044] Although human Fc regions are preferred for use in humans,
Fc regions from other sources may be used, such as mouse, rat,
hamster, rabbit, monkey and the like. For pre-clinical testing,
both the Fc region and the phosphatidylserine binding protein can
be selected from the same animal species in which the pre-clinical
testing is to be conducted, e.g., a mouse Fc region and a mouse
.beta.2GPI polypeptide.
[0045] In the art, antibodies are typically depicted as "Y" shaped
molecules, made from four polypeptides--two heavy chains and two
light chains. The antigen binding or "variable" region, which gives
the antibody its specificity for binding antigen, resides in the
ends of the light and heavy chains Treating an antibody with a
protease can cleave this region, producing separate Fab ("fragment
antigen binding") and Fc (fragment crystallizable) regions, domains
or fragments. Accordingly, the term "Fc" is used in the art to mean
an antibody region, domain or fragment "without an antigen binding
region, domain or fragment". This is the meaning intended in the
present application, such that the "Fc region" of the constructs is
an antibody region, domain or fragment "that does not comprise an
antigen binding region, domain or fragment", i.e., that does not
specifically bind to an antigen. Thus, the invention provides
constructs with effector functions that are not complicated by
targeting issues.
[0046] The constant regions determine the mechanism used to destroy
antigen, i.e., contain determinants of effector function.
Antibodies are divided into five major classes, IgM, IgG, IgA, IgD
and IgE, based on their constant region structure and immune
function. The heavy-chain constant domains that correspond to the
difference classes of immunoglobulins are termed .mu., .gamma.,
.alpha., .delta. and .epsilon., respectively. Several of these are
further divided into subclasses or isotypes, such as IgG1, IgG2,
IgG3, and IgG4. The subunit structures of different classes of
immunoglobulins are well known. The constant regions of heavy
chains .gamma., .alpha. and .delta. have three domains, whereas the
constant region of heavy chains .mu. and .epsilon. have four
domains.
[0047] In the constructs of the invention, it is preferred to use
an antibody Fc region that has an antibody hinge and antibody heavy
chain constant domains C.sub.H2 and C.sub.H3. The antibody hinge
may be important for structural considerations, such as
flexibility, which may facilitate formation of a dimer when two or
more phosphatidylserine binding proteins, such as two or more
.beta.2GPI polypeptides, are attached. The hinge region and hinge
link or lower hinge region may also provide effector functions.
Although it is not required, the Fc region of the constructs may
also comprise at least one of antibody heavy chain constant domains
C.sub.H1 or C.sub.H4.
[0048] In light of the effector functions provided, such as
complement activation (C1q binding), Fc Receptor binding, and the
ability to effectively stimulate processes such as ADCC and cell
lysis (Bruggemann et al., 1987; Riechmann et al., 1988; Clark,
1997; Padlan, 1994; each specifically incorporated herein by
reference), it is currently preferred that the antibody Fc region
is an Fc region from a human IgG1 (.gamma.1) or human IgG3
(.gamma.3) antibody. For a mouse antibody, the Fc region is
preferably an Fc region from a mouse IgG2a (.gamma.2a) or mouse
IgG2b (.gamma.2b) antibody.
[0049] The antibody Fc region will be attached, linked or
conjugated to the at least a first phosphatidylserine binding
protein, such as a .beta.2GPI polypeptide, by any operative means.
For example, by a direct covalent bond, such as via a chemical
cross-linker, or wherein the antibody Fc region and the
phosphatidylserine binding protein are prepared by recombinant
expression as a fusion protein. Indirect attachment may be used,
such as avidin:biotin and other such linkages.
[0050] The Fc region and the phosphatidylserine binding protein are
operatively attached or conjugated, such that the Fc region and
phosphatidylserine binding protein each function sufficiently as
intended after attachment or conjugation. For example,
"operatively" attached means that the Fc region substantially
retains the desired effector functions, and the phosphatidylserine
binding protein substantially retains desired properties,
particularly phosphatidylserine binding where desired, or
anti-angiogenic activity.
[0051] In certain embodiments, "operatively attaching" will
comprise attaching the phosphatidylserine binding proteins to the
Fc region in a manner effective to permit the phosphatidylserine
binding proteins to form a dimer when attached, particularly where
the phosphatidylserine binding proteins are two or more .beta.2GPI
polypeptides. In other embodiments, operatively attaching will
produce clustering of phosphatidylserine binding proteins.
[0052] Where the chosen phosphatidylserine binding protein is a
.beta.2GPI polypeptide, and/or where dimerization on the Fc region
is particularly desired, it is currently preferred to use the Fc
region as the N-terminal portion of the construct and to attach the
phosphatidylserine binding proteins or .beta.2GPI polypeptides to
become the C-terminal portion of the construct (FIG. 16).
[0053] However, other schemes for operatively attaching are
contemplated, such that the phosphatidylserine binding proteins or
.beta.2GPI polypeptides become the C-terminal portion of the
construct, i.e., are inserted in place of an antigen binding region
or in place of a C.sub.H1 domain and an antigen binding region. In
such embodiments, it may be preferred to use a peptide or chemical
linker that provides additional flexibility to the resultant
construct, such as, e.g., a peptide linker with four glutamine
residues and one serine residue (G4S flexible linker).
[0054] After operatively attaching the two or more components, the
resultant construct will exhibit desired biological properties.
Exemplary desired biological properties in the construct as a whole
include, binding phosphatidylserine; stimulating host effector
functions; targeting phosphatidylserine on activated cells, such as
activated, dividing, injured or apoptotic endothelial cells, tumor
cells or virally infected cells; localizing to target sites, such
as tumor blood vessels and tumor cells; and exerting therapeutic
effects, such as anti-cancer and/or anti-viral effects.
[0055] The biological properties of the resultant construct will
also preferably include desired safety features. For example, the
construct will preferably not significantly damage quiescent cells,
significantly inhibit coagulation reactions in vitro, cause
significant thrombosis in vivo or have significant lupus
anticoagulant activities.
[0056] The present invention thus provides new receptorbody and
betabody constructs effective in the treatment of cancer, viral
infections and other diseases. These constructs include those that
effectively bind phosphatidylserine targets in disease and enhance
their destruction by increasing host effector functions.
[0057] Additional Conjugates, Compositions and Kits:
[0058] Although highly effective alone, the present invention
nonetheless also provides further conjugates, compositions and
related methods in which the constructs, receptorbodies and
betabodies of the invention are further attached to additional
biological, diagnostic and therapeutic agents. Such "additional
conjugates" of the invention are "trifunctional agents", as they
have the three properties of binding phosphatidylserine,
stimulating host effector functions and delivering the attached
biological, diagnostic or therapeutic agent to the desired
target.
[0059] In the following descriptions of the conjugates,
compositions, pharmaceuticals, combinations, cocktails, kits, first
and second medical uses and all methods in accordance with this
invention, the "constructs" include all the constructs,
receptorbodies and betabodies of the primary invention described
above.
[0060] In the additional conjugate embodiments, the invention still
further provides new categories of conjugates, compositions, kits
and methods in which the original construct, receptorbody or
betabody is operatively attached to an "additional", further or
exogenous biological, diagnostic or therapeutic agent. The
"exogenous" biological, diagnostic or therapeutic agent is "an
agent distinct from the Fc domain already present in the original
construct".
[0061] The additional or exogenous "biological agent" need not
directly be a therapeutic or diagnostic agent. For example, as the
invention can be used in connection with prodrugs, including ADEPT
embodiments, the biological agent may be an agent, preferably an
enzyme, which cleaves a substantially inactive prodrug to release a
substantially active drug. Such agents and enzymes are described
below in relation to the prodrug and ADEPT method embodiments.
[0062] As to "diagnostic agents", preferred diagnostic agents for
attachment are in vivo diagnostic, imaging or detectable agent
agents. Such diagnostic conjugates may be used in imaging
pre-apoptotic and apoptotic cells in a range of diseases, in
combined tumor imaging and treatment, and in methods of using the
invention as a surrogate marker to monitor chemotherapy. Preferred
diagnostic agents include an X-ray detectable compound, a
radioactive ion, a nuclear magnetic spin-resonance isotope and a
CEST or paraCEST agent.
[0063] Suitable detectable labels include 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, mercuryl.sup.97,
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 (In), vanadium (II) or
ytterbium (III); or rhodamine or fluorescein.
[0064] Regarding "therapeutic agents", certain preferred
therapeutic agents are cytotoxic, cytostatic, anticellular and
anti-cancer agents. A construct, receptorbody or betabody of the
invention may therefore be linked to at least a first
chemotherapeutic agent, anti-angiogenic agent, apoptosis-inducing
agent, anti-tubulin drug, antibiotic, radioisotope or
coagulant.
[0065] Within the cytotoxic agents, currently preferred are ricin,
gelonin, abrin, diphtheria, pseudomonas and pertussis toxins. Of
the cytokines and chemokines, currently preferred agents are IL-2,
IL-12, TNF-.alpha., interferon-.alpha. (IFN-.alpha.), IFN-.beta.,
IFN-.gamma., and LEC (liver-expressed chemokine). V-type ATPase
inhibitors are also currently preferred, such as salicylihalamide,
concanamycin or bafilomycin, as are protein synthesis inhibitors,
such as psymberin, pederin, irciniastatin A.
[0066] Taxol, docetaxel, paclitaxel, cisplatin, gemcitabine, a
combretastatin, doxorubicin and adriamycin are currently preferred
anti-cancer agents. Arsenic radioisotopes are also currently
preferred as additional or exogenous agents. In terms of
coagulants, truncated Tissue Factor is currently preferred.
[0067] A construct, receptorbody or betabody of the invention may
also be further operatively attached to an anti-viral agent or
drug, such as a nucleoside reverse transcriptase inhibitor, a
non-nucleoside reverse transcriptase inhibitor or a protease
inhibitor. AZT, cidofovir and ribavirin are currently preferred as
exogenous anti-viral agents.
[0068] Again, in the additional conjugate embodiments, the term
"conjugate" is used to define the operative association of the
original construct, receptorbody or betabody and the additional
agent, and is not intended to refer solely to any type of operative
association, and is particularly not limited to chemical
"conjugation". Recombinant fusion proteins may again be used. So
long as the phosphatidylserine binding protein, Fc region and
additional attached agent(s) function sufficiently as intended,
particularly when delivered to a target site in vivo, any mode of
attachment will be suitable.
[0069] Where a .beta.2GPI polypeptide is used as the
phosphatidylserine binding protein of a construct, it will
generally be preferred not to attach any additional agent in
.beta.2GPI domain V, or at least not within the lipid binding
region(s) of domain V. In certain embodiments, such as those
exemplified in FIG. 16, it is therefore preferred for simplicity to
operatively attach any additional agent to the N-terminal portion
of the construct, i.e., towards the hinge or C.sub.H2 area.
[0070] The invention also provides compositions comprising a
biologically effective amount of at least a first construct,
receptorbody or betabody. Preferred compositions are
"pharmaceutical compositions" comprising, in a pharmaceutically
acceptable carrier, a biologically or therapeutically effective
amount of at least a first construct, receptorbody or betabody of
the invention, or a conjugate thereof. These compositions are
intended for pharmaceutical, pharmacological, therapeutic, medical
and veterinary uses, preferably for use in treating cancer and
viral infections.
[0071] The pharmaceutical compositions include those formulated for
parenteral administration, such as for intravenous administration,
or for administration as a liposome or as an aerosol. The aerosol
formulations are particularly suitable for treating viral
infections. The "biologically or therapeutically effective amounts"
in the pharmaceutical compositions are amounts effective for
treating a disease or disorder, particularly amounts effective for
treating cancer or a viral infection.
[0072] Although uniquely effective, the various constructs,
receptorbodies or betabodies of the invention, and conjugates
thereof, and the related methods of the invention, can also be used
to advantage in combination with other agents and therapies to
provide combined, compositions, pharmaceuticals and kits of the
invention and related combined treatment methods. In further
embodiments, therefore, the invention further provides particular
combined compositions, methods and kits, e.g. for cancer and
anti-viral treatment, which have been selected to work surprisingly
well together, as explained in more detail herein.
[0073] Aspects of the invention thus further include compositions,
pharmaceutical compositions, combinations, mixtures, medicaments
and/or medicinal cocktails of agents, comprising at least a first
construct, receptorbody or betabody of the invention, or a
conjugate thereof, in combination with a biologically or
therapeutically effective amount of at least a second biological
agent. All such combinations preferably comprise "combined
biologically or therapeutically effective amounts", such as a
combined amount effective to treat a disease, such as to treat
cancer or a viral infection.
[0074] In the compositions, the "at least a second biological
agent" will often be a diagnostic or therapeutic agent, but it need
not be. For example, the second biological agent may be a component
of a pharmaceutical composition such as a dispersion agent or an
absorption delaying agent. Other biological agents, such as agents
for making conjugates and prodrugs for use in prodrug and ADEPT
methods, and diagnostic agents, are preferably maintained in
combination, but separately, from the first composition of the
invention and are therefore discussed below in reference to the
kits of the invention. "In combination, but separately" means in
close confinement together, but not part of the same composition,
such as not part of the same solution or pharmaceutical
composition.
[0075] As to the "at least a second therapeutic agent", the term
"second" is in reference to the construct, receptorbody or betabody
of the invention, or conjugate thereof, being the "first"
therapeutic agent.
[0076] Where the invention is intended for use in cancer treatment,
the at least a second therapeutic agent will preferably be "at
least a second, distinct anti-cancer agent". The second,
anti-cancer agents for combined use may be radiotherapeutic,
chemotherapeutic, anti-angiogenic or apoptosis-inducing agents,
cytokines or antibodies or an antibody-therapeutic agent constructs
that bind to a tumor cell, an intracellular antigen released from a
necrotic tumor cell or to a component of tumor vasculature (i.e.,
anti-cancer immunotoxins or coaguligands). The term
"chemotherapeutic agent", as used herein, includes genes, vectors,
antisense constructs and ribozymes.
[0077] Certain preferred second, anti-cancer agents for combined
use are those that complement or enhance the therapeutic effect of
the first construct, receptorbody or betabody of the invention, or
conjugate thereof, and/or those selected for a particular tumor
type or patient. "Therapeutic agents that complement or enhance the
therapeutic effect" include radiotherapeutic agents, vascular
permeability enhancing agents, anti-angiogenic agents,
apoptosis-inducing agents, certain cytokines, anti-tumor cell
immunotoxins, as well as selected chemotherapeutic agents.
Currently preferred "selected chemotherapeutic agents" are
chemotherapeutic agents with anti-angiogenic effects, as in Table
E; chemotherapeutic agents that induce apoptosis, as in Table F;
calcium flux inducing agents, inflammatory cytokines,
H.sub.2O.sub.2, thrombin, and anti-tubulin drugs from the
combretastatin family. Doxorubicin, etoposide and actinomycin-D are
further preferred, with docetaxel being most preferred.
[0078] Where the invention is intended for use in viral treatment,
the at least a second therapeutic agent will preferably be "at
least a second, distinct anti-viral agent". The second, anti-viral
agents for combined use may be selected from any anti-viral agent
or drug available at the time of practicing the invention,
including the range of anti-viral agents and drugs described herein
for attachment to the constructs of the invention. By way of
example, anti-retroviral drugs such as NTRIs, non-nucleoside RT
inhibitors and protease inhibitors, anti-viral agents as set forth
in Table G, and preferably, AZT or cidofovir.
[0079] The invention further provides a liposome, lipid carrier,
complex, mixture, supramolecular structure multimolecular aggregate
or lipid-based drug delivery system comprising at least a first
construct, receptorbody or betabody of the invention, or a
conjugate thereof. The liposome or liposome-like composition may be
in the form of a monolayer, bilayer, multimolecular aggregate,
vesicle, helix, disc, tube, fiber, torus, hexagonal phase, gel
phase, liquid-crystalline phase, liquid-crystalline multimolecular
aggregate, micelle, reverse micelle, microemulsion, emulsion,
microreservoir, oil globule, fat globule, wax globule and/or
colloidal particle.
[0080] Liposomes or liposome-like compositions generally comprise
an "outer membrane" or bulk aqueous phase and "central core" or
inner aqueous phase. In preferred embodiments, the liposome or
liposome-like composition is a stealthed liposome, lipid carrier,
complex, mixture, supramolecular structure multimolecular aggregate
or lipid-based drug delivery system. "Stealthed" liposomes and
liposome-like compositions comprise a biologically effective amount
of at least a first stealthing agent in operative association with
the outer membrane. A "stealthing agent" is a component that
increases the biological half life of a liposome or liposome-like
composition when operatively associated with the outer membrane of
the liposome or liposome-like composition. In "operative
association", the outer membrane of the liposome or liposome-like
composition is preferably "coated" with the one or more stealthing
agents.
[0081] Effective stealthing agents include a range of biocompatible
hydrophilic polymers, such as polyamines, polylactic acid,
polyglycolic acid, polylactic-polyglycolic acid (PLGA),
polypeptides and related materials. A preferred stealthing agent is
polyethylene glycol (PEG) component, wherein the resulting
stealthed liposomes are termed "PEGylated liposomes".
[0082] Preferred liposomes of the invention are stealthed or
PEGylated liposomes wherein at least a first construct,
receptorbody or betabody of the invention, or a conjugate thereof,
is operatively associated with the outer membrane of the liposome,
preferably where the liposome is "coated" therewith.
[0083] Particularly preferred liposomes are such "coated" and
stealthed or PEGylated liposomes wherein at least a first
therapeutic agent, such as an anti-viral agent or preferably an
anti-cancer agent, is operatively associated with the liposome or
dispersed within the liposomal formulation. Preferably, the
therapeutic, anti-viral or anti-cancer agent is operatively
associated with or maintained within the central core of the
liposome. Exemplary anti-cancer agents are radionuclide(s) and
chemotherapeutic agents, such as anti-tubulin drugs, docetaxel and
paclitaxel, with docetaxel being preferred.
[0084] Further embodiments of the invention concern kits
comprising, in at least a first composition or container, at least
a first construct, receptorbody or betabody of the invention, or a
conjugate thereof, in combination with a biologically or
therapeutically effective amount of at least a second biological
agent, component or system.
[0085] The "second biological agents, components or systems" are
not limited to therapeutic or diagnostic agents. For example,
second biological agents, components or systems may comprise
components for modification of the construct and/or for attaching
other agents. Certain preferred second biological agents,
components or systems are prodrugs or components for making and
using prodrugs, including components for making the prodrug itself
and components for adapting the constructs of the invention to
function in such prodrug or ADEPT embodiments.
[0086] The at least a "second diagnostic agent, component or
system" may be a diagnostic agent, component or system directly or
indirectly detectable by an in vitro diagnostic test. "Directly
detectable in vitro reporter agents" include radiolabels, reporter
agents detectable by immunofluorescence and luciferase. "Indirectly
detectable in vitro reporter agents" function in conjunction with
further exogenous agent(s), such as detectable enzymes that yield a
colored product on contact with a chromogenic substrate. These
include "secondary antibodies", which are attached to a direct or
indirect detectable agent, such a radiolabel or enzyme, and
"secondary and tertiary antibody detection systems" in which the
tertiary antibody is attached to the detectable agent.
[0087] Preferred diagnostic kits of the invention are those
comprising a diagnostic agent, component or system detectable by in
vivo diagnosis or imaging. An advantage of the imaging embodiments
of the invention is that the same construct can be used for imaging
and treatment. The invention therefore provides kits and
medicaments that comprise: [0088] (a) a first pharmaceutical
composition comprising a diagnostically effective amount of a
construct, receptorbody or betabody of the invention, operatively
attached to a detectable label or diagnostic agent; and [0089] (b)
a second pharmaceutical composition comprising a therapeutically
effective amount of a construct, receptorbody or betabody of the
invention, preferably a therapeutically effective amount of the
same construct, receptorbody or betabody used in the first
pharmaceutical composition.
[0090] For use in therapeutic embodiments, the kits will comprise
"at least a second therapeutic agent". Preferably, such kits
comprise a combined biologically or therapeutically effective
amount of at least the two specified agents, such as combined
amounts effective to inhibit proliferation or viral replication, or
to treat a disease such as cancer or a viral infection.
[0091] In terms of cancer treatment, the kits of the invention
include antibodies for use in combination with prodrugs and ADEPT.
In such compositions, the construct, receptorbody or betabody is
"modified to provide a converting or enzymatic capacity".
Preferably, the construct, receptorbody or betabody is operatively
associated with, preferably covalently linked or conjugated to, at
least a first converting agent or enzyme capable of converting at
least one prodrug to the active form of the drug.
[0092] The enzymatic or enzyme-conjugated construct, receptorbody
or betabody will combined with an initially separate formulation of
the "prodrug". The prodrug will be an inactive or weakly active
form of a drug that is that is converted to the active form of the
drug on contact with the enzymatic capacity, converting function or
enzyme associated with the construct, receptorbody or betabody of
the invention.
[0093] Accordingly, kits are provided that comprise, preferably in
separate compositions and/or containers: [0094] (a) a biologically
effective amount of at least a first construct, receptorbody or
betabody of the invention, wherein the construct, receptorbody or
betabody is operatively associated with, covalently linked or
conjugated to, at least a first enzyme; and [0095] (b) a
biologically effective amount of at least a first substantially
inactive prodrug that is converted to a substantially active drug
by the enzyme associated with, linked to or conjugated to the
construct, receptorbody or betabody.
[0096] Suitable enzymes that cleave a substantially inactive
prodrug to release a substantially active drug include
arylsulfatase, serratia protease, thermolysin, subtilisin, a
carboxypeptidase, a cathepsin, D-alanylcarboxypeptidase,
.beta.-galactosidase, neuraminidase, .beta.-lactamase, penicillin
amidase and cytosine deaminase.
[0097] Other than prodrugs, the at least a second, anti-cancer
agent may be any of the second, anti-cancer agents described above
in relation to the combined anti-cancer compositions of the
invention. For treating viral infections, the at least a second,
anti-viral agent may also be any of the second, anti-viral agents
described above in relation to the combined anti-viral compositions
of the invention. However, the "kits" may comprise the at least two
recited the agents "in combination, but separately", thus providing
even more flexibility in the selection of agents.
[0098] The kits of the invention may therefore comprise combined
biologically or therapeutically effective amounts of at least the
two specified agents within a single container or container means,
or within distinct containers or container means. The kits may also
comprise instructions for using the biological and therapeutic
agents included therein. Imaging components may also be included in
combination, but separately with the therapeutic kits.
[0099] Tumor Treatment and Related Methods:
[0100] The present invention provides a number of methods and uses
for a construct, receptorbody or betabody of the invention, or a
conjugate thereof. Concerning all methods, the terms "a" and "an"
are 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 or inhalations, may be
used, up to and including multiple injections or inhalations.
[0101] Various useful in vitro methods and uses are provided that
have important biological implications. Thus provided are methods
of, and uses in, binding phosphatidylserine, which generally
comprise effectively contacting a composition comprising
phosphatidylserine with at least a first construct, receptorbody or
betabody of the invention, or a conjugate thereof. The "contacting"
is under conditions effective to allow the formation of bound
complexes, and any complexes so formed are detected.
[0102] The detection methods and uses may be used in connection
with biological samples, e.g., in diagnostics for apoptosis, tumors
and virally infected cells, and diagnostic kits based thereon are
also provided. These methods and kits provide specific and credible
uses for the constructs, receptorbodies, betabodies and conjugates
of the present invention. For example, annexin V is currently used
in methods and kits to detect apoptotic cells. However, annexin V
binds to phosphatidylethanolamine as well as phosphatidylserine,
whereas the constructs, receptorbodies, betabodies and conjugates
of the present invention bind to phosphatidylserine with no
significant, or preferably no detectable, binding to
phosphatidylethanolamine. Thus, the constructs of the invention are
better able to specifically detect phosphatidylserine in detection
methods and diagnostic assays.
[0103] The invention further provides many useful in vivo methods
and uses. For example, methods for tumor vascular targeting, tumor
imaging and treatment based upon localization to
phosphatidylserine, which is an accessible and stably targetable
marker of tumor vasculature. The constructs, receptorbodies and
betabodies of the invention, and conjugates thereof, specifically
localize to the vasculature of solid tumors upon administration to
an animal with a tumor. Thus, translocation of phosphatidylserine
to the surface of tumor vascular endothelial cells occurs, at least
in a significant part, independently of complete apoptosis and cell
death, such that phosphatidylserine is exposed on morphologically
intact vascular endothelial cells.
[0104] The methods and uses can be performed in vitro and in vivo,
in the latter case, wherein the tissues or cells are located within
an animal and the construct, receptorbody or betabody of the
invention, or conjugate thereof, is administered to the animal.
Where tissues or cells are maintained ex vivo, the present
invention has utility in drug discovery programs. In vitro
screening assays, with reliable positive and negative controls, are
useful as a first step in the development of drugs. Where the
tissues or cells are located within an animal or patient, the
composition is administered to the animal as a form of therapy.
[0105] Anti-angiogenic and anti-vascular therapies are provided in
terms of animals and patients that have, or are at risk for
developing, any disease or disorder characterized by undesired,
inappropriate, aberrant, excessive and/or pathological angiogenesis
or vascularization. The methods and uses of the present invention
are particularly intended for use in animals and patients that
have, or are at risk for developing, any form of vascularized
tumor; macular degeneration, including age-related macular
degeneration; arthritis, including rheumatoid arthritis;
atherosclerosis and atherosclerotic plaques; diabetic retinopathy
and other retinopathies; thyroid hyperplasias, including Grave's
disease; hemangioma; neovascular glaucoma; and psoriasis. In
certain embodiments, the use of an Fc region operatively attached
to a .beta.2GPI polypeptide that comprises nicked domain V will be
preferred for use in inhibiting angiogenesis.
[0106] As disclosed in U.S. Pat. Nos. 5,712,291 and 6,524,583,
specifically incorporated herein by reference, each of the
foregoing treatment groups are by no means exhaustive of the types
of conditions that are to be treated by the present invention. U.S.
Pat. Nos. 5,712,291 and 6,524,583 are incorporated herein by
reference for certain specific purposes, including the purpose of
identifying a number of other conditions that may be effectively
treated once a defined category of compounds have been disclosed
and claimed; and the purpose of showing that the treatment of other
diseases is enabled by data from only a single model system.
[0107] In addition to the treatment of vascular diseases, important
and unified aspects of the present invention are compositions and
methods for treating cancer. Such methods comprise administering to
an animal or patient that has, or is at risk for developing,
cancer, a biologically or therapeutically effective amount of at
least a first construct, receptorbody or betabody of the invention,
or a conjugate thereof.
[0108] The cancer treatment methods and uses of the invention are
suitable for treating all forms of cancer, including animals and
patients that have, or are at risk for developing, a vascularized
solid tumor, a metastatic tumor or metastases from a primary tumor.
The cancer treatment methods of the invention do not rely solely on
exerting anti-vascular effects, such as by targeting
phosphatidylserine exposed on the luminal surface of tumor blood
vessel endothelial cells, as the constructs of the invention can
also target phosphatidylserine exposed on the surface of tumor
cells. The methods of the invention preferably exert an anti-cancer
effect without causing significant thrombotic complications.
[0109] Either the unconjugated construct, receptorbody or betabody
of the original invention, or an additional conjugate thereof, may
be used in the cancer treatment aspects of the invention. As to the
use of immunoconjugates, the invention provides methods for
delivering selected diagnostic or therapeutic agents to tumors.
Such embodiments comprise administering to an animal or patient
having a tumor a biologically effective amount of at least a first
conjugate in which a diagnostic or therapeutic agent is operatively
attached to a construct, receptorbody or betabody of the
invention.
[0110] The invention also provides tumor diagnostic, prognostic,
imaging and related methods using a construct, receptorbody or
betabody of the invention to detect pre-apoptotic and apoptotic
cells. Such methods can be used as a surrogate marker to monitor
the progress of other treatment, particularly chemotherapy, or to
form an image of a tumor prior to treatment.
[0111] The use of the invention as a surrogate marker to monitor
the progress of cancer treatment, particularly chemotherapy,
comprises: [0112] (a) subjecting an animal or patient with a tumor
to at least a first treatment designed to exert an anti-tumor
effect; and [0113] (b) subsequently administering to the same
animal or patient a diagnostically effective amount of at least a
first construct, receptorbody or betabody of the invention,
operatively attached to a detectable label or diagnostic agent,
thereby forming a detectable image of the tumor, preferably an
image of pre-apoptotic or apoptotic tumor cells or tumor vascular
endothelial cells within the tumor; and preferably [0114] (c)
analyzing the detectable image of the tumor, preferably the image
of the pre-apoptotic or apoptotic tumor cells or tumor vascular
endothelial cells within the tumor, thereby assessing the progress
or effectiveness of the at least a first treatment designed to
exert an anti-tumor effect.
[0115] The combined imaging and cancer treatment methods comprise:
[0116] (a) forming an image of a tumor by administering to an
animal or patient having a tumor a diagnostically minimal or
effective amount of at least a first construct, receptorbody or
betabody of the invention, operatively attached to a detectable
label or diagnostic agent, thereby forming a detectable image of
the tumor; and [0117] (b) subsequently administering to the same
animal or patient a therapeutically optimized or effective amount
of at least a first construct, receptorbody or betabody of the
invention, or a conjugate thereof, thereby causing an anti-tumor
effect.
[0118] Within the cancer treatment methods of the invention, the
invention further provides prodrug treatment methods, which
generally comprise: [0119] (a) administering to an animal or
patient with a tumor a first pharmaceutical composition comprising
a first construct, receptorbody or betabody of the invention,
operatively associated with, covalently linked or conjugated to, at
least a first enzyme; wherein the construct, receptorbody or
betabody localizes to the tumor after administration and [0120] (b)
subsequently administering to the animal or patient, after an
effective time period, at least a second pharmaceutical composition
comprising a biologically effective amount of at least one
substantially inactive prodrug; wherein the prodrug is converted to
a substantially active drug by the enzyme associated with, linked
to or conjugated to the construct, receptorbody or betabody of the
invention localized within the tumor.
[0121] The present invention further provides a range of
combination cancer treatment methods, comprising administering to
an animal or patient with cancer a therapeutically effective
combined amount of at least a first construct, receptorbody or
betabody of the invention, or a conjugate thereof, and at least a
second, distinct therapeutic or anti-cancer agent.
[0122] Generally speaking, the at least a second anti-cancer agent
may be administered to the animal or patient before, during or
after administration of the construct, receptorbody or betabody of
the invention, or conjugate thereof. The at least a second
anti-cancer agent may be administered to the animal or patient
"substantially simultaneously" with the construct, receptorbody or
betabody of the invention, or conjugate thereof; such as from a
single pharmaceutical composition or from two pharmaceutical
compositions administered closely together.
[0123] Alternatively, the at least a second anti-cancer agent may
be administered to the animal or patient at a time sequential to
the administration of the construct, receptorbody or betabody of
the invention, or conjugate thereof. "At a time sequential", as
used herein, means "staggered", such that the at least a second
anti-cancer agent is administered to the animal or patient at a
time distinct to the administration of construct, receptorbody or
betabody of the invention, or conjugate thereof.
[0124] In sequential administration, 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". The at
least a second anti-cancer agent may be administered to the animal
or patient at a biologically effective time prior to the construct,
receptorbody or betabody of the invention, or conjugate thereof, or
at a biologically effective time subsequent to that
therapeutic.
[0125] Any therapeutic or anti-cancer agent may be used as the
second, therapeutic or anti-cancer agent in the combined cancer
treatment methods of the invention, including any of the
therapeutic or anti-cancer agents described above in relation to
the anti-cancer compositions and kits of the invention. Preferred
agents are those that complement or enhance the therapeutic effects
of the construct, receptorbody or betabody of the invention, or
conjugate thereof, such as vascular permeability enhancing agents,
anti-angiogenic agents, apoptosis-inducing agents, calcium flux
inducing agents, inflammatory cytokines, antibodies and
immunotoxins to tumor cells and necrotic tumor cells,
chemotherapeutic agents from Table E or Table F, a combretastatin,
doxorubicin, etoposide and actinomycin-D.
[0126] Docetaxel is a particularly preferred agent for use in
combination therapy. Docetaxel may be administered separately to
the construct, receptorbody or betabody of the invention, or
conjugate thereof, either before or afterwards. As to simultaneous
administration, docetaxel may be given in separate or the same
formulations, optionally within a liposome or stealthed liposome,
and preferably within the core of a stealthed liposome coated with
a construct, receptorbody or betabody of the invention.
[0127] Treating Viral Infections:
[0128] In another overall embodiment, the invention further
provides an important new class of compositions and methods for
inhibiting viral replication, infection and spread for use in
treating viral infections and diseases. These methods are based on
the use of a construct, receptorbody or betabody of the invention,
whether conjugated to an anti-viral agent or not. Importantly, a
construct, receptorbody or betabody of the invention will exert an
anti-viral effect without attachment to any additional agent. If
attachment to additional agents is desired, cytotoxic and other
agents will be effective in anti-viral treatment, as well as
classic anti-viral agents. Such constructs and conjugates are
therefore broadly applicable in the treatment of a range of viral
infections and associated diseases.
[0129] In a first instance, the anti-viral methods of the invention
concern contacting a composition comprising, or population of cells
or tissue(s) that contains or is suspected to contain, a virally
infected cell with a biologically effective amount of at least a
first construct, receptorbody or betabody of the invention, or a
conjugate thereof. The virally infected cell is preferably a
eukaryotic cell, such as an animal cell, and preferably a mammalian
or human cell.
[0130] The anti-viral methods and uses can be performed in vitro
and in vivo. In the in vitro embodiments, the methods have
important utilities. For example, in drug discovery programs for
the development of anti-viral drugs or combinations thereof, as
well as in the delineation of further information on viral
infection, replication and spread. The in vitro anti-viral methods
may also be used in purging viruses from biological samples, such
as cell populations and tissue cultures for laboratory use, from
samples, tissues, seeds, plant parts and plants for agricultural
use, and from blood and tissue samples for therapeutic use.
[0131] In the in vivo methods, where the cells, populations or
tissues are located within an animal, the construct, receptorbody
or betabody of the invention, or a conjugate thereof, is
administered to the animal as anti-viral therapy. A construct,
receptorbody or betabody of the invention, or a conjugate thereof,
may bind to phosphatidylserine exposed on the surface of
virally-infected cells, or may bind to phosphatidylserine exposed
on the surface of viral particles.
[0132] In all cases, the compositions, methods and uses inhibit one
or more steps or stages necessary for a productive or ongoing viral
infection, including inhibiting viral entry. Preferably, the
compositions, methods and uses inhibit viral replication and/or
spread, such as inhibiting one or more steps of viral
transcription, translation, assembly, packaging and/or egress
within or from an infected host cell, such as a mammalian or human
cell. The invention therefore preferably limits or substantially
confines viral infections to initially infected cells and cell
populations, thus substantially inhibiting or preventing the
subsequent or ongoing infection of additional host cells or
tissues.
[0133] The anti-viral treatment methods of the invention preferably
concern administering to an animal or patient having, suspected of
having or at risk for developing a viral infection or associated
disease a biologically effective amount of at least a first
construct, receptorbody or betabody of the invention, or a
conjugate thereof. The conjugates may be operatively attached to a
V-type ATPase inhibitors, such as salicylihalamide, concanamycin or
bafilomycin; a protein synthesis inhibitor, such as psymberin,
pederin, irciniastatin A; a ricin, gelonin, abrin, diphtheria,
pseudomonas or pertussis toxin; or to at least a second, distinct
anti-viral agent. Suitable anti-viral agents for attachment include
those set forth in Table G, such as AZT or cidofovir.
[0134] As the invention inhibits one or more steps or stages
necessary for productive or ongoing infection common to all
viruses, the anti-viral methods and uses of the invention are
suitable for treating all viruses, both enveloped and non-enveloped
viruses, including those that infect plants, animals, vertebrates,
mammals and human patients. The invention is suitable for treating
all viruses that infect vertebrates, as listed herein in Table H,
particularly humans, and particularly viruses that are pathogenic
in animals and humans. The viral infections and associated and
resultant diseases that can be treated by the invention include
those viruses and diseases set forth in Table J, as exemplified by
treating CMV, RSV, arenavirus and HIV infections, and the diseases
hepatitis, influenza, pneumonia, Lassa fever and AIDS. Treating
enveloped viruses is particularly preferred.
[0135] The anti-viral treatment methods of the invention may also
be used in combination with other therapeutics and diagnostics. The
combined treatment methods comprise administering to an animal or
patient with a viral infection a therapeutically effective combined
amount of at least a first construct, receptorbody or betabody of
the invention, or a conjugate thereof, and at least a second,
distinct therapeutic or anti-viral agent.
[0136] The at least a "second, distinct" anti-viral agent is in
reference to the construct, receptorbody or betabody of the
invention, or a conjugate thereof, being the "first" anti-viral
agent. The at least a second anti-viral agent may be administered
to the animal or patient during administration of, or substantially
simultaneously with, the first anti-viral agent of the invention;
or before or after, i.e., sequential to the administration of the
first anti-viral agent of the invention.
[0137] Any therapeutic or anti-viral agent may be used as the
second therapeutic or anti-viral agent in the combined anti-viral
treatment methods of the invention, including any of the anti-viral
agents described above in relation to the anti-viral conjugates,
compositions and kits of the invention.
[0138] The foregoing cancer and anti-viral treatment methods and
uses will often involve the administration of a pharmaceutically
effective composition to the animal or patient systemically, such
as by transdermal, intramuscular, intravenous injection and the
like. For treating viral infections, particularly respiratory viral
infections, delivery to the lung is another preferred embodiment,
as may be achieved using an aerosol. However, any route of
administration that allows the therapeutic agent to localize to the
site of the tumor or viral infection will be acceptable. Therefore,
other suitable routes of delivery include oral, rectal, nasal,
topical, and vaginal. For uses and methods for the treatment of
arthritis, e.g., intrasynovial administration may be employed, as
described for other immunological agents in U.S. Pat. No.
5,753,230, specifically incorporated herein by reference. For
conditions associated with the eye, ophthalmic formulations and
administration are contemplated.
[0139] "Administration", as used herein, means provision or
delivery of a construct, receptorbody or betabody of the invention,
or a conjugate thereof, in an amount(s) and for a period of time(s)
effective to exert a therapeutic effect. The passive administration
of proteinaceous therapeutics is generally preferred, in part, for
its simplicity and reproducibility.
[0140] However, the term "administration" is herein used to refer
to any and all means by which the therapeutics are delivered.
"Administration" therefore includes the provision of cells that
produce the construct, receptorbody or betabody of the invention,
or conjugates thereof, in an effective manner. 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
administration will still generally be preferred, as this
represents a non-invasive method that allows the dose to be closely
monitored and controlled.
[0141] The therapeutic methods and uses of the invention also
extend to the provision of nucleic acids that encode a construct,
receptorbody or betabody of the invention, or a conjugate thereof,
in a manner effective to result in expression in vivo. 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. Viral vectors
may be used, such as comprised within a recombinant retrovirus,
herpes simplex virus (HSV), adenovirus, adeno-associated virus
(AAV), cytomegalovirus (CMV), and the like. Liposomes and stealthed
liposomes will be preferred for use in some embodiments.
[0142] The pharmaceutical compositions and treatment methods of the
invention employ "therapeutically effective amounts" of a
construct, receptorbody or betabody of the invention, or a
conjugate thereof. The "therapeutic effects" and consequent
"therapeutically effective amounts" are measured by different
parameters in cancer treatment vs. anti-viral treatment.
[0143] In cancer treatment, the amounts of the agents are effective
to kill or specifically kill at least a portion of tumor cells,
tumor or intratumoral vascular endothelial cells; to induce
apoptosis or specifically induce apoptosis in at least a portion of
tumor cells, tumor or intratumoral vascular endothelial cells; to
promote coagulation or specifically promote coagulation in at least
a portion of tumor or intratumoral blood vessels; to occlude or
destroy, or specifically occlude or destroy at least a portion of
blood transporting vessels of the tumor; to induce necrosis or
specifically induce necrosis in at least a portion of a tumor;
and/or to induce tumor regression or remission upon administration
to an animal or patient.
[0144] In treating viral infections and related diseases, the
amounts of the agents are effective to inhibit one or more
requirements for ongoing viral infection, such as viral entry, and
preferably, viral replication, egress and spread from the infected
host cells. The amounts may also kill or remove at least a portion
of the virally infected cells in a manner that counteracts viral
replication, spread and ongoing infection. Overall, the amounts of
the agents are effective to reduce, significantly reduce or
eradicate the viral infection upon administration to an animal or
patient.
[0145] The terms "preferentially" and "specifically", as used
herein, mean that the construct, receptorbody or betabody of the
invention, or a conjugate thereof, achieve anti-cancer or
anti-viral effects that are substantially confined to the disease
site, and do not substantially cause 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.
BRIEF DESCRIPTION OF THE DRAWINGS
[0146] 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. The U.S. file of this patent contains at least
one drawing executed in color. Copies of this patent with color
drawing(s) will be provided by the Patent and Trademark Office upon
request and payment of the necessary fee.
[0147] FIG. 1. Leukocytes infiltrate the tumor in mice treated with
the 3G4 antibody. Nude mice bearing MDA-MB-231 orthotopic tumors
were treated 3 times a week with 100 .mu.g/dose 3G4 antibody or
with the same dose of an isotype-matched, control antibody. At the
conclusion of treatment, animals were perfused and tumors were
snap-frozen, cut and stained to detect leukocytes. The leukocytes
infiltrating the tumor are shown in the figure.
[0148] FIG. 2A, FIG. 2B, FIG. 2C and FIG. 2D. DNA and amino acid
sequences of the complementarity determining regions (CDRs) of the
3G4 antibody and the 2aG4 antibody. DNA and amino acid sequences
for the heavy (FIG. 2A; SEQ ID NO:1 and SEQ ID NO:2) and light
(FIG. 2B; SEQ ID NO:3 and SEQ ID NO:4) chains of the 3G4 antibody
are presented, and the restriction sites in the DNA sequences are
shown. The leader sequence is distinguished from the mature
protein, which begins as shown by the first arrow in each of FIG.
2A and FIG. 2B. Exemplary means of grafting each variable sequence
with a human constant region are set forth, wherein the first part
of the respective human constant region sequences (SEQ ID NO:7 and
SEQ ID NO:8) is shown by the second arrow in each of FIG. 2A and
FIG. 2B. The amino acid sequences of the IgG2a heavy chain and the
3G4 Light Chain (C.sub..kappa.) are represented by SEQ ID NO:10 and
SEQ ID NO:11, respectively, as shown in FIG. 2C and FIG. 2D.
[0149] FIG. 3. Inhibition of binding of 3G4 antibody to immobilized
PS using competing phospholipid liposomes. Phospholipid-coated
microtiter plates were treated with 3G4 at concentrations ranging
from 0.016 to 33 nM in 10% bovine serum. The bound antibody was
detected using goat anti-mouse IgG-HRP. Competition assays with
liposomes prepared from various phospholipids demonstrate that
anionic phospholipids can compete with 3G4 (6.7 nM) binding to PS.
Bars represent SD of triplicate measurements.
[0150] FIG. 4. Binding of 3G4 to phospholipids. Phospholipid-coated
microtiter plates were treated with 3G4 at concentrations ranging
from 0.016 to 33 nM in 10% bovine serum. The bound antibody was
detected using goat anti-mouse IgG-HRP. 3G4 specifically bound to
anionic phospholipids including PS, PI, PA and CL, but not to
neutral lipids, including PE, PC and SM.
[0151] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E. Induction of
3G4 binding to intact HUVEC and MDA-MB-435 cells by H.sub.2O.sub.2
treatment as shown by FACS (FIG. 5A) and immunohistochemistry (FIG.
5B, FIG. 5C, FIG. 5D and FIG. 5E). FIG. 5A, HUVEC or MDA-MB-435
cells were treated with H.sub.2O.sub.2 (200 .mu.M) for 1 h at
37.degree. C. The cells were washed and detached from the culture
dish with trypsin. Cells were stained with 3G4 (solid line) or
control mouse IgG.sub.3 (BBG3) (dotted line) and were analyzed by
cytofluorometry using a FACS. The instrument was gated on intact
cells (propidium iodide negative). FIG. 5A, top left, HUVEC; top
right, H.sub.2O.sub.2-treated HUVEC; lower left, MDA-MB-435; lower
right, H.sub.2O.sub.2-treated MDA-MB-435. FIG. 5B, FIG. 5C, FIG. 5D
and FIG. 5E, the morphology of 3G4 binding to intact,
non-permeablized H.sub.2O.sub.2-treated HUVEC was determined by
treating adherent cells with H.sub.2O.sub.2 as above, washing the
cells and staining them with 3G4 or control mouse IgG.sub.3, BBG3,
followed by FITC-labeled goat anti-mouse IgG antibody (green). The
cells were then fixed with paraformaldehyde and permeabilized. The
cytoskeleton was stained with Texas-red labeled phalloidin (red)
and nuclei were counterstained with DAPI (blue). 3G4 bound to
discrete regions of the plasma membrane, having the appearance of
membrane blebs. FIG. 5B, HUVEC stained with BBG3; FIG. 5C, HUVEC
stained with 3G4; FIG. 5D, MDA-MB-435 cells stained with BBG3; and
FIG. 5E, MDA-MB-435 cells stained with 3G4. Cells not treated with
H.sub.2O.sub.2 were not stained by 3G4. Scale bar represents 50
.mu.m.
[0152] FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D. Antibodies to
phosphatidylserine and anionic phospholipids cause monocytes to
bind to tumor blood vessels and macrophages to infiltrate tumors.
SCID mice bearing orthotopic human breast tumors were treated i.p.
with 100 .mu.g of control antibodies BBG3 (FIG. 6A and FIG. 6C) or
3G4 antibodies (FIG. 6B and FIG. 6D) three times a week for two
weeks. Tumors were MDA-MB-435 (FIG. 6A and FIG. 6B) or MDA-MB-231
(FIG. 6C and FIG. 6D). Frozen tumor sections were prepared. Mouse
macrophages and monocytes were detected with rat anti-mouse M1/70
(Mac-1) antibody followed by FITC-labeled anti-rat IgG (green).
Anti-F4/80 and anti-Fc.gamma.R antibodies gave coincident staining
patterns with anti-M1/70 antibody. Blood vessels were detected with
hamster anti-mouse CD31 followed by Texas-red labeled anti-hamster
IgG (red). Nuclei were visualized with DAPI (blue). FIG. 6A, tumor
from mouse treated with control BBG3 showing sparse infiltration by
macrophages. FIG. 6B, tumor from mouse treated with 3G4 showing
abundant macrophage infiltration. FIG. 6C, control tumor from mouse
treated with BBG3 showing absence of monocytes attaching to vessel.
FIG. 6D, tumor from mouse treated with 3G4 showing monocytes
attaching to the luminal surface of tumor vascular endothelium
(arrows). Scale bars in FIG. 6A and FIG. 6B represent 50 .mu.m and
in FIG. 6C and FIG. 6D represent 10 .mu.m.
[0153] FIG. 7. The silent and inflammatory phases of phagocytosis.
From tumor treatment studies using the 3G4 antibody, it is proposed
that antibodies such as 3G4 cause a blockade of PS signaling from
PS-expressing tumor endothelial cells. Normally, PS on the tumor
endothelial cells would suppress inflammatory responses by
macrophages that bind to the tumor vessels and tumor cells (silent
phase). When antibodies such as 3G4 are present, the antibodies
bind to PS such that the PS receptor on the macrophage does not
have a binding partner. The macrophage then secretes TNF-.alpha.,
IL-1 and other inflammatory cytokines that directly damage tumor
endothelium and recruit further host cells into the tumor
(inflammatory phase).
[0154] FIG. 8. The F(ab').sub.2 fragment of the 3G4 antibody is as
effective as 3G4 as an anti-tumor agent. Mice were inoculated with
tumors on day 1, and treated on day 9 with the 3G4 antibody, the
F(ab').sub.2 fragment of the 3G4 antibody or an isotype-matched
control antibody (BBG3). The tumors in the control animals
continued to grow rapidly (.box-solid.), whereas in mice treated
with either the 3G4 antibody ( ) or the F(ab').sub.2 fragment of
the 3G4 antibody (), tumor growth was significantly slowed, with
the F(ab').sub.2 fragment being at least as effective as 3G4.
[0155] FIG. 9A and FIG. 9B. 3G4 binding to PS-coated microtiter
plates is serum-dependent. FIG. 9A, the 3G4 antibody was purified
from cells grown in bovine serum-containing media (SCM) or
serum-free media (SFM). A microtiter plate was coated with PS and
blocked in 1% OVA. Serial dilutions of 3G4 were performed in 10%
fetal bovine serum (FBS) or 1% ovalbumin from chicken egg white
(OVA). FIG. 9A, the microtiter plate was coated with PS and blocked
in 1% OVA. Serial dilutions of 3G4 SFM were performed in 10% serum
from the species mouse, rat, human and bovine, as indicated.
[0156] FIG. 10A and FIG. 10B. 3G4 binds the plasma protein
.beta.2GPI (FIG. 10A) and binds at .beta.2GPI domain II (FIG. 10B).
FIG. 10A, a microtiter plate was coated with human .beta.2GPI
(h.beta.2GPI) purified from human plasma and blocked in 1% OVA.
Serial dilutions of a commercial mouse anti-human .beta.2GPI
(anti-.beta.2GPI or ".alpha.-.beta.2GPI"), 3G4 SFM, and a control
mouse IgG (mIgG) were performed in 1% OVA. FIG. 10B, the wells of a
microtiter plate were coated with recombinant full-length
H.beta.2GPI (domain I-V) or h.beta.2GPI peptides absent domain I
(II-V), absent domains I & II (III-V), absent domains I-III
(IV-V) or absent domains I-IV (V). The plate was blocked in 1% OVA
and serial dilutions of 3G4 SFM were performed in 1% OVA.
[0157] FIG. 11. ch3G4 and .beta.2GPI must be present simultaneously
to bind endothelial cells (EC) with exposed PS. ABAE cells were
incubated for 30 min with 200 .mu.M lysophosphatidylchloine (LPC)
in DMEM+10% normal mouse serum (MS), plus (i) purified h.beta.2GPI,
(ii) ch3G4, or (iii) ch3G4+h.beta.2GPI simultaneously. Cells were
then washed and incubated for 30 min with (i) ch3G4, (ii)
h.beta.2GPI, or (iii) DMEM+10% MS, respectively. Finally, cells
were washed, fixed, and stained with fluorescent markers to detect
binding of ch3G4. ch3G4 and h.beta.2GPI were used at a
concentration of 2 .mu.g/ml. The pixel area of ch3G4 binding was
quantified using MetaVue software. Values are relative to the
binding of ch3G4 under condition (i), which was set to one.
[0158] FIG. 12A and FIG. 12B. The lipid binding region of
.beta.2GPI is required to mediate binding of ch3G4 to endothelial
cells with exposed PS. FIG. 12A, ABAE cells were incubated with
ch3G4 plus (i) a non-lipid binding form of .beta.2GPI (nicked
h.beta.2GPI) or (ii) intact h.beta.2GPI. The incubations were
performed in the presence or absence of 200 .mu.M LPC in DMEM+10%
MS for 30 min. Cells were then washed, fixed, and stained with
fluorescent markers to detect binding of ch3G4. ch3G4, h.beta.2GPI,
and nicked h.beta.2GPI were used at a concentration of 2 .mu.g/ml.
The pixel area of ch3G4 binding was quantified using MetaVue
software. Values are relative to the binding of ch3G4 under
condition (i) no LPC, which was set to one. FIG. 12B, the wells of
a microtiter plate were coated with h.beta.2GPI or nicked
h.beta.2GPI and blocked in 1% OVA. Serial dilutions of ch3G4 or a
control mIgG were performed in 1% OVA.
[0159] FIG. 13. Excess ch3G4 inhibits binding of ch3G4/.beta.2GPI
complexes to endothelial cells with exposed PS. ABAE cells were
incubated for 30 min with 200 .mu.M LPC, 40 nM purified
h.beta.2GPI, and a titer of ch3G4 in DMEM+10% MS. Cells were then
washed, fixed, and stained with fluorescent markers to detect
binding of ch3G4. The pixel area of ch3G4 binding was quantified
using MetaVue software. Values are relative to the binding of 320
pM ch3G4, which was set to one.
[0160] FIG. 14A, FIG. 14B and FIG. 14C. ch3G4 divalency is required
for .beta.2GPI-mediated binding to endothelial cells with exposed
PS. FIG. 14A, ABAE cells were incubated for 30 min with 20 nM 3G4,
3G4 F(ab').sub.2, or 3G4 Fab' monomer in the presence or absence of
200 .mu.M LPC in DMEM+10% FBS. Cells were then washed, fixed, and
stained with fluorescent markers to detect binding of 3G4 or 3G4
fragments. The pixel area of antibody binding was quantified using
MetaVue software. Values are relative to the binding of 3G4 in the
absence of LPC, which was set to one. FIG. 14B, ABAE cells were 30
min with 200 .mu.M LPC, 40 nM purified h.beta.2GPI, and a titer of
3G4 Fab' monomer in DMEM+10% MS. Cells were then washed, fixed, and
stained with fluorescent markers to detect binding of 3G4 Fab'. The
pixel area of 3G4 Fab' binding was quantified using MetaVue
software. Values are relative to the binding of 2 nM 3G4 Fab',
which was set to one. FIG. 14C, ABAE cells were incubated for 30
min with 200 .mu.M LPC, 40 nM purified h.beta.2GPI, 20 nM ch3G4,
and a titer of 3G4 Fab' monomer in DMEM+10% MS. Cells were then
washed, fixed, and stained with fluorescent markers to detect
binding of ch3G4. The pixel area of ch3G4 binding was quantified
using MetaVue software. Values are relative to the binding of ch3G4
without competing 3G4 Fab', which was set to 100.
[0161] FIG. 15. Schematic representation of the
.beta.2GPI-dependent binding of the 3G4 antibody and the
human-mouse chimeric antibody counterpart, termed bavituximab, to a
PS surface. .beta.2GPI is shown with its five domains (I, II, III,
IV and V). Antibody binding to PS surfaces, including PS exposed on
cell membranes, is mediated by binding to domain II of
.beta.2GPI.
[0162] FIG. 16. Schematic representation of an exemplary
Fc-.beta.2GPI construct. In this construct, the variable regions of
an antibody have been removed, but are not replaced by .beta.2GPI.
Rather, .beta.2GPI is attached at the C-terminus of the C.sub.H3
domains of the Fc region. The figure shows the disulphide-bonded
hinge region, two C.sub.H2 and C.sub.H3 domains of the Fc region
from mouse IgG.sub.2a operatively attached to two mouse .beta.2GPI
proteins. In the counterpart human construct, the Fc region from
human IgG.sub.1 would be operatively attached to two human
.beta.2GPI proteins. The Fc region from human IgG.sub.3 would also
be preferred for use with two human .beta.2GPI proteins. In the
exemplary Fc-.beta.2GPI construct depicted, mouse .beta.2GPI is
shown with all five domains (I-V), although other mouse or human
constructs could readily be made without all five domains, so long
as the lipid binding region of domain V of .beta.2GPI is
maintained.
[0163] FIG. 17. Plasmid map of the Fc-m.beta.2GPI construct. The
first Fc-.beta.2GPI construct prepared, termed "Fc-m.beta.2GPI",
was generated from three units: (1) the signal sequence of the 3G4
light chain amplified by PCR from 2aG4 construct; (2) a mouse IgG2a
Fc region containing the hinge, C.sub.H2, C.sub.H3, amplified by
PCR from 2aG4 construct; and (3) mouse .beta.2GPI amplified by
RT-PCR from commercially obtained mouse liver RNA. The plasmid map
is shown. The sequence of the plasmid is provided as SEQ ID
NO:25.
[0164] FIG. 18A. Fc-m.beta.2GPI amino acid sequence. The signal
peptide ("mIgG.kappa. signal seq") MDMRAPAQILGFLLLLFPGTRCLR, which
is cleaved, is represented by SEQ ID NO:18. Position 1 indicates
the start of the mature Fc-m.beta.2GPI protein after the signal
peptide has been cleaved. The amino acid sequence of the mature
Fc-m.beta.2GPI is SEQ ID NO:19. The hinge, C.sub.H2 and C.sub.H3
domains of the Fc region are indicated, as are domains I, II, III,
IV and V of mouse .beta.2GPI. The Cys residues involved in
intra-domain disulfide bond formation in .beta.2GPI are shown in
bold and shading. The amino acids in underlined italics are part of
the positively charged region involved in recognition of anionic
phospholipids. The amino acids in double underlined italics are
part of the hydrophobic loop required for binding to lipid
membranes. The sequence of amino acids beginning with those
involved in recognition of anionic phospholipids and concluding
with those that are part of the hydrophobic loop required for
binding to lipid membranes, KNKEKKCSYTVEAHCRDGTIEIPSCFKEHSSLAFWK,
is SEQ ID NO:20.
[0165] FIG. 18B. Amino acid sequences for human a heavy chain
constant region and human .beta.2GPI to prepare human Fc-.beta.2GPI
(Fc-h.beta.2GPI). The human IgG.sub.1 heavy chain constant region
(Accession number P01857; SEQ ID NO:21) is presented starting with
the C.sub.H1 domain, which may be deleted. In a human
Fc-h.beta.2GPI, the hinge (hinge start matches position 1 in FIG.
18A), human C.sub.H2 and human C.sub.H3 domains are followed by
human .beta.2GPI (Accession number 1C1ZA; SEQ ID NO:22), shown to
include domains I, II, III, IV and V. The amino acid sequence of an
Fc-h.beta.2GPI is SEQ ID NO:23. The domain structure of human
.beta.2GPI closely matches that of mouse .beta.2GPI (FIG. 18A),
including the location of the Cys residues involved in intra-domain
disulfide bond formation (bold and shaded), amino acids that are
part of the positively charged region involved in recognition of
anionic phospholipids (underlined italics) and amino acids that are
part of the hydrophobic loop required for binding to lipid
membranes (double underlining italics). The sequence of amino acids
beginning with those involved in recognition of anionic
phospholipids and concluding with those that are part of the
hydrophobic loop required for binding to lipid membranes,
KNKEKKCSYTEDAQCIDGTIEVPKCFKEHSSLAFWK, is SEQ ID NO:24.
[0166] FIG. 19. Expression of Fc-m.beta.2GPI and capture by
anti-mouse IgG. The Fc-m.beta.2GPI construct was transfected into
CHO cells with FuGENE 6 reagent. Two days later, the cell
supernatant was harvested and assayed. In the expression assay, a
microtiter plate was coated with anti-mouse IgG and blocked with 1%
OVA. Serial dilutions of the cell culture supernatants were
performed in 1% OVA. #8 and #18 are two different clones of
Fc-m.beta.2GPI. Binding was detected with ch3G4 and anti-human
IgG-HRP. The vector is the negative control, which is the
baseline.
[0167] FIG. 20. Fc-m.beta.2GPI binds PS on PS-coated plates. In
this binding assay, a microtiter plate was coated with PS and
blocked with 1% OVA. Serial dilutions of Fc-m.beta.2GPI cell
culture supernatants were performed in 1% OVA. #8 and #18 are two
different clones of Fc-m.beta.2GPI. Binding was detected with
anti-mouse IgG-HRP.
[0168] FIG. 21. Fc-m.beta.2GPI binds anionic phospholipids. This
assay measures Fc-m.beta.2GPI binding to phospholipids in 10% FBS.
The microtiter plates were coated with various lipids, as
indicated, and blocked with 1% OVA. Serial dilutions of
Fc-m.beta.2GPI cell culture supernatant were performed in 1% OVA.
Binding was detected with anti-mouse IgG-HRP. Fc-m.beta.2GPI binds
to the anionic phospholipids PS, PA, PI and PG, but not to the
neutral lipids, PC and SM.
[0169] FIG. 22A, FIG. 22B and FIG. 22C. Fc-m.beta.2GPI detects PS
exposed on naturally apoptotic ABAE cells. 2.times.10.sup.4 ABAE
cells were seeded onto 8-well glass chamber slides in DMEM+10% FBS
overnight at 37.degree. C. The next day, cells were washed and
incubated with 2aG4 (FIG. 22A), supernatant from Fc-m.beta.2GPI
transfected CHO cells (FIG. 22B), or mock transfected cells
("control supernatant", FIG. 22C). 2aG4 and Fc-m.beta.2GPI were
detected with a FITC-labeled secondary antibody (green). The
cytoskeletons and nuclei were counter-stained with Texas
Red-labeled phalloidin and DAPI (blue), respectively. Arrows
highlight apoptotic cells.
[0170] FIG. 23A and FIG. 23B. Fc-m.beta.2GPI detects PS exposed on
LPC-treated ABAE cells. 2.times.10.sup.4 ABAE cells were seeded
onto 8-well glass chamber slides in DMEM+10% FBS overnight at
37.degree. C. The next day, cells were washed and incubated
supernatant from Fc-m.beta.2GPI transfected CHO cells.
Fc-m.beta.2GPI was detected with a FITC-labeled secondary antibody
(green). The cytoskeletons and nuclei were counter-stained with
Texas Red-labeled phalloidin and DAPI (blue), respectively. FIG.
23A and FIG. 23B each depict binding of Fc-m.beta.2GPI, seen as
small pinpoints of green staining.
[0171] FIG. 24A, FIG. 24B, FIG. 24C and FIG. 24D. An artificial
dimeric .beta.2GPI construct binds endothelial cells with exposed
PS. ABAE cells were incubated for 30 min with 200 .mu.M LPC in
DMEM+10% FBS plus purified h.beta.2GPI-monomer (FIG. 24A),
h.beta.2GPI-dimer (FIG. 24B), or a mutant h.beta.2GPI-dimer unable
to bind lipid (FIG. 24C). Cells were then washed and incubated with
anti-.beta.2GPI to detect h.beta.2GPI monomers and dimers. The
anti-.beta.2GPI antibody does not recognize bovine .beta.2GPI;
therefore, the presence of 10% FBS did not inhibit detection of
h.beta.2GPI-monomers or h.beta.2GPI-dimers. Cells were then washed,
fixed and stained with fluorescent markers. The cytoskeleton
appears red, nuclei appear blue, and h.beta.2GPI-monomers and
h.beta.2GPI-dimers appear green. FIG. 24D, the binding area of
h.beta.2GPI-monomers and h.beta.2GPI-dimers was quantified using
MetaVue software. All values are relative to the binding of
h.beta.2GPI-dimers to non-LPC treated cells, which was set to
one.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0172] 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
(Denekamp, 1990; Dvorak et al., 1991).
[0173] 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, resulting in an avalanche of tumor cell death.
Antibody-toxin and antibody-coagulant constructs, examples of VTA
which selectively destroy and/or occlude tumor blood vessels, have
already been used to great effect in the specific targeting and
destruction of tumor vasculature, resulting in tumor necrosis
(Burrows et al., 1992; Burrows and Thorpe, 1993; WO 93/17715; WO
96/01653; Huang et al., 1997; each incorporated herein by
reference).
[0174] VTAs exert their primary action on the pre-existing blood
vessels of solid tumors, and differ from anti-angiogenic agents
that prevent new blood vessel formation. There are numerous
advantages of VTAs over other cancer therapies. First, a single
vessel provides the nutrition for and facilitates removal of waste
products of metabolism from hundreds or thousands of tumor cells,
and only has to be damaged at one point to block blood flow
upstream and downstream. VTAs are thus particularly effective on
established tumors. Second, endothelial cell killing, although one
useful mechanism, is not required. A change of shape or local
initiation of blood coagulation can be sufficient. Third, the
endothelial cell is adjacent to the blood stream, ensuring adequate
drug delivery. Fourth, the target is a normal diploid cell that is
unlikely to acquire genetic mutations that render it drug
resistant. Fifth, a surrogate marker of biological activity, i.e.,
blood flow, is measurable.
[0175] Sixth, temporary effects on vascular function may be
sufficient for significant anti-tumor effects. Studies indicate
that over 99% of tumor cells in vivo can be killed during a 2 hour
period of ischemia. Finally, unlike angiogenesis inhibitors, VTAs
only require intermittent administration to synergize with
conventional treatments, rather than chronic administration over
months or years.
[0176] Cytotoxic VTAs are described in the following patents: U.S.
Pat. Nos. 5,660,827, 5,776,427, 5,855,866, 5,863,538, 5,965,132,
6,004,554, 6,051,230, 6,261,535 and 6,451,312, each incorporated
herein by reference. Where antibodies, growth factors or other
binding ligands are used to specifically deliver a coagulant to the
tumor vasculature, such agents are termed "coaguligands".
Coaguligand VTAs are described in the following patents: U.S. Pat.
Nos. 6,093,399, 6,004,555, 5,877,289 and 6,036,955, each
incorporated herein by reference.
[0177] 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; Edgington 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).
[0178] 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. 6,093,399, 6,004,555, 5,877,289
and 6,036,955). A coaguligand is thus created that selectively
thromboses tumor vasculature.
[0179] Truncated TF 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. 6,093,399,
6,004,555, 5,877,289 and 6,036,955).
[0180] 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, pleiotropin or
endosialin (U.S. Pat. No. 5,855,866 5,877,289; 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).
[0181] 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, 6,051,230 and 6,004,555). Fibronectin
isoforms are ligands that bind 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 (Camemolla et al., 1989) specifically binds to
tumor-associated fibronectin isoforms.
[0182] 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).
[0183] 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, 6,051,230, 6,004,555 and 6,093,399). 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).
[0184] 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).
[0185] Certain of the data presented herein even further supplement
those provided in U.S. Pat. Nos. 5,855,866, 5,877,289, 6,051,230,
6,004,555 and 6,093,399, and show the selective induction of
thrombosis and tumor infarction resulting from administration of an
anti-VCAM-1tTF 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.
[0186] Using a covalently-linked anti-VCAM-1tTF 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, 6,051,230, 6,004,555 and 6,093,399, where the inventions
were shown to be most useful in larger solid tumors.
[0187] 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.
[0188] 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 a different approach to
tumor vasculature destruction.
A. Discovery of Naked Anti-Phosphatidylserine Antibodies for Tumor
Treatment
[0189] 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.
[0190] 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"). Despite extensive research, a clear
molecular explanation for the prothrombotic nature of the tumor
environment could not be discerned until recently.
[0191] After detailed analyses of many 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 phosphatidylserine 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 demonstrated.
[0192] 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-1tTF
coaguligand action, both in vitro and in vivo.
[0193] The lack of thrombotic effect of the anti-VCAM-1 coaguligand
on normal heart and lung vessels was thus explained, at least in
part: the absence of phosphatidylserine, means that the normal
vessels lack a procoagulant surface upon which coagulation
complexes can assemble. In the absence of surface PS,
anti-VCAM-1tTF 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.
[0194] In addition to delineating the tumor-specific thrombotic
effects of anti-VCAM-1 coaguligands, the specific expression of
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. The PS expression
plays a significant role in the prothrombotic state of tumor
vasculature.
[0195] 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 other aminophospholipids
had potential as targets for therapeutic intervention. The
inventors therefore developed tumor vasculature targeting and
treatment methods based on targeting the aminophospholipids
phosphatidylserine and phosphatidylethanolamine (PE).
[0196] Once the discovery of phosphatidylserine as a specific
marker of tumor vasculature had been proven, the inventors began to
develop a range of phosphatidylserine-targeted immunotoxins and
coaguligands for use in tumor treatment. As explained in U.S. Pat.
No. 6,406,693, whilst investigating the potential of
phosphatidylserine targeting in the context of delivering a toxin
or coagulant to the tumor vasculature, the inventors
serendipitously found that naked anti-PS antibodies had a
destructive effect on tumor vasculature in vivo in the absence of
any additional effector moiety. The ability of
anti-aminophospholipid antibodies to both specifically localize to
tumor vasculature and to exert a concomitant destructive effect,
leading to tumor necrosis, was most unexpected.
[0197] These discoveries gave rise to tumor treatment using
unconjugated or "naked" antibodies that bind to phosphatidylserine,
as described in U.S. Pat. No. 6,406,693, incorporated herein by
reference. Although anti-tumor effects in art-accepted animal
models are demonstrated in U.S. Pat. No. 6,406,693, and extended
herein, the ability of aminophospholipids to act as safe and
effective targetable markers of tumor vasculature could not have
been predicted from studies previous to U.S. Pat. No.
6,406,693.
B. Extensive Tumor Treatment with Anti-Phosphatidylserine
Antibodies
[0198] Phosphatidylserine is normally segregated to the inner
surface of the plasma membrane bilayer in different cells (Gaffet
et al., 1995; Julien et al., 1995) and this lipid segregation
creates an asymmetric transbilayer (Williamson and Schlegel, 1994).
The inventors earlier demonstrated that PS is translocated to the
surface of tumor vascular endothelial cells and that this occurs,
at least in significant part, independently of apoptotic or other
cell-death mechanisms (U.S. Pat. No. 6,406,693). Thus, PS surface
expression in the tumor environment is not a consequence of cell
death, nor does it trigger immediate cell destruction. Despite PS
exposure being detected consistently on intact vascular endothelial
cells in various solid tumors, the tumor vascular endothelium is
not frankly apoptotic, but is morphologically sound (although
different to that in normal tissues) and metabolically active. This
is important for therapeutic methods based on PS targeting, meaning
that PS translocation to the outer membrane in tumor vascular
endothelial cells is sufficiently stable for PS to serve as a
targetable entity for successful therapy (using either naked
antibodies or therapeutic conjugates).
[0199] Through the development of biological tools with exquisite
specificity for different phospholipids, the present inventors
identified that anionic phospholipids are also upregulated on tumor
vascular endothelial cells. Anionic phospholipids are thus specific
and stable markers of tumor vasculature, permitting therapeutic
intervention using both naked antibodies and immunoconjugates that
bind to anionic phospholipids.
[0200] Anionic phospholipids are largely absent from the surface of
resting mammalian cells under normal conditions.
Phosphatidylserine, which is the most abundant anionic phospholipid
of the plasma membrane, is tightly segregated to the internal
leaflet of the plasma membrane in most cell types under normal
conditions (Williamson and Schlegel, 1994; Zwaal and Schroit,
1997). Phosphatidylinositol (PI), another major anionic
phospholipid, is also predominantly situated in the internal
leaflet of the plasma membrane (Calderon and DeVries, 1997). The
minor anionic phospholipids, phosphatidic acid (PA) and
phosphatidylglycerol (PG), have only been examined in a few cells
types, but they also appear to be mainly situated in the internal
leaflet of the plasma membrane (Hinkovska-Galcheva et al., 1989).
Cardiolipin (CL), another anionic phospholipid, is present in the
mitochondrial membrane and is absent from the plasma membrane
(Daum, 1985).
[0201] The neutral phospholipids are also asymmetrically
distributed in the plasma membrane. The neutral aminophospholipid,
phosphatidylethanolamine (PE) is predominately on the internal
leaflet. The choline-containing neutral phospholipids,
phosphatidylcholine (PC) and sphingomyelin (SM), are predominantly
on the external leaflet.
[0202] PS asymmetry is maintained by an ATP-dependent transporter,
aminophospholipid translocase (Mg.sup.2+ ATPase), which catalyzes
the transport of aminophospholipids from the external leaflet to
the internal leaflet of the plasma membrane (Seigneuret and Devaux,
1984). Loss or collapse of PS asymmetry results from the outward
movement of these phospholipids in the plasma membrane and is
caused either by inhibition of the translocase (Bitbol et al.,
1987; Comfurius et al., 1990), activation of PS transporters and/or
activation of scramblase enzymes (Zhao et al., 1998) or the ABC-1
floppase (Hamon et al., 2000), Ca.sup.2+ dependent enzymes that
transport all lipids bidirectionally. Sphingomyelinase might also
be activated to generate ceramide, which facilitates transbilayer
lipid translocation (Contreras et al., 2003).
[0203] Loss of PS asymmetry is observed under different
pathological and physiological conditions, including cell injury,
programmed cell death and apoptosis (Blankenberg et al., 1998;
Bombeli et al., 1997), cell aging (Herrmann and Devaux, 1990),
activation of platelets (Rote et al., 1993; Zwaal et al., 1989),
injury (Boyle et al., 1996) and malignant transformation (Sugimura
et al., 1994). Exposure of PS also plays a role in intercellular
fusion of myoblasts (Sessions and Horwitz, 1981) and trophoblasts
(Adler et al., 1995), cell migration (Vogt et al., 1996) and cell
degranulation (Demo et al., 1999). Endothelial cells externalize PS
in response to increased Ca.sup.2+ fluxes induced by thrombin (Qu
et al., 1996), calcium ionophore or phorbol esters (Julien et al.,
1997), hyperlipidemia (Lupu et al., 1993), and non-lytic
concentrations of complement proteins C5b-9 (Christiansen et al.,
1997). Spontaneous PS exposure has been also observed in malignant
cells in the absence of exogenous activators or cell injury (Utsugi
et al., 1991).
[0204] Several major consequences follow membrane PS exposure.
Phagocytic macrophages recognize, attach and eliminate PS-positive
senescent and apoptotic cells (McEvoy et al., 1986; Tait and Smith,
1999). PS also mediates attachment of T lymphocytes to
thrombin-activated endothelial cells (Qu et al., 1996). The
complement system is activated by PS and contributes to the lysis
of PS-positive cells (Test and Mitsuyoshi, 1997). Finally, PS
exposure contributes to a procoagulant shift on the endothelium
(Williamson and Schlegel, 1994; Bombeli et al., 1997) by providing
a negatively charged lipid surface for assembly and activation of
coagulation complexes (Bevers et al., 1985; Dachary-Prigent et al.,
1996). The prothrombotic character of the tumor endothelium has
long been recognized (Donati and Falanga, 2001).
[0205] The inventors realized that injury and activation of tumor
endothelium are caused by: 1) tumor-derived cytokines, such as
interleukin-1 and tumor necrosis factor, which activate the
endothelium and induce expression of cell adhesion molecules
(Shaughnessy et al., 1989; Orr et al., 2000); 2) reactive oxygen
species (ROS) generated by leukocytes that adhere to the
endothelium (Orr et al., 2000); and 3) ROS generated by tumor cells
themselves as a byproduct of metabolism (Shaughnessy et al., 1989;
Soares et al., 1994) or as a result of exposure to hypoxia followed
by reoxygenation (Zulueta et al., 1995). These observations
suggested that Ca.sup.2+ fluxes might be generated by these
stresses within the tumor endothelium that, in turn, cause exposure
of PS, through activation of scramblase or inhibition of
aminophospholipid translocase.
[0206] To detect cell surface anionic phospholipids, the inventors
generated a new monoclonal antibody, 9D2, which reacts with anionic
but not neutral phospholipids. 9D2 thus differentiates from general
aminophospholipid binding agents, as it binds to the anionic
aminophospholipid, phosphatidylserine, but not to the neutral
aminophospholipid, phosphatidylethanolamine (PE). The 9D2 antibody
is also more specific for anionic phospholipids than is the natural
ligand, annexin V, which strongly binds to PE, in addition to
anionic phospholipids (Blankenberg et al., 1998).
[0207] As detailed in the present application, the inventors found
that 9D2 and annexin V localize specifically to tumor endothelium
after intravenous injection to mice bearing various types of solid
tumors. This finding validates the inventors' hypothesis that
phosphatidylserine and anionic phospholipids routinely become
exposed on the surface of tumor vascular endothelium and can be
used as target molecules for tumor therapy (and imaging).
[0208] One of the major findings to emerge from the present
inventors is that phosphatidylserine and anionic phospholipids are
exposed on the surface of tumor endothelium (Example VI; Ran and
Thorpe, 2002; Ran et al., 2002b). This phenomenon was demonstrated
using two independent reagents that bind selectively to anionic
phospholipids: a monoclonal antibody, 9D2, developed by the
inventors particularly to validate this point, and annexin V.
[0209] 9D2 antibody and annexin V bind with high affinity and
specificity to phosphatidylserine and anionic phospholipids
adsorbed to plastic, as liposomes, or presented on the membrane
surface of activated or apoptotic endothelial cells in vitro. 9D2
binds strongly to PS, PA and CL, but more weakly to PI and PG.
Annexin V binds to PE in addition to PS, CL, PA, PI and PG, as
found previously (Andree et al., 1990; Schlaepfer et al., 1987;
Boustead et al., 1993; Blackwood and Ernst, 1990). Recognition of
phosphatidylserine and anionic phospholipids by 9D2 antibody was
identical in the presence and absence of serum, indicating that
binding does not require serum co-factors. Binding of 9D2 to
anionic phospholipids, did not require Ca.sup.2+ ions, whereas the
binding of annexin V did require Ca.sup.2+.
[0210] Cross-blocking studies on PS-coated plates showed that 9D2
and annexin V do not block each other's binding to PS. This
indicates that the two reagents recognize different epitopes on the
PS molecule, or, more likely, differently packed forms of PS.
Annexin V is thought to bind to planar PS surfaces, whereas anti-PS
antibodies are thought to bind to hexagonally packed PS (Rauch and
Janoff, 1990). Both forms are probably present on PS-coated plates.
These practical cross-blocking studies (Example VI) also serve to
show that antibodies which effectively compete for binding to
anionic phospholipids, i.e., bind to essentially the same epitope,
can be readily identified once a reference antibody (e.g. 9D2) is
provided.
[0211] The present application also shows that 9D2 antibody and
annexin V specifically localize to tumor vessels, and to tumor
cells in and around necrotic regions of all tumors examined in vivo
(Example VI). Between 15 and 40% of blood vessels in the tumors had
phosphatidylserine-positive endothelium. In contrast, none of the
blood vessels in normal tissues had detectable externalized anionic
phospholipids. The PS-expressing tumor endothelial cells are
viable. They lack markers of apoptosis (active caspase-3, TUNEL),
are morphologically intact and metabolically active, and the
vessels are functional at transporting blood and solutes.
[0212] The specificity of staining of tumor vasculature by 9D2 was
demonstrated by: 1) the lack of tumor vessel staining by control
rat IgM; 2) the blocking of 9D2 or annexin V binding to
H.sub.2O.sub.2-treated endothelial cells in vitro by liposomes
prepared from anionic phospholipids, but not neutral phospholipids;
3) the finding that extraction of phospholipids from tumor sections
with detergents or organic solvents abolished staining; and 4) the
lack of localization of either 9D2 or annexin V to the quiescent
endothelium in normal organs.
[0213] The main anionic phospholipid that is localized by 9D2 or
annexin V on tumor vasculature is phosphatidylserine, as this is
the most abundant anionic phospholipid and its exposure on the cell
surface is regulated by environmental influences or injury. To
examine the mechanism of exposure of anionic phospholipids on tumor
endothelial cells, a series of studies was performed in which
endothelial cells in vitro were treated with various factors and
conditions known to be present in the tumor microenvironment
(Example VII). Hypoxia followed by re-oxygenation, acidity, and
thrombin increased PS exposure on viable endothelial cells to
between 10 and 22% of the level seen when all cells are apoptotic.
Inflammatory cytokines (TNF.alpha. and IL-1) also caused a weak but
definite induction of PS exposure.
[0214] These findings are consistent with the possibility that, in
tumors, exposure of phosphatidylserine on the vascular endothelium
is induced by hypoxia/reoxygenation in combination with
inflammatory cytokines, thrombin and acidity. Although the precise
mechanism does not need to be understood to practice the present
invention, ROS may be generated by tumor cells as a bi-product of
metabolism or in response to hypoxia (Zulueta et al., 1995).
Cytokines released by tumor cells may induce leukocytes adhesion
molecules on the endothelium that mediate adherence of activated
macrophages, polymorphonuclear cells and platelets to tumor
endothelium and further secretion of ROS. The ROS may then induce
PS translocation through oxidation of thiol-containing transport
molecules or peroxidation of lipids (Herrmann and Devaux, 1990),
possibly by causing an influx of Ca.sup.2+ or release of Ca.sup.2+
from intracellular stores (Wang and Joseph, 2000). Indeed,
peroxides have been shown to induce PS-exposure on viable
endothelial cells in vitro by a mechanism that relates to
glutathione oxidation and/or lipid peroxidation, not apoptosis (van
Gorp et al., 1999).
[0215] Exposure of PS and other anionic phospholipids in part
explains the procoagulant status of tumor endothelium that has long
been recognized (Donati and Falanga, 2001). The anionic
phospholipids provide the surface upon which coagulation factors
concentrate and assemble (Bevers et al., 1985; Dachary-Prigent et
al., 1996). It also provides an attachment site for circulating
macrophages (McEvoy et al., 1986), T lymphocytes (Qu et al., 1996)
and polymorphonuclear cells that assists in leukocyte infiltration
into tumors.
[0216] In further studies detailed herein, the inventors generated
and characterized the monoclonal antibody termed 3G4, which is
directed against phosphatidylserine and anionic phospholipids. This
antibody is also shown to localize specifically to vascular
endothelial cells in tumors, reduce tumor vascularity and plasma
volume and to retard tumor growth.
[0217] The 3G4 antibody binds with high affinity to anionic
phospholipids absorbed to plastic, and on the surface of activated
or apoptotic cells in the presence of serum or .beta.2-glycoprotein
I. The binding pattern of 3G4 on cells was indistinguishable from
that of annexin A5 or the 9D2 antibody against anionic
phospholipids. All three reagents bound to clusters of plasma
membrane resembling membrane blebs, consistent with other
observations on endothelial cells treated with H.sub.2O.sub.2 (van
Gorp et al., 1999). Like 9D2, 3G4 recognizes all anionic
phospholipids tested, including synthetic phospholipids having
saturated fatty acids, which are resistant to oxidation, and
lysophospholipids.
[0218] Unlike 9D2, 3G4 binding to anionic phospholipids was
partially inhibited in the complete absence of serum and restored
when .beta.2-glycoprotein I was added. 3G4 recognizes thus an
epitope in lipid-.beta.2-glycoprotein I complexes. Irrespective,
3G4 is shown to be safe when administered to animals, and not to be
associated with pathological effects reported in the literature for
antibodies associated with anti-phospholipid syndrome(s) (APS).
[0219] 3G4 localized specifically to tumor vessels and to tumor
cells in and around necrotic regions of tumors after injection into
mice bearing orthotopic human breast MDA-MB-435 tumors. An average
of 40.+-.10% of vessels were bound by 3G4. Staining patterns were
similar to those reported herein using 9D2 and annexin A5. Vascular
endothelium in normal tissues was unstained. In this regard, 3G4
differs from other antibodies that recognize tumor vessel markers.
Most tumor vessel markers are present on vessels in the ovary, a
site of physiological angiogenesis, or in the kidney and pancreatic
islets where vessels have high permeability (Thorpe, 2004).
[0220] Phosphatidylserine is the anionic phospholipid primarily
detected by 3G4. PS is the most abundant anionic phospholipid and
the one whose exposure is best known to be regulated by
environmental conditions or injury (Zwaal and Schroit, 1997;
Balasubramanian and Schroit, 2003). In vivo, the exposed PS on
tumor vessels is probably complexed with serum components, such as
.beta.2-glycoprotein I, and 3G4 probably binds to these complexes.
As noted throughout the present studies, the PS-positive tumor
vessels in untreated mice appear to be intact and functional. They
transport blood and are perfusible. The vascular endothelium of
PS-positive vessels does not display markers of advanced apoptosis
(active caspase 3, TUNEL), is morphologically intact and is
metabolically active, as judged by co-expression of the rapidly
turned over protein, VCAM-1.
[0221] Treatment with 3G4 retarded tumor growth in various murine
models, including established (0.6-0.7 cm diameter) orthotopic
human MDA-MB-231 and MDA-MB-435 breast cancers, large (1 cm
diameter) subcutaneous L540 human Hodgkin's tumors, and small
syngeneic Meth A fibrosarcomas. 3G4 treatment resulted in 75%, 65%,
50% and 90% retardation of growth of these tumors, respectively.
Other studies in the present application demonstrate that these
tumors are nourished by vasculature with exposed anionic
phospholipids.
[0222] The antitumor effect of 3G4 is mediated, at least in part,
through damage to tumor vasculature. Histological examination of
orthotopic MDA-MB-231 tumors from mice treated with 3G4 revealed a
marked reduction in the vascular density and plasma content of the
tumors. Localization of 3G4 to tumor vessels preceded macrophage
binding to tumor vessels, impairment of vascular function and the
development of necrosis. The vascular shutdown and pattern of
necrosis are consistent with the primary effect being on tumor
vessels. Central necrosis of tumors with survival of a peripheral
rim of tumor cells was observed. This pattern of tumor cell killing
is characteristic of VTAs (U.S. Pat. No. 5,855,866; Thorpe, 2004).
It is thought that VTAs are most effective against vessels in the
interior of the tumor because high interstitial pressure in these
regions contributes to vascular collapse. In contrast, many
direct-acting tumor therapies are most effective against the
rapidly dividing tumor cells in the well-oxygenated periphery of
the tumor. The inventors therefore expect that combining 3G4 with
antiproliferative antitumor therapies will to lead to additive or
even synergistic antitumor activity, as has been observed with
other VTAs in experimental solid tumors (U.S. Pat. No. 5,855,866;
Burrows and Thorpe, 1993; Siemann et al., 2002; Siim et al.,
2004).
[0223] As with the other antibodies used herein, 3G4 therapy is
well-tolerated in tumor-bearing animals treated repeatedly with the
therapeutic dose (4 mg/kg in mice, three times a week). The mice
retained normal physical signs, coagulation parameters, bone marrow
cellularity, white blood cell counts and histology. Manifestations
of APS were not observed, in contrast to those observed with
anticardiolipin antibodies (Matzinger, 1998; Fadok et al., 1998;
Fadok et al., 2001a;b). Despite effects of high concentrations of
3G4 in partially inhibiting phospholipid-dependent coagulation
pathways, a substantial safety margin exists between the
therapeutic dose and the dose that prolongs coagulation times in
vivo.
[0224] The inventors have considered the question as to whether PS
becomes exposed on vascular endothelium in nonmalignant lesions
(e.g., atherosclerotic lesions, sites of inflammation), where
cytokines, hypoxia and ROS might induce PS translocation (Moldovan
et al., 1994). Is this occurred, it is possible this could lead to
some toxicity with an anti-PS antibody, making it advisable to
exclude patients with these conditions from treatment. However,
other studies of the inventors showed that treatment of
atherosclerotic rabbits with a chimeric version of the 3G4 antibody
did not exacerbate aortic atherosclerotic lesions.
[0225] Vascular targeting agents employing drugs or coagulants have
been shown to be highly effective, and sometimes curative, in mice
with large solid tumors (Huang et al., 1997; Nilsson et al., 2001;
U.S. Pat. Nos. 5,660,827, 5,776,427, 5,855,866, 5,863,538,
5,965,132, 6,004,554, 6,051,230, 6,261,535, 6,093,399, 6,004,555,
5,877,289 and 6,036,955). The present inventors provide naked
antibodies and vascular targeting agents directed against
phosphatidylserine for use in targeting tumor vasculature in the
diagnosis and treatment of cancer in man.
[0226] Although a precise molecular understanding of how naked
antibodies directed against phosphatidylserine function in tumor
treatment is not necessary in order to practice the treatment, the
inventors have contemplated several mechanisms that may account for
the observed endothelial cell killing. The favored mechanisms
(particularly for the 3G4 antibody described herein) are Fc
domain-mediated immune effector functions, such as
antibody-dependent cellular cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC) and antibody mediated
phagocytosis. Cell-mediated cytotoxicity, complement-mediated lysis
and/or apoptosis, antibody-induced cell signaling and/or
disturbances to the cytoskeleton may also be involved.
[0227] Binding of intact antibodies against phosphatidylserine,
particularly 3G4, to the vascular endothelial cell surface means
that the Fc portions of the antibodies protrude into the vessel
lumen. As antibody Fc fragments activate the complement pathway,
the observed cellular destruction may be a result of
complement-directed lysis. Antibody binding thus activates the
complement-dependent coagulation cascade, causing multi-component
complexes to assemble and, ultimately, to generate a lytic complex
that permeabilizes the target cell. "Complement-activated ADCC" may
also be operating in the destruction, in which complement binds to
the antibody-coated target cell, and in which cells, such as
neutrophils, having receptors for complement, lyse the target
cell.
[0228] As the naked or unconjugated antibodies, including the
antigen binding fragments thereof, bind to phosphatidylserine at
the surface of the tumor vascular endothelial cells, they will form
an antibody coating on the luminal surface. This may function to
attract immune effector cells, such as cytotoxic T cells and/or
natural killer (NK) cells, which will then exert a cell-mediated
cytotoxic effect on the vascular endothelial cells.
[0229] Antibody binding to phosphatidylserine may also induce
apoptosis in the tumor vascular endothelial cells. Although there
are no known reports of antibody binding to PS actually inducing
apoptosis (rather than PS being a marker resulting from apoptosis),
the inventors consider this to be another possible mechanism for
the observed anti-tumor effects.
[0230] It is also possible that antibody binding to
phosphatidylserine at the surface of tumor vascular endothelial
cells may cause disturbances in the cytoskeletal organization of
the cell. As the cytoskeleton plays a role in the organization of
surface membranes, and as antibody binding may disturb (or further
disturb) the membrane, binding of antibodies to phosphatidylserine
may transmit changes to cytoskeletal proteins that interact with
the bilayer. It is already known that the spatial organization of
cytoskeletal proteins controls membrane stability and cell shape,
and it is possible that perturbation of some cytoskeletal
equilibrium may have far-reaching consequences on cell
integrity.
[0231] A further mechanism of operation of the invention may be
that antibody binding to phosphatidylserine at the endothelial cell
surface may initiate signal transduction by, as yet, undefined
pathways. Antibody binding may also disturb known signal
transduction pathways, e.g., by altering the conformation and/or
interactions of membrane receptors, signal transduction proteins,
membrane channels, and the like. Signals for cell destruction
(apoptosis) may be initiated or mimicked, and/or
preservation/homeostatic signals may be inhibited.
[0232] Although of scientific interest, determining the exact
nature of the vascular destruction achieved by the naked antibodies
to phosphatidylserine is not necessary to practice the treatment.
Given that the administration of these antibodies is shown to
advantageously result in specific anti-tumor effects in vivo, the
treatment can be utilized irrespective of the molecular mechanism
that underlies this phenomenon. The use of naked antibodies that
bind to phosphatidylserine thus represents an important advance in
tumor therapy, providing advantages in preparation and cost.
C. Detailed Analysis of the 3G4 Antibody
[0233] As shown herein, 3G4 is an effective and well-tolerated
anti-tumor agent, which acts by homing to phosphatidylserine on
tumor blood vessels, and causing host cell-mediated antitumor
effects. Since PS is the same molecule in the human and mouse, and
has the same cellular distribution, regulation and induction by ROS
in both species (Balasubramanian and Schroit, 2003; Whitworth et
al., 1990), these studies further support the use of antibodies and
other constructs that bind to phosphatidylserine to treat cancer in
man. Indeed, chimeric and humanized versions of 3G4 have already
been prepared for this and other purposes.
[0234] Antibodies and other ligands that bind to phosphatidylserine
can thus be used for the targeting, imaging and/or treatment of
tumor blood vessels. Phosphatidylserine is attractive as tumor
target vessels for several reasons: it is abundant (PS is present
at 3.times.10.sup.6 molecules per cell); it is on the luminal
surface of tumor endothelium, which is directly accessible for
binding by vascular targeting agents in the blood; it is present on
a major percentage of tumor endothelial cells in diverse solid
tumors; and it is essentially absent from endothelium in all normal
tissues.
[0235] The 3G4 antibody has been shown to localize specifically to
vascular endothelial cells in tumors, reduce tumor vascularity and
plasma volume and to retard tumor growth (the present examples; Ran
et al., 1998; Ran and Thorpe, 2002; Ran et al., 2002b; Ran et al.,
2005; Huang et al., 2005).
[0236] Ongoing studies have shown that 3G4 treatment inhibits
growth of murine tumor allografts and human tumor xenografts (the
present examples; Ran et al., 2005), including orthotopic human
breast tumors (Example XX; Huang et al., 2005) and orthotopic human
pancreatic tumors (Example XX; Beck et al., 2005). 3G4 also
inhibits metastatic spread and growth of these tumors (Example XX;
Huang et al., 2005; Beck et al., 2005). When used in combination,
3G4 enhances the therapeutic efficacy of the chemotherapeutic drugs
docetaxel and gemcitabine for treatment of breast and pancreatic
tumors, respectively (Example XX; Huang et al., 2005; Beck et al.,
2005).
[0237] As with the other antibodies used herein, 3G4 therapy is
well-tolerated in tumor-bearing animals treated repeatedly with the
therapeutic dose (4 mg/kg in mice, three times a week). The mice
retained normal physical signs, coagulation parameters, bone marrow
cellularity, white blood cell counts and histology. Despite effects
of very high concentrations of 3G4 in partially inhibiting
phospholipid-dependent coagulation pathways, a substantial safety
margin exists between the therapeutic dose and the dose that
prolongs coagulation times in vivo. 3G4 is thus an effective and
well-tolerated anti-tumor agent, which acts by homing to anionic
phospholipids on tumor blood vessels and causing host cell-mediated
antitumor effects. 3G4 is not associated with pathogenic effects
reported in the literature for antibodies associated with
anti-phospholipid syndrome(s).
[0238] Anti-phospholipid syndrome(s) (APS) are associated with
autoantibodies termed "anti-cardiolipin" antibodies and "lupus
anticoagulant antibodies". These syndromes are associated with a
predisposition towards venous and arterial thromboemboli,
thrombocytopenia and a number of neurological syndromes. The
anti-phospholipid antibodies in these patients are thus "pathogenic
antibodies". Such anti-phospholipid antibodies in the human
population occur in systemic lupus erythematosus (Branch et al.,
1987; Staub et al., 1989; Drouvalakis and Buchanan, 1998; Smimov et
al., 1995; Rauch et al., 1986; Rauch and Janoff, 1990) and are
associated with recurrent pregnancy loss (Rote et al., 1995; Rote,
1996; Vogt et al., 1996; 1997; Katsuragawa et al., 1997).
[0239] Although described for years as "anti-phospholipid
antibodies" and "anti-PS antibodies", such pathogenic antibodies in
fact recognize protein cofactors that bind to cardiolipin, PS or
both, not the phospholipids themselves (Galli et al., 1990; 1993;
McNeil et al., 1990; Rote, 1996). There is considerable
heterogeneity in the pathogenic antibodies. Certain
anti-cardiolipin antibodies have been reported to recognize
particular regions on .beta.2-glycoprotein I, whereas lupus
anticoagulant antibodies recognize prothrombin. Similarly, anti-PE
antibodies that occur in disease states bind to PE in combination
with proteins, such as low and high molecular weight kininogen
(HK), prekallikrein and factor XI (Sugi and McIntyre, 1995; 1996a;
1996b).
[0240] In selecting antibodies for administration as therapeutics,
it was thus thought that such antibodies should be identified on
the basis of not binding to phosphatidylserine in combination with
protein cofactors, but rather "true" anti-phosphatidylserine
antibodies should be sought (WO 2004/006847).
[0241] However, further in vitro studies of the 3G4 antibody
revealed that, unlike 9D2, binding of 3G4 to phosphatidylserine and
anionic phospholipids was partially inhibited in the complete
absence of serum. Moreover, binding was found to be restored when
.beta.2-glycoprotein I (.beta.2GPI) was added. This prompted the
inventors to believe that 3G4 recognizes an epitope in
lipid-.beta.2GPI complexes, such that the interaction between 3G4
and PS is dependent on .beta.2GPI (U.S. provisional application
Ser. No. 60/646,333, filed Jan. 24, 2005; Ran et al., 2005). The
inventors reasoned that PS exposed on tumor vessels in vivo is
probably complexed with serum components, such as .beta.2GPI, and
that 3G4 probably binds to these complexes.
[0242] The potential for 3G4 to bind to a PS-.beta.2GPI complex was
very surprising, not least because 3G4 has been shown to be safe
when administered to animals in numerous studies, and not to be
associated with pathogenic effects reported in the literature for
antibodies associated with APS, which antibodies are known to bind
to lipid-serum protein complexes, including PS-.beta.2GPI
complexes. No manifestations of APS have been observed in any 3G4
treatment, in contrast to those observed with anticardiolipin
antibodies against .beta.2GPI (Matzinger, 1998; Fadok et al., 1998;
Fadok et al., 2001a;b). Mice treated with 3G4 at high doses for
prolonged periods showed no changes in coagulation capability, yet
mice respond with APS when injected with anticardiolipin or lupus
anticoagulant antibodies.
[0243] The present invention resolves this discrepancy, elucidates
the interaction of 3G4 and .beta.2GPI required for binding to
endothelial cells with exposed PS, and explains how 3G4 can bind to
a PS-.beta.2GPI complex without succumbing to the toxicities
associated with previously known pathogenic antibodies.
[0244] As shown in Example XXX, the interaction between 3G4 and PS
is dependent on .beta.2GPI. Although .beta.2GPI binds anionic
phospholipids weakly under physiological conditions, the present
invention shows that 3G4 greatly enhances the binding of .beta.2GPI
to PS-positive endothelial cells. The data show that divalent
3G4/.beta.2GPI complexes are required for enhanced binding, since
3G4 Fab' fragments do not bind endothelial cells with exposed PS.
It is also demonstrated that an artificial dimeric .beta.2GPI
construct binds to endothelial cells with exposed PS without the
need for 3G4. Together, these data suggest that 3G4 targets
PS-positive cells, including tumor endothelial cells, by increasing
the affinity of .beta.2GPI for PS via the formation of a divalent
3G4/.beta.2GPI complex.
[0245] Example XXX also shows that 3G4 binds to domain II of
.beta.2GPI. This is important, as antibodies from APS patients that
recognize .beta.2GPI domain II are not pathogenic. In contrast, a
significant recent study demonstrated that pathogenic
anti-.beta.2GPI antibodies isolated from patients with APS commonly
recognize domain I of .beta.2GPI (de Laat et al., 2005a). The
ability of the 3G4 antibody to bind PS-.beta.2GPI complexes without
binding .beta.2GPI domain I is important in the lack of toxicity
that the antibody exhibits. .beta.2GPI can also interact with the
apolipoprotein E receptor 2' (apoER2') (Lutters et al., 2003).
Another recent study indicated that domain V of .beta.2GPI
interacts with apoER2' and is involved in the activation of
platelets, which causes increased platelet deposition to collagen
(van Lummel et al., 2005).
[0246] Accordingly, by binding neither domain I nor domain V, the
3G4 antibody can bind to a PS-.beta.2GPI complex and yet show no
toxicity following extensive toxicological studies performed in a
variety of animal models. In light of these findings, other
antibodies that bind to PS-.beta.2GPI complexes can now be made and
used safely in the treatment of various conditions, such as cancer
and viral infections. The selection of antibodies that do not
significantly bind to .beta.2GPI domain I is currently the primary
requirement, and the selection of antibodies that do not
significantly bind to .beta.2GPI domain I or to .beta.2GPI domain V
is envisioned to provide additional advantages.
[0247] In review, the new studies have characterized the
interaction between the 3G4 antibody and its main anionic
phospholipid target, phosphatidylserine. The data demonstrate that
the interaction between 3G4 and PS is serum-dependent. .beta.2GPI
was identified as the serum factor required to mediate the 3G4-PS
interaction.
[0248] 3G4 was originally generated by immunizing mice with murine
endothelial cells treated with H.sub.2O.sub.2 to induce PS exposure
(Example IV; Ran et al., 2005). The cells were grown in
FBS-containing media and likely injected with a small amount of
bovine .beta.2GPI, leading to the production of anti-PS/.beta.2GPI
antibodies. Initial antibody screens for reactivity with PS were
performed in the presence of bovine serum. Later screens were
performed in the absence of serum, but as indicated herein,
antibody purified from SCM is co-purified with the .beta.2GPI
antigen. Therefore, "purified" antibody can bind PS in the absence
of serum due the presence of contaminating bovine .beta.2GPI. Only
when 3G4 was grown and purified from serum-free media was the
serum-dependence identified. Other groups have reported similar
concerns regarding obtaining .beta.2GPI free preparations of
anti-.beta.2GPI antibodies (Roubey et al., 1995). Therefore, it is
quite possible that many so-called "antiphospholipid antibodies"
reported to bind phospholipids directly, actually recognize serum
proteins with affinity for phospholipids.
[0249] .beta.2GPI is a 50-kDa glycoprotein that is present in
plasma at a concentration of .about.200 .mu.g/mL (4 .mu.M) (Cleve
et al., 1969). The protein is a member of the complement control
protein (CCP) family (Steinkasserer et al., 1991). .beta.2GPI has
five CCP repeats in which the first four domains are regular
repeats consisting of .about.0.60 amino acids. The fifth domain
differs from the other four domains as it has 82 amino acids,
including a cluster of positively charged amino acids (282-287) and
a conserved hydrophobic region (311-317) responsible for binding of
.beta.2GPI to anionic phospholipids (Steinkasserer et al., 1992;
Hunt and Krilis, 1994; Sheng et al., 1996; Mehdi et al., 2000; each
specifically incorporated herein by reference).
[0250] There is little known about the normal biological function
of .beta.2GPI. Proposed functions include facilitating apoptotic
cell clearance (Balasubramanian et al., 1997b; Balasubramanian et
al., 2005), modulation of platelet function (Nimpf et al., 1985;
Nimpf et al., 1987), and inhibition of coagulation (Nimpf et al.,
1986; Schousboe, 1985); however, no definitive phenotype has been
observed in mice or patients with .beta.2GPI deficiency (Miyakis et
al., 2004; Yasuda et al., 2000).
[0251] In contrast, .beta.2GPI is well known for its involvement in
the autoimmune disorder APS, where it has been identified as a
plasma co-factor required for binding of so-called antiphospholipid
(aPL) antibodies to anionic phospholipid surfaces (de Laat et al.,
2004a; Bevers et al., 2004). Antiphospholipid antibodies are known
to interact with a variety of serum proteins, but aPL antibodies
recognizing .beta.2GPI correlate most strongly with the clinical
symptoms of APS (de Laat et al., 2004b). Therefore, the interaction
between .beta.2GPI, anti-.beta.2GPI antibodies, and anionic
phospholipid membrane surfaces has been studied extensively (Bevers
et al., 2004).
[0252] The present studies also examined the characteristics of 3G4
binding to PS-coated microtiter plates and to endothelial cells
with exposed PS. Using a live-cell binding assay, it was determined
that neither 3G4 nor .beta.2GPI bind endothelial cells with exposed
PS unless both molecules are present simultaneously (Example XXX).
This observation is consistent with the ELISA-based findings
demonstrating that 3G4 depends on .beta.2GPI for binding to anionic
lipid surfaces. Furthermore, this observation supports reports that
.beta.2GPI has low affinity for anionic phospholipid membranes
under physiological conditions (Willems et al., 1996; Bevers et
al., 2004; Bevers et al., 2005), and suggests that the affinity is
greatly enhanced by the presence of 3G4.
[0253] The data also demonstrate that 3G4 enhances the affinity of
.beta.2GPI for anionic phospholipid surfaces via the formation of
divalent .beta.2GPI complexes. When the .beta.2GPI concentration is
held constant and the 3G4 concentration is increased, binding of
3G4/.beta.2GPI to endothelial cells with exposed PS peaks at a 3G4
to .beta.2GPI ratio of 2:1. At higher concentrations of 3G4, the
binding decreases. The bell-shaped curve suggests competition
between monovalent and divalent complexes, leading to a decrease in
overall 3G4/.beta.2GPI complex binding. A bell-shaped relationship
has been reported for anti-.beta.2GPI antibodies in a thrombosis
model (Jankowski et al., 2003). In other studies, 3G4 Fab fragments
did not bind endothelial cells with exposed PS in the presence of
.beta.2GPI, demonstrating that monovalent 3G4/.beta.2GPI complexes
cannot bind anionic phospholipid surfaces.
[0254] It is also demonstrated that artificial dimeric .beta.2GPI
constructs can bind endothelial cells with exposed PS without the
need for 3G4. Thus, anti-.beta.2GPI antibodies increase the
affinity of .beta.2GPI for anionic phospholipid surfaces by
crosslinking two .beta.2GPI molecules, forming a divalent
.beta.2GPI complex.
[0255] Importantly, although 3G4/.beta.2GPI complexes interact with
PS much like aPL antibodies from patients with APS, 3G4 has not
caused clinical manifestations of APS in any animal model tested.
The mechanism(s) by which aPL cause APS remains largely unknown.
Some studies suggest that aPL/.beta.2GPI can bind and activate
resting endothelial cells (which do not expose PS), causing them
adopt a more thrombogenic phenotype (Simantov et al., 1995;
Pierangeli et al., 1999). More recent studies demonstrate that
.beta.2GPI and aPL/.beta.2GPI complexes cannot bind and fully
activate endothelial cells unless they are already "pre-activated"
(Chen et al., 2004). Pre-activation causes endothelial cells to
expose PS, providing the appropriate anionic phospholipid surface
for binding of aPL/.beta.2GPI complexes.
[0256] 3G4 does not bind resting ABAE cells or HUVECs in a
live-cell assay. Furthermore, 3G4 does not activate resting HUVECs
or HUVECs pre-activated with low concentrations of LPS. Recent
studies report a high correlation between the presence of aPL
antibodies recognizing domain I of .beta.2GPI in patient sera and
clinical symptoms of APS (de Laat et al., 2005), and a
conformational change may be required for antibody binding (de Laat
et al., 2005b). Antibodies recognizing the other domains of
.beta.2GPI are also detected in patient sera, but do not correlate
with pathogenesis. Importantly, the present invention demonstrates
that 3G4 recognizes domain II of .beta.2GPI, which likely explains
the lack of endothelial cell activation in vitro and the lack of
toxicity in vivo.
[0257] Domain II of mouse .beta.2GPI differs from domain II of rat,
dog, cow, chimp and human .beta.2GPI at only 7 of 60 amino acids.
Therefore, the inability of 3G4 to bind PS in the presence of mouse
serum or to detect murine .beta.2GPI by immunoblot is unexpected,
especially in light of the documented anti-tumor activity of 3G4 in
mice (the present examples; Ran et al., 2005; Huang et al., 2005;
Beck et al., 2005). Importantly, these studies were performed using
3G4 purified from SCM. As shown herein, 3G4 SCM is able to bind PS,
due to co-purification of bovine .beta.2GPI. When 3G4 SCM was run
on an SDS-PAGE gel, transferred to membrane support, and probed for
the presence of bovine .beta.2GPI, a band of 50 kDa was clearly
visible. Furthermore, 3G4 present in sera collected from mice
following injection with 3G4 SCM binds PS. Therefore, bovine
.beta.2GPI is able to mediate binding of 3G4 to PS in mouse plasma
and likely contributed to the anti-tumor effect of 3G4. All tumor
studies performed in mice using 3G4 should be supplemented with
bovine or human .beta.2GPI to ensure targeting of tumor endothelial
cells with exposed PS.
[0258] The identification of .beta.2GPI as an important co-factor
required for the interaction between 3G4 and PS greatly enhances
the understanding of this unique tumor vascular targeting agent.
3G4 targets tumor endothelial cells with exposed PS by enhancing
the affinity of .beta.2GPI for anionic phospholipid surfaces via
the formation of dimeric .beta.2GPI complexes. In addition to
clarifying these properties, the present studies now permit the
development of third generation antibodies that bind to
aminophospholipids and anionic phospholipids in complexes with
serum proteins, without duplicating the properties of pathogenic
antibodies.
[0259] In summary, preferred "third generation" or "non-pathogenic
PS-.beta.2GPI antibodies" can now be selected that bind to PS and
.beta.2GPI at a position other than within .beta.2GPI domain I, or
other than within .beta.2GPI domain I or domain V. In light of the
data herein the most preferred antibodies are currently those that
bind .beta.2GPI within domain II. A range of non-pathogenic
PS-.beta.2GPI antibodies can be prepared following the methodology
and screening techniques described herein, e.g., using the sequence
information in FIG. 18A and FIG. 18B and the binding assays in
Example XXX. Such antibodies and immunoconjugates thereof can be
advantageously used in the safe and effective treatment of viral
infections and in treating cancer and other diseases.
D. ReceptorBodies and BetaBodies
[0260] The foregoing detailed analyses of the 3G4 antibody, and
particularly the interaction of 3G4 with .beta.2GPI, which
contrasts with that set forth in WO 2004/006847, also contributed
to the inventors' development of the receptorbodies of the present
invention. Importantly, this invention also provides a range of
constructs, termed "receptorbodies", which bind to
phosphatidylserine, and evoke host effector functions. Aspects of
the development of the receptorbodies are described below.
[0261] Data are presented herein to show that targeting tumor
vasculature with antibodies to phosphatidylserine is effective in
tumor treatment. Antibodies to phosphatidylserine are also shown to
be effective in treating viral infections. Considering the tumor
studies as an example, it is shown herein that stress conditions in
the tumor microenvironment induce phosphatidylserine exposure on
tumor vascular endothelium (Example VII, Example II, Example V and
Example VI). The exposed phosphatidylserine provide a selective
marker of tumor vasculature for imaging and therapy (Example IX,
Example X, Example XI and Example XX).
[0262] In conjunction with the tumor treatment, antibodies to
phosphatidylserine damage blood vessels within tumors and cause
leukocyte infiltration (e.g., FIG. 1). Moreover, administration of
such antibodies recruits macrophages into tumors. For example, in
tumor treatment studies using the 3G4 antibody, extensive binding
of blood monocytes to tumor vascular endothelium and profuse
infiltration of macrophages into the tumor interstitium were seen
(e.g., Example XXVII; FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D).
[0263] The infiltration of macrophages into the treated tumor,
taken together with the finding that 3G4 enhances the rate of
phagocytosis of PS-expressing cells by bone-marrow derived mouse
macrophages in vitro by 5-fold in an Fc-dependent fashion, are
consistent with antibodies such as 3G4 provoking macrophage
cytotoxicity towards tumor vessels or tumor cells. Also, 3G4 does
not directly inhibit the proliferation of PS-expressing endothelial
cells or tumor cells, or mediate complement lysis of the cells in
vitro, suggesting that the antibody is not directly cytotoxic.
[0264] The inventors therefore propose two feasible mechanisms of
macrophage-mediated damage to tumor vessels or tumor cells. First,
antibodies such as 3G4 bind to complexes of anionic phospholipids,
mainly phosphatidylserine, and serum proteins on tumor vessels and
tumor cells. The antibodies then stimulate the binding of monocytes
and macrophages via Fey receptors, thereby enhancing
antibody-dependent cellular cytotoxicity (ADCC) and
antibody-dependent macrophage phagocytosis. Activated macrophages
have long been recognized as having direct tumoricidal activity
(Whitworth et al., 1990). In support of this, Manfredi et al.
(1998) have reported that anti-phospholipid antibodies can
facilitate opsonization of PS-expressing cells by scavenger
macrophages with massive induction of TNF-.alpha. secretion.
Although macrophages have PS receptors and can bind to, and engulf,
PS-expressing cells (Balasubramanian and Schroit, 2003; Utsugi et
al., 1991; Fadok et al., 2001a), PS exposure alone is insufficient
to stimulate engulfment (Devitt et al., 2003).
[0265] Second, antibodies such as 3G4 may block PS-mediated
"quiescence" signals from PS-expressing tumor endothelial cells,
which normally would suppress inflammatory responses by macrophages
that bind to the tumor vessels and tumor cells (FIG. 7). Analogous
mechanisms are thought to explain the lack of inflammatory response
of macrophage-lineage cells to apoptotic cells (Henson et al.,
2001; Matzinger, 1998; Gallucci and Matzinger, 2001; Fadok et al.,
2001b). Accordingly, antibodies such as 3G4 may evoke tumor vessel
damage by provoking macrophages to secrete TNF-.alpha., IL-1 and
other inflammatory cytokines that directly damage tumor endothelium
(FIG. 7) and recruit further host cells into the tumor (Fadok et
al., 1998).
[0266] Ideally, each of the above possible mechanisms should be
reconciled with the proposal that tumor-associated macrophages can
induce tumor angiogenesis, which promotes tumor growth (e.g.,
Sunderkotter et al., 1994). The inventors therefore reason that
there is a balance between the proangiogenic effects of macrophages
and their direct cytotoxic effects on tumor vessels and tumor cells
that is determined by local conditions (e.g., hypoxia, TGF-.beta.)
in the tumor microenvironment (Breier et al., 2002). It is
currently envisioned that antibodies such as 3G4 alter the tumor
microenvironment in a manner that favors a direct cytotoxic
response from macrophages.
[0267] Considering phosphatidylserine biology, and optionally in
light of the data presented herein concerning macrophage behaviour
in tumor treatment, the inventors therefore provide a range of
advantageous constructs or "receptorbodies" that bind to
phosphatidylserine, and optionally to other anionic phospholipids,
and related conjugates and methods of use.
[0268] Such a construct or receptorbody of the invention will
typically comprise a binding protein or polypeptide, receptor,
ligand or peptide that recognizes and binds to phosphatidylserine,
and optionally to other anionic phospholipids, which binding
protein or polypeptide, receptor, ligand or peptide is linked to an
antibody or immunoglobulin Fc region or domain. The Fc region
modifies the binding protein or polypeptide, receptor, ligand or
peptide to increase its biological half life, but more importantly,
provides the resultant construct with the ability to stimulate host
effector functions to enhance disease treatment.
[0269] The receptorbodies of the invention may be used in any of
the embodiments in which antibodies against phosphatidylserine may
be used. Such receptorbodies therefore have multiple applications,
including binding phosphatidylserine on tumor blood vessels and
tumor cells, leading to host cell mediated anti-vascular and
anti-cellular effects on the tumor, and binding phosphatidylserine
on viruses or virally infected cells, leading to an anti-viral
effect in animals.
[0270] One of the advantages of these aspects of the invention is
that the receptorbodies use natural phosphatidylserine binding
proteins, polypeptides or receptors to achieve specific binding.
Although the use of antibodies against phosphatidylserine is herein
shown to be effective in many treatment embodiments, the
receptorbodies could have higher avidity or specificity than
anti-PS antibodies. By dimerizing phosphatidylserine binding
proteins or polypeptides, particularly .beta.2GPI, onto an Fc
immunoglobulin region, the constructs achieve better avidity,
stability, longer in vivo half lives, and possibly superior
localization to phosphatidylserine-expressing tissues, as well as
stimulating host effector functions in vivo. Anti-PS antibodies are
herein shown to be both effective and safe when administered to
animals, including monkeys. Likewise, the receptorbodies containing
the natural ligands will be effective and safe and will not cause
antiphospholipid syndromes. Accordingly, the receptorbodies of the
present invention are simple, human, specific and safe
therapeutics, which may be used to treat a range of tumors and
viral infections.
[0271] D1. Phosphatidylserine Binding Proteins
[0272] The binding protein or polypeptide, receptor, ligand or
peptide in the receptorbodies may be any one of several
phosphatidylserine binding proteins, including cell-derived
receptors, factor V, prothrombin (factor II), and such like. The
use of .beta.2-glycoprotein I is much preferred.
[0273] Certain factors in the coagulation cascade also bind
phosphatidylserine and can thus be used in the receptorbodies of
the invention. For example, factor V and prothrombin (factor II)
bind phosphatidylserine. In the prothrombinase complex, binding of
factor Va to an anionic lipid surface promotes Ca.sup.2+-dependent
binding of factor Xa, which converts prothrombin to thrombin. In
both complexes, phosphatidylserine is the most effective anionic
phospholipid. The phosphatidylserine binding proteins Protein C,
Protein S, Factor II/IIa, Factor V/Va, Factor VII/VIIa, Factor
IX/IXa and Factor X/Xa may thus be used.
[0274] Fadok et al. (2000, specifically incorporated herein by
reference) reported the cloning of a PS receptor that directly
binds apoptotic cells. This receptor is expressed in many cells
types, including macrophages, fibroblasts and epithelial cells.
Studies using antibodies and PS vesicles indicated that the
receptor does recognize phosphatidylserine on the cell surface.
Hence, this is a PS-specific receptor, "the PS receptor" or "PSr"
(Accession Number AF304118). This receptor is present on
phagocytes, such as macrophages, and also on other cell types. The
PS receptor of Fadok et al. (2000, specifically incorporated herein
by reference) may therefore be used in the receptorbodies of the
invention.
[0275] Other phosphatidylserine binding proteins that could be used
in the receptorbodies of the invention are described in
Balasubramanian and Schroit, 2003 (specifically incorporated herein
by reference), in particular, see Balasubramanian and Schroit,
2003, Table 2, and the references cited therein, which are all
specifically incorporated herein by reference. These
phosphatidylserine binding proteins include, for example, CD14;
integrins, such as .alpha.5.beta.3 (vitronectin, CD51/CD61) and
.alpha.5.beta.3/CD36; several scavenger receptors, such as SRB
(CD36), SRC (LOX-1, SRCL), SRD (CD68, macrosialin) and PSOX;
complement receptors, such as CR3 (CD11b/CD18) (Accession Number NM
000632), CR4 (CD11c/CD18) (Accession Number NM 000887), and CD93
(cClqr, calreticulin)/CD91 (.alpha.2-macroglobulin receptor); and
other receptors such as the Mer/Gas 6 phagocyte recognition
partners for PS-expressing apoptotic cells (Accession Number
AH010001).
[0276] Although annexins, such as annexin V, may be used as a
phosphatidylserine binding protein in a receptorbody, their use is
not a universal part of the present invention. Therefore, in
certain embodiments, the present invention provides constructs in
which the phosphatidylserine binding protein is not an annexin.
Nonetheless, the following information is provided for use in those
embodiments in which an annexin is contemplated. In addition, the
technical information in the annexin patent documents specifically
incorporated herein by reference, e.g., regarding expression and
conjugation, may be used to support the other Fc-phosphatidylserine
binding protein constructs of the invention.
[0277] At least nine members of the annexin family have been
identified in mammalian tissues (Annexin I through Annexin IX).
Currently preferred amongst these is annexin V (also known as
PAP-I). The protein and DNA sequences for annexins are known in the
art, facilitating the ready production of recombinant fusion
proteins for use in these aspects of the invention.
[0278] 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 I fragment.
[0279] 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). 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).
[0280] WO 91/07187 provides natural, synthetic or genetically
prepared derivatives and analogues of `annexine` (annexin).
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.
[0281] 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.
[0282] U.S. Pat. No. 6,197,278 is specifically incorporated herein
by reference for purposes including further enabling and providing
written description support for annexins in a generic sense, their
safe and effective administration in vivo and imaging embodiments.
To the extent that they clearly describe appropriate annexin
starting materials for preparing 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 useful
recombinant expression vectors.
[0283] U.S. Pat. No. 5,632,986 is also 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.
[0284] 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 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 so long as the mutein or second generation annexin
molecule does not contain substantially lower affinity for
aminophospholipid. The same reasoning applies to proteins other
than annexin.
[0285] 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 the Fc regions
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.
These agents may also be used with proteins other than
annexins.
[0286] 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.
[0287] U.S. Pat. Nos. 6,312,694, 6,783,760 and 6,818,213 are each
specifically incorporated herein by reference for purposes
including further enabling and providing written description
support for phosphatidylserine binding proteins in a generic sense,
and their safe and effective administration in vivo. These patents
disclose binding ligand compositions in which a therapeutic agent
is operatively attached to a targeting agent that binds to
phosphatidylserine, preferably phosphatidylserine exposed on the
luminal surface of blood vessels of a vascularized tumor, and
cancer treatment methods using such binding ligands and
combinations thereof. Although these patents do not teach or
suggest phosphatidylserine binding proteins linked to an Fc region,
their disclosures concerning phosphatidylserine binding proteins,
attached therapeutic agents and safe and effective in vivo
treatment methods are specifically incorporated herein by
reference.
[0288] U.S. Pat. Nos. 6,312,694, 6,783,760 and 6,818,213
particularly concern binding ligands in which a therapeutic agent
is operatively attached to at least a first phosphatidylserine
binding protein. Although the proteins in these patents are linked
to therapeutic agents and are used to deliver the therapeutic
agents to the tumor vasculature without an Fc region, and hence do
not elicit host cell effector functions, these patents are
specifically incorporated herein by reference for purposes
including further enabling and describing protein conjugates and
their safe and effective administration in vivo, including to treat
cancer. Using the information in these patents and the teaching in
the present disclosure, a range of receptorbodies can thus be
prepared in which a phosphatidylserine binding protein, or a
phosphatidylserine-binding fragment thereof, is now linked to an Fc
region or domain.
[0289] .beta.2-glycoprotein I (.beta.2GPI) is the currently
preferred phosphatidylserine binding protein for use in the
receptorbodies of the invention. .beta.2GPI, also known as
apoplipoprotein H, is a 50 kDa plasma glycoprotein that binds
negatively charged phospholipids through its C terminal (Wurm,
1984, specifically incorporated herein by reference). The following
accession numbers are provided for human B2GPI (NP 000033, AAP72014
and ICIZA) and for mouse B2GPI (AAH53338, NP 038503, CAA69401,
BAB2721). In vitro and in vivo evidence indicates that .beta.2GPI
plays a role in the clearance of PS-expressing apoptotic cells
(Chonn et al., 1995; Balasubramanian et al., 1997; Balasubramanian
and Schroit, 1998). .beta.2GPI interactions with phagocytes are
electrostatic in nature and protein glycosylation is not critical
for phagocyte recognition. Accordingly, there are no significant
obstacles to recombinant production of .beta.2GPI for use in
receptorbodies.
[0290] When using a .beta.2GPI protein, polypeptide or peptide in
the invention, the resulting Fc-.beta.2GPI construct is termed a
"betabody". Betabodies will preferably include a lipid binding
region from domain V of .beta.2GPI, as shown herein in FIG. 18A and
FIG. 18B. Constructs containing all of domain V may be preferred to
ensure lipid binding. Domain V may be used alone, or with one or
more of the other four domains. Although using the full length
.beta.2GPI protein will be convenient, betabodies that do not
contain domain I of .beta.2GPI may be preferred in certain aspects,
as antibodies from patients with APS commonly recognize domain I of
.beta.2GPI (de Laat et al., 2005a).
[0291] Other preferred betabodies are those in which two .beta.2GPI
polypeptides are dimerized onto an Fc region. Such betabodies are
shown herein to be effective in binding to phosphatidylserine on
plates and exposed on cell surfaces. However, other means of
preparing dimerized and multimerized betabodies are provided, such
as nanoparticles, liposomes and other molecular scaffolds.
[0292] D2. Fc Regions
[0293] The Fc region will be attached, linked or conjugated to the
phosphatidylserine binding protein or polypeptide, receptor, ligand
or peptide so that the desired activity of the resultant
receptorbody is not substantially destroyed by attaching the Fc
region. Any of the conjugation or linker technologies known in the
art may be employed, such as, e.g., those described in the relevant
section herein. If desired, fusion proteins can be created using
molecular biological techniques, which are now standard practice to
those of ordinary skill in the art, as again exemplified herein.
Thus, the receptorbody can be made as a chemical conjugate or as a
fusion protein.
[0294] Within the heavy chains of an immunoglobulin, the amino
terminal domains are the variable domains (V.sub.H). The variable
domains of the heavy and light chains function together in antigen
binding. In the heavy chains, the variable domains are followed by
three constant domains: C.sub.H1, C.sub.H2, and the carboxy
terminal C.sub.H3. In certain antibodies, a fourth constant domain,
C.sub.H4, is present. A short stretch, the switch, connects the
heavy chain variable and constant regions. The hinge connects
C.sub.H2 and C.sub.H3 (the Fc fragment) to the remainder of the
antibody (the Fab fragments).
[0295] The nature of the constant region determines various
mechanisms of action other than antigen binding. Antibodies are
divided into five major classes, IgM, IgG, IgA, IgD and IgE, based
on their constant region structure and immune function. The
heavy-chain constant domains that correspond to the difference
classes of immunoglobulins are termed .mu., .gamma., .alpha.,
.delta. and .epsilon., and respectively.
[0296] In light of the effector functions provided by the Fc
regions (Bruggemann et al., 1987; Riechmann et al., 1988; Clark,
1997; Padlan, 1994; each specifically incorporated herein by
reference), currently preferred Fc regions are human IgG1
(.gamma.1) and human IgG3 (.gamma.3) for clinical use, and mouse
IgG2a (.gamma.2a) and mouse IgG2b (.gamma.2b) for pre-clinical
testing in mice. .gamma.2 is believed to be silent and thus would
not be chosen to provide effector functions, although it is still a
useful human protein for use as a dimerization domain. Although
chosen for effector functions, the Fc piece of immunoglobulins also
contributes to prolonged half-life, which can result from the
active readsorption of antibodies within the kidney.
[0297] Although recombinant expression of Fc regions and binding
constructs is convenient, such fragments can also be obtained by
proteolysis of the whole immunoglobulin, preferably by the
non-specific thiol protease, papain. Papain digestion yields two
identical antigen-binding fragments, termed "Fab fragments", each
with a single antigen-binding site, and the residual "Fc
fragment".
[0298] Papain should 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.
[0299] In certain embodiments, two Fc fragments may be used,
preferably two human Fc fragments. In such aspects, two binding
proteins, receptors or ligands can be combined with the two Fc
fragments to prepare another type of divalent receptorbody. Human
Fc fragments will be preferred to make receptorbodies for human
administration. Indeed, this is an advantage of the invention, as a
totally human therapeutic construct results. Thus, Fc-.beta.2GPI
constructs include completely human betabody therapeutics.
[0300] D3. Other Constructs and Carriers
[0301] Although linking an Fc region to a phosphatidylserine
binding protein or polypeptide is the preferred embodiment of the
receptorbody invention, phosphatidylserine binding proteins may
also be linked to inert carriers to impart longevity to the
biologically active molecules. As such, the present invention
further provides constructs comprising at least a first
aminophospholipid binding protein, including .beta.2GPI,
operatively attached to at least a first inert carrier.
[0302] As known in the art, carrier proteins can be used to
increase biological half life, and exemplary proteins are albumins
and globulins, such as neutravidin and streptavidin. Non-protein
carriers can also be used to increase biological half life, such as
natural or synthetic polymers, including polysaccharides and PEG.
Any such inert carrier may be attached to any of the
phosphatidylserine binding proteins described herein.
[0303] Other aspects of the present invention concern compositions
of .beta.2GPI dimers prepared by means other than using an Fc
region, and various methods of use. The invention particularly
provides methods of treating cancer and methods of treating viral
infections, comprising administering to an animal in need thereof a
therapeutically effective amount of a .beta.2GPI dimer.
[0304] Still further aspects of the invention are liposomes
comprising .beta.2GPI polypeptides, whether or not in dimeric form,
and including .beta.2GPI polypeptides other than those attached to
an Fc region. As such, the invention concerns a nanoparticle,
liposome, lipid carrier, complex, mixture, supramolecular structure
multimolecular aggregate or lipid-based drug delivery system
comprising at least a first .beta.2GPI polypeptide. The .beta.2GPI
polypeptide may be a dimer, but it need not be. The .beta.2GPI
polypeptide may, or may not, be attached to an additional
therapeutic agent. Such liposomes will preferably be stealthed
liposomes, and may contain a therapeutic agent in the liposome
core.
[0305] Yet further aspects of the present invention concern
compositions of .beta.2GPI polypeptides (other than .beta.2GPI
polypeptides attached to an Fc region) where the .beta.2GPI
polypeptide is operatively attached to at least a first therapeutic
agent, and various methods of use. Particularly preferred are
compositions wherein a .beta.2GPI polypeptide is operatively
attached to at least a first cytokine, such as TNF.alpha., IL-2 or
IFN.alpha.. In certain embodiments, the .beta.2GPI polypeptide will
be a dimer and dimeric .beta.2GPI polypeptide will be operatively
attached to at least a first therapeutic agent, such as a cytokine.
Any such .beta.2GPI-cytokine constructs or conjugates are
particularly contemplated for use in treating diseases, including
viral infections.
[0306] The invention thus further provides methods of treating
cancer and methods of treating viral infections, comprising
administering to an animal in need thereof a therapeutically
effective amount of a construct comprising a .beta.2GPI polypeptide
operatively attached to at least a first cytokine, preferably to
TNF.alpha., IL-2 or IFN.alpha.. The .beta.2GPI polypeptide may be a
dimeric .beta.2GPI polypeptide.
E. Conjugates of Additional Agents
[0307] The constructs and receptorbodies of the invention may also
be used to deliver attached therapeutic agents to specific targets,
such as tumor and intratumoral vasculature, tumor cells, virally
infected cells and viral particles. Although not concerning
constructs with Fc regions, U.S. Pat. Nos. 6,312,694, 6,783,760 and
6,818,213 are each specifically incorporated herein by reference
for their general teaching regarding aminophospholipid-binding
proteins attached to therapeutic agents. As the constructs and
receptorbodies of the present invention may also be used in safe
and effective anti-viral therapy, these constructs may also be
advantageously linked to a range of known anti-viral agents.
[0308] In these aspects of the invention, any construct,
receptorbody or betabody of the invention can be used to prepare a
conjugate. Agents for use in such conjugates preferably include an
anticellular or cytotoxic agent, cytokine, chemokine, V-type ATPase
inhibitor, protein synthesis inhibitor, chemotherapeutic agent,
anti-angiogenic agent, apoptosis-inducing agent, anti-tubulin drug,
radioisotope, coagulant or anti-viral agent. In the anti-viral
conjugates, there is no requirement to use a second anti-viral
agent as the attached agent.
[0309] E1. Anti-Cellular and Cytotoxic Agents
[0310] For certain applications, the therapeutic agents will be
cytotoxic or pharmacological agents, particularly cytotoxic,
cytostatic or otherwise anti-cellular agents having the ability to
kill or suppress the growth or cell division of cells, particularly
tumor endothelial cells, tumor cells or virally infected cells.
[0311] Exemplary anti-cellular agents include chemotherapeutic
agents, as well as cytotoxins. Chemotherapeutic agents that may be
used include: hormones, such as steroids; antimetabolites, 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 a construct in a
manner that will allow its targeting, internalization, release
and/or presentation to blood components at the site of the targeted
cells, such as endothelial cells.
[0312] 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 and shown to function
pharmacologically, including doxorubicin, daunomycin, methotrexate,
vinblastine, neocarzinostatin, macromycin, trenimon and
.alpha.-amanitin.
[0313] 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 certain aspects of 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.
[0314] A wide variety of cytotoxic agents are known that may be
conjugated. 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.
[0315] The well-known 1992 toxin book, "Genetically Engineered
Toxins", edited by Arthur E. Frankel, including the appendix, which
includes the primary amino acid sequences of a large number of
toxins, is specifically incorporated herein by reference, for
purposes of further describing and enabling the use of toxins in
targeted constructs.
[0316] Of the toxins, the use of gelonin and ricin A chains are
preferred. The use of gelonin as the effector or toxin portion of
immunoconjugates that bind to markers expressed, accessible to
binding, adsorbed or localized on intratumoral blood vessels of a
vascularized tumor is described in U.S. Pat. No. 6,051,230,
specifically incorporated herein by reference, and in U.S. Pat. No.
6,451,312, which particularly concerns gelonin linked to VEGF as a
targeting agent.
[0317] As to ricin A chains, a further preferred toxin moiety 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.
[0318] 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.
[0319] 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.
[0320] E2. Cytokines
[0321] Cytokines and chemokines are particular examples of agents
for linking to a construct, receptorbody or betabody of the present
invention. A range of cytokines may be used, including IL-3, IL-4,
IL-5, IL-7, IL-8, IL-9, IL-11, IL-13, TGF-.beta., M-CSF, G-CSF,
TNF.beta., LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF,
IFN-.alpha., IFN-.beta.. More preferred cytokines include
IL-1.alpha., IL-1.beta., IL-2, IL-6, IL-10, GM-CSF, IFN-.gamma.,
monocyte chemoattractant protein-1 (MCP-1), platelet-derived growth
factor-BB (PDGF-BB) and C-reactive protein (CRP) and the like.
Particularly preferred examples are TNF.alpha., TNF.alpha.
inducers, IL-2, IL-12, IFN-.alpha., IFN-.beta., IFN-.gamma. and
LEC.
[0322] TNF.alpha. increases vascular permeability. This agent is
contemplated for attachment to a construct, receptorbody or
betabody of the invention, particularly where the resultant
conjugate is used in combination therapy for the treatment of
cancer. The construct will deliver the attached TNF.alpha. to the
tumor environment, and the enhanced vascular permeability cause in
the tumor will facilitate the penetration of a second anti-cancer
agent into the tumor, thus amplifying the overall anti-tumor
effect.
[0323] IL-12, for example, may be attached to a construct,
receptorbody or betabody and used to redirect host defenses to
attack the tumor vessels. The chemokine LEC (liver-expressed
chemokine, also known as NCC-4, HCC-4, or LMC) is another preferred
component (Giovarelli et al., 2000). LEC is chemotactic for
dendritic cells, monocytes, T cells, NK cells and neutrophils and
can therefore improve host-mediated anti-tumor responses.
[0324] E3. Coagulation Factors
[0325] A construct, receptorbody or betabody of the invention may
be linked to a component that is capable of directly or indirectly
stimulating coagulation, to form a coaguligand. U.S. Pat. Nos.
6,093,399, 6,004,555, 5,877,289 and 6,036,955 are specifically
incorporated herein by reference for purposes of further describing
the operative attachment of coagulants to form coaguligands.
[0326] A construct, receptorbody or betabody of the invention 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.
[0327] 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.
[0328] U.S. Pat. Nos. 5,504,067, 6,156,321, 6,132,729 and 6,132,730
are 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.
[0329] 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.
[0330] 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.
[0331] 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.
[0332] Such membrane insertion sequences may be located either at
the N-terminus or the C-terminus of the TF 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
TF 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.
[0333] 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.
[0334] 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.
[0335] 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 VIIa at low levels in plasma. Upon administration of such
a Factor VII activation mutant 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.
[0336] 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).
[0337] As detailed in U.S. Pat. Nos. 6,093,399, 6,004,555,
5,877,289 and 6,036,955, 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.
[0338] 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.
[0339] 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.
[0340] 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.
[0341] .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.
[0342] 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.
[0343] E4. Anti-Tubulin Drugs
[0344] A range of drugs exert their effects via interfering with
tubulin activity. As tubulin functions are essential to mitosis and
cell viability, certain "anti-tubulin drugs" are powerful
chemotherapeutic agents. "Anti-tubulin drug(s)", as used herein,
means any agent, drug, prodrug or combination thereof that inhibits
cell mitosis, preferably by directly or indirectly inhibiting
tubulin activities necessary for cell mitosis, preferably tubulin
polymerization or depolymerization.
[0345] Some of the more well known and currently preferred
anti-tubulin drugs for use with the present invention are
colchicine; taxanes, such as taxol; vinca alkaloids, such as
vinblastine, vincristine and vindescine; and combretastatins. Other
suitable anti-tubulin drugs are cytochalasins (including B, J, E),
dolastatin, auristatin PE, paclitaxel, ustiloxin D, rhizoxin,
1069C85, colcemid, albendazole, azatoxin and nocodazole.
[0346] As described in U.S. Pat. Nos. 5,892,069, 5,504,074 and
5,661,143, each specifically incorporated herein by reference,
combretastatins are estradiol derivatives that generally inhibit
cell mitosis. Exemplary combretastatins that may be used in
conjunction with the invention include those based upon
combretastatin A, B and/or D and those described in U.S. Pat. Nos.
5,892,069, 5,504,074 and 5,661,143. Combretastatins A-1, A-2, A-3,
A-4, A-5, A-6, B-1, B-2, B-3 and B-4 are exemplary of the foregoing
types.
[0347] U.S. Pat. Nos. 5,569,786 and 5,409,953, are incorporated
herein by reference for purposes of describing the isolation,
structural characterization and synthesis of each of combretastatin
A-1, A2, A-3, B-1, B-2, B-3 and B-4 and formulations and methods of
using such combretastatins to treat neoplastic growth. Any one or
more of such combretastatins may be used in conjunction with the
present invention.
[0348] Combretastatin A-4, as described in U.S. Pat. Nos.
5,892,069, 5,504,074, 5,661,143 and 4,996,237, each specifically
incorporated herein by reference, may also be used herewith. U.S.
Pat. No. 5,561,122 is further incorporated herein by reference for
describing suitable combretastatin A-4 prodrugs, which are
contemplated for combined use with the present invention.
[0349] U.S. Pat. No. 4,940,726, specifically incorporated herein by
reference, particularly describes macrocyclic lactones denominated
combretastatin D-1 and `Combretastatin D-2`, each of which may be
used in combination with the compositions and methods of the
present invention. U.S. Pat. No. 5,430,062, specifically
incorporated herein by reference, concerns stilbene derivatives and
combretastatin analogues with anti-cancer activity that may be used
in combination with the present invention.
[0350] E5. Anti-Angiogenic Agents
[0351] Anti-angiogenic agents are useful for attachment to a
construct, receptorbody or betabody of the invention. Many
anti-cancer agents have an anti-angiogenic effect as part of their
mechanism of action. Any one or more of such agents described for
use in combination therapies, including those in Table E, may also
be conjugated to a construct, receptorbody or betabody of the
invention, as described herein. Certain other agents have been
discovered, designed or selected to have an anti-angiogenic effect
as a primary mechanism of action. Examples of such agents are
described below, any of which may also be used to prepare a
conjugate or used separately in combination therapy with the
invention.
[0352] Numerous tyrosine kinase inhibitors useful for the treatment
of angiogenesis, as manifest in various diseases states, are now
known. These include, for example, the
4-aminopyrrolo[2,3-d]pyrimidines of U.S. Pat. No. 5,639,757,
specifically incorporated herein by reference, which may also be
used in combination with the present invention. Further examples of
organic molecules capable of modulating tyrosine kinase signal
transduction via the VEGFR2 receptor are the quinazoline compounds
and compositions of U.S. Pat. No. 5,792,771, which is specifically
incorporated herein by reference for the purpose of describing
further combinations for use with the present invention in the
treatment of angiogenic diseases.
[0353] Compounds of other chemical classes have also been shown to
inhibit angiogenesis and may be used in combination with the
present invention. For example, steroids such as the angiostatic
4,9(11)-steroids and C21-oxygenated steroids, as described in U.S.
Pat. No. 5,972,922, specifically incorporated herein by reference,
may be employed in combined therapy. U.S. Pat. Nos. 5,712,291 and
5,593,990, each specifically incorporated herein by reference,
describe thalidomide and related compounds, precursors, analogs,
metabolites and hydrolysis products, which may also be used in
combination with the present invention to inhibit angiogenesis. The
compounds in U.S. Pat. Nos. 5,712,291 and 5,593,990 can be
administered orally. Further exemplary anti-angiogenic agents that
are useful in connection with combined therapy are listed in Table
B. Each of the agents listed therein are exemplary and by no means
limiting.
TABLE-US-00001 TABLE B 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
[0354] Certain preferred components for use in inhibiting
angiogenesis are angiostatin, endostatin, vasculostatin, canstatin
and maspin. The protein named "angiostatin" 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.
[0355] 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.
[0356] 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.
[0357] 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).
[0358] 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. 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.
[0359] Endostatin appears to be an even more powerful
anti-angiogenesis and anti-tumor agent although its biology is 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 thought to bind an unidentified
endothelial cell surface receptor that mediates its effect.
[0360] U.S. Pat. No. 5,854,205, to Folkman and O'Reilly,
specifically incorporated herein by reference, concerns endostatin
and its use as an inhibitor of endothelial cell proliferation and
angiogenesis. The endostatin protein corresponds to a C-terminal
fragment of collagen type XVIII, and the protein can be isolated
from a variety of sources. U.S. Pat. No. 5,854,205 also teaches
that endostatin can have an amino acid sequence of a fragment of
collagen type XVIII, a collagen type XV, or BOVMPE 1 pregastric
esterase. Combinations of endostatin with other anti-angiogenic
proteins, particularly angiostatin, are also described by U.S. Pat.
No. 5,854,205, such that the combined compositions are capable of
effectively regressing the mass of an angiogenesis-dependent
tumor.
[0361] 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 with this
invention.
[0362] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also
be used in 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.
[0363] 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.
[0364] Interferons and metalloproteinase inhibitors are two other
classes of naturally occurring angiogenic inhibitors that can be
delivered according to 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..
[0365] 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 the treatment
protocols of 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.
[0366] There are a number of pharmacological agents that inhibit
angiogenesis, any one or more of which may be used as part of 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.
[0367] 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.
[0368] 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.
[0369] 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.
[0370] Further specific angiogenesis inhibitors may be delivered to
tumors using the tumor targeting methods of the present invention.
These include, but are 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
polyamide 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.
[0371] Compositions comprising an antagonist of an
.alpha..sub.v.beta..sub.3 integrin may also be used to inhibit
angiogenesis as part of 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.
[0372] As angiopoietins are ligands for Tie2, other methods of
therapeutic intervention based upon altering signaling through the
Tie2 receptor can also be used in combination herewith. For
example, a soluble Tie2 receptor capable of blocking Tie2
activation (Lin et al., 1998a) can be employed. 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., 1998a).
[0373] The angiopoietins, in common with the members of the VEGF
family, are growth factors specific for vascular endothelium (Davis
and Yancopoulos, 1999; Holash et al., 1999; incorporated herein by
reference). The angiopoietins first described were a naturally
occurring receptor activator or agonist, angiopoietin-1 (Ang-1),
and a naturally occurring receptor antagonist, angiopoietin-2
(Ang-2), both of which act by means of the endothelial cell
tyrosine kinase receptor, Tie2.
[0374] 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 (like
Ang-2), whereas angiopoietin-4 appears to function as an agonist
(like Ang-1) (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).
[0375] Whereas VEGF is necessary for the early stages of vascular
development, angiopoietin-1 is generally required for the later
stages of vascularization. VEGF thus acts to promote endothelial
cell differentiation, proliferation and primitive vessel formation.
Angiopoietin-1 acts, via the Tie2 receptor, to promote maintenance
and stabilization of mature vessels. Angiopoietin-1 is thus a
maturation or stabilization factor, thought to convert immature
vessels to immature vessels by promoting interactions between
endothelial cells and surrounding support cells (Holash et al.,
1999).
[0376] E6. Apoptosis-Inducing Agents
[0377] The present invention may also be used to deliver agents
that induce apoptosis in any cell, including tumor cells, tumor
vascular endothelial cells and virally infected cells. Many
anti-cancer agents have, as part of their mechanism of action, an
apoptosis-inducing effect. Any one or more of such agents described
for use in combination therapies, including those in Table F, may
also be conjugated to a construct, receptorbody or betabody of the
invention, as described herein. Certain other agents have been
discovered, designed or selected to have an apoptosis-inducing
effect as a primary mechanism. Examples of such agents are
described below, any of which may also be used to prepare a
conjugate or used separately in combination therapy with the
invention.
[0378] 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, delivery 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.
[0379] 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.
[0380] Other oncogenes that inhibit apoptosis or programmed cell
death 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-xl, Mcl-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).
[0381] Other compositions that may be delivered by a construct,
receptorbody or betabody of the present invention 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.
[0382] 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 the antibodies of the present invention (as supported by U.S.
Pat. No. 5,587,459; incorporated herein by reference).
[0383] E7. Anti-Viral Agents
[0384] As PS and other anionic phospholipids become exposed on
virally infected cells, a construct, receptorbody or betabody of
the invention may also be linked to any one or more anti-viral
agents. Exemplary anti-viral agents for linking to a construct,
receptorbody or betabody include those in Table G. Such anti-viral
agents may also be used separately in the combination anti-viral
therapies of the invention.
[0385] In addition to so-called classic anti-viral agents, other
DNA/RNA inhibitors may also be attached to form an anti-viral
therapeutic. Exemplary anti-viral agents are listed in Table G, any
one or more of which may be attached to prepare an anti-viral
conjugate of the invention, or can be used separately in the
anti-viral combination therapies of the invention.
TABLE-US-00002 TABLE G Common Disease-Causing Viruses and
Anti-Viral Drugs Disease-Causing Exemplary Viruses Drug Categories
Anti-Viral Drugs Herpes virus Cidofovir, acyclovir, penciclovir
(famciclovir), gancyclovir (ganciclovir), deoxyguanosine,
foscarnet, idoxuridine, trifluorothymidine, vidarabine, sorivudine
Retroviruses Nucleoside reverse Zidovudine, didanosine,
transcriptase (RT) zalcitabine, lamivudine, inhibitors stavudine,
abacavir, multinucleoside resistance A, multinucleoside resistance
B Non-nucleoside RT Nevirapine, delavirdine, inhibitors efavirenz,
Adefovir Dipivoxil Protease Inhibitors Indinavir, ritonavir,
saquinavir, nelfinavir, amprenavir Cell cycle phase Hydroxyurea
(Hydrea .TM., specific Bristol Myers-Squibb) antineoplastic
Hepatitis B Deoxycytosine triphosphate, lamivudine triphosphate,
emticitabine triphosphate, adefovir diphosphate, penciclovir
triphosphate, lobucavir triphosphate Hepatitis C Interferon alpha,
ribavirin Influenza Amantadine, rimantadine, A and B zanamivir,
oseltamivir
[0386] Within the range of anti-viral agents and drugs, AZT and
cidofovir are currently preferred. Irrespective of the chosen
anti-viral drug, the anti-viral conjugate will bind to macrophages
in the lungs, to virally infected cells and may also bind to virus
particles. Depending on the linker or conjugation technology used,
the anti-viral drug may be released at the surface of the target
cell and then be taken up into the cell. Preferably, the conjugate
itself is taken up into the cell, such as a macrophage or virally
infected cell. Uptake can either occur naturally or can be
virus-mediated. Once inside the cell, as with an antibody
conjugate, hydrolysis of the linker releases the active anti-viral
agent.
[0387] Other linkages containing biologically labile bonds can be
used, such as, e.g., disulfide, acid labile, enzymatically
cleavable or hydrolysable. Accordingly, any biologically-releasable
or selectively hydrolyzable bond described for use in linking to
therapeutic agents can be used in connection with the anti-virals
of the present invention.
F. Biologically Functional Equivalents
[0388] Equivalents, or even improvements, of a construct,
receptorbody or betabody can now be made, generally using the
materials provided above as a starting point. Modifications and
changes may be made in the structure of such a construct,
receptorbody or betabody 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. These
considerations also apply to toxins, anti-angiogenic agents,
apoptosis-inducing agents, coagulants and the like.
[0389] 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 the antibodies
or therapeutic agents (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.
TABLE-US-00003 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
[0390] 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.
[0391] 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.
[0392] 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).
[0393] 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.
[0394] 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).
[0395] 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.
G. Conjugation
[0396] An antibody Fc region is operatively attached, associated
with or conjugated to at least a first phosphatidylserine binding
protein to provide a construct, receptorbody or betabody of the
invention. Such a construct, receptorbody or betabody may be
further conjugated or attached to, e.g., anti-cellular and
cytotoxic agents, coagulants and anti-viral agents.
[0397] Although covalent linkages are preferred, other means of
operative attachment may also be used. For example, any linked
construct may be generated using avidin:biotin bridges. In addition
to the knowledge available to those of ordinary skill in the art,
co-owned U.S. Pat. No. 6,093,399 is specifically incorporated
herein by reference for purposes of even further describing and
enabling the use of avidin:biotin in the operative attachment of
targeting agents to biological and therapeutic agents.
[0398] Any two or three agents may also be joined by a second
binding region, preferably an antibody or antigen binding region
thereof. This is exemplified by coaguligands wherein the targeting
agent is linked to the coagulant via a second binding region (U.S.
Pat. Nos. 6,093,399, 6,004,555, 5,877,289, and 6,036,955, each
specifically incorporated herein by reference), which have been
made and used successfully in the treatment of cancer.
[0399] Immunoconjugate technology is now generally known in the
art. However, certain advantages may be achieved through the
application of certain preferred technology, both in the
preparation and purification for subsequent clinical
administration. Additionally, while numerous types of
disulfide-bond containing linkers are known that can be
successfully employed in conjugation, 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"
may be preferred, due to their greater stability in vivo, thus
preventing release prior to binding at the site of action.
[0400] Each type of cross-linker, as well as how the cross-linking
is performed, will tend to vary the pharmacodynamics of the
resultant conjugate. One may desire 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 the cross-linking
reagent used and the structures that are cross-linked, will be of
some significance.
[0401] Depending on the specific agents to be conjugated, it may be
necessary or desirable to provide a peptide spacer operatively
attaching the agents. Certain peptide spacers are capable of
folding into a disulfide-bonded loop structure. Proteolytic
cleavage within the loop would then yield a heterodimeric
polypeptide wherein the agents are linked by only a single
disulfide bond. An example of such a toxin is a Ricin A-chain
toxin.
[0402] When certain other toxin compounds are utilized, a
non-cleavable peptide spacer may be provided to operatively attach
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.
[0403] A variety of chemotherapeutic and other pharmacological
agents have now been successfully conjugated 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
a-amanitin has been described. These attachment methods can be
adapted for use herewith.
[0404] Any covalent linkage should ideally be made at a site
distinct from the functional site(s). The compositions are thus
"linked" in any operative manner that allows each region to perform
its intended function without significant impairment, in
particular, so that the resultant construct still binds to the
intended PS and so that the attached agent(s) substantially
maintains biological activity and/or recovers biological activity
when released from the construct.
[0405] Attachment of biological agents via carbohydrate moieties on
Fc regions is also contemplated. Glycosylation, both O-linked and
N-linked, naturally occurs in antibodies. Recombinant antibodies
can be modified to recreate or create additional glycosylation
sites if desired, which is simply achieved by engineering the
appropriate amino acid sequences (such as Asn-X-Ser, Asn-X-Thr,
Ser, or Thr) into the primary sequence of the antibody.
[0406] G1. Biochemical Cross-Linkers
[0407] In additional to the general information provided above,
certain preferred biochemical cross-linkers may be used.
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 C.
TABLE-US-00004 TABLE C 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
[0408] 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 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.
[0409] 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, carbamate, or alkylating groups may be used for
binding or cross-linking.
[0410] 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.
[0411] 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 in conjugation. 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.
[0412] One of the most preferred cross-linking reagents 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 conjugates of this invention.
[0413] 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-hydroxysuccinimidyl group reacts
with primary amino groups and the phenylazide (upon photolysis)
reacts non-selectively with any amino acid residue.
[0414] 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.
[0415] Once conjugated, the conjugate is separated from
unconjugated 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.
[0416] G2. Biologically Releasable Linkers
[0417] Although it is preferred that any linking moiety will have
reasonable stability in blood, to prevent substantial release of
the attached therapeutic agent before targeting to the disease,
e.g., tumor site, in certain aspects, the use of
biologically-releasable bonds and/or selectively cleavable spacers
or linkers is contemplated. "Biologically-releasable bonds" and
"selectively cleavable spacers or linkers" still have reasonable
stability in the circulation.
[0418] A construct, receptorbody or betabody of the invention may
thus be linked to one or more therapeutic or second agents via a
biologically-releasable bond. "Biologically-releasable bonds" or
"selectively hydrolyzable bonds" include all linkages that are
releasable, cleavable or hydrolyzable only or preferentially under
certain conditions. This includes disulfide and trisulfide bonds
and acid-labile bonds, as described in U.S. Pat. Nos. 5,474,765 and
5,762,918, each specifically incorporated herein by reference.
[0419] The use of an acid sensitive spacer for attachment of a
therapeutic agent to a construct, receptorbody or betabody of the
invention is particularly contemplated. In such embodiments, the
therapeutic agents are released within the acidic compartments
inside a cell. It is contemplated that acid-sensitive release may
occur extracellularly, but still after specific targeting,
preferably to the tumor site or virally infected cell. Certain
currently preferred examples include antibodies linked to
colchicine or doxorubicin via an acid sensitive spacer. Attachment
via carbohydrate moieties of antibodies is also contemplated. In
such embodiments, the therapeutic agent are released within the
acidic compartments inside a cell.
[0420] A construct, receptorbody or betabody may also be
derivatized to introduce functional groups permitting the
attachment of the therapeutic agents through a biologically
releasable bond. A construct, receptorbody or betabody may thus be
derivatized to introduce side chains terminating in hydrazide,
hydrazine, primary amine or secondary amine groups. Therapeutic
agents may be conjugated through a Schiff's base linkage, a
hydrazone or acyl hydrazone bond or a hydrazide linker (U.S. Pat.
Nos. 5,474,765 and 5,762,918, each specifically incorporated herein
by reference).
[0421] Also as described in U.S. Pat. Nos. 5,474,765 and 5,762,918,
each specifically incorporated herein by reference, a construct,
receptorbody or betabody may be operatively attached to a
therapeutic agent through one or more biologically releasable bonds
that are enzyme-sensitive bonds, including peptide bonds, esters,
amides, phosphodiesters and glycosides.
[0422] Certain aspects of the invention concern the use of peptide
linkers that include at least a first cleavage site for a peptidase
and/or proteinase that is preferentially located within a disease
site, particularly within the tumor environment. The delivery of
the attached therapeutic agent thus results in cleavage
specifically within the disease site or tumor environment,
resulting in the specific release of the active therapeutic agent.
Certain peptide linkers will include a cleavage site that is
recognized by one or more enzymes involved in remodeling.
[0423] Peptide linkers that include a cleavage site for urokinase,
pro-urokinase, plasmin, plasminogen, TGF.beta., staphylokinase,
Thrombin, Factor IXa, Factor Xa or a metalloproteinase, such as an
interstitial collagenase, a gelatinase or a stromelysin, are
particularly preferred. U.S. Pat. Nos. 6,004,555, 5,877,289, and
6,093,399 are specifically incorporated herein by reference for the
purpose of further describing and enabling how to make and use
immunoconjugates comprising biologically-releasable bonds and
selectively-cleavable linkers and peptides. U.S. Pat. No. 5,877,289
is particularly incorporated herein by reference for the purpose of
further describing and enabling how to make and use
immunoconjugates that comprise a selectively-cleavable peptide
linker that is cleaved by urokinase, plasmin, Thrombin, Factor IXa,
Factor Xa or a metalloproteinase, such as an interstitial
collagenase, a gelatinase or a stromelysin, within a tumor
environment.
[0424] Currently preferred selectively-cleavable peptide linkers
are those that include a cleavage site for plasmin or a
metalloproteinase (also known as "matrix metalloproteases" or
"MMPs"), such as an interstitial collagenase, a gelatinase or a
stromelysin. Additional peptide linkers that may be advantageously
used in connection with the present invention include, for example,
plasmin cleavable sequences, such as those cleavable by
pro-urokinase, TGF.beta., plasminogen and staphylokinase; Factor Xa
cleavable sequences; MMP cleavable sequences, such as those
cleavable by gelatinase A; collagenase cleavable sequences, such as
those cleavable by calf skin collagen (.alpha.1(I) chain), calf
skin collagen (.alpha.2(I) chain), bovine cartilage collagen
(.alpha.1(II)chain), human liver collagen (.alpha.1(III) chain),
human .alpha..sub.2M, human PZP, rat .alpha..sub.1M, rat
.alpha..sub.2M, rat .alpha..sub.1I.sub.3(2J), rat
.alpha..sub.1I.sub.3(27J), and the human fibroblast collagenase
autolytic cleavage sites. In addition to the knowledge available to
those of ordinary skill in the art, the text and sequences from
Table B2 in co-owned U.S. Pat. Nos. 6,342,219, 6,524,583, 6,342,221
and 6,416,758, are specifically incorporated herein by reference
for purposes of even further describing and enabling the use of
such cleavable sequences.
[0425] G3. Fusion Proteins and Recombinant Expression
[0426] A construct, receptorbody or betabody can be prepared as a
fusion protein using molecular biological techniques. Any fusion
protein may be designed and made using any construct, receptorbody
or betabody and second therapeutic agents disclosed herein and
those known in the art. The fusion protein technology is readily
adaptable to prepare fusion proteins with other modifications, such
as linkage via a selectively cleavable peptide sequence, and such
like.
[0427] 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).
[0428] 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. 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".
[0429] To obtain a so-called "recombinant" version of a construct,
receptorbody or betabody, the vector 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
expression.
[0430] A construct, receptorbody or betabody of the invention 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
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.
[0431] 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.
[0432] A recombinantly produced construct, receptorbody or betabody
may be purified and formulated for human administration.
Alternatively, nucleic acids encoding a construct, receptorbody or
betabody 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.
[0433] 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.
[0434] 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.
[0435] 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.
[0436] 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 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.
H. Pharmaceutical Compositions
[0437] The therapeutic agents of the present invention will
generally be formulated as pharmaceutical compositions. The
pharmaceutical compositions will comprise a biologically or
therapeutically effective amount of at least a first therapeutic
agent of the invention, 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.
[0438] 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. Veterinary
uses are equally included within the invention and
"pharmaceutically acceptable" formulations include formulations for
both clinical and/or veterinary use.
[0439] 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.
[0440] "Unit dosage" formulations are those containing a dose or
sub-dose of the administered ingredient adapted for a particular
timed delivery. For example, exemplary "unit dosage" formulations
are those containing a daily dose or unit or daily sub-dose or a
weekly dose or unit or weekly sub-dose and the like.
[0441] H1. Injectable Formulations
[0442] The therapeutic agents of the invention will often be
formulated for parenteral administration, particularly for tumor
treatment, 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 an antibody,
immunoconjugate or peptide conjugate 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.
[0443] 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.
[0444] The therapeutic agents can be formulated into a sterile
aqueous composition in a neutral or salt form. Solutions of
therapeutic 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.
[0445] 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.
[0446] 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.
[0447] Prior to or upon formulation, the therapeutic agents 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 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.
[0448] 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 ingredient, plus any additional desired ingredient
from a previously sterile-filtered solution thereof.
[0449] Suitable pharmaceutical compositions in accordance with the
invention will generally include an amount of the therapeutic agent
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. For human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biological Standards. Upon formulation, the therapeutic agents will
be administered in a manner compatible with the dosage formulation
and in such amount as is therapeutically effective.
[0450] H2. Sustained Release Formulations
[0451] 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, topical formulations, liposomal forms
and the like. The type of form for administration will be matched
to the disease or disorder to be treated.
[0452] Pharmaceutical "slow release" capsules or "sustained
release" compositions or preparations 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 agents in accordance with the present invention. The
slow release formulations are typically implanted in the vicinity
of the disease site, for example, at the site of a tumor or viral
infection.
[0453] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing
therapeutic agents, which matrices are in the form of shaped
articles, e.g., films or microcapsule. Examples of
sustained-release matrices include polyesters; hydrogels, for
example, poly(2-hydroxyethyl-methacrylate) or poly(vinylalcohol);
polylactides, e.g., U.S. Pat. No. 3,773,919; copolymers of
L-glutamic acid and .gamma. ethyl-L-glutamate; non-degradable
ethylene-vinyl acetate; degradable lactic acid-glycolic acid
copolymers, such as the Lupron Depot.TM. (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide
acetate); and poly-D-(-)-3-hydroxybutyric acid.
[0454] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated antibodies remain in the body for a long time, they
may denature or aggregate as a result of exposure to moisture at
37.degree. C., thus reducing biological activity and/or changing
immunogenicity. Rational strategies are available for stabilization
depending on the mechanism involved. For example, if the
aggregation mechanism involves intermolecular S--S bond formation
through thio-disulfide interchange, stabilization is achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives,
developing specific polymer matrix compositions, and the like.
[0455] H3. Liposomes and Nanocapsules
[0456] In certain embodiments, liposomes and/or nanoparticles may
also be employed with the therapeutic agents. The formation and use
of liposomes is generally known to those of skill in the art, as
summarized below. The present invention provides particular
combinations of antibodies, liposomes and chemotherapeutic agents,
which are described below. In addition, a liposomal formulation may
be used as a routine component of any of the therapeutic agents of
the overall invention.
[0457] 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.
[0458] 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.
[0459] 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.
[0460] 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.
[0461] H4. Ophthalmic Formulations
[0462] Many diseases of the eye, particularly those having an
angiogenic component, can be treated by the present invention. For
example ocular neovascular disease, age-related macular
degeneration, diabetic retinopathy, retinopathy of prematurity,
corneal graft rejection, neovascular glaucoma, retrolental
fibroplasias and other diseases associated with corneal
neovascularization or retinal/choroidal neovascularization, as
described hereinbelow.
[0463] The therapeutic agents of the present invention may thus be
advantageously employed in the preparation of pharmaceutical
compositions suitable for use as ophthalmic solutions, including
those for intravitreal and/or intracameral administration. For the
treatment of any of the foregoing or other disorders the
therapeutic agents are administered to the eye or eyes of the
subject in need of treatment in the form of an ophthalmic
preparation prepared in accordance with conventional pharmaceutical
practice, see for example "Remington's Pharmaceutical Sciences"
15th Edition, pages 1488 to 1501 (Mack Publishing Co., Easton,
Pa.).
[0464] The ophthalmic preparations will contain a therapeutic agent
in a concentration from about 0.01 to about 1% by weight,
preferably from about 0.05 to about 0.5% in a pharmaceutically
acceptable solution, suspension or ointment. Some variation in
concentration will necessarily occur, depending on the particular
compound employed, the condition of the subject to be treated and
the like, and the person responsible for treatment will determine
the most suitable concentration for the individual subject. The
ophthalmic preparation will preferably be in the form of a sterile
aqueous solution containing, if desired, additional ingredients,
for example preservatives, buffers, tonicity agents, antioxidants
and stabilizers, nonionic wetting or clarifying agents,
viscosity-increasing agents and the like.
[0465] Suitable preservatives for use in such a solution include
benzalkonium chloride, benzethonium chloride, chlorobutanol,
thimerosal and the like. Suitable buffers include boric acid,
sodium and potassium bicarbonate, sodium and potassium borates,
sodium and potassium carbonate, sodium acetate, sodium biphosphate
and the like, in amounts sufficient to maintain the pH at between
about pH 6 and pH 8, and preferably, between about pH 7 and pH 7.5.
Suitable tonicity agents are dextran 40, dextran 70, dextrose,
glycerin, potassium chloride, propylene glycol, sodium chloride,
and the like, such that the sodium chloride equivalent of the
ophthalmic solution is in the range 0.9 plus or minus 0.2%.
[0466] Suitable antioxidants and stabilizers include sodium
bisulfite, sodium metabisulfite, sodium thiosulfite, thiourea and
the like. Suitable wetting and clarifying agents include
polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol.
Suitable viscosity-increasing agents include dextran 40, dextran
70, gelatin, glycerin, hydroxyethylcellulose,
hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum,
polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone,
carboxymethylcellulose and the like. The ophthalmic preparation
will be administered topically to the eye of the subject in need of
treatment by conventional methods, for example in the form of drops
or by bathing the eye in the ophthalmic solution.
[0467] H5. Topical Formulations
[0468] In the broadest sense, formulations for topical
administration include those for delivery via the mouth (buccal)
and through the skin. "Topical delivery systems" also include
transdermal patches containing the ingredient to be administered.
Delivery through the skin can further be achieved by iontophoresis
or electrotransport, if desired.
[0469] Formulations suitable for topical administration in the
mouth include lozenges comprising the ingredients in a flavored
basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
ingredient to be administered in a suitable liquid carrier.
[0470] Formulations suitable for topical administration to the skin
include ointments, creams, gels and pastes comprising the
ingredient to be administered in a pharmaceutical acceptable
carrier. The formulation of therapeutic agents for topical use,
such as in creams, ointments and gels, includes the preparation of
oleaginous or water-soluble ointment bases, will be well known to
those in the art in light of the present disclosure. For example,
these compositions may include vegetable oils, animal fats, and
more preferably, semisolid hydrocarbons obtained from petroleum.
Particular components used may include white ointment, yellow
ointment, cetyl esters wax, oleic acid, olive oil, paraffin,
petrolatum, white petrolatum, spermaceti, starch glycerite, white
wax, yellow wax, lanolin, anhydrous lanolin and glyceryl
monostearate. Various water-soluble ointment bases may also be
used, including glycol ethers and derivatives, polyethylene
glycols, polyoxyl 40 stearate and polysorbates.
[0471] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising, for example, cocoa
butter or a salicylate. Formulations suitable for vaginal
administration may be presented as pessaries, tampons, creams,
gels, pastes, foams or spray formulations containing in addition to
the active ingredient such carriers as are known in the art to be
appropriate.
[0472] H6. Nasal Formulations
[0473] Local delivery via the nasal and respiratory routes is
contemplated for treating various conditions, particularly for use
in the anti-viral treatment methods of the present invention. These
delivery routes are also suitable for delivering agents into the
systemic circulation. Formulations of active ingredients in
carriers suitable for nasal administration are therefore also
included within the invention, for example, nasal solutions,
sprays, aerosols and inhalants. Where the carrier is a solid, the
formulations include a coarse powder having a particle size, for
example, in the range of 20 to 500 microns, which is administered,
e.g., by rapid inhalation through the nasal passage from a
container of the powder held close up to the nose.
[0474] Suitable formulations wherein the carrier is a liquid are
useful in nasal administration. Nasal solutions are usually aqueous
solutions designed to be administered to the nasal passages in
drops or sprays and are prepared so that they are similar in many
respects to nasal secretions, so that normal ciliary action is
maintained. Thus, the aqueous nasal solutions usually are isotonic
and slightly buffered to maintain a pH of 5.5 to 6.5. In addition,
antimicrobial preservatives, similar to those used in ophthalmic
preparations, and appropriate drug stabilizers, if required, may be
included in the formulation. Various commercial nasal preparations
are known and include, for example, antibiotics and antihistamines
and are used for asthma prophylaxis.
[0475] Inhalations and inhalants are pharmaceutical preparations
designed for delivering a drug or compound into the respiratory
tree of a patient. A vapor or mist is administered and reaches the
affected area. This route can also be employed to deliver agents
into the systemic circulation. Inhalations may be administered by
the nasal or oral respiratory routes. The administration of
inhalation solutions is only effective if the droplets are
sufficiently fine and uniform in size so that the mist reaches the
bronchioles.
[0476] Another group of products, also known as inhalations, and
sometimes called insufflations, comprises finely powdered or liquid
drugs that are carried into the respiratory passages by the use of
special delivery systems, such as pharmaceutical aerosols, that
hold a solution or suspension of the drug in a liquefied gas
propellant. When released through a suitable valve and oral
adapter, a metered does of the inhalation is propelled into the
respiratory tract of the patient. Particle size is of major
importance in the administration of this type of preparation. It
has been reported that the optimum particle size for penetration
into the pulmonary cavity is of the order of 0.5 to 7 .mu.m. Fine
mists are produced by pressurized aerosols and hence their use in
considered advantageous.
I. Binding, Functional and Screening Assays
[0477] Although the present invention has significant utility in
animal and human treatment regimens, it also has many other
specific and credible uses, including practical uses in many in
vitro embodiments. Certain of these uses are related to the
specific binding properties of a construct, receptorbody or
betabody. In that each of the constructs of the invention include
at least one protein that binds to PS or an anionic phospholipid,
they may be used in a variety of binding embodiments, including
useful binding assays.
[0478] The presence of an Fc region or an attached agent, where
relevant, although providing advantageous properties, does not
negate the utility of the first region in any binding assay.
Suitably useful binding assays thus include those commonly employed
in the art, such as in immunoblots, Western blots, dot blots, RIAs,
ELISAs, immunohistochemistry, fluorescent activated cell sorting
(FACS), immunoprecipitation, affinity chromatography, and the like,
as further described herein.
[0479] Certain standard binding assays are those in which an
antigen is immobilized onto a solid support matrix, e.g.,
nitrocellulose, nylon or a combination thereof, such as in
immunoblots, Western blots, ELISAs and related assays. Other
important assays are those using cells, wherein the components of
the present invention can be used to assay for cells with PS or
anionic phospholipids at the cell surface. Such assays can be
applied in pre-clinical testing, e.g., regarding the design of
drugs, testing the mechanism of action and/or selecting therapeutic
agents for combined use.
[0480] Further in vitro assays are useful in the diagnosis of
diseases connected with aberrant cell activation and/or apoptosis,
wherein testing for the presence of PS or anionic phospholipids at
the cell surface would be particularly useful. The constructs of
the invention may thus be used in conjunction with both
fresh-frozen and formalin-fixed, paraffin-embedded tissue blocks in
immunohistochemistry; in fluorescent activated cell sorting, flow
cytometry or flow microfluorometry.
[0481] They constructs of the invention have further practical uses
in immunoprecipitation, antigen purification embodiments, such as
affinity chromatography, and in many other binding assays that will
be known to those of skill in the art given the information
presented herein.
[0482] Yet further practical uses of the present constructs are as
controls in functional assays, including many in vitro and ex vivo
assays and systems. As the binding and functional properties of a
construct, receptorbody or betabody of the invention are
particularly specific, as disclosed herein, such "control" uses are
actually extremely valuable. The assays that benefit from such a
practical application of the present invention include, for
example, assays concerning detection of PS or anionic phospholipids
at the cell surface.
[0483] These assays systems can also be developed into in vitro or
ex vivo drug screening assays, wherein the present provision of
biological materials with well defined properties is particularly
important. For example, in using the constructs of the present
invention as positive controls in the selection of small molecules
that have similar, equivalent or improved binding properties, e.g.,
in drug screening and development.
[0484] The binding assays and systems of the invention can also be
developed into in vitro or ex vivo drug screening assays, wherein
the present provision of biological materials with well defined
properties, is particularly important. For example, in using the
constructs of the present invention as positive controls in the
selection of small molecules that have similar, equivalent or
improved binding properties, e.g., in drug screening and
development.
J. Diagnostic and Therapeutic Kits
[0485] This invention also provides diagnostic and therapeutic kits
comprising at least a first construct, receptorbody or betabody of
the present invention, for use in treatment methods, combined
treatment methods and/or in imaging and treatment embodiments. Such
kits will generally contain, in at least a first suitable container
(or container means), a pharmaceutically acceptable formulation of
at least one construct, receptorbody or betabody of the invention.
The kits may include written or electronic instructions for use,
e.g. in pre-clinical, clinical and/or veterinary embodiments.
[0486] The kits may also contain other compositions,
pharmaceutically acceptable formulations and second biological and
therapeutic agents, including those for combined therapy and/or for
diagnostic and imaging. For example, such kits may contain any one
or more of a range of chemotherapeutic, radiotherapeutic or
anti-angiogenic agents, anti-tumor cell, anti-tumor vasculature or
anti-tumor stroma antibodies, immunotoxins or coaguligands,
anti-viral agents and/or diagnostic components or agents. Written
or electronic instructions for use in combined therapy and/or for
diagnosis and imaging may also be included.
[0487] The kits may have a single container that contains the first
construct, receptorbody or betabody, 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 premixed, either in a molar equivalent combination, or with
one component in excess of the other. Alternatively, the primary
therapeutic agent of the invention and the second biological or
therapeutic agent, such as a second anti-cancer or anti-viral
agent, kit may be maintained separately within distinct containers
of the kit prior to administration to a patient.
[0488] Diagnostic components will most often be maintained in at
least a second container, distinct from the other or first
container that comprises the one or more therapeutic agents. The
diagnostic kits may include labeled antibodies or peptides that
bind to PS, or any other agent suitable for diagnosing the disease
to be treated. The kits may include diagnostic agents for use in
vitro, for use in vivo, or both such agent. The kits may include
written or electronic instructions for use, e.g. in pre-clinical,
clinical and/or veterinary diagnostic embodiments.
[0489] For immunodetection in vitro, a construct, receptorbody or
betabody may be bound to a solid support, such as a well of a
microtitre plate. The immunodetection kits preferably comprise at
least a first immunodetection reagent. The immunodetection reagents
of the kit may take any one of a variety of forms, including those
detectable labels that are associated with or linked to the given
antibody, such as used in vivo. Detectable labels that are
associated with or attached to a secondary binding ligand are also
contemplated. Exemplary secondary ligands are those secondary
antibodies that have binding affinity for the first antibody.
[0490] Further suitable immunodetection reagents for use in the
present kits include the two-component reagent that comprises a
secondary antibody that has binding affinity for the first
antibody, along with a third antibody that has binding affinity for
the second antibody, the third antibody being linked to a
detectable label. A number of exemplary labels are known in the art
and all such labels may be employed in connection with the present
invention. These kits may contain antibody-label conjugates either
in fully conjugated form, in the form of intermediates, or as
separate moieties to be conjugated by the user of the kit. The
imaging kits will preferably comprise a targeting agent or antibody
that is already attached to an in vivo detectable label. However,
the label and attachment means could be separately supplied.
[0491] Either form of diagnostic kit may further comprise control
agents, such as suitably aliquoted biological compositions, whether
labeled or unlabeled, as may be used to prepare a standard curve
for a detection assay. The components of the kits may be packaged
either in aqueous media or in lyophilized form.
[0492] 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. The solvent may also be provided in another
container within the kit.
[0493] The containers of the therapeutic and diagnostic kits will
generally include at least one vial, test tube, flask, bottle,
syringe or other container or container means, into which the
therapeutic and any other desired agent are placed and, preferably,
suitably aliquoted. As at least two separate components are
preferred, the kits will preferably include at least two such
containers. The kits may also comprise a third or fourth container
for containing a sterile, pharmaceutically acceptable buffer or
other diluent.
[0494] The kits may also contain a means by which to administer the
therapeutic agents 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 formulations 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.
K. Immunodetection and Imaging
[0495] The present invention further provides in vitro and in vivo
diagnostic and imaging methods. Such methods are applicable for use
in generating diagnostic, prognostic and/or imaging information,
e.g., related to angiogenic diseases and viral infections, and
preferably related to tumor treatment and imaging methods. The
methods of the invention include in vitro diagnostic tests, e.g.,
wherein the samples can be obtained non-invasively and preferably
tested in high throughput assays and/or where the clinical
diagnosis in ambiguous and confirmation is desired. In the field of
in vivo diagnostics and imaging, a construct, receptorbody or
betabody of the invention is linked to one or more detectable
agents and used to form an image of an angiogenic site or tumor,
optionally as a first step prior to treatment.
[0496] K1. Immunodetection Methods and Kits
[0497] The invention thus concerns immunodetection methods for
binding, purifying, quantifying or otherwise generally detecting PS
and anionic phospholipids, e.g., for use in diagnosing activated
and apoptotic cells and associated diseases. A construct,
receptorbody or betabody of the present invention may be employed
to detect PS and anionic phospholipids in vivo (see below), in
isolated issue samples, biopsies or swabs and/or in homogenized
tissue samples. Such immunodetection methods have evident
diagnostic utility, but also have applications to non-clinical
samples, such as in the titering of antigen samples, and the
like.
[0498] The steps of various useful immunodetection methods have
been described in the scientific literature, such as, e.g.,
Nakamura et al., 1987, specifically incorporated herein by
reference. In general, the immunobinding methods include obtaining
a sample suspected of containing PS or anionic phospholipids,
preferably cells suspected of having PS at the cell surface, and
contacting the sample with a construct, receptorbody or betabody of
the invention, under conditions effective to allow the formation of
immune complexes. Any immune complexes formed during the binding
process are then detected and preferably quantified.
[0499] The sample analyzed may be a cell sample, such as cells
exposed to certain test conditions in the laboratory. The sample
may also be a biological sample from an animal or patient, e.g.,
one suspected of having a disease associated with activation or
apoptosis of one or more cell types. Such a sample may be a tissue
section or specimen, a biopsy, a swab or smear test sample, a
homogenized tissue extract or separated or purified forms of
such.
[0500] Contacting the chosen biological sample with a construct,
receptorbody or betabody under conditions effective and for a
period of time sufficient to allow the formation of immune
complexes (primary immune complexes) is generally a matter of
simply adding a construct, receptorbody or betabody to the sample
and incubating the mixture for a period of time long enough for the
construct, receptorbody or betabody to form immune complexes with,
i.e., to bind to, any PS or anionic phospholipids present. After
this time, the sample composition, such as a tissue section or
ELISA plate, will generally be washed to remove any
non-specifically bound species, allowing only those specifically
bound within the primary immune complexes to be detected.
[0501] The detection of immunocomplex formation is well known in
the art and may be achieved through the application of numerous
approaches. These methods are generally based upon the detection of
a label or marker, such as any radioactive, fluorescent, biological
or enzymatic tags or labels known in the art. U.S. patents
concerning the use of such labels include U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and
4,366,241, each incorporated herein by reference. The use of
enzymes that generate a colored product upon contact with a
chromogenic substrate are generally preferred. Secondary binding
ligands, such as a second antibody or a biotin/avidin ligand
binding arrangement, may also be used, as is known in the art.
[0502] A construct, receptorbody or betabody employed in the
detection may themselves be linked to a detectable label, wherein
one would then simply detect this label, thereby allowing the
amount of the primary immune complexes in the composition to be
determined.
[0503] Preferably, the primary immune complexes are detected by
means of a second binding ligand that has binding affinity for a
construct, receptorbody or betabody of the invention. In such
cases, the second binding ligand may be linked to a detectable
label. The second binding ligand is itself often an antibody, and
may thus be termed a "secondary" antibody. The primary immune
complexes are contacted with the labeled, secondary binding ligand,
or antibody, under conditions effective and for a period of time
sufficient to allow the formation of secondary immune complexes.
The secondary immune complexes are then generally washed to remove
any non-specifically bound labeled secondary antibodies or ligands,
and the remaining label in the secondary immune complexes is then
detected.
[0504] Further methods include the detection of primary immune
complexes by a two step approach. A second binding ligand, such as
an antibody, that has binding affinity for the first antibody is
used to form secondary immune complexes, as described above. After
washing, the secondary immune complexes are contacted with a third
binding ligand or antibody that has binding affinity for the second
antibody, again under conditions effective and for a period of time
sufficient to allow the formation of immune complexes (tertiary
immune complexes). The third ligand or antibody is linked to a
detectable label, allowing detection of the tertiary immune
complexes thus formed. This system may provide for signal
amplification if desired.
[0505] Clinical diagnosis or monitoring may be applied to patients
with a variety of diseases, particularly those associated with
increased PS or anionic phospholipid exposure at the cell surface.
The detection of PS or anionic phospholipid, or an increase in the
levels of PS or anionic phospholipid, in comparison to the levels
in a corresponding biological sample from a normal subject, is
indicative of a patient with such a disease.
[0506] However, as is known to those of skill in the art, such a
clinical diagnosis would not likely be made on the basis of this
method in isolation. Those of skill in the art are very familiar
with differentiating between significant expression of a biomarker,
which represents a positive identification, and low level or
background expression of a biomarker. Indeed, background expression
levels are often used to form a "cut-off" above which increased
staining will be scored as significant or positive.
[0507] K2. In Vivo Imaging
[0508] The present invention provides a variety of in vivo
diagnostic and imaging embodiments. Certain aspects of the
invention concern new and surprisingly effective compositions for
in vivo diagnosis and imaging. For example, a construct,
receptorbody or betabody of the invention is linked to an in vivo
detectable agent to form an immunodiagnostic conjugate of the
invention. The resultant immunodiagnostics may now be used in any
previously described diagnostic or imaging embodiment connected
with the detection of PS or an anionic phospholipid.
[0509] In this regard, immunodiagnostics comprising a construct,
receptorbody or betabody of the invention, may be used 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. Such compositions of the invention
may also be used in imaging activated platelets, e.g., in
conditions such as abscesses, restenosis, inflammation of joints
and in hemostatic disorders, such as arterial, coronary, venous and
cerebral thrombosis and the like. The immunodiagnostic compositions
of the invention may also be used in detecting apoptotic cells, as
may be used in the diagnosis and imaging of a variety of diseases
in which increased or inappropriate apoptosis occurs.
[0510] The in vivo imaging compositions and methods of the
invention can be used in imaging per se, or in pre-imaging a site
in the body to form a reliable image prior to treatment.
Preferably, the imaging is tumor imaging. These compositions and
methods can also be applied to imaging and diagnosis of other
diseases or conditions associated with PS and anionic
phospholipids, such those involving cell activation and/or
apoptosis, including angiogenic diseases, atherosclerosis, viral
infections, and other such conditions in which an internal image is
desired for diagnostic or prognostic purposes or to design
treatment.
[0511] In these embodiments, a construct, receptorbody or betabody
of the invention is operatively attached, linked 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. In conjugates for in vivo diagnostic protocols or
"imaging methods", the labels can be detected using non-invasive
methods.
[0512] Many appropriate imaging agents are known in the art, as are
methods for their attachment to 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.
[0513] 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.
[0514] 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.
[0515] 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.
[0516] A radioactively labeled construct, receptorbody or betabody
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).
[0517] A construct, receptorbody or betabody 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. A construct, receptorbody or betabody
according to the invention may be labeled with technetium-.sup.99
by a 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. Direct
labeling techniques are also suitable, 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.
[0518] Any of the foregoing type of detectably labeled binding
ligands may be used in the imaging aspects of the invention, either
for imaging alone or to form an image of a disease site or tumor
prior to treatment. Either way, the methods generally comprise
administering to an animal or patient a diagnostically effective
amount of a construct, receptorbody or betabody that is conjugated
to a marker that is detectable by non-invasive methods. The binding
ligand-marker conjugate is allowed sufficient time to localize and
bind to cells expressing PS or anionic phospholipids in the disease
site, such as the tumor or tumor vasculature. The patient is then
exposed to a detection device to identify the detectable marker,
thus forming an image of the disease site or tumor.
[0519] 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 detectably labeled
constructs into the blood of an individual, and means for
determining the distribution of the detectably labeled agent
extracorporally, e.g., using a gamma scintillation camera or by
magnetic resonance measurement.
[0520] 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 antibody- or binding ligand-conjugate per
patient is contemplated to be useful.
[0521] K3. Surrogate Marker for Cancer Therapy
[0522] In regard to the in vivo diagnostic and imaging, the present
invention further provides compositions and methods for use as a
surrogate marker for cancer therapy. Such embodiments concern the
use of a construct, receptorbody or betabody of the invention
linked to an in vivo detectable agent.
[0523] Many anti-cancer therapies in current use induce apoptosis
and necrosis. Anionic phospholipids, particularly PS, are markers
of pre-apoptotic and apoptotic cells. Therefore, imaging with a
suitable construct, receptorbody or betabody can be used to
identify pre-apoptotic and apoptotic cells and thus provide
information regarding the progress of the therapy. This is what is
meant by a "surrogate marker for cancer therapy", as used
herein.
[0524] The use of a construct, receptorbody or betabody of the
invention provides particular advantages as a surrogate marker for
cancer therapy. For example, the ability to identify pre-apoptotic
cells is a particular advantage. The specificity will also provide
more meaningful imaging data for the physician. Also, the safety
profile is impressive and provides advantages over annexin, for
example, as annexin suffers from drawbacks associated with
coagulation.
[0525] Accordingly, any of the in vivo diagnostic and imaging
methods described above may be adapted for prognostic use as a
surrogate marker for cancer therapy simply by use in a patient
undergoing cancer therapy.
L. Tumor Treatment
[0526] Important aspects of the present invention concern the
treatment of malignancies, tumors and vascularized tumors. This
includes tumors in which angiogenesis is more or less important and
tumors having prothrombotic blood vessels. The treatment of benign
tumors is included in the invention, such as acoustic neuroma,
neurofibroma, trachoma, pyogenic granulomas and BPH. The treatment
of blood-born tumors, such as leukemias, and various acute or
chronic neoplastic diseases of the bone marrow is also
encompassed.
[0527] The present invention is broadly applicable to the treatment
of any malignant tumor, whether having a vascular component or not.
Tumors for treatment include 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,
glioblastomas, neuroblastomas, and the like.
[0528] The present invention is contemplated for use in the
treatment of any patient that presents with a solid tumor. In
general, the invention can be used to treat tumors of all sizes,
including those about 0.3-0.5 cm and upwards, tumors of greater
than 0.5 cm in size and patients presenting with tumors of between
about 1.0 and about 2.0 cm in size, although tumors up to and
including the largest tumors found in humans may also be
treated.
[0529] 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
these aspects 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 a construct, receptorbody or betabody of the
invention is 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
treatments.
[0530] The guidance provided herein regarding the 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 cancer. A
further consideration is the fact that the assault on the tumor
provided by the antibody therapy 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.
[0531] 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 tertiary therapeutic agents, particularly
chemotherapeutics and anti-tumor cell immunotoxins. As the present
invention includes within its modes of action the targeting and
destruction of tumor vasculature, and as the vasculature is
substantially or entirely the same in all solid tumors, it will be
understood that the present 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.
The data presented herein is compelling as it shows impressive
results in a wide range of different tumor models.
[0532] Therapeutically effective doses are readily determinable
using data from an animal model, as shown in the studies detailed
herein, and from clinical data using a range of therapeutic agents.
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 agents that
give beneficial anti-tumor effects with minimal toxicity.
[0533] In terms of tumor therapy, bearing in mind the attendant
safety benefits associated with the overall invention, one may
refer to the scientific and patent literature on the success of
using other anti-vascular therapies. By way of example, U.S. Pat.
Nos. 5,855,866; 5,877,289; 5,965,132; 6,051,230; 6,004,555;
5,776,427; 6,004,554; 6,036,955; and 6,093,399 are incorporated
herein by reference for the purpose of further describing the use
of such agents as may be applied to those of the present invention.
U.S. Pat. Nos. 6,312,694, 6,783,760, 6,818,213 and 6,406,693 are
further specifically incorporated herein by reference for guidance
on dosing and treatment using unconjugated antibodies to PS and
related immunoconjugates.
[0534] 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 agents, doses or
combinations.
[0535] Any dose, combined method or medicament that results in any
consistently detectable anti-tumor effect, including detectable
tumor vasculature regression, thrombosis and/or destruction and
tumor necrosis, will still define a useful invention. Regressive,
thrombotic, destructive and necrotic effects should preferably 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.
[0536] It will also be understood that even in such circumstances
where the anti-tumor effects of the 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. 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.
[0537] In designing appropriate doses of a construct, receptorbody
or betabody 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.
[0538] For example, in taking the successful doses of therapeutics
used in the mouse studies, and applying standard calculations based
upon mass and surface area, effective doses of agents 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.
[0539] Accordingly, using this information, the inventors
contemplate that useful low doses 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 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 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. 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 per patient
will be preferred. However, any particular range using any of the
foregoing recited exemplary doses or any value intermediate between
the particular stated ranges is contemplated.
[0540] Notwithstanding the 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. It will thus be
understood that lower doses may be more appropriate in combination
with certain agents, and that high doses can still be tolerated,
particularly given the enhanced safety of the present constructs.
The use of human constructs and human effectors renders the present
invention even safer for clinical use, further reducing the chances
of significant toxicity or side effects in healthy tissues.
[0541] 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 antibody, 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.
[0542] 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 the tumor cells
themselves begin to die within about 24 to 72 hours. Widespread
tumor necrosis is generally observed in the next about 48-96 hours,
up to and including greater than 60% necrosis being observed.
[0543] 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.
[0544] Patients chosen for the first 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.
[0545] Certain advantages will be found in the use of an indwelling
central venous catheter with a triple lumen port. The therapeutics
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.
[0546] The 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 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.
[0547] 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 construct, 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 therapeutics to be
evaluated.
[0548] 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.
[0549] 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.
[0550] 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.
M. Combination Tumor Therapies
[0551] The 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 treatment of the invention, its
combination with the present invention is contemplated.
[0552] Combination therapy for non malignant diseases is also
contemplated. A particular example of such is benign prostatic
hyperplasia (BPH), which may be treated in combination other
treatments currently practiced in the art. For example, targeting
of immunotoxins to markers localized within BPH, such as PSA.
[0553] In connection solid tumor treatment, the present invention
may be used in combination with classical approaches, such as
surgery, chemotherapy, radiotherapy, cytokine therapy,
anti-angiogenesis and the like. The invention therefore provides
combined therapies in which a construct, receptorbody or betabody
is used simultaneously with, before, or after surgery or radiation
treatment; or is administered to patients with, before, or after
conventional chemotherapeutic or radiotherapeutic agents,
cytokines, anti-angiogenic agents, apoptosis-inducing agents,
targeted immunotoxins or coaguligands or such like. Many examples
of suitable therapeutic agents have been described above in
connection with the conjugate aspects of the present invention. Any
of the agents initially described for use as one part of a
therapeutic conjugate may also be used separately, in the
combination therapies of the present invention.
[0554] 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.
[0555] The general use of combinations of substances in cancer
treatment is well known. 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.
[0556] When one or more agents are used in combination with a
construct, receptorbody or betabody of the present invention, 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.
[0557] M1. Selection of Second Anti-Cancer Agents
[0558] The "primary therapeutic agent" of the present invention, as
used herein, is a construct, receptorbody or betabody or conjugate
thereof. The "secondary therapeutic agents", as used herein, are
second, distinct therapeutic agents or anti-cancer agents, i.e.,
therapeutic agents or anti-cancer agents "other than" the primary
therapeutic agent. Any secondary therapeutic agent may be used in
the combination therapies of the present invention. Also, secondary
therapeutic agents or "second anti-cancer agents" may be selected
with a view to achieving additive, greater than additive and
potentially synergistic effects, according to the following
guidance.
[0559] To practice combined anti-tumor therapy, one would simply
administer to an animal or patient a construct, receptorbody or
betabody of the present invention in combination with another,
i.e., a second, distinct anti-cancer agent in a manner effective to
result in their combined anti-tumor actions within the animal or
patient. The agents would therefore be provided in amounts
effective and for periods of time effective to result in their
combined presence within the tumor or tumor vasculature and their
combined actions in the tumor environment. To achieve this goal,
the primary therapeutics of the present invention and the second,
distinct anti-cancer agents may be administered to the animal
substantially simultaneously, either in a single composition, or as
two distinct compositions using different administration
routes.
[0560] Alternatively, a construct, receptorbody or betabody of the
present invention may precede, or follow, the second, distinct
anti-cancer agent by, e.g., intervals ranging from minutes to
weeks. In certain embodiments where the primary therapeutics of the
present invention and the second, distinct anti-cancer agents 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 each agent 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.
[0561] The secondary therapeutic agents for separately timed
combination therapies may be selected based upon certain criteria,
including those discussed below. However, a preference for
selecting one of more second, distinct anti-cancer agents for prior
or subsequent administration does not preclude their use in
substantially simultaneous administration if desired.
[0562] Second, distinct anti-cancer agents selected for
administration "prior to" the primary therapeutic agents of the
present invention, and designed to achieve increased and
potentially synergistic effects, include agents that induce the
expression of aminophospholipids or anionic phospholipids within
the tumor vasculature. For example, agents that stimulate localized
calcium production, activate membrane transporters that move PS and
other phospholipids to the outer surface of the plasma membrane,
injure the tumor endothelium, cause preapoptotic changes and/or
induce apoptosis in the tumor endothelium will generally result in
increased aminophospholipid and anionic phospholipid expression.
Examples of such agents are docetaxel and paclitaxol. The
aminophospholipids and anionic phospholipids can then be targeted
using a construct, receptorbody or betabody of the invention, thus
amplifying the overall therapeutic effect, and also giving
increased attack via host effectors (complement, ADCC,
antibody-mediated phagocytosis, CDC).
[0563] Drugs that have selectivity for angiogenic, remodeling or
activated endothelial cells, such as are present in tumor blood
vessels, but not in normal resting blood vessels, can also be used
to selectively causes exposure of PS and other phospholipids on the
surface of tumor endothelial cells. Examples of such agents are
combretastatins and docetaxel. This again would lead to increased
antibody binding and enhanced initiation of host effector
mechanisms.
[0564] Second, distinct anti-cancer agents selected for
administration "subsequent to" the primary therapeutic agents of
the present invention, and designed to achieve increased and
potentially synergistic effects, include agents that benefit from
the effects of the primary therapeutic agent. The construct,
receptorbody or betabody of the present invention will cause tumor
destruction. Accordingly, effective second, distinct anti-cancer
agents for subsequent administration include anti-angiogenic
agents, which inhibit metastasis; agents targeting necrotic tumor
cells, such as antibodies specific for intracellular antigens that
become accessible from malignant cells in vivo (U.S. Pat. Nos.
5,019,368, 4,861,581 and 5,882,626, each specifically incorporated
herein by reference); and chemotherapeutic agents and anti-tumor
cell immunoconjugates, which attack any tumor cells that may
survive at the periphery.
[0565] In some situations, it may 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 primary therapeutic agent of the present invention, and
another treatment was intended to prevent micrometastasis or tumor
re-growth, such as the administration of an anti-angiogenic agent.
Anti-angiogenics should be administered at a careful time after
surgery, however, to allow effective wound healing. Anti-angiogenic
agents may then be administered for the lifetime of the
patient.
[0566] It is also envisioned that more than one administration of
either the primary therapeutic agent or the second, distinct
anti-cancer agent will be utilized. The primary therapeutic agent
and the second, distinct anti-cancer agent may be administered
interchangeably, on alternate days or weeks; or a sequence of one
agent treatment may be given, followed by a sequence of the other
treatment. 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.
[0567] Whether administered substantially simultaneously or
sequentially, the construct, receptorbody or betabody and
therapeutics of the present invention may be administered in
combination with one or more chemotherapeutic agents or drugs.
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 primary
therapeutic agents of the invention.
[0568] Most cancer chemotherapeutic drugs are selective for
dividing, oxygenated cells. These have advantages in combined
therapy as the chemotherapeutic drug acts on different targets from
the primary therapeutic agents of the invention, leading to a more
complete anti-vascular or anti-tumor effect. For example,
chemotherapeutic drugs are selectively active against the rapidly
dividing, oxygenated tumor cells in the tumor periphery, whereas
the agents of the invention act primarily on vessels or tumor cells
in the `stressed` tumor core, where activating reactive oxygen
species are abundant. Anti-angiogenic drugs that are selective for
well-oxygenated, angiogenic vessels in the tumor periphery would
also be effective in combination, as the agents of the invention
act on the relatively hypoxic, quiescent vessels in the tumor
core.
[0569] By inducing the formation of thrombi in tumor vessels, the
primary therapeutic agents of the present invention can also
enhance the action of the chemotherapeutic drugs 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.
[0570] Further drugs for combined use in the present invention are
those that act on cells that are "sensitized" to the drug by the
action of the primary therapeutic agent, such that reduced doses of
the second drug are needed to achieve its anti-tumor effect. For
example, this could occur where a major component of the second
drug's action is exerted on tumor vessels and the agents of the
invention sensitize the cells to the drug. The same is true where
the primary therapeutic agent of the invention sensitizes tumor
cells to a second drug, either directly or through stimulation of
cytokine release.
[0571] Other suitable second anti-cancer agents for combination
therapy are those that further enhance the activity of host
effector cells, e.g., by selectively inhibiting the activity of
immunosuppressive components of the immune system. Such agents
enable the primary therapeutic agents of the invention, which
stimulate attack by effector cells as part of their mechanism, to
work more aggressively. An example of such an agent is
docetaxel.
[0572] Although an understanding of the precise mechanism(s) of
action of the primary therapeutic agents is not necessary to
practice the treatment of the invention, data and reasoned
deductions concerning such mechanisms can be used to select
particular second anti-cancer agents for combined use in the
present invention. The effectiveness of the chosen combination
therapy, in turn, supports the original data and proposed
mechanisms of action, and also leads to preferred categories of
second anti-cancer agents for practicing combination therapy.
[0573] Drugs that induce apoptosis are preferred for use in the
combination therapies. Docetaxel, for example, induces apoptosis
and therefore PS exposure by binding to microtubules and disrupting
cell mitosis (Hotchkiss et al., 2002). Treatment of endothelial
cells, which line tumor blood vessels, and tumor cells with
docetaxel at subclinical concentrations is herein shown to induce
PS expression at the cell surface, as demonstrated by strong
binding of the 3G4 antibody in vitro.
[0574] The present inventors have also determined that the
anti-tumor effects of the invention include Fc domain-mediated
augmentation of immune effector functions, as shown by increased
antibody-mediated phagocytosis. Therefore, other Fc domain-mediated
functions will occur, such as ADCC, CDC, stimulation of cytokine
production, and such mechanisms in combination. This is also
relevant to docetaxel, as other studies have shown that the
treatment of breast cancer patients with docetaxel leads to
increases in serum IFN-.gamma., IL-2, IL-6 and GM-CSF cytokine
levels, augmenting the anti-tumor immune responses in these
patients by enhancing the activity of natural killer (NK) and
lymphokine activated killer (LAK) cells (Tsavaris et al.,
2002).
[0575] Therefore, the inventors reasoned that docetaxel will both
induce PS expression and binding of the administered construct,
receptorbody or betabody, and also enhances the activities of
immune effectors, which mediate anti-tumor effects. Based upon the
foregoing considerations, the inventors have shown that combination
of the 3G4 antibody with docetaxel was significantly superior to
either docetaxel or 3G4 alone in mice bearing orthotopic MDA-MB-435
human breast cancer xenografts (Example XX).
[0576] Accordingly, docetaxel and other chemotherapeutic agents
that induce apoptosis are preferred agents for use in the
combination treatments of the present invention. Combinations of a
construct, receptorbody or betabody with chemotherapeutics or drugs
that induce apoptosis, such as docetaxel, should synergistically
attack tumor vasculature endothelial cell and tumor cell
compartments, leading to not only significantly enhanced treatment
efficacy but also lower toxicity. These combinations are
contemplated for use in breast cancer treatment, particularly the
combination of metronomic chemotherapy using docetaxel with an
antibody of the present invention.
[0577] M2. Endotoxin
[0578] Endotoxin and detoxified endotoxin derivatives may be used
in the combination treatment, preferably at low doses (PCT
Publication No. WO 03/028840, specifically incorporated herein by
reference). Various detoxified endotoxins are available, which are
preferred for use in animals and particularly for use in humans.
Detoxified and refined endotoxins, and combinations thereof, are
described in U.S. Pat. Nos. 4,866,034; 4,435,386; 4,505,899;
4,436,727; 4,436,728; 4,505,900, each specifically incorporated
herein by reference.
[0579] The non-toxic derivative monophosphoryl lipid A (MPL) is one
example of a detoxified endotoxin that may be used in the present
invention. MPL is known to be safe for humans; clinical trials
using MPL as an adjuvant have shown 100 .mu.g/m.sup.2 to be safe
for human use, even on an outpatient basis.
[0580] M3. Cytokines
[0581] Cytokine therapy has proven to be an effective partner for
combined therapeutic regimens. Various cytokines may be employed in
the combined approaches of the present invention. 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).
[0582] M4. TNF.alpha. and Inducers of TNF.alpha.
[0583] TNF.alpha. and inducers of TNF.alpha. may also be used in
combination with the present invention. TNF.alpha. increases
vascular permeability, and is therefore useful in facilitating the
penetration of anti-cancer agents into the tumor. Although
localization is by no means a problem when targeting PS and anionic
phospholipids, as in the present invention, the combined use of
TNF.alpha. can facilitate access of other chemotherapeutics and
immunoconjugates to the tumor, and even increase binding of the
antibodies of the invention to far distant tumor cells.
[0584] Low levels of endotoxin, Rac1 antagonists, such as an
attenuated or engineered adenovirus, DMXAA (and FAA), CM101 and
thalidomide may also be used. Rac1 antagonists may be used in the
combined treatment of the present invention, as about 5000 DNA
particles per cell cause TNF upregulation independent of CD14
(Sanlioglu et al., 2001). CM101, thalidomide and DMXAA can also be
used in combination herewith, at standard or reduced doses.
[0585] M5. Chemotherapeutics
[0586] Irrespective of the underlying mechanism(s), a variety of
chemotherapeutic agents may be used in the combined treatment
methods disclosed herein. Chemotherapeutic agents contemplated for
combined use include, e.g., tamoxifen, taxol, vinblastine,
etoposide (VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin,
actinomycin-D, mitomycin C, combretastatin(s), more particularly
docetaxel (taxotere), cisplatin (CDDP), cyclophosphamide,
doxorubicin, methotrexate, paclitaxel and vincristine, and
derivatives and prodrugs thereof.
[0587] As will be understood by those of ordinary skill in the art,
appropriate doses of chemotherapeutic agents include those already
employed in clinical therapies wherein the chemotherapeutics are
administered alone or in combination with other chemotherapeutics.
However, lower doses are now possible due to the advantages
provided by the present invention. 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.
[0588] Further useful agents include compounds that interfere with
DNA replication, mitosis, chromosomal segregation and/or tubulin
activity. Such chemotherapeutic compounds include adriamycin, also
known as doxorubicin, etoposide, verapamil, podophyllotoxin(s),
combretastatin(s) 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.
[0589] 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.
[0590] Exemplary chemotherapeutic agents that are useful in
connection with combined therapy are listed in Table D. 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-00005 TABLE D 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
[0591] M6. Anti-Angiogenics
[0592] 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 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. New evidence, however, shows that angiogenesis is
important in certain normal situations, such as in adrenal tissue,
prostate and ovary. The therapeutic agents of the present
invention, in which anti-angiogenesis is not the only mode of
action, thus have advantages over prominent anti-angiogenic
therapies, such as antibody A4.6.1 (Brem, 1998; Baca et al., 1997;
Presta et al., 1997), in that desirable or "physiological"
angiogenesis will not be inhibited when using the present
invention.
[0593] Uncontrolled (persistent and/or unregulated) angiogenesis is
related to various disease states, and occurs during tumor
development and metastasis. 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.
[0594] Despite the new evidence that angiogenesis is required in
some normal tissues, anti-angiogenic therapies are still important
in the treatment of tumors and other diseases. Anti-angiogenic
therapies are therefore intended for use in the combination
treatments of the present invention. The combination of a low,
relatively frequent dose of a therapeutic agent of the present
invention in combination with an agent that inhibits angiogenesis
is particularly contemplated. Exemplary anti-angiogenic agents that
are useful in connection with combined therapy are listed above (in
connection with immunoconjugates). Any one or more of such agents,
including those in Table B, may be used in combination therapy with
the invention. Angiostatin, endostatin, vasculostatin, canstatin
and maspin are currently preferred.
[0595] Many known anti-cancer agents also have an anti-angiogenic
effect as part of their mechanism of action. These agents, as
exemplified by those in Table E, are particularly contemplated for
use in the combination therapy aspects of the present invention
(they may also be conjugated to a construct, receptorbody or
betabody of the invention, as described above).
TABLE-US-00006 TABLE E Anti-Cancer Agents with Anti-Angiogenic
Activity Class or Type of Agent Examples Alkylators
Cyclophosphamide, edelfosine, estramustine, melphalan
Antimetabolites Fluorouracil, methotrexate, mercaptopurine, UFT,
tegafur, uracil, cytarabine Anti-Tumor Bleomycin, daunorubicin,
doxorubicin, epirubicin, Antibiotics mitomycin, mitoxantrone
Topoisomerase Camptothecin, irinotecan, etoposide, topotecan
Inhibitors Taxanes Docetaxel, paclitxael Vinca Alkaloids
Vinblastine, vincristine Miscellaneous Cisplatin, octreotide
[0596] In addition, the antibody LM609 against the
.alpha..sub.v.beta..sub.3 integrin also induces tumor regressions
and may be used in combination therapies. 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.
[0597] Apoptosis of the angiogenic endothelium by LM609 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.
[0598] Antibodies to angiogenin may also be employed, as described
in U.S. Pat. No. 5,520,914, specifically incorporated herein by
reference. As FGF is connected with angiogenesis, FGF inhibitors
may also be used. Certain examples are the compounds having
N-acetylglucosamine alternating in sequence with 2-O-sulfated
uronic acid as their major repeating units, including
glycosaminoglycans, such as archaran sulfate. Such compounds are
described in U.S. Pat. No. 6,028,061, specifically incorporated
herein by reference, and may be used in combination herewith.
[0599] M7. VEGF Inhibitors
[0600] VEGF is a multifunctional cytokine that is induced by
hypoxia and oncogenic mutations. VEGF is a primary stimulant of the
development and maintenance of a vascular network in embryogenesis.
It functions as a potent permeability-inducing agent, an
endothelial cell chemotactic agent, an endothelial survival factor,
and endothelial cell proliferation factor. Its activity is required
for normal embryonic development, as targeted disruption of one or
both alleles of VEGF results in embryonic lethality.
[0601] The use of one or more VEGF inhibition methods is a
preferred aspect of the combination therapies of the present
invention. The recognition of VEGF as a primary stimulus of
angiogenesis in pathological conditions has led to various methods
to block VEGF activity. Any of the VEGF inhibitors developed may
now be advantageously employed herewith. Accordingly, any one or
more of the following neutralizing anti-VEGF antibodies, soluble
receptor constructs, antisense strategies, RNA aptamers and
tyrosine kinase inhibitors designed to interfere with VEGF
signaling may thus be used.
[0602] Suitable agents include neutralizing antibodies (Kim et al.,
1992; Presta et al., 1997; Sioussat et al., 1993; Kondo et al.,
1993; Asano et al., 1995), soluble receptor constructs (Kendall and
Thomas, 1993; Aiello et al., 1995; Lin et al., 1998; Millauer et
al., 1996), tyrosine kinase inhibitors (Siemeister et al., 1998),
antisense strategies, RNA aptamers and ribozymes against VEGF or
VEGF receptors (Saleh et al., 1996; Cheng et al., 1996). Variants
of VEGF with antagonistic properties may also be employed, as
described in WO 98/16551. Each of the foregoing references are
specifically incorporated herein by reference.
[0603] Blocking antibodies against VEGF will be preferred in
certain embodiments, particularly for simplicity. Monoclonal
antibodies against VEGF have been shown to inhibit human tumor
xenograft growth and ascites formation in mice (Kim et al., 1993;
Mesiano et al., 1998; Luo et al., 1998a; 1998b; Borgstrom et al.,
1996; 1998; each incorporated herein by reference). The antibody
A4.6.1 is a high affinity anti-VEGF antibody capable of blocking
VEGF binding to both VEGFR1 and VEGFR2 (Kim et al., 1992; Wiesmann
et al., 1997; Muller et al., 1998; Keyt et al., 1996; each
incorporated herein by reference). A4.6.1 has recently been
humanized by monovalent phage display techniques and is currently
in Phase I clinical trials as an anti-cancer agent (Brem, 1998;
Baca et al., 1997; Presta et al., 1997; each incorporated herein by
reference).
[0604] Alanine scanning mutagenesis and X-ray crystallography of
VEGF bound by the Fab fragment of A4.6.1 showed that the epitope on
VEGF that A4.6.1 binds is centered around amino acids 89-94. This
structural data demonstrates that A4.6.1 competitively inhibits
VEGF from binding to VEGFR2, but inhibits VEGF from binding to
VEGFR1 most likely by steric hindrance (Muller et al., 1998; Keyt
et al., 1996; each incorporated herein by reference)
[0605] A4.6.1 may be used in combination with the present
invention. However, a new antibody termed 2C3 (4545) is currently
preferred, which selectively blocks the interaction of VEGF with
only one of the two VEGF receptors. 2C3 inhibits VEGF-mediated
growth of endothelial cells, has potent anti-tumor activity and
selectively blocks the interaction of VEGF with VEGFR2 (KDR/Flk-1),
but not VEGFR1 (FLT-1). In contrast to A4.6.1, 2C3 allows specific
inhibition of VEGFR2-induced angiogenesis, without concomitant
inhibition of macrophage chemotaxis (mediated by VEGFR1), and is
thus contemplated to be a safer therapeutic. U.S. Pat. Nos.
6,342,219, 6,342,221, 6,416,758 and 6,416,758, are specifically
incorporated herein by reference for the purposes of even further
describing the 2C3 antibody and its uses in anti-angiogenic therapy
and VEGF inhibition.
[0606] M8. Apoptosis-Inducing Agents
[0607] The therapeutic agents of the present invention are also
preferably combined with treatment methods that induce apoptosis in
any cells within the tumor, including tumor cells and tumor
vascular endothelial cells. Exemplary agents that induce apoptosis
are listed above (in connection with immunoconjugates). Any one or
more of such apoptosis-inducing agents may be used in the
combination therapies of the present invention, without being
linked to an antibody of the invention.
[0608] Many known anti-cancer agents also have an
apoptosis-inducing effect as part of their mechanism of action.
These agents, as exemplified by those in Table F, are particularly
contemplated for use in the combination therapy aspects of the
present invention (they may also be conjugated to an antibody of
the invention, as described above).
TABLE-US-00007 TABLE F Anti-Cancer Agents that Induce Apoptosis
Class or Type of Agent Examples Antimetabolites Cytarabine,
fludarabine, 5-fluoro-29-deoxyuridine, gemcitabine, hydroxyurea,
methotrexate DNA Cross- Chlorambucil, cisplatin, Linking Agents
cyclophosphamide, nitrogen mustard Intercalating Adriamycin
(doxorubicin), Agents mitixantrone Topoisomerase Etoposide,
teniposide II Poisons Microtubule- Colcemid, colchicine, Directed
Agents docetaxel, vincristine Kinase Inhibitors Flavopiridol,
staurosporine, STI571 (CPG 57148B), UCN-01 (7-hydroxystaurosporine)
Farnesyl Transferase L-739749, L-744832 Inhibitors Hormones
Glucocorticoids, fenretinide DNA Fragmenting Bleomycin Agents
Hormone Antagonists Tamoxifen, finasteride, LHRH antagonists
Biologicals TNF-.alpha., TRAIL, anti-CD20 Protein Synthesis
L-asparaginase, cycloheximide, Inhibitors puromycin, diphtheria
toxin Topoisomerase Camptothecin, toptecan II Poisons
[0609] M9. Immunotoxins and Coaguligands
[0610] The present invention may also be used in combination with
other immunotoxins or coaguligands in which the targeting portion
is directed to a marker of tumor cells, tumor vasculature or tumor
stroma. Any targeting agent for use in targeting to a tumor cell,
tumor vasculature or tumor stroma may be used in these embodiments.
In the immunotoxins, the attached agents include anti-cellular or
cytotoxic agents, cytokines, radiotherapeutic agents,
anti-angiogenic agents, apoptosis-inducing agents and anti-tubulin
drugs. In the coaguligands, the attached agents are coagulants.
U.S. Pat. Nos. 5,855,866, 5,965,132, 6,261,535, 6,051,230,
6,451,312 (immunotoxins), U.S. Pat. Nos. 6,093,399, 6,004,555,
5,877,289, and 6,036,955 (coaguligands) are specifically
incorporated herein by reference to exemplify such constructs.
[0611] M10. ADEPT and Prodrug Therapy
[0612] A construct, receptorbody or betabody of the present
invention may also be used in conjunction with prodrugs, wherein
the construct, receptorbody or betabody is operatively associated
with a prodrug-activating component, such as a prodrug-activating
enzyme, which converts a prodrug to the more active form only upon
contact with the antibody. This technology is generally termed
"ADEPT", and is described in, e.g., WO 95/13095; WO 97/26918, WO
97/24143, and U.S. Pat. Nos. 4,975,278 and 5,658,568, each
specifically incorporated herein by reference.
[0613] The term "prodrug", as used herein, refers to a precursor or
derivative form of a biologically or pharmaceutically active
substance that exerts reduced cytotoxic or otherwise anticellular
effects on targets cells, including tumor vascular endothelial
cells, in comparison to the parent drug upon which it is based.
Preferably, the prodrug or precursor form exerts significantly
reduced, or more preferably, negligible, cytotoxic or anticellular
effects in comparison to the "native" or parent form. "Prodrugs"
are capable of being activated or converted to yield the more
active, parent form of the drug.
[0614] The technical capability to make and use prodrugs exists
within the skill of the ordinary artisan. Willman et al. (1986) and
Stella et al. (1985) are each specifically incorporated herein by
reference for purposes of further supplementing the description and
teaching concerning how to make and use various prodrugs. Exemplary
prodrug constructs that may be used in the context of the present
invention include, but are not limited to, phosphate-containing
prodrugs (U.S. Pat. No. 4,975,278), thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-based prodrugs (U.S.
Pat. Nos. 5,660,829; 5,587,161; 5,405,990; WO 97/07118), D-amino
acid-modified prodrugs, glycosylated prodrugs (U.S. Pat. Nos.
5,561,119; 5,646,298; 4,904,768, 5,041,424),
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs (U.S. Pat. No. 4,975,278),
optionally substituted phenylacetamide-containing prodrugs, and
even 5-fluorocytosine (U.S. Pat. No. 4,975,278) and 5-fluorouridine
prodrugs and the like, wherein each of the patents are specifically
incorporated herein by reference.
[0615] The type of therapeutic agent or cytotoxic drug that can be
used in prodrug form is virtually limitless. The more cytotoxic
agents will be preferred for such a form of delivery, over, e.g.,
the delivery of coagulants, which are less preferred for use as
prodrugs. All that is required in forming the prodrug is to design
the construct so that the prodrug is substantially inactive and the
"released" or activated drug has substantial, or at least
sufficient, activity for the intended purpose.
[0616] Various improvements on the original prodrugs are also known
and contemplated for use herewith, as disclosed in WO 95/03830; EP
751,144 (anthracyclines); WO 97/07097 (cyclopropylindoles); and WO
96/20169. For example, prodrugs with reduced Km are described in
U.S. Pat. No. 5,621,002, specifically incorporated herein by
reference, which may be used in the context of the present
invention. Prodrug therapy that be conducted intracellularly is
also known, as exemplified by WO 96/03151, specifically
incorporated herein by reference, and can be practiced
herewith.
[0617] For use in ADEPT, the agent that activates or converts the
prodrug into the more active drug is operatively attached to an
antibody of the invention. The antibody thus localizes the prodrug
converting capability within the angiogenic or tumor site, so that
active drug is only produced in such regions and not in circulation
or in healthy tissues.
[0618] Enzymes that may be attached to the antibodies of the
invention to function in prodrug activation include, but are not
limited to, alkaline phosphatase for use in combination with
phosphate-containing prodrugs (U.S. Pat. No. 4,975,278);
arylsulfatase for use in combination with sulfate-containing
prodrugs (U.S. Pat. No. 5,270,196); peptidases and proteases, such
as serratia protease, thermolysin, subtilisin, carboxypeptidase
(U.S. Pat. Nos. 5,660,829; 5,587,161; 5,405,990) and cathepsins
(including cathepsin B and L), for use in combination with
peptide-based prodrugs; D-alanylcarboxypeptidases for use in
combination with D-amino acid-modified prodrugs;
carbohydrate-cleaving enzymes such as .beta.-galactosidase and
neuraminidase for use in combination with glycosylated prodrugs
(U.S. Pat. Nos. 5,561,119; 5,646,298); .beta.-lactamase for use in
combination with .beta.-lactam-containing prodrugs; penicillin
amidases, such as penicillin V amidase (U.S. Pat. No. 4,975,278) or
penicillin G amidase, for use in combination with drugs derivatized
at their amino nitrogens with phenoxyacetamide or phenylacetamide
groups; and cytosine deaminase (U.S. Pat. Nos. 5,338,678;
5,545,548) for use in combination with 5-fluorocytosine-based
prodrugs (U.S. Pat. No. 4,975,278), wherein each of the patents are
specifically incorporated herein by reference.
N. Antibody-Coated Liposomes and Therapeutics
[0619] Liposomal formulations are often used in therapeutics and
pharmaceuticals. However, the biodistribution of liposomes in
initial studies meant that such formulations were not widely
applicable for use in humans. Liposomes are rapidly taken up by the
phagocytic cells of the reticuloendothelial system (RES), including
the circulating mononuclear phagocytic cells and those located in
the liver and spleen. Thus, the blood circulation half-lives can be
as short as a few minutes.
[0620] The technology of "stealth or stealthed" liposomes and
formulations was thus developed, which allows liposomes to evade
uptake by the RES and circulate for longer (Hristova and Needham,
1993). A preferred agent for use in stealthing liposomes is
polyethylene glycol (PEG), and the resultant liposomes are also
termed PEGylated liposomes. Other stealthing agents include
poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) conjugates
(Woodle et al., 1994). A range of improved stealthed liposomes are
described in U.S. Pat. No. 6,284,267, specifically incorporated
herein by reference, which may be used in combination with the
present invention.
[0621] Liposomes smaller in diameter than the average diameter of
the fenestrae in capillaries leak out from the circulation. The
average diameter of the fenestrae in rapidly growing tumors is
larger than in normal tissues and therefore liposomes smaller than
about 100 nm in diameter migrate into tumors. Stealth liposomes
have thus been proposed for use in delivering cytotoxic agents to
tumors in cancer patients. A range of drugs have been incorporated
into stealth liposomes, including cisplatin (Rosenthal et al.,
2002), TNF.alpha. (Kim et al., 2002), doxorubicin (Symon et al.,
1999) and adriamycin (Singh et al., 1999), each reference being
specifically incorporated herein by reference. However, recent
reports have indicated unexpected low efficacy of stealth liposomal
doxorubicin and vinorelbine in the treatment of metastatic breast
cancer (Rimassa et al., 2003).
[0622] The present invention provides improved stealthed liposome
formulations, overcoming various of the drawbacks in the art, in
which the stealthed liposomes are functionally associated or
"coated" with a construct, receptorbody or betabody of the
invention. A divalent construct is not required in these aspects of
the invention.
[0623] Any stealthed liposome may form the basis of the new
liposomal formulations, and preferably a PEGylated liposome will be
employed. The stealthed liposomes are "coated", i.e., operatively
or functionally associated with a construct, receptorbody or
betabody. The operative or functional association is made such that
the construct, receptorbody or betabody retains the ability to
specifically bind to the target PS or anionic phospholipid, thereby
delivering or targeting the stealthed liposome and any contents
thereof to PS-positive cells, such as tumor cells and tumor
vascular endothelial cells.
[0624] The coated stealthed liposomes of the invention may be used
alone. Preferably, however, such liposomes will also contain one or
more second therapeutic agents, such as anti-cancer or
chemotherapeutic agents (the first therapeutic agent being the
antibody itself). The second therapeutic agents are generally
described as being within the "core" of the liposome. Any one or
more of the second, anti-cancer or chemotherapeutic agents known in
the art and/or described herein for conjugation, or for combination
therapies, may be used in the antibody-coated stealthed liposomes
of the invention. For example, any chemotherapeutic or
radiotherapeutic agent, cytokine, anti-angiogenic agent or
apoptosis-inducing agent. Currently preferred within the
chemotherapeutic agents are anti-tubulin drugs, docetaxel and
paclitaxel.
[0625] Moreover, the antibody-coated stealthed liposomes of the
invention may also be loaded with one or more anti-viral drugs for
use in treating viral infections and diseases. As with the
anti-cancer agents, any one or more of the second, anti-viral drugs
known in the art and/or described herein for conjugation to
antibodies, or for combination therapies, may be used in the
antibody-coated stealthed liposomes of the invention. Cidofovir and
AZT are currently preferred examples.
O. Anti-Vascular, Anti-Angiogenic and Other Therapies
[0626] The present invention may also be used in the treatment of
other diseases in which aberrant vasculature is involved, including
diseases and disorders having prothrombotic blood vessels. Although
not the only therapeutic mechanism, the construct, receptorbody or
betabody of the present invention may also be used to treat animals
and patients with aberrant angiogenesis, such as that contributing
to a variety of diseases and disorders.
[0627] Whether based upon anti-angiogenesis, prothrombotic
vasculature or other anti-vascular mechanisms, the present
invention may thus be used to treat prevalent and/or clinically
important diseases outside the field of cancer, including
arthritis, rheumatoid arthritis, psoriasis, atherosclerosis,
diabetic retinopathy, age-related macular degeneration, Grave's
disease, vascular restenosis, including restenosis following
angioplasty, arteriovenous malformations (AVM), meningioma,
hemangioma and neovascular glaucoma. Other targets for intervention
include angiofibroma, atherosclerotic plaques, corneal graft
neovascularization, hemophilic joints, hypertrophic scars,
osler-weber syndrome, pyogenic granuloma retrolental fibroplasia,
scleroderma, trachoma, vascular adhesions, synovitis, dermatitis,
various other inflammatory diseases and disorders, and even
endometriosis. Further diseases and disorders that are treatable by
the invention, and the unifying basis of such disorders, are set
forth below.
[0628] One prominent disease in which aberrant vasculature and
angiogenesis is involved is rheumatoid arthritis, wherein the blood
vessels in the synovial lining of the joints undergo angiogenesis.
In addition to forming new vascular networks, the endothelial cells
release factors and reactive oxygen species that lead to pannus
growth and cartilage destruction. The factors involved in
angiogenesis may actively contribute to, and help maintain, the
chronically inflamed state of rheumatoid arthritis. Factors
associated with angiogenesis also have a role in osteoarthritis,
contributing to the destruction of the joint. Various factors,
including VEGF, have been shown to be involved in the pathogenesis
of rheumatoid arthritis and osteoarthritis.
[0629] Another important example of a disease involving aberrant
vasculature and angiogenesis is ocular neovascular disease. This
disease is characterized by invasion of new blood vessels into the
structures of the eye, such as the retina or cornea. It is the most
common cause of blindness and is involved in approximately twenty
eye diseases. In age-related macular degeneration, the associated
visual problems are caused by an ingrowth of chorioidal capillaries
through defects in Bruch's membrane with proliferation of
fibrovascular tissue beneath the retinal pigment epithelium.
Angiogenic damage is also associated with diabetic retinopathy,
retinopathy of prematurity, corneal graft rejection, neovascular
glaucoma and retrolental fibroplasia.
[0630] Other diseases associated with corneal neovascularization
that can be treated according to the present invention include, but
are not limited to, epidemic keratoconjunctivitis, Vitamin A
deficiency, contact lens overwear, atopic keratitis, superior
limbic keratitis, pterygium keratitis sicca, sjogrens, acne
rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid
degeneration, chemical burns, bacterial ulcers, fungal ulcers,
Herpes simplex infections, Herpes zoster infections, protozoan
infections, Kaposi sarcoma, Mooren ulcer, Terrien's marginal
degeneration, mariginal keratolysis, rheumatoid arthritis, systemic
lupus, polyarteritis, trauma, Wegeners sarcoidosis, Scleritis,
Steven's Johnson disease, periphigoid radial keratotomy, and
corneal graph rejection.
[0631] Diseases associated with retinal/choroidal
neovascularization that can be treated according to the present
invention include, but are not limited to, diabetic retinopathy,
macular degeneration, sickle cell anemia, sarcoid, syphilis,
pseudoxanthoma elasticum, Pagets disease, vein occlusion, artery
occlusion, carotid obstructive disease, chronic uveitis/vitritis,
mycobacterial infections, Lyme's disease, systemic lupus
erythematosis, retinopathy of prematurity, Eales disease, Bechets
disease, infections causing a retinitis or choroiditis, presumed
ocular histoplasmosis, Bests disease, myopia, optic pits, Stargarts
disease, pars planitis, chronic retinal detachment, hyperviscosity
syndromes, toxoplasmosis, trauma and post-laser complications.
[0632] Other diseases that can be treated according to the present
invention include, but are not limited to, diseases associated with
rubeosis (neovascularization of the angle) and diseases caused by
the abnormal proliferation of fibrovascular or fibrous tissue
including all forms of proliferative vitreoretinopathy, whether or
not associated with diabetes.
[0633] Chronic inflammation also involves aberrant vasculature and
pathological angiogenesis. Such disease states as ulcerative
colitis and Crohn's disease show histological changes with the
ingrowth of new blood vessels into the inflamed tissues.
Bartonellosis, a bacterial infection found in South America, can
result in a chronic stage that is characterized by proliferation of
vascular endothelial cells.
[0634] Another pathological role associated with aberrant
vasculature and angiogenesis is found in atherosclerosis. The
plaques formed within the lumen of blood vessels have been shown to
have angiogenic stimulatory activity. There is particular evidence
of the pathophysiological significance of angiogenic markers, such
as VEGF, in the progression of human coronary atherosclerosis, as
well as in recanalization processes in obstructive coronary
diseases. The present invention provides an effective treatment for
such conditions.
[0635] One of the most frequent angiogenic diseases of childhood is
the hemangioma. In most cases, the tumors are benign and regress
without intervention. In more severe cases, the tumors progress to
large cavernous and infiltrative forms and create clinical
complications. Systemic forms of hemangiomas, the hemangiomatoses,
have a high mortality rate. Therapy-resistant hemangiomas exist
that cannot be treated with therapeutics currently in use, but are
addressed by the invention.
[0636] Angiogenesis is also responsible for damage found in
hereditary diseases such as Osler-Weber-Rendu disease, or
hereditary hemorrhagic telangiectasia. This is an inherited disease
characterized by multiple small angiomas, tumors of blood or lymph
vessels. The angiomas are found in the skin and mucous membranes,
often accompanied by epistaxis (nosebleeds) or gastrointestinal
bleeding and sometimes with pulmonary or hepatic arteriovenous
fistula.
[0637] Angiogenesis is also involved in normal physiological
processes such as reproduction and wound healing. Angiogenesis is
an important step in ovulation and also in implantation of the
blastula after fertilization. Prevention of angiogenesis according
to the present invention could be used to induce amenorrhea, to
block ovulation or to prevent implantation by the blastula. In
wound healing, excessive repair or fibroplasia can be a detrimental
side effect of surgical procedures and may be caused or exacerbated
by angiogenesis. Adhesions are a frequent complication of surgery
and lead to problems such as small bowel obstruction. This can also
be treated by the invention.
[0638] Each of the foregoing diseases and disorders, along with all
types of tumors, are also contemplated for treatment according to
the present invention. U.S. Pat. No. 5,712,291 is specifically
incorporated herein by reference to further demonstrate the
knowledge in the art that once the inhibition of angiogenesis has
been shown using a particular agent, the treatment of an extensive
range of diseases associated with aberrant angiogenesis using that
and like agents can reasonably be carried out. U.S. Pat. No.
6,524,583 is also specifically incorporated herein by reference for
similar purposes and to particularly demonstrate that this
principle applies to the inhibition of angiogenesis and the
treatment of angiogenic diseases using targeted therapeutics.
[0639] The invention further provides compositions and methods for
use in treating other diseases in which PS or anionic phospholipids
play a role. For example, as PS is involved in cell adhesion,
inflammatory responses and septic shock, a construct, receptorbody
or betabody can be used in the treatment of inflammation and septic
shock.
[0640] Anionic phospholipids, particularly PS, are also involved in
sickle cell anaemia, in particular, as part of the clearance
mechanism. A construct, receptorbody or betabody can therefore be
used to treat or ameliorate sickle cell anaemia. A construct,
receptorbody or betabody of the invention can also be used to treat
a parasitic disease and to treat malaria.
P. Anti-Viral Treatment Methods
[0641] The present invention further provides a range of
constructs, optionally conjugated to anti-viral agents, for use in
treating viral infections. The treatment regimens, and particularly
the doses, are generally as described above for the cancer
treatment aspects of the present invention, which adaptability is
an advantage of the invention overall. Although an understanding of
the particular mechanism(s) of action is not necessary to practice
the anti-viral treatment of the invention, certain of the reasons
underlying the viral treatment, as supported by the working
examples herein, are as follows.
[0642] The most important mechanisms are believed to be connected
with viral replication and activation of the host cell. During
viral infection, the virus activates the cell during its
replication process inside the cell. This process of cell
activation is necessary for viral replication, as shown for herpes
viruses, hepatitis C and HIV-1. Viral progression activates gene
expression, both viral and host. For example, the replication of
Pichinde virus and Machupo virus is inhibited by actinomycin D late
in the replication cycle, indicating that host cell gene
transcription is needed for completion of viral replication.
[0643] The activation of the host cell by the virus causes the cell
to externalize anionic phospholipids, such PS. In particular, the
inventors reason that viral activation causes Ca.sup.2+ fluxes into
the cell, which activate scramblase, externalizing anionic
phospholipids, particularly PS. Constructs and conjugates that bind
anionic phospholipids, preferably PS, then bind and interfere with
the activation process, preventing the virus from being able to
replicate properly.
[0644] The present examples show that the invention acts late in
the process of viral infection, blocking viral maturation or
egress. The inventors' studies show that the inhibitory effect of
the agents of the invention is widely applicable, as it has been
shown to operate on viruses that use different egression
mechanisms. For example, the present examples demonstrate block of
herpes virus (CMV), which escapes from Golgi-derived exocytotic
vesicles, and block of arenavirus (Pichinde virus) and
paramyxovirus (RSV), which bud out directly from the plasma
membrane.
[0645] Virally infected cells externalize anionic phospholipids,
particularly PS, which are normally intracellular, i.e., confined
to the inner surface of plasma membrane. During escape of the
virus, phospholipids redistribute at the site of escape,
accommodating membrane bending during viral budding or exocytosis
from the plasma membrane, and anionic phospholipids and
aminophospholipids are externalized during this process. The
constructs and conjugates of the invention can thus bind to the
externalized anionic phospholipids, particularly PS, and block the
escape of the virus from the infected cell. Binding of the
constructs of the invention to virally infected cells is also shown
in the present examples.
[0646] The constructs and conjugates of the invention may further
bind to the externalized anionic phospholipids, particularly PS,
and interfere with one or more signaling pathways necessary for
viral gene expression and/or replication.
[0647] Moreover, enveloped virions themselves likely have anionic
phospholipids, such as PS, on their external surface. Since viruses
lack a translocase to maintain or restore phospholipid asymmetry,
continued exposure of phospholipids such as PS is expected. The
constructs and conjugates of the invention may thus cause
opsonization, complement binding, phagocytosis by host cells such
as macrophages and clearance of free virus particles.
[0648] In a further aspect of the invention, viruses likely need
anionic phospholipids for infection and/or syncitia formation. The
constructs and conjugates of the invention may further block these
aspects of the viral life cycle by binding to anionic
phospholipids.
[0649] According to the foregoing insights, and in light of the
present examples, the spectrum of viral treatment for the present
invention extends to any virus, whether enveloped or not, DNA or
RNA. As the anionic phospholipid- and PS-binding constructs and
conjugates of the invention at least in part block viral
replication inside the cell, and/or prevent escape of virus from
cells, the invention is not limited to the treatment of enveloped
viruses alone, nor to any particular virus, which is an important
advantage. For example, work published subsequent to the invention
reports that annexin V and PS vesicles can inhibit HIV-1 infection
of macrophages, but cannot inhibit HIV-1 infection of T cells or
inhibit other viruses, such as vesicular stomatitis virus G and
amphotropic murine leukemia virus (Callahan et al., 2003).
[0650] Naturally, the constructs and conjugates of the invention do
act on enveloped viruses, particularly those viruses that have
anionic phospholipids, particularly PS, on the outer surface of the
envelope, wherein the constructs and conjugates cause viral
clearance and/or inhibiting viral entry of target cells.
[0651] An important aspect of the present invention is therefore
that it is universally applicable, being suitable for the treatment
of recombinant, engineered and synthetic viruses, e.g., created as
part of bio-terrorism. Indeed, the invention is not limited to the
treatment of animals and humans. As the categories of hosts found
in the virus taxa include algae, archaea, bacteria, fungi,
invertebrates, mycoplasma, plants, protozoa, spiroplasma and
vertebrates, the invention can be used to inhibit viral infection
and replication in any such setting, including in viruses of
agricultural importance. The treatment of viral infection and
associated diseases in vertebrates is currently preferred, and any
one or more of the viruses in Table H, which infect vertebrate
animals, may be inhibited, and the resultant infection treated,
using the present invention.
TABLE-US-00008 TABLE H Viruses of Vertebrates Family Genus Type
Species Adenoviridae Mastadenovirus Human adenovirus 2
Aviadenovirus Fowl adenovirus 1 African Swine African swine fever
Fever-like Viruses virus Arenaviridae Arenavirus Lymphocytic
choriomeningitis virus Arterivirus Equine arteritis virus
Astroviridae Astrovirus Human astrovirus 1 Birnaviridae
Aquabirnavirus Infectious pancreatic necrosis virus Avibirnavirus
Infectious bursal disease virus Bunyaviridae Bunyavirus Bunyamwera
virus Hantavirus Hantaan virus Nairovirus Nabrobi sheep disease
virus Phlebovirus Sandfly fever Sicilian virus Caliciviridae
Calicivirus Vesicular exanthema of swine virus Circoviridae
Circovirus Chicken anemia virus Coronaviridae Coronavirus Avian
infectious bronchitis virus Torovirus Berne virus Deltavirus
Hepatitis delta virus Filoviridae Filovirus Marburg virus
Flaviviridae Flavivirus Yellow fever virus Pestivirus Bovine
diarrhea virus Hepatitis C - Hepatitis C virus like viruses
Hepadnaviridae Orthophepadnavirus Hepatitis B virus Avihepadnavirus
Duck hepatitis B virus Herpesviridae Subfamily Simplexvirus Human
herpesvirus 1 Alphaherpesvirinae Varicellovirus Human herpesvirus 3
Subfamily: Cytomegalovirus Human herpesvirus 5 Betaherpesvirinae
Muromegalovirus Mouse cytomegalo- virus 1 Subfamily Roseolovirus
Human herpesvirus 6 Gammaherpesvirinae Lymphocryptovirus Human
herpesvirus 4 Rhadinovirus Ateline herpesvirus 2 Iridoviridae
Ranavirus Frog virus 3 Lymphocystivirus Flounder virus Goldfish
virus - Goldfish virus 1 like viruses Orthomyxoviridae
Influenzavirus A, B Influenza A virus Influenzavirus C Influenza C
virus Thogoto-Like viruses Thogoto virus Papovaviridae Polyomavirus
Murine polyomavirus Papillomavirus Cottontail rabbit papillomavirus
(Shope) Paramyxoviridae Parayxovirus Human parainfluenza Subfamily
virus 1 Paramyxovirinae Morbillivirus Measles virus Rubulavirus
Mumps virus Subfamily Pneumovirus Human respiratory Pneumovirinae
syncytial virus Parvoviridae Parvovirus Mice minute virus Subfamily
Erythovirus B19 virus Parovirinae Dependovirus Adeno-associated
virus 2 Picornaviridae Enterovirus Poliovirus 1 Rhinovirus Human
rhinovirus 1A Hepatovirus Hepatitis A virus Cardiovirus
Encephalomyocarditis virus Aphthovirus Foot-and-mouth disease virus
O Poxviridae Orthopoxvirus Vaccinia virus Subfamily Parapoxyvirus
Orf virus Chordopoxvirinae Avipoxvirus Fowlpox virus Capripoxvirus
Sheeppox virus Leporipoxvirus Myxoma virus Suipoxvirus Swinepox
virus Molluscipoxvirus Molluscum contagiosum virus Yatapoxvirus
Yaba monkey tumor virus Reoviridae Orthoreovirus Reovirus 3
Orbivirus Bluetongue virus 1 Rotavirus Simian rotavirus SA11
Coltivirus Colorado tick fever virus Aquareovirus Golden shiner
virus Retroviridae Mammalian type Mouse mammary B retroviruses
tumor virus Mammalian type Murine leukemia C retroviruses virus
Avian type C Avian leukosis retroviruses virus Type D retroviruses
Mason-Pfizer monkey virus Blv-htlv Bovine leukemia retroviruses
virus Lentivirus Human immuno- deficiency virus 1 Spumavirus Human
spumavirus Rhabdoviridae Vesiculovirus Vesicular stomatitis Indiana
virus Lyssavirus Rabies virus Ephemerovirus Bovine ephemeral fever
Togaviridae Alphavirus Sindbis virus Rubivirus Rubella virus
[0652] The use of the invention in treating viral infections and
associated diseases in mammals is preferred, particularly in terms
of valuable or valued animals, such as racehorses and domestic
pets, and animals and birds used to directly produce (e.g., meat)
or indirectly produce (e.g., milk and eggs) food for human
consumption. In addition to human treatment, exemplary embodiments
of the invention include the treatment of horses, dogs, cats and
the like; the treatment of cows, pigs, boar, sheep, goat, buffalo,
bison, llama, deer, elk, and other large animals, as well as their
young, including calves and lambs.
[0653] The treatment of humans is particularly preferred, whether
for naturally occurring viruses or for those created by
bioterrorism. In terms of naturally occurring viruses and the
resultant diseases, the invention is again unlimited in its
applications. Accordingly, any one or more of the viruses in Table
J may be inhibited using the present invention, and the resultant
infections and diseases thus treated.
TABLE-US-00009 TABLE J Viral Diseases in Humans Disease Virus Type
of Virus AIDS Human Retrovirus Immunodeficiency Virus (HIV)
Bronchiolitis and Respiratory Paramyxovirus viral pneumonia
syncytial virus Bronchiolitis Parainfluenza virus Paramyxovirus
Cervical cancer Human papilloma virus Papovavirus Chicken pox
Varicella Zoster virus Herpesvirus Dengue Dengue virus Flavivirus
Ebola Ebola virus Filovirus hemorrhagic fever Genital Herpes Herpes
Simplex Herpesvirus virus-2 Hantavirus Hantavirus Bunyavirus
hemorrhagic fever Hepatitis Hepatitis A Picornavirus Hepatitis B
Hepadavirus Hepatitis C Flavivirus Hepatitis D Deltavirus Hepatitis
E Calcivirus Influenza Influenza viruses Orthomyxovirus A, B and C
Junin Argentinian Junin virus Arenavirus Hemorrhagic Fever Lassa
hemorrhagic Lassa virus Arenavirus fever Machupo hemorrhagic
Machupo virus Arenavirus fever Measles Rubeola virus Paramyxovirus
Mononucleosis Epstein Barr virus Herpesvirus CMV disease (viral
Cytomegalovirus Herpesvirus pneumonia, mononucleosis like syndrome)
Severe Acute Human coronavirus Coronavirus Respiratory Syndrome
(SARS) Shingles Varicella zoster Herpesvirus virus Smallpox Variola
virus Poxvirus Yellow fever Yellow fever virus Flavivirus West Nile
Disease West Nile virus Western equine Western EE virus Togavirus
encephalitis Pneumonia, Hepatitis, Adenovirus Adenovirus acute
respiratory disease Gastroenteritis Rotavirus Rotavirus
Encephalitis Semliki Forest virus Alphavirus Cowpox Vaccinia virus
Poxvirtts Encephalitis Venezuelan EE Alphavirus Meningitis,
encephalitis, Lymphocytic Arenavirus meningoencephalitis
choriomeningitis Venezuelan Guanarito virus Arenavirus hemorrhagic
fever Rift valley fever Rift valley fever Bunyavirus (hemorrhagic
fever, virus encephalitis) Marburg Hemorrhagic fever Marburg virus
Filovirus Tick borne encephalitis Tick borne Flavivirus
encephalitis virus (TBEV) Encephalitis Hendra virus Paramyxovirus
Encephalitis Nipah virus Paramyxovirus Crimean-Congo Crimean-Congo
Bunyavirus hemorrhagic fever hemorrhagic fever virus Brazilian
Sabia virus Arenavirus hemorrhagic fever
[0654] The invention is particularly contemplated for use in the
treatment of CMV related diseases such as viral pneumonia,
mononucleosis like syndrome, and associated congenital
malformations (deafness and mental retardation); respiratory
diseases, such as those caused by RSV, including bronchiolitis and
viral pneumonia, influenza, the common cold and SARS; AIDS;
hepatitis; cancers associated with viral infections; mononucleosis;
and smallpox.
[0655] In other embodiments, the inventors particularly contemplate
the inhibition of arenaviruses, which are pathogenic in man. The
arenaviruses include the Old World viruses responsible for Lassa
fever (Lassa virus) and lymphocytic choriomeningitis (LCMV). Lassa
fever is endemic in West Africa, affecting up to 300,000 people
annually and causing up to 3000 deaths. Infection with Lassa fever
leads to fever and malaise within about 10 days. Abdominal pain,
nausea, vomiting and diarrhea are common. Pharyngitis and cough may
develop. Neurological symptoms are usually mild. Vascular leak
syndromes, such as edema and pleural effusions, are present in more
severe cases. Bleeding is seen about one quarter of patients. The
disease can cause changes in the cardiovascular system that
culminate in shock and death.
[0656] Arenaviruses also include and the antigenically-distinct New
World viruses responsible for Argentine hemorrhagic fever (Junin
virus), Bolivian hemorrhagic fever (Machupo virus) and Venezuelan
hemorrhagic fever (Guanarito virus). All of these viruses are on
the CDC Category A list of potential bioterrorism weapons.
[0657] The doses that are suitable for the anti-tumor embodiments
are also suitable for the anti-viral treatments. Similarly,
multiple administration may be used for chronic infections, and
high doses may be used for acute infections. Any suitable route of
administration may be employed, again as disclosed for the cancer
treatment aspects, including IV, IM, SC, as an aerosol to lungs or
airways and such like.
[0658] The therapeutics provided by the invention are valuable
agents having broad-spectrum anti-viral activity. In addition to
being effective against a large number of potentially lethal
viruses, the agents can also be administered after exposure to the
virus, even in settings where the exact nature of the virus is not
known. Thus, the anti-viral therapeutics of the present invention
do not require a prolonged period of time between identification of
the pathogen and delivery of the therapy, in marked contrast with
the time and expense entailed by the development, production or
delivery of specific vaccines.
[0659] 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
Tumor Treatment with Anti-VCAM-1-tTF Coaguligand
[0660] The present example shows the specific coagulation of tumor
vasculature in vivo that results following the administration of a
tumor vasculature-targeted coagulant ("coaguligand") to
tumor-bearing animals and the resultant anti-tumor effects. In this
coaguligand, an antibody directed to VCAM-1 (vascular endothelial
adhesion molecule-1, VCAM-1) is used as a targeting agent to
deliver truncated Tissue Factor (tTF), a modified form of a human
coagulant, to tumor vasculature.
[0661] The MK2.7 hybridoma, secreting a rat IgG.sub.1 antibody
against murine VCAM-1, was obtained from the American Type Culture
Collection (ATCC, Rockville, Md.; ATCC CRL 1909). The R187
hybridoma, secreting a rat IgG.sub.1 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.
[0662] 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. Overall, VCAM-1 expression was observed on 20-30% of
total tumor blood vessels stained by the anti-endoglin antibody, MJ
7/18, used as a positive control. Constitutive vascular expression
of VCAM-1 was found in heart and lungs in both tumor-bearing and
normal animals. Strong stromal staining was observed in testis
where VCAM-1 expression was strictly extravascular.
[0663] 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.
Anti-VCAM-1 antibody was detected on endothelium of tumor, heart
and lung. 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.
[0664] An anti-VCAM-1tTF conjugate or "coaguligand" was prepared
using truncated tissue factor (tTF). Intravenous administration of
the anti-VCAM-1tTF coaguligand induces selective thrombosis of
tumor blood vessels, as opposed to vessels in normal tissues, in
tumor-bearing mice.
[0665] The anti-VCAM-1tTF 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. 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.
[0666] 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. 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.
[0667] The thrombotic action of anti-VCAM-1tTF 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.
[0668] In addition to the thrombosis of tumor blood vessels, this
study also shows that intravenous administration of the
anti-VCAM-1tTF coaguligand does not induce thrombosis of blood
vessels in normal organs. Despite expression of VCAM-1 on vessels
in the heart and lung of normal or L540 tumor-bearing mice,
thrombosis did not occur after anti-VCAM-1tTF 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.
[0669] 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.
[0670] 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.
[0671] The anti-tumor activity of anti-VCAM-1tTF 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. Mean
tumor volume of anti-VCAM-1tTF 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.
Example II
Phosphatidylserine Expression on Tumor Blood Vessels
[0672] To explain the lack of thrombotic effect of anti-VCAM-1tTF
on VCAM-1 positive vasculature in heart and lungs, certain of the
inventors developed a concept of differential aminophospholipid and
anionic phospholipid, e.g. PS and PE, localization between normal
and tumor blood vessels. Specifically, they hypothesized that
endothelial cells in normal tissues segregate aminophospholipids
and anionic phospholipids, e.g. PS and PE, to the inner surface of
the plasma membrane phospholipid bilayer, where PS is unable to
participate in thrombotic reactions; whereas endothelial cells in
tumors translocate aminophospholipids and anionic phospholipids to
the external surface of the plasma membrane, where PS 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.
[0673] The inventors' model of aminophospholipid and anionic
phospholipid 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. Aminophospholipid and anionic phospholipid expression
at the tumor endothelial cell surface is thus sufficiently stable
to allow aminophospholipids and anionic phospholipids, e.g. PS and
PE, to serve as targetable entities for therapeutic
intervention.
[0674] To confirm the hypothesis that tumor blood vessel
endothelium expresses PS on the luminal surface of the plasma
membrane, the inventors used the following immunohistochemical
study to determine the distribution of anti-PS antibody after
intravenous injection into L540 tumor bearing mice.
A. Methods
[0675] Anti-PS and anti-cardiolipin antibodies, both mouse
monoclonal IgM antibodies, were produced and characterized by Rote
et al. (1993, incorporated herein by reference) as described in
Example IV. The major reactivity of 3SB is with PS, but it also has
reactivity with the anionic phospholipid, phosphatidic acid, a
relatively minor component of the plasma membrane also tightly
segregated to the internal leaflet in normal cells.
[0676] 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.
[0677] 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.
[0678] 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
[0679] This immunohistochemical study showed that 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.
[0680] 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.
[0681] 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 III
Annexin V Blocks Coaguligand Activity
[0682] The present example provides further evidence of the role of
surface PS expression in coaguligand activity from studies using
the high affinity PS binding ligand, annexin V, to block PS
function in vitro and in vivo.
A. Annexin V Blocks Coaguligand Activation of Factor X In Vitro
[0683] The ability of Annexin V to affect Factor Xa formation
induced by coaguligand was determined by a chromogenic assay.
IL-1.alpha.-stimulated bEnd.3 cells were incubated with
anti-VCAM-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. Each treatment was performed in duplicate and
repeated at least twice.
[0684] 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-1tTF bound to bEnd.3 cells to generate factor Xa in
vitro.
[0685] Annexin V added to permeabilized cells preincubated with
anti-VCAM-1tTF inhibited the formation of factor Xa in a
dose-dependent manner. 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%. 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.
B. Annexin V Blocks Coaguligand Activity In Vivo
[0686] 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 and 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-1tTF; d) 100 .mu.g of Annexin V followed 2
hours later by 40 .mu.g of anti-VCAM-1tTF.
[0687] 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 was counted and the percentage of thrombosis was
calculated.
[0688] Annexin V also blocks the activity of the anti-VCAM-1tTF
coaguligand in vivo. Groups of tumor-bearing mice were treated with
one of the control or test reagents. The mice were given (a)
saline; (b) 100 .mu.g of Annexin V; (c) 40 .mu.g of anti-VCAM-1tTF
coaguligand; or (d) 100 .mu.g of Annexin V followed 2 hours later
by 40 .mu.g of anti-VCAM-1tTF coaguligand. Identical results were
obtained in both mice per group.
[0689] 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.
[0690] In accordance with other data presented herein, 40 .mu.s of
anti-VCAM-1tTF 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.
[0691] 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 IV
Generating Antibodies to Aminophospholipids, Anionic Phospholipids
and Complexes
[0692] This example describes an immunization protocol designed by
the inventors in light of their observations on aminophospholipid
and anionic phospholipid translocation in tumor vascular
endothelial cells, and discovered to function well in the
generation of antibodies against aminophospholipids and anionic
phospholipids. A number of antibodies reactive with
aminophospholipids and anionic phospholipids, such as PS and PE,
were obtained. In the present and following examples, for
simplicity, antibodies reactive with PS can be termed "anti-PS
antibodies", although the binding of certain of these antibodies is
not restricted to PS but extends to certain other
aminophospholipids and anionic phospholipids as shown herein.
A. Immunization Protocol
[0693] To present aminophospholipids and anionic phospholipids to
the immune system as stronger immunogens, the aminophospholipids
and anionic phospholipids were formulated as
aminophospholipid-positive and anionic phospholipid-positive cells.
The membrane-inserted aminophospholipids and anionic phospholipids,
surrounded by other membrane components, have a better conformation
and clearance rate for raising antibodies.
[0694] The intent is to immunize immunocompetent animals with
autologous cells expressing aminophospholipids and anionic
phospholipids, as exemplified in this instance by PS, wherein the
animals would not produce antibodies against all self surface
antigens, but would recognize membrane-exposed phospholipids, e.g.
PS, as a foreign element. The procedure is applicable to the use of
any standard laboratory animals, such as immunocompetent BALB/c
mice and Lewis rats, with any aminophospholipid-positive or anionic
phospholipid-positive cells.
[0695] BALB/c mice and mouse endothelioma cells, bEnd.3
(immortalized mouse (BALB/c strain) endothelial cells), were first
chosen. bEnd.3 were cultured in 10% DMEM with 9 ml/500 ml HEPES
Buffer, in 10% CO.sub.2 incubator. The bEnd.3 cells were expanded
in T175 TC flasks until the desired number of cells were obtained.
Typically, each flask at .about.70-80% confluency has about
3.times.10.sup.6 cells, and each mouse should receive from
1.times.10.sup.6 to 20.times.10.sup.6 cells, up to 1.times.10.sup.7
cells.
[0696] bEnd.3 cells are treated with 50 .mu.M to 200 .mu.M of
hydrogen peroxide for 1 or 2 hours at 37.degree. C. to expose
anionic phospholipids, such as PS, before immunization. The stock
of H.sub.2O.sub.2 is [9.8M]; 30% (v/v). This is diluted 1:1000,
then 0.4 ml is add into the T175 TC flask with 40 ml media to a
final concentration of 100 .mu.M H.sub.2O.sub.2. The cells were
maintained for 1 hour at 37.degree. C. To harvest, the cells were
washed 3.times. with warm PBS, +10 mM EDTA, to remove all BSA or
serum protein in the medium. The cells were removed with gentle
trypsin treatment, washed and centrifuged for 5 minutes at 1000
rpm. The supernatant was aspirated and the cells resuspended in
DMEM without additives to the appropriate volume (each mouse
receives about 1.times.10.sup.7 cells in 200 .mu.l) and kept on
ice.
[0697] Cells treated in this manner were injected (200 .mu.l of
cell suspension) into each mouse IP using 1 ml syringe and 23 gauge
needle. Mice were immunized from three to seven times at intervals
of 3 to 4 weeks. Immune sera were collected by bleeding the mice
ten days after each boost, starting from the second boost. The
serum titer for anti-PS was tested by ELISA.
[0698] These immunizations with autologous PS-positive cells did
not result in unrestricted production of autoantibodies, but were
limited to the production of antibodies reactive with PS, reactive
with PS in combination with other aminophospholipids and anionic
phospholipids and/or reactive with PS in combination with serum
proteins.
[0699] In another study, female Lewis rats were immunized with
bEnd.3 endothelial cells that had been treated with 200 .mu.M of
hydrogen peroxide for 2 h. The treatment caused translocation of
anionic phospholipids to the external surface in 70-90% of cells as
detected by .sup.125I-labeled annexin V. Treated cells were washed,
detached and counted. Two million cells were suspended in sterile
PBS and injected 5 times i.p., with the interval of 3 wk between
injections. The titer of polyclonal antibodies to anionic
phospholipids was determined 2 days after each immunization.
B. High Titer Antisera
[0700] Mice with extremely high titers of antibodies reactive with
anionic phospholipids such as PS were obtained (Table 1). The mice
did not show any signs of toxicity. Although this immunization
protocol was more effective in mice than rats overall, immunization
of rats was effective and produced the 9D2 antibody (see
below).
TABLE-US-00010 TABLE 1 Anti-PS IgG Antibody Generation Number of
Mice per Group Titer Range (% of total) 1:100-1:1,000 2/30 (6.66%)
1:1000-1:10,000 5/30 (16.6%) 1:10,000-1:100,000 18/30 (60%)
1:100,000-1,000,000 5/30 (16.6%)
[0701] In further immunizations, various mice were immunized three
times with hydrogen peroxide-treated bEnd.3 cells and the serum was
tested 54 days after the first immunization. IgG antibodies
reactive with PS within serum were detected with an anti-mouse IgG,
Fc specific secondary antibody, and IgM antibodies within serum
were detected with an anti-mouse IgG mu specific secondary
antibody. A number of effective antisera with IgG and IgM
antibodies reactive with PS were obtained using this immunization
protocol, of which the antisera with IgG antibodies were generally
more effective.
[0702] These methods can now be used to generate further particular
anti-PS antibodies, e.g., including those screened for effectively
competition with the 3G4 antibody described below. Typically, when
the IgG titer of the desired antisera for PS reaches >200,000,
but PC titer is <50,000, fusion can be performed to generate the
monoclonal antibody.
[0703] Also, these methods are not limited to initial cell
treatment with H.sub.2O.sub.2, as other methods to induce
expression of aminophospholipids and anionic phospholipids can be
used. For example, treatment with TNF and actinomycin D is another
useful method. In one case, subconfluent (.about.85% confluence)
bEnd. 3 cells were treated with 10 ng/ml mouse TNF and 1 .mu.g/ml
actinomycin D for 16 hrs at 37.degree. C. in the incubator. The
cells were then taken through the immunization procedure as
outlined above. Treatment with the membrane disrupting agent,
lysophosphatidylcholine (LPC) may also be used to induce PS
exposure.
C. IgG and IgM Monoclonal Antibodies
[0704] Hybridomas were obtained by fusing splenocytes from
immunized animals with myeloma partner P3X63AG8.653 cells (ATCC,
Rockville, Md.).
[0705] An important aspect of the inventors' technique to prepare
monoclonal antibodies useful in tumor treatment is the selection
strategy, which involves screening to select antibodies that bind
to aminophospholipids or anionic phospholipids, but not to neutral
phospholipids. Another important aspect is to select antibodies
that do not cause or significantly contribute to anti-phospholipid
syndrome.
[0706] The strategy to isolate monoclonal antibodies reactive with
PS, for example, involved screening hybridoma supernatants on
PS-coated plates using an anti-mouse IgG, Fc gamma specific
secondary antibody. Screening was first conducted against four
phospholipids (PS, phosphatidylserine; PE,
phosphatidylethanolamine; CL, cardiolipin; and PC,
phosphatidylcholine), as well as bEnd3 cells. Clones reactive with
the neutral phospholipid, PC were discarded, as were clones
non-reactive with bEnd3 cells. High binding anti-PS clones were
selected. The wells that had PS only reactivity, or strong
preference for PS were sub-cloned first, and wells that exhibited
PS reactivity in combination with binding to other anionic
phospholipids were sub-cloned second.
[0707] In certain in the following studies, mouse monoclonal IgM
antibodies termed 3SB, D11 and BA3, produced as described by Rote
et al. (1993), were also included. The 3SB antibody is described in
the literature as an anti-PS antibody and the D11 antibody is
described in the literature as an anti-cardiolipin (anti-CL)
antibody. Details of the generation and characterization of these
antibodies were reported by Rote et al. (1993, incorporated herein
by reference).
[0708] The isotype of each selected hybridoma generated by the
inventors was determined. As antibodies of IgG class have numerous
advantages over IgM, including typically higher affinity, lower
clearance rate in vivo and simplicity of purification, modification
and handling, their generation was particularly desired. To focus
on wells with homogeneous IgG isotype, wells containing IgM or a
mixture of different Igs were discarded or re-cloned. Sub-cloning
of highly positive clones was repeated three to four times.
[0709] The isotype of representative IgG and IgM antibodies, as
determined by ELISA, is shown in Table 2. The inventors initially
termed the 3G4 antibody "F3-G4", before changing the designation to
3G4. This does not reflect any change in biological material. The
serum dependence or independence of the antibodies is also set
forth in Table 2 (see also, Example XXX).
TABLE-US-00011 TABLE 2 Isotype and Serum-Dependence of Anti-PS
Antibodies Serum Name Origin Species/Isotype cofactor? 3SB Rote et
al., 1993 Mouse IgM kappa None D11 N. Rote Mouse IgM kappa BA3 Rote
et al., 1993 Mouse IgM kappa 3G4 This study Mouse IgG.sub.3 kappa
Yes, .beta.2-glycoprotein I 2aG4 This study Mouse IgG.sub.2a Yes
Ch3G4 This study Human chimeric IgG.sub.1 Yes 9D2 This study Rat
IgM kappa No P2D9 This study Mouse IgG.sub.3 1B12 This study Mouse
IgG.sub.1 kappa 1B9 This study Mouse IgG.sub.1 kappa Yes 3B10 This
study Mouse IgG.sub.3 kappa Yes, at least .beta.2-glycoprotein I
2G7 This study Mouse IgG.sub.1 kappa Yes 7C5 This study Mouse
IgG.sub.1 kappa Yes 3F8 This study Mouse IgG.sub.3 Annexin No V
D. ELISA Protocol and Initial Antibody Characterization
[0710] The antibodies were studied further by ELISA and compared to
3SB and D11. The anti-PS ELISA used in the present studies example
is conducted as follows. Unless particular differences are
specified, this is the format of the ELISA used throughout the
studies of the present application. Other types of ELISA were later
developed and results from those studies are set forth in Example
XXX.
[0711] The ELISA of the present example is exemplified using the
antigen PS (P-6641 25 mg 10 mg/ml (solvent is Chloroform:MeOH 95:5)
in 2.5 ml bottle). Other phospholipids can be used using the same
protocol. The PS (or other phospholipids) stock solution should be
aliquoted and stored in an airtight container at -30.degree. C. The
preferred 96 well plates are Dynatech U bottom Immulon 1 (from
Dynatech Labs, Cat#011-010-3550).
[0712] The standard blocking buffer used in the present example is
10% bovine serum dissolved in PBS. Other blocking solutions are
suitable, but any detergents should be excluded from block and wash
solutions. The primary antibody is the test sample or admixture.
The preferred secondary antibody is goat, anti-mouse IgG-HRP. The
developing solutions are: 10 ml of 0.2M Na.sub.2PO.sub.4, 10 ml of
0.1M citric acid, one 10 mg tablet of OPD, and 10 .mu.l of hydrogen
peroxide. The stop solution is 0.18 M H.sub.2SO.sub.4.
[0713] The protocol of the present example entails coating 96-well
plate with PS as follows: dilute the PS stock solution in n-hexane
to 10 .mu.g/ml and mix well. Add 50 .mu.l to each well and allow
this to evaporate for one hour. Add 200 .mu.l of 10% serum (or
other blocking buffer) to each well, cover and maintain at room
temperature for 2 hours or overnight at 4.degree. C. Wash the plate
three times with PBS. Add the primary antibody (dilute in blocking
buffer) and incubate for 2 hours at 37.degree. C. Wash three times
with PBS. Add 100 .mu.l/well of secondary antibody (typically goat,
anti-mouse IgG-HRP or other appropriate secondary antibody) and
incubate for 1 hour at 37.degree. C. Wash the plate three times
with PBS. Develop the ELISA by adding 100 .mu.l of developing
solution to each of the wells, develop for 10 minutes, then add 100
.mu.l of stop solution to each plate and read the O.D. at 490
nm.
[0714] The following results are presented for 9D2, 1B12, 3G4 and
1B9. The affinity of these antibodies for PS was determined and
compared to 3SB. Certain of the relative affinities of the new
antibodies are much improved compared to 3SB (Table 3).
TABLE-US-00012 TABLE 3 Relative Affinity of Anti-PS Antibodies
EC.sub.50 Binding vs. 3SB EC.sub.50 Affinity vs. 3SB Name
(.mu.g/ml).sup.1 (-fold increased) (nM).sup.2 (-fold increased) 3SB
0.468 1 0.518 1 D11 >40.0 0.011 >44.4 0.011 9D2 0.104 4.50
0.115 4.50 1B12 0.312 1.50 2.07 0.25 3G4 0.040 11.7 0.266 1.94 1B9
0.019 24.6 0.126 4.11 Annexin V.sup.3 0.100 4.68 2.77 0.18
.sup.1Based on dilutions of Tissue Culture supernatants;
concentration of IgG and IgM were determined by sandwich ELISA
using either anti-mouse or rat Igs as capturing Antibodies. All
clones secrete in average 10 to 15 .mu.g/ml of Ig. .sup.2MW used
for conversion: IgM - 900 kDa, IgG - 150 kDa, Annexin V - 36 kDa
.sup.3Affinity of Annexin V to PS is in the range of 0.1 nM to 1
nM. The value in this table represents binding of commercial
biotinylated Annexin V detected by streptavidin-HRP using the same
ELISA conditions as for anti-PS antibodies.
[0715] The specificity of the antibodies was determined by ELISA
using plates coated with the following phospholipids: PS,
phosphatidylserine; PE, phosphatidylethanolamine; PI,
phosphatidylinositol; PA, phosphatidic acid; PG,
phosphatidylglycerol; PC, phosphatidylcholine; CL, cardiolipin; and
SM, sphingomyelin. The specificity profiles of 9D2, 1B12, 3G4 and
1B9, as compared to 3SB and D11, are shown in Table 4.
TABLE-US-00013 TABLE 4 Phospholipid Specificity of Anti-PS
Antibodies Relative Strength of Name Reactivity on ELISA.sup.1,2
3SB PS = PA >> CL, PI, PE, PG D11 CL = PA >> PS, PI,
PE, PG 3G4 PS = PA = PI = PG = CL >> PE 2aG4 PS = PA = PI =
PG = CL >> PE Ch3G4 PS = PA = PI = PG = CL >> PE 9D2 PA
> PS = CL > PG = PI >> PE P2D9 PS > PE = CL (No PC)
1B12 PS = PA > CL > PE = PI, PG 3B10 PS = PA = PI >> PE
1B9 PS only 2G7 PS only 7C5 PS only 3F8 PS > PE > CL (little
or no PC) Annexin V PS = PE = PI = PA > CL > PG .sup.1The
symbol > indicates at least 2-fold difference in binding to
various phospholipids tested at identical antibody concentration.
.sup.2The symbol >> indicates at least 10-fold difference in
binding to various phospholipids tested at identical antibody
concentration.
[0716] The 1B9, 2G7 and 7C5 antibodies behave essentially the same.
These antibodies recognize only PS and require serum or serum
proteins for binding to PS. The binding of 1B9, 2G7 and 7C5 to
various phospholipids in the present example was assayed only in
the presence of 10% bovine serum, whereas binding of the other
antibodies was tested either in the absence or in the presence of
serum. 3SB is essentially devoid of reactivity with
phosphatidylethanolamine and phosphatidylinositol, as well as
phosphatidylcholine and sphingomyelin (Table 4).
E. Further Antibody Characterization
[0717] The reactivity of the 3G4 antibody with plastic-immobilized
phospholipids was further tested. Phospholipids were dissolved in
n-hexane to a concentration of 50 .mu.g/ml. 100 .mu.l of this
solution was added to wells of 96-well microtiter plates. After
evaporation of the solvent in air, the plates were blocked for 2 h
with 10% FBS diluted in DPBS containing 2 mM Ca.sup.2+ (binding
buffer). The 3G4 antibody was diluted in the binding buffer in the
presence of 10% bovine serum at an initial concentration of 33 nM.
Serial two-fold dilutions were prepared in the plates (100 .mu.l
per well). The plates were then incubated for 2 hr. at room
temperature. After washing, HRP goat anti-mouse IgG (diluted
1:1000) was used to detect 3G4. Secondary reagents were detected by
using chromogenic substrate OPD followed by reading plates at 490
nm using a microplate reader (Molecular Devices, Palo Alto,
Calif.).
[0718] Specificity of phospholipid recognition was further
confirmed by competition assays with various liposomes. Liposomes
were prepared from solutions of 5 mg of a single phospholipid (PS,
PI, PC, CL, PA) in chloroform. The solutions were dried under
nitrogen to form a thin layer in a round-bottomed glass flask. 10
ml of Tris buffer (0.1 M, pH 7.4) were then added and the flask was
sonicated five times for 2 min. 3G4 (6.6 nM) was pre-incubated with
200 .mu.g/ml of liposome solution for 1 h at room temperature. The
mixture was added to phospholipid-coated plates. The ability of 3G4
to bind to an immobilized phospholipid in the presence or absence
of the different liposomes was determined as described above.
[0719] The ongoing studies showed that 3G4 is a mouse IgG.sub.3
.kappa. monoclonal antibody that specifically recognizes anionic
phospholipids. It binds strongly to ELISA plates coated with
anionic phospholipids (PS, PA, CL, PI) in the presence of 5% bovine
serum (FIG. 4) or human serum. Half-maximal binding was observed
with 3G4 at concentrations of 0.2 to 0.4 nM (FIG. 4). 3G4 does not
bind neutral phospholipids (PE, PC and SM) in ELISA. Control mouse
IgG.sub.3 monoclonal antibodies of irrelevant specificity did not
bind. Binding was blocked by liposomes prepared from anionic
phospholipids, but not from liposomes prepared from neutral
phospholipids (FIG. 3).
[0720] 3G4 bound to ELISA plates coated with synthetic PS, PA and
CL having saturated (non-oxidizable) dipalmitoyl side chains and to
lysophosphatidic acid having a single fatty acid side chain.
Binding was unaffected by the presence of 5 mM EDTA, showing that
binding is not dependent on Ca.sup.2+.
[0721] In the studies of the present example, in the absence of
serum, binding to ELISA plates coated with anionic phospholipids
was reduced by 90%. Full binding was restored in the presence of 1
mg/ml human .beta.2-glycoprotein I. Binding was unaffected by
prothrombin, protein protein C, protein S, oxidized LDL, HMW
kininogen, LMW kininogen, factor XII, tissue plasminogen activator
or annexin A5. Thus, the 3G4 antibody has now been discovered to
recognize anionic phospholipids in the presence of serum or
.beta.2-glycoprotein I. Results from further studies relating to
this characterization are presented herein in Example XXX.
[0722] PS is the most abundant anionic phospholipid of the plasma
membrane and is tightly segregated to the internal leaflet of the
plasma membrane in normal cells under normal conditions. PS is an
aminophospholipid. PE is also an aminophospholipid, but PE is
neutral, not anionic. Other than being a neutral aminophospholipid,
PE behaves similarly to PS and is normally tightly segregated to
the internal leaflet of the plasma membrane.
[0723] PI is another major anionic phospholipid of the plasma
membrane, which is further tightly segregated to the internal
leaflet in normal cells under normal conditions. PA and PG are
minor anionic phospholipids of the plasma membrane
(Hinkovska-Galcheva et al., 1989), which are also normally
segregated to the internal leaflet. CL is an anionic phospholipid
present in mitochondrial membranes, and typically absent from the
plasma membrane.
[0724] PC and SM are choline-containing, neutral phospholipids of
the plasma membrane. Each of PC and SM are predominantly located on
the external leaflet under normal conditions.
[0725] In keeping with the inventors' model for differential
aminophospholipid and anionic phospholipid expression between
normal and tumor blood vessels, none of the antibodies developed
using the selected protocol reacted with the neutral phospholipids,
PC and SM. The 1B9 antibody was specific for PS, whereas 9D2, 1B12
and 3G4 bound to anionic phospholipids and aminophospholipids with
the preferences shown in Table 4. The 9D2 antibody is also
described in Example VI.
Example V
Externalized Phosphatidylserine is a Global Marker of Tumor Blood
Vessels
[0726] The present example shows that the exposure of PS occurs on
endothelial cells in each of ten different solid tumors growing in
mice and is not limited to the L540 tumor model described in
Example II.
[0727] Externalized PS in vivo was detected by injecting a
monoclonal antibody directed against PS intravenously into mice
bearing various types of human or murine tumors. Anti-PS antibodies
are shown to bind specifically to vascular endothelium in all ten
different tumor models. Vascular endothelium in normal organs
derived from the same mice were unstained. An isotype-matched
control monoclonal antibody did not localize to either tumor or
normal cells. Apoptotic cells were also identified
immunohistochemically, wherein very few endothelial cells in tumors
expressed markers of apoptosis.
[0728] The present example therefore shows that vascular
endothelial cells in tumors but not in normal vessels externalize
PS. Most of the tumor endothelial cells having exposed PS were not
apoptotic. PS is thus an abundant and accessible marker of tumor
vasculature that can be used for tumor vessel imaging and
therapy.
A. L540, H358 and HT29 Tumors
[0729] The anti-PS antibody used in these studies was the mouse
monoclonal IgM antibody termed 3SB (Example IV, Rote et al., 1993).
3SB mainly binds to PS, but also reacts with PA, a relatively minor
anionic phospholipid with a distribution like PS. The anti-CL
antibody used was the mouse monoclonal IgM antibody termed D11
(Example IV, Rote et al., 1993).
[0730] PS exposure on tumor and normal vascular endothelium was
first examined in three animal tumor models: L540 human Hodgkin's
lymphomas, NCI H358 human non-small cell lung carcinoma (NSCLC) and
HT29 human colorectal carcinomas. To grow the tumors in vivo,
2.times.10.sup.6 cells were injected into the right flank of SCID
mice and tumors allowed to reach 0.8-1.2 cm in diameter.
[0731] 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-CL antibodies. 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 for preparation of cryosections. Mouse IgM was detected
using goat anti mouse IgM (.mu. specific)-HRP conjugate followed by
development with carbazole. At least 10 random fields per section
were examined at .times.40 magnification and the average percentage
of positive vessels was calculated.
[0732] 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. In this first study, 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.
[0733] No vessel staining was observed with anti-PS antibodies in
any normal tissues. In the kidney, tubules were stained in both
anti-PS and anti-CL recipients, and this relates to the secretion
of IgM through this organ. Anti-CL antibodies were not detected in
any tumors or normal tissues, except kidney. These findings
indicate that only tumor endothelium exposes PS to the outer site
of the plasma membrane.
B. Small and Large L540 Tumors
[0734] To estimate the time at which tumor vasculature loses the
ability to segregate PS to the inner side of the membrane, anti-PS
localization was examined in L540 tumors ranging in volume from 140
to 1,600 mm.sup.3.
[0735] 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 5). 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 5).
TABLE-US-00014 TABLE 5 PS Externalization Detected in Mid and Large
Sized Tumors Tumor Size No. Positive % PS-Positive (mm.sup.3)
Tumors/Total* Vessels/Total.dagger. 350-800.sup. 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.
C. L540, H358, HT29, Colo26, B16 and 3LL Tumors
[0736] Using the same anti-PS (3 SB) and anti-CL (D11) antibodies,
PS exposure on tumor and normal vascular endothelium was examined
in further studies using an additional three animal tumor models
(six in total): L540 human Hodgkin's lymphomas, NCI H358 human
non-small cell lung carcinoma (NSCLC), HT29 human colorectal
carcinomas, Colo26 mouse colon carcinomas, B16 mouse melanomas and
3LL mouse lung tumors.
[0737] In these studies, tumors were grown subcutaneously in SCID
mice and allowed to reach a volume of 0.4-0.7 cm.sup.3. Three or
more mice were used per group. Anti-PS or anti-CL mouse IgM
antibodies (30 .mu.s/mouse) were injected intravenously in 200
.mu.l of saline. Thirty minutes later, the mice were sacrificed,
exsanguinated and their blood circulation perfused with heparinized
saline. Major organs and tumors were harvested and snap-frozen for
preparation of cryosections. Mouse IgM was detected using goat anti
mouse IgM (.mu. specific)-HRP conjugate followed by development
with carbazole.
[0738] Serial sections of tumor were stained with a monoclonal
antibody, MECA 32, directed against a pan-endothelial marker of
mouse vessels. PS-positive vessels were identified morphologically
and by their coincident staining with anti-mouse IgM and MECA 32.
At least 10 random fields per section (0.317 mm.sup.2/field) were
examined in blinded fashion by two independent observers. The
percentage of MECA 32-positive vessels that stained positively for
PS was calculated. Three tumors of each type were examined in each
of two separate studies. The mean values and standard errors (SE)
were calculated. Inter-tumor variation in the number of total and
PS-positive vessels in each group was approximately 10%.
[0739] All six tumors in this study contained PS-positive vessels
(Table 6A). Detection of PS by 3SB was specific since no staining
of tumor endothelium was observed with the anti-CL antibody (Table
6A). No vascular localization of anti-PS or anti-CL antibodies was
observed in normal organs other than the kidneys (tubule staining
in both anti-PS and anti-CL recipients reflects secretion of IgM
through this organ).
TABLE-US-00015 TABLE 6A Specific Localization of Anti-PS Antibodies
to Tumor Vessels Tissue Anti-PS* Anti-CL L540 tumor 19.3 .+-. 3.3 0
H358 tumor 15.6 .+-. 4.1 0 HT29 tumor 4.2 .+-. 1.6 0 B16 tumor 40.6
.+-. 5.4 0 3LL tumor 5.3 .+-. 3.7 0 Colo 26 tumor 12.4 .+-. 2.4 0
Adrenal 0 0 Brain 0 0 Heart 0 0 Kidney .sup. 0 .sup..dagger. .sup.
0 .sup..dagger. Intestine 0 0 Liver 0 0 Lung 0 0 Pancreas 0 0
Spleen 0 0 Testis 0 0 *The results are presented as the mean
(.+-.SE) percentage of PS-positive vessels of MECA 32-stained
vessels per field of 0.317 mm.sup.2. Six tumors of each type were
analyzed. The average number of MECA 32-positive vessels per 0.317
mm.sup.2 field was 25, 21, 17, 18, 27 and 22 .+-. 10% vessels for
L540, H358, HT29, B16, 3LL and Colo 26 tumors, respectively
.sup..dagger. Non-antigen specific tubular staining was visible in
both anti-PS and anti-CL recipients.
[0740] In these studies, the percentage of PS-positive vessels
ranged from 10% in Colo 26 tumors to 40% in B16 tumors. Anti-PS IgM
was present on the luminal surface of capillaries and venules in
all regions of the tumors. PS-positive vessels appeared to be
particularly prevalent in and around regions of necrosis. Positive
vessels usually did not show morphological abnormalities that were
apparent by light microscopy. Occasional vessels located in
necrotic areas showed morphological signs of deterioration. Anti-PS
antibody (but not anti-CL antibody) also localized to necrotic and
apoptotic tumor cells.
[0741] These controlled studies demonstrate that PS is consistently
exposed on the luminal surface of vascular endothelial in various
tumors, but not in normal tissues, and that the tumor vasculature
expression is not model-specific.
D. The Majority of PS-Positive Tumor Vessels are not Apoptotic
[0742] A double labeling technique was used to identify apoptotic
endothelial cells in tumor sections. Endothelial cells were
identified with the pan-endothelial cell marker, MECA 32. Apoptotic
cells were identified immunohistochemically using two independent
markers: an active form of caspase-3, which identifies cytosolic
changes in dying cells (Krajewska et al., 1997), and fragmented
DNA, which identifies cells having nuclear alterations (Gavrieli et
al., 1992).
[0743] Active caspase-3 was detected by a rabbit anti-caspase-3
specific antibody (R&D, Minneapolis, Minn.) followed by
incubation with anti-rabbit IgG conjugated to alkaline phosphatase
(AP, Pierce, Rockford, Ill.). Other tumor sections were analyzed by
Tunel assay (ApopTag.TM. kit, Oncor, Md.) using
anti-digoxigenin-alkaline phosphatase conjugate as a detecting
reagent. Sections were double stained for apoptosis markers (pink)
and the endothelial cell marker, MECA 32 (brown). Both colors were
clearly visible on the same cells, if markers of endothelial cells
and apoptotic cells coincided.
[0744] Endothelial cells in five out of six types of tumors (HT29,
H358, B16, Colo 26, L540) did not display either of the apoptosis
markers (Table 7). The sixth type of tumor, 3LL, displayed a few
apoptotic endothelial cells that were located in necrotic areas. In
contrast, apoptotic malignant cells were common in all types of
tumors. The percentage of apoptotic tumor cells ranged from 1-2% in
L540 tumors to 12.6-19.6% in 3LL tumors.
TABLE-US-00016 TABLE 7 Expression of Apoptotic Markers in Tumors
Active caspase-3 Tunel assay Tumor cells Tumor Tumor cells Tumor
Tumor type (% of total)* vessels (% of total) vessels 3LL 19.8 .+-.
4.3 <1.0 .sup..dagger. 12.6 .+-. 3.6 0 HT29 13.7 .+-. 2.3 0 7.8
.+-. 2.5 0 H358 5.8 .+-. 2.0 0 4.3 .+-. 1.6 0 Colo 26 5.3 .+-. 1.5
0 4.1 .+-. 1.5 0 B16 4.2 .+-. 1.8 0 3.5 .+-. 1.6 0 L540 2.3 .+-.
1.0 0 1.6 .+-. 0.5 0 *The percentage of tumor cells or tumor blood
vessels that were positive for either caspase-3 or Tunel was
determined in ten high power fields per section. The fields were
randomly selected along two perpendicular directions from the edges
through the center of the tumor. The mean (.+-.SE) of the
percentage of positive cells or vessels in tumors from 6 mice is
presented. .sup..dagger. Occasional vessels (1 of >100) in the
necrotic area of 3LL tumor displayed both markers of apoptosis.
E. MDA-MB-231 and Meth A Tumors
[0745] PS exposure on tumor vascular endothelium was also examined
in MDA-MB-231 human breast tumors growing in mice and in mouse Meth
A fibrosarcoma growing subcutaneously. The antibody used in these
studies was the 9D2 antibody, generated as described in Example IV,
which is reactive with anionic phospholipids.
[0746] As described in detail in Example VI, 9D2 localized to tumor
vessels in L540, NCI-H358 and B16 tumors, as well as in models of
MDA-MB-231 breast tumor growing orthotopically in the mammary fat
pads of SCID mice and mouse Meth A fibrosarcoma growing
subcutaneously. 9D2 localized to tumor vessels in all of five
tumors. Vascular endothelium in the tumors showed distinct membrane
staining. 9D2 antibody also localized to the membrane and cytosol
of necrotic and apoptotic tumor cells. No vascular localization of
9D2 antibody was observed in 9 of the 10 normal organs that were
examined, with non-specific staining of the tubules in the kidney
being observed.
[0747] Double-staining studies were also performed in which mice
bearing orthotopic MDA-MB-231 breast tumors were injected i.v. with
biotinylated 9D2 antibody and frozen sections later stained with
FITC-conjugated MECA32 (Example VI). About 40% of MECA 32-positive
vessels bound 9D2.
F. MD-MBA-435 Tumors
[0748] In a further breast cancer model, PS exposure on tumor
vascular endothelium was examined in MDA-MB-435 human breast cancer
cells growing in mice. The antibody used in these studies is a
chimeric version of the 3G4 antibody (ch3G4). The 3G4 antibody
generation is described in Example IV, and the production of the
chimeric 3G4 antibody is detailed in Example XIX. The localization
of ch3G4 to tumor vascular endothelium in the MDA-MB-435 model is
described in more detail in Example XIX.
[0749] Briefly, tumors were established using MD-MBA-435s cells and
biotinylated versions of the chimeric 3G4 antibody and a control
IgG of irrelevant specificity were administered. Tumor sections
were stained with Cy3-conjugated streptavidin to detect the
biotinylated proteins. Double staining with the MECA 32 antibody
followed by FITC-tagged anti-rat IgG secondary antibody was
conducted to detect vascular endothelium. This detection method
labeled the biotinylated proteins and the vascular endothelium
using red and green, so that biotinylated proteins bound to the
endothelium appear yellow in a converged image. This study showed
specific localization of the chimeric 3G4 antibody to tumor
vascular endothelium.
[0750] In similar studies, the ability of 3G4 to localize
selectively to tumor blood vessels in vivo was determined by
injecting the antibody i.p. or i.v. and exsanguinating the mice 1
h, 6 h or 24 h later. Frozen sections of tumor and normal tissues
were stained for the presence of mouse immunoglobulin. SCID mice
that had been confirmed as having no detectable circulatory
immunoglobulin were used to avoid background staining. In these
studies, sections were counterstained with anti-mouse CD31 to
detect vascular endothelium and the images were merged. Coincidence
of staining between localized 3G4 and CD31 was taken as evidence of
specific localization. Coincident staining appeared yellow, unless
dominated by a particularly intense green or red fluorescence in
that region. The antigen specificity of vessel localization was
confirmed by the lack of endothelial staining in tumors from mice
injected with the isotype-matched control antibodies, BBG3.
[0751] In these studies, 3G4 localized to an average of 40.+-.10%
of tumor blood vessels after i.p. or i.v. injection into mice
bearing orthotopic MDA-MB-435 breast tumors, as determined by the
merged images. Localization to tumor vessels after i.p. injection
of 3G4 was visible 1 hr. after injection and was maximal by 6 hr.
after injection, whereas i.v. injected 3G4 gave maximal staining
within 1 hr. after injection. Labeled vessels were visible in all
regions of the tumors, but were particularly abundant in and around
regions of necrosis. In the larger vessels, heterogeneity of PS
exposure within a single vessel was sometimes observed. Regions
where 3G4 had leaked into the tumor interstitium were also visible
around the endothelium of some vessels. Tumor cells in and around
regions of tumor necrosis were stained. No staining of necrotic
tumor cells was observed in tumors from mice injected with the
control antibody, BBG3, indicating that the localization to
necrotic tumor cells in mice injected with 3G4 was
antigen-specific.
[0752] Localization of 3G4 to vascular endothelium in normal
tissues was not observed in mice injected i.v. with 3G4 or control
antibody (BBG3) 4 hr. earlier. Normal tissues examined were: heart,
lung, liver, gallbladder, esophagus, stomach, pancreas, duodenum,
cecum, rectum, kidney, adrenal gland, spleen, brain, eye, salivary
gland and ovary. Non-vascular components of these normal tissues
were also unstained.
G. RIP-Tag Tumors
[0753] For the tenth model, PS exposure on tumor vascular
endothelium was examined in a "RIP-Tag" transgenic mouse model
(RIP1-Tag 2) of multistage carcinogenesis. In this transgenic mouse
model, every mouse develops islet tumors of the pancreas by 12-14
weeks of age as a result of expression of the SV40 T antigen (Tag)
oncogene in insulin-producing beta-cells. Tumors develop in
multiple stages from hyper-proliferative islets, and require an
angiogenic switch in order to progress towards malignancy. Matrix
metalloprotinase-9 controls the angiogenic switch (REF).
[0754] 9D2 localization studies were conducted in the RIP1-Tag2
model in collaboration with Dr. Donald McDonald, Professor of
Pathology at UCSF. 9D2 was injected intravenously into RIP1-Tag2
mice starting at 10 weeks of age, when all mice have small, highly
vascularized, solid tumors. Double staining of thick (80 .mu.m)
tumor sections was performed to identify localized 9D2 and CD31 in
tumors and normal pancreas. Approximately 50% of vessels (CD31
positive) in pancreatic tumors had localized 9D2, whereas vessels
in normal islets were unstained. Mice injected with control rat IgM
had weak and infrequent staining of tumor vessels. Some leakage of
9D2 and control rat IgM into extravascular tissues beyond the
endothelium was also apparent.
H. Summary of Tumor Localization Studies
[0755] The inventors have now studied the localization of various
anti-PS antibodies to tumor blood vessels in mice bearing many
different tumors. The combined results from such studies are
summarized below in Table 6B. Anti-PS antibodies localize to tumor
blood vessels in all tumors, whereas no vascular localization is
observed in normal organs (Table 6B).
TABLE-US-00017 TABLE 6B Localization of Anti-PS Antibodies to Tumor
and Normal Vessels Tissues Localization Tumor Tissues L540
Hodgkin's + + H358 NSCLC + + HT29 colon + + + Colo 26 colon + + B16
melanoma + + + 3LL lung + + + MDA-MB-231 + + + MDA-MB-435 + + +
Rip-Tag + + + Normal Tissues Adrenal - Brain - Heart - Kidney -
Intestine - Liver - Lung - Pancreas - Spleen - Testis -
[0756] The present example therefore confirms that vascular
endothelial cells in tumors externalize PS and anionic
phospholipids to their luminal surface, where they can be bound by
anti-PS antibodies in vivo. PS is absent from the external surface
of vascular endothelial cells in normal tissues, indicating that
PS-recognizing antibodies, annexin V and other ligands can be used
for delivering cytotoxic drugs, coagulants and radionuclides for
the selective imaging or destruction of vessels in solid
tumors.
[0757] PS-positive tumor endothelium appeared, for the most part,
to be viable in the tumors used in this study. It does not display
markers of apoptosis, it is morphologically intact and
metabolically active, as indicated by its expression of VCAM-1,
E-selectin and other rapidly turned-over proteins. Although often
regarded as an indicator of apoptosis, PS exposure has been
observed in several types of viable cells, including malignant
cells (Rao et al., 1992), (Utsugi et al., 1991) activated platelets
(Rote et al., 1993), and embryonic trophoblasts at various stages
of migration, matrix invasion and fusion (Adler et al., 1995).
[0758] Lack of correlation between PS exposure and commitment to
cell death has been also shown on pre-apoptotic B lymphoma cells
that restore PS asymmetry and grow normally after removal of the
pro-apoptotic stimulus (Hammill et al., 1999). In normal viable
cells, PS exposure is probably triggered by surface events, such as
ligand-receptor interactions, that induce Ca.sup.2+ fluxes into the
cells (Dillon et al., 2000). Ca.sup.2+ fluxes activate scramblase
(Zhao et al., 1998) and simultaneously inhibit aminophospholipid
translocase (Comfurius et al., 1990).
[0759] PS on tumor vessels is attractive as a target for cancer
imaging or therapy for several reasons: it is abundant
(approximately 3.times.10.sup.6 molecules per cell); it is on the
luminal surface of tumor endothelium, which is directly accessible
for binding by vascular targeting agents in the blood; it is
present on a high percentage of tumor endothelial cells in diverse
solid tumors, and it is absent from endothelium in all normal
tissues examined to date. Unconjugated antibodies, vascular
targeting agents and imaging agents directed against PS on tumor
vasculature can therefore be used for the detection and treatment
of cancer in man.
Example VI
Anionic Phospholipids are Exposed on the Surface of Tumor Blood
Vessels
[0760] Anionic phospholipids are largely absent from the external
leaflet of the plasma membrane of mammalian cells under normal
conditions. Exposure of phosphatidylserine, for example, on the
cell surface occurs during apoptosis, necrosis, cell injury, cell
activation and malignant transformation. The present example shows
that anionic phospholipids are upregulated on tumor vasculature in
vivo, as demonstrated by localization of both a specific antibody
and a natural ligand that binds to anionic phospholipids.
[0761] A monoclonal antibody, 9D2, which specifically recognizes
anionic phospholipids, was injected into mice bearing a variety of
orthotopic or ectopic tumors. Other mice received annexin V, a
natural ligand that binds to anionic phospholipids. Both 9D2 and
annexin V specifically localized to vascular endothelium in all
tumors and also to tumor cells in and around regions of necrosis.
Between 15 and 40% of endothelial cells in tumor vessels were
stained. No localization was detected on normal endothelium.
[0762] Various factors and tumor-associated conditions known to be
present in the tumor microenvironment were examined for their
ability to cause exposure of anionic phospholipids in cultured
endothelial cells, as judged by 9D2 and annexin V binding.
Hypoxia/reoxygenation, acidity, thrombin and inflammatory cytokines
all induced exposure of anionic phospholipids. Hydrogen peroxide
was also a strong inducer. Combined treatment with inflammatory
cytokines and hypoxia/reoxygenation had greater than additive
effects. The demonstrated exposure of anionic phospholipids on
tumor endothelium in vivo is thus likely to be caused by injury and
activation by cytokines and reactive oxygen species. Irrespective
of the mechanism, anionic phospholipids are markers of tumor
vessels that can now be used for tumor vessel targeting, imaging
and therapy.
A. Materials and Methods
1. Materials
[0763] Na.sup.125I was obtained from Amersham (Arlington Heights,
Ill.). Dulbecco's modified Eagle's tissue culture medium 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). L-.alpha.-phosphatidylserine,
L-.alpha.-phosphatidylcholine, cardiolipin,
L-.alpha.-phosphatidylethanolamine, L-.alpha.-phosphatidylinositol,
sphingomyelin, phosphatidic acid, phosphatidylglycerol,
O-phenylenediamine, hydrogen peroxide and thrombin were from Sigma
(St. Louis, Mo.). Flat bottom plates with 24 wells were obtained
from Falcon (Becton Dickinson and Co., Lincoln Park, N.J.).
[0764] Recombinant hepatocyte growth factor (HGF or scatter factor)
and actinomycin D was from Calbiochem (San Diego, Calif.).
Recombinant murine interleukin-1 alpha, beta and tumor necrosis
factor alpha (TNF .alpha.) were purchased from R&D Systems
(Minneapolis, Minn.). Interferon of Universal Type I (hybrid
protein that substitutes for all types of interferons) was
purchased from PBL Biomedical Laboratories (New Brunswick, N.J.).
Recombinant human vascular endothelial growth factor 121 (VEGF),
human platelet-derived growth factor-BB, interleukin-6 (IL-6),
interleukin-8 (IL-8), interleukin-10 (IL-10) and human fibroblast
growth factor-2 (FGF-2) were purchased from PeproTech (Rocky Hill,
N.J.).
2. Antibodies
[0765] MECA 32, a pan mouse endothelial cell antibody, was obtained
from Dr. E. Butcher (Stanford University, CA) and served as a
positive control for immunohistochemical studies. Details of this
antibody have been published (Leppink et al., 1989). Rabbit
anti-rat immunoglobulin, rat-anti mouse immunoglobulin and
goat-anti mouse and anti-rat secondary antibodies conjugated to
horseradish peroxidase (HRP) were purchased either from Daco
(Carpinteria, Calif.) or from Jackson Immunoresearch Labs (West
Grove, Pa.).
[0766] The 9D2 antibody used in these studies was generated as
described in Example IV. 9D2 is a rat monoclonal antibody reactive
with anionic phospholipids. Further characterization of the
phospholipid specificity of 9D2 is given in the results section of
this example.
3. Cells
[0767] L540Cy Hodgkin lymphoma cells, derived from a patient with
end-stage disease, were provided by Prof. V. Diehl (Koln, Germany).
NCI-H358 human non-small cell lung carcinoma was provided by Dr.
Adi Gazdar (Southwestern Medical Center, Dallas, Tex.). Meth A
mouse fibrosarcoma and MDA-MB-231 human breast carcinoma were
obtained from American Type Cell Collection (Rockville, Md.). The
mouse brain endothelioma line, bEnd.3, was provided by Prof. Werner
Risau (Max Plank Institution, Munich, Germany) and was maintained
in DMEM with 10% FBS. Adult bovine aortic endothelial (ABAE) cells
were purchased from Clonetics (San Diego, Calif.; Walkerville,
Md.). ABAE cells were maintained in DMEM with 10% serum and 2 ng/ml
of bFGF.
4. Tissue Culture
[0768] bEnd.3, ABAE cells and all tumor cells except L540Cy
lymphoma 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. L540Cy cells were maintained in RPMI 1640 containing
the same additives. Cells were sub-cultured once a wk.
Trypsinization of bEnd.3 cells was performed using 0.125% trypsin
in PBS containing 0.2% EDTA. For in vitro studies, endothelial
cells were seeded at a density of 10.times.10.sup.3 cells/ml in 1
ml of culture medium in 24 well plates and incubated 48-96 h before
being used in the assays. Medium was refreshed 24 h before each
study.
5. Reactivity with Plastic-Immobilized Phospholipids
[0769] Phospholipids were dissolved in n-hexane to a concentration
of 50 .mu.g/ml. 100 .mu.l of this solution was added to wells of
96-well microtiter plates. After evaporation of the solvent in air,
the plates were blocked for 2 h with 10% fetal bovine serum diluted
in DPBS containing 2 mM Ca.sup.2+ (binding buffer).
[0770] 9D2 antibody or annexin V were diluted in the binding buffer
in the presence of 10% serum at an initial concentration of 6.7 nM.
Serial two-fold dilutions were prepared in the plates (100 .mu.l
per well). The plates were then incubated for 2 h at room
temperature. The plates were washed and the 9D2 and annexin V were
detected by goat anti-rat IgM conjugated to HRP and rabbit
anti-human annexin V followed by goat anti-rabbit IgG conjugated to
HRP (all diluted 1:1000), respectively. Secondary reagents were
detected by using chromogenic substrate OPD followed by reading
plates at 490 nm using a microplate reader (Molecular Devices, Palo
Alto, Calif.).
[0771] The specificity of the 9D2 antibody binding was validated by
using control rat IgM of irrelevant specificity (Pharmingen, San
Diego, Calif.). The specificity of annexin V binding to
phospholipids, which is Ca.sup.2+-dependent, was determined by
diluting the reagent in the DPBS containing 5 mM EDTA. Additional
negative controls consisted of washing the plates with the binding
buffer containing 0.2% of a detergent Tween 20. This treatment
extracts lipids, thus removing the phospholipid that was absorbed
to plastic. Neither 9D2 antibody nor annexin V bound to
detergent-washed plates.
6. Detection of Anionic Phospholipids on the Surface of Cultured
Endothelial Cells
[0772] Endothelial cells were grown until they reached
approximately 70% confluence. To induce PS exposure, cells were
treated with H.sub.2O.sub.2 (200 .mu.M) for 1 h at 37.degree. C.
Control and treated slides were washed with DPBS containing
Ca.sup.2+ and Mg.sup.2+ and fixed with 0.25% of glutaraldehyde
diluted in the same buffer. Excess aldehyde groups were quenched by
incubation with 50 mM of NH.sub.4Cl for 5 min. To examine the
effect of detergents and organic solvents on detection of
phospholipids, some slides were pre-incubated with acetone (5 min)
or with PBS containing 1% (v/v) Triton.TM. X-100.
[0773] Cells were washed with DPBS (containing Ca.sup.2+, Mg.sup.2+
and 0.2% (w/v) gelatin) and incubated with 1 .mu.g/ml of
biotinylated annexin V (Pharmingen, San Diego, Calif.) or with 1
.mu.g/ml of 9D2 antibody. After 2 h of incubation, cells were
washed with 0.2% gelatin buffer and were incubated with
streptavidin-HRP (1:500 dilution). Rat IgM of irrelevant
specificity and streptavidin alone were used as negative controls
in these studies. 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 15 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 490 nm. Alternatively, PS-positive cells
were detected by addition of carbazole substrate, resulting in
insoluble red-brownish precipitate. Each study was performed in
duplicate and repeated at least twice.
7. Inhibition of 9D2 and Annexin V Binding to Phospholipids by
Liposomes
[0774] The specificity of phospholipid recognition was further
confirmed by competition assays with various liposomes. Liposomes
were prepared from solutions of 5 mg of a single phospholipid in
chloroform. The solutions were dried under nitrogen to form a thin
layer in a round-bottomed glass flask. Ten ml of Tris buffer (0.1
M, pH 7.4) were then added and the flask was sonicated five times
for 2 min. 9D2 or annexin V (6.66 nM) were pre-incubated with 200
.mu.g/ml of liposomal solution for 1 h at room temperature. The
mixture was added to phospholipid-coated plates or endothelial cell
monolayers. The ability of 9D2 to bind to an immobilized
phospholipid or cell surface in the presence or absence of the
different liposomes was determined as described above.
8. Competition of 9D2 and Annexin V for Binding to Immobilized
PS
[0775] Biotinylated 9D2 antibody and annexin V were prepared by
incubating purified proteins with a 10-fold molar excess of
N-hydroxysuccinimide biotin (Sigma, MO) for 1 h at room
temperature. Free biotin was removed by dialysis against PBS. The
biotinylation procedure did not impair the PS-binding capacity of
either protein. For competition studies, unmodified and
biotinylated proteins were premixed with a 10-fold molar excess of
unmodified proteins. The mixtures were then added to PS-coated
plates. Bound reagents were detected by streptavidin-HRP conjugate
diluted 1:1000. The binding to PS of each reagent in the absence of
a competitor was taken as the 100% value.
9. Growth of Subcutaneously Implanted Tumors
[0776] For localization studies, 2.times.10.sup.7 L540 human
Hodgkin's lymphoma cells or 1.times.10.sup.7 cells of other tumor
types were injected subcutaneously into the right flank of SCID
mice (Charles River, Wilmington, Mass.). Tumors were allowed to
reach a volume of 0.4-0.7 cm.sup.3. A minimum of three animals per
group was used. Studies were replicated at least three times.
10. Orthotopic Model of Human MDA-MB-231 Breast Carcinoma
[0777] Female nu/nu or SCID mice were purchased from Charles River.
MDA-MB-231 human mammary carcinoma cells were implanted into the
mammary fat pad according to a published protocol (Price, 1996).
Briefly, mice were anesthetized and a 5-mm incision was made in the
skin over the lateral thorax. The mammary pad was exposed to ensure
the correct site for injection of 1.times.10.sup.7 MDA-MB-231 cells
re-suspended in 0.1 ml of saline.
11. Detection of Anionic Phospholipids in Tumor Bearing Mice In
Vivo
[0778] Immunohistochemical techniques, in which 9D2 or annexin V
are applied directly to sections of frozen tissues, do not
discriminate between anionic phospholipids on the inner leaflet and
the outer leaflet of the plasma membrane. To detect
externally-positioned phospholipids, methods were performed
essentially as previously described (Example V; Ran et al., 1998).
Tumor-bearing SCID mice were injected intravenously with either 50
.mu.g of 9D2 or biotinylated 9D2 antibody or 100 .mu.g of
biotinylated annexin V. Sixty min later mice were sacrificed and
their blood circulation was exsanguinated and perfused with
heparinized saline as previously described (Burrows et al., 1992).
All major organs and tumor were harvested and snap-frozen for
preparation of cryosections.
[0779] Sections were blocked with PBS containing 10% serum. To
prevent loss of phospholipids during slide processing, detergents
and organic solvents were omitted from blocking and washing
buffers. Rat IgM was detected using goat anti rat IgM specific)-HRP
conjugate followed by development with carbazole or DAB (Fries et
al., 1993). Biotinylated reagents were detected by streptavidin
conjugated to HRP.
[0780] Tumor sections derived from mice injected with saline or rat
IgM of irrelevant specificity served as negative controls.
Additional controls consisted of incubating the slides in 1% Triton
solution or in acetone for 10 min. These treatments extract
phospholipids. No signal was detected under these conditions. The
number of positive vessels per high power field was determined at
magnification of .times.100. At least 10 fields per section were
examined and the average percentage of positive vessels was
calculated. Staining of the sections by this method for the
presence of 9D2 or annexin V detects cells having externalized
anionic phospholipids that were accessible for binding by the
reagents in vivo.
12. Identification and Quantification of PS-Positive Tumor
Vessels
[0781] Structures with localized 9D2 antibody or annexin V were
identified as blood vessels by morphological appearance on
DAB-stained sections and by co-incident staining with the
pan-endothelial cell marker, MECA 32 on serial sections of frozen
tissues. Quantification on DAB-stained sections was done by
counting vessels stained by MECA 32, 9D2 or annexin V in serial
sections of a tumor. Six slides of each tumor type derived from 6
mice injected with 9D2 antibody, control rat 10\4 or annexin V were
examined. At least 10 random fields per section (0.317
mm.sup.2/field) were scored in blinded fashion by two independent
observers. The mean numbers and standard errors of vessels stained
by 9D2, annexin V or MECA 32 were calculated. The mean number of
9D2 or annexin V-positive vessels determined in each tumor type
group was compared to the mean number of MECA 32-positive vessels
in the same tumor group. The percentage of 9D2 or annexin
V-positive vessels was calculated.
[0782] In further studies, mice bearing MDA-MB-231 tumors (0.3-0.7
cm.sup.3 in volume) were injected intravenously with 50 .mu.g of
biotinylated 9D2, control IgM or annexin V (six mice per group).
Biotinylated reagents were first incubated with streptavidin-Cy3
conjugate, washed in PBS, then incubated with MECA 32 antibody
followed by FITC-tagged anti-rat IgG secondary antibody. Single
images, taken with appropriate filters for Cy3 (red) and FITC
(green) fluorescence respectively, were captured by digital camera
and transferred to a computer. Images of 10 random fields (0.317
mm.sup.2/field) demonstrating yellow color (a product of merged
green and red fluorescence) were superimposed with the aid of
Metaview software. The same method was used to analyze tumors from
mice injected with control rat IgM or saline. The percentage of
vessels with localized 9D2 or annexin V was calculated as follows:
mean number of yellow vessels per field divided by mean number of
green (total) vessels multiplied by 100.
B. Results
1. Phospholipid Specificity of 9D2 Antibody and Annexin V
[0783] The 9D2 antibody specifically recognized anionic
phospholipids (PS, PA, CL, PI, PG) and had no significant
reactivity with neutral phospholipids (PE, PC and SM) in ELISA
(Table 8). The order of strength of binding of 9D2 to phospholipids
in ELISA was PA>PS=CL>PG=PI. The binding was antigen-specific
since no binding was observed with several control rat IgM of
irrelevant specificity. Binding of 9D2 to any of the anionic
phospholipids adsorbed to ELISA plates was blocked by liposomes
prepared from any of the anionic phospholipids, but not by
liposomes prepared from any of the neutral phospholipids.
TABLE-US-00018 TABLE 8 Phospholipid Specificity of 9D2 and Annexin
V Abundance and location Phospholipid in the plasma membrane
EC.sub.50 of binding (pM) Name Type under normal conditions.sup.a
9D2 Annexin V PS Anionic Major PL (15%), 12 100 amino-PL located on
inner side PA Anionic PL Minor PL (less than 1%) 2 100 PG Anionic
PL Minor PL (less than 1%) 100 250 PI Anionic PL Major PL (7%),
mainly 100 50 located on the inner side CL Anionic PL Absent from
the plasma 15 130 membrane PE Neutral Major PL (22%), mainly
>8000 100 amino-PL located on inner side SM Neutral Major PL
(9%), located >8000 >8000 choline-PL on the outer side PC
Neutral Major PL (46%), located >8000 >8000 choline-PL on the
outer side .sup.aPercentage of total phospholipids, taken from
Fridrikkson, et al., 1999. Percentages may vary for different cell
types.
[0784] Annexin V also bound to anionic phospholipids, but its
binding was less specific than that of 9D2 in that it also bound
strongly to the neutral phospholipid, PE. The order of strength of
binding of annexin V to phospholipids in ELISA was
PI>PS=PE=PA=CL>PG (Table 8). These findings for annexin V are
consistent with earlier data (Andree et al., 1990).
[0785] The binding of 9D2 was unaffected by the presence of 5 mM
EDTA, showing it did not require Ca.sup.2+ for binding to anionic
phospholipids. In contrast, the binding of annexin V to anionic
phospholipids was abolished in the presence of 5 mM EDTA, as
expected from its known dependence on Ca.sup.2+ for binding to
anionic phospholipids or PE (Schlaepfer et al., 1987; Blackwood and
Ernst, 1990).
[0786] Neither 9D2 nor annexin V bound to ELISA plates that had
been coated with phospholipids but then washed with 0.2% Tween in
saline, confirming that their binding was to the absorbed
phospholipids. 9D2 and annexin V did not bind detectably to
heparin, heparan sulfate or to double or single stranded DNA.
2. 9D2 Antibody and Annexin V do not Cross-Block Each Other's
Binding to PS
[0787] To examine whether 9D2 antibody and annexin V compete for
binding to PS, cross-blocking studies were performed using
biotinylated proteins on PS-coated plates. Binding of biotinylated
9D2 antibody and annexin V was blocked by a 10-fold molar excess of
unmodified 9D2 and annexin V, respectively (Table 9). However,
unmodified annexin V did not affect the ability of biotinylated 9D2
to bind to the PS plate. Likewise, addition of unmodified 9D2
antibody did not alter the ability of biotinylated annexin V to
bind to the PS plate (Table 9).
TABLE-US-00019 TABLE 9 9D2 and Annexin V Do Not Cross-Block Binding
to PS Binding PS-binding protein Competitor .sup.a (% Control)
.sup.b Biotinylated annexin V Annexin V 8% Biotinylated 9D2 Annexin
V 93% Biotinylated annexin V 9D2 95% Biotinylated 9D2 9D2 5% .sup.a
Annexin V or 9D2 antibody were pre-mixed in 10-fold molar excess
over the biotinylated reagents. Binding of biotinylated reagents to
PS on microtiter plates was detected by streptavidin-HRP. .sup.b
Reactivity of biotinylated reagents in the absence of a competitor
was taken as 100%. The mean values of triplicate determinations are
presented. SD was less than 10% of the mean value.
[0788] These results indicate that 9D2 antibody and annexin V do
not cross-block each other binding to PS-coated plates, either
because they recognize different epitopes on the PS molecule or
different conformations of PS adsorbed on plastic.
3. Binding to Externalized Anionic Phospholipids on Cell
Surfaces
[0789] The binding of 9D2 antibody and annexin V to cell surfaces
was examined using mouse bEnd.3 endothelioma cells or bovine ABAE
cells. Neither 9D2 nor annexin V bound to non-permeabilized
monolayers of either cell type under quiescent conditions. This
indicates that the majority of anionic phospholipids of the plasma
membrane are normally sequestered to the cytosolic domain. In
contrast, strong staining was observed when cells were
pre-incubated with TNF.alpha. and actinomycin D under conditions
that caused apoptosis in 90-100% of the endothelial cells.
[0790] To confirm that 9D2 and annexin V were binding to
phospholipids on cell surfaces, H.sub.2O.sub.2-treated bEnd.3 cells
were incubated with 9D2 antibody or annexin V in the presence or
absence of various competing liposomes. Anionic phospholipids
become exposed on non-apoptotic, viable bEnd.3 cells when they are
pre-treated with a sub-toxic concentration (100-200 .mu.M) of
H.sub.2O.sub.2 (Ran et al., 2002a).
[0791] The binding of 9D2 antibody to H.sub.2O.sub.2-treated bend.3
cells was inhibited by liposomes containing anionic phospholipids
but not by liposomes containing neutral phospholipids. The
magnitude of inhibition of 9D2 binding to cells varied in the order
PA>PS>CL>PG>PI, in close agreement with the results
obtained using plastic-immobilized phospholipids. Similarly, the
binding of annexin V to H.sub.2O.sub.2-treated cells was blocked by
liposomes containing PS, PA, PE, CL and, to a lesser extent, PI and
PG. Liposomes containing SM or PC did not block annexin V binding
to cells, all in agreement with the results obtained using
plastic-immobilized phospholipids.
[0792] These results confirm that 9D2 binds to anionic
phospholipids in the H.sub.2O.sub.2-treated endothelial cells,
whereas annexin V binds to PE in addition of anionic
phospholipids.
4. Detection of Externalized Anionic Phospholipids on Cells In
Vivo
[0793] Direct immunohistochemical techniques, in which 9D2 or
annexin V are applied directly to sections of frozen tissues, do
not discriminate between anionic phospholipids on the inner leaflet
and the outer leaflet of the plasma membrane. To detect
externally-positioned phospholipids, 9D2 and annexin V were
injected intravenously into tumor-bearing mice and localization to
tumor vessels was determined by indirect immunohistochemistry.
[0794] Mice bearing various types of solid tumors were injected
intravenously with 9D2 antibody or biotinylated annexin V, and one
hour later, were exsanguinated and the tumors and normal tissues
were removed and frozen sections were prepared. Frozen sections of
tissues were cut and stained with HRP-labeled anti-rat IgM or with
HRP-labeled streptavidin to determine to which cells the 9D2 and
annexin V had bound after injection. Blood vessels were identified
morphologically, and from their positive staining by the
pan-endothelial cell antibody, MECA 32, on serial sections.
5. Biodistribution of 9D2 Antibody and Annexin V in Tumor Bearing
Mice
[0795] 9D2 antibody and annexin V localized to tumor vessels in all
of five tumors included in this study (Table 10). The tumors were:
human MDA-MB-231 breast tumor growing orthotopically in the mammary
fat pads of SCID mice; human L540 Hodgkin's tumor growing
subcutaneously; human NCI-H358 NSCLC growing subcutaneously; mouse
B16 melanoma growing subcutaneously and mouse Meth A fibrosarcoma
growing subcutaneously.
TABLE-US-00020 TABLE 10 Specific Localization of 9D2 and Annexin V
to Tumor Vessels Rat IgM Tissue 9D2 Antibody.sup.a control Annexin
V.sup.b Tumors MDA-MB-231 40.6 .+-. 5.4 -- 45.3 .+-. 5.6 L540cy
19.3 .+-. 3.3 -- 16.7 .+-. 3.9 NCI-H358 15.6 .+-. 4.1 -- ND B16
23.4 .+-. 4.5 -- 21.3 .+-. 6.6 Meth A 25.7 .+-. 6.8 -- ND Normal
Adrenal -- -- -- Brain -- -- -- Heart -- -- -- Kidney .sup. --
.sup.c .sup. -- .sup.c -- Intestine -- -- -- Liver -- -- -- Lung --
-- -- Pancreas -- -- -- Spleen -- -- -- Testis -- -- --
.sup.aLocalization of 9D2 antibody and rat IgM control in tumor
bearing mice was determined by injecting the antibody (50 .mu.g),
perfusing the blood circulation of the mice with saline and
detecting the antibody on sections of the tissues by using an
anti-mouse IgM - peroxidase conjugate. The results are presented as
the mean (.+-.SE) percentage of PS-positive vessels of MECA
32-stained vessels per field of 0.317 mm.sup.2. Six samples of each
type were analyzed. The mean number of MECA 32-positive vessels per
0.317 mm.sup.2 field was 23, 25, 21, 18 and 19 .+-. 10 vessels for
MDA-MB-231, L540cy, H358, B16 and Meth A tumors, respectively
.sup.bLocalization of annexin V was determined by injecting
biotinylated annexin V followed by detection on frozen sections
using streptavidin-peroxidase conjugate. .sup.c Non-antigen
specific tubular staining was visible in both 9D2 and control
antibody recipients.
[0796] 9D2 and annexin V gave essentially the same patterns of
staining. Localization of the 9D2 antibody to tumor vessels was
specific since no staining of tumor endothelium was observed with
rat IgM of irrelevant specificity. Presumably, leakage of the
control rat IgM out of tumor vessels occurred to some extent, but
the staining of extravascular IgM was too diffuse or too weak to
discern by indirect immunohistochemistry.
[0797] No vascular localization of 9D2 antibody or annexin V was
observed in nine of the ten normal organs that were examined (Table
10). In the kidney, staining of tubules was observed that appeared
not to be antigen specific. Tubules were stained in both 9D2 and
control rat IgM recipients, presumably because of secretion of IgM
or its metabolites through this organ. The ovaries, a site of
physiological angiogenesis, were not examined.
[0798] The percentage of 9D2 and annexin V positive vessels ranged
from 40% in MDA-MB-231 tumors to 15% in H358 tumors. Anionic
phospholipid-positive vessels were present on the luminal surface
of capillaries and vessels in all regions of the tumors, but were
particularly prevalent in and around regions of necrosis. Most
anionic phospholipid-positive vessels did not show morphological
abnormalities that were apparent by light microscopy. Occasional
vessels, particularly those located in necrotic areas, showed
morphological signs of deterioration. 9D2 antibody and annexin V
also localized to necrotic and apoptotic tumor cells, whereas
localization of the control IgM was not detectable.
[0799] These findings demonstrate that anionic phospholipids are
present on the luminal surface of vascular endothelial cells in
various tumors but not in normal tissues.
6. Double Staining Studies
[0800] Double staining studies were also performed in which mice
bearing orthotopic MDA-MB-231 breast tumors were injected
intravenously with biotinylated 9D2 antibody, biotinylated control
IgM or biotinylated annexin V. One hour later, the mice were
exsanguinated, and their tumors were removed and frozen sections
were cut. The tumor sections were then stained with Cy3-conjugated
streptavidin to detect the biotinylated proteins and with
FITC-conjugated MECA32 to detect vascular endothelium. This
detection method labeled the biotinylated proteins and the vascular
endothelium by red and green. Where the biotinylated proteins are
bound to the endothelium, the converged image appears yellow.
[0801] In these studies, the biotinylated 9D2 and annexin V
appeared mostly to be bound to the vascular endothelium, because
their staining patterns converged with that of MECA 32. About 40%
of MECA 32 positive vessels bound 9D2 and annexin V, in close
agreement with the results obtained by indirect
immunohistochemistry. However, leakage of the biotinylated proteins
into the tumor interstitium was detected by double staining,
whereas it was not apparent by indirect immunohistochemistry.
[0802] Biotinylated proteins were visible outside the vascular
endothelium around a minority (about 5%) of vessels. In tumors from
mice that had been injected with biotinylated rat IgM of irrelevant
specificity, the biotinylated IgM had also leaked into the tumor
interstitium around a similar percentage (about 5%) of vessels, but
mostly appeared not to be bound by the vascular endothelium.
Presumably, the detection of extravasated 9D2 and annexin V by the
double staining technique, but not by the indirect
immunohistochemistry technique, reflects the greater sensitivity of
the former technique and the greater precision with which two
staining patterns can be compared. Non-injected control tumors were
completely unstained by streptavidin-Cy3, indicating that red
fluorescence corresponds to a localized protein.
Example VII
Anionic Phospholipid Membrane Translocation in a Tumor
Environment
[0803] The discovery of aminophospholipids and anionic
phospholipids as in vivo surface markers unique to tumor vascular
endothelial cells prompted the inventors to further investigate the
effect of a tumor microenvironment on the translocation and outer
membrane expression of such molecules. The present example shows
that exposing endothelial cells in vitro to certain conditions that
mimic those in a tumor duplicates the earlier observed
aminophospholipid and anionic phospholipid surface expression in
intact, viable cells.
A. Materials and Methods
1. Iodination of Annexin V
[0804] Recombinant human annexin V was purified from E. coli
transformed with ET12a-Panionic phospholipid1 plasmid (obtained
from Dr. J. Tait, University of Washington, Seattle). The purity of
the protein and the binding to PS were confirmed on SDS-PAGE and on
PS-coated plastic, respectively. Rabbit polyclonal,
affinity-purified anti-annexin V antibodies were used to detect
annexin V bound to PS. Annexin V was radiolabeled with .sup.125I
using Chloramine T as described by Bocci (1964). The specific
activity was approximately 1.times.10.sup.6 cpm per .mu.g of
protein, as measured by a Bradford assay (1976).
2. Endothelial Cell Treatment
[0805] Endothelial cells were treated with cytokines or growth
factors at the concentrations listed in Table 11. All reagents were
diluted in medium containing 10% serum and incubated with the cells
at 37.degree. C. for 24 h.
[0806] To study the effect of hypoxia, cells were seeded on 24 well
plates and were incubated in a humidified normoxic atmosphere (21%
O.sub.2, 5% CO.sub.2) for 48 h before being transferred to a
humidified hypoxic atmosphere (1% O.sub.2, 5% CO.sub.2, 94%
N.sub.2) in a sealed chamber (Billups Rothenberg Inc., Del Mar,
Ca). Cells were incubated in a hypoxic chamber for 24 h at
37.degree. C. and were then returned to a normoxic environment for
4 h at 37.degree. C. The cells were compared to a parallel culture
from an identical passage, seeded on the same day and maintained
entirely under normoxic conditions. In some studies, IL-1.alpha.
(10 ng/ml) and TNF.alpha. (20 ng/ml) were added to the medium
before transfer to the hypoxic chamber.
[0807] To examine the effect of an acidic microenvironment, cells
were exposed to the growth medium lacking bicarbonate, which was
adjusted to different pHs (ranging between 7.3 and 6.2) with the
required amount of HCl. Cells were incubated at 37.degree. C. in
the absence of CO.sub.2. It was confirmed that culture media held
the assigned pH during the 24 h period of culture. These
experimental conditions were not toxic to either bovine or mouse
endothelial cells and had no effect on cell morphology or viability
of the attached monolayer.
3. Detection of PS on Cultured Endothelial Cells by
.sup.125I-Labeled Annexin V
[0808] After treatment with the reagents described above, treated
and control cells were incubated with 7.1 pmoles of
.sup.125I-labeled annexin V (200 .mu.l/well) in the 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. Non-specific binding was determined in the presence of 5
mM EDTA and was subtracted from experimental values. The results
were expressed as net pmoles of cell-bound annexin V, normalized
per 1.times.10.sup.6 cells.
[0809] Maximal binding of annexin V was determined on cells
simultaneously treated with actinomycin D and TNF.alpha. (50 ng/ml
of each component). As has been previously reported, these agents
cause apoptosis and PS exposure in 90-100% of endothelial cells
(Lucas et al., 1998). Basal binding of .sup.125I-annexin V to
untreated cells was determined in the presence of medium with 10%
serum. The amount of .sup.125I-annexin V that bound to the
untreated cultures was subtracted from that in the treated
cultures. Exposure of PS was calculated according to the following
formula: cell-bound annexin V (pmoles) under experimental
conditions divided by maximal annexin V binding (pmoles),
multiplied by 100. Each study was performed in duplicate and was
performed at least three times. Mean values were calculated. The SE
of the mean values from three separate experiments was less than
5%.
4. Detection of PS on Cultured Endothelial Cells and MDA-MB-435
Tumor Cells
[0810] HUVEC cells and tumor cells were grown on 8 well chamber
slides to approximately 70% confluence. To induce PS exposure,
cells were treated with H.sub.2O.sub.2 (200 .mu.M) in serum-free
media for 1 h at 37.degree. C. Cells were washed with DPBS and
incubated with 2 .mu.g/ml 3G4 antibody diluted in serum-free media
for 1 h at room temperature. After gentle washing with DPBS, the
cells were fixed with 4% (v/v) paraformaldehyde in PBS for 15
min.
[0811] To co-stain the cytoskeleton with Texas Red labeled
phalloidin (Molecular Probes, Eugene, Oreg.), cells were
permeabilized with 0.1% Triton-X100 in PBS for 5 min. Texas Red
labeled phalloidin (1:50 diluted in PBS containing 1% BSA) and
FITC-labeled goat anti-mouse antibody (1:200 diluted in PBS
containing 1% BSA) were incubated for 1 h at room temperature. Cell
nuclei were counterstained with DAPI. Mouse IgG.sub.3 of irrelevant
specificity and secondary antibody alone were used as negative
controls in these studies. Each study was performed in duplicate
and repeated at least twice.
[0812] In other studies, H.sub.2O.sub.2-treated cells were detached
with 0.25% trypsin, washed, suspended in ice cold DMEM containing
0.05% w/v sodium azide and 2 .mu.g/ml 3G4 for 1h. The cell pellets
were washed with PBS containing 1% BSA and suspended in the same
buffer containing FITC-labeled goat anti-mouse antibody (1:200
diluted) for 30 min. After washing three times, the cell pellets
were suspended in PBS containing 1% BSA and 0.05% w/v sodium azide.
For live/dead discrimination, propidium iodide was added before
FACS analysis.
B. Results
[0813] 1. Induction by H.sub.2O.sub.2
[0814] 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.
[0815] Exposing endothelial cells to H.sub.2O.sub.2 at high
concentrations causes PS translocation in .about.90% cells.
However, this is 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.
[0816] Using lower concentrations of H.sub.2O.sub.2 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.
[0817] 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 high H.sub.2O.sub.2 concentrations were detached,
floating and had disrupted cytoskeletons.
[0818] 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).
[0819] 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).
[0820] 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.
[0821] 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 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. Induction by Thrombin
[0822] Thrombin was also 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.
[0823] 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 I).
3. Other Agents of Oxidative Stress
[0824] Mouse bEnd.3 or bovine ABAE cells in vitro were treated for
24 h with various concentrations of factors and conditions that are
present in the microenvironment of many tumors (Lichtenbeld et al.,
1996; Harris et al., 1996), such as hypoxia/reoxygenation,
thrombin, acidity, inflammatory cytokines and hydrogen peroxide
(Table 11).
[0825] Externalization of PS and anionic phospholipids was
quantified by measuring .sup.125I-annexin V binding. The amount of
annexin V binding was compared with that of cells in which
apoptosis of 90-100% of cells had been induced by combined
treatment with actinomycin D and TNF-.alpha.. Actinomycin D and
TNF-.alpha. induced the binding of 6.2 pmoles of annexin V per
10.sup.6 cells (3.8.times.10.sup.6 molecules of annexin V per cell)
on both cell types, in good agreement with literature reports (Rao
et al., 1992). This value was taken as the maximal level of
externalized anionic phospholipids.
TABLE-US-00021 TABLE 11 Induction of PS by Recreating Tumor
Environment .sup.125I-Annexin V (% of Max binding) Treatment
Concentration ABAE CELLS bEnd.3 cells Medium with N/A 0 0 10% serum
Actinomycin 50 ng/ml each 100 100 D + TNF .alpha. VEGF 20 ng/ml 0 0
FGF-2 20 ng/ml 0 0 Scatter factor 40 ng/ml 0 0 TGF .beta..sub.1 20
ng/ml 0 0 PDGF-BB 20 ng/ml 0 0 IL-10 20 ng/ml 0 0 IL-8 20 ng/ml 0 0
IL-6 20 ng/ml 0 0 IL-1.alpha. 10 ng/ml 6.4 7.5 IL-1.beta. 10 ng/ml
5.8 5.5 Interferon 40 ng/ml 8.6 2.8 TNF.alpha. 20 ng/ml 7.4 13.7
Thrombin 50 nM 8.8 17.4 Hypoxia 1% O.sub.2 15.0 to 17.5 22.5
Hypoxia + IL-1.alpha. Same as above 26.0 31.0 Hypoxia + TNF.alpha.
Same as above 33.0 36.0 pH 6.6 N/A 20.2 18.9 Hydrogen 200 .mu.M
95.5 98.4 peroxide
[0826] In Table 11, the concentrations of cytokines, growth factors
and thrombin used were selected from literature values to have
maximal stimulatory effect on cultured endothelial cells. These
concentrations did not cause toxicity over the period of the test
(24 h) as judged by morphological appearance, a lack of detachment,
and a lack of uptake of trypan blue. The concentration of
H.sub.2O.sub.2 employed was the maximal concentration that did not
cause cytotoxicity under the chosen conditions.
[0827] The basal binding of .sup.125I-annexin V was determined in
the presence of growth medium alone. Maximal PS exposure was
determined after induction of apoptosis by the combined treatment
with actinomycin D and TNF .alpha.. Average of duplicates from
three separate studies is presented. Standard error was less than
5%.
[0828] Untreated cells were largely devoid of externalized PS, as
judged by annexin V or anti-PS (9D2) antibody binding (Table 11).
The basal binding in the presence of growth medium alone was 0.44
and 0.68 pmoles of .sup.125I-annexin V for ABAE and bEnd.3 cells,
respectively. This corresponds to approximately 7.1% and 10.9% of
the maximal binding for ABAE and bEnd.3 cells, respectively, which
correlated well with the finding that approximately 10% of cells
bound biotinylated annexin V under the same conditions.
[0829] VEGF, HGF, FGF, TGF.beta..sub.1, PDGF, IL-6, IL-8 and IL-10
did not increase binding of .sup.125I-annexin V above the basal
level for untreated cells (Table 11), neither did GM-CSF.
Inflammatory mediators (IL-1.alpha., IL-1.beta., TNF.alpha. and
interferon) caused a small but reproducible increase in PS and
anionic phospholipid translocation that ranged from 5 to 8% of the
maximal level for ABAE cells and from 3 to 14% for bEnd3 cells.
[0830] Hypoxia/reoxygenation, thrombin or acidic external
conditions (pH 6.8-6.6) induced a moderately high externalization
of PS and anionic phospholipid that ranged from 8 to 20% of the
maximal level for ABAE cells and from 17 to 22% of the maximal
level for bend.3 cells. The largest increase in PS and anionic
phospholipid translocation was observed after treatment with 100 to
200 .mu.M of hydrogen peroxide. This treatment caused nearly
complete (95%) externalization of PS in both cell types as judged
by .sup.125I-annexin V binding (Table 11). More than 70% of ABAE
and bEnd.3 cells bound biotinylated annexin V, as judged
immunohistochemically.
[0831] Endothelial cells in which PS and anionic phospholipid
translocation was generated by treatment with
hypoxia/reoxygenation, thrombin, acidity, TNF.alpha., IL-1 or
H.sub.2O.sub.2 remained attached to the matrix during time period
of the assay (24 h), retained cell-cell contact and retained their
ability to exclude trypan blue dye. Normal PS and anionic
phospholipid orientation was restored 24 to 48 h later in the
majority of the cells after the inducing-factor was removed, or the
culture conditions were returned to normal. These results indicate
that mild oxidative stress, created by direct application of
H.sub.2O.sub.2 or indirectly by hypoxia/reoxygenation, acidity,
thrombin, or inflammatory cytokines, triggers a transient
translocation of PS and anionic phospholipids on viable endothelial
cells.
4. Combined Effects of Inflammatory Cytokines and
Hypoxia/Reoxygenation
[0832] Enhanced PS and anionic phospholipid exposure was observed
when ABAE and bEnd.3 cells were subjected to hypoxia/re-oxygenation
in the presence of IL-1.alpha. or TNF.alpha.. In the absence of the
cytokines, hypoxia/reoxygenation increased PS-exposure by ABAE
cells to 15%-17.5% of the maximum level for cells treated with
apoptotic concentrations of actinomycin D and TNF.alpha.. In the
presence of sub-toxic concentrations of IL-1.alpha. or TNF.alpha.,
hypoxia/reoxygenation increased anionic phospholipid-exposure to
26% and 33% respectively of the maximum (Table 11). Comparison with
the effect of cytokines in the absence of hypoxia/reoxygenation
indicates that the combination of cytokines and
hypoxia/reoxygenation had a greater than additive effects on
PS-exposure. Similar effects were observed on bEnd.3 cells.
[0833] Therefore, in the tumor environment, the exposure of PS and
anionic phospholipids induced by hypoxia/re-oxygenation may be
amplified by inflammatory cytokines and possibly by such other
stimuli as acidity and thrombin. Neutrophils could play a role in
this process.
[0834] These in vitro studies shed light on the mechanism of PS
exposure on tumor endothelial cells in vivo. They show that various
factors induce PS exposure on endothelial cells without causing
cytotoxicity, which mimics the situation in tumors in vivo. Hypoxia
followed by reoxygenation, acidity, and thrombin most increased PS
exposure on viable endothelial cells. Inflammatory cytokines
(TNF.alpha. and IL-1.alpha.) also caused a weak but definite
induction of PS exposure.
[0835] These conditions are likely to be the major inducing stimuli
in tumors in vivo because: i) PS positive endothelium is prevalent
in and around regions of necrosis where hypoxia, acidity,
thrombosed blood vessels, and infiltrating host leukocytes are
commonly observed; ii) the finding that hypoxia/reoxygenation
amplifies the weak PS-exposing activity of TNF.alpha. and IL-1 on
endothelial cells in vitro correlates with the situation in vivo in
tumors where hypoxia and cytokine-secreting tumor and host cells
co-exist; iii) hypoxia/reoxygenation and thrombin have been
reported to generate reactive oxygen species (ROS) in endothelial
cells through activation of NADPH oxidase-like membrane enzyme
(Zulueta et al., 1995). ROS produced by malignant cells might
contribute to endothelial cell injury (Shaughnessy et al., 1989).
Hydrogen peroxide was the most powerful inducer of PS exposure on
cultured endothelial cells found in the present study, providing
indirect support for the involvement of ROS.
[0836] Externalized PS provides a negative phospholipid surface
upon which coagulation factors concentrate and assemble. This may
contribute to the procoagulant status on the tumor endothelium that
has long been recognized. PS also provides an attachment site for
circulating macrophages (McEvoy et al., 1986), T lymphocytes (Qu et
al., 1996) and polymorphonuclear cells that assist in leukocyte
infiltration into tumors. Adherence of activated macrophages,
polymorphonuclear cells and platelets to PS on tumor endothelium
may lead to further secretion of reactive oxygen species and
further amplification of PS exposure.
5. Antibody Binding to H.sub.2O.sub.2-Treated HUVEC and MDA-MB-435
Cells
[0837] The binding of the 3G4 antibody to H.sub.2O.sub.2-treated
and untreated HUVEC and MDA-MB-435 cells was analyzed by flow
cytometry (FIG. 5A). The H.sub.2O.sub.2 treatment conditions were
established as set forth above to induce exposure of anionic
phospholipids on the external surface of the plasma membrane.
[0838] Neither cell type bound detectable levels of 3G4 before
treatment with H.sub.2O.sub.2. After treatment with H.sub.2O.sub.2,
the mean fluorescence intensity of cells stained with 3G4 followed
by FITC-anti-mouse IgG was approximately 10-fold greater than that
of cells treated with BBG3 followed by the secondary reagent.
H.sub.2O.sub.2-treated cells did not stain with propidium iodide,
indicating that their outer membranes were intact. 3G4 binding was
blocked by liposomes prepared from anionic phospholipids, but not
by liposomes prepared from neutral phospholipids, indicating that
the 3G4 was binding to cellular anionic phospholipids.
[0839] To determine the distribution of the 3G4 antibody on the
cell surface, H.sub.2O.sub.2-treated HUVEC and MDA-MB-435 cells
were stained with 3G4 by indirect immunofluorescence and examined
using fluorescence microscopy (FIG. 5B, FIG. 5C, FIG. 5D and FIG.
5E). The 3G4 antibody stained discrete regions of the plasma
membrane of H.sub.2O.sub.2-treated HUVEC and MDA-MB-435 cells. The
stained regions of cell membrane had the appearance of small
surface blebs (FIG. 5C, FIG. 5E) similar to the "membrane blebs"
observed on endothelial cells treated with H.sub.2O.sub.2 (Hastie
et al., 1997; van Gorp et al., 2002). The H.sub.2O.sub.2-treated
cells were not stained by the control antibody, BBG3 (FIG. 5B, FIG.
5D), showing that the binding of 3G4 to the cells was
antigen-specific. Identical staining patterns were observed with
FITC-labeled annexin A5. The 3G4-positive H.sub.2O.sub.2-treated
cells did not show morphological signs of nuclear condensation when
examined 1 hr. after addition of H.sub.2O.sub.2, consistent with
reports that peroxide-induced membrane blebbing in endothelial
cells is related to glutathione oxidation, not apoptosis, and can
be reversible (van Gorp et al., 2000).
[0840] These findings therefore indicate that the 3G4 antibody
binds to anionic phospholipids that are normally absent from the
surface of HUVEC or MDA-MB-435 cells, and that become exposed on
the cell surface when the cells are treated with
H.sub.2O.sub.2.
Example VIII
Anti-Tumor Effects of Annexin Conjugates
[0841] The surprising finding that aminophospholipids and anionic
phospholipids are stable markers of tumor vasculature means that
antibody-therapeutic agent constructs can be used in cancer
treatment. In addition to using antibodies as targeting agents,
annexins, and other specific binding proteins, can 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
[0842] 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
[0843] 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 IX
Anti-Tumor Effects of 3SB Anti-PS Antibodies
[0844] The present example shows the anti-tumor effects of anti-PS
antibodies using syngeneic and xenogeneic tumor models. The 3SB
antibody used in this study binds to PS (and PA), but is
essentially devoid of reactivity with PE. This anti-PS antibody
caused tumor vascular injury, accompanied by thrombosis, and tumor
necrosis.
[0845] The effects of anti-PS antibodies were first examined in
syngeneic and xenogeneic tumor models using the 3SB antibody. 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.
[0846] Tumor-bearing mice (4 animals per group) were injected i.p.
with 20 .mu.g of 3SB 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 I. Mice
were sacrificed when tumors had reached 2 cm.sup.3, or earlier if
tumors showed signs of necrosis or ulceration.
[0847] The growth of both syngeneic and xenogeneic tumors was
effectively inhibited by treatment with 3SB anti-PS antibodies.
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.
[0848] Quantitatively, the 3SB anti-PS antibody treatment inhibited
tumor growth by up to 60% of control tumor volume in mice bearing
large Colo 26 and L540 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.
[0849] 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.
[0850] 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 X
Anti-Tumor Effects of Antibody (9D2) Against Anionic
Phospholipids
[0851] This example demonstrates the effects of the 9D2 antibody,
which binds to PS and other anionic phospholipids, in anti-tumor
studies in vivo.
[0852] A high dose (>150 .mu.g) of the rat antibody that binds
to anionic phospholipids, 9D2, was injected into nude mice bearing
H358 tumors. Immunolocalization studies shows that it strongly
localized to tumor endothelium (4+), although some low level,
non-specific binding of 9D2 by normal vessels was observed due to
the high dose (as would be observed for a control IgM antibody of
irrelevant specificity).
[0853] When 9D2 was injected i.p. into a SCID mouse with an L540
tumor for ascites production, the tumor became necrotic and
collapsed. Upon injection of a control antibody (MK 2.7, rat IgG)
into a SCID mouse with an L540 tumor, no similar effects were
observed.
[0854] The effect of the 9D2 anti-PS antibody on the growth of L540
tumors in vivo was then determined more precisely. Treatment was
started when tumors reached 200-250 .mu.l (day 0). From day 0 to
day 7, mice were injected i.p. with .about.150 .mu.g of IgM (200
.mu.l supernatant) or 200 .mu.l of 10% DMEM. From day 7 to day 22,
mice were injected i.p. with .about.300 .mu.g of IgM (400 .mu.l
supernatant) or 400 .mu.l of 10% DMEM. Day 22 was the last day of
treatment and the mice were sacrificed.
[0855] As shown in Table 12, from days 10 to 22, tumor growth is
generally inhibited by about 40% to 50%. At the end of the study,
only 4 mice in the treated group have tumors larger than 2000 .mu.l
in volume, in contrast to 9/9 in the control group.
TABLE-US-00022 TABLE 12 Effects of Anti-PS Antibodies on L540
Tumors In Vivo Day after Average Tumor Number of mice with start of
the Volume (.mu.l) % tumor volume >2000 .mu.l treatment Control
Treated Inhibition Control Treated 0 341 320 6.2 0 0 1 464 325 10.8
0 0 3 412 415 0 0 0 7 687 455 33.8 0 0 10 904 544 39.9 1/9 0 13 945
545 42.4 1/9 0 15 1373 685 50.1 4/9 1/10 17 1426 842 41.0 4/9 4/10
20 1992 987 50.5 6/9 4/10 22 2560 1365 53.3 9/9 4/10
[0856] In another in vivo study, the effects of the rat anti-PS
antibody on the growth of L540 tumors in CB17 SCID mice were
followed for 45 days after tumor cell injections. These
tumor-bearing mice were treated with 300 .mu.g of anti-PS antibody
daily, i.p. or with 300 .mu.l of 10% DMEM daily, i.p., as a
control. Various parameters of tumor treatment were markedly better
in the treated group in comparison to those of the controls (Table
13).
TABLE-US-00023 TABLE 13 Effects of Anti-PS Antibodies on L540
Tumors In Vivo Other parameters Control Treated % Regressed
tumors.sup.1 0 40% (60 days post treatment) % Regressed
tumors.sup.1 0 20% (90 days post treatment) Average volume of 537
.+-. 30 366 .+-. 56 secondary tumors (.mu.l).sup.2 .sup.1Tumors too
small to measure in treated mice at indicated times (60 vs. 90
days) after treatment .sup.2Metastases in lymph nodes
[0857] In a further study, the 9D2 antibody was injected
intraperitoneally at a dose of 100 .mu.g 3 times per week to mice
with L540 tumors. The tumor size was measured with calipers twice a
week. The anti-tumor effects in comparison to the control group
were marked.
Example XI
Anti-Tumor Effects of Anti-PS Antibody 3G4
[0858] The present example demonstrates additional anti-tumor
effects using the anti-PS antibody 3G4 in syngeneic and xenogeneic
tumor models. The 3G4 antibody used in this study is an IgG
antibody that binds to PS and other anionic phospholipids (Example
IV).
A. Protocols for Animal Tumor Studies
[0859] The effects of 3G4 was examined in syngeneic and xenogeneic
tumor models. The general protocol for the animal tumor treatment
studies is conducted as follows. Unless particular differences are
specified, this is the protocol used throughout the studies of the
present application.
[0860] The animals are obtained from Charles Rivers Laboratories.
The mice are 4-5 weeks, female, C.B-17 SCID or Fox Chase SCID mice.
Mice are housed in autoclaved caging, sterile food and water, with
sterile handling. All procedures performed in laminar flow hoods.
Mice are acclimated 1 week and then ear-tagged and a blood sample
(approximately 75-100 .mu.l) taken from the tail vein to check for
leakiness by ELISA. Any mice that fail the leakiness ELISA test
should not be used for test procedures. Mice are injected
orthotopically with tumor cells into mammary fat pad (MFP) or
subcutaneously into the right flank 2-3 days post ear-tagging and
blood sample removal.
[0861] In the orthotopic model, 1.times.10.sup.7 cells in 0.1 ml
DMEM are typically injected into MFP of anesthetized mice. Mice are
anesthetized with 0.075 ml of mouse cocktail injected IP. The mouse
cocktail is 5 ml Ketamine (100 mg/ml); 2.5 ml Xylazine (20 mg/ml);
1 ml Acepromazine (10 mg/ml); 11 ml sterile water. Dosage is 0.1 ml
per 20-30 grams body weight via the IP route for a duration of 30
minutes.
[0862] Once the mouse is anesthetized, as measured by no response
to toe/foot pinch, the mouse is laid on its left side and wiped
with 70% ethanol just behind the head and around the right
forearm/back area. A 2-3 mm incision is made just behind the right
forearm (lateral thorax), which reveals a whitish fat pad when the
skin flap is raised. 0.1 ml of cells are injected into the fat pad
using a 1 ml syringe and a 27-gauge needle, producing a bleb in the
fat pad. The incision is closed using a 9 mm sterile wound clip.
The mouse is returned to its cage and observed until it has wakened
from anesthesia and is mobile. Post-operative health status is
determined, and if any signs of distress are observed, the animal
is given acetaminophen (0.24 mg/ml)+codeine (0.024 mg/ml) in the
drinking water. The wound clip is removed after 1 week. This method
is used so that the cells are accurately placed into the selected
site and not into the subcutaneous region. Tumors will be
approximately 200 .mu.l in volume (L.times.W.times.W) in 14-15 days
and the take rate is essentially 100%.
[0863] In the subcutaneous model, mice are typically injected with
1.times.10.sup.7 cells in 0.2 ml. Mice are not anesthetized, but
are restrained using a steady grip of mouse skin exposing the right
flank. A 1 ml syringe with a 23 gauge needle is used to inject
1.times.10.sup.7 cells in 200 .mu.l, just under the skin of the
mice and a bleb will be seen. It is not unusual to observe a small
amount of fluid leak from the injection site. A twisting motion may
be used when withdrawing the needle from the subcutaneous injection
to reduce this leakage. Tumor volume is measured by
L.times.W.times.H.
[0864] In the perfusion protocol, mice are injected IV with 1000 U
of heparin in 0.2 ml saline. Mice are then be sedated by injecting
the mouse IP with 0.1 ml mouse cocktail. Once the mouse is sedated
enough, as measured by no reflex when toe/foot is pinched, the
thoracic cavity is opened to expose the heart and lungs. A 30 gauge
needle attached to tubing and perfusion pump is inserted into the
left ventricle. The right ventricle is snipped so that blood can
drip out. Saline is pumped through for 12 minutes at a speed of 1
ml per minute. At the end of the perfusion, the needle and tubing
are removed. Tissues are removed for further studies, either
immunohistochemistry or pathology.
B. Tumor Treatment Results
[0865] For the syngeneic model, Meth A mouse fibrosarcoma tumor
cells were used. In one xenogeneic model, human MDA-MB-231 breast
tumor cells were seeded into the mammary fat pad. In another
xenogeneic model, a large human Hodgkin's lymphoma L540 xenograft
was established by injecting cells and allowing the tumor to grow
to a size of over 500 mm.sup.3 before treatment. Tumor-bearing mice
(10 animals per group) were injected i.p. with 100 .mu.g of 3G4
anti-PS antibody (IgG) as opposed to control. Treatment was
repeated 3 times a week. Animals were monitored twice a week for
tumor measurements.
[0866] The growth of both syngeneic and xenogeneic tumors was
effectively inhibited by treatment with 3G4 anti-PS antibodies. The
antibodies caused tumor vascular injury, localized thrombosis and
tumor necrosis.
[0867] The treatment of the syngeneic, Meth A tumor cells was
particularly successful, and the treatment of the human MDA-MB-231
breast tumor cells growing in the mammary fat pad also produced
tumor regressions. Even in mice bearing large L540 tumors, known to
be resistant to necrosis, the 3G4 antibody treatment inhibited
tumor growth in comparison to control. No retardation of tumor
growth was found in control mice. No toxicity was observed in mice
treated with anti-PS antibodies.
[0868] Tumors were also established using MD-MBA-435s cells and
treated as described above. The growth of these tumors was also
effectively inhibited by treatment with the 3G4 antibody. The
treatment of large L540 tumors, MDA-MB-231 and MD-MBA-435s tumor
cells for 60 days was also effective. The antibodies caused tumor
vascular injury, thrombosis and necrosis and retarded tumor growth,
with no evidence of toxicity.
[0869] MD-MBA-435s lucerifase cells were obtained from Dr. Angels
Sierra Jimenez, Barcelona, Spain and were grown in 10% DMEM. Mice
were injected with tumor cells as described as above, and at 2
weeks post injection, the tumors were measured and the volume
recorded. Treatment of mice with tumors of similar average volumes
(200 mm.sup.3) was performed using the 3G4 antibody and the
chimeric 3G4 antibody, produced as described in Example XIX, versus
control. Treatment was initiated by IP injection (800 .mu.g) at day
15 and continued with injections of 200 .mu.g every two to three
days until the final injection of 400 .mu.g at day 35. Tumor
volumes and mouse body weights were measured on injection days.
Mice were sacrificed and perfused with saline for 12 minutes. The
organs and tumor were removed, snap-frozen in liquid nitrogen and
the tumor sectioned for immunohistochemistical analysis.
[0870] This study showed that both the 3G4 antibody and the
chimeric 3G4 antibody effectively retarded tumor growth as opposed
to control.
Example XII
Anti-Viral Effects of Anti-PS Antibodies Against CMV
[0871] Surprisingly switching fields from tumor vasculature to
viral infections, the inventors next reasoned that antibodies to
aminophospholipids and anionic phospholipids would also likely
exert an anti-viral effect. The present example indeed shows this
to be true, first using the 3G4 antibody in the treatment of
cytomegalovirus (CMV) infection.
A. Methods
1. Treatment of CMV-Infected Cells In Vitro
[0872] Confluent monolayers of human diploid foreskin fibroblasts
(HHF-R2) in 6-well plates were infected with human CMV AD169
expressing green fluorescent protein (GFP) at an MOI=0.01 as
previously described (Bresnahan et al., 1996). Briefly, the cells
were incubated with virus in a total volume of 1 ml per well at
37.degree. C. for 90 minutes. During the infection, the plates were
gently rocked every 30 minutes. Following the infection, the cell
supernatant was removed and DMEM/10% FBS/pen-strep (2 ml per well)
was added to each well.
[0873] Dilutions of 3G4 or the isotype matched control antibody
GV39G (100 .mu.g/ml and 50 .mu.g/ml) were added to the wells. The
infected cells were incubated at 37.degree. C. for a total of 19
days. The medium and antibody in each well was replaced every 3
days. On day 19, the cells and supernatants from each well were
harvested and frozen at -80.degree. C. until the plaque assays were
carried out.
2. Fluorescent Microscopy
[0874] The recombinant CMV expresses GFP under the control of the
SV40 promoter. Hence, infected cells appear green under a
fluorescent microscope. In these studies, the antibody treated
CMV-infected cells were observed under a fluorescent microscope at
days 2, 3 and 9.
3. Plaque Assays
[0875] The plaque assays were carried out using standard protocols.
Briefly, the frozen cells cell suspensions were thawed quickly at
37.degree. C. and centrifuged to remove debris at 1000 rpm for 1
minute. Different dilutions of the cell supernatants were added to
sub-confluent monolayers of HHF-R2 cells in 6-well plates and the
cells incubated at 37.degree. C. for 90 minutes (the plates were
gently rocked every 30 minutes). Following the infection, the cell
supernatants were removed and replaced with 2 ml of DMEM/10% FBS.
On day 4, the supernatant in each well was removed and the cells
overlayed with 0.01% low melting point agarose/DMEM/10% FBS. The
plates were incubated at 37.degree. C. for a total of 14 days
post-infection. On day 14, the infected monolayers were fixed with
10% buffered formalin and stained with methylene blue to visualize
the plaques.
B. Results
1. 3G4 Inhibits Viral Spread of CMV
[0876] To investigate whether 3G4 has an inhibitory effect on CMV
infection and replication, confluent human fibroblasts were
pretreated with 3G4 before CMV was added at a low m.o.i. The CMV
used in these studies expresses green fluorescent protein (GFP).
Hence, infected cells appear green when observed under a
fluorescence microscope.
[0877] On day 3 of treatment, with both 50 .mu.g/ml and 100
.mu.g/ml of antibody, there are single infected cells both in
untreated wells and in wells treated with 3G4 or isotype-matched
control antibody, GV39G. Thus, treating the fibroblasts with 3G4
does not appear to significantly inhibit the entry of the virus
into the cells.
[0878] On day 9, however, there is a dramatic difference in the
number of infected cells in 3G4-treated vs. control, GV39G-treated
wells. While the virus has spread to approximately 80% of the
monolayer in the control wells, the virus is restricted to the
original singly-infected cell in the 3G4-treated wells. Hence, 3G4
limits the spread of CMV from the original infected cell to the
surrounding cells. This inhibition of viral spread is observed when
cells are treated with 100 .mu.g/ml and 50 .mu.g/ml.
2. Viral Inhibition is Antibody Concentration-Dependent
[0879] In order to determine what concentration of 3G4 is necessary
for the anti-viral effect at a low m.o.i., infected cells were
treated with different concentrations of 3G4 and the control
antibody, GV39G. The complete inhibition of cell-to-cell spread is
observed with 3G4 at 100 .mu.g/ml and 50 .mu.g/ml. When the cells
were treated with 25, 12.5 and 6.25 .mu.g/ml of 3G4, there are
increasing numbers of GFP positive CMV-infected cells. Although 3G4
does not totally prevent viral spread from the primary infected
cells at these lower concentrations, it still has a meaningful
anti-viral effect, since fewer GFP-positive CMV-infected cells are
seen in the 3G4-treated well as compared to GV39G-treated, control
wells.
3. Quantification of Viral Load at a Low M.O.I.
[0880] The anti-viral effect of 3G4 was quantitated by carrying out
plaque assays to determine the viral load following
antibody-treatment. The controls included untreated cells, the
GV39G antibody and an additional antibody control using the C44
antibody, a mouse IgG2a isotype antibody to colchicine.
[0881] Treatment of infected cells (m.o.i.=0.01 pfu/cell) with 100
.mu.g/ml of 3G4 resulted in a dramatic 6 log.sub.10 decrease in
viral titer as compared to control, GV39G-treated cells. This
inhibition translates into an approximately 99.9999% inhibition of
viral replication. At a concentration of 50 .mu.g/ml, treatment
with 3G4 results in a 3.5 log.sub.10 decrease in viral titer as
compared to GV39G-treatment. Using 3G4 at 25 .mu.g/ml and 12.5
.mu.g/ml, the results are still dramatic, and even at 6.25 .mu.g/ml
an inhibitory effect is still observed.
4. Quantification of Viral Load at a High M.O.I.
[0882] 3G4 treatment of fibroblasts infected at a high m.o.i. of 3
also results in a dramatic reduction in viral titer. At 100
.mu.g/ml, treatment with 3G4 resulted in a 5 log.sub.10 decrease in
viral titer as compared to control, GV39G-treated cells. At 50
.mu.g/ml, 3G4 inhibited viral replication by 3 logs when compared
to GV39G.
5. Inhibition of Replication at a Late Stage
[0883] To determine which stage of the CMV replicative cycle is
blocked by 3G4, a timed addition study was performed. For this, 3G4
was added to fibroblasts infected at a high m.o.i. at different
time points after the infection. The viral load (in both the cells
and supernatant) was quantified using a standard plaque assay.
[0884] Addition of 3G4 up to 24 hours after infection resulted in a
5-6 log.sub.10 decrease in viral titer. However, when addition of
3G4 was delayed to 48 hours, the inhibitory effect of 3G4 was
reduced to 2 log.sub.10 and when addition was delayed to 72 to 96
hours, the inhibitory effect was further reduced. This shows that
3G4 interferes with a late stage of CMV replication that occurs
between 24-48 hours after infection. Thus, 3G4 does not
significantly interfere with infection or with immediate early or
early gene expression. It rather acts later in the viral
replication cycle, e.g., on late gene expression, viral DNA
synthesis, viral packaging or egress.
Example XIII
Anti-Viral Effects of Anti-PS Antibodies Against RSV
[0885] In addition to the dramatic anti-viral effects against CMV
shown in Example XII, the present example demonstrates the use of
three different anti-PS antibodies in the inhibition of Respiratory
Syncitial Virus (RSV) replication.
A. Methods
1. Treatment of RSV-Infected Cells In Vitro
[0886] A-549 cells were grown to 100% confluence in three Costar
12-well tissue culture plates. 200 .mu.L of minimum essential Eagle
medium was added to all wells. Anti-phospholipid antibody (Ab) was
added (100 .mu.g in 100 .mu.L) to 9 wells of each plate and 30 min.
later cells in 6 of those initial 9 wells were infected with an MOI
of 1 with RSV long strain in a volume of 100 uL. The three
remaining wells were left as non-infected, antibody-treated wells.
The three other wells with no antibody were infected with RSV at
the same MOI as described above.
[0887] Each plate was used to test the three different antibodies:
3G4, 3SB and 1B9 (Example N). Cells were incubated in 5% CO.sub.2
at 40.degree. C. for 2 hours and then 600 .mu.L of medium was added
to complete 1 mL volume in each well. An A-549 cell plate was kept
in the same conditions, as control. Supernatants were collected at
4, 24 and 72 hours after infection. At each time point, four wells
from each plate were sampled: one well with only-Ab treated cells,
two wells had Ab-treated/RSV-infected cells and one well had
RSV-infected only cells. The samples were frozen at -80 until the
plaque assay.
2. Plaque Assays
[0888] The plaque assays were carried out as previously described
(Kisch et al., 1963; Graham et al., 1988). Briefly, the frozen
cells cell suspensions were thawed quickly at 37.degree. C. Three
10-fold dilutions were made from the undiluted cell supernatants:
10.sup.-1, 10.sup.-2, and 10.sup.-3. 100 .mu.L of each dilution
plus the undiluted sample were inoculated into 80% confluent Hep-2
cell line plates, all in triplicates. Plates were placed in the 5%
CO.sub.2, 40.degree. C. incubator for 5 days. On the 5.sup.th day,
the plates were developed and stained with hematoxylin and eosin to
reveal the plaques in each well. The plaques were counted using a
dissecting microscope to calculate the RSV viral load in pfu
(plaque forming units)/mL.
B. Results
[0889] Treatment of RSV-infected cells with either 3SB or 1B9
resulted in a log decrease in viral replication. The anti-viral
effect was even more pronounced when the infected cells were
treated with 3G4. Treatment with 3G4 resulted in a 2 log.sub.10
decrease in viral titer. The inhibition was lower than seen with
CMV, most likely because the concentration of 3G4 was low (25-50
.mu.g/ml).
Example XIV
Single Chain Anti-PS Antibodies
[0890] Given the many uses of anti-PS antibodies described herein,
including as anti-tumor agents alone, as targeting agents for
delivering attached therapeutic agents to tumors, and as anti-viral
agents, the present example describes techniques suitable for
generating single chain (scFv) anti-PS antibodies, i.e., wherein
the V.sub.H and V.sub.L domains are present in a single polypeptide
chain, generally joined by a peptide linker.
A. Preparation of the Phage Antibody Library
[0891] The secondary stock of the bacterial library (about
1.times.10.sup.10 clones) was inoculated into 100 ml 2.times.TY
containing 100 .mu.g/ml ampicillin and 1% glucose. It was grown
with shaking at 37.degree. C. until the OD at 600 nm was 0.5.
[0892] M13K07 helper phage was added at 10.sup.13 pfu and incubated
without shaking in a 37.degree. C. water bath for 30 min. The
infected cells were centrifuged at 3,500 g for 10 min. The pellet
was resuspended in 200 ml of 2.times.TY containing 100 .mu.g/ml
ampicillin and 75 .mu.g/ml kanamycin and incubated with shaking at
30.degree. C. overnight.
[0893] The culture was centrifuged at 10,800 g for 10 min. 1/5
volume PEG/NaCl was added to the supernatant, mixed well and left
for 1 hr at 4.degree. C. It was then centrifuged at 10,800 g for 30
min. The pellet was resuspended in 40 ml PBS and 8 ml PEG/NaCl was
added. It was mixed and left for 20 min at 4.degree. C. It was then
centrifuged at 10,800 g for 10 min and the supernatant aspirated.
The pellet was resuspended in 2 ml 10% human serum and centrifuged
at 11,600 g for 10 min in a microcentrifuge to remove most of the
remaining bacterial debris.
[0894] To pre-pan, the phage antibody library in 10% human serum
was added to the PC coated dish and incubated for 60 min at room
temperature.
B. Selection on Biotinylated Liposomes
[0895] 20 .mu.mol phosphatidylinositol and 20 .mu.mol biotinylated
phosphatidylserine were dissolved in 10 ml hexane. This solution
was dried to a thin layer on the surface of a flask using a
rotating evaporator. 2 ml PBS was added and bath sonicated
4.degree. C. for 30 minutes.
[0896] 100 .mu.l phage scFv and 100 .mu.l biotinylated liposomes
were then mixed in the presence of 10% human serum and gently
rotated for one hour at room temperature. Blocking was done with
100 .mu.l streptavidin M-280 dynabeads by adding 600 .mu.l 2.5%
casein/0.5% BSA for 30 min at room temperature. The beads were
separated from the blocking buffer with a MPC-E (Magnetic Particle
Concentrator from Dynal) for 4-5 min.
[0897] The beads were resuspended in 100 .mu.l PBS. 100 .mu.l of
blocked streptavidin Dynabeads was added to the phage bound to the
biotinylated antigen and gently rotated for 15 min at room
temperature. Separation was achieved with a MPC-E for 5 minutes and
the supernatant poured off. It was washed five times with 1 ml PBS.
For each wash, the beads were resuspended and brought down with a
MPC-E.
[0898] Finally, the phage was eluted from the beads by resuspending
in 300 .mu.l 100 mM triethalamine for 30 mins. 150 .mu.l 1 M Tris
pH=7.4 was added for neutralization. The beads were separated again
with the MPC-E.
[0899] 150 .mu.l of the phage supernatant was used to infect 10 ml
TG1 bacteria in log phase. The 10 ml culture was shaken in the
presence of 20 .mu.g/ml ampicillin at 37.degree. C. for one hour.
Ampicillin was added to the final concentration of 50 .mu.g/ml and
shaken for another hour. 10.sup.13 pfu M13 helper phage was added
to this culture, transferred to 100 ml 2TY medium containing 100
.mu.g/ml ampicillin and shaken at 37.degree. C. for one hour.
Kanamycin was added to the final concentration of 100 .mu.g/ml and
shaken at 30.degree. C. overnight.
[0900] The phage preparation procedure was repeated and the
selection procedure repeated another 3 to 4 times.
C. Monoclonal Single Chain Antibody ELISA
[0901] Individual HB2151 colonies from the plates (after 4 rounds
of selection) were inoculated into 500 .mu.l 2.times.TY containing
100 .mu.g/ml ampicillin and 1% glucose in 96-well plates and grown
with shaking (300 rpm.) overnight at 37.degree. C. 5 .mu.l from
this plate were transferred to a second 96-well plate containing
500 .mu.l 2.times.TY containing 100 .mu.g/ml ampicillin per well
and grown shaking at 37.degree. C. for 3 hr (OD600=0.9).
[0902] To each well was added 50 .mu.l 2.times.TY containing 100
.mu.g/ml ampicillin, 10 mM IPTG (final concentration is 1 mM),
which was grown with shaking overnight at 30.degree. C. It was
centrifuged at 1,800 g for 10 min and 100 .mu.l of the supernatant
used in the following ELISA.
[0903] 96 well plates (DYNEX IMMULON.RTM.1B) were coated with PS
dissolved in ethanol at a concentration of 10 .mu.g/ml (P6641 10
mg/ml solvent was Chloroform:MeOH 95:5). 10 .mu.g/ml PC was coated
in the same way. These plates were evaporated at 4.degree. C. in
the cold room. 250 .mu.l 2.5% casein was added to each well, and
the plates were covered and blocked at 37.degree. C. for 1
hour.
[0904] Wells were rinsed 3 times with PBS, 100 .mu.l/well 10% human
serum and 100 .mu.l/well supernatant containing soluble scFv was
added and incubated for 60 min at 37.degree. C. The solution was
discarded and washed 6 times with PBS. 100 .mu.l 9E10 in 5%
casein/0.5% BSA-PBS (1:5000 dilution) was added to each well,
incubated at 37.degree. C. for 1 hour and washed 6 times with PBS.
100 .mu.l HRP-goat-anti-mouse antibody (1:10000 dilution) was added
to each well, incubated at 37.degree. C. for 1 hour and washed 5
times with PBS. 100 .mu.l 0.05% OPD was added to each well and
developed for 5 minutes. 100 .mu.l 0.18 M H.sub.2SO.sub.4 was added
to stop the reaction and read at O.D. 490.
[0905] Antigen-positive clones were streaked on 2.times.TYAG plates
and grown overnight at 30.degree. C. Positive single colonies were
picked into 3 ml 2.times.TYAG media and grown 12 hours at
37.degree. C. Plasmids were extracted and scFv gene inserts checked
by enzyme digestion and PCR. The ones with the correct size inserts
were sequenced.
[0906] The colonies with the correct size inserts were grown into
100 ml 2.times.TYAG media and shaken at 37.degree. C. OD 600=0.5.
These were transferred into 900 ml 2.times.TYA and grown until OD
600=0.9. 1 M IPTG was added to a final concentration of 1 mM and
shaken at 30.degree. C. overnight. The supernatant was checked
using the same ELISA method as previously. The scFv protein was
purified from the periplasmic fraction using Ni.sup.++-agarose
affinity chromatography.
D. Results
[0907] After 4 rounds of panning, the following clones gave
promising ELISA signal on PS plates and have the correct size
insert: 3E5, 3A2, G5, C8, E4 and 4D5. These have been subcloned,
wherein E4 gave 5 positive subclones and 4D5 gave 5 positive
subclones (Table 14).
TABLE-US-00024 TABLE 14 ELISA on PS Plate 0.099 0.107 0.118 0.115
0.100 0.094 0.084 0.086 0.166 0.164 0.102 0.191 0.113 0.106 0.127
0.150 0.128 0.097 0.078 0.087 0.190 0.144 0.102 0.154 0.122 0.115
0.117 0.112 0.105 0.097 0.085 0.088 0.230 0.071 0.168 0.150 0.107
0.108 0.121 0.123 0.107 0.101 0.083 0.085 0.191 0.246 0.186 0.150
0.138 0.121 0.114 0.131 0.100 0.096 0.082 0.079 0.183 0.187 0.275
0.171 0.118 0.115 0.116 0.132 0.099 0.094 0.082 0.086 0.185 0.073
0.208 0.102 0.111 0.176 0.126 0.118 0.096 0.087 0.123 0.087 0.144
0.226 0.112 0.126 0.102 0.107 0.131 0.125 0. 089 0.102 0.082 0.084
0.188 0.073 0.142 0.151 3E5 3A2 G5 C8 E4 4D5
[0908] Once the positive clones were identified, they were
sequenced. The ScFv nucleic acid and protein sequence of clone 3A2
is set forth in SEQ ID NO:5 and SEQ ID NO:6, respectively. The
positive clones were grown up on a large scale and the scFv
purified using Nickel agarose affinity chromatography. The purified
scFv has been obtained using Phast-gel electrophoresis.
Example XV
Synthesis of PE-Binding Peptide Derivatives
[0909] The present example concerns the design and synthesis of
exemplary PE-binding peptide derivatives and conjugates for use in
treating tumors and viral diseases. The structures for exemplary
duramycin derivatives result from the following description.
A. DLB
[0910] 0.5 mg (0.25 .mu.mole) of duramycin dissolved in 0.387 ml
0.1M NaHCO.sub.3 in water was added to 0.113 mg (0.25 .mu.mole) of
NHS-LC-Biotin (Sigma). The reaction mixture was incubated at room
temperature for 1 hr and then at 4.degree. C. overnight. The sample
was loaded onto a silica column, washed with 0.1% trifluoroacetic
acid (TFA), eluted with 0.1% TFA and 70% CH.sub.3CN. The eluant was
collected and concentrated by centrifugation. The total yield was
0.5 mg.
B. DIB
[0911] 0.5 mg (0.25 .mu.mole) of duramycin dissolved in 0.286 ml of
0.1M NaHCO.sub.3 in water was added to 0.034 mg (0.25 .mu.mole) of
2-iminothiolane hydrochloride (2-IT). The mixture was incubated at
room temperature for 1 hr. 0.13 mg (0.26 .mu.mole) of
iodoacetyl-LC-Biotin (Pierce) was added and the reaction incubated
at room temperature for 1 hr and at 4.degree. C. overnight. The
sample was loaded onto a silica column, washed with 0.1% TFA,
eluted with 0.1% TFA and 70% CH.sub.3CN. The eluant was collected
and concentrated by centrifugation. The total yield was 0.5 mg.
C. (DLB).sub.4NA
[0912] 1.9 mg (0.94 .mu.mole) duramycin was dissolved in 0.5 ml of
0.1 M NaHCO.sub.3 in water. To this, 0.4 mg (0.88 .mu.mole)
NHS-LC-Biotin (Sigma) in 200 .mu.l dimethylformamide (DMF) was
added. The mixture was incubated at room temperature for 4 hr. 10
mg (0.17 .mu.mole) neutravidin (NA) in 1 ml was added to the
reaction mixture, which was incubated at room temperature for 2 hr
and then at 4.degree. C. overnight. The reaction mixture was then
loaded onto a G-25 column (volume 50 ml) in PBS buffer. The
fractions were collected and analyzed by SDS PAGE (phast gel).
Protein-containing fractions (7-16) were pooled together,
sterilized by filtration through a 0.22 .mu.m filter and the
concentration determined by measuring absorption at 280 nm. The
total yield was 5.1 mg.
[0913] The sample was then fractionated by FPLC. Three peaks were
collected that corresponded to the following: peak 1:
[(DLB).sub.4NA].sub.3 (fractions 17-23); peak 2:
[(DLB).sub.4].sub.2 (fractions 24 33) and peak 3: (DLB).sub.4NA
(fractions 35-48). All the samples were sterilized by filtration
through a 0.22 .mu.m filter. The final yields obtained were: 0.34
mg of [(DLB).sub.4NA].sub.3; 0.59 mg of [(DLB).sub.4].sub.2 and
1.41 mg of (DLB).sub.4NA.
D. (DLB).sub.4NA-F
[0914] 0.61 mg of (DLB).sub.4NA in PBS buffer was added to 0.005 mg
N-hydroxysuccinimidyl fluorescein (NHS-Fluorescein) (Sigma) in DMF.
The mixture was incubated at room temperature for 1 hr. The
reaction mixture was then fractionated on a PD10 column (10 ml).
(DLB).sub.4NA-F was eluted in the protein-containing fractions (3
and 4), which were pooled together and sterilized by filtration
through a 0.22 .mu.m filter. The total yield was 0.5 mg.
E. (DIM).sub.n HIgG
[0915] Human IgG (HIgG) was first purified as follows: 1.3 ml HIgG
(that included 100 mg/ml HIgG, 22.5 mg/ml glycine and 3 mg/ml
albumin in borate buffer with 1 mM EDTA, pH 9) was applied to an
FPLC (S200, 250 ml) column. The fractions were collected and
analyzed by SDS PAGE on a phast gel. Fractions containing monomeric
IgG (21-32) were pooled together and sterilized by filtration
through a 0.22 .mu.m filter. The total yield as determined by
absorption at 280 nm was 111 mg.
[0916] Purified HIgG (55 mg in 13 ml of borate buffer, pH 9) was
added to 1.003 mg in 0.5 ml of SMCC (Pierce) in DMF. The mixture
was incubated at room temperature for 1 hr. At the same time,
another reaction mixture containing 6 mg duramycin (3 .mu.mole;
dissolved in 0.5 ml 0.1M NaHCO.sub.3) and 0.413 mg 2-IT (3
.mu.mole; in 0.1M NaCO.sub.3) was incubated at room temperature for
1 hr. After completion of the reactions, the two reaction mixtures
were combined and incubated at room temperature for 2 hr and at
4.degree. C. overnight. The reaction products were analyzed by SDS
PAGE on a phast gel. The reaction products were loaded onto an FPLC
column in borate buffer, pH 9. The FPLC fractions corresponding to
trimer (5-14), dimer (15-24), and monomer (25-37) were pooled and
sterilized by filtration through a 0.22 .mu.m filter. The total
yield of monomer was 54.6 mg. Five to seven duramycin groups were
attached to each molecule of HigG.
F. (DIM).sub.n HIgG-F
[0917] 1 mg (0.7 ml) of (DIM).sub.nHIgG was added to 5 .mu.l of
NHS-Fluorescein in DMF. The reaction mixture was incubated at room
temperature for 1 hr and desalted on a PD-10 column.
Protein-containing fractions (2-3) were pooled and sterilized by
filtration through a 0.22 .mu.m filter. The total yield was 0.9
mg.
G. (DIM).sub.n HIgG-B and [(DIM).sub.nHIgG].sub.2-B
[0918] To synthesize biotinylated derivatives of
[(DIM).sub.nHIgG].sub.2, 0.66 mg (1 ml) of [(DIM).sub.nHIgG].sub.2
was added to 8 .mu.l of 1 mg/ml of NHS-LC-Biotin (Pierce) in DMF.
The mixture was incubated at room temperature for 1 hr. The
reaction mixture was then desalted on a PD-10 column.
Protein-containing fractions (3 and 4) were pooled and sterilized
by filtration through a 0.22 .mu.m filter. The final yield was 0.46
mg.
[0919] The biotinylation of the monomer (DIM).sub.nHIgG was
performed in the same manner. Briefly, 1.06 mg (0.75 ml) of
(DIM).sub.nHIgG were added to 12 .mu.l of 1 mg/ml NHS-LC-Biotin in
DMF. After incubation at room temperature for 1 hr, the reaction
product was desalted on a PD-10 column. Protein-containing
fractions (3 and 4) were pooled and sterilized by filtration
through a 0.22 .mu.m filter. The final yield was 0.62 mg.
H. (DIB).sub.4NA
[0920] 2 mg (0.99 .mu.mole) of duramycin were dissolved in 0.5 ml
0.1M NaHCO.sub.3 and added to 0.136 mg (0.99 .mu.mole) of 2-IT. The
reaction mixture was incubated at room temperature for 1 hr.
Following this, 0.483 mg (0.95 .mu.mole) of iodoacetyl-LC-Biotin
(Pierce) was added and the reaction mixture incubated at room
temperature for 1 hr. 10 mg (0.17 .mu.mole) of neutravidin in 1 ml
of H.sub.2O was added and incubated at 4.degree. C. overnight. The
reaction mixture was fractionated by FPLC. Three different peaks
were collected and pooled: [(DIB).sub.4NA].sub.3 (fractions 17-23);
[(DIB).sub.4NA].sub.2 (fractions 24-33); and (DIB).sub.4NA
(fractions 35-48). All the samples were sterilized by filtration
through a 0.22 .mu.m filter. The total yields obtained were 0.87 mg
of [(DIB).sub.4NA].sub.3; 1.25 mg of [(DIB).sub.4NA].sub.2; and
1.83 mg of (DIB).sub.4NA.
I. (DIB).sub.4NA-B
[0921] 0.023 mg (0.3 .mu.mole) of (DIB).sub.4NA was added to 0.9
.mu.g of NHS-LC-Biotin (Pierce). The reaction was incubated at room
temperature for 1 hr and then desalted on a PD-10 column. The total
yield was 0.04 mg.
J. DS-1
[0922] 5 mg (2.5 .mu.mole) of duramycin dissolved in 0.5 ml of 0.1M
NaHCO.sub.3 in water was added to 0.319 mg (2.6 .mu.mole) of 1,3
propane sultone. The mixture was incubated at 4.degree. C.
overnight. The sample was loaded onto a silica column, washed with
0.1% TFA, eluted with 0.1% TFA and 70% CH.sub.3CN. The eluant was
collected and concentrated by centrifugation under reduced
pressure. The total yield was 5 mg.
K. DS-2
[0923] 1 mg (0.497 .mu.mole) of duramycin dissolved in 0.3 ml of
0.1M NaHCO.sub.3 in water was added to 0.072 mg (0.523 .mu.mole) of
2-IT. The reaction mixture was incubated at room temperature for 1
hr. 0.125 mg (0.49 .mu.mole) of SBF-Chloride (Pierce) was added.
The reaction mixture was incubated at room temperature for 1 hr and
4.degree. C. overnight. The peptide was purified on a silica
column. The eluant was collected and concentrated by centrifugation
under reduced pressure. The total yield was 1 mg.
L. DS-3
[0924] 1 mg (0.497 .mu.mole) of duramycin dissolved in 0.4 ml of
0.1M NaHCO.sub.3 in water was added to 0.109 mg (0.592 .mu.mole) of
2-sulfobenzoic acid cyclic anhydride. The reaction was incubated at
room temperature for 1 hr and 4.degree. C. overnight. The peptide
was purified on a silica column. The eluant was collected and
concentrated by centrifugation under reduced pressure. The total
yield was 1 mg.
M. DS-4
[0925] 0.25 mg (0.124 .mu.mole) of duramycin dissolved in 0.5 ml of
0.1 M NaHCO.sub.3 in water was added to 0.017 mg (0.124 .mu.mole)
of 2-IT. The reaction mixture was incubated at room temperature for
1 hr. The mixture was then added to 0.049 mg (0.124 .mu.mole)
Ellman's reagent. The mixture was incubated at room temperature for
2 hr and overnight at 4.degree. C. 250 .mu.l of 1 mg/ml of
4-Amino-5-hydroxy-2,7-naphthalene disulfonic acid mono-sodium salt
hydrate was added to 100 .mu.l of 1 mg/ml 2-IT. The reaction was
incubated at room temperature for 1 hr. 50 .mu.l of this reaction
mixture was added to the previous reaction and incubated at room
temperature for 1 hr. The peptide was purified on a silica column.
The eluant was collected and concentrated by centrifugation under
reduced pressure.
N. DS-5
[0926] 5 mg (2.5 .mu.mole) of duramycin dissolved in 0.5 ml of 0.1M
NaHCO.sub.3 in water was added to 0.356 mg (2.6 .mu.mole) of 1,3
butane sultone. The mixture was incubated at 4.degree. C.
overnight. The sample was loaded onto a silica column, washed with
0.1% TFA, eluted with 0.1% TFA and 70% CH.sub.3CN. The eluant was
collected and concentrated by centrifugation under reduced
pressure. The total yield was 5 mg.
O. DC-1
[0927] 0.25 mg (0.124 .mu.mole) of duramycin dissolved in 0.5 ml of
0.1M NaHCO.sub.3 in water was added to 0.017 mg (0.124 .mu.mole) of
2-IT. The reaction mixture was incubated at room temperature for 1
hr. The mixture was then added to 0.049 mg (0.124 .mu.mole)
Ellman's reagent. The mixture was incubated at room temperature for
2 hr and overnight at 4.degree. C. The peptide was purified on a
silica column. The eluant was collected and concentrated by
centrifugation under reduced pressure.
Example XVI
Duramycin Derivatives Specifically Bind PE
[0928] The present example shows that the duramycin derivatives
synthesized in Example XV are specific for PE and can therefore be
used as designed, by linking to cell-impermeant, targeting or
anti-viral agents and use in the treatment of tumors and viral
diseases.
[0929] To test the specificity of the duramycin derivatives,
particularly the binding to PE in preference to other
phospholipids, a series of competition ELISAs were performed. The
ability of the duramycin derivatives to compete with either DIB or
DLB for binding to PE was tested in the following method.
[0930] PE and PC were dissolved separately in ethanol. The final
concentration was 5 .mu.g/ml. 100 .mu.l was added to each well of
96 well ELISA plates (DYNEX IMMULON.RTM.1B). These plates were
evaporated at 4.degree. C. in a cold room. 250 .mu.l 2.5% casein
was added to each well, covered and blocked at 37.degree. C. for 1
hour. The blocking buffer was discarded and 100 .mu.l 2.5% casein
added to each well. The duramycin compound was added as a serial
dilution across the plate, such as (DIM)nHIgG, (DIB)4NA, (DLB)4NA,
DS, duramycin and DIB.
[0931] The (DIM)nHIgG starting concentration was 1.4 mg/ml, the
(DIB)4NA starting concentration was 800 .mu.g/ml, and the (DLB)4NA
starting concentration was 800 .mu.g/ml. These were incubated at
37.degree. C. for 1 hour and washed 5 times with PBS. 100 .mu.l
HRP-streptavidin (1:5000 dilution) was added to each well,
incubated at 37.degree. C. for 1 hour and washed 5 times with PBS.
100 .mu.l 0.05% OPD was added to each well and developed for 5
minutes. 100 .mu.l 0.18 M H2SO4 was added to stop the reaction and
read at O.D. 490.
[0932] The resultant data was tabulated and then plotted
graphically. Increasing concentrations of the duramycin derivatives
decrease absorbance at 490 nm, showing that the duramycin
derivatives compete with DIB and DLB for binding to
phosphatidylethanolamine.
[0933] The phospholipid binding profiles of duramycin constructs
were confirmed using further ELISAs. The respective test lipids PS,
PE, PI, CL, PC, PG, SM, and cholesterol were dissolved separately
in ethanol and used to coat ELISA plates. Duramycin compounds were
added as serial dilutions across the plates. After incubation and
washing steps, a secondary detection reagent was added to each well
and reactivity determined using the colorimetric assay as described
above.
[0934] Representative phospholipid binding profiles for the
duramycin biotin derivatives, DIB and DLB were plotted. It was
shown that DIB and DLB are specific for PE, with binding to each of
PS, PI, CL, PC, PG and SM being negligible or undetectable.
(DIM).sub.nHIgG-B and [(DIM).sub.nHIgG].sub.2-B had essentially the
same binding profile as DLB. Although minimal binding to PS was
observed at high concentrations of DIB, this is not meaningful in
the context of this study, as binding to PS was undetectable at DIB
concentrations that were saturating and half maximal for PE
binding. Therefore, the duramycin constructs specifically bind to
phosphatidylethanolamine.
[0935] It was also shown that serum has no significant effect on PE
binding by duramycin derivatives. This is exemplified by binding of
the duramycin biotin derivative, DLB to PE-coated ELISA plates in
the presence and absence of serum (BSA), wherein the binding
profiles show no significant differences.
Example XVII
Anti-Viral Effects of PE-Binding Peptide Derivatives
[0936] In addition to the anti-viral effects mediated by anti-PS
antibodies, as shown in Example XII and Example XIII, the present
example demonstrates the anti-viral effects of peptide derivatives
that specifically bind to the other common aminophospholipid,
PE.
A. Methods
1. Treatment of CMV-Infected Cells In Vitro
[0937] Confluent monolayers of human diploid foreskin fibroblasts
(HHF-R2) in 6-well plates were infected with human CMV AD169
expressing green fluorescent protein (GFP) at an MOI=0.01 as
described in Example XII (Bresnahan et al., 1996). The cells were
incubated with virus in a total volume of 1.5 ml per well at
37.degree. C. for 90 minutes. During the infection, the plates were
gently rocked every 30 minutes. Following the infection, the cell
supernatant was removed and DMEM/10% FBS/pen-strep (2 ml per well)
was added to each well.
[0938] Different dilutions of duramycin derivatives (DLB).sub.4NA,
(DIM).sub.nHIgG, DS-1, DS-2, DS-3 and DC-1 were added to the wells
before the addition of the virus, and following infection. The
infected cells were incubated at 37.degree. C. for a total of 14
days. The medium and duramycin derivative in each well were
replaced every 3 days.
2. Fluorescent Microscopy
[0939] As in Example XII, the recombinant CMV expresses GFP under
the control of the SV40 promoter. Hence, infected cells appear
green under a fluorescent microscope. In these studies, the
CMV-infected cells treated with the duramycin derivatives were
observed under a fluorescent microscope at days 4 and 6.
B. Results
[0940] On day 4, there are single infected GFP-positive green cells
in untreated wells and wells treated with (DLB).sub.4NA and
(DIM).sub.nHIgG. Thus, treatment of HHF-R2 cells with these
duramycin derivatives does not appear to inhibit the entry of the
virus into the cells. There is some preliminary evidence that the
duramycin derivatives DS-1, DS-2 and DS-3 inhibit viral entry into
the cells.
[0941] On day 6 after treatment with (DLB).sub.4NA and
(DIM).sub.nHIgG, there is a marked difference in the number of
infected GFP-positive cells in untreated vs. the duramycin
derivative treated wells. By day 6, the virus has spread from the
single infected cell seen on day 4 surrounding cells in the
untreated wells. However, on day 6 in the wells treated with
(DLB).sub.4NA and (DIM).sub.nHIgG, the virus is limited to the
original singly infected cell.
[0942] Accordingly, (DLB).sub.4NA and (DIM).sub.nHIgG limit the
spread of CMV from the original infected cell to the surrounding
cells. This inhibition of viral spread is observed when cells were
treated with different concentrations of (DLB).sub.4NA (100
.mu.g/ml and 50 .mu.g/ml) and (DIM).sub.nHIgG (200 .mu.g/ml and 100
.mu.g/ml).
Example XVIII
Advantages of 3G4 Antibody
[0943] The 3G4 antibody developed by the inventors' unique
protocol, as described in Example IV, has many advantages over the
anti-PS antibodies in the literature, including the prominent
anti-PS antibody, 3SB (Rote et al. (1993). The present example
describes certain of those advantages.
A. Class and Specificity
[0944] 3G4 is an IgG antibody, whereas 3SB is IgM. Antibodies of
IgG class have numerous advantages over IgM, including higher
affinity, lower clearance rate in vivo and simplicity of
purification, modification and handling. A comparison of the PS
binding of the IgM antibody, 3SB, with 3G4 and another IgG antibody
was plotted.
[0945] 3G4 reacts strongly with the anionic phospholipids PS, PA,
PI, PG and CL with approximately the same intensity, and binds to
the aminophospholipid, PE less strongly. It has no reactivity with
PC and SM and has the binding specificity profile:
PS=PA=PI=PG>CL>>PE (Example IV; Table 4). 3G4 does not
bind detectably to heparin, heparan sulfate or to double or single
stranded DNA, nor to cellular proteins extracted from bEnd.3 cells
on Western blots. The binding of 3G4 is unaffected by the presence
of 5 mM EDTA, showing that Ca.sup.2+ is not require for 3G4 binding
to anionic phospholipids. 3G4 did not bind to ELISA plates that had
been coated with phospholipids but then washed with 0.2% Tween 20
in saline, confirming that the binding was to the absorbed
phospholipid.
[0946] The epitope recognized by 3G4 appears to lie within the
phosphoglycerol core of the anionic phospholipids, which is the
same in phospholipids from all mammalian species. The antibody thus
reacts with both mouse and human phospholipids, which is important
for pre-clinical and clinical development. 3G4 is more specific for
anionic phospholipids than the natural ligand, annexin V. Unlike
3G4, annexin V also binds strongly to neutral phospholipids in
physiological concentrations of Ca.sup.2+.
[0947] The specificity of 3G4 for anionic phospholipids was
confirmed by assays in which liposomes formed from different
phospholipids were used to compete for 3G4 binding to immobilized
PS. Liposomes were prepared from solutions of 5 mg of a single
phospholipid in chloroform. The solutions were dried under nitrogen
to form a thin layer in a round-bottomed glass flask. Ten ml of
Tris buffer (0.1 M, pH 7.4) were then added and the flask was
sonicated five times for 2 min. The 3G4 antibody (0.1 .mu.g/ml) was
added to either buffer or different phospholipid liposomes and
pre-incubated for 30 minutes at room temperature. The mixture was
added to PS-coated plates (after standard blocking), incubated for
1 hour, washed and the secondary antibody added. After 1 hour, the
plates were washed and developed for 5 minutes using OPD.
[0948] As shown in Example IV, 3G4 binds to PS, PA, PI, PG and CL
when immobilized and binds to immobilized PE to a lesser degree,
but does not bind to immobilized PC. The ability of 3G4 to bind to
immobilized PS in the presence or absence of the different
liposomes is shown in FIG. 3. Results from these liposome
competition studies show that binding of 3G4 to PS adsorbed to
ELISA plates was blocked by liposomes prepared from PS, PA, PI and
CL, but that liposomes prepared from PE and PC did not result in a
detectable reduction in 3G4 binding (FIG. 3). Also, SM liposomes
were not inhibitory.
B. Inhibition of Cell Proliferation
[0949] 3G4 binds to activated, dividing, injured, apoptotic and
malignant cells that externalize PS and other anionic
phospholipids. The 3G4 antibody inhibits the proliferation of
endothelial cells in vitro, and shows a marked selective inhibition
of dividing endothelial cells as opposed to quiescent cells.
[0950] The effect of the anti-PS antibodies 3G4, 9D2, 3B10, 1B9,
2G7, 7C5 and 3SB on the growth of bEnd.3 cells in vitro was
determined. bEnd.3 cells (10,000/well) were seeded in 48 well
plates and allowed to attach. 20% DMEM alone (control) or 20% DMEM
containing the antibodies (20 .mu.g to 40 .mu.g total IgG per well)
was added 4 hours after seeding. Each clone was tested on two
separate plates in triplicates. Cells were detached 48 and 96 hours
later, the cell count was determined in each well and the average
cell number per treatment was calculated.
[0951] The 3G4 and 9D2 antibodies were particularly effective,
followed by 3SB and 3B10, with 1B9, 2G7 and 7C5 having less
inhibitory effects. Each of the antibodies show a selective
inhibition of dividing (subconfluent) endothelial cells as opposed
to quiescent (confluent) cells. In comparative studies, 3G4 showed
the greatest inhibitory effect, followed by 9D2, each of which were
more inhibitory than 3SB.
C. Anti-Tumor Effects
[0952] 3G4 binds to the surface of tumor vascular endothelial cells
in vivo. When injected intravenously into mice bearing various
tumors, 3G4 specifically and consistently localized to the tumor,
but not to normal organs. Staining was observed on tumor vascular
endothelium, necrotic areas and individual malignant cells. There
are multiple binding sites for 3G4 in tumors, which allows
simultaneous targeting of both tumor endothelial and tumor
cells.
[0953] 3G4 suppresses angiogenesis and tumor growth in vivo and
shows no detectable organ toxicity in tumor-bearing mice. In
initial studies, 3G4 has shown impressive anti-tumor effects in
syngeneic and xenogeneic tumor models, wherein the antibody causes
tumor vascular injury, decrease in vascularity and tumor necrosis
(Example XI). Regressions of established tumors have been observed
in 30% to 50% of the animals treated.
[0954] The anti-angiogenic and vascular targeting effects of the
3G4 antibody have been observed in repeated studies. Analyses of
tumor sections from nude mice bearing MDA-MB-231 orthotopic tumors
treated with 3G4 revealed anti-angiogenic effects in all treated
tumors, as opposed to control antibodies. The control tumor showed
no signs of necrosis and is highly vascularized, as demonstrated by
the pan-endothelial cell marker, CD31, detected on tumor blood
vessels. In contrast, tumors from the mice treated with 3G4 have 80
to 90% necrosis and almost complete disappearance of CD31-positive
structures, indicating that the treatment produced a substantial
anti-angiogenic effect.
[0955] Another component of the anti-cancer activity of 3G4 is the
induction of tumor vascular damage. This is illustrated by blood
vessels in the control tumors being well perfused, morphologically
intact and surrounded by viable dividing tumor cells, whereas the
blood vessels in the 3G4-treated animals are frequently observed to
have a disintegrating endothelial layer and are blocked by the
detached endothelial cells and, likely, by host cells that are
attracted to the denuded vessels. The vessels in the 3G4-treated
tumor clearly show loss of function, as indicated by the
pre-necrotic layer of surrounding tumor cells. These studies also
showed that treatment with 3G4 causes leukocyte infiltration into
tumors (FIG. 1).
[0956] In these studies, the 3G4 treatment of mice bearing
orthotopic MDA-MB-231 tumors also decreased the plasma volume and
reduced the vascular density in the tumors. A 60% percentage
reduction was observed in the total tumor plasma volume of
3G4-treated mice as compared with BBG3-treated mice, as judged by
the reduction in plasma marker, FITC-dextran. The mean number of
CD-31 positive vessels per square millimeter in tumors from
3G4-treated mice was 50.+-.15 as compared with 160.+-.20 in tumors
from BBG3-treated mice, representing a reduction in tumor
vascularity of about 70% after 3G4 treatment.
[0957] In summary, the histological examination following the
treatment of orthotopic MDA-MB-231 tumors using 3G4 shows: 1)
disintegration of vascular endothelium in about 50% of vessels in
the tumor; 2) attachment of leukocytes to tumor endothelium and
infiltration of mononuclear cells into the tumor interstitium; 3)
occlusion of tumor vessels by platelet aggregates and red cells; 4)
a 70% reduction in microvascular density in tumors from 3G4 treated
vs. untreated mice; and 5) central necrosis of the tumors, with
survival of a peripheral rim of tumor cells, typical of a VTA.
Thus, a primary anti-tumor action of the 3G4 antibody is exerted
through effects on tumor vasculature. Other mechanisms,
particularly antibody-dependent cellular cytotoxicity directed
against the tumor cells themselves, likely contributes to the
anti-tumor effect. This is important, and may permit killing of
more tumor cells, including those in the peripheral rim.
[0958] In follow-up studies, the effect of 3G4 on tumor growth has
been examined in other murine models, including syngeneic (mouse
Meth A fibrosarcoma), subcutaneous xenografts (L540 human Hodgkin's
lymphoma) and orthotopic tumors (human MDA-MB-231 breast cancer and
human MDA-MB-435 breast cancer). Treatment of mice with 3G4
antibody resulted in 90%, 65% and 50% and 70% growth retardation of
these tumors, respectively. Both small (0.1 cm diameter) and
well-established (0.3 cm diameter, 200 mm.sup.3) tumors were
inhibited alike. Anti-PS treatment induced long-term complete
remissions in 50% of Meth A-bearing mice and 30% of mice with
MBA-MD-231 tumors. 3G4 has the highest inhibitory effect in
immunocompetent mice. The orthotopic models of human breast tumors
(MDA-MB-231 and MDA-MB-435), in which human breast tumors are grown
in the mammary fat pads of mice, are important as these are
practical and realistic models of human breast cancer growing
within the breast of humans.
D. Safety Profile
[0959] The 3G4 antibody is different to anti-phospholipid
antibodies described in the literature. Typically,
anti-phospholipid antibodies are regarded as pathogenic antibodies
that interfere with the coagulation cascade. They inhibit
coagulation reactions in vitro and cause thrombosis in vivo. In
contrast, 3G4, 9D2 and like antibodies are therapeutic antibodies
without pathogenic effects.
1. Coagulation
[0960] An important aspect of the 3G4, 9D2 and like antibodies
stems from the inventors' ability to prepare antibodies that are
not linked to anti-phospholipid syndrome or associated
pathologies.
[0961] In studies of blood coagulation in vitro, a weak inhibition
of Tissue Factor (TF)-induced coagulation was observed using high
doses of 3G4 antibody. In other studies using lower doses,
recalcified plasma from 3G4 treated mice coagulated at the same
rate as did recalcified plasma from BBG3 treated mice in the
presence of tissue factor. Also, the addition of 100 .mu.g/ml of
3G4 to cells plus tissue factor in vitro did not affect the
generation rate of coagulation Factor Xa in proplex (extrinsic
coagulation pathway).
[0962] Despite the weak inhibition of TF-induced coagulation using
high antibody levels in vitro, the 3G4 antibody has been tested in
vivo and does not cause thrombotic complications in normal or
tumor-bearing mice (e.g., see Example XI). The 3G4 antibody has
also been tested in monkeys in vivo and no significant side effects
have been observed.
2. Other Indicators of Low or No Toxicity
[0963] The first evidence that 3G4 has no or low toxicity in mice
came from the finding that 3G4 grows as a hybridoma in mice without
evidence of toxicity. Also, when 1 mg of purified 3G4 was injected
intraperitoneally, no toxicity was observed.
[0964] Systematic in vivo studies have now been conducted in which
groups of three 8 week old BALB/c mice were injected IP with 100
.mu.g of purified 3G4 or with an isotype-matched control IgG.sub.3
(BBG3) three times a week for 2 to 4 weeks. No physical signs of
toxicity have been observed, and no histopathological signs of
organ toxicity or morphological abnormalities have been detected in
sections of major organs removed from 3G4-treated mice. The
following parameters were specifically examined.
[0965] In terms of bodyweight, 3G4-treated mice gained weight at
the same rate as BBG3 treated mice. No weight loss was observed in
the earlier studies. There were no physical signs of toxicity, e.g.
hair loss, loss of appetite, etc., and physical activity was normal
compared with control animals.
[0966] No evidence of hematologic toxicity was identified compared
with control animals. Peripheral blood composition was normal
(based upon measurements of complete blood counts with
differentials); evaluation of erythrocyte morphology showed no
evidence of intravascular hemolysis (i.e., absence of
schistocytes); all blood coagulation parameters (PT, APTT, D-dimer)
were normal. There are no changes in blood cell counts, including
red cells, platelets, white cells, absolute lymphocyte counts or
absolute neutrophil counts.
[0967] Bone marrow cellularity and composition were normal. To
analyze bone marrow cellularity, paraffin sections of bone marrow
derived from 3G4 or BBG3-treated mice (six injections, 100 .mu.g)
were examined for total cellularity and cellular composition.
Marrows in the treated animals were essentially completely cellular
(as would be expected for a young mammal). Erythroid, granulocytic,
lymphocytic progenitors and megakaryocytes were present in normal
numbers. Other organ toxicity was absent, as assessed by
post-mortem histologic examination of the lung, liver, heart,
brain, intestine, stomach and kidney.
[0968] In summary, no instance of toxicity has been observed in
more than 200 mice treated with high doses of 3G4 (0.1 mg) three
times a week for 2-4 weeks, or in rats. Even when doses as high as
2 mg were given, no signs of toxicity were seen. Mice retain normal
physical signs, bone marrow cellularity, white blood cell counts,
histology and coagulation functions. In further studies, groups of
five non-tumor bearing mice given a single i.p. injection of 2 mg
3G4, or repeated i.p. injections of 0.5 mg 3G4 daily for 14 days (7
mg total dose), showed no physical signs of toxicity.
[0969] Blood clearance kinetic studies have also been conducted in
mice. 3G4 was radioiodinated using the Bolton Hunter reagent and
was injected intravenously into mice (25 g). Samples of blood were
removed via the tail vein at various later time points. The blood
clearance rate of 3G4 was typical of a mouse IgG in the mouse. The
half-life in the .alpha.-phase of clearance was 3 hours while that
in the .beta.-phase was 5 days. Volume of distribution was normal
(100 ml/kg). These studies indicate that 3G4 does not interact with
normal host tissues, leading to its accelerated clearance.
[0970] The humanized 3G4 antibody has also been administered to
atherosclerotic rabbits and shown to be safe.
3. Monkey Safety Studies
[0971] The humanized 3G4 antibody (see Example XIX, below) has also
been administered to monkeys in safety studies and no significant
side effects have been observed. Humanized 3G4 antibody was
administered IV as a single bolus at up to 100 mg/kg to cynomolgus
monkeys. This is 100 times the calculated therapeutic dose (1
mg/kg).
[0972] No adverse effect level (NOAEL) was approximately 10
mg/kg/week in repeat dosing. There was a transient prolongation in
APTT and PT at doses of 10-100 mg/kg. There were no significant
changes in blood cell counts, including white blood cells, red
blood cells and platelets, or other clinical chemistries.
E. Anti-Viral Effects
[0973] The 3G4 antibody also exerts significant anti-viral effects.
As shown in seen in Example XIII, the treatment of RSV-infected
cells with 3G4 was superior to the effect observed using 3SB. These
results therefore highlight another advantage of the 3G4 antibody
over the prominent anti-PS antibody in the literature, 3SB (Rote et
al. (1993).
[0974] The 3G4 antibody is also shown to be very effective in
inhibiting CMV, both in vitro (Example XII) and in enhancing the
survival of mice infected with mCMV in vivo (Example XXI). In
addition, the 3G4 antibody is further shown to inhibit Pichinde
virus infection, the infectious agent of Lassa fever (Example
XXIV). The cell surface PS exposure herein shown to follow viral
infection, and the ability of the 3G4 antibody to bind to cells
infected with Vaccinia virus (Example XXIII), shows that the 3G4
antibody has enormous potential as a broad spectrum anti-viral
agent.
Example XIX
3G4 Antibody, CDR Sequences, Chimera and Related Construct
[0975] The 3G4 antibody thus possesses the combined properties of
an anti-angiogenic, anti-tumor vascular and anti-viral agent. The
inhibitory activities of 3G4 on cell division, angiogenesis, tumor
growth and viral infectivity, taken together with lack of apparent
toxicity, show broad therapeutic indications for this antibody,
including in the treatment of angiogenic disorders, cancer,
diabetes and viral infections.
[0976] Antibodies recognizing substantially the same epitope as the
3G4 antibody can be generated for use in one or more of the
anti-angiogenic, anti-tumor vascular and anti-viral therapies,
e.g., by immunization and confirmed by antibody competition
studies. Antibodies that bind to essentially the same epitope as
the 3G4 antibody can also be generated from a knowledge of the 3G4
antibody sequences provided herein. The present example provides
the sequences of the complementarity determining regions (CDRs) of
the 3G4 antibody and the use of the sequence information.
A. 3G4 Antibody Sequences
[0977] The original sequences of the antibody variable regions were
obtained by RACE from the hybridoma that produces the 3G4 antibody
and the sequences verified. The nucleic acid and amino acid
sequences of the variable region of the heavy chain (Vh) of the 3G4
antibody CDR1-3 are represented by SEQ ID NO:1 and SEQ ID NO:2,
respectively.
[0978] SEQ ID NO:1 and SEQ ID NO:2 include part of the mouse leader
sequence and constant chain sequences, as shown in FIG. 2A. The
leader sequence is represented by amino acids 1 through 19 of SEQ
ID NO:2, and the mature protein begins as shown by the arrow in
FIG. 2A. Sufficient complementarity determining region sequence
information is included by the sequence of the mature protein up to
the sequence portion concluding VSS, after which the amino acids
are not essential for antigen binding. As such, the BstEII site in
the nucleic acid sequence can be used as a convenient site to
prepare a functional mouse variable region, e.g., for use in
grafting onto a human constant region (FIG. 2A).
[0979] In practice, the 3G4-2BVH sequence has been grafted onto a
human .gamma.1 constant region at the BstEII site using a Lonza pEE
vector. The resultant product contains the mouse leader sequence
and its VH is joined to the human CH1 sequence in the manner shown
in FIG. 2A, wherein ASTLGPSVFPLAPSSKSTSG (SEQ ID NO:7) represents
the first part of the human CH1 sequence.
[0980] The nucleic acid and amino acid sequences of the variable
region of the light chain (V.kappa.) of the 3G4 antibody CDR1-3 are
represented by SEQ ID NO:3 and SEQ ID NO:4, respectively. SEQ ID
NO:3 and SEQ ID NO:4 again include part of the mouse leader
sequence and constant chain sequences, as shown in FIG. 2B. The
leader sequence is amino acids 1 through 22 of SEQ ID NO:4, and the
mature protein begins as shown by the arrow in FIG. 2B. Sufficient
complementarity determining region sequence information is included
by the sequence of the mature protein up to the sequence portion
concluding TVF, after which the amino acids are not essential for
antigen binding. As such, the BbsI site in the nucleic acid
sequence can be used as a convenient site to prepare a functional
mouse variable region, e.g., for use in grafting onto a human
constant region (FIG. 2B).
[0981] In practice, the 3G4-2BVL sequence has been grafted onto a
human .kappa. constant region at the BbsI site using a Lonza pEE
vector. The resultant product contains the mouse leader sequence
and its VL is joined within the human CL1 sequence in the manner
shown in FIG. 2B, wherein IFPPSDEQLKSGTAS (SEQ ID NO:8) represents
the first part of the human .kappa. constant region sequence.
B. Generation and Characterization of 3G4 Human Chimeric
Antibody
[0982] The chimeric construct containing the murine complementarity
determining regions and the human constant regions has been
produced (ch3G4) and shown to behave essentially the same as the
original murine antibody.
[0983] The murine 3G4 antibody was converted into a human-mouse
chimeric antibody (Avanir (Xenerex) Biosciences, San Diego,
Calif.). The murine V.sub.H was cloned and grafted onto the human
.gamma..sub.1 constant region at the BstEII site of the Lonza 2BVH
vector. The murine V.sub.K was cloned and grafted onto the human K
constant region at the BbsI site of the Lonza 2BVL vector. The
sequences were verified. The entire construct was expressed in CHO
cells and purified. The human-mouse chimeric ("humanized") antibody
has been termed bavituximab.
[0984] The resultant ch3G4 bound at least as did well as the murine
3G4 to phospholipid-coated ELISA plates. The in vitro binding
profile of chimeric 3G4 to the panel of phospholipids is shown in
FIG. 4, wherein binding to PS, PA, CL and PI is shown to be
similar. The binding was antigen-specific since no binding was
observed with control antibodies of irrelevant specificity. In
certain studies, an apparently greater binding of chimeric 3G4 vs.
the 3G4 antibody was observed; this may be due to superior binding
of the secondary antibody.
C. Anti-Tumor Effects of the Humanized Antibody
[0985] In vivo, ch3G4 localizes to tumor vascular endothelium and
exerts anti-tumor effects. The anti-tumor effects of ch3G4 in
MDA-MB-435 human breast cancer cells growing in mice is described
in Example XI. Treatment of mice with MDA-MB-435 tumors using the
chimeric antibody effectively retarded tumor growth as opposed to
control.
[0986] Localization of ch3G4 was examined in MDA-MB-435 human
breast cancer cells growing in mice. Mice were injected
intravenously with biotinylated ch3G4 or control IgG of irrelevant
specificity. One hour later, the mice were exsanguinated, and their
tumors were removed and frozen sections were cut. Biotinylated
reagents were first incubated with streptavidin-Cy3 conjugate,
washed in PBS, then incubated with MECA 32 antibody followed by
FITC-tagged secondary antibody. Single images, taken with
appropriate filters for Cy3 (red) and FITC (green) fluorescence
respectively, were captured by digital camera and transferred to a
computer. Converged images demonstrating yellow color (a product of
merged green and red fluorescence) were superimposed with the aid
of Metaview software.
[0987] In this double staining method, the biotinylated proteins
and the vascular endothelium are labeled by red and green. Where
the biotinylated proteins are bound to the endothelium, the
converged image appears yellow. Biotinylated ch3G4 binds to the
tumor vascular endothelium, because the staining patterns converges
with that of MECA 32.
[0988] Bavituximab has also been radiolabeled and shown to home to
syngeneic prostatic tumors in rats. Reduced tumor perfusion was
also observed in tumor-bearing rats treated with bavituximab.
D. Generation and Characterization of Recombinant IgG2a Isotype of
3G4
[0989] The human chimera of the murine 3G4 antibody (ch3G4) is a
human IgG.sub.1 isotype (hIgG.sub.1). The murine IgG homolog of
ch3G4 requires a mouse IgG.sub.2a isotype (mIgG.sub.2a). This
construct was made and tested, and shown to behave essentially the
same as the parent antibody.
[0990] Lonza expression vectors pEE12.4 & pEE6.4 were obtained
from Lonza Biologics via an agreement with Peregrine
Pharmaceuticals Inc. Use of the vectors, transfection, and
screening of transfected NS0 mouse myeloma cells was conducted
according to Lonza Biologics' "Operating Procedures for use with:
NS0 Myeloma cells". Briefly, the 3G4 light chain coding sequence
was amplified by RT-PCR from total RNA isolated from the 3G4
hybridoma cell line. RT-PCR primers were designed such that the
amplified fragment contained XmaI and EcoRI restriction enzyme
sites on either end of the amplified product for cloning into the
Lonza pEE12.4 vector.
[0991] The variable region of the 3G4 heavy chain was amplified by
RT-PCR from total RNA isolated from the 3G4 hybridoma cell line.
Primers were designed such that the amplified fragment contained
HindIII and XmaI restriction enzyme sites on either end of the
amplified product for cloning into the Lonza pEE6.4 vector. The
murine IgG2a constant region was amplified by PCR from a plasmid
vector provided by Dr. Shozo Izui. PCR primers were designed with
BstII and EcoRI restriction enzyme sites at either end of the
amplification product for cloning into the pEE6.4+3G4VH vector.
Importantly, the BstEII site was designed to be in-frame with the
3G4 VH variable region sequence upstream. The heavy and light chain
constructs were combined into a single double gene vector (12.4 3G4
IgG2a) by cutting both vectors with SalI and NotI. The heavy and
light chain coding regions were verified by sequencing at the UT
Southwestern sequencing core facility.
[0992] The 12.4 3G4 IgG2a vector was transfected into NS0 cells by
electroporation. Following transfection, the NS0 cells were diluted
and plated into 96-well plates in media lacking glutamine. Only
cells transfected with the construct (which contains the glutamine
synthease gene for positive selection) can grow in the absence of
glutamine. Over the next two months, various transfectants were
identified screened for antibody secretion by PS-ELISA. Those
transfectants secreting the highest amounts of antibody were grown
in large culture to generate purified antibody. The antibodies were
purified using the same purification protocol as used for the 3G4
antibody.
[0993] The amino acid sequences of the IgG2a heavy chain and the
3G4 Light Chain (C.sub..kappa.) are represented by SEQ ID NO:10 and
SEQ ID NO:11, respectively, as shown in FIG. 2C and FIG. 2D.
[0994] As expected, purified 2aG4 antibodies migrate on an SDS-PAGE
gel as a 150 kDa band, and bind to PS in PS-ELISA with an affinity
and specificity essentially the same as for 3G4. 2aG4 thus binds to
anionic phospholipids with the same profile as 3G4 (see Table 4,
above).
[0995] The mouse IgG.sub.2, isotype was generated mainly to provide
a better model for the human chimera, ch3G4 (bavituximab). The
mouse IgG.sub.2, isotype is also thought to be more stable than the
mouse IgG.sub.3, the current isotype of the murine 3G4 antibody.
The 2aG4 antibody may generate a stronger anti-tumor effect than
the 3G4 IgG.sub.3, via enhanced ADCC.
[0996] The 2aG4 antibody has been tested in a preliminary study in
a mouse WiDr (colon carcinoma) model, along with the 3G4 and ch3G4
antibodies and the C44 antibody as a control. Treatment was started
on day 5, when the tumors were small (10 mice per group). 100 .mu.g
of each antibody was injected 3.times./week and tumor growth
monitored. All three of the test antibodies slowed tumor growth to
essentially the same degree.
Example XX
3G4 Antibody in Combination Therapies, Including Docetaxel
[0997] The present example concerns combination therapies for tumor
treatment using the 3G4 antibody and the chemotherapeutic drug,
docetaxel. These agents are designed to attack tumor vasculature
endothelial cell and tumor cell compartments, leading to
synergistic treatment with lower toxicity. The results showed that
this combination therapy did indeed significantly enhance treatment
efficacy.
A. Fc Domain-Mediated Anti-Tumor Effects
[0998] The 3G4 antibody was tested for inhibitory effects on tumor
cells in vitro. No direct inhibitory effect on tumor cells was
observed. Therefore, it is likely that the anti-tumor effects of
the 3G4 antibody include Fc domain-mediated augmentation of immune
effector functions, such as antibody mediated phagocytosis, ADCC,
CDC and stimulation of cytokine production, or these mechanisms
combined.
[0999] The effects of 3G4 on the phagocytosis of PS-positive cells
by macrophages have been evaluated. Fluorescent tumor cells were
treated with H.sub.2O.sub.2 to induce PS exposure. Treated and
untreated cells were then harvested and contacted with the 3G4
antibody or a control antibody (BBG). Mouse bone marrow macrophages
were then added, and the ability of the macrophages to phagocytose
the fluorescent tumor cells was analyzed using a fluorescent
microscope.
[1000] It was determined that 3G4 could increase the phagocytosis
of PS-positive cells by macrophages by more than three fold. This
finding supports the inventors' reasoning that the Fc domain of the
3G4 antibody contributes to the anti-tumor effects of the antibody.
That is, the Fc domain activates host immune effector functions,
which then exert anti-tumor effects. The 3G4 antibody should
therefore enhance the lytic activity of NK cells, leading to more
effective ADCC.
B. Docetaxel Induces PS Exposure on Endothelial Cells and Tumor
Vessels
[1001] The induction of PS exposure on endothelial cells by
subclinical concentrations of docetaxel was examined in vitro by
FACS analysis. Human umbilical vein endothelial cells (HUVEC) and
human microvessel endothelial cells (HMVEC) were treated with 10 nM
of docetaxel for 24 hrs and examined by FACS. Both treated HUVEC
and HMVEC showed significant increase in 3G4 binding as compared to
untreated cells. Docetaxel incubations for 48 and 72 hrs were also
conducted.
[1002] Treatment of HUVEC cells in vitro with concentrations of
docetaxel at 20 pM also caused anionic phospholipids to be
externalized without inducing apoptosis.
C. Docetaxel Induces PS Exposure on Tumor Cells
[1003] The in vitro induction of PS exposure by subclinical
concentrations of docetaxel was also examined by FACS analysis
using a panel of tumor cell lines. Mouse lewis lung carcinoma 3LL,
mouse colon carcinoma Colo26 and human breast cancer MDA-MB-435
cells were treated with 10 nM of docetaxel for 24 hrs and examined
by FACS. All tumor cell lines tested showed significant increase in
3G4 binding as compared with untreated cells. Docetaxel incubations
for 48 and 72 hrs were also conducted. Mouse melanoma B16 and mouse
firbrosarcoma Meth A tumor cell lines were further examined and
also showed significant increase in 3G4 binding as compared with
untreated cells.
[1004] Human breast cancer MDA-MB-231 cells were treated with 10 nM
of docetaxel for 24 hrs and incubated with either the chimeric 3G4
antibody (ch3G4) or control, human IgG and analyzed by FACS. These
results show that the significant increase in antibody binding is
antigen-specific and that the chimeric antibody behaves like the
parent 3G4 antibody.
D. Docetaxel Induces PS Exposure on Tumor Vessels
[1005] Results from controlled studies also showed that docetaxel
increases PS exposure on tumor vessels. In such studies, docetaxel
treatment of mice increased the percentage of tumor vessels that
expose anionic phospholipids from 35% to 60%. No induction of PS
was observed on vessels in normal tissues even after systemic
treatment with docetaxel.
E. Synergistic Tumor Treatment with 3G4 and Docetaxel
[1006] The inventors have thus shown that the treatment of
endothelial cells and tumor cells with docetaxel at subclinical
concentration significantly increases 3G4 binding. They have also
shown that the 3G4 antibody facilitates macrophage-mediated
phagocytosis of tumor cells on which PS is exposed at the surface.
The increased 3G4 binding mediated by docetaxel should therefore
augment the phagocytosis of tumor cells and other anti-tumor
effects mediated by the Fc domain of the 3G4 antibody, such as
increasing the lytic activity of NK cells, leading to more
effective ADCC. Studies of others have also shown that treatment of
breast cancer patients with docetaxel leads to an increase in serum
IFN-.gamma., IL-2, IL-6 and GM-CSF cytokine levels and enhancement
of NK and LAK cell activity (Tsavaris et al., 2002).
[1007] The anti-tumor effect of the combined therapy of 3G4 with
docetaxel was therefore examined in an orthotopic model in SCID
mice bearing human MDA-MB-435 breast carcinoma. Mice bearing
orthotopic MDA-MB-435 human breast tumor were treated i.p. with 3G4
alone (100 .mu.g/dose), docetaxel alone (10 mg/kg), or 3G4 in
combination with docetaxel (100 .mu.g/dose and 10 mg/kg,
respectively), for three weeks, with administration 3 times a week.
Treatment started 6 days after tumor cell implantation.
[1008] These studies showed that treatment of mice bearing
orthotopic MDA-MB-435 human breast tumors with 3G4 plus docetaxel
inhibited tumor growth by 93%. Treatment of mice bearing
disseminated MDA-MB-435 tumors with 3G4 plus docetaxel reduced the
average number of tumor colonies in the lungs by 93% and half the
animals did not develop tumors. In both tumor models, the antitumor
effect of the combination was statistically superior (p<0.01) to
that of docetaxel or 3G4 alone. Combination therapy reduced the
tumor vessel density and plasma volume in tumors to a greater
extent than did the individual drugs. The combination therapy was
no more toxic to the mice than was docetaxel alone. These results
indicate that, as an adjuvant therapy, 3G4 could enhance the
therapeutic efficacy of docetaxel in breast cancer patients.
F. 364 Enhances the Activity of Cisplatin Against Drug-Resistant
Breast Tumors
[1009] The 3G4 antibody also enhances the activity of cisplatin
against drug-resistant breast tumors in animals. Mice were
inoculated with drug-resistant breast tumors and treated after day
20. Treatment groups were cisplatin alone, the 3G4 antibody alone,
an isotype-matched control antibody (BBG3) or cisplatin in
combination with the 3G4 antibody.
[1010] The tumors in the control animals continued to grow rapidly.
In mice treated with either cisplatin alone or the 3G4 antibody
alone, tumor growth was slowed. The combination treatment group
(cisplatin and 3G4 antibody) showed the best anti-tumor response.
Thus, antibodies such as 3G4 enhance the effectiveness of
chemotherapeutic drugs, such as cisplatin, even against
drug-resistant tumors.
G. Combination Therapy of Pancreatic Cancer
[1011] There is an urgent need for improved treatments for
pancreatic cancer. In humans, the overall long-term survival for
patients with pancreatic cancer is less than 4%. Approximately
70-80% of pancreatic cancer patients fail therapy due to metastases
to the liver, and there is currently no effective therapy for
metastatic disease.
[1012] Pan02 mouse pancreatic adenocarcinoma cells were injected
into the pancreas of immunocompetent C57BL/6 mice. Treatment was
started on day 5 after tumor cell injection. Treatment groups were
PBS as a control, the 3G4 antibody alone (100 .mu.g), gemcitabine
alone (3.5 mg), or the 3G4 antibody in combination with gemcitabine
(100 .mu.g and 3.5 mg, respectively). Gemcitabine is a pyrimidine
anti-metabolite. The animals were sacrificed at day 22 after tumor
cell injection, and the tumor weight determined.
[1013] In these studies, both gemcitabine and the 3G4 antibody
showed a significant anti-tumor effect. Animals treated with
gemcitabine in combination with the 3G4 antibody showed a
significantly enhanced anti-tumor effect over each agent alone,
without adverse effects, e.g., on weight loss. The nodal,
peritoneal and liver metastases were also significantly reduced in
treatment with either gemcitabine or the 3G4 antibody alone. Again,
animals treated with gemcitabine in combination with the 3G4
antibody showed significantly reduced nodal, peritoneal and liver
metastases when compared to each agent alone. Importantly, in
animals treated with the combination of gemcitabine and the 3G4
antibody, there were no detectable metastases to the liver. This is
significant, as in humans, metastases to the liver are the most
frequent cause of death following diagnosis of pancreatic
cancer.
[1014] These studies also showed significant infiltration of
macrophages into the tumor in the combination treatment group.
Microvessel density was reduced upon treatment with the 3G4
antibody alone and with the 3G4 antibody in combination with
gemcitabine.
H. Treatment of Lung Cancer with Radiation and 3G4
[1015] In studies of mice with lung cancer, 10 Gy focal irradiation
induced PS exposure on tumor blood vessel endothelium, as shown in
double-labeling studies. In the treatment phase, both irradiation
and the 3G4 antibody alone showed an anti-tumor effect. Again, the
combination of treatment modalities, i.e., irradiation and the 3G4
antibody, showed the greatest anti-tumor effect.
I. 3G4-Targeting of Apoptotic Tumor Cells to Fc.gamma.R on
Dendritic Cells
[1016] Tumors from mice treated with 3G4 plus docetaxel also
contained unusual amount of lymphocytes, as compared to control
tumors. Although this phenomenon could represent typical
chemoattraction of immune cells by disintegrating tumor cells, it
could also reflect activation of the immune system by 3G4 mediated
through Fc binding to Fc.gamma.R on immune effector cells.
[1017] To characterize the effects of 3G4 and docetaxel
administration on the intratumoral immune cell infiltrate, the
types of cells present in these infiltrates can be identified by
immunostaining of frozen sections and/or paraffin sections of tumor
tissues using antibodies directed against specific markers of
macrophages, neutrophils, granulocytes, NK cells and activated
lymphocytes (Pharmingen, San Diego, Calif.). The extent, phenotype,
and activation status of this infiltrate can be graded. Cytokine
production by infiltrating immune cells, including IL-2 and INF,
can also be analyzed via immunohistochemical techniques. Serum
cytokine levels can be evaluated by ELISA and intracellular
staining can be used to identify the specific cellular compartments
responsible for cytokine production. The effects of infiltrating
immune cells on tumor cell proliferation and apoptosis can thus be
systematically evaluated.
[1018] In light of the foregoing data, the inventors further
contemplate methods enhancing the potency of immunotherapy of
breast cancer by 3G4-mediated targeting of apoptotic tumor cells to
the Fc gamma receptor (Fc(.gamma.)R) on dendritic cells. Efficient
antigen presentation, which induces effective cellular and humoral
immune responses, is important for the development of tumor
vaccines and immunotherapies. Dendritic cells (DC) are the most
potent antigen-presenting cells (APC) that prime cytotoxic T
lymphocytes against tumor-associated antigens. Improvement of tumor
antigen presentation by dendritic cells (DCs) should lead to
develop more potent tumor vaccines.
[1019] Antigenic presentation by Fc(.gamma.)R receptor-mediated
internalization of DCs can be enhanced up to 1,000-fold compared
with fluid phase antigen pinocytosis. Apoptotic tumor cells (ATC)
are an excellent source of antigens for dendritic cell loading
because multiple tumor specific antigens (both known and unknown)
can be efficiently presented to naive T cells, making the
occurrence of immune escape variants less likely due to the lock of
certain epitopes. In animal studies, DCs pulsed with ATCs have been
shown to produce potent anti-tumor immunity in vitro and in vivo.
However, recent data has demonstrated that ATCs alone were somewhat
inefficient for activating anti-tumor immunity, possibly because of
their insufficient uptake and inability to induce DC
maturation.
[1020] Recent studies have also demonstrated that ATC-immune
complex, formed by binding of anti-tumor antibody to apoptotic
tumor cells, can be targeted to Fc(.gamma.)R on DC. Compared with
ATCs alone, ATC-immune complexes were more efficiently internalized
by DC, more efficient in inducing DC activation and maturation, and
more importantly, ATC-immune complexes can significantly enhance
both MHC I and II-restricted antigen presentation, therefore induce
potent anti-tumor T helper and CTL immunity.
[1021] The inventors therefore envision using the anti-PS
antibodies of the present invention to enhance both hormonal and
cellular anti-tumor immunity, and boost the efficacy of ATC based
DC tumor vaccines. As PS is a universal and the most abundant
specific marker of apoptotic tumor cells, the panel of antibodies
of the invention, particularly 3G4, can bind to PS on ATCs. The
inventors have already demonstrated that 3G4 can enhance DC uptake
of apoptotic tumor cells by 300% through Fc(.gamma.)R mediated
internalization of 3G4-ATC complexes. By enhancing the uptake of
ATC by DC mediated through Fc(.gamma.)R, it is therefore reasoned
that 3G4 and like antibodies can greatly enhance both MHC I and II
restricted antigen presentation, induce both potent hormonal and
cellular anti-tumor immunity, and boost the efficacy of ATC based
DC tumor vaccines. This can be demonstrated by establishing the
efficacy of DC loaded with 3G4-ATC immune complexes in the
induction of T hl, CTL and antibody response in vivo, and by
determining the potency of anti-tumor immunity induced by
immunization of DC loaded with 3G4-ATC immune complexes in
vivo.
Example XXI
Anti-PS Antibodies Treat CMV Infections In Vivo
[1022] Following the anti-viral effects against CMV in vitro shown
in Example XII, the present example demonstrates the enhanced
survival of mice infected with the murine version of the CMV virus,
mCMV.
[1023] Balb/C mice (6 week old, five mice per group) were infected
i.p. with 5.times.10.sup.5 pfu of mCMV RVG102. The mice were
treated i.p. on day 1 with the 3G4 antibody (1 mg/mouse), or the
human-mouse chimeric antibody, ch3G4 described above (1 mg/mouse).
Untreated mice served as the control. The mice were treated every
four days thereafter with 0.5 mg/mouse of antibody or chimeric
antibody until day 26. The mice were monitored for survival past 90
days post infection.
[1024] Treatment with both the parent and chimeric forms of the 3G4
antibody resulted in increased survival of the mCMV-infected mice.
Mice treated with 3G4 or ch3G4 had 100% and 80% survival,
respectively, as compared to untreated mice, wherein only 25% of
the mice survived the infection.
Example XXII
PE-Binding Peptide Derivative Treats CMV Infection In Vivo
[1025] In addition to the in vitro anti-viral effects against CMV
shown in Example XVII, this example demonstrates that the
duramycin-biotin derivative, DLB increased survival of mice
infected with mCMV.
[1026] Balb/C mice (6 week old, five mice per group) were infected
i.p. with 5.times.10.sup.5 pfu of mCMV RVG102. The mice were
treated i.p. on day 1 and every four days with 20 .mu.g/mouse of
the duramycin derivative, DLB. Untreated mice served as the
control. The mice were monitored for survival past 90 days post
infection.
[1027] Treatment with the duramycin-biotin derivative, DLB enhanced
survival of the mCMV-infected mice. Mice treated with DLB had 100%
survival, as compared to untreated mice, wherein only 25% of the
mice survived the infection.
Example XXIII
Anti-PS Antibodies Bind to Virally Infected Cells
[1028] The present example shows that viral infection induces PS
exposure at the cell surface and that anti-PS antibodies bind to
virally infected cells. Cells infected with Vaccinia virus become
PS-positive, as shown by increased binding of the chimeric 3G4
antibody to the cell surface demonstrated in FACS analyses.
[1029] U937 cells were infected with trypsinized Vaccinia virus at
a high m.o.i of 2. Briefly, Vaccinia virus was treated with an
equal volume of 0.25 mg/ml trypsin for 30 minutes at 37.degree. C.
The virus was added to U937 cells in a total volume of 0.5 ml.
After 1.5 hr, fresh medium was added to the cells and the cells
were incubated in a T25 flask at 37.degree. C. for 2 days.
Uninfected cells served as the controls.
[1030] Infected and uninfected U937 cells were stained with a
primary antibody, either with the chimeric 3G4 antibody (ch3G4) or
with human IgG (HIgG) as a control. The cells were washed, blocked
with normal mouse serum and then stained with the primary antibody
for 45 minutes on ice. After three washes, the cells were stained
with a 1:400 dilution of goat anti-human FITC-conjugated secondary
antibody and were analyzed on a FACScan.
[1031] Results from the FACS analyses show that there is a
significant shift with ch3G4 on U-937 cells infected with Vaccinia
virus, as compared to that obtained on uninfected U937 cells. This
study therefore shows that infection of cells with Vaccinia virus
leads to PS exposure on the cell surface and that the chimeric
version of the anti-PS antibody, 3G4 is capable of binding to these
virally infected cells.
Example XXIV
Anti-Viral Effects of Anti-PS Antibodies Against Pichinde Virus
[1032] In addition to the anti-viral effects against CMV and RSV,
the present example further shows that anti-PS antibodies inhibit
Pichinde virus infection in vitro. Pichinde virus is New World
arenavirus, which is non-pathogenic in man, and is used in an
animal model for Lassa fever.
[1033] Confluent monolayers of Vero cells were treated with the 3G4
antibody or an isotype-matched control antibody, GV39G, after
infection with Pichinde virus at a low m.o.i. of 0.01 pfu/cell.
Briefly, the cells were incubated with virus in a total volume of 1
ml per well at 37.degree. C. for 90 minutes. During the infection,
the plates were gently rocked every 30 minutes. Following the
infection, the cell supernatant was removed and DMEM/10%
FBS/pen-strep was added to each well (2 ml per well). On day 2, the
cells were harvested with trypsin and allowed to adhere to Biocoat
chamber slides. They were fixed and stained with polyclonal rabbit
anti-PIC serum followed by a biotin-conjugated goat anti-rabbit
secondary (secondary antibody alone produced no staining). The
number of infected cells per field of 100 cells was counted.
[1034] In cells treated with 3G4, the virus is restricted to single
cells that stain a dark red, numbering about one in about a hundred
cells. These are probably the cells that were originally infected
by the virus, as was seen with CMV (Example XII). However, in cells
treated with the control, GV39G antibody, the virus has spread and
infected all the cells.
[1035] This pattern of inhibition of viral replication is similar
to that observed when 3G4 was used to treat CMV-infected human
fibroblasts. Thus, the anti-PS antibody, 3G4 effectively and
quantifiably prevents the spread of Pichinde virus from cell to
cell.
Example XXV
Tumor Treatment Using PE-Binding Peptide Derivative
[1036] Further to the anti-viral effects of duramycin derivatives,
both in vitro and in vivo, the present example demonstrates the
localization of duramycin derivatives to tumor vasculature and
associated anti-tumor effects.
A. Tumor Treatment with Duramycin-HuIgG Conjugate
[1037] Human IgG (HIgG) was first purified as described in Example
XV. Purified HIgG was linked to duramycin using the SIAB linker,
and the resultant (D-SIAB).sub.nHIgG conjugate purified.
[1038] Mouse fibrosarcoma cell-line MethA was grown, harvested at
log phase and resuspended in DPBS. Approximately 10.sup.6 MethA
tumor cells were injected subcutaneously in the middle dorsum of
6-8 week old BALB/c male mice. 5 days after implantation, the mice
were randomly separated into two groups (n=15). From day 10, one
group received 150 .mu.g Duramycin-HuIgG conjugate by
intraperitoneal injection for consecutive 2 weeks. The other group
received the same amount of HuIgG as a control. Tumor volumes were
measured twice a week and were calculated using the formula
1/2ab.sup.2, (where "a" is the long axis and "b" the short axis of
the tumor). Mice were sacrificed when the tumors reached a size of
approximately 1400 mm.sup.3.
[1039] The duramycin-HIgG conjugate inhibited MethA tumor growth in
BALB/c mice at the dose of 150 .mu.g/day, as compared to the human
IgG control.
B. Duramycin-HuIgG Conjugate Localizes to Tumor Vasculature
[1040] Using the same MethA mouse tumor model as above, when the
tumor size reach 500 mm.sup.3, 100 .mu.g (D-SIAB).sub.nHIgG in 100
.mu.l PBS was injected through the tail vein. The same amount of
human IgG was injected as a control. After 4 hours, mice was
euthanized and perfused with normal saline for 5 minutes and 1%
paraformadehyde for 10 minutes. The tumor and other major organs
were dissected and frozen in liquid nitrogen. After embedding in
OCT, tissue was cryosected in 10 .mu.m section and placed on
silanized slides. After fixing in cold acetone for 10 minutes,
slides were stained with peroxidase labeled goat anti human IgG to
detect the biodistribution of duramycin-HuIgG. Meca32 and
peroxidase labeled goat anti-rat IgG were used to detect blood
vasculature of tissue.
[1041] This study showed that the duramycin-HIgG conjugate
localized to the tumor vasculature in the treated animals.
Example XXVI
Biodistribution and Properties of Duramycin Conjugates
[1042] The present example demonstrates the lack of toxicity of
cell-impermeant duramycin derivatives in vitro, the biodistribution
of duramycin derivatives administered in vivo and the ability of
duramycin-antibody conjugates to increase the phagocytosis of
apoptotic cells by macrophages.
A. Duramycin-Biotin Conjugates are not Cytotoxic
[1043] The duramycin derivatives and conjugates of the invention
are designed to minimize the non-specific toxic effects of the
parent duramycin molecule. In many examples, this is achieved by
linking duramycin to a cell impermeant group (Example XV).
[1044] The biotinylated duramycin construct DLB was prepared as
described in Example XV. The unmodified duramycin compound and DLB
were tested for cytotoxic effects on HUVEC using an MTT assay.
Whilst the unmodified duramycin showed dose-dependent toxicity, DLB
was non-toxic, matching the untreated control.
B. Localization of Duramycin-Biotin Conjugate to Macrophages in
Lung
[1045] The human breast cancer cell line MDA-MB-435 was grown,
harvested at log phase, and resuspended in DPBS. Approximately
10.sup.7 cells were injected into the mammary fat pad of 6-8 week
old female ethylic nude mice. 100 .mu.g duramycin-biotin in 100
.mu.l PBS was injected through the tail vein. After 4 hours, mice
was euthanized and perfused with normal saline for 5 minutes and 1%
paraformadehyde for 10 minutes. Major organs, including heart,
lung, liver, kidney, brain, intestine, testes and spleen were
dissected and frozen in liquid nitrogen. After embedding in OCT,
tissue was cryosected in 10 .mu.m sections and placed on silanized
slides. After fixing in cold acetone for 10 minutes, slides were
stained with Cy3 labeled streptavidin to detect the biodistribution
of the duramycin-biotin construct. Meca32 and FITC labeled goat
anti rat IgG were used to detect blood vasculature of tissue.
[1046] The intravenous injection of the duramycin-biotin conjugate
into nude mice bearing MDA-MB-435 tumors resulted in the deposition
of drug in the tumor cells, renal tubules and in the macrophages in
the lung. There was minimal deposition in liver and no detectable
distribution in brain, intestine, testes. The localization to
macrophages in the lung can be exploited in the anti-viral
embodiments of the invention.
C. Duramycin-Antibody Conjugate Enhances Phagocytosis of Apoptotic
Cells
[1047] The ability of a duramycin-antibody conjugate
(duramycin-C44, DuC44) to increase the phagocytosis of apoptotic
cells was next investigated.
[1048] Macrophages were isolated and cultured from mouse bone
marrow. The medium used for the isolation, culture, and stimulation
of BM macrophages was DMEM containing 2 mM glutamine, 0.37% (w/v)
NaHCO.sub.3, 10% (v/v) heat-inactivated FCS, and 0.5 ng/ml mouse
GM-CSF. Bone marrow cells were flushed asceptically from the
dissected femurs with jet of complete medium directed through a
25-gauge needle. The cells were then adjusted to a density of
approximately 3.times.10.sup.5 cells/ml of complete medium, and
were distributed in 0.5 ml aliquots into 8 well chamber slides.
[1049] Cells were incubated for 1 hour at 37.degree. C. in 5% CO2,
in a humidified chamber to allow macrophages to adhere and spread.
Nonadherent cells were removed by adding 5 ml of warmed PBS to each
well, resuspending nonadherent cells by moderately tapping the
plate, and flicking the slides to discard the nonadherent cells.
This washing was performed a total of three times. The cells were
maintained at 37.degree. C. under a 7.5% (v/v) CO.sub.2 atmosphere
for 5 days. The complete medium was changed every other day until
the cells were used.
[1050] The following method was used to label HL-60 target cells
with a fluorescent cell tracer. A 10 mM CFDA SE stock solution was
prepared immediately prior to use by dissolving the contents of one
vial dye in 90 .mu.L of the DMSO and diluting in PBS to 10 .mu.M.
Centrifugation was used to obtain a HL-60 cell pellet and the
supernatant aspirated. The cells were resuspend in CFDA/PBS and
incubated at 37.degree. C. for 15 minutes. The samples was
centrifuged and the supernatant aspirated. The cells were resuspend
in media and incubated for another 30 minutes. The cell viability
and fluorescence were confirmed to be over 95%.
[1051] In this phagocytosis assay, labeled HL-60 cells were exposed
to UV 254 nm for 5 minutes and incubated at 37.degree. C. for one
hour to induce apoptosis. 10.sup.4 apoptotic HL-60 cells were
incubated with macrophages for one hour. Duramycin-C44 conjugate
was included at the concentration of 10 .mu.g/ml. The same
concentration of mouse antibody BBG3 was used as a negative
control, and the 3G4 antibody was also included for comparison.
Hoechst 33342 was added in media in the last 45 minutes at the
concentration of 10 .mu.g/ml.
[1052] Slides were washed with PBS 3 times, and fixed in 4%
paraformadehyde for 15 minutes. The slides were stained with rat
anti-mouse CD11 antibody (CD11 is a macrophage marker), diluted in
0.2% gelatin for one hour, washed and stained with Texas red
labeled goat anti-rat secondary antibody.
[1053] The cells were analyzed under the fluorescence microscope.
Macrophages are identified as red cells, due to the CD11 marker.
Macrophages that have phagocytosed apoptotic cells are identified
as green cells, due to the fluorescent tracer in the target cells.
Red and green cells are counted and the phagocytosis is quantified
as the percent phagocytes positive for uptake.
[1054] This study shows that the duramycin-antibody conjugate,
DuC44 enhanced phagocytosis of apoptotic HL-60 cells by
macrophages. Thus, the duramycin portion is binding to the surface
of the apoptotic cells, permitting the protruding antibody portion
of the conjugate to be recognized by the macrophages. The
duramycin-antibody conjugate thus functioned similarly to the 3G4
antibody. As expected, an (Fab).sub.2 fragment of the 3G4 antibody,
lacking the Fc region, did not induce phagocytosis above control
levels.
[1055] As the earlier study showed duramycin-biotin conjugates to
localize to macrophages in the lung following administration in
vivo, the stimulation of macrophage-mediated phagocytosis of
apoptotic cells shown in the present study has important
implications for the therapeutic uses of the present invention,
such as in treating pulmonary viral infections.
Example XXVII
Infiltration of Macrophages During Tumor Treatment
[1056] As shown in the foregoing studies, antibodies to
aminophospholipids and anionic phospholipids are effective in tumor
treatment. For example, the 3G4 antibody specifically localizes to
tumor vessels, causes tumor vessel destruction and retards tumor
growth in multiple mouse models without causing toxicity (Example
XI; Example XVIII). The present example shows that 3G4 treatment of
animals with tumors also causes mononuclear leukocytes to bind to
the tumor vascular endothelium and induces macrophages to
infiltrate into the tumor.
A. Methods
[1057] Groups of 8-10 female SCID mice were injected subcutaneously
with 2.times.10.sup.7 L540 cells or orthotopically with
1.times.10.sup.7 MDA-MB-435 or MDA-MB-231 cells. BALB/c mice were
injected subcutaneously with 1.times.10.sup.6 Meth A cells. Tumors
were allowed to grow to an average diameter of 0.8-1 cm (L540),
0.6-0.7 cm (MDA-MB-435), 0.5-0.7 cm (MDA-MB-231), or <0.1 cm
(Meth A). The mice were then treated i.p. with 100 .mu.g 3G4
antibody or control antibody (BBG3) three times a week for 2-3
weeks. Animals were monitored three times a week for tumor size and
body weight. Mice were sacrificed when tumors in control mice
reached a diameter of 1.5-2 cm.
B. Results
[1058] 3G4 treatment of mice bearing orthotopic MDA-MB-435 and
MDA-MB-231 tumors caused mononuclear leukocytes to bind to the
tumor vascular endothelium and infiltrate into the tumor
interstitium (FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D). In this
figure, the red staining shows endothelium, the green staining
shows macrophages, and the blue staining shows nuclei.
[1059] Often vessels in 3G4-treated tumors were packed with
mononuclear leukocytes (FIG. 6D), whereas only occasional
leukocytes were seen adhering to vessels in tumors from control,
BBG3-treated mice (FIG. 6C). Mononuclear leukocytes infiltrated
into the interstitium of tumors from 3G4-treated mice in strikingly
greater numbers than they did in BBG3-treated mice (compare FIG. 6B
and FIG. 6A).
[1060] Almost all (>90%) of the cells that adhered to tumor
vessels and infiltrated into the tumors expressed the
monocytes/macrophage markers F4/80, M1/70 (CD11b, Mac-1) and
Fc.gamma.R (FIG. 6A, FIG. 6B, FIG. 6C and FIG. 6D). The cells
lacked the neutrophil marker, Ly-6G, the NK cell marker, Ly-49, and
the dendritic cell marker, CD11c. Lymphocyte markers were absent,
as expected of SCID mice. Taken together, the results indicate that
the cells that were adhering to tumor vessels and that were
infiltrating into the tumor interstitium were monocytes and
macrophages respectively.
Example XXVIII
F(ab').sub.2 Fragments are Effective in Tumor Treatment
[1061] Antibodies to aminophospholipids and anionic phospholipids,
such as the 3G4 antibody, are effective in tumor treatment (Example
XI; Example XVIII). The present example shows that the F(ab').sub.2
fragment of the 3G4 antibody is as effective as the intact 3G4
antibody in tumor treatment.
[1062] In controlled studies mice with tumors were treated (on day
9) with either the 3G4 antibody, the F(ab').sub.2 fragment of the
3G4 antibody or an isotype-matched control antibody (BBG3). The
tumors in the control animals continued to grow rapidly, whereas in
mice treated with either the 3G4 antibody or the F(ab').sub.2
fragment of the 3G4 antibody, tumor growth was significantly slowed
(FIG. 8). The F(ab').sub.2 fragment was at least as effective as
the intact antibody (FIG. 8), and the anti-tumor effect of the
F(ab').sub.2 fragment may have been underestimated in these
studies.
[1063] This supports the proposal that antibodies such as 3G4
enhance the ability of monocytes and macrophages to mount an
inflammatory response against PS-expressing tumor vasculature and
tumor cells. Antibodies such as 3G4 may prevent PS on tumor
vasculature from silencing host inflammatory responses, thereby
permitting macrophages to secrete inflammatory cytokines, such as
TNF-.alpha., IL-1 and others, directly damaging tumor endothelium
and recruiting further host cells into the tumor (FIG. 7).
Example XXIX
Annexin V Dimer Localizes to Tumor Vessels Better than Monomer
[1064] Example XXVII and Example XXVIII concern the role of
macrophages in tumor treatment using antibodies to
aminophospholipids and anionic phospholipids. From such data and
other information, the inventors developed the receptorbody aspects
of the overall invention. As supportive of such embodiments, an
annexin V homodimer was generated and found to localize to tumor
vessels better than the corresponding annexin V monomer.
Example XXX
Co-Binding of 3G4 and .beta.2GPI to PS
[1065] The present example demonstrates that the interaction
between the 3G4 antibody and PS is dependent on the plasma protein,
.beta.2-glycoprotein I (.beta.2GPI). 3G4 binds to .beta.2GPI at
domain II, which is not linked to pathogenic antibodies isolated
from patients with APS (which commonly recognize .beta.2GPI domain
I). The data show that divalent 3G4/.beta.2GPI complexes are
required for enhanced PS binding, including to PS-positive cells,
since 3G4 Fab' fragments do not have this activity. The divalent
3G4/.beta.2GPI binding to PS-positive cells supports the use of the
Fc-.beta.2GPI construct of the invention, which are also dimers, to
target PS-positive cells and thus treat diseases such as cancer and
viral infections.
A. Materials and Methods
1. Materials
[1066] Dulbecco's modified Eagle's medium (DMEM) and trypsin/EDTA
were obtained from Mediatech, Inc. (Herndon, Va.). Fetal bovine
serum (FBS), normal human serum, normal rat serum and normal mouse
serum were obtained from Biomeda (Foster City, Calif.). Fresh human
plasma was obtained from Carter Blood Care (Dallas, Tex.).
Serum-free Hybridoma Media, Synthechol NS0 supplement,
L-.alpha.-phosphatidylserine (PS), bovine serum albumin (BSA), and
ovalbumin from chicken egg white (OVA) and were obtained from Sigma
Chemical Co. (St. Louis, Mo.). DEAE cellulose, heparin-Sepharose,
and Hybond-P membranes were obtained from Amersham Biosciences
(Buckinghamshire, UK).
1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine
[lysophosphatidylchloine (LPC)] was obtained from Avanti Polar
Lipids (Alabaster, Ala.). Ninety six-well Immulon-1B and -2HB
microtiter plates were obtained from Thermo Lab Systems (Franklin,
Mass.). Tris-HCl gradient SDS-PAGE gels and an Opti-4CN Substrate
kit were obtained from Biorad (Hercules, Calif.). Eight-well glass
chamber slides were obtained from BD Biosciences (Bedford,
Mass.).
2. Antibodies
[1067] The 3G4 mouse monoclonal antibody, which was raised to bind
the anionic phospholipid PS, is the antibody described in Example
IV and other examples herein (see also, Ran et al., 2005), 3G4 was
produced originally in hybridoma supernatant, but was subsequently
converted to a mouse IgG2a isotype (Example XIX, D) and is now
produced in the NS0 mouse myeloma cell line.
[1068] NS0 cells were cultured in DMEM supplemented with 10% FBS or
serum-free Hybridoma Media with Synthechol NS0 supplement. A human
IgG I chimeric version of 3G4 (ch3G4) has also been generated
(Example XIX, B) and is produced under serum-free conditions by
Peregrine Pharmaceuticals, Inc. (Tustin, Calif.). This antibody has
been termed bavituximab.
[1069] The mouse anti-human .beta.2GPI (anti-.beta.2GPI or
.alpha.-.beta.2GPI) mAb was obtained from USBiological (Swampscott,
Mass.). A hybridoma secreting C44, a colchicine-specific mouse
IgG2a mAb, was obtained from the American Type Culture Collection
(Rockville, Md.) and used as a control for 3G4 and anti-2GPI.
Rituximab (human IgG1 chimeric mAb) was obtained from the UT
Southwestern pharmacy and used as a control for ch3G4. All
antibodies produced in culture supernatants were purified as
described in the previous examples (see also, Ran et al., 2003,
specifically incorporated herein by reference). All secondary
antibodies were obtained from Jackson Immunoresearch Labs (West
Grove, Pa.).
3. Preparation of Antibody Fragments
[1070] 3G4 F(ab').sub.2 was generated by incubation with the
protease pepsin. 3G4 Fab and control Fab 7H11 (anti-adenovirus)
were generated by incubation with the protease papain. All antibody
cleavage products were purified by FPLC, and verified by
SDS-PAGE.
4. Purification of .beta.2GPI from Human Plasma
[1071] Human .beta.2GPI (h.beta.2GP1) was purified from human
plasma essentially as described previously (Polz et al., 1980; Wurm
et al., 1984; each specifically incorporated herein by reference).
Briefly, perchloric acid (70%) was added to pooled plasma to a
final concentration of 1.57% (v/v). The precipitate was discarded
and the supernatant was adjusted to pH 7.5 with saturated
Na.sub.2CO.sub.3, followed by extensive dialysis against 50 mM
Tris, pH 8.0. This material was applied to a DEAE cellulose column
equilibrated with 50 mM Tris, pH 8.0 to remove contaminants. The
DEAE column flow-through was then applied to a heparin-Sepharose
affinity column equilibrated with 50 mM Tris, pH 8.0, and bound
proteins were eluted using 1.0 M NaCl. Finally, the .beta.2GPI
preparation was dialyzed against PBS and purified further by
protein A/G to remove contaminating IgG. The final preparation
contained a homogeneous band at 50 kDa, as shown by non-reduced
SDS/PAGE and commassie staining.
5. Construction and Expression of .beta.2GPI and .beta.2GPI
Domains
[1072] To generate pure recombinant full-length and deleted forms
of .beta.2GPI, the yeast shuttle expression vector pPIC6.alpha.A
(Invitrogen) and host strain Mut.sup.+X-33 (Invitrogen) were used.
The expression vector contains the 5' promoter and the 3'
transcription termination sequences of the alcohol (methanol)
oxidase gene (AOX1). The vector also has a yeast a mating factor
signal sequence downstream of the AOX1 promoter to which foreign
cDNA can be fused for secretion of recombinant heterologous protein
into the culture medium. Expression in P. pastoris provides
glycosylation and disulfide bond formation similar to that in
mammalian cells.
[1073] To generate the expression constructs, the following five
expression constructs were made using human .beta.2GPI cDNA:
TABLE-US-00025 (1) The entire coding region of .beta.2GPI cDNA
without its cognate signal peptide (domain 1-5), 5' primer; (SEQ ID
NO: 12) 5'-GGAATTCGGACGGACCTGTCCCAAGC-3'; (2) Domain 1 deleted
(domain 2-5), 5' primer; (SEQ ID NO: 13)
5'-GGAATTCGTATGTCCTTTTGC-3'; (3) Domain 1 & 2 deleted (domain
3-5), 5' primer, (SEQ ID NO: 14) 5'-GGAATTCGCTCCCATCATCTGC-3'; (4)
Domain 1-3 deleted (domain 4-5), 5' primer, (SEQ ID NO: 15)
5'-GGAATTCGTAAAATGCCCATTC-3'; and (5) Domain 5 only, 5' primer,
(SEQ ID NO: 16) 5'-GGAATTCGCATCTTGTAAAGTAC-3'.
[1074] A common 3' primer, 5'-TTCTAGATTAGCATGGCTTTAC-3' (SEQ ID
NO:17) was used for PCR of all fragments. PCR amplified fragments
were inserted in-frame between the EcoR1 and XbaI restriction sites
of pPIC.alpha.A, directly downstream from the .alpha. mating factor
signal sequence. A Stop codon was introduced at the end of each
fragment to prevent fusion of the recombinant proteins to a c-myc
epitope or a His tag at the C-terminus. Plasmid constructs were
propagated in E. coli in presence of 100 .mu.g/ml blasticidin and
verified by restriction analysis and nucleotide sequencing.
Recombinant proteins expressed by constructs (1), (2), (3), (4),
and (5) encoded proteins of approximately 36, 29, 24, 16, and 9
kDa, respectively, before glycosylation.
[1075] For the transformation and screening of expression clones,
the recombinant plasmid constructs were linearized with restriction
enzyme SacI, purified, and 10 .mu.g was used to transform host
strain X-33 by the spheroplasts method (Invitrogen). Transformants
for each of these constructs were selected on YPD (Yeast extract
Peptone Dextrose Medium) plates containing 400 .mu.g/ml blasticidin
for 4 days. Several clones for each of these constructs were
restreaked on YPD plates with 400 .mu.g/ml blasticidin to determine
the true integrants. Ten clones of each construct were then
streaked on Minimal Dextrose (MD) and Minimal Methanol (MM) plates.
Five clones of each construct, growing equally well on both MD and
MM plates, were then grown in liquid MD and MM medium for 24, 48,
72, 96 and 120 hours. Supernatants and pellets for each clone at
each time point were analyzed by Western blot using anti-human
.beta.2GPI polyclonal antibody. Clones that showed highest
expression of the protein in supernatant were further used for
large-scale preparation.
[1076] For the large scale purification of the recombinant
proteins, recombinant proteins were produced using culture
conditions recommended by Invitrogen. A starter culture of each
clone was cultured in 5 ml of buffered minimal glycerol-complex
medium (BMGY) at 30.degree. C. with vigorous shaking overnight.
Cells were collected, used to inoculate 25 ml of BMGY, and grown
for 2 days. Cells from the 25 ml culture were then used to
inoculate 1 L of buffered minimal methanol-complex (BMMY) medium
(1.0% methanol). Culture was continued for 4 days at 30.degree. C.
with vigorous shaking and 100% methanol was added every 24 hours
(final concentration of 1.0%) to maintain protein expression.
Culture medium was clarified by centrifugation (4000.times.g, 15
min) and supernatant was dialyzed for 2 days at 4.degree. C. in 50
mM Tris buffer before being applied to a DEAE-sephacel column
equilibrated with 50 mM Tris buffer. Flow through solution was
collected and applied to a heparin-sepharose column. .beta.2GPI was
eluted from heparin-sepharose column with 1 M NaCl, dialyzed
against 50 mM Tris buffer, concentrated using Amicon concentrator,
and analyzed by Western blot. The N-terminus of each protein was
sequenced to confirm cleavage of the a-factor leader sequence.
Protein yields varied from 10 mg/L (full-length .beta.2GPI) to 25
mg/L (.beta.2GPI domain V).
6. Preparation of "Nicked" h.beta.2GPI
[1077] Nicked h.beta.2GPI was prepared from intact h.beta.2GPI
purified from human plasma as described above. h.beta.2GPI was
incubated with plasmin-coated beads at 37.degree. C. for 17 hrs.
The beads were removed by centrifugation and the supernatant
containing the cleaved protein was recovered. Western blotting of
the purified product indicated the nicked .beta.2GPI preparations
were plasmin-free and did not contain plasmin autoproteolytic
products (no reactivity with anti-plasmin or anti-angiostatin
antibodies). N-terminal sequence analysis revealed two N-termini
that corresponded to the N-terminus of .beta.2GPI and a new
sequence generated at the Lys317/Thr318 cleavage site.
7. Anti-PS ELISA
[1078] The assay was performed as follows (see also, Ran et al.,
2005, specifically incorporated herein by reference). PS-coated
Immunlon 1B microtiter plates were blocked overnight in 1% OVA
(w/v). The following day, serial 2-fold dilutions of 3G4 purified
from serum-containing or serum-free supernatant were prepared from
an initial concentration of 13.33 nM. Dilutions were performed in
1% OVA or 10% non-heat inactivated sera from cow, human, rat or
mouse. Plates were incubated for 1 hr. at 37.degree. C. and binding
of 3G4 was detected. All ELISA studies were performed at least
three times.
8. Anti-h.beta.2GPI ELISA
[1079] The assay was performed as described above with the
following modifications. h.beta.2GPI, nicked h.beta.2GPI, or
recombinant h.beta.2GPI peptides were coated on 96-well Immunlon
2HB microtiter plates overnight at a concentration of 10 .mu.g/ml.
Plates were then blocked in 1% OVA for 1 hr. at room temperature.
3G4, ch3G4, or anti-.beta.2GPI were diluted in 1% OVA to an initial
concentration of 13.33 nM and serial 2-fold dilutions were
prepared. Plates were incubated for 1 hr. at 37.degree. C. and
antibody binding was detected. All ELISA studies were performed at
least three times.
9. Western Blot
[1080] Protein samples were heated to 95.degree. C. for 5 min in
non-reducing SDS sample buffer. The samples were then loaded onto a
Tris-HCl 4-15% gradient SDS-PAGE gel and separated using a Mini
Protean II apparatus (Biorad). Separated proteins were transferred
to a PVDF membrane and blocked overnight in 3% BSA (w/v). Membranes
were probed with anti-.beta.2GPI, 3G4, or control mouse IgG diluted
to 1 .mu.g/ml in 3% BSA, washed thoroughly, and incubated with
peroxidase-labeled goat anti-mouse IgG. Membranes were developed
using an Opti-4CN Substrate kit.
10. Induction and Detection of PS Exposure on Endothelial Cells
[1081] Adult bovine aortic endothelial (ABAE) cells were maintained
in DMEM supplemented with 10% FBS and 2 mM L-glutamine. ABAE cells
were removed from subconfluent cultures by brief exposure to 0.25%
trypsin/0.02% EDTA, and 8-well chamber slides were seeded with
2.times.10.sup.4 cells/well. Following overnight culture, cells
were washed gently with PBS and treated with 200 .mu.M
lysophosphatidylcholine (LPC) to induce PS exposure. LPC-treatment
was performed in the presence of 3G4, ch3G4, or control IgG for 30
min at 37.degree. C. in either 10% FBS or 10% normal mouse serum
(MS). If LPC-treatment was performed in 10% MS, h.beta.2GPI was
added as a co-factor because 3G4/ch3G4 cannot bind PS in MS (see
Results).
[1082] PS exposure was determined by immunofluorescence staining.
Cells were washed thoroughly in PBS, fixed in 4% paraformaldehyde
(w/v), and incubated with a biotin-conjugated anti-mouse secondary
antibody. Next, cells were incubated with FITC-conjugated
streptavidin (Jackson Immunoresearch) to detect antibody binding.
Cells were then permeablized with 0.1% Triton-X100 in PBS and
counterstained with Texas Red-conjugated phalloidin (Molecular
Probes, Eugene, Oreg.), and 4', 6-diamidino-2-phenylindole (DAPI;
Molecular Probes). Images were captured using a Coolsnap digital
camera (Photometrics, Tucson, Ariz.) mounted on a Nikon microscope
and processed with MetaVue software (Universal Imaging Corporation,
Downingtown, Pa.).
11. Quantification of Antibody Binding to ABAE Cells
[1083] The area of antibody binding was determined using MetaVue
image analysis software, which is able to quantify the number of
illuminated pixels in an image. Images of FITC fluorescence were
used to quantify antibody binding. Corresponding images of DAPI
fluorescence were used to normalize the FITC images for the number
of cells present in the field. A small FITC/DAPI ratio indicates a
small antibody binding area, whereas a large FITC/DAPI ratio
indicates a large binding area. The FITC/DAPI ratios were used to
determine increases or decreases in antibody binding area relative
to a basal amount of antibody binding under the selected
conditions. Five images at 200.times. magnification were used for
each analysis. Data are presented as average relative FITC/DAPI
ratios with error bars representing the standard deviation.
B. Results
1. 3G4 Requires a Serum Factor to Bind PS-Coated Microtiter
Plates
[1084] The 3G4 antibody purified from serum-containing media (SCM)
or serum-free media (SFM) binds to PS-coated microtiter plates when
serial dilutions are performed in 10% FBS (FIG. 9A, solid lines).
In contrast, when serial dilutions are performed in 1% OVA (which
lacks bovine serum proteins) 3G4 purified from SFM no longer binds
PS (FIG. 9A, dashed line, .box-solid.). This finding indicates a
factor present in bovine serum mediates the interaction between 3G4
and PS.
[1085] Interestingly, 3G4 purified from SCM binds PS weakly when
serial dilutions are performed in 1% OVA (FIG. 9A, dashed line,
.tangle-solidup.). This suggests purification of 3G4 grown in SCM
does not completely remove the serum protein required to mediate
the interaction between 3G4 and PS. Therefore, studies described
below were performed using 3G4 purified from SFM.
2. 3G4 Binding to PS in Sera from Different Species
[1086] To determine whether sera from other mammalian species can
mediate the interaction between 3G4 and PS, serial dilutions of 3G4
were performed in 10% mouse, rat, human or other sera. 3G4 bound PS
in the presence of rat and human serum, much like in the presence
of bovine serum (FIG. 9B). However, 3G4 did not bind PS in the
presence of mouse serum (FIG. 9B). In other studies, 3G4 bound PS
in the presence of hamster, ferret, guinea pig, rabbit and monkey
serum. Therefore, the serum protein epitope recognized by 3G4 is
conserved among all mammalian species tested except mouse.
3. 3G4 Binds the Serum Glycoprotein .beta.2GPI
[1087] In the late 1980's, a cohort of patients suffering venous
and arterial thromboses, thrombocytopenia and/or recurrent
pregnancy loss was shown to have circulating anti-phospholipid
(aPL) antibodies (Hughes et al., 1986; Deleze et al., 1989). These
patients were described as having the "Anti-phospholipid Syndrome"
(APS). In the early 1990s, it was shown that many so-called aPL
antibodies do not recognize phospholipids directly, but bind serum
proteins with affinity for phospholipids instead (Galli et al.,
1990; McNeil et al., 1990). Therefore, a panel of human serum
proteins known to interact with anionic phospholipids was screened
for 3G4 reactivity.
[1088] In terms of .beta.2GPI, human .beta.2GPI (h.beta.2GPI) was
coated on a microtiter plate and incubated with mouse anti-human
.beta.2GPI antibody (anti-.beta.2GPI), 3G4, or a control mouse
IgG2a of irrelevant specificity (control mIgG). As expected,
anti-.beta.2GPI bound to h.beta.2GPI, while the control mIgG did
not (FIG. 10A). 3G4 also bound strongly to the h.beta.2GPI coated
plate (FIG. 10A).
[1089] To determine whether .beta.2GPI is the only serum protein
recognized by 3G4, purified h.beta.2GPI and 10% human serum were
run on an SDS-PAGE gel and transferred to a membrane support for
immunoblot. 3G4 detected the 50-kDa purified h.beta.2GPI and a
single band of similar size in human serum. Importantly, the 3G4
immunoblot was virtually identical to a blot generated using the
anti-.beta.2GPI antibody. The control mIgG antibody did not detect
any protein. Together, these data indicate that 3G4 binds serum
protein .beta.2GPI.
[1090] Other human serum proteins known to interact with anionic
phospholipids were tested for 3G4 reactivity in ELISAs. Equal
amounts of the particular protein were coated on microtiter plates,
blocked in 1% OVA, and incubated with a serial dilution of 3G4.
Plates were washed thoroughly and antibody binding was detected
with a peroxidase-labeled secondary detection antibody. All studies
included positive and negative control antibodies, which worked as
expected. The results of the immunoblot and ELISA studies are shown
in Table 15, which thus identifies .beta.2GPI as the co-binding
factor for 3G4.
TABLE-US-00026 TABLE 15 3G4 Reactivity with Serum Proteins that
interact with Phospholipids Serum Protein 3G4 Reactivity annexin V
- beta2-glycoprotein I + factor XII - kininogen - oxidized LDL -
protein C - protein S - prothrombin - tPA -
4. 3G4 Binds .beta.2GPI at Domain II
[1091] .beta.2GPI has five domains, of which the fifth domain is
responsible for binding to anionic phospholipids. To determine
which domain of .beta.2GPI is recognized by the 3G4 antibody,
recombinant human .beta.2GPI constructs were generated with
different domain structures and tested alongside recombinant
full-length h.beta.2GPI. These domain constructs were made by
serial truncations from the N-terminus, and so lack each of the
N-terminal domains in turn, as follows: recombinant full-length
h.beta.2GPI contains domains I-V; h.beta.2GPI from which domain I
has been deleted contains domains II-V; h.beta.2GPI from which
domains I and II have been deleted contains domains III-V;
h.beta.2GPI from which domains I, II and III have been deleted
contains domains IV-V; and h.beta..beta.2GPI from which domains I,
II, III and IV have been deleted contains domain V only.
[1092] Equal amounts of the full-length h.beta.2GPI and each of the
above h.beta.32 GPI domain constructs were coated on microtiter
plates and incubated with a serial dilution of 3G4. This study
showed that only h.beta.2GPI constructs containing domain II of
.beta.2GPI (domains I-V and domains II-V) were detected by 3G4
(FIG. 10B). When domain I was deleted, 3G4 bound equally well to
domains II-V (FIG. 10B). Thus, 3G4 binds to .beta.2GPI at domain
II.
[1093] The finding that 3G4 binds to .beta.2GPI at domain II is
important in light of the information known about pathogenic
antibodies isolated from patients with APS. Pathogenic
anti-.beta.2GPI antibodies isolated from patients with APS commonly
recognize domain I of .beta.2GPI (de Laat et al., 2005a).
Anti-.beta.2GPI antibodies from APS patients that recognize domain
II are not often pathogenic. This likely explains the lack of
toxicity associated with 3G4 following extensive toxicological
studies performed in a variety of animal models.
5. Co-Binding of 3G4 and .beta.2GPI to Cells with Exposed PS
[1094] Characterizing the binding of anti-.beta.2GPI antibodies
using .beta.2GPI-coated microtiter plates has been controversial
due to inconsistencies observed depending on the type or even the
lot of microtiter plate (de Laat et al., 2004a). To verify the
above findings under more physiological conditions, a live-cell
binding assay was developed. This assay detects and measures
antibody binding to endothelial cell membrane surfaces following
treatment with the membrane disrupting agent,
lysophosphatidylcholine (LPC) to induce PS exposure.
[1095] In this assay, ABAE cells were incubated with 3G4 or control
mIgG in DMEM+10% FBS in the presence or absence of 200 .mu.M LPC
for 30 min. Cells were then washed, fixed, and stained with
fluorescent markers to visualize binding of antibody to the cell
surface. The pixel area of 3G4 or mIgG binding was quantified using
MetaVue software. All values were relative to the binding of 3G4 to
non-LPC treated cells, which was set to one.
[1096] When 3G4 is added to ABAE cell culture media under normal
conditions, no binding to the cells is observed. However, when ABAE
cells are incubated with 3G4 in the presence of LPC, numerous
pinpoints of 3G4 binding are readily detectable. LPC is known to
induce temporary membrane distortions (Kogure et al., 2003), which
likely cause a loss of membrane asymmetry and exposure of PS.
[1097] Quantification showed that the area of 3G4 binding increased
greater than 500-fold upon LPC-treatment, while binding of a
control mIgG remained undetectable. Similar results were obtained
previously, when 3G4 and the PS-binding molecule annexin V were
shown to bind endothelial cells following induction of PS exposure
with H.sub.2O.sub.2 (Example VI; Example VII; see also, Ran et al.,
2005).
[1098] Importantly, LPC-treated ABAE cells were not stained by the
membrane impermeant dyes propidium iodide or DAPI, indicating 3G4
bound PS on the cell surface, not the inner leaflet of the plasma
membrane.
[1099] To determine whether .beta.2GPI is required for binding of
3G4 to endothelial cells with exposed PS, the live-cell binding
assay was performed in media containing 10% MS rather than 10% FBS
to prevent interference from bovine .beta.2GPI. As demonstrated
above, 3G4 does not bind PS in the presence of MS. Furthermore, 3G4
did not detect any protein in 10% MS by immunoblot, indicating that
3G4 does not recognize murine .beta.2GPI.
[1100] For this study, a human chimeric 3G4 (ch3G4) was used to
prevent background caused by detection of murine IgG present in MS.
When ABAE cells were incubated with ch3G4 in the presence of 10% MS
and LPC, no antibody binding was detected (FIG. 11). In contrast,
addition of purified h.beta.2GPI to the binding reaction supported
widespread binding of ch3G4 (FIG. 11), demonstrating that ch3G4
binding is dependent upon h.beta.2GPI. In all situations, ch3G4
binding was dependent upon LPC treatment and no binding was
detected using a control human IgG of irrelevant specificity.
[1101] Interestingly, when ABAE cells were incubated with
h.beta.2GPI in the presence of 10% MS and LPC, washed thoroughly,
then incubated with ch3G4 to detect binding of h.beta.2GPI, very
little ch3G4 binding was detected (FIG. 11). This finding indicates
that h.beta.2GPI does not bind endothelial cells with exposed PS in
the absence of ch3G4, and is consistent with reports that
.beta.2GPI has a low affinity for anionic phospholipid membrane
surfaces under physiological conditions (Willems et al., 1996;
Bevers et al., 2005). Together, these data show that ch3G4 and
h.beta.2GPI must be present simultaneously to bind ABAE cells with
exposed PS, suggesting ch3G4 enhances the affinity of .beta.2GPI
for anionic phospholipids surfaces.
6. The Lipid Binding Region of .beta.2GPI is Required for
Co-Binding of 3G4
[1102] To confirm that the lipid binding region of .beta.2GPI is
required for co-binding of .beta.2GPI and 3G4 (and ch3G4) to
endothelial cells with exposed PS and other anionic phospholipids,
the live-cell binding assay was performed using "nicked"
h.beta.2GPI. Nicked h.beta.2GPI is unable to bind PS and other
anionic phospholipids due to plasmin-mediated cleavage within the
lipid binding region of domain V (Hunt et al., 1993; Hunt and
Krilis, 1994).
[1103] When ABAE cells are incubated with ch3G4 and h.beta.2GPI or
nicked h.beta.2GPI in the absence of LPC, no ch3G4 binding is
detected (FIG. 12A). In the presence of LPC, h.beta.2GPI is able to
mediate binding of ch3G4 to ABAE cells with exposed PS, while
nicked h.beta.2GPI is not able to mediate binding (FIG. 12A). The
lack of binding in the live-cell assay is not due to an inability
of ch3G4 to bind nicked h.beta.2GPI, since ch3G4 binds nicked
h.beta.2GPI as well as h.beta.2GPI when equal amounts of protein
are coated on microtiter plates (FIG. 12B). These findings
demonstrate that the ch3G4/h.beta.2GPI complex detects PS and other
anionic phospholipids exposed on ABAE cells following LPC-treatment
through the lipid binding region of h.beta.2GPI domain V.
7. Antibody Divalency is Required for Co-Binding of .beta.2GPI
[1104] The data presented above suggest that 3G4 detects PS and
anionic phospholipids by enhancing the affinity of .beta.2GPI for
anionic phospholipids. Interestingly, anti-.beta.2GPI antibodies
isolated from many APS patients are known to increase the affinity
of .beta.2GPI for anionic phospholipid surfaces (Bevers et al.,
2004). Furthermore, the phenomenon is dependent upon the formation
of a divalent antibody-.beta.2GPI complex.
[1105] To determine whether divalency is required for binding of
3G4/.beta.2GPI complexes to PS and anionic phospholipids,
LPC-treated ABAE cells were incubated with purified h.beta.2GPI and
varying amounts of ch3G4. The area of ch3G4 binding increased in a
concentration-dependent manner to a peak at an antibody to
.beta.2GPI ratio of 2:1 (FIG. 13). Further increases in ch3G4
concentration caused a concentration-dependent decrease in binding.
The bell shaped relationship between the concentration of ch3G4 and
binding to endothelial cells with exposed PS suggests the formation
of divalent ch3G4/.beta.2GPI complexes on the membrane surface. At
very high antibody concentration, competition between monovalent
and divalent complexes causes a decrease in the amount of
ch3G4/.beta.2GPI complex bound to the LPC-treated ABAE cells.
[1106] To further examine whether divalency is required for binding
to endothelial cells with exposed PS, 3G4 F(ab').sub.2 and 3G4 Fab'
monomers were generated and used in live-cell binding assays with
intact 3G4. As expected, intact 3G4 bound to LPC-treated ABAE
cells, but not to untreated cells (FIG. 14A). An equivalent
concentration of 3G4 F(ab').sub.2 also bound to LPC-treated ABAE
cells, but binding of 3G4 Fab' was negligible (FIG. 14A). The
apparent decrease in binding of 3G4 F(ab').sub.2 relative to 3G4
(FIG. 14A) is likely due to lost binding of the polyclonal
secondary antibody to Fc epitopes missing on 3G4 F(ab').sub.2. No
binding of 3G4 Fab' was detectable on ABAE cells even at a
concentration of 2 .mu.M (FIG. 14B), which is 1,000-fold above the
concentration required to bind .beta.2GPI coated on microtiter
plates.
[1107] Moreover, 3G4 Fab' inhibited ch3G4/.beta.2GPI binding to
LPC-treated ABAE cells in a concentration-dependent manner (FIG.
14C), while a control Fab' of irrelevant specificity did not. The
ability of 3G4 Fab' to inhibit ch3G4 binding confirms that 3G4 Fab'
is able to bind .beta.2GPI and that monomeric 3G4 Fab'/.beta.2GPI
complexes cannot bind endothelial cells with exposed PS. These data
support the hypothesis that divalent 3G4/.beta.2GPI complexes are
required to bind anionic phospholipid surfaces.
[1108] As shown in FIG. 10B, the 3G4 antibody binds to .beta.2GPI
at domain II, and as shown in FIG. 12A and FIG. 12B, the lipid
binding region of .beta.2GPI domain V is required for co-binding of
3G4 (and ch3G4) and .beta.2GPI to PS exposed on endothelial cells.
In addition, as demonstrated here, antibody divalency is required
for the co-binding of 3G4 (and ch3G4) and .beta.2GPI to the exposed
PS. Accordingly, the inventors have presented a model of antibody
and .beta.2GPI co-binding to PS exposed on the outer surfaces of
membranes, such as occurs on activated endothelial cells, tumor
vascular endothelial cells and tumor cells, as well as on virally
infected cells (FIG. 15).
Example XXXI
Preparation and Characterization of an Fc-.beta.2GPI
[1109] This example concerns the preparation and characterization
of an exemplary Fc-.beta.2GPI construct. In this construct, the Fc
region of mouse IgG.sub.2a was attached to full length mouse
.beta.2GPI (domains I-V) to create murine Fc-.beta.2GPI or
Fc-m.beta.2GPI. The mouse IgG.sub.2a Fc is used both as a
dimerization domain and to provide effector functions. The
resultant dimeric Fc-.beta.2GPI construct has various uses,
including targeting tumor blood vessels and virally infected cells,
each of which have exposed PS. An advantage of using an Fc region
is that the Fc-.beta.2GPI construct can mark the targeted cells for
attack by the host immune system.
A. Preparation
[1110] Although a variety of Fc-.beta.2GPI constructs have been
designed, the exemplary Fc-.beta.2GPI (Fc-m.beta.2GPI) construct of
the present example uses a murine Fc region as the N-terminus and
domains I-V of murine .beta.2GPI as the C-terminus. The
Fc-.beta.2GPI thus contains the disulphide-bonded hinge region and
two C.sub.H2 and C.sub.H3 domains of the Fc region from mouse
IgG.sub.2a, with the C-terminal end of each C.sub.H3 domain being
linked to the N-terminal end of two full length mouse .beta.2GPI
proteins (FIG. 16).
[1111] To prepare an expression plasmid, the signal sequence of the
3G4 light chain was amplified by PCR from the 2aG4 construct; a
mouse IgG2a Fc region containing the hinge, C.sub.H2 and C.sub.H3
domains was amplified by PCR from the 2aG4 construct; and mouse
.beta.2GPI was amplified by RT-PCR from commercially obtained mouse
liver RNA. The resultant Fc-m.beta.2GPI protein sequence is shown
in FIG. 18A. The mIgGK signal sequence (SEQ ID NO:18) is cleaved,
leaving the mature Fc-m.beta.2GPI as SEQ ID NO:19 (FIG. 18A).
[1112] The plasmid map is shown in FIG. 17 and the entire sequence
of the plasmid is provided as SEQ ID NO:25. The Fc-m.beta.2GPI
construct was transfected into CHO cells with FuGENE 6 reagent. Two
days later, the supernatant was harvested and used in several
assays.
[1113] A counterpart human Fc-.beta.2GPI (Fc-h.beta.2GPI) can be
prepared using the sequence information in FIG. 18B, which provides
an exemplary amino acid sequence for a human IgG.sub.1 heavy chain
constant region (Accession number P01857; SEQ ID NO:21) and the
entire sequence for human .beta.2GPI (Accession number 1C1ZA; SEQ
ID NO:22), including the location of domains I, II, III, IV and V.
The human hinge, C.sub.H2 and C.sub.H3 domains would be attached to
domains I, II, III, IV and V of human .beta.2GPI to provide the
Fc-h.beta.2GPI of SEQ ID NO:23.
B. Fc-m.beta.2GPI Is a Dimer
[1114] The chosen system was effective to express Fc-m.beta.2GPI.
FIG. 19 shows results from a capture assay using two different
clones of Fc-m.beta.2GPI (#8 and #18), in which the Fc-m.beta.2GPI
cell culture supernatants bind to anti-mouse IgG.
[1115] Samples of the Fc-m.beta.2GPI protein were subjected to gel
electrophoresis. Under non-reducing gel conditions, a band of the
expected size for a dimer was detected (apparent Mw on the gel of
150 kDa). Under reducing conditions, bands of a smaller size were
detected (apparent Mw on the gel .about.90-100 kDa).
C. Fc-m.beta.2GPI Binds PS and Cells with Exposed PS
[1116] Fc-m.beta.2GPI was able to bind to PS on PS-coated plates.
Using a microtiter plate coated with PS, the Fc-m.beta.2GPI cell
culture supernatants (#8 and #18) were determined to bind to PS in
a concentration-dependent manner (FIG. 20).
[1117] In common with the 3G4 antibody and the chimeric version
(ch3G4 or bavituximab), Fc-m.beta.2GPI binding is not limited to
PS, but extends to other anionic phospholipids. Results from
phospholipid binding assays (in 10% FBS) show that Fc-m.beta.2GPI
cell culture supernatants bind to the anionic phospholipids PS, PA,
PI and PG, but not to the neutral lipids, PC and SM.
[1118] Fc-m.beta.2GPI is also capable of binding PS exposed at the
cell surface. Cells undergoing apoptosis expose PS at the outer
surface. In studies using ABAE cells undergoing apoptosis,
Fc-m.beta.2GPI was found to stain the apoptotic cells (FIG. 22A,
FIG. 22B and FIG. 22C). The arrows highlight apoptotic cells, which
are rounded and have dense nuclei.
[1119] Cells can also be induced to present PS at the cell surface
without undergoing apoptosis. When ABAE cells are incubated in the
presence of LPC, temporary membrane distortions occur, causing a
loss of membrane asymmetry and PS exposure. Using LPC-treated ABAE
cells, Fc-m.beta.2GPI was also found to bind to PS exposed at the
cell surface, seen as small pinpoints of green staining (FIG. 23A
and FIG. 23B). No pinpoint staining was seen on non-LPC treated
cells or using control supernatant.
Example XXXII
Dimeric .beta.2GPI Binds to Endothelial Cells with Exposed PS
[1120] Following the results described in Example XXX, and to
further validate the information set forth in Example XXXI, the
present example provides data to show that dimeric .beta.2GPI binds
to endothelial cells with exposed PS in the absence of the 3G4
antibody.
[1121] In these studies, an artificial .beta.2GPI dimer is used,
which was generated by fusing h.beta.2GPI to the C-terminus of the
apple 4 dimerization domain of factor XI (Lutters et al., 2001).
This .beta.2GPI does not have the advantages provided by attachment
to an Fc region, as described earlier in the present application.
Nonetheless, binding of this dimer to cells with exposed PS
validates the use of Fc-.beta.2GPI dimers to bind to PS-positive
cells.
[1122] ABAE cells were treated with LPC to induce PS exposure and
incubated with purified h.beta.2GPI, h.beta.2GPI-dimer, or a mutant
h.beta.2GPI-dimer containing a disrupted lipid binding domain. As
expected, binding of the monomeric h.beta.2GPI to LPC-treated ABAE
cells was negligible (FIG. 24A). In contrast, the h.beta.2GPI-dimer
bound strongly to LPC-treated cells (FIG. 24B), but not to non
LPC-treated cells (FIG. 24D). Binding of the h.beta.2GPI-dimer was
also found to be dependent upon a functional lipid binding domain
(FIG. 24C). These data indicate that divalent .beta.2GPI constructs
bind to cells with exposed PS.
[1123] The Fc-.beta.2GPI dimers of the present invention will also
bind to cells with exposed PS, such as tumor vascular endothelial
cells, tumor cells, virally infected cells and viral particles.
However, the Fc-.beta.2GPI constructs will have the additional
advantage that the Fc region will promote host effector functions
to enhance treatment of disease. Additional agents may also be
attached to the Fc-.beta.2GPI constructs, such as toxins,
cytokines, radioisotopes, drugs and such like, to deliver a
therapeutic payload to the PS-positive target cells.
[1124] 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
251519DNAMus musculus 1atgggatgga cctggatctt tattttaatc ctgtcagtaa
ctacaggtgt ccactctgag 60gtccagctgc agcagtctgg acctgagctg gagaagcctg
gcgcttcagt gaagctatcc 120tgcaaggctt ctggttactc attcactggc
tacaacatga actgggtgaa acagagccat 180ggaaagagcc ttgaatggat
tggacatatt gatccttact atggtgatac ttcctacaac 240cagaagttca
ggggcaaggc cacattgact gtagacaaat cctccagcac agcctacatg
300cagctcaaga gcctgacatc tgaggactct gcagtctatt actgtgtaaa
ggggggttac 360tacgggcact ggtacttcga tgtctggggc gcagggacca
cggtcaccgt ctcctcagct 420acaacaacag ccccatctgt ctatcccttg
gtcccgggcg gatcccccgg gctgcaggaa 480ttcgatatca agcttatcga
taccgtcgac ctcgagggg 5192152PRTMus musculus 2Met Gly Trp Thr Trp
Ile Phe Ile Leu Ile Leu Ser Val Thr Thr Gly1 5 10 15Val His Ser Glu
Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys 20 25 30Pro Gly Ala
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45Thr Gly
Tyr Asn Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60Glu
Trp Ile Gly His Ile Asp Pro Tyr Tyr Gly Asp Thr Ser Tyr Asn65 70 75
80Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala
Val 100 105 110Tyr Tyr Cys Val Lys Gly Gly Tyr Tyr Gly His Trp Tyr
Phe Asp Val 115 120 125Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser
Ala Thr Thr Thr Ala 130 135 140Pro Ser Val Tyr Pro Leu Val Pro145
1503435DNAMus musculus 3atggacatga gggctcctgc acagattttg ggcttcttgt
tgctcttgtt tccaggtacc 60agatgtgaca tccagatgac ccagtctcca tcctccttat
ctgcctctct gggagaaaga 120gtcagtctca cttgtcgggc aagtcaggac
attggtagta gcttaaactg gcttcagcag 180ggaccagatg gaactattaa
acgcctgatc tacgccacat ccagtttaga ttctggtgtc 240cccaaaaggt
tcagtggcag taggtctggg tcagattatt ctctcaccat cagcagcctt
300gagtctgaag attttgtaga ctattactgt ctacaatatg ttagttctcc
tcccacgttc 360ggtgctggga ccaagctgga gctgaaacgg gctgatgctg
caccaactgt cttcatcttc 420gggcggatcc cccgg 4354144PRTMus musculus
4Met Asp Met Arg Ala Pro Ala Gln Ile Leu Gly Phe Leu Leu Leu Leu1 5
10 15Phe Pro Gly Thr Arg Cys Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser 20 25 30Leu Ser Ala Ser Leu Gly Glu Arg Val Ser Leu Thr Cys Arg
Ala Ser 35 40 45Gln Asp Ile Gly Ser Ser Leu Asn Trp Leu Gln Gln Gly
Pro Asp Gly 50 55 60Thr Ile Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu
Asp Ser Gly Val65 70 75 80Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly
Ser Asp Tyr Ser Leu Thr 85 90 95Ile Ser Ser Leu Glu Ser Glu Asp Phe
Val Asp Tyr Tyr Cys Leu Gln 100 105 110Tyr Val Ser Ser Pro Pro Thr
Phe Gly Ala Gly Thr Lys Leu Glu Leu 115 120 125Lys Arg Ala Asp Ala
Ala Pro Thr Val Phe Ile Phe Gly Arg Ile Pro 130 135
1405783DNAArtificialSynthetic Oligonucleotide 5gcccagccgg
ccatggccga ggtgcagctg gtggagtctg ggggaggcgt ggtccagcct 60gggaggtccc
tgagactctc ctgtgcagcc tctggattca ccttcagtag ctatggcatg
120cactgggtcc gccaggctcc aggcaagggg ctggagtggg tggcagttat
atcatatgat 180ggaagtaata aatactatgc agactccgtg aagggccgat
tcaccatctc cagagacaat 240tccaagaaca cgctgtatct gcaaatgaac
agcctgagag ctgaggacac ggccgtgtat 300tactgtgcaa gattgcatgc
tcagacttgg ggccaaggta ccctggtcac cgtctcgagt 360ggtggaggcg
gttcaggcgg aggtggctct ggcggtagtg cacttcagtc tgtgctgacg
420cagccgcctt cagtgtctgc ggccccagga cagaaggtca ccatctcctg
ctctggaagc 480agctccgaca tggggaatta tgcggtatcc tggtaccagc
agctcccagg aacagccccc 540aaactcctca tctatgaaaa taataagcga
ccctcaggga ttcctgaccg attctctggc 600tccaagtctg gcacctcagc
caccctgggc atcactggcc tctggcctga ggacgaggcc 660gattattact
gcttagcatg ggataccagc ccgcggaatg tattcggcgg agggaccaag
720ctgaccgtcc taggtgcggc cgcacatcat catcaccatc acggggccgc
agaacaaaaa 780ctc 7836261PRTArtificialSynthetic Polypeptide 6Ala
Gln Pro Ala Met Ala Glu Val Gln Leu Val Glu Ser Gly Gly Gly1 5 10
15Val Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
20 25 30Phe Thr Phe Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro
Gly 35 40 45Lys Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser
Asn Lys 50 55 60Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn65 70 75 80Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp 85 90 95Thr Ala Val Tyr Tyr Cys Ala Arg Leu His
Ala Gln Thr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser
Gly Gly Gly Gly Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Ser Ala
Leu Gln Ser Val Leu Thr Gln Pro Pro Ser 130 135 140Val Ser Ala Ala
Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser145 150 155 160Ser
Ser Asp Met Gly Asn Tyr Ala Val Ser Trp Tyr Gln Gln Leu Pro 165 170
175Gly Thr Ala Pro Lys Leu Leu Ile Tyr Glu Asn Asn Lys Arg Pro Ser
180 185 190Gly Ile Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser
Ala Thr 195 200 205Leu Gly Ile Thr Gly Leu Trp Pro Glu Asp Glu Ala
Asp Tyr Tyr Cys 210 215 220Leu Ala Trp Asp Thr Ser Pro Arg Asn Val
Phe Gly Gly Gly Thr Lys225 230 235 240Leu Thr Val Leu Gly Ala Ala
Ala His His His His His His Gly Ala 245 250 255Ala Glu Gln Lys Leu
260720PRTHomo sapiens 7Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly 20815PRTHomo sapiens
8Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser1 5 10
15919PRTStreptomyces cinnamoneusMISC_FEATURE(11)..(18)Xaa = Abu
9Ala Lys Gln Ala Ala Ala Phe Gly Pro Phe Xaa Phe Val Ala Asp Gly1 5
10 15Asn Xaa Lys10468PRTMus musculus 10Met Gly Trp Thr Trp Ile Phe
Ile Leu Ile Leu Ser Val Thr Thr Gly1 5 10 15Val His Ser Glu Val Gln
Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys 20 25 30Pro Gly Ala Ser Val
Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe 35 40 45Thr Gly Tyr Asn
Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu 50 55 60Glu Trp Ile
Gly His Ile Asp Pro Tyr Tyr Gly Asp Thr Ser Tyr Asn65 70 75 80Gln
Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser 85 90
95Thr Ala Tyr Met Gln Leu Lys Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110Tyr Tyr Cys Val Lys Gly Gly Tyr Tyr Gly His Trp Tyr Phe
Asp Val 115 120 125Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala
Lys Thr Thr Ala 130 135 140Pro Ser Val Tyr Pro Leu Ala Pro Val Cys
Gly Asp Thr Thr Gly Ser145 150 155 160Ser Val Thr Leu Gly Cys Leu
Val Lys Gly Tyr Phe Pro Glu Pro Val 165 170 175Thr Leu Thr Trp Asn
Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe 180 185 190Pro Ala Val
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr 195 200 205Val
Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala 210 215
220His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Glu Pro Arg Gly
Pro225 230 235 240Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
Pro Asn Leu Leu 245 250 255Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
Lys Ile Lys Asp Val Leu 260 265 270Met Ile Ser Leu Ser Pro Ile Val
Thr Cys Val Val Val Asp Val Ser 275 280 285Glu Asp Asp Pro Asp Val
Gln Ile Ser Trp Phe Val Asn Asn Val Glu 290 295 300Val His Thr Ala
Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr305 310 315 320Leu
Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser 325 330
335Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro
340 345 350Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala
Pro Gln 355 360 365Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
Lys Lys Gln Val 370 375 380Thr Leu Thr Cys Met Val Thr Asp Phe Met
Pro Glu Asp Ile Tyr Val385 390 395 400Glu Trp Thr Asn Asn Gly Lys
Thr Glu Leu Asn Tyr Lys Asn Thr Glu 405 410 415Pro Val Leu Asp Ser
Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg 420 425 430Val Glu Lys
Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val 435 440 445Val
His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg 450 455
460Thr Pro Gly Lys46511236PRTMus musculus 11Met Asp Met Arg Ala Pro
Ala Gln Ile Leu Gly Phe Leu Leu Leu Leu1 5 10 15Phe Pro Gly Thr Arg
Cys Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 20 25 30Leu Ser Ala Ser
Leu Gly Glu Arg Val Ser Leu Thr Cys Arg Ala Ser 35 40 45Gln Asp Ile
Gly Ser Ser Leu Asn Trp Leu Gln Gln Gly Pro Asp Gly 50 55 60Thr Ile
Lys Arg Leu Ile Tyr Ala Thr Ser Ser Leu Asp Ser Gly Val65 70 75
80Pro Lys Arg Phe Ser Gly Ser Arg Ser Gly Ser Asp Tyr Ser Leu Thr
85 90 95Ile Ser Ser Leu Glu Ser Glu Asp Phe Val Asp Tyr Tyr Cys Leu
Gln 100 105 110Tyr Val Ser Ser Pro Pro Thr Phe Gly Ala Gly Thr Lys
Leu Glu Leu 115 120 125Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile
Phe Pro Pro Ser Ser 130 135 140Glu Gln Leu Thr Ser Gly Gly Ala Ser
Val Val Cys Phe Leu Asn Asn145 150 155 160Phe Tyr Pro Lys Asp Ile
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu 165 170 175Arg Gln Asn Gly
Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp 180 185 190Ser Thr
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr 195 200
205Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr
210 215 220Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys225 230
2351226DNAArtificialSynthetic Oligonucleotide 12ggaattcgga
cggacctgtc ccaagc 261321DNAArtificialSynthetic Oligonucleotide
13ggaattcgta tgtccttttg c 211422DNAArtificialSynthetic
Oligonucleotide 14ggaattcgct cccatcatct gc
221522DNAArtificialSynthetic Oligonucleotide 15ggaattcgta
aaatgcccat tc 221623DNAArtificialSynthetic Oligonucleotide
16ggaattcgca tcttgtaaag tac 231722DNAArtificialSynthetic
Oligonucleotide 17ttctagatta gcatggcttt ac 221824PRTMus musculus
18Met Asp Met Arg Ala Pro Ala Gln Ile Leu Gly Phe Leu Leu Leu Leu1
5 10 15Phe Pro Gly Thr Arg Cys Leu Arg 2019561PRTMus musculus 19Glu
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro1 5 10
15Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
20 25 30Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys
Val 35 40 45Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser
Trp Phe 50 55 60Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
His Arg Glu65 70 75 80Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
Leu Pro Ile Gln His 85 90 95Gln Asp Trp Met Ser Gly Lys Glu Phe Lys
Cys Lys Val Asn Asn Lys 100 105 110Asp Leu Pro Ala Pro Ile Glu Arg
Thr Ile Ser Lys Pro Lys Gly Ser 115 120 125Val Arg Ala Pro Gln Val
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 130 135 140Thr Lys Lys Gln
Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro145 150 155 160Glu
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 165 170
175Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
180 185 190Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg
Asn Ser 195 200 205Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn
His His Thr Thr 210 215 220Lys Ser Phe Ser Arg Thr Pro Gly Lys Thr
Gly Gly Arg Ile Cys Pro225 230 235 240Lys Pro Asp Asp Leu Pro Phe
Ala Thr Val Val Pro Leu Lys Thr Ser 245 250 255Tyr Asp Pro Gly Glu
Gln Ile Val Tyr Ser Cys Lys Pro Gly Tyr Val 260 265 270Ser Arg Gly
Gly Met Arg Arg Phe Thr Cys Pro Leu Thr Gly Met Trp 275 280 285Pro
Ile Asn Thr Leu Arg Cys Val Pro Arg Val Cys Pro Phe Ala Gly 290 295
300Ile Leu Glu Asn Gly Ile Val Arg Tyr Thr Ser Phe Glu Tyr Pro
Lys305 310 315 320Asn Ile Ser Phe Ala Cys Asn Pro Gly Phe Phe Leu
Asn Gly Thr Ser 325 330 335Ser Ser Lys Cys Thr Glu Glu Gly Lys Trp
Ser Pro Asp Ile Pro Ala 340 345 350Cys Ala Arg Ile Thr Cys Pro Pro
Pro Pro Val Pro Lys Phe Ala Leu 355 360 365Leu Lys Asp Tyr Arg Pro
Ser Ala Gly Asn Asn Ser Leu Tyr Gln Asp 370 375 380Thr Val Val Phe
Lys Cys Leu Pro His Phe Ala Met Ile Gly Asn Asp385 390 395 400Thr
Val Met Cys Thr Glu Gln Gly Asn Trp Thr Arg Leu Pro Glu Cys 405 410
415Leu Glu Val Lys Cys Pro Phe Pro Pro Arg Pro Glu Asn Gly Tyr Val
420 425 430Asn Tyr Pro Ala Lys Pro Val Leu Leu Tyr Lys Asp Lys Ala
Thr Phe 435 440 445Gly Cys His Glu Thr Tyr Lys Leu Asp Gly Pro Glu
Glu Ala Glu Cys 450 455 460Thr Lys Thr Arg Thr Trp Ser Phe Leu Pro
Thr Cys Arg Glu Ser Cys465 470 475 480Lys Leu Pro Val Lys Lys Ala
Thr Val Leu Tyr Gln Gly Met Arg Val 485 490 495Lys Ile Gln Glu Gln
Phe Lys Asn Gly Met Met His Gly Asp Lys Ile 500 505 510His Phe Tyr
Cys Lys Asn Lys Glu Lys Lys Cys Ser Tyr Thr Val Glu 515 520 525Ala
His Cys Arg Asp Gly Thr Ile Glu Ile Pro Ser Cys Phe Lys Glu 530 535
540His Ser Ser Leu Ala Phe Trp Lys Thr Asp Ala Ser Glu Leu Thr
Pro545 550 555 560Cys2036PRTMus musculus 20Lys Asn Lys Glu Lys Lys
Cys Ser Tyr Thr Val Glu Ala His Cys Arg1 5 10 15Asp Gly Thr Ile Glu
Ile Pro Ser Cys Phe Lys Glu His Ser Ser Leu 20 25 30Ala Phe Trp Lys
3521329PRTHomo sapiens 21Ala Ser Thr Lys Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70
75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu225 230 235 240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly 32522326PRTHomo sapiens
22Gly Arg Thr Cys Pro Lys Pro Asp Asp Leu Pro Phe Ser Thr Val Val1
5 10 15Pro Leu Lys Thr Phe Tyr Glu Pro Gly Glu Glu Ile Thr Tyr Ser
Cys 20 25 30Lys Pro Gly Tyr Val Ser Arg Gly Gly Met Arg Lys Phe Ile
Cys Pro 35 40 45Leu Thr Gly Leu Trp Pro Ile Asn Thr Leu Lys Cys Thr
Pro Arg Val 50 55 60Cys Pro Phe Ala Gly Ile Leu Glu Asn Gly Ala Val
Arg Tyr Thr Thr65 70 75 80Phe Glu Tyr Pro Asn Thr Ile Ser Phe Ser
Cys Asn Thr Gly Phe Tyr 85 90 95Leu Asn Gly Ala Asp Ser Ala Lys Cys
Thr Glu Glu Gly Lys Trp Ser 100 105 110Pro Glu Leu Pro Val Cys Ala
Pro Ile Ile Cys Pro Pro Pro Ser Ile 115 120 125Pro Thr Phe Ala Thr
Leu Arg Val Tyr Lys Pro Ser Ala Gly Asn Asn 130 135 140Ser Leu Tyr
Arg Asp Thr Ala Val Phe Glu Cys Leu Pro Gln His Ala145 150 155
160Met Phe Gly Asn Asp Thr Ile Thr Cys Thr Thr His Gly Asn Trp Thr
165 170 175Lys Leu Pro Glu Cys Arg Glu Val Lys Cys Pro Phe Pro Ser
Arg Pro 180 185 190Asp Asn Gly Phe Val Asn Tyr Pro Ala Lys Pro Thr
Leu Tyr Tyr Lys 195 200 205Asp Lys Ala Thr Phe Gly Cys His Asp Gly
Tyr Ser Leu Asp Gly Pro 210 215 220Glu Glu Ile Glu Cys Thr Lys Leu
Gly Asn Trp Ser Ala Met Pro Ser225 230 235 240Cys Lys Ala Ser Cys
Lys Leu Pro Val Lys Lys Ala Thr Val Val Tyr 245 250 255Gln Gly Glu
Arg Val Lys Ile Gln Glu Lys Phe Lys Asn Gly Met Leu 260 265 270His
Gly Asp Lys Val Ser Phe Phe Cys Lys Asn Lys Glu Lys Lys Cys 275 280
285Ser Tyr Thr Glu Asp Ala Gln Cys Ile Asp Gly Thr Ile Glu Val Pro
290 295 300Lys Cys Phe Lys Glu His Ser Ser Leu Ala Phe Trp Lys Thr
Asp Ala305 310 315 320Ser Asp Val Lys Pro Cys 32523557PRTHomo
sapiens 23Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro 20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val 50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln 85 90 95Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala 100 105 110Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 115 120 125Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150
155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr 165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr 180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe 195 200 205Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 210 215 220Ser Leu Ser Leu Ser Pro Gly
Gly Arg Thr Cys Pro Lys Pro Asp Asp225 230 235 240Leu Pro Phe Ser
Thr Val Val Pro Leu Lys Thr Phe Tyr Glu Pro Gly 245 250 255Glu Glu
Ile Thr Tyr Ser Cys Lys Pro Gly Tyr Val Ser Arg Gly Gly 260 265
270Met Arg Lys Phe Ile Cys Pro Leu Thr Gly Leu Trp Pro Ile Asn Thr
275 280 285Leu Lys Cys Thr Pro Arg Val Cys Pro Phe Ala Gly Ile Leu
Glu Asn 290 295 300Gly Ala Val Arg Tyr Thr Thr Phe Glu Tyr Pro Asn
Thr Ile Ser Phe305 310 315 320Ser Cys Asn Thr Gly Phe Tyr Leu Asn
Gly Ala Asp Ser Ala Lys Cys 325 330 335Thr Glu Glu Gly Lys Trp Ser
Pro Glu Leu Pro Val Cys Ala Pro Ile 340 345 350Ile Cys Pro Pro Pro
Ser Ile Pro Thr Phe Ala Thr Leu Arg Val Tyr 355 360 365Lys Pro Ser
Ala Gly Asn Asn Ser Leu Tyr Arg Asp Thr Ala Val Phe 370 375 380Glu
Cys Leu Pro Gln His Ala Met Phe Gly Asn Asp Thr Ile Thr Cys385 390
395 400Thr Thr His Gly Asn Trp Thr Lys Leu Pro Glu Cys Arg Glu Val
Lys 405 410 415Cys Pro Phe Pro Ser Arg Pro Asp Asn Gly Phe Val Asn
Tyr Pro Ala 420 425 430Lys Pro Thr Leu Tyr Tyr Lys Asp Lys Ala Thr
Phe Gly Cys His Asp 435 440 445Gly Tyr Ser Leu Asp Gly Pro Glu Glu
Ile Glu Cys Thr Lys Leu Gly 450 455 460Asn Trp Ser Ala Met Pro Ser
Cys Lys Ala Ser Cys Lys Leu Pro Val465 470 475 480Lys Lys Ala Thr
Val Val Tyr Gln Gly Glu Arg Val Lys Ile Gln Glu 485 490 495Lys Phe
Lys Asn Gly Met Leu His Gly Asp Lys Val Ser Phe Phe Cys 500 505
510Lys Asn Lys Glu Lys Lys Cys Ser Tyr Thr Glu Asp Ala Gln Cys Ile
515 520 525Asp Gly Thr Ile Glu Val Pro Lys Cys Phe Lys Glu His Ser
Ser Leu 530 535 540Ala Phe Trp Lys Thr Asp Ala Ser Asp Val Lys Pro
Cys545 550 5552436PRTHomo sapiens 24Lys Asn Lys Glu Lys Lys Cys Ser
Tyr Thr Glu Asp Ala Gln Cys Ile1 5 10 15Asp Gly Thr Ile Glu Val Pro
Lys Cys Phe Lys Glu His Ser Ser Leu 20 25 30Ala Phe Trp Lys
35259546DNAMus musculus 25agcttcgcga cgtacgttcg aacccgggcc
gccaccatgg acatgagggc tcctgcacag 60attttgggct tcttgttgct cttgtttcca
ggtaccagat gcctaaggga gcccagaggg 120cccacaatca agccctgtcc
tccatgcaaa tgcccaggta agtcactaga ccagagctcc 180acccgggaga
atggtaagtg ctgtaaacat ccctgcacta gaggataagc catgtacaga
240tccatttcca tctctcctca tcagcaccta acctcttggg tggaccatcc
gtcttcatct 300tccctccaaa gatcaaggat gtactcatga tctccctgag
ccccatagtc acatgtgtgg 360tggtggatgt gagcgaggat gacccagatg
tccagatcag ctggtttgtg aacaacgtgg 420aagtacacac agctcagaca
caaacccata gagaggatta caacagtact ctccgggtgg 480tcagtgccct
ccccatccag caccaggact ggatgagtgg caaggagttc aaatgcaagg
540tcaacaacaa agacctccca gcgcccatcg agagaaccat ctcaaaaccc
aaaggtgaga 600gctgcagcct gactgcatgg gggctgggat gggcataagg
ataaaggtct gtgtggacag 660ccttctgctt cagccatgac ctttgtgtat
gtttctaccc tcacagggtc agtaagagct 720ccacaggtat atgtcttgcc
tccaccagaa gaagagatga ctaagaaaca ggtcactctg 780acctgcatgg
tcacagactt catgcctgaa gacatttacg tggagtggac caacaacggg
840aaaacagagc taaactacaa gaacactgaa ccagtcctgg actctgatgg
ttcttacttc 900atgtacagca agctgagagt ggaaaagaag aactgggtgg
aaagaaatag ctactcctgt 960tcagtggtcc acgagggtct gcacaatcac
cacacgacta agagcttctc tcggactccg 1020ggtaaaaccg ggggacggat
ctgtccgaag ccggatgacc taccatttgc tacggttgtc 1080cccttaaaga
catcctacga ccctggggag cagattgtct actcctgcaa gccaggctac
1140gtgtccaggg gagggatgag acggtttacc tgtcctctca caggaatgtg
gcccatcaac 1200accctgagat gtgtccccag agtatgtcct ttcgctggaa
tcttagaaaa tggaattgta 1260cgctacacga gttttgaata tcccaagaac
atcagttttg cttgtaaccc tgggtttttt 1320ctgaatggga ccagctcatc
taagtgcacg gaggaaggaa aatggagccc agatattcct 1380gcttgtgctc
gcatcacctg cccgccacca ccagttccaa agtttgcact ccttaaggat
1440tataggcctt cagctgggaa caactctttg tatcaggaca cagtggtctt
taaatgcttg 1500ccacactttg ccatgatcgg aaatgacaca gtcatgtgca
cagaacaagg aaactggacc 1560cgattgccag aatgcctgga agtaaaatgt
cccttccctc cgaggccaga aaatgggtat 1620gtgaattatc ctgcaaagcc
ggtgcttcta tataaggata aagccacatt tggttgccat 1680gagacataca
agctggacgg cccagaagaa gcggaatgta ccaagacgag aacttggtcc
1740ttcttgccga cctgtagaga gtcttgcaaa ctccccgtta agaaagccac
cgtgctgtac 1800caagggatga gggtgaagat ccaggaacag tttaagaatg
ggatgatgca tggcgacaaa 1860attcacttct actgcaaaaa caaagagaag
aagtgcagct acactgtgga ggctcattgc 1920agagatggca ctatcgagat
tccctcgtgc ttcaaggagc acagttctct ggctttctgg 1980aaaacggatg
catcagaact gacaccgtgc tgaagtcgaa ttcattgatc ataatcagcc
2040ataccacatt tgtagaggtt ttacttgctt taaaaaacct cccacacctc
cccctgaacc 2100tgaaacataa aatgaatgca attgttgttg ttaacttgtt
tattgcagct tataatggtt 2160acaaataaag caatagcatc acaaatttca
caaataaagc atttttttca ctgcattcta 2220gttgtggttt gtccaaactc
atcaatgtat cttatcatgt ctggcggccg cgacctgcag 2280gcgcagaact
ggtaggtatg gaagatccct cgagatccat tgtgctggcg gtaggcgagc
2340agcgcctgcc tgaagctgcg ggcattccca gtcagaaatg agcgccagtc
gtcgtcggct 2400ctcggcaccg aagtgctatg attctccgcc agcatggctt
cggccagtgc gtcgagcagc 2460gcccgcttgt tcctgaagtg ccagtaaagc
gccggctgct gaacccccaa ccgttccgcc 2520agtttgcgtg tcgtcagacc
gtctacgccg acctcgttca acaggtccag ggcggcacgg 2580atcactgtat
tcggctgcaa ctttgtcatg cttgacactt tatcactgat aaacataata
2640tgtccaccaa cttatcagtg ataaagaatc cgcgccagca caatggatct
cgaggtcgag 2700ggatctctag aggatcctct acgccggacg catcgtggcc
ggcatcaccg gcgccacagg 2760tgcggttgct ggcgcctata tcgccgacat
caccgatggg gaagatcggg ctcgccactt 2820cgggctcatg agcgcttgtt
tcggcgtggg tatggtggca ggccccgtgg ccgggggact 2880gttgggcgcc
atctccttgc atgcaccatt ccttgcggcg gcggtgctca acggcctcaa
2940cctactactg ggctgcttcc taatgcagga gtcgcataag ggagagcgtc
gacctcgggc 3000cgcgttgctg gcgtttttcc ataggctccg cccccctgac
gagcatcaca aaaatcgacg 3060ctcaagtcag aggtggcgaa acccgacagg
actataaaga taccaggcgt ttccccctgg 3120aagctccctc gtgcgctctc
ctgttccgac cctgccgctt accggatacc tgtccgcctt 3180tctcccttcg
ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
3240gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
ccgaccgctg 3300cgccttatcc ggtaactatc gtcttgagtc caacccggta
agacacgact tatcgccact 3360ggcagcagcc actggtaaca ggattagcag
agcgaggtat gtaggcggtg ctacagagtt 3420cttgaagtgg tggcctaact
acggctacac tagaagaaca gtatttggta tctgcgctct 3480gctgaagcca
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
3540cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
aaaaaggatc 3600tcaagaagat cctttgatct tttctacggg gtctgacgct
cagtggaacg aaaactcacg 3660ttaagggatt ttggtcatga gattatcaaa
aaggatcttc acctagatcc ttttaaatta 3720aaaatgaagt tttaaatcaa
tctaaagtat atatgagtaa acttggtctg acagttacca 3780atgcttaatc
agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
3840ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
gccccagtgc 3900tgcaatgata ccgcgagacc cacgctcacc ggctccagat
ttatcagcaa taaaccagcc 3960agccggaagg gccgagcgca gaagtggtcc
tgcaacttta tccgcctcca tccagtctat 4020taattgttgc cgggaagcta
gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt 4080tgccattgct
acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
4140cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
aagcggttag 4200ctccttcggt cctccgatcg ttgtcagaag taagttggcc
gcagtgttat cactcatggt 4260tatggcagca ctgcataatt ctcttactgt
catgccatcc gtaagatgct tttctgtgac 4320tggtgagtac tcaaccaagt
cattctgaga atagtgtatg cggcgaccga gttgctcttg 4380cccggcgtca
atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
4440tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
gatccagttc 4500gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
tttactttca ccagcgtttc 4560tgggtgagca aaaacaggaa ggcaaaatgc
cgcaaaaaag ggaataaggg cgacacggaa 4620atgttgaata ctcatactct
tcctttttca atattattga agcatttatc agggttattg 4680tctcatgagc
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg
4740cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
tgacattaac 4800ctataaaaat aggcgtatca cgaggccctg atggctcttt
gcggcaccca tcgttcgtaa 4860tgttccgtgg caccgaggac aaccctcaag
agaaaatgta atcacactgg ctcaccttcg 4920ggtgggcctt tctgcgttta
taaggagaca ctttatgttt aagaaggttg gtaaattcct 4980tgcggctttg
gcagccaagc tagatccggc tgtggaatgt gtgtcagtta gggtgtggaa
5040agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
tagtcagcaa 5100ccaggtgtgg aaagtcccca ggctccccag caggcagaag
tatgcaaagc atgcatctca 5160attagtcagc aaccatagtc ccgcccctaa
ctccgcccat cccgccccta actccgccca 5220gttccgccca ttctccgccc
catggctgac taattttttt tatttatgca gaggccgagg 5280ccgcctcggc
ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
5340tttgcaaaaa gctagcttgg ggccaccgct cagagcacct tccaccatgg
ccacctcagc 5400aagttcccac ttgaacaaaa acatcaagca aatgtacttg
tgcctgcccc agggtgagaa 5460agtccaagcc atgtatatct gggttgatgg
tactggagaa ggactgcgct gcaaaacccg 5520caccctggac tgtgagccca
agtgtgtaga agagttacct gagtggaatt ttgatggctc 5580tagtaccttt
cagtctgagg gctccaacag tgacatgtat ctcagccctg ttgccatgtt
5640tcgggacccc ttccgcagag atcccaacaa gctggtgttc tgtgaagttt
tcaagtacaa 5700ccggaagcct gcagagacca atttaaggca ctcgtgtaaa
cggataatgg acatggtgag 5760caaccagcac ccctggtttg gaatggaaca
ggagtatact ctgatgggaa cagatgggca 5820cccttttggt tggccttcca
atggctttcc tgggccccaa ggtccgtatt actgtggtgt 5880gggcgcagac
aaagcctatg gcagggatat cgtggaggct cactaccgcg cctgcttgta
5940tgctggggtc aagattacag gaacaaatgc tgaggtcatg cctgcccagt
gggaactcca 6000aataggaccc tgtgaaggaa tccgcatggg agatcatctc
tgggtggccc gtttcatctt 6060gcatcgagta tgtgaagact ttggggtaat
agcaaccttt gaccccaagc ccattcctgg 6120gaactggaat ggtgcaggct
gccataccaa ctttagcacc aaggccatgc gggaggagaa 6180tggtctgaag
cacatcgagg aggccatcga gaaactaagc aagcggcacc ggtaccacat
6240tcgagcctac gatcccaagg ggggcctgga caatgcccgt ggtctgactg
ggttccacga 6300aacgtccaac atcaacgact tttctgctgg tgtcgccaat
cgcagtgcca gcatccgcat 6360tccccggact gtcggccagg agaagaaagg
ttactttgaa gaccgcggcc cctctgccaa 6420ttgtgacccc tttgcagtga
cagaagccat cgtccgcaca tgccttctca atgagactgg 6480cgacgagccc
ttccaataca aaaactaatt agactttgag tgatcttgag cctttcctag
6540ttcatcccac cccgccccag agagatcttt gtgaaggaac cttacttctg
tggtgtgaca 6600taattggaca aactacctac agagatttaa agctctaagg
taaatataaa atttttaagt 6660gtataatgtg ttaaactact gattctaatt
gtttgtgtat tttagattcc aacctatgga 6720actgatgaat gggagcagtg
gtggaatgcc tttaatgagg aaaacctgtt ttgctcagaa 6780gaaatgccat
ctagtgatga tgaggctact gctgactctc aacattctac tcctccaaaa
6840aagaagagaa aggtagaaga ccccaaggac tttccttcag aattgctaag
ttttttgagt 6900catgctgtgt ttagtaatag aactcttgct tgctttgcta
tttacaccac aaaggaaaaa 6960gctgcactgc tatacaagaa aattatggaa
aaatattctg taacctttat aagtaggcat 7020aacagttata atcataacat
actgtttttt cttactccac acaggcatag agtgtctgct 7080attaataact
atgctcaaaa attgtgtacc tttagctttt taatttgtaa aggggttaat
7140aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata
ccacatttgt 7200agaggtttta cttgctttaa aaaacctccc acacctcccc
ctgaacctga aacataaaat 7260gaatgcaatt gttgttgtta acttgtttat
tgcagcttat aatggttaca aataaagcaa 7320tagcatcaca aatttcacaa
ataaagcatt tttttcactg cattctagtt gtggtttgtc 7380caaactcatc
aatgtatctt atcatgtctg gatctagctt cgtgtcaagg acggtgactg
7440cagtgaataa taaaatgtgt gtttgtccga aatacgcgtt ttgagatttc
tgtcgccgac 7500taaattcatg tcgcgcgata gtggtgttta tcgccgatag
agatggcgat attggaaaaa 7560tcgatatttg aaaatatggc atattgaaaa
tgtcgccgat gtgagtttct gtgtaactga 7620tatcgccatt tttccaaaag
tgatttttgg gcatacgcga tatctggcga tagcgcttat 7680atcgtttacg
ggggatggcg atagacgact ttggtgactt gggcgattct gtgtgtcgca
7740aatatcgcag tttcgatata ggtgacagac gatatgaggc tatatcgccg
atagaggcga 7800catcaagctg gcacatggcc aatgcatatc gatctataca
ttgaatcaat attggccatt 7860agccatatta ttcattggtt atatagcata
aatcaatatt ggctattggc cattgcatac 7920gttgtatcca tatcataata
tgtacattta tattggctca tgtccaacat taccgccatg 7980ttgacattga
ttattgacta gttattaata gtaatcaatt acggggtcat tagttcatag
8040cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
gctgaccgcc 8100caacgacccc cgcccattga cgtcaataat gacgtatgtt
cccatagtaa cgccaatagg 8160gactttccat tgacgtcaat gggtggagta
tttacggtaa actgcccact tggcagtaca 8220tcaagtgtat catatgccaa
gtacgccccc tattgacgtc aatgacggta aatggcccgc 8280ctggcattat
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt
8340attagtcatc gctattacca tggtgatgcg gttttggcag tacatcaatg
ggcgtggata 8400gcggtttgac tcacggggat ttccaagtct ccaccccatt
gacgtcaatg ggagtttgtt 8460ttggcaccaa aatcaacggg actttccaaa
atgtcgtaac aactccgccc cattgacgca 8520aatgggcggt aggcgtgtac
ggtgggaggt ctatataagc agagctcgtt tagtgaaccg 8580tcagatcgcc
tggagacgcc atccacgctg ttttgacctc catagaagac accgggaccg
8640atccagcctc cgcggccggg aacggtgcat tggaacgcgg attccccgtg
ccaagagtga 8700cgtaagtacc gcctatagag tctataggcc cacccccttg
gcttcttatg catgctatac 8760tgtttttggc ttggggtcta tacacccccg
cttcctcatg ttataggtga tggtatagct 8820tagcctatag gtgtgggtta
ttgaccatta ttgaccactc ccctattggt gacgatactt 8880tccattacta
atccataaca tggctctttg ccacaactct ctttattggc tatatgccaa
8940tacactgtcc ttcagagact gacacggact ctgtattttt acaggatggg
gtctcattta 9000ttatttacaa attcacatat acaacaccac cgtccccagt
gcccgcagtt tttattaaac 9060ataacgtggg atctccacgc gaatctcggg
tacgtgttcc ggacatgggc tcttctccgg 9120tagcggcgga gcttctacat
ccgagccctg ctcccatgcc tccagcgact catggtcgct 9180cggcagctcc
ttgctcctaa cagtggaggc cagacttagg cacagcacga tgcccaccac
9240caccagtgtg ccgcacaagg ccgtggcggt agggtatgtg tctgaaaatg
agctcgggga 9300gcgggcttgc accgctgacg catttggaag acttaaggca
gcggcagaag aagatgcagg 9360cagctgagtt gttgtgttct gataagagtc
agaggtaact cccgttgcgg tgctgttaac 9420ggtggagggc agtgtagtct
gagcagtact cgttgctgcc gcgcgcgcca ccagacataa 9480tagctgacag
actaacagac tgttcctttc catgggtctt ttctgcagtc accgtccttg 9540acacga
9546
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