U.S. patent application number 13/811485 was filed with the patent office on 2013-07-11 for anti-despr inhibitors as therapeutics for inhibition of pathological angiogenesis and tumor cell invasiveness and for molecular imaging and targeted delivery.
This patent application is currently assigned to Trustee of Boston University. The applicant listed for this patent is Victoria L.M. Herrera, Nelson Ruiz-Opaz. Invention is credited to Victoria L.M. Herrera, Nelson Ruiz-Opaz.
Application Number | 20130177500 13/811485 |
Document ID | / |
Family ID | 44533112 |
Filed Date | 2013-07-11 |
United States Patent
Application |
20130177500 |
Kind Code |
A1 |
Ruiz-Opaz; Nelson ; et
al. |
July 11, 2013 |
ANTI-DESPR INHIBITORS AS THERAPEUTICS FOR INHIBITION OF
PATHOLOGICAL ANGIOGENESIS AND TUMOR CELL INVASIVENESS AND FOR
MOLECULAR IMAGING AND TARGETED DELIVERY
Abstract
Provided herein are novel compositions comprising anti-DEspR
antibodies and fragments thereof, including fully human, composite
engineered human, humanized, monoclonal, and polyclonal anto-DEspR
antibodies and fragments thereof, and methods of their use in a
variety of therapeutic applications. The compositions comprising
the anti-DEspR antibodies and fragments thereof described herein
are useful in diagnostic and imaging methods, such as
DEspR-targeted molecular imaging of angiogenesis, and for companion
diagnostic and/or in vivo-non invasive imaging and/or
assessments.
Inventors: |
Ruiz-Opaz; Nelson;
(Westwood, MA) ; Herrera; Victoria L.M.;
(Westwood, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ruiz-Opaz; Nelson
Herrera; Victoria L.M. |
Westwood
Westwood |
MA
MA |
US
US |
|
|
Assignee: |
Trustee of Boston
University
Boston
MA
|
Family ID: |
44533112 |
Appl. No.: |
13/811485 |
Filed: |
July 22, 2011 |
PCT Filed: |
July 22, 2011 |
PCT NO: |
PCT/US11/45056 |
371 Date: |
March 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61367206 |
Jul 23, 2010 |
|
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Current U.S.
Class: |
424/1.49 ;
424/139.1; 424/178.1; 424/9.1; 424/9.5; 530/387.9; 530/391.1;
530/391.3; 530/391.7; 604/22 |
Current CPC
Class: |
C07K 16/2863 20130101;
C07K 16/2869 20130101; A61P 19/10 20180101; C07K 2317/34 20130101;
A61K 47/55 20170801; A61P 9/00 20180101; A61P 3/10 20180101; A61P
9/10 20180101; A61P 25/00 20180101; A61K 38/18 20130101; C07K
2317/24 20130101; G01N 33/57496 20130101; A61K 2039/507 20130101;
A61P 9/08 20180101; A61P 27/02 20180101; C07K 2317/76 20130101;
A61P 15/00 20180101; A61P 43/00 20180101; C07K 16/28 20130101; A61M
37/0092 20130101; A61P 17/00 20180101; A61P 25/28 20180101; C07K
2317/21 20130101; C07K 16/30 20130101; A61K 49/221 20130101; A61P
19/02 20180101; A61P 27/06 20180101; G01N 2800/7028 20130101; A61K
2039/505 20130101; A61P 25/14 20180101; A61P 35/00 20180101; A61P
3/04 20180101 |
Class at
Publication: |
424/1.49 ;
530/387.9; 530/391.7; 530/391.3; 530/391.1; 424/139.1; 424/178.1;
424/9.5; 424/9.1; 604/22 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61M 37/00 20060101 A61M037/00; A61K 49/22 20060101
A61K049/22 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government Support under
Contract No. RR025771 awarded by the National Institutes of Health.
The Government has certain rights in the invention.
Claims
1-73. (canceled)
74. An isolated anti-DEspR antibody or antibody fragment thereof
that specifically binds to human DEspR (dual endothelin/VEGF signal
peptide receptor) expressed on a living cell, wherein the antibody,
or antibody fragment thereof, is a composite fully human monoclonal
antibody or antibody fragment thereof and wherein the antibody or
antibody fragment thereof binds to a tumor cell.
75. The anti-DEspR antibody or antibody fragment thereof of claim
74, wherein the antibody or antibody fragment thereof specifically
binds to residues 1-9 of SEQ ID NO:1.
76. The anti-DEspR antibody or antibody fragment thereof of claim
74, wherein the antibody or antibody fragment thereof comprises the
complementarity determining regions (CDRs) of a variable heavy
(V.sub.H) chain amino acid sequence comprising a sequence of SEQ ID
NO: 4 and the CDRs of a variable light (V.sub.L) chain amino acid
sequence comprising a sequence of SEQ ID NO: 9.
77. A pharmaceutical composition comprising an isolated anti-DEspR
antibody or antibody fragment thereof that specifically binds to
human DEspR (dual endothelin/VEGF signal peptide receptor)
expressed on a living cell, wherein the antibody, or antibody
fragment thereof, is a composite fully human monoclonal antibody or
antibody fragment thereof and wherein the antibody or antibody
fragment thereof binds to a tumor cell.
78. A pharmaceutical composition comprising an isolated anti-DEspR
antibody or antibody fragment thereof that specifically binds to
human DEspR (dual endothelin/VEGF signal peptide receptor)
expressed on a living cell, wherein the antibody, or antibody
fragment thereof, is a composite fully human monoclonal antibody or
antibody fragment thereof and wherein the antibody or antibody
fragment thereof binds to a tumor cell and is conjugated to a
chemotherapeutic agent, a toxin, a radioactive isotope, a small
molecule, an siRNA, a nanoparticle, or a microbubble.
79. A pharmaceutical composition comprising an antibody that
specifically binds to human VEGF signal peptide (VEGFsp).
80. A method of inhibiting angiogenesis in a subject having a
disease or disorder dependent or modulated by angiogenesis, the
method comprising administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising: an isolated anti-DEspR antibody or antibody fragment
thereof that specifically binds to human DEspR (dual
endothelin/VEGF signal peptide receptor) expressed on a living
cell, wherein the antibody, or antibody fragment thereof, is a
composite fully human monoclonal antibody or antibody fragment
thereof and wherein the antibody or antibody fragment thereof binds
to a tumor cell; an isolated anti-DEspR antibody or antibody
fragment thereof that specifically binds to human DEspR expressed
on a living cell, wherein the antibody, or antibody fragment
thereof, is a composite fully human monoclonal antibody or antibody
fragment thereof and wherein the antibody or antibody fragment
thereof binds to a tumor cell and is conjugated to a
chemotherapeutic agent, a toxin, a radioactive isotope, a small
molecule, an siRNA, a nanoparticle, or a microbubble; or an
antibody that specifically binds to human VEGF signal peptide
(VEGFsp).
81. The method of claim 80, wherein the disease or disorder
dependent or modulated by angiogenesis is cancer, vascularization
of the vaso vasorum, age-related macular degeneration, carotid
artery disease, diabetic retinopathy, rheumatoid arthritis,
neurodegenerative disorder, Alzheimer's disease, obesity,
endometriosis, psoriasis, atherosclerosis, ocular
neovascularization, neovascular glaucoma, osteoporosis, and
restenosis.
82. A method of inhibiting tumor cell invasiveness in a subject
having a cancer or a tumor, the method comprising administering to
a subject in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising: an isolated anti-DEspR
antibody or antibody fragment thereof that specifically binds to
human DEspR (dual endothelin/VEGF signal peptide receptor)
expressed on a living cell, wherein the antibody, or antibody
fragment thereof, is a composite fully human monoclonal antibody or
antibody fragment thereof and wherein the antibody or antibody
fragment thereof binds to a tumor cell; an isolated anti-DEspR
antibody or antibody fragment thereof that specifically binds to
human DEspR expressed on a living cell, wherein the antibody, or
antibody fragment thereof, is a composite fully human monoclonal
antibody or antibody fragment thereof and wherein the antibody or
antibody fragment thereof binds to a tumor cell and is conjugated
to a chemotherapeutic agent, a toxin, a radioactive isotope, a
small molecule, an siRNA, a nanoparticle, or a microbubble; or an
antibody that specifically binds to human VEGF signal peptide
(VEGFsp).
83. A method of inhibiting tumor growth in a subject in need
thereof by inhibiting DEspR expression and/or function in a tumor
cell, the method comprising administering to a subject in need
thereof a therapeutically effective amount of a pharmaceutical
composition comprising: an isolated anti-DEspR antibody or antibody
fragment thereof that specifically binds to human DEspR (dual
endothelin/VEGF signal peptide receptor) expressed on a living
cell, wherein the antibody, or antibody fragment thereof, is a
composite fully human monoclonal antibody or antibody fragment
thereof and wherein the antibody or antibody fragment thereof binds
to a tumor cell; an isolated anti-DEspR antibody or antibody
fragment thereof that specifically binds to human DEspR expressed
on a living cell, wherein the antibody, or antibody fragment
thereof, is a composite fully human monoclonal antibody or antibody
fragment thereof and wherein the antibody or antibody fragment
thereof binds to a tumor cell and is conjugated to a
chemotherapeutic agent, a toxin, a radioactive isotope, a small
molecule, an siRNA, a nanoparticle, or a microbubble; or an
antibody that specifically binds to human VEGF signal peptide
(VEGFsp).
84. A method of killing a tumor cell, a tumor initiating cell, a
cancer stem-like cell, or a cancer stem cell, comprising contacting
the cell with a DEspR (dual endothelin/VEGF signal peptide
receptor) inhibitor that binds DEspR on the cell or that binds
VEGFsp and inhibits VEGFsp binding to DEspR, thereby inhibiting
DEspR expression and/or function in the cell.
85. The method of claim 84, wherein the DEspR inhibitor binds to
and kills a cancer stem cell.
86. The method of claim 84, wherein the DEspR inhibitor is a
pharmaceutical composition comprising an isolated anti-DEspR
antibody or antibody fragment thereof that specifically binds to
human DEspR expressed on a living cell, wherein the antibody, or
antibody fragment thereof, is a composite fully human monoclonal
antibody or antibody fragment thereof and wherein the antibody or
antibody fragment thereof binds to a tumor cell.
87. A method of imaging a tumor cell, a tumor initiating cell, a
cancer stem-like cell, or a cancer stem cell, comprising contacting
the cell with an agent that binds specifically human DEspR (dual
endothelin/VEGF signal peptide receptor).
88. The method of claim 87, wherein the agent comprises an antibody
or VEGFsp.
89. The method of claim 88, wherein the imaging is molecular
imaging via targeting DEspR, wherein the agent is an antibody or
VEGFsp conjugated to a targeting moiety, and further comprising
determining the presence or absence of the antibody or VEGFsp
conjugated to the targeting moiety using molecular imaging.
90. The method of claim 89, wherein the molecular imaging is
contrast-enhanced ultrasound imaging, MRI (magnetic resonance
imaging), near infrared imaging, or photoacoustics imaging.
91. A method for enhancing delivery of a therapeutic agent via
DEspR (dual endothelin/VEGF signal peptide receptor)-targeted
sonoporation, the method comprising delivering an effective amount
of a pharmaceutical composition comprising a DEspR inhibitor and
the therapeutic agent using targeted ultrasound delivery to a
subject in need thereof, wherein delivery of the therapeutic agent
is enhanced relative to delivering the therapeutic agent in the
absence of the pharmaceutical composition comprising the DEspR
inhibitor.
92. The method of claim 91, wherein the therapeutic agent is a
chemotherapeutic agent, a small molecule, a peptide, or an aptamer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit under 35 U.S.C. .sctn.119(e)
of U.S. Provisional Patent Application Ser. No. 61/367,206 filed on
23 Jul. 2010, the contents of which are incorporated herein by
reference in their entirety.
FIELD OF THE INVENTION
[0003] This invention relates to monoclonal antibodies against
DEspR, and their use as therapeutics in the inhibition of
pathological angiogenesis and tumor cell invasiveness, as well as
diagnostic agents and targeting agents for molecular imaging and
targeted delivery of other therapeutic agents.
BACKGROUND
[0004] The establishment of a critical role of the angiogenic
switch in tumorigenesis has made the rationale behind the
development of anti-angiogenesis therapy clear (Hanahan &
Weinberg 2007). Unfortunately, the ability to attain long-term
efficacy of anti-angiogenesis therapy for all cancer-types, in
order to reduce cancer to a dormant, chronic manageable disease
without increasing morbidity from side effects, has not yet been
achieved (Loges et al. 2010, Ferrara 2009, Abdollahi & Folkman
2009, Bergers & Hanahan 2008).
[0005] Cumulative observations indicate that all three FDA-approved
VEGF pathway inhibitors (anti-VEGFbevacizumab or Avastin,
AntiVEGFR2 sunitinib, and sorafanib) result in only transitory
improvements in the form of tumor stasis or shrinkage, and only for
certain cancers despite most, if not all cancer types exhibiting
pathological angiogenesis (Carmeliet 2005; Bergers and Hanahan
2008). Moreover, while anti-VEGF pathway therapies have reduced
primary tumor growth and metastasis in preclinical studies
(Crawford & Ferrara 2008), recent mouse tumor model studies
have reported that sunitinib and an anti-VEGFR2 antibody, DC101,
increased metastasis of tumor cells despite inhibition of primary
tumor growth and increased overall survival in some cases (Ebos et
al. 2009, Paez-Ribes et al. 2009). Addressing this
"antiangiogenesis therapy conundrum," cumulative observations have
suggested several mechanisms of evasive and intrinsic resistances
(Loges et al. 2010, Ferrara 2009, Abdollahi & Folkman 2009,
Bergers & Hanahan 2008) such as: a) activation and/or
upregulation of alternative pro angiogenic pathways, b) recruitment
of bone marrow-derived pro-angiogenic cells, c) increased pericyte
coverage for the tumor vasculature, attenuating the need for VEGF
signaling; d) activation and enhancement of invasion and metastasis
to provide access to normal tissue vasculature without obligate
neovascularization; [for intrinsic resistance]: e) pre-existing
multiplicity of redundant pro-angiogenic signals; f) pre-existing
inflammatory cell-mediated vascular protection; g) tumor
hypovascularity; and h) invasive and metastatic co-option of normal
vessels without requisite angiogenesis (Bergers and Hanahan
2008).
SUMMARY OF THE INVENTION
[0006] Described herein are novel compositions comprising
anti-DEspR antibodies and fragments thereof, including fully human,
composite engineered human, humanized, monoclonal, and polyclonal
anto-DEspR antibodies and fragments thereof, and methods of their
use in a variety of applications, including, but not limited to: 1)
anti-angiogenesis therapies and anti-tumor cell invasiveness
relevant for treatment of cancer, 2) anti-angiogenesis approaches
relevant to treatment of those vascular diseases where pathological
angiogenesis plays a role in pathogenesis or progression such as in
carotid artery disease, vasa vasorum neovascularization (thus
impacting stroke), and vulnerable plaque neovascularization (thus
impacting, for example, heart disease), and 3) pro-autophagy
approaches pertinent to neurodegenerative diseases wherein
increased autophagy can prevent the accumulation of toxic products
or misfolded proteins or abnormal proteins as in Alzheimer's
disease, Huntington's disease etc.
[0007] In addition, the compositions comprising the anti-DEspR
antibodies and fragments thereof described herein are useful in
diagnostic and imaging methods, such as DEspR-targeted molecular
imaging of angiogenesis, which can be used, for example, in
monitoring response to therapy, in vivo detection of tumor
"angiogenic switch" or vascular mimicry. The compositions
comprising the anti-DEspR antibodies and fragments thereof are
useful for novel companion diagnostic and/or in vivo-non invasive
imaging and/or assessments. Additionally, the value-added benefit
of targeted delivery of therapeutic agents using the compositions
comprising the anti-DEspR antibodies and fragments thereof is
especially important in cancer wherein maximal efficacy is needed
with minimal systemic toxicity. Notably, such diagnostics provide
novel approaches for anti-angiogenic therapies for use in
personalized medicine. Accordingly, the compositions comprising the
anti-DEspR antibodies and fragments thereof described herein
comprise targeting tools and/or modules for target-specific
delivery of therapeutics, in forms such as toxins, drugs, small
molecules, peptides, fusion proteins, chimeric proteins,
nanoparticles, DNA, siRNA, etc., as well as for combinatorial
target-specific diagnostics and therapeutics, termed herein as
"theranostics."
[0008] Accordingly, provided herein, in some aspects are isolated
anti-DEspR antibodies or antibody fragments thereof that
specifically bind to DEspR (dual endothelin/VEGF signal peptide
receptor) and reduce or inhibit DEspR biological activity.
[0009] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
thereof specifically binds to DEspR comprising the amino acid
sequence of SEQ ID NO:1. In some embodiments of these aspects, the
antibody or antibody fragment thereof specifically binds to an
epitope of DEspR comprising residues 1-9 of SEQ ID NO:1. In some
embodiments of these aspects, the antibody or antibody fragment
thereof specifically binds to an epitope of DEspR consisting
essentially of residues 1-9 of SEQ ID NO: 1. In some embodiments of
these aspects, the antibody or antibody fragment thereof
specifically binds to an epitope of DEspR consisting of residues
1-9 of SEQ ID NO: 1.
[0010] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
thereof specifically binds to DEspR at a VEGF signal peptide
(VEGFsp) binding site. In some such embodiments, the VEGF signal
peptide comprises the amino acid sequence of SEQ ID NO:2. In some
such embodiments, the VEGF signal peptide consists essentially of
the amino acid sequence of SEQ ID NO:2. In some such embodiments,
the VEGF signal peptide consists of the amino acid sequence of SEQ
ID NO:2.
[0011] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a monoclonal antibody
or antibody fragment thereof.
[0012] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises a variable heavy (VH) chain amino acid sequence
comprising a sequence of SEQ ID NO: 4.
[0013] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises a variable light (VL) chain amino acid sequence
comprising a sequence of SEQ ID NO: 9.
[0014] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises a variable heavy (VH) chain amino acid sequence
comprising a sequence of SEQ ID NO: 4 and a variable light (VL)
chain amino acid sequence comprising a sequence of SEQ ID NO:
9.
[0015] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a humanized antibody
or antibody fragment thereof.
[0016] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises one or more heavy chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
and SEQ ID NO: 7. In some such embodiments, one or more heavy chain
CDR regions consist essentially of a sequence selected from the
group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
In some such embodiments, one or more heavy chain CDR regions
consist of a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
[0017] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises one or more light chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11,
and SEQ ID NO: 12. In some such embodiments, one or more light
chain CDR regions consist essentially of a sequence selected from
the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID
NO: 12. In some such embodiments, one or more light chain CDR
regions consist of a sequence selected from the group consisting of
SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
[0018] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
comprises one or more heavy chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
and SEQ ID NO: 7, and one or more light chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In some such embodiments,
the one or more heavy chain CDR regions consist essentially of a
sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID
NO: 6, and SEQ ID NO: 7. In some such embodiments, the one or more
heavy chain CDR regions consist of a sequence selected from the
group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
In some such embodiments, the one or more light chain CDR regions
consist essentially of a sequence selected from the group
consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In
some such embodiments, the one or more light chain CDR regions
consist of a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
[0019] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a composite antibody
or antibody fragment thereof. In some such embodiments, the
anti-DEspR composite antibody or antibody fragment comprises one or
more heavy chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7. In some such embodiments, the anti-DEspR composite antibody or
antibody fragment comprises one or more heavy chain CDR regions
consisting essentially of a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some
such embodiments, the anti-DEspR composite antibody or antibody
fragment comprises one or more heavy chain CDR regions consisting
of a sequence selected from the group consisting of SEQ ID NO: 5,
SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments, the
anti-DEspR composite antibody or antibody fragment comprises one or
more light chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID
NO: 12. In some such embodiments, the anti-DEspR composite antibody
or antibody fragment comprises one or more light chain CDR regions
consisting essentially of a sequence selected from the group
consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12. In
some such embodiments, the anti-DEspR composite antibody or
antibody fragment comprises one or more light chain CDR regions
consists of a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12.
[0020] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a composite antibody
or antibody fragment thereof comprising a variable heavy (VH) chain
amino acid sequence selected from the group consisting of SEQ ID
NO: 13-SEQ ID NO: 17. In some embodiments of these aspects and all
such aspects described herein, the anti-DEspR antibody is a
composite antibody or antibody fragment thereof consisting
essentially of a variable heavy (VH) chain amino acid sequence
selected from the group consisting of SEQ ID NO: 13-SEQ ID NO: 17.
In some embodiments of these aspects and all such aspects described
herein, the anti-DEspR antibody is a composite antibody or antibody
fragment thereof consisting of a variable heavy (VH) chain amino
acid sequence selected from the group consisting of SEQ ID NO:
13-SEQ ID NO: 17.
[0021] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a composite antibody
or antibody fragment thereof comprising a variable light (VL) chain
amino acid sequence selected from the group consisting of SEQ ID
NO: 18 and SEQ ID NO: 19. In some embodiments of these aspects and
all such aspects described herein, the anti-DEspR antibody is a
composite antibody or antibody fragment thereof consisting
essentially of a variable light (VL) chain amino acid sequence
selected from the group consisting of SEQ ID NO: 18 and SEQ ID NO:
19. In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody is a composite antibody
or antibody fragment thereof consisting of a variable light (VL)
chain amino acid sequence selected from the group consisting of SEQ
ID NO: 18 and SEQ ID NO: 19.
[0022] In other embodiments of these aspects, the anti-DEspR
antibody or antibody fragment thereof is an antibody expressed or
produced by hybridomas 7C5C55 or G12E8.
[0023] In some embodiments of these aspects, the anti-DEspR
antibody or antibody fragment thereof displays a similar binding
pattern to the binding pattern displayed by an antibody expressed
or produced by hybridomas 7C5B2, 7C5C55, or G12E8. In some
embodiments of these aspects, the anti-DEspR antibody or antibody
fragment thereof displays a similar avidity to the avidity
displayed by an antibody expressed or produced by hybridomas 7C5B2,
7C5C55, or G12E8. In some embodiments of these aspects, the
anti-DEspR antibody or antibody fragment thereof binds to the same
epitope(s) as those epitope(s) bound by an antibody expressed or
produced by hybridomas 7C5B2, 7C5C55, or G12E8.
[0024] In some embodiments of these aspects, the anti-DEspR
antibody or antibody fragment thereof comprises an amino acid
sequence of one or more CDRs of an antibody expressed or produced
by hybridomas 7C5C55 or G12E8. In some embodiments of these
aspects, the anti-DEspR antibody or antibody fragment thereof has
one or more biological characteristics of a monoclonal antibody
expressed or produced by hybridoma 7C5B2, 7C5C55, or G12E8. In some
embodiments of these aspects, the anti-DEspR antibody or antibody
fragment thereof specifically binds to an epitope of DEspR that is
bound by an antibody expressed or produced by hybridoma 7C5B2,
7C5C55, or G12E8.
[0025] In some embodiments of these aspects and all such aspects
described herein, the antibody fragment is a Fab fragment, a Fab'
fragment, a Fd fragment, a Fd' fragment, a Fv fragment, a dAb
fragment, a F(ab')2 fragment, a single chain fragment, a diabody,
or a linear antibody.
[0026] In some embodiments of these aspects and all such aspects
described herein, the anti-DEspR antibody or antibody fragment
thereof further comprises an agent conjugated to the anti-DEspR
antibody or antibody fragment thereof to form an immunoconjugate
specific for DEspR. In some such embodiments, the agent conjugated
to the antibody or antibody fragment thereof is a chemotherapeutic
agent, a toxin, a radioactive isotope, a small molecule, an siRNA,
a nanoparticle, or a microbubble.
[0027] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR, and a pharmaceutically acceptable carrier.
[0028] In some aspects, provided herein are methods of inhibiting
angiogenesis in a subject having a disease or disorder dependent or
modulated by angiogenesis, comprising administering to a subject in
need thereof a therapeutically effective amount of a pharmaceutical
composition comprising any of the anti-DEspR antibodies or antibody
fragments thereof described herein that specifically binds to
DEspR. In some embodiments of these aspects and all such aspects
described herein, the disease or disorder dependent or modulated by
angiogenesis is a cancer or a tumor. In some embodiments of these
aspects and all such aspects described herein, the disease or
disorder dependent or modulated by angiogenesis is selected from
the group consisting of age-related macular degeneration, carotid
artery disease, diabetic retinopathy, rheumatoid arthritis,
neurodegenerative disorder, Alzheimer's disease, obesity,
endometriosis, psoriasis, atherosclerosis, ocular
neovascularization, neovascular glaucoma, osteoporosis, and
restenosis.
[0029] In some aspects, provided herein are methods of inhibiting
tumor cell invasiveness in a subject having a cancer or a tumor,
comprising administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising any of the anti-DEspR antibodies or antibody fragments
thereof described herein that specifically binds to DEspR. In some
embodiments of these aspects and all such aspects described herein,
the method further comprises the administration of one or more
chemotherapeutic agents, angiogenesis inhibitors, cytotoxic agents,
or anti-proliferative agents.
[0030] In some aspects, provided herein are methods of inhibiting
tumor growth and reducing tumor size or tumor metastasis in a
subject having a tumor or metastasis by inhibiting DEspR expression
and/or function in a cell, comprising administering to a subject in
need thereof a therapeutically effective amount of a pharmaceutical
composition comprising any of the anti-DEspR antibodies or antibody
fragments thereof described herein that specifically binds to
DEspR. In some embodiments of these aspects and all such aspects
described herein, the DEspR expression and/or function is inhibited
in a tumor cell, a tumor initiating cell, a cancer stem-like cell,
a cancer stem cell, a metastatic tumor cell, an endothelial
progenitor cell, an inflammatory cell, a tumor stromal cell, a
tumor vasculature cell, or any combination thereof. In some such
embodiments, the tumor vasculature cell is an endothelial cell, a
pericyte, a smooth muscle cell, an adventitial cell, or any
combination thereof.
[0031] In some aspects, provided herein are methods of inhibiting
tumor resistance and tumor recurrence in a subject by inhibiting
DEspR expression and/or function in a cell, the methods comprising
administering to a subject in need thereof a therapeutically
effective amount of a pharmaceutical composition comprising any of
the anti-DEspR antibodies or antibody fragments thereof described
herein that specifically binds to DEspR. In some embodiments of
these aspects and all such aspects described herein, the DEspR
expression and/or function is inhibited in a tumor cell, a tumor
initiating cell, a cancer stem-like cell, a cancer stem cell, a
metastatic tumor cell, or any combination thereof.
[0032] In some aspects, provided herein are methods of inhibiting
cancer progression through promotion of autophagy of a cancer cell
by inhibiting DEspR expression and/or function in a tumor cell, the
methods comprising administering to a subject in need thereof a
therapeutically effective amount of a pharmaceutical composition
comprising any of the anti-DEspR antibodies or antibody fragments
thereof described herein that specifically binds to DEspR. In some
embodiments of these aspects and all such aspects described herein,
the DEspR expression and/or function is inhibited in a tumor cell,
a tumor initiating cell, a cancer stem-like cell, a cancer stem
cell, a metastatic tumor cell, or any combination thereof.
[0033] In some aspects, provided herein are methods of promoting
autophagy or a reduction in accumulation of intracellular noxious
substances or pathogens by inhibiting DEspR expression and/or
function in a cell, the methods comprising administering to a
subject in need thereof a therapeutically effective amount of a
pharmaceutical composition comprising any of the anti-DEspR
antibodies or antibody fragments thereof described herein that
specifically binds to DEspR. In some embodiments of these aspects
and all such aspects described herein, the subject has Alzheimer's
disease or Huntington's disease.
[0034] In some aspects, provided herein are methods of molecular
imaging via targeting DEspR, the methods comprising administering
an effective amount of a pharmaceutical composition comprising any
of the anti-DEspR antibodies or antibody fragments thereof
described herein that specifically binds to DEspR conjugated to a
targeting moiety, and determining the presence or absence of the
anti-DEspR antibodies or antibody fragments thereof conjugated to
the targeting moiety using molecular imaging. In some embodiments
of these aspects and all such aspects described herein, the
molecular imaging is contrast-enhanced ultrasound imaging, MRI
(magnetic resonance imaging), near infrared imaging, or
photoacoustics imaging. In some embodiments of these aspects and
all such aspects described herein, the targeting moiety is an
antibody, a DEspR-binding peptide ligand, a small molecule, a
nanoparticle, a polymer, an aptamer, or any combination
thereof.
[0035] In some aspects, provided herein are methods of stratifying
or classifying a tumor via determination of DEspR expression, the
methods comprising contacting a cell with any of the anti-DEspR
antibodies or antibody fragments thereof described herein that
specifically binds to DEspR, and determining whether the anti-DEspR
antibody or antibody fragment thereof binds to the cell after said
contacting, such that binding of the DEspR antibody or antibody
fragment thereof to the cell indicates that the cell expresses
DEspR. In some embodiments of these aspects and all such aspects
described herein, the cell is a tumor cell, an endothelial cell, a
pericyte, a smooth muscle cell, an adventitial cell, a tumor
stromal cell, or any combination thereof. In some such embodiments,
the tumor stromal cell is a fibroblast, a myofibroblast, an
inflammatory cell, a stellate cell, or any combination thereof. In
some embodiments of these aspects and all such aspects described
herein, the cell being contacted is in a tissue biopsy, a
paraffin-embedded section, or a frozen section.
[0036] In some aspects, provided herein are methods for enhancing
delivery of a therapeutic agent via DEspR-targeted sonoporation,
the methods comprising delivering an effective amount of a
pharmaceutical composition comprising any of the anti-DEspR
antibodies or antibody fragments thereof described herein that
specifically binds to DEspR and a therapeutic agent using targeted
ultrasound delivery, to a subject in need thereof, such that
delivery of the therapeutic agent is enhanced or increased relative
to delivering the therapeutic agent in the absence of the
pharmaceutical composition comprising any of the anti-DEspR
antibodies or antibody fragments thereof described herein. In some
embodiments of these aspects and all such aspects described herein,
the therapeutic agent is a chemotherapeutic agent, a small
molecule, a peptide, or an aptamer.
[0037] Also provided herein, in some aspects, are method for
reducing toxicity of a therapeutic agent via DEspR-targeted
sonoporation, the methods comprising delivering an effective amount
of a pharmaceutical composition comprising any of the anti-DEspR
antibodies or antibody fragments thereof described herein that
specifically binds to DEspR and a therapeutic agent using targeted
ultrasound delivery to a subject in need thereof, such that
toxicity of the therapeutic agent is reduced relative to delivering
the therapeutic agent in the absence of the pharmaceutical
composition comprising any of the anti-DEspR antibodies or antibody
fragments thereof described herein. In some embodiments of these
aspects and all such aspects described herein, the therapeutic
agent is a chemotherapeutic agent, a small molecule, a peptide, or
an aptamer.
[0038] In some aspects, provided herein are methods for combining
DEspR-targeted molecular imaging and DEspR-targeted delivery of a
therapeutic agent. These methods comprise administering to a
subject an effective amount of a therapeutic agent and a
pharmaceutical composition comprising any of the anti-DEspR
antibodies or antibody fragments thereof described herein
conjugated to a targeting moiety, and determining the presence or
absence of the anti-DEspR antibodies or antibody fragments thereof
described herein conjugated to the targeting moiety using molecular
imaging. In some embodiments of these aspects and all such aspects
described herein, the molecular imaging is contrast-enhanced
ultrasound imaging, MRI (magnetic resonance imaging), near infrared
imaging, or photoacoustics imaging. In some embodiments of these
aspects and all such aspects described herein, the therapeutic
agent is a chemotherapeutic agent, a small molecule, a peptide, or
an aptamer.
[0039] In other aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in inhibiting angiogenesis in a subject having a
disease or disorder dependent or modulated by angiogenesis. In some
embodiments of these aspects and all such aspects described herein,
the disease or disorder dependent or modulated by angiogenesis is a
cancer or a tumor. In some embodiments of these aspects and all
such aspects described herein, the disease or disorder dependent or
modulated by angiogenesis is selected from the group consisting of
age-related macular degeneration, carotid artery disease, diabetic
retinopathy, rheumatoid arthritis, neurodegenerative disorder,
Alzheimer's disease, obesity, endometriosis, psoriasis,
atherosclerosis, ocular neovascularization, neovascular glaucoma,
osteoporosis, and restenosis.
[0040] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in inhibiting tumor cell invasiveness in a subject
having a cancer or a tumor. In some embodiments of these aspects
and all such aspects described herein, the pharmaceutical
compositions further comprise one or more chemotherapeutic agents,
angiogenesis inhibitors, cytotoxic agents, or anti-proliferative
agents.
[0041] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in inhibiting tumor growth and reducing tumor size
or tumor metastasis by inhibiting DEspR expression and/or function
in a cell in a subject in need thereof. In some embodiments of
these aspects and all such aspects described herein, the DEspR
expression and/or function is inhibited in a tumor cell, a tumor
initiating cell, a cancer stem-like cell, a cancer stem cell, a
metastatic tumor cell, an endothelial progenitor cell, an
inflammatory cell, a tumor stromal cell, a tumor vasculature cell,
or any combination thereof. In some such embodiments, the tumor
vasculature cell is an endothelial cell, a pericyte, a smooth
muscle cell, an adventitial cell, or any combination thereof.
[0042] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in inhibiting tumor resistance and tumor
recurrence by inhibiting DEspR expression and/or function in a cell
in a subject in need thereof. In some embodiments of these aspects
and all such aspects described herein, the DEspR expression and/or
function is inhibited in a tumor cell, a tumor initiating cell, a
cancer stem-like cell, a cancer stem cell, a metastatic tumor cell,
or any combination thereof.
[0043] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in inhibiting cancer progression through promotion
of autophagy of a cancer cell by inhibiting DEspR expression and/or
function in a tumor cell in a subject in need thereof. In some
embodiments of these aspects and all such aspects described herein,
the DEspR expression and/or function is inhibited in a tumor cell,
a tumor initiating cell, a cancer stem-like cell, a cancer stem
cell, a metastatic tumor cell, or any combination thereof.
[0044] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in promoting autophagy or a reduction in
accumulation of intracellular noxious substances or pathogens by
inhibiting DEspR expression and/or function in a subject in need
thereof. In some embodiments of these aspects and all such aspects
described herein, the subject has Alzheimer's disease or
Huntington's disease.
[0045] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in enhancing delivery of a therapeutic agent via
DEspR-targeted sonoporation using targeted ultrasound delivery to a
subject in need thereof. In some embodiments of these aspects and
all such aspects described herein, the therapeutic agent is a
chemotherapeutic agent, a small molecule, a peptide, or an
aptamer.
[0046] In some aspects, provided herein are pharmaceutical
compositions comprising any of the anti-DEspR antibodies or
antibody fragments thereof described herein that specifically binds
to DEspR for use in reducing toxicity of a therapeutic agent via
DEspR-targeted sonoporation using targeted ultrasound delivery to a
subject in need thereof. In some embodiments of these aspects and
all such aspects described herein, the therapeutic agent is a
chemotherapeutic agent, a small molecule, a peptide, or an
aptamer.
DEFINITIONS
[0047] A "DEspR antagonist" refers to a molecule capable of
neutralizing, blocking, inhibiting, abrogating, reducing or
interfering with DEspR activities including its binding to
endothelin-1 or VEGFsp. DEspR antagonists include anti-DEspR
antibodies and antigen-binding fragments thereof, receptor
molecules and derivatives that bind specifically to DEspR thereby
inhibiting, preventing, or sequestering its binding to its ligands,
such as VEGFsp and endothelin-1.
[0048] The term "antibody" is used in the broadest sense and
includes monoclonal antibodies (including full length or intact
monoclonal antibodies), polyclonal antibodies, multivalent
antibodies, multispecific antibodies (e.g., bispecific antibodies),
and antibody fragments (see below) so long as they exhibit the
desired biological activity and specificity.
[0049] As used herein, the term "target" refers to a biological
molecule (e.g., peptide, polypeptide, protein, lipid, carbohydrate)
to which a polypeptide domain which has a binding site can
selectively bind. The target can be, for example, an intracellular
target (e.g., an intracellular protein target) or a cell surface
target (e.g., a membrane protein, a receptor protein). Preferably,
a target is a cell surface target, such as a cell surface
protein.
[0050] The term "specificity" refers to the number of different
types of antigens or antigenic determinants to which an antibody or
antibody fragment thereof as described herein can bind. The
specificity of an antibody or antibody fragment thereof can be
determined based on affinity and/or avidity. The affinity,
represented by the equilibrium constant for the dissociation
(K.sub.D) of an antigen with an antigen-binding protein, is a
measure of the binding strength between an antigenic determinant
and an antigen-binding site on the antigen-binding protein, such as
an antibody or antibody fragment thereof: the lesser the value of
the K.sub.D, the stronger the binding strength between an antigenic
determinant and the antigen-binding molecule. Alternatively, the
affinity can also be expressed as the affinity constant (K.sub.A),
which is 1/K.sub.D). As will be clear to the skilled person,
affinity can be determined in a manner known per se, depending on
the specific antigen of interest. Accordingly, an antibody or
antibody fragment thereof as defined herein is said to be "specific
for" a first target or antigen compared to a second target or
antigen when it binds to the first antigen with an affinity (as
described above, and suitably expressed, for example as a K.sub.D
value) that is at least 10 times, such as at least 100 times, and
preferably at least 1000 times, and up to 10000 times or more
better than the affinity with which said amino acid sequence or
polypeptide binds to another target or polypeptide.
[0051] Avidity is the measure of the strength of binding between an
antigen-binding molecule (such as an antibody or antibody fragment
thereof described herein) and the pertinent antigen. Avidity is
related to both the affinity between an antigenic determinant and
its antigen binding site on the antigen-binding molecule, and the
number of pertinent binding sites present on the antigen-binding
molecule. Typically, antigen-binding proteins (such as an antibody
or antibody fragment thereof described herein) will bind to their
cognate or specific antigen with a dissociation constant (K.sub.D
of 10.sup.-5 to 10.sup.-12 moles/liter or less, and preferably
10.sup.-7 to 10.sup.-12 moles/liter or less and more preferably
10.sup.-8 to 10.sup.-12 moles/liter (i.e., with an association
constant (K.sub.A) of 10.sup.5 to 10.sup.12 liter/moles or more,
and preferably 10.sup.7 to 10.sup.12 liter/moles or more and more
preferably 10.sup.8 to 10.sup.12 liter/moles). Any K.sub.D value
greater than 10.sup.-4 mol/liter (or any K.sub.A value lower than
10.sup.4 M.sup.-1) is generally considered to indicate non-specific
binding. The K.sub.D for biological interactions which are
considered meaningful (e.g., specific) are typically in the range
of 10.sup.-1.degree. M (0.1 nM) to 10.sup.-5 M (10000 nM). The
stronger an interaction is, the lower is its K.sub.D. Preferably, a
binding site on an antibody or antibody fragment thereof described
herein will bind to the desired antigen with an affinity less than
500 nM, preferably less than 200 nM, more preferably less than 10
nM, such as less than 500 pM. Specific binding of an
antigen-binding protein to an antigen or antigenic determinant can
be determined in any suitable manner known per se, including, for
example, Scatchard analysis and/or competitive binding assays, such
as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich
competition assays, and the different variants thereof known per se
in the art; as well as other techniques as mentioned herein.
[0052] Accordingly, as used herein, "selectively binds" or
"specifically binds" refers to the ability of an antibody or
antibody fragment thereof described herein to bind to a target,
such as a molecule present on the cell-surface, with a K.sub.D
10.sup.-5 M (10000 nM) or less, e.g., 10.sup.-6 M, 10.sup.-7 M,
10.sup.-8 M, 10.sup.-9 M, 10.sup.-10 M, 10.sup.-11 M, 10.sup.-12 M,
or less. Specific binding can be influenced by, for example, the
affinity and avidity of the polypeptide agent and the concentration
of polypeptide agent. The person of ordinary skill in the art can
determine appropriate conditions under which the polypeptide agents
described herein selectively bind the targets using any suitable
methods, such as titration of a polypeptide agent in a suitable
cell binding assay.
[0053] As described herein, an "antigen" is a molecule that is
bound by a binding site on a polypeptide agent, such as an antibody
or antibody fragment thereof. Typically, antigens are bound by
antibody ligands and are capable of raising an antibody response in
vivo. An antigen can be a polypeptide, protein, nucleic acid or
other molecule. In the case of conventional antibodies and
fragments thereof, the antibody binding site as defined by the
variable loops (L1, L2, L3 and H1, H2, H3) is capable of binding to
the antigen. The term "antigenic determinant" refers to an epitope
on the antigen recognized by an antigen-binding molecule, and more
particularly, by the antigen-binding site of said molecule.
[0054] As used herein, an "epitope" can be formed both from
contiguous amino acids, or noncontiguous amino acids juxtaposed by
tertiary folding of a protein. Epitopes formed from contiguous
amino acids are typically retained on exposure to denaturing
solvents, whereas epitopes formed by tertiary folding are typically
lost on treatment with denaturing solvents. An epitope typically
includes at least 3, and more usually, at least 5, about 9, or
about 8-10 amino acids in a unique spatial conformation. An
"epitope" includes the unit of structure conventionally bound by an
immunoglobulin V.sub.H/V.sub.L pair. Epitopes define the minimum
binding site for an antibody, and thus represent the target of
specificity of an antibody. In the case of a single domain
antibody, an epitope represents the unit of structure bound by a
variable domain in isolation. The terms "antigenic determinant" and
"epitope" can also be used interchangeably herein.
[0055] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that can be present in minor amounts. Monoclonal
antibodies are highly specific, being directed against a single
antigen. Furthermore, in contrast to polyclonal antibody
preparations that typically include different antibodies directed
against different determinants (epitopes), each monoclonal antibody
is directed against a single determinant on the antigen. The
modifier "monoclonal" is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the
invention can be made by the hybridoma method first described by
Kohler et al., Nature 256:495 (1975), or can be made by recombinant
DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal
antibodies" can also be isolated from phage antibody libraries
using the techniques described in Clackson et al., Nature
352:624-628 (1991) or Marks et al., J. Mol. Biol. 222:581-597
(1991), for example. A monoclonal antibody can be of any species,
including, but not limited to, mouse, rat, goat, rabbit, and human
monoclonal antibodies.
[0056] The term "antibody fragment," as used herein, refer to a
protein fragment that comprises only a portion of an intact
antibody, generally including an antigen binding site of the intact
antibody and thus retaining the ability to bind antigen. Examples
of antibody fragments encompassed by the present definition
include: (i) the Fab fragment, having V.sub.L, C.sub.L, V.sub.H and
C.sub.H1 domains; (ii) the Fab' fragment, which is a Fab fragment
having one or more cysteine residues at the C-terminus of the
C.sub.H1 domain; (iii) the Fd fragment having V.sub.H and C.sub.H1
domains; (iv) the Fd' fragment having V.sub.H and C.sub.H1 domains
and one or more cysteine residues at the C-terminus of the CH1
domain; (v) the Fv fragment having the V.sub.L and V.sub.H domains
of a single arm of an antibody; (vi) the dAb fragment (Ward et al.,
Nature 341, 544-546 (1989)) which consists of a V.sub.H domain;
(vii) isolated CDR regions; (viii) F(ab').sub.2 fragments, a
bivalent fragment including two Fab' fragments linked by a
disulphide bridge at the hinge region; (ix) single chain antibody
molecules (e.g., single chain Fv; scFv) (Bird et al., Science
242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883
(1988)); (x) "diabodies" with two antigen binding sites, comprising
a heavy chain variable domain (V.sub.H) connected to a light chain
variable domain (V.sub.L) in the same polypeptide chain (see, e.g.,
EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising
a pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1)
which, together with complementary light chain polypeptides, form a
pair of antigen binding regions (Zapata et al. Protein Eng.
8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).
[0057] An "Fv" fragment is an antibody fragment which contains a
complete antigen recognition and binding site. This region consists
of a dimer of one heavy and one light chain variable domain in
tight association, which can be covalent in nature, for example in
scFv. It is in this configuration that the three CDRs of each
variable domain interact to define an antigen binding site on the
surface of the V.sub.H-V.sub.L dimer. Collectively, the six CDRs or
a subset thereof confer antigen binding specificity to the
antibody. However, even a single variable domain (or half of an Fv
comprising only three CDRs specific for an antigen) has the ability
to recognize and bind antigen, although usually at a lower affinity
than the entire binding site.
[0058] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), which is also available
on the world wide web, and is expressly incorporated herein in its
entirety by reference. The "EU index as in Kabat" refers to the
residue numbering of the human IgG1 EU antibody.
[0059] As used herein, "antibody variable domain" refers to the
portions of the light and heavy chains of antibody molecules that
include amino acid sequences of Complementarity Determining Regions
(CDRs; i.e., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
V.sub.H refers to the variable domain of the heavy chain. V.sub.L
refers to the variable domain of the light chain. According to the
methods used in this invention, the amino acid positions assigned
to CDRs and FRs can be defined according to Kabat (Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md., 1987 and 1991)). Amino acid numbering of antibodies
or antigen binding fragments is also according to that of
Kabat.
[0060] As used herein, the term "Complementarity Determining
Regions" (CDRs), i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region can comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e., about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some instances, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop. For example, the CDRH1 of the heavy
chain of antibody 4D5 includes amino acids 26 to 35.
[0061] Framework regions" (hereinafter FR) are those variable
domain residues other than the CDR residues. Each variable domain
typically has four FRs identified as FR1, FR2, FR3 and FR4. If the
CDRs are defined according to Kabat, the light chain FR residues
are positioned at about residues 1-23 (LCFR1), 35-49 (LCFR2), 57-88
(LCFR3), and 98-107 (LCFR4) and the heavy chain FR residues are
positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94
(HCFR3), and 103-113 (HCFR4) in the heavy chain residues. If the
CDRs comprise amino acid residues from hypervariable loops, the
light chain FR residues are positioned about at residues 1-25
(LCFR1), 33-49 (LCFR2), 53-90 (LCFR3), and 97-107 (LCFR4) in the
light chain and the heavy chain FR residues are positioned about at
residues 1-25 (HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113
(HCFR4) in the heavy chain residues. In some instances, when the
CDR comprises amino acids from both a CDR as defined by Kabat and
those of a hypervariable loop, the FR residues will be adjusted
accordingly. For example, when CDRH1 includes amino acids H26-H35,
the heavy chain FR1 residues are at positions 1-25 and the FR2
residues are at positions 36-49.
[0062] The "Fab" fragment contains a variable and constant domain
of the light chain and a variable domain and the first constant
domain (C.sub.H1) of the heavy chain. F(ab').sub.2 antibody
fragments comprise a pair of Fab fragments which are generally
covalently linked near their carboxy termini by hinge cysteines
between them. Other chemical couplings of antibody fragments are
also known in the art.
[0063] "Single-chain Fv" or "scFv" antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, wherein these domains are
present in a single polypeptide chain. Generally the Fv polypeptide
further comprises a polypeptide linker between the V.sub.H and
V.sub.L domains, which enables the scFv to form the desired
structure for antigen binding. For a review of scFv, see Pluckthun
in The Pharmacology of Monoclonal Antibodies, Vol 113, Rosenburg
and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994).
[0064] The term "diabodies" refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0065] The expression "linear antibodies" refers to the antibodies
described in Zapata et al., Protein Eng., 8(10):1057-1062 (1995).
Briefly, these antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0066] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, as well
as fragments of such antibodies, so long as they exhibit the
desired biological activity (U.S. Pat. No. 4,816,567; and Morrison
et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
[0067] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that are engineered or designed to comprise
minimal sequence derived from non-human immunoglobulin. For the
most part, humanized antibodies are human immunoglobulins
(recipient antibody) in which residues from a hypervariable region
of the recipient are replaced by residues from a hypervariable
region of a non-human species (donor antibody) such as mouse, rat,
rabbit or nonhuman primate having the desired specificity,
affinity, and capacity. In some instances, Fv framework region (FR)
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Furthermore, humanized antibodies can comprise
residues which are not found in the recipient antibody or in the
donor antibody. These modifications are made to further refine
antibody performance. In general, the humanized antibody will
comprise substantially all of at least one, and typically two,
variable domains, in which all or substantially all of the
hypervariable loops correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin sequence. The humanized antibody
optionally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). As used
herein, a "composite human antibody" is a specific type of
engineered or humanized antibody.
[0068] A "human antibody," "non-engineered human antibody," or
"fully human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human and/or has been made using any of the techniques for making
human antibodies as disclosed herein. This definition of a human
antibody specifically excludes a humanized antibody comprising
non-human antigen-binding residues. Human antibodies can be
produced using various techniques known in the art. In one
embodiment, the human antibody is selected from a phage library,
where that phage library expresses human antibodies (Vaughan et al.
Nature Biotechnology 14:309-314 (1996): Sheets et al. Proc. Natl.
Acad. Sci. 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol.
Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581
(1991)). Human antibodies can also be made by introducing human
immunoglobulin loci into transgenic animals, e.g., mice in which
the endogenous mouse immunoglobulin genes have been partially or
completely inactivated. Upon challenge, human antibody production
is observed, which closely resembles that seen in humans in all
respects, including gene rearrangement, assembly, and antibody
repertoire. This approach is described, for example, in U.S. Pat.
Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425;
5,661,016, and in the following scientific publications: Marks et
al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368:
856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et
al., Nature Biotechnology 14: 845-51 (1996); Neuberger, Nature
Biotechnology 14: 826 (1996); Lonberg and Huszar, Intern. Rev.
Immunol. 13:65-93 (1995). Alternatively, the human antibody can be
prepared via immortalization of human B lymphocytes producing an
antibody directed against a target antigen (such B lymphocytes can
be recovered from an individual or can have been immunized in
vitro). See, e.g., Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J. Immunol.,
147 (1):86-95 (1991); and U.S. Pat. No. 5,750,373.
[0069] An "affinity matured" antibody is one with one or more
alterations in one or more CDRs thereof which result an improvement
in the affinity of the antibody for antigen, compared to a parent
antibody which does not possess those alteration(s). Preferred
affinity matured antibodies will have nanomolar or even picomolar
affinities for the target antigen. Affinity matured antibodies are
produced by procedures known in the art. Marks et al.
Bio/Technology 10:779-783 (1992) describes affinity maturation by
V.sub.H and V.sub.L domain shuffling. Random mutagenesis of CDR
and/or framework residues is described by: Barbas et al. Proc Nat.
Acad. Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155
(1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et
al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol.
Biol. 226:889-896 (1992).
[0070] A "functional antigen binding site" of an antibody is one
which is capable of binding a target antigen. The antigen binding
affinity of the antigen binding site is not necessarily as strong
as the parent antibody from which the antigen binding site is
derived, but the ability to bind antigen must be measurable using
any one of a variety of methods known for evaluating antibody
binding to an antigen. Moreover, the antigen binding affinity of
each of the antigen binding sites of a multivalent antibody herein
need not be quantitatively the same. For multimeric antibodies, the
number of functional antigen binding sites can be evaluated using
ultracentrifugation analysis as described in Example 2 of U.S.
Patent Application Publication No. 20050186208. According to this
method of analysis, different ratios of target antigen to
multimeric antibody are combined and the average molecular weight
of the complexes is calculated assuming differing numbers of
functional binding sites. These theoretical values are compared to
the actual experimental values obtained in order to evaluate the
number of functional binding sites.
[0071] As used herein, a "blocking" antibody or an antibody
"antagonist" is one which inhibits or reduces biological activity
of the antigen it binds. For example, a DEspR-specific antagonist
antibody binds DEspR and inhibits the ability of DEspR to, for
example, bind VEGFsp and induce angiogenesis, to induce vascular
endothelial cell proliferation or to induce vascular permeability.
In certain embodiments, blocking antibodies or antagonist
antibodies completely inhibit the biological activity of the
antigen.
[0072] Unless indicated otherwise, the expression "multivalent
antibody" is used throughout this specification to denote an
antibody comprising three or more antigen binding sites. For
example, the multivalent antibody is engineered to have the three
or more antigen binding sites and is generally not a native
sequence IgM or IgA antibody.
[0073] An antibody having a "biological characteristic" of a
designated antibody is one which possesses one or more of the
biological characteristics of that antibody which distinguish it
from other antibodies that bind to the same antigen.
[0074] In order to screen for antibodies which bind to an epitope
on an antigen bound by an antibody of interest, a routine
cross-blocking assay such as that described in Antibodies, A
Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and
David Lane (1988), can be performed.
[0075] A "species-dependent antibody" is one which has a stronger
binding affinity for an antigen from a first mammalian species than
it has for a homologue of that antigen from a second mammalian
species. Normally, the species-dependent antibody "binds
specifically" to a human antigen (i.e., has a binding affinity
(K.sub.D) value of no more than about 1.times.10.sup.-7 M,
preferably no more than about 1.times.10.sup.-8 M and most
preferably no more than about 1.times.10.sup.-9 M) but has a
binding affinity for a homologue of the antigen from a second
nonhuman mammalian species which is at least about 50 fold, or at
least about 500 fold, or at least about 1000 fold, weaker than its
binding affinity for the human antigen. The species-dependent
antibody can be any of the various types of antibodies as defined
above, but typically is a humanized or human antibody.
[0076] As used herein, "antibody mutant" or "antibody variant"
refers to an amino acid sequence variant of the species-dependent
antibody wherein one or more of the amino acid residues of the
species-dependent antibody have been modified. Such mutants
necessarily have less than 100% sequence identity or similarity
with the species-dependent antibody. In one embodiment, the
antibody mutant will have an amino acid sequence having at least
75% amino acid sequence identity or similarity with the amino acid
sequence of either the heavy or light chain variable domain of the
species-dependent antibody, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, and most
preferably at least 95%. Identity or similarity with respect to
this sequence is defined herein as the percentage of amino acid
residues in the candidate sequence that are identical (i.e., same
residue) or similar (i.e., amino acid residue from the same group
based on common side-chain properties, see below) with the
species-dependent antibody residues, after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity. None of N-terminal, C-terminal, or internal
extensions, deletions, or insertions into the antibody sequence
outside of the variable domain shall be construed as affecting
sequence identity or similarity.
[0077] To increase the half-life of the antibodies or polypeptide
containing the amino acid sequences described herein, one can
attach a salvage receptor binding epitope to the antibody
(especially an antibody fragment), as described, e.g., in U.S. Pat.
No. 5,739,277. For example, a nucleic acid molecule encoding the
salvage receptor binding epitope can be linked in frame to a
nucleic acid encoding a polypeptide sequence described herein so
that the fusion protein expressed by the engineered nucleic acid
molecule comprises the salvage receptor binding epitope and a
polypeptide sequence described herein. As used herein, the term
"salvage receptor binding epitope" refers to an epitope of the Fc
region of an IgG molecule (e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3,
or IgG.sub.4) that is responsible for increasing the in vivo serum
half-life of the IgG molecule (e.g., Ghetie et al., Ann. Rev.
Immunol. 18:739-766 (2000), Table 1). Antibodies with substitutions
in an Fc region thereof and increased serum half-lives are also
described in WO00/42072, WO 02/060919; Shields et al., J. Biol.
Chem. 276:6591-6604 (2001); Hinton, J. Biol. Chem. 279:6213-6216
(2004)). In another embodiment, the serum half-life can also be
increased, for example, by attaching other polypeptide sequences.
For example, antibodies or other polypeptides useful in the methods
of the invention can be attached to serum albumin or a portion of
serum albumin that binds to the FcRn receptor or a serum albumin
binding peptide so that serum albumin binds to the antibody or
polypeptide, e.g., such polypeptide sequences are disclosed in
WO01/45746. In one embodiment, the serum albumin peptide to be
attached comprises an amino acid sequence of DICLPRWGCLW (SEQ ID
NO:3). In another embodiment, the half-life of a Fab is increased
by these methods. See also, Dennis et al. J. Biol. Chem.
277:35035-35043 (2002) for additional serum albumin binding peptide
sequences.
[0078] A "chimeric DEspR receptor protein" is a DEspR molecule
having amino acid sequences derived from at least two different
proteins, at least one of which is a DEspR protein. In certain
embodiments, the chimeric DEspR protein is capable of binding to
and inhibiting the biological activity of DEspR.
[0079] An "isolated" antibody is one that has been identified and
separated and/or recovered from a component of its natural
environment. Contaminant components of its natural environment are
materials that would interfere with diagnostic or therapeutic uses
for the antibody, and can include enzymes, hormones, and other
proteinaceous or nonproteinaceous solutes. In certain embodiments,
the antibody will be purified (1) to greater than 95% by weight of
antibody as determined by, for example, the Lowry method, and most
preferably more than 99% by weight, (2) to a degree sufficient to
obtain at least 15 residues of N-terminal or internal amino acid
sequence by use of a spinning cup sequenator, or (3) to homogeneity
by SDS-PAGE under reducing or nonreducing conditions using
Coomassie blue or, silver stain. Isolated antibody includes the
antibody in situ within recombinant cells since at least one
component of the antibody's natural environment will not be
present. Ordinarily, however, isolated antibody will be prepared by
at least one purification step.
[0080] By "fragment" is meant a portion of a polypeptide, such as
an antibody or antibody fragment thereof, or nucleic acid molecule
that contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, or more of the entire length of the reference
nucleic acid molecule or polypeptide. A fragment can contain 10,
20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, or
more nucleotides or 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120,
140, 160, 180, 190, 200 amino acids or more.
[0081] An "anti-angiogenesis agent" or "angiogenesis inhibitor"
refers to a small molecular weight substance, a polynucleotide, a
polypeptide, an isolated protein, a recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or undesirable vascular permeability,
either directly or indirectly. It should be understood that the
anti-angiogenesis agent includes those agents that bind and block
the angiogenic activity of the angiogenic factor or its receptor.
For example, an anti-angiogenesis agent is an antibody or other
antagonist to an angiogenic agent as defined throughout the
specification or known in the art, e.g., but are not limited to,
antibodies to VEGF-A or to the VEGF-A receptor (e.g., KDR receptor
or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such as
Gleevec.TM. (Imatinib Mesylate). Anti-angiogensis agents also
include native angiogenesis inhibitors, e.g., angiostatin,
endostatin, etc. See, e.g., Klagsbrun and D'Amore, Annu. Rev.
Physiol., 53:217-39 (1991); Streit and Detmar, Oncogene,
22:3172-3179 (2003) (e.g., Table 3 listing anti-angiogenic therapy
in malignant melanoma); Ferrara & Alitalo, Nature Medicine
5:1359-1364 (1999); Tonini et al., Oncogene, 22:6549-6556 (2003)
(e.g., Table 2 listing known antiangiogenic factors); and Sato.
Int. J. Clin. Oncol., 8:200-206 (2003) (e.g., Table 1 lists
anti-angiogenic agents used in clinical trials).
[0082] The term "anti-cancer therapy" refers to a therapy useful in
treating cancer. Examples of anti-cancer therapeutic agents
include, but are not limited to, e.g., surgery, chemotherapeutic
agents, growth inhibitory agents, cytotoxic agents, agents used in
radiation therapy, anti-angiogenesis agents, apoptotic agents,
anti-tubulin agents, and other agents to treat cancer, such as
anti-HER-2 antibodies (e.g., Herceptin.RTM.), anti-CD20 antibodies,
an epidermal growth factor receptor (EGFR) antagonist (e.g., a
tyrosine kinase inhibitor), HER1/EGFR inhibitor (e.g., erlotinib
(Tarceva.RTM.)), platelet derived growth factor inhibitors (e.g.,
Gleevec.TM. (Imatinib Mesylate)), a COX-2 inhibitor (e.g.,
celecoxib), interferons, cytokines, antagonists (e.g., neutralizing
antibodies) that bind to one or more of the following targets
ErbB2, ErbB3, ErbB4, PDGFR-beta, BlyS, APRIL, BCMA or VEGF
receptor(s), TRAIL/Apo2, and other bioactive and organic chemical
agents, etc. Combinations thereof are also included in the
invention.
[0083] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents the function of cells and/or
causes destruction of cells. The term is intended to include
radioactive isotopes (e.g. At.sup.211, I.sup.131, I.sup.125,
Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153, Bi.sup.212, P.sup.32
and radioactive isotopes of Lu), chemotherapeutic agents, and
toxins such as small molecule toxins or enzymatically active toxins
of bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof.
[0084] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include, but are not limited to, alkylating agents such as thiotepa
and CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gamma1I and calicheamicin omegall (see, e.g., Agnew,
Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elformithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofuran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., TAXOL.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), ABRAXANE.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
GEMZAR.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); lapatinib (Tykerb.RTM.); inhibitors of PKC-alpha,
Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva.RTM.)) and VEGF-A that
reduce cell proliferation and pharmaceutically acceptable salts,
acids or derivatives of any of the above.
[0085] Also included in this definition are anti-hormonal agents
that act to regulate or inhibit hormone action on tumors such as
anti-estrogens and selective estrogen receptor modulators (SERMs),
including, for example, tamoxifen (including NOLVADEX.RTM.
tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen,
trioxifene, keoxifene, LY117018, onapristone, and FARESTON
toremifene; aromatase inhibitors that inhibit the enzyme aromatase,
which regulates estrogen production in the adrenal glands, such as,
for example, 4(5)-imidazoles, aminoglutethimide, MEGASE.RTM.
megestrol acetate, AROMASIN.RTM. exemestane, formestanie,
fadrozole, RIVISOR.RTM. vorozole, FEMARA.RTM. letrozole, and
ARIMIDEX.RTM. anastrozole; and anti-androgens such as flutamide,
nilutamide, bicalutamide, leuprolide, and goserelin; as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog);
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in abherant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; ribozymes such as a VEGF expression inhibitor (e.g.,
ANGIOZYME.RTM. ribozyme) and a HER2 expression inhibitor; vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.
vaccine, LEUVECTIN.RTM. vaccine, and VAXID.RTM. vaccine;
PROLEUKIN.RTM. rIL-2; LURTOTECAN.RTM. topoisomerase 1 inhibitor;
ABARELIX.RTM. rmRH; and pharmaceutically acceptable salts, acids or
derivatives of any of the above.
[0086] A "growth inhibitory agent" as used herein refers to a
compound or composition which inhibits growth of a cell in vitro
and/or in vivo. Thus, the growth inhibitory agent can be one which
significantly reduces the percentage of cells in S phase. Examples
of growth inhibitory agents include agents that block cell cycle
progression (at a place other than S phase), such as agents that
induce G1 arrest and M-phase arrest. Classical M-phase blockers
include the vincas (vincristine and vinblastine), TAXOL.RTM., and
topo II inhibitors such as doxorubicin, epirubicin, daunorubicin,
etoposide, and bleomycin. Those agents that arrest G1 also spill
over into S-phase arrest, for example, DNA alkylating agents such
as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,
methotrexate, 5-fluorouracil, and ara-C. Further information can be
found in The Molecular Basis of Cancer, Mendelsohn and Israel,
eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and
antineoplastic drugs" by Murakami et al. (WB Saunders:
Philadelphia, 1995), especially p. 13.
[0087] The term "prodrug" as used in this application refers to a
precursor or derivative form of a pharmaceutically active substance
that is less cytotoxic to tumor cells compared to the parent drug
and is capable of being enzymatically activated or converted into
the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer
Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382,
615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A
Chemical Approach to Targeted Drug Delivery," Directed Drug
Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press
(1985). The prodrugs described herein include, but are not limited
to, phosphate-containing prodrugs, thiophosphate-containing
prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,
D-amino acid-modified prodrugs, glycosylated prodrugs,
.beta.-lactam-containing prodrugs, optionally substituted
phenoxyacetamide-containing prodrugs or optionally substituted
phenylacetamide-containing prodrugs, 5-fluorocytosine and other
5-fluorouridine prodrugs which can be converted into the more
active cytotoxic free drug. Examples of cytotoxic drugs that can be
derivatized into a prodrug form for use in this invention include,
but are not limited to, those chemotherapeutic agents described
above.
[0088] By "radiation therapy" is meant the use of directed gamma
rays or beta rays to induce sufficient damage to a cell so as to
limit its ability to function normally or to destroy the cell
altogether. It will be appreciated that there will be many ways
known in the art to determine the dosage and duration of treatment.
Typical treatments are given as a one time administration and
typical dosages range from 10 to 200 units (Grays) per day.
[0089] By "reduce or inhibit" is meant the ability to cause an
overall decrease preferably of 20% or greater, 30% or greater, 40%
or greater, 45% or greater, more preferably of 50% or greater, of
55% or greater, of 60% or greater, of 65% or greater, of 70% or
greater, and most preferably of 75%, 80%, 85%, 90%, 95%, or
greater. Reduce or inhibit can refer to, for example, the symptoms
of the disorder being treated, the presence or size of metastases
or micrometastases, the size of the primary tumor, the presence or
the size of the dormant tumor, or the size or number of the blood
vessels in angiogenic disorders.
[0090] The term "intravenous infusion" refers to introduction of a
drug into the vein of an animal or human subject over a period of
time greater than approximately 5 minutes, preferably between
approximately 30 to 90 minutes, although, according to the
invention, intravenous infusion is alternatively administered for
10 hours or less. The term "intravenous bolus" or "intravenous
push" refers to drug administration into a vein of an animal or
human such that the body receives the drug in approximately 15
minutes or less, preferably 5 minutes or less.
[0091] The term "subcutaneous administration" refers to
introduction of a drug under the skin of an animal or human
subject, preferable within a pocket between the skin and underlying
tissue, by relatively slow, sustained delivery from a drug
receptacle. The pocket can be created by pinching or drawing the
skin up and away from underlying tissue.
[0092] The term "subcutaneous infusion" refers to introduction of a
drug under the skin of an animal or human subject, preferably
within a pocket between the skin and underlying tissue, by
relatively slow, sustained delivery from a drug receptacle for a
period of time including, but not limited to, 30 minutes or less,
or 90 minutes or less. Optionally, the infusion can be made by
subcutaneous implantation of a drug delivery pump implanted under
the skin of the animal or human subject, wherein the pump delivers
a predetermined amount of drug for a predetermined period of time,
such as 30 minutes, 90 minutes, or a time period spanning the
length of the treatment regimen.
[0093] The term "subcutaneous bolus" refers to drug administration
beneath the skin of an animal or human subject, where bolus drug
delivery is preferably less than approximately 15 minutes, more
preferably less than 5 minutes, and most preferably less than 60
seconds. Administration is preferably within a pocket between the
skin and underlying tissue, where the pocket is created, for
example, by pinching or drawing the skin up and away from
underlying tissue.
[0094] A "disorder" is any condition that would benefit from
treatment with, for example, an antibody described herein. This
includes chronic and acute disorders or diseases including those
pathological conditions which predispose the mammal to the disorder
in question. Non-limiting examples of disorders to be treated
herein include cancer; benign and malignant tumors; leukemias and
lymphoid malignancies; neuronal, glial, astrocytal, hypothalamic
and other glandular, macrophagal, epithelial, stromal and
blastocoelic disorders; and inflammatory, angiogenic and
immunologic disorders.
[0095] The word "label" when used herein refers to a detectable
compound or composition which is conjugated directly or indirectly
to the polypeptide. The label can be itself be detectable (e.g.,
radioisotope labels or fluorescent labels) or, in the case of an
enzymatic label, can catalyze chemical alteration of a substrate
compound or composition which is detectable.
[0096] By "subject" is meant a mammal, including, but not limited
to, a human or non-human mammal, such as a bovine, equine, canine,
ovine, or feline. Preferably, the subject is a human. Patients are
also subjects herein.
BRIEF DESCRIPTION OF THE FIGURES
[0097] FIG. 1 shows that DEspR is a key angiogenesis player in
embryonic development using DEspR (formerly called Dear, deposited
in Gen Bank as Dear) null or knockout (Dear.sup.-/-) mice.
[0098] FIG. 2 shows that DEspR contributes to adult tissue
vascularity as seen in adult haplo-deficient (+/-) mice exhibiting
decreased tissue vascularity using power Doppler analysis.
[0099] FIGS. 3A-3E show that DEspR and VEGFsp are detected by
immunostaining in umbilical vein endothelial cells (HUVECs) (FIGS.
3A-3C) and microvascular endothelial cells (HMECS) under both basal
and angiogenic tube-formation conditions. Importantly, inhibition
of angiogenesis neovessel tube length is seen using both anti-DEspR
(Ab1) and anti-VEGFsp (Ab2) antibodies in HUVECs (FIG. 3D) and
HMECs (FIG. 3E) angiogenesis assays. (Tukey's all pairwise multiple
comparison P<0.001 for both HUVECs and HMECs). Similar findings
were observed for other angiogenesis parameters including neovessel
branching and inter-connections made.
[0100] FIGS. 4A-4D demonstrate that DEspR and VEGFsp are also
detected in tumor cells, with colocalization of VEGFsp and DEspR in
the cell membrane and nuclear membrane using immunostaining. DEspR
cell-membrane and nuclear-membrane expression are detected in
multiple tumor cell types, indicating that anti-DEspR therapy is
effective for different cancer types. DEspR expression is detected
in human lung non-small cell ca NCI-H727, lung giant cell tumor
TIB-223/GCT; breast adenoca MDA-MB-231 (FIGS. 4A-4C) and
MDA-MB-468, bladder ca 253J BV, colon adenoca SW480, hepatocellular
ca, HEP3B, melanoma SK-MEL-2, osteosarcoma MG-63, ovarian adenoca
HTB-161/NIH:OVCA R3, prostate adeno ca PC-3 mm2, and pancreatic ca
CRL-1469/PANC-1 (FIG. 4D).
[0101] FIGS. 5A-5C show that DEspR expression was not detected in
HCI-H292 lung mucoepidermoid ca, and HEPG2 hepatocellular ca (FIG.
5A), and CCL-86/Raji Burkitt's lymphoma, thus showing specificity
of positive observations. Findings in NCI-727 lung ca cells (FIG.
5B) are corroborated on tumor-section immunostaining of Gr.III lung
adenoca (FIG. 5C).
[0102] FIGS. 6A-6B show that in contrast to control (C) and
pre-immune ab treatment (PI), DEspR-inhibition via anti-human DEspR
antibody treatment inhibits tumor cell invasiveness in two cell
lines tested, metastatic breast tumor MDA-MB-231 and pancreatic
adenocarcinoma PANC-1 cell lines.
[0103] FIG. 7 shows that anti-DEspR treated rats (.quadrature.)
exhibited minimal tumor growth compared with mock-treated controls
(.box-solid.), two-tailed t-test *P<0.05; **P<0.001.
[0104] FIGS. 8A-8D show, using immunohistochemical analysis, that
DEspR is expressed in mammary tumors cells (FIG. 8A) similar to
MDA-MB-231 breast cancer cells, with no expression in normal breast
tissue (FIG. 8B). In addition, residual tumors in treated rats
exhibited normalization of blood vessels (FIG. 8C) in contrast to
mock-treated tumors which showed disrupted endothelium in tumor
vessels with encroachment of tumor cells into the lumen (FIG.
8D).
[0105] FIG. 9 shows characterization of selected monoclonal
antibodies. Monoclonal antibodies 2E4A8, 2E4B11, 2E4H10, 5G12E8,
7C5B2, 7C5C5, 8E7D11, 8E2F6, E2G4 and 8E7F8 were tested by indirect
ELISA using standard procedures. Serial dilutions from supernatants
containing monoclonal antibodies at 1 .mu.g/ml were tested as
follows: 1=1/2; 2=1/4; 3=1/8; 4=1/16; 5=1/32; 6=1/64; 7=1/128;
8=1/256; 9=1/512; 10=1/1024; 11=1/2048 and 12=1/4096.
[0106] FIG. 10 shows Western blot analysis of monoclonal antibodies
tested. To ascertain specificity, low- (5G12E8), mid- (2E4H6), and
high-affinity (7C5B2) monoclonal antibodies were tested as well as
the subclone supernatant, and the subsequent purified antibody. The
anti-human DEspR monoclonal antibodies are specific for the
predicted 10 kD protein for human DEspR. Western blot analysis was
performed using total cellular protein isolated from Cos1 human
DEspR-transfected cells as antigen, primary antibody comprised
purified antibody and subclone supernatant of 3 selected clones,
10% gel concentration in order to detect the expected 10 kD
molecular weight protein of human DEspR. Nitrocellulose (PIERCE)
with a transfer buffer of 3.07 g Tris, 14.4 g Glycine, 200 ml
methanol, 800 ml dH.sub.2O were used. HRP-anti mouse polyvalent
immunoglobulins (Sigma #0412) were used at 1:100,000; ECL reagent
(SuperSignal West Femto Kit #34094), Stain reagent Kodak RP-X-Omat,
and x-film (Kodak X-film #XBT-1). The Western blot results
demonstrate specificity of anti-human DEspR monoclonal antibodies
regardless of relative affinity, thus identifying more than one
successful anti-human DEspR monoclonal antibody. The results
indicate that the monoclonal antibody clone with highest relative
affinity and specificity is clone 7C5B2.
[0107] FIGS. 11A-11C show inhibition of different parameters of
angiogenesis by monoclonal antibody 7C5B2 and a polyclonal antibody
preparation to DEspR. 7C5B2 monoclonal antibody was shown to
immunostain HUVECs undergoing tube formation, pancreatic adenoca
PANC-1, and breast cancer MDA-MB-231 cells. FIG. 11A shows mean
number of branchpoints as a measure of neovessel complexity, and
total length of tubes as a measure of neovessel density is shown in
FIG. 11B. FIG. 11C shows concentration-dependent inhibition of in
vitro serum-induced HUVEC tubulogenesis by monoclonal antibody
7C5B2. HUVEC (human umbilical vein endothelial cells) were grown
onto Matrigel-coated wells in basal medium supplemented with 2% FBS
(control), or 2% FBS+monoclonal antibody 7C5B2 (0.05-500 nM). The
percentage of serum-induced tubulogenesis was determined as the
difference between HUVECs grown in control conditions and the
indicated monoclonal antibody 7C5B2-supplemented media. The % of
the total tube length per well and the total number of branching
points per well in the in vitro tube formation assay is presented.
Data are shown as mean.+-.standard error. Each experimental
condition was performed in five replica wells. EC.sub.50 for total
tube length=4.34.+-.0.45 nM; EC.sub.50 for # branching
points=3.97.+-.0.51 nM.
[0108] FIGS. 12A-12C demonstrate that a monoclonal antibody 7C5B2
inhibits tumor cell invasiveness in MDA-MB-231 human breast cancer
(FIG. 12A) and PANC-1 pancreatic cancer (FIG. 12B) cell lines
(P<0.001*, <0.01*). FIG. 12C shows dose response curve of
inhibition of MDA-MB-231 cell invasion by monoclonal antibody 7C5B2
(EC.sub.50=3.55.+-.0.32 nM). Data, mean.+-.standard error of 5
replicates. *P<0.001,**P<0.01 (one way ANOVA, all pairwise
multiple comparison Tukey's Test).
[0109] FIGS. 13A-13D show effects of an anti-human DEspR monoclonal
antibody 7C5B2 (IgG2b isotype) on in vitro serum-induced HUVEC
tubulogenesis (established in vitro angiogenesis assay). HUVECs
(human umbilical vein endothelial cells) were grown onto
Matrigel-coated wells in basal medium supplemented with 2% FBS
(control C1), or 2% FBS+pre-immune IgG isotype control for
polyclonal anti-hDESPR antibody (500 nM, control C2), or 2%
FBS+IgG2b isotype control for anti-hDESPR mAB (500 nM, C3 control)
or 2% FBS+polyclonal anti-hDEspR (500 nM, P) or 2% FBS+monoclonal
antibody 7C5B2 (500 nM, M). Quantitative analysis of the mean
number of tubes formed per well is shown in FIG. 13A, the mean
number of branching points per well is shown in FIG. 13B, the mean
number of connections per well is shown in FIG. 13C and the mean
total tube length in mm per well is shown in FIG. 13D, using the in
vitro tube formation assay. Data are shown as mean.+-.standard
error. Each experimental condition was performed in five replica
wells. Statistically significant differences (as compared with
respective control conditions), are indicated as follows:
*P<0.001 (one way ANOVA followed by all pairwise multiple
comparison Tukey Test).
[0110] FIGS. 14A-14B show immunohistochemical analysis of human
tumor tissue-arrays comprised of core biopsy specimens representing
tumors and normal tissue on the same slide using an anti-human
DEspR 7C5B2 monoclonal antibody. Conditions that optimized
specificity and sensitivity of detection were first tested using
formalin-fixed, paraffin embedded core biopsy sections.
Double-immunofluorescence experiments were performed to evaluate
human DEspR expression and CD133 expression, with the latter
serving as a marker for cancer stem cells. Antigen-retrieval was
performed using anti-human DEspR monoclonal antibody at 1:10, and
commercially available anti-CD133 monoclonal antibody at 1:20
dilutions. Representative immunohistochemical analysis of human
tumor tissue-arrays using anti-human DEspR 7C5B2 monoclonal
antibody detected increased expression of hDEspR (Alexa-568red) in
stage II-lung cancer tumor cells, as shown in FIG. 14A. Some tumor
cells were immunostained double-positive for both human DEspR and
CD133, while other tumor cells immunostained only for CD133. These
observations demonstrate that human DEspR is also present in
CD133-positive cancer stem cells, as well as CD133-negative tumor
cells. As shown in FIG. 14B, in contrast, a normal lung specimen
does not exhibit any immunostaining for human DEspR or CD133.
[0111] FIGS. 15A-15B show that there is minimal DEspR expression in
normal human pancreas (FIG. 15B), with .alpha.-smooth muscle actin
serving as positive control, while, in contrast, stage IV
pancreatic cancer tumor cells and tumor blood vessels exhibits
increased DEspR expression (FIG. 15A).
[0112] FIGS. 16A-16D demonstrate DEspR-targeted ultrasound
molecular imaging and show that a DEspR-specific antibody (FIG.
16A) detects DEspR+ endothelial lesions (FIG. 16B) and vasa vasorum
angiogenesis (FIG. 16C). Quantitation of contrast intensity is done
using integrated VisualSonics Micro-imaging System software (FIG.
16D) and demonstrates increased contrast intensity in DEspR+
carotid artery endothelium and vasa vasorum, in contrast to both
low contrast intensity in DEspR(-) endothelium and vasa vasorum,
and isotype-microbubble controls. P<0.0001, ANOVA and pairwise
multiple comparison. Anti-DEspR-antibody is biotinylated and
coupled to streptavidin-PEG coated commercially available
microbubbles for ultrasound analysis and imaging.
[0113] FIGS. 17A-17F show immunohistochemical analysis of DEspR
expression in human breast tissue using an anti-DEspR monoclonal
antibody (FIGS. 17A-17C) normal; Grade-1, T1 invasive ductal
carcinoma (FIGS. 17D-17F). FIG. 17A shows normal breast tissue:
3.times.-overlay of DEspR, aSMA and DAPI nuclear stain detects aSMA
expression in mammary myoepithelial cells but no expression of
DEspR in epithelial cells and microvessels. FIG. 17B shows
2.times.-immunofluorescence overlay of DEspR and DAPI nuclear stain
and confirms absence of DEspR expression in normal breast tissue.
FIG. 17C is a 4.times.-overlay of DEspR, aSMA, DAPI
immunofluorescence and diffusion contrast imaging (DIC) that
delineates tissue morphology, expression of aSMA. and
non/minimal-expression of DEspR in normal mammary epithelium and
endothelium. FIG. 17D is a 3.times.-Overlay of DAPI, aSMA and DEspR
immunofluorescence in Gr.I-T1 invasive ductal carcinoma that
detects DEspR expression in vascular endothelium, and
co-localization with aSMA in mammary tissue. FIG. 17E is a
2.times.-overlay of DAPI and DEspR of breast cancer shown in panel
17D that highlights DEspR expression. FIG. 17F is a
4.times.-overlay of DAPI, aSMA, DEspR, DIC to elucidate DEspR
spatial expression with tissue morphology of epithelial cells and
microvessels. bar=20 microns.
[0114] FIGS. 18A-18F show monoclonal antibody immunohistochemical
analysis of DEspR expression in normal pancreatic tissue (FIGS.
18A-18C) normal; and Grade-3, T3 pancreatic ductal carcinoma (FIGS.
18D-18F). FIG. 18A shows that normal pancreatic tissue, with a
3.times.-overlay of DEspR, aSMA and DAPI nuclear stain, detects
minimal DEspR expression in microvessels. FIG. 18B shows a
4.times.-immunofluorescence overlay of DEspR, aSMA, DAPI, with DIC
imaging of tissue morphology. FIG. 18C (left) shows a
3.times.-overlay of DEspR, aSMA, DAPI immunofluorescence; (right)
shows a 4.times.-overlay of DEspR, aSMA, DAPI and diffusion
contrast imaging (DIC) for tissue morphology that shows aSMA
expression and non/minimal-expression of DEspR in normal
endothelium. FIG. 18D shows that 3.times.-overlay of DAPI, aSMA and
DEspR immunofluorescence in Gr.3-T3 pancreatic ductal carcinoma
detects DEspR expression in vascular endothelium, and
co-localization with aSMA. FIG. 18E shows a 2.times.-overlay of
DAPI and DEspR of the image shown in FIG. 19D and highlights DEspR
expression. FIG. 18F shows a 3.times.-overlay of DAPI, aSMA, DEspR,
that shows increased DEspR expression in pancreatic ductal
carcinoma cells. bar=20 microns.
[0115] FIGS. 19A-19E show representative contrast enhanced
ultrasound (CEU)-images with contrast intensity signals (CIS)
depicted. FIG. 19A shows MB.sub.D DEspR-targeted molecular imaging
in transgenic rat-R1 demonstrating CEU-positive imaging and the
characteristic drop in CIS-peak after acoustic disruption (|). FIG.
19B shows subsequent isotype-microbubble (MB.sub.C) imaging in
transgenic rat-R1 showing low peak CIS-levels and `flat-line
pattern of CIS pre- and post-destruction indicating CIU-negative
imaging. FIG. 19C shows MB.sub.D DEspR-targeted molecular imaging
in non-transgenic rat-R2 demonstrating CEU-negative imaging similar
to MB.sub.C CEU-negative imaging.
[0116] FIG. 19D shows a graph of CIS-differences (A) among
different study groups as notated distinguishing CEU-positive
imaging in Tg MB.sub.D CEU+ group from the other CEU-negative
groups.
[0117] FIG. 20E shows a graph of CIS-difference between all
transgenic rats (Tg+) and non-transgenic rats (nonTg). Hatched bar
represents a threshold between MB.sub.D-infused CEU+ and
MB.sub.D-infused CEU-transgenic rats. Blood pool, CEU-image 1
minute after bolus injection of MBs, demonstrating equivalent
MB-infusion among different rats and minimal contrast-intensity
signals from movement artifacts. 1-Pre, pre-acoustic destruction
CEU-images obtained 4-minutes after bolus infusion, in order to
allow MB-adherence to target, if any, and to document minimal, if
any, circulating MBs in the lumen. Image corresponds to #1 on
CIS-plot. 2-Post, CEU-image after acoustic destruction
corresponding to #2 on scatter plot. CIS-plot, scatter plot of
contrast-intensity signals (CIS) in representative regions of
interest (encircled in aqua). #1, CIS detected pre-acoustic
destruction; #2, CIS detected post-acoustic destruction (2). Black
line and following gap mark period of acoustic destruction in
CIS-scatter plots. MB.sub.D, DEspR-targeted microbubble; MB.sub.C,
control isotype-targeted microbubble; Tg, transgenic rat; nonTg,
nontransgenic control rat; CEU+, CEU positive imaging; CEU-, CEU
negative imaging, .DELTA. Contrast Intensity, pre-/post-destruction
CIS-difference; ***, P<0.0001.
[0118] FIGS. 20A-20H depict representative MB.sub.D-specific
contrast enhanced ultrasound (CEU)-positive images depicting
complex pattern of acoustic destruction of adherent
MB.sub.D-microbubbles in a transgenic rat, R3. FIG. 20A shows
representative CEU-image documenting blood pool of circulating
MB.sub.Ds filling carotid artery lumen one-minute after bolus
infusion. CCA, common carotid artery; ECA, external carotid artery;
ICA, internal carotid artery; *, CCA bifurcation. FIGS. 20B-20D
show scatter plots of contrast-intensity signals marked with
same-dashed blocks to refer to corresponding regions of interest
(ROI) in panel-20E. (20B) white solid line; (20C), white hatched
line; (20D) white dotted line ROIs. FIG. 20E shows representative
CEU-image that corresponds to #1 on scatter plots b,c,d documenting
adherent DEspR-targeted microbubbles (MB.sub.D) just prior to
pre-acoustic destruction (black line). Adherent MB.sub.Ds are seen
in the three ROIs encircled white solid line, white hatched line,
and white dotted line. FIG. 20F shows representative CEU-image
corresponding to #2 on scatter plots b-d showing a post-acoustic
destruction dip in signal intensity compared to levels in #1 in the
different ROIs respectively. FIG. 20G shows representative
CEU-image corresponding to #3 on scatter plots b-d showing a
post-acoustic destruction secondary peak in contrast intensity
signals in the different ROIs. FIG. 20H shows representative
CEU-image corresponding to #4 on scatter plots documenting the
decline in contrast-intensity signals approaching baseline levels
observed in isotype control or MB.sub.D-infused CEU-negative images
and demonstrating low background CIS levels.
[0119] FIGS. 21A-21H depict representative histological and
fluorescence immunostaining analysis of carotid arteries with
DEspR-positive molecular imaging corresponding to rat-R1 (panels
21A-21D), and rat-R3 in (panels 21E-21H). FIG. 21A shows Masson
trichrome stained section of carotid artery endothelium. FIGS.
21B-212C show differential interference contrast (DIC) image
overlaid with fluorescence immunostaining for DEspR expression and
DAPI nuclear stain. FIG. 21D shows control isotype-ab
immunostaining and DAPI nuclear stain overlaid with DIC image of
endothelium. FIG. 21E shows carotid artery Masson trichrome-stained
section showing increased adventitial vasa vasorum neovessels.
Boxed area is shown in higher magnification in FIG. 21F documenting
rbc-filled vasa vasorum. FIG. 21G shows fluorescence immunostaining
detects DEspR-positive expression in vasa vasorum and surrounding
cells. FIG. 21H shows double immunostaining with .alpha.-SMA and
DEspR detects .alpha.SMA co-expression in DEspR-positive
neovessels. .fwdarw., adherent DEspR-targeted microbubble MB.sub.D;
white arrowheads point to vasa vasorum neovessel in panels 21G and
21H; m, media; bar=10-microns panels 21A-21D, 21F; 20 microns
panels 21E, 21G, 21H.
[0120] FIGS. 22A-22E depict representative fluorescence
immunostaining analysis of carotid arteries from rats exhibiting
MB.sub.D-specific CEU-positive imaging (22B, 22C) and CEU-negative
imaging (22D, 22E). FIG. 22A shows scatter dot plot of
pre-destruction CIS-peak levels highlighting a threshold (hatched
bar) between MB.sub.D-specific CEU-positive (CEU+) and CEU-negative
(CEU-) imaging. FIG. 22B shows DEspR-positive immunostaining of
carotid artery endothelium and expanded vasa vasorum;
.alpha.SMA-positive immunostaining in smooth muscle cells (SMCs) in
the media. Some vasa vasorum neovessels are double-immunostained
for DEspR and .alpha.SMA. FIG. 22C shows corresponding DIC-image
shows structural layers of carotid artery and vasa vasorum. FIG.
22D shows representative minimal to no DEspR-expression in rat
carotid artery exhibiting CEU-negative imaging (shown here, nonTg
rat-R2). Similar images obtained for CEU-negative transgenic rat
carotid arteries. .alpha.SMA-immunostaining detects expression in
SMCs in the media. Low levels of .alpha.SMA-immunostaining in the
medial indicates synthetic SMC phenotype in both carotid arteries,
consistent with hypertensive remodeling. FIG. 22D shows
corresponding DIC-image shows structural layers of carotid artery
and adventitia with no vasa vasorum expansion. Bar=20 microns (22B,
22C), 10 microns (22D, 22E). m, media; adv, adventitia; white small
arrow, endothelium; white large arrow, vasa vasorum.
[0121] FIGS. 23A-23G depicts phase contrast-fluorescence microscopy
analysis of anti-humanDEspR-targeted microbubbles (MB.sub.D)
binding to human endothelial cells, HUVECs, in vitro. Increasing
DEspR-targeted microbubbles (MB.sub.D) to cell ratio (23A)
8.times., (23B) 80.times., and (23C) 800.times.. (23D) Isotype
control (MB.sub.C) at 800.times.; (23E) non-targeted control
MB.degree. at 800.times.. (23F) % of HUVECs with bound MBs
(.box-solid.) and no MB binding (.quadrature.). FIG. 23G shows
number of MBs (mean+/-sem) per bound cell with increasing MB to
cell ratio: MB.sub.D compared with isotype control MB.sub.C and
control non-targeted MB.degree.. ***, ANOVA P<0.0001.
[0122] FIGS. 24A-24F show DEspR expression in liver (24A-24C) and
pancreas (24D-24F) non-cancerous and cancerous tissues. (24A)
Adjacent normal liver tissue; (24B, 24C) hepatic carcinoma T-2 from
two patients; (24D), adjacent normal pancreatic tissue; (24E, 24F)
pancreatic ductal carcinoma Grade III-IV from two patients. Black
arrow, microvessels; DAB-detection of DEspR-positive immunostaining
with color-intensity roughly proportional to expression;
hematoxylin nuclear counterstain. Bar, 20 microns.
[0123] FIGS. 25A-25F show DEspR expression in a human tissue array:
stomach (25A-25C) and breast (25D-25F) non-cancerous and cancerous
tissues. (25A) Adjacent normal stomach tissue; (25B) stomach
adenocarcinoma T-3, (25C) stomach adenocarcinoma metastasis to
lung; (25D) adjacent normal breast tissue with fibrosis; (25E)
breast medullary carcinoma T-2; (25F) breast tumor metastasis to
lymphnode. Black arrow, vascular endothelium; DAB-detection of
DEspR-positive immunostaining with color-intensity roughly
proportional to expression; hematoxylin nuclear counterstain. Bar,
20 microns.
[0124] FIGS. 26A-26F show DEspR expression in lung and colon
non-cancerous and cancerous tissue. (26A) adjacent normal lung;
(26B) Gr-I lung adenocarcinoma; (26C), Gr.III,T2 lung
adenocarcinoma; (26D) adjacent normal colon; (26E, 26F) colon
adenocarcinoma Gr.III-IV, T2. white arrow, endothelium; black arrow
.fwdarw., DEspR-immunostaining of nuclear membrane in cancer cells
DAB-detection of DEspR-immunostaining with color-intensity roughly
proportional to expression; hematoxylin nuclear counterstain. Bar,
20 microns 26A-26C; 25 microns 26D; 10 microns 26E, 26F.
[0125] FIGS. 27A-27F show DEspR expression in different tissue-type
cancer cell lines. (27A) non-small cell lung cancer cell line,
#NCI-H727; (27B) colon carcinoma, SW480 Duke's type B; (27C)
pancreatic carcinoma, PANC-1; (27D) breast adenocarcinoma
metastasis, MDA-MB-231; (27E) bladder carcinoma 253J BV; (27F)
prostate adenocarcinoma PC-3 mm2. .fwdarw., DEspR-immunostaining of
nuclear membrane in cancer cells, DAB-detection of
DEspR-immunostaining with color-intensity roughly proportional to
expression; hematoxylin nuclear counterstain. Bar, 20 microns
A-F.
[0126] FIGS. 28A-28B show characterization of a human-specific
anti-DEspR monoclonal antibody. (28A) Analysis by indirect ELISA of
10 candidate monoclonal antibody clones is shown. Serial dilutions
from supernatants containing mAbs at 1 .mu.g/ml were tested as
follows: 1=1/2; 2=1/4; 3=1/8; 4=1/16; 5=1/32; 6=1/64; 7=1/128;
8=1/256; 9=1/512; 10=1/1024; 11=1/2048 and 12=1/4096. white
diamond, selected Mab 7c5b2 clone, open symbols, all others. (28B)
Western blot analysis of purified Mabs (lanes 1-3), and "super
clone" supernatants (lanes 4-6), with PBS serving as control (lane
7) are depicted. Selected 7C5B2 Mab in lanes 1 and 4 (diamond).
Double immunostaining of HUVECs with anti-DEspR Mab-immunostaining
and anti-VEGFsp immunostaining was performed and colocalization of
DEspR and VEGFsp determined.
[0127] FIGS. 29A-29C demonstrate that DEspR inhibition via
monoclonal antibody decreases angiogenesis in in vitro HUVECs
assay. DEspR immunostaining of HUVECs using anti-DEspR Mab was
performed. (29A) Dose response curve to anti-DEspR Mab inhibition
of angiogenesis measuring total tube length per well
(.largecircle.) with EC.sub.50=4.34+/-0.45 nM; and number of tube
branch points ( ) with EC..sub.50 3.97+/-0.51 nM. (29B) Analysis of
total tube length changes upon DEspR-inhibition via anti-DEspR
polyclonal (Pab) and monoclonal (Mab) antibodies compared to
control untreated cells (30C), pre-immune serum (PI) and IgG2b
isotype (Iso) controls for Pab and Mab, respectively. (29C)
Analysis of mean number (#) of branchpoints inhibited by Pab and
Mab anti-DEspR ab-inhibition compared with controls (C, PI, Iso).
Data expressed as mean+/-sem; 4 replicates; *, P<0.01 (ANOVA
followed by all pairwise multiple comparison Tukey test).
[0128] FIGS. 30A-30C demonstrate that DEspR inhibition via
monoclonal antibody decreases angiogenesis in in vitro HUVECs
assay. DEspR-positive immunostaining of MDA-MB-231 breast cancer
cells and PANC-1 pancreatic cancer cell line via anti-DEspR Mab was
performed. (30A) Dose response curve to increasing DEspR-inhibition
via anti-DEspR Mab of MDA=MB-231 breast cancer cell invasiveness
(black), EC.sub.50=3.55+/-0.32 nM. (30B-30C) Analysis of cell
invasiveness inhibited by anti-DEspR Mab inhibition compared to
control untreated cells, and IgG2b isotype control for MDA-MB-231
breast cancer cells (31B), and PANC-1 pancreatic cell line (31C).
All data shown as mean+/-sem of 4 replicates; *, P<0.01; **,
P<0.001 (1-way ANOVA followed by all pairwise multiple
comparison Tukey Test).
[0129] FIGS. 31A-31F show immunohistochemical analysis of DEspR
expression in human breast tissue. (31A-31C) normal; (31D-31F)
Grade-1, T1 invasive ductal carcinoma. 31A. Normal breast tissue:
3.times.-overlay of DEspR, aSMA and DAPI nuclear stain detects aSMA
expression in mammary myoepithelial cells but no expression of
DEspR in epithelial cells (white triangular arrowhead .fwdarw.) and
microvessels (white rounded arrowhead). 31B,
2.times.-immunofluorescence overlay of DEspR and DAPI nuclear stain
confirms absence of DEspR expression in normal breast tissue. 31C,
4.times.-overlay of DEspR, aSMA, DAPI immunofluorescence and
diffusion contrast imaging (DIC) delineates tissue morphology,
expression of aSMA. and non/minimal-expression of DEspR in normal
mammary epithelium and endothelium. 31D, 3.times.-Overlay of DAPI,
aSMA and DEspR immunofluorescence in Gr.I-T1 invasive ductal
carcinoma detects DEspR expression in vascular endothelium, and
co-localization with aSMA in mammary tissue. 31E, 2.times.-overlay
of DAPI and DEspR of breast cancer shown in panel d highlights
DEspR expression. 31F, 4.times.-overlay of DAPI, aSMA, DEspR, DIC
elucidate DEspR spatial expression with tissue morphology. (white
triangular arrowhead .fwdarw.), epithelial cells; (white rounded
arrowhead), microvessels. DEspR-positive; aSMA-positive; DAPI
nuclear stain; colocalization of aSMA and DEspR; bar=20
microns.
[0130] FIGS. 32A-32F demonstrate immunohistochemical analysis of
DEspR expression in pancreatic tissue using anti-DEspR Mab.
(32A-32C) normal; (32D-32F) Grade-3, T3 pancreatic ductal
carcinoma. (32A) Normal pancreatic tissue: 3.times.-overlay of
DEspR, aSMA and DAPI nuclear stain detects minimal DEspR expression
in microvessels seen better in panel (32B)
4.times.-immunofluorescence overlay: DEspR, aSMA, DAPI, with DIC
imaging of tissue morphology. (32C) left: 3.times.-overlay of
DEspR, aSMA, DAPI immunofluorescence; right: 4.times.-overlay of
DEspR, aSMA, DAPI and diffusion contrast imaging (DIC) for tissue
morphology shows aSMA expression and non/minimal-expression of
DEspR in normal endothelium. (32D) 3.times.-overlay of DAPI, aSMA
and DEspR immunofluorescence in Gr.3-T3 pancreatic ductal carcinoma
detects DEspR expression in vascular endothelium, and
co-localization with aSMA. (32E) 2.times.-overlay of DAPI and DEspR
of image shown in panel 32D highlights DEspR expression. (32F)
3.times.-overlay of DAPI, aSMA, DEspR, shows increased DEspR
expression in pancreatic ductal carcinoma cells. (white .fwdarw.),
epithelial cells; (white rounded arrowhead, microvessels.
DEspR-positive; aSMA-positive; DAPI nuclear stain; colocalization
of aSMA and DEspR; bar=20 microns.
[0131] FIG. 33 demonstrates 1% agarose gel separation of RT-PCR
products of antibody obtained from the 7C5B2 hybridoma. Gel was
stained with SYBR.RTM. Safe DNA gel stain (Invitrogen cat. no.
533102) and photographed over ultraviolet light. Size marker (L) is
GeneRuler.TM. 1 Kb Plus (Fermentas cat. no. SM1331). RT-PCR was
performed using degenerate primer pools for murine signal sequences
with constant region primers for each of IgGVH, IgMVH, Ig.kappa.VL
and Ig.lamda.VL.
[0132] FIG. 34 shows the variable heavy chain amino acid (SEQ ID
NO: 4) and nucleotide (SEQ ID NO: 3) sequence of the 7C5B2
antibody. CDR definitions and protein sequence numbering according
to Kabat.
[0133] FIG. 35 shows the variable light chain amino acid (SEQ ID
NO: 9) and nucleotide (SEQ ID NO: 8) sequence of a composite 7C5B2
antibody. CDR definitions and protein sequence numbering according
to Kabat.
[0134] FIG. 36 shows an exemplary variable heavy chain amino acid
(SEQ ID NO: 13) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0135] FIG. 37 shows an exemplary variable heavy chain amino acid
(SEQ ID NO: 14) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0136] FIG. 38 shows an exemplary variable heavy chain amino acid
(SEQ ID NO: 15) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0137] FIG. 39 shows an exemplary variable heavy chain amino acid
(SEQ ID NO: 16) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0138] FIG. 40 shows an exemplary variable heavy chain amino acid
(SEQ ID NO: 17) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0139] FIG. 41 shows an exemplary variable light chain amino acid
(SEQ ID NO: 18) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
[0140] FIG. 42 shows an exemplary variable light chain amino acid
(SEQ ID NO: 19) and nucleotide sequence of a composite anti-DEspR
humanized 7C5B2 antibody generated using the methods described
herein. CDR definitions and protein sequence numbering according to
Kabat.
DETAILED DESCRIPTION
[0141] Provided herein are novel compositions comprising anti-DEspR
antibodies and DEspR-binding fragments thereof, and methods of
their use in anti-angiogenesis and anti-tumor proliferation and
invasiveness therapies, such as the treatment of cancer, as well as
the treatment of those vascular diseases where pathological
angiogenesis plays a role, such as in carotid artery disease, vasa
vasorum neovascularization (thus impacting, for example, stroke),
and vulnerable plaque neovascularization (thus impacting, for
example, heart disease). In addition, the compositions comprising
the anti-DEspR antibodies and DEspR-binding fragments thereof
described herein are useful in assessment and imaging methods, such
as companion diagnostics for determining DEspR expression in tumor
biopsies to identify likely responders for personalized medicine
approaches, DEspR-targeted molecular imaging of angiogenesis, which
can be used, for example, in serial monitoring of response(s) to
therapy, in vivo detection of tumor "angiogenic switch," or
vascular mimicry. Further, such diagnostics provide novel
approaches for anti-angiogenic therapies for use in personalized
medicine applications. Furthermore, the compositions comprising the
anti-DEspR antibodies and DEspR-binding fragments thereof described
herein are useful as targeting moieties for other diagnostic and
therapeutic compositions, in combination with delivery agents such
as nanoparticles, polyplexes, microparticles, etc.
[0142] Therapies targeting the VEGF and VEGFR2 receptor pathways,
such as bevacizumab, sunitinib, and sorafanib treatments, have
recently been shown to have only transitory benefits, and appear to
promote or induce a feedback angiogenic response, such that 10-fold
increases in VEGF levels have been detected following anti-VEGF
treatment (Willett et al., 2005 and Carmelie et al. 2005), and
exacerbations of metastasis have been seen after VEGFR2 inhibitor
(sunitinib) treatments.
[0143] In contrast, the inventors have discovered another
angiogenesis arm of the VEGF system, based on their discovery that
key, non-redundant, and distinct roles are played by interaction of
the VEGF signal peptide (VEGFsp) with its receptor "DEspR" or "dual
endothelin-1/VEGFsp receptor". The inventors have found that: a) a
DEspR null mutation leads to E10.5-E12.5 embryonic lethality due to
abnormal embryonic vasculogenesis and angiogenesis (Herrera et al.
2005); b) VEGFsp binds DEspR with high affinity, equal to what is
observed for ET1 binding (Herrera et al. 2005); c) DEspR
antibody-mediated inhibition in a rat mammary tumor model and DEspR
haplo-deficiency in DEspR+/- mice reduces tumor growth in vivo
(Herrera et al. 2005); d) VEGFsp stimulates adult rat aortic ring
angiogenesis (Decano et al., 2010); and e) DEspR mediates adult
angiogenesis and its expression is increased during carotid
atherosclerotic vasa vasorum neovascularization, as described
herein.
[0144] As described herein, the inventors further demonstrate that:
a) DEspR expression is increased in several human cancer tumor
vessels in both males and females (e.g., breast, lung, liver,
bladder, pancrease, stomach, esophagus, colon, etc.), and
surprisingly, also in a variety of tumor cells, including breast,
lung, glioblastoma, bladder, melanoma, and pancreatic tumor cells,
using tumor tissue arrays and tumor cell-line arrays respectively;
as well as in cancer stem cells or cancer stem-like cells or tumor
initiating cells; b) DEspR and VEGFsp are colocalized in both
nuclear and cell membranes in cultured tumor cells; c) VEGFsp
stimulates both tumor cell proliferation and invasiveness; and that
d) DEspR inhibition via polyclonal and monoclonal anti-human DEspR
antibodies potently suppress angiogenesis and tumor cell
invasiveness, and reduce tumor growth rate and decrease tumor size
significantly.
DEspR
[0145] The dual endothelin-1/VEGF signal peptide activated receptor
(DEspR), formerly DEAR was originally cloned from a Dahl
salt-sensitive hypertensive rat brain cDNA library and was shown to
be a single transmembrane receptor coupled to a Ca2+-mobilizing
transduction pathway binding endothelin-1 (ET-1) and angiotensin-II
(Ang II) with equivalent affinities (Ruiz-Opazo N. et al. (1998),
Molecular characterization of a dual Endothelin-1/Angiotensin II
Receptor. Mol. Med. 4: 96-108). Subsequent molecular studies
elucidated that the mouse ortholog does not interact with AngII but
binds ET-1 and the vascular endothelial growth factor signal
peptide (VEGFsp) with equal affinities instead. DEspR.sup.-/-
double mutant deficiency in mice resulted in embryonic lethality
due to impaired vasculogenesis, abnormal angiogenesis and vascular
network formation. DEspR.sup.-/- embryos also showed abnormal
neurogenesis marked by a hyperconvoluted neuroepithelium and
dysregulated neural tube differentiation from the telencephalon to
myelencephalon (Herrera V L M, et al., (2005), Embryonic lethality
in Dear gene deficient mice: new player in angiogenesis. Physiol.
Genomics 23: 257-268.). This phenotype is strikingly opposite to
the proapoptotic effects observed in the developing neural tube in
VEGF.sup.+/- deficient mice, although abnormalities in
vasculogenesis and angiogenesis are similar (Herrera V L M, et al.,
(2005)).
[0146] Accordingly, the term "DEspR," as used herein, refers to the
85-amino acid dual endothelin/VEGF signal peptide receptor (DEspR)
having the human amino acid native sequence of:
MTMFKGSNEMKSRNWGSITCIICFTCVGSQLSMSSSKASNFSGPLQLYQRELEIFIVLTDVP
NYRLIKENSHLHTTIVDQGRTV (SEQ ID NO:1), as described by, e.g.,
Glorioso et al. 2007, together with naturally occurring allelic,
splice variants, and processed forms thereof.
[0147] As used herein a DEspR "native sequence" or DEspR "wild-type
sequence" polypeptide comprises a polypeptide having the same amino
acid sequence as a DEspR polypeptide derived from nature. Thus, a
native sequence polypeptide can have the amino acid sequence of
naturally-occurring polypeptide from any mammal. Such native
sequence polypeptide can be isolated from nature or can be produced
by recombinant or synthetic means. The term "native sequence"
polypeptide specifically encompasses naturally-occurring truncated
or secreted forms of the polypeptide (e.g., an extracellular domain
sequence), naturally-occurring variant forms (e.g., alternatively
spliced forms) and naturally-occurring allelic variants of the
polypeptide.
[0148] A DEspR polypeptide "variant" means a biologically active
DEspR polypeptide having at least about 80% amino acid sequence
identity with a native sequence of a DEspR polypeptide. Such
variants include, for instance, polypeptides wherein one or more
amino acid residues are added, or deleted, at the N- or C-terminus
of the polypeptide. Ordinarily, a variant has at least about 80%
amino acid sequence identity, more preferably at least about 90%
amino acid sequence identity, and even more preferably at least
about 95% amino acid sequence identity with the native sequence
polypeptide.
[0149] DEspR is part of the G protein coupled receptor family, and
binds to endothelin-1 and to VEGF signal peptide (VEGFsp). VEGFsp
has the human sequence MNFLLSWVHWSLALLLYLHHAKWSQA (SEQ ID NO:2).
Typically, as used herein, DEspR refers to human DEspR. The term
"DEspR" is also used to refer to truncated forms or fragments of
the polypeptide comprising specific amino acids sequences of the
85-amino acid human dual endothelin/VEGF signal peptide receptor.
Reference to any such forms of DEspR can be identified in the
application, e.g., by "DEspR (1-9)."
DEspR Antagonists & Anti-DEspR Antibodies
[0150] Provided herein are compositions and methods comprising
DEspR antagonists that are capable of neutralizing, blocking,
inhibiting, abrogating, reducing, or interfering with DEspR
activities including its binding to endothelin-1 or VEGFsp. DEspR
antagonists include, but are not limited to, anti-DEspR antibodies
and antigen-binding fragments thereof, receptor molecules, small
molecules, nanoparticles, polyplex combinations and derivatives
thereof that bind specifically to DEspR thereby inhibiting,
preventing, or sequestering its binding to its ligands, such as
VEGFsp and endothelin-1.
Anti-DEspR Antibodies and Antibody Production
[0151] Accordingly, in some aspects, provided herein is an
anti-DEspR antibody or antibody fragment thereof that is specific
for a DEspR target, where the anti-DEspR antibody or antibody
fragment thereof specifically binds to the DEspR target and reduces
or inhibits DEspR biological activity. In some embodiments, the
DEspR is human DEspR. In some embodiments, the DEspR target
comprises an amino acid sequence of SEQ ID NO:1 or an allelic or
splice variant thereof.
[0152] As used herein, an "anti-DEspR antibody" refers to an
antibody that binds to DEspR with sufficient affinity and
specificity. The antibody selected will normally have a binding
affinity for DEspR, for example, the antibody can bind human DEspR
with a K.sub.D value between 10.sup.-5 M to 10.sup.-10 M. As used
herein, "selectively binds" or "specifically binds" refers to the
ability of an anti-DEspR antibody or antibody fragment thereof
described herein to bind to DEspR, with a K.sub.D 10.sup.-5 M
(10000 nM) or less, e.g., 10.sup.-6 M, 10.sup.7M, 10.sup.-8M,
10.sup.-9 M, 10.sub.-10 M, 10.sup.-11 M, 10.sup.-12 M, or less.
[0153] Antibody affinities can be determined, for example, by a
surface plasmon resonance based assay (such as the BIAcore assay
described in PCT Application Publication No. WO2005/012359);
enzyme-linked immunoabsorbent assay (ELISA); and competition assays
(e.g. RIA's), for example. In certain aspects described herein, an
anti-DEspR antibody can be used as a therapeutic agent in targeting
and interfering with diseases or conditions where DEspR activity is
involved. Also, the anti-DEspR antibody can be subjected to other
biological activity assays, e.g., in order to evaluate its
effectiveness as a therapeutic, or its effectiveness as a
diagnostic aid, etc. Such assays are known in the art and depend on
the target antigen and intended use for the antibody. Examples
include the HUVEC inhibition assay; tumor cell growth inhibition
assays (as described in WO 89/06692, for example);
antibody-dependent cellular cytotoxicity (ADCC) and
complement-mediated cytotoxicity (CDC) assays (U.S. Pat. No.
5,500,362); and agonistic activity or hematopoiesis assays (see WO
95/27062). Other biological activity assays that can be used to
assess an anti-DEspR antibody are described herein in the Examples
section.
[0154] Thus, anti-DEspR antibodies or antibody fragments thereof
that are useful in the compositions and methods described herein
include any antibodies or antibody fragments thereof that bind with
sufficient affinity and specificity to DEspR, i.e., are specific
for DEspR, and can reduce or inhibit the biological activity of
DEspR.
[0155] Accordingly, in some aspects, provided herein is an
anti-DEspR antibody or antibody fragment thereof that binds to
DEspR and inhibits DEspR biological activity or blocks interaction
of DEspR with VEGFsp. In some embodiments of these aspects and all
such aspects described herein, the VEGFsp has a sequence comprising
the sequence of SEQ ID NO: 2. In some embodiments of these aspects
and all such aspects described herein, the anto-DEspR antibody or
antibody fragment thereof is specific for an epitope of DEspR
comprising an extracellular portion of DEspR. In some embodiments
of these aspects and all such aspects described herein, the
anti-DEspR antibody or antibody fragment thereof is specific for an
epitope of DEspR comprising amino acids 1-9 of SEQ ID NO: 1.
[0156] Further description and examples of anti-DEspR antibodies
and antibody fragments thereof useful with the compositions and
methods described herein, as well as methods of making and
characterizing the same, are provided below:
Polyclonal Antibodies
[0157] Polyclonal antibodies are preferably raised in animals by
multiple subcutaneous (sc) or intraperitoneal (ip) injections of
the relevant antigen, e.g., DEspR(1-9) and an adjuvant. It can be
useful, in some embodiments, to conjugate the relevant antigen to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sup.1N.dbd.C.dbd.NR, where R and R.sup.1 are
different alkyl groups.
[0158] Animals can be immunized against the antigen, immunogenic
conjugates, or derivatives by combining, e.g., 100 .mu.g or 5 .mu.g
of the protein or conjugate (for rabbits or mice, respectively)
with 3 volumes of Freund's complete adjuvant and injecting the
solution intradermally at multiple sites. One month later the
animals are boosted with 1/5 to 1/10 the original amount of peptide
or conjugate in Freund's complete adjuvant by subcutaneous
injection at multiple sites. Seven to 14 days later the animals are
bled and the serum is assayed for antibody titer. Animals are
boosted until the titer plateaus. Preferably, the animal is boosted
with the conjugate of the same antigen, but conjugated to a
different protein and/or through a different cross-linking reagent.
Conjugates also can be made in recombinant cell culture as protein
fusions. Also, aggregating agents such as alum are suitably used to
enhance the immune response.
Monoclonal Antibodies
[0159] Preferably, anti-DEspR antibodies or antibody fragments
thereof for use with the compositions and methods described herein
are anti-DEspR monoclonal antibodies or fragments thereof. The term
"monoclonal antibody" refers to an antibody obtained from a
population of substantially homogeneous antibodies, i.e., the
individual antibodies comprising the population are identical
except for possible naturally occurring mutations that can be
present in minor amounts. Monoclonal antibodies are highly
specific, being directed against a single antigen. Furthermore, in
contrast to polyclonal antibody preparations that typically include
different antibodies directed against different determinants
(epitopes), each monoclonal antibody is directed against a single
determinant on the antigen. Various methods for making monoclonal
antibodies specific for DEspR as described herein are available in
the art. For example, the monoclonal antibodies can be made using
the hybridoma method first described by Kohler et al., Nature,
256:495 (1975), or by recombinant DNA methods (U.S. Pat. No.
4,816,567). "Monoclonal antibodies" can also be isolated from phage
antibody libraries using the techniques described in Clackson et
al., Nature 352:624-628 (1991) or Marks et al., J. Mol. Biol.
222:581-597 (1991), for example.
[0160] The term anti-DEspR "antibody fragment" refers to a protein
fragment that comprises at least an antigen binding site of the
intact antibody and thus retaining the ability to bind antigen.
Examples of antibody fragments encompassed by the term antibody
fragment include: (i) the Fab fragment, having V.sub.L, C.sub.L,
V.sub.H and C.sub.H1 domains; (ii) the Fab' fragment, which is a
Fab fragment having one or more cysteine residues at the C-terminus
of the C.sub.H1 domain; (iii) the Fd fragment having V.sub.H and
C.sub.H1 domains; (iv) the Fd' fragment having V.sub.H and C.sub.H1
domains and one or more cysteine residues at the C-terminus of the
CH1 domain; (v) the Fv fragment having the V.sub.L and V.sub.H
domains of a single arm of an antibody; (vi) the dAb fragment (Ward
et al., Nature 341, 544-546 (1989)) which consists of a V.sub.H
domain; (vii) isolated CDR regions; (viii) F(ab').sub.2 fragments,
a bivalent fragment including two Fab' fragments linked by a
disulphide bridge at the hinge region; (ix) single chain antibody
molecules (e.g., single chain Fv; scFv) (Bird et al., Science
242:423-426 (1988); and Huston et al., PNAS (USA) 85:5879-5883
(1988)); (x) "diabodies" with two antigen binding sites, comprising
a heavy chain variable domain (V.sub.H) connected to a light chain
variable domain (V.sub.L) in the same polypeptide chain (see, e.g.,
EP 404,097; WO 93/11161; and Hollinger et al., Proc. Natl. Acad.
Sci. USA, 90:6444-6448 (1993)); (xi) "linear antibodies" comprising
a pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1)
which, together with complementary light chain polypeptides, form a
pair of antigen binding regions (Zapata et al. Protein Eng.
8(10):1057-1062 (1995); and U.S. Pat. No. 5,641,870).
[0161] In the hybridoma method of making an anti-DEspR monoclonal
antibody, a mouse or other appropriate host animal, such as a
hamster or macaque monkey, is immunized as described herein to
elicit lymphocytes that produce or are capable of producing
antibodies that will specifically bind to the DEspR protein or
fragment thereof used for immunization. Alternatively, lymphocytes
can be immunized in vitro. Lymphocytes then are fused with myeloma
cells using a suitable fusing agent, such as polyethylene glycol,
to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles
and Practice, pp. 59-103 (Academic Press, 1986)).
[0162] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0163] Preferred myeloma cells are those that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these, preferred myeloma cell lines are murine
myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center,
San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)).
[0164] Culture medium in which hybridoma cells are growing is
assayed for production of monoclonal antibodies directed against
the antigen. Preferably, the binding specificity of monoclonal
antibodies produced by hybridoma cells is determined by
immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay
(ELISA).
[0165] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells can be grown in vivo as
ascites tumors in an animal.
[0166] The monoclonal antibodies secreted by the subclones are
suitably separated from the culture medium, ascites fluid, or serum
by conventional immunoglobulin purification procedures such as, for
example, protein A-Sepharose, hydroxylapatite chromatography, gel
electrophoresis, dialysis, or affinity chromatography.
[0167] DNA encoding the monoclonal antibodies can be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
monoclonal antibodies). The hybridoma cells serve as a preferred
source of such DNA. Once isolated, the DNA can be placed into
expression vectors, which are then transfected into host cells such
as E. coli cells, simian COS cells, Chinese hamster ovary (CHO)
cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells. Recombinant production of
antibodies is described in more detail below.
Anti-DEspR Hybridomas and Monoclonal Antibodies Thereof
[0168] In certain aspects described herein, anti-DEspR monoclonal
antibodies include, but are not limited to, the monoclonal
anti-DEspR antibody 7C5B2 produced or expressed by the hybridoma
7C5B2 described herein, and referred to as the "7C5B2 antibody,"
and derivatives or antigen-binding fragments thereof, including,
for example, a "7C5B2 variable heavy chain," or a "7C5B2" variable
light chain.
[0169] As described herein, the 7C5B2 hybridoma produces a
monoclonal antibody, termed herein as the "7C5B2 anti-DEspR
antibody" or "7C5B2 antibody," that is highly specific for DEspR
and can potently inhibit DEspR biological activity. The biological
characteristics of the 7C5B2 anti-DEspR antibody render it
particularly useful for the compositions and methods described
herein, including therapeutic and diagnostic applications.
Accordingly, sequence analysis of the 7C5B2 antibody was performed,
as described herein, to identify the heavy and light chain variable
domain sequences, and complementarity determining region (CDR)
sequences, of the 7C5B2 antibody for use in the compositions and
methods described herein.
[0170] Throughout the present specification and claims, the
numbering of the residues in an immunoglobulin heavy chain is that
of the EU index as in Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991), which is also available
on the world wide web, and is expressly incorporated herein in its
entirety by reference. The "EU index as in Kabat" refers to the
residue numbering of the human IgG1 EU antibody.
[0171] As used herein, "antibody variable domain" refers to the
portions of the light and heavy chains of antibody molecules that
include amino acid sequences of Complementarity Determining Regions
(CDRs; i.e., CDR1, CDR2, and CDR3), and Framework Regions (FRs).
V.sub.H refers to the variable domain of the heavy chain. V.sub.L
refers to the variable domain of the light chain. According to the
methods used herein, the amino acid positions assigned to CDRs and
FRs can be defined according to Kabat (Sequences of Proteins of
Immunological Interest (National Institutes of Health, Bethesda,
Md., 1987 and 1991)). Amino acid numbering of antibodies or antigen
binding fragments is also according to that of Kabat.
[0172] As used herein, the term "Complementarity Determining
Regions" (CDRs), i.e., CDR1, CDR2, and CDR3) refers to the amino
acid residues of an antibody variable domain the presence of which
are necessary for antigen binding. Each variable domain typically
has three CDR regions identified as CDR1, CDR2 and CDR3. Each
complementarity determining region can comprise amino acid residues
from a "complementarity determining region" as defined by Kabat
(i.e., about residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the
light chain variable domain and 31-35 (H1), 50-65 (H2) and 95-102
(H3) in the heavy chain variable domain; Kabat et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service,
National Institutes of Health, Bethesda, Md. (1991)) and/or those
residues from a "hypervariable loop" (i.e., about residues 26-32
(L1), 50-52 (L2) and 91-96 (L3) in the light chain variable domain
and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the heavy chain
variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917
(1987)). In some embodiments, a complementarity determining region
can include amino acids from both a CDR region defined according to
Kabat and a hypervariable loop.
[0173] The nucleotide sequence encoding the V.sub.H or variable
domain of the heavy chain of the 7C5B2 antibody, as obtained by
sequence analysis of sequences obtained from the 7C5B2 hybridoma,
is: C A G G T G C A A C T G A A G G A G T C A G G A C C T G G C C T
G G T G G C G C C C T C A C A G A G C C T G T C C A T T A C C T G C
A C T G T C T C T G G G T T C T C A T T A A C C A G C T A T G A T A
T A A G C T G G A T T C G C C A G C C A C C A G G A A A G G G T C T
G G A G T G G C T T G G A G T A A T A T G G A C T G G T G G A G G C
A C A A A T T A T A A T T C A G C T T T C A T G T C C A G A C T G A
G C A T C A G C A A G G A C A A C T C C A A G A G C C A A G T T T T
C T T A A A A A T G A A C A G T C T G C A A A C T G A T G A C A C A
G C C A T A T A T T A C T G T G T A A G A G A T C G G G A T T A C G
A C G G G T G G T A C T T C G A T G T C T G G G G C G C A G G G A C
C A C G G T C A C C G T C T C C T C A (SEQ ID NO: 3).
[0174] The corresponding amino acid of the V.sub.H domain of the
7C5B2 antibody is: Q V Q L K E S G P G L V A P S Q S L S I T C T V
S G F S L T S Y D I S W I R Q P P G K G L E W L G V I W T G G G T N
Y N S A F M S R L S I S K D N S K S Q V F L K M N S L Q T D D T A I
Y Y C V R D R D Y D G W Y F D V W G A G T T V T V S S (SEQ ID NO:
4).
[0175] The 10 amino acid complementarity determining region 1 or
CDR1 of the V.sub.H domain of the 7C5B2 antibody is: G F S L T S Y
D I S (SEQ ID NO: 5). The 16 amino acid CDR2 of the V.sub.H domain
of the 7C5B2 antibody is: V I W T G G G T N Y N S A F M S (SEQ ID
NO: 6). The 11 amino acid CDR2 of the V.sub.H domain of the 7C5B2
antibody is: D R D Y D G W Y F D V (SEQ ID NO: 7).
[0176] The nucleotide sequence encoding the V.sub.L or variable
domain of the light chain of the 7C5B2 antibody, as obtained by
sequence analysis of sequences obtained from the 7C5B2 hybridoma,
is: G A T G T T T T G A T G A C C C A A A C T C C A C T C T C C C T
G C C T G T C A G T C T T G G A G A T C A A G C C T C C A T C T C T
T G C A G A T C T A G T C A G A G C A T T G T A C A T A G T A A T G
G A A A C A C C T A T T T A G A A T G G T A C C T G C A G A A A C C
A G G C C A G T C T C C A A A G C T C C T G A T C T A C A A A G T T
T C C A A C C G A T T T T C T G G G G T C C C A G A C A G G T T C A
G T G G C A G T G G A T C A G G G A C A G A T T T C A C A C T C A A
G A T C A G C A G A G T G G A G G C T G A G G A T C T G G G A G T T
T A T T A C T G C T T T C A A G G T T C A C A T G T T C C G T A C A
C G T T C G G A G G G G G G A C C A A G C T G G A A A T A A A A
(SEQ ID NO: 8).
[0177] The corresponding amino acid of the V.sub.L domain of the
7C5B2 antibody is: D V L M T Q T P L S L P V S L G D Q A S I S C R
S S Q S I V H S N G N T Y L E W Y L Q K P G Q S P K L L I Y K V S N
R F S G V P D R F S G S G S G T D F T L K I S R V E A E D L G V Y Y
C F Q G S H V P Y T F G G G T K L E I K (SEQ ID NO: 9).
[0178] The 16 amino acid complementarity determining region 1 or
CDR1 of the V.sub.L domain of the 7C5B2 antibody is: R S S Q S I V
H S N G N T Y L E (SEQ ID NO: 10). The 7 amino acid CDR2 of the
V.sub.L domain of the 7C5B2 antibody is: K V S N R F S (SEQ ID NO:
11). The 9 amino acid CDR2 of the V.sub.L domain of the 7C5B2
antibody is: F Q G S H V P Y T (SEQ ID NO: 12).
[0179] As shown in Table 1, sequence analysis of the heavy and
light chain variable regions of the 7C5B2 antibody indicates strong
homology to human germline sequences:
TABLE-US-00001 TABLE 1 Antibody Sequence Analysis.sup.a H Chain L
Chain CDR 1 Length 10aa 16aa CDR 2 Length 16aa 7aa CDR 3 Length
11aa 9aa Closest Human IGHV4-59*01 (64%) IGKV2-30*01 (82%)
Germline.sup.b Closest Human FW1.sup.b IGHV4-31*01 (84%)
IGKV2-30*01 (78%) Closest Human FW2.sup.b IGHV4-61*01 (93%)
IGKV2-40*01 (93%) Closest Human FW3.sup.b IGHV3-66*01 (60%)
IGKV2-30*01 (97%) Closest Human J.sup.b IGHJ6 (91%) IGKJ2 (90%)
.sup.aCDR definitions and sequence numbering according to Kabat
.sup.bGermline ID(s) indicated followed by % homology
[0180] Accordingly, in some embodiments of the aspects provided
herein, the heavy and/or light chain variable domain(s) sequence(s)
of the 7C5B2 antibody, i.e., SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:
8, and/or SEQ ID NO: 9 can be used to generate, for example,
humanized antibodies, as described elsewhere herein.
[0181] In some aspects, monoclonal antibodies that specifically
bind to DEspR are provided having one or more biological
characteristics of the 7C5B2 monoclonal antibody. As used herein,
an antibody having a "biological characteristic" of a designated
antibody, such as the 7C5B2 antibody, is one that possesses one or
more of the biological characteristics of that antibody which
distinguish it from other antibodies that bind to the same
antigen.
[0182] Accordingly, in some such embodiments of these aspects,
having a biological characteristic of the 7C5B2 monoclonal antibody
can include having an ED.sub.50 value (i.e., the dose
therapeutically effective in 50% of the population) at or around
the ED.sub.50 value of the 7C5B2 antibody for the given population;
having an EC.sub.50 value (i.e., the dose that achieves a
half-maximal inhibition of a given parameter or phenotype) at or
around the EC.sub.50 value of the 7C5B2 antibody for a given
parameter or phenotype. The effects of any particular dosage can be
monitored by a suitable bioassay. For example, in some embodiments
of these aspects, the given parameter or phenotype to be inhibited
by the antibody that specifically binds to DEspR and has one or
more biological characteristics of the 7C5B2 antibody can include,
but is not limited to, the mean total tube number in an in vitro
tubulogenesis assay, the mean total tube length in an in vitro
tubulogenesis assay, the mean number of branching points in an in
vitro tubulogenesis assay, the mean number of vessel connections in
an in vitro tubulogenesis assay, and/or tumor cell
invasiveness.
[0183] Accordingly, in those embodiments where the phenotype to be
inhibited is mean total tube length, as measured using an in vitro
tubulogenesis assay, the EC.sub.50 value of the monoclonal antibody
having a biological characteristic of the 7C5B2 monoclonal antibody
is 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or
less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1
nM or less. In some such embodiments, the EC.sub.50 value of the
monoclonal antibody is in the range of 3.0-5.0 nM, in the range of
3.1-4.9 nM, in the range of 3.2-4.8 nM, in the range of 3.3-4.7 nM,
in the range of 3.4-4.6 nM, in the range of 3.5-4.5 nM, in the
range of 3.6-4.4 nM, in the range of 3.7-4.3 nM, in the range of
3.8-4.2 nM, or in the range of 3.9-4.1 nM. In some embodiments, the
EC.sub.50 value for inhibiting mean total tube length of the
monoclonal antibody having a biological characteristic of the 7C5B2
monoclonal antibody is in the range of 3.8 nM-4.8 nM.
[0184] For example, in those embodiments where the phenotype to be
inhibited is number of branch points, as measured using an in vitro
tubulogenesis assay, the EC.sub.50 value of the monoclonal antibody
having a biological characteristic of the 7C5B2 monoclonal antibody
is 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or
less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, or 1
nM or less. In some such embodiments, the EC.sub.50 value of the
monoclonal antibody is in the range of 3.0-5.0 nM, in the range of
3.1-4.9 nM, in the range of 3.2-4.8 nM, in the range of 3.3-4.7 nM,
in the range of 3.4-4.6 nM, in the range of 3.5-4.5 nM, in the
range of 3.6-4.4 nM, in the range of 3.7-4.3 nM, in the range of
3.8-4.2 nM, or in the range of 3.9-4.1 nM. In some embodiments, the
EC.sub.50 value for inhibiting total number of branch points of the
monoclonal antibody having a biological characteristic of the 7C5B2
monoclonal antibody is in the range of 3.4 nM-4.5 nM, in the range
of 3.5 nM-4.4 nM, in the range of 3.6 nM-4.3 nM, in the range of
3.7 nM-4.2 nM, in the range of 3.8 nM-4.1 nM, in the range of 3.9
nM-4.0 nM.
[0185] For example, in those embodiments where the phenotype to be
inhibited is tumor cell invasiveness, as measured in vitro, the
EC.sub.50 value of the monoclonal antibody having a biological
characteristic of the 7C5B2 monoclonal antibody is 10 nM or less, 9
nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less,
4 nM or less, 3 nM or less, 2 nM or less, or 1 nM or less. In some
such embodiments, the EC.sub.50 value of the monoclonal antibody is
in the range of 3.0-5.0 nM, in the range of 3.1-4.9 nM, in the
range of 3.2-4.8 nM, in the range of 3.3-4.7 nM, in the range of
3.4-4.6 nM, in the range of 3.5-4.5 nM, in the range of 3.6-4.4 nM,
in the range of 3.7-4.3 nM, in the range of 3.8-4.2 nM, or in the
range of 3.9-4.1 nM. In some embodiments, the EC.sub.50 value for
inhibiting tumor cell invasiveness of the monoclonal antibody
having a biological characteristic of the 7C5B2 monoclonal antibody
is in the range of 3.2 nM-3.9 nM, in the range of 3.3 nM-3.8 nM,
3.4 nM-3.7 nM, or in the range of 3.5 nM-3.6 nM.
[0186] In some embodiments of the aspects described herein,
anti-DEspR antibodies for use in the compositions and methods
described herein include monoclonal antibodies that bind to the
same epitope or epitopes of DEspR as the monoclonal anti-DEspR
7C5B2 antibody.
[0187] In other aspects described herein, anti-DEspR antibodies for
use in the compositions and methods described herein include: the
monoclonal anti-DEspR antibody 7C5C5 produced or expressed by the
hybridoma 7C5C5 described herein, referred to as the "7C5C5
antibody," and derivatives or fragments thereof; monoclonal
antibodies that bind to the same epitope or epitopes of DEspR as
the monoclonal anti-DEspR 7C5C5 antibody; the monoclonal anti-DEspR
antibody 5G12E8 produced or expressed by the hybridoma5G12E8
described herein, referred to as the "5G12E8 antibody," and
derivatives or fragments thereof; monoclonal antibodies that bind
to the same epitope or epitopes of DEspR as the monoclonal
anti-DEspR 5G12E8 antibody; and monoclonal antibodies produced by
hybridomas 2E4A8, 2E4B11, 2E4H10, 8E7D11, 8E2F6, E2G4 and
8E7F8.
[0188] In addition to generation and production via hybridomas,
antibodies or antibody fragments that specifically bind DEspR can
be isolated from antibody phage libraries generated using the
techniques described in McCafferty et al., Nature, 348:552-554
(1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et
al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of
murine and human antibodies, respectively, using phage libraries.
Subsequent publications describe the production of high affinity
(nM range) human antibodies by chain shuffling (Marks et al.,
Bio/Technology, 10:779-783 (1992)), as well as combinatorial
infection and in vivo recombination as a strategy for constructing
very large phage libraries (Waterhouse et al., Nuc. Acids. Res.,
21:2265-2266 (1993)). Thus, these techniques are viable
alternatives to traditional monoclonal antibody hybridoma
techniques for isolation of monoclonal antibodies.
[0189] The DNA sequences encoding the antibodies or antibody
fragment that specifically bind DEspR also can be modified, for
example, by substituting the coding sequence for human heavy- and
light-chain constant domains in place of the homologous murine
sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl.
Acad. Sci. USA, 81:6851 (1984)), or by covalently joining to the
immunoglobulin coding sequence all or part of the coding sequence
for a non-immunoglobulin polypeptide, as also described elsewhere
herein.
[0190] Such non-immunoglobulin polypeptides can be substituted for
the constant domains of an antibody, or they can be substituted for
the variable domains of one antigen-combining site of an antibody
to create a chimeric bivalent antibody comprising one
antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
Humanized and Human Antibodies
[0191] Provided herein, in some aspects, are humanized anti-DEspR
antibodies for use in the compositions and methods described
herein. Humanized forms of non-human (e.g., murine) antibodies
refer to chimeric antibodies that contain minimal sequence derived
from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which
residues from a hypervariable region of the recipient are replaced
by residues from a hypervariable region of a non-human species
(donor antibody) such as mouse, rat, rabbit or nonhuman primate
having the desired specificity, affinity, and capacity. In some
instances, Fv framework region (FR) residues of the human
immunoglobulin are replaced by corresponding non-human residues.
Furthermore, humanized antibodies can comprise residues that are
not found in the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, a humanized antibody can comprise substantially all of at
least one, and typically two, variable domains, in which all or
substantially all of the hypervariable loops correspond to those of
a non-human immunoglobulin and all or substantially all of the FR
regions are those of a human immunoglobulin sequence. The humanized
antibody optionally also can comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin. For further details, see Jones et al., Nature
321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988);
and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).
[0192] A humanized antibody has one or more amino acid residues
introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as "import"
residues, which are typically taken from an "import" variable
domain. Humanization can be essentially performed following the
method of Winter and co-workers (Jones et al., Nature, 321:522-525
(1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et
al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or
CDR sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567) where substantially less than an intact
human variable domain has been substituted by the corresponding
sequence from a non-human species. In practice, humanized
antibodies are typically human antibodies in which some CDR
residues and possibly some FR residues are substituted by residues
from analogous sites in rodent antibodies. In some embodiments,
humanized antibodies comprising one or more variable domains
comprising the amino acid sequence of the variable heavy (SEQ ID
NO: 4) and/or variable light (SEQ ID NO: 9) chain domains of the
murine anti-DEspR antibody 7C5B2, are provided.
[0193] Accordingly, in some embodiments of the aspects described
herein, one or more heavy and/or one or more light chain CDR
regions of a humanized anti-DEspR antibody or antibody fragment
thereof comprises a sequence of the 7C5B2 antibody described
herein. In some such embodiments, the one or more variable heavy
chain CDR regions comprises a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some
such embodiments, the one or more variable light chain CDR regions
comprises a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some such embodiments,
the one or more variable heavy chain CDR regions comprises a
sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7, and the one or more variable light chain
CDR regions comprises a sequence selected from the group consisting
of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[0194] In some embodiments of the aspects described herein, a
humanized anti-DEspR monoclonal antibody comprises mutated human
IgG1 framework regions and one or more heavy and/or one or more
light chain CDR regions from the murine anti-human DEspR monoclonal
antibody 7C5B2, described herein, that blocks binding of human
DEspR to its ligands. In some such embodiments, the one or more
variable heavy chain CDR regions comprises a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
7. In some such embodiments, the one or more variable light chain
CDR regions comprises a sequence selected from the group consisting
of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some such
embodiments, the one or more variable heavy chain CDR regions
comprises a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and the one or more variable
light chain CDR regions comprises a sequence selected from the
group consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
12.
[0195] In some embodiments, a humanized anti-DEspR monoclonal
antibody comprises mutated human IgG4 framework regions and one or
more heavy and/or one or more light chain CDR regions from the
murine anti-human DEspR monoclonal antibody 7C5B2, described
herein, that blocks binding of human DEspR to its ligands. In some
such embodiments, the one or more variable heavy chain CDR regions
comprises a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some such embodiments, the
one or more variable light chain CDR regions comprises a sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11,
or SEQ ID NO: 12. In some such embodiments, the one or more
variable heavy chain CDR regions comprises a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
7, and the one or more variable light chain CDR regions comprises a
sequence selected from the group consisting of SEQ ID NO: 10, SEQ
ID NO: 11, or SEQ ID NO: 12.
[0196] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is very important to
reduce antigenicity. According to the so-called "best-fit" method,
the amino acid sequences of the variable heavy and light chain
domains of a rodent antibody, such as that of the 7C5B2 antibody
(SEQ ID NO: 4 and SEQ ID NO: 9, receptively), are screened against
the entire library of known human variable-domain sequences. The
human sequence which is closest to that of the rodent is then
accepted as the human framework (FR) for the humanized antibody
(Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol.
Biol., 196:901 (1987)). Another method uses a particular framework
derived from the consensus sequence of all human antibodies of a
particular subgroup of light or heavy chains. The same framework
can be used for several different humanized antibodies (Carter et
al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J.
Immunol., 151:2623 (1993)).
[0197] It is further important that antibodies be humanized with
retention of high affinity for the antigen and other favorable
biological properties, for example, the anti-angiogenic properties
of the anti-DEspR antibody 7C5B2 described herein. To achieve this
goal, according to a preferred method, humanized antibodies are
prepared by a process of analysis of the parental sequences and
various conceptual humanized products using three-dimensional
models of the parental and humanized sequences. Three-dimensional
immunoglobulin models are commonly available and are familiar to
those skilled in the art. Computer programs are available which
illustrate and display probable three-dimensional conformational
structures of selected candidate immunoglobulin sequences.
Inspection of these displays permits analysis of the likely role of
the residues in the functioning of the candidate immunoglobulin
sequence, i.e., the analysis of residues that influence the ability
of the candidate immunoglobulin to bind its antigen. In this way,
FR residues can be selected and combined from the recipient and
import sequences so that the desired antibody characteristic, such
as increased affinity for the target antigen(s), is achieved. In
general, the CDR residues are directly and most substantially
involved in influencing antigen binding.
[0198] Exemplary humanized antibodies and affinity matured variants
thereof directed against the VEGF antigen are described in, for
example, U.S. Pat. No. 6,884,879 issued Feb. 26, 2005.
[0199] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993); and Duchosal et al. Nature 355:258 (1992).
[0200] Alternatively, phage display technology (McCafferty et al.,
Nature 348:552-553 (1990)) can be used to produce human antibodies
and antibody fragments in vitro, from immunoglobulin variable (V)
domain gene repertoires from unimmunized donors. According to this
technique, antibody V domain genes are cloned in-frame into either
a major or minor coat protein gene of a filamentous bacteriophage,
such as M13 or fd, and displayed as functional antibody fragments
on the surface of the phage particle. Because the filamentous
particle contains a single-stranded DNA copy of the phage genome,
selections based on the functional properties of the antibody also
result in selection of the gene encoding the antibody exhibiting
those properties. Thus, the phage mimics some of the properties of
the B-cell. Phage display can be performed in a variety of formats;
for their review see, e.g., Johnson, Kevin S, and Chiswell, David
J., Current Opinion in Structural Biology 3:564-571 (1993). Several
sources of V-gene segments can be used for phage display. Clackson
et al., Nature, 352:624-628 (1991) isolated a diverse array of
anti-oxazolone antibodies from a small random combinatorial library
of V genes derived from the spleens of immunized mice. A repertoire
of V genes from unimmunized human donors can be constructed and
antibodies to a diverse array of antigens (including self-antigens)
can be isolated essentially following the techniques described by
Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al.,
EMBO J. 12:725-734 (1993). See, also, U.S. Pat. Nos. 5,565,332 and
5,573,905.
[0201] Human antibodies can also be generated by in vitro activated
B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275).
Design & Generation of Composite Human Antibodies
[0202] In some embodiments of the aspects described herein,
composite human antibody technology that generates de-immunized
100% engineered human antibodies at the outset can be used to
prepare humanized composite anti-DEspR antibodies for use in the
compositions and methods described herein, using, for example, a
technology as described by Antitope.
[0203] Briefly, as used herein, "composite human antibodies"
comprise multiple sequence segments ("composites") derived from
V-regions of unrelated human antibodies that are selected to
maintain monoclonal antibody sequences critical for antigen binding
of the starting murine precursor anti-human DEspR monoclonal
antibody, such as 7C5B2 antibody, and which have all been filtered
for the presence of potential T-cell epitopes using "in silico
tools" (Holgate & Baker, 2009). The close fit of human sequence
segments with all sections of the starting antibody V regions and
the elimination of CD4+ T cell epitopes from the outset allow this
technology to circumvent immunogenicity in the development of `100%
engineered human` therapeutic antibodies while maintaining optimal
affinity and specificity through the prior analysis of sequences
necessary for antigen-specificity (Holgate & Baker 2009).
[0204] As described herein, structural models of mouse anti-hDEspR
antibody V regions were produced using Swiss PDB and analysed in
order to identify important "constraining" amino acids in the V
regions that were likely to be essential for the binding properties
of the antibody. Residues contained within the CDRs (using Kabat
definition) together with a number of framework residues were
considered to be important. Both the V.sub.H and V.sub.L (V.sub.K)
sequences of anti-hDEspR, as described herein as SEQ ID NO: 4 and
SEQ ID NO: 9, comprise typical framework residues and the CDR1,
CDR2, and CDR3 motifs are comparable to many murine antibodies, as
described elsewhere herein.
[0205] From the above analysis, it was determined that composite
human sequences of anti-hDEspR can be created with a wide latitude
of alternatives outside of CDRs but with only a narrow menu of
possible alternative residues within the CDR sequences. Analysis
indicated that corresponding sequence segments from several human
antibodies could be combined to create CDRs similar or identical to
those in the murine sequences. For regions outside of and flanking
the CDRs, a wide selection of human sequence segments were
identified as possible components of novel anti-DEspR composite
human antibody V regions for use with the compositions and methods
described herein (see, for example, Table 1).
[0206] Based upon these analyses, a large preliminary set of
sequence segments that could be used to create novel anti-DEspR
composite human antibody variants were selected and analysed using
iTope.TM. technology for in silico analysis of peptide binding to
human MHC class II alleles (Perry et al 2008), and using the
TCED.TM. (T Cell Epitope Database) of known antibody
sequence-related T cell epitopes (Bryson et al 2010). Sequence
segments that were identified as significant non-human germline
binders to human MHC class II or that scored significant hits
against the TCED.TM. were discarded. This resulted in a reduced set
of segments, and combinations of these were again analysed, as
above, to ensure that the junctions between segments did not
contain potential T cell epitopes. Selected segments were then
combined to produce heavy and light chain V region sequences for
synthesis.
[0207] Accordingly, provided herein are variable heavy and light
chain sequences for use in anti-DEspR composite human antibody or
engineered human antibody production. In some embodiments, an
anti-DEspR composite human antibody can comprise a variable heavy
(V.sub.H) chain amino acid sequence selected from the group
consisting of: Q V Q L Q E S G P G L V K P S Q T L S L T C T V S G
F S L T S Y D I S W I R Q P P G K G L E W L G V I W T G G G T N Y N
S A F M S R L T I S K D N S K S T V Y L Q M N S L R A E D T A I Y Y
C V R D R D Y D G W Y F D V W G Q G T T V T V S S (SEQ ID NO: 13);
Q V Q L Q E S G P G L V K P S Q T L S L T C T V S G F S L T S Y D I
S W I R Q P P G K G L E W L G V I W T G G G T N Y N S A F M S R L T
I S K D N S K N T V Y L Q M N S L R A E D T A I Y Y C V R D R D Y D
G W Y F D V W G Q G T T V T V S S(SEQ ID NO: 14); V Q L Q E S G P G
L V K P S Q T L S L T C T V S G F S L T S Y D I S W I R Q P P G K G
L E W L G V I W T G G G T N Y N S A F M S R F T I S K D N S K N T V
Y L Q M N S L R A E D T A I Y Y C V R D R D Y D G W Y F D V W G Q G
T T V T V S S(SEQ ID NO: 15); Q V Q L Q E S G P G L V K P S Q T L S
L T C T V S G F S L T S Y D I S W I R Q P P G K G L E W L G V I W T
G G G T N Y N S A F M S R L T I S K D N S K N T V Y L Q M N S L R A
E D T A V Y Y C V R D R D Y D G W Y F D V W G Q G T T V T V S S(SEQ
ID NO: 16); and Q V Q L Q E S G P G L V K P S Q T L S L T C T V S G
F S L T S Y D I S W I R Q P P G K G L E W L G V I W T G G G T N Y N
S A F M S R F T I S K D N S K N T V Y L Q M N S L R A E D T A V Y Y
C V R D R D (SEQ ID NO: 17).
[0208] In some embodiments, an anti-DEspR composite human antibody
can comprise a variable light (V.sub.L) chain amino acid sequence
selected from the group consisting of: D V L M T Q S P L S L P V T
L G Q P A S I S C R S S Q S I V H S N G N T Y L E W Y L Q K P G Q S
P Q L L I Y K V S N R F S G V P D R F S G S G S G T D F T L K I S R
V E A E D V G V Y Y C F Q G S H V P Y T F G Q G T K L E I K (SEQ ID
NO: 18) and D V V M T Q S P L S L P V T L G Q P A S I S C R S S Q S
I V H S N G N T Y L E W Y L Q K P G Q S P Q L L I Y K V S N R F S G
V P D R F S G S G S G T D F T L K I S R V E A E D V G V Y Y C F Q G
S H V P Y T F G Q G T K L E I K (SEQ ID NO: 19).
[0209] In some embodiments, an anti-DEspR composite human antibody
can comprise a heavy chain CDR1 region comprising an amino acid
sequence of SEQ ID NO: 5. In some embodiments, an anti-DEspR
composite human antibody can comprise a heavy chain CDR2 region
comprising an amino acid sequence of SEQ ID NO: 6. In some
embodiments, an anti-DEspR composite human antibody can comprise a
heavy chain CDR3 region comprising an amino acid sequence of SEQ ID
NO: 7.
[0210] In some embodiments, an anti-DEspR composite human antibody
can comprise a light chain CDR1 region comprising a sequence of SEQ
ID NO: 10. In some embodiments, an anti-DEspR composite human
antibody can comprise a light chain CDR2 region comprising an amino
acid sequence of SEQ ID NO: 11. In some embodiments, an anti-DEspR
composite human antibody can comprise a light chain CDR3 region
comprising an amino acid sequence of SEQ ID NO: 12.
Antibody Fragments
[0211] In some embodiments of the aspects described herein, an
antibody specific for DEspR, such as, for example the anti-DEspR
7C5B2 antibody; an anti-DEspR antibody comprising one or more heavy
chain CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; an
anti-DEspR antibody comprising one or more light chain CDR regions
comprises a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; an anti-DEspR composite
human antibody comprising a variable heavy (V.sub.H) chain amino
acid sequence selected from the group consisting of SEQ ID NO: 4
and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite human
antibody comprising a variable light (V.sub.L) chain amino acid
sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID
NO: 18, and SEQ ID NO: 19 can be treated or processed into an
antibody fragment thereof.
[0212] Various techniques have been developed and are available for
the production of antibody fragments. Traditionally, these
fragments were derived via proteolytic digestion of intact
antibodies (see, e.g., Morimoto et al., Journal of Biochemical and
Biophysical Methods 24:107-117 (1992) and Brennan et al., Science,
229:81 (1985)). However, these fragments can now be produced
directly by recombinant host cells. For example, antibody fragments
can be isolated from the antibody phage libraries discussed above.
Alternatively, Fab'-SH fragments can be directly recovered from E.
coli and chemically coupled to form F(ab').sub.2 fragments (Carter
et al., Bio/Technology 10:163-167 (1992)). According to another
approach, F(ab').sub.2 fragments can be isolated directly from
recombinant host cell culture. Other techniques for the production
of antibody fragments will be apparent to the skilled practitioner.
In other embodiments, the antibody fragment of choice is a single
chain Fv fragment (scFv). See WO 93/16185.
[0213] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a Fab fragment comprising
V.sub.L, C.sub.L, V.sub.H and C.sub.H1 domains. Fab fragments
comprise a variable and constant domain of the light chain and a
variable domain and the first constant domain (C.sub.H1) of the
heavy chain. In some such embodiments, the V.sub.H domain is
selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:
13-SEQ ID NO: 17. In some such embodiments, the V.sub.H domain
comprises one or more heavy chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
and SEQ ID NO: 7. In some such embodiments, the V.sub.L domain is
selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18,
and SEQ ID NO: 19. In some such embodiments, the V.sub.L domain
comprises one or more light chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11,
or SEQ ID NO: 12.
[0214] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a Fab' fragment, which is a Fab
fragment having one or more cysteine residues at the C-terminus of
the C.sub.H1 domain.
[0215] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a Fd fragment comprising
V.sub.H and C.sub.H1 domains. In some such embodiments, the V.sub.H
domain is selected from the group consisting of SEQ ID NO: 4 and
SEQ ID NO: 13-SEQ ID NO: 17. In some such embodiments, the V.sub.H
domain comprises one or more heavy chain CDR regions comprising a
sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID
NO: 6, and SEQ ID NO: 7.
[0216] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a Fd' fragment comprising
V.sub.H and C.sub.H1 domains and one or more cysteine residues at
the C-terminus of the C.sub.H1 domain. In some such embodiments,
the V.sub.H domain is selected from the group consisting of SEQ ID
NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. In some such embodiments,
the V.sub.H domain comprises one or more heavy chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7.
[0217] Single-chain Fv or scFv antibody fragments comprise the
V.sub.H and V.sub.L domains of antibody, such that these domains
are present in a single polypeptide chain. Generally, a Fv
polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains, which enables the scFv to form the
desired structure for antigen binding. For a review of scFv, see
Pluckthun in The Pharmacology of Monoclonal Antibodies, Vol 113,
Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315
(1994). Accordingly, in some embodiments of the aspects described
herein, a human DEspR-specific antibody fragment is a Fv fragment
comprising the V.sub.L and V.sub.H domains of a single arm of an
antibody. In some such embodiments, the V.sub.H domain is selected
from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID
NO: 17. In some such embodiments, the V.sub.H domain comprises one
or more heavy chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7. In some such embodiments, the V.sub.L domain is selected from
the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO:
19. In some such embodiments, the V.sub.L domain comprises one or
more light chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
12.
[0218] The term diabodies refers to small antibody fragments with
two antigen-binding sites, which fragments comprise a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain (V.sub.H and
V.sub.L). By using a linker that is too short to allow pairing
between the two domains on the same chain, the domains are forced
to pair with the complementary domains of another chain and create
two antigen-binding sites. Diabodies are described more fully in,
for example, EP 404,097; WO 93/11161; and Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993).
[0219] Accordingly, in some embodiments of the aspects described
herein, a human DEspR-specific antibody fragment is a diabody
comprising two antigen binding sites, comprising a heavy chain
variable domain (V.sub.H) connected to a light chain variable
domain (V.sub.L) in the same polypeptide chain. In some such
embodiments, the V.sub.H domain is selected from the group
consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. In some
such embodiments, the V.sub.H domain comprises one or more heavy
chain CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some
such embodiments, the V.sub.L domain is selected from the group
consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO: 19. In
some such embodiments, the V.sub.L domain comprises one or more
light chain CDR regions comprising a sequence selected from the
group consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
12.
[0220] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a dAb fragment comprising a
V.sub.H domain. In some such embodiments, the V.sub.H domain is
selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:
13-SEQ ID NO: 17. In some such embodiments, the V.sub.H domain
comprises one or more heavy chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6,
and SEQ ID NO: 7.
[0221] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment comprises isolated CDR regions. In
some such embodiments, the isolated CDR region comprises one or
more heavy chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO:
7. In some such embodiments, the isolated CDR region comprises one
or more light chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:
12.
[0222] In some embodiments of the aspects described herein, the
human DEspR-specific antibody fragment is a F(ab).sub.2 fragment,
which comprises a bivalent fragment comprising two Fab' fragments
linked by a disulphide bridge at the hinge region.
[0223] Linear antibodies refers to the antibodies as described in
Zapata et al., Protein Eng., 8(10):1057-1062 (1995). Briefly, these
antibodies comprise a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1) which, together with
complementary light chain polypeptides, form a pair of antigen
binding regions. Linear antibodies can be bispecific or
monospecific.
[0224] In some embodiments of the aspects described herein, a human
DEspR-specific antibody fragment is a linear antibody comprising a
pair of tandem Fd segments (V.sub.H-C.sub.H1-V.sub.H-C.sub.H1)
which, together with complementary light chain polypeptides, form a
pair of antigen binding regions. In some such embodiments, the
V.sub.H domain is selected from the group consisting of SEQ ID NO:
4 and SEQ ID NO: 13-SEQ ID NO: 17. In some such embodiments, the
V.sub.H domain comprises one or more heavy chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such embodiments,
the V.sub.L domain is selected from the group consisting of SEQ ID
NO: 9, SEQ ID NO: 18, and SEQ ID NO: 19. In some such embodiments,
the V.sub.L domain comprises one or more light chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12.
[0225] In other embodiments of these aspects, a human
DEspR-specific antibody fragment has specificity for the same
epitope as the monoclonal anti-DEspR antibody 7C5B2, described
herein, and produced by hybridoma 7C5B2.
[0226] Some further examples of DEspR-inhibiting antibodies are
described in PCT/US2005/041594, the contents of which are
incorporated herein by reference in their entirety.
Other Amino Acid Sequence Modifications
[0227] In some embodiments of the aspects described herein, amino
acid sequence modification(s) of the antibodies or antibody
fragments thereof specific for DEspR described herein are
contemplated. For example, it can be desirable to improve the
binding affinity and/or other biological properties of the
antibody. Amino acid sequence variants of the antibody are prepared
by introducing appropriate nucleotide changes into the antibody
nucleic acid, or by peptide synthesis. Such modifications include,
for example, deletions from, and/or insertions into and/or
substitutions of, residues within the amino acid sequences of the
antibody. Any combination of deletion, insertion, and substitution
is made to arrive at the final construct, provided that the final
construct possesses the desired characteristics, e.g., binding
specificity, inhibition of biological activity. The amino acid
changes also can alter post-translational processes of the
antibody, such as changing the number or position of glycosylation
sites.
[0228] A useful method for identification of certain residues or
regions of the antibody that are preferred locations for
mutagenesis is called "alanine scanning mutagenesis" as described
by Cunningham and Wells Science, 244:1081-1085 (1989). Here, a
residue or group of target residues are identified (e.g., charged
residues such as arg, asp, his, lys, and glu) and replaced by a
neutral or negatively charged amino acid (most preferably alanine
or polyalanine) to affect the interaction of the amino acids with
antigen. Those amino acid locations demonstrating functional
sensitivity to the substitutions then are refined by introducing
further or other variants at, or for, the sites of substitution.
Thus, while the site for introducing an amino acid sequence
variation is predetermined, the nature of the mutation per se need
not be predetermined. For example, to analyze the performance of a
mutation at a given site, ala scanning or random mutagenesis is
conducted at the target codon or region and the expressed antibody
variants are screened for the desired activity.
[0229] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include antibody with an N-terminal
methionyl residue or the antibody fused to a cytotoxic polypeptide.
Other insertional variants of the antibody molecule include the
fusion to the N- or C-terminus of the antibody to an enzyme (e.g.
for ADEPT) or a polypeptide which increases the serum half-life of
the antibody.
[0230] Another type of variant is an amino acid substitution
variant. These variants have at least one amino acid residue in the
antibody molecule replaced by a different residue. The sites of
greatest interest for substitutional mutagenesis include the
hypervariable regions, but FR alterations are also contemplated for
use in the antibodies or antibody fragments thereof specific for
DEspR described herein.
[0231] Substantial modifications in the biological properties of
the antibodies or antibody fragments thereof specific for DEspR are
accomplished by selecting substitutions that differ significantly
in their effect on maintaining (a) the structure of the polypeptide
backbone in the area of the substitution, for example, as a sheet
or helical conformation, (b) the charge or hydrophobicity of the
molecule at the target site, or (c) the bulk of the side chain.
Amino acids can be grouped according to similarities in the
properties of their side chains (in A. L. Lehninger, in
Biochemistry, second ed., pp. 73-75, Worth Publishers, New York
(1975)): (1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro
(P), Phe (F), Trp (W), Met (M); (2) uncharged polar: Gly (G), Ser
(S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q); (3) acidic: Asp
(D), Glu (E); (4) basic: Lys (K), Arg (R), His (H).
[0232] Alternatively, naturally occurring residues can be divided
into groups based on common side-chain properties: (1) hydrophobic:
Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys,
Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; (6)
aromatic: Trp, Tyr, Phe. Non-conservative substitutions will entail
exchanging a member of one of these classes for another class.
[0233] Any cysteine residue not involved in maintaining the proper
conformation of the antibodies or antibody fragments thereof
specific for DEspR also can be substituted, generally with serine,
to improve the oxidative stability of the molecule and prevent
aberrant crosslinking Conversely, cysteine bond(s) can be added to
the antibody to improve its stability (particularly where the
antibody is an antibody fragment such as an Fv fragment).
[0234] A particularly preferred type of substitutional variant
involves substituting one or more hypervariable region residues of
a parent antibody (e.g., the monoclonal anti-DEspR antibody 7C5B2,
or a humanized or human antibody or antibody fragment thereof
specific for DEspR, as provided herein). Generally, the resulting
variant(s) selected for further development will have improved
biological properties relative to the parent antibody from which
they are generated. A convenient way for generating such
substitutional variants involves affinity maturation using phage
display. Briefly, several hypervariable region sites (e.g., 6-7
sites) are mutated to generate all possible amino substitutions at
each site. The antibody variants thus generated are displayed in a
monovalent fashion from filamentous phage particles as fusions to
the gene III product of M13 packaged within each particle. The
phage-displayed variants are then screened for their biological
activity (e.g. binding affinity) as herein disclosed. In order to
identify candidate hypervariable region sites for modification,
alanine scanning mutagenesis can be performed to identify
hypervariable region residues contributing significantly to antigen
binding.
[0235] Alternatively, or additionally, it can be beneficial to
analyze a crystal structure of the antigen-antibody complex to
identify contact points between the antibody or antibody fragments
thereof specific for DEspR and human DEspR. Such contact residues
and neighboring residues are candidates for substitution according
to the techniques elaborated herein. Once such variants are
generated, the panel of variants is subjected to screening as
described herein and antibodies or antibody fragments thereof with
superior properties in one or more relevant assays can be selected
for further development.
[0236] Another type of amino acid variant of the antibody alters
the original glycosylation pattern of the antibody. By altering is
meant deleting one or more carbohydrate moieties found in the
antibody, and/or adding one or more glycosylation sites that are
not present in the antibody.
[0237] Glycosylation of antibodies is typically either N-linked or
O-linked N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tripeptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tripeptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-aceylgalactosamine, galactose, or xylose to a hydroxyamino
acid, most commonly serine or threonine, although 5-hydroxyproline
or 5-hydroxylysine can also be used.
[0238] Addition of glycosylation sites to the antibodies or
antibody fragments thereof specific for DEspR is accomplished by
altering the amino acid sequence such that it contains one or more
of the above-described tripeptide sequences (for N-linked
glycosylation sites). The alteration can also be made by the
addition of, or substitution by, one or more serine or threonine
residues to the sequence of the original antibody (for O-linked
glycosylation sites).
[0239] Where the antibody comprises an Fc region, the carbohydrate
attached thereto can be altered. For example, antibodies with a
mature carbohydrate structure that lacks fucose attached to an Fc
region of the antibody are described in US Pat Appl No US
2003/0157108 A1, Presta, L. See also US 2004/0093621 A1 (Kyowa
Hakko Kogyo Co., Ltd). Antibodies with a bisecting
N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc
region of the antibody are referenced in WO03/011878, Jean-Mairet
et al. and U.S. Pat. No. 6,602,684, Umana et al. Antibodies with at
least one galactose residue in the oligosaccharide attached to an
Fc region of the antibody are reported in WO97/30087, Patel et al.
See, also, WO98/58964 (Raju, S.) and WO99/22764 (Raju, S.)
concerning antibodies with altered carbohydrate attached to the Fc
region thereof.
[0240] In some embodiments, it can be desirable to modify the
antibodies or antibody fragments thereof specific for DEspR
described herein with respect to effector function, e.g., so as to
enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This can
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody or antibody fragment thereof.
Alternatively or additionally, cysteine residue(s) can be
introduced in the Fc region, thereby allowing interchain disulfide
bond formation in this region. The homodimeric antibody thus
generated can have improved internalization capability and/or
increased complement-mediated cell killing and antibody-dependent
cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med.
176:1191-1195 (1992) and Shopes, B. J. Immunol. 148:2918-2922
(1992). Homodimeric antibodies with enhanced anti-tumor activity
can also be prepared using heterobifunctional cross-linkers as
described in Wolff et al. Cancer Research 53:2560-2565 (1993).
Alternatively, an antibody can be engineered which has dual Fc
regions and can thereby have enhanced complement lysis and ADCC
capabilities. See Stevenson et al. Anti-Cancer Drug Design
3:219-230 (1989).
[0241] For example, WO00/42072 (Presta, L.) describes antibodies
with improved ADCC function in the presence of human effector
cells, where the antibodies comprise amino acid substitutions in
the Fc region thereof. Preferably, the antibody with improved ADCC
comprises substitutions at positions 298, 333, and/or 334 of the Fc
region (Eu numbering of residues). Preferably the altered Fc region
is a human IgG1 Fc region comprising or consisting of substitutions
at one, two or three of these positions. Such substitutions are
optionally combined with substitution(s) which increase C1q binding
and/or CDC.
[0242] Antibodies with altered C1q binding and/or complement
dependent cytotoxicity (CDC) are described in WO99/51642, U.S. Pat.
No. 6,194,551B1, U.S. Pat. No. 6,242,195B1, U.S. Pat. No.
6,528,624B1 and U.S. Pat. No. 6,538,124 (Idusogie et al.). The
antibodies comprise an amino acid substitution at one or more of
amino acid positions 270, 322, 326, 327, 329, 313, 333 and/or 334
of the Fc region thereof (Eu numbering of residues).
[0243] To increase the serum half life of the antibody specific for
DEspR described herein, one can incorporate a salvage receptor
binding epitope into the antibody (especially an antibody fragment)
as described in U.S. Pat. No. 5,739,277, for example. As used
herein, the term "salvage receptor binding epitope" refers to an
epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0244] Antibodies with improved binding to the neonatal Fc receptor
(FcRn), and increased half-lives, are described in WO00/42072
(Presta, L.) and US2005/0014934A1 (Hinton et al.). These antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. For example, the Fc
region can have substitutions at one or more of positions 238, 250,
256, 265, 272, 286, 303, 305, 307, 311, 312, 314, 317, 340, 356,
360, 362, 376, 378, 380, 382, 413, 424, 428 or 434 (Eu numbering of
residues). The preferred Fc region-comprising antibody variant with
improved FcRn binding comprises amino acid substitutions at one,
two or three of positions 307, 380 and 434 of the Fc region thereof
(Eu numbering of residues). In one embodiment, the antibody has
307/434 mutations.
[0245] Engineered antibodies specific for DEspR with three or more
(preferably four) functional antigen binding sites are also
contemplated (US Appln No. US2002/0004587 A1, Miller et al.).
[0246] Nucleic acid molecules encoding amino acid sequence variants
of the antibody are prepared by a variety of methods known in the
art. These methods include, but are not limited to, isolation from
a natural source (in the case of naturally occurring amino acid
sequence variants) or preparation by oligonucleotide-mediated (or
site-directed) mutagenesis, PCR mutagenesis, and cassette
mutagenesis of an earlier prepared variant or a non-variant version
of the antibody.
Immunoconjugates
[0247] In some embodiments of these aspects, immunoconjugates
comprising the antibody and antibody fragments specific for DEspR
described herein are conjugated to an agent such as a
chemotherapeutic agent, toxin (e.g. an enzymatically active toxin
of bacterial, fungal, plant or animal origin, or fragments
thereof), a small molecule, an siRNA, a nanoparticle, a targeting
agent (e.g., a microbubble), or a radioactive isotope (i.e., a
radioconjugate) can be used. Such immunoconjugates can be used, for
example, in diagnostic, theranostic, or targeting methods.
[0248] Chemotherapeutic agents useful in the generation of such
immunoconjugates are described herein. Enzymatically active toxins
and fragments thereof which can be used include diphtheria A chain,
nonbinding active fragments of diphtheria toxin, exotoxin A chain
(from Pseudomonas aeruginosa), ricin A chain, abrin A chain,
modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin
proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S),
momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin and the tricothecenes. A variety of
radionuclides are available for the production of radioconjugate
antibodies. Examples include .sup.212Bi, .sup.131I, .sup.131In,
.sup.90Y and .sup.186Re.
[0249] Conjugates of the antibodies specific for DEspR described
herein and a cytotoxic agent can be made using any of a variety of
bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCL), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutareldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as tolyene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al. Science 238: 1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026.
[0250] In other embodiments, the DEspR-specific antibody or
antibody fragment thereof can be conjugated to a "receptor" (such
as, for example, streptavidin) for utilization in tumor
pretargeting wherein the antibody-receptor conjugate is
administered to the subject, followed by removal of unbound
conjugate from the circulation using a clearing agent and then
administration of a "ligand" (e.g. avidin) which is conjugated to a
cytotoxic agent (e.g. a radionucleotide). In some embodiments, the
DEspR-specific antibody or antibody fragment thereof can be
conjugated to biotin, and the biotin conjugated antibody or
antibody fragment thereof can be further conjugated or linked to a
streptavidin-bound or -coated agent, such as a streptavidin-coated
microbubble, for use in, for example, molecular imaging of
angiogenesis.
Immunoliposomes
[0251] The antibodies and antibody fragments thereof specific for
DEspR described herein can also be formulated as immunoliposomes.
Liposomes containing the antibody are prepared by methods known in
the art, such as described in Epstein et al., Proc. Natl. Acad.
Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA,
77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.
Liposomes with enhanced circulation time are disclosed in U.S. Pat.
No. 5,013,556.
[0252] Particularly useful liposomes can be generated, for example,
by the reverse phase evaporation method with a lipid composition
comprising phosphatidylcholine, cholesterol and PEG-derivatized
phosphatidylethanolamine (PEG-PE). Liposomes are extruded through
filters of defined pore size to yield liposomes with the desired
diameter. Fab' fragments of the antibody of the invention can be
conjugated to the liposomes as described in Martin et al. J. Biol.
Chem. 257: 286-288 (1982) via a disulfide interchange reaction. A
chemotherapeutic agent is optionally contained within the liposome.
See Gabizon et al. J. National Cancer Inst. 81(19)1484 (1989)
[0253] The hybridoma cell lines 7C5B2, 7C5C5, and 5G12E8 are being
maintained and stored.
Compositions and Therapeutic & Diagnostic Uses of Anti-DEspR
Antibodies and Fragments Thereof
[0254] Certain aspects described herein are based, in part, on the
discovery by the inventors that DEspR plays contributes to adult
tissue vascularity, as well as playing a critical role in
angiogenesis during embryonic development, and further that DEspR
is surprisingly expressed in certain tumor cells, cancer stem cells
or stem-like cells, or tumor initiating cells, as well as in
tumor-surrounding blood vessels' endothelial cells, pericytes, and
smooth muscle cells. The inventors further discovered that
inhibition of DEspR, using DEspR-specific inhibitors, such as the
anti-DEspR antibodies and antibody fragments thereof described
herein, can inhibit a variety of parameters that characterize tumor
metastasis, including cell invasiveness, tumor growth, such as
tumor volume or tumor mass, as well as parameters that characterize
angiogenesis, including neovessel tube length, neovessel branching,
and formation of vessel interconnections. The anti-DEspR antibodies
and antibody fragments thereof described herein are further highly
suitable for antibody-target sonoporation and demonstrate enhanced
penetration and efficacy when administered using, for example,
ultrasound methods. In addition, the inventors have determined that
DEspR serves as a diagnostic marker for a variety of disease
conditions.
Anti-Angiogenic Therapeutics and Treatments
[0255] Angiogenesis is a process of tissue vascularization that
involves both the growth of new developing blood vessels into a
tissue (neo-vascularization) and co-opting of existing blood
vessels to a target site. Blood vessels are the means by which
oxygen and nutrients are supplied to living tissues and waste
products are removed from living tissue. Angiogenesis can be a
critical biological process. For example, angiogenesis is essential
in reproduction, development and wound repair. Conversely,
inappropriate angiogenesis can have severe negative consequences.
For example, it is only after solid tumors are vascularized as a
result of angiogenesis that the tumors have a sufficient supply of
oxygen and nutrients that permit it to grow rapidly and
metastasize.
[0256] Where the growth of new blood vessels is the cause of, or
contributes to, the pathology associated with a disease, inhibition
of angiogenesis, using the compositions and methods described
herein, can reduce the deleterious effects of the disease.
Non-limiting examples include tumors, carotid artery disease,
rheumatoid arthritis, diabetic retinopathy, inflammatory diseases,
restenosis, and the like. Where the growth of new blood vessels is
required to support growth of a deleterious tissue, inhibition of
angiogenesis, using the compositions and methods described herein,
can reduce the blood supply to the tissue and thereby contribute to
reduction in tissue mass based on blood supply requirements.
Non-limiting examples include growth of tumors where
neovascularization is a continual requirement in order that the
tumor growth beyond a few millimeters in thickness, and for the
establishment of solid tumor metastases. Another example is
coronary plaque enlargement.
[0257] There are a variety of diseases or disorders in which
angiogenesis is believed to lead to negative consequences, referred
to as pathological angiogenesis, or diseases or disorders dependent
or modulated by angiogenesis, including but not limited to,
inflammatory disorders such as immune and non-immune inflammation,
chronic articular rheumatism and psoriasis, disorders associated
with inappropriate or inopportune invasion of vessels such as
diabetic retinopathy, neovascular glaucoma, restenosis, capillary
proliferation in atherosclerotic plaques and osteoporosis, and
cancer associated disorders, such as solid tumors, solid tumor
metastases, angiofibromas, retrolental fibroplasia, hemangiomas,
Kaposi sarcoma and the like cancers which require
neovascularization to support tumor growth. In a preferred
embodiment of the aspects described herein, the methods are
directed to inhibiting angiogenesis in a subject with cancer.
[0258] The antibodies and antibody fragments specific for DEspR
described herein, such as, for example the anti-DEspR 7C5B2
antibody; an anti-DEspR antibody comprising one or more heavy chain
CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; an
anti-DEspR antibody comprising one or more light chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; an anti-DEspR composite
human antibody comprising a variable heavy (V.sub.H) chain amino
acid sequence selected from the group consisting of SEQ ID NO: 4
and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite human
antibody comprising a variable light (V.sub.L) chain amino acid
sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID
NO: 18, and SEQ ID NO: 19, and fragments thereof, can be used in
compositions and methods of antiangiogenic therapy. These
antiangiogenic therapies can be used as novel cancer treatment
strategies aimed at inhibiting existing tumor blood vessels and
development of tumor blood vessels required for providing nutrients
to support tumor growth. Because angiogenesis is involved in both
primary tumor growth and metastasis, the antiangiogenic treatments
using the antibodies and antibody fragments specific for DEspR
described herein are capable of inhibiting the neoplastic growth of
tumor at the primary site, as well as preventing micro- and
macro-metastasis of tumors at the secondary sites, therefore
allowing attack of the tumors by other therapeutics.
[0259] Additionally, the antibodies and antibody-fragments specific
for DEspR described herein, such as, for example the anti-DEspR
7C5B2 antibody; an anti-DEspR antibody comprising one or more heavy
chain CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; an
anti-DEspR antibody comprising one or more light chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; an anti-DEspR composite
human antibody comprising a variable heavy (V.sub.H) chain amino
acid sequence selected from the group consisting of SEQ ID NO: 4
and SEQ ID NO: 13-SEQ ID NO: 17; or an anti-DEspR composite human
antibody comprising a variable light (V.sub.L) chain amino acid
sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID
NO: 18, and SEQ ID NO: 19, and fragments thereof, can be used in
methods of antimetastasis therapy. Such antimetastasis therapies
provide novel cancer treatment strategies aimed at inhibiting
concurrent inhibition of tumor vascularization and tumor cell
invasiveness for treatment and/or inhibition of micrometastasis and
macrometastasis, as further described herein. Furthermore, since
DEspR is also expressed in tumor cells, including cancer stem
cells, as demonstrated herein, immunoconjugates of DEspR specific
antibodies or antibody fragments thereof, as described herein, can
be generated by conjugation to any agent such as a toxin, cytotoxic
or pro-apoptotic agent, and can further inhibit tumor growth by
directly targeting/killing tumor cells and cancer stem cells.
[0260] Accordingly, angiogenesis-dependent diseases and disorders
that can be treated using the methods and compositions comprising
antibodies and antibody fragments specific for DEspR described
herein, such as, for example, the anti-DEspR 7C5B2 antibody; an
anti-DEspR antibody comprising one or more heavy chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7; an anti-DEspR antibody
comprising one or more light chain CDR regions comprising a
sequence selected from the group consisting of SEQ ID NO: 10, SEQ
ID NO: 11, or SEQ ID NO: 12; an anti-DEspR composite human antibody
comprising a variable heavy (V.sub.H) chain amino acid sequence
selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO:
13-SEQ ID NO: 17; or an anti-DEspR composite human antibody
comprising a variable light (V.sub.L) chain amino acid sequence
selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18,
and SEQ ID NO: 19, and fragments thereof, are those diseases and
disorders affected by vascular growth. In other words, an
"angiogenesis-dependent disease or disorder" refers to those
diseases or disorders that are dependent on a rich blood supply and
blood vessel proliferation for the diseases' pathological
progression (e.g., metastatic tumors), or diseases or disorders
that are the direct result of aberrant blood vessel proliferation
(e.g., diabetic retinopathy and hemangiomas).
[0261] Non-limiting examples of angiogenesis-dependent diseases or
disorder that can be treated using the compositions and methods
described herein include abnormal vascular proliferation, ascites
formation, psoriasis, age-related macular degeneration, thyroid
hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis,
carotid artery disease, vaso vasorum neovascularization, vulnerable
plaque neovascularization, neurodegenerative disorders, Alzheimer's
disease, obesity, pleural effusion, atherosclerosis, endometriosis,
diabetic/other retinopathies, ocular neovascularizations such as
neovascular glaucoma and corneal neovascularization, disorders
associated with inappropriate or inopportune invasion of vessels
such as diabetic retinopathy, macular degeneration, neovascular
glaucoma, restenosis, capillary proliferation in atherosclerotic
plaques and osteoporosis, and cancer associated disorders, such as
solid tumors, solid tumor metastases, angiofibromas, retrolental
fibroplasia, hemangiomas, Kaposi sarcoma, cancers which require
neovascularization to support tumor growth, etc.
[0262] Accordingly, described herein are methods of inhibiting
angiogenesis in a tissue of a subject or individual having a
disease or disorder dependent or modulated by angiogenesis, where
the disease or disorder can be treated by the inhibition of
angiogenesis. Generally, the methods comprise administering to the
subject a therapeutically effective amount of a composition
comprising an angiogenesis-inhibiting amount of a DEspR inhibitor.
In some embodiments, the methods further comprises selecting or
diagnosing a subject having or at risk for a disease or disorder
modulated by angiogenesis.
[0263] In some embodiments of these methods and all such methods
described herein, the DEspR inhibitor is an antibody or antibody
fragment thereof. Accordingly, in some aspects, an anti-DEspR
antibody or antibody-fragment thereof that is specific for a DEspR
target is provided, where the anti-DEspR antibody or
antibody-fragment thereof specifically binds to the DEspR target
and reduces or inhibits DEspR biological activity, thus inhibiting
angioegensis in the subject having a disease or disorder dependent
on angiogenesis.
[0264] In some such embodiments, the DEspR is human DEspR. In some
such embodiments, the DEspR target has a sequence comprising SEQ ID
NO:1 or an allelic variant thereof. In some such embodiments of
these methods, an antibody or antibody fragment thereof that
specifically binds to DEspR and inhibits DEspR biological activity
blocks interaction of DEspR with VEGFsp. In some such embodiments,
the VEGFsp has a sequence comprising the sequence of SEQ ID NO:2.
In some such embodiments, the antibody or antibody fragment thereof
is specific for an epitope of DEspR comprising an extracellular
portion of DEspR. In some embodiments, the antibody or antibody
fragment thereof is specific for an epitope of DEspR comprising
amino acids 1-9 of SEQ ID NO:1.
[0265] In some such embodiments of these compositions and methods
for inhibiting angiogenesis, the anti-DEspR antibody or
antibody-fragment thereof is the anti-DEspR 7C5B2 antibody or
fragment thereof. In some such embodiments, the anti-DEspR antibody
or antibody-fragment thereof comprises one or more heavy chain CDR
regions comprising a sequence selected from the group consisting of
SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. In some such
embodiments, the anti-DEspR antibody or antibody-fragment thereof
comprises one or more light chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11,
or SEQ ID NO: 12. In some such embodiments, the anti-DEspR antibody
or antibody-fragment thereof comprises one or more heavy chain CDR
regions comprising a sequence selected from the group consisting of
SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7, and one or more light
chain CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In
some such embodiments, the anti-DEspR antibody or antibody-fragment
thereof comprises a variable heavy (V.sub.H) chain amino acid
sequence selected from the group consisting of SEQ ID NO: 4 and SEQ
ID NO: 13-SEQ ID NO: 17. In some such embodiments, the anti-DEspR
antibody or antibody-fragment thereof comprises a variable light
(V.sub.L) chain amino acid sequence selected from the group
consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO: 19, and
fragments thereof.
[0266] In other embodiments of these compositions and methods for
inhibiting angiogenesis, monoclonal anti-DEspR antibodies or
antibody fragments thereof that specifically bind to DEspR are
provided having one or more biological characteristics of the 7C5B2
monoclonal antibody. In some such embodiments, having a biological
characteristic of the 7C5B2 monoclonal antibody can include having
an ED.sub.50 value (i.e., the dose therapeutically effective in 50%
of the population) at or around the ED.sub.50 value of the 7C5B2
antibody for the given population; or having an EC.sub.50 value
(i.e., the dose that achieves a half-maximal inhibition of a given
parameter or phenotype) at or around the EC.sub.50 value of the
7C5B2 antibody for a given parameter or phenotype. For example, in
some embodiments of these aspects, the given parameter or phenotype
to be inhibited by the antibody that specifically binds to DEspR
can include, but is not limited to, the mean total tube number in
an in vitro tubulogenesis assay, the mean total tube length in an
in vitro tubulogenesis assay, the mean number of branching points
in an in vitro tubulogenesis assay, the mean number of vessel
connections in an in vitro tubulogenesis assay, and tumor cell
invasiveness.
[0267] In some embodiments these compositions and methods for
inhibiting angiogenesis, a humanized anti-DEspR monoclonal antibody
or antibody fragment thereof is provided for use in the
compositions and methods for inhibiting angiogenesis as described
herein. In some embodiments, one or more variable heavy chain CDR
regions of the humanized anti-DEspR antibody or antibody fragment
thereof comprises a sequence selected from the group consisting of
SEQ ID NO: 5-SEQ ID NO: 7. In some embodiments, one or more
variable light chain CDR regions of the humanized anti-DEspR
antibody or antibody fragment thereof comprises a sequence selected
from the group consisting of SEQ ID NO: 10-SEQ ID NO: 12. In some
such embodiments, the anti-DEspR antibody or antibody-fragment
thereof comprises one or more heavy chain CDR regions comprising a
sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID
NO: 6, or SEQ ID NO: 7, and one or more light chain CDR regions
comprising a sequence selected from the group consisting of SEQ ID
NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12. In some embodiments, the
humanized anti-DEspR monoclonal antibody comprises mutated human
IgG1 framework regions and antigen-binding
complementarity-determining regions (CDRs) selected from the group
consisting of SEQ ID NO: 5-SEQ ID NO: 7 and the group consisting of
SEQ ID NO: 10-SEQ ID NO: 12, that blocks binding of human DEspR to
its ligands. In some embodiments, the humanized anti-DEspR antibody
comprises mutated human IgG4 framework regions and antigen-binding
complementarity-determining regions (CDRs) from the selected from
the group consisting of SEQ ID NO: 5-SEQ ID NO: 7 and the group
consisting of SEQ ID NO: 10-SEQ ID NO: 12, that blocks binding of
human DEspR to its ligands.
[0268] In other embodiments of these aspects, the anti-DEspR
antibody is an antibody fragment having specificity for the same
epitope as the monoclonal anti-DEspR antibody 7C5B2, described
herein, and produced by hybridoma 7C5B2. In some such embodiments,
the anti-DEspR antibody is an antibody fragment comprising one or
more variable heavy chain CDR sequences selected from the group
consisting of SEQ ID NO: 5-SEQ ID NO: 7 and/or one or more variable
light chain CDR sequences selected from the group consisting of SEQ
ID NO: 10-SEQ ID NO: 12 of the 7C5B2 monoclonal antibody. In some
embodiments, the antibody fragment is a Fab fragment. In some
embodiments, the anti-DEspR antibody fragment is a Fab' fragment.
In some embodiments, the anti-DEspR antibody fragment is a Fd
fragment. In some embodiments, the anti-DEspR antibody fragment is
a Fd' fragment. In some embodiments, the antibody fragment is a Fv
fragment. In some embodiments, the anti-DEspR antibody fragment is
a dAb fragment. In some embodiments, the anti-DEspR antibody
fragment comprises isolated CDR regions. In some embodiments, the
anti-DEspR antibody fragment is a F(ab').sub.2 fragment. In some
embodiments, the anti-DEspR antibody fragment is a single chain
antibody molecule. In some embodiments, the anti-DEspR antibody
fragment is a diabody comprising two antigen binding sites. In some
embodiments, the anti-DEspR antibody fragment is a linear antibody
comprising a pair of tandem Fd segments
(V.sub.H-C.sub.H1-V.sub.H-C.sub.H1).
[0269] Accordingly, in some aspects, the disease or disorder
dependent or modulated by angiogenesis is cancer, where the rapidly
dividing neoplastic cancer cells require an efficient blood supply
to sustain their continual growth of the tumor. Inhibition of
angiogenesis or tumor cell invasiveness or a combination thereof
using the compositions and therapeutic methods described herein at
the primary tumor site and secondary tumor site serve to prevent
and limit metastasis and progression of disease.
[0270] Accordingly, in some aspects, provided herein are methods to
treat a subject having or at risk for a cancer or tumor comprising
administering an effective amount of an anti-DEspR antibody or
antibody fragment thereof. In some such embodiments of these
methods for treating cancer, the anti-DEspR antibody or antibody
fragment thereof is the anti-DEspR 7C5B2 antibody or fragment
thereof. In some such embodiments, the anti-DEspR antibody or
antibody-fragment thereof comprises one or more heavy chain CDR
regions comprising a sequence selected from the group consisting of
SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7. In some such
embodiments, the anti-DEspR antibody or antibody-fragment thereof
comprises one or more light chain CDR regions comprising a sequence
selected from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11,
and SEQ ID NO: 12. In some such embodiments, the anti-DEspR
antibody or antibody-fragment thereof comprises one or more heavy
chain CDR regions comprising a sequence selected from the group
consisting of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 and one
or more light chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID
NO: 12. In some such embodiments, the anti-DEspR antibody or
antibody-fragment thereof comprises a variable heavy (V.sub.H)
chain amino acid sequence selected from the group consisting of SEQ
ID NO: 4 and SEQ ID NO: 13-SEQ ID NO: 17. In some such embodiments,
the anti-DEspR antibody or antibody-fragment thereof comprises a
variable light (V.sub.L) chain amino acid sequence selected from
the group consisting of SEQ ID NO: 9, SEQ ID NO: 18, and SEQ ID NO:
19, and fragments thereof.
[0271] In some embodiments, the methods can further comprise first
selecting or diagnosing the subject having or at risk for a cancer
or tumor. In some such embodiments, the diagnosis of the subject
can comprise administering to the subject an anti-DEspR antibody or
antibody fragment thereof coupled to a label, for example, a
radioactive label, or a label used for molecular imaging, as
described elsewhere herein. In such embodiments, detection of the
labeled anti-DEspR antibody or antibody fragment is indicative of
the subject having a cancer or tumor.
[0272] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Included in this definition are benign
and malignant cancers, as well as dormant tumors or
micrometastases. Accordingly, the terms "cancer" or "tumor" as used
herein refers to an uncontrolled growth of cells which interferes
with the normal functioning of the bodily organs and systems,
including cancer stem cells and tumor vascular niches. A subject
that has a cancer or a tumor is a subject having objectively
measurable cancer cells present in the subject's body. Included in
this definition are benign and malignant cancers, as well as
dormant tumors or micrometastases. Cancers which migrate from their
original location and seed vital organs can eventually lead to the
death of the subject through the functional deterioration of the
affected organs. Hematopoietic cancers, such as leukemia, are able
to out-compete the normal hematopoietic compartments in a subject,
thereby leading to hematopoietic failure (in the form of anemia,
thrombocytopenia and neutropenia) ultimately causing death.
[0273] By "metastasis" is meant the spread of cancer from its
primary site to other places in the body. Cancer cells can break
away from a primary tumor, penetrate into lymphatic and blood
vessels, circulate through the bloodstream, and grow in a distant
focus (metastasize) in normal tissues elsewhere in the body.
Metastasis can be local or distant. Metastasis is a sequential
process, contingent on tumor cells breaking off from the primary
tumor, traveling through the bloodstream, and stopping at a distant
site. At the new site, the cells establish a blood supply and can
grow to form a life-threatening mass. Both stimulatory and
inhibitory molecular pathways within the tumor cell regulate this
behavior, and interactions between the tumor cell and host cells in
the distant site are also significant.
[0274] Metastases are most often detected through the sole or
combined use of magnetic resonance imaging (MRI) scans, computed
tomography (CT) scans, blood and platelet counts, liver function
studies, chest X-rays and bone scans in addition to the monitoring
of specific symptoms.
[0275] Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include, but are not limited
to, basal cell carcinoma, biliary tract cancer; bladder cancer;
bone cancer; brain and CNS cancer; breast cancer; cancer of the
peritoneum; cervical cancer; choriocarcinoma; colon and rectum
cancer; connective tissue cancer; cancer of the digestive system;
endometrial cancer; esophageal cancer; eye cancer; cancer of the
head and neck; gastric cancer (including gastrointestinal cancer);
glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial
neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver
cancer; lung cancer (e.g., small-cell lung cancer, non-small cell
lung cancer, adenocarcinoma of the lung, and squamous carcinoma of
the lung); lymphoma including Hodgkin's and non-Hodgkin's lymphoma;
melanoma; myeloma; neuroblastoma; glioblastoma; oral cavity cancer
(e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic
cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal
cancer; cancer of the respiratory system; salivary gland carcinoma;
sarcoma; skin cancer; squamous cell cancer; stomach cancer;
testicular cancer; thyroid cancer; uterine or endometrial cancer;
cancer of the urinary system; vulval cancer; as well as other
carcinomas and sarcomas; as well as B-cell lymphoma (including low
grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic
(SL) NHL; intermediate grade/follicular NHL; intermediate grade
diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic
NHL; high grade small non-cleaved cell NHL; bulky disease NHL;
mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome.
[0276] In other aspects, the compositions and methods described
herein are used in the treatment or inhibition or imaging of
artherosclerotic plaques and atherosclerosis. Atherosclerosis is
the most common form of vascular disease and is a disorder of large
arteries that underlies most coronary artery disease, aortic
aneurysm, cerebrovascular disease and arterial disease of lower
extremities (Libby, in "The Principles of Internal Medicine", 15th
ed., Braunward et al. (editors), Saunders, Philadelphia, Pa., 2001,
pp. 1377-1382.). The pathogenesis of atherosclerosis occurs as a
reaction to injury (Libby, in "The Principles of Internal
Medicine", 15th ed., Braunward et al. (editors), Saunders,
Philadelphia, Pa., 2001, pp. 1377-1382.). The injury to the
endothelium can be subtle, resulting in a loss of the ability of
the cells to function normally. Examples of types of injury to the
endothelium include hypercholesterolemia and mechanical stress
(Ross, 1999, N. Engl. J. Med., 340:115).
[0277] The process of atherosclerosis involves inflammation, and
white blood cells (e.g., lymphocytes, monocytes, and macrophages)
are often present throughout the development of atherosclerosis.
Atherosclerosis begins when monocytes are activated and move out of
the bloodstream into the wall of an artery. There, they are
transformed into foam cells, which collect cholesterol and other
fatty materials. In time, these fat-laden foam cells accumulate and
form atheromas in the lining of the artery's wall, causing a
thickening and hardening of the wall. Atheromas can be scattered
throughout medium-sized and large arteries, but usually form where
the arteries branch. Treatment of and diagnosis of atherosclerosis
is important because it often leads to heart disease and can also
cause stroke or other vascular problems such as claudication.
[0278] Accordingly, in some embodiments of the aspects described
herein, pathological angiogenesis in atherosclerotic plaques and in
the vasa vasorum of atherosclerotic arteries (coronary and carotid
artery disease) is considered a risk and/or causal factor for
vulnerable plaque progression and disruption. Thus, in some such
embodiments, a subject having an angiogenic disorder to be treated
using the compositions and methods described herein can have or be
at risk for atherosclerosis. As used herein, "atherosclerosis"
refers to a disease of the arterial blood vessels resulting in the
hardening of arteries caused by the formation of multiple
atheromatous plaques within the arteries. Atherosclerosis can be
associated with other disease conditions, including but not limited
to, coronary heart disease events, cerebrovascular events, acute
coronary syndrome, and intermittent claudication. For example,
atherosclerosis of the coronary arteries commonly causes coronary
artery disease, myocardial infarction, coronary thrombosis, and
angina pectoris. Atherosclerosis of the arteries supplying the
central nervous system frequently provokes strokes and transient
cerebral ischemia. In the peripheral circulation, atherosclerosis
causes intermittent claudication and gangrene and can jeopardize
limb viability. Atherosclerosis of an artery of the splancnic
circulation can cause mesenteric ischemia. Atherosclerosis can also
affect the kidneys directly (e.g., renal artery stenosis). Also,
persons who have previously experienced one or more non-fatal
atherosclerotic disease events are those for whom the potential for
recurrence of such an event exists.
[0279] Sometimes these other diseases can be caused by or
associated with other than atherosclerosis. Therefore, in some
embodiments, one first diagnoses that atherosclerosis is present
prior to administering the compositions described herein to the
subject. A subject is "diagnosed with atherosclerosis" or "selected
as having atherosclerosis" if at least one of the markers of
symptoms of atherosclerosis is present. In one such embodiment, the
subject is "selected" if the person has a family history of
atherosclerosis or carries a known genetic mutation or polymorphism
for high cholesterol. In one embodiment, a subject is diagnosed by
measuring an increase level of C-reactive protein (CRP) in the
absence of other inflammatory disorders. In other embodiments,
atherosclerosis is diagnosed by measuring serum levels of
homocysteine, fibrinogen, lipoprotein (a), or small LDL particles.
Alternatively a computed tomography scan, which measures calcium
levels in the coronary arteries, can be used to select a subject
having atherosclerosis. In one embodiment, atherosclerosis is
diagnosed by an increase in inflammatory cytokines. In one
embodiment, increased interleukin-6 levels is used as an indicator
to select an individual having atherosclerosis. In other
embodiments, increased interleukin-8 and/or interleukin-17 level is
used as an indicator to select an individual having
atherosclerosis.
[0280] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in age-related macular degeneration. It is known, for example, that
VEGF contributes to abnormal blood vessel growth from the choroid
layer of the eye into the retina, similar to what occurs during the
wet or neovascular form of age-related macular degeneration.
Macular degeneration, often called AMD or ARMD (age-related macular
degeneration), is the leading cause of vision loss and blindness in
Americans aged 65 and older. New blood vessels grow
(neovascularization) beneath the retina and leak blood and fluid.
This leakage causes permanent damage to light-sensitive retinal
cells, which die off and create blind spots in central vision or
the macula. Accordingly, encompassed in the methods disclosed
herein are subjects treated for age-related macular degeneration
with anti-angiogenic therapy.
[0281] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in a subject having diabetic retinopathy, where abnormal blood
vessel growth is associated with diabetic eye diseases and diabetic
macular edema. When normal blood vessels in the retina are damaged
by tiny blood clots due to diabetes, a chain reaction is ignited
that culminates in new blood vessel growth. However, the backup
blood vessels are faulty; they leak (causing edema), bleed and
encourage scar tissue that detaches the retina, resulting in severe
loss of vision. Such growth is the hallmark of diabetic
retinopathy, the leading cause of blindness among young people in
developed countries. Therefore, encompassed in the methods
disclosed herein are subjects treated for diabetic retinopathy
and/or diabetic macular edema.
[0282] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in a subject having rheumatoid arthritis. Rheumatoid arthritis (RA)
is characterized by synovial tissue swelling, leukocyte ingress and
angiogenesis, or new blood vessel growth. The expansion of the
synovial lining of joints in rheumatoid arthritis (RA) and the
subsequent invasion by the pannus of underlying cartilage and bone
necessitate an increase in the vascular supply to the synovium, to
cope with the increased requirement for oxygen and nutrients.
Angiogenesis is now recognized as a key event in the formation and
maintenance of the pannus in RA (Paleolog, E. M., Arthritis Res.
2002; 4 Suppl 3:S81-90; Afuwape A O, Histol Histopathol. 2002;
17(3):961-72). Even in early RA, some of the earliest histological
observations are blood vessels. A mononuclear infiltrate
characterizes the synovial tissue along with a luxuriant
vasculature. Angiogenesis is integral to formation of the
inflammatory pannus and without angiogenesis, leukocyte ingress
could not occur (Koch, A. E., Ann. Rheum. Dis. 2000, 59 Suppl
1:165-71). Disruption of the formation of new blood vessels would
not only prevent delivery of nutrients to the inflammatory site, it
could also reduce joint swelling due to the additional activity of
VEGF, a potent proangiogenic factor in RA, as a vascular
permeability factor. Anti-VEGF hexapeptide RRKRRR (dRK6) can
suppress and mitigate the arthritis severity (Seung-Ah Yoo, et.
al., 2005, supra). Accordingly, encompassed in the methods
disclosed herein are subjects having or being treated for
rheumatoid arthritis.
[0283] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in Alzheimer's disease. Alzheimer's disease (AD) is the most common
cause of dementia worldwide. AD is characterized by an excessive
cerebral amyloid deposition leading to degeneration of neurons and
eventually to dementia. The exact cause of AD is still unknown. It
has been shown by epidemiological studies that long-term use of
non-steroidal anti-inflammatory drugs, statins, histamine
H2-receptor blockers, or calcium-channel blockers, all of which are
cardiovascular drugs with an anti-angiogenic effects, seem to
prevent Alzheimer's disease and/or influence the outcome of AD
patients. Therefore, AD angiogenesis in the brain vasculature can
play an important role in AD. In Alzheimer's disease, the brain
endothelium secretes the precursor substrate for the beta-amyloid
plaque and a neurotoxic peptide that selectively kills cortical
neurons. Moreover, amyloid deposition in the vasculature leads to
endothelial cell apoptosis and endothelial cell activation which
leads to neovascularization. Vessel formation could be blocked by
the VEGF antagonist SU 4312 as well as by statins, indicating that
anti-angiogenesis strategies can interfere with endothelial cell
activation in AD (Schultheiss C., el. al., 2006; Grammas P., et.
al., 1999) and can be used for preventing and/or treating AD.
Accordingly, encompassed in the methods disclosed herein are
subjects being treated for Alzheimer's disease.
[0284] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in ischemic regions in the brain, which can contribute to edema,
leaky neovessels, and predispose a subject to hemorrhagic
transformation after an ischemic stroke event, thus worsening the
morbidity and mortality risk from the stroke event. Inhibition of
leaky angiogenic neovessels using the compositions and methods
described herein can reduce neurologic deficits from an ischemic
stroke event, as well as prevent the progression to hemorrhagic
stroke. Currently, there is no therapy for ischemic hemorrhagic
transformation nor effective therapies to reduce the neurologic
deficits from stroke.
[0285] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in obesity. Adipogenesis in obesity involves interplay between
differentiating adipocytes, stromal cells, and blood vessels. Close
spatial and temporal interrelationships between blood vessel
formation and adipogenesis, and the sprouting of new blood vessels
from preexisting vasculature was coupled to adipocyte
differentiation. Adipogenic/angiogenic cell clusters can
morphologically and immunohistochemically be distinguished from
crown-like structures frequently seen in the late stages of adipose
tissue obesity. Administration of anti-vascular endothelial growth
factor (VEGF) antibodies inhibited not only angiogenesis but also
the formation of adipogenic/angiogenic cell clusters, indicating
that the coupling of adipogenesis and angiogenesis is essential for
differentiation of adipocytes in obesity and that VEGF is a key
mediator of that process. (Satoshi Nishimura et. al., 2007,
Diabetes 56:1517-1526). It has been shown that the angiogenesis
inhibitor, TNP-470 was able to prevent diet-induced and genetic
obesity in mice (Ebba Brakenhielm et. al., Circulation Research,
2004; 94:1579). TNP-470 reduced vascularity in the adipose tissue,
thereby inhibiting the rate of growth of the adipose tissue and
obesity development. Accordingly, encompassed in the methods
disclosed herein are subjects suffering from obesity.
[0286] In other aspects, the compositions and methods described
herein are used in blocking or inhibiting angiogenesis that occurs
in endometriosis. Excessive endometrial angiogenesis is proposed as
an important mechanism in the pathogenesis of endometriosis (Healy,
D L., et. al., Hum Reprod Update. 1998 September-October;
4(5):736-40). The endometrium of patients with endometriosis shows
enhanced endothelial cell proliferation. Moreover there is an
elevated expression of the cell adhesion molecule integrin v.beta.3
in more blood vessels in the endometrium of women with
endometriosis when compared with normal women. The U.S. Pat. No.
6,121,230 described the use of anti-VEGF agents in the treatment of
endometriosis and is patent is incorporated hereby reference.
Accordingly, encompassed in the methods disclosed herein are
subjects having or being treated for endometriosis.
[0287] As described herein, any of a variety of tissues, or organs
comprised of organized tissues, can support angiogenesis in disease
conditions including skin, muscle, gut, connective tissue, joints,
bones and the like tissue in which blood vessels can invade upon
angiogenic stimuli.
[0288] The individual or subject to be treated as described herein
in various embodiments is desirably a human patient, although it is
to be understood that the methods are effective with respect to all
mammals, which are intended to be included in the term "patient" or
"subject". In this context, a mammal is understood to include any
mammalian species in which treatment of diseases associated with
angiogenesis is desirable. The terms "subject" and "individual" are
used interchangeably herein, and refer to an animal, for example a
human, recipient of the DEspR-specific antibodies and antibody
fragments described herein. For treatment of disease states which
are specific for a specific animal such as a human subject, the
term "subject" refers to that specific animal. The terms "non-human
animals" and "non-human mammals" are used interchangeably herein,
and include mammals such as rats, mice, rabbits, sheep, cats, dogs,
cows, pigs, and non-human primates. The term "subject" also
encompasses any vertebrate including but not limited to mammals,
reptiles, amphibians and fish. However, advantageously, the subject
is a mammal such as a human, or other mammals such as a
domesticated mammal, e.g. dog, cat, horse, and the like, or
production mammal, e.g. cow, sheep, pig, and the like are also
encompassed in the term subject.
Modes of Administration
[0289] The DEspR-specific antagonist agents, such as anti-DEspR
antibodies or antibody fragments thereof, described herein can be
administered to a subject in need thereof by any appropriate route
which results in an effective treatment in the subject. As used
herein, the terms "administering," and "introducing" are used
interchangeably and refer to the placement of an anti-DEspR
antibody or antibody fragment thereof into a subject by a method or
route which results in at least partial localization of such agents
at a desired site, such as a site of inflammation or cancer, such
that a desired effect(s) is produced.
[0290] In some embodiments, the anti-DEspR antibody or antibody
fragment thereof is administered to a subject having an angiogenic
disorder to be inhibited by any mode of administration that
delivers the agent systemically or to a desired surface or target,
and can include, but is not limited to, injection, infusion,
instillation, and inhalation administration. To the extent that
anti-DEspR antibodies or antibody fragments thereof can be
protected from inactivation in the gut, oral administration forms
are also contemplated. "Injection" includes, without limitation,
intravenous, intramuscular, intraarterial, intrathecal,
intraventricular, intracapsular, intraorbital, intracardiac,
intradermal, intraperitoneal, transtracheal, subcutaneous,
subcuticular, intraarticular, sub capsular, subarachnoid,
intraspinal, intracerebro spinal, and intrasternal injection and
infusion. In preferred embodiments, the anti-DEspR antibodies or
antibody fragments thereof for use in the methods described herein
are administered by intravenous infusion or injection.
[0291] The phrases "parenteral administration" and "administered
parenterally" as used herein, refer to modes of administration
other than enteral and topical administration, usually by
injection. The phrases "systemic administration," "administered
systemically", "peripheral administration" and "administered
peripherally" as used herein refer to the administration of the
bispecific or multispecific polypeptide agent other than directly
into a target site, tissue, or organ, such as a tumor site, such
that it enters the subject's circulatory system and, thus, is
subject to metabolism and other like processes.
[0292] The DEspR-specific antagonists described herein are
administered to a subject, e.g., a human subject, in accord with
known methods, such as intravenous administration as a bolus or by
continuous infusion over a period of time, by intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
Local administration, for example, to a tumor or cancer site where
angiogenesis is occurring, is particularly desired if extensive
side effects or toxicity is associated with the use of the DEspR
antagonist. An ex vivo strategy can also be used for therapeutic
applications in some embodiments. Ex vivo strategies involve
transfecting or transducing cells obtained from a subject with a
polynucleotide encoding a DEspR antagonist. The transfected or
transduced cells are then returned to the subject. The cells can be
any of a wide range of types including, without limitation,
hematopoietic cells (e.g., bone marrow cells, macrophages,
monocytes, dendritic cells, T cells, or B cells), fibroblasts,
epithelial cells, endothelial cells, keratinocytes, or muscle
cells.
[0293] In some embodiments, when the DEspR-specific antagonist is
an anti-DEspR antibody or antibody fragment thereof, the antibody
or antibody fragment thereof is administered by any suitable means,
including parenteral, subcutaneous, intraperitoneal,
intrapulmonary, and intranasal, and, if desired for local
immunosuppressive treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration. In
some embodiments, the antibody or antibody fragment thereof is
suitably administered by pulse infusion, particularly with
declining doses of the antibody. Preferably the dosing is given by
injections, most preferably intravenous or subcutaneous injections,
depending in part on whether the administration is brief or
chronic.
[0294] In some embodiments, the DEspR-specific antagonist compound
is administered locally, e.g., by direct injections, when the
disorder or location of the tumor permits, and the injections can
be repeated periodically. The DEspR-specific antagonist can also be
delivered systemically to the subject or directly to the tumor
cells, e.g., to a tumor or a tumor bed following surgical excision
of the tumor, in order to prevent or reduce local recurrence or
metastasis, for example of a dormant tumor or micrometastases.
Administration by Sonoporation
[0295] Antibody-targeted sonoporation methods are contemplated for
use in some embodiments of the methods for inhibiting angiogenesis
described herein, in order to enhance the efficacy and potency of
the therapeutic compositions comprising anti-DEspR antibodies and
antibody fragments thereof provided herein.
[0296] The inventors have discovered that DEspR-targeted
sonoporation of pharmaceutical compositions comprising anti-DEspR
monoclonal antibodies and antibody fragments provides surprisingly
enhanced reduction of tumor growth and metastases, indicating
enhanced penetration and delivery of the compositions, and enhances
delivery to sites of pathological angiogenesis, and to tumor cells,
and tumor initiating cells or cancer stem cells or cancer stem-like
cells. Further, the inventors have discovered that sonoporation of
anti-DEspR antibodies and antibody fragments thereof in combination
with other therapeutic agents, such as small molecule compounds or
other drug compounds, can be used to enhance delivery of the other
therapeutic agents, thus providing a means of targeted and enhanced
delivery.
[0297] Accordingly, in some embodiments of the methods of
inhibiting angiogenesis described herein, anti-DEspR antibodies and
antibody fragments thereof are administered to a subject in need
thereof by sonoporation.
[0298] As used herein, "sonoporation" refers to the use of sound,
preferably at ultrasonic frequencies, or the interaction of
ultrasound with contrast agents (e.g., stabilized microbubbles) for
temporarily modifying the permeability of cell plasma membranes,
thus allowing uptake of large molecules, such as therapeutic
agents. The membrane permeability caused by the sonoporation is
transient, leaving the agents trapped inside the cell after the
ultrasound exposure. Sonoporation employs acoustic cavitation of
microbubbles to enhance delivery of large molecules.
[0299] Accordingly, in some embodiments of the methods, therapeutic
anti-DEspR agents, such as the anti-DEspR antibodies and antibody
fragments thereof described herein, mixed with ultrasound contrast
agents, such as microbubbles, can be injected locally or
systemically into a subject in need of treatment for an angiogenic
disorder, and ultrasound can be coupled and even focused into the
defined area, e.g., tumor site, to achieve targeted delivery of the
anti-DEspR antibodies and antibody fragments thereof described
herein. Additionally, the anti-DEspR antibody or antibody fragment
thereof is known to target the tumor vessel endothelium, thus
directing the sonoporation to areas of increased DEspR expression
in tumor endothelial cells. In addition to the operator-determined
focused ultrasound, anti-DEspR targeting of a microbubble can be
used to target the sonoporation-mediated enhanced entry of any
therapeutic agent, including antiDEspR monoclonal antibody per se,
into said targeted cancerous areas.
[0300] In some embodiments, the methods use focused ultrasound
methods to achieve targeted delivery of the anti-DEspR antibodies
and antibody fragments thereof described herein. As used herein,
HIFU or "High Intensity Focused Ultrasound" refers to a
non-invasive therapeutic method using high-intensity ultrasound to
heat and destroy malignant or pathogenic tissue without causing
damage to overlying or surrounding health tissue. Typically, HIFU
has been used in tissue ablation techniques, whereby the biological
effects of HIFU treatment, including coagulative necrosis and
structural disruption, can be induced in a tissue requiring
ablation, such as a solid tumor site. However, as described in
Khaibullina A. et al., J Nucl Med. 2008 February; 49(2):295-302,
and WO2010127369, the contents of which are herein incorporated in
their entireties by reference, HIFU can also be used as a means of
delivery of therapeutic agents, such as antibodies or antibody
fragments thereof.
[0301] Methods using contrast-enhanced ultrasound (CEUS) are also
contemplated for use with anti-DEspR inhibiting agents described
herein. Contrast-enhanced ultrasound (CEUS) refers to the
application of ultrasound contrast medium and ultrasound contrast
agents to traditional medical sonography. Ultrasound contrast
agents refer to agents that rely on the different ways in which
sound waves are reflected from interfaces between substances. This
can be the surface of a small air bubble or a more complex
structure. Commercially available contrast media include gas-filled
microbubbles that are administered intravenously to the systemic
circulation. Microbubbles have a high degree of echogenicity, i.e.,
the ability of an object to reflect the ultrasound waves. The
echogenicity difference between the gas in the microbubbles and the
soft tissue surroundings of the body is immense, and enhances the
ultrasound backscatter, or reflection of the ultrasound waves, to
produce a unique sonogram with increased contrast due to the high
echogenicity difference. Contrast-enhanced ultrasound can be used
with the compositions and methods described herein to image a
variety of conditions and disorders, such as angiogenesis dependent
disorders, as described herein
[0302] A variety of microbubble contrast agents are available for
use with the compositions and methods described herein.
Microbubbles can differ in their shell makeup, gas core makeup, and
whether or not they are targeted.
[0303] The microbubble shell material determines how easily the
microbubble is taken up by the immune system. A more hydrophilic
shell material tends to be taken up more easily, which reduces the
microbubble residence time in the circulation. This reduces the
time available for contrast imaging. The shell material also
affects microbubble mechanical elasticity. The more elastic the
material, the more acoustic energy it can withstand before
bursting. Example of materials used in current microbubble shells
include albumin, galactose, lipid, and polymers, as described in
Lindner, J. R. 2004. Microbubbles in medical imaging: current
applications and future directions. Nat Rev Drug Discov. 3: 527-32,
the contents of which are herein incorporated by reference in their
entireties.
[0304] The microbubble gas core is an important part of the
ultrasound contrast microbubble because it determines the
echogenicity. When gas bubbles are caught in an ultrasonic
frequency field, they compress, oscillate, and reflect a
characteristic echo--this generates the strong and unique sonogram
in contrast-enhanced ultrasound. Gas cores can be composed of, for
example, air, or heavy gases like perfluorocarbon, or nitrogen.
Heavy gases are less water-soluble so they are less likely to leak
out from the microbubble to impair echogenicity. Therefore,
microbubbles with heavy gas cores are likely to last longer in
circulation.
[0305] Regardless of the shell or gas core composition, microbubble
size are typically fairly uniform. They can lie within in a range
of 1-4 micrometres in diameter. That makes them smaller than red
blood cells, which allows them to flow easily through the
circulation as well as the microcirculation.
[0306] Targeting ligands that bind to receptors characteristic of
angiogenic disorders, such as DEspR, can be conjugated to
microbubbles, enabling the microbubble complex to accumulate
selectively in areas of interest, such as diseased or abnormal
tissues. This form of molecular imaging, known as targeted
contrast-enhanced ultrasound, will only generate a strong
ultrasound signal if targeted microbubbles bind in the area of
interest. Targeted contrast-enhanced ultrasound has many
applications in both medical diagnostics and medical therapeutics.
Microbubbles targeted with an agent that binds to DEspR, such as an
anti-DEspR antibody or antibody fragment thereof, are injected
systemically in a small bolus. These DEspR-targeted microbubbles
can travel through the circulatory system, eventually finding their
respective targets and binding specifically. Ultrasound waves can
then be directed on the area of interest. If a sufficient number of
DEspR-targeted microbubbles have bound in the area, their
compressible gas cores oscillate in response to the high frequency
sonic energy field. The DEspR-targeted microbubbles also reflect a
unique echo that is in stark contrast to the surrounding tissue due
to the orders of magnitude mismatch between microbubble and tissue
echogenicity. The ultrasound system converts the strong
echogenicity into a contrast-enhanced image of the area of
interest, revealing the location of the bound DEspR-targeted
microbubbles. Detection of bound microbubbles can then show that
the area of interest is expressing DEspR, which can be indicative
of a certain disease state, or identify particular cells in the
area of interest. In addition, targeted sonoporation can be done at
the site where DEspR-targeted microbubbles are attached, thus
achieving targeted delivery of any therapeutic agent (drug, siRNA,
DNA, small molecule) encapsulated in or carried on the echogenic
microbubble.
[0307] Accordingly, in some embodiments of the methods described
herein, an anti-DEspR antibody or antibody fragment thereof, such
as, for example, an anti-DEspR 7C5B2 antibody or fragment thereof,
an anti-DEspR antibody or antibody-fragment thereof comprising one
or more heavy chain CDR regions comprising a sequence selected from
the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO:
7; an anti-DEspR antibody or antibody-fragment thereof comprising
one or more light chain CDR regions comprising a sequence selected
from the group consisting of SEQ ID NO: 10, SEQ ID NO: 11, or SEQ
ID NO: 12; an anti-DEspR antibody or antibody-fragment comprising a
variable heavy (V.sub.H) chain amino acid sequence selected from
the group consisting of SEQ ID NO: 4 and SEQ ID NO: 13-SEQ ID NO:
17; and/or an anti-DEspR antibody or antibody-fragment thereof
comprising a variable light (V.sub.L) chain amino acid sequence
selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 18,
and SEQ ID NO: 19, is administered to a subject in need of
treatment for an angiogenic disorder, such as for example, cancer,
using a targeted ultrasound delivery. In some such embodiments, the
targeted ultrasound delivery comprises using microbubbles as
contrast agents to which an anti-DEspR antibody or antibody
fragment thereof. In some such embodiments, the targeted ultrasound
is HIFU.
Pharmaceutical Formulations
[0308] For the clinical use of the methods described herein,
administration of the DEspR antagonists, such as the anti-DEspR
antibodies or antibody fragments thereof described herein, can
include formulation into pharmaceutical compositions or
pharmaceutical formulations for parenteral administration, e.g.,
intravenous; mucosal, e.g., intranasal; ocular, or other mode of
administration. In some embodiments, the anti DEspR antibodies or
antibody fragments thereof described herein can be administered
along with any pharmaceutically acceptable carrier compound,
material, or composition which results in an effective treatment in
the subject. Thus, a pharmaceutical formulation for use in the
methods described herein can contain an anti-DEspR antibody or
antibody fragment thereof as described herein in combination with
one or more pharmaceutically acceptable ingredients.
[0309] The phrase "pharmaceutically acceptable" refers to those
compounds, materials, compositions, and/or dosage forms which are,
within the scope of sound medical judgment, suitable for use in
contact with the tissues of human beings and animals without
excessive toxicity, irritation, allergic response, or other problem
or complication, commensurate with a reasonable benefit/risk ratio.
The phrase "pharmaceutically acceptable carrier" as used herein
means a pharmaceutically acceptable material, composition or
vehicle, such as a liquid or solid filler, diluent, excipient,
solvent, media, encapsulating material, manufacturing aid (e.g.,
lubricant, talc magnesium, calcium or zinc stearate, or steric
acid), or solvent encapsulating material, involved in maintaining
the stability, solubility, or activity of, an anti-DEspR antibody
or antibody fragment thereof. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation and not injurious to the patient. The terms
"excipient", "carrier", "pharmaceutically acceptable carrier" or
the like are used interchangeably herein.
[0310] The anti-DEspR antibodies or antibody fragments thereof
described herein can be specially formulated for administration of
the compound to a subject in solid, liquid or gel form, including
those adapted for the following: (1) parenteral administration, for
example, by subcutaneous, intramuscular, intravenous or epidural
injection as, for example, a sterile solution or suspension, or
sustained-release formulation; (2) topical application, for
example, as a cream, ointment, or a controlled-release patch or
spray applied to the skin; (3) intravaginally or intrarectally, for
example, as a pessary, cream or foam; (4) ocularly; (5)
transdermally; (6) transmucosally; or (79) nasally. Additionally,
an anti-DEspR antibody or antibody fragment thereof can be
implanted into a patient or injected using a drug delivery system.
See, for example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol.
24: 199-236 (1984); Lewis, ed. "Controlled Release of Pesticides
and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Pat. No.
3,773,919; and U.S. Pat. No. 35 3,270,960.
[0311] Therapeutic formulations of the DEspR-specific antagonist
agents, such as anti-DEspR antibodies or antibody fragments
thereof, described herein can be prepared for storage by mixing a
DEspR-specific antagonist having the desired degree of purity with
optional pharmaceutically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed. (1980)), in the form of lyophilized formulations or
aqueous solutions. Acceptable carriers, excipients, or stabilizers
are nontoxic to recipients at the dosages and concentrations
employed, and include buffers such as phosphate, citrate, and other
organic acids; antioxidants including ascorbic acid and methionine;
preservatives (such as octadecyldimethylbenzyl ammonium chloride;
hexamethonium chloride; benzalkonium chloride, benzethonium
chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as
methyl or propyl paraben; catechol; resorcinol; cyclohexanol;
3-pentanol; and m-cresol); low molecular weight (less than about 10
residues) polypeptides; proteins, such as serum albumin, gelatin,
or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone; amino acids such as glycine, glutamine,
asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose,
or dextrins; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g. Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., PLURONICS.TM. or
polyethylene glycol (PEG). Exemplary lyophilized anti-VEGF antibody
formulations are described in WO 97/04801, expressly incorporated
herein be reference.
[0312] Optionally, but preferably, the formulations comprising the
compositions described herein contain a pharmaceutically acceptable
salt, typically, e.g., sodium chloride, and preferably at about
physiological concentrations. Optionally, the formulations of the
invention can contain a pharmaceutically acceptable preservative.
In some embodiments the preservative concentration ranges from 0.1
to 2.0%, typically v/v. Suitable preservatives include those known
in the pharmaceutical arts. Benzyl alcohol, phenol, m-cresol,
methylparaben, and propylparaben are examples of preservatives.
Optionally, the formulations of the invention can include a
pharmaceutically acceptable surfactant at a concentration of 0.005
to 0.02%.
[0313] The therapeutic formulations of the compositions comprising
DEspR-specific antagonists, such as anti-DEspR antibodies and
antibody fragments thereof, described herein can also contain more
than one active compound as necessary for the particular indication
being treated, preferably those with complementary activities that
do not adversely affect each other. For example, in some
embodiments, it can be desirable to further provide antibodies
which bind to EGFR, VEGF (e.g. an antibody which binds a different
epitope on VEGF), VEGFR, or ErbB2 (e.g., Herceptin.TM.)
Alternatively, or in addition, the composition can comprise a
cytotoxic agent, cytokine, growth inhibitory agent and/or VEGFR
antagonist. Such molecules are suitably present in combination in
amounts that are effective for the purpose intended.
[0314] The active ingredients of the therapeutic formulations of
the compositions comprising DEspR-specific antagonists described
herein can also be entrapped in microcapsules prepared, for
example, by coacervation techniques or by interfacial
polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly-(methylmethacylate) microcapsules,
respectively, in colloidal drug delivery systems (for example,
liposomes, albumin microspheres, microemulsions, nano-particles and
nanocapsules) or in macroemulsions. Such techniques are disclosed
in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980).
[0315] In some embodiments, sustained-release preparations can be
used. Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
DEspR-specific antagonist, such as an anti-DEspR antibody, in which
the 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 (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and y 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. 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 can denature or aggregate as a
result of exposure to moisture at 37.degree. C., resulting in a
loss of biological activity and possible changes in immunogenicity.
Rational strategies can be devised for stabilization depending on
the mechanism involved. For example, if the aggregation mechanism
is discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization can be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
[0316] The therapeutic formulations to be used for in vivo
administration, such as parenteral administration, in the methods
described herein can be sterile, which is readily accomplished by
filtration through sterile filtration membranes, or other methods
known to those of skill in the art.
Dosages and Duration
[0317] The DEspR-specific antagonists described herein, such as
anti-DEspR antibodies and antibody fragments thereof, are
formulated, dosed, and administered in a fashion consistent with
good medical practice. Factors for consideration in this context
include the particular disorder being treated, the particular
subject being treated, the clinical condition of the individual
subject, the cause of the disorder, the site of delivery of the
agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "therapeutically effective amount" of the DEspR-specific
antagonist to be administered will be governed by such
considerations, and refers to the minimum amount necessary to
ameliorate, treat, or stabilize, the cancer; to increase the time
until progression (duration of progression free survival) or to
treat or prevent the occurrence or recurrence of a tumor, a dormant
tumor, or a micrometastases. The DEspR-specific antagonist is
optionally formulated with one or more additional therapeutic
agents currently used to prevent or treat cancer or a risk of
developing a cancer. The effective amount of such other agents
depends on the amount of VEGF-specific antagonist present in the
formulation, the type of disorder or treatment, and other factors
discussed above. These are generally used in the same dosages and
with administration routes as used herein before or about from 1 to
99% of the heretofore employed dosages.
[0318] Depending on the type and severity of the disease, about 1
.mu.g/kg to 100 mg/kg (e.g., 0.1-20 mg/kg) of a DEspR-specific
antagonist is an initial candidate dosage for administration to a
subject, whether, for example, by one or more separate
administrations, or by continuous infusion. A typical daily dosage
might range from about 1 .mu.g/kg to about 100 mg/kg or more,
depending on the factors mentioned above. Particularly desirable
dosages include, for example, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, and 15
mg/kg. For repeated administrations over several days or longer,
depending on the condition, the treatment is sustained until, for
example, the cancer is treated, as measured by the methods
described above or known in the art. However, other dosage regimens
can be useful. In one non-limiting example, if the DEspR-specific
antagonist is an anti-DEspR antibody or antibody fragment thereof,
the anti-DEspR antibody or antibody fragment thereof is
administered once every week, every two weeks, or every three
weeks, at a dose range from about 5 mg/kg to about 15 mg/kg,
including but not limited to 5 mg/kg, 7.5 mg/kg, 10 mg/kg or 15
mg/kg. The progress of using the methods described herein can be
easily monitored by conventional techniques and assays.
[0319] The duration of a therapy using the methods described herein
will continue for as long as medically indicated or until a desired
therapeutic effect (e.g., those described herein) is achieved. In
certain embodiments, the DEspR-specific antagonist therapy, such as
a DEspR-specific antibody or antibody fragment described herein is
continued for 1 month, 2 months, 4 months, 6 months, 8 months, 10
months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20
years, or for a period of years up to the lifetime of the
subject.
Efficacy of the Treatment
[0320] The efficacy of the treatment methods for cancer comprising
therapeutic formulations of the compositions comprising the
DEspR-specific antagonists described herein can be measured by
various endpoints commonly used in evaluating cancer treatments,
including but not limited to, tumor regression, tumor weight or
size shrinkage, time to progression, duration of survival,
progression free survival, overall response rate, duration of
response, and quality of life. Because the DEspR-specific
antagonists, e.g., anti-DEspR antibodies and antibody fragments
thereof, described herein target the tumor vasculature, cancer
cells, and some cancer stem cell subsets, they represent a unique
class of multi-targeting anticancer drugs, and therefore can
require unique measures and definitions of clinical responses to
drugs. For example, tumor shrinkage of greater than 50% in a
2-dimensional analysis is the standard cut-off for declaring a
response. However, the anti-DEspR-antibodies or antibody fragments
thereof described herein can cause inhibition of metastatic spread
without shrinkage of the primary tumor, or can simply exert a
tumoristatic effect. Accordingly, novel approaches to determining
efficacy of an anti-angiogenic therapy should be employed,
including for example, measurement of plasma or urinary markers of
angiogenesis, and measurement of response through molecular
imaging, using, for example, an DEspR-antibody or antibody fragment
conjugated to a label, such as a microbubble. In the case of
cancers, the therapeutically effective amount of the DEspR-antibody
or antibody fragment thereof can reduce the number of cancer cells;
reduce the tumor size; inhibit (i.e., slow to some extent and
preferably stop) cancer cell infiltration into peripheral organs;
inhibit (i.e., slow to some extent and preferably stop) tumor
metastasis; inhibit, to some extent, tumor growth; and/or relieve
to some extent one or more of the symptoms associated with the
disorder. To the extent the DEspR-antibody or antibody fragment
thereof can prevent growth and/or kill existing cancer cells, it
can be cytostatic and/or cytotoxic. For cancer therapy, efficacy in
vivo can, for example, be measured by assessing the duration of
survival, duration of progression free survival (PFS), the response
rates (RR), duration of response, and/or quality of life.
[0321] In other embodiments, described herein are methods for
increasing progression free survival of a human subject susceptible
to or diagnosed with a cancer. Time to disease progression is
defined as the time from administration of the drug until disease
progression or death. In a preferred embodiment, the combination
treatment of the invention using a DEspR-specific antagonist, such
as an anti-DEspR antibody or antibody fragment thereof, and one or
more chemotherapeutic agents significantly increases progression
free survival by at least about 1 month, 1.2 months, 2 months, 2.4
months, 2.9 months, 3.5 months, preferably by about 1 to about 5
months, when compared to a treatment with chemotherapy alone. In
another embodiment, the methods described herein significantly
increase response rates in a group of human subjects susceptible to
or diagnosed with a cancer who are treated with various
therapeutics. Response rate is defined as the percentage of treated
subjects who responded to the treatment. In one embodiment, the
combination treatment described herein using a DEspR-specific
antagonist, such as an anti-DEspR antibody or antibody fragment
thereof, and one or more chemotherapeutic agents significantly
increases response rate in the treated subject group compared to
the group treated with chemotherapy alone.
[0322] As used herein, the terms "treat," "treatment," "treating,"
or "amelioration" refer to therapeutic treatments, wherein the
object is to reverse, alleviate, ameliorate, inhibit, slow down or
stop the progression or severity of a condition associated with, a
disease or disorder. The term "treating" includes reducing or
alleviating at least one adverse effect or symptom of a condition,
disease or disorder associated with a chronic immune condition,
such as, but not limited to, a chronic infection or a cancer.
Treatment is generally "effective" if one or more symptoms or
clinical markers are reduced. Alternatively, treatment is
"effective" if the progression of a disease is reduced or halted.
That is, "treatment" includes not just the improvement of symptoms
or markers, but also a cessation of at least slowing of progress or
worsening of symptoms that would be expected in absence of
treatment. Beneficial or desired clinical results include, but are
not limited to, alleviation of one or more symptom(s), diminishment
of extent of disease, stabilized (i.e., not worsening) state of
disease, delay or slowing of disease progression, amelioration or
palliation of the disease state, and remission (whether partial or
total), whether detectable or undetectable. The term "treatment" of
a disease also includes providing relief from the symptoms or
side-effects of the disease (including palliative treatment).
[0323] For example, in some embodiments, the methods described
herein comprise administering an effective amount of the anti-DEspR
antibodies or antibody fragments thereof described herein to a
subject in order to alleviate a symptom of a cancer, or other such
disorder characterized by excess or unwanted angiogenesis. As used
herein, "alleviating a symptom of a cancer" is ameliorating or
reducing any condition or symptom associated with the cancer. As
compared with an equivalent untreated control, such reduction or
degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%,
90%, 95%, or 100% as measured by any standard technique. Ideally,
the cancer is completely cleared as detected by any standard method
known in the art, in which case the cancer is considered to have
been treated. A patient who is being treated for a cancer is one
who a medical practitioner has diagnosed as having such a
condition. Diagnosis can be by any suitable means. Diagnosis and
monitoring can involve, for example, detecting the level of cancer
cells in a biological sample (for example, a tissue or lymph node
biopsy, blood test, or urine test), detecting the level of a
surrogate marker of the cancer in a biological sample, detecting
symptoms associated with the specific cancer, or detecting immune
cells involved in the immune response typical of such a cancer
infections.
[0324] The term "effective amount" as used herein refers to the
amount of an anti-DEspR antibody or antibody fragment thereof
needed to alleviate at least one or more symptom of the disease or
disorder, and relates to a sufficient amount of pharmacological
composition to provide the desired effect, i.e., inhibit the
formation of new blood vessels. The term "therapeutically effective
amount" therefore refers to an amount of an anti-DEspR antibody or
antibody fragment thereof using the methods as disclosed herein,
that is sufficient to effect a particular effect when administered
to a typical subject. An effective amount as used herein would also
include an amount sufficient to delay the development of a symptom
of the disease, alter the course of a symptom disease (for example
but not limited to, slow the progression of a symptom of the
disease), or reverse a symptom of the disease. Thus, it is not
possible to specify the exact "effective amount". However, for any
given case, an appropriate "effective amount" can be determined by
one of ordinary skill in the art using only routine
experimentation.
[0325] Effective amounts, toxicity, and therapeutic efficacy can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD.sub.50 (the
dose lethal to 50% of the population) and the ED.sub.50 (the dose
therapeutically effective in 50% of the population). The dosage can
vary depending upon the dosage form employed and the route of
administration utilized. The dose ratio between toxic and
therapeutic effects is the therapeutic index and can be expressed
as the ratio LD.sub.50/ED.sub.50. Compositions and methods that
exhibit large therapeutic indices are preferred. A therapeutically
effective dose can be estimated initially from cell culture assays.
Also, a dose can be formulated in animal models to achieve a
circulating plasma concentration range that includes the IC.sub.50
(i.e., the concentration of the anti-DEspR antibody or antibody
fragment thereof), which achieves a half-maximal inhibition of
symptoms) as determined in cell culture, or in an appropriate
animal model. Levels in plasma can be measured, for example, by
high performance liquid chromatography. The effects of any
particular dosage can be monitored by a suitable bioassay. The
dosage can be determined by a physician and adjusted, as necessary,
to suit observed effects of the treatment.
Combination Antiangiogenic Therapies
[0326] In other embodiments, the methods provided for inhibiting
angiogenesis in a tissue of a subject or individual having a
disease or disorder dependent or modulated by angiogenesis by
administering to the subject a therapeutically effective amount of
a composition comprising an angiogenesis-inhibiting amount of an
anti-DEspR inhibitor, such as an anti-DEspR antibody or antibody
fragment thereof, can further comprise administration one or more
additional treatments such as angiogenic inhibitors, chemotherapy,
radiation, surgery, or other treatments known to those of skill in
the art to inhibit angiogenesis.
[0327] In some embodiments, the methods described herein further
comprise administration of a combination of at least one
DEspR-specific antagonist, such an anti-DEspR antibody or antibody
fragment thereof, with one or more additional anti-cancer
therapies. Examples of additional anti-cancer therapies include,
without limitation, surgery, radiation therapy (radiotherapy),
biotherapy, immunotherapy, chemotherapy, or a combination of these
therapies. In addition, cytotoxic agents, anti-angiogenic and
anti-proliferative agents can be used in combination with the
DEspR-specific antagonist.
[0328] In certain aspects of any of the methods and uses, the
invention provides treating cancer by administering effective
amounts of an anti-DEspR antibody and one or more chemotherapeutic
agents to a subject susceptible to, or diagnosed with, locally
recurrent or previously untreated cancer. A variety of
chemotherapeutic agents can be used in the combined treatment
methods and uses of the invention. An exemplary and non-limiting
list of chemotherapeutic agents contemplated for use in the methods
described herein is provided under "Definition," or described
herein.
[0329] In some embodiments, the methods described herein comprise
administration of a DEspR-specific antagonist with one or more
chemotherapeutic agents (e.g., a cocktail) or any combination
thereof. In certain embodiments, the chemotherapeutic agent is for
example, capecitabine, taxane, anthracycline, paclitaxel,
docetaxel, paclitaxel protein-bound particles (e.g., Abraxane.TM.)
doxorubicin, epirubicin, 5-fluorouracil, cyclophosphamide or
combinations thereof therapy. As used herein, combined
administration includes simultaneous administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities. Preparation and
dosing schedules for such chemotherapeutic agents can be used
according to manufacturers' instructions or as determined
empirically by the skilled practitioner. Preparation and dosing
schedules for chemotherapy are also described in Chemotherapy
Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md.
(1992). Accordingly, in some embodiments, the chemotherapeutic
agent can precede, or follow administration of the DEspR-specific
antagonist or can be given simultaneously therewith.
[0330] In some other embodiments of the methods described herein,
other therapeutic agents useful for combination tumor therapy with
the DEspR antagonists, such as antibodies, of the invention include
antagonists of other factors that are involved in tumor growth,
such as EGFR, ErbB2 (also known as Her2), ErbB3, ErbB4, or TNF. In
some embodiments, it can be beneficial to also administer one or
more cytokines to the subject. In some embodiments, the DEspR
antagonist is co-administered with a growth inhibitory agent. For
example, the growth inhibitory agent can be administered first,
followed by the DEspR antagonist. However, simultaneous
administration or administration of the DEspR antagonist first is
also contemplated. Suitable dosages for the growth inhibitory agent
are those presently used and can be lowered due to the combined
action (synergy) of the growth inhibitory agent and DEspR
antagonist.
[0331] Examples of additional angiogenic inhibitors that can be
used in combination with the DEspR inhibitors, such as anti-DEspR
antibodies and antibody fragments thereof, described herein
include, but are not limited to: direct angiogenesis inhibitors,
Angiostatin, Bevacizumab (Avastin.RTM.), Arresten, Canstatin,
Combretastatin, Endostatin, NM-3, Thrombospondin, Tumstatin,
2-methoxyestradiol, cetuximab (Erbitux.RTM.), panitumumab
(Vectibix.TM.), trastuzumab (Herceptin.RTM.) and Vitaxin; and
indirect angiogenesis inhibitors: ZD1839 (Iressa), ZD6474, OS1774
(Tarceva), CI1033, PKI1666, IMC225 (Erbitux), PTK787, SU6668,
SU11248, Herceptin, and IFN-.alpha., CELEBREX.RTM. (Celecoxib),
THALOMID.RTM. (Thalidomide), and IFN-.alpha..
[0332] In some embodiments, the additional angiogenesis inhibitors
for use in the methods described herein include but are not limited
to small molecule tyrosine kinase inhibitors (TKIs) of multiple
pro-angiogenic growth factor receptors. The three TKIs that are
currently approved as anti-cancer therapies are erlotinib
(Tarceva.RTM.), sorafenib (Nexavar.RTM.), and sunitinib
(Sutent.RTM.).
[0333] In some embodiments, the angiogenesis inhibitors for use in
the methods described herein include but are not limited to
inhibitors of mTOR (mammalian target of rapamycin) such as
temsirolimus (Toricel.TM.), bortezomib (Velcade.RTM.), thalidomide
(Thalomid.RTM.), and Doxycyclin,
[0334] In other embodiments, the angiogenesis inhibitors for use in
the methods described herein include one or more drugs that target
the VEGF pathway. Bevacizumab (Avastin.RTM.) was the first drug
that targeted new blood vessels to be approved for use against
cancer. It is a monoclonal antibody that binds to VEGF, thereby
blocking VEGF from reaching the VEGF receptor (VEGFR). Other drugs,
such as sunitinib (Sutent.RTM.) and sorafenib (Nexavar.RTM.), are
small molecules that attach to the VEGF receptor itself, preventing
it from being turned on. Such drugs are collectively termed VEGF
inhibitors. As the VEGF/VPF protein interacts with the VEGFRs,
inhibition of either the ligand VEGF, e.g. by reducing the amount
that is available to interact with the receptor; or inhibition of
the receptor's intrinsic tyrosine kinase activity, blocks the
function of this pathway. This pathway controls endothelial cell
growth, as well as permeability, and these functions are mediated
through the VEGFRs.
[0335] Accordingly, as described herein, "VEGF inhibitors" for use
as angiogenesis inhibitors include any compound or agent that
produces a direct or indirect effect on the signaling pathways that
promote growth, proliferation and survival of a cell by inhibiting
the function of the VEGF protein, including inhibiting the function
of VEGF receptor proteins. These include any organic or inorganic
molecule, including, but not limited to modified and unmodified
nucleic acids such as antisense nucleic acids, RNAi agents such as
siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and
antibodies that inhibit the VEGF signaling pathway. The siRNAs are
targeted at components of the VEGF pathways and can inhibit the
VEGF pathway. Preferred VEGF inhibitors, include for example,
AVASTIN.RTM. (bevacizumab), an anti-VEGF monoclonal antibody of
Genentech, Inc. of South San Francisco, Calif., VEGF Trap
(Regeneron/Aventis). Additional VEGF inhibitors include CP-547,632
(3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin
1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide
hydrochloride; Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.),
SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New
York, N.Y.), ZD-6474 (AstraZeneca), ZD4190 which inhibits VEGF-R2
and -R1 (AstraZeneca), CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC
412 (Novartis), AEE788 (Novartis), AZD-2171), NEXAVAR.RTM. (BAY
43-9006, sorafenib; Bayer Pharmaceuticals and Onyx
Pharmaceuticals), vatalanib (also known as PTK-787, ZK-222584:
Novartis & Schering: AG), MACUGEN.RTM. (pegaptanib octasodium,
NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), 1M862 (glufanide
disodium, Cytran Inc. of Kirkland, Wash., USA), VEGFR2-selective
monoclonal antibody DC101 (ImClone Systems, Inc.), angiozyme, a
synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron
(Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,
Sirna Therapeutics, San Francisco, Calif.) Caplostatin, soluble
ectodomains of the VEGF receptors, Neovastat (terna Zentaris Inc;
Quebec City, Calif.), ZM323881 (CalBiochem. CA, USA), pegaptanib
(Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer and
combinations thereof.
[0336] VEGF inhibitors are also disclosed in U.S. Pat. Nos.
6,534,524 and 6,235,764, both of which are incorporated in their
entirety. Additional VEGF inhibitors are described in, for example
in WO 99/24440 (published May 20, 1999), International Application
PCT/IB99/00797 (filed May 3, 1999), in WO 95/21613 (published Aug.
17, 1995), WO 99/61422 (published Dec. 2, 1999), U.S. Pat. Publ.
No. 20060094032 "siRNA agents targeting VEGF", U.S. Pat. No.
6,534,524 (discloses AG13736), U.S. Pat. No. 5,834,504 (issued Nov.
10, 1998), WO 98/50356 (published Nov. 12, 1998), U.S. Pat. No.
5,883,113 (issued Mar. 16, 1999), U.S. Pat. No. 5,886,020 (issued
Mar. 23, 1999), U.S. Pat. No. 5,792,783 (issued Aug. 11, 1998),
U.S. Pat. No. 6,653,308 (issued Nov. 25, 2003), WO 99/10349
(published Mar. 4, 1999), WO 97/32856 (published Sep. 12, 1997), WO
97/22596 (published Jun. 26, 1997), WO 98/54093 (published Dec. 3,
1998), WO 98/02438 (published Jan. 22, 1998), WO 99/16755
(published Apr. 8, 1999), and WO 98/02437 (published Jan. 22,
1998), WO 01/02369 (published Jan. 11, 2001); U.S. Provisional
Application No. 60/491,771 piled Jul. 31, 2003); U.S. Provisional
Application No. 60/460,695 (filed Apr. 3, 2003); and WO 03/106462A1
(published Dec. 24, 2003). Other examples of VEGF inhibitors are
disclosed in International Patent Publications WO 99/62890
published Dec. 9, 1999, WO 01/95353 published Dec. 13, 2001 and WO
02/44158 published Jun. 6, 2002.
[0337] In other embodiments, the angiogenesis inhibitors for use in
the methods described herein include anti-angiogenic factors such
as alpha-2 antiplasmin (fragment), angiostatin (plasminogen
fragment), antiangiogenic antithrombin III, cartilage-derived
inhibitor (CDI), CD59 complement fragment, endostatin (collagen
XVIII fragment), fibronectin fragment, gro-beta (a C-X-C
chemokine), heparinases heparin hexasaccharide fragment, human
chorionic gonadotropin (hCG), interferon alpha/beta/gamma,
interferon inducible protein (IP-10), interleukin-12, kringle 5
(plasminogen fragment), beta-thromboglobulin, EGF (fragment), VEGF
inhibitor, endostatin, fibronection (45 kD fragment), high
molecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of
HGF, PF-4, serpin proteinase inhibitor 8, TGF-beta-1,
thrombospondin-1, prosaposin, p53, angioarrestin, metalloproteinase
inhibitors (TIMPs), 2-Methoxyestradiol, placental ribonuclease
inhibitor, plasminogen activator inhibitor, prolactin 16 kD
fragment, proliferin-related protein (PRP), retinoids,
tetrahydrocortisol-S transforming growth factor-beta (TGF-b),
vasculostatin, and vasostatin (calreticulin fragment). pamidronate
thalidomide, TNP470, the bisphosphonate family such as
amino-bisphosphonate zoledronic acid. bombesin/gastrin-releasing
peptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol A
M, et. al., British Journal of Cancer (2004) 90, 245-252),
anti-VEGF peptide RRKRRR (dRK6) (Seung-Ah Yoo, J. Immuno, 2005,
174: 5846-5855).
[0338] Thus, in connection with the administration of a DEspR
inhibitor, such as anti-DEspR antibodies and antibody fragments
thereof, a compound which inhibits angiogenesis indicates that
administration in a clinically appropriate manner results in a
beneficial effect for at least a statistically significant fraction
of patients, such as improvement of symptoms, a cure, a reduction
in disease load, reduction in tumor mass or cell numbers, extension
of life, improvement in quality of life, or other effect generally
recognized as positive by medical doctors familiar with treating
the particular type of disease or condition.
[0339] Examples of additional DEspR inhibitors include, but are not
limited to, molecules which block the binding of VEGFsp, ET-1
and/or other ET-1 or VEGFsp-like ligands to DEspR, compounds which
interfere with downstream signaling events of DEspR, or other
compounds or agents that inhibit activation of the receptor. Such
compounds can bind to DEspR and prevent binding of VEGFsp, ET-1 or
other mimetic ligands. Other inhibitors including small molecules
that bind to the DEspR domain that binds to VEGFsp, soluble DEspR
receptors, peptides containing the DEspR ET-1 and/or VEGFsp binding
domains, etc. are also contemplated.
[0340] The compositions described herein can also contain more than
one active compound as necessary for the particular indication
being treated, and these active compounds are preferably those with
complementary activities that do not adversely affect each other.
For example, it can be desirable to further provide antibodies or
antagonists that bind to EGFR, VEGF, VEGFR, or ErbB2 (e.g.,
Herceptin.TM.). Alternatively, or in addition, the composition can
comprise a cytotoxic agent, cytokine, growth inhibitory agent
and/or VEGFR antagonist. Such molecules are suitably present in
combination in amounts that are effective for the purpose
intended.
[0341] In certain aspects of any of the methods and uses described
herein, other therapeutic agents useful for combination cancer
therapy with the antibody of the invention include other
anti-angiogenic agents. Many anti-angiogenic agents have been
identified and are known in the arts, including those listed by
Carmeliet and Jain (2000). In some embodiments, the DEspR
antagonist, such as a humanized anti-DEspR antibody or antibody
fragment thereof described herein is used in combination with a
VEGF antagonist or a VEGF receptor antagonist such as VEGF
variants, soluble VEGF receptor fragments, aptamers capable of
blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, low
molecule weight inhibitors of VEGFR tyrosine kinases and any
combinations thereof. Alternatively, or in addition, two or more
anti-DEspR antagonists can be co-administered to the subject.
[0342] For the treatment of diseases, as described herein, the
appropriate dosage of DEspR-specific antagonists will depend on the
type of disease to be treated, as defined above, the severity and
course of the disease, whether the DEspR-specific antagonist is
administered for preventive or therapeutic purposes, previous
therapeutic indications, the subject's clinical history and
response to the DEspR-specific antagonist, and the discretion of
the attending physician. The DEspR-specific antagonist is suitably
administered to the subject at one time or over a series of
treatments. In a combination therapy regimen, the DEspR-specific
antagonist and the one or more anti-cancer therapeutic agents
described herein are administered in a therapeutically effective or
synergistic amount. As used herein, a therapeutically effective
amount is such that co-administration of a DEspR specific
antagonist and one or more other therapeutic agents, or
administration of a composition described herein, results in
reduction or inhibition of the cancer as described herein. A
therapeutically synergistic amount is that amount of a
DEspR-specific antagonist and one or more other therapeutic agents
necessary to synergistically or significantly reduce or eliminate
conditions or symptoms associated with a particular disease.
[0343] The DEspR-specific antagonist and the one or more other
therapeutic agents can be administered simultaneously or
sequentially in an amount and for a time sufficient to reduce or
eliminate the occurrence or recurrence of a tumor, a dormant tumor,
or a micrometastases. The DEspR-specific antagonist and the one or
more other therapeutic agents can be administered as maintenance
therapy to prevent or reduce the likelihood of recurrence of the
tumor.
[0344] As will be understood by those of ordinary skill in the art,
the appropriate doses of chemotherapeutic agents or other
anti-cancer agents will be generally around those already employed
in clinical therapies, e.g., where the chemotherapeutics are
administered alone or in combination with other chemotherapeutics.
Variation in dosage will likely occur depending on the condition
being treated. The physician administering treatment will be able
to determine the appropriate dose for the individual subject.
[0345] In addition to the above therapeutic regimes, the subject
can be subjected to radiation therapy.
[0346] In certain embodiments of any of the methods, uses and
compositions described herein, the administered DEspR antibody is
an intact, naked antibody. However, in some embodiments, the DEspR
antibody can be conjugated with a cytotoxic agent. In certain
embodiments of any of the methods and uses, the conjugated DEspR
antibody and/or DEspR antibody fragment thereof is/are internalized
by the cell, resulting in increased therapeutic efficacy of the
conjugate in killing the cancer cell to which it binds. In some
embodiments, the cytotoxic agent conjugated to the DEspR antibody
and/or DEspR antibody fragment thereof targets or interferes with
nucleic acid in the cancer cell. Examples of such cytotoxic agents
include maytansinoids, calicheamicins, ribonucleases and DNA
endonucleases, and are further described elsewhere herein.
[0347] Unless otherwise defined herein, scientific and technical
terms used in connection with the present application shall have
the meanings that are commonly understood by those of ordinary
skill in the art to which this disclosure belongs. It should be
understood that this invention is not limited to the particular
methodology, protocols, and reagents, etc., described herein and as
such can vary. The terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention, which is defined solely
by the claims. Definitions of common terms in immunology, and
molecular biology can be found in The Merck Manual of Diagnosis and
Therapy, 18th Edition, published by Merck Research Laboratories,
2006 (ISBN 0-911910-18-2); Robert S. Porter et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Science
Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.),
Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8); Immunology by Werner Luttmann, published by
Elsevier, 2006. Definitions of common terms in molecular biology
are found in Benjamin Lewin, Genes IX, published by Jones &
Bartlett Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory
Manual (2 ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., USA (1989); Davis et al., Basic Methods in Molecular
Biology, Elsevier Science Publishing, Inc., New York, USA (1986);
or Methods in Enzymology: Guide to Molecular Cloning Techniques
Vol. 152, S. L. Berger and A. R. Kimmerl Eds., Academic Press Inc.,
San Diego, USA (1987); Current Protocols in Molecular Biology
(CPMB) (Fred M. Ausubel, et al. ed., John Wiley and Sons, Inc.),
Current Protocols in Protein Science (CPPS) (John E. Coligan, et.
al., ed., John Wiley and Sons, Inc.) and Current Protocols in
Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and
Sons, Inc.), which are all incorporated by reference herein in
their entireties.
[0348] As used herein, the term "comprising" means that other
elements can also be present in addition to the defined elements
presented. The use of "comprising" indicates inclusion rather than
limitation.
[0349] As used herein the term "consisting essentially of" refers
to those elements required for a given embodiment. The term permits
the presence of additional elements that do not materially affect
the basic and novel or functional characteristic(s) of that
embodiment of the invention.
[0350] The term "consisting of" refers to compositions, methods,
and respective components thereof as described herein, which are
exclusive of any element not recited in that description of the
embodiment.
[0351] Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. As used in this specification and the appended claims,
the singular forms "a," "an," and the include plural references
unless the context clearly dictates otherwise. Thus for example,
references to "the method" includes one or more methods, and/or
steps of the type described herein and/or which will become
apparent to those persons skilled in the art upon reading this
disclosure and so forth.
[0352] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages can mean.+-.1%.
[0353] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such can vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0354] All patents and other publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that could be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0355] This invention is further illustrated by the following
examples which should not be construed as limiting.
EXAMPLES
Example 1
Development of Novel Anti-Human Dual Endothelin-1/VEGFsp Receptor
(Anti-hDEspR) Monoclonal Antibody Treatments as Inhibitors of Tumor
Angiogenesis and Tumor Cell Invasiveness
[0356] DEspR is a key angiogenesis player in embryonic development
as seen in DEspR.sup.-/- knockout mice (Herrera et al. 2005), and
contributes to adult tissue vascularity as seen in adult
haplo-deficient (+/-) mice exhibiting decreased tissue vascularity
shown by power Doppler analysis (FIG. 2).
[0357] Based on the association of tumor invasion and metastasis
with intrinsic and evasive resistance to VEGF-targeted therapies,
the combination of anti-invasive and anti-metastatic drugs with
anti-angiogenesis therapies is important to analyze (Bergers and
Hanahan 2008). This new therapeutic mandate for anti-cancer
therapies can be addressed through a novel therapy comprising
DEspR-inhibition, since DEspR and VEGFsp expression are detected in
human endothelial cells, increased in tumor vessels, detected in
cancer cells in tumor tissue arrays and in different established
metastatic cancer cell lines, and since inhibition of DEspR
decreases both angiogenesis and tumor cell invasiveness using
corresponding established in vitro assays, as shown herein.
[0358] DEspR and VEGFsp were detected by immunostaining in
umbilical vein endothelial cells (HUVECs) and microvascular
endothelial cells (HMECS) in both basal and angiogenic
tube-formation conditions (FIGS. 3A-3E). Importantly, inhibition of
angiogenesis neovessel tube length was seen using both anti-DEspR
(Ab1) and anti-VEGFsp (Ab2) antibodies in HUVECs (FIG. 3D) and
HMECs (FIG. 3E) angiogenesis assays (Tukey's pairwise multiple
comparison P<0.001 for both HUVECs and HMECs). Similar findings
were observed using other angiogenesis parameters, such as
neovessel branching and inter-connections made. Equally important,
DEspR and VEGFsp were also detected in tumor cells, with
colocalization of VEGFsp and DEspR in the cell membrane and nuclear
membrane. Representative immunostaining is shown in FIGS.
3A-3C.
[0359] DEspR cell-membrane and nuclear-membrane expression were
detected in multiple tumor cell types, indicating that anti-DEspR
therapy is effective for different cancer types. Briefly, DEspR
expression was detected in human lung non-small cell ca NCI-H727,
lung giant cell tumor TIB-223/GCT; breast adenoca MDA-MB-231 (FIGS.
4A-4C) & MDA-MB-468, bladder ca 253J BV, colon adenoca SW480,
hepatocellular ca HEP3B, melanoma SK-MEL-2, osteosarcoma MG-63,
ovarian adenoca HTB-161/NIH:OVCA R3, prostate adeno ca PC-3 mm2,
and pancreatic ca CRL-1469/PANC-1 (FIG. 4D). DEspR expression was
not detected in HCI-H292 lung mucoepidermoid ca, and HEPG2
hepatocellular ca (FIG. 5A), and CCL-86/Raji Burkitt's lymphoma,
thus showing specificity of positive observations. Findings in
NCI-727 lung ca cells (FIG. 5B) were corroborated on tumor-section
immunostaining of Gr.III lung adenoca (FIG. 5C).
[0360] As shown in FIGS. 6A-6B, in contrast to control (C) and
pre-immune ab treatment (PI), DEspR-inhibition via anti-humanDEspR
antibody treatment inhibits tumor cell invasiveness in two cell
lines tested, metastatic breast tumor MDA-MB-231 and pancreatic
adenocarcinoma PANC-1 cell lines. The ability to target both tumor
angiogenesis and tumor cell invasiveness through DEspR inhibition
can more effectively address combined angiogenesis-metastasis
phenotypes seen in aggressive tumors and in evasive resistance to
current anti-VEGF therapies.
[0361] In vivo proof has also been demonstrated in an
irradiation-induced mammary tumor model in immunocompetent rats
using anti-ratDEspR antibody (Herrera et al. 2005). As shown in
FIG. 7, anti-DEspR treated rats exhibited minimal tumor growth
compared with mock-treated controls.
[0362] Concordantly, immunohistochemical analysis of mammary tumors
showed DEspR expression in tumor cells (FIG. 8A) similar to human
MDA-MB231 breast cancer cells, with no expression in normal breast
tissue (FIG. 8B). Importantly, residual tumors in treated rats
exhibited normalization of blood vessels (FIG. 8C) in contrast to
mock-treated tumors which showed disrupted endothelium in tumor
vessels with encroachment of tumor cells into the lumen (FIG.
8D).
[0363] Clinically, the addition of VEGFsp/DEspR-targeted
anti-angiogenic therapies to current VEGF/VEGFR2-targeted therapies
can additively or synergistically lead to the desired endpoint of
increasing overall survival in cancer patients. Given that there
are several VEGF/VEGFR2 therapies already in the clinics, the
translational development of anti-DEspR therapy as described herein
is done in order to provide this addition.
[0364] Logistically, the experiments described herein demonstrate
successful development of precursor polyclonal anti-rat DEspR
antibodies (FIGS. 7 and 8A-8D; Herrera et al. 2005) and polyclonal
anti-human DEspR ab (FIGS. 5A-5C and 6A-6B; Glorioso et al. 2007)
that exhibit robust affinity, specificity and functionality.
[0365] There are key advantages for selecting the human monoclonal
antibody therapy approaches described herein for DEspR-targeted
anti-angiogenesis therapy and target-specific molecular imaging.
Humanized/all human monoclonal antibody therapies (Ab-Rx) are a
rapidly growing class of human therapeutics (Carter 2006) and have
a relatively high success rate at 18-24% compared to new chemical
entities, including small-molecule agents at 5% (Imai & Takaoka
2006).
[0366] We have developed and validated a murine monoclonal antibody
specific for human-DEspR, termed herein as the 7C5B2 antibody,
using a 9-amino acid (aa)-long epitope located in the extracellular
amino-terminal end of hDEspR (Glorioso et al., 2007).
[0367] Briefly, mice were immunized with a KLH-conjugated antigenic
peptide comprising the NH.sub.2-terminal 9 amino acids of hDEspR,
i.e., DEspR(1-9). After four injections, sera were collected for
screening of antibody titer using free antigenic peptide as
antigen. The mouse exhibiting the best titer was used for fusion
experiments. Supernatants of fused clones were screened by ELISA
using free antigenic peptide as antigen. All positive clones were
transferred onto 24-well plate and re-tested/confirmed by ELISA.
The 10 best clones were selected for further testing, which
comprised the candidate monoclonal antibodies, anti-hDEspR
monoclonal antibody. Relative affinities of prospective monoclonal
antibodies were determined by ELISA using the supernatant from 10
best clones identified.
[0368] Analysis of relative monoclonal antibody affinity for
antigenic hDEspR 9-aa peptide identified clones 7C5C5 and 7C5B2 as
the monoclonal antibodies with strongest affinity. These two were
selected for expansion and subsequent large-scale production based
upon their higher affinity for the antigenic peptide.
[0369] To ascertain specificity, low- (5G12E8), mid- (2E4H6), and
high-affinity (7C5B2) monoclonal antibodies were tested for western
blot analysis by testing the subclone supernatant, and the
subsequent purified antibody. Candidate anti-hDEspR monoclonal
antibodies were specific for the predicted 10 kD protein for
hDEspR. Western blot analysis was done using total cellular protein
isolated from Cos1 hDEspR-transfected cells as antigen, primary
antibody comprised purified antibody and subclone supernatant of 3
selected clones, 10% gel concentration in order to detect the
expected 10 kD molecular weight protein of hDEspR. Nitrocellulose
(PIERCE) was used with a transfer buffer of 3.07 g Tris, 14.4 g
Glycine, 200 ml methanol, 800 nil dH20. HRP-anti mouse polyvalent
immunoglobulins were used (Sigma #0412) 1:100,000; ECL reagent
(SuperSignal West Femto Kit #34094), Stain reagent Kodak RP-X-Omat,
and x-film (Kodak X-film #XBT-1).
[0370] The Western blot results demonstrated specificity of
anti-hDEspR monoclonal antibody regardless of relative affinity,
and identified more than one successful anti-hDEspR monoclonal
antibody. Of the antibodies tested, the monoclonal antibody clone
with highest relative affinity and specificity was clone 7C5B2.
[0371] The top candidate anti-hDEspR monoclonal antibodies were
tested for inhibition of angiogenesis parameters in order to
identify candidate anti-hDEspR mAb-Rxtic as anti-angiogenic using
established in vitro assays.
[0372] To assess anti-angiogenic properties specific to human
cells, commercially available, pre-validated established
angiogenesis assays based on human umbilical vein cells (HUVECs)
were used. Multiple in vitro-assay angiogenesis parameters were
monitored, such as number of angiogenic tubes formed, ability of
"neovessels" or tubes to branch (# branch points), ability of said
neovessel branches to connect and form complex connections (#
branch=connections), and robustness of angiogenesis represented by
neovessel tube length (tube length in mm). Purified 7C5B2
anti-DESPR monoclonal antibody's ability to inhibit HUVECS
angiogenic capacity in vitro was assessed accordingly.
[0373] An optimal effective concentration of anti-hDEspR 7C5B2
monoclonal antibody that can inhibit >80% of neovessel tube
length and number of branch points was first assessed. This optimal
inhibitor concentration for anti-angiogenesis efficacy was found to
be 500 nM of the anti-hDEspR 7C5B2 monoclonal antibody. This
concentration was then used in a series of experiments to evaluate
other in vitro parameters of angiogenesis.
[0374] The anti-hDEspR 7C5B2 monoclonal antibody effectively
inhibited different in vitro parameters of angiogenesis, such as
number of neovessel tubes formed, branch points, branch connections
and tube length. The anti-hDEspR 7C5B2 monoclonal antibody worked
as well if not better than a previously validated polyclonal
antibody, thus validating its potential as a monoclonal
therapeutic.
[0375] The anti-hDEspR 7C5B2 monoclonal antibody was also tested
for specific binding to tumor vessel endothelium and/or tumor cells
in human cancer tissue arrays. The anti-hDEspR 7C5B2 monoclonal
antibody was evaluated in immunohistochemical analyses of human
tumor tissue-arrays comprised of core biopsy specimens representing
tumors and normal tissue on the same slide. Conditions that
optimized specificity and sensitivity of detection using
formalin-fixed, paraffin embedded core biopsy sections were tested.
Double-immunofluorescence experiments were performed in order to
evaluate hDEspR expression and CD133 expression, with the latter
serving as a marker for putative cancer stem cells.
Antigen-retrieval was performed and used anti-hDEspR monoclonal
antibody at 1:10, and commercially available anti-CD133 mAb at 1:20
dilution.
[0376] As shown in FIGS. 14A-14B, representative
immunohistochemical analysis of human tumor tissue-arrays using
anti-hDEspR 7C5B2 monoclonal antibody detected increased expression
of hDEspR in stage II-lung cancer tumor cells (FIG. 14A). Some
tumor cells are double immunostain-positive for both hDEspR and
CD133, with other tumor cells immunostained for CD133. These
observations demonstrate that hDEspR is also present in postulated
CD133-positive cancer stem cells, as well as CD133-negative tumor
cells. In contrast, normal lung specimen does not exhibit any
immunostaining for hDEspR or CD133 (FIG. 14B). In addition,
increased DEspR expression was observed in a variety of CD133+
cancer stem cell subsets, as detected by immunofluorescence with a
combination of anti-DEspR, anti-CD133 and anti-CXCR4 monoclonal
antibodies, including NBC mda-mb-231 cells, pancreatic ductal
adenoca Panc1 cells, glioblastoma cells, and breast cancer cells.
Accordingly, in some embodiments, the compositions and methods
described herein can be used in targeted treatments for tumor
resistance and/or recurrence by targeting cancer stem cells or
cancer initiating cells.
[0377] Accordingly, to summarize, this murine antibody "7C5B2"
exhibited high affinity binding by ELISA to the 9 aa-long epitope
(FIG. 9), demonstrates specificity by western blot (FIG. 10),
immunostains HUVECs undergoing tubeformation (FIGS. 3A-3E), and
pancreatic adenoca PANC-1, and breast cancer MDA-MB-231 cells.
[0378] We demonstrated functional efficacy in vitro by showing that
both the polyclonal (Pab) and monoclonal anti-DEspR 7C5B2, specific
for human DEspR, inhibit different parameters of angiogenesis in
HUVECs (FIGS. 10A-10C): mean number of branchpoints as a measure of
neovessel complexity (FIG. 10A), and total length of tubes as a
measure of neovessel density (FIG. 10B). Dose response curve for
inhibition (FIG. 10C) showed equivalent robustness to inhibit both
angiogenesis parameters. Importantly, murine 7C5B2 also inhibits
tumor cell invasiveness in MDA-MB-231 human breast cancer and
PANC-1 pancreatic cancer cell lines.
[0379] This murine anti-human DEspR monoclonal antibody 7C5B2 is
thus shown to have high affinity, specificity, and functionality
serves as the starting antibody for the development of anti-DEspR
composite de-immunized all human antibodies, as described
herein.
[0380] Accordingly, described herein are the development,
characterization, and in vitro efficacy testing of anti-hDEspR
composite de-immunized all human monoclonal antibody (cdHMAb) for
use as novel antibody therapies aimed at addressing evasive and
intrinsic resistances to current anti-VEGF/VEGFR2 antiangiogenic
therapies.
[0381] We have selected Antitope's Composite Human Antibody
technology to generate anti-hDEspR deimmunized human monoclonal
antibodies for antibody therapeutics (Antitope, 2010). This
technology generates de-immunized 100% human antibodies at the
outset, in contrast to non-deimmunized human antibodies derived
from phage and transgenic mice technologies. Briefly, composite
human antibodies comprise multiple sequence segments ("composites")
derived from V-regions of unrelated human antibodies are selected
to maintain monoclonal antibody sequences critical for antigen
binding of the starting murine precursor anti-human DEspR
monoclonal antibody, and are filtered for the presence of potential
T-cell epitopes using proprietary "in silico tools" (Holgate &
Baker 2009). The close fit of human sequence segments with all
sections of the starting antibody V regions and the elimination of
CD4+ T cell epitopes from the outset circumvent immunogenicity in
the development of `100% human` therapeutic antibodies while
maintaining optimal affinity and specificity through the prior
analysis of sequences necessary for antigen-specificity (Holgate
& Baker 2009). Immunogenicity can hinder clinical applications
of 100% human monoclonal antibodies (Chester et al. 2009).
[0382] Briefly, "composite human antibodies" comprise multiple
sequence segments ("composites") derived from V-regions of
unrelated human antibodies that are selected to maintain monoclonal
antibody sequences critical for antigen binding of the starting
murine precursor anti-human DEspR monoclonal antibody, such as
7C5B2 antibody, and which have all been filtered for the presence
of potential T-cell epitopes using "in silico tools" (Holgate &
Baker, 2009). The close fit of human sequence segments with all
sections of the starting antibody V regions and the elimination of
CD4+ T cell epitopes from the outset allow this technology to
circumvent immunogenicity in the development of `100% human`
therapeutic antibodies while maintaining optimal affinity and
specificity through the prior analysis of sequences necessary for
antigen-specificity (Holgate & Baker 2009).
[0383] As described herein, structural models of mouse anti-hDEspR
antibody V regions were produced using Swiss PDB and analysed in
order to identify important "constraining" amino acids in the V
regions that were likely to be essential for the binding properties
of the antibody. Residues contained within the CDRs (using Kabat
definition) together with a number of framework residues were
considered to be important. Both the V.sub.H and V.sub.L (V.sub.K)
sequences of anti-hDEspR, as described herein as SEQ ID NO: 4 and
SEQ ID NO: 9, comprise typical framework residues and the CDR1,
CDR2, and CDR3 motifs are comparable to many murine antibodies.
[0384] From the above analysis, it was determined that composite
human sequences of anti-hDEspR can be created with a wide latitude
of alternatives outside of CDRs but with only a narrow menu of
possible alternative residues within the CDR sequences. Analysis
indicated that corresponding sequence segments from several human
antibodies could be combined to create CDRs similar or identical to
those in the murine sequences. For regions outside of and flanking
the CDRs, a wide selection of human sequence segments were
identified as possible components of novel anti-DEspR composite
human antibody V regions for use with the compositions and methods
described herein (see, for example, Table 1).
[0385] Based upon these analyses, a large preliminary set of
sequence segments that could be used to create novel anti-DEspR
composite human antibody variants were selected and analysed using
iTope.TM. technology for in silico analysis of peptide binding to
human MHC class II alleles (Perry et al 2008), and using the
TCED.TM. (T Cell Epitope Database) of known antibody
sequence-related T cell epitopes (Bryson et al 2010). Sequence
segments that were identified as significant non-human germline
binders to human MHC class II or that scored significant hits
against the TCED.TM. were discarded. This resulted in a reduced set
of segments, and combinations of these were again analysed, as
above, to ensure that the junctions between segments did not
contain potential T cell epitopes. Selected segments were then
combined to produce heavy and light chain V region sequences for
synthesis. Exemplary heavy chain V region sequences provided herein
and generated using the above-described methods include SEQ ID NO:
13-SEQ ID NO: 17. Exemplary heavy chain V region sequences provided
herein and generated using the above-described methods include SEQ
ID NO: 18-SEQ ID NO: 19.
[0386] In vitro efficacy of the antibodies described herein are
assessed by examining dose response-inhibition of angiogenesis of
HUVECs (human umbilical vein cells) and HMECs (adult human
microvascular endothelial cells) in angiogenesis assays (see FIGS.
3A-3E, 10A-10C), which in some embodiments are set-up with
co-cultured cancer cells, such as PANC-1 and MDA-MB-231, and in
some embodiments in normoxia and hypoxia (2%02) conditions. Both
HUVECs and HMECs are used for the following reasons: HUVECs is the
standard in the field, but as these cells are umbilical vein
derived, and adult microvascular endothelial cells (HMECs) are also
used. In addition, angiogenesis is assessed with co-cultured cancer
cells, in addition to the fetal bovine serum that is usually added
in angiogenesis assays, in order to better simulate angiogenic
factors that cancer cells produce which contribute to evasive and
intrinsic resistance.
[0387] In some embodiments, since hypoxia is one of the triggers
for angiogenesis, and one of the contributing factors suspected of
underlying evasive resistance to current anti-VEGF therapies, in
vitro efficacy assays are conducted in normoxia and in 2%02
hypoxia. Composite deimmunized monoclonal antibody-mediated
inhibition of tumor cell invasiveness in vitro is analyzed using
MDA-MB-231 and PANC-1 cells and by using established quantitative
assays. These are also done in normoxia and 2% O2-hypoxia
conditions, to test a more aggressive tumor cell phenotype known to
be associated with hypoxia.
[0388] The effects of anti-hDEspR inhibition are compared to
controls, which can include untreated controls, isotype controls,
murine precursor anti-hDEspR monoclonal antibody controls, and
bevacizumab controls. Each point for angiogenesis and tumor cell
invasiveness assays are done using at least 5 replicates.
Furthermore, for the top 2 candidate-leads, dose response curve
inhibition responses are also performed, where each dosage is
studied using at least 5 replicates.
[0389] Assays can be analyzed by one way ANOVA and multiple
pairwise comparison to assess significant changes. Mean levels of
%-inhibition from control by each candidate lead (e.g., 5-10) are
used to rank them according to different assays, and the highest
ranked two identifies the top-2 leads corresponding to best
inhibitor of angiogenesis and tumor cell invasiveness in both for
example, normoxia and hypoxia conditions, and in both, for example,
MDA-MB-231 and PANC-1 cancer cell lines respectively.
[0390] Tumor array analysis is done to corroborate specificity and
sensitivity of each to detect tumor cells and tumor neovessels in
tissue arrays of human biopsy core samples form different cancer
tissue types. This is performed on a tissue array panel
representing solid tumors from brain, pancreas, lung, breast,
ovarian, prostate, bladder, colon, stomach. Results are analyzed
for specificity given the same immunochemistry conditions used in
validation of the murine precursor anti-hDEspR Mab-H1. As shown,
there is minimal DEspR expression in normal human pancreas, whereas
in stage IV pancreatic cancer exhibits increased DEspR expression
in pancreatic tumor cells and tumor blood vessels. The composite
deimmunized monoclonal antibody candidate leads are ranked and the
top-2 that have the best detection of tumor cells and tumor
neovessels with optimal signal to noise ratio in tumor tissue array
immunohistochemistry are determined. This can be compared to
tumor-array immunostaining observations obtained with the murine
precursor anti-hDEspR Mab.
[0391] In addition to de-immunizing the antibodies described herein
using in silico screening of T-cell epitopes to minimize and reduce
immunogenicity, the composite humanized anti-hDEspR composite
deimmunized monoclonal antibodies are tested in vitro for
immunogenicity in order to select for the least immunogenic
composite all human Mab. Immunogenicity screening can be performed
using a representative of 50 donors, which has proven to correlate
with clinical observations (Baker & Jones 2007).
[0392] Immunogenicity testing, along with the other in vitro assays
of specificity and efficacy allows for the selection of a top
anti-hDEspR lead, based on a combination of factors, including best
affinity (ELISA), specificity (western blot analysis), in vitro
efficacy (inhibition of angiogenesis and tumor cell invasiveness)
and lowest immunogenicity. A priori ascertainment of low
immunogenicity by elimination of T cell epitopes in the composite
antibody humanization process, and low immunogenicity ascertainment
by using ex vivo T cell assay technology are important
translational research steps, since high immunogenicity limits ab
therapeutic efficacy (Iwai & Takaoka 2006) despite
target-specificity and total humanization as has been discussed in
clinical studies for Infliximab, Alemtuzumab (review by Baker &
Jones 2007).
[0393] The top composite deimmunized monoclonal antibodies leads
are tested for in vivo efficacy by testing anti-DEspR-mediated
inhibition of tumor growth, angiogenesis and metastasis in
established human cancer cell line xenograft and metastasis models
in immuno-compromised mice. Cancer tissue types representative of
evasive resistance (breast cancer) and intrinsic resistance
(pancreatic cancer) as observed in published reports are also
tested. For example, MDA-MB-231 breast cancer and PANC-1 pancreatic
carcinoma cell lines are used, since both can be used to generate
xenograft and metastasis spleen-infusion models. For MDA-MB-231
orthotopic and metastasis models nude mice are used (Oh et al.
2009, Roland et al. 2009). For PANC-1 xenograft subcutaneous models
nude mice are used as described (Zheng et al. 2008) and NOG mice
for PANC-1 metastasis model as described (Suemizu et al. 2007).
[0394] Through the strategic use of anti-humanDEspR-specific (e.g.,
composite deimmunized monoclonal antibody primary lead) and
anti-human-VEGF-specific (bevacizumab) antibodies, and a
murine-DEspR-specific Mab, 1) efficacy of anti-DEspR therapy
compared with anti-VEGF therapy alone can be assessed, and 2)
determination of synergistic efficacy using a combination of
anti-DEspR and anti-VEGF antibodies.
[0395] Treatment in xenograft models begin when tumors are 200-300
mm in size to simulate clinical cancer therapy scenarios. To assess
anti-DEspR therapy efficacy in metastasis models, a sustained
treatment regimen begun 5 days after the intrasplenic infusion of
cancer cells is assessed, as described (Oh et al. 2009). To assess
whether anti-DEspR therapy induces increased risk for metastasis
observed with sunitinib (Ebos et al. 2009), Ebos's experiment are
performed, whereby anti-murineDEspR Mab is infused daily for 7
doses beginning 7 days prior to cancer cell infusion. 250 ug is
used for each antibody-therapeutic given IP 2.times./week as
described for bevacizumab (Roland et al. 2009), and 3.times. per
week for anti-DEspR (Herrera et al. 2005).
[0396] Treatment outcomes are assessed by multifaceted parameters:
serial imaging of tumor volume and tumor angiogenesis for
orthotopic mammary and subcutaneous pancreatic tumors by, for
example, high-resolution Vevo770 ultrasound imaging and power
Doppler analysis. Overall survival is determined, and at this
endpoint, repeat ultrasound imaging and histological analysis of
tumor size and angiogenesis is done, along with histological
analysis of malignancy phenotype: nuclear grade, tumor cell
invasion of stroma, tumor cell vascular mimicry, loss of integrity
of tumor neovessels and macrophage infiltrates.
[0397] Heterozygous DEspR+/- mice live beyond 1 year and breed,
which is in contrast to VEGF+/- haplodeficiency which is embryonic
lethal at E11.5. However, since adverse effects have been observed
in patients on anti-VEGF (bevacizumab) and anti-VEGFR2 (sunitinib,
sorafanib) therapies, the anti-humanDEspR-specific antibodies
described herein are also tested for these effects. Analysis of
parameters of potential adverse effects are done in PANC-1 and
MDA-MB-231 xenograft models treated with cdHMAb-H1 and mDEspR-Mab.
For example, potential a) cardiotoxicity can be monitored by serial
non-invasive ultrasound cardiac function analysis; b) hypertension
can be monitored by tail cuff BP; c) bowel perforation can be
monitored on post-mortem anatomical inspection at endpoint; d)
bleeding, thrombosis can be monitored by examination and vascular
ultrasound and Doppler flow analysis, and e) toxicity screen can be
performed, such as liver function tests, renal function tests,
complete blood count, blood chemistries at endpoint of study. These
parameters are compared in mock-treated age-matched tumor model
controls.
Analysis of Molecular Imaging of Tumor Angiogenesis and Tumor Cell
Vascular Mimicry Changes in Response to Therapy by
Contrast-Enhanced Ultrasound Imaging of DEspR-Targeted Neovessels
Compared with VEGFR2-Targeted Tumor Neovessels.
[0398] Molecular imaging of angiogenesis in tumors has been
demonstrated by contrast-enhanced ultrasound imaging using
anti-VEGFR2 antibody-directed microbubbles with imaging and
contrastenhanced analysis done using the VisualSonics Vevo770
high-resolution ultrasound system (Willmann et al. 2007). We have
used this same system to detect anti-DEspR antibody-directed
microbubbles in carotid artery disease vasa vasorum angiogenesis in
a transgenic rat atherosclerotic model associated with carotid
artery disease progression and stroke risk (Decano et al. 2010). As
shown in FIGS. 16A-16D, DEspR-targeted molecular imaging (16A)
detects DEspR+ endothelial lesions (16B) and vasa asorum
angiogenesis (16C). Quantitation of contrast intensity is done
using integrated software (16D).
[0399] DEspR-targeted molecular imaging is used to test composite
deimmunized monoclonal antibodies as the targeting module for
molecular imaging applicable to xenograft tumor cell vascular
mimicry, and microbubbles are confined to the vascular lumen.
MouseDEspR-specific molecular imaging using composite deimmunized
monoclonal antibodies as described herein is performed in order to
monitor mouse-derived tumor angiogenesis, and is compared to
VEGFR2-specific molecular imaging. The observations described
herein provide proof that composite deimmunized monoclonal
antibodies specific for DEspR can serve as the targeting module for
molecular imaging of tumor cell vascular mimicry in a mouse model;
that molecular imaging of DEspR expression provides a translatable
diagnostic in vivo imaging modality to assess tumor angiogenesis,
and that comparative analysis of DEspR-specific molecular imaging
provides new insight into the differential contribution of tumor
cell vascular mimicry and tumor angiogenesis.
[0400] Both MDA-MB-231 xenograft orthotopic and PANC-1 xenograft
heterotopic tumor models, as well as a PANC-1 intrasplenic-infusion
liver metastasis model are used for molecular imaging experiments.
Isotype-antibody molecular imaging is used as a control to
demonstrate specificity of DEspR-positive molecular imaging.
Identical conditions are followed for anti-DEspR and anti-VEGFR2
molecular imaging in order to validate comparative analysis. For
example, a composite deimmunized monoclonal antibody can be used to
target tumor cell vascular mimicry; an anti-DEspR composite
deimmunized monoclonal antibody can be used to target mouse
neovessel formation monoclonal antibody in human xenograft tumors;
anti-VEGFR2 can be used as a comparative benchmark, and an isotype
antibody can be used as a negative control.
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Example 2
Molecular Imaging of Vasa Vasorum Neovascularization Via
DEspR-Targeted Contrast-Enhanced Ultrasound Micro-Imaging in
Transgenic Atherosclerosis Rat Model
[0426] Given that carotid vasa vasorum neovascularization is
associated with increased risk for stroke and cardiac events, the
in vivo study described herein was designed to investigate
molecular imaging of carotid artery vasa vasorum neovascularization
via target-specific contrast-enhanced ultrasound (CEU)
micro-imaging. Accordingly, molecular imaging was performed in male
transgenic rats with carotid artery disease (CAD) and
non-transgenic controls using DEspR (dual endothelin1/VEGFsp
receptor)-targeted microbubbles (MB.sub.D) and the Vevo770
micro-imaging system and CEU-imaging software.
[0427] It was found that DEspR-targeted CEU-positive imaging
exhibited significantly higher contrast intensity signal
(CIS)-levels and pre-/post-destruction CIS-differences in 7/13
transgenic rats, in contrast to significantly lower CIS-levels and
differences in control isotype-targeted microbubble (MB.sub.C)--CEU
imaging (n=8) and in MB.sub.D CEU-imaging of 5/5 non-transgenic
control rats (P<0.0001). Ex vivo immunofluorescence analysis
demonstrated binding of MB.sub.D to DEspR-positive endothelial
cells, and association of DEspR-targeted increased contrast
intensity signals with DEspR expression in vasa vasorum neovessel
and intimal lesions. In vitro analysis demonstrated dose-dependent
binding of MB.sub.D to DEspR-positive human endothelial cells with
increasing % cells bound and number of MB.sub.D per cell, in
contrast to MB.sub.C or non-labeled microbubbles (P<0.0001).
[0428] The dual endothelin-1 (ET1)/vascular endothelial growth
factor-signal peptide (VEGFsp) receptor or DEspR (formerly dear
gene as deposited in GenBank) [1] plays a key role in developmental
angiogenesis deduced from the embryonic lethal phenotype exhibited
by despr.sup.-/- knockout mice due to absent embryonic and
extraembryonic angiogenesis, aborted dorsal aorta vasculogenesis,
and abnormal cardiac development [2]. While exhibiting similar
abnormal vasculogenesis and angiogenesis phenotypes with
VEGF.sup.+/- haploinsufficient mice, despr.sup.-/- null mice
exhibit distinct neural tube phenotypes [2-4]. Consistent with its
role in developmental angiogenesis, DEspR inhibition results in
decreased tumor angiogenesis and tumor growth in adult rat mammary
tumors and mouse melanomas [2].
[0429] Development of target-specific contrast enhanced
ultrasonography (CEU)-imaging, herein referred to as "molecular
imaging" of vascular disease neovascularization is important since
carotid artery vasa vasorum neovascularization is associated with
increased risk for stroke [5,6]. However, successful molecular
imaging of vasa vasorum neovessels has not been reported, although
detection by non-targeted CEU-imaging has [7]. On the other hand,
successful molecular imaging in different disease models detecting
different targets [8,9] has shown the potential of molecular
imaging in different disease contexts, such as .alpha.v.beta.3 in
tumor and hind limb ischemia angiogenesis [10,11], VEGFR2 in tumor
angiogenesis [12], ICAM-1 in transplant rejection [13], L-selectin
in malignant lymphnodes [14], and ICAM-1 and VCAM-1 in
atherosclerosis [15], P-selectin in myocardial ischemia [16,17],
GIIb/IIIa and fibrinogen in thrombosis [18,19]. Molecular imaging
of vascular disease neovascularization in studies targeting
VEGFR2-, ICAM-1 and VCAM-1 did not detect vasa vasorum neovessels
in a hyperlipidemic rabbit model of injury-induced vascular
neovascularization[9,20].
[0430] Demonstrated herein is molecular imaging of DEspR in carotid
artery lesions and expanded vasa vasorum neovessels in
transgenic-hyperlipidemic, hypertensive carotid artery disease rat
model.
Materials and Methods
[0431] Animals.
[0432] In order to facilitate molecular imaging studies of
pathological angiogenesis in vascular lesions or in expanded vasa
vasorum neovessels, a carotid artery disease rat model with
hypertension-atherosclerosis as risk factors, the Tg25[hCETP]
Dahl-S rat model, Tg25, transgenic for human cholesteryl ester
transfer protein which develops accelerated stroke [21] or
later-onset coronary heart disease, was selected [22]. 4-month old
transgenic male rats (n=13) projected to be around early-midpoint
along the disease course of stroke [21] or coronary atherosclerosis
phenotype [22], were studied for DEspR-targeted molecular imaging
(n=13). MB.sub.D-infused non-transgenic, non-atherosclerotic
littermates were studied as negative biological controls (n=5).
Isotype-specific MB.sub.C-infused transgenic rats (n=8), with the
following subgroups: 4 transgenic rats which exhibited
MB.sub.D-specific CEU-positive imaging, and 4 de novo transgenic
rats, were studied concurrently as negative imaging controls.
[0433] Target-Specific CEU-Molecular Imaging.
[0434] The Vevo770 high resolution ultrasound system with contrast
mode software, and streptavidin-coated "target ready" MicroMarker
microbubbles (VisualSonics Inc, Canada) previously validated for
molecular imaging of VEGFR2 on tumor angiogenesis in mice was used
[12]. To target the microbubble to rat DEspR-positive endothelial
cells, target ready-MicroMarker microbubbles were linked to
biotinylated anti-DEspR antibody (MB.sub.D) via streptavidin-biotin
coupling. For control, target ready-MicroMarker microbubbles were
linked to biotinylated, isotype-antibody (MB.sub.C). Each bolus
comprised of 3-4.times.10.sup.8 microbubbles in 200-microliters
saline, infused into the rat tail vein over 8-seconds.
[0435] CEU-imaging of rat carotid arteries comprised a sequence of
steps aimed at optimizing MB-target binding, eliminating
confounders, and ascertaining reproducible CEU-imaging. Baseline
images of the carotid artery were first obtained and immobilized
the scanhead to maintain the optimal B-mode view of the common,
external, and internal carotid arteries in one 2D image. One minute
after MB bolus infusion, the MB blood pool was documented by B-mode
imaging for all rats to ascertain MB infusion and to demonstrate
absence of contrast intensity in surrounding tissue. A wait of 4-5
minutes was taken to allow MB.sub.D adherence to DEspR-positive
endothelial targets [12], and to allow clearance of unbound
circulating microbubbles [23]. Clearance of most circulating MBs
facilitates detection of increased contrast intensity signals due
to adherent MBs validated for detection using the Vevo770 imaging
system [23]. Adherent MBs were defined by the loss of
contrast-intensity upon acoustic destruction performed using
pre-set Contrast Enhanced software (VisualSonics, Inc, Canada) as
described [12].
[0436] Four regions of interest (ROI) on the carotid artery were
monitores: the common carotid artery, bifurcation, external and
internal carotid arteries. Quantitation of contrast intensity
signals (CIS) resulting from backscatter of adherent
targeted-microbubbles was done using contrast-enhanced analysis
program validated for the Vevo770 imaging platform (VisualSonics
Inc, Canada) detecting pre- and post-acoustic disruption contrast
intensity signals. The contra-lateral carotid artery was checked
immediately, and the same CEU-imaging protocol followed. After a
20-minute interval to allow complete clearance of any residual MBs,
a pre-set destruction sequence was performed for subsequent
CEU-imaging with isotype-specific MB.sub.Cs following identical
procedures. For quantitative comparative analyses, the difference
in contrast intensity signals between pre- and post-acoustic
destruction, CIS-difference, as well as their respective
pre-destruction CIS-peak levels were studied for each carotid
artery per rat.
[0437] Histology and Immunofluorescence Staining of Rat Carotid
Arteries.
[0438] After CEU-imaging, carotid arteries were collected en bloc
preserving the surrounding tissue around the common (CCA), external
(ECA) and internal (ICA) carotid arteries including the carotid
artery bifurcation. The ECA was cut longer than the ICA to be able
to distinguish the two. Longitudinal serial sections were obtained
per carotid artery (50-100 sections) and staining every 10th slide
with Masson-trichrome allowed proper orientation and site-specific
analyses corresponding to ROIs in CEU-imaging. The flanking serial
sections to MT-stained slides of interest were then immunostained.
Double immunofluorescence staining was done on deparaffinized
sections via sequential antigen retrieval, treatment to reduce
background, blocking, incubation with primary antibody at 4.degree.
C. overnight, secondary antibody incubation overnight at 4.degree.
C. with AlexaFluor 568 goat anti-mouse IgG and AlexaFluor 488 goat
anti-rabbit IgG, washing, and mounting using Prolong Gold with DAPI
(Invitrogen, CA). Negative controls were run using rabbit-isotype
antibody for anti-rat DEspR antibody. A Zeiss Axioskop2plus
microscope was used for fluorescence imaging and differential
interference contrast (DIC) photomicroscopy to provide
morphological information overlay to immunostained sections. Low
2.5.times. magnification was used for proper orientation and
site-specific identification along the carotid artery.
[0439] In Vitro Analysis of MB.sub.D and DEspR-Positive Endothelial
Cell Interactions
[0440] Human-specific DEspR-targeted MB.sub.Ds were made following
identical procedures for rat-specific DEspR molecular imaging with
the exception of the use of a anti-humanDEspR monoclonal antibody.
Fixed numbers of human umbilical vein endothelial cells (HUVECs)
were seeded onto IBIDI perfusion 6-lane .mu.-slide VI (ibidiGmbH,
Germany). After 24 hours, MB.sub.D-type microbubbles were infused
at the following MB-cell ratios: 8.times., 80.times., and
800.times.. Negative controls comprised of 800.times.MB.sub.Cs and
800.times. non-targeted microbubbles, MB.sub.Ds. These were all
infused at 20 dynes/cm.sup.2 shear stress 1-way flow on the same
6-lane micro-flow chamber slide. After 45 minutes of incubation,
DAPI nuclear staining was performed and excess MBs were washed with
HUVECs media at same shear stress. Phase contrast and
epifluorescence microscopy was performed in 6 random high power
fields. Cells and microbubbles were documented by photomicroscopy
and counted as to percent cells with bound MB, and number of MBs
per cell. We compared MB.sub.D, MB.sub.C and non-targeted
microbubbles MB.sub.O.
[0441] Statistical Analysis.
[0442] Values are expressed as mean.+-.S.E.M. Data were analyzed
with Prism 5 statistics software (GraphPad Software Inc, CA). Where
applicable, nonparametric ANOVA and Dunn's multiple comparison
tests or ANOVA and Tukey's multiple pairwise comparison tests were
used. For two group comparison, nonparametric Kruskal Wallis test
was performed using Prism5 (GraphPad Software Inc, CA).
Results
[0443] DEspR-Targeted Molecular Imaging of Carotid Artery.
[0444] Given the need for detecting vascular disease-associated
angiogenesis in carotid artery disease [5,6], DEspR was tested to
determine whether it can serve as an endothelial target for
contrast enhanced ultrasonographic (CEU)-imaging of pathological
angiogenesis in carotid artery disease lesions or vasa vasorum
neovascularization. The Tg25 rat model of carotid artery disease
was used, comparing 4-month old male Tg25 rats projected to be at
midpoint of atherosclerotic disease course [21, 22], with
age-matched non-transgenic male littermates. Compared to coronary
artery disease, investigation of carotid artery disease provides a
tactical experimental system with less movement artifacts.
[0445] Using the Vevo770 ultrasound contrast-enhanced imaging
system and DEspR-targeted microbubbles (MB.sub.D) compared with
control isotype-microbubbles (MB.sub.C), MB.sub.D-specific
CEU-positive imaging was detected in different regions-of-interest
(ROI) along the common carotid artery (CCA), carotid artery
bifurcation, proximal internal and/or external carotid arteries in
7/13 transgenic rats. MB.sub.D-specific CEU-positive imaging was
defined as stably increased contrast intensity signals detected
after circulating microbubbles have cleared, and which decreased
upon acoustic destruction (FIG. 19A). The peak pre-destruction
contrast intensity signals and the differences in
pre-/post-destruction contrast intensity signals (CIS-differences)
were significantly higher in MB.sub.D-specific CEU-positive images
(FIG. 19A, Table 2) compared with CEU-imaging observed in isotype
MB.sub.S-infused rats (FIG. 19B) and in MB.sub.D-infused
non-transgenic control rats (n=5), with the latter two empirically
defining CEU-negative imaging. Notably, of the 7 transgenic rats
exhibiting MB.sub.D-specific CEU-positive imaging, four exhibited
CEU-positive imaging in both carotid arteries, while three
exhibited CEU-negative imaging on the contra-lateral carotid
artery, suggesting selectivity of MB.sub.D-specific CEU-positive
imaging and concordant with specificity (Table 2). Moreover, six
transgenic rats exhibited CEU-negative imaging with low peak
contrast intensity signals, "flat-line" pre-/post-destruction
CIS-plot pattern, and minimal CIS-differences (FIGS. 19D, 19E,
Table 2) similar to CEU-negative imaging observed in
MB.sub.S-control rats (FIG. 19B) and in MB.sub.D-infused
non-transgenic controls (FIG. 19C).
[0446] Altogether, these observations provide compelling evidence
that MB.sub.D-based CEU-positive images are specific and due to
adherent MB.sub.Ds in said carotid arteries. Statistical analysis
by one way analysis of variance (ANOVA) and post-hoc multiple
comparison testing establish that the CIS-differences of
MB.sub.D-specific CEU-positive imaging are significantly higher,
P<0.0001, compared to each CEU-negative imaging study group,
respectively (Table 2, FIG. 19D). Interestingly, since CEU-positive
imaging is detected only in transgenic rats, and with 54% of
transgenic rats exhibiting MB.sub.D-specific CEU-positive imaging
at 4 months of age equivalent to an early-midpoint of the typical
model disease course in males [21, 22], average CIS-differences are
significantly different (P<0.0001) between transgenic rats and
their non-transgenic controls (FIG. 19E). With 7/13 transgenic rats
exhibiting CEU-positive imaging, and 6/13 exhibiting CEU-negative
imaging upon MB.sub.D infusion, a sub-grouping of transgenic rats
based on MB.sub.D CEU-imaging CIS-differences at the 4-month
midpoint of the disease course is apparent (FIG. 19E).
[0447] Interestingly, the CIS-plots of three transgenic rats with
the highest MB.sub.D-specific CIS-differences exhibited the
expected post-acoustic destruction drop in signal intensity but had
secondary peaks of contrast intensity signals followed subsequently
by decline to low/baseline levels (FIGS. 20A-20H). This
post-acoustic destruction/disruption pattern is consistent with a
particular sequence of microbubble events: microbubble
fragmentation accounting for the drop, residual microbubble
acoustic stimulation accounting for the secondary peak, followed by
acoustically driven diffusion accounting for the subsequent steady
decline to baseline levels.
[0448] Histological Analysis Detects MB.sub.D-Microbubbles on
DEspR-Positive Endothelial Cells.
[0449] Unexpectedly, Masson-trichrome stained histological analysis
detected a few microbubbles still attached to endothelial cells or
within intimal lesions (FIG. 21A) obtained from R1:MB.sub.D rat
with CEU-positive imaging shown. Corresponding DEspR-immunostaining
on the adjacent serial section confirmed adherence of
MB.sub.D-microbubbles to DEspR-positive endothelial cells (FIGS.
21B, 21C). Immunostaining with isotype antibody confirms
specificity of DEspR-positive immunostaining (FIG. 21D).
Altogether, these observations corroborate MB.sub.D-binding and
specificity of MB.sub.D-binding to DEspR-positive endothelium.
Survival of PEG-coated Target-ready MicroMarker microbubbles
(VisualSonics, Inc., Canada) through PBS-buffered 4%
paraformaldehyde fixation, paraffin embedding and deparaffinization
parallels our observation that PEG-based biomaterials survive
fixation, paraffin embedding, deparaffinization and Masson
trichrome staining [24].
[0450] Histological analysis of R3:MB.sub.D rat shown in FIGS.
20A-20H also detected increased endothelial DEspR-positive
expression and luminal endothelial pathology, as well as marked
carotid vasa vasoral expansion by neovascularization (FIG. 21E,
21F) with DEspR-positive expression in vasa vasorum neovessel (FIG.
21G). Double-immunofluorescence immunostaining with DEspR and
.alpha.-smooth muscle actin (.alpha.SMA) detected some
co-localization of DEspR+.alpha.SMA-positive immunostaining in
carotid artery vasa vasorum (FIG. 21H).
[0451] Increased DESPR-Expression is Associated with DEspR
Positivemolecular Imaging.
[0452] To determine whether increased level and/or area of
DEspR-expression is associated with MB.sub.D-specific CEU-positive
imaging defined by higher CIS-differences (FIG. 19D) and higher
pre-destruction CIS-peak levels (FIG. 22A), double
immunofluorescence-staining was performed with anti-DEspR and
anti-.alpha.-smooth muscle alpha actin (.alpha.SMA) antibodies, the
latter serving as a positive control for immunostaining of vascular
smooth muscle cells in the media. Serial sections from
representative rats were analyzed (n=3/group) with
MB.sub.D-specific bilateral CEU-positive imaging, MB.sub.D-infused
bilateral CEU-negative imaging, and with one-sided
CEU-positive/CEU-negative imaging. Analysis of immunofluorescence
and differential-interference contrast (DIC)-microscopy showed that
MB.sub.D-specific CEU-positive imaging is associated with DEspR+
expression in carotid intimal lesions, vasa vasorum
neovascularization and DEspR+ expression in vasa vasorum neovessels
(FIGS. 21B, 21C, 22B, 22C, Table 2). In contrast, rat carotid
arteries exhibiting MB.sub.D-CEU-negative molecular imaging were
associated with minimal, if any, DEspR+ endothelial expression
(FIG. 22D, Table 2). Low levels of .alpha.aSMA expression in
carotid media smooth muscle cells (SMCs) compared with the expanded
vasa vasorum were also noted (FIG. 22A), due, without wishing to be
bound or limited by a theory, most likely to the synthetic state of
SMCs in these hypertensive rats, since .alpha.SMA expression is
deinduced in synthetic or proliferating SMCs [25]. These
observations link MB.sub.D-specific CEU-positive imaging in this
rat model with increased DEspR expression intensity and area in
both intimal lesions and vasa vasorum neovessel density.
[0453] In Vitro Analysis of Dose-Response MB.sub.D-Adherence to
DEspR Positive Endothelial Cells.
[0454] In order to further dissect MB.sub.D interactions with
DEspR-positive cells, the dose-response of MB.sub.D adherence in
vitro was tested. In order to avail of standardized primary
cultures of endothelial cells and to gain translational insight
into molecular imaging in humans, human umbilical vein endothelial
cells (HUVECs) which express DEspR in proliferating and
pro-angiogenesis culture conditions as detected by a human-specific
anti-DEspR monoclonal antibody were used. Using increasing number
of MB.sub.Ds from 8.times., 80.times., and 800.times.MB.sub.D to
cell ratio, it was observed that HUVECs are increasingly bound by
MB.sub.Ds being 100% bound at 80.times.MB.sub.D:cell ratio (FIGS.
23A-23C), in contrast to 800.times.MB.sub.Cs (FIG. 23D) and
non-targeted MB.sub.Ds (FIG. 23E) which bound 6.8% and 8.2% of
HUVECs respectively (FIG. 23F). Moreover, analysis of number of MBs
bound per cell after a 45-minute incubation and wash at flow rates
with aortic-like shear stress of >20 dyne/cm.sup.2 revealed
significant differences in number of MBs bound per cell increasing
from 8.times., 80.times. to 800.times. as follows: 2.3, 17 and 49
MBs/cell, with only 0.6 and 1.1 MB/cell for non-targeted MBs and
isotype MB.sub.Cs (ANOVA P<0.0001). These observations reflect
the relative stability and specificity of the MB-cell interaction.
Importantly, cell toxicity was not observed upon contact of MB with
cells even at high-dose 800.times.MB.sub.Ds.
[0455] Although VEGFR2-targeted molecular imaging of tumor
angiogenesis has been reported [12], previous VEGFR2-targeted
molecular imaging of vasa vasorum neovascularization was not
successful, along with other vascular adhesion molecule targets,
leading authors of these reports to suggest that vasa vasoral flow
might be a technical hurdle for target-specific CEU-molecular
imaging [9]. Accordingly, the molecular imaging of DEspR-positive
endothelial cells in carotid artery disease demonstrated herein
(FIGS. 19A-22E) provide novel research and diagnostic tools for in
vivo molecular imaging of carotid artery disease endothelium and
expanded vasa vasorum. Without wishing to be bound or limited by
theory, given optimal ultrasound imaging parameters, the likely
factors for differential success in target-specific CEU-molecular
imaging could be differences in molecular thresholds defined by the
level and/or area of expression of the target, and/or in technical
thresholds defined by density and size of, as well as flow in
target vessel(s). These thresholds must be surpassed concurrently
for detectable targeted CEU-positive imaging or molecular imaging.
More specifically, the level of DEspR expression, the degree of
luminal endothelial pathology, and the density of vasa vasorum
neovascularization, along with the larger size of the rat carotid
artery disease model used here, comprise factors contributing to
successful DEspR-targeted CEU-positive imaging of carotid artery
vasa vasorum in the Tg25 rat model of carotid artery disease, in
contrast to the negative molecular imaging results targeting VEGFR2
reported for vasa vasorum neovascularization in a carotid artery
injury-induced mouse model [9]. Furthermore, differences between
CEU-positive transgenic rats from CEU-negative transgenic rats
reveal a putative threshold for CIS-differences (FIG. 19E) and
pre-disruption CIS-peak levels (FIG. 22A). This observed threshold
for CEU-positive imaging provides evidence that DEspR-targeted
CEU-positive imaging can be a non-invasive biomarker for
pathological angiogenesis, and have predictive value for disease
progression.
[0456] Surpassing the molecular and technical threshold for
successful detection of target-specific molecular imaging is
concordant with the principle that reflectivity is directly
proportional to the concentration of the microbubbles themselves
[26]. More specifically, greater DEspR-expression and greater
density of DEspR-positive endothelial cells, be it at the lumen or
in vasa vasorum, can translate to greater concentration of bound
microbubbles in the methods described herein. This in turn, without
wishing to be bound or limited by theory, is expected to translate
to greater reflectivity and detection levels since microbubble-cell
binding does not dampen microbubble reflectivity in contrast to
leukocyte engulfment of microbubble [27]. After clearance of most
circulating microbubbles and prior to acoustic disruption, stable
binding of target-specific microbubbles exhibits a relatively
stable contrast-intensity level that is significantly greater than
negative or background contrast-intensity (FIG. 20d, ANOVA
P<0.0001). Since high-frequency imaging can induce microbubble
fragmentation or gas diffusion per se, a slight decline could also
be observed prior to acoustic disruption, without wishing to be
bound or limited by theory. However, upon acoustic disruption a
drop in contrast-intensity due to fragmentation is observed to
confirm microbubble binding (FIGS. 19A-19E). Acoustic fragmentation
may not be complete due, without wishing to be bound or limited by
theory, to microbubble interaction in high-density ROIs which could
dampen microbubble resonance [28], or from inability of
microbubbles within microvessels to reach 10-fold
diameter-fluctuation that underlies acoustic fragmentation [29].
Furthermore, incomplete fragmentation with gas release and
relatively low flow, as would be expected in vasa vasorum compared
to carotid artery lumen, without wishing to be bound or limited by
theory, could account for the secondary peak observed in rat-R3
followed by slow decline back to baseline levels. The secondary
peak is likely not due to refill because at this experimental time
point there is minimal, if any, circulating microbubbles (FIGS.
19A-19E, 20A-20H). The fact that rat-R3 reached higher
contrast-intensity levels than rat-R1 suggests greater microbubble
concentration, which can also dampen acoustic destruction due to
inter-microbubble interactions [28]. Notably, while acoustic
fragmentation corroborates microbubble binding, the pattern of
acoustic fragmentation or diffusion can also provide further
insight into microbubble concentration, as well as binding site
vessel-caliber and flow. This provides a novel, alternative
molecular imaging paradigm to that reported for mouse aortic root
atherosclerosis [30]. While CEU-imaging in the current set-up is
successful, in other embodiments, non-linear imaging of adherent
microbubbles can be used to provide greater sensitivity and/or
improved quantitation as observed for intravascular ultrasound for
vasa vasorum flow imaging [31].
[0457] The detection of dose-dependent increase in % cells targeted
by MB.sub.Ds and dose-dependent increase in number of MBs per cell
(FIGS. 23A-23G), gives insight into the stable interaction,
kinetics, specificity and non-toxicity of DEspR-targeted MB-cell
interactions. More importantly, given that in vitro studies were
performed using human endothelial cells and human-specific
anti-DEspR monoclonal antibody for targeting, that MB-cell coupling
withstood a high shear stress wash after 45 minutes and did not
elicit cell toxicity on contact, these in vitro observations of
MB.sub.D-cell interactions demonstrate DEspR-targeted molecular
imaging of pathological angiogenesis as a useful therapeutic and
diagnostic tool.
[0458] Altogether, comparative analysis of molecular imaging
contrast-intensity levels, histological confirmation of
microbubble-to-endothelium binding, immunostaining confirmation
that DEspR-positive molecular imaging is associated with
DEspR-positive endothelial cell expression, and concordant patterns
of bound microbubble behavior after acoustic destruction,
demonstrate that target-specific molecular imaging of carotid
endothelium and vasa vasorum neovascularization in carotid artery
disease rat model is feasible using the methods and reagents
described herein that target DEspR. The identification of DEspR as
a successful target for in vivo molecular imaging of vasa vasorum
neovascularization and carotid artery disease lesions can
facilitate the longitudinal study of vasa vasorum
neovascularization and endothelial changes in carotid artery
disease progression in animal models. Along with the in vitro
observations of MB.sub.D-HUVECs stable binding, the data
demonstrate the use of molecular imaging techniques described
herein in the earlier detection of pathophysiological changes in
cardiovascular disease for estimations of risk for disease
progression and complications.
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TABLE-US-00002 [0490] TABLE 2 DEspR-targeted molecular imaging in
transgenic rat model of carotid artery disease Tg 25+
Non-transgenic Rat groups: 4 m-old male MB.sub.D Contrast enhanced
CEU(+) CEU (-) CEU (-) image # rats: both carotid 4 6 5 arteries #
rats: one carotid artery 3* 3* -- Contrast intensity signal .DELTA.
MB.sub.D (n = 18 rats) 89.96 .+-. 11.0*** 2.2 .+-. 0.9 2.0 .+-. 0.8
MB.sub.C (n = 8 rats) 1.9 .+-. 0.7 ND ND Histopathology: Intimal
lesions, plaque (+) +/- (-) Vasa vasorum expansion (+) +/- (-)
Immunostaining: DEspR (+): in vasa +/- (-) vasorum, initimal
lesions Values are group means .+-. sem; #, number; .DELTA., delta
or difference; (+), present; (-), absent; +/-, low to no
expression; *same 3 rats; ***ANOVA and Tukey's multiple pairwise
comparison P < 0.0001. CAD, carotid artery disease; m, month;
MB.sub.D, DEspR-targeted microbubble; MB.sub.C, isotype-targeted
microbubble.
Example 3
Dual Endothelin-1/VEGFsp Receptor (DEspR) in Cancer: Target for
Dual Anti-Angiogenesis/Anti-Tumor Cell Invasiveness Therapy
[0491] The development of intrinsic and extrinsic resistance to
current anti-VEGF/VEGFR2 therapies have been observed. As described
herein, DEspR expression is found to be increased in primary and
metastatic tumor .alpha.SMA-positive and .alpha.SMA-negative
vascular endothelium, and in tumor cell- and nuclear-membranes of
different human cancer tissue types and cell lines. Further,
DEspR-inhibition using the human-specific anti-DEspR antibody
treatments described herein decreased human endothelial cell
angiogenesis and tumor cell invasiveness. Further, it was found
that ligand-specific DEspR signaling-profiles are distinct from
VEGF/VEGFR2's. Accordingly, described herein are data demonstrating
targeting of DEsPR for dual tumor-cell and endothelial deliveries,
and for dual anti-angiogenesis/anti-invasiveness therapies.
Introduction
[0492] Although the critical role of the angiogenic switch in
cancer pathogenesis has been recognized [1], anti-angiogenesis
therapies directed at vascular endothelial growth factor and/or its
receptor, VEGF/VEGFR2-centric anti-angiogenesis therapies, alone or
in combination with other anti-cancer therapies, have not attained
the hoped-for treatment goal of long-term efficacy such that cancer
is reduced to a dormant, chronic manageable disease [2-5].
Cumulative observations have shows that all three FDA-approved VEGF
pathway inhibitors (anti-VEGF bevacizumab or Avastin, AntiVEGFR2
sunitinib, and sorafanib) result in significant but transitory
improvements in the form of tumor stasis or shrinkage, and only for
certain cancers despite most, if not all cancer types exhibiting
pathological angiogenesis [2,6]. Moreover, while anti-VEGF pathway
therapies have reduced primary tumor growth and metastasis in
preclinical studies [7], recent mouse tumor model studies report
that sunitinib and an anti-mouseVEGFR2 antibody, DC101, increased
metastasis of tumor cells despite inhibition of primary tumor
growth and increased overall survival in some cases [8,9].
Cumulative observations implicate several mechanisms of intrinsic
and evasive resistance, such as, without wishing to be bound or
limited by theories, pre-existing multiplicity of redundant
pro-angiogenic signals; upregulation of alternative pro-angiogenic
pathways, recruitment of bone marrow-derived pro-angiogenic cells,
increased pericyte coverage for the tumor vasculature obviating the
need for VEGF signaling, and invasive and metastatic co-option of
normal vessels without requisite angiogenesis [2-5]. Additionally,
10-fold increase in VEGF levels have been detected upon bevacizumab
anti-VEGF therapy in humans [10] and upon anti-VEGFR2 ab-therapy in
mice [11], which could, without wishing to be bound or limited by a
theory, contribute to evasive resistance.
[0493] Both VEGF and VEGFsp (vascular endothelial growth factor
signal peptide) originate from the same propeptide, and a 10-fold
`rebound` increase in VEGF could, without wishing to be bound or
limited by a theory, also result in a concomitant 10-fold increase
in VEGFsp, thus resulting in a 10-fold increase in VEGFsp's
post-cleavage function of activating its receptor, the dual
endothelin1/VEGFsp receptor or DEspR, formerly called Dear and
deposited in GenBank as Dear [12]. DEspR knockout mouse exhibits
arrested vasculogenesis and absent angiogenesis resulting in
E10.5-E12.5 day embryonic lethality [13]. Concordantly,
DEspR-haploinsufficiency resulted in decreased syngeneic melanoma
tumor growth, and anti-DEspR antibody inhibition decreased tumor
growth and tumor angiogenesis in rats with irradiation-induced
mammary tumors [13]. Furthermore, DEspR's other ligand is
endothelin-1 (ET1) [12], and all other known ET1 receptors, ETa and
ETb, do not exhibit an embryonic lethal angiogenic phenotype in
their respective knockout mouse models [14,15, 16.].
[0494] Described herein are novel anti-angiogenic strategies using
anti-human DEspR ab-inhibition and characterizing the murine
precursor of an anti-DEspR antibody therapeutic. It was found that
DEspR is upregulated in some solid tumor cells and tumor vascular
endothelium, and that human-specific anti-DEspR polyclonal and
monoclonal antibodies inhibit human endothelial cell tube formation
and tumor cell invasiveness in vitro, and that DEspR utilizes
ligand-specific signaling pathways known to mediate angiogenesis
and cancer cell invasiveness.
Materials and Methods
[0495] Cell Lines and Antibody Development
[0496] MDA-MB-231 and PANC-1 cells were obtained from American Type
Culture Collection (Rockville, Md.). MDA-MB-231 cells were
maintained in DMEM media (Sigma Chemical, St. Louis, Mo.)
supplemented with 10% FBS, L-glutamine, penicillin, and
streptomycin (GPS). PANC-1 cells were maintained in DMEM (Sigma
Chemical, St. Louis, Mo.) with high glucose, 10% FBS and GPS. Human
umbilical vein endothelial cells, HUVECs, were obtained from
Cascade Biologics, Inc., and maintained in Endothelial Growth
Media-2 (EGM-2) containing 2% FBS and GPS. Monoclonal antibody
development was custom performed by ProMab Biotechnologies, Inc
(Richmond, Calif.) using a nine amino-acid DEspR NH.sub.2-terminal
peptide, M.sub.1TMFKGSNE.sub.9 of hDEspR as antigen. Screening of
hybridoma supernatants and initial characterization of candidate
monoclonal antibodies were performed by ELISA using free
hDEspR-antigenic peptide as antigen.
[0497] Monoclonal Antibody Characterization by ELISA and Western
Blot Analysis.
[0498] The M.sub.1TMFKGSNE.sub.9 antigenic peptide was coated
directly on wells of a microtiter plate. Appropriate dilutions of
primary antibodies were incubated at 37.degree. C. for 1 hr. The
wells were then incubated with HRP labeled anti-IgG (SIGMA cat
#A0168) at 1:9000 at 37.degree. C. for 1 hr. The reactions were
visualized by the addition of 3,3'5,5'-tetramethylbenzidine
substrate (incubation at 37.degree. C. for 10 min) and read
spectrophotometrically at 450 nm. Western blot analysis was done as
described [17] using equal amounts of whole cell protein extract
(40 .mu.g) from Cos1 cell transfectants stably expressing hDEspR
[17] and corresponding candidate monoclonal antibodies raised
against hDEspR specific synthetic peptide. Immunoreactive hDEspR
(10 kDa polypeptide) was detected by chemiluminescence using the
ECL Western Detection kit (GE Healthcare).
[0499] HUVEC Tube Formation Assay for Angiogenesis.
[0500] Validated 2.sup.nd passage human umbilical vein endothelial
cells--HUVECs (Cascade Biologics, Oregon) were obtained and
cultured until the 4th passage and were then harvested at 80%
confluence using mild trypsinization. The cell pellet was then
washed twice in serum free media (basal media) containing M-200
(Cascade Biologics, Oregon) 1 .mu.g/ml hydrocortisone, 10 ng/ml
EGF, 3 ng/ml bFGF and 10 .mu.g/ml heparin. Cells were then
resuspended in this serum free media and seeded at 20,000 cells per
well (100 .mu.L) onto a 96 well plate Angiogenesis System:
Endothelial Cell Tube Formation Matrigel Matrix (BD Biosciences,
MA). Different angiogenic and anti-angiogenic conditions were
assayed in quadruplicate as indicated using basal media alone or
with one or more of the following: 2% FBS, 20 nM VEGF, 20 nM
VEGFsp, 20 nM ET1. Antibodies used for inhibition were all affinity
purified and used in the following concentrations: 500 nM
anti-hDEspR polyclonal antibody (Pab), 500 nM anti-hDEspR 7C5B2
monoclonal antibody (Mab), 500 nM anti-VEGFsp Pab, and for
corresponding isotype controls either 500 nM preimmune IgG (75
.mu.g/ml) for Pab, and 500 nM IgG2b for anti-hDEspR Mab. Different
experimental conditions were tested in quadruplicate as follows:
basal media alone (BM), BM with 2% FBS; BM with 20 nM VEGF; BM with
20 nM VEGFsp; BM with 20 nM ET1; BM with 20 nM VEGF and 500 nM (75
m/ml) pre-immune IgG; BM with 20 nM VEGF and 500 nM anti-VEGFsp; BM
with 20 nM VEGF and 500 nM anti-hDEspR; BM with 20 nM VEGFsp and
500 nM anti-hDEspR; BM with 20 nM ET1 and 500 nM anti-hDEspR; BM
with 2% FBS and 500 nM anti-VEGFsp; and BM with 2% FBS plus 500 nM
anti-hDEspR. In other experiments increasing concentrations of
anti-hDEspR 7C5B2 mAb (0.05-500 nM) were tested. HUVECs were then
incubated in different conditions as specified at 37.degree. C. for
16 hours; after which, resulting angiogenic tube formations were
viewed under the microscope and images of .about.70% of the well
(central parts) were taken for analysis. Various parameters were
measured for each angiogenic condition using ImageJ
(NIH--http://rsb.info.nih.gov/ij/) namely total tube length,
average tube length, average tube thickness, number of branch
points defined as cluster of cells possessing tube-like extensions
measuring more than 2.times. the length of the cell aggregates,
number of connections defined as 3 or more connections between
tube-like structures in series or parallel and number of closed
polygons bounded by the tubular structures.
[0501] Invasion Assay.
[0502] MDA-MB-231 and PANC-1 cell invasion assays were performed as
described [18] using the BD Bio-Coat Matrigel invasion assay system
(BD Biosciences, Franklin Lakes, N.J.). MDA-MB-231 and PANC-1 cells
were suspended in growth media and seeded onto pre-coated transwell
chambers (3.times.10.sup.4 cells/well). The transwell chambers were
then placed into 24-well plates, to which basal medium only or
basal medium containing various concentration of antibodies were
added. Cells were incubated for 16 hr and the invading cells were
fixed and stained with Diff-Quick stain. The number of invading
cells per well were counted under the microscope. Each condition
was assessed in four replicates.
[0503] Immunostaining of Tumor Tissue Arrays and Tumor Cells.
[0504] Human cancer cell line-array DEspR immunostaining was
custom-performed by Pantomics, Inc. using our in-house polyclonal
human-specific anti-DEspR antibody. Tumor tissue arrays were
obtained from Pantomics, Inc. and immunostained for DEspR using
polyclonal and monoclonal anti-hDEspR antibodies at 1:20 after
demonstration of concentration-dependent immunostaining 1:10, 1:50,
1:100. Deoxyaminobenzidine immunostaining was done using the
polyclonal antibody as described [13]. Double immunofluorescence
staining was done on deparaffinized sections via the following
steps: antigen retrieval, treatment to reduce background, blocking,
incubation with primary antibody at 4.degree. C. overnight,
secondary antibody incubation overnight at 4.degree. C. with
AlexaFluor 568 goat anti-mouse IgG and AlexaFluor 488 goat
anti-rabbit IgG, washing, and mounting using Prolong Gold with DAPI
(Invitrogen). Negative controls were run using rabbit-isotype
antibody for anti-rat DEspR antibody. A Zeiss Axioskop2plus
microscope was used for fluorescence imaging and
photomicroscopy.
[0505] Multiplex Analysis of Signaling Proteins by
Ab-Microarray.
[0506] Analysis of ligand-dependent modulation of different
signaling pathways by DEspR was custom performed by Kinexus Corp.
(Kinexus, Canada) utilizing the Kinex.TM. Antibody Microarray
System spanning 506 phosphoprotein-specific antibodies in
duplicates or multiple replicates, as well as 740 pan-specific
antibodies of signaling molecules. The effects of ET1- and
VEGFsp-DEspR activation were analyzed on multiplex signaling
pathways after 30 minutes of ligand-treatment (ET1, 10 nM; VEGFsp,
10 nM), compared with the respective non-activated DEspR in
non-treated controls, using Cos1-hDEspR permanent cell
transfectants. All fluorescent signals were normalized to
background. Data are presented as percentage change from control (%
CFC), or change detected after 30 minutes of ET1 or
VEGFsp-treatment compared with non-treated transfectant-matched
controls respectively. The %
CFC=[Treated.sup.Ave-Control.sup.Ave]/Control.sup.Ave.times.100.
Although % CFC>25% is suggested as a significant difference,
only values exhibiting >50% CFC and with % error range between
duplicates less than 20% for both test and control samples were
presented. The % error range=[Duplicate.sup.n-Average]/Average. A %
error >20% was accepted if the % CFC remained >50% using the
lesser of the duplicates in calculating % CFC.
[0507] Statistical Analysis.
[0508] One way analysis of variance (ANOVA) followed by all
pairwise multiple comparison Tukey test were performed after
ascertaining normality using SigmaStat 2.03 software package. A
P<0.05 was considered statistically significant.
Results
[0509] DEspR Expression is Increased in Human Tumor Cells and Tumor
Vessels.
[0510] DEspR-specific expression patterns were investigated in
human cancer tissues and cells. Tumor tissue array analysis was
performed using a human-specific anti-DEspR polyclonal-antibody
[17]. Concordant with rat irradiation-induced mammary tumor model
observations of rat-specific anti-DEspR antibody [13]
immunostaining, immunohistochemical analysis of DEspR expression in
human tumor tissue arrays detected increased DEspR expression in
thin-walled tumor vascular endothelium in hepatic, pancreatic
(FIGS. 24A-24F), stomach, breast (FIGS. 25A-25F), colon and lung
(FIGS. 26A-26F) cancer, compared with vascular endothelium in
normal tissue biopsy cores respectively be it arterial or
microvascular endothelium (FIGS. 24A-24F, 25A-25F, and 26A-26F).
Notably, vascular endothelium in stomach cancer metastatic foci in
the lung (FIG. 25C) and breast cancer metastatic foci in lymph node
(FIG. 25F) also exhibit increased DEspR immunostaining. Moreover,
pancreatic (FIGS. 24E, 24F), stomach (FIGS. 25B, 25C), breast (FIG.
25E), lung (FIG. 26C) and colon (FIG. 26E, 26F) tumor cells exhibit
increased DEspR expression with sub-cellular localization in the
cell membrane, cytoplasm and nuclear membrane. This increased DEspR
expression in tumor neovessels and tumor cells demonstrated herein
indicate that that DEspR plays a role in both tumor
neovascularization and in tumorigenesis.
[0511] To further confirm expression in tumor cells
DEspR-immunostaining of cancer cell-array testing different types
of previously characterized, established cancer cell lines was next
performed (Table 3). In contrast to a few cell lines tested with
minimal if any DEspR expression, several cancer cell lines exhibit
DEspR expression with nuclear membrane DEspR expression associated
with high-nuclear grade (Table 3, FIGS. 27A-27F). Representative
photomicrographs demonstrate tumor cell expression with strongest
DEspR-immunostaining in nuclear membranes of most tumor cells, but
not all. The selective nuclear membrane immunostaining (FIGS.
27A-27F) confirms specificity of DEspR immunostaining, along with
negative immunostaining of some cancer cell lines (Table 3).
Importantly, these observations are concordant with the
observations in cancer tissue sections described herein (FIGS.
24A-24F, 25A-25F, and 26A-26F). Nuclear membrane localization
indicates that DEspR can play a role in crosstalk between the cell
membrane and nuclear membrane, beyond receptor-mediated signal
transduction.
[0512] High-Affinity Anti-hDEspR Monoclonal Antibody Generated
Against N-Terminal 9-Aa Extra-Cellular Domain.
[0513] In order to investigate anti-DEspR inhibition as an
anti-angiogenic strategy, a human-specific anti-DEspR monoclonal
antibody was developed using a 9-aa peptide spanning the N-terminal
extracellular domain of human DEspR identical to the strategy use
to develop the human-specific anti-DEspR polyclonal antibody used
in DEspR immunostaining (FIGS. 24A-24F, 25A-25F, 26A-26F, and
27A-27F) [17]. From 67 hybridoma clones, a preliminary screen
identified top ten candidate monoclonal antibody hybridoma clones
which were then analyzed for affinity to the 9-aa peptide
N-terminal domain by indirect ELISA (FIG. 28A). Analysis of
specificity by Western blot analysis of mab-mediated binding to
hDEspR protein (10 kDa) isolated from Cos1-hDEspR transfectants in
contrast to control non-transfected Cos1 cells identified hybridoma
clone 7C5B2. As shown in FIG. 28B, 7C5B2 anti-hDEspR monoclonal
antibody hybridoma clone exhibited specificity as both "super
clone" supernatant and purified monoclonal antibody. Isotyping of
7C5B2 showed that this monoclonal antibody belongs to the murine
IgG2b isotype class of antibodies.
[0514] Co-Localization of DEspR and its Ligand, VEGFsp in Human
Umbilical Vascular Endothelial Cells (HUVECs).
[0515] Analysis of receptor-ligand co-localization by double
immunostaining in HUVECs showed specific detection of DEspR on
endothelial cell membrane cultured in pro-angiogenesis conditions
using the anti-hDEspR monoclonal antibody. Double immunostaining
detected co-localization of DEspR with its ligand VEGFsp using an
anti-VEGFsp polyclonal antibody, thus demonstrating that
anti-hDEspR monoclonal antibody specifically targets DEspR.
Anti-DEspR polyclonal antibody also gave identical results.
[0516] Anti-DEspR Inhibition by Anti-hDEspR Polyclonal Antibody and
7C5B2 Monoclonal Antibody Decrease Angiogenesis.
[0517] The effects of 7C5B2 monoclonal antibody inhibition of DEspR
on angiogenesis using established in vitro HUVECs-based
angiogenesis assays was then assessed. It was first showed that
7C5B2 monoclonal antibody detects cell-membrane DEspR expression in
tubes/"neovessels" formed by HUVECs in pro-angiogenesis conditions,
thus validating the use of this angiogenesis assay system. Next,
two established parameters of in vitro angiogenesis were analyzed,
total tube length and branching of neovessel-tubes formed by HUVECs
in pro-angiogenesis conditions. Using varying doses of 7C5B2
monoclonal antibody from 0.05 to 500 nM, concentration dependence
of angiogenesis inhibition is demonstrated for both total tube
length and number of branch-points, and identifies 500 nM 7C5B2
monoclonal antibody as the full-strength inhibitory dose (FIG.
29A). This dose was then applied to repeat independent inhibition
experiments comparing the newly developed 7C5B2 monoclonal antibody
with the previously characterized anti-hDEspR polyclonal antibody.
Compared to non-treated controls, and pre-immune and
IgG2b-isotype-specific negative controls for polyclonal antibody
and 7C5B2 monoclonal antibody respectively, 500 nM anti-hDEspR
antibody inhibited angiogenesis, measured as total tube length and
mean number of branchpoints, significantly (ANOVA with all pairwise
multiple comparison Tukey test, P<0.01). Other angiogenesis
parameters, number of tubes and branch-interconnections were also
significantly inhibited. Concordantly, a polyclonal anti-VEGFsp
antibody also inhibited angiogenesis in HUVECs.
[0518] Analysis of Anti-hDEspR 7C5B2 Monoclonal Antibody
Immunostaining and Inhibition of Tumor Cell Invasiveness.
[0519] Having shown that DEspR inhibition reduces angiogenesis, the
efficacy of 7C5B2 monoclonal antibody-mediated anti-DEspR
inhibition on tumor cell invasiveness was next assessed since DEspR
is detected in different tumor cell lines (FIGS. 27A-27F) and
cancer tissues (FIGS. 24A-24F, 25A-25F, and 26A-26F). Two cancer
cell lines representing aggressive breast cancer and pancreatic
cancer, MDA-MB-231 and PANC-1 cancer cell lines respectively, were
examined. Immunostaining with 7C5B2 monoclonal antibody detected
nuclear- and cell-membrane DEspR expression in both cell lines, as
well as cytoplasmic expression. Functional analysis detected
concentration dependent inhibition of tumor cell invasiveness from
0.05 to 500 nM 7C5B2 monoclonal antibody, with an EC50 of
3.55.+-.0.32 nM. Using 500 nM 7C5B2 monoclonal antibody, DEspR
inhibition was observed in both MDA-MB-231 (FIG. 30B) and PANC1
(FIG. 30C) cells, compared to control non-treated cells and
IgG2b-isotype treated cells respectively (ANOVA followed by all
pairwise multiple comparison test, P<0.001 and P<0.01
respectively). These observations indicate dual effects of DEspR
inhibition on both angiogenesis (FIG. 29B-29C) and tumor cell
invasiveness (FIG. 30B-30C).
[0520] Anti-hDEspR 7C5B2 Monoclonal Antibody-Immunostaining of
Tumor Vascular Endothelium and Tumor Cells.
[0521] Having shown efficacy of DEspR-inhibition on angiogenesis
and tumor cell invasiveness, 7C5B2 monoclonal
antibody-immunostaining in breast and pancreatic cancer tissues in
contrast to normal was next evaluated to confirm increased DEspR
expression in tumor vascular endothelium and tumor cells as
detected using anti-hDEspR polyclonal antibody (FIGS. 24A-24F,
25A-25F, and 26A-26F), as well as to delineate DEspR-targeting
profile of 7C5B2 monoclonal antibody.
[0522] Double immunostaining of DEspR and alpha smooth muscle actin
(.alpha.SMA), to track microvascular pericytes and cancer tissue
stromal myofibroblasts, detected minimal DEspR expression in normal
breast tissue blood vessels and mammary epithelial cells, and
normal .alpha.SMA expression in mammary myoepithelial cells and
arteriolar smooth muscle cells highlighting minimal to no DEspR
expression (FIGS. 31A-31C). In contrast, in a representative breast
cancer tissue sections of ductal invasive carcinoma, double
immunostaining detected prominent DEspR expression in tumor
microvascular endothelium, in microvessels and arterioles
co-expressing .alpha.SMA, as well as in ductal carcinoma epithelial
cells (FIGS. 31D-31F). Increased tumor vascularization is also
noted compared to non-cancer `normal` control tissue (FIGS.
31A-31C).
[0523] Similarly, in normal pancreas, minimal DEspR expression is
detected in microvessels (FIGS. 32A-32C), and in arterial
endothelium in contrast to strong .alpha.SMA expression in arterial
media smooth muscle cells (FIG. 32C). In contrast, DEspR expression
is increased in pancreatic cancer .alpha.SMA-negative microvascular
and .alpha.SMA-positive microvascular and arteriolar endothelium
(FIGS. 32D-32E). As observed in breast cancer epithelial cells and
in PANC-1 cancer cell line, pancreatic cancer ductal carcinoma
epithelial cells exhibit marked DEspR-positive immunostaining (FIG.
32F).
[0524] Phosphoproteome Analysis of DEspR Signal Transduction.
[0525] Using a phosphoprotein-specific antibody-array,
ligand-specific signal transduction pathways activated by DEspR
upon binding to its dual ligands, ET1 and VEGFsp respectively in
permanent Cos1-cell DEspR transfectants were identified (Table 4).
Cos1 cells were used as these cells do not have endogenous DEspR,
ET1 or VEGFR2 expression. Non-treated and treated Cos1-DEspR
transfectants were compared. As shown in Table 4, regardless of
ligand, DEspR's phosphoproteome (limited to signaling
phosphoproteins with >50% CFC) activates signaling pathways
known to be involved in mechanisms of angiogenesis, tumor cell
invasiveness or metastasis. Additionally, some DEspR-phosphorylated
signaling molecules for either ET1 or VEGFsp-activation of DEspR
have been directly linked to either neuronal or hematopoietic stem
cells, with some also implicated in cancer stem cell renewal such
as ERK1/2, FAK, Met, PKC-alpha, SHP2, Smad, STAT1, and STAT3 (Table
3). It is noted herein that DEspR's phosphoproteome overlaps with
VEGFR2/VEGF for some signaling molecules like FAK, ERK1/2, Raf,
PKC.alpha. [19]. However, the collective signaling complexes of
DEspR/ET1 and DEspR/VEGFsp (Table 3) are quite distinct from that
described for VEGFR2/VEGFa [19], thus confirming non-redundant
angiogenesis roles as deduced from null mutant abnormal
angiogenesis phenotypes for DEspR [13] and VEGF [20,21] with
identical embryonic lethality between embryonic E10.5 and E12.5
days, although VEGFR2 or Flk1 null mutants died earlier between
E8.5-E9.5 days [22].
Discussion
[0526] DEspR as a Novel Target for Anti-Tumor Vascularization
Therapy.
[0527] The detection of increased DEspR expression in tumor
vascular endothelium, in contrast to normal tissue-matched
controls, detection of DEspR expression in both .alpha.SMA-negative
capillaries/microvessels and .alpha.SMA-positive arterioles and
arteries in the tumor stroma, and successful inhibition of
angiogenesis through DEspR-inhibition all demonstrate that DEspR is
a novel target for therapies aimed at both tumor angiogenesis and
at existing or `mature` tumor microvasculature. More specifically,
targeting DEspR on .alpha.SMA-positive microvessels can address
anti-VEGF therapy-resistant tumors which are thought, without
wishing to be limited or bound by a theory, to have a stromal
vasculature no longer dependent on VEGF due their `maturation` as
marked by .alpha.SMA-positive pericyte sheath or non-dependent on
VEGF due to "cooption of existing" microvasculature [2].
Furthermore, combined targeting DEspR along with anti-VEGF
therapies can address the expected concomitant 10-fold increase in
VEGFsp that accompanies the observed 10-fold increase in VEGF upon
anti-VEGF therapy [10], since VEGF and VEGFsp originate from a
common propeptide.
[0528] Insights from the Ligand-Specific DEspR Phosphoproteome.
[0529] Given that hypoxia inducible factor-1 alpha (HIF1.alpha.)
stabilization induces VEGF, and hence VEGFsp, in hypoxia,
phosphorylation of BRCA1 and induction of PCNA expression by
VEGFsp-DEspR activation (Table 3), indicates that DEspR can
contribute to the needed DNA repair response activated in hypoxia
[24], thus allowing DEspR-positive endothelial and cancer cells to
proliferate despite the hypoxic microenvironment, rather than
undergo hypoxia-induced cell cycle arrest and apoptosis [24,25].
The hepatocyte growth factor receptor, MET, is induced upon
ET1/DEspR stimulation and Smad1/5/9 is phosphorylated upon
DEspR/VEGFsp activation, thus indicating a mechanism for crosstalk
and/or redundancy among VEGFsp/DEspR, MET/HGF, and TGF.beta./Smad
pathways pertinent to angiogenesis in endothelial cells and
invasiveness in cancer cells. Importantly, DEspR phosphorylates
BRCA1 and STAT3 both of which have been shown to stabilize
HIF1.alpha., and along with Raf1, lead to the induction of VEGF,
and hence VEGFsp. Furthermore, the phosphorylation of BRCA1 [26] by
VEGFsp/DEspR and STAT3 by both ET1/DEspR and VEGFsp/DEspR, can both
lead to DEspR-mediated stabilization of HIF1-alpha without the need
for hypoxia, leading to constitutive HIF1-.alpha. mediated
pro-angiogenic and pro-DNA repair response which can contribute to
tumor resistance to conventional therapy.
[0530] DEspR Inhibition as Target for Dual
Anti-Angiogenesis/Anti-Cancer Cell Invasiveness Treatment
Paradigm.
[0531] In addition to expression on tumor vascular endothelium,
DEspR is expressed in solid tumor epithelial cells seen in both
established cancer cell lines and histology sections of breast,
pancreatic, lung, stomach, bladder and colon cancers (FIGS.
24A-24F, 25A-25F, 26A-26F, and 27A-27F). Just as anti-DEspR
inhibition reduces in vitro angiogenesis (FIGS. 28A-28B), 7C5B2
monoclonal antibody-inhibition decreases tumor cell invasiveness in
two aggressive cancer cell lines, breast cancer cell line
MDA-MB-231 (and -468) and pancreatic cancer cell line PANC-1 (FIGS.
29A-29C). Thus, targeting DEspR as a receptor involved in both
angiogenesis and tumor cell invasiveness via anti-hDEspR monoclonal
antibody-inhibition using the compositions and methods described
herein provides a robust new anti-tumor therapy, and demonstrates
the use of the anti-hDEspR 7C5B2 monoclonal antibody described
herein as an anti-hDEspR monoclonal antibody-therapeutic
precursor.
[0532] Furthermore, dual-targeting of angiogenesis and metastasis
mechanisms comprise novel methods for next-generation anti-cancer
treatment strategies [2]. The data described herein demonstrate
that targeting DEspR is can be used to achieve a dual-treatment
paradigm. The increased expression in both pancreatic tumor
neovessel and tumor cells, along with the inhibition of
angiogenesis and pancreatic cancer cell line PANC-1
cell-invasiveness by anti-DEspR inhibition altogether indicate that
anti-DEspR therapy can provide a new treatment approach for
pancreatic cancer. The combinatorial anti-angiogenesis and
anti-invasiveness caused by DEspR-inhibition, as shown herein, as
well as targeting DEspR for dual tumor endothelial and tumor cell
targeted-delivery, can be used, in some embodiments, as a
therapeutic basis for next generation dual
anti-tumor/anti-angiogenesis cancer therapies and methods thereof
[2].
TABLE-US-00003 TABLE 3 Tumor array analysis of DEspR expression in
different cancers and cancer cell lines. Cancer tumor vascular
Representative Cancer cell lines tissue-type endothelium vs cancer
DEspR-positive (n) normal types DEspR-negative Bladder (23) 17/23
Adenocarcinoma *253J BV (74%) Squamous cell ca Transitional cell ca
Breast (36) 34/36 Invasive ductal ca *MDA-MB-231 (94%) Adenoca
*MDA-MB-468 Medullary ca Invasive lobular ca Colon (6) 5/6 Adenoca
*SW480 Liver (35) 24/35 Hepatocellular ca HEP3B (68%) Clear cell ca
HEPG2 Bile duct ca Lung (2) 2/2 Adenocarcinoma *NCI-H627 NCI-H292
Pancreas (6) 6/6 Ductal carcinoma *PANC-1 Stomach (2) Primary and
in Adenocarcinoma na metastasis to lung *nuclear membrane
immunostaining; ca, carcinoma; cancer cell line nomenclature based
on ATCC; na, not available on cell-line array, n, number of biopsy
cores on tissue array.
TABLE-US-00004 TABLE 4 Phosphoproteome of hDEspR upon ET1 and
VEGFsp stimulation respectively. ET1 VEGFsp Pro- Pro- Pro- Protein
Name Symbol P*-Site (% CFC) (% CFC) Angiogenesis Cancer Stem cell
Breast cancer type 1 BRCA1 S1497 32 82 [26] [27] susceptibility
protein Cyclin-dependent protein- CDK1/2 T14/Y15 53 -16 [28] serine
kinase 1/2 Y15 281 -57 Extracellular regulated ERK1/2 T202 + Y204;
135 -25 [29, 30] [31-33] NSC: [34] protein-serine kinase 1/2 T185 +
Y187 CSC: [35] (p44/p42 MAP kinases) Focal adhesion protein- FAK
S722 55 -38 [36, 37] Metastasis: NSC: [34] tyrosine kinase S732 62
-11 [37, 38] CSC: [37] Panspecific 205 0 Hepatocyte growth factor
Met Panspecific 384 0 [39-41] Metastasis: [43] receptor-tyrosine
kinase [42]; Resistance: [39] Proliferating cell nuclear PCNA
Panspecific -47 119 [44] antigen Protein-serine kinase C- PKCa
T638/T641 137 -17 [45, 46] alpha Protein-serine kinase C- PKCe
Panspecific 103 -29 [47-50] [50] NSC: epsilon [34]; CSC: [50] Raf1
proto-oncogene- Raf1 S259 12 63 [51] [52] encoded protein-serine
kinase SH2 domain-containing Shc1 Y349, 9 97 [53-55] [53, 56, 57]
transforming protein 1 or Y350 ShcA Protein-tyrosine SHP2 S576 14
97 [58-60] [58, 61, 62] [61, 63-65] phosphatase 1D SMA- and mothers
against Smad S463 + S465/ 18 147 [30] [66] HSC: [67]
decapentaplegic homologs 1/5/9 S465 + S467 1/5/9 Src
proto-oncogene- Src Y529 -20 73 [47] [68-70] encoded
protein-tyrosine Y418 -11 174 kinase Signal transducer and STAT1
S727 86 123 Metastasis, CSC: [72] activator of transcription 1 Y701
95 557 invasiveness: [71] Signal transducer and STAT3 S727 133 126
[73-75] [74] NSC: [76] activator of transcription 3 Invasiveness:
[75] CSC, cancer stem cell; ET1, endothelin 1; hDEspR, human dual
endothelin-1/vascular endothelial growth factor-signal peptide
receptor; NSC, neural stem cell; VEGFsp, vascular endothelial
growth factor-signal peptide; % CFC, percentage change in treated
vs non-treated control averages: % CFC = [Treated -
Control]/Control ave] .times. 100. Phospho-site, phosphorylation
site detected with phosphorylated site-specific antibodies. Data
represent >50% CFC taken from mean of treated vs control
non-treated duplicates (A, B) with % error range <20%. % error
range = [Treated.sub.A - ave]/ave .times. 100. Kinexus antibody
array: phosphoprotein-specific ab to detect phosphorylation
changes, and panspecific antibodies to detect expression
changes.
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Example 4
7C5B2 Antibody Sequencing and hDEspR Composite Human Antibody
Variant Generation
[0609] Described herein are sequencing results obtained from the
monoclonal antibody expressed by the murine hybridoma 7C5B2
(anti-hDEspR), in which the heavy and light chain V-region (V.sub.H
and V.sub.L) sequences of the 7C5B2 antibody have been determined
and exemplary anti-hDEspR composite human antibody variants have
been designed.
[0610] From viable frozen hybridoma cell pellets, RNA was extracted
and PCR amplification of antibody specific transcripts was
performed after reverse transcription of mRNA. The nucleotide and
amino acid sequences of the antibody heavy and light chain
V-regions were determined and the sequence data was analyzed. Fully
humanized antibodies were then designed using Composite Human
Antibody.TM. technology, as described herein.
Methods and Results
RNA Extraction, RT-PCR and Cloning
[0611] RNA was extracted from a cell pellet using an
RNAqueous.RTM.-4PCR kit (Ambion cat. no. AM1914). RT-PCR was
performed using degenerate primer pools for murine signal sequences
with constant region primers for each of IgGVH, IgMVH, Ig.kappa.VL
and Ig.lamda.VL. Heavy chain V-region RNA was amplified using a set
of six degenerate primer pools (HA to HF) and light chain V-region
mRNA was amplified using a set of eight degenerate primer pools,
seven for the .kappa. cluster (KA to KG) and one for the .lamda.
cluster (LA).
[0612] For the heavy chain V-region, amplification products of the
expected size were obtained from the IgGVH reverse transcription
primer and primer pool HC. For the light chain V-region,
amplification products were obtained from the Ig.kappa.VL reverse
transcription primer and light chain primer pools KB, KC, KD, and
KG (FIG. 33). The PCR products from each of the above pools were
purified and cloned into a `TA` cloning vector (pGEM (R)-T Easy,
Promega cat. no. A1360). Six VH and 24 V.kappa. clones were
sequenced.
[0613] A single functional VH gene was identified in five clones
from pool HC and a single functional V.kappa. gene sequence was
identified in six clones from primer pool KG. The twelve clones
from primer pools KB and KC were found to contain an aberrant
transcript (GenBank accession number M35669) normally associated
with the hybridoma fusion partner SP2/0 and the six clones from
pool KD were found to not contain a functional V.kappa.
transcript.
Chimeric Antibody
[0614] VH and V.kappa. (pool KG) genes were PCR amplified using
primers that introduced flanking restriction enzyme sites for
cloning into Antitope's VH and V.kappa. chain expression vectors.
The VH region was cloned using MluI and HindIII sites, and the
V.kappa.s region were cloned using BssHII and BamHI restriction
sites. All constructs were confirmed by sequencing.
[0615] The chimeric antibody constructs were transiently
transfected into HEK293 cells using calcium phosphate precipitation
The transient transfections were incubated for three days prior to
harvesting supernatants.
Sequence Analysis
[0616] Analysis of sequences obtained from the hybridoma 7C5B2 is
summarized in Table 1. The heavy and light chain V-regions show
good homology to their closest human germline sequences (64% and
82%, respectively) and the individual framework sequences have
close homologues in the human germline database.
Design of Composite Human Antibodies
Design of Composite Human Antibody.TM. Variable Region
Sequences
[0617] Structural models of the mouse anti-hDEspR 7C5B2antibody V
regions were produced using Swiss PDB and analysed in order to
identify important "constraining" amino acids in the V regions that
were likely to be essential for the binding properties of the
antibody. Residues contained within the CDRs (using Kabat
definition) together with a number of framework residues were
considered to be important. Both the VH and V.kappa. sequences of
anti-hDEspR contain typical framework residues and the CDR 1, 2 and
3 motifs are comparable to many murine antibodies.
[0618] From the above analysis, it was considered that composite
human sequences of anti-hDEspR could be created with a wide
latitude of alternatives outside of CDRs but with only a narrow
menu of possible alternative residues within the CDR sequences.
Analysis indicated that corresponding sequence segments from
several human antibodies could be combined to create CDRs similar
or identical to those in the murine sequences. For regions outside
of and flanking the CDRs, a wide selection of human sequence
segments were identified as components of the novel Composite Human
Antibody.TM. V regions described herein (see Table 1).
Design of Variants
[0619] Based upon the above analysis, a large preliminary set of
sequence segments that could be used to create anti-hDEspR
Composite Human Antibody.TM. variants were selected and analysed
using iTope.TM. technology for in silico analysis of peptide
binding to human MHC class II alleles (Perry et al 2008), and using
the TCED.TM. (T Cell Epitope Database) of known antibody
sequence-related T cell epitopes (Bryson et al 2010). Sequence
segments that were identified as significant non-human germline
binders to human MHC class II or that scored significant hits
against the TCED.TM. were discarded. This resulted in a reduced set
of segments, and combinations of these were again analysed, as
above, to ensure that the junctions between segments did not
contain potential T cell epitopes.
[0620] Selected segments were then combined to produce heavy and
light chain V region sequences for synthesis. For anti-hDEspR, five
VH chains (SEQ ID NO: 13-SEQ ID NO: 17) and two V.kappa. chains
(SEQ ID NO: 18 and SEQ ID NO: 19) were designed with sequences as
detailed herein.
Sequence CWU 1
1
27185PRTHomo sapiens 1Met Thr Met Phe Lys Gly Ser Asn Glu Met Lys
Ser Arg Trp Asn Trp 1 5 10 15 Gly Ser Ile Thr Cys Ile Ile Cys Phe
Thr Cys Val Gly Ser Gln Leu 20 25 30 Ser Met Ser Ser Ser Lys Ala
Ser Asn Phe Ser Gly Pro Leu Gln Leu 35 40 45 Tyr Gln Arg Glu Leu
Glu Ile Phe Ile Val Leu Thr Asp Val Pro Asn 50 55 60 Tyr Arg Leu
Ile Lys Glu Asn Ser His Leu His Thr Thr Ile Val Asp 65 70 75 80 Gln
Gly Arg Thr Val 85 226PRTHomo sapiens 2Met Asn Phe Leu Leu Ser Trp
Val His Trp Ser Leu Ala Leu Leu Leu 1 5 10 15 Tyr Leu His His Ala
Lys Trp Ser Gln Ala 20 25 3357DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 3cag gtg caa ctg aag gag tca gga cct ggc ctg gtg
gcg ccc tca cag 48Gln Val Gln Leu Lys Glu Ser Gly Pro Gly Leu Val
Ala Pro Ser Gln 1 5 10 15 agc ctg tcc att acc tgc act gtc tct ggg
ttc tca tta acc agc tat 96Ser Leu Ser Ile Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Ser Tyr 20 25 30 gat ata agc tgg att cgc cag cca
cca gga aag ggt ctg gag tgg ctt 144Asp Ile Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 gga gta ata tgg act ggt
gga ggc aca aat tat aat tca gct ttc atg 192Gly Val Ile Trp Thr Gly
Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 tcc aga ctg agc
atc agc aag gac aac tcc aag agc caa gtt ttc tta 240Ser Arg Leu Ser
Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe Leu 65 70 75 80 aaa atg
aac agt ctg caa act gat gac aca gcc ata tat tac tgt gta 288Lys Met
Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95
aga gat cgg gat tac gac ggg tgg tac ttc gat gtc tgg ggc gca ggg
336Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val Trp Gly Ala Gly
100 105 110 acc acg gtc acc gtc tcc tca 357Thr Thr Val Thr Val Ser
Ser 115 4119PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 4Gln Val Gln Leu Lys Glu
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln 1 5 10 15 Ser Leu Ser Ile
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile
Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45
Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50
55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Phe
Leu 65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr
Tyr Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp
Val Trp Gly Ala Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
510PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 5Gly Phe Ser Leu Thr Ser Tyr Asp Ile
Ser 1 5 10 616PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 6Val Ile Trp Thr Gly Gly Gly
Thr Asn Tyr Asn Ser Ala Phe Met Ser 1 5 10 15 711PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 7Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val 1 5 10
8336DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 8gat gtt ttg atg acc caa act cca
ctc tcc ctg cct gtc agt ctt gga 48Asp Val Leu Met Thr Gln Thr Pro
Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 gat caa gcc tcc atc tct
tgc aga tct agt cag agc att gta cat agt 96Asp Gln Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aat gga aac acc
tat tta gaa tgg tac ctg cag aaa cca ggc cag tct 144Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cca aag
ctc ctg atc tac aaa gtt tcc aac cga ttt tct ggg gtc cca 192Pro Lys
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
gac agg ttc agt ggc agt gga tca ggg aca gat ttc aca ctc aag atc
240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80 agc aga gtg gag gct gag gat ctg gga gtt tat tac tgc ttt
caa ggt 288Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 tca cat gtt ccg tac acg ttc gga ggg ggg acc aag
ctg gaa ata aaa 336Ser His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys 100 105 110 9112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 9Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val
Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val
Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser
His Val Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
110 1016PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 10Arg Ser Ser Gln Ser Ile
Val His Ser Asn Gly Asn Thr Tyr Leu Glu 1 5 10 15 117PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Lys Val Ser Asn Arg Phe Ser 1 5 129PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Phe Gln Gly Ser His Val Pro Tyr Thr 1 5
13119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 13Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr
Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
14119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 14Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr
Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
15119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 15Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr
Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
16119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 16Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
17119PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 17Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 Asp Ile Ser Trp
Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 Gly Val
Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60
Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65
70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr
Cys Val 85 90 95 Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115
18112PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 18Asp Val Leu Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr
Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe
Gln Gly 85 90 95 Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 19112PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 19Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val
Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr
Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Lys
Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser
His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110 209PRTHomo sapiens 20Met Thr Met Phe Lys Gly Ser Asn Glu 1 5
21357DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polynucleotide" 21cag gtg cag ctg cag gag agc
ggc cct ggc ctg gtg aag cct agc cag 48Gln Val Gln Leu Gln Glu Ser
Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 acc ctg agc ctg acc
tgc acc gtg agc ggc ttc agc ctg acc agc tac 96Thr Leu Ser Leu Thr
Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 gac atc agc
tgg atc aga cag cct cct ggc aag ggc ctg gag tgg ctg 144Asp Ile Ser
Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 ggc
gtg atc tgg acc ggc ggc ggc acc aac tac aac agc gcc ttc atg 192Gly
Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55
60 agc aga ctg acc atc agc aag gac aac agc aag agc acc gtg tac ctg
240Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Lys Ser Thr Val Tyr Leu
65 70 75 80 cag atg aac agc ctg aga gcc gag gac acc gcc atc tac tac
tgc gtg 288Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr
Cys Val 85 90 95 aga gac aga gac tac gac ggc tgg tac ttc gac gtg
tgg ggc cag ggc 336Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val
Trp Gly Gln Gly 100 105 110 acc acc gtg acc gtg agc agc 357Thr Thr
Val Thr Val Ser Ser 115 22357DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 22cag gtg cag ctg cag gag agc ggc cct ggc ctg gtg
aag cct agc cag 48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln 1 5 10 15 acc ctg agc ctg acc tgc acc gtg agc ggc
ttc agc ctg acc agc tac 96Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Ser Tyr 20 25 30 gac atc agc tgg atc aga cag cct
cct ggc aag ggc ctg gag tgg ctg 144Asp Ile Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 ggc gtg atc tgg acc ggc
ggc ggc acc aac tac aac agc gcc ttc atg 192Gly Val Ile Trp Thr Gly
Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 agc aga ctg acc
atc agc aag gac aac agc aag aac acc gtg tac ctg 240Ser Arg Leu Thr
Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 cag atg
aac agc ctg aga gcc gag gac acc gcc atc tac tac tgc gtg 288Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95
aga gac aga gac tac gac ggc tgg tac ttc gac gtg tgg ggc
cag ggc 336Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val Trp Gly
Gln Gly 100 105 110 acc acc gtg acc gtg agc agc 357Thr Thr Val Thr
Val Ser Ser 115 23357DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polynucleotide" 23cag gtg cag ctg
cag gag agc ggc cct ggc ctg gtg aag cct agc cag 48Gln Val Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 acc ctg
agc ctg acc tgc acc gtg agc ggc ttc agc ctg acc agc tac 96Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30
gac atc agc tgg atc aga cag cct cct ggc aag ggc ctg gag tgg ctg
144Asp Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45 ggc gtg atc tgg acc ggc ggc ggc acc aac tac aac agc gcc
ttc atg 192Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala
Phe Met 50 55 60 agc aga ttc acc atc agc aag gac aac agc aag aac
acc gtg tac ctg 240Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn
Thr Val Tyr Leu 65 70 75 80 cag atg aac agc ctg aga gcc gag gac acc
gcc atc tac tac tgc gtg 288Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Ile Tyr Tyr Cys Val 85 90 95 aga gac aga gac tac gac ggc tgg
tac ttc gac gtg tgg ggc cag ggc 336Arg Asp Arg Asp Tyr Asp Gly Trp
Tyr Phe Asp Val Trp Gly Gln Gly 100 105 110 acc acc gtg acc gtg agc
agc 357Thr Thr Val Thr Val Ser Ser 115 24357DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 24cag gtg cag ctg cag gag agc ggc cct ggc ctg gtg
aag cct agc cag 48Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Gln 1 5 10 15 acc ctg agc ctg acc tgc acc gtg agc ggc
ttc agc ctg acc agc tac 96Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Phe Ser Leu Thr Ser Tyr 20 25 30 gac atc agc tgg atc aga cag cct
cct ggc aag ggc ctg gag tgg ctg 144Asp Ile Ser Trp Ile Arg Gln Pro
Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45 ggc gtg atc tgg acc ggc
ggc ggc acc aac tac aac agc gcc ttc atg 192Gly Val Ile Trp Thr Gly
Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met 50 55 60 agc aga ctg acc
atc agc aag gac aac agc aag aac acc gtg tac ctg 240Ser Arg Leu Thr
Ile Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 cag atg
aac agc ctg aga gcc gag gac acc gcc gtg tac tac tgc gtg 288Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Val 85 90 95
aga gac aga gac tac gac ggc tgg tac ttc gac gtg tgg ggc cag ggc
336Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe Asp Val Trp Gly Gln Gly
100 105 110 acc acc gtg acc gtg agc agc 357Thr Thr Val Thr Val Ser
Ser 115 25357DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 25cag gtg cag ctg cag
gag agc ggc cct ggc ctg gtg aag cct agc cag 48Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 acc ctg agc
ctg acc tgc acc gtg agc ggc ttc agc ctg acc agc tac 96Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30 gac
atc agc tgg atc aga cag cct cct ggc aag ggc ctg gag tgg ctg 144Asp
Ile Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40
45 ggc gtg atc tgg acc ggc ggc ggc acc aac tac aac agc gcc ttc atg
192Gly Val Ile Trp Thr Gly Gly Gly Thr Asn Tyr Asn Ser Ala Phe Met
50 55 60 agc aga ttc acc atc agc aag gac aac agc aag aac acc gtg
tac ctg 240Ser Arg Phe Thr Ile Ser Lys Asp Asn Ser Lys Asn Thr Val
Tyr Leu 65 70 75 80 cag atg aac agc ctg aga gcc gag gac acc gcc gtg
tac tac tgc gtg 288Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys Val 85 90 95 aga gac aga gac tac gac ggc tgg tac ttc
gac gtg tgg ggc cag ggc 336Arg Asp Arg Asp Tyr Asp Gly Trp Tyr Phe
Asp Val Trp Gly Gln Gly 100 105 110 acc acc gtg acc gtg agc agc
357Thr Thr Val Thr Val Ser Ser 115 26336DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polynucleotide" 26gac gtg ctg atg acc cag agc cct ctg agc ctg cct
gtg acc ctg ggc 48Asp Val Leu Met Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Leu Gly 1 5 10 15 cag cct gcc agc atc agc tgc aga agc agc
cag agc atc gtg cac agc 96Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Ile Val His Ser 20 25 30 aac ggc aac acc tac ctg gag tgg
tac ctg cag aag cct ggc cag agc 144Asn Gly Asn Thr Tyr Leu Glu Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 cct cag ctg ctg atc tac
aag gtg agc aac aga ttc agc ggc gtg cct 192Pro Gln Leu Leu Ile Tyr
Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 gac aga ttc agc
ggc agc ggc agc ggc acc gac ttc acc ctg aag atc 240Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 agc aga
gtg gag gcc gag gac gtg ggc gtg tac tac tgc ttc cag ggc 288Ser Arg
Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95
agc cac gtg cct tac acc ttc ggc cag ggc acc aag ctg gag atc aag
336Ser His Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 27336DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polynucleotide" 27gac gtg gtg atg acc
cag agc cct ctg agc ctg cct gtg acc ctg ggc 48Asp Val Val Met Thr
Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 cag cct gcc
agc atc agc tgc aga agc agc cag agc atc gtg cac agc 96Gln Pro Ala
Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 aac
ggc aac acc tac ctg gag tgg tac ctg cag aag cct ggc cag agc 144Asn
Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40
45 cct cag ctg ctg atc tac aag gtg agc aac aga ttc agc ggc gtg cct
192Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60 gac aga ttc agc ggc agc ggc agc ggc acc gac ttc acc ctg
aag atc 240Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Lys Ile 65 70 75 80 agc aga gtg gag gcc gag gac gtg ggc gtg tac tac
tgc ttc cag ggc 288Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr
Cys Phe Gln Gly 85 90 95 agc cac gtg cct tac acc ttc ggc cag ggc
acc aag ctg gag atc aag 336Ser His Val Pro Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 110
* * * * *
References