U.S. patent application number 14/793065 was filed with the patent office on 2015-10-22 for methods and compositions for modulation of olfml3 mediated angiogenesis.
This patent application is currently assigned to Research Development Foundation. The applicant listed for this patent is Research Development Foundation. Invention is credited to Philippe HAMMEL, Beat A. IMHOF, Marijana MILJKOVIC-LICINA.
Application Number | 20150299306 14/793065 |
Document ID | / |
Family ID | 46599027 |
Filed Date | 2015-10-22 |
United States Patent
Application |
20150299306 |
Kind Code |
A1 |
IMHOF; Beat A. ; et
al. |
October 22, 2015 |
METHODS AND COMPOSITIONS FOR MODULATION OF OLFML3 MEDIATED
ANGIOGENESIS
Abstract
The present invention relates to antibodies against specific
domains of Olfml3 and the use of such in angiogenesis. In
particular aspects, angiogenesis-related conditions, such as
cancer, can be treated by the composition comprising the Olfml3
antagonists.
Inventors: |
IMHOF; Beat A.; (Geneva,
CH) ; MILJKOVIC-LICINA; Marijana; (Geneva, CH)
; HAMMEL; Philippe; (Geneva, CH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Research Development Foundation |
Carson City |
NV |
US |
|
|
Assignee: |
Research Development
Foundation
Carson City
NV
|
Family ID: |
46599027 |
Appl. No.: |
14/793065 |
Filed: |
July 7, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14158463 |
Jan 17, 2014 |
9096662 |
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14793065 |
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13557660 |
Jul 25, 2012 |
8663637 |
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14158463 |
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61664491 |
Jun 26, 2012 |
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61515669 |
Aug 5, 2011 |
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Current U.S.
Class: |
424/130.1 ;
435/71.1 |
Current CPC
Class: |
C07K 2317/34 20130101;
A61K 2039/505 20130101; A61P 9/00 20180101; C07K 16/18 20130101;
A61P 27/02 20180101; C07K 2317/76 20130101; A61P 1/00 20180101;
A61P 29/00 20180101; A61P 1/18 20180101; A61P 35/00 20180101; A61P
35/02 20180101; C07K 16/2896 20130101; A61P 9/10 20180101; A61P
19/02 20180101; C07K 2317/73 20130101; C07K 2317/20 20130101 |
International
Class: |
C07K 16/18 20060101
C07K016/18 |
Claims
1-18. (canceled)
19. A method of producing an antibody comprising administering to a
subject a peptide comprising (i) an epitope within amino acid
positions 86-99 of SEQ ID NO:1 (human Olfml3 protein), or (ii) an
epitope within amino acid positions 390-403 of SEQ ID NO:1 (human
Olfml3 protein).
20. The method of claim 19, further comprising obtaining a B-cell
expressing said antibody.
21. The method of claim 20, further comprising immortalizing said
B-cell.
22. The method of claim 21, further comprising culturing said
immortalized B-cell to produce a monoclonal antibody.
23. The method of claim 19, wherein the antibody recognizes amino
acid positions 86-99.
24. The method of claim 19, wherein the antibody recognizes amino
acid positions 390-403.
25. The method of claim 20, wherein said subject is a mouse.
26. The method of claim 25, further comprising humanizing said
mouse antibody.
27. The method of claim 20, wherein said subject is a human.
28. The method of claim 19, further comprising obtaining polyclonal
sera reactive with said peptide from said subject.
29. The method of claim 28, wherein said polyclonal sera is human
polyclonal sera.
Description
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/158,463, filed Jan. 17, 2014, which is a
divisional of U.S. patent application Ser. No. 13/557,660, filed
Jul. 25, 2012, now U.S. Pat. No. 8,663,637, which claims benefit of
priority to U.S. Provisional Application Ser. No. 61/515,669, filed
Aug. 5, 2011 and U.S. Provisional Application Ser. No. 61/664,491,
filed Jun. 26, 2012, the entire contents of each of which are
hereby incorporated by reference.
[0002] The present invention relates generally to the fields of
molecular biology and oncology. More particularly, it concerns
compositions comprising binding molecules for Olfml3, an
angiogenesis modulator, and associated methods of treating
angiogenesis-related conditions.
DESCRIPTION OF RELATED ART
[0003] Angiogenesis is a multi-step cellular process of capillary
sprouting and formation of neo-vasculature from preexisting blood
vessels. The complex process involves disassembly of endothelial
junctions, followed by endothelial cells detachment, proliferation
and migration as well as subsequent re-establishment of
intercellular and cell-matrix contact. As such it requires
coordinated actions of a variety of vascular cell adhesion
molecules and growth factors originating from endothelial cells
themselves or neighboring mural cells. Indeed, angiogenesis is a
tightly tuned process regulated by pro- and anti-angiogenic factors
(Folkman, 1995).
[0004] Numerous studies have demonstrated that excessive
angiogenesis influences significantly various disease states
including tumor growth, ischemic cardiovascular pathologies or
chronic inflammatory diseases (Carmeliet, 2003; Carmeliet, 2005;
Gariano and Gardner, 2005).
[0005] From vascular mediated pathologies, tumor-associated
angiogenesis is the most extensively studied. It was first
postulated that tumors cannot grow further than a size of 2-3
mm.sup.3 in the absence of neovascularization (Folkman, 1971).
Therefore, angiogenesis is a prerequisite for tumor growth and
blocking this process can prevent further proliferation of tumor
cells. Furthermore, prevention of angiogenesis targets normal
tissue and does not escape therapy by mutagenesis as seen with
tumor cells. It is thus expected that anti-angiogenic therapy be
better sustained in keeping tumor growth under control than any
other treatment directly addressing tumor cells. Despite the fact
that vascular endothelial cell growth factor (VEGF), fibroblast
growth factor (FGF) and other pro-angiogenic molecules are
indispensable for vessel formations (Hanahan, 1997; Yancopoulos et
al., 2000), the complete molecular and cellular mechanisms
governing tumor-associated angiogenesis are poorly understood.
[0006] In addition, diseases complicated by vascular leakage and/or
neovascularization in the eye are responsible for the vast majority
of visual morbidity and blindness in developed countries. Retinal
neovascularization occurs in ischemic retinopathies such as
diabetic retinopathy and is a major cause of visual loss in working
age patients (Klein et al., 1984). Choroidal neovascularization
occurs as a complication of age-related macular degeneration and is
a major cause of visual loss in elderly patients (Ferris et al.,
1984). Improved treatments are needed to reduce the high rate of
visual loss, and their development is likely to be facilitated by
greater understanding of the molecular pathogenesis of ocular
neovascularization.
[0007] Therefore, there remains a need to develop novel methods for
targeting novel vascular molecules expressed and/or secreted by
angiogenic cells.
SUMMARY OF THE INVENTION
[0008] Olfml3 protein is discovered to be a proangiogenic,
endothelial cell-derived factor that interacts with BMP4 and
promotes tumor angiogensis. Specific Olfml3 inhibitors may be
useful for angiogensis inhibition, especially in pathological
angiogenic conditions. In accordance with certain aspects of the
present invention, there may be provided a method of inhibiting
angiogenesis in a subject having an angiogenic condition. The
method may comprise administering to the subject a composition
comprising an antibody or a nucleic acid encoding the antibody,
wherein the antibody recognizes and binds to at least one amino
acid sequence on an Olfml3 protein. For example, the amino acid
sequence may be defined by (i) amino acid positions 86-403, (ii)
amino acid positions 86-99, (iii) amino acid positions 114-143, or
(iv) amino acid positions 390-403 of SEQ ID NO:1 (human Olfml3
protein) or SEQ ID NO:3 (mouse Olfml3 protein). In a particular
aspect, the antibody may inhibit the binding of an Olfml3 protein
to BMP4 protein.
[0009] In a certain aspect, the subject has tumor. The antibody may
reduce the number of pericytes in vessels associated with the
tumor. In a further aspect, the antibody may reduce the tumor
size.
[0010] In a further aspect, there may be provided a method of
inhibiting angiogenesis in a cell comprising inhibiting the binding
of Olfml3 protein to BMP4 protein. For example, the binding of
Olfml3 protein to BMP4 protein may be inhibited through a
polypeptide that binds to Olfml3 at a position which BMP4 protein
normally binds to thereby inhibit the binding of BMP4 thereto. The
cell may be located in a subject having an angiogenic condition.
The method may further comprise administering to the subject a
composition comprising an antibody that inhibits the binding
between Olfml3 protein and BMP4 protein, or a nucleic acid encoding
the antibody. For example, the antibody may recognize and bind to
an amino acid sequence defined by (i) amino acid positions 86-403,
(ii) amino acid positions 86-99, (iii) amino acid positions
114-143, or (iv) amino acid positions 390-403 of SEQ ID NO:1 (human
Olfml3) or SEQ ID NO:3 (mouse Olfml3).
[0011] The antibody may be a monoclonal antibody, a polyclonal
antibody, a chimeric antibody, an affinity matured antibody, a
humanized antibody, a human antibody or an antibody fragment.
Particularly, the antibody is a monoclonal antibody, polycolonal
antibody or a humanized antibody. The antibody fragment may be Fab,
Fab', Fab'-SH, F(ab').sub.2, or scFv.
[0012] For medical or clinical applications, the antibody may be
attached to an agent to be delivered to an angiogenic cell or
targeted to an Olfml3-expressing cell. The agent may be a cytotoxic
agent, a cytokine, an anti-angiogenic agent, a chemotherapeutic
agent, a diagnostic agent, an imaging agent, a radioisotope, a
pro-apoptosis agent, an enzyme, a hormone, a growth factor, a
peptide, a protein, an antibiotic, an antibody or fragment thereof,
an imaging agent, an antigen, a survival factor, an anti-apoptotic
agent, a hormone antagonist, a virus, a bacteriophage, a bacterium,
a liposome, a microparticle, a magnetic bead, a microdevice, a
cell, a nucleic acid or an expression vector.
[0013] There may also be provided a pharmaceutical composition
comprising one or more nucleic acids or the antibody described
above in a pharmaceutically acceptable carrier, for example, a
pharmaceutical composition comprising the antibody or fragment and
a pharmaceutically acceptable carrier or a pharmaceutical
composition comprising one or more nucleic acids described above
and a pharmaceutically acceptable carrier.
[0014] The pharmaceutical composition of the present invention may
further comprise a lipid component, which is believed to likely
give the nucleic acid or antibody an improved stability, efficacy
and bioavailability, with perhaps even reduced toxicity. The lipid
component may form a liposome, but this is not believed to be
required. In certain aspects, the composition further comprises
cholesterol or polyethyleneglycol (PEG).
[0015] Exemplary lipids include, but are not limited to,
1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), egg
phosphatidylcholine ("EPC"), dilauryloylphosphatidylcholine
("DLPC"), dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl
phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoyl
phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoyl
phosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoyl
phosphatidylcholine ("SPPC"), dimyristyl phosphatidylcholine
("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
palmitoyloeoyl phosphatidylcholine ("POPC"),
lysophosphatidylcholine, dilinoleoylphosphatidylcholine
distearoylphophatidylethanolamine ("DSPE"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), palmitoyloeoyl
phosphatidylethanolamine ("POPE"),lysophosphatidylethanolamine,
phosphatidylserine, phosphatidylglycerol, dimyristoyl
phosphatidylserine ("DMPS"), dipalmitoyl phosphatidylserine
("DPPS"), brain phosphatidylserine ("BPS"),
dilauryloylphosphatidylglycerol ("DLPG"),
dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"),
dioleoylphosphatidylglycerol ("DOPG"), cholesterol or
polyethyleneglycol (PEG).
[0016] It is contemplated that the Olfml3 inhibitory molecules, the
antibody or the composition described above may be used in the
treatment of any disease or disorder in which angiogenesis plays a
role, which will be referred to generally as an
angiogenesis-related condition. It is contemplated that the
invention will find applicability in any such disorder in subjects
such as humans or animals. Exemplary angiogenesis-related
conditions include an ocular neovascularization, an arterio-venous
malformation, coronary restenosis, peripheral vessel restenosis,
glomerulonephritis, rheumatoid arthritis, pancreatitis, a bowel
disease, an ischemic cardiovascular pathology, or a chronic
inflammatory disease.
[0017] In the case of cancer, exemplary angiogenic cancers include
breast cancer, lung cancer, prostate cancer, ovarian cancer, brain
cancer, liver cancer, cervical cancer, colorectal cancer, renal
cancer, skin cancer, head and neck cancer, bone cancer, esophageal
cancer, bladder cancer, uterine cancer, lymphatic cancer, stomach
cancer, pancreatic cancer, testicular cancer, lymphoma, or
leukemia. Ocular neovascularization disorders may include macular
degeneration (e.g., age-related macular degeneration (AMD), corneal
graft rejection, corneal neovascularization, retinopathy of
prematurity (ROP) and diabetic retinopathy.
[0018] Embodiments discussed in the context of methods and/or
compositions of the invention may be employed with respect to any
other method or composition described herein. Thus, an embodiment
pertaining to one method or composition may be applied to other
methods and compositions of the invention as well.
[0019] As used herein the terms "encode" or "encoding" with
reference to a nucleic acid are used to make the invention readily
understandable by the skilled artisan; however, these terms may be
used interchangeably with "comprise" or "comprising"
respectively.
[0020] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. The use of the term "or" in the claims is used to
mean "and/or" unless explicitly indicated to refer to alternatives
only or the alternatives are mutually exclusive, although the
disclosure supports a definition that refers to only alternatives
and "and/or." As used herein "another" may mean at least a second
or more.
[0021] Throughout this application, the term "about" is used to
indicate that a value includes the inherent variation of error for
the device, the method being employed to determine the value, or
the variation that exists among the study subjects.
[0022] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0024] FIGS. 1A-1B. Differential expression of Olfml3 mRNA in
angiogenic (t.End.1V.sup.high) versus resting (t.End.1V.sup.low)
endothelial cells. FIG. 1A. Validation of data obtained by
microarray analysis using quantitative real-time reverse
transcription-polymerase chain reaction (RT-PCR). Bars represent
the quantity of the Olfml3 mRNA (relative units) in total RNA
isolates from t.End.1V.sup.high and t.End.1V.sup.high cells. Values
for each sample were normalized to values of mouse .beta.-actin,
.beta.-tubulin and/or EEF1A genes according to the GeNorm method
(Vandesompele et al., 2002). Relative values from individual
experiments were averaged and plotted with standard deviation (SD)
as error bars. The statistical analysis was performed using the
paired t-test (p=0.00918). FIG. 1B. Western blotting shows the
differential expression of Olfml3 protein in angiogenic
(t.End.1V.sup.high) versus resting (t.End.1V.sup.low) endothelial
cells.
[0025] FIGS. 2A-2C. In vivo expression of Olfml3 in angiogenic
tissues. FIG. 2A. Immunostaining on human placental villi at a term
pregnancy. Parafine sections were incubated with the rabbit
anti-Olfml3 antibody detected using a biothinylated anti-rabbit
antibody (brown). Staining illustrates that Olfml3 is expressed by
endothelial cells of placental angiogenic vessels. H&E:
hematoxylin and eosin stain; by: blood vessels. Bars corresponds to
20.times. (upper panels) and 40.times. magnification (lower
panels). FIG. 2B. Left panel: Double in situ mRNA hybridization on
angiogenic vessels (arrowheads) immigrated into bFGF-loaded
matrigel plugs (mg). Cryosections were incubated with 013
anti-sense RNA probes (green) and PECAM-1 anti-sense RNA probes
(red). Double labeling illustrates that Olfml3-expressing cells are
PECAM-1 positive (merge). No staining observed in the control,
cryosections incubated with Olfml3 and Pecam1 sense probes (lower
panel). Right panel: Double immunofluorescence on angiogenic
vessels immigrated into bFGF-loaded matrigel plugs (mg).
Cryosections were incubated with rabbit anti-Olfml3 antibody
detected by donkey anti-rabbit IgG (red) and rat anti-PECAM-1
antibody detected by donkey anti-rat IgG (green). Double labeling
illustrates that Olfml3-expressing cells are PECAM-1 positive
(merge). No staining observed in the control, cryosections
incubated with Olfml3 preimmune sera (lower panel). FIG. 2C. Left
panel: Double in situ mRNA hybridization on angiogenic vessels
(arrowheads) migrated into LLC1 tumors. Cryosections were incubated
with Olfml3 RNA probes (green) and PECAM-1 RNA probes (red). Double
labeling illustrates that Olfml3-expressing cells are PECAM-1
positive (merge). Surrounding pericytes express low levels of
Olfml3 as well (stars). No staining observed in the controls
incubated with sense probes (lowest panel). Right panel: Double
immunofluorescence on angiogenic vessels migrated into LLC1 tumors.
Cryosections were incubated with rabbit anti-Olfml3 antibody (red)
and rat anti-PECAM-1 antibody (green). Double labeling illustrates
that Olfml3-expressing cells are PECAM-1 positive (merge).
Pericytes indicated by stars. TO-PRO dye was used for nuclear
staining (blue, B-C). Representative single-frame confocal images
are shown. Bars correspond to 20 .mu.m (B and C, upper and lower
panels) and 10 .mu.m (B and C, middle panels).
[0026] FIGS. 3A-3B. Olfml3 is secreted but remains in vicinity of
endothelial cells associated with immature .alpha.-SMA-positive
mural cells. FIG. 3A. Triple immunofluorescence on angiogenic
vessels in LLC1 tumors. Cryosections were incubated with rabbit
anti-Olfml3 detected by donkey anti-rabbit IgG (light blue), rat
anti-PECAM-1 detected by donkey anti-rat IgG (green) and mouse
anti-.alpha.-SMA detected by goat anti-mouse IgG.sub.2a. (red).
Triple labeling illustrates that Olfml3-expressing endothelial cell
(light blue, arrowhead) are PECAM-1 positive (green) and covered by
immature, .alpha.-SMA-positive mural cells (red). FIG. 3B. Triple
immunofluorescence on angiogenic vessels in LLC1 tumors.
Cryosections were incubated with rabbit anti-Olfml3 detected by
donkey anti-rabbit IgG (light blue), rat anti-PECAM-1 detected by
donkey anti-rat IgG (green) and mouse anti-NG2 detected by donkey
anti-mouse IgG (red). Triple labeling illustrates that PECAM-1
positive endothelial cells (green) that are covered by mature,
NG2-positive mural cells (red) express Olfml3 at very low level
(light blue). DAPI nuclear counterstain was used (blue; A-C).
Representative single-frame confocal images are shown. Bars
correspond to 20 .mu.m (upper and lower panels) and 10 .mu.m
(middle panels).
[0027] FIGS. 4A-4E. Delayed wound healing of Olfml3-silenced
t.End.1V.sup.high cells. FIG. 4A. Examples of monolayer cultures of
t.End.1V.sup.high cells silenced for either mock; control siRNA
(ctrl siRNA, 0.5 .mu.M), or Olfml3 (Olfml3 siRNA 3, 0.5 .mu.M).
Confluent cell monolayers were wounded with a pipette tip (yellow
area), and wounded areas were illustrated using an imaging program
(yellow area). Cells at the edge of the wound migrated into the
wounded area, shown after 16 hours (violet area). FIG. 4B. Distance
of migration (.mu.m) was calculated. The progress of wound closure
was significantly delayed in the Olfml3-siRNA-silenced cells
compared with mock or control siRNA-treated cells. Bars represent
means .+-.SD of nine independent culture wells; in total, 3
experiments were performed. FIG. 4C. Reduced migratory ability of
Olfml3-silenced t.End.1V.sup.high cells was rescued by coating of
recombinant Olfml3 protein (+) in vitro, when compared to the
t.End.1V.sup.high cells cultured on non-coated control plates (-).
FIG. 4D. Reduced migratory ability of Olfml3-silenced
t.End.1V.sup.high cells was rescued by addition of recombinant
Olfml3 protein in vitro, when compared to the t.End.1V.sup.high
cells cultured on control plates (-). FIG. 4E. When coated on
culture plates, recombinant Olfml3 promoted t.End.1V.sup.high cell
migration in the concentration-dependent manner (1-5 ng/.mu.l),
when compared to non-coating control (no coat). The statistical
analysis using one-way ANOVA with Bonferroni post hoc test was
performed.
[0028] FIGS. 5A-5C. Silencing of Olfml3 in t.End.1V.sup.high cells
attenuates the initiation and the final steps angiogenesis in
vitro. FIG. 5A. In 3D fibrin gels, control siRNA-treated
t.End.1V.sup.high cells first send out spikes after 24 h of culture
(ctrl siRNA, arrows, 24-32 h). This process continues by sprouting,
cell-cell contact formation, which leads to branching of the
proliferating cells forming a polygonal network (ctrl siRNA,
arrows, 48-72 h). Olfml3 siRNA 3 (0.5 .mu.M) delayed (arrowheads)
sprout formation by 48 h (arrowheads). FIG. 5B. Quantification of
sprout-forming t.End.1V.sup.high cells at early time points (24 h
and 32 h) of sprouting assay. Olfml3-siRNA (0.5 .mu.M) treated
t.End.1V.sup.high cells reduced the total number of sprout-forming
cells. The mean and standard deviation of three experiments is
shown. The statistical analysis using one-way ANOVA with Bonferroni
post hoc test was performed. FIG. 5C. Total length of vascular
cords representing the capillary-like network was quantified using
MetaMorph software. Length and complexity of the vascular network
(cords) of Olfml3-silenced cells is reduced in comparison to
control siRNA-transfected cells (top panel) at 72 h. Measurement of
total length of the vascular network after Olfml3 silencing (Olfml3
siRNA 3, 0.5 .mu.M) compared to mock- and control siRNA (ctrl
siRNA, 0.5 .mu.M)-transfected cells. Error bars =SD. The
statistical analysis using one-way ANOVA with Bonferroni post-hoc
test was performed.
[0029] FIGS. 6A-6F. Treatment of mice with the anti-Olfml3
antibodies reduces tumor growth. C57BL6/J mice were injected
subcutaneously (s.c.) with Lewis lung carcinoma cells (LLC1) into
the flank. Mice received intraperitoneal (i.p.) injections of
either control total rabbit IgG (ctrl IgG), or affinity-purified
anti-Olfml3 every third day (50 .mu.g). FIG. 6A. Macroscopic
aspects of 9-days-old tumors grown in mice treated with control,
total rabbit IgG or anti-Olfml3 antibody. Bar represent 0.5 cm.
Mice treated with anti-Olfml3 antibodies showed reduced tumor
weight compared to controls (FIG. 6B). n=5 ; two tumors per mouse),
p<0.05. Statistical differences compared to control group,
calculated by one-way ANOVA tests with Bonferroni post-hoc test.
FIG. 6C. Macroscopic aspects of 9-days-old tumors grown in mice
treated with control, total rabbit IgG or Olfml3 antibody
affinity-purified against either Olfml3 peptide a or b. FIG. 6D.
Mice treated with either anti-Olfml3 antibody showed reduced tumor
weight compared with controls. Control IgG (n=3 ; two tumors per
mouse), anti-Olfml3 A (n=4 ; two tumors per mouse), anti-Olfml3 B
(n=4 ; two tumors per mouse). p<0.01 and p<0.001,
respectively. Statistical differences compared to control group,
calculated by one-way ANOVA tests with Bonefferoni post-hoc test.
Scale bar represents 0.5 cm. FIG. 6E. Immunofluorescence analysis
of LLC1 tumors treated with control (ctrl IgG) or Olfml3 antibodies
(anti-Olfml3) using anti-PECAM-1 antibody (green). DAPI was used as
a nuclear counterstain (blue). Representative single-frame confocal
images are shown (63.times. magnification). Bars correspond to 20
.mu.m. FIG. 6F. Quantification of the vascularization level between
control (ctrl IgG) and anti-Olfml3 treated tumors was measured as a
ratio of the PECAM-1 (green) to DAPI nuclear stain (blue) cells.
p<0.01. In each group, quantification was determined by 10
fields in the three different planes per tumor, followed by
averaging the values for 10 tumors.
[0030] FIGS. 7A-7D. Recombinant Olfml3 binds recombinant BMP4. FIG.
7A. Binding of recombinant Olfml3-FLAG to the recombinant BMP4 was
detected by enzyme linked immunosorbent assay (ELISA) using FLAG
(M2) antibody. The Olfml3-FLAG specifically recognized BMP4 but not
BMP1 or BMP9 in a dose-dependent fashion (0.1-1 ng/.mu.l). Negative
control was human JAM-C-FLAG recognized by the anti-JAM-C antibody
D33. FIG. 7B. Immobilized Olfml3 FLAG-tagged protein on M2 beads
binds recombinant BMP4 (21 kDa). Silver-stained SDS gel: lane 1)
MW--molecular weight marker, lane 2) directly loaded BMP4 (BMP4);
lane 3) pull down of recombinant BMP4 by M2 beads ; lane 4) pull
down using Olfml3 FLAG-tagged protein and recombinant BMP4 by M2
beads (M2 +Olfml3-FLAG+BMP4). FIG. 7C. Blocking of the binding
specificity of the rOlfml3-FLAG to recombinant BMP4 protein was
detected by enzyme linked immunosorbent assay (ELISA) using Olfml3
antibodies. Binding of Olfml3-FLAG to recombinant BMP4 could be
reduced up to 50% using Olfml3 antibody against Olfml3 peptides
A+B, A or B (86-99 and 390-403, respectively). FIG. 7D. Blocking of
Olfml3-FLAG binding to recombinant BMP4 protein was detected by
ELISA using Olfml3 antibodies. Binding of Olfml3-FLAG to
recombinant BMP4 could be reduced using Olfml3 antibody against
peptides A+B and commercial antibodies against peptide D (Abcam,
114-143), while commercial antibodies against peptide S (Sigma,
46-60) showed no blocking effect.
[0031] FIGS. 8A-8B. Olfml3-BMP4 interaction stimulates ERK1/2
phosphorylation. FIG. 8A. HUVEC were treated with VEGF (50 ng/ml),
BMP4 (50 ng/ml) or VEGF+Olfml3 (50 ng/ml +100 ng/ml) for 35 min.
Densitometric analysis showed significant increase of ERK tyrosin
phosphorylation in HUVECs treated with both BMP4 and Olfml3
together but not in control cells or cells treated with single
growth factors. Total ERK serves as a loading control. Each graph
value represents the mean of three determinations; error bars, SD.
Statistical differences compared to control group, calculated by
one-way ANOVA tests with Bonfferoni post-hoc test. FIG. 8B. Effects
of VEGF (50 ng/ml) and BMP4 (50 ng/ml) on the Olfml3 protein
expression in HUVECs. HUVEC were cultured for 24 h in the absence
or presence of VEGF and BMP4 and subjected to Western blotting
using Olfml3 antibody. Quantitative values of Olfml3 protein
expression were normalized by the amounts of .beta.-actin protein,
and results were given as relative density of the Olfml3/Actin
protein ratio. Each value represents the mean of three
determinations; error bars, SD. Statistical differences compared to
control group, calculated by one-way ANOVA tests with Bonefferoni
post-hoc test.
[0032] FIG. 9. Validation of Olfml3 down-regulation using siRNAs
Inhibition of Olfml3 expression in t.End.1V.sup.high cells using
three siRNA sequences (Olfml3 siRNA 1, 2 and 3). Transfection of
Olfml3-targeted and control (ctrl siRNA and GAPDH) siRNAs at the
concentration of 0.5 .mu.M was carried out using Nucleofector
technology (Amaxa). At 24 hours post-transfection, expression of
target and control genes were analyzed by qPCR. The values were
normalized to the expression levels of mouse .beta.-actin,
.beta.-tubulin and EEF1A. Abbreviations: nh siRNA, non homologous
siRNA; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNAs,
small interfering RNAs; qPCR, quantitative polymerase chain
reaction.
[0033] FIG. 10. Detection of Olfml3 mRNA in mouse MyEnd
microvascular myocardial endothelial cells, LMEC primary lung
microvascular endothelial cells, lung tissue and LLC1 Lewis lung
carcinoma cells. Expression of Olfml3 and control genes were
analyzed by qPCR. The values were normalized to the expression
levels of mouse .beta.-actin, .beta.-tubulin, and EEF1A.
[0034] FIG. 11. Total length of vascular cords representing the
capillary-like network quantified using MetaMorph software. Length
and complexity of the vascular network (cords) of Olfml3-silenced
cells (bottom panel) is reduced compared with control
siRNA-transfected cells (top panel) at both 56 and 72 h.
[0035] FIGS. 12A-12D. Peptide sequences used for generation of the
Olfml3 antibodies. FIG. 12A. Comparison of human and mouse Olfml3
sequences showed complete homology in the protein regions used for
generation of the Olfml3 antibodies. Olfml3 peptide A comprises
epitopes in the coiled-coil domain of Olfml3 and peptide B in the
Olfactomedin-like domain. FIG. 12B. Peptide sequences used for
generation of the Olfml3 antibodies. Mouse sequences were identical
with human Olfml3. FIGS. 12C-12D. Immunoreactivity of the rabbit
anti-serum 928 009 against the peptides A (FIG. 12C; left panel)
and B (FIG. 12D; right panel).
[0036] FIG. 13. HEK293e cells suspension in the serum-free media
was used to produce Olfml3 FLAG-tagged protein in the soluble form.
The protein was affinity purified on an anti-FLAG affinity column
and eluted with FLAG peptide. Shown are Western blotting of
affinity-purified protein after immunoreactions with anti-FLAG (M2)
or anti-Olfml3 antibodies. A unique 54 kDa Olfml3 band appears in
either condition.
[0037] FIG. 14. Pull down of BMP4 by immobilized Olfml3-FLAG
protein. BMP4 was identified in an SDS gel slice by tandem mass
spectrometry and displayed as a table using Scaffold software.
Detailed results detect BMP4 and false-positive proteins
(keratins). Analysis of the peptide and spectra evidence supported
the identification of mouse BMP4 as a binding partner of
recombinant Olfml3.
[0038] FIGS. 15A-E. Increased Olfml3 expression in tumor
endothelial cells and pericytes. FIG. 15A, in situ mRNA
hybridization of LLC1 tumors with Olfml3 (green) and PECAM-1 (red)
RNA probes shows Olfml3 expression on tumor vessels (arrows) and
vessel-associated pericytes (insets, stars). No staining with sense
Olfml3 RNA probe (sense). Olfml3-expressing endothelial cells
(anti-sense) are PECAM-1.sup.+ (overlay). Pericytes express Olfml3
but not PECAM-1 (insets, stars). Bars correspond to 30 .mu.m and 5
.mu.m (insets). FIG. 15B, Olfml3 (red) and PECAM-1 (green)
immunostaining of LLC 1 tumors shows Olfml3 expression on tumor
vessels (arrows) and accompanying pericytes (overlay/inset, stars).
Pericytes express Olfml3 but not PECAM-1 (insets, stars). Bars
correspond to 30 and 10 .mu.m (inset). FIG. 15C, Olfml3 (light
blue), PECAM-1 (green) and .alpha.-SMA (red) immunostaining of LLC
1 tumors shows Olfml3 expression on tumor vessels and accompanying
pericytes (arrows). No Olfml3 staining on .alpha.-SMA.sup.- cells
(stars). FIG. 15D, Olfml3 (light blue), PECAM-1 (green) and NG2
(red) immunostaining of LLC1 tumors shows Olfml3 expression on
tumor vessels and accompanying pericytes (arrows). DAPI-nuclear
counterstain (blue) (overlays; FIGS. 15A-D). Bars correspond to 20
.mu.m (FIGS. 15C, D). FIG. 15E, relative Olfml3 mRNA levels in
activated R-SMCs versus resting S-SMCs quantified by RT-qPCR. Error
bars represent .+-.SD (2 experiments, each group in triplicates);
***P<0.001.
[0039] FIGS. 16A-F. Effects of Olfml3 targeting and rOlfml3-FLAG on
t.End.1V.sup.high cell migration and sprouting. FIG. 16A, Top: in
vitro migration assays using mock, control siRNA (ctrl siRNA, 0.5
.mu.M) or Olfml3 siRNA-treated (Olfml3 siRNA, 0.5 .mu.M)
t.End.1V.sup.high cells. Confluent cell monolayers were wounded
(yellow area). Cells migrated into the wounded area after 16 hours
(violet area). Bottom: quantification of migration distance (.mu.m)
of mock, control- or Olfml3 siRNA-treated t.End.1V.sup.high cells.
FIG. 16B, rescued migratory ability of Olfml3-silenced
t.End.1V.sup.high cells on rOlfml3-FLAG-coated plates (1 .mu.g/mL)
when compared with non-coated control (0 .mu.g/mL). FIG. 16C,
coated rOlfml3-FLAG promotes t.End.1V.sup.high cell migration in a
concentration-dependent manner (1-5 .mu.g/mL) compared with
non-coated control (0 .mu.g/mL). FIG. 16D, in vitro
t.End.1V.sup.high sprouting assays in 3D-fibrin gels. Control
siRNA-treated cells start sprouting after 24 hours (arrows) to form
a vascular-like network (32-72 hours). Targeting Olfml3 delays
sprouting (arrowheads) by 32 hours (arrows). Bar corresponds to 10
.mu.m. FIG. 16E, quantification of sprout-forming t.End.1V.sup.high
cells at early-time points (24, 32 hours). Olfml3 targeting (0 13
siRNA) reduces the total number of sprouting cells compared with
mock or control siRNA-treated cells. FIG. 16F, quantification of
total length of vascular-like network of t.End.1V.sup.high cells
treated with mock, control or Olfml3 siRNAs, normalized to total
number of cells/condition. At later time points (48, 72 hours),
targeting Olfml3 reduces the length of the vascular-like network
compared with controls. Error bars represent .+-.s.d. (5
experiments; each group in triplicates; FIGS. 16A-C, E, F).
*P<0.05; **P<0.01; ***P<0.001; ns--non significant (FIGS.
16A-C, E, F).
[0040] FIGS. 17A-F. Inhibitory effects of anti-Olfml3 antibodies on
tumor growth and vascularization. FIG. 17A, 9-day-old LLC1 tumors
in mice treated with rabbit IgG (control), or anti-Olfml3.sup.A+B.
Bar represents 1 cm. FIG. 17B, reduced tumor weight in mice treated
with anti-Olfml3.sup.A+B compared with control IgG-treated tumors.
Error bars represent .+-.SEM (3 experiments; 4-5 mice/group; 2
tumors/mouse). *P<0.05. FIG. 17C, 9-day-old tumors in mice
treated with rabbit IgG (control), anti-Olfml3.sup.A or
anti-Olfml3.sup.B. Bar represents 1 cm. FIG. 17D, reduced tumor
weight in mice treated with either anti-Olfml3.sup.A or
anti-Olfml3.sup.B compared with control IgG-treated tumors. Error
bars represent .+-.SEM (2 experiments; 4-5 mice/group; 2
tumors/mouse). *P<0.05 **P<0.01, ns--non significant. FIG.
17E, representative confocal images compare the dense vasculature
(PECAM-1, green) of tumors under baseline condition (control) and
pruned vasculature after treatments with anti-Olfml3.sup.A or
anti-Olfml3.sup.B. DAPI-nuclear counterstain (blue). Bars
correspond to 20 .mu.m. FIG. 17F, relative vascular area in tumors
treated with total IgG (control), anti-Olfml3.sup.A or
anti-Olfml3.sup.B measured as a ratio of the total pixel count of
PECAM-1 to DAPI. Ten individual images at three planes analyzed in
8-10 tumors/group. Error bars represent .+-.SEM (2 experiments; 4-5
mice/group; 2 tumors/mouse). **P<0.01; ***P<0.001.
[0041] FIGS. 18A-D. Anti-Olfml3 antibody tumor treatment inhibits
pericyte association with vessels. FIG. 18A, top: the abundance of
pericytes (.alpha.-SMA, red) in LLC1 tumors under baseline
condition (control) and after treatment with anti-Olfml3.sup.A or
anti-Olfml3.sup.B. Bottom: insets of top panels at higher
magnification. FIG. 18B, quantification of pericyte area in LLC1
tumors treated under baseline condition (control) or with
anti-Olfml3.sup.A or anti-Olfml3.sup.B. Relative .alpha.-SMA.sup.+
area measured as a ratio of the total pixel count of .alpha.-SMA
(red) to DAPI (blue). FIG. 18C, top: the abundance of pericytes
(NG2, red) in tumors under baseline condition (control) and after
treatment with anti-Olfml3.sup.A or anti-Olfml3.sup.B. Bottom:
insets of top panels at higher magnification. DAPI (blue)-nuclear
counterstain (FIGS. 18A, C). FIG. 18D, relative NG2.sup.+ area in
tumors treated under baseline condition (control) or with
anti-Olfml3.sup.A or anti-Olfml3.sup.B measured as a ratio of the
total pixel count of NG2 (red) to DAPI (blue). Ten individual
images at three planes analyzed in 8-10 tumors/group (FIGS. 18B,
D). Error bars represent .+-.SEM (2 experiments; 4-5 mice/group; 2
tumors/mouse; FIGS. 18B, D). **P<0.01; ***P<0.001; ns-non
significant (FIGS. 18B, D). Bars correspond to 20 .mu.m (top
panels; FIGS. 18A, C) and 10 .mu.m (bottom panels; FIGS. 18A,
C).
[0042] FIGS. 19A-E. Recombinant Olfml3 binds rBMP4. FIG. 19A,
binding of rOlfml3-FLAG to rBMP4 detected by ELISA using FLAG (M2)
antibody. The rOlfml3-FLAG specifically recognizes BMP4 but not
BMP1 or BMP9 in a dose-dependent manner (0.1-1 .mu.g/mL). Human
JAM-C-FLAG-negative control (0.1 .mu.g/mL). FIG. 19B, immobilized
rOlfml3-FLAG on M2-beads binds rBMP4. Silver-stained SDS gel: left,
input of rBMP4 loaded for comparison (rBMP4; 21 kDa); middle,
pull-down of rBMP4 by M2-beads; right, pull-down of rOlfml3-FLAG
and rBMP4 by M2-beads (arrow). FIG. 19C, Olfml3 domains relative to
anti-Olfml3.sup.A, anti-Olfml3.sup.B and commercial
anti-Olfml3.sup.C epitope regions. FIG. 19D, blocking of
rOlfml3-FLAG binding to rBMP4 by anti-Olfml3.sup.A+B (A+B),
anti-Olfml3.sup.A (A) or anti-Olfml3.sup.B (B). FIG. 19E, blocking
of rOlfml3-FLAG binding to rBMP4 by anti-Olfml3.sup.A+B, but not by
anti-Olfml3.sup.C. Error bars represent .+-.SD (5 experiments; each
group in triplicates; FIGS. 19D, E). *P<0.05; ***P<0.001;
ns--non significant (FIGS. 19D, E).
[0043] FIGS. 20A-D. Olfml3 activates the canonical SMAD1/5/8
pathway. FIG. 20A, Olfml3 induces nuclear translocation of
SMAD1/5/8. SMAD1 (red) immunostaining compares SMAD1 cytoplasmic
localization under baseline conditions (control) with SMAD1 nuclear
translocation in HUVECs treated with rOlfml3-FLAG (Olfml3; 100
ng/mL); rBMP4 (BMP4; 50 ng/mL), or combination (Olfml3+BMP4) for 15
minutes. FIG. 20B, Olfml3 induces SMAD1/5/8 phosphorylation in
HUVECs. PhosphoSMADl/5/8 immunostaining (red) compares low levels
of phoshoSMADl/5/8 under control conditions (control: FLAG peptide)
and high levels of phosphoSMADl/5/8 in HUVECs treated with
rOlfml3-FLAG (Olfml3; 100 ng/mL), rBMP4 (BMP4; 50 ng/mL) or
combination (Olfml3+BMP4) for 15 min. Olfml3 does not induce
phosphoSMADl/5/8 in the presence of anti-Olfml3A+B
(Olfml3+anti-Olfml3) compared with control (Olfml330 IgG).
FITC-phalloidin staining (green) allows visualization of the cell
scaffolds (FIGS. 20A, B). DAPI (blue)-nuclear counterstain (FIGS.
20A, B). Scale bars represent 10 .mu.m (FIGS. 20A, B). FIG. 20C,
quantification of the intensity of nuclear phosphoSMADl/5/8
signals. The combination of the rOlfml3-FLAG and rBMP4
(Olfml3+BMP4) shows an additive effect on SMAD1/5/8
phosphorylation. Mean nuclear intensity was measured from 5-10
random fields/group in 2 experiments. ***P<0.001; ns--non
significant. FIG. 20D, prolonged effect on SMAD1/5/8
phosphorylation using both recombinant proteins (Olfml3+BMP4),
compared to the effect of rOlfml3-FLAG alone. HUVECs were treated
with control (0 min); rOlfml3-FLAG (100 ng/mL) or rOlfml3-FLAG and
rBMP4 (Olfml3+BMP4; 100 and 50 ng/mL, respectively) for 15, 30 and
45 minutes and blotted with pSMAD1/5/8 and SMAD1 antibodies.
[0044] FIGS. 21A-C. Olfml3 is upregulated in angiogenic
endothelium. FIG. 21A, relative Olfml3 mRNA levels in angiogenic
endothelial cells that form aggressive hemangiomas in vivo
(t.End.1Vhigh) (Hanahan and Weinberg, 2011; Potente et al., 2011),
when compared with their resting counterparts (t.End.1Vlow),
quantified by RT-qPCR. Bars represent mean .+-.SD (3 experiments,
each group in triplicates); **P<0.01. FIG. 21B, Olfml3
expression in angiogenic blood vessels (bv) of FGF2-loaded matrigel
plugs (mp). Representative confocal images of Olfml3 (red) and
PECAM-1 (green) immunostaining of matrigel plugs are shown. White
dotted lines indicate the margins of matrigel plugs.
Olfml3-expressing endothelial cells are PECAM-1+ (overlay). Scale
bar represents 20 .mu.m. FIG. 21C, relative Olfml3 expression
levels in primary lung microvascular endothelial cells (LMECs),
lung tissue and LLC1 tumor cells. Expression of mouse Olfml3 and
reference genes were analyzed by real-time qPCR. LMECs were used as
a negative and lung tissue as a positive control for Olfml3
expression (Crawford and Ferrara, 2009). LLC1 tumor cells did not
express Olfml3 mRNA. The values were normalized to the expression
levels of mouse .beta.-actin, .beta.-tubulin, and EEF1A, according
to the GeNorm method (Carmeliet and Jain, 2011). Error bars
represent .+-.SD (2 experiments; each condition in triplicates);
*P<0.05.
[0045] FIGS. 22A-B. Validation of Olfml3 down-regulation after
siRNA delivery and production of rOlfml3 FLAG-tagged protein. FIG.
22A, inhibition of Olfml3 expression in t.End.Vlhigh cells after
transfection of three Olfml3 siRNAs (Olfml3 siRNA 1, 2, and 3),
compared with transfection using control siRNAs: siRNA
non-homologous to any known mouse gene (ctrl siRNA) or GAPDH siRNA.
At 24 hours after transfection, expression of target and reference
genes were analyzed by RT-qPCR. The values were normalized to the
expression levels of mouse .beta.-actin, .beta.-tubulin, and EEF1A,
according to the GeNorm method (Carmeliet and Jain, 2011). Olfml3
siRNA 3 silenced >85% of the Olfml3 mRNA in t.End.V1high cells
after transfection and was used for all subsequent experiments.
Abbreviations: GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
siRNAs, small interfering RNAs; RT-qPCR, real-time quantitative
polymerase chain reaction. Bars represent mean .+-.SD (3
experiments, each condition in triplicates); *P<0.05 ;
**P<0.01 ; ***P<0.001; ns--non significant. FIG. 22B, HEK293e
cell suspension in serum-free media was used to produce rOlfml3
FLAG-tagged protein in the soluble form. The protein was
affinity-purified on an anti-FLAG affinity column and eluted with
FLAG peptides. Shown are Western blots of affinity-purified protein
after immunoreactions with 1, anti-FLAG (M2) or 2, anti-Olfml3A+B
antibody. A unique 54-kDa Olfml3 band appears in either
condition.
[0046] FIG. 23. In vitro t.End.1V.sup.high sprouting assays in 3D
fibrin gels. Length and complexity of the vascular-like network of
Olfml3-silenced t.End.1V.sup.high cells (Olfml3 siRNA, right
panels) are reduced compared with mock (mock, left) or control
siRNA-treated cells (ctrl siRNA, middle) at 48 and 72 hours of
t.End.1V.sup.high sprouting in 3D fibrin gels.
[0047] FIGS. 24A-C. Structural domains of mouse Olfml3 protein and
peptide sequences used for generation of anti-Olfml3 A+B and its
immunoreactivity. FIG. 24A, peptide sequences used for generation
of anti-Olfml3A+B: peptide A (red, 86-99 aa) comprises epitopes in
the coiled-coil domain (orange, 25-101 aa) and peptide B (blue,
390-403 aa) comprises epitopes in the olfactomedin-like domain
(green, 134-401 aa). FIG. 24B, Comparison of human and mouse Olfml3
protein sequences showed complete homology in the protein regions
used for generation of anti-Olfml3 A+B. FIG. 24C, immunoreactivity
of the rabbit anti-Olfml3A+B antibody (serum) against Olfml3
peptide A (left panel) and Olfml3 peptide B (right panel).
[0048] FIGS. 25A-B Inhibitory effects of rat monoclonal antibodies
against human Olfml3 on tumor growth. FIG. 25A, 9-day-old LLC1
tumors in mice treated with rat IgG.sub.2B (isotype control), 9F8BO
(anti-Olfml3.sup.B) or 46A9BO (anti-Olfml3.sup.B) antibodies. Bar
corresponds to 1 cm. FIG. 25B, reduced tumor weight in mice treated
with 9F8BO (anti-Olfml3.sup.B) or 46A9BO (anti-Olfml3.sup.B)
antibodies compared with control IgG.sub.2B-treated tumors. Error
bars represent .+-.SEM (1 experiment; 5 mice/group; 2
tumors/mouse). *P<0.05.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0049] In clinical trials, beneficial effects of anti-angiogenic
drugs were so far reached with antibodies against VEGF in the
context of colon and breast carcinomas. However, it was less
successful with other tumors for which alternate factors may be
involved. Thus, other molecules involved in angiogenesis should be
identified and used in combination with the growth factors.
Specific targeting of vascular molecules expressed and/or secreted
by angiogenic endothelial cells might be useful.
[0050] The present invention is based, in part, on the finding that
Olfml3 exhibits proangiogenic function in tumors possibly mediated
through the modulation of BMP4 signaling in vascular endothelial
cells. Olfml3 is found to be a novel angiogenic regulator. To study
its function in angiogenesis, Olfml3 is identified in the Examples
as a binding partner of BMP4, a growth factor known for its
proangiogenic activity in cancer progression. Binding of Olfml3 to
BMP4 enhances BMP4 signaling to the Extracellular Signal-Regulated
Kinase 1/2 (ERK1/2) cascade and stimulates endothelial cell
proliferation, migration and sprouting. Thus, Olfml3 is an
endothelial cell-derived proangiogenic factor and provides an
alternative target for modulating tumor angiogenesis. Without
wishing to be bound by theory, method are provided herein by
targeting at least one of the Olfml3 domains that mediate the
binding between Olfml3 protein and BMP4 protein. Further
embodiments and advantages of the invention are described
below.
I. Olfml3 Binding Molecules
[0051] In certain embodiments, an antibody or binding molecule that
binds to at least a particular portion of Olfml3 protein and
inhibits Olfml3 activity in angiogenesis and methods for treatment
of diseases using such an antibody or binding molecule are
contemplated. For example, the particular portion of Olfml3
targeted may be a part of one or more BMP4-binding domains on
Olfml3. In a particular aspect, the particular portion may be an
amino acid sequence defined by (i) amino acid positions 86-403,
(ii) amino acid positions 86-99, (iii) amino acid positions
114-143, or (iv) amino acid positions 390-403 of SEQ ID NO:1 (human
Olfml3) or SEQ ID NO:3 (mouse Olfml3). In a further aspect, the
particular portion may be a functional variant that has an amino
acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%
identical to the amino acid sequence defined as above.
[0052] This is the first report describing Olfml3 as a BMP4 agonist
that promotes endothelial cell functions, at least in part, through
the binding to BMP4 and stimulation of BMP4 signaling. Two
mechanisms that interfere with BMP activation and signaling have
been proposed. The first is the intracellular regulation of BMP
cleavage into the secreted, active forms while the second involves
binding of the extracellular BMPs to different BMP
agonist/antagonist. Depending on the nature of BMP interacting
molecules, BMP receptor binding activity may be activated or
inhibited (Umulis et al., 2009). According to the data, the
Olfml3-BMP4 interaction leads to an activated BMP4 complex. Olfml3
likely keeps the BMP4-receptor interactions and kinetics within a
physiologically useful range. Olfml3 binds to BMP4 and possibly
increases the affinity of BMP4 for its receptors, accounting for
the activation of BMP4 signaling that the inventors observed in
cultured endothelial cells. Indeed, when either BMP4 or Olfml3 is
absent, induction of BMP4 signaling is suppressed. Another
possibility is that Olfml3 may also promote BMP4 activity by
dislodging the growth factor from a putative BMP antagonist in the
extracellular space, as it was shown for pro-BMP activity of
Twisted gastrulation (Twseg1) (Oelgeschlager et al., 2000). This
mode of action is apparently different from the dose-dependent
BMPER activity during regulation of BMP4 signaling in endothelial
cells (Zhang et al., 2007; Serpe et al., 2008). At low molar
concentration, BMPER presents BMP4 to its receptors and activates
BMP4 signaling. At high molar concentrations, BMP4 binds
preferentially to BMPER and it is not available for the receptor
binding, hence inhibiting BMP4 signaling. Additional studies are
needed to elucidate definitive Olfml3 mode of action to stabilize
BMP4 and potentiate its signaling in endothelial cells.
[0053] Olfactomedin-like protein 3 (Olfml3) is a protein that in
humans is encoded by the Olfml3 gene. The inventors used the
t.End.1V.sup.high angiogenic and t.End.1V.sup.low resting cell
lines to identify novel molecules differentially expressed and
associated with angiogenesis. Among the identified new
angiogenesis-associated genes, which fulfill the criteria described
above they identified the mouse Olfml3 gene (olfactomedin-like
3).
[0054] The Olfactomedin-like 3 (Olfml3) gene encodes a secreted,
extracellular protein, also known as ONT1 in Xenopus and chicken,
mONT3 in mice, and HNOEL-iso or hOLF44 in humans (Zeng et al.,
2004; Sakuragi et al., 2006; Inomata et al., 2008; Ikeya et al.,
2005). Olfml3 belongs to a large family of olfactomedin
domain-containing proteins with distinct roles in neurogenesis,
neural crest formation, dorso-ventral patterning, cell cycle
regulation, and tumorigenesis (reviewed in Tomarev et al., 2009).
Together with Olfactomedin-like 1 (Olfml1), Olfml3 forms the
Olfactomedin-like subfamily VII (Ikeya et al., 2005; Tomarev et
al., 2009). Olfml3 is preferentially expressed in human placenta
and secreted in the extracellular compartment, suggesting a
possible Olfml3 function in the extracellular matrix-related
processes during placental development (Zheng et al., 2004). This
secreted protein contains a putative signal peptide and a
coiled-coil domain at the N-terminus and Olfactomedin-like domain
at the C-terminus.
[0055] The olfactomedin-like (ONT) subfamily is distinct from the
olfactomedin (OLF) subfamily consisting of well-characterized
members such as olfactomedin. The phylogenetic analysis revealed
the olfactomedin-like domains are highly conserved among this
subfamily of olfactomedin-like proteins with more than 90% homology
in the mouse, rat and human counterparts of ONT3 (Olfml3) and at
lesser extent (64%) in the chicken cONT1 (Olfml3). However, the
homology of the Olfactomedin-like domains to the Olfactomedin
domains of noelin, tiarin or other olfactomedin family members is
as low as 30% (Ikeya et al., 2005).
[0056] Bone morphogenetic protein 4 (BMP4) belongs to the BMP2/4
subgroup of the BMP family, sharing 92% homology with BMP2 (Celeste
et al., 1990). BMP2 stimulates angiogenesis in developing tumors
(Langenfeld and Langenfeld, 2004; Raida et al., 2005), through
recruitment of endothelial progenitor cells and triggering tumor
stromal cells to produce and secrete proangiogenic factors such as
VEGF and P1GF (Raida et al., 2006). During embryonic development,
BMP4 is critical for the induction of the mesoderm, endothelial
progenitor cell differentiation and vasculogenesis (e.g. blood
vessel formation) (Astorga and Carlsson, 2007; Winnier et al.,
1995). Additionally, BMP4 regulates ocular angiogenesis through
stimulation of VEGF secretion by retinal pigment epithelial cells
(Valdimarsdottir et al., 2002; Vogt et al., 2006). Endothelial
progenitor cells from human blood produce and secret both BMP2 and
BMP4, which then promote neovascularization (Smadja et al., 2008).
In mouse embryonic stem cells, proangiogenic effects of BMP4 are
mediated through the activation of the VEGF/VEGF receptor 2
(VEGFR2), angiopoietin-1/Tie2 and Smad signaling pathways (Suzuki
et al., 2008). The extracellular signal-regulated kinase 1/2
(ERK1/2) signaling pathway is shown to be a central regulator for
BMP4 signal transduction, leading to capillary sprouting in human
umbilical vein endothelial cells (HUVECs; Zhou et al., 2007).
Additionally, BMP4 also acts as a chemo-attractant for endothelial
cells migrating to the tumor and promotes tumor cell migration and
invasion (Rothhammer et al., 2007).
[0057] The activities of BMPs are tightly regulated through a
family of cysteine-knot proteins (Balemans and Van Hul, 2002;
Rosen, 2006; Walsh et al., 2010). The reactivation of previously
quiescent expression of BMP binding proteins can contribute to
tumor progression. It has been reported that BMP binding proteins
posses both anti- and pro-angiogenic activities during normal and
pathological conditions (reviewed in (Moreno-Miralles et al.,
2009).
[0058] In certain aspects, methods and compositions may be provided
to inhibit the binding between Olfml3 and BMP4, for example, by
molecules that specifically block the Olfml3 binding site for BMP4.
Such molecules may be an antibody, a synthetic peptide or a small
molecule. The antibody may be selected from the group consisting of
a chimeric antibody, an affinity matured antibody, a polyclonal
antibody, a monoclonal antibody or a humanized antibody, and a
human antibody. In a particular example, the antibody is a
monoclonal antibody or a humanized antibody. In another example,
the antibody is a polyclonal antibody.
[0059] In one embodiment, the antibody is a chimeric antibody, for
example, an antibody comprising antigen binding sequences from a
non-human donor grafted to a heterologous non-human, human or
humanized sequence (e.g., framework and/or constant domain
sequences). In one embodiment, the non-human donor is a mouse. In
one embodiment, an antigen binding sequence is synthetic, e.g.,
obtained by mutagenesis (e.g., phage display screening, etc.). In
one embodiment, a chimeric antibody of the invention has murine V
regions and human C region. In one embodiment, the murine light
chain V region is fused to a human kappa light chain. In one
embodiment, the murine heavy chain V region is fused to a human
IgG1 C region.
[0060] Examples of antibody fragments suitable for the present
invention include, without limitation: (i) the Fab fragment,
consisting of VL, VH, CL and CH1 domains; (ii) the "Fd" fragment
consisting of the VH and CH1 domains; (iii) the "Fv" fragment
consisting of the VL and VH domains of a single antibody; (iv) the
"dAb" fragment, which consists of a VH domain; (v) isolated CDR
regions; (vi) F(ab')2 fragments, a bivalent fragment comprising two
linked Fab fragments; (vii) single chain Fv molecules ("scFv"),
wherein a VH domain and a VL domain are linked by a peptide linker
which allows the two domains to associate to form a binding domain;
(viii) bi-specific single chain Fv dimers (see U.S. Pat. No.
5,091,513) and (ix) diabodies, multivalent or multispecific
fragments constructed by gene fusion (US Patent App. Pub.
20050214860). Fv, scFv or diabody molecules may be stabilized by
the incorporation of disulphide bridges linking the VH and VL
domains. Minibodies comprising a scFv joined to a CH3 domain may
also be made (Hu et al, 1996).
[0061] A polyclonal antibody for a particular domain of Olfml3
protein may be provided in certain aspects. Animals may be
inoculated with an antigen, such as a particular portion of Olfml3
protein, in order to produce antibodies specific for the particular
portion of Olfml3 protein. Such an antigen may be bound or
conjugated to another molecule to enhance the immune response. As
used herein, a conjugate is any peptide, polypeptide, protein or
non-proteinaceous substance bound to an antigen that is used to
elicit an immune response in an animal. Antibodies produced in an
animal in response to antigen inoculation comprise a variety of
non-identical molecules (polyclonal antibodies) made from a variety
of individual antibody producing B lymphocytes. A polyclonal
antibody is a mixed population of antibody species, each of which
may recognize a different epitope on the same antigen. Given the
correct conditions for polyclonal antibody production in an animal,
most of the antibodies in the animal's serum will recognize the
collective epitopes on the antigenic compound to which the animal
has been immunized. This specificity is further enhanced by
affinity purification to select only those antibodies that
recognize the antigen or epitope of interest.
[0062] A monoclonal antibody is a single species of antibody
wherein every antibody molecule recognizes the same epitope because
all antibody producing cells are derived from a single B-lymphocyte
cell line. Hybridoma technology involves the fusion of a single B
lymphocyte from a mouse previously immunized with an Olfml3 antigen
with an immortal myeloma cell (usually mouse myeloma). This
technology provides a method to propagate a single
antibody-producing cell for an indefinite number of generations,
such that unlimited quantities of structurally identical antibodies
having the same antigen or epitope specificity (monoclonal
antibodies) may be produced. However, in therapeutic applications a
goal of hybridoma technology is to reduce the immune reaction in
humans that may result from administration of monoclonal antibodies
generated by the non-human (e.g. mouse) hybridoma cell line.
[0063] Methods have been developed to replace light and heavy chain
constant domains of the monoclonal antibody with analogous domains
of human origin, leaving the variable regions of the foreign
antibody intact. Alternatively, "fully human" monoclonal antibodies
are produced in mice transgenic for human immunoglobulin genes.
Methods have also been developed to convert variable domains of
monoclonal antibodies to more human form by recombinantly
constructing antibody variable domains having both rodent and human
amino acid sequences. In "humanized" monoclonal antibodies, only
the hypervariable CDR is derived from mouse monoclonal antibodies,
and the framework regions are derived from human amino acid
sequences. It is thought that replacing amino acid sequences in the
antibody that are characteristic of rodents with amino acid
sequences found in the corresponding position of human antibodies
will reduce the likelihood of adverse immune reaction during
therapeutic use. A hybridoma or other cell producing an antibody
may also be subject to genetic mutation or other changes, which may
or may not alter the binding specificity of antibodies produced by
the hybridoma.
[0064] It is possible to create engineered antibodies, using
monoclonal and other antibodies and recombinant DNA technology to
produce other antibodies or chimeric molecules which retain the
antigen or epitope specificity of the original antibody, i.e., the
molecule has a binding domain. Such techniques may involve
introducing DNA encoding the immunoglobulin variable region or the
CDRs of an antibody to the genetic material for the framework
regions, constant regions, or constant regions plus framework
regions, of a different antibody. See, for instance, U.S. Pat. Nos.
5,091,513, and 6,881,557, which are incorporated herein by this
reference.
[0065] By known means as described herein, polyclonal or monoclonal
antibodies, antibody fragments and binding domains and CDRs
(including engineered forms of any of the foregoing), may be
created that are specific to Olfml3 protein, one or more of its
respective epitopes, or conjugates of any of the foregoing, whether
such antigens or epitopes are isolated from natural sources or are
synthetic derivatives or variants of the natural compounds.
[0066] Antibodies may be produced from any animal source, including
birds and mammals. Preferably, the antibodies are ovine, murine
(e.g., mouse and rat), rabbit, goat, guinea pig, camel, horse, or
chicken. In addition, newer technology permits the development of
and screening for human antibodies from human combinatorial
antibody libraries. For example, bacteriophage antibody expression
technology allows specific antibodies to be produced in the absence
of animal immunization, as described in U.S. Pat. No. 6,946,546,
which is incorporated herein by this reference. These techniques
are further described in: Marks (1992); Stemmer (1994); Gram et al.
(1992); Barbas et al. (1994); and Schier et al. (1996).
[0067] Methods for producing polyclonal antibodies in various
animal species, as well as for producing monoclonal antibodies of
various types, including humanized, chimeric, and fully human, are
well known in the art and highly predictable. Methods for producing
these antibodies are also well known and predictable. For example,
the following U.S. patents and patent applications provide enabling
descriptions of such methods and are herein incorporated by
reference: U.S. Patent Application Nos. 2004/0126828 and
2002/0172677; and U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;
3,996,345; 4,196,265; 4,275,149; 4,277,437; 4,366,241; 4,469,797;
4,472,509; 4,606,855; 4,703,003; 4,742,159; 4,767,720; 4,816,567;
4,867,973; 4,938,948; 4,946,778; 5,021,236; 5,164,296; 5,196,066;
5,223,409; 5,403,484; 5,420,253; 5,565,332; 5,571,698; 5,627,052;
5,656,434; 5,770,376; 5,789,208; 5,821,337; 5,844,091; 5,858,657;
5,861,155; 5,871,907; 5,969,108; 6,054,297; 6,165,464; 6,365,157;
6,406,867; 6,709,659; 6,709,873; 6,753,407; 6,814,965; 6,849,259;
6,861,572; 6,875,434; and 6,891,024. All patents, patent
application publications, and other publications cited herein and
therein are hereby incorporated by reference in the present
application.
[0068] It is fully expected that antibodies to particular domains
of Olfml3 will have the ability to neutralize or counteract the
effects of the Olfml3, possibly through the binding to BMP4,
regardless of the animal species, monoclonal cell line or other
source of the antibody. Certain animal species may be less
preferable for generating therapeutic antibodies because they may
be more likely to cause allergic response due to activation of the
complement system through the "Fc" portion of the antibody.
However, whole antibodies may be enzymatically digested into "Fc"
(complement binding) fragment, and into antibody fragments having
the binding domain or CDR. Removal of the Fc portion reduces the
likelihood that the antigen antibody fragment will elicit an
undesirable immunological response and, thus, antibodies without Fc
may be preferential for prophylactic or therapeutic treatments. As
described above, antibodies may also be constructed so as to be
chimeric, partially or fully human, so as to reduce or eliminate
the adverse immunological consequences resulting from administering
to an animal an antibody that has been produced in, or has
sequences from, other species.
II. Lipid Preparations
[0069] In certain aspects, the present invention provides methods
and compositions for associating an inhibitory antibody with a
lipid and/or liposome. The inhibitory antibody may be encapsulated
in the aqueous interior of a liposome, interspersed within the
lipid bilayer of a liposome, attached to a liposome via a linking
molecule that is associated with both the liposome and the
polynucleotide, entrapped in a liposome, complexed with a liposome,
dispersed in a solution containing a lipid, mixed with a lipid,
combined with a lipid, contained as a suspension in a lipid,
contained or complexed with a micelle, or otherwise associated with
a lipid. The liposome or liposome/antibody associated compositions
of the present invention are not limited to any particular
structure in solution. For example, they may be present in a
bilayer structure, as micelles, or with a "collapsed" structure.
They may also simply be interspersed in a solution, possibly
forming aggregates which are not uniform in either size or
shape.
[0070] Lipids are fatty substances which may be naturally occurring
or synthetic lipids. For example, lipids include the fatty droplets
that naturally occur in the cytoplasm as well as the class of
compounds which are well known to those of skill in the art which
contain long-chain aliphatic hydrocarbons and their derivatives,
such as fatty acids, alcohols, amines, amino alcohols, and
aldehydes. An example is the lipid dioleoylphosphatidylcholine
(DOPC).
[0071] "Liposome" is a generic term encompassing a variety of
unilamellar, multilamellar, and multivesicular lipid vehicles
formed by the generation of enclosed lipid bilayers or aggregates.
Liposomes may be characterized as having vesicular structures with
a phospholipid bilayer membrane and an inner aqueous medium.
Multilamellar liposomes have multiple lipid layers separated by
aqueous medium. They form spontaneously when phospholipids are
suspended in an excess of aqueous solution. The lipid components
undergo self-rearrangement before the formation of closed
structures and entrap water and dissolved solutes between the lipid
bilayers (Ghosh and Bachhawat, 1991). However, certain aspects of
the present invention also encompass compositions that have
different structures in solution than the normal vesicular
structure. For example, the lipids may assume a micellar structure
or merely exist as non-uniform aggregates of lipid molecules. Also
contemplated are lipofectamine-nucleic acid complexes.
[0072] Liposome-mediated polynucleotide delivery and expression of
foreign DNA in vitro has been very successful. Wong et al. (1980)
demonstrated the feasibility of liposome-mediated delivery and
expression of foreign DNA in cultured chick embryo, HeLa and
hepatoma cells. Nicolau et al. (1987) accomplished successful
liposome-mediated gene transfer in rats after intravenous
injection.
[0073] In certain embodiments of the invention, the lipid may be
associated with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the lipid may be complexed or employed in conjunction
with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al.,
1991). In yet further embodiments, the lipid may be complexed or
employed in conjunction with both HVJ and HMG-1. In that such
expression vectors have been successfully employed in transfer of a
polynucleotide in vitro and in vivo, then they are applicable for
the present invention.
[0074] Exemplary lipids include, but are not limited to,
dioleoylphosphatidylycholine ("DOPC"), egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DMPC"),
dipalmitoylphosphatidylcholine ("DPPC"),
distearoylphosphatidylcholine ("DSPC"), 1-myristoyl-2-palmitoyl
phosphatidylcholine ("MPPC"), 1-palmitoyl-2-myristoyl
phosphatidylcholine ("PMPC"), 1-palmitoyl-2-stearoyl
phosphatidylcholine ("PSPC"), 1-stearoyl-2-palmitoyl
phosphatidylcholine ("SPPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"),
distearoylphosphatidylglycerol ("DSPG"), distearoyl sphingomyelin
("DS SP"), distearoylphophatidylethanolamine ("DSPE"),
dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl
phosphatidylethanolamine ("DPPE"), dimyristoyl phosphatidylserine
("DMPS"), dipalmitoyl phosphatidylserine ("DPPS"), brain
phosphatidylserine ("BPS"), brain sphingomyelin ("BSP"),
dipalmitoyl sphingomyelin ("DPSP"), dimyristyl phosphatidylcholine
("DMPC"), 1,2-distearoyl-sn-glycero-3-phosphocholine ("DAPC"),
1,2-diarachidoyl-sn-glycero-3-phosphocholine ("DBPC"),
1,2-dieicosenoyl-sn-glycero-3-phosphocholine ("DEPC"),
dioleoylphosphatidylethanolamine ("DOPE"), palmitoyloeoyl
phosphatidylcholine ("POPC"), palmitoyloeoyl
phosphatidylethanolamine ("POPE"), lysophosphatidylcholine,
lysophosphatidylethanolamine, dilinoleoylphosphatidylcholine,
phosphatidylcholines, phosphatidylglycerols,
phosphatidylethanolamines, cholesterol.
[0075] Liposomes and lipid compositions in certain aspects of the
present invention can be made by different methods. The size of the
liposomes varies depending on the method of synthesis. A liposome
suspended in an aqueous solution is generally in the shape of a
spherical vesicle, and may have one or more concentric layers of
lipid bilayer molecules. Each layer consists of a parallel array of
molecules represented by the formula XY, wherein X is a hydrophilic
moiety and Y is a hydrophobic moiety. In aqueous suspension, the
concentric layers are arranged such that the hydrophilic moieties
tend to remain in contact with an aqueous phase and the hydrophobic
regions tend to self-associate. For example, when aqueous phases
are present both within and without the liposome, the lipid
molecules may form a bilayer, known as a lamella, of the
arrangement XY-YX. Aggregates of lipids may form when the
hydrophilic and hydrophobic parts of more than one lipid molecule
become associated with each other. The size and shape of these
aggregates will depend upon many different variables, such as the
nature of the solvent and the presence of other compounds in the
solution.
[0076] Lipids suitable for use according to the present invention
can be obtained from commercial sources. For example, dimyristyl
phosphatidylcholine ("DMPC") can be obtained from Sigma Chemical
Co., dicetyl phosphate ("DCP") can be obtained from K & K
Laboratories (Plainview, N.Y.); cholesterol ("Chol") can be
obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol
("DMPG") and other lipids may be obtained from Avanti Polar Lipids,
Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or
chloroform/methanol can be stored at about -20.degree. C.
Chloroform may be used as the only solvent since it is more readily
evaporated than methanol.
[0077] Liposomes within the scope of the present invention can be
prepared in accordance with known laboratory techniques. In certain
embodiments, liposomes are prepared by mixing liposomal lipids, in
a solvent in a container (e.g., a glass, pear-shaped flask). The
container will typically have a volume ten-times greater than the
volume of the expected suspension of liposomes. Using a rotary
evaporator, the solvent may be removed at approximately 40.degree.
C. under negative pressure. The solvent may be removed within about
5 minutes to 2 hours, depending on the desired volume of the
liposomes. The composition can be dried further in a desiccator
under vacuum. Dried lipids can be hydrated at approximately 25-50
mM phospholipid in sterile, pyrogen-free water by shaking until all
the lipid film is resuspended. The aqueous liposomes can be then
separated into aliquots, each placed in a vial, lyophilized and
sealed under vacuum.
[0078] Liposomes can also be prepared in accordance with other
known laboratory procedures: the method of Bangham et al. (1965),
the contents of which are incorporated herein by reference; the
method of Gregoriadis (1979), the contents of which are
incorporated herein by reference; the method of Deamer and Uster
(1983), the contents of which are incorporated by reference; and
the reverse-phase evaporation method as described by Szoka and
Papahadjopoulos (1978). The aforementioned methods differ in their
respective abilities to entrap aqueous material and their
respective aqueous space-to-lipid ratios.
[0079] Dried lipids or lyophilized liposomes may be dehydrated and
reconstituted in a solution of inhibitory peptide and diluted to an
appropriate concentration with a suitable solvent (e.g., DPBS). The
mixture may then be vigorously shaken in a vortex mixer.
Unencapsulated nucleic acid may be removed by centrifugation at
29,000 g and the liposomal pellets washed. The washed liposomes may
be resuspended at an appropriate total phospholipid concentration
(e.g., about 50-200 mM). The amount of nucleic acid or antibody
encapsulated can be determined in accordance with standard methods.
After determination of the amount of nucleic acid or antibody
encapsulated in the liposome preparation, the liposomes may be
diluted to appropriate concentrations and stored at 4.degree. C.
until use.
III. Treatment of Diseases
[0080] Certain aspects of the present invention can be used to
prevent or treat a disease or disorder associated with Olfml3
mediated angiogenesis. Functioning of Olfml3 may be reduced or
enhanced by any suitable drugs to stimulate or prevent
angiogenesis. Such exemplary substances can be an anti-Olfml3
antibody or a nucleic acid encoding such an antibody, particularly
an antibody recognizes and binds to specific domains of Olfml3,
soluble Olfml3 receptors or blocking small molecules.
[0081] "Treatment" and "treating" refer to administration or
application of a therapeutic agent to a subject or performance of a
procedure or modality on a subject for the purpose of obtaining a
therapeutic benefit of a disease or health-related condition. For
example, a treatment may include administration of a
pharmaceutically effective amount of a nucleic acid that inhibits
the expression of a gene that encodes a Olfml3 and a lipid for the
purposes of minimizing the growth or invasion of a tumor, such as a
colorectal cancer.
[0082] A "subject" refers to either a human or non-human, such as
primates, mammals, and vertebrates. In particular embodiments, the
subject is a human.
[0083] The term "therapeutic benefit" or "therapeutically
effective" as used throughout this application refers to anything
that promotes or enhances the well being of the subject with
respect to the medical treatment of this condition. This includes,
but is not limited to, a reduction in the frequency or severity of
the signs or symptoms of a disease. For example, treatment of
cancer may involve, for example, a reduction in the size of a
tumor, a reduction in the invasiveness of a tumor, reduction in the
growth rate of the cancer, or prevention of metastasis. Treatment
of cancer may also refer to prolonging survival of a subject with
cancer.
[0084] Certain aspects of the present invention may be used to
treat any condition or disease associated with increased or
decreased expression of Olfml3. For example, the disease may be an
angiogenesis-related condition or disease. Angiogenesis-related
condition or disease is a consequence of an imbalanced angiogenic
process resulting in an excessive amount of new blood vessels or
insufficient number of blood vessels.
[0085] In certain embodiments, the present methods can be used to
inhibit angiogenesis which is non-pathogenic; i.e., angiogenesis
which results from normal processes in the subject. Examples of
non-pathogenic angiogenesis include endometrial neovascularization,
and processes involved in the production of fatty tissues or
cholesterol. Thus, the invention provides a method for inhibiting
non-pathogenic angiogenesis, e.g., for controlling weight or
promoting fat loss, for reducing cholesterol levels, or as an
abortifacient.
[0086] The present methods can also inhibit angiogenesis which is
associated with an angiogenic disease; i.e., a disease in which
pathogenicity is associated with inappropriate or uncontrolled
angiogenesis. For example, most cancerous solid tumors generate an
adequate blood supply for themselves by inducing angiogenesis in
and around the tumor site. This tumor-induced angiogenesis is often
required for tumor growth, and also allows metastatic cells to
enter the bloodstream.
[0087] Other angiogenic diseases include diabetic retinopathy,
age-related macular degeneration (ARMD), psoriasis, rheumatoid
arthritis and other inflammatory diseases. These diseases are
characterized by the destruction of normal tissue by newly formed
blood vessels in the area of neovascularization. For example, in
ARMD, the choroid is invaded and destroyed by capillaries. The
angiogenesis-driven destruction of the choroid in ARMD eventually
leads to partial or full blindness. The angiogenesis-related
conditions also include ocular neovascularization, arterio-venous
malformations, coronary restenosis, peripheral vessel restenosis,
glomerulonephritis, rheumatoid arthritis, ischemic cardiovascular
pathologies, or chronic inflammatory diseases.
[0088] Exemplary eye angiogenic diseases to be treated or prevented
also include choroidal neovascularization (CNV) due to any cause
including but not limited to age-related macular degeneration,
ocular histoplasmosis, pathologic myopia, and angioid streaks. It
also applies to retinal neovascularization of any cause including
but not limited to proliferative diabetic retinopathy, retinal vein
occlusions, and retinopathy of prematurity. It also applies to iris
neovascularization and corneal neovascularization of any
causes.
[0089] The neovascularization may also be neovascularization
associated with an ocular wound. For example, the wound may the
result of a traumatic injury to the globe, such as a corneal
laceration. Alternatively, the wound may be the result of
ophthalmic surgery. In some embodiments, the methods of the present
invention may be applied to prevent or reduce the risk of
proliferative vitreoretinopathy following vitreoretinal surgery,
prevent corneal haze following corneal surgery (such as corneal
transplantation and laser surgery), prevent closure of a
trabeculectomy, prevent or substantially slow the recurrence of
pterygii, and so forth.
[0090] The neovascularization may be located either on or within
the eye of the subject. For example, the neovascularization may be
corneal neovascularization (either located on the corneal
epithelium or on the endothelial surface of the cornea), iris
neovascularization, neovascularization within the vitreous cavity,
retinal neovasculization, or choroidal neovascularization. The
neovascularization may also be neovascularization associated with
conjunctival disease.
[0091] The cancer may specifically be of the following histological
type, though it is not limited to these: neoplasm, malignant;
carcinoma; carcinoma, undifferentiated; giant and spindle cell
carcinoma; small cell carcinoma; papillary carcinoma; squamous cell
carcinoma; lymphoepithelial carcinoma; basal cell carcinoma;
pilomatrix carcinoma; transitional cell carcinoma; papillary
transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant;
cholangiocarcinoma; hepatocellular carcinoma; combined
hepatocellular carcinoma and cholangiocarcinoma; trabecular
adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in
adenomatous polyp; adenocarcinoma, familial polyposis coli; solid
carcinoma; carcinoid tumor, malignant; branchiolo-alveolar
adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;
acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma;
clear cell adenocarcinoma; granular cell carcinoma; follicular
adenocarcinoma; papillary and follicular adenocarcinoma;
nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma;
endometroid carcinoma; skin appendage carcinoma; apocrine
adenocarcinoma; sebaceous adenocarcinoma; ceruminous
adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma;
papillary cystadenocarcinoma; papillary serous cystadenocarcinoma;
mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring
cell carcinoma; infiltrating duct carcinoma; medullary carcinoma;
lobular carcinoma; inflammatory carcinoma; paget's disease,
mammary; acinar cell carcinoma; adenosquamous carcinoma;
adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian
stromal tumor, malignant; thecoma, malignant; granulosa cell tumor,
malignant; androblastoma, malignant; sertoli cell carcinoma; leydig
cell tumor, malignant; lipid cell tumor, malignant; paraganglioma,
malignant; extra-mammary paraganglioma, malignant;
pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic
melanoma; superficial spreading melanoma; malignant melanoma in
giant pigmented nevus; epithelioid cell melanoma; blue nevus,
malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant;
myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma;
embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal
sarcoma; mixed tumor, malignant; mullerian mixed tumor;
nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma,
malignant; brenner tumor, malignant; phyllodes tumor, malignant;
synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal
carcinoma; teratoma, malignant; struma ovarii, malignant;
choriocarcinoma; mesonephroma, malignant; hemangiosarcoma;
hemangioendothelioma, malignant; kaposi's sarcoma;
hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma;
juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma,
malignant; mesenchymal chondrosarcoma; giant cell tumor of bone;
ewing's sarcoma; odontogenic tumor, malignant; ameloblastic
odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma;
pinealoma, malignant; chordoma; glioma, malignant; ependymoma;
astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma;
astroblastoma; glioblastoma; oligodendroglioma;
oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;
ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory
neurogenic tumor; meningioma, malignant; neurofibrosarcoma;
neurilemmoma, malignant; granular cell tumor, malignant; malignant
lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant
lymphoma, small lymphocytic; malignant lymphoma, large cell,
diffuse; malignant lymphoma, follicular; mycosis fungoides; other
specified non-hodgkin's lymphomas; malignant histiocytosis;
multiple myeloma; mast cell sarcoma; immunoproliferative small
intestinal disease; leukemia; lymphoid leukemia; plasma cell
leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid
leukemia; basophilic leukemia; eosinophilic leukemia; monocytic
leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid
sarcoma; and hairy cell leukemia.
[0092] Nonetheless, it is also recognized that certain aspects of
the present invention may also be used to treat a non-cancerous
disease (e.g., a fungal infection, a bacterial infection, a viral
infection, and/or a neurodegenerative disease).
[0093] In certain embodiments, Olfml3 protein or peptide is
contemplated to treat angiogenesis-related conditions in a subject
in need of angiogenesis. Insufficient angiogenesis is related to a
large number of diseases and conditions, such as cardiovascular
diseases, transplantation, aneurisms and delayed wound healing.
Therapeutic angiogenesis is aimed at stimulating new blood vessel
growth. The concept of such a therapy is based on the premise that
the inherent potential of vascularization in a vascular tissue can
be utilized to promote the development of new blood vessels under
the influence of the appropriate angiogenic molecules.
[0094] In certain aspect, the Olfml3 antibodies may be used to
reduce pericytes, particularly in vessels associated with tumor or
tumor vessels. Pericytes are critical regulators of vascular
morphogenesis and function. Shortly after endothelial tubes form,
they become associated with mural cells. These cells provide
structural support to the vessels and are important regulators of
blood flow. Pericytes constitute a heterogeneous population of
cells. Several functions of pericytes during angiogenesis have been
proposed, including sensing the presence of angiogenic stimuli,
depositing or degrading extracellular matrix and controlling
endothelial cell proliferation and differentiation in a paracrine
fashion. In certain diseases such as diabetic retinopathy,
pericytes may be the primary affected vascular cells, which lead to
secondary effects on the endothelial cells.
IV. Pharmaceutical Preparations
[0095] Where clinical application of a composition containing an
inhibitory antibody is undertaken, it will generally be beneficial
to prepare a pharmaceutical composition appropriate for the
intended application. This will typically entail preparing a
pharmaceutical composition that is essentially free of pyrogens, as
well as any other impurities that could be harmful to humans or
animals. One may also employ appropriate buffers to render the
complex stable and allow for uptake by target cells.
[0096] The phrases "pharmaceutical or pharmacologically acceptable"
refer to molecular entities and compositions that do not produce an
adverse, allergic or other untoward reaction when administered to
an animal, such as a human, as appropriate. The preparation of a
pharmaceutical composition comprising a inhibitory antibody or
additional active ingredient will be known to those of skill in the
art in light of the present disclosure, as exemplified by Remington
(2005), incorporated herein by reference. Moreover, for animal
(e.g., human) administration, it will be understood that
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biological
Standards.
[0097] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art. A pharmaceutically acceptable carrier is particularly
formulated for administration to a human, although in certain
embodiments it may be desirable to use a pharmaceutically
acceptable carrier that is formulated for administration to a
non-human animal but which would not be acceptable (e.g., due to
governmental regulations) for administration to a human. Except
insofar as any conventional carrier is incompatible with the active
ingredient, its use in the therapeutic or pharmaceutical
compositions is contemplated.
[0098] The actual dosage amount of a composition of the present
invention administered to a patient or subject can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0099] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
to about 1000 mg/kg/body weight (this such range includes
intervening doses) or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range
from the numbers listed herein, a range of about 5 .mu.g/kg/body
weight to about 100 mg/kg/body weight, about 5 microgram/kg/body
weight to about 500 milligram/kg/body weight, etc., can be
administered.
[0100] A gene expression inhibitor may be administered in a dose of
1-100 (this such range includes intervening doses) or more .mu.g or
any number in between the foregoing of nucleic acid per dose. Each
dose may be in a volume of 1, 10, 50, 100, 200, 500, 1000 or more
.mu.l or ml or any number in between the foregoing.
[0101] Solutions of therapeutic compositions can be prepared in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0102] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the
like.
[0103] Examples of non-aqueous solvents are propylene glycol,
polyethylene glycol, vegetable oil and injectable organic esters
such as ethyloleate. Aqueous carriers include water,
alcoholic/aqueous solutions, saline solutions, parenteral vehicles
such as sodium chloride, Ringer's dextrose, etc. Intravenous
vehicles include fluid and nutrient replenishers. Preservatives
include antimicrobial agents, anti-oxidants, chelating agents and
inert gases. The pH and exact concentration of the various
components the pharmaceutical composition are adjusted according to
well known parameters.
[0104] In particular embodiments, the compositions of the present
invention are suitable for application to mammalian eyes. For
example, the formulation may be a solution, a suspension, or a gel.
In some embodiments, the composition is administered via a
biodegradable implant, such as an intravitreal implant or an ocular
insert, such as an ocular insert designed for placement against a
conjunctival surface. In some embodiments, the therapeutic agent
coats a medical device or implantable device.
[0105] In preferred aspects the formulation of the invention will
be applied to the eye in aqueous solution in the form of drops.
These drops may be delivered from a single dose ampoule which may
preferably be sterile and thus rendering bacteriostatic components
of the formulation unnecessary. Alternatively, the drops may be
delivered from a multi-dose bottle which may preferably comprise a
device which extracts preservative from the formulation as it is
delivered, such devices being known in the art.
[0106] In other aspects, components of the invention may be
delivered to the eye as a concentrated gel or similar vehicle which
forms dissolvable inserts that are placed beneath the eyelids.
[0107] Additional formulations are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders.
[0108] The therapeutic compositions of the present invention may
include classic pharmaceutical preparations. Administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Topical administration may be particularly advantageous
for the treatment of skin cancers, to prevent chemotherapy-induced
alopecia or other dermal hyperproliferative disorder.
Alternatively, administration may be by orthotopic, intradermal,
subcutaneous, intramuscular, intraperitoneal or intravenous
injection. Such compositions would normally be administered as
pharmaceutically acceptable compositions that include
physiologically acceptable carriers, buffers or other excipients.
For treatment of conditions of the lungs, or respiratory tract,
aerosol delivery can be used. Volume of the aerosol is between
about 0.01 ml and 0.5 ml.
[0109] An effective amount of the therapeutic composition is
determined based on the intended goal. For example, one skilled in
the art can readily determine an effective amount of the antibody
of the invention to be administered to a given subject, by taking
into account factors such as the size and weight of the subject;
the extent of the neovascularization or disease penetration; the
age, health and sex of the subject; the route of administration;
and whether the administration is regional or sysemic. The term
"unit dose" or "dosage" refers to physically discrete units
suitable for use in a subject, each unit containing a
predetermined-quantity of the therapeutic composition calculated to
produce the desired responses discussed above in association with
its administration, i.e., the appropriate route and treatment
regimen. The quantity to be administered, both according to number
of treatments and unit dose, depends on the protection or effect
desired.
[0110] Precise amounts of the therapeutic composition also depend
on the judgment of the practitioner and are peculiar to each
individual. Factors affecting the dose include the physical and
clinical state of the patient, the route of administration, the
intended goal of treatment (e.g., alleviation of symptoms versus
cure) and the potency, stability and toxicity of the particular
therapeutic substance.
V. Combination Treatments
[0111] In certain embodiments, the compositions and methods of the
present invention involve an inhibitor of expression of Olfml3, or
construct capable of expressing an inhibitor of Olfml3 expression,
or an antibody or an antibody fragment against Olfml3 to inhibit
its activity in angiogenesis, in combination with a second or
additional therapy. Such therapy can be applied in the treatment of
any disease that is associated with increased expression or
activity of Olfml3. For example, the disease may be an
angiogenesis-related disease.
[0112] The methods and compositions including combination therapies
enhance the therapeutic or protective effect, and/or increase the
therapeutic effect of another anti-angiogenesis, anti-cancer or
anti-hyperproliferative therapy. Therapeutic and prophylactic
methods and compositions can be provided in a combined amount
effective to achieve the desired effect, such as the killing of a
cancer cell and/or the inhibition of cellular hyperproliferation.
This process may involve contacting the cells with both an
inhibitor of gene expression and a second therapy. A tissue, tumor,
or cell can be contacted with one or more compositions or
pharmacological formulation(s) including one or more of the agents
(i.e., inhibitor of gene expression or an anti-cancer agent), or by
contacting the tissue, tumor, and/or cell with two or more distinct
compositions or formulations, wherein one composition provides 1)
an inhibitor of gene expression; 2) an anti-cancer agent, or 3)
both an inhibitor of gene expression and an anti-cancer agent.
Also, it is contemplated that such a combination therapy can be
used in conjunction with a chemotherapy, radiotherapy, surgical
therapy, or immunotherapy.
[0113] An inhibitor of gene expression and/or activity may be
administered before, during, after or in various combinations
relative to an anti-cancer treatment. The administrations may be in
intervals ranging from concurrently to minutes to days to weeks. In
embodiments where the inhibitor of gene expression is provided to a
patient separately from an anti-cancer agent, one would generally
ensure that a significant period of time did not expire between the
time of each delivery, such that the two compounds would still be
able to exert an advantageously combined effect on the patient. In
such instances, it is contemplated that one may provide a patient
with the inhibitor of gene expression therapy and the anti-cancer
therapy within about 12 to 24 or 72 h of each other and, more
particularly, within about 6-12 h of each other. In some situations
it may be desirable to extend the time period for treatment
significantly where several days (2, 3, 4, 5, 6 or 7) to several
weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between respective
administrations.
[0114] In certain embodiments, a course of treatment will last 1-90
days, or more (this such range includes intervening days). It is
contemplated that one agent may be given on any day of day 1 to day
90 (this such range includes intervening days) or any combination
thereof, and another agent is given on any day of day 1 to day 90
(this such range includes intervening days) or any combination
thereof. Within a single day (24-hour period), the patient may be
given one or multiple administrations of the agent(s). Moreover,
after a course of treatment, it is contemplated that there is a
period of time at which no anti-cancer treatment is administered.
This time period may last 1-7 days, and/or 1-5 weeks, and/or 1-12
months or more (this such range includes intervening days),
depending on the condition of the patient, such as their prognosis,
strength, health, etc.
[0115] Various combinations may be employed. For the example below
an inhibitor of gene expression therapy is "A" and an anti-cancer
therapy is "B":
TABLE-US-00001 A/B/AB/A/BB/B/AA/A/BA/B/BB/A/AA/B/B/BB/A/B/B B/B/B/A
B/B/A/B A/A/B/B A/B/A/BA/B/B/AB/B/A/A B/A/B/A B/A/A/B
A/A/A/BB/A/A/AA/B/A/AA/A/B/A
[0116] Administration of any compound or therapy of the present
invention to a patient will follow general protocols for the
administration of such compounds, taking into account the toxicity,
if any, of the agents. Therefore, in some embodiments there is a
step of monitoring toxicity that is attributable to combination
therapy. It is expected that the treatment cycles would be repeated
as necessary. It also is contemplated that various standard
therapies, as well as surgical intervention, may be applied in
combination with the described therapy.
[0117] In specific aspects, it is contemplated that a standard
therapy will include antiangiogenic therapy, chemotherapy,
radiotherapy, immunotherapy, surgical therapy or gene therapy and
may be employed in combination with the inhibitor of gene
expression therapy, anticancer therapy, or both the inhibitor of
gene expression therapy and the anti-cancer therapy, as described
herein. [0118] A. Antiangiogenic Therapy
[0119] The skilled artisan will understand that additional
antiangiogenic therapies may be used in combination or in
conjunction with methods of the invention. For example additional
antiangiogenic therapies may antagonize the VEGF and/or FGF
signaling pathway. Thus, in some cases and additional therapy may
comprise administration an antibody that binds to VEGF, a VEGF
receptor, FGF or an FGF receptor. In certain specific aspects,
methods and compositions of the invention may be used in
conjunction with AVASTIN.RTM. (bevacizumab), LUCENTIS.RTM.
(ranibizumab), MACUGEN.RTM. (pegaptanib sodium) or an
anti-inflammatory drug. Thus, in certain specific cases there is
provided a therapeutic composition comprising an anti-Olfml3
composition and bevacizumab or pegaptanib sodium in a
pharmaceutically acceptable carrier.
[0120] In still further aspects a gene that regulates angiogenesis
may be delivered in conjunction with the methods of the invention.
For example, in some aspects, a gene that regulates angiogenesis
may be a tissue inhibitor of metalloproteinase, endostatin,
angiostatin, endostatin XVIII, endostatin XV, kringle 1-5, PEX, the
C-terminal hemopexin domain of matrix metalloproteinase-2, the
kringle 5 domain of human plasminogen, a fusion protein of
endostatin and angiostatin, a fusion protein of endostatin and the
kringle 5 domain of human plasminogen, the monokine-induced by
interferon-gamma (Mig), the interferon-alpha inducible protein 10
(IP10), a fusion protein of Mig and IP10, soluble FLT-1 (fins-like
tyrosine kinase 1 receptor), and kinase insert domain receptor
(KDR) gene. In certain specific aspects, such an angiogenic
regulator gene may be delivered in a viral vector such as the
lentiviral vectors described in U.S. Pat. No. 7,122,181,
incorporated herein by reference. [0121] B. Chemotherapy
[0122] A wide variety of chemotherapeutic agents may be used in
accordance with the present invention. The term "chemotherapy"
refers to the use of drugs to treat cancer. A "chemotherapeutic
agent" is used to connote a compound or composition that is
administered in the treatment of cancer. These agents or drugs are
categorized by their mode of activity within a cell, for example,
whether and at what stage they affect the cell cycle.
Alternatively, an agent may be characterized based on its ability
to directly cross-link DNA, to intercalate into DNA, or to induce
chromosomal and mitotic aberrations by affecting nucleic acid
synthesis. Most chemotherapeutic agents fall into the following
categories: alkylating agents, antimetabolites, antitumor
antibiotics, mitotic inhibitors, and nitrosoureas.
[0123] Examples of chemotherapeutic agents include alkylating
agents such as thiotepa and 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 omegaI 1; dynemicin,
including dynemicin A; bisphosphonates, such as clodronate; an
esperamicin; as well as neocarzinostatin chromophore and related
chromoprotein enediyne antibiotic chromophores, aclacinomysins,
actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin,
carabicin, carminomycin, carzinophilin, chromomycinis,
dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, doxorubicin (including
morpholino-doxorubicin, cyanomorpholino-doxorubicin,
2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin
C, mycophenolic acid, nogalarnycin, 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, 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 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;
PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran;
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; taxoids, e.g.,
paclitaxel and docetaxel gemcitabine; 6-thioguanine;
mercaptopurine; platinum coordination complexes such as cisplatin,
oxaliplatin and carboplatin; vinblastine; platinum; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine;
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase
inhibitor RFS 2000; difluoromefihylornithine (DMFO); retinoids such
as retinoic acid; capecitabine; carboplatin,
procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein
transferase inhibitors, transplatinum, and pharmaceutically
acceptable salts, acids or derivatives of any of the above.
[0124] 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, raloxifene, droloxifene,
4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone,
and toremifene; aromatase inhibitors that inhibit the enzyme
aromatase, which regulates estrogen production in the adrenal
glands, such as, for example, 4(5)-imidazoles, aminoglutethimide,
megestrol acetate, exemestane, formestanie, fadrozole, vorozole,
letrozole, and 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 and a HER2
expression inhibitor; vaccines such as gene therapy vaccines and
pharmaceutically acceptable salts, acids or derivatives of any of
the above. [0125] C. Radiotherapy
[0126] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves, proton beam irradiation (U.S. Pat. Nos.
5,760,395 and 4,870,287) and UV-irradiation. It is most likely that
all of these factors affect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0127] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing, for example, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing. [0128] D.
Immunotherapy
[0129] In the context of cancer treatment, immunotherapeutics,
generally, rely on the use of immune effector cells and molecules
to target and destroy cancer cells. Trastuzumab (Herceptin.TM.) is
such an example. The immune effector may be, for example, an
antibody specific for some marker on the surface of a tumor cell.
The antibody alone may serve as an effector of therapy or it may
recruit other cells to actually affect cell killing. The antibody
also may be conjugated to a drug or toxin (chemotherapeutic,
radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.)
and serve merely as a targeting agent. Alternatively, the effector
may be a lymphocyte carrying a surface molecule that interacts,
either directly or indirectly, with a tumor cell target. Various
effector cells include cytotoxic T cells and NK cells. The
combination of therapeutic modalities, i.e., direct cytotoxic
activity and inhibition or reduction of ErbB2 would provide
therapeutic benefit in the treatment of ErbB2 overexpressing
cancers.
[0130] Another immunotherapy could also be used as part of a
combined therapy with gene silencing therapy discussed above. In
one aspect of immunotherapy, the tumor cell must bear some marker
that is amenable to targeting, i.e., is not present on the majority
of other cells. Many tumor markers exist and any of these may be
suitable for targeting in the context of the present invention.
Common tumor markers include carcinoembryonic antigen, prostate
specific antigen, urinary tumor associated antigen, fetal antigen,
tyrosinase (p97), gp68, TAG-72, HMFG, Sialyl Lewis Antigen, MucA,
MucB, PLAP, estrogen receptor, laminin receptor, erb B and p155. An
alternative aspect of immunotherapy is to combine anticancer
effects with immune stimulatory effects. Immune stimulating
molecules also exist including: cytokines such as IL-2, IL-4,
IL-12, GM-CSF, gamma-IFN, chemokines such as MIP-1, MCP-1, IL-8 and
growth factors such as FLT3 ligand. Combining immune stimulating
molecules, either as proteins or using gene delivery in combination
with a tumor suppressor has been shown to enhance anti-tumor
effects (Ju et al., 2000). Moreover, antibodies against any of
these compounds can be used to target the anti-cancer agents
discussed herein.
[0131] Examples of immunotherapies currently under investigation or
in use are immune adjuvants e.g., Mycobacterium bovis, Plasmodium
falciparum, dinitrochlorobenzene and aromatic compounds (U.S. Pat.
Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998;
Christodoulides et al., 1998), cytokine therapy, e.g., interferons
.alpha., .beta. and .gamma.; IL-1, GM-CSF and TNF (Bukowski et al.,
1998; Davidson et al., 1998; Hellstrand et al., 1998) gene therapy,
e.g., TNF, IL-1, IL-2, p53 (Qin et al., 1998; Austin-Ward and
Villaseca, 1998; U.S. Pat. Nos. 5,830,880 and 5,846,945) and
monoclonal antibodies, e.g., anti-ganglioside GM2, anti-HER-2,
anti-p185 (Pietras et al., 1998; Hanibuchi et al., 1998; U.S. Pat.
No. 5,824,311). It is contemplated that one or more anti-cancer
therapies may be employed with the gene silencing therapies
described herein.
[0132] In active immunotherapy, an antigenic peptide, polypeptide
or protein, or an autologous or allogenic tumor cell composition or
"vaccine" is administered, generally with a distinct bacterial
adjuvant (Ravindranath and Morton, 1991; Morton et al., 1992;
Mitchell et al., 1990; Mitchell et al., 1993).
[0133] In adoptive immunotherapy, the patient's circulating
lymphocytes, or tumor infiltrated lymphocytes, are isolated in
vitro, activated by lymphokines such as IL-2 or transduced with
genes for tumor necrosis, and readministered (Rosenberg et al.,
1988; 1989). [0134] E. Surgery
[0135] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative, and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0136] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
microscopically controlled surgery (Mohs' surgery). It is further
contemplated that certain aspects of the present invention may be
used in conjunction with removal of superficial cancers,
precancers, or incidental amounts of normal tissue.
[0137] Upon excision of part or all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well. [0138] F. Other Agents
[0139] It is contemplated that other agents may be used in
combination with certain aspects of the present invention to
improve the therapeutic efficacy of treatment. These additional
agents include immunomodulatory agents, agents that affect the
upregulation of cell surface receptors and GAP junctions,
cytostatic and differentiation agents, inhibitors of cell adhesion,
agents that increase the sensitivity of the hyperproliferative
cells to apoptotic inducers, or other biological agents.
Immunomodulatory agents include tumor necrosis factor; interferon
alpha, beta, and gamma; IL-2 and other cytokines; F42K and other
cytokine analogs; or MIP-1, MIP-lbeta, MCP-1, RANTES, and other
chemokines It is further contemplated that the upregulation of cell
surface receptors or their ligands such as Fas/Fas ligand, DR4 or
DR5/TRAIL (Apo-2 ligand) would potentiate the apoptotic inducing
abilities of the present invention by establishment of an autocrine
or paracrine effect on hyperproliferative cells. Increase of
intercellular signaling by elevating the number of GAP junctions
would increase the anti-hyperproliferative effects on the
neighboring hyperproliferative cell population. In other
embodiments, cytostatic or differentiation agents can be used in
combination with certain aspects of the present invention to
improve the anti-hyperproliferative efficacy of the treatments.
Inhibitors of cell adhesion are contemplated to improve the
efficacy of the present invention. Examples of cell adhesion
inhibitors are focal adhesion kinase (FAKs) inhibitors and
Lovastatin. It is further contemplated that other agents that
increase the sensitivity of a hyperproliferative cell to apoptosis,
such as the antibody c225, could be used in combination with
certain aspects of the present invention to improve the treatment
efficacy.
[0140] There have been many advances in the therapy of cancer
following the introduction of cytotoxic chemotherapeutic drugs.
However, one of the consequences of chemotherapy is the
development/acquisition of drug-resistant phenotypes and the
development of multiple drug resistance. The development of drug
resistance remains a major obstacle in the treatment of such tumors
and therefore, there is an obvious need for alternative approaches
such as gene therapy.
[0141] Another form of therapy for use in conjunction with
chemotherapy, radiation therapy or biological therapy includes
hyperthermia, which is a procedure in which a patient's tissue is
exposed to high temperatures (up to 106.degree. F.). External or
internal heating devices may be involved in the application of
local, regional, or whole-body hyperthermia. Local hyperthermia
involves the application of heat to a small area, such as a tumor.
Heat may be generated externally with high-frequency waves
targeting a tumor from a device outside the body. Internal heat may
involve a sterile probe, including thin, heated wires or hollow
tubes filled with warm water, implanted microwave antennae, or
radiofrequency electrodes.
[0142] A patient's organ or a limb is heated for regional therapy,
which is accomplished using devices that produce high energy, such
as magnets. Alternatively, some of the patient's blood may be
removed and heated before being perfused into an area that will be
internally heated. Whole-body heating may also be implemented in
cases where cancer has spread throughout the body. Warm-water
blankets, hot wax, inductive coils, and thermal chambers may be
used for this purpose.
[0143] Hormonal therapy may also be used in conjunction with
certain aspects of the present invention or in combination with any
other cancer therapy previously described. The use of hormones may
be employed in the treatment of certain cancers such as breast,
prostate, ovarian, or cervical cancer to lower the level or block
the effects of certain hormones such as testosterone or estrogen.
This treatment is often used in combination with at least one other
cancer therapy as a treatment option or to reduce the risk of
metastases.
VI. Kits and Diagnostics
[0144] In various aspects of the invention, a kit is envisioned
containing therapeutic agents and/or other therapeutic and delivery
agents. In some embodiments, the present invention contemplates a
kit for preparing and/or administering a therapy of the invention.
The kit may comprise one or more sealed vials containing any of the
pharmaceutical compositions of the present invention. In some
embodiments, the lipid is in one vial, and the Olmlf3 inhitory
molecule component is in a separate vial. The kit may include, for
example, at least one inhibitor of Olfml3 function, such as an
Olfml3 domain-specific antibody, one or more lipid component, as
well as reagents to prepare, formulate, and/or administer the
components of the invention or perform one or more steps of the
inventive methods. In some embodiments, the kit may also comprise a
suitable container means, which is a container that will not react
with components of the kit, such as an eppendorf tube, an assay
plate, a syringe, a bottle, or a tube. The container may be made
from sterilizable materials such as plastic or glass.
[0145] The kit may further include an instruction sheet that
outlines the procedural steps of the methods set forth herein, and
will follow substantially the same procedures as described herein
or are known to those of ordinary skill. The instruction
information may be in a computer readable media containing
machine-readable instructions that, when executed using a computer,
cause the display of a real or virtual procedure of delivering a
pharmaceutically effective amount of a therapeutic agent.
EXAMPLES
[0146] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1--Differential Olfml3 Gene Expression in Angiogenic Versus
Resting Endothelial Cells
[0147] To mimic molecular and functional properties of endothelial
cells during tumor angiogenesis, the inventors previously isolated
two subpopulations of an endothelioma cell line with molecular
characteristics of angiogenic (t.End.1V.sup.high) and resting
(t.End.1V.sup.low) states (Aurrand-Lions et al., 2004). The
t.End.1V.sup.high cells express high levels of the integrin
.alpha.V.beta.3 and do not endocytose acetylated low-density
lipoprotein (Ac-LDL), while t.End.1V.sup.low cells express low
levels of .alpha.V.beta.3 integrin and efficiently take up Ac-LDL.
In contrast, t.End.1V.sup.high cells show increased migration and
form capillary-like structures in three dimensional (3D) fibrin
gels (Aurrand-Lions et al., 2004). Therefore, t.End.1V.sup.high and
t.End.1V.sup.low cells appeared to be suitable cellular
representatives of angiogenic and resting endothelial cells. These
cells were exploited as a starting point for the transcriptomic
profiling using Affymetrix mouse 430 Gene Chip arrays. Data
analysis resulted in more than 3500 differentially expressed genes
with 1700 genes 2-fold (P.ltoreq.0.05) over-expressed in
t.End.1V.sup.high cells (Miljkovic-Licina et al., 2009). To focus
on novel genes that are relevant for angiogenesis, the microarray
dataset was compared with a published dataset of genes upregulated
after angiogenic activation of HUVEC by vMIP-II (viral macrophage
inflammatory protein II) a chemokine with described proangiogenic
activity (Cherqui et al., 2007). This comparison identified 38
genes, of which several were already implicated in the regulation
of angiogenesis, validating the biological relevance of the
experimental approach. The transcriptomic profiling further yielded
several new candidate genes without known proangiogenic activity
(Miljkovic-Licina et al., 2009). One of the most promising
candidate genes in this category was Olfml3 (NM.sub.--133859),
since its expression was remarkably upregulated in
t.End.1V.sup.high cells (30 fold). Upregulation of Olfml3 gene
expression was validated through quantitative real-time PCR
analysis using total RNA isolated from angiogenic and resting cells
(FIG. 1A) and Western blotting (FIG. 1B). Subsequent analysis
focused on the characterization of Olfml3, as a novel
differentially expressed angiogenic cell-derived factor.
Example 2--Expression of Olfml3 in Angiogenic Tissues
[0148] In mouse embryos, high levels of Olfml3 transcripts are
detected during early embryogenesis in the presumptive vasculogenic
regions (Ikeya et al., 2005; Drake and Fleming, 2000). In humans,
the highest levels of Olfml3 transcripts are found in placenta but
also in a few highly vascularized organs, such as heart and liver
albeit at lower levels (Zheng et al., 2004). To determine
localization of Olfml3 expression in these organs, the inventors
analyzed the Olfml3 expression pattern using in situ mRNA
hybridization or immunostaining Interestingly, Olfml3 expression
was strongly upregulated in capillaries and large vessels of human
placenta, which is a tissue characterized by continuous
angiogenesis (Khankin et al., 2010) (FIG. 2A). This unique
expression pattern suggested that high levels of Olfml3 expression
in placental vasculature might be associated with vascular growth
and remodeling. Therefore, the inventors induced de novo formation
of blood vessels in wild-type mice by subcutaneous injection of
bFGF-loaded matrigel. After eight days, the vascularized plugs were
harvested and in situ mRNA hybridization or immunostaining were
performed on frozen samples. Double labeling using antisense RNA
probes for Olfml3 and the endothelial marker PECAM-1 revealed
robust endothelial specific expression of Olfml3 in these
angiogenic blood vessels (FIG. 2B, left panel). Subsequently, the
inventors were able to detect Olfml3 expression in angiogenic tumor
vessels using subcutaneous Lewis Lung Carcinoma (LLC1) grafts in wt
mice (FIG. 2C, left panel). These results were expanded by Olfml3
protein detection using double immunostaining for Olfml3 and
platelet/endothelial cell adhesion molecule-1 (PECAM-1) in the
bFGF-loaded matrigel plugs or in the LLC1 tumor tissue (FIGS.
2B-2C, right panels). Of interest, Olfml3 protein was enriched
along the vessel wall of an angiogenic tumor vessel subset (FIG.
2C, right panel). To further characterize perivascular Olfml3
expression, the LLC 1 tumors were triple stained using specific
antibodies for Olfml3, PECAM-1 and the mural cell markers,
.alpha.-smooth muscle actin (.alpha.-SMA) or NG2 chondroitin
sulfate proteoglycan. Immunofluorescence microscopy revealed that
Olfml3 expression was not only found on PECAM-1-positive
endothelial cells but also overlapped with .alpha.-SMA-positive
mural cells (FIG. 3A) and to a lesser extent, with NG2-positive
pericytes (FIG. 3C). These observations indicate that expression of
Olfml3 by tumor vessel-associated mural cells cannot be excluded,
despite the fact that these cells were not shown to be positive for
Olfml3 transcripts (FIGS. 2B-2C, left panels). The
Olfml3-expressing tumor vessels were predominantly covered by
.alpha.-SMA-positive mural cells (FIG. 3A), while none or partial
overlapping of the Olfml3-expressing vessels was observed with
NG2-positive pericytes (FIG. 3B). Intense .alpha.-SMA staining
along with the reduced NG2 expression in the tumor-associated
pericytes generally reflects a phenotype of more immature, highly
angiogenic tumor blood vessels (Morikawa et al., 2002; Gerhardt et
al., 2003). Therefore, these data suggest that Olfml3 expression
and secretion are mainly associated with immature, highly
angiogenic tumor vessels.
Example 3--Olfml3 is Required for Endothelial Cell Migration
[0149] To characterize the functions of Olfml3 in the extracellular
compartment surrounding angiogenic cells, the inventors first
tested whether Olfml3 mediates endothelial cell migration, an
essential step of angiogenesis. As the t.End.1V.sup.high cells can
migrate efficiently in wound healing assays (Aurrand-Lions et al.,
2004), the inventors silenced Olfml3 expression in these cells and
tested their migratory capacities in this assay. Three siRNAs were
designed, of which siRNA 3 silenced >90% of the Olfml3 message
in t.End.1V.sup.high cells (FIG. 9). As for controls, the inventors
used mock-transfected t.End.1V.sup.high cells as well as cells
transfected with either GAPDH siRNA or a control siRNA
non-homologous to any known mouse genes (ctrl siRNA) (FIG. 9). The
t.End.1V.sup.high cells transfected with siRNA 3 displayed a
significantly decreased migration in wound healing assays in which
the rate of cell migration into a denuded area was monitored (FIGS.
4A-4B). Mock or control siRNA transfection had no effect on
t.End.1V.sup.high cell migration. Furthermore, reduced migratory
ability of silenced t.End.1V.sup.high cells was partly compensated
by coating or adding recombinant Olfml3 protein in vitro (FIGS.
4C-4D). In addition, when coated on culture plates, recombinant
Olfml3 protein promoted t.End.1V.sup.high cell migration in a
concentration-dependent manner (FIG. 4E). These data suggest that
Olfml3 promotes migration of endothelial cells, a prerequisite for
angiogenesis.
Example 4--Olfml3 is Required for Endothelial Sprouting In
Vitro
[0150] Because the inventors observed Olfml3 involvement in
endothelial cell migration, the inventors next examined whether
Olfml3 is required for sprouting of endothelial cells, a subsequent
step of angiogenesis. As the t.End.1V.sup.high cells efficiently
form a capillary-like network of ramified cords in 3D fibrin gels
(Aurrand-Lions et al., 2004; Pepper et al., 1996), the inventors
used these cells to perform the endothelial sprout formation assay
in vitro (FIGS. 5A-5C). In this assay, endothelial cells sprout in
3D fibrin gels and organize into structures morphologically similar
to capillaries (Montesano et al., 1990; Pepper et al., 1996).
Sprout formation starts with individual endothelial cells sending
out filopodia-like protrusions (spikes) within 24 hours after cell
seeding (FIG. 5A, 24 h panels). These spikes then initiate contacts
with other cells in the vicinity, align and start forming
capillary-like structures (FIG. 5A, 32-72 h panels). Using
Olfml3-silenced t.End.1V.sup.high cells, the inventors observed a
severe reduction of the number of spike-forming cells during the
first 24-32 h (FIG. 5B) compared to mock or control siRNA-treated
cells. At later time points (between 56-72 h), the inventors
observed reduced a total length of the vascular network in the
Olfml3-silenced cells when compared with the mock-transfected or
control cells (FIG. 5C and FIG. 11). These results suggest that
abrogation of Olfml3 delays endothelial sprout formation further
demonstrating its key role in angiogenesis.
Example 5--Anti-Olfml3 Antibodies Reduce Tumor Growth In Vivo
[0151] The highly abundant Olfml3 expression in angiogenic tumor
vessels (FIGS. 2A-2C, FIGS. 3A-3B) and its ability to promote
endothelial migration and sprouting in vitro (FIGS. 4A-4E, FIGS.
5A-5C) prompted us to test whether Olfml3 is able to promote tumor
angiogenesis in vivo. To test this hypothesis and to determine
which structural domain of Olfml3 is necessary for its potential
proangiogenic effect, the inventors generated rabbit anti-Olfml3
antibodies specific for two 13 amino acid long peptides comprising
epitopes in the coiled-coil or the Olfactomedin-like domain of
murine Olfml3 (peptide A or B respectively) (FIG. 12A). The
sequence comparison of Olfml3 peptides used to generate these
antibodies revealed no homology with the other members of the
Olfactomedin protein family (data not shown), while both peptides
were identical to the human Olfml3 protein sequence (FIG. 12B). The
rabbit anti-Olfml3 antisera recognized specifically the Olfml3
peptides A and B (FIG. 12C) as well as mouse recombinant Olfml3
protein (FIG. 13).
[0152] The anti-Olfml3 antibodies were first affinity-purified
against both Olfml3 peptides and subsequently used for in vivo
treatment of mice bearing LLC1 tumors. Tumor cells were injected
subcutaneously and the anti-Olfml3 antibodies were given i.p. every
72 hours. At day 9, animals were sacrificed and the tumors excised.
The anti-Olfml3 antibody treatments significantly decreased the
tumor weight when compared to control rabbit IgG treated tumors
(FIGS. 6A-6B). In order to determine which Olfml3 structural domain
might be necessary for this effect, the inventors affinity-purified
the anti-Olfml3 antibodies against either the Olfml3 peptide A or
peptide B and used them for the LLC1 tumor treatment. Both
antibodies significantly reduced tumor growth with no significant
difference observed between either target (FIGS. 6C-6D). Because
LLC1 tumor cells do not express Olfml3 (FIG. 2C and FIG. 10.), the
reduction in tumor growth after anti-Olfml3 treatment was likely
due to reduced angiogenesis. Indeed, the rate of the tumor
vascularization measured by staining of the endothelial PECAM-1,
was significantly decreased (26%) in the anti-Olfml3 treated tumors
compared to the control antibody-treated tumors (FIGS. 6E-6F). This
observation confirms the hypothesis that Olfml3 promotes tumor
angiogenesis.
Example 6--Molecular Mechanism of Olfml3-Mediated Angiogenesis
[0153] Previous studies have shown that ONT1, a Xenopus homologue
of Olfml3, interacts with the BMP1/Tolloid-class proteinases and
Chordin, a BMP antagonist, through the coiled-coil and the
olfactomedin-like domain, respectively (Inomata, 2008). The
inventors sought to investigate possible Olfml3 interactions with
other BMP family members, particularly those with prominent
proangiogenic activity in tumors such as BMP4. The inventors
produced recombinant Olfml3 FLAG-tagged protein (rOlfml3-FLAG),
purified from the total cell lysates and analyzed its expression by
Western blotting, using anti-FLAG and anti-Olfml3 antibodies (FIG.
13). The rOlfml3-FLAG was then used for interaction studies using
different BMPs using ELISA assays. The inventors found that
rOlfml3-FLAG specifically binds recombinant BMP4 but not rBMP1 or
rBMP9 (FIG. 7A) and confirmed that rOlfml3-FLAG
co-immunoprecipitates with mouse recombinant BMP4 (FIG. 7B). Mass
spectrometry analysis confirmed the identity of BMP4, as a binding
partner of Olfml3 protein (FIG. 14). To map the BMP4-binding
regions on the Olfml3 protein, four different anti-Olfml3
antibodies raised against non-overlapping Olfml3 peptide sequences,
were used for the interaction studies (FIGS. 7C-7D). Three of the
antibodies blocked Olfml3-BMP4 interaction and defined two binding
domains on the Olfml3 protein, corresponding to the coiled-coil
(Olfml3 peptide A) and the Olfactomedin-like domain (Olfml3 peptide
B and D) (FIGS. 7C-7D). Therefore, the inventors demonstrate that
both Olfml3 protein domains are equally required for the
interaction with recombinant BMP4 protein. In contrast, Xenopus
ONT1 binds BMP1 exclusively through the coiled-coil domain and it
does not bind to BMP4 (Inomata et al., 2008). The results define a
novel interaction between mouse Olfml3 and BMP4, a potent
proangiogenic growth factor. The question arises whether the
Olfml3-BMP4 interaction complex is needed to potentiate the
proangiogenic effect of BMP4.
Example 7--Olfml3-BMP4 Interaction Promotes BMP4 Signaling in
Endothelial Cells
[0154] BMP4 mediates a cellular response in endothelium through the
activation of the ERK1/2 signaling pathway, thus regulating
critical endothelial functions such as proliferation or tube
formation in HUVECs (Langenfeld and Langenfeld, 2004; Zhou et al.,
2007). Since the inventors demonstrated that BMP4 directly
interacts with Olfml3 (FIGS. 7A-7D), the inventors sought to
investigate the possible effect of this interaction in the
induction of the ERK1/2 signaling in HUVECs. The level of the
ERK1/2 phosphorylation in HUVECs treated with BMP4 in combination
with Olfml3 was 5 fold increased compared to phosphorylation of
untreated cells or those stimulated with BMP4 or Olfml3
individually (FIG. 8A). Moreover, the synergistic effect of BMP4
and Olfml3 on the ERK1/2 phosphorylation was higher than that
following VEGF stimulation (FIG. 8A). This demonstrates that Olfml3
may act as an enhancer of BMP4-ERK1/2 signaling in HUVECs,
suggesting that Olfml3-associated angiogenesis may occur, at least
in part, through the activation of endothelial cells via this
particular signaling cascade.
[0155] BMP4 induced activation of ERK1/2 signaling leads to
increased BMP4 gene expression forming an autocrine feedback loop
(Zhou et al., 2007). To check if Olfml3 gene expression is also
under the BMP4 control, HUVECs were stimulated with BMP4 during 24
h, and upregulation of Olfml3 protein expression was detected after
this period (FIG. 8B). Notably, BMP4-induced expression of Olfml3
was equally upregulated as Olfml3 expression induced by VEGF
stimulation of HUVECs. These data suggest that Olfml3 expression is
driven by angiogenic growth factors and it amplifies effects these
factors have on endothelial cells.
Example 8--Dual Expression of Olfml3 in Tumor Endothelium and
Accompanying Pericytes
[0156] Following the transcriptome and histological analyses,
Olfml3 expression was found to be restricted to angiogenic
endothelial cells (t.End.1V.sup.high) and vessels undergoing
angiogenesis in matrigel plugs (FIGS. 21A-B). To evaluate Olfml3
expression in tumor angiogenic vessels, Lewis Lung Carcinoma (LLC1)
cells were s.c. implanted in wild-type mice (FIG. 15). Transcripts
of Olfml3 were detected in LLC1 tumor endothelium (PECAM-1.sup.+)
and accompanying pericytes (PECAM-1.sup.-) (FIG. 15A). Tumor cells
themselves did not express Olfml3 mRNA (FIG. 21C). Double staining
of tumors for Olfml3 and PECAM-1 revealed that Olfml3 protein is
enriched in the extracellular space of endothelial cells and
pericytes of a subset of tumor vessels (FIG. 15B). To validate
vascular-specific Olfml3 expression, tumors were triple stained for
Olfml3, PECAM-1, and the pericyte markers .alpha.-smooth muscle
actin (.alpha.-SMA) or nerve/glial antigen-2 (NG2), respectively
(FIGS. 15C, D). Olfml3 expression was detected in both
.alpha.-SMA.sup.+ and NG-2.sup.+ pericytes, while was absent from
.alpha.-SMA.sup.- pericytes (FIG. 15C). In order to determine
whether Olfml3 is produced by pericytes on established, resting
tumor vessels or de novo forming vessels, two different types of
smooth muscle cells having pericyte-like characteristics (Brisset
et al., 2007) were isolated. The actively proliferating and
migrating cells (R-SMCs) expressed higher levels of Olfml3 compared
with resting counterparts (S-SMCs) (FIG. 15E). Therefore, Olfml3
expression may correlate with the activation state of both
endothelial cells and pericytes, implying a potential functional
importance of Olfml3 during activation and maturation phases of
angiogenesis.
Example 9--Autocrine Effects of Olfml3 on Endothelial Cells
[0157] To define the Olfml3-dependent vascular functions, the
inventors first tested whether Olfml3 mediates endothelial cell
migration. As t.End.1V.sup.high cells migrate efficiently in wound
healing assays (Aurrand-Lions et al., 2004; Miljkovic-Licina et
al., 2009), the inventors investigated the consequences of Olfml3
gene silencing (FIG. 22A) on the migration of t.End.1V.sup.high
cells in this assay. The Olfml3-silenced t.End.1V.sup.high cells
displayed a significantly decreased migration rate into the denuded
area (FIG. 16A). Olfml3 silencing did not significantly affect
endothelial cell proliferation (data not shown). This reduced
migratory ability of Olfml3-silenced cells was partly compensated
when recombinant Olfml3 FLAG-tagged protein (rOlfml3-FLAG) (FIG.
22B) was coated on plates (FIG. 16B). In addition, rOlfml3-FLAG
promoted t.End.1V.sup.high cell migration in a
concentration-dependent manner (FIG. 16C). These data identified
Olfml3 as a novel autocrine regulator of endothelial cell
migration.
[0158] The pro-migratory action of Olfml3 on t.End.1V.sup.high
cells suggested that Olfml3 might also exert an effect on
endothelial cell sprouting. As t.End.1V.sup.high cells efficiently
form a capillary-like network of ramified cords in
three-dimensional fibrin gels (Aurrand-Lions et al., 2004), the
inventors used this assay to study the effect of Olfml3 depletion
on t.End.1V.sup.high cell sprouting (FIGS. 16D-F). Compared with
mock- or control siRNA-treated t.End.1V.sup.high cells (FIG. 16D),
the number of Olfml3-silenced cells that initialized sprout
protrusions at early time points (24-32 hours) was significantly
decreased (FIG. 16D, E). In addition, total length of the vascular
network in Olfml3-silenced cells was reduced drastically at later
time points (72 hours) (FIG. 16F and FIG. 23). These findings
suggest that abrogation of Olfml3 was sufficient to attenuate
endothelial migration and sprouting, further supporting its
potential role in angiogenesis.
Example 10--Anti-Olfml3 Antibodies Reduce LLC1 Tumor Growth and
Angiogenesis
[0159] In order to test whether Olfml3 promotes tumor angiogenesis
in vivo, the inventors generated rabbit anti-Olfml3 antibodies by
injecting simultaneously two 13-aa long peptides comprising
epitopes in the coiled-coil (peptide A) and the olfactomedin-like
domains (peptide B) (FIG. 24A). Both peptides are identical in the
mouse and human Olfml3 protein sequences (FIG. 24B). The
anti-Olfml3 antibodies recognized the peptides A and B,
respectively (FIG. 24C) as well as rOlflm3-FLAG (FIG. 22B).
[0160] The Olfml3 antibodies were affinity-purified against both
Olfml3 peptides (anti-Olfml3.sup.A+B) and evaluated for the ability
to block tumor growth and angiogenesis in the LLC1 mouse model.
Treatment with anti-Olfml3.sup.A+B antibodies significantly
decreased the tumor weight compared with control rabbit
immunoglobulin G (IgG) treatment (FIGS. 17A, B). To determine which
Olfml3 structural domain might be necessary for this effect, the
inventors affinity-purified the Olfml3 antibodies against either
the Olfml3 peptide A (anti-Olfml3.sup.A) or peptide B
(anti-Olfml3.sup.B) and used them for the LLC1 tumor treatment.
Both antibodies significantly reduced tumor growth by 38% and 52%
respectively with no significant difference observed between either
treatment (FIGS. 17C, D). The rate of tumor vascularization
measured by PECAM-1 staining was significantly decreased by
treatment with either anti-Olfml3.sup.A or anti-Olfml3.sup.B (FIGS.
17E, F). The antibodies showed different efficacy of reducing tumor
vascularization. Anti-Olfml3.sup.B reduced tumor vascularization by
58%, whereas anti-Olfml3.sup.A had smaller but significant effect
(30%), suggesting that both structural domains of the protein are
necessary for its pro-angiogenic activity. However, when the two
Olfml3 antibodies were co-injected, no synergistic inhibition of
tumor vascularization was observed. These findings confirmed the
hypothesis that Olfml3 promotes tumor angiogenesis, whereas
blocking its function leads to reduced angiogenesis and tumor
growth.
Example 11--Impaired Pericyte Coverage of Tumor Vessels After
Anti-Olfml3 Treatment
[0161] Endothelial cell survival correlates with the extent of
pericyte coverage in tumor vessels (Franco et al., 2011). As Olfml3
was co-expressed in tumor endothelial cells and accompanying
pericytes (FIG. 1), the inventors investigated whether anti-Olfml3
antibodies affect pericyte coverage of tumor vessels using the
pericyte marker .alpha.-SMA as the readout. Tumor blood vessels of
control-treated mice exhibited abundant .alpha.-SMA.sup.+
pericytes, while treatment with anti-Olfml3.sup.A or
anti-Olfml3.sup.B dramatically reduced .alpha.-SMA immunoreactivity
by 61.5 and 63%, respectively (FIGS. 18A, B). The observed effect
could reflect a decrease in .alpha.-SMA expression by pericytes or
a loss in the number of pericytes. To distinguish between these two
possibilities, the inventors stained tumors for NG2, another
pericyte marker (FIG. 18C). Numerous NG2.sup.+ pericytes were
observed under control conditions (FIGS. 18C, D). Following
treatment with anti-Olfml3.sup.A or anti-Olfml3.sup.B, however, NG2
immunoreactivity decreases substantially, by 67% and 78%,
respectively (FIGS. 18C, D). These supporting observations indicate
that the reduction in .alpha.-SMA immunoreactivity reflects a
decrease in pericytes number rather than a decrease in .alpha.-SMA
protein expression per cell. Therefore, targeting Olfml3 with its
blocking antibodies decreases the pericyte coverage in tumor
vessels, implying Olfml3 involvement in the maturation of de
novo-forming vasculature.
Example 12--Olfml3 is a BMP4-Binding Protein
[0162] Previous studies have shown that Xenopus Olfml3 interacts
with BMP1 and chordin through the coiled-coil and olfactomedin-like
domains, respectively (Inomata et al., 2008). The inventors
therefore investigated a possible interaction of Olfml3 with BMPs
known as either pro- or anti-angiogenic cues within the tumor
microenvironment (David et al., 2009). The inventors used
rOlfml3-FLAG for interaction studies with three different BMPs in
enzyme-linked immunosorbent assays. rOlfml3-FLAG specifically bound
recombinant BMP4 (rBMP4) but not rBMP1 or rBMP9 (FIG. 19A), and
rOlfml3-FLAG co-immunoprecipitated with rBMP4 (FIG. 19B). To map
the BMP4-binding regions on the Olfml3 protein, anti-Olfml3.sup.A,
anti-Olfml3.sup.B and a commercial antibody raised against a
distinct Olfml3 peptide (Olfml3 peptide C) were used for binding
studies (FIG. 19C). Both anti-Olfml3.sup.A and anti-Olfml3.sup.B
antibodies blocked the interaction of rOlfml3-FLAG with rBMP4 (FIG.
19D). The third high-affinity antibody, targeting a non-overlapping
epitope in the coiled-coiled domain, did not block Olfml3-BMP4
interaction (FIG. 19E). These results suggest that the coiled-coil
(peptide A) and the olfactomedin-like domain (peptide B) are
equally required for the interaction with BMP4, confirming the
previous hypothesis of a single ligand for the two Olfml3 domains.
The results define a novel interaction between mouse Olfml3 and
BMP4, a potent pro-angiogenic growth factor.
Example 13--Olfml3 Activates Canonical SMAD1/5/8 Signaling Pathway
in HUVECs
[0163] As BMP4 directly binds to Olfml3 (FIG. 19), the inventors
sought to investigate the possible effect of this interaction in
BMP4 downstream signaling. HUVECs were treated with rOlfml3-FLAG
and/or BMP4 and subsequently both nuclear translocation of SMAD1
and phosphorylation of SMAD1/5/8 as readouts of the BMP4 pathway
activity were analyzed (FIG. 20). rOlfml3-FLAG alone induced
nuclear translocation of SMAD1 after 15 minutes (FIG. 20A).
Likewise, nuclear translocation of SMAD1 was observed in
BMP4-treated HUVECs (FIG. 20A). Upon challenge of HUVECs with
rOlfml3-FLAG or BMP4, Smad1/5/8 proteins were phosphorylated
rapidly (FIGS. 20B-D), whereas SMAD1/5/8 phosphorylation was not
observed in untreated control cells (data not shown) or cells
treated with the FLAG peptide (FIGS. 20B, C). In the presence of
anti-Olfml3.sup.A+B antibodies, the ability of Olfml3 to induce
SMAD1/5/8 phoshorylation is lost (FIGS. 20B, C). Of interest,
Olfml3 and BMP4 showed additive effects on pSMAD1/5/8
phosphorylation when combined (FIGS. 20B-D). While SMAD1/5/8
phosphorylation reached a maximum after 15 minutes of rOlfml3-FLAG
exposure in HUVECs (FIG. 20D), rOlfml3-FLAG and BMP4 exposure gave
rise to an increased and prolonged effect on SMAD1/5/8
phosphorylation in time course experiments (FIGS. 20C, D). These
findings demonstrate that Olfml3 alone or in a complex with BMP4
acts as an enhancer of the SMAD1/5/8 signaling pathway in
HUVECs.
Example 14--Anti-Olfml3 Monoclonal Antibodies Reduce Tumor
Growth
[0164] Two rat monoclonal antibodies that recognize peptide B
(390-403 aa of human/mouse Olfml3) reduced tumor growth by half
(FIGS. 25A-B). The mice were injected with LLC1 tumor cells and
treated with novel anti-Olfml3 monoclonal antibodies over several
days. Two out of three tested anti-Olfml3 peptide B monoclonal
antibodies substantially reduced the size of the tumor and the
number of blood vessels as the tumor tissue appears white
(anaemic). Interestingly there is a non-functional
anti-Olfml3.sup.B mAb against peptide B that can serve as control.
This also suggests that a subdomain of peptide B represents the
active epitope.
Example 15--Materials and Methods
[0165] Cell Lines and Culture
[0166] t.End.1V.sup.high cells were maintained as described
previously (Aurrand-Lions et al., 2004). Lewis lung carcinoma cells
(LLC1; European Collection of Cell Cultures) were cultured in DMEM
(Life Technologies), supplemented with 10% FBS. Smooth muscle cells
(SMCs) were isolated from the media of porcine carotid artery using
enzymatic digestion (S-SMCs) or tissue explanation (R-SMCs) as
described previously (Brisset et al., 2007). HUVECs were isolated
freshly and cultured in EGM-2 Bulletkit (Lonza).
[0167] Tumor Model
[0168] All studies were conducted in accordance with the ethical
approval and recommendations of the Veterinary Office of Geneva
state, according to the Swiss federal law. To generate an
implantation tumor model, a suspension of 0.5.times.10.sup.6 LLC1
tumor cells in 100 .mu.L PBS was implanted subcutaneously into the
flank of female C56BL/6J mice (8-10 weeks old). Mice were then
treated with 25-50 .mu.g of control, total rabbit IgG; 50 .mu.g of
anti-Olfml3.sup.A+B affinity-purified against both Olfml3 peptides,
and 25 .mu.g of anti-Olfml3.sup.A or anti-Olfml3.sup.B
affinity-purified against each peptide i.p. every third day
starting from day 1. When tumors reached an average size of 1 cm,
mice were sacrificed and tumors were harvested for evaluation of
tumor growth.
[0169] In Situ mRNA Hybridization
[0170] The digoxigenin- and fluorescein-labeled (Roche) RNA probes
were prepared after PCR amplification of mouse PECAM-1 and Olfml3
genes as described in Supplementary Methods. In situ mRNA
hybridization was performed on frozen sections of LLC1 tumors as
previously described (Miljkovic-Licina et al., 2009).
[0171] Immunohistochemistry
[0172] HUVECs were grown on glass slides and immunohistochemistry
was performed as detailed in Supplementary Methods. LLC1 tumors
were processed for and stained by immunohistochemistry as
previously described (Miljkovic-Licina et al., 2009). Samples were
incubated with: rabbit anti-Olfml3.sup.A+B serum, rat monoclonal
anti-PECAM-1 (Piali et al., 1993), mouse anti-.alpha.-SMA (Brisset
et al., 2007) or mouse monoclonal anti-NG2 (clone 132.38;
Millipore). Quantification of relative vascular and pericyte areas
was performed using Metamorph6.0 (Molecular Devices). Ten
individual images at three section planes were analyzed in 8-10
tumors/group (4-5 mice/group) in 2-3 independent experiments.
Relative vascular and pericyte area were measured as the ratios of
the total pixel counts of PECAM-1, .alpha.-SMA or NG2 to DAPI
staining
[0173] In Vitro Wound Healing Assay
[0174] Transient transfection of t.End.1V.sup.high cells was
performed using Amaxa.TM. Nucleofector (Lonza) with Stealth.TM.
Select siRNAs (Life Technologies) as described in Supplementary
Methods. The efficiency of Olfml3 silencing in t.End.1V.sup.high
cells was evidenced by RT-qPCR. Transfected t.End.1V.sup.high cells
(1.5.times.10.sup.4) were seeded onto matrigel- or
rOlfml3-FLAG-coated (BD Biosciences) plates and in vitro wound
healing assays were performed as described previously
(Miljkovic-Licina et al., 2009).
[0175] In Vitro Sprouting Assay
[0176] Transfected t.End.1V.sup.high cells (1.2.times.10.sup.4
cells/gel) were seeded in suspension into fibrin gels (Pepper et
al., 1996) and in vitro sprouting assays were performed as
described previously (Miljkovic-Licina et al., 2009).
[0177] Enzyme-Linked Immunosorbent Assay (ELISA)
[0178] Maxisorb immunoplates (Nunc) were coated overnight at
4.degree. C. with rBMP4 (2 .mu.g/mL). Wells were washed, blocked
with 1% BSA, and incubated with rOlfml3-FLAG at 0.5 .mu.g/mL in PBS
containing 0.05% Tween 20 and 0.5% BSA. Biotinylated M2 antibody (2
.mu.g/mL) was added. Bound M2 was detected using streptavidin-HRP
(Jackson Immunoresearch Laboratories) and substrate Reagent Pack
(R&D Systems). Optical densities at 450 nm were read using a
kinetic microplate reader and SoftMAXPro (Molecular Devices).
[0179] Pull-Down Assay of rBMP4 by rOlfml3-FLAG
[0180] rBMP4 (R&D Systems) was incubated at 4.degree. C. with
anti-FLAG M2-Agarose beads (Sigma-Aldrich) loaded with or without
rOlfml3-FLAG (1 .mu.g) in TBS, 0.1% NP-40, 0.05% BSA. Beads were
eluted with non-reducing SDS sample buffer. Samples were further
subjected to SDS-PAGE and silver staining was performed using
SilverQuest staining kit (Invitrogen).
[0181] Western Blotting
[0182] HUVECs were serum-starved in OptiMEM (Invitrogen) and 50
ng/mL rBMP4 (R&D Systems) and/or 50 ng/mL of rOlfml3-FLAG were
added. Cells were lysed with lysis buffer [50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 10 mM MgCl2 and 0.5% Triton X-100] containing a
cocktail of protease and phosphatase inhibitors (Sigma-Aldrich).
Blots were incubated with anti-phosphoSMADl/5/8 or anti-SMAD1 (Cell
Signaling Technology) and revealed using the HRP-labeled
anti-rabbit antibodies (Jackson ImmunoResearch Laboratories),
visualized using an enhanced chemiluminescence system and a
quantitative imaging system Fujifilm LAS4000Mini (Fujifilm).
[0183] In Situ mRNA Hybridization
[0184] The digoxigenin (DIG)- and fluorescein (FLUO)-labeled
(Roche) RNA probes were prepared after PCR amplification of mouse
PECAM-1 and Olfml3 genes using corresponding forward and reverse
primers containing the T7 polymerase binding site (underscored) for
sense or antisense RNA probes, as follows:
TABLE-US-00002 PECAM-1-sense-for1, (SEQ ID NO: 5)
5'-CTAATACGACTCACTATAGGGATGCTCCTGGCTCTGGGACTC-3';
PECAM1-sense-rev1, (SEQ ID NO: 6) 5'-TGCAGCTGGTCCCCTTCTATG-3';
PECAM-1-antisense-for1, (SEQ ID NO: 7) 5'-ATG CTC CTG GCT CTG GGA
CTC-3'; PECAM-1-antisense-rev1, (SEQ ID NO: 8) 5'-CTA ATA CGA CTC
ACT ATA GGG TGC AGC TGG TCC CCT TCT ATG)-3'; Olfml3-sense-for1,
(SEQ ID NO: 9) 5'-CTA ATA CGA CTC ACT ATA GGGAGT GCT CCT CTG CTG
CTC CTC-3'; Olfml3-sense-rev1, (SEQ ID NO: 10) 5'-CGT GTC GTT CTG
GGT GCC GTC-3'; Olfml3-antisense-for1, (SEQ ID NO: 11) 5'-AGT GCT
CCT CTG CTG CTC CTC-3'; and mOlfml-3-antisense-rev1, (SEQ ID NO:
12) 5'-CTA ATA CGA CTC ACT ATA GGG CGT GTC GTT CTG GGT GCC
GTC-3'.
[0185] siRNA Delivery
[0186] The following chemically modified duplex siRNAs were
engaged: three siRNAs directed against non-overlapping regions of
the mouse Olfml3 gene (OLFML3MSS235376, OLFML3MSS235377, and
OLFML3MSS235378, named as Olfml3 siRNA 1, 2, and 3, respectively),
a siRNA against mouse GAPDH, and a non-targeting negative control
siRNA (ctrl siRNA) (Stealth.TM. Select Technology; Life
Technologies). Single siRNAs or combinations of two siRNAs were
transfected in the t.End.1V.sup.high cells at the concentration of
0.5 .mu.M using Amaxa.TM. Nucleofector technology (Lonza). The
efficiency of Olfml3 silencing in t.End.1V.sup.high cells was
evidenced by real-time qPCR 24-72 h after transfection.
[0187] Quantitative Real-Time PCR
[0188] Total RNA was extracted from following cells:
t.End1.V.sup.high, t.End1.V.sup.low, LLC1, LMEC, R-SMC, S-SMC and
murine lung tissue using the RNeasy Mini Kit (Qiagen). The purified
RNA was quantified at 260 nm and RNA quality was evaluated by
capillary electrophoresis on an Agilent 2100 Bioanalyzer (Agilent
Technologies). Total RNA was reverse transcribed using the cDNA
synthesis kit (Roche). Primers used for real-time qPCR were as
follows: mouse Olfml3_for1, 5'-GCTGTCTATGCCACTCGAGATG-3' (SEQ ID
NO:13) (forward) and Olfml3_rev1, 5'-TGTGTCAAGTGTCTGTGGGTCTAA-3'
(SEQ ID NO:14) (reverse); human Olfml3_for1,
5'-GTCTATGCCACCCGGGAGGAT-3' *SEQ ID NO:15) (forward) and
Olfml3_rev1, 5'-TGTGTCCAGTGTCTGTGGATCTAA-3' (SEQ ID NO:16).
Reactions were performed in triplicate with the Power SYBR Green
PCR kit and primers were assayed on an ABI Prism 7900 HT (Applied
Biosystems). Raw threshold cycle (ct) values were obtained using
SDS2.2 software (Applied Biosystems) and the normalization factor
and fold changes were calculated using three mouse reference genes
(.beta.-actin, .beta.-tubulin and EEF1A1) or a porcine GAPDH
reference gene, according to the GeNorm method (Carmeliet and Jain,
2011).
[0189] Cloning Strategy for Production of Recombinant Olfml3
Protein Tagged With a FLAG Sequence
[0190] The full-length Olfml3 cDNA was obtained by PCR performed on
the pCMV-SPORT6 vector (Invitrogen) containing the Olfml3 clone
(ID3485412) from the MGC cDNA library (NIH). The Olfml3 PCR
fragment was cloned into the pcDNA3.1 (Invitrogen) vector
containing a FLAG sequence, where a FLAG sequence was inserted
downstream to and in-frame with the Olfml3 coding sequence. The
Olfml3-FLAG PCR fragment was then inserted into the pcDNA3.3 TOPO
TA vector (Invitrogen). The plasmid was multiplied in DH5a
Escherichia coli, purified using EndoFree Plasmid maxi preparation
(Qiagen), and used for production of the recombinant protein.
[0191] Production and Purification of Mouse Recombinant Olfml3-FLAG
Tagged Protein (rOlfml3-FLAG)
[0192] The expression vector pcDNA3.3 TOPO TA (Invitrogen) with
Olfml3-FLAG sequence was used for transient transfection of human
HEK-293 cell line in a serum-free suspension, as described
previously (Folkman, 2007). The cell culture supernatants were
collected and rOlfml3-FLAG was affinity-purified using anti-FLAG M2
agarose beads (Sigma-Aldrich), eluted with FLAG peptide (100
.mu.g/mL; Sigma-Aldrich). Next, 0.1 .mu.g of rOlfml3-FLAG was
subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE), blotted on nitrocellulose, and revealed
either with biotinylated FLAG antibody in combination with
streptavidin-HRP conjugate or rabbit anti-Olfml13 antibody revealed
with the anti-rabbit HRP labeled antibodies (Jackson ImmunoResearch
Laboratories), visualized using an enhanced chemiluminescence
system (GE Healthcare).
[0193] Generation of Rabbit Anti-mouse Olfml3 Polyclonal and
monoclonal rat anti-human/mouse Antibodies
[0194] Polyclonal antibodies against mouse Olfml3 were generated by
immunizing rabbits with Olfml3 synthetic peptide A (86-99) and
peptide B (390-403) (Covalab, France). The rabbit sera were then
tested for reactivity. Antibodies were purified from serum by
affinity chromatography against peptide A and B or each peptide
separately. Fischer rats were immunized with human peptide B mixed
with Titermax adjuvant (Sigma) and LN B cells and splenocytes were
fused to Sp2/0 cells. Hybridomas were then selected in
HAT-containing medium and resistant clones screened by ELISA for
the production of mAbs against peptide B and recombinant Olfml3.
Antibodies recognized human and mouse peptides.
[0195] FGF2-Loaded Matrigel Plug Assay
[0196] Eight-week-old female C57BL6/J mice were injected
subcutaneously with matrigel (400 .mu.L per animal, BD Biosciences)
supplemented with the angiogenic growth factor FGF2 (500 ng/mL per
animal; Peprotech) into C57BL/6J mice. After 8 days, the plugs were
excised and prepared for immunohistological evaluation.
[0197] Immunohistochemistry
[0198] HUVECs grown to 80% confluence on glass slides were serum
starved for 2 h in OptiMEM (Invitrogen) and treated with 50 ng/mL
of BMP4 (R&D Systems) and/or 100 ng/mL of rOlfml3-FLAG for
indicated times. As controls, HUVECs were incubated with 500 ng/mL
of FLAG peptide (Sigma-Aldrich) and 100 ng/mL of rOlfml3-FLAG in
the presence of rabbit total IgG or anti-Olfml3.sup.A+B serum.
HUVECs were fixed in 4% para-formaldehyde for 20 min at room
temperature, washed in phosphate buffered saline (PBS) and
permeabilized in 0.1% sodium citrate, 0.1% Triton X-100 for 2 min
on ice. Cells were then washed in PBS and saturated in 1% bovine
serum albumin and 2% donkey serum in PBS for 1 h at room
temperature. For detection, glass slides were incubated with:
rabbit anti-SMAD1 or anti-phosphoSMAD1/5/8 antibodies (Cell
Signaling Technology), for 1 h at room temperature. Unbound
antibodies were removed using 0.1% Tween 20 in PBS. Rabbit
anti-SMAD1 or anti-phosphoSMAD1/5/8 antibodies were detected using
donkey anti-rabbit IgG coupled to rhodamine (Jackson ImmunoResearch
Laboratories). Samples were stained for FITC-Phalloidin
(Sigma-Aldrich) and mounted as described above. Quantification of
nuclear phosphoSMAD1/5/8 staining was quantified using Metamorph6.0
software (Molecular Devices) and mean intensity was measured from
at least five random microscopic fields for each group in three
independent experiments.
[0199] Statistical Analysis
[0200] All data are presented as means .+-.standard deviation (SD)
unless indicated otherwise. For comparisons of two means, Student's
t-test (2-sided, paired) was used. For multiple mean comparisons,
one-way or two-way ANOVA followed by the Bonferroni's test was
used. All statistical computations were done using GraphPadPrism.
Results were considered statistically significant at P<0.05.
[0201] All of the methods disclosed and claimed herein can be made
and executed without undue experimentation in light of the present
disclosure. While the compositions and methods of this invention
have been described in terms of preferred embodiments, it will be
apparent to those of skill in the art that variations may be
applied to the methods and in the steps or in the sequence of steps
of the method described herein without departing from the concept,
spirit and scope of the invention. More specifically, it will be
apparent that certain agents which are both chemically and
physiologically related may be substituted for the agents described
herein while the same or similar results would be achieved. All
such similar substitutes and modifications apparent to those
skilled in the art are deemed to be within the spirit, scope and
concept of the invention as defined by the appended claims.
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Sequence CWU 1
1
161406PRTHomo sapiens 1Met Gly Pro Ser Thr Pro Leu Leu Ile Leu Phe
Leu Leu Ser Trp Ser 1 5 10 15 Gly Pro Leu Gln Gly Gln Gln His His
Leu Val Glu Tyr Met Glu Arg 20 25 30 Arg Leu Ala Ala Leu Glu Glu
Arg Leu Ala Gln Cys Gln Asp Gln Ser 35 40 45 Ser Arg His Ala Ala
Glu Leu Arg Asp Phe Lys Asn Lys Met Leu Pro 50 55 60 Leu Leu Glu
Val Ala Glu Lys Glu Arg Glu Ala Leu Arg Thr Glu Ala 65 70 75 80 Asp
Thr Ile Ser Gly Arg Val Asp Arg Leu Glu Arg Glu Val Asp Tyr 85 90
95 Leu Glu Thr Gln Asn Pro Ala Leu Pro Cys Val Glu Phe Asp Glu Lys
100 105 110 Val Thr Gly Gly Pro Gly Thr Lys Gly Lys Gly Arg Arg Asn
Glu Lys 115 120 125 Tyr Asp Met Val Thr Asp Cys Gly Tyr Thr Ile Ser
Gln Val Arg Ser 130 135 140 Met Lys Ile Leu Lys Arg Phe Gly Gly Pro
Ala Gly Leu Trp Thr Lys 145 150 155 160 Asp Pro Leu Gly Gln Thr Glu
Lys Ile Tyr Val Leu Asp Gly Thr Gln 165 170 175 Asn Asp Thr Ala Phe
Val Phe Pro Arg Leu Arg Asp Phe Thr Leu Ala 180 185 190 Met Ala Ala
Arg Lys Ala Ser Arg Val Arg Val Pro Phe Pro Trp Val 195 200 205 Gly
Thr Gly Gln Leu Val Tyr Gly Gly Phe Leu Tyr Phe Ala Arg Arg 210 215
220 Pro Pro Gly Arg Pro Gly Gly Gly Gly Glu Met Glu Asn Thr Leu Gln
225 230 235 240 Leu Ile Lys Phe His Leu Ala Asn Arg Thr Val Val Asp
Ser Ser Val 245 250 255 Phe Pro Ala Glu Gly Leu Ile Pro Pro Tyr Gly
Leu Thr Ala Asp Thr 260 265 270 Tyr Ile Asp Leu Ala Ala Asp Glu Glu
Gly Leu Trp Ala Val Tyr Ala 275 280 285 Thr Arg Glu Asp Asp Arg His
Leu Cys Leu Ala Lys Leu Asp Pro Gln 290 295 300 Thr Leu Asp Thr Glu
Gln Gln Trp Asp Thr Pro Cys Pro Arg Glu Asn 305 310 315 320 Ala Glu
Ala Ala Phe Val Ile Cys Gly Thr Leu Tyr Val Val Tyr Asn 325 330 335
Thr Arg Pro Ala Ser Arg Ala Arg Ile Gln Cys Ser Phe Asp Ala Ser 340
345 350 Gly Thr Leu Thr Pro Glu Arg Ala Ala Leu Pro Tyr Phe Pro Arg
Arg 355 360 365 Tyr Gly Ala His Ala Ser Leu Arg Tyr Asn Pro Arg Glu
Arg Gln Leu 370 375 380 Tyr Ala Trp Asp Asp Gly Tyr Gln Ile Val Tyr
Lys Leu Glu Met Arg 385 390 395 400 Lys Lys Glu Glu Glu Val 405
21852DNAHomo sapiens 2caggagagaa ggcaccgccc ccaccccgcc tccaaagcta
accctcgggc ttgaggggaa 60gaggctgact gtacgttcct tctactctgg caccactctc
caggctgcca tggggcccag 120cacccctctc ctcatcttgt tccttttgtc
atggtcggga cccctccaag gacagcagca 180ccaccttgtg gagtacatgg
aacgccgact agctgcttta gaggaacggc tggcccagtg 240ccaggaccag
agtagtcggc atgctgctga gctgcgggac ttcaagaaca agatgctgcc
300actgctggag gtggcagaga aggagcggga ggcactcaga actgaggccg
acaccatctc 360cgggagagtg gatcgtctgg agcgggaggt agactatctg
gagacccaga acccagctct 420gccctgtgta gagtttgatg agaaggtgac
tggaggccct gggaccaaag gcaagggaag 480aaggaatgag aagtacgata
tggtgacaga ctgtggctac acaatctctc aagtgagatc 540aatgaagatt
ctgaagcgat ttggtggccc agctggtcta tggaccaagg atccactggg
600gcaaacagag aagatctacg tgttagatgg gacacagaat gacacagcct
ttgtcttccc 660aaggctgcgt gacttcaccc ttgccatggc tgcccggaaa
gcttcccgag tccgggtgcc 720cttcccctgg gtaggcacag ggcagctggt
atatggtggc tttctttatt ttgctcggag 780gcctcctgga agacctggtg
gaggtggtga gatggagaac actttgcagc taatcaaatt 840ccacctggca
aaccgaacag tggtggacag ctcagtattc ccagcagagg ggctgatccc
900cccctacggc ttgacagcag acacctacat cgacctggca gctgatgagg
aaggtctttg 960ggctgtctat gccacccggg aggatgacag gcacttgtgt
ctggccaagt tagatccaca 1020gacactggac acagagcagc agtgggacac
accatgtccc agagagaatg ctgaggctgc 1080ctttgtcatc tgtgggaccc
tctatgtcgt ctataacacc cgtcctgcca gtcgggcccg 1140catccagtgc
tcctttgatg ccagcggcac cctgacccct gaacgggcag cactccctta
1200ttttccccgc agatatggtg cccatgccag cctccgctat aacccccgag
aacgccagct 1260ctatgcctgg gatgatggct accagattgt ctataagctg
gagatgagga agaaagagga 1320ggaggtttga ggagctagcc ttgttttttg
catctttctc actcccatac atttatatta 1380tatccccact aaatttcttg
ttcctcattc ttcaaatgtg ggccagttgt ggctcaaatc 1440ctctatattt
ttagccaatg gcaatcaaat tctttcagct cctttgtttc atacggaact
1500ccagatcctg agtaatcctt ttagagcccg aagagtcaaa accctcaatg
ttccctcctg 1560ctctcctgcc ccatgtcaac aaatttcagg ctaaggatgc
cccagaccca gggctctaac 1620cttgtatgcg ggcaggccca gggagcaggc
agcagtgttc ttcccctcag agtgacttgg 1680ggagggagaa ataggaggag
acgtccagct ctgtcctctc ttcctcactc ctcccttcag 1740tgtcctgagg
aacaggactt tctccacatt gttttgtatt gcaacatttt gcattaaaag
1800gaaaatccac tgctaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa
18523406PRTMus musculus 3Met Gly Pro Ser Ala Pro Leu Leu Leu Leu
Phe Phe Leu Ser Trp Thr 1 5 10 15 Gly Pro Leu Gln Gly Gln Gln His
His Leu Val Glu Tyr Met Glu Arg 20 25 30 Arg Leu Ala Ala Leu Glu
Glu Arg Leu Ala Gln Cys Gln Asp Gln Ser 35 40 45 Ser Arg His Ala
Ala Glu Leu Arg Asp Phe Lys Asn Lys Met Leu Pro 50 55 60 Leu Leu
Glu Val Ala Glu Lys Glu Arg Glu Thr Leu Arg Thr Glu Ala 65 70 75 80
Asp Ser Ile Ser Gly Arg Val Asp Arg Leu Glu Arg Glu Val Asp Tyr 85
90 95 Leu Glu Thr Gln Asn Pro Ala Leu Pro Cys Val Glu Leu Asp Glu
Lys 100 105 110 Val Thr Gly Gly Pro Gly Ala Lys Gly Lys Gly Arg Arg
Asn Glu Lys 115 120 125 Tyr Asp Met Val Thr Asp Cys Ser Tyr Thr Val
Ala Gln Val Arg Ser 130 135 140 Met Lys Ile Leu Lys Arg Phe Gly Gly
Ser Val Gly Leu Trp Thr Lys 145 150 155 160 Asp Pro Leu Gly Pro Ala
Glu Lys Ile Tyr Val Leu Asp Gly Thr Gln 165 170 175 Asn Asp Thr Ala
Phe Val Phe Pro Arg Leu Arg Asp Phe Thr Leu Ala 180 185 190 Met Ala
Ala Arg Lys Ala Ser Arg Ile Arg Val Pro Phe Pro Trp Val 195 200 205
Gly Thr Gly Gln Leu Val Tyr Gly Gly Phe Leu Tyr Tyr Ala Arg Arg 210
215 220 Pro Pro Gly Gly Pro Gly Gly Gly Gly Glu Leu Glu Asn Thr Leu
Gln 225 230 235 240 Leu Ile Lys Phe His Leu Ala Asn Arg Thr Val Val
Asp Ser Ser Val 245 250 255 Phe Pro Ala Glu Ser Leu Ile Pro Pro Tyr
Gly Leu Thr Ala Asp Thr 260 265 270 Tyr Ile Asp Leu Ala Ala Asp Glu
Glu Gly Leu Trp Ala Val Tyr Ala 275 280 285 Thr Arg Asp Asp Asp Arg
His Leu Cys Leu Ala Lys Leu Asp Pro Gln 290 295 300 Thr Leu Asp Thr
Glu Gln Gln Trp Asp Thr Pro Cys Pro Arg Glu Asn 305 310 315 320 Ala
Glu Ala Ala Phe Val Ile Cys Gly Thr Leu Tyr Val Val Tyr Asn 325 330
335 Thr Arg Pro Ala Ser Arg Ala Arg Ile Gln Cys Ser Phe Asp Ala Ser
340 345 350 Gly Thr Leu Ala Pro Glu Arg Ala Ala Leu Ser Tyr Phe Pro
Arg Arg 355 360 365 Tyr Gly Ala His Ala Ser Leu Arg Tyr Asn Pro Arg
Glu Arg Gln Leu 370 375 380 Tyr Ala Trp Asp Asp Gly Tyr Gln Ile Val
Tyr Lys Leu Glu Met Lys 385 390 395 400 Lys Lys Glu Glu Glu Val 405
41736DNAMus musculus 4agagctaacg ggctggaggg aaagaggccg aatgcacaca
ctcctctggc cccacttaag 60gctgccatgg ggcccagtgc tcctctgctg ctcctcttct
ttttgtcatg gacgggaccc 120cttcagggac agcagcacca ccttgtggag
tacatggaac gccgactagc tgccttagag 180gaacggctgg cccaatgcca
ggatcagagt agtcggcatg ctgccgagct tcgggacttc 240aaaaacaaga
tgttgcctct cctggaggtg gcagagaagg agcgggagac cctcagaact
300gaagcagact ccatctcagg aagagtggac cgtcttgaaa gggaggtaga
ctatctggag 360acacagaacc cagctttgcc ctgtgtagag ctggatgaga
aggtgactgg aggtcctgga 420gccaaaggca agggccgaag aaatgagaaa
tacgatatgg tgacggactg tagctacaca 480gtcgctcagg tgaggtcaat
gaagatcctg aagcggtttg gtggttcagt tggcctatgg 540accaaggatc
cgctggggcc agcagagaag atctacgtgt tagacggcac ccagaacgac
600acggcttttg tcttcccaag gctgcgtgac ttcacccttg ccatggctgc
ccggaaagct 660tcccgaattc gggtgccctt cccctgggta ggcacggggc
agctggtgta cggtggcttc 720ctttattatg ctcgaaggcc tcctggagga
cctggagggg gtggtgaatt ggagaacact 780ctgcagctga tcaaatttca
cttggcaaac cgaacagtgg tggatagctc agtgttccct 840gcagagagcc
tgataccccc ctacggcctg acagcagata catatatcga cctggcagct
900gatgaggagg gcctgtgggc tgtctatgcc actcgagatg atgacaggca
tttgtgtcta 960gccaagttag acccacagac acttgacaca gagcagcagt
gggacacacc atgtcccaga 1020gagaacgcag aggctgcgtt tgtcatctgt
gggaccctgt acgttgtcta taacacccgc 1080cctgccagta gggctcgtat
tcagtgttcc ttcgatgcca gtggtactct cgcccctgaa 1140agggcagcac
tctcctattt tccacgccga tatggtgccc atgccagcct tcgctataac
1200ccccgtgagc gccagctgta tgcctgggat gatggctacc agattgtcta
caaattggag 1260atgaagaaga aggaggagga agtttaagca gctagccttg
tgctcttgat tcttatgccc 1320agacatttat attcctgtga gctctcctgc
agttcatcct tcaaaacgaa ggccagtggt 1380ggtagctcat ataccctaat
ttctaaagga caaccaaatt ctcaagcccc tctgttttat 1440gcagaactcc
agatcctggg tagcatttta gaactgaaca gcaaacaaac accctaaatc
1500ttcactcctg ccttatgtcc acaaagttta gttccaaact cagagccctg
tcctttggag 1560agggtcaacc ccagacagca ggcgacagca ttcttgccct
cagtatgacc gaagggagag 1620aactcagaga caaagctgcc ctccctccct
tccccctcca gtgtagggga gaatggggct 1680ttccccacat cactttgtat
ggtaacagtt tgcattaaaa ggaaaaccca ccattc 1736541DNAArtificial
SequenceSynthetic primer 5ctaatacgac tcactatagg gatgctcctg
gctctgggac t 41621DNAArtificial SequenceSynthetic primer
6tgcagctggt ccccttctat g 21721DNAArtificial SequenceSynthetic
primer 7atgctcctgg ctctgggact c 21842DNAArtificial
SequenceSynthetic primer 8ctaatacgac tcactatagg gtgcagctgg
tccccttcta tg 42942DNAArtificial SequenceSynthetic primer
9ctaatacgac tcactatagg gagtgctcct ctgctgctcc tc 421021DNAArtificial
SequenceSynthetic primer 10cgtgtcgttc tgggtgccgt c
211121DNAArtificial SequenceSynthetic primer 11agtgctcctc
tgctgctcct c 211242DNAArtificial SequenceSynthetic primer
12ctaatacgac tcactatagg gcgtgtcgtt ctgggtgccg tc
421322DNAArtificial SequenceSynthetic primer 13gctgtctatg
ccactcgaga tg 221424DNAArtificial SequenceSynthetic primer
14tgtgtcaagt gtctgtgggt ctaa 241521DNAArtificial SequenceSynthetic
primer 15gtctatgcca cccgggagga t 211624DNAArtificial
SequenceSynthetic primer 16tgtgtccagt gtctgtggat ctaa 24
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