U.S. patent application number 13/477181 was filed with the patent office on 2013-06-20 for method of inhibiting angiogenesis or invasion or formation of metastases.
This patent application is currently assigned to INSERM. The applicant listed for this patent is He Lu, Claudine Soria, Veronique Trochon. Invention is credited to He Lu, Claudine Soria, Veronique Trochon.
Application Number | 20130158502 13/477181 |
Document ID | / |
Family ID | 8865951 |
Filed Date | 2013-06-20 |
United States Patent
Application |
20130158502 |
Kind Code |
A1 |
Trochon; Veronique ; et
al. |
June 20, 2013 |
METHOD OF INHIBITING ANGIOGENESIS OR INVASION OR FORMATION OF
METASTASES
Abstract
A method of treating a mammal with a tumor by decreasing
intratumoral vessels associated with tumor or inhibiting formation
of new intratumoral vessels includes injecting tumor with
therapeutically effective amount of expression plasmid including
polynucleotide coding for therapeutic peptide consisting of SEQ ID
NO: 2 absent any operably linked coding sequence, wherein
polynucleotide sequence is operably linked to promoter or
expression control sequence, followed by application of electric
pulses to site of injection in tumor or injecting muscle of mammal
with therapeutically effective amount of expression plasmid
including polynucleotide coding for therapeutic peptide consisting
of SEQ ID NO: 2 absent any operably linked coding sequence, wherein
polynucleotide sequence is operably linked to promoter or
expression control sequence, followed by applying electric pulses
to site of injection in muscle of mammal, whereby expression of SEQ
ID NO: 2 decreases intratumoral vessels associated with tumor, or
formation of new intratumoral vessels therein.
Inventors: |
Trochon; Veronique; (Paris,
FR) ; Lu; He; (Epinay-sur-Seine, FR) ; Soria;
Claudine; (Taverny, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Trochon; Veronique
Lu; He
Soria; Claudine |
Paris
Epinay-sur-Seine
Taverny |
|
FR
FR
FR |
|
|
Assignee: |
INSERM
Paris Cedex
FR
|
Family ID: |
8865951 |
Appl. No.: |
13/477181 |
Filed: |
May 22, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10764628 |
Jan 26, 2004 |
8207137 |
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13477181 |
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PCT/FR2002/002691 |
Jul 26, 2002 |
|
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10764628 |
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Current U.S.
Class: |
604/501 |
Current CPC
Class: |
A61P 17/06 20180101;
A61K 38/00 20130101; A61P 29/00 20180101; A61P 9/10 20180101; C12N
9/6489 20130101; A61P 35/00 20180101; A61N 1/306 20130101; A61K
48/00 20130101; A61P 27/00 20180101 |
Class at
Publication: |
604/501 |
International
Class: |
A61N 1/30 20060101
A61N001/30 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 26, 2001 |
FR |
01/10015 |
Claims
1. A method of treating a mammal with a tumor by decreasing
intratumoral vessels associated with the tumor or inhibiting
formation of new intratumoral vessels comprising: a) injecting the
tumor with a therapeutically effective amount of an expression
plasmid comprising a polynucleotide coding for a therapeutic
peptide consisting of SEQ ID NO: 2 absent any operably linked to a
promoter or an expression control sequence, followed by application
of electric pulses to a site of the injection in the tumor; or b)
injecting a muscle of the mammal with a therapeutically effective
amount of an expression plasmid comprising a polynucleotide coding
for a therapeutic peptide consisting of SEQ ID NO: 2 absent any
operably linked coding sequence, wherein the polynucleotide
sequence is operably linked to a promoter or an expression control
sequence, followed by applying electric pulses to a site of the
injection in the muscle of the mammal, whereby expression of SEQ ID
NO: 2 decreases intratumoral vessels associated with the tumor, or
formation of new intratumoral vessels therein.
2. The method according to claim 1, wherein said polynucleotide
sequence consists of SEQ ID NO: 1.
3. The method according to claim 1, wherein said expression plasmid
coding for the therapeutic peptide consisting of SEQ ID NO: 2 is
administered by injection in said muscle of the mammal with a tumor
followed by application of electric pulses to the site of injection
in said muscle of the mammal with a tumor.
4. A method of treating a mammal with metastases by decreasing
intratumoral vessels associated with the metastases or inhibiting
formation of new intratumoral vessels comprising: a) injecting a
muscle of the mammal with a therapeutically effective amount of an
expression plasmid comprising a polynucleotide coding for a
therapeutic peptide consisting of SEQ ID NO: 2 absent any operably
linked coding sequence, wherein the polynucleotide sequence is
operably linked to a promoter or an expression control sequence; b)
followed by applying electric pulses to a site of the injection in
the muscle, whereby expression of SEQ ID NO: 2 decreases
intratumoral vessels associated with the metastases or formation of
new intratumoral vessels.
5. The method according to claim 4, wherein said polynucleotide
sequence consists of SEQ ID NO: 1.
6. The method according to claim 4, wherein said expression plasmid
coding for the therapeutic peptide consisting of SEQ ID NO: 2 is
administered by injection in said muscle of the mammal with
metastases followed by application of electric pulses to the site
of injection in said muscle of the mammal with metastases.
Description
RELATED APPLICATIONS
[0001] This is a continuation of U.S. Ser. No. 10/764,628, filed
Jan. 26, 2004, which is a continuation of International Application
No. PCT/FR02/02691, with an international filing date of Jul. 26,
2002 (WO 03/009866, published Feb. 6, 2003), which is based on
French Patent Application No. 01/10015, filed Jul. 26, 2001.
TECHNICAL FIELD
[0002] Our technology relates to a method of inhibiting
angiogenesis or invasion or formation of metastases implicated in
numerous pathologies such as cancer, inflammatory diseases,
atherosclerosis and pathological angiogenesis of the retina.
SUMMARY
[0003] We provide a method of inhibiting angiogenesis or invasion
or formation of metastases in a mammal including administering a
therapeutically effective amount of an active agent selected from
the group consisting of a protein substance including all or part
of a disintegrin domain of an adamalysin or a derivative thereof, a
nucleic acid molecule including a polynucleotide sequence coding
all or part of the disintegrin domain of an adamalysin or a
derivative thereof to the mammal.
[0004] We also provide methods of treating cancer, inflammatory
diseases, atherosclerosis, macular degeneration and psoriasis in
mammals including administering a therapeutically effective amount
of an active agent selected from the group consisting of a protein
substance including all or part of a disintegrin domain of an
adamalysin or a derivative thereof and a nucleic acid molecule
including a polynucleotide sequence coding all or part of the
disintegrin domain of an adamalysin or a derivative thereof to the
mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0005] Other advantages and characteristics of our technology will
become clear from the description below pertaining to the
preparation AMEP and its in vitro and in vivo antiangiogenic,
anti-invasive and antimetastatic activity, and in which reference
will be made to the attached drawings in which:
[0006] FIG. 1 represents: in FIG. 1a, the visualization of the
fusion protein (glutathione-S transferase-AMEP) in SDS-PAGE after
purification. A single band is visible after staining with
Coomassie blue at 36 kDa. In FIG. 1b, a Western blot of purified
AMEP. Visualization of a single band at 10 kDa by an
anti-disintegrin antiserum performed in the rabbit.
[0007] FIG. 2 shows the effect of AMEP on the adhesion of CPAE to
fibrinogen (30 .mu.g/ml), vitronectin (10 .mu.g/ml) and fibronectin
(40 .mu.g/ml). The cells were pretreated for 24 h with AMEP or the
14-amino-acid fragment prior to the adhesion test (description in
Materials and methods). The experiments were performed in
triplicate and repeated three times. The results are expressed in
percentage in relation to the control (mean.+-.SEM).
[0008] FIG. 3 shows the effect of AMEP on the morphology and
migration of endothelial cells. The migration front is represented
on the photographs B, D, E, respectively, control conditions, AMEP
at 5 .mu.g/ml and 10 .mu.g/ml (phase-contrast microscopy).
[0009] FIG. 4 represents the dose-dependent inhibition of the
migration of CPAE by AMEP. The position of the migration front of
the cells was measured every day over a 3-day period. The results
are the mean of five experiments and are expressed as percentage in
relation to the control: mean.+-.SEM.
[0010] FIG. 5 represents the dose-dependent inhibition of the
proliferation of CPAE by AMEP. The cells were cultured for 48 h in
the presence of AMEP or a fragment of this domain containing the
sequence RGDC and incubated with 1 .mu.Ci of tritiated thymidine
for 18 h. The incorporated radioactivity was then measured. The
results are the mean of five experiments and are expressed in
percentage in relation to the control: mean.+-.SEM.
[0011] FIG. 6 shows the effect of AMEP on the formation of
capillary structures using HMEC-1. Cytodex beads covered with
endothelial cells were incorporated in a fibrin gel in the absence
of AMEP (photographs A, B) and in the presence of AMEP (5 .mu.g/ml)
(photographs C, D).
[0012] FIG. 7 shows the effect of AMEP on the formation of
capillaries in fibrin gel using CPAE in the presence of 5 .mu.g/ml
(B) and 10 .mu.g/ml (C, E) of AMEP in relation to the control (A,
D).
[0013] FIG. 8 shows the inhibition of tumor growth by AMEP. Each
point of the curve represents the volume of the tumor measured on a
nude mouse. The experiment comprised five animals for the control
group as well as five animals for the AMEP group, treated for 14
days. The histograms represent the mean tumor volume of 5 mice for
each group. This figure is representative of 3 distinct
experiments.
[0014] FIG. 9 shows the inhibition of the number of pulmonary
metastases by AMEP using melanoma cells. Each point represents the
number of metastases counted in the lung of a C57B1/6 mouse. The
experiment presented comprised 12 animals for each group. The
histogram shows the mean of the number of metastases in the control
and treated group. This experiment is representative of two
distinct experiments.
[0015] FIG. 10 shows a photographic representation of the
inhibition of the number of pulmonary metastases (black spots) of a
C57B1/6 mouse lung treated with AMEP in comparison with a control
mouse lung.
DETAILED DESCRIPTION
[0016] We demonstrate the anti-angiogenic, anti-invasive and
anti-metastatic functions of a fragment of a molecule present on
human endothelial cells. We also disclose the use of an adamalysin
fragment constituted by all or part of the disintegrin domain, more
particularly, the use of the disintegrin domain of metargidin
(Kratzschmar et al., 1996) also referred to below as "AMEP" which
stands for "antiangiogenic metargidin peptide."
[0017] Compared to other antiangiogenic substances that have been
described in the literature, this fragment: [0018] simultaneously
inhibits all of the stages of angiogenesis: migration and
proliferation of endothelial cells, their adhesion to different
matrix substrates and formation of capillary structures, and [0019]
induces an apoptosis of the endothelial cells.
[0020] Furthermore, in an unexpected manner, this fragment has the
capacity, on the one hand, to inhibit the invasion of cancerous
cells and, on the other hand, to prevent formation of metastases,
notably of cells that express the integrin alpha v beta 3 at their
surface.
[0021] Angiogenesis designates a morphogenetic process by which new
blood capillaries are formed by budding of existing vessels in
response to a stimulation. During angiogenesis in vivo, the
neocapillaries are born from capillaries or postcapillary venules,
but not from arteries, arterioles or veins. Thus, an angiogenic
factor is a molecule that enables initiation and/or maintenance of
angiogenesis, such as, for example FGF2. An antiangiogenic factor
is, thus, a molecule that inhibits angiogenesis by acting on one or
more key stages of angiogenesis.
[0022] Adhesions consist of the capacity of cells to attach
themselves to an extracellular matrix. This phenomenon involves
numerous adhesion molecules present at the surface of the
cells.
[0023] The migration of cells causes the intervention of enzymes
which enable the cells to degrade the compounds of the matrix as
well as the adhesion molecules which provide for anchoring the
cells to the matrix. Moreover, the dynamic architecture of the
cytoskeleton enables the cells to alternate the periods of adhesion
and detachment indispensable for motility.
[0024] Proliferation is a phenomenon which relates to the division
of cells over time.
[0025] Apoptosis consists of the intrinsic capacity of normal cells
to trigger their own suicide according to a complex program
referred to as cell death. Anoikis is a form of induced apoptosis
in normal cells resulting from a loss of their adhesion to the
substrate.
[0026] Invasion is an excessive multiplication of a class of
anatomic elements which leads to the replacement by these elements
of adjacent elements.
[0027] Metastasis is a focus of cancerous cells related to a
preexisting cancer, referred to as primary, but developed remotely
from this primary focus without continuity with it. The
dissemination of these secondary foci takes place via lymphatic or
hematic routes.
[0028] The development of a tumor and its dissemination in various
organs depends on intra- and perivascular vascularization, also
called angiogenesis (Folkman, 1984). Targeting the angiogenic
process is a new therapeutic approach and represents a revolution
in the treatment of cancer. Numerous antiangiogenic molecules are
presently involved in clinical trials.
[0029] Vitaxin is humanized anti-integrin alpha v beta 3 antibody
which induces an inhibition of the proliferation of endothelial
cells as well as a proapoptotic effect (Brooks et al., 1994; Hammes
et al., 1996). However, it does not modify their migration.
Integrin alpha v beta 3 is an adhesion molecule expressed
preferentially by the endothelial cells of neovessels and certain
cancerous cells. It interacts with certain compounds of the
extracellular matrix notably vitronectin, fibronectin, laminin
collagen IV and the fibrin inducing the adhesion and migration of
endothelial cells. The major role of integrin alpha v beta 3 in
angiogenesis has been described in detail (review, Eliceiri and
Cheresh, 2000).
[0030] Marimastat blocks the proteolytic activity of the
metalloproteinases and thereby inhibits the migration of
endothelial cells, but has no effect on their proliferation. The
metalloproteinases (MMPs) belong to the large family of enzymes
that enable degradation of the group of compounds of the
extracellular matrix indispensable for migration of endothelial
cells. Other enzymes, belonging to the serine proteases, also
participate in cell migration such as urokinase plasminogen
activator (uPA) when it is bound to its receptor anchored to the
membrane surface (u-PAR) and plasmin.
[0031] This group of drugs has the goal of blocking angiogenesis,
but their action is limited to one or two stages of this process in
contrast to AMEP which is multipotent. Thus, AMEP blocks not only
the set of angiogenic functions of the integrin alpha v beta 3,
against which it is directed initially, i.e., proliferation and
adhesion of endothelial cells, but, surprisingly, it also induces a
complete inhibition of migration of these cells as well as an
inhibition of formation of capillary structures. Its originality
is, thus, based on its proapoptotic effect on these cells
independent of a modification of their cell cycle.
[0032] Moreover, in an unexpected manner, AMEP possesses both
noteworthy anti-invasive and antimetastatic capacities.
[0033] The adamalysins, also referred to as ADAM for "a disintegrin
and metalloprotein" or MDC for "metalloprotein-rich,
disintegrin-rich and cysteine-rich protein" are a family of
proteins anchored in the plasma membrane of cells. The structure
common to the group of 29 adamalysins comprises: [0034] a
metalloproteinases domain the protease catalytic activity of which
is zinc dependent, [0035] a disintegrin domain, and [0036] a domain
rich in cysteine and in EGF type repetition (Wolfsberg et al.,
1995).
[0037] It should be noted, however, that out of the group of
adamalysins, only about ten have a metalloproteinase domain
possessing a catalytic activity. The physiological role of the
different adamalysins is extremely varied: regulation of cell
adherence, release of a ligand, activation of a receptor, cell
fusion (review, Primakoff and Myles, 2000). However, the mode of
action of these molecules remains unknown.
[0038] AMEP should be differentiated from the snake disintegrins
which have been described in the literature from two points of
view: [0039] the snake disintegrins exert a limited action on the
different stages of angiogenesis. For example, accutin (Yeh et al.,
1998) inhibits the adhesion of endothelial cells to different
components of the matrix and induces their apoptosis whereas
salmosin (Kang et al., 1999) induces an inhibition of the adhesion
of endothelial cells and of their proliferation induced by
FGF2.
[0040] AMEP also has the advantage of being of human origin and,
consequently, not having the antigenic character of the snake
disintegrins which exhibit an immunogenic character that prevents
them from being used as drugs in long-term therapy required in
anticancer treatment.
[0041] Thus, we provide a drug for inhibiting angiogenesis or
invasion and/or formation of metastases of an active agent selected
from among a protein substance comprising or constituted by all or
part of the disintegrin domain of an adamalysin or a derivative
thereof, a nucleic acid molecule comprising or constituted by a
polynucleotide sequence coding all or part of the disintegrin
domain of an adamalysin or a derivative thereof.
[0042] The adamalysin is advantageously metargidin. Consequently,
the invention pertains most particularly to a protein substance
comprising or constituted in part or entirely by the disintegrin
domain of metargidin the amino acid sequence of which is
represented in SEQ ID NO. 2 or a derivative thereof.
[0043] The AMEP fragment is remarkable in that it is capable of
inhibiting tumoral invasion, formation of metastases and all of the
stages of angiogenesis, i.e., both the migration and proliferation
of endothelial cells--in contrast to the assumptions of certain
authors (Zhang et al., 1998) whose opinion was that the disintegrin
domain by binding the integrin alpha v beta 3 could be implicated
solely in the homotypic aggregation of endothelial cells during
angiogenesis. Moreover, the inhibitory action of AMEP on
angiogenesis is observed in the absence of any addition of
angiogenic factors.
[0044] The necessity of using exogenous angiogenic factors is seen,
in contrast, when demonstrating the antiangiogenic effect of
anti-alpha v beta 3 antibodies. They inhibit angiogenesis solely
after induction by FGF2, an angiogenic factor indispensable for
maintaining angiogenesis (Klein et al., 1993). The therapeutic
value of AMEP--compared to other peptides possessing the RGD
sequence described as simple inhibitors of the adhesion of
endothelial cells (Kostesky and Artemjev, 2000)--is also based on
its spectrum of action.
[0045] We provide the disintegrin domain of an adamalysin, more
particularly, metargidin and derivatives thereof. Such derivatives
constitute functional equivalents having antiangiogenic,
anti-invasive and/or antimetastatic properties that one skilled in
the art can determine from this disclosure and, more particularly,
from the models and tests presented in the experimental part below.
The derivatives can be fragments of truncated form, sequences
modified by deletion, addition, suppression or replacement of one
or more amino acids. The derivatives can also be fragments
corresponding to said derivatives constituted by chemically
modified amino acids, these modifications making the derivatives
more stable. The disclosure also pertains to polynucleotide
sequences coding for said derivatives.
[0046] Our technology thus also pertains most especially to a
nucleic acid molecule comprising a polynucleotide sequence coding
all or part of the disintegrin domain of metargidin the sequence in
SEQ ID NO. 1 or a derivative thereof. The coding sequence of this
domain is constituted by 276 nucleotides (Met-420 to Glu-511).
[0047] The sequence is advantageously placed under the control of
regulation sequences of its expression. Such a nucleic acid
molecule is, for example, a vector such as: [0048] an expression
plasmid coding for the antiangiogenic fragment AMEP or a derivative
irrespective of the transfer technique, [0049] an expression
plasmid coding for a protein of fusion between the fragment or a
derivative and protein domain facilitating purification (pGEX type
plasmid) or facilitating tissue targeting, [0050] a plasmid or
other type of expression vector coding for the AMEP fragment or a
derivative, specific of a host organism other than a bacterium, for
example, a baculovirus, in an insect cell or a plasmid in a
eukaryote cell.
[0051] Such a nucleic acid molecule can be used in gene therapy or
cell therapy protocols comprising administering the molecule or
cells transformed by the molecule to an individual in a manner to
express all or part of the disintegrin domain at the level of a
site to be treated.
[0052] Such a nucleic acid molecule is also useful for preparing
the protein substance of the invention. Thus, human AMEP was
synthesized from bacteria and eukaryote cells transformed with a
plasmid coding for AMEP. More precisely, Escherichia coli (clone
DH5 alpha) was used as bacterial production system and the muscle
tibia cranial as eukaryote production system, but yeast or any
other production system could be used.
[0053] The demonstration of the inhibitory action of AMEP on all of
the stages of angiogenesis (migration, proliferation, adhesion,
apoptosis of the endothelial cells and the formation of capillary
type structures) and on the tumoral invasion and the formation
metastases makes it possible to offer a new antitumor drug in the
treatment of cancers. In fact, in contrast to the other inhibitors
of angiogenesis described in the literature to date, AMEP exerts an
intrinsic anti-invasive, antimetastatic and antiangiogenic activity
which is multipotent and exceptional. It inhibits both migration
and proliferation of endothelial cells of different origins
(macrovascular or microvascular, transformed or not transformed) as
well as adhesion of cells (on fibrinogen, vitronectin and
fibronectin) and formation of capillary type structures in
three-dimensional models in vitro. A proapoptotic affect of AMEP
has also been demonstrated. In vivo, AMEP blocks tumor growth by
inhibiting formation of blood vessels and metastatic dissemination,
in particular, of cells expressing the integrin alpha v beta 3.
[0054] We thus offer a new method for treating and/or preventing
cancer pathologies in general as well as diseases in which
angiogenesis contributes to the pathogenesis of the diseases such
as inflammatory diseases, psoriasis, atherosclerosis, macular
degeneration and the like.
[0055] The active agent is combined in the drugs with any
pharmaceutically acceptable vehicle known in the art and suitable
for the mode of administration employed. Thus, the drugs can be
administered: [0056] alone, via the systemic, local or oral route
or as an implant; [0057] by cell or gene therapy; [0058] in
combination with other active principles; [0059] in any
pharmaceutical form whatsoever, such as, for example, a
nanoparticle form.
[0060] The description below uses conventional molecular biology
techniques described in the literature, such as, for example:
Sambrook, Fritsch and Maniatis, Molecular Cloning; A Laboratory
Manual, Second Edition (1989), Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. (Sambrook et al., 1989); DNA Cloning: A
Practical Approach, Volumes I and II (D. N. Glover ed., 1985; B.
Perdal, A Practical Guide to Molecular Cloning (1984); F. M.
Ausubel et al. (editors) Current Protocols in Molecular Biology,
John Wiley and Sons, Inc. (1994), all the disclosures of which are
all incorporated by reference.
[0061] Thus, the term "nucleic acid" is understood to mean a
chimeric compound comprising subunits linked covalently and called
nucleotides. The nucleic acids include ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA), both of which can appear as a single
or double strand. The DNAs include cDNA (complementary), genomic
DNA, synthetic DNA and semisynthetic DNA. The sequence of
nucleotides or nucleic acids that code for a protein is called a
"sense" sequence. A "recombinant DNA molecule" is understood to
mean a DNA molecule which has been subjected to manipulation by
molecular biology techniques.
[0062] The term "DNA coding sequence" is understood to mean a
sequence of double-strand DNA which is transcribed and translated
into polypeptides in a cell in vitro or in vivo when it is under
the control of suitable regulatory sequences. Initiation of the
coding sequence is determined by an initiator codon at 5'
aminoterminal and the end of the translation by a stop codon at 3'
carboxyterminal. A polyadenylation signal (termination of the
transcription) would generally be located at 3' of the coding
sequence. The sequences coding the transcription and translation
are regulatory RNA sequences such as promoters and stimulators and,
thus, enable expression of a coding sequence in a host cell.
[0063] A "promoting" sequence is an RNA region capable of linking
the RNA polymerase in the cell and initiating transcription of the
coding sequence.
[0064] A "coding" sequence is under the control of transcriptional
and translational sequences in the cell when the RNA polymerase
transcribes the sequence coding mRNA (messenger), which is then
translated into protein.
[0065] An "expression plasmid" is an extrachromosomal, circular
double-strand DNA molecule that comprises regulatory sequences
between which a structure gene (DNA sequence corresponding to the
desired protein) is inserted. It replicates itself independently of
bacteria.
[0066] An "expression vector" comprises a nucleic acid molecule and
at a minimum an origin of independent replication and an inducible
promoter and which can be introduced specifically into host cells
such as in bacteria or eukaryote cells. Into this vector can be
inserted a coding sequence called an "insert" and corresponding to
the desired protein or peptide.
I--Method
1) Cell Culture
[0067] CPAE cells (calf pulmonary artery endothelial cells) were
provided by Dr. J. Badet (Eukaryote Cell Biotechnology Laboratory,
University of Creteil, France). These cells were cultured in an MEM
medium supplemented by 20% of fetal calf serum (FCS), 2 mM of
L-glutamine, 100 IU/ml of penicillin and 10 .mu.g/ml of
streptomycin (Gibco, Paisley, UK). All of the media referred to
below ("complete medium") contained the same concentrations of
penicillin/streptomycin and L-glutamine as that used for the CPAE
cells. They are used in passages 12-20. The HMEC-1 cells (human
microvasculature endothelial cells) were provided by Dr. Ades
(Centers for Disease Control and Prevention, Atlanta, Ga.) who
established this cell line by transfecting human dermal endothelial
cells with the gene SV40 and the large antigen T. The HMEC-1 cells
were cultured in complete MCDB 131 medium (Sigma, St Louis, Mo.)
supplemented with 10% of FCS, 10 ng/ml of EGF (Biomedical Products
Collaborative) and 1 .mu.g/ml of hydrocortisone (Sigma). The HUVEC
cells (human umbilical cord vein endothelial cells) were extracted
in the umbilical cord laboratory according to the method described
by Jaffe et al. The cords were subjected to controlled digestion by
0.2% collagenase A (Boehringer GmbH, Mannheim, Germany). The
primary cells were cultured in complete M199 medium supplemented by
20% of FCS, 75 mM HEPES, 3.7 mM of sodium bicarbonate pH 7.5 and 5
.mu.g/ml of fungizone (Life Technologies, France). The HMVEC-d
cells (dermal microvasculature endothelial cells, Biowhittaker
Europe, Belgium) were cultured from passage 4 to 6 in the complete
medium supplied by the manufacturer (EGM-2MV) supplemented by 10%
FCS. The C51 cells (murine colon cancer) were cultured in complete
RPMI medium+10% FCS. The 3T3 cells (murine tumor fibroblasts) were
cultured in complete DNEM medium+10%, the MDA MB 231 cells (human
breast cancer cells) use the complete DMEM medium with the addition
of 10% of FCS as do the cancerous fibroblast cells 3T3. The B16F10
cells (murine melanoma cells) were cultured in DMEM medium+10% FCS
and 1.5 g/l of sodium bicarbonate.
[0068] The anti-disintegrin antibodies were obtained after
inoculation in the rabbit of an AMEP fragment according to the
method described by Herren et al. (Neosysteme, France). This
fragment was constituted by 12 amino acids and contained the
sequence RGDC; its molecular weight was 1.4 kDa.
2) Construction and Synthesis of Human AMEP in a Bacterial
System
[0069] A fusion protein of AMEP with glutathione S-transferase
(GST) was prepared. The 276-nucleotide cDNA fragment of SEQ ID NO.
1 which codes for AMEP (Met-420 to Glu-511) was amplified by
polymerase chain reaction (PCR). This cDNA was subcloned in the
plasmid pGEX-6P at the level of the BamH1 site (Amersham Pharmacia
Biotech). Synthesis of the fusion protein GST-AMEP was induced in
Escherichia coli DH5 .alpha. by
isopropyl-1-thio-.beta.-D-galactopyranoside (1 mM) as described by
Smith and Johnson, 1988. In brief, after lysis of the bacteria with
1% Triton X100 followed by sonication, the GST-disintegrin was
purified on affinity chromatography using glutathione-agarose and
eluted with reduced glutathione (5 mM Tris, HCl) (ph 8.0)
containing 5 mM of reduced glutathione Sigma (final pH 7.5),
prepared as needed). A single band corresponding to the molecular
weight of this fusion protein (36 kDa) was detected in SDS-PAGE
(FIG. 1a).
[0070] We cleaved the AMEP of GST using a specific protease: the
"PreScision.TM. protease" which is itself a protein coupled to a
GST (Amersham, Buckinghamshire), according to the Amersham
instructions to test the activity of the peptide corresponding to
AMEP on the different angiogenesis models in vitro.
[0071] The AMEP was then purified by affinity chromatography using
glutathione-agarose on a column according to the Sigma
instructions. The AMEP was constituted of 91 amino acids and its
estimated molecular mass was 9.7 kDa.
[0072] The purity of the AMEP was analyzed by Western blot (FIG.
1b) and high performance liquid chromatography (HPLC). The protein
concentration was determined by BCA test (Pierce, Perbio, Science,
France).
3) Western Blot
[0073] One hundred micrograms of purified AMEP were deposited on an
electrophoresis gel constituted by 12% polyacrylamide-SDS and
transferred onto a nitrocellulose membrane (Schleicher and
Schuell). The membrane was saturated for 1 h with a TBS buffer
(Tris buffer solution: Tris 10 mM pH 7.5; NaCl 200 mM/Tween (0.05%)
containing 10% milk then incubated with a polyclonal rabbit serum
at the dilution 1:1000 directed against AMEP (Neosystem, France) or
with an anti-GST antibody (Amersham). After five washings in
TBS/Tween, the membrane was incubated for 1 h with a suitable
secondary antibody coupled with peroxidase at the dilution of
1:2000 according to the manufacture's instructions (DAKO). The
membrane was washed five times in the same rinsing buffer and
detection of the signal was performed according to the ECL
chemoluminescence method (Amersham).
4) Adhesion of the Fibrinogen to Vitronectin and Fibronectin
[0074] First technique: punctual incubation of the endothelial
cells with AMEP during the test.
[0075] The CPAE were detached from the culture plates by incubation
with 1.5 mM EDTA and resuspended at the rate of 5.times.10.sup.5
cells/ml in the adhesion buffer (140 mM NaCl, 10 mM Hepes, 5 mM
glucose, 5.4 mM KCl, 2 mM CaCl.sub.2, 1 mM MgCl.sub.2, 1 mM
MnCl.sub.2, pH 7.4). Then, 100 .mu.l of cell suspension was brought
into the presence of AMEP (5 .mu.g/ml) at ambient temperature then
introduced into the wells of a 96-well plate (Greiner, D. Dutcher)
that had been incubated in advance for one night at 4.degree. C.
with 50 .mu.l of purified fibrinogen (40 .mu.g/ml in PBS; Kabi), 10
.mu.g/ml of vitronectin (Sigma); 30 .mu.g/ml of fibronectin (Sigma)
and 1% of BSA (bovine serum albumin) as negative control. After 20
minutes of incubation of the CPAE cells at 37.degree. C., the plate
was washed twice with 200 .mu.l of adhesion buffer and the
nonspecific sites were saturated with PBS supplemented with 1% of
BSA for 1 hour at 37.degree. C. The plate was then washed again
twice with the adhesion buffer containing 1% of BSA. The
nonadherent cells were eliminated by washing the wells three times
with 200 .mu.l of adhesion buffer plus 1% of BSA. Measurement of
the phosphatase activity of the cells enabled quantification of the
adherent cells. In brief, 100 .mu.l of paranitrophenol phosphate
(Sigma) at 3 mg/ml in the acetate buffer pH containing 0.1% of X100
Triton was added to the wells and incubated for 2 h at 37.degree.
C. The reaction was stopped by the addition of 1N NaOH. The release
of paranitrophenol, which indicates the number of adherent cells,
was measured after reading of the absorbance at 405 nm with an
ELISA reader (Titertek Twinreader). Each experiment was performed
three times.
[0076] Second technique: the CPAE cells were cultured for 24 h in
the presence of AMEP at the final concentration of 5 .mu.g/ml
before being detached. The remainder of the protocol was identical
to the that described above with the exception that no additional
incubation of the cells with AMEP was performed.
5) Model of Endothelial Cell Migration
[0077] The migration model was implemented in 24-well plates. 1.5%
agarose was dissolved in the culture medium to form a gel in the
wells. Half of an agarose cylinder was then inserted in a well. The
CPAE cells were added to the free space left in the well and
cultured until their confluence. The piece of agarose was then
removed to allow the cells to migrate and AMEP was added, at the
desired concentrations, to the culture medium. Then, a transparent
graph paper was placed under the plate to determine the migration
rate of the cells using an ocular micrometer under reverse
microscope. The experiments were performed in duplicate and
repeated three times. The results were expressed in percentage in
relation to the control.
6) Model of Cellular Proliferation
[0078] The cells were cultured at the rate of 20,000 cells per well
(96-well plate, Greiner) in the complete medium. After 24 h, the
cells were cultured in a medium containing a concentration lower by
half than that corresponding to the complete medium to induce the
cell in phase G0/G1 of proliferation for 24 supplementary hours.
The cells were then cultured for 30 h with complete medium in the
presence or without the presence of AMEP. Tritiated thymidine (1
.mu.Ci per well) was then added to the cells and incubated for 18
hours. Incorporation of the tritiated thymidine by the cells was
quantified by filter paper according to the protocol described for
the use of a Skatron (Skatron, Lier, Norway). Radioactivity was
then determined by counting after addition of scintillation liquid.
The results were expressed as a percentage in relation to the
control.
7) Analysis of Apoptosis and Cell Cycle (Use of Hoechst 33342) and
Quantification of Early Apoptosis (Use of Annexin V)
[0079] Technique of specific vital staining of DNA with Hoechst
33342 (Sigma).
[0080] The endothelial cells (CPAE) were trypsinated and the cell
suspension was adjusted to 110.sup.6 cells/ml. Hoechst 33342 dye
was added at the rate of 20 .mu.g/ml and the cells were incubated
for three minutes at ambient temperature under agitation. The
percentage of apoptotic cells was analyzed by a flow cytometer
(FACS). The cells were then incubated for 30 minutes in darkness
(37.degree. C.) and analysis of the cell cycle was performed.
[0081] Technique using annexin V (R and D system): A residue of
1.10.sup.6 endothelial cells (CPAE) was resuspended in 1 ml of
reaction buffer (100 .mu.A of 10.times. binding buffer (100 mM
Hepes/NaOH pH 7.4, 1.5 M NaCl, 50 mM KCl, 10 mM MgCl.sub.2, 18 mM
CaCl.sub.2), 100 .mu.l of propidium iodide (initial concentration
50 .mu.g/ml), 10 .mu.l of annexin V-FITC and 790 .mu.l of deionized
water. The suspension was incubated for 15 minutes in darkness at
ambient temperature. The percentage of apoptotic cells was analyzed
by flow cytometry.
8) Formation of Capillary Structures in Two Models of Fibrin Gel
Angiogenesis
[0082] One of the models used the aggregates of CPAE cells
according to the method described by Pepper et al., 1991. In brief,
10,000 CPAE were aggregated for 24 hours on 2% agarose on a 96-well
plate. Three aggregates collected and incorporated in a fibrin gel:
purified fibrinogen (3 mg/ml; Kabi) was dialyzed against MEM medium
then mixed with 20% of FCS, 1% of L-glutamine, 1% of
penicillin/streptomycin, 2 .mu.M of aprotinin and AMEP at the
desired concentration. Human thrombin (1 IU/ml; Sigma) was then
added to obtain a fibrin gel to the surface of which was added the
complete medium supplemented by aprotinin (2 .mu.M), with this
medium being changed every three days. Formation of capillary
structures could be observed after 24 hours of culture. These
capillaries were photographed under reverse microscope and the size
of these structures was measured on the photographs. The
statistical analysis (Mann-Whitney method) made it possible to
determine whether the size of these structures was different.
[0083] The second model used beads on the order of 150 .mu.m
according to the technique described by Nehl et al. The cells used
were HMEC-1 since CPAE could not be used in this model. The HMEC-1
cells cannot, however, be employed in the previously described
model. The HMEC-1 cells adhere to the Cytodex 3 beads (Sigma) when
the cells are incubated with the beads in the complete medium for 4
h at 37.degree. C. The beads were then resuspended in a large
volume of complete medium to have 30 cells/beads and agitated for 5
minutes every 30 minutes at 30 rpm for 12 h, followed by a
continuous culture at the same rate for 4 days. When the entire
surface of the beads was covered by cells, the beads were
centrifuged at 800 g for 5 minutes to concentrate and incorporate
them in a fibrin gel the same as that described for the preceding
model, and the same as the methods of quantification and analysis
of the results. In contrast to the model used for the CPAE, the
capillary structures only appeared after 3 days of culture at
37.degree. C.
9) Preparation of the Plasmids for Electrotransfer
[0084] The cDNA of AMEP was subcloned at the Eco RV site of the
vector pBi (Clonetech, Palo Alto, Calif., USA). The expression of
the gene of interest in this vector was under the dependence of a
promoter responding to tetracycline in an expression system
involving the eukaryote gene Tet-On. The Tet-On vector expresses
the transactivator rtTA (reverse tetracycline transcriptional
activator) and the vector Tet-tTS expresses the silencer tTS
(tetracycline transcriptional silencer). The purifications of the
plasmids were performed in a manner such that no endotoxin was
present (Maxi endo free Kit, Quiagen). The purified plasmid DNA was
dissolved in endotoxin-free sterile 0.9% NaCl at the desired
concentration.
10) Electrotransfer of the Gene Coding for Human AMEP in the Muscle
of Nude and C57B1/6 mice
[0085] 20 .mu.g of plasmid pBi-AMEP, 10 .mu.g of plasmid Tet-off
and 20 .mu.g of plasmid Tet-on were dissolved in 30 .mu.l of
sterile 0.9% NaCl and injected into the tibia cranial muscle of
nude or C57B1/6 mice aged 8 weeks and previously anesthetized by
intraperitoneal inoculation of pentobarbital as described by Mir et
al. (Mir et al., 1999). In brief, 8 electric shocks of 200 V/cm
were applied for 20 ms at a frequency of 1 Hz, by means of an
electrode to the mouse paw and containing two steel plates. The
electrode was connected to an electropulsator PS-15 (Jouan, St
Herblain, France). The same plasmids not containing the AMEP gene
constituted the negative control.
11) Athymic Murine Model of Tumor Growth (MDA MB 231 Cells)
[0086] The MDA-MB-231 cells, which had been previously cultured to
80% confluence, were detached, washed and resuspended in PBS at the
rate of 2010.sup.6 cells/ml. Two hundred microliters of cell
suspension were injected subcutaneously in the backs of 8-week-old
nude mice which had been previously treated as described above. The
measurements of two diameters of a tumor enabled calculation of its
volume according to the mathematical formula (sum of the two
diameters divided by 2).sup.3/0.52. When the tumors reached a
volume of 18 mm.sup.3, doxycycline (stable analog of tetracycline)
(Sigma-Aldrich, Saint Quentin Fallavier, France) at 200 .mu.g/ml
was added to the mice's drinking water and supplemented with 5% of
sucrose to induce expression of AMEP in the muscles of the mice.
The size of the tumors was monitored for 14 days after
induction.
12) Quantification of the Tumoral Angiogenesis
(Immunohistochemistry and Image Analysis of the Subcutaneous Solid
Tumors)
[0087] The tumor tissues were fixed in ethanol. Sections of 5 .mu.m
were prepared in paraffin. The endogenous peroxidase was
extinguished by 3% of H.sub.2O.sub.2 for 10 minutes so that the
sections could be used in immunohistochemistry. After washing the
sections with distilled water then saturation with 1:10 Optimax
serum (BioGenex, San Ramon, Calif.) for 10 minutes, the slides were
incubated for 1 h with an anti-CD31 rat antibody (endothelial cell
adhesion molecule) at 1:50. After two washings with Optimax for 4
minutes, the slides were incubated with an anti-rat goat polyclonal
antibody coupled to biotin (1/50) followed by two 4-minute washings
with Optimax. The slides were then treated with a DAB chromogenic
substrate for 10 minutes, washed with distilled water,
counterstained with Mayer's hematoxylin and mounted on Pertex. All
of the slides were immunotagged and counterstained on the same day
to ensure a standardized intensity of the tagging.
[0088] For each animal, a representative histological sample of the
sections tagged with CD31 was subjected to image analysis using an
AxiophotZeiss microscope (Germany) and a Sony 3CCD camera
(resolution 768.times.576 pixels). Selection of an
enlargement.times.100 allowed digitization of the totality of the
sample. Only the tumor tissue was taken into account, with the
necrotic and fibrinous zones being excluded. For each sample, the
totality of the surface--or 8 contiguous fields if the size of the
sample was too large--was digitized. The images were analyzed with
a specific Linux-based program producing a quantitative index from
0 to 255. The digitized color images were transformed into
different levels of gray. A theoretical image composed solely of
brown-red vessels would correspond to an index of 255, whereas an
image lacking in vessels (stained blue in its entirety) would be
associated with an index of 0. A value comprised between 0 and 255
was associated with each pixel of the image and the mean of these
values was obtained for each image. The final index for each animal
was the result of the calculation of the mean value of 8 contiguous
fields.
13) Syngenic Metastatic Tumor Model (Pulmonary Metastases, B16F10
Cells)
[0089] Doxycycline (Sigma-Aldrich) at 200 .mu.g/ml was added to the
drinking water of C57B1/6 mice to induce expression of AMEP in the
muscles of the mice three days before injection of B16F10 mouse
melanoma cells. These cells were first cultured up to 50%
confluence, detached, washed and resuspended in PBS at the rate of
410.sup.6 cells/ml. One hundred microliters of cell suspension were
injected intravenously in the retro-orbital sinus of the mice. The
mice were sacrificed 7 days after the transplant of the cells, the
cells were collected and a counting of the pulmonary metastases of
black color was performed under a binocular loupe.
II--Results
1) Inhibition of the Adhesion of Endothelial Cells by AMEP on
Fibronectin, Vitronectin and Fibrinogen
[0090] The two adhesion techniques described in the Methods section
were performed in the presence of AMEP or of a 1.4-kDa fragment of
AMEP constituted of 12 amino acids with the sequence RGDC
(Neosystem, France). This peptide ("1.4-kDa peptide") was used to
determine whether the mode of action of AMEP differs from that of a
control RGD peptide.
[0091] When the endothelial cells were incubated punctually for 30
minutes with the 1.4-kDa peptide (1 .mu.g/ml) prior to the adhesion
test, a strong reduction in the adhesion of the cells was observed
on vitronectin (49.+-.1.2% of inhibition) and on fibrinogen
(50.+-.2.4% of inhibition). This result is not surprising in that
this fragment blocks the interaction of the alpha v beta 3
integrins present at the surface of the endothelial cells at their
privileged substrates. Under these same conditions, no significant
effect of AMEP on adhesion of the endothelial cells at these
substrates was detected at a comparable molar concentration (10
.mu.g/ml, not shown).
[0092] In contrast, when the cells were preincubated for 24 h with
AMEP or with the 1.4-kDa peptide at the same molar concentration as
described above, the results obtained were reversed: AMEP inhibited
adhesion of the endothelial cells on vitronectin, fibronectin and
fibrinogen by 30% for the three substrates, whereas the 1.4-kDa
peptide had no significant effect (FIG. 2).
2) Inhibition of the Migration of Endothelial Cells by AMEP
[0093] FIG. 3 shows the appearance of endothelial cells (CPAE)
after addition of AMEP at 5 .mu.g/ml (C, D) and 10 .mu.g/ml (E, F)
compared to the control (absence of the domain: A, B) in our
migration model. A very clear morphological change in the cells is
observed in the presence of AMEP: the endothelial cells form long
pseudopodia and cohesion of the cells with each other deteriorates
with numerous cells becoming detached (10 .mu.g/ml). This
phenomenon is even more visible at the migration front of the cells
(D, F). The effect of AMEP on the displacement rate of these cells
is dose dependent (2-10 .mu.g/ml) with a complete inhibition of
cell migration at 10 .mu.g/ml (FIG. 4) in contrast to the 1.4-kDa
peptide (1 .mu.g/ml) which does not induce any inhibitory
effect.
3) Effect of AMEP on Cell Proliferation
[0094] In contrast to the 12-amino-acid fragment (1.4-kDa peptide),
which has no effect on the proliferation of endothelial cells no
matter what concentration is employed (1-100 .mu.g/ml, not shown),
AMEP strongly inhibits their proliferation (reduction of 40%)
beginning at 2 .mu.g/ml (FIG. 5). This effect is maximal at 5
.mu.g/ml with 60% inhibition of proliferation (identical percentage
at 10 .mu.g/ml). It should be noted that the polyclonal rabbit
serum, previously used in Western blot (Neosystem, France),
directed against AMEP, inhibits in a comparable manner
proliferation of these cells (65.+-.0.1%).
[0095] To analyze the specificity of action of AMEP, we studied its
effect on the proliferation of primary endothelial cells from the
macrovasculature or microvasculature as well as cancer cells known
to possess or not to possess the integrin alpha v beta 3 at their
surface, one of the known targets of AMEP. The results presented in
Table 1 below show that AMEP inhibits in a comparable manner
different types of endothelial cells, whereas it has no effect on
proliferation of cancer cells of diverse origin (fibroblastic,
breast, colon) which have little or no integrin alpha v beta 3 at
their surface (MDA MB 231, C51, 3T3, respectively). A noteworthy
effect of AMEP is advantageously observed on the proliferation of
cells expressing the integrin alpha v beta 3.
TABLE-US-00001 TABLE 1 Cell type Endothelial cells Cancer cells
Bovine Human Human Murine Murine Human CPAE HMVEC-d HMVEC 3T3 C51
MDA-MB-231 B16F10 % inhibition of cell 60.4 .+-. 3.2 54.2 .+-. 2.1
52.6 .+-. 3.1 9.1 .+-. 2.2 0.5 .+-. 1.1 17.5 .+-. 0.5 74.3 .+-. 7.0
proliferation by AMEP (5 .mu.g/ml)
[0096] Table 1 shows the effect of AMEP on the proliferation of
endothelial and cancer cells. The experiments were repeated five
time (mean.+-.SEM). The values shown represent the percentage of
inhibition of proliferation of the indicated cells by AMEP used at
the rate of 5 .mu.g/ml compared to the control (absence of AMEP)
performed under the same conditions.
4) Demonstration of the Proapoptotic Activity of AMEP on
Endothelial Cells
[0097] Two techniques were employed to determine the percentage of
apoptotic cells. One technique used Hoechst 33342 to keep the cells
alive and monitor the phases of the cell cycle. The technique using
annexin-V enabled determination of early apoptosis of the
endothelial cells (visualization of the serine phosphatidyls at
their surface). The results obtained are comparable, i.e., a
percentage of apoptotic cells multiplied by 3 in the presence of
AMEP (Table 2 below). In contrast, contrary to most molecules that
induce apoptosis, no modification of the cell cycle was found with
Hoechst 33342 in the presence of AMEP (not shown).
TABLE-US-00002 TABLE 2 Hoechst 33342 Annexin-V method method % of
apoptotic Control 3.9 .+-. 1.1 4.6 .+-. 0.9 cells AMEP 5 .mu.g/ml
12.6 .+-. 3.7 12.8 .+-. 2.5
[0098] Table 2 shows the percentage of cells in apoptosis. The flow
cytometry analyses were performed according to the two methods
described in the Materials and methods section. The experiments
were performed three times. Mean.+-.SEM.
5) AMEP Inhibits the Formation of Capillary Structures in Two
Fibrin Gel Angiogenesis Models
[0099] Endothelial cells of the microvasculature are more suitable
for studying angiogenesis which is why we used HMEC-1 cells (Nehls
and Herrmann, 1995). These cells were cultured on beads and then
incorporated in a fibrin gel (FIG. 6, control: A, B). The
inhibitory effect of AMEP (5 .mu.g/ml) on formation of capillary
structures was observed after three days of culture (C) and became
spectacular after 10 days with a 90% reduction in the size of the
tubes (D). Since the prior studies were intended to determine the
effects of AMEP on different stages of angiogenesis employing CPAE
as endothelial cells, we wanted to verify the effect of this domain
on another model of angiogenesis. These cells can only be used in
the model using beads because their morphology is not adapted.
Aggregation of CPAE was the sole means of studying the effect of
AMEP on angiogenesis in vitro. The capillary structures appeared 24
h after incorporation of the aggregates in the fibrin gel. FIG. 7
brings together photographs taken after 3 days of incubation. The
addition of AMEP at 5 .mu.g/ml (B) or 10 .mu.g/ml (C, E) induced a
disorganization of capillary structures compared to the control (A,
D) leading to the death of the endothelial cells (not shown). Under
the control conditions, an increase in the length of the structures
was observed up to 6 days of incubation (not shown).
6) Inhibition of Tumor Growth and Tumor Angiogenesis by AMEP on
Nude Mice.
[0100] Production of AMEP in nude mice was obtained after
electrotransfer of the gene coding for AMEP in the mouse muscle,
followed by induction of its expression by doxycycline.
[0101] As shown in FIG. 8, the tumor volume of the AMEP group is
markedly smaller than that of the control group with a confirmed
inhibition that reached 78% after 14 days of treatment. A similar
percentage of inhibition was observed after only 7 days of
treatment. Quantification of the intratumoral angiogenesis was
implemented on the sections of these tumors. The results presented
in Table 3 (below) show that the powerful inhibitory effect of AMEP
on tumor growth is correlated with a significant inhibition of the
number of vessels within the tumors treated with AMEP of 53.4%.
[0102] The results show that AMEP acts powerfully on the in vivo
tumor models not expressing the integrin alpha v beta 3.
[0103] Table 3 below shows the statistical index of tumor
vascularization obtained after digitization and computerized image
analysis of a representative sample of tumor sections originating
from mice expressing or not expressing AMEP.
TABLE-US-00003 TABLE 3 Control group Group treated with AMEP Mean
2.96 1.38 Variance 0.28 0.14
7) Inhibition of the Formation of Pulmonary Metastases by AMEP on
Syngenic Mice
[0104] Production of AMEP in the muscle of C57B1/6 mice was induced
by doxycycline. An exceptional inhibition of the number of
pulmonary metastases of 74.2% after 7 days of treatment was
observed in the group of mice treated by AMEP compared to the
control group (FIGS. 9 and 10).
[0105] Endothelial cells are activated and acquire an angiogenic
phenotype during the angiogenic process. They then possess at their
surface the integrin alpha v beta 3 and metargidin (molecules
undetectable on endothelial cells stemming from mature vessels)
(Herren et al., 1997).
[0106] The set of results obtained show that AMEP possesses an
antiangiogenic activity that is greater than that of the 1.4-kDa
peptide. Given that both AMEP and the 1.4-kDa peptide possess an
RGD sequence implicated in bonding endothelial cells to alpha v
beta 3 integrins, we believe that the action of AMEP is not limited
to blocking the functions of the alpha v beta 3 integrin. AMEP
appears to possess its own activity which could be linked to
modifications of the signalization at the cellular level (message
that could be transported by the integrin alpha v beta 3 and/or
metargidin).
[0107] Cell adhesion is a phenomenon which intervenes in cell
migration. However, we believe that inhibition of adhesion is not
the sole mechanism responsible for inhibition of migration of
endothelial cells. Thus, 10 .mu.g/ml of AMEP is sufficient to
totally block migration of endothelial cells, whereas, at this same
concentration, the inhibitory effect of AMEP on adhesion is only
partial.
[0108] This exceptional inhibitory activity of AMEP on the key
stages of angiogenesis is reinforced by its antiproliferative
effect (up to 60% inhibition of endothelial cells). It is
noteworthy that inhibition of proliferation of endothelial cells
induced by AMEP is not associated with a detectable modification of
the cell cycle.
[0109] During the final stage of angiogenesis, the cells are
organized into tubes which anastomose together to enable formation
of a vascular lumen. We have recreated this phenomenon in vitro
with microvasculature endothelial cells (HMEC-1) and
macrovasculature endothelial cells (CPAE) by means of the two
techniques described in the Materials section. In a remarkable
manner, total inhibition of the formation of capillary structures
was observed in the model using HMEC-1 in the presence of AMEP. In
contrast to that which we had observed until now with other
inhibitors of angiogenesis, AMEP induces a lethal disorganization
of the CPAE tubes previously formed on an early basis (12 h). The
CPAE have a proliferation and migration rate higher than that of
the HMEC-1 (not shown), which can explain the duality of the effect
of AMEP on the two angiogenesis models.
[0110] These research studies also show that inhibition of tumoral
angiogenesis in vivo by AMEP leads to an exceptional inhibition of
tumor growth, even for tumors known to not express the integrin
alpha v beta 3. Moreover, a pronounced antimetastatic effect of
AMEP in a pulmonary metastasis model was demonstrated.
[0111] Finally, the treatment of endothelial cells with 10 .mu.g/ml
of AMEP induced an augmentation of the number of dead endothelial
cells (floating in the culture medium) that could be seen with the
phase-contrast microscope (FIG. 3). Nevertheless, the effect of
this product on apoptosis (phenomenon quantifiable on still living
cells) was modest (augmentation by a factor of 3). AMEP, thus, has
the particular characteristic of associating a powerful
antiangiogenic effect with an induction of cell death by a
phenomenon recently described under the name of anoikis (Frisch,
2000; Zhu et al., 2001).
[0112] The innovative aspect of AMEP, thus, is based on its
capacity to inhibit the stages of angiogenesis including the
migration and proliferation of endothelial cells, which
differentiates it from other molecules discovered to date (cf.
angiostatin which inhibits the proliferation of endothelial cells,
O'Reilly et al., 1994; Wu et al., 1997; Sim et al., 1997) or
endostatin which also inhibits their proliferation as well their
migration, but solely when this migration is induced by an
angiogenic factor such as VEGF or bFGF (O'Reilly et al., 1998, Sim
et al., 2000; Yamaguchi et al., 1999). Moreover, the respective
effects of AMEP on migration and angiogenesis in vitro are
spectacular: substantially complete shutdown of the mobility of the
endothelial cells and absence of formation of capillary
structures.
[0113] Unexpectedly, the powerful inhibitory effects of AMEP,
synthesized in the form of recombinant protein in bacteria, on the
total set of in vitro experiments described were confirmed by the
results obtained in vivo performed with AMEP synthesized de novo in
mammals. The inhibitory effect of AMEP on tumor growth in the
athymic model is advantageously seen in relation to a diminution of
the number of intratumoral vessels, a direct consequence of the
inhibition by AMEP, in all stages of angiogenesis in vitro.
Moreover, the anti-invasive effect of AMEP on formation of
pulmonary metastases using melanoma cells correlated with the
particular antiproliferative effect of AMEP on these same cells in
vitro.
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Sequence CWU 1
1
21276DNAHomo sapiensCDS(1)..(276)Coding sequence for the
disintegrin domain of the metargidin 1atg gct gct ttc tgc gga aat
atg ttt gtg gag ccg ggc gag cag tgt 48Met Ala Ala Phe Cys Gly Asn
Met Phe Val Glu Pro Gly Glu Gln Cys1 5 10 15gac tgt ggc ttc ctg gat
gac tgc gtc gat ccc tgc tgt gat tct ttg 96Asp Cys Gly Phe Leu Asp
Asp Cys Val Asp Pro Cys Cys Asp Ser Leu 20 25 30acc tgc cag ctg agg
cca ggt gca cag tgt gca tct gac gga ccc tgt 144Thr Cys Gln Leu Arg
Pro Gly Ala Gln Cys Ala Ser Asp Gly Pro Cys 35 40 45tgt caa aat tgc
cag ctg cgc ccg tct ggc tgg cag tgt cgt cct acc 192Cys Gln Asn Cys
Gln Leu Arg Pro Ser Gly Trp Gln Cys Arg Pro Thr 50 55 60aga ggg gat
tgt gac ttg cct gaa ttc tgc cca gga gac agc tcc cag 240Arg Gly Asp
Cys Asp Leu Pro Glu Phe Cys Pro Gly Asp Ser Ser Gln65 70 75 80tgt
ccc cct gat gtc agc cta ggg gat ggc gag taa 276Cys Pro Pro Asp Val
Ser Leu Gly Asp Gly Glu 85 90291PRTHomo sapiens 2Met Ala Ala Phe
Cys Gly Asn Met Phe Val Glu Pro Gly Glu Gln Cys1 5 10 15Asp Cys Gly
Phe Leu Asp Asp Cys Val Asp Pro Cys Cys Asp Ser Leu 20 25 30Thr Cys
Gln Leu Arg Pro Gly Ala Gln Cys Ala Ser Asp Gly Pro Cys 35 40 45Cys
Gln Asn Cys Gln Leu Arg Pro Ser Gly Trp Gln Cys Arg Pro Thr 50 55
60Arg Gly Asp Cys Asp Leu Pro Glu Phe Cys Pro Gly Asp Ser Ser Gln65
70 75 80Cys Pro Pro Asp Val Ser Leu Gly Asp Gly Glu 85 90
* * * * *