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Cancer Patent Abstract
Use of an antagonist compound of protein ESM-1 for the production
of a drug for the treatment of a cancer.
Cancer Patent Claims
The invention claimed is:
1. A method of inhibiting or blocking the tumorigenic capacity
of ESM-1 protein in a cancer patient, wherein said method comprises
administering to the patient a pharmaceutical composition comprising
(i) a monoclonal antibody produced by the hybridoma line deposited
at the Collection National de Cultures de Microorganismes of the
Institut Pasteur under accession No. I-1941, also named MEP08, in
an amount that inhibits or blocks the tumorigenic capacity of ESM-1
protein in vivo, and (ii) one or more pharmaceutically acceptable
vehicles.
Cancer Patent Description
SCOPE OF THE INVENTION
The present invention relates to the fields of prevention and/or
treatment of cancers.
STATE OF THE ART
Despite huge financial and human investments, cancer remains one
of the major causes of death.
Cancer is frequently a disease associated with defects in the system
of intracellular signaling. Normal cells respond to numerous extracellular
signals by proliferating, differentiating or more generally changing
their metabolic activity. Such signals are received on the surface
of the cells and converted by a system of signal transduction proteins
into a message recognized by the cell. This message is responsible
for subsequent cell regulation phenomena.
Metastasis is the formation of a secondary tumour colony at a site
distant from the initial tumour. It represents a multi-step process
for which the tumoral invasion is an early event. The tumour cells
escape locally across the barrier tissues, such as the basal membrane
of the epithelium, and reach the interstitial stroma, from which
they gain access to the blood vessels or the lymph canals before
subsequent dissemination. After having invaded the endothelial layer
of the vascular wall, the circulating tumour cells are carried around
by the blood circulation and are stopped in the precapillary venules
of the target organ by adhesion to the lumen surfaces of the endothelial
cell, or are exposed to the basal membranes. The tumour cells leave
the vascular wall and enter into the parenchyma of the organ. Finally,
the tumour cell, after extravasation, multiplies in a different
tissue from that in which it originated.
It has been shown that some cancers are caused by defects associated
with genes responsible for the transduction of the signal. Such
genes are called oncogenes. These oncogenes may lead to an overexpression
of one or more signal transduction proteins inducing an abnormal
cell proliferation. The defective signals may be linked to various
mechanisms.
Some anticancer therapies aim to inhibit the expression or the
bioavailability of the oncogenic proteins responsible for the proliferation
of the cancer cells, such as the proteins of the MAP kinase family
or the products of certain oncogenes such as c-myc.
Protein ESM-1 is a polypeptide of 184 amino acids secreted by the
endothelial cells and which was described for the first time by
LASSALLE et al. (1996). The messenger RNAs coding for protein ESM-1
are mainly found in the endothelial cells and in pulmonary and renal
tissues. The expression of the gene coding for ESM-1 is regulated
by the cytokines. TNF-.alpha. and L'IL-1.beta. induce an increase
in the expression of the ESM-1 gene in the endothelial cells of
the human umbilical vein, while .gamma.-Interferon reduces its expression.
A high level of circulating protein ESM-1 has been found in patients
presenting a systemic inflammatory syndrome, such as septic shock
(BECHARD et al., 2000).
Current hospital treatment for cancer predominantly makes use of
radiation and/or chemotherapeutic agents, such as vinblastine or
adriamycine. However, the widely known undesirable effects of such
treatments render these strategies very difficult for the patient
to support.
The object of the present invention is to supply anti-cancer compounds
which overcome the disadvantages of the methods of therapeutic treatment
of cancer in the state of the art.
SUMMARY OF THE INVENTION
A first object of the invention consists of the use of an antagonist
compound of protein ESM-1 for the production of a drug for the treatment
of a cancer.
According to a first embodiment, an antagonist compound of the
invention is an antibody specifically binding to protein ESM-1.
According to a second embodiment, an antagonist compound used in
the scope of the invention is a peptide of at least 10 amino acids
of a modified protein ESM-1 and which contains the amino acid grouping
Ala(134)-Ala(135).
According to a third embodiment, an antagonist compound of protein
ESM-1 consists of an antisense oligonucleotide hybridizing with
the cDNA coding for ESM-1.
A further object of the invention consists of an antagonist compound
of protein ESM-1, chosen from among the antagonist compounds defined
above.
The invention also relates to a pharmaceutical composition intended
for the treatment of cancer comprising an antagonist compound of
protein ESM-1.
Another object of the invention consists of a method for preventing
cancer comprising a step in which an antagonist compound of protein
ESM-1 is administered.
The invention also concerns a method for the therapeutic treatment
of cancer comprising a step in which an antagonist compound of protein
ESM-1 is administered.
DETAILED DESCRIPTION OF THE INVENTION
It has been shown for the first time according to the invention
that protein ESM-1 is secreted in humans in the form of a proteoglycan
of the chondroitin/dermatan sulfate type and that the secreted protein
ESM-1 is able to stimulate in vitro the mitogenic activity of the
factor HGF/SF (Hepatocyte growth factor/scatter factor).
HGF/SF is an important factor in the appearance of renal multicystic
dysplasias and in the appearance of hyperproliferation of the renal
tubules and has also been associated with the development of carcinomas
of the breast, kidneys and lungs and also the development of malignant
melanomas.
It has also been shown according to the invention that transfected
human renal epithelial cells expressing protein ESM-1 have a strong
tumoral potential and cause the appearance of a renal carcinoma
in vivo in mice. It has also been shown that antibodies directed
against protein ESM-1 were able to inhibit the development of a
renal tumour in vivo and that a peptide antagonist of protein ESM-1
had the same anti-tumoral activity.
In addition, an increase of the serum level of protein ESM-1 in
patients with a broncho-pulmonary carcinoma has been shown according
to the invention
In consequence, a first object of the invention consists of the
use of an antagonist compound of protein ESM-1 for the production
of a drug for the prevention and/or treatment of cancer.
GENERAL DEFINITIONS
The expressions "protein ESM-1" or "polypeptide
of ESM-1", in the context of the invention, include a polypeptide
of 184 amino acids referenced as sequence SEQ ID N.sup.o1 in the
list of sequences, and also a polypeptide of 165 amino acids identical
to the polypeptide of sequence SEQ ID N.sup.o1 in which the 19 amino
acids of the N-terminal end corresponding to the signal peptides
are absent, this polypeptide of 165 amino acids comprising the secreted
form of the polypeptide of sequence SEQ ID N.sup.o1. Also included
in the definition of "protein ESM-1" and "polypeptide
of ESM-1" respectively are a glycopeptide of 184 amino acids
of sequence SEQ ID N.sup.o1 and a polypeptide of 165 amino acids
corresponding to the sequence running from the amino acid in position
20 to the amino acid in position 184 of the sequence SEQ ID N.sup.o1
whose serine residue in position 137 has been modified by O-glycosylation,
the O-glycosylated forms of the protein ESM-1 being also designated
"glycopeptides" in the present description. The ESM-1
glycopeptide preferably has the serine residue in position 137 which
is O-glycosylated by a chondroitin/dermatan sulfate group.
By "antagonist compound" of protein ESM-1, should be
understood according to the invention a compound able significantly
to reduce the bioavailability of protein ESM-1 compared to target
molecules onto which protein ESM-1 naturally fixes. An antagonist
compound of protein ESM-1 may reduce the bioavailability of these
proteins by reducing the probability of the binding of protein ESM-1
to the target molecules of the organism onto which it naturally
fixes. An antagonist compound according to the invention may reduce
the bioavailability of protein ESM-1 by inhibiting or blocking the
transcription of the gene coding for ESM-1, by inhibiting or blocking
the translation of the corresponding messenger RNA, by modifying
the intracellular maturation of protein ESM-1, for example by affecting
the enzymatic process leading to its glycosylation, or by inhibiting
or blocking the secretion of the mature protein ESM-1.
A first object of the invention consists of the use of an antagonist
compound of protein ESM-1 for the production of a drug for the treatment
of a cancer.
An antagonist compound of protein ESM-1 may be of any type, polypeptide,
saccharide, or any organic or inorganic compound causing the reduction
of the bioavailability of protein ESM-1 compared to the target molecules
onto which this protein fixes.
Antagonist Compounds of Protein ESM-1 of the Antibody Type
A first family of preferred antagonist compounds of ESM-1 according
to the invention is composed of antibodies specifically binding
to protein ESM-1.
It has been shown according to the invention that antibodies directed
specifically against protein ESM-1 are able to inhibit or block
the tumorigenic power of this protein. Anti-ESM-1 antibodies thus
constitute antagonist compounds of major therapeutic value.
By "antibody" in the context of the invention, should
be understood in particular polyclonal or monoclonal antibodies
or their fragments (for example the fragments Fab or F(ab)'.sub.2)
or any polypeptide containing a domain of the initial antibody recognizing
protein ESM-1.
Monoclonal antibodies may be prepared from a hybridoma according
to the technique described by KOHLER and MIELSTEIN (1975).
They may also be antibodies directed against ESM-1 or a fragment
of this protein produced by the trioma technique or the hybridoma
technique described by KOZBOR et al. (1983).
They may also be single chain Fv antibody fragments (ScFv) such
as those disclosed in the U.S. Pat. No. 4,9476,778 or by MARTINEAU
et al. (1998).
Anti-ESM-1 antibodies according to the invention also comprise
fragments of antibodies obtained using phage banks such as described
by RIDDER et al. (1995) or human antibodies such as described by
REINMANN et al. (1997) or by LEGER O J, et al., 1997.
They may also be anti-ESM-1 antibodies produced according to the
techniques described by BECHARD et al. (2000). The antibodies described
by BECHARD et al. (2000) are monoclonal antibodies secreted by hybrdoma
lines prepared from mouse spleen cells previously immunized against
the C-terminal fragment of molecular weight 14 kD of ESM-1 which
has been produced in Escherichia coli, in other words a nonglycosylated
fragment of protein ESM-1. By epitope mapping, BECHARD et al. (2000)
were able to classify the monoclonal antibodies produced by different
hybridoma lines according to the region of protein ESM-1 recognized
by them.
A first preferred family of antibodies according to the invention
which comprises antagonist compounds of protein ESM-1 are the monoclonal
antibodies specifically recognizing the region running from the
proline residue in position 79 up to the cysteine residue in position
99 of sequence SEQ ID N.sup.o1, this region representing the antigenic
determinant D1. They are preferably monoclonal antibodies produced
by the hybridoma line deposited at the Collection Nationale de Cultures
de Microorganismes of the Institut Pasteur (CNCM) under the access
number N.sup.oI-1944, also named antibody MEP21.
Other preferred monoclonal antibodies are those specifically binding
to the part of protein ESM-1 contained between the glycine residue
in position 159 and the arginine residue in position 184 of sequence
SEQ ID N.sup.o1 which is the region comprising the antigenic determinant
D3. Specific preferred monoclonal antibodies of the antigenic determinant
D3 may be obtained from the hybridoma line I-1943 (MEP19), deposited
on 19 Nov. 1997 at the Collection Nationale de Cultures des Micro-organismes
of the Institut Pasteur (CNCM).
Other preferred monoclonal antibodies according to the invention
are the monoclonal antibodies specifically binding to the region
contained between the serine residue in position 119 and the valine
residue in position 139 of protein ESM-1 of sequence SEQ ID NO:1,
this region being defined as the antigenic determinant D2 of protein
ESM-1. Preferred monoclonal antibodies specifically binding to antigenic
determinant D2 of ESM-1 may be obtained from the hybridoma line
MEP08 deposited on 19 Nov. 1997 at the Collection Nationale de Cultures
de Micro-organismes of the Institut Pasteur (CNCM), at 28 Rue du
Docteur Roux, F-75724,Paris, Cedex 15 under Accession No. I-1941.
Other monoclonal antibodies of interest constituting antagonist
compounds of protein ESM-1, within the scope of the invention, are
the monoclonal antibodies specifically directed against the N-terminal
part of protein ESM-1. The preferred monoclonal antibodies directed
against the N-terminal part of protein ESM-1 may be obtained from
the hybridoma line MEC15 deposited at the Collection Nationale de
Cultures des Micro-organismes of the INSTITUT PASTEUR (CNCM) on
17 Oct. 2000 under the access number I-2572.
According to a preferred embodiment, the anti-ESM1 antibodies having
the best antagonist activities against ESM-1 are chosen from among
the antibodies specifically recognizing the epitopes localized in
the region around the phenylalanine residue in position 115. They
are in particular the antibodies specifically binding to the region
contained between the serine residue in position 119 and the valine
residue in position 139 of protein ESM-1 of sequence SEQ ID N.sup.o1,
such as the monoclonal antibody MEP08 described above.
It has been shown according to the invention that the monoclonal
antibody MEP08 is able to inhibit the pro-tumoral activity of protein
ESM-1 on the formation of tumours caused by the proliferation of
human cells of renal origin in mice.
Polypeptide Antagonists of Protein ESM-1
It has been shown according to the invention that the region containing
the antigenic determinant D2 of protein ESM-1 is important for the
pro-tumoral activity of protein ESM-1.
In particular, the applicant has synthesized a polypeptide derived
from protein ESM-1 in which the phenylalanine residues in positions
134 and 135 of sequence SEQ ID N.sup.o1, in other words the residues
in positions 115 and 116 of the secreted protein ESM-1, have been
replaced by two alanine residues. The applicant has shown that this
modified polypeptide was not able to induce tumours in mice. Such
a modified polypeptide could thus compete with protein ESM-1, produced
at a high level in cancer patients, for its potentializing action
with growth factors such as HGF/SF or growth factors FGF-2 and FGF-7.
The antagonist compounds of protein ESM-1 include polypeptides
with a length of at least 10 consecutive amino acids of sequence
SEQ ID N.sup.o1, which includes a sequence of amino acids running
from the amino acid in position 119 up to the amino acid in position
139 of sequence SEQ ID N.sup.o1, such an antagonist polypeptide
of ESM-1 containing at least one substitution of an amino acid,
compared to the sequence corresponding to protein ESM-1.
An antagonist polypeptide of protein ESM-1 such as defined above
preferably has at the most 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 30, 35, 40, 45 or 50 consecutive amino acids
of sequence SEQ ID N.sup.o1 and at least one substitution of amino
acids, compared to sequence SEQ ID n.sup.o1.
An antagonist polypeptide of protein ESM-1, such as defined above,
contains at the most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions
of an amino acid, compared to the sequence SEQ ID N.sup.o1, the
number of substitutions of amino acids being adapted as a function
of the length of the polypeptide, it being understood that the number
of substitutions of amino acids compared to the sequence SEQ ID
N.sup.o1 in an antagonist polypeptide according to the invention
is at the most 25% of the amino acids contained in the sequence
of this antagonist polypeptide, preferably at most 20%, 15% and
more preferably at most 10% of the number of amino acids contained
in the sequence of the antagonist polypeptide of ESM-1.
A substitution of amino acids, compared to the sequence SEQ ID
N.sup.o1, in an antagonist polypeptide according to the invention
is s preferably a "non-conservative" substitution. By
"non-conservative" substitution should be understood the
substitution of an amino acid residue by an amino acid of a different
class.
Amino acids are conventionally classified according to the following
classes: non polar amino acids (hydrophobic): alanine, leucine,
isoleucine, valine, proline, phenylalanine, tryptophan and methionine;
amino acids containing aromatic rings: phenylalanine, tryptophan
and tyrosine; neutral polar amino acids: glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine; positively charged
amino acids (basic): arginine, lysine and histidine); negatively
charged amino acids (acid): aspartic acid and glutamic acid.
A preferred type of substitution of amino acids for the preparation
of an antagonist polypeptide of protein ESM-1 according to the invention
is the substitution of an amino acid containing an aromatic ring
by an amino acid not containing an aromatic ring.
An antagonist polypeptide of protein ESM-1 according to the invention
preferably contains a substitution of the phenylalanine residues
in positions 134 and 135 of SEQ ID N.sup.o1 by two amino acid residues,
identical or different, not containing an aromatic ring.
Such a preferred antagonist polypeptide of protein ESM-1 is a polypeptide
of at least 10 consecutive amino acids of sequence SEQ ID N.sup.o1,
such as defined above, in which the phenylalanine residues in positions
134 and 135 have been replaced by two alanine residues.
According to a first embodiment, an antagonist polypeptide of protein
ESM-1 according to the invention may be prepared by conventional
chemical synthesis techniques, either in homogenous solution or
in the solid phase.
As an illustration, an antagonist polypeptide of protein ESM-1
may be prepared by the homogeneous solution technique described
by HOUBEN WEIL (1974) or by the solid phase synthesis technique
described by MERRIFIELD (1965a; 1965b) and MERRIFIELD 1965b.
An antagonist polypeptide of protein ESM-1 according to the invention
may also be prepared by genetic recombination.
In order to produce an antagonist polypeptide of protein ESM-1
such as defined above, a method may be used comprising the steps
of:
a) inserting a nucleic acid coding for the antagonist polypeptide
of protein ESM-1 in an appropriate expression vector;
b) culturing, in an appropriate culture medium, a host cell previously
transformed or transfected with the recombinant expression vector
of step a);
c) recovering the culture medium or lysing the host cell, for example
by sonication or osmotic shock;
d) separating and purifying from said culture medium or cell lysates
obtained in step c), said antagonist polypeptide;
e) if appropriate, characterizing the recombinant antagonist polypeptide
thus produced.
The antagonist polypeptides according to the invention may be characterized
by fixation on an immunoaffinity chromatography column on which
the antibodies directed against this polypeptide or against a fragment
of it have previously been immobilized.
According to another embodiment, an antagonist polypeptide of ESM-1
may be purified by passage over an appropriate series of chromatography
columns, according to methods known to a person skilled in the art
and described for example by AUSUBEL F. et al. (1989).
Antagonist Compounds of Protein ESM-1 of the Antisense Oligonucleotide
Type.
Another preferred family of antagonist compounds of protein ESM-1
aiming to reduce the bioavailability of protein ESM-1 secreted in
patients at risk or in patients having already developed tumours
are compounds able to inhibit or block the expression of the gene
coding for ESM-1 in humans.
Such antagonist compounds of protein ESM-1 may be antisense polynucleotides.
The antagonist compounds of protein ESM-1 according to the invention
thus include an antisense polynucleotide able to hybridize specifically
to a given region of the gene coding for protein ESM-1 and able
to inhibit or to block its transcription and/or its translation.
The sequence of the human ESM-1 gene is referenced under the access
number AJ401 1091 and AJ401 1092 in the database Genbank.
An antisense polynucleotide according to the invention preferably
contains a sequence complementary to a sequence localized in the
region of the 5'-end of the DNA of the ESM-1 gene, and more preferably
close to the initiation codon of the translation (ATG) of the ESM-1
gene.
According to a second preferred embodiment, an antisense polynucleotide
according to the invention contains a sequence complementary to
one of the sequences localized at the exon/intron junctions of the
ESM-1 gene and preferably sequences corresponding to a splicing
site.
A preferred antisense polynucleotide according to the invention
contains at least 15 consecutive nucleotides of the cDNA coding
for ESM-1 having the nucleotide sequence SEQ ID N.sup.o2.
For the purposes of the present invention, a first polynucleotide
is considered as being "complementary" to a second polynucleotide
when each base of the first nucleotide is paired with the complementary
base of the second polynucleotide whose direction is inversed. The
complementary bases are A and T (or A and U), and C and G.
In general, an antisense polynucleotide according to the invention
has at least 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400,
500, 1000 or 2000 consecutive nucleotides of the cDNA of ESM-1 of
sequence SEQ ID N.sup.o2.
As an illustration, a preferred antisense polynucleotide according
to the invention consists of a nucleic acid of complementary sequence
to the nucleic acid of the cDNA of ESM-1 of sequence SEQ ID N.sup.o2.
An antisense polynucleotide comprising an antagonist compound of
protein ESM-1 according to the invention may be prepared by any
suitable method well known to a person skilled in the art, including
cloning and the action of a restriction enzyme or by chemical synthesis
according to techniques such as the phosphodiester method of NARANG
et al. (1979) or of BROWN et al. (1979), the diethylphosphoramidite
method of BEAUCAGE et al. (1980) or the solid support technique
disclosed in the European patent n.sup.oEP-0 707 592.
In general, antisense polynucleotides must have a length and a
melting point sufficient to allow the formation of an intracellular
duplex hybrid having sufficient stability to inhibit the expression
of the mRNA of ESM-1. Strategies to construct antisense polynucleotides
are in particular described by GREEN et al. (1986) and IZANT and
WEINTRAUB (1984).
Methods for construction of antisense polynucleotides are also
described by ROSSI and al (1991) and in the PCT applications N.sup.oWO
947/23.026, WO 95/04141, WO 92/L18.522 and in the European patent
application n.sup.o EP 0 572 287.
Other methods for the use of antisense polynucleotides are for
example those described by SCZAKIEL et al. (1995) or those disclosed
in the PCT application N.sup.oWO 95/24,223.
A skilled person may advantageously refer to the methods of production
and use of antisense polynucleotides inhibiting or blocking the
expression of genes associated with the development of cancers,
such as the techniques disclosed in the U.S. Pat. No. 5,582,986
which discloses antisense oligonucleotides for inhibiting the ras
gene, the technique described by HOLT et al. (1988) which describes
antisense oligonucleotides specifically hybridizing with messenger
RNAs of the oncogene c-myb or the technique described by WICKSTRON
et al. (1988) which describes antisense oligonucleotides specifically
hybridizing with the messenger RNA of the gene c-myc.
Other techniques for using antisense polynucleotides usable by
a skilled person are those of SALE et al. (1995) and that of GAO
et al. (1996).
Method for Selecting an Antagonist Compound of Protein ESM-1
An antagonist compound of protein ESM-1 according to the invention
may be selected by a person skilled in the art for its capacity
to inhibit the development of a tumour induced by protein ESM-1
in vivo.
According to a first embodiment, a method for selecting an antagonist
compound of protein ESM-1 comprises the following steps:
a) injecting an animal with cells able to form tumours in the presence
of protein ESM-1, said cells being transfected or transformed by
a nucleic acid able to express protein ESM-1 in vivo;
b) administering to this animal a candidate antagonist compound
of protein ESM-1;
c) comparing the formation of tumours in a first animal such as
obtained after step b) and in a second animal such as obtained after
step a); and
d) selecting the candidate compound able to inhibit or block the
formation of tumours in the first animal.
The animal used in the selection method above is preferably a non-human
mammal, advantageously a rodent, and more preferably a rat, guinea
pig or mouse.
In a particular embodiment of the method, this includes a step
e) consisting of sacrificing the first and the second animal.
Advantageously, the cell line able to form tumours in the animal
in the presence of protein ESM-1 is the line HEK 293 (ATCC N.sup.oCRL
1573).
According to a further embodiment, an antagonist compound of protein
ESM-1 according to the invention may be selected according to a
method using the demonstration of the fixation of a candidate compound
onto protein ESM-1. Such a method of selection of a candidate antagonist
compound of protein ESM-1 comprises the following steps:
a) supplying a polypeptide consisting of protein ESM-1 or a peptide
fragment of this protein;
b) placing said polypeptide in contact with the candidate compound
to be tested;
c) detecting the complexes formed between said polypeptide and
the candidate compound;
d) selecting the candidate compounds fixing onto the polypeptide
consisting of protein ESM-1 or a peptide fragment of this protein.
By "fragment" of protein ESM-1, should be understood
a polypeptide containing at least 20, preferably at least 30, 35,
40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130,
140 or 150 consecutive amino acids of the polypeptide ESM-1 of sequence
SEQ ID N.sup.o1 and containing the sequence running from the proline
residue in position 133 up to the valine residue in position 138
of SEQ ID N.sup.o1.
The invention also relates to a kit for selecting a candidate antagonist
compound of protein ESM-1, this kit comprising:
a) a purified preparation of a polypeptide consisting of protein
ESM-1 or of a fragment of this protein;
b) where appropriate, means of detection of a complex formed between
the polypeptide and the candidate compound to be tested.
The method of detection of a complex formed between the polypeptide
derived from protein ESM-1 and the candidate compound may be performed
by various techniques, such as microdialysis coupled with an HPLC
method as described by WANG et al. (1997) or affinity capillary
electrophoresis as described by BOUSH et al. (1997).
A candidate compound may be of any type, and particularly the final
product of a combinatorial chemistry method.
A. Candidate Compounds Obtained from Peptides Banks
A candidate antagonist compound of protein ESM-1 may be selected
according to the method above as an expression product of a DNA
insert contained in a phage vector according to the technique described
by PARMLEY & SMITH (1988). In this type of peptide bank, the
DNA inserts code for peptides of 8 to 20 amino acids in length,
as is described by OLDENBURG K R et al. (1992), VALADON P et al.
(1996), LUCAS A H (1994), WESTERINK (1995), FELICI et al. (1991).
According to this particular embodiment, the recombinant phages
expressing a protein able to fix onto the polypeptide consisting
of protein ESM-1 or a fragment of it are retained and the complex
formed between protein ESM-1 or a fragment of it and the recombinant
phage may be subsequently immunoprecipitated by an anti-ESM-1 monoclonal
or polyclonal antibody.
B. Candidate Compound Obtained by Competition Experiments
The candidate antagonist compounds of protein ESM-1 may also be
selected by the fact that they fix onto protein ESM-1, or onto a
polypeptide fragment of it, in competition with a previously selected
antagonist compound of protein ESM-1 such as one of the anti-ESM-1
antibodies described above, and particularly the monoclonal antibody
secreted by the hybridoma line MEPOB deposited on 19 Nov. 1997 at
the CNCM under the access number 1-1941.
Such competition experiments are for example described in the article
by BECHARD et al. (2000).
C. Candidate Antagonist Compounds of Protein ESM-1 Selected by
Affinity Chromatography.
Proteins or other molecules of any type able to fix onto protein
ESM-1, or to a polypeptide fragment of this protein, may be selected
by using affinity columns on which protein ESM-1 or a fragment of
it have previously been immobilized, for example by conventional
techniques, including the chemical coupling of protein ESM-1 or
a fragment of it with the matrix of a column such as of agarose,
or AffiGel.RTM.. A solution containing the candidate compound to
be tested is placed in contact with the chromatographic support
on which protein ESM-1 or a peptide fragment of it has been immobilized.
The compounds retained on the affinity column are positively selected.
D. Candidate Compounds Selected by Optical Biocaptor Techniques
A candidate antagonist compound of protein ESM-1 may also be selected
by using an optical biocaptor such as described by EDWARDS and LEATHERBARROW
(1997). This technique allows the detection of interactions between
molecules in real time without the necessity of using marked molecules.
This technique is based on SPR (Surface Plasmon Resonance). Briefly,
the candidate compound to be tested is fixed onto a surface, such
as a carboxymethyidextran matrix. A light ray is directed onto the
part of the surface which does not contain the sample to be tested
and is reflected by this surface. The SPR phenomenon causes a reduction
in the intensity of the reflected light with a specific association
between the angle of the reflected light and the wavelength of the
light ray. The fixation of the candidate compound causes a change
in the refractive index of the surface, the change in the refractive
index being detected as a modification of the SPR signal.
Such a detection method by optical biocaptor may also permit the
selection of candidate compounds which enter into competition with
another ligand for the fixation onto protein ESM-1 or a peptide
fragment of it.
For example, a candidate antagonist compound of protein ESM-1 includes
compounds able to inhibit the fixation of an anti-ESM-1 antibody
onto protein ESM-1, to inhibit the fixation of factor HGF-SF or
factors FGF-2 and FGF-7 onto protein ESM-1 or a peptide fragment
of this protein.
Thus, according to a further embodiment, the invention relates
to a method of selection of an antagonist compound of protein ESM-1
characterized in that it comprises the following steps:
a) Placing protein ESM-1 or a peptide fragment of it in contact
with:
(i) an antagonist compound of protein ESM-1 fixing onto protein
ESM-1; and
(ii) a candidate compound to be tested;
b) in a separate step from step a), but optionally simultaneously
with it, placing protein ESM-1 or a peptide fragment of it in contact
with an antagonist compound of protein ESM-1 fixing onto protein
ESM-1;
c) detecting the respective quantity of antagonist compound of
protein ESM-1 fixed after each of steps a) and b); and
d) selecting the candidate compound which enters into competition
with the antagonist compound for the fixation onto protein ESM-1.
An antagonist compound of ESM-1 for the use of the selection method
above is preferably an anti-ESM-1 antibody or a peptide antagonist
compound such as defined above in the present description.
In a first embodiment of a method for selecting an antagonist compound
of ESM-1 from a candidate compound, said method comprises the following
steps:
1) selecting, among the candidate compounds, the compounds which
fix onto protein ESM-1 or onto a peptide fragment of this protein;
2) administering a compound selected in step 1) to an animal and
determining the capacity of this compound to inhibit, in this animal,
the development of tumours induced by protein ESM-1;
3) selecting the compounds which inhibit the development of tumours
determined in step 2) as antagonist compounds of protein ESM-1.
Step 1) preferably consists of the use of a selection method of
a candidate compound fixing onto protein ESM-1 or onto a peptide
fragment of this protein, chosen from among the methods detailed
in the present description.
Step 2) preferably consists of the use of a selection method of
a candidate compound in vivo such as is detailed in the description.
In a particular embodiment of the method, this also contains a
step 4) consisting of sacrificing the animal.
Pharmaceutical Composition of the Invention.
A further object of the invention is a pharmaceutical composition
for the treatment and/or prevention of a cancer containing an antagonist
compound of protein ESM-1.
Pharmaceutical Composition Containing an Antagonist Compound of
the Antibody Type or of the Peptide Type According to the Invention.
According to a first embodiment, a pharmaceutical composition according
to the invention contains a therapeutically effective quantity of
an anti-ESM-1 antibody or of a peptide antagonist compound derived
from ESM-1, in combination with one or more pharmaceutically compatible
vehicles. The pharmaceutical compositions according to the invention
include those suitable for topical, oral, rectal, nasal or parenteral
(including intramuscular, subcutaneous and intravenous) administration
or in a form suitable for administration by inhalation or insufflation.
The pharmaceutical compositions according to the invention may be
presented in the form of unit doses and may be prepared by any method
well known to a person skilled in the art of pharmaceutical medicine
All the methods include a step consisting of combining the antagonist
compound comprising the active principle of the composition with
a liquid vehicle or a finely divided solid vehicle and, if necessary,
forming the product, for example in the form of tablets or capsules.
For oral administration, a pharmaceutical composition according
to the invention is preferably presented in the form of dose units
such as tablets, capsules or hard capsules. When it is presented
in a form contained in a pressurized container, the pharmaceutical
composition may contain a propellant such as dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide
or other appropriate gases. In the case of a pressurized aerosol,
the dose unit may be provided with a valve able to supply a given
quantity of the pharmaceutical composition.
According to another embodiment, the pharmaceutical composition
according to the invention may be in the form of a dry powder composition
for administration by inhalation or insufflation, for example in
the form of a mixture of a powder of the antagonist compound and
of a suitable base powder, such as lactose or starch. The powder
composition may be presented in a dose unit, for example in s the
form of capsules or dispensers from which the powder may be administered
using an inhaler or insufflator device.
A solid pharmaceutically acceptable vehicle compatible with a pharmaceutical
composition according to the invention includes substances such
as flavouring agents, lubricants, solubilizing agents, suspension
agents, fillers, compression auxiliaries, binders or dispersion
agents as well as encapsulating materials. In the powders, the vehicle
is a finely divided solid which is in admixture with the antagonist
compound of ESM-1 also in a finely divided form. In the tablets,
the active principle antagonist of ESM-1 is mixed with a vehicle
having suitable compression properties and compacted into the desired
form and size. The powders and tablets preferably contain less than
99% of the active principle. The preferred solid vehicles are for
example calcium phosphate, magnesium stearate, talc, sugars, lactose,
dextrin, starch, gelatine, cellulose, polyvinylpyrrolidone and the
ionexchange resins.
Liquid vehicles are used to prepare a pharmaceutical composition
according to the invention in the form of a solution, a suspension,
an emulsion, a syrup, an elixir and a pressurized composition. The
active principle antagonist of protein ESM-1 may be dissolved or
suspended in a pharmaceutically acceptable vehicle such as water,
an organic solvent, or a mixture of the two or pharmaceutically
acceptable oils or fats. The liquid vehicle may contain other pharmaceutically
acceptable additives such as solubilizing agents, emulsifiers, buffers,
preservatives, sweeteners, flavouring agents, suspension agents,
thickening agents, colorants, viscosity regulators, stabilizers
or osmo-regulators. Illustrative examples of liquid vehicles for
oral and parenteral administration include water, alcohols, (including
monohydric and polyhydric alcohols such as the glycols), oils such
as coconut oil or fractionated peanut oil. For parenteral administration,
the vehicle may also be an ester such as ethyl oleate and isopropyl
myristate. Liquid pharmaceutical compositions in the form of sterile
solutions or suspensions may be used for intramuscular, intraperitoneal
or subcutaneous injection.
A pharmaceutical composition according to the invention preferably
contains from 1 to 1000 mg of antagonist compounds of protein ESM-1
per dose unit, and preferably from 10 to 500 mg of antagonist compound
of protein ESM-1 per dose unit.
The present invention also concerns a method of treatment and/or
prevention of a cancer comprising a step during which a pharmaceutical
composition such as defined above is administered to a patient having
need of such treatment.
Pharmaceutical Composition Containing an Antagonist Compound of
Protein ESM-1 of the Antisense Polynucleotide Type.
Also forming part of the invention are pharmaceutical compositions
containing a therapeutically effective quantity of an antagonist
compound of protein ESM-1 of the antisense polynucleotide type as
defined in the present description in addition to methods of treatment
and/or prevention of a cancer comprising the administration to a
patient having need of such a treatment of a pharmaceutical composition
containing an antisense polynucleotide such as defined above.
An antisense oligonucleotide according to the invention may be
administered by any means, either local or systemic.
The local administration of an antisense polynucleotide of the
invention, for example in the tumour, may be performed by the administration
of the antisense polynucleotide directly into the tumour or into
the tissue surrounding the tumour so that the oligonucleotide can
migrate to, and where appropriate enter into, the tumour cells.
For example, the antisense polynucleotides may be injected using
a syringe. The injection may be intramuscular, intravenous, intraperitoneal
or subcutaneous. The antisense polynucleotide may be administered
to the liver via the hepatic portal vein. Similarly, the antisense
polynucleotide may be administered to the lung using an inhalation
device.
Other means of administration of an antisense polynucleotide may
be used. For example, the antisense polynucleotides may administered
systemically after their insertion into an expression vector. The
term "expression vector" includes a plasmid, a virus or
any other vehicle known in the state of the art to ensure the expression
of an antisense polynucleotide.
For the use of vectors suitable for the recombinant expression
of an antisense polynucleotide, a person skilled in the art may
advantageously use the vectors pMSXND described by LEE and NATHANS
(1988), eukaryotic virus vectors, such as those described by GLUZMAN
(1982), or the adenoviruses and adeno-associated viruses such as
those described in the U.S. Pat. Nos. 5,173,414 and 5,354,678 or
an expression system including an expression vector described by
MOXHAM et al. (1993).
The expression vector preferably contains a promoter allowing the
production of the antisense polynucleotide in an animal, preferably
a mammal, and preferably in humans, such as the polyhedrin promoter.
The expression vector may be suitable for the targeted expression
of the antisense polynucleotide at the site of the tumour, for example
by placing the nucleic acid coding for the antisense polynucleotides
under the control of a promoter specific to certain cells, such
as the epithelial cells or the endothelial cells. An example of
such a promoter is the viral promoter designated NuNTV which is
specifically useful in the treatment of breast cancers. Other examples
of such specific promoters are milk protein promoters such as .beta.-lactoglobulin,
.alpha.-casein and .beta.-casein.
The therapeutically effective quantity of an antisense polynucleotide
of the invention may be determined as the quantity necessary for
a significant reduction of the translation of protein ESM-1 at the
systemic or local level.
It will be clear to a person skilled in the art that the therapeutically
effective concentration of the antisense potynucleotide varies with
the choice of the mode of administration. For example, if the antisense
polynucleotide is administered by injection to a mammal, the dose
unit comprises a syringe containing an effective quantity of the
antisense polynucleotide. An effective quantity of the antisense
polynucleotide for a systemic administration is between 0.01 mg/kg
and 50 mg/kg administered once or twice per day. A therapeutically
effective quantity of an antisense polynucleotide according to the
invention included in a pharmaceutical composition is generally
between 10.sup.4 and 10.sup.11 molecules of antisense polynucleotide
per administration and preferably between 10.sup.5 and 10.sup.10
molecules of DNA per administration.
However, different dosage protocols may be used according to (i)
the individual capacity of the antisense polynucleotide to inhibit
the expression of protein ESM-1, (ii) the severity or extent of
the disease, or (iii) the pharmacokinetic behaviour of the antisense
polynucleotide used.
The antisense polynucleotide may be combined with a pharmaceutically
acceptable vehicle or an excipient. Examples of excipients include
fillers, binders, dispersion agents, lubricants, according to the
type of administration and the forms of dosage. Preferred forms
of dosage include liquid solutions, advantageously physiologically
compatible buffers such as HANK's or RINGER solutions. In addition,
the antisense polynucleotides according to the invention may be
formulated in a solid form then redissolved or resuspended immediately
before use. This includes lydphilized forms and liposomes containing
such antisense polynucleotides.
An antisense polynucleotide of the invention may also be systemically
administered by the transmucosal, transdermal or oral routes. For
the transmucosal or transdermal routes of administration, penetrating
agents may be used in formulation such as bile salts or derivatives
of fusidic acid.
The present invention also relates to a method of treatment and/or
prevention of a cancer comprising a step of administration, to a
patient having need of such treatment, of a pharmaceutical composition
such as defined above containing an antagonist compound of ESM-1
of the antisense polynucleotide type.
In general, any of the pharmaceutical compositions of the invention
such as defined above and containing a therapeutically effective
quantity of an antagonist compound of protein ESM-1 is useful in
the prevention and/or treatment of a cancer.
As a non-limiting illustration, a pharmaceutical composition according
to the invention is useful for the prevention and/or treatment of
cancers such as cancers of the respiratory tracts, broncho-pulmonary
cancers, breast cancers, cancers of the colon and renal cancers
as well as cancers of the digestive system.
The present invention is in addition illustrated, without in any
way being limited, by the following examples and figures.
FIGURES
FIG. 1 illustrates Western Blot immunoblotting gels and colorations
of ESM-1 on SDS-PAGE gel.
Each immunoblotting gel was revealed with the anti-ESM-1 monoclonal
antibody MEP14. The second anti-mouse antibody marked with horseradish
peroxidase was purified by affinity and gave negative results when
used alone.
FIG. 1A. Immunoblotting gel of protein ESM-1 from different cell
types expressing this protein.
The immuno-precipitation of ESM-1 from cell culture supernatants
SVI (1), 293-ESM(2) and CHO-ESM(3) was performed with the antibody
MEP19 when this is indicated, or with a control antibody. The arrows
show the band specific to ESM-1. The native form of ESM-1 is represented
by a diffuse band around 50 kD.
FIG. 1B. Absence of detection of purified protein ESM-1 with coomassie
blue.
5 .mu.g of protein ESM-1 purified from SVI cells was loaded onto
an SDS-PAGE gel at 15% and coloured with coomassie blue in order
to detect the peptide part of the molecule. The arrows show the
absence of detection of ESM-1.
FIG. 1C. Detection of purified protein ESM-1 with alcian blue.
5 .mu.g of protein ESM-1 purified from SVI cells was loaded onto
an SDS-PAGE gel at 15% and revealed with alcian blue in order to
detect the glycan part of the molecule. The arrow shows protein
ESM-1.
FIG. 2 illustrates the apparent molecular weight of the peptide
and glycan parts of ESM-1.
FIG. 2A Analysis by mutation of the site of fixation of O-glycosylation.
Two presumed O-glycosylation sites (threonine 120 and serine 137)
were substituted by an alanine residue by directed mutagenesis.
The wild-type protein ESM-1 (VT), the ESM-1 T120A and S137A mutants,
and negative controls (MOCK) were transfected in 293 cells and the
cell culture supernatants and cell lysates were analysed by immunoblotting
(Western-Blot) using monoclonal antibody MEP14. The arrows show
the specific bands,
FIG. 2B. Effect of a treatment with proteinase K on ESM-1.
Protein ESM-1 purified from SVI (1) and 293-ESM cells (2) was digested
by proteinase K and loaded onto an SDS-PAGE gel at 15% The upper
arrow shows the wild type of untreated protein ESM-1 and the lower
arrow shows protein ESM-1 digested by proteinase K.
FIG. 3 illustrates the effects of specific chondroitinases on ESM-1.
FIG. 3A. Treatment of purified wild-type protein ESM-1 with chondroitinase
ABC.
Secreted protein ESM-1 was purified by ion-exchange chromatography,
followed by immunoaffinity chromatography from cell culture supernatants
of SVI(1), 293ESM(2) and from human plasma (3), then digested or
not by chondroitinase ABC. 50 ng of the digested protein were loaded
onto an SDS-PAGE gel at 15% then analysed by immunoblotting (Western-Blot).
The upper arrow shows the undigested forms of ESM-1 and the lower
arrow the digested forms of ESM-1.
FIG. 3B. Treatment of purified wild-type protein ESM-1 with chondroitinase
B.
Protein ESM-1 purified from cell culture supernatants of SVI (1)
and 293-ESM(2) was digested or not by the chondroitinase. The proteins
were loaded onto SDS-PAGE gel at 15%. The upper arrow shows the
different undigested forms of ESM-1 around 50 kD and the lower arrow
shows the different forms of digested ESM-1, around 22 kD.
FIG. 3C. Treatment of purified wild-type protein ESM-1 with chondroitinase
AC.
Protein ESM-1 purified from cell culture supernatants of HUVEC
(1) and 293-ESM(2) was digested by chondroitinase AC and loaded
onto SDS-PAGE gel at 15%. The upper arrow shows the different undigested
forms of ESM-1 around 502 kD and the lower arrow shows the different
forms of digested ESM-1, around 22 kD.
FIG. 3D. Treatment of purified wild-type protein ESM-1 with chondroitinase
C.
Protein ESM-1 purified from cell culture supernatants of HUVEC
(1) and 293-ESM (2) was digested or not by chondroitinase C and
loaded onto SDS-PAGE gel at 15%. The upper arrow shows the different
undigested forms of ESM-14 around 52 kD and the lower arrow shows
the different forms of digested ESM-1 around 22 kD.
FIG. 4 illustrates the effects of purified wild-type protein ESM-1
on the coagulation time in the presence of thrombin. The delay and
the reduction of thrombin production can be seen for heparinized
plasma and also for the four other curves (plasma rich in platelets
or PRP: open diamonds; plasma rich in platelets+ESM-1 at 0.2 mg/ml:
solid squares plasma rich in platelets+ESM-1 00.5 mg/ml: solid triangle;
plasma rich in platelets+ESM-1 at 1 mg/ml: solid circle; plasma
rich in platelets+heparin: open circle).
FIG. 5 illustrates the biological activity of the proteoglycan
ESM-1 on the proliferation of 293 cells induced by factor HGF/SF.
The stimulation of the incorporation of .sup.3H-thymidine by 293
cells induced by factor HGF/SF was studied. The cells were sown
at 1.times.10.sup.4 cells per well in a DMEM medium supplemented
with transferin and insulin and HGF/SF at 50 ng per ml before addition
of different molecules. The bars represent the percentage increase
in .sup.3H-thymidine incorporation (mean+/-s.d. of triple samples
of a representative experiment) in the presence of the additions
shown of serum, different forms of ESM-1 at 2.5 mg/ml and decorin
at 2.5 mg/ml. The background noise level of .sup.3H-thymidine incorporation
in the presence of HGF/SF was generally between 7.000 and 8.000
cpm per well. The results presented are similar to those obtained
in three other separate experiments.
FIG. 6 illustrates a study of twelve responses of the different
forms of ESM-1 and of decorin on the mitogenic activity induced
by factor HGF/SF. The simulation of DNA synthesis by 293 cells was
studied in the presence of HGF/SF at 50 nanograms per ml alone or
in the presence of different concentrations of wild-type protein
ESM-1/WT (open square), of mutant non-glycosylated protein ESM/S137A
(solid circle), of the GAG chain derived from protein ESM/WT (solid
square) or of decorin (open circle). The mean values of triplicate
measurements of .sup.3H-thymidine incorporation obtained in one
experiment among three independent experiments are shown in FIG.
6. The results are expressed in cpm. The standard deviations were
approximately 10%.
FIG. 7 illustrates the tumorigenic power of protein ESM-1. Two
batches of more than 10 mice received control HEK cells or cells
transfected with a vector coding for the cDNA of wild-type protein
ESM-1 (ESM/WT). On FIG. 7A, the percentage of tumours macroscopically
visible at the eighth week at the injection point and whose tumoral
volume was more than 1 cm.sup.3 is shown as ordinate. FIG. 7B illustrates
the kinetics of appearance of the tumours in mice having received
transfected HEK expressing glycosylated protein ESM-1 (ESM/WT).
The number of weeks after injection of the cells is given on the
abscissa. The mean tumoral volume, expressed in cm.sup.3, is given
on the ordinate.
FIG. 8 illustrates the production of ESM-1 by tumours induced in
mice.
FIG. 8A represents the serum level of protein ESM-1 found in the
two batches of mice, at the eighth week following the injection
of the cells. The abscissa shows respectively the batch of mice
having received the control HEK cells and the batch of mice having
received the HEK cells expressing glycosylated protein ESM-1 (ESM/WT).
The serum level of ESM-1 found, expressed in nanogram/ml, is given
on the ordinate.
FIG. 8B illustrates the kinetics of the serum levels of ESM-1 measured
by ELISA, for the mice of the batch having received the HEK cells
transfected with a DNA coding for the glycosylated protein ESM-1
(ESM/WT). The abscissa shows the number of weeks following the injection
of the transfected cells The serum level of protein ESM-1, expressed
in nanogram/ml, is given on the ordinate.
FIG. 9 illustrates the tumorigenic activity of the different forms
of protein ESM-1.
FIG. 9A illustrates the appearance of tumours in different batches
of mice, the mice having received respectively control HEK cells,
HEK cells transfected with a cDNA coding for glycosylated protein
ESM-1 (ESM/WT), cells transfected with nonglycosylated protein ESM-1
(ESM/S137A) and HEK cells transfected with a cDNA coding for protein
ESM-1 replaced in positions 134 and 135 (ESM/F115A, F116A). The
ordinate shows the percentage of tumours macroscopically visible
at the eighth week at the point of injection whose tumoral volume
was greater than 1 cm.sup.3.
FIG. 9B illustrates the serum level of ESM-1 in the different identical
batches of mice. The serum level of ESM-1, expressed in nanogram/ml,
is given on the ordinate.
FIG. 10 illustrates the inhibiting effect of the monoclonal antibody
MEP08 on the pro-tumoral activity of protein ESM-1.
The injection of MEP-08 antibodies increased the survival of mice
from the HEK ESM/WT group. The monoclonal antibodies MEP-08 were
injected intraperitoneally at a dose of 400 .mu.g from the second
following the inoculation of the HEK/ESM-WT cells. The injections
were repeated weekly for 12 weeks. A control antibody, MEP-14, was
used under the same conditions. The mice were sacrificed when their
tumoral volume was greater than 6 cm.sup.3. (n>8 mice in each
group). The figure shows the percentage of surviving mice in each
of the groups.
EXAMPLES
Example 1
Post-translational Modification of the Secreted Form of Protein
ESM-1
A. Materials and Methods
A.1 Cell Culture and Materials
CHO cells were cultured in a culture medium MAM.alpha. (Gibco BRL,
Life Technologies, France) supplemented with 10% foetal calf serum.
Human endothelial cells transfected by the virus SV40, the SVI cells
described by LASSALLE P et al. (1992), were cultured in a medium
RPMI 1640 containing 2 mM of L-glutamine and 10% foetal calf serum.
Human embryo kidney cells, the cells of the line 293, were cultured
in a medium DMEM from Dulbecco with 10% foetal calf serum. The human
embryo kidney cells, the cells of the line 293, used for the proliferation
test were cultured in modified EAGLE medium from Dulbecco (Gibco
BRL) supplemented with insulin at 10 mg/ml and transferin at 10
mg/ml. The proteinase and chondroitinase ABC were commercially available
from Boehringer Mannheim. Chondroitinases B, AC and C are marketed
by Sigma. Human factor HGF/SF is marketed by R & D and decorin
by Sigma. Anti-ESM-1 monoclonal antibodies were produced and purified
as described by BECHARD et al. (2000).
A.2 Development of Cell Lines Expressing ESM-1.
The complete cDNA coding for ESM-1 was directed, purified and inserted
into the expression vector pcDNA3 (marketed by Invitrogen) between
the XhoI and HindlIII sites. The vector constructions were transfected
in the cell lines CHO and 293 in the presence of lipofectamine (GIBCO
BRL), then selected on G418 (1000 .mu.g/ml for the CHO line and
300 .mu.g/ml for the 293 line). The cell lines which had been transfected
stably were obtained by limiting dilution and the cells thus selected
were designated respectively CHO-ESM and 2936-ESM.
A.3 Determination of the Site of O-lycosylation of ESM-1 by Mutation
Analysis.
2 Potential sites of Olycosylation had been predicted using the
software NET 0 glyc:0 Prediction Serveur.
The serine residue in position 137 (SEQ ID N.sup.o1) and the threonine
in position 120 (SEQ ID N.sup.o1) were substituted by an alanine
residue. The O-glycosylation mutants were produced by PCR using
the mutagenesis kit Quick Change according to the manufacturer's
recommendations (Stratagene).
The mutant cDNAs were confirmed by sequencing (sequencer ABI prism
377 from Applied Biosystems). The 293 cells were then transfected
with the vectors into which the mutant cDNAs had been inserted to
obtain the transitory and stable transfectants, respectively the
293-ESM/S 137A and 293-ESM/T120A.
A.4 Purification of the Proteoglycan ESM-1 Chondroitin/dermatan
Sulfate.
The cell culture supernatants were adjusted to pH8, then passed
over a column of DEAE-Sepharose (Pharmacia), washed with a Tris
buffer 50 mM (pH8), 0.2 M NaCl, then eluted with a buffer Tris 50
mM (pH8), 0.8 M NaCl.
The eluates were adjusted to 50 mM Tris (pH8), 0.5 M NaCl and passed
over an affinity column. The affinity column was composed of anti-ESM-1
monoclonal antibodies (produced by the hybridoma line MEC4) immobilized
on a Affigel Hz hydrazide gel, according to the manufacturer's recommendations
(Biorad).
After a washing step with a Tris buffer 50 mM (pH8), M NaCl 0.5,
protein ESM-1 was eluted with a solution of 3M MgCl.sub.2, concentrated
and dialysed against the same buffer on an ultrafree 30 device (millipore).
The eluted material was then quantified by immunodetection with
anti-ESM-1 antibodies, and checked on SDS-PAGE using a coloration
with coomassie blue or alcian blue.
The purification of protein ESM-1 from human plasma was performed
according to the following protocol.
800 ml of plasma supplied by the blood transfusion agency (Lille,
France) were precipitated with a 60% ammonium sulfate solution and
dialysed against a Tris buffer 50 mM (pH 8), 0.5 M NaCl. The precipitated
and dialysed plasma extract was then passed over a 50 ml pre-column
of the Affigel type (Biorad) before a passage over an anti-ESM-1
immunoaffinity column. The protein ESM-1 fixed on the immunoaffinity
column was recovered as described below.
The non-glycosylated form of ESM-1 (ESM/S137A) was purified in
a single step by chromatography and immunoaffinity. The degree of
purity of glycosylated protein ESM-1 (ESM/WT) and of the non-glycosylated
protein replaced on serine 137 (ESM/S137A) was checked by FPLC.
The purified material was free from endotoxins, as proved by the
results of a limulus amebocyte lysate test (BIOwhitaker).
A.5 Immunoprecipitation, Immunoblotting and Sequencing.
The size of the different forms of ESM-1 was determined by immunoprecipitation
and immunoblotting from cell culture supernatants and cell lysates.
The cells were lysed in a buffer containing 0.5% of NP40, a cocktail
of anti-proteases (Boehringer Mannheim, Germany) in PBS for 30 minutes
at 4.degree. C. with agitation.
The lysates were then centrifuged at 10.000 g for 15 min in order
to obtain the clarified cell lysates.
The culture supernatants were filtered over a filter having a pore
diameter of 0.45 mm.
1 .mu.g of ESM-1 monoclonal antibody produced by the hybridoma
line MEP19 or 1 .mu.g anti-ICAM-1 monoclonal antibody (clone 164B)
was added to the clarified lysate or to the cell culture supernatant
and incubated overnight at 4.degree. C. with agitation.
50 .mu.l of an anti-mouse immunoglobulin conjugated with agarose
beads (sigma) were added at 4.degree. C. over 90 min, before centrifugation
and washing with a lysis buffer and washing in PBS.
The beads were resuspended in 20 and 40 .mu.l of SDS-PAGE buffer
for 5 min, centrifuged, and the supernatants were analysed.
The samples were subjected to electrophoresis on SDS-PAGE gel,
then transferred onto a nitrocellulose membrane using standard procedures.
After a blocking step, the membranes were incubated for one hour
with 1 .mu.l of an ESM-1 monoclonal antibody produced by the hybridoma
line MEP14, washed, then incubated for 1 hour with an anti-Fc mouse
secondary antibody conjugated with horseradish peroxidase (marketed
by SIGMA). After several washings revelation was performed using
the detection kit ECL marketed by Amersham.
For amino acid sequence analysis, purified protein ESM-1 was subjected
to electrophoresis on SDS-PAGE gel, then electrotransferred onto
a polyvinylidene difluoride membrane (PVDF) marketed by MILLIPORE,
then coloured using 0.1% coomassie blue. The protein band at 50
kD was excised from the membrane and the N-terminal sequence was
determined by EDMAN degradation on a protein sequencer of type ABI
473A.
A.6 Digestion of the Peptide Part of ESM-1 by Proteinase K
In order to determine the size of the glycosaminoglycan, purified
protein ESM-1 was digested with proteinase K with an enzyme:ESM-1
ratio of 1:50 (w/w) in a Tris buffer 10 mM, pH8, in the presence
or absence of 0.1% SDS at 56.degree. C. for 3 hours. A quantity
of bovine serum albumin (BSA) 10 times?? greater than that of the
protein ESM-1 was digested by proteinase K in order to verify its
complete degradation. The samples were analysed on a 12% SDS-PAGE
gel, followed by coloration with coomassie blue and alcian blue.
A.7 Digestion of ESM-1 by Chondroitinases ABC, B, AC and C
In order to analyse the nature of the substitution of the glycosaminoglycan,
purified protein ESM-1 was digested with several chondroitinases:
chondroitinases ABC (0.5 units/mg in buffer 100 mM TrisHCl, pH 8.
30 mM sodium acetate, pH 5.2 at 37.degree. C. for 45 min), chondroitinase
B (200 units/mg in buffer 20 mM Tris-HCl, 50 mM NaCl, 4 mM CaCl.sub.2,
0.01% BSA, pH 7.5 at 25.degree. C. for two hours), chondroitinase
AC (one unit per ml in buffer 250 mM Tris HCl, 75 mM sodium acetate,
pH 7.3 at 37.degree. C. for two hours), chondroitinases C (80-120
units/ml in buffer 50 mM Tris HCl , pH 8 at 25.degree. C. for 3
hours). The samples were analysed by immunoblotting.
A.8 Anti-coagulant Activity.
The control plasma poor in platelets (PPP) was prepared from blood
in the presence of the anticoagulant sodium citrate (30 mM), by
centrifugation at 2500 g for 15 min. All the reagents were marketed
by STAGO Diagnostica (France). Three parameters were evaluated,
by adding protein ESM-1, buffer or heparin to the platelet-poor
plasma.
a) APTT (Activated Partial Thromboplastin Time): this parameter
explores the intrinsic route of blood coagulation (FI, FII, FV,
FVIII, FIX, FX, FXI, FXII). The deficit or inhibition of one of
these factors increases the coagulation time of the mixture PPP
reagent, cephalin, activator, CaCl.sub.2.
b) TCT (Thrombin Clotting Time): this parameter is analysed on
a mixture of platelet-poor plasma (PPP) in the presence of thrombin.
With a standard concentration of thrombin, the coagulation time
of the plasma is constant. Defects in the formation of fibrin induce
an increase in the coagulation time.
c) anti-Xa activity: the anti-Xa activity of heparin or other inhibitors
acting on factor FXa is detected by a competitive test. The sample
studied (PPP+ESM-1, +buffer or +heparin) is mixed with factor Fxa
and a specific chromogenic substrate of factor Fxa. The final coloration
is inversely proportional to the concentration of inhibitor.
A.9 Test of Thrombin Generation
This sensitive global test can detect defects in plasma or platelets
inducing a delay or a reduction in thrombin generation. A plasma
rich in platelets (PRP) was prepared from blood in the presence
of sodium citrate by centrifugation at 150 g for 10 min. The thrombin
generation test was performed, for each of the subjects, in samples
in the absence of ESM-1, with non-fractionated calcium heparinate
(0.5 Ul of anti-Xa/ml) or with 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml
of ESM-1 (final concentration).
The protein ESM-1 was added 10 min before the test.
At 37.degree. C., 1 ml of plasma was mixed with 1 ml of CaCl.sub.2
and a chronometer was started. Aliquot fractions of 0.1 ml were
taken from the reaction mixture each minute for 1 min.
The clot formed in the reaction mixture was regularly removed.
The aliquot fractions were mixed with 0.2 ml of fibrinogen (Sigma,
4/1000 in an Owren buffer at 37.degree. C. and the coagulation time
was measured for each of the aliquot fractions).
The thrombin formed in the reaction mixture acts of the forminogens,
inducing the formation of fibrin. The coagulation activity was maximal
between 4 and 8 min then reduced due to the neutralization of the
thrombin by the anti-thrombin.
A.10 Analytical Chromatography by Gel Filtration
50 .mu.g of purified glycosylated ESM-1 (ESM/WT) and of purified
non glycosylated ESM-1 (ESM/S137A) in 50 mM Tris buffer, pH 8.5,
0.5 M NaCl were separated by liquid chromatography on a Superdex
200 column (for ESM/WT) or Superdex 75 (for ESM/S137A) marketed
by Pharmacia, using the chromatography system Biorad Biologic Chromatography
System with a flow of 1 ml/min.
As standard, the following calibration kit of high and low molecular
weight (Pharmacia Biotech) was used, ribonuclease A (bovine pancreas,
13.7 kD), ovalbumin (43 kD) albumin (bovine serum, 67 kd), aldolase
(rabbit muscle, 158 kD), ferrtine (horse spleen, 440 kD), thiroglobulin
(bovine thyroid, 669 kD).
The molecular weight standards were separated using a buffer identical
to that used for the proteins ESM-1 and the separation was performed
immediately after the separation of the proteins ESM/WT and ESM/S137A.
The elution time of the standard proteins was used to draw a standard
linear curve, Kav=f(log MR) in order to determine the apparent molecular
weights of the proteins ESM/WT and ESM/S137A respectively.
Fractions of 1 ml were collected an protein ESM-1 was detected
using a specific immunoenzymatic test (ELISA).
B. Results
B.1 Post-translational Modifications of the Secreted Form of Protein
ESM-1 Produced by Endothelial Cells and by Established Cell Lines.
In order to determine whether ESM-1 was matured as a secreted molecule,
as suggested by the presence of an N-terminal amino acid sequence
predicted as a signal peptide, protein ESM-1 was purified from the
cell line 293-ESM.
The N-terminal sequence of the 50 kD form indicated that the signal
peptide of 19 amino acids was cleaved at the predicted site, resulting
in a mature polypeptide of ESM-1 of 165 amino acids beginning at
the tryptophan residue in position 20 of sequence SEQ ID N.sup.o1,
the N-terminal sequence being WSNNYAVD-P.
ESM-1 was immunoprecipitated from culture supernatants of HUVEC,
SV1, 293-ESM and CHO-ESM cells, the analysed by immunoblotting.
It had previously been shown that in HUVEC cells supernatants,
ESM-1 migrated in the form of a diffuse band at around 50 kD.
A band similar in size was observed with the supernatants of SV1,
293-ESM and CHO-ESM cells (FIG. 1A).
The molecular weight found was larger than the predicted molecular
weight. This result suggested that the secreted form of ESM-1 had
undergone post-translational modifications. The fact that purified
protein ESM-1 was better coloured on SDS-PAGE gel with alcian blue
than with coomassie blue suggested that ESM-1 was glycosylated (FIGS.
1B, 1C) rather than oligomerized across the disulfide bridge, because
reductive conditions did not modify the apparent molecular weight
of ESM-1.
B.2. The Serine Residue in Position 137 (SEQ ID N.sup.o1) is the
Site of O-glycosylation of ESM-1.
A computer analysis of the potential glycosylation sites identified
three putative sites of Olycosylation, respectively on the serine
in position 16, on the threonine in position 120 and on the serine
in position 127, but no site of N-glycosylation.
The threonine residue in position 120 and the serine residue in
position 137 were replaced by an alanine residue.
These mutants were transitorily expressed in the 293 cells.
Protein ESM-1 was then immunoprecipitated from cell lysates and
culture supernatants, and analysed by immunoblotting.
Protein ESM/T120A migrated at 50 kD, at a position similar to the
apparent molecular weight of the wild form of ESM-1 (ESM/WT), as
shown on FIG. 2A.
In contrast, protein ESM/S137A migrated at 22 kD corresponding
to the intracellular form of ESM-1 (FIG. 2A), a molecular weight
compatible with the predicted molecular weight of ESM-1.
The immunoprecipitations performed from transitorily transfected
COS and CHO cells gave the same results, showing that only the serine
residue in position 137 constituted a site of glycoconjugation in
all the cell models studied.
In order to determine the length of the glycosaminoglycan (GAG)
of ESM-1, the peptide part of ESM-1 was completely digested by proteinase
K.
The treatment by proteinase K caused a change in the molecular
weight from 50 kD to 25-30 kD (FIG. 2B). These results show that
the band of apparent molecular weight at 50 kD is compatible with
the presence of a polypeptide of 22 kD which is glycoconjugated
on the serine in position 137 by a GAG chain of a mean size of 25-30
kD.
B.3 The GAG Chain of ESM-1 is Sensitive to Chondroitinase ABC
In order to characterize the GAG chain of ESM-1, protein ESM-1
was first digested by chondroitinase ABC. The treatment by chondroitinase
ABC reduced the molecular weight of secreted protein ESM-1 to 22
kD (FIG. 3A), suggesting that the carbohydrate of ESM-1 is a chain
of the chondroitin type.
The profile is similar with protein ESM-1 purified from 293-ESM
cells and from the human endothelial cell line SVI. Because protein
ESM-1 circulates in the blood, we also studied the behaviour of
protein ESM-1 purified, from human plasma. The results showed a
single principal band of 50 kD, which had a molecular weight of
22 kD after treatment with chondroitinase ABC, as for all the other
cell lines studied (FIG. 3A). Thus protein ESM-1 is a soluble proteoglycan
containing a single chondroitin sulfate chain.
B.4 The GAG Chain of ESM-1 is a Heterogeneous Chondroitin/dermatan
Sulfate Chain.
In order better to determine the type of saccharidic unit which
constituted the GAG chain of ESM-1, several specific enzymes were
used, such as chondroitinases B, AC and C.
The treatment with chondroitinase B reduced the apparent molecular
weight from 50 kD to 22 kD (FIG. 3B).
A similar profile was observed after treatment of ESM-1 by chondroitinases
AC and C (FIGS. 3C, D).
These different enzymatic treatments showed that the GAG chain
of ESM-1 contained different component units including a type of
amino sugar, N-acetylgalactosamine, coupled to a differently sulfated
iduronic or glucuronic acid.
These different units alternated in the chain, and were present
at the beginning of the chain, close to the N-terminal disulfated
dissacharides which persisted in the protein part after digestion
by the chondroitinase, because all the treatments with chondroitinase
lead to the same reduced apparent molecular weights of 22 kD.
B.5 Biological Activity of the Soluble ESM-1 Proteoglycan on Coagulation
Because protein ESM-1 is a secreted as a proteoglycan of the chondroitin/dermatan
sulfate type by endothelial cells, and the dermatan sulfate shows
effects on thrombin generation in vitro DELORME et al., (1996) and
on coagulation, the anticoagulant potential of ESM-1 was verified
using the parameters APTT, TCT, anti-Xa activity and on thrombin
generation.
The results are given in table 1 below.
TABLE-US-00001 TABLE 1 Biological activity of the ESM-1 proteoglycan
on coagulation Activity APTT (dry) TCT (dry) Anti-Xa (UI/ml) PPP
+ buffer 30.6 16.5 0 PPP + ESM-1 (0.2 .mu.g/ml) 30.8 17.5 0 PPP
+ ESM-1 (0.5 .mu.g/ml) 31 18.8 0 PPP + ESM-1 (1 .mu.g/ml) 31.8 20.7
0 PPP + heparin 89 39 0.45
The results in table 1 show that protein ESM-1 at different significant
doses from 0.2 mg/ml to 1 mg/ml did not change the different parameters
tested.
The parameters APTT, TCT and anti-Xa activity were similar for
plasma poor in platelets (PPP) with the buffer or with protein ESM-1.
In the positive controls, the APTT, TCT and anti-Xa activities
were higher for PPP in the presence of heparin.
In addition, protein ESM-1 did not have an inhibitor effect on
the thrombin generation test; no difference was observed according
to concentrations of 0.2 mg/ml, 0.5 mg/ml and 1 mg/ml of ESM-1 compared
to the control buffer, while heparin induced a delay in the formation
of thrombin (FIG. 4).
Example 2
Effect of Protein ESM-1 onto the Mitogenic Activity of Factor HGF/SF
A. Materials and Methods
The activity of stimulation of proliferation was determined by
measuring the incorporation of .sup.3H thymidine by 293 cells.
The 293 cells were sown at a concentration of 1.times.10.sup.4
cells per well in 96-well microplates of type TPP and maintained
for 24 hours in DMEM culture medium supplemented with transferin
and insulin.
The human recombinant HGF/SF was diluted in PBS containing 0.1%
bovine serum albumin and added in water to 3 identical wells in
order to obtain a final concentration of 50 ng/ml.
The recombinant proteins ESM/WT, ESM/S137A, the purified GAG chain
derived from ESM-1 and decorin were added alone or in combination
with factor HGF/SF at doses of from 1 ng/ml to 2.5 .mu.g/ml, simultaneously
with the addition of HGF/SF.
After 96 hours of culture, the cells were incubated with 0.5 .mu.Ci
of .sup.3H thymidine per well for 16 hours and incorporation of
.sup.3H thymidine was determined using a scintillation counter of
the type Topcount Microplate Scintillation Counter (Packard).
The tests were performed on batches of three identical wells.
The cell viability was measured using the MTT reduction test.
B. Results
The effect of protein ESM-1 on the activity of factor HGF/SF was
studied.
The incorporation of .sup.3H-thymidine by. 293 cells was measured
in the presence of HGF/SF at 50 ng/ml alone or in combination with
different quantities of ESM/WT.
In a first batch of experiments, it was observed that HGF/SF alone
at 50 ng/ml induced a proliferation of the 293 cells at a level
equal to about 45% of the proliferation induced by the serum, while
protein ESM/WT alone did not stimulate the proliferation of 293
cells.
In contrast, when it was combined with factor HGF/SF, protein ESM/WT
considerably increased the proliferation of 293 cells induced by
HGF/SF with an increase of 162.3%, when the protein was tested at
a concentration of 2.5 .mu.g/ml (FIG. 5).
This increase effect of protein ESM-1 on HGF/SF activity was dependent
on the dose of ESM-1 and began to be significant at a dose of 10
ng/ml (FIG. 6).
In addition, the effect of protein ESM/WT was compared to the effect
of decorin, another proteoglycan of the type chondroitin sulfate/dermatan
sulfate, on the mitogenic activity of factor HGF/SF. In contrast
to protein ESM/WT, decorin showed no activity of increasing the
proliferation of 293 cells induced by factor HGF/SF (FIGS. 5, 6).
These results showed that protein ESM-1 had a specific effect on
the mitogenic activity of factor HGF/SF.
In order to examine the respective involvement of the protein part
of ESM-1 and of the GAG chain on the activity of increasing the
mitogenic effect, the incorporation of .sup.3H-thymidine by 293
cells in the presence of HGF/SF supplemented with different concentrations
of non-glycosylated ESM/S137A and of the GAG chain derived from
of ESM-1 was measured.
The non-glycosylated form of ESM-1 was incapable of inducing a
proliferation of the 293 cells, either in the presence or absence
of factor HGFSF (FIG. 5), even when it was used at high concentration.
In contrast, the GAG chain purified from ESM-1 considerably increased
the proliferation of 293 cells induced by factor HGF/SF, with a
factor of increase close to 96.6%, compared to factor HGF/SF alone
(FIG. 5). The pro-mitogenic effect of the GAG chain was less than
that observed with the wild form of protein ESM-1, but this effect
was nevertheless dependent on the dose of GAG chain added (FIG.
6).
The results given above clearly show that protein ESM/WT increases
the proliferation of 293 cells induced by factor HGF/SF and that
this pro-mitogenic activity is specific and due to the GAG chain
of the chondroitin sulfate/dermatan sulfate type of ESM-1.
In general, factor HGF/SF is expressed during the critical early
periods of human organogenesis from 6 to 13 weeks of gestation.
The organs which express the HGF/SF gene are particularly the liver,
metanephric kidney, intestine and lung, each of these organs developing
by inductive interaction between the mesenchyma and the epithelium.
In addition, factor HGF/SF is an important factor in human renal
multcystic dysplasia (TAKAYAMA et al., 1997) and in the appearance
of malformation and hyperproliferation in the tubules. The results
presented above show that protein ESM-1 significantly increases
the proliferation of the cells of the embryonic kidney in the presence
of HGF/SF while the nonglycosylated form of protein ESM-1 has no
effect. In addition, the GAG chain isolated from protein ESM-1 is
able to mimic the effects of the glycosylated protein ESM/WT. These
results clearly show that the biological activity of ESM-1 on the
function of factor HGF/SF is principally mediated by its GAG chain.
It may be noted that decorin, another proteoglycan of the chondroitin
sulfate/dermatan sulfate type secreted by endothelial cells and
which is able to fix onto factor HGF/SF (CELLA et al., 1992) has
no effect on the activity of HGFSF. These comparisons show a specificity
of action of protein ESM-1 on the activity of factor HGF/SF requiring
a composition of the GAG chain different from the GAG chain of the
proteoglycans belonging to the family of proteoglycans with small
leucine-rich repeats.
In the kidney, protein ESM-1 is selectively detected in the distal
tubules, a result which may be associated with the observation of
a preferential localization of factor HGF/SF in the same part of
the nephron in situations of human renal multicystic dysplasia (WEIDNER
et al., 1993). These results indicate an application of protein
ESM-1 in pathological disorders depending on factor HGF/SF, which
has also been shown as being associated with the development of
cancers of the breast (RAHIMI et al., 1998), kidney (NATALI and
al, (1996)) and lung (OTSUKA et al., 1998) and also in malignant
melanomas (SIEGFRIED et al., 1998). Thus, factor HGF/SF is likely
to favorize the extension of hyperplasia and to generate cells with
an invasive phenotype. Protein ESM-1 is likely to be involved in
these phenomena of deregulated mitogenic activities of factor HGF/SF.
Example 3
Preparation of an Antagonist Compound of Protein ESM-1 of the Antibody
Type
In order to obtain anti-ESM-1 monoclonal antibodies directed against
the N-terminal region of protein ESM-1 rich in cysteine residues,
the native form of protein ESM-1 produced by the CHO cell line transfected
by an expression vector containing a DNA insert coding for protein
ESM-1 was purified.
The cDNA of ESM-1 was inserted into the eukaryotic expression vector
pcDNA3 (In vitrogen) then transfected in CHO cells with lipofectamine
(GIBGO) according to the manufacturer's recommendation. 48 Hours
after the transfection the cells were transplanted in the presence
of a selection agent (G418, Gibco) at a dose of 1000 microgram/ml).
After two weeks of selection, the CHO cells resistant to G418 were
cloned by limiting dilution. The clones expressing ESM-1 were then
selected and named CHO-ESM (deposited at the CNCM).
For the production, the CHO-ESM cells were cultured in suspension
in a medium without foetal calf serum (medium CHO SFM II, Gibco).
The supernatant was adjusted to pH 8 and passed over a DEAE-sepharose
column (Pharmacia). The column was washed with a buffer 50 mM Tris,
pH 8, 0.2 M NaCl. The ESM-1 molecule was eluted in a buffer 50 mM
Tris, pH 8, 1 M NaCl. The eluate was then diluted 1:4 in a buffer
50 mM Tris, pH 8 and incubated in the presence of anti-ESM-1 monoclonal
antibody (MEC4) immobilized on agarose (Biorad). After incubation
overnight at 4.degree. C. with agitation the agarose beads were
washed with buffer 50 mM Tris, pH 8, 0.2 M NaCl. ESM-1 was eluted
with 3 M MgCl.sub.2. The eluate was concentrated and dialysed in
buffer 50 mM Tris, pH 8, 0.5 M NaCl and stored at -70.degree. C.
Balb/C mice were immunized by injection of 10 .mu.m of purified
recombinant protein ESM-1 per mouse, according to a standard immunization
protocol in the presence of Freund's adjuvant.
The hybridoma cells secreting the anti-ESM-1 monoclonal antibodies
were obtained by fusion, screening and sub-cloning according to
the technique described by BECHARD et al. (2000).
Five hybridoma cell clones were obtained and generically designated
MEC (Mouse Monoclonal Antibody to ESM-1 produced by CHO Cells).
Four of the hybridomas selected were of isotypes IgG1, k, respectively
the hybridomas designated MEC4, MEC5, MEC15 and MEC36.
One of the hybridomas was of isotype IgM,k, the hybridoma MEC11.
The hybrdoma cell clones were cultured in culture medium in the
absence of serum and the anti-ESM-1 antibodies were purified by
chromatography on a column of protein G-Sepharose marketed by Pharmacia
(UPPSALA, Sweden).
Example 4
Preparation of an Antagonist Compound of Protein ESM-1 of the Polypeptide
Type
The directed mutagenesis was performed with the kit marketed by
STRATAGENE under the reference Site-directed quick mutagenesis kit,
used according to the recommendations of the manufacturer.
Briefly, a pair of forward and reverse primers of strictly complementary
sequences were synthesized, these primers comprising the nucleotides
coding for the mutated amino acid(s), or the complementary nucleotides,
these nucleotides being localized in the centre of the sequence
of the primers which also comprise about 10 to 15 consecutive nucleotides
complementary to the sequence to be amplified both on the 5' and
the 3' side of the central nucleotides.
After amplification by PCR, the amplified polynucleotides coding
for the mutant protein ESM-1 were inserted into the vector pcDNA3.
The following pairs of primers respectively were used:
a) For protein ESM-1 F115A
TABLE-US-00002 Forward primer: 5'-GCC TGA AAT TCC CCG CCT TCC AAT
ATT CAG-3'. (SEQ ID N.sup.o 3) Reverse primer: 5'-CTG AAT ATT GGA
AGG CGG GGA ATT TCA GGC-3'. (SEQ ID N.sup.o 4)
b) For protein ESM-1 F116A
TABLE-US-00003 Forward primer: 5'-CCT GAA ATT CCC CTT CGC CCA ATA
TTC AGT AAC C-3'. (SEQ ID N.sup.o 5) Reverse primer: 5'-GGT TAC
TGA ATA TTG CGC GAA GGG GAA TTT CAGT G-3'. (SEQ ID N.sup.o 6)
c) For protein ESM-1 F115 F116A
TABLE-US-00004 Forward primer: 5'- CCT GAA ATT CCC CGC CGC CCA
ATA TTC AGT AAC C-3'. (SEQ ID N.sup.o 7) Reverse primer: 5'- GGT
TAC TGA ATA TTG GGC GGC GGG GAA TTT CAG G-3'-. (SEQ ID N.sup.o 8)
Example 5
Pro-tumorigenic Activity of Glycosylated Protein ESM-1.
A. Materials and Methods
A.1. Cell lines : HEK T. HEK ESM/WT, HEK ESM/S137A, HEK ESM/69,
HEK ESM/71, HEK ESM/73.
The cell line HEK ESM/WT transfected stably with the cDNA coding
for the wild form of ESM-1 (ESM/WT) was used. Four other cell lines
were obtained by transfection with the cDNA coding for the purified
forms of ESM-1 obtained by directed mutagenesis of the wild type.
The first of these, named HEK ESM/S137A, expressed the mutant non-glycosylated
protein ESM-1, where an alanine has replaced serine 137, the major
site of O-glycosylation. The three other lines expressed a glycosylated
form of ESM-1 whose protein part has been mutated. They were lines
HEK ESM/F115A (replacement of the phenylalanine in position 134,
HEK ESM/71 (replacement of the phenylalanine in position 135) and
HEK ESM/F115A, F116A (double deletion/replacement 134-135).
Thus, six cell line producing different forms of ESM-1 were used:
control HEK, not secreting ESM-1; Wild form of ESM-1: HEK ESM/WT;
Deglycosylated form of ESM-1: HEK ESM/S137A; Glycosylated forms
whose protein part has been mutated in the region 115-116; HEK-ESM/69,
HEK-ESM/71, HEK ESM/73. A2. Murine Model of Xenogenic Tumours
The mice used were of type SCID (Severe Combined IMMUNO Deficiency).
They were more precisely mice C.B.17 Scid/scid supplied by the animal
service of the Institut Pasteur of Lille. These mice had a recessive
autosomal mutation in their recombination system (Blunt., 1995).
This mutation causes the production of non-functional immunoglobulins
and T cell receptors (TcR) and B 5BcR) As a result, they do not
possess functional T and B lymphocytes; these mice therefore tolerate
non-self and represent a model of choice for the development of
xenogenic tumours. The SCID mice used were young male mice aged
from 3 to 5 weeks. For each of them, an intra-peritoneal injection
of anti-ascialo GM-1 antibodies (100 .mu.g per mouse diluted in
200 .mu.l of RPMI) was performed 24 hours before injection with
the different cell lines. These were rabbit polyclonal antibodies
(Wako Pure Chemical Industries, Ltd) directed specifically against
the asialo GM-1 antigen expressed by NK cells. Previous work has
shown that the use of these antibodies in mudne models neutralizes
the cytotoxic effect of the NK cells and encourages the tumoral
grafting (Mather G et al. (1994).
Four batches of mice (10 to 15 mice per group) anaesthetized with
ether, were then injected subcutaneously in the back. Each mouse
received 1 million cells diluted in 200 .mu.l of RPMI. The injection
of these cells defined the first day of the experiment (D0). For
each mouse, macroscopic inspection of the point of injection in
order to observe the appearance of a possible tumour, as well as
measurement of body weight, was performed weekly. A blood sample
(about 500 .mu.l per mouse) was weekly taken from the 5th week onwards,
in order to determine the serum levels of ESM-1 by an ELISA test
(BECHARD D) et al., 2000). An anatomo-pathological examination was
performed on each mouse.
B. Results
B.1. Induction of Tumours in Mice by Glycosylated Protein ESM-1.
HEK cells were transfected with a vector possessing an insert containing
the cDNA coding for glycosylated wild protein ESM-1, designated
ESM/WT. The HEK cells were injected subcutaneously into SCID mice
aged 5 weeks. Each mouse had previously received an intraperitoneal
injection of anti-asialo GN-1 antibodies.
The percentage of tumours having a volume greater than 1 cm.sup.3
observed in the mice at the eighth week following the injection
of the transfected HEK cells was analysed.
The results are given on FIG. 7.
On FIG. 7A, it can be observed that the injection of control HEK
cells did not induce the appearance of tumours in mice In contrast,
the HEK cells transfected with a DNA coding for glycosylated protein
ESM-1 induced numerous macroscopically visible tumours, of which
about 95% had a tumoral volume greater than 1 cm.sup.3.
FIG. 7B illustrates the kinetics of appearance of tumours in mice
which had received transfected HEK cells transfected with a DNA
coding for glycosylated protein ESM-1. It can be observed that the
mean tumoral volume, expressed in cm.sup.3, increased continuously
from the fourth week following the injection of the transfected
HEK cells.
The experimental results presented in FIG. 7 clearly show that
glycosylated protein ESM-1 has a pro-tumoral activity.
The serum levels of protein ESM-1 were also measured in mice having
received control HEK cells and mice having received HEK cells transfected
with cDNA coding for protein ESM-1.
The results are shown on FIG. 8.
The results illustrated in FIG. 8A show that protein ESM-1 was
not found in the serums of mice having received control HEK cells.
In contrast, a serum level of 40 to 50 nanograms per ml was found
in mice having received HEK cells transfected with cDNA coding for
protein ESM-1 at the eighth week following injection of the cells.
The kinetics of the serum levels of ESM-1 in mice having received
transfected HEK cells expressing glycosylated protein ESM-1 (ESM/IT)
were also analysed.
The results are given in FIG. 8B.
It can be observed that a detectable quantity of protein ESM-1
was found in the serum of mice from the fifth week following the
injection of cells and that the serum level increased rapidly and
continuously from the fifth to the twelfth week following injection
of the cells.
The experimental results illustrated in FIG. 8 show that the tumours
which developed in mice having received transfected HEK cells produce
protein ESM-1. In addition, the quantity of protein ESM-1 produced
in the circulation follows the kinetics of development of the tumours
in the mice.
Example 6
Pro-tumorigenic Activity of Different Forms of Protein ESM-1
A. Materials and Methods
The materials and methods used in this example are identical to
those described for example 5.
B. Results
The HEK cells were transfected by vectors possessing a DNA insert
coding respectively for the nonglycosylated wild form of ESM-1 (ESM/WT),
a non-glycosylated form of ESM-1 (ESM/S137A) and a glycosylated
form of ESM-1 mutated at the phenylalanine residues in positions
134 and 135 which have both been replaced by an alanine residue
(ESM/73). The different transfected cells were injected subcutaneously
into SCID mice aged 5 weeks and having previously received anti-asialo
GM-1 antibodies.
The percentage of tumours macroscopically visible having a tumoral
volume greater than 1 am.sup.3 in the different batches of mice
was analysed. The results are shown on FIG. 9A.
The results of FIG. 9A show that only the glycosytated protein
ESM-1 is able to induce tumours in mice. Neither the non-glycosylated
ESM-1 nor the glycosylated ESM-1 mutated at the phenylalanine residues
in positions 134 and 135 induced the development of tumours in SCID
mice.
The serum levels of protein ESM-1 circulating in the different
batches of mice were also measured. The results are given in FIG.
9B.
The results on FIG. 9B show that detectable levels of serum protein
ESM-1 could be measured, at the eighth week following injection
of the cells, only in the mice having received the HEK cells expressing
glycosylated protein ESM-1 (ESM/WT).
Neither the mice injected with cells expressing the non-glycosylated
protein ESM-1 (ESM/S137A), nor the mice having received the glycosylated
and mutated protein ESM-1 HEK-ESM/F115A, F116A) produced protein
ESM-1.
The overall results presented in this example confirm the pro-tumorigenic
activity of glycosylated protein ESM-1.
The results also show that the non-glycosylated forms of protein
ESM-1 or the mutant forms of protein ESM-1 can behave as antagonists
of this protein and possess preventive and/or curative power with
regard to cancerous pathologies.
Example 7
Determination of Circulating Protein ESM-1 in Patients Suffering
from Broncho-pulmonary Cancers at Different Stages of Development
A. Materials and Method
The immunodetection test consisted of an immuno-enzymatic test
of the "sandwich" type whose general characteristics are
identical to those described by BECHARD et al. (2000).
The anti-ESM-1 monoclonal antibody produced by the hybridoma lines
MEP14 (CNCM N.sup.oI-1942) was diluted to a concentration of 5 .mu.g/ml
in a carbonate buffer 0.1 M, pH 95, and adsorbed overnight at +4.degree.
C. on a 96-well plate (plate E.I.A./R.I.A., Costar, Cambridge, Mass.,
USA).
The plate was saturated for one hour at laboratory temperature
with a volume of 200 .mu.l/well of PBS buffer containing 0.1% of
bovine serum albumin and 5 mM of EDTA, then washed twice with an
ELISA buffer (the PBS buffer above supplemented with 0.1% Tween
20).
A calibration was performed with protein ESM-1 purified according
to the technique described by BECHARD et al. (2000).
The blood samples were serially diluted (1:2 to 1:128), in an ELISA
buffer and incubated on an ELISA plate for one hour at laboratory
temperature.
The wells were washed three times with an ELISA buffer, then incubated
for 1 hour at laboratory temperature with a second monoclonal antibody
directed against ESM-1, the antibody MEC15 (CNCM N.sup.oI-2572)
at a concentration of 0.1 .mu.g/ml in 100 .mu.l of buffer per well.
After three washings, a biotinylated rat monoclonal antibody rat
directed against mouse IgG1 (marketed by PHARMINGEN) diluted in
an ELISA buffer was added and left to incubate this second antibody
for one hour.
After three washings in the ELISA buffer, the wells were incubated
with a streptavidine-peroxidase conjugate at a dilution 1:10.000
v/v (marketed by ZYMED).
After 30 minutes of incubation-with the streptavidine-peroxidase
conjugate, three washings of each well were performed with an ELISA
buffer, then two washings in a PBS buffer.
The streptavidine-peroxidase conjugate was revealed with the substrate
TMB marketed by SIGMA (Saint-Louis, Mo., USA) in the presence of
255 .mu.l of H.sub.2O.sub.2 for 30'.
The revelation reaction was stopped by addition of a volume of
100 .mu.l of H.sub.2SO.sub.4 2N.
The plate was read using a spectrophotometer (anthos labtec LP40.
France) at a wavelength of 405 nanometres.
The plasma or serum concentration of protein ESM-1 was calculated
from the optical density measurements and expressed in nanograms
per ml.
B. Results
The concentration of protein ESM-1 circulating in the serum of
different patients -with broncho-pulmonary cancer at different stages
development, respectively at stage I, II, IIIA, IIIB and IV according
to the international classification TNM defined below:
T=size of the tumour (T1:<1 cm; T2: between 1 and 3 cm; T3:>3
cm.
N=ganglion nodule (NO if not invaded; N1 if invaded).
M=metastasis at distance (MO if no metastasis; M if metastasis).
The patients suffering from cancer at stage I had a serum concentration
of protein ESM-1 of 1.43+/-0.76 nanograms/ml (n=3).
The patients suffering from a bronchopulmonary cancer at stage
If had a serum concentration of protein ESM-1 of 0.72+/-0.39 nanograms/ml
(n=3).
The patients suffering from a bronchopulmonary cancer at stage
IIIA had a concentration of circulating protein ESM-1 of 0.9+/-0.53
nanograms/ml (n=2).
The patients suffering from a bronchopulmonary cancer at stage
IIIB had a concentration of circulating protein ESM-1 of 3.1+/-2.17
nanograms/ml (n=3).
The patients suffering from a bronchopulmonary cancer at stage
IV had a concentration of circulating protein ESM-1 of 3.1+/-1.91
nanograms/ml (n=11).
The results given above show that the serum levels of protein ESM-1
increase as a function of the stage of development of the cancer.
A clear relation is thus demonstrated between the level of production
of protein ESM-1 in the blood circulation and the severity of a
cancer in a patient.
Example 8
Anti-tumoral Activity of an Antagonist Compound of ESM-1 of the
Antibody Type
A. Materials and Method
MEP08 monoclonal antibodies were injected intraperitoneally at
a dose of 400 .mu.g from the second following the inoculation of
HEK/ESM-WT cells. The injections were repeated weekly for 12 weeks.
A control antibody, MEP-14, was used under the same conditions.
The mice were sacrificed when their tumoral volume was greater than
6 cm.sup.3. (n>8 mice in each group). The figure shows the percentage
of surviving mice in each of the groups.
B. Results
To the extent that the phenylalanine in position 115 is necessary
for tumoral development, it comprises a new therapeutic target.
For this reason the anti-ESM-1 monoclonal antibodies MEP-08, produced
by the hybridoma line MEP-08 deposited at the CNCM under the n.sup.oI-1941,
directed specifically against this region, were produced and injected
into the group of HEK-ESM/WT mice. The object was to study the role
of the peptide of ESM-1 in the tumoral development and to evaluate
a possible therapeutic effect. In order to eliminate an anti-tumoral
effect depending on the fragment Fc of the antibody (reaction of
ADCC), a control antibody of the same isotype but recognizing a
different epitope was used in parallel and under the same conditions.
FIG. 10 shows that the early injections of MEP-08 antibodies significantly
increased the survival of the mice by nearly 60% while the MEP-14
antibodies had no effect. These first results show that this is
a specific action linked to the fragment Fab of the antibody directed
specifically against the phenylalanine in position 115 and confirm
the involvement of the peptide in the tumoral growth. It is surprising
to observe that this effect on the survival reduces when the antibodies
are administered later.
Whichever week the injections begin, the antibodies can delay or
prevent the tumoral growth. This anti-tumoral effect remains more
pronounced when the antibodies are used earlier. |