EP1335745A1 - Utilisation d'un compose antagoniste de la proteine esm-1 pour la fabrication d'un medicament pour le traitement d'un cancer - Google Patents
Utilisation d'un compose antagoniste de la proteine esm-1 pour la fabrication d'un medicament pour le traitement d'un cancerInfo
- Publication number
- EP1335745A1 EP1335745A1 EP01993475A EP01993475A EP1335745A1 EP 1335745 A1 EP1335745 A1 EP 1335745A1 EP 01993475 A EP01993475 A EP 01993475A EP 01993475 A EP01993475 A EP 01993475A EP 1335745 A1 EP1335745 A1 EP 1335745A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- esm
- protein
- compound
- antagonist
- antagonist compound
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates to the field of cancer prevention and / or treatment.
- Cancer is frequently a disease linked to deficits in the intracellular signaling system. Normal cells respond to many extracellular signals by proliferating, differentiating or more generally by altering their metabolic activity.
- Such signals are received on the surface of cells and converted by a signal transduction protein system into a message recognized by the cell. This message is responsible for phenomena of subsequent cellular regulation.
- Metastasis is the formation of a tumor colony
- Tumor cells locally escape through tissue barriers, such as the basement membrane of the epithelium, and reach the interstitial stroma, from which they access blood vessels
- the circulating tumor cells are carried into the bloodstream and are stopped in the precapillary venules of the target organ by adhesion to the lumen surfaces of the endothelial cell, or are
- the tumor cells leave the vascular wall and enter the parenchyma of the organ. Finally, the tumor cell, after extravasation, multiplies in a tissue different from that from which it originates.
- a number of cancers have been shown to be caused by defects associated with genes responsible for signal transduction. Such genes are called encogenes. Oncogenes can lead to overexpression of one or more signal transducing proteins inducing abnormal cell proliferation. Defective signals can be linked to various mechanisms. Certain anticancer therapies aim to inhibit the expression or the bioavailability of oncogenic proteins responsible for the proliferation of cancer cells, such as proteins of the MAP kinase family or products of certain oncogenes such as c-myc.
- the ESM-1 protein is a polypeptide of 184 amino acids secreted by endothelial cells and which was described for the first time by LASSALLE et al. (1996).
- the messenger RNAs coding for the protein ESM-1 are mainly found in endothelial cells and in pulmonary and renal tissues.
- the expression of the gene encoding ESM-1 is regulated by cytokines. TNF- ⁇ and IL-1 ⁇ induce an increase in the expression of the ESM-1 gene in endothelial cells of the human umbilical vein, while Interferon- ⁇ decreases its expression.
- ESM-1 protein A large amount of circulating ESM-1 protein has been found in patients with systemic inflammatory syndrome, such as septic shock (BECHARD et al., (2000).
- the object of the present invention is to provide anti-cancer compounds which would make it possible to overcome the disadvantages of State of the art methods of therapeutic treatment of cancer.
- a first object of the invention consists in the use of a compound antagonist of the ESM-1 protein for the manufacture of a medicament for the treatment of cancer.
- an antagonist compound of the invention is an antibody specifically binding to the protein ESM-1.
- an antagonist compound used in the context of the invention is a peptide of at least 10 amino acids of the modified ESM-1 protein and which comprises the chain of amino acids Ala (134) - Ala (135).
- an antagonist compound of the ESM-1 protein consists of an antisense oligonucleotide hybridizing with the cDNA coding for ESM-1.
- Another object of the invention consists of an antagonist compound of the ESM-1 protein, chosen from the antagonist compounds defined above.
- the invention also relates to a pharmaceutical composition intended for the treatment of cancer comprising an antagonist compound of the protein ESM-1.
- Another subject of the invention consists of a method of preventing cancer comprising a step during which a compound antagonist of the protein ESM-1 is administered.
- the invention also relates to a method of therapeutic treatment of cancer comprising a step during which a compound antagonist of the protein ESM-1 is administered to a patient.
- the protein ESM-1 is secreted in humans in the form of a proteoglycan of the chondroitin / dermatan sulfate type and that the protein Secreted ESM-1 is capable of stimulating the mitogenic activity of factor HGF / SF in vitro (for “Hepatocyte growth factor / scatter factor”).
- HGF / SF is an important factor in the onset of renal multicystic dysplasia 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 lung but also in the development of malignant melanomas.
- transfected human renal epithelial cells expressing the protein ESM-1 have a high tumor potential and cause the appearance of renal carcinoma in vivo in mice.
- Antibodies against the ESM-1 protein have also been shown to be capable of inhibiting the development of a renal tumor in vivo and that an antagonist peptide of the ESM-1 protein has the same anti-tumor activity.
- an increase in the serum ESM-1 protein level has been shown according to the invention in patients with bronchopulmonary carcinoma.
- a first object of the invention consists in the use of an antagonist compound of the ESM-1 protein for the manufacture of a medicament for the prevention and / or treatment of cancer.
- ESM-1 protein or “ESM-1 polypeptide”, within the meaning of the invention, include a polypeptide of 184 amino acids referenced as the sequence SEQ ID No. 1 of the sequence listing, as well as a polypeptide of 165 amino acids identical to the polypeptide of sequence SEQ ID No. 1 in which the 19 amino acids of the N-terminal corresponding to the signal peptides are absent, this polypeptide of 165 amino acids constituting the secreted form of the polypeptide of sequence SEQ ID N 1. Also included in the definition of “ESM-1 protein” “ESM-1 polypeptide” is a glycopeptide of 184 amino acids of sequence SEQ ID No.
- the ESM-1 glycopeptide has the serine residue at position 137 which is O-glycosylated by a chondroitin / dermatan sulfate motif.
- an antagonist compound of the protein ESM-1 is meant according to the invention a compound capable of significantly reducing the bioavailability of the protein ESM-1 with respect to target molecules on which which the protein ESM-1 is naturally fixes.
- An ESM-1 protein antagonist compound can reduce the bioavailability of these proteins by reducing the likelihood of binding of the ESM-1 protein to target molecules in the body to which it naturally binds.
- An antagonist compound according to the invention can reduce the bioavailability of the 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 altering the intracellular processing of the ESM-1 protein, for example by affecting the enzymatic process leading to its glycosylation, or by inhibiting or blocking the secretion of the mature ESM-1 protein.
- a first object of the invention consists in the use of a compound antagonist of the ESM-1 protein for the manufacture of a medicament for the treatment of cancer.
- An ESM-1 protein antagonist compound may be of any kind, polypeptide, saccharide, or any organic or inorganic compound making it possible to reduce the bioavailability of the ESM-1 protein with respect to the target molecules on which this protein is fixed.
- a first family of ESM-1 antagonist compounds preferred according to the invention consists of antibodies specifically binding to the ESM-1 protein. It is shown according to the invention that antibodies directed specifically against the protein ESM-1 are capable of inhibiting or blocking the tumorigenicity of this protein. Anti-ESM-1 antibodies therefore constitute antagonistic compounds of great therapeutic value.
- antibody within the meaning of the invention is meant in particular polyclonal or monoclonal antibodies or fragments (for example the Fab or F (ab) ' 2 fragments or any polypeptide comprising a domain of the initial antibody recognizing the protein ESM-1.
- Monoclonal antibodies can be prepared from a hybridoma using the technique described by KOHLER and MIELSTEIN (1975).
- ESM-1 or a fragment of this protein produced by the trioma technique or also the hybridoma technique described by KOZBOR et al. (1983).
- Anti-ESM-1 antibodies according to the invention can also be fragments of single chain Fv antibody (ScFv) such as those described in US Patent No. 0 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 as described by RIDDER et al. (1995) or humanized antibodies as described by REINMANN et al. (1997) or also by LEGER OJ, 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.
- a first preferred family of antibodies according to the invention which constitute antagonist compounds of the ESM-1 protein are the monoclonal antibodies specifically recognizing the region going from the praline residue in position 79 to the cysteine residue in position 99 of the sequence SEQ ID No. 1, this region representing the antigenic determinant D1. They are preferably monoclonal antibodies produced by the hybridoma line deposited on November 19, 1997 with the National Collection of Cultures of Microorganisms of the Pasteur Institute (CNCM) under the access number N ° l -1944, also called MEP21 antibody.
- CNCM National Collection of Cultures of Microorganisms of the Pasteur Institute
- preferred monoclonal antibodies are they which specifically bind to the part of the ESM-1 protein between the glycine residue at position 159 and the arginine residue at position 184 of the sequence SEQ ID No. 1 which is the region constituting the determinant D3 antigen.
- Preferred monoclonal antibodies specific for the antigenic determinant D3 can be obtained from the hybridoma line 1-1943 (MEP19), deposited on November 19, 1997 with the National Collection of Cultures of Microorganisms of the Institut Pasteur (CNCM ).
- Other preferred monoclonal antibodies according to the invention are the monoclonal antibodies binding specifically with the region between the serine residue at position 119 and the valine residue at position 139 of the protein ESM-1 of sequence SEQ ID No.
- the antigenic determinant D2 of the protein ESM-1 is defined as the antigenic determinant D2 of the protein ESM-1.
- the preferred monoclonal antibodies specifically binding to the antigenic determinant D2 of ESM-1 can be obtained from the hybridoma line MEP08 deposited on November 19, 1997 with the National Collection of Cultures of Microorganisms of the Institut Pasteur (CNCM) under the access number N ° 1-1941.
- Other monoclonal antibodies of interest constituting antagonist compounds of the ESM-1 protein are the monoclonal antibodies directed specifically against the N-terminal part of the ESM-1 protein.
- the preferred monoclonal antibodies directed against the N-terminal part of the ESM-1 protein can be obtained from the hybridoma line MEC15 deposited in the Collection National Culture of Microorganisms of the INSTITUT PASTEUR (CNCM) on October 17, 2000 under the access number I-2572.
- the anti-ESM1 antibodies having the best antagonistic activities vis-à-vis ESM-1 are chosen from antibodies specifically recognizing epitopes located in the region surrounding the phenylalanine residue at position 115. It s acts in particular antibodies which bind specifically with the region between the serine residue at position 119 and the valine residue at position 139 of the protein ESM-1 of sequence SEQ ID No. 1, such as the monoclonal antibody MEP08 described above above.
- the monoclonal antibody MEP08 was capable of inhibiting the pro-tumor activity of the protein ESM-1 on the formation of tumors caused by the proliferation of human cells of renal origin in mice .
- the region covering the antigenic determinant D2 of the protein ESM-1 is important for the pro-tumor activity of the protein ESM-1.
- the applicant has synthesized a polypeptide derived from the ESM-1 protein in which the phenylalanine residues in positions 134 and 135 of the sequence SEQ ID No. 1, that is to say the residues in positions 115 and 116 of the secreted protein ESM-1, have been replaced by two alanine residues.
- This modified polypeptide is incapable of inducing tumors in mice.
- Such a modified polypeptide can therefore compete with the protein ESM-1 produced at high level in cancer patients for its potentiating action with growth factors such as HGF / SF or even growth factors FGF-2 and FGF-7 .
- ESM-1 protein antagonist compounds include polypeptides of at least 10 consecutive amino acids in sequence SEQ ID NO: 1, which include an amino acid sequence ranging from amino acid at position 119 to with the amino acid in position 139 of the sequence SEQ ID No. 1, such a polypeptide ESM-1 antagonist comprising at least one substitution of an amino acid, with respect to the corresponding sequence of the ESM-1 protein.
- an ESM-1 protein antagonist polypeptide as defined above has at 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 the sequence SEQ ID No 1 and at least one substitution of amino acids, compared to the sequence SEQ ID No 1.
- An ESM-1 protein antagonist polypeptide comprises at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, relative to the sequence SEQ ID No 1, the number of amino acid substitutions being adapted as a function of the length of the polypeptide, it being understood that the number of amino acid substitutions relative to the sequence SEQ ID No 1 in an antagonistic polypeptide according to the invention is at most 25% of the amino acids included in the sequence of this antagonistic polypeptide, preferably at most 20%, 15% and preferably at most 10% of the number of amino acids included in the ESM-1 antagonist polypeptide sequence.
- an amino acid substitution, with respect to the sequence SEQ ID No. 1, in an antagonistic polypeptide according to the invention is a so-called “non-conservative” substitution.
- non-conservative substitution is meant the substitution of an amino acid residue with an amino acid of a distinct class.
- Amino acids are classically classified according to the following classes:
- - non-polar (hydrophobic) amino acids alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan and methionine;
- - amino acids containing aromatic rings phenylalanine, tryptophan and tyrosine
- - polar neutral amino acids glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine;
- an ESM-1 protein antagonist polypeptide for the preparation of an ESM-1 protein antagonist polypeptide according to the invention is the substitution of an amino acid containing an aromatic ring with an amino acid not containing an aromatic ring.
- an antagonist polypeptide of the ESM-1 protein according to the invention comprises a substitution of the phenylalanine residues in positions 134 and 135 of SEQ ID No. 1 with two amino acid residues, identical or different, not containing an aromatic cycle.
- Such a preferred ESM-1 protein antagonist polypeptide is a polypeptide of at least 10 consecutive amino acids of the sequence SEQ ID No.
- an antagonist polypeptide of the ESM-1 protein according to the invention can be prepared by conventional techniques of chemical synthesis, either in homogeneous solution or in solid phase.
- an ESM-1 protein antagonist polypeptide can be prepared by the homogeneous solution technique described by HOUBEN WEIL (1974) or the solid phase synthesis technique described by MERRIFIELD (1965a; 1965b) and MERRIFIELD 1965b.
- An ESM-1 protein antagonist polypeptide according to the invention can also be prepared by genetic recombination.
- a method can be implemented comprising the steps of: a) inserting a nucleic acid coding for the ESM-1 protein antagonist polypeptide in a vector appropriate expression; b) cultivating, in an appropriate culture medium, a host cell previously transformed or transfected with the recombinant expression vector of step a); c) recovering the conditioned culture medium or lysing the host cell, for example by sonication or by osmotic shock; d) separating and purifying from said culture medium or also from the cell lysates obtained in step c), said antagonist polypeptide; e) where appropriate, characterize the recombinant antagonist polypeptide thus produced.
- the antagonistic polypeptides according to the invention can be characterized by fixation on an immunoaffinity chromatography column on which the antibodies directed against this polypeptide or against a fragment of the latter have been immobilized beforehand.
- an ESM-1 antagonist polypeptide can be purified by passage through an appropriate series of chromatography columns, according to methods known to those skilled in the art is described for example in AUSUBEL F. et al. (1989).
- ESM-1 antagonist compounds aimed at reducing the bioavailability of the secreted ESM-1 protein in patients at risk or in patients who have already developed tumors are compounds capable of inhibiting or blocking expression of the gene encoding ESM-1 in humans.
- ESM-1 protein antagonist compounds can be antisense polynucleotides.
- the antagonist compounds of the ESM-1 protein according to the invention thus include an antisense polynucleotide capable of hybridizing specifically to a determined region of the gene coding for the protein ESM-1 and capable of inhibiting or blocking 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 Genbank database.
- an antisense polynucleotide according to the invention comprises a sequence complementary to a sequence localized in the region of the 5 ′ end of the DNA of the ESM-1 gene, and so completely preferred is the proximity of the translation initiation codon (ATG) of the ESM-1 gene.
- ATG translation initiation codon
- an antisense polynucleotide according to the invention comprises a sequence complementary to one of the sequences located at the exon / intron junctions of the ESM-1 gene and preferably sequences corresponding to a site of splicing.
- a preferred antisense polynucleotide according to the invention comprises at least 15 consecutive nucleotides of the cDNA coding for ESM-1 having the nucleotide sequence SEQ ID No. 2.
- a first polynucleotide is considered to be “complementary” to a second polynucleotide when each base of the first nucleotide is paired with the base complementary to the second polynucleotide whose orientation is reversed.
- the complementary bases are A and T (or A and U), and C and G.
- 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 No. 2.
- a preferred antisense polynucleotide according to the invention consists of a nucleic acid of sequence complementary to the nucleic acid of the cDNA of ESM-1 of sequence SEQ ID No. 2.
- An antisense polynucleotide constituting an antagonist compound of the ESM-1 protein according to the invention can be prepared by any suitable method well known to those skilled in the art, including by cloning and restriction enzyme action or by direct chemical synthesis. according to techniques such as the phosphodiester method of NARANG et al. (1979) or BROWN et al. (1979), the diethylphosphoramidite method of BEAUCAGE et al. (1980) or the solid support technique described in European patent No. EP-0 707 592.
- the antisense polynucleotides must have a sufficient length and melting temperature to allow the formation of an intracellular duplex hybrid having sufficient stability to inhibit expression of the ESM-1 mRNA.
- Strategies for constructing the antisense polynucleotides are notably described by GREEN et al. (1986) and IZANT and WEINTRAUB (1984).
- antisense polynucleotides are for example those described by SCZAKIEL et al. (1995) or those described in PCT application No. WO 95 / 24.223.
- Those skilled in the art can 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 described in US Patent No. 5,582,986 which discloses oligonucleotides antisense for the inhibition of the ras gene, the technique described by HOLT et al.
- an ESM-1 protein antagonist compound according to the invention can be selected by a person skilled in the art for its ability to inhibit the development of a tumor induced by the ESM-1 protein in vivo.
- a method for selecting an antagonist compound of the ESM-1 protein comprises the following steps: a) injecting into an animal cells capable of forming tumors in the presence of the ESM-1 protein, said cells being transfected or transformed with a nucleic acid capable of expressing the ESM-1 protein in vivo; b) administering to this animal a candidate antagonist compound of the ESM-1 protein; c) comparing the formation of tumors in a first animal as obtained at the end of step b) and in a second animal as obtained at the end of step a); and d) selecting the candidate compound capable of inhibiting or blocking the formation of tumors in the first animal.
- the animal used in the above selection process is a non-human mammal, advantageously a rodent, and very preferably a rat, a guinea
- step e) consisting in sacrificing the first and the second animal.
- the cell line capable of forming tumors in animals in the presence of the protein ESM-1 is the line
- HEK 293 ATCC N ° CRL 1573.
- a protein antagonist compound in another aspect, a protein antagonist compound
- ESM-1 according to the invention can be selected according to a method making it possible to demonstrate the binding of a candidate compound to the protein ESM-1.
- a method for selecting a candidate antagonist compound for the ESM-1 protein comprises the following steps: a) providing a polypeptide consisting of the ESM-1 protein or a peptide fragment of this protein; b) bringing said polypeptide into 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 which bind to the polypeptide consisting of the ESM-1 protein or of a fragment of this protein.
- fragment of the ESM-1 protein is meant a polypeptide comprising 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 ESM-1 polypeptide of sequence SEQ ID No. 1 and comprising the sequence going from the praline residue in position 133 to the valine residue in position 138 of SEQ ID N ° 1.
- the invention also relates to a kit or kit for the selection of a candidate antagonist compound of the ESM-1 protein, this kit or kit comprising: a) a purified preparation of a polypeptide consisting of the ESM-1 protein or a fragment of this protein; b) where appropriate, means for detecting a complex formed between the polypeptide and the candidate compound to be tested.
- the method for detecting a complex formed between the polypeptide derived from the ESM-1 protein and the candidate compound can be carried out by various techniques, such as by microdialysis coupled to an HPLC method as described by WANG et al. (1997) or capillary affinity electrophoresis as described by BOUSH et al. (1997).
- a candidate compound can be of any kind, and in particular the final product of a combinatorial chemistry process.
- a candidate antagonist compound of the ESM-1 protein can be selected according to the above method as an expression product of a DNA insert contained in a phage vector according to the technique described by PARMLEY & SMITH (1988) .
- the DNA inserts encode peptides of 8 to 20 amino acids in length, as described by OLDENBURG KR et al. (1992), VALADON P et al. (1996), LUCAS AH (1994), WESTERINK (1995), FELICI et al. (1991).
- the recombinant phages expressing a protein capable of binding to the polypeptide consisting of the protein ESM-1 or a fragment thereof is retained and the complex formed between the protein ESM-1 or a fragment of this and the recombinant phage can be subsequently immunoprecipitated by a monoclonal or polyclonal antibody anti-ESM-1.
- the candidate antagonist compounds of the ESM-1 protein can also be selected in that they bind to the ESM-1 protein, or to a polypeptide fragment thereof, in competition with an antagonist compound of the ESM-1 protein previously selected such as one of the anti-ESM-1 antibodies described above, and in particular the monoclonal antibody secreted by the hybridoma line MEP08 deposited on November 19, 1997 with the CNCM under the access number 1-1941.
- an antagonist compound of the ESM-1 protein previously selected such as one of the anti-ESM-1 antibodies described above, and in particular the monoclonal antibody secreted by the hybridoma line MEP08 deposited on November 19, 1997 with the CNCM under the access number 1-1941.
- Such competitive experiences are for example described in the article by BECHARD et al. (2000).
- Proteins or other molecules of any kind capable of binding to the ESM-1 protein, or a polypeptide fragment of this protein can be selected using affinity columns on which the ESM-1 protein has been immobilized or a fragment of the latter, for example using conventional techniques, including chemical coupling of the protein ESM-1 or a fragment of the latter to the matrix of a column such as agarose, or Affigel®.
- a solution containing the candidate compound to be tested is brought into contact with the chromatographic support on which the ESM-1 protein or a peptide fragment thereof is immobilized. The compounds retained on the affinity column are selected positively.
- ESM-1 protein antagonist candidate compound can also be selected using an optical biosensor as described by EDWARDS and LEATHERBARROW (1997).
- This technique allows the detection of interactions between molecules in real time without requiring the use of labeled molecules.
- This technique is based on the surface plasmon resonance (SPR for “Surface Plasmon Resonance”). Briefly, the candidate compound to be tested is attached to a surface, such as a matrix of carboxymethyldextran. A light ray is directed on the end 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 binding of the candidate compound causes a change in the refractive index of the surface, the change in refractive index being detected as a modification of the SPR signal.
- Such an optical biosensor detection method can also make it possible to select the candidate compounds which compete with another ligand for binding to the ESM-1 protein or a peptide fragment of the latter.
- a candidate antagonist compound of the protein ESM-1 includes the compounds capable of inhibiting the binding of an anti-ESM-1 antibody to the protein ESM-1, of inhibiting the binding of factor HGF-SF or else factors FGF-2 and FGF-7 on the protein ESM-1 or a peptide fragment of the latter.
- the invention relates to a process for the selection of an antagonist compound of the ESM-1 protein, characterized in that it comprises the following steps: a) bringing the ESM-1 protein into contact or a peptide fragment of the latter in the presence of:
- step a) a candidate compound to be tested; b) in a step separate from step a), but possibly simultaneous with the latter, bringing the ESM-1 protein or a peptide fragment of the latter with an antagonist compound of the ESM-1 protein binding to the ESM-1 protein; c) detecting the respective amount of the antagonist compound of the ESM-1 protein fixed at the end of each of steps a) and b); and d) selecting the candidate compound which competes with the antagonist compound for binding to the ESM-1 protein.
- an ESM-1 antagonist compound for carrying out the above selection method is an anti-ESM-1 antibody or else a peptide antagonist compound as defined above in the present description.
- a method for selecting an ESM-1 antagonist compound from a candidate compound comprises the following steps:
- step 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 tumors induced by the ESM-1 protein; 3) select the compounds inhibiting the development of tumors determined in step 2) as antagonist compounds of the protein ESM-1.
- Step 1) preferably consists of implementing a process for selecting a candidate compound which binds to the ESM-1 protein or a fragment thereof, chosen from the processes detailed in the present description.
- Step 2) preferably consists of implementing a process for selecting a candidate compound in vivo as detailed in the description. In a particular embodiment of the method, it also comprises a step 4) consisting in sacrificing the animal.
- Pharmaceutical composition of the invention preferably consists of implementing a process for selecting a candidate compound in vivo as detailed in the description. In a particular embodiment of the method, it also comprises a step 4) consisting in sacrificing the animal.
- Another subject of the invention consists of a pharmaceutical composition for the treatment and / or prevention of cancer comprising a compound antagonist of the protein ESM-1.
- composition comprising an antagonist compound of the antibody type or of the peptide type according to the invention.
- a pharmaceutical composition according to the invention comprises a therapeutically effective amount 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 administration (including intramuscular, subcutaneous and intravenous) or also in a form suitable for administration by inhalation or insufflation.
- the pharmaceutical compositions according to the invention can be presented in the form of dosage units and can be prepared by any method well known to those skilled in the art in the pharmaceutical pharmaceutical field. All the methods include a step consisting in associating the agonist compound constituting the active principle of the composition with a liquid vehicle or a finely divided solid vehicle and, if necessary, shaping the product, for example. in the form of tablets or capsules.
- a pharmaceutical composition according to the invention is preferably presented in the form of dosage units such as capsules, tablets or capsules.
- the pharmaceutical composition may include a propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases.
- a propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases.
- the dosing unit can be provided with a valve capable of releasing a given amount of the pharmaceutical composition.
- 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 powder of the antagonist compound and of a suitable base powder, such as lactose or starch.
- the powder composition can be presented in dosage units, for example in the form of capsules or cartridges from which the powder can be administered using an inhaler or insufflator device.
- a pharmaceutically compatible solid vehicle of a pharmaceutical composition according to the invention includes substances such as flavoring agents, lubricants, solubilizers, suspending agents, bulking agents, compression aids, binders or agents disintegrants as well as encapsulation materials.
- the vehicle is a finely divided solid which is mixed with the antagonist compound of ESM-1 also in the finely divided form.
- the antagonistic active principle of ESM-1 is mixed with a vehicle having suitable compression properties and compacted in the desired shape and size.
- the powders and tablets preferably contain less than 99% of the active principle.
- Preferred solid vehicles are for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone and ion exchange 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 antagonist active principle of the protein ESM-1 can be dissolved or suspended in a pharmaceutically acceptable vehicle such as water, an organic solvent, a mixture of the two or pharmaceutically acceptable oils or fats.
- the liquid vehicle may contain other pharmaceutically acceptable additives such as agents solubilizers, emulsifiers, buffers, preservatives, sweeteners, aromatics, suspending agents, thickening agents, coloring agents, viscosity regulators, stabilizers or osmo-regulators.
- liquid vehicles for oral and parenteral administration include water, alcohols (including monohydric and polyhydric alcohols such as glycols), oils such as coconut oil or fractionated peanut oil.
- the vehicle can also be an ester such as ethyl oleate and isopropyl myristate.
- Liquid pharmaceutical compositions in the form of sterile solutions or suspensions can be used for intramuscular, intraperitoneal or subcutaneous injection.
- a pharmaceutical composition according to the invention comprises from 1 to 1000 mg of antagonist compounds of the ESM-1 protein per dosage unit, and preferably from 10 to 500 mg of the antagonist compound of the ESM-1 protein per unit of dosage dosage.
- the present invention also relates to a method of treatment and / or prevention of cancer comprising a step during which a pharmaceutical composition as defined above is administered to a patient in need of such treatment.
- composition comprising an antagonist compound of the ESM-1 protein of the antisense polynucleotide type.
- compositions containing a therapeutically effective amount of a protein antagonist compound ESM-1 of the antisense polynucleotide type as defined in the present description as well as methods of treatment and / or prevention of a cancer comprising the administration to a patient in need of such treatment of a pharmaceutical composition comprising an antisense polynucleotide as defined above.
- An antisense oligonucleotide according to the invention can be administered by any means, whether local or systemic.
- the local administration of an antisense polynucleotide of the invention can be carried out by the administration of the antisense polynucleotide directly at the level of the tumor or at the level of the tissue surrounding the tumor and allowing the oligonucleotide to migrate and, where appropriate, to enter the tumor cells.
- antisense polynucleotides can be injected using a syringe. The injection can be intramuscular, intravenous, intraperitoneal or subcutaneous.
- the antisense polynucleotide can be administered to the liver through the hepatic portal vein system. Likewise, the antisense polynucleotide can be administered to the lung using an inhalation device.
- an antisense polynucleotide can be administered systemically after their insertion into an expression vector.
- expression vector encompasses a plasmid, a virus or another vehicle known in the art for ensuring the expression of an antisense polynucleotide.
- vectors suitable for the recombinant expression of an antisense polynucleotide may advantageously use pMSXND vectors described by LEE and NATHANS (1988), eukaryotic viral vectors, such as those described by GLUZMAN (1982), or adenoviruses and adeno-associated viruses such as those described in US patents 5, 173,414 and 5,354,678 or also an expression system including the expression vector described by MOXHAM et al. (1993).
- the expression vector contains a promoter allowing the production of the antisense polynucleotide in an animal, preferably a mammal, and more preferably in humans, such as the polyhedrin promoter.
- the expression vector can be adapted to the targeted expression of the antisense polynucleotide at the tumor site, for example by placing the nucleic acid coding for the antisense polynucleotides under the control of a promoter specific for certain cells, such as epithelial cells or endothelial cells.
- a promoter is the designated viral promoter NuNTV which is useful specifically in the treatment of breast cancer.
- the therapeutically effective amount of an antisense polynucleotide of the invention can be determined as the amount necessary for a significant reduction in the translation of the protein ESM-1 at the systemic or local level.
- the concentration of the therapeutically effective antisense polynucleotide varies with the choice of the form of administration.
- the dosing unit consisting of a syringe containing an effective amount of the antisense polynucleotide.
- An effective amount of the antisense polynucleotide for systemic administration is between 0.01 mg / kg to 50 mg / kg administered once or twice a day.
- a therapeutically effective amount of an antisense polynucleotide according to the invention included in a pharmaceutical composition is generally between 10 4 and 10 11 molecules of antisense polynucleotide per administration and preferably between 10 5 and 10 10 DNA molecules per administration.
- assay protocols can be used depending on (i) the individual ability of the antisense polynucleotide to inhibit expression of the ESM-1 protein, (ii) the severity or extent of the disease, or (iii) behavior pharmacokinetics of the antisense polynucleotide used.
- the antisense polynucleotide can be associated with a pharmaceutically compatible vehicle or with an excipient.
- excipients include bulking agents, binders, disintegrants, lubricants, depending on the nature of the administration and the dosage forms.
- Preferred dosage forms consist of liquid solutions, preferably physiologically compatible buffers such as HANK's or RINGER solutions.
- the antisense polynucleotides according to the invention can be formulated in solid form and then redissolved or resuspended immediately before use. According to encompasses lyophilized forms and liposomes containing such antisense polynucleotides.
- An antisense polynucleotide of the invention can also be administered systemically by the transmucosal, transdermal or oral routes.
- penetrating agents can be used in the formulation such as bile salts or fusidic acid derivatives.
- the present invention also relates to a method of treatment and / or prevention of cancer comprising a step of administration to a patient, in need of such treatment, a pharmaceutical composition as defined above comprising a compound ESM-1 antagonist of the antisense polynucleotide type .. ...
- any of the pharmaceutical compositions of the invention as defined above and comprising a therapeutically effective amount of an ESM-1 protein antagonist compound is useful in the prevention and / or treatment of cancer.
- a pharmaceutical composition according to the invention is useful for the prevention and / or treatment of cancers such as cancers of the respiratory tract, bronchopulmonary cancers, breast cancers, colon cancers and renal cancers as well as cancers of the digestive tract.
- cancers such as cancers of the respiratory tract, bronchopulmonary cancers, breast cancers, colon cancers and renal cancers as well as cancers of the digestive tract.
- FIG. 1 illustrates immunoblotting gels (“Western-Blot”) and stains of ESM-1 on SDS-PAGE gel. Each immunoblock gel was revealed with the anti-ESM-1 MEP14 monoclonal antibody. The second anti-mouse antibody labeled with horseradish peroxidase has been affinity purified and gives negative results when used alone.
- Figure 1A ESM-1 protein immunoblot gel of different cell types expressing this protein.
- ESM-1 Immunoprecipitation of ESM-1 from culture supernatants of cells SVI (1), 293-ESM (2) and CHO-ESM (3) was carried out with the antibody MEP19 when indicated, or with a control antibody.
- the arrows indicate the specific band of ESM-1.
- the native form of ESM-1 is represented by a diffuse band around 50 kD.
- FIG. 1B Absence of detection of the purified protein ESM-1 with coomassie blue.
- FIG. 1C Detection of the protein ESM-1 purified with alkian blue.
- FIG. 2 illustrates the apparent molecular weight of the peptide and glycan bodies of ESM-1.
- FIG. 1A Analysis by mutation of the O-glycosylation binding site.
- the wild ESM-1 protein (WT for “Wild-Type”), the ESM-1 mutants T120A and S137A) as well as the negative controls (MOCK) were transfected into 293 cells and the cell culture supernatants as well as the cell lysates were analyzed by immunoblotting (Western-Blot) using the monoclonal antibody MEP14. The arrows indicate the specific bands.
- FIG. 2B Effect of proteinase K treatment on ESM-
- the ESM-1 protein purified from SVI (1) and 293-ESM (2) cells was digested with proteinase K and loaded onto a 15% SDS-PAGE gel.
- the upper arrow indicates the wild form of the untreated ESM-1 protein and the lower arrow indicates the proteinase K digested ESM-1 protein.
- Figure 3 illustrates the effects of specific chondroitinases on ESM-1.
- FIG. 3A Treatment of purified wild protein ESM-1 with chondroitinase ABC.
- the secreted ESM-1 protein was purified by ion exchange chromatography, followed by immunoaffinity chromatography from supernatants from SVI (1), 293-ESM (2) cell cultures and from of human plasma (3), then digested or not by chondroitinase ABC. 50 ng of the digested protein were loaded onto a 15% SDS-PAGE gel and then analyzed by immunoblotting (Western-Blot). The upper arrow indicates undigested forms of ESM-1 and the lower arrow indicates digested forms of ESM-1.
- Figure 3B Treatment of purified wild-type ESM-1 protein with chondroitinase B.
- ESM-1 protein purified from culture supernatants of SVI (1) and 293-ESM (2) cells were digested or not by chondroitinase. Proteins were loaded onto a 15% SDS-PAGE gel.
- the upper arrow indicates the different forms of ESM-1 no digested around 50 kD and the lower arrow indicates the different forms of the digested ESM-1 protein, around 22 kD.
- Figure 3C Treatment of purified wild protein ESM-1 with chondroitinase AC.
- the protein ESM-1 purified from culture supernatants of cells HUVEC (1) and 293-ESM (2) have been fired ??? with chondroitinase AC and were loaded onto a 15% SDS-PAGE gel.
- the upper arrow indicates the different forms of undigested ESM-1 around 502 kD and the lower arrow indicates the different digested forms of the ESM-1 protein around 22 kD.
- Figure 3D Treatment of wild ESM-1 protein. purified with chondroitinase C.
- the protein ESM-1 purified from culture supernatants from HUVEC (1) and 293-ESM (2) cells were digested or not by chondroitinase C and were loaded onto a 15% SDS-PAGE gel.
- the upper arrow indicates the different undigested forms of ESM-14 around 50 kD and the lower arrow indicates the different digested forms of the ESM-1 protein around 22 kD.
- FIG. 4 illustrates the effects of the purified wild-type ESM-1 protein on the coagulation time in the presence of thrombin.
- the delay and reduction in thrombin formation can be noted for the heparin plasma as well as for the other four curves (plasma rich in platelet or PRP: empty diamond; plasma rich in platelet + ESM-1 at 0.2 mg / ml : full square; plasma rich in platelet + ESM-1 0 0.5 mg / ml: solid triangle; plasma rich in platelet + ESM-1 at 1 mg / ml: full circle; plasma rich in platelet + heparin: empty circle) .
- FIG. 5 illustrates the biological activity of the proteoglycan ESM-1 on the proliferation of 293 cells induced by the HGF / SF factor.
- the stimulation of the incorporation of 3 H-thymidine by the 293 cells induced by the HGF / SF factor was studied.
- the cells were seeded at 1 ⁇ 10 4 cells per well in DMEM medium added transferin and insulin and HGF / SF at 50 ng per ml before adding different molecules.
- the bars represent the percentage increase in the incorporation of 3 H-thymidine (mean +/- SEM of samples in triplicate from a representative experiment) in the presence of the indicated additions of serum, of the different forms of ESM-1 to 2.5 mg / ml and decorin at 2.5 mg / ml.
- the background noise of incorporation of 3 H-thymidine 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 decorin on the mitogenic activity induced by the HGF / SF factor.
- ESM-1 / WT empty square
- ESM / S137A solid circle
- GAG chain derived from the protein ESM / WT solid square
- decorin empty circle
- FIG. 7 illustrates the tumorigenicity of the protein ESM-1.
- Two batches of more than 10 mice received HEK control cells or HEK cells transfected with a vector coding for the cDNA of the wild-type ESM-1 protein (ESM / WT).
- ESM / WT wild-type ESM-1 protein
- FIG. 7A the percentage of macroscopically visible tumors at the eighth week at the injection site and whose tumor volume is greater than 1 cm 3 is represented on the ordinate.
- FIG. 7B illustrates the kinetics of appearance of tumors in mice having received transfected HEK cells expressing the glycosylated protein ESM-1 (ESM / WT). The number of weeks after the injection of the cells is shown on the abscissa. The ordinate shows the average tumor volume, expressed in cm 3 .
- 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. On the abscissa are shown respectively the batch of mice having received the control HEK cells and the batch of mice having received the HEK cells expressing the glycosylated protein ESM-1 (ESM / WT). The serum ESM-1 level found, expressed in nanograms / ml, is represented 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 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 ESM-1 protein level, expressed in nanograms / ml, is represented on the ordinate.
- FIG. 9 illustrates the tumorigenic activity of different forms of the protein ESM-1.
- FIG. 9A illustrates the appearance of tumors in different batches of mice, the mice having respectively received the HEK control cells, the HEK cells transfected with a cDNA coding for the glycosylated protein ESM-1 (ESM / WT), the cells transfected with the non-glycosylated ESM-1 protein (ESM / S137A) and the HEK cells transfected with a cDNA coding for the ESM-1 protein mutated at positions 134 and 135 (ESM / F1 15A, F116A).
- the ordinate shows the percentage of macroscopically visible tumors at the eighth week at the injection site, the tumor volume of which is greater than 1 cm 3 .
- FIG. 9B illustrates the serum level of ESM-1 in the different identical batches of mice.
- the serum ESM-1 level, expressed in nanograms / ml, is represented on the ordinate.
- FIG. 10 illustrates the inhibitory effect of the monoclonal antibody MEP08 on the pro-tumor activity of the protein ESM-1.
- the injection of MEP-08 antibodies increases the survival of the mice of the HEK ESM / WT group.
- the MEP-08 monoclonal antibodies are injected intraperitoneally at a dose of 400 ⁇ g from the second following inoculation of the HEK / ESM-WT cells. The injections are repeated weekly for 12 weeks.
- a control antibody, MEP-14 is used under the same conditions.
- the mice are sacrificed when their tumor volume is greater than 6 cm 3 , (n> 8 mice in each group). The figure represents the percentage of live mice in each of the groups.
- CHO cells were cultured in a MAM ⁇ culture medium (Gibco BRL, Life Technologies, France) supplemented with 10% fetal calf serum.
- Human endothelial cells transfected with the SV40 virus, the SV1 cells described by LASSALLE P et al. (1992) were cultured in RPMI 1640 medium containing 2 mM L-glutamine and 10% fetal calf serum.
- Human embryonic kidney cells, cells of line 293, were cultured in DMEM medium from Dulbecco with 10% fetal calf serum.
- the human embryonic renal cells, cells of line 293, used for the proliferation test were cultured in medium of EAGLE modified by Dulbecco (Gibco BRL) supplemented with insulin at 10 mg / ml and with transferin at 10 mg / ml.
- Proteinase and chondroitinase ABC are marketed by Boehringer Mannheim.
- Chondroitinases B, AC and C are marketed by Sigma Human HGF / SF factor is marketed by R & D and decorin is marketed by Sigma.
- Anti-ESM-1 monoclonal antibodies were produced and purified as described by BECHARD et al. (2000).
- the complete cDNA encoding ESM-1 was directed, purified and inserted into the expression vector pcDNA3 (marketed by Invitrogen) between the Xhol and Hindllll sites.
- the vector constructs were transfected into CHO and 293 cell lines in the presence of lipofectamine (GIBCO BRL), then selected on G418 (1000 ⁇ g / ml for the CHO line and 300 ⁇ g / ml for the line 293).
- Stably transfected cell lines were obtained by limiting dilution and the cells thus selected were designated CHO-ESM and 2936-ESM respectively
- the serine residue at position 137 (SEQ ID No. 1) and the threonine at position 120 (SEQ ID No. 1) were substituted with an alanine residue.
- O-glycosylation mutants were produced by PCR using the Quick kit
- the mutated cDNAs were confirmed by sequencing (ABI prism 377 sequencer from the company Applied Biosystems. Then the 293 cells were transfected with the vectors into which the mutant cDNAs were inserted to obtain transient transfectants and stable transfectants, respectively the lines. 293-ESM / S 137A and 293-ESM / T120A.
- the cell culture supernatants were adjusted to pH8, then passed over a DEAE-Sepharose column (Pharmacia), washed with a 50 mM Tris buffer (pH8), 0.2 MNaCI, then eluted in a 50 mM Tris buffer (pH8 ), 0.8 M Nacl.
- the eluates were adjusted to 50 mM Tris (pH8), 0.5 MNaCI and passed through an affinity column.
- the affinity column consists of anti-ESM-1 monoclonal antibodies (produced by the hybridoma line MEC4) immobilized on an Affigel Hz hydrazide gel, according to the manufacturer's recommendations (Biorad).
- the ESM-1 protein was eluted with a 3M solution of MgCI 2 , concentrated and dialyzed against the same buffer on an ultrafree device 30 ( millipore).
- the eluted material was then quantified by immunodetection with anti-ESM-1 antibodies, and checked on SDS-PAGE using a staining with coomassie blue or alkian blue.
- the purification of the ESM-1 protein from human plasma was carried out according to the following protocol.
- ESM-1 The non-glycosylated form of ESM-1 (ESM / S137A) was purified in a single step by chromatography and immunoaffinity. The degree of purity of the glycosylated protein ESM-1 (ESM / WT) and of the serine 137 non-glycosylated protein (ESM / S137A) was checked by FPLC. The purified material is free of endotoxins, as evidenced by the results of the limulus amebocyte lysate test (BlOwhitaker).
- ESM-1 The size of the different forms of ESM-1 was determined by immunoprecipitation and immuno-imprinting 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 ° C with shaking. Then, the lysates were centrifuged at 10,000g for 15 min to obtain clarified cell lysates.
- the culture supernatants were filtered through a filter having a pore diameter of 0.45 mm.
- 1 ⁇ g of the ESM-1 monoclonal antibody produced by the MEP19 hybridoma line or 1 ⁇ g of anti-ICAM-1 monoclonal antibody (clone 164B) was added to the clarified lysate or to the cell culture supernatant and incubated for one overnight at 4 ° C with stirring.
- the beads were resuspended in 20 and 40 ⁇ l of SDS-PAGE buffer for 5 min, centrifuged, and the supernatants were analyzed.
- the samples were subjected to SDS-PAGE gel electrophoresis, then transferred to a nitrocellulose membrane according to standard procedures.
- the membranes were incubated for one hour with a monoclonal antibody ESM-1 produced by the hybridoma line MEP14 at 1 ⁇ l, washed and then incubated for I hour with a secondary anti-mouse Fc antibody conjugated to horseradish peroxidase ("Horse Radish peroxidase”) (sold by the company Sigma).
- Horse Radish peroxidase horseradish peroxidase
- washes were carried out before revelation using the ECL detection kit sold by the company Amersham.
- the purified ESM-1 protein was subjected to electrophoresis on an SDS-PAGE gel, then electrotransfered onto a polyvinylidene difluoride (PVDF) membrane sold by the company Millipore, then stained. using 0.1% coomassie blue.
- PVDF polyvinylidene difluoride
- the protein band at 50 kD was excised from the membrane and the N-terminal sequence was carried out by degradation of automated EDMAN on a protein sequencer of the ABI 473A type.
- ESM-1 ratio equal to 1: 50 (weight: weight) in a 10 mM Tris buffer, pH8, in the presence or in the absence of 0.1% SDS at 56 ° C for 3 hours.
- BSA bovine serum albumin
- the purified ESM-1 protein was digested with several chondroitinases: ABC chondroitinases (0.5 unit / mg in 100 mM TrisHCI buffer, pH 8, 30 mM acetate sodium, pH 5.2 at 37 ° C for 45 min), chondroitinase B (200 units / mg in 20 mM Tris-HCl buffer, 50 mM Nacl, 4 mM CaCl 2 , 0.01% BSA, pH 7.5 at 25 ° C for two hours), chondroitinase AC (one unit per ml in 250 mM Tris Hcl buffer, 75 mM sodium acetate, pH 7.3 at 37 ° C for two hours) chondroitinases C (80-120 units / ml in 50 mM Tris HCl buffer, pH 8 at 25 ° C for 3 hours). The samples were analyzed by immunoblotting.
- ABC chondroitinases 0.5 unit / mg in 100 mM TrisHCI buffer, pH
- the control plasma poor in platelets was prepared from blood in the presence of the anticoagulant sodium citrate (30 mM), by centrifugation at 2500 g for 15 min. All reagents are marketed by STAGO Diagnostica (France). Three parameters were evaluated, by adding the ESM-1 protein, buffer or heparin to the plasma poor in platelets: a) APTT (for “Activated Partialo Thromboplastin Time”): this parameter explores the intrinsic pathway of blood coagulation (FI, Wire, FV, FVIII, FIX, FX, FXI, FXII). The deficit or inhibition of one of these factors increases the coagulation time of the PPP reactive mixture, cephalin, activator, CACI 2 .
- TCT for “Thrombin Clotting Time”: this parameter is analyzed on a mixture of plasma poor in platelets (PPP) in the presence of thrombin. With a standard concentration of thrombin, the plasma coagulation time is constant. Defects in fibrin formation induce an increase in clotting time.
- anti-Xa activity the anti-Xa activity of heparin or of other inhibitors acting on the factor FXa is detected by a competitive test.
- the studied sample PPP + ESM-1, + buffer or + heparin
- factor Fxa a chromogenic substrate specific for factor Fxa. The final coloration is inversely proportional to the concentration of inhibitor.
- This sensitive global test can detect plasma or platelet defects inducing a delay or a reduction in the generation of thrombin.
- a platelet rich plasma PRP
- PRP platelet rich plasma
- the thrombin generation test was carried out, for each of the subjects, in samples in the absence of ESM-1, with unfractionated calcium heparinate (0.5 IU of anti-Xa / ml) or with 0.2mg / ml, 0.5 mg / ml and 1 mg / ml ESM-1 (final concentration).
- the ESM-1 protein was added 10 min before the test. At 37 ° C, 1 ml of plasma was mixed with 1 ml of CaCl 2 and the stopwatch was started. 0.1 ml aliquots were taken up from the reaction mixture every minute for 1 min.
- the clot formed in the reaction mixture is regularly removed.
- the aliquots were mixed with 0.2 ml of fibrinogen (Sigma, 4/1000 in Owren buffer at 37 ° C and the coagulation time was measured for each of the aliquots).
- the thrombin formed in the reaction mixture acts on the forminogens, inducing the formation of fibrin.
- the coagulation activity was maximum between 4 and 8 min and then decreased due to the neutralization of the thrombin by the anti-thrombin.
- ESM-1 glycosylated (ESM / WT) and non-glycosylated ESM-1 (ESM / S137A) purified in 50 mM Tris buffer, pH 8.5, 0.5 MNAcl were separated by liquid chromatography on a Superdex 200 column (for ESM / WT) or Superdex 75 column (for ESM / S137A) sold by Pharmacia, using the Biorad Biologie Chromatography System with a flow rate of 1 ml / min.
- ribonuclease A from bovine pancreas, 13.7 kD
- ovalbumin 43 kD
- albumin bovine serum, 67 kd
- aldolase rabbit muscle, 158 kD
- ferritin horse spleen, 440 kD
- thiroglobulin bovine thyroid, 669 kD
- the molecular weight standards were separated using a buffer identical to that used for the ESM-1 proteins and the separation was carried out immediately after the separation of the ESM / WT and ESM / S137A proteins.
- ESM-1 protein was detected using a specific enzyme-linked immunosorbent assay (ELISA).
- ELISA enzyme-linked immunosorbent assay
- ESM-1 Post-translational modifications of the secreted form of the ESM-1 protein produced by endothelial cells and by established cell lines.
- the 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 ESM-1 polypeptide of 165 amino acids starting at the tryptophan residue in position 20 of the sequence SEQ ID No. 1, the N-terminal sequence being "WSNNYAVD-P".
- ESM-1 was immunoprecipitated from the cell culture supernatants of HUVEC, SV1, 293-ESM and CHO-ESM cells, then analyzed by immunoblotting.
- ESM-1 The molecular weight found is more important than the predicted molecular weight. This result suggested that the secreted form of ESM-1 had undergone post-translational modifications. The fact that the purified ESM-1 protein was better stained on the SDS-PAGE gel with alkian blue than with coomassie blue suggested that ESM-1 was glycosylated (FIGS. 1B, 1C) rather than oligomerized across the bridge disulfide, since reducing conditions do not change the apparent molecular weight of ESM-1.
- the serine residue at position 137 (SEQ ID No. 1) is the O-glycosylation site of ESM-1.
- a computer analysis of the potential glycosylation sites made it possible to identify three putative sites of O-glycosylation, respectively for the serine in position 16, on the threonine in position
- the threonine residue at position 120 and the serine residue at position 137 have been mutated and replaced by an alanine residue. These mutants were transiently expressed in 293 cells.
- the ESM-1 protein was then immunoprecipitated from cell lysates and culture supernatants, and analyzed by immunoblotting.
- ESM / T120A protein migrates at 50 kD, at a position similar to the apparent molecular weight of the wild form of ESM-1 (ESM / WT), as shown in Figure 2A.
- ESM / S137A protein migrates to 22 kD corresponding to the intracellular form of ESM-1 ( Figure 2A), a molecular weight compatible with the predicted molecular weight of ESM-1.
- glycosaminoglycan (GAG) of ESM-1 In order to determine the length of the glycosaminoglycan (GAG) of ESM-1, the peptide body of ESM-1 was completely digested by proteinase K.
- Proteinase K treatment induces a change in molecular weight from 50 kD to 25-30 kD (Figure 2B). These results show that the band of apparent molecular weight at 50. kD is compatible with the presence of a 22 kD polypeptide which is glycoconjugated on the serine at position 137 by a GAG chain with an average size of 25-30 kD.
- the profile is similar with the protein ESM-1 purified from 293-ESM cells as well as from the human endothelial cell line SVI. Because the protein ESM-1 circulates in the blood, not a also studied the behavior of the protein ESM-1 purified from human plasma. The results make it possible to observe a single main band of 50 kD, which has a molecular weight of 22 kD after treatment with chondroitinase ABC, as for all the other cell lines studied (FIG. 3A). Thus, the ESM-1 protein is a soluble proteoglycan containing a single chain of chondroitin sulfate.
- the GAG chain of ESM-1 is a heterogeneous chain of chondroitin / dermatan sulfate.
- chondroitinases B Treatment with chondroitinase B reduces the apparent molecular weight from 50 kD to 22 kD ( Figure 3B).
- GAG chain of ESM-1 contains different composite units containing a type of amino sugar, N-acetylgalactosamine, coupled to differently sulphated iduronic or glucuronic acid.
- ESM-1 protein is secreted as a proteoglycan of the chondroitin / dermatan sulfate type by endothelial cells and because dermatan sulfate has effects on the generation of thrombin in vitro DELORME et al., (1996) and on coagulation, the potential anticoagulant activity of ESM-1 has been verified on the parameters APTT, TCT, anti-Xa activity and on the generation of thrombin.
- the APTT, TCT and anti-Xz activity parameters are similar for platelet-poor plasma (PPP) with the buffer or with the protein ESM-1. In the positive controls, the APTT, TCT and anti-Xa activities are greater for the PPP in the presence of heparin.
- the ESM-1 protein has no inhibitory effect on the thrombin generation test: there is no difference according to the concentrations of 0.2 mg / ml, 0.5 mg / ml and 1 mg / ml of ESM-1 relative to the buffer control, while heparin induces a delay in the formation of thrombin (FIG. 4).
- the proliferation-stimulating activity was determined by measuring the incorporation of 3 H thymidine by the 293 cells.
- the 293 cells were seeded at the concentration of 1 ⁇ 10 4 cells per well in 96-well microplates of the TPP type and were maintained for 24 hours in the DMEM culture medium supplemented with transferin and insulin.
- the recombinant human HGF / SF was diluted in PBS containing 0.1% bovine serum albumin and added with water from 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 the factor HGF / SF at doses ranging from 1 ng / ml to 2.5 ⁇ g / ml, simultaneously with the addition of HGF / SF.
- HGF / SF alone at 50 ng / ml induces proliferation of 293 cells at a level equal to approximately 45% of the proliferation induced by serum, while the protein ESM / WT alone did not stimulate the proliferation of 293 cells.
- the effect of the protein ESM / WT was compared with the effect of decorin, another proteoglycan of the chondroitin sulfate / dermatan sulfate type, on the mitogenic activity of the HGF / SF factor.
- decorin does not show any activity for increasing the proliferation of 293 cells induced by the HGF / SF factor (FIGS. 5, 6).
- ESM-1 The non-glycosylated form of ESM-1 is incapable of inducing proliferation of 293 cells, whether in the presence or in the absence of the factor HGFSF (FIG. 5), even when it is used in high concentration.
- the GAG chain purified from ESM-1 considerably increases the proliferation of 293 cells induced by the HGF / SF factor, with an increase factor close to 96.6%, compared to the HGF / SF factor alone (FIG. 5).
- the pro-mitogenic effect of the GAG chain is less than that observed with the wild form of the protein ESM-1, but this effect is nevertheless dependent on the dose of GAG chain added (FIG. 6).
- the HGF / SF factor is expressed during the critical early periods of human organogenesis of 6 to 13 weeks of gestation. Organs that express the HGF / SF gene are notably the liver, the metanephric kidney, the intestine and the lung, each of these organs developing by inductive interaction between the mesenchyme and the epithelium.
- the HGF / SF factor is an important factor in human renal multicystic dysplasia (TAKAYAMA et al., 1997) as well as in the appearance of malformation and hyperproliferation in the tubules.
- ESM-1 protein significantly increases the proliferation of embryonic kidney cells in the presence of HGF / SF while the non-glycosylated form and of the ESM-1 protein has no effect.
- GAG chain isolated from the ESM-1 protein is capable of mimicking the effects of the glycosylated protein ESM / WT.
- the protein ESM-1 is selectively detected in the distal tubules, the result which can be associated with the observation of the preferential localization of the HGF / SF factor in the same part, of the nephron in situations of renal multicystic dysplasia human (WEIDNER et al., 1993).
- WEIDNER et al. 1993
- These results indicate an application of the protein ESM-1 in pathological disorders dependent on the factor HGF / SF, which has also been shown to be associated in the development of cancers of the breast (RAHIMI et al., 1998), of the kidney (NATALI et al, (1996)) and lung (OTSUKA et al., 1998) but also in malignant melanomas (SIEGFRIED et al., 1998).
- the HGF / SF factor is likely to promote the extension of hyperplasia and to generate cells with an invasive phenotype.
- the ESM-1 protein is likely to be involved in these phenomena of deregulated mitogenic activities of the HGF / SF factor.
- the native form of the ESM-1 protein produced by the CHO cell line transfected with a vector d the expression containing a DNA insert coding for the protein ESM-1 has been purified.
- the ESM-1 cDNA is inserted into the eukaryotic expression vector pcDNA3 (In vitrogen) and then transfected into CHO cells with lipofectamine (GIBCO) according to the manufacturer's recommendations. 48 hours after transfection, the cells are subcultured in the presence of a selection agent (G418, Gibco) at a dose of 1000 micrograms / ml). After two weeks of selection, the G418-resistant CHO cells are given by limiting dilution. Clones expressing ESM-1 are then selected and called CHO-ESM (deposited at the CNCM). For production, CHO-ESM cells are cultured in suspension in a conditioned medium without fetal calf serum (CHO SFM II medium, Gibco).
- the supernatant is adjusted to pH 8 and weighed on a DEAE-sepharose column (Pharmacia).
- the column is washed with a 50 mM Tris buffer, pH 8, 0.2 M NaCl.
- the ESM-1 molecule is eluted in a 50 mM Tris buffer, pH 8, 1 M NaCl.
- the eluate is then diluted 1: 4 in a 50 mM Tris buffer, pH 8 and incubated in the presence of anti-ESM-1 monoclonal antibody (MEC4) immobilized on agarose (Biorad). After an overnight incubation at 4 ° C. with agitation, the agarose beads are washed with the 50 mM Tris buffer, pH 8, 0.2 M NaCl.
- ESM-1 is elected with 3M MgCI 2 .
- the eluate is concentrated and dialyzed in the 50 mM Tris buffer, pH 8, 0.5 M NaCl and stored at -70 ° C.
- Balb / C mice were immunized by injection of 10 ⁇ m of recombinant protein ESM-1 purified by mouse, according to a standard immunization protocol in the presence of Freund's adjuvant.
- Hybridoma cells secreting anti-ESM-1 monoclonal antibodies were obtained by fusion, screening and subcloning according to the technique described by BECHARD et al. (2000).
- hybridoma Five cell clones of hybridoma were obtained and were generically designated MEC ("Mouse Monoclonal Antibody to ESM-1 produced by CHO Cells"). Four of the hybridomas selected are isotypes lgG1, k respectively the hybridomas designated MEC4, MEC5, MEC15 and MEC36.
- hybridomas is the IgM.k isotype, the MEC11 hybridoma.
- the cell clones of hybridomas were cultured in culture medium in the absence of serum and the anti-ESM-1 antibodies were purified by column chromatography on protein G-Sepharose sold by the company Pharmacia (UPSALA, Sweden) .
- Directed mutagenesis was carried out with the kit marketed by the company STRATAGENE under the reference “Site-directed quick mutagenesis kit", which was used according to the manufacturer's recommendations.
- a pair of round-trip primers of strictly complementary sequences are synthesized, these primers comprising the nucleotides coding for the mutated amino acid (s), or the complementary nucleotides, these nucleotides being located at the center of the sequence of the primers which also include approximately 10 to 15 consecutive nucleotides complementary to the sequence to be amplified both 5 'and 3' of the central nucleotides.
- the amplified polynucleotides encoding the mutated ESM-1 protein are inserted into the vector pCDNA3.
- Primer go: 5'-GCC TGA AAT TCC CCG CCT TCC AAT ATT CAG-3 '(SEQ ID N ° 3).
- the cell line named HEK ESM / WT was transfected stably with the cDNA encoding the wild form of ESM-1 (ESM / WT).
- ESM / WT The cell line named HEK ESM / WT was transfected stably with the cDNA encoding the wild form of ESM-1 (ESM / WT).
- ESM / WT Four other cell lines are obtained by transfection with cDNA encoding purified forms of ESM-1 obtained by site-directed mutagenesis from the wild form.
- the first of these, called HEK ESM / S137A expresses the mutated, non-glycosylated protein ESM-1, where an alanine is substituted for serine 137, the major site of O-glycosylation.
- the other three lines expressing a glycosylated form of ESM-1 whose protein part is mutated.
- ESM-1 HEK ESM / WT
- ESM-1 HEK ESM / S137A
- mice used are of the SCID (Severe Combined IMMUNO Deficiency) type. More specifically, they are C.B.17 Scid / scid mice supplied by the animal facility of the Institut Pasteur in Lille. These mice have an autosomal recessive mutation in their recombination system (Blunt., 1995). This mutation results in the production of immunoglobulins and non-functional T cell receptors (TcR) and B 5BcR). As a result, they do not have functional T and B lymphocytes; these mice therefore tolerate non-self and represent a model. of choice for the development of xenogenic tumors.
- the SCID mice used are male and young mice since they are 3 to 5 weeks old.
- anti-ascialo GM-1 antibodies 100 ⁇ g per mouse diluted in 200 ⁇ l of RPMI
- rabbit polyclonal antibodies (Wako Pure Chemical Industries, Ltd) directed specifically against the asialo GM-1 antigen expressed by NK cells.
- mice Four batches of mice (10 to 15 mice per group) anesthetized with ether are then injected, subcutaneously at the back. Each mouse receives 1 million cells diluted in 200 ⁇ l of RPMI. The injection of these cells defines the first day of the experiment (Jo). For each mouse, macroscopic monitoring of the injection site in search of the appearance of a possible tumor as well as a measurement of the body weight are carried out weekly. From the 5th week, the mice are bled (approximately 500 ⁇ l per mouse), once a week, in order to measure the serum levels of ESM-1 by a ELISA test (BECHARD D et al., 2000). For each mouse, an anatomo-pathological examination is carried out.
- HEK cells were transfected with a vector having an insert containing the cDNA coding for the glycosylated wild-type protein ESM-1, designated ESM / WT.
- HEK cells are injected subcutaneously into 5 week old SCID mice. Each mouse receives. previously injected anti-asialo GN-1 antibody intraperitoneally. The percentage of tumors with a volume greater than 1 cm 3 observed in the mice at the eighth week following the injection of the transfected HEK cells was analyzed.
- FIG. 7A it is observed that the injection of HEK control cells does not induce the appearance of tumors in the mouse.
- HEK cells transfected with DNA encoding the glycosylated protein ESM-1 induce many macroscopically visible tumors, of which approximately 95% of them have a tumor volume greater than 1 cm 3 .
- FIG. 7B illustrates the kinetics of appearance of tumors in mice which have received HEK cells transfected with DNA coding for the glycosylated protein ESM-1. It can be observed that the average tumor volume, expressed in cm 3 , increases continuously from the fourth week following the injection of the transfected HEK cells.
- the experimental results presented in FIG. 7 clearly demonstrate that the glycosylated protein ESM-1 has a pro-tumor activity.
- ESM-1 protein serum levels were also measured in mice having received control HEK cells and in mice having received HEK cells transfected with the cDNA encoding protein ESM-1. The results are shown in Figure 8.
- FIG. 8A show that the ESM-1 protein is not found in the sera of the mice having received the HEK control cells. On the contrary, a serum level of 40 to 50 nanograms per ml is found in mice having received HEK cells transfected with the cDNA coding for the protein ESM-1 at the eighth week following the injection of the cells.
- the kinetics of the serum ESM-1 levels were also analyzed in the mice having received the transfected HEK cell expressing the glycosylated protein ESM-1 (ESM / WT).
- the experimental results illustrated in FIG. 8 show that the tumors which have grown in mice having received the transfected HEK cells produce the protein ESM-1.
- the amount of ESM-1 protein produced in the circulation follows the kinetics of tumor development in mice.
- the HEK cells were transfected respectively by vectors having a DNA insert coding respectively for the 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 each been replaced by an alanine residue (ESM / 73).
- the different transfected cells were injected subcutaneously into 5-week-old SCID mice having previously received anti-asialo GM-1 antibodies.
- Glycosylated ESM-1 is capable of inducing tumors in mice. Neither the non-glycosylated ESM-1 protein nor the glycosylated but mutated ESM-1 protein on the phenylalanine residues at positions 134 and 135 are capable of inducing the development of tumors in SCID mice.
- the serum level of circulating ESM-1 protein was also analyzed in the different batches of mice. The results are shown in Figure 9B.
- FIG. 9B show that detectable levels of serum ESM-1 protein can be measured, at the eighth week following the injection of the cells, only in mice having received HEK cells expressing the glycosylated ESM-1 protein ( ESM / WT).
- mice injected with cells expressing the non-glycosylated protein ESM-1 ESM / S137A
- mice having received the HEK cells expressing the glycosylated and mutated protein ESM-1 HEK-ESM / F115A, F116A do not produce protein ESM-1.
- the immuno-detection test consists of an immuno-enzymatic test of the “sandwich” type, the general characteristics of which are identical to that described by BECHARD et al. (2000).
- the anti-ESM-1 monoclonal antibody produced by the MEP14 hybridoma line (CNCM No. l-1942) was diluted to a concentration of 5 ⁇ g / ml in 0.1 M carbonate buffer, pH 9.5, and adsorbed overnight at + 4 ° C on a 96-well plate (EIA / R.IA plate, Costar, Cambridge, MA, USA).
- the plate was saturated for one hour at laboratory temperature with a volume of 200 ⁇ l / well of PBS buffer containing 0.1% bovine serum albumin and 5 mM EDTA, then washed twice with ELISA buffer (PBS buffer above with 0.1% Tween 20 added).
- ELISA buffer PBS buffer above with 0.1% Tween 20 added.
- the blood samples were diluted serially (1: 2 to 1: 128) in ELISA buffer and incubated on an ELISA plate for one hour at laboratory temperature.
- the wells were washed three times with ELISA buffer and then incubated for 1 hour at laboratory temperature with a second monoclonal antibody directed against ESM-1, the antibody MEC15 (CNCM No. 1-2572) at the concentration of 0, 1 ⁇ g / ml in 100 ⁇ l of buffer per well.
- a biotinylated rat monoclonal antibody directed against mouse lgG1 was added (marketed by PHARMINGEN) diluted in ELISA buffer and allowed to incubate this second antibody for one hour.
- the streptavidin-peroxidase conjugate is revealed with the TMB substrate sold by the SIGMA company (Saint-Louis, MO, USA) in the presence of 255 ⁇ l of H 2 0 2 for 30 '.
- the revelation reaction is stopped by the addition of a volume of 100 ⁇ l of 2N H 2 SO 4 .
- the plate is read using a spectrophotometer (anthos labtec LP40, France) at the wavelength of 405 nanometers.
- the concentration of the plasma or serum ESM-1 protein is calculated from the optical density measurements and expressed in nanograms per ml.
- the concentration of circulating ESM-1 protein in the serum of different patients with bronchopulmonary cancer was measured at different stages of development, respectively in stages I, II, IIIA, IIIB and IV according to the international TNM classification which is defined below. -after:
- T tumor size (T1: ⁇ 1 cm; T2: between 1 and 3 cm; T3:> 3 cm.
- N lymph node (NO if not invaded; N1 if invaded).
- M distant metastasis (MO if no meastasis; M if metastasis).
- EXAMPLE 8 Anti-tumor activity of an ESM-1 antagonist compound of the antibody type.
- the monoclonal antibodies MEP-08 are injected intraperitoneally at a dose of 400 ⁇ g from the second following inoculation of the HEK ESM-WT cells. The injections are repeated weekly for 12 weeks. A control antibody, MEP-14, is used under the same conditions. The mice are sacrificed when their tumor volume is greater than 6 cm 3 , (n> 8 mice in each group). The figure represents the percentage of live mice in each of the groups.
- FIG. 10 shows that early injections of MEP-08 antibodies significantly increase mouse survival by almost 60% while MEP-14 antibodies have no effect.
- These first results show that it is a specific action linked to the Fab fragment of the antibody directed specifically against phenylalanine at position 115 and confirm the involvement of the peptide in tumor growth. It is surprising to find that this effect on survival decreases when antibodies are administered in a delayed manner. No matter which week the injections start, antibodies can delay or prevent tumor growth. This anti-tumor effect remains more pronounced when the antibodies are used early.
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FR0014422 | 2000-11-09 | ||
FR0014422A FR2816214B1 (fr) | 2000-11-09 | 2000-11-09 | Utilisation d'un compose antagoniste de la proteine esm-1 pour la fabrication d'un medicament pour la prevention et/ou le traitement d'un cancer |
PCT/FR2001/003475 WO2002038178A1 (fr) | 2000-11-09 | 2001-11-08 | Utilisation d'un compose antagoniste de la proteine esm-1 pour la fabrication d'un medicament pour le traitement d'un cancer |
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US (1) | US7306797B2 (fr) |
EP (1) | EP1335745A1 (fr) |
JP (1) | JP4121852B2 (fr) |
AU (1) | AU2002218342A1 (fr) |
CA (1) | CA2429404A1 (fr) |
FR (1) | FR2816214B1 (fr) |
WO (1) | WO2002038178A1 (fr) |
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EP1565552A4 (fr) * | 2002-07-01 | 2006-05-10 | Pharmacia Corp | Gene esm-1 exprime differentiellement dans l'angiogenese, antagonistes de dernier et methodes d'utilisation correspondantes |
US20070197452A1 (en) * | 2006-02-17 | 2007-08-23 | Mclaurin Joanne | Treatment of amyloid-related diseases |
JP2011502111A (ja) * | 2007-10-24 | 2011-01-20 | アンスティチュ パストゥール ドゥ リール | 非グリカン化ポリペプチドの癌治療での使用 |
TWI772927B (zh) * | 2015-03-31 | 2022-08-01 | 德商英麥提克生物技術股份有限公司 | 用於腎細胞癌(rcc)免疫治療的新型肽和肽組合物和支架 |
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US5747280A (en) * | 1995-06-05 | 1998-05-05 | Human Genome Sciences, Inc. | Human vascular IBP-like growth factor |
AU710568B2 (en) * | 1994-12-09 | 1999-09-23 | Human Genome Sciences, Inc. | Human vascular IBP-like growth factor |
US6670328B1 (en) | 1997-06-24 | 2003-12-30 | Institut Pasteur De Lille | Proteins and peptides derived from protein ESM-1 and their uses in the treatment and diagnosis of diseases linked to leukocyte migration |
FR2775691B1 (fr) * | 1998-03-05 | 2000-12-15 | Pasteur Institut | Anticorps monoclonaux specifiques de la proteine esm-1, et utilisation de ces anticorps pour la detection de la proteine esm-1 |
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2000
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2001
- 2001-11-08 WO PCT/FR2001/003475 patent/WO2002038178A1/fr active Application Filing
- 2001-11-08 CA CA002429404A patent/CA2429404A1/fr not_active Abandoned
- 2001-11-08 EP EP01993475A patent/EP1335745A1/fr not_active Withdrawn
- 2001-11-08 US US10/416,203 patent/US7306797B2/en not_active Expired - Fee Related
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AU2002218342A1 (en) | 2002-05-21 |
JP4121852B2 (ja) | 2008-07-23 |
US20040234526A1 (en) | 2004-11-25 |
US7306797B2 (en) | 2007-12-11 |
CA2429404A1 (fr) | 2002-05-06 |
FR2816214A1 (fr) | 2002-05-10 |
WO2002038178A1 (fr) | 2002-05-16 |
FR2816214B1 (fr) | 2005-10-21 |
JP2004518638A (ja) | 2004-06-24 |
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