EP0763121A1 - VERFAHREN ZUR KREBSBEHANDLUNG DURCH REGULIERUNG DES p53-PROTEINS - Google Patents

VERFAHREN ZUR KREBSBEHANDLUNG DURCH REGULIERUNG DES p53-PROTEINS

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Publication number
EP0763121A1
EP0763121A1 EP95920973A EP95920973A EP0763121A1 EP 0763121 A1 EP0763121 A1 EP 0763121A1 EP 95920973 A EP95920973 A EP 95920973A EP 95920973 A EP95920973 A EP 95920973A EP 0763121 A1 EP0763121 A1 EP 0763121A1
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EP
European Patent Office
Prior art keywords
lys
glu
ser
calpain
asp
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EP95920973A
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English (en)
French (fr)
Inventor
Jean-Marie Blanchard
Serge Carillo
Marc Peichaczyk
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Aventis Pharma SA
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Rhone Poulenc Rorer SA
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Publication of EP0763121A1 publication Critical patent/EP0763121A1/de
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/8139—Cysteine protease (E.C. 3.4.22) inhibitors, e.g. cystatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates to a new method for the treatment of cancers. More particularly, it relates to a method of treating cancers by regulating the cellular levels of the p53 protein. It also relates to gene therapy vectors making it possible to regulate the p53 protein, as well as the pharmaceutical compositions containing them.
  • the elucidation of the catabolism of oncogenic and anti-oncogenic proteins represents a major challenge in terms of anti-cancer control insofar as it suggests, in the case of oncogenic proteins, the possibility of accelerating their degradation and therefore to annihilate their action, in the case of tumor suppressors, to inhibit their degradation and therefore to increase their anti-proliferative or anti-tumor effect, in the case of mutated proteins, to potentiate their antigenic presentation by the molecules of the Major Histocompatibility Complex and thus stimulate a tumor-specific immune response, and, in cases where the strong expression of the oncogene or anti-oncogene is capable of inducing programmed cell death, the possibility of stabilizing these proteins to trigger the apoptotic process.
  • the p53 protein was classified as a nuclear oncogene since it could, in transfection experiments, lengthen the life of rodent cells in culture as well as cooperate with oncogenes activated as ras to transform cells into primary culture.
  • the genes used in these first experiments were mutated and led to the expression of variant p53 proteins characterized by a gain in function.
  • the p53 protein at least in its wild form, is a transcription factor which negatively regulates growth and cell division and which, in certain situations, is capable of inducing apoptosis (Yonish-Rouach et al., Nature, 352, 345-347, 1991).
  • p53 has been suggested be a "genome keeper".
  • the wild-type p53 protein is subject to complex regulation which involves the control of its synthesis and catabolism as well as that of its intracellular localization and its post-translational modifications (see the journals cited above).
  • the wild-type p53 protein is extremely unstable with a half-life of a few minutes.
  • certain mutated proteins which accumulate at high level in tumors have a significantly extended half-life. Little has been clearly established regarding the degradation of p53. In fact, neither the intracellular degradation sites, nor the number and nature of the catabolic pathways used, nor the peptide motifs marking p53 for its degradation are known.
  • the present invention results in part from the demonstration that the p53 proteins are substrates for calcium-dependent proteases: calpains. It more particularly results from the demonstration that the p53 proteins are specifically degraded by m-calpain or ⁇ -calpain.
  • the present invention constitutes the first demonstration of a mechanism for regulating the cellular levels of the p53 proteins and thus offers a new particularly effective and specific approach for modulating the levels of this protein in pathological situations such as in particular certain cancers.
  • the present invention describes a new approach for the treatment of cancers, based on the use of compounds which modulate the activity of calpains on the p53 proteins, which make it possible either to activate the degradation of mutated p53 proteins, to block their tumorigenic effect and / or to increase the presentation of immunogenic peptides, ie to stabilize the wild-type p53 protein, to counterbalance the tumorigenic effect of mutated proteins expressed in tumors and / or to induce apoptosis of tumor cells.
  • a first object of the invention therefore resides in the use of a compound capable of modulating the activity of calpain for the preparation of a pharmaceutical composition intended for the treatment of cancers.
  • Calpains are ubiquitous enzymes found in most mammalian cells (for review, see Croall and deMartino, Physiol. Rev., 71, 813-847, 1991). They are essentially cytoplasmic but they can penetrate into the nucleus thanks to the destruction of the nuclear envelope during mitosis or following certain stimuli. As indicated above, the proteolytic activity of calpains is dependent on the presence of calcium.
  • the compounds capable of modulating the activity of calpain within the meaning of the present invention can be of several types.
  • inhibitor compounds capable of inhibiting the activity of calpain on the p53 proteins. These compounds are particularly advantageous since they can be used to inhibit, at least in part, the degradation of the wild-type p53 protein. These compounds therefore make it possible to intracellularly stabilize the wild-type p53 protein and to counterbalance the effect of the mutated forms.
  • protease inhibitors leupeptin, aprotinin, PMSF, etc.
  • calcium chelators EGTA, EDTA, etc.
  • Calpastatin is a known inhibitor of calpains.
  • a particularly advantageous embodiment of the present invention consists in transferring into the tumors a vector carrying all or part of the sequence coding for calpastatin. This approach is particularly suitable for the treatment of cancers always presenting a wild p53 allele, such as colonic or bronchial carcinomas for example.
  • Different fragments or derivatives of calpastatin can be used in the context of the present invention.
  • Such fragments or derivatives can be any molecule obtained from the sequence SEQ ID No. 1 by modification (s) of genetic and / or chemical nature, retaining the capacity to inhibit at least in part the activity of a calpain .
  • modification of a genetic and / or chemical nature is meant any mutation, deletion, substitution, addition, and / or modification of one or more nucleotides. Such modifications can be carried out for different purposes, and in particular that of preparing suitable sequences to expression in a particular type of vector or host, that of reducing the size of the sequence to facilitate their cell penetration, that of increasing the inhibition activity, or, in a particularly advantageous manner, of increasing the selectivity of the inhibitor with respect to the activity of calpains on the degradation of the wild-type p53 protein.
  • Such modifications can be made, for example, by in vitro mutagenesis, by introduction of additional elements or synthetic sequences, or by deletions or substitutions of the original elements.
  • a derivative as defined above When a derivative as defined above is produced, its activity as an inhibitor of the activity of calpains on the p53 proteins can be demonstrated in several ways, and in particular by bringing said inhibitor and the various forms into contact of p53 proteins, then by detecting the degradation products obtained (see examples 1 to 3). Any other technique known to those skilled in the art can obviously be used for this purpose.
  • all or part of calpastatin is used as an inhibitor, or a nucleic acid coding for all or part of calpastatin.
  • a peptide is used comprising all or part of the sequence SEQ ID No. 1 or a derivative thereof.
  • any derivative compound of sequence SEQ ID No. 1 or 2 capable of specifically or preferentially inhibiting the degradation of the wild-type p53 protein by calpain is used.
  • the compounds capable of modulating the activity of calpain on the p53 proteins within the meaning of the present invention may also be derivatives of calpain capable of specifically or preferentially degrading the mutated p53 proteins.
  • Such derivatives are also very advantageous since they make it possible to activate the degradation of the mutated p53 proteins, to block their tumorigenic effect and / or to increase the presentation of immunogenic peptides, without significantly affecting the cellular levels of wild-type p53 protein.
  • Such derivatives can be obtained from calpain, by structural modification (s) of genetic and / or chemical nature. The capacity of the derivatives thus obtained to specifically or preferentially degrade the mutated p453 proteins can then be demonstrated as described in Examples 1 to 3.
  • the modulators used in the context of the invention are proteins or polypeptides, or nucleic acid sequences coding for these polypeptides or proteins.
  • the modulator compounds are proteins or polypeptides specific for inhibiting the activity of calpain on the wild-type p53 protein or forms of calpains, modified or not, for specifically degrading the mutated p53 proteins.
  • the invention lies in the possibility of expressing, in cancer cells having both a wild-type p53 allele and a mutated p53 allele, nucleic sequences coding for calpain inhibitors, such as calpastatin or part calpastatin, or forms of calpains, modified or not, to specifically degrade mutated p53 proteins.
  • nucleic acid sequence used in the context of the present invention can be administered as it is, in the form of naked DNA according to the technique described in application WO 90/11092. It can also be administered in complex form, for example with DEAE-dextran (Pagano et al., J. Virol. 1 (1967) 891), with nuclear proteins (Kaneda et al., Science 243 (1989) 375) , with lipids
  • the sequence used in the context of the invention is part of a vector.
  • the use of such a vector in fact makes it possible to improve the administration of the nucleic acid in the cells to be treated, and also to increase its stability in said cells, which makes it possible to obtain a lasting therapeutic effect.
  • the vector used can be of various origins, since it is capable of transforming animal cells, preferably human cancer cells.
  • a viral vector which can be chosen from adenoviruses, retroviruses, adeno-associated viruses (AAV) or herpes virus.
  • AAV adeno-associated viruses
  • the present invention also relates to any recombinant virus comprising, inserted into its genome, a nucleic acid coding for a compound capable of modulating the activity of calpain.
  • viruses used in the context of the invention are defective, that is to say that they are unable to replicate autonomously in the infected cell.
  • the genome of the defective viruses used in the context of the present invention is therefore devoid of at least the sequences necessary for the replication of said virus in the infected cell. These regions can be either eliminated (in whole or in part), or made non-functional, or substituted by other sequences and in particular by the sequence coding for the calpain modulator.
  • the defective virus nevertheless retains the sequences of its genome which are necessary for the packaging of the viral particles.
  • adenoviruses various serotypes, whose structure and properties vary somewhat, have been characterized.
  • serotypes it is preferred to use, within the framework of the present invention, human adenoviruses of type 2 or 5 (Ad 2 or Ad 5) or adenoviruses of animal origin (see application FR 93 05954).
  • adenoviruses of animal origin which can be used in the context of the present invention, mention may be made of adenoviruses of canine, bovine, murine origin (example: Mavl, Beard et al., Virology 75 (1990) 81), ovine, porcine , avian or even simian (example: after-sales service).
  • the adenovirus of animal origin is a canine adenovirus, more preferably a CAV2 adenovirus [Manhattan strain or A26 / 61 (ATCC VR-800) for example].
  • adenoviruses of human or canine or mixed origin are used.
  • the defective adenoviruses of the invention comprise the ITRs, a sequence allowing the encapsidation and the sequence coding for the calpain modulator.
  • the El gene and at least one of the E2, E4, L1-L5 genes are non-functional.
  • the viral gene considered can be made non-functional by any technique known to those skilled in the art, and in particular by total suppression, substitution, partial deletion, or addition of one or more bases in the gene or genes considered. Such modifications can be obtained in vitro (on isolated DNA) or in situ, for example, by means of genetic engineering techniques, or by treatment with mutagenic agents.
  • the defective recombinant adenoviruses according to the invention can be prepared by any technique known to those skilled in the art (Levrero et al., Gene 101 (1991) 195, EP 185 573; Graham, EMBO J. 3 (1984) 2917). In particular, they can be prepared by homologous recombination between an adenovirus and a plasmid carrying among others the DNA sequence coding for the calpain modulator. Homologous recombination occurs after co-transfection of said adenovirus and plasmid in an appropriate cell line.
  • the cell line used must preferably (i) be transformable by said elements, and (ii), contain the sequences capable of complementing the part of the genome of the defective adenovirus, preferably in integrated form to avoid the risks of recombination.
  • a line mention may be made of the human embryonic kidney line 293 (Graham et al., J. Gen. Virol. 36 (1977) 59) which contains in particular, integrated into its genome, the left part of the genome an Ad5 adenovirus (12%).
  • Strategies for the construction of vectors derived from adenoviruses have also been described in applications Nos. FR 93 05954 and FR 93 08596.
  • the adenoviruses which have multiplied are recovered and purified according to conventional techniques of molecular biology, as illustrated in the examples.
  • AAV adeno-associated viruses
  • AAVs vectors derived from AAVs for gene transfer in vitro and in vivo has been described in the literature (see in particular WO 91/18088; WO 93/09239; US 4,797,368, US 5,139,941, EP 488,528). These applications describe various constructs derived from AAVs, in which the rep and / or cap genes are deleted and replaced by a gene of interest, and their use for transferring in vitro (onto cells in culture) or in vivo (directly into an organism ) said gene of interest.
  • the defective recombinant AAVs according to the invention can be prepared by co-transfection, in a cell line infected with a virus.
  • human helper for example an adenovirus
  • a plasmid containing the sequence coding for the modulator of calpains bordered by two inverted repeat regions (ITR) of AAV and of a plasmid carrying the packaging genes (rep and cap genes) of AAV.
  • ITR inverted repeat regions
  • rep and cap genes packaging genes
  • a defective recombinant adenovirus or retrovirus indeed have properties which are particularly advantageous for the transfer of genes into tumor cells.
  • the sequence coding for the calpain modulator is placed under the control of signals allowing its expression in tumor cells.
  • these are heterologous expression signals, that is to say signals different from those naturally responsible for the expression of the modulator.
  • They may in particular be sequences responsible for the expression of other proteins, or synthetic sequences.
  • they may be promoter sequences of eukaryotic or viral genes.
  • they may be promoter sequences originating from the genome of the cell which it is desired to infect.
  • they may be promoter sequences originating from the genome of a virus, including the virus used.
  • these expression sequences can be modified by adding activation, regulation sequences or allowing tissue-specific expression. It may in fact be particularly advantageous to use expression signals which are active specifically or mainly in tumor cells, so that the DNA sequence is not expressed and does not produce its effect until the virus has effectively infected a tumor cell.
  • the invention relates to a defective recombinant virus comprising a cDNA sequence coding for a calpain modulator under the control of a viral promoter, preferably chosen from LTR-RSV and the CMV promoter. Still in a preferred embodiment, the invention relates to a defective recombinant virus comprising a DNA sequence coding for a calpain modulator under the control of a promoter allowing predominant expression in tumor cells. Expression is considered to be predominant within the meaning of the invention when, even if residual expression is observed in other cell types, the expression levels are higher in tumor cells.
  • the present invention also relates to any pharmaceutical composition comprising one or more defective recombinant viruses as described above.
  • These pharmaceutical compositions can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, etc. administration.
  • the pharmaceutical compositions of the invention contain a pharmaceutically acceptable vehicle for an injectable formulation, especially for direct injection into the patient's tumor.
  • injectable formulations especially for direct injection into the patient's tumor.
  • They may in particular be sterile, isotonic solutions, or dry compositions, in particular lyophilized, which, by addition as appropriate of sterilized water or physiological saline, allow the constitution of injectable solutes.
  • Direct injection into the patient's tumor is advantageous because it allows the therapeutic effect to be concentrated in the affected tissues.
  • the doses of defective recombinant virus used for injection can be adapted as a function of various parameters, and in particular as a function of the viral vector, of the mode of administration used, of the pathology concerned or also of the duration of the treatment sought.
  • the recombinant adenoviruses according to the invention are formulated and administered in the form of doses of between 10 ⁇ and 10 ⁇ 4 pfu / ml, and preferably 10 à to 10 p pfu / ml.
  • the term pfu ( "plaque forming unit") corresponds to the infectious power of a virus solution, and is determined by infection of an appropriate cell culture, and measures, generally after 48 hours, the number of plaques of infected cells.
  • pfu titer of a viral solution are well documented in the literature Concerning retroviruses, the compositions according to the invention can directly comprise the producer cells, with a view to their implantation.
  • the present invention is particularly suitable for the treatment of cancers in which mutated forms of p53 are observed. More specifically, the The present invention is particularly advantageous for the treatment of cancers in which the wild and mutated alleles of p53 are present.
  • canccers are in particular colorectal cancers, breast cancers, lung cancers, gastric cancers, esophageal cancers, B lymphomas, ovarian cancers, bladder cancers, etc.
  • Figure 1 Study of the regulation of the p53 protein by calpain.
  • the reaction is carried out in a final volume of 30 ⁇ l, including 1 coming from the translation mixture, line 1: T0; line 2: 30 min in the presence of ImM Calcium + 20 ⁇ g ml Calpain; line 4: 30 min in the presence of ImM Calcium + 20 ⁇ g / ml Calpain + 0.5 mg / ml calpastatin; line 5: 30 min in the presence of ImM Calcium + 20 ⁇ g / ml Calpain + 10 mM EGTA; lane 6: PBS; lane 7: PBS + calcium; line 8: PBS + calpastatin.
  • the pBR322, pUC and phage plasmids of the M13 series are of commercial origin (Bethesda Research Laboratories).
  • the DNA fragments can be separated according to their size by electrophoresis in agarose or acrylamide gels, extracted with phenol or with a phenol / chloroform mixture, precipitated with ethanol and then incubated in the presence of the DNA ligase from phage T4 (Biolabs) according to the supplier's recommendations.
  • the filling of the protruding 5 ′ ends can be carried out by the Klenow fragment of DNA Polymerase I of E. coli (Biolabs) according to the supplier's specifications.
  • the destruction of the protruding 3 ′ ends is carried out in the presence of the DNA polymerase of phage T4 (Biolabs) used according to the manufacturer's recommendations.
  • the destruction of the protruding 5 ′ ends is carried out by gentle treatment with nuclease SI.
  • Mutagenesis directed in vitro by synthetic oligodeoxynucleotides can be carried out according to the method developed by Taylor et al. [Nucleic Acids Res. 13 . (1985) 8749-8764] using the kit distributed by Amersham.
  • the enzymatic amplification of DNA fragments by the technique known as
  • PCR Polymerase-catalyzed Chain Reaction, Saiki R.K. et al., Science 230 (1985) 1350-1354; Mullis K.B. and Faloona F.A., Meth. Enzym. 155 (1987) 335-350] can be performed using a "DNA thermal cycler" (Perkin Elmer Cetus) according to the manufacturer's specifications. Verification of the nucleotide sequences can be carried out by the method developed by Sanger et al. [Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5477] using the kit distributed by Amersham.
  • This example shows that the addition of m-calpain to the rabbit reticulocyte lysate induces the degradation of the wild-type p53 protein as well as that of certain mutated forms.
  • This example also shows that inhibitors of calpains are capable of inhibiting the degradation of p53 and therefore of modulating the activity of this protein.
  • Example 1.1 Use of calpain inhibitors to modulate the levels of p53 proteins:
  • m-calpain has been shown to induce degradation of the p53 proteins.
  • various compounds were introduced into the medium to test their capacity to inhibit the activity of calpain.
  • the results obtained show that the addition of a calcium chelator (EGTA) as well as a specific inhibitor peptide of calpains (derived from a physiological inhibitor, calpastatin; Maki et al., J. Biol. Chem ., 254, 18866-18869, 1989) are capable of inhibiting the degradation of p53 proteins induced by exogenous calpain.
  • EGTA calcium chelator
  • a specific inhibitor peptide of calpains derived from a physiological inhibitor, calpastatin; Maki et al., J. Biol. Chem ., 254, 18866-18869, 1989
  • cytoplasmic extracts of Daudi human lymphoblastoid cells. or Jurkat.
  • the cytoplasmic extracts were prepared as follows: The cells (accessible to ATCC) were cultured in DMEM medium supplemented with 10% fetal calf serum.
  • the cells were then harvested, washed in PBS buffer, then incubated for 5 min in hypotonic lysis buffer without detergent (HEPES 20 mM pH 7.5; KOAc 10 mM; MgOAc 1.5 mM; 2 ml for 5.10 8 cells) .
  • the lysis was completed using a Dounce homogenizer, then verified under a microscope.
  • the nuclei were then removed by centrifugation at 2000 g for 5 min, and the supernatants were centrifuged at 10,000 g for 1 hour (Beckman SW60).
  • the cytoplasmic extracts were then aliquoted at 5 to 12 mg / ml.
  • calpain inhibitors to modulate p53 protein levels: Calcium chelation by EGTA, as well as the use of a whole range of protease inhibitors (leupeptin, aprotinin, soybean trypsin inhibitor and PMSF) and above all the peptide calpastatin show that the degradation of these proteins is dependent on the calpains of the cytoplasmic extract, and that various compounds capable of modulating the activity of calpains can be used to regulate the levels of protein p53.
  • protease inhibitors leupeptin, aprotinin, soybean trypsin inhibitor and PMSF
  • Examples 1 and 2 show that calpains can induce the degradation of p53 in complex reaction mixtures. These experiments do not however exclude that under the conditions used the calpains activate secondary proteases which are those which really act on p53.
  • the following experiment was carried out: (1) the wild-type p53 proteins of mice and of humans neo-synthesized in the rabbit reticulocyte lysate were incubated for 30 minutes in the presence of cytoplasmic extract of Daudi cells as well as in the presence of calcium to activate the calpains as in Example 2, (2) of the protein p53 was then added to the reaction mixture and the reaction was continued for 30 minutes under permissive conditions (same reaction conditions) or no (addition of either EGTA to chelate calcium, or of peptide calpastatin) for calpains.
  • This example describes the construction of a recombinant adenovirus comprising a nucleic acid sequence coding for calpastatin.
  • This adenovirus is constructed by homologous recombination between the defective adenovirus Ad-dll324 and a plasmid carrying the sequence SEQ ID No. 1 under the control of the RSV promoter.
  • the plasmid SEQ ID No. 1 comprises the sequence coding for calpastatin under the control of the LTR-RSV promoter, as well as regions of the adenovirus allowing homologous recombination. It is constructed by inserting the sequence
  • the plasmid pAd.RSV ⁇ Gal contains, in the orientation 5 '-> 3',
  • the PvuII fragment corresponding to the left end of the Ad5 adenovirus comprising: the ITR sequence, the origin of replication, the packaging signals and the E1A amplifier;
  • the vector described in 4.1. is linearized and cotransfected with a deficient adenoviral vector, in helper cells (line 293) providing in trans the functions encoded by the El regions (El A and E11B) of adenovirus.
  • the recombinant adenovirus is obtained by homologous in vivo recombination between the mutant adenovirus Ad-dll324 (Thimmappaya et al., Cell 31 (1982) 543) and the vector described in Example 4.1., According to the following protocol : the plasmid SEQ ID No. 1 and the adenovirus Ad-dll324, linearized by the enzyme Clal, are co-transfected in line 293 in the presence of calcium phosphate, to allow homologous recombination. The recombinant adenoviruses thus generated are then selected by purification on a plate.
  • the DNA of the recombinant adenovirus is amplified in the cell line 293, which leads to a culture supernatant containing the non-purified recombinant defective adenovirus having a titer of approximately 10 ⁇ 10 pfu ml.
  • the viral particles are purified by centrifugation on a cesium chloride gradient according to known techniques (see in particular Graham et al., Virology 52 (1973) 456).
  • the adenovirus obtained can be stored at -80 ° C in 20% glycerol.
  • NAME RHONE-POULENC RORER S.A.
  • CAC AAT AAA AAA GCA GTT TCC AGA TCA GCT GAA CAG CAG CCA TCA GAG 240
  • GCT GCT CCA CCC CAA GAG AAG AAA AGA AAG GTG GAG AAG GAT ACA ATG 816 Ala Ala Pro Pro Gin Glu Lys Lys Arg Lys Val Glu Lys Asp Thr Met 260 265 270 AGT GAT CAA GCA CTC GAG GCT CTG TCG GCT TCA CTG GGC ACC CGG CAA 864 Ser Asp Gin Ala Leu Glu Ala Leu Ser Ala Ser Leu Gly Thr Arg Gin 275 280 285
  • GAG AAA CCA TCT AAG CCA ACT GAA AAG ACA GAA GAA TCT AAG GCC
  • GCT 1152 Glu Lys Pro Ser Lys Pro Thr Glu Lys Thr Glu Glu Ser Lys Ala Ala 370 375 380
  • GCT CCA GCT CCT GTG TCG GAG GCT GTG TCT CGG ACC TCC ATG TGT AGT 1200
  • ORGANISM homo sapiens
  • CHARACTERISTIC
  • GTC ACA ATT CCT CCA AAA TAT AGG GAA CTA TTG GCT AAA AAG GAA GGG 192 Val Thr Ile Pro Pro Lys Tyr Arg Glu Leu Leu Ala Lys Lys Glu Gly 745 750 755 ATC ACA GGG CCT CCT GCA GAC TCT TCA AAA CCC ATA GGG CCA GAT GAT 240 Ile Thr Gly Pro Pro Ala Asp Ser Ser Lys Pro Ile Gly Pro Asp Asp Asp 760 765 770 775
  • GCT GGA AAG AAA ACT GAA AAA GAG GAA TCT ACA GAA GTT TTA AAA GCT 336 Ala Gly Lys Lys Thr Glu Lys Glu Glu Ser Thr Glu Val Leu Lys Ala 795 800 805

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EP95920973A 1994-05-31 1995-05-22 VERFAHREN ZUR KREBSBEHANDLUNG DURCH REGULIERUNG DES p53-PROTEINS Withdrawn EP0763121A1 (de)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
FR9406583 1994-05-31
FR9406583A FR2720277B1 (fr) 1994-05-31 1994-05-31 Méthode de traitement des cancers par régulation de la protéine P53.
PCT/FR1995/000670 WO1995033060A1 (fr) 1994-05-31 1995-05-22 Methode de traitement des cancers par regulation de la proteine p53

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AU (1) AU714209B2 (de)
CA (1) CA2190293C (de)
FI (1) FI120501B (de)
FR (1) FR2720277B1 (de)
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DE19518488C1 (de) * 1995-05-19 1996-10-10 Progen Biotechnik Gmbh Autoantigen, geeignet zur Feststellung einer Thromboseneigung
EP0799892A3 (de) * 1996-04-05 1998-08-12 Takeda Chemical Industries, Ltd. Calpain, seine Herstellung und Verwendung
US6232454B1 (en) 1998-02-27 2001-05-15 Incyte Genomics, Inc. Human proteinase molecules
CA2351587A1 (en) * 1998-11-18 2000-05-25 Canji, Inc. Viral vectors with late transgene expression
AU2001247922A1 (en) * 2000-03-31 2001-10-15 Parker Hughes Institute Calpain inhibitors in cancer treatment

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NO964772D0 (no) 1996-11-11
AU714209B2 (en) 1999-12-23
FI964783A0 (fi) 1996-11-29
FR2720277B1 (fr) 1996-07-12
CA2190293A1 (fr) 1995-12-07
NO321411B1 (no) 2006-05-08
FI964783L (fi) 1996-11-29
FI120501B (fi) 2009-11-13
NO964772L (no) 1996-11-11
WO1995033060A1 (fr) 1995-12-07
AU2620695A (en) 1995-12-21
JPH10500978A (ja) 1998-01-27
CA2190293C (fr) 2011-06-28
FR2720277A1 (fr) 1995-12-01

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