EP1100916A2 - Zusammensetzung zur durchführung eines zytotoxischen und insbesondere antitumoralen oder antiviralen behandlung in einem säugetier - Google Patents

Zusammensetzung zur durchführung eines zytotoxischen und insbesondere antitumoralen oder antiviralen behandlung in einem säugetier

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Publication number
EP1100916A2
EP1100916A2 EP00942167A EP00942167A EP1100916A2 EP 1100916 A2 EP1100916 A2 EP 1100916A2 EP 00942167 A EP00942167 A EP 00942167A EP 00942167 A EP00942167 A EP 00942167A EP 1100916 A2 EP1100916 A2 EP 1100916A2
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EP
European Patent Office
Prior art keywords
polypeptide
nucleic acid
vector
composition according
activity
Prior art date
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EP00942167A
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English (en)
French (fr)
Inventor
Etienne Regulier
Philippe Erbs
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Transgene SA
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Transgene SA
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Classifications

    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/195—Chemokines, e.g. RANTES
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2013—IL-2
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/217—IFN-gamma
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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

  • composition intended for the implementation of a cytotoxic treatment, in particular antitumor or antiviral, in a mammal
  • the present invention relates to a cytotoxic composition
  • a cytotoxic composition comprising a first nucleic acid sequence coding for all or part of a MIP chemokine and a second nucleic acid sequence coding for all or part of a polypeptide having at least one cytotoxic activity, in particular antitumor or antiviral.
  • the present invention is particularly useful in the context of the implementation of a treatment by gene therapy of proliferative or infectious diseases.
  • Recent research in the cancer field has proposed adapting gene therapy protocols to tumor therapy.
  • one can for example cite the work of Meneguzzi et al., 1991, Nirology, 181, 61-69 relating to immunization against tumor cells using a recombinant vaccinia vector expressing the genes E6 and E7 of the human papilloma virus type 16.
  • Mention may also be made of the content of French patent FR 92/03120 relating to the use of a recombinant adenovirus expressing a cytokine in the context of anti-tumor gene therapy.
  • Cytokines are molecules naturally produced following antigenic stimulation or an inflammatory reaction (Gillis and Williams, 1998, Curr. Opin. Immunol., 10, 501-503) whose usefulness in the context of treatment of certain cancers has been shown in particular by Oettger (Curr. Opin. Immunol., 1991, 3, 699-705).
  • Oettger a mechanism for preventing the production of cytokines at tumor sites after intra-tumor administration of recombinant viral vectors.
  • this anti-tumor response although encouraging, does not allow the definitive disappearance of the tumor cells, and consequently the implementation of a satisfactory anti-tumor treatment.
  • Chemokines are a subclass of the cytokine family. They are distinguished from other cytokines by their chemo-attractive property, in particular during the natural processes of chemotaxis, and in particular of attraction of the cells of the immune system towards the tissues in which the inflammation or infection takes place, as well as their properties anti-angiogenic. Chemokines are proteins of low molecular weight (between 8 and 10 kd), of small size (from 70 to 80 amino acids) whose amino acid sequences have a low homology rate (varying from 10 to 70% depending on the chemokines considered ) allowing to define about 50 different chemokines to date. These chemokines can nevertheless be subdivided into 4 main families relating to the position of the cysteine residues which they contain.
  • compositions whose various constituents are chosen so as to obtain a synergistic effect of their respective activities and of the improved properties of said constituents. More particularly, such compositions make it possible to inhibit or delay cell proliferation by inducing the specific death of cells, in particular tumor cells, better presentation of the antigens and / or stimulation of the immune cells of the host organism.
  • the present invention provides an advantageous and effective alternative to the techniques of the prior art, in particular for treating human or animal cancer.
  • the invention relates firstly to a composition intended for the implementation of a cytotoxic treatment, for example anti-tumor or antiviral, or any applications requiring cell death, in a mammal comprising: (i) a nucleic acid sequence encoding for all or part of a MIP chemokine,
  • nucleic acid sequence coding for all or part of a polypeptide having at least one cytotoxic activity, said nucleic acid sequences being placed under the control of the elements necessary for their expression in a host cell of said mammal .
  • the preferred MIP chemokine is the MIP 1 type chemokine, and more particularly selected from the group consisting of the chemokines MlPl ⁇ and MlPl ⁇ , the properties of which have been demonstrated by Wolpe et al, 1988, J Exp. Med, 167, 570-581.
  • MIP 1 the nucleic acid and peptide sequences of which are described in Obaru et al. 1986, J. Biochem. 99, 885-894, the content of which is incorporated by reference into the present application, is produced by T lymphocytes and monocytes. It allows chemo-attraction of eosinophils and T lymphocytes during respiratory tract infections; monocytes and neutrophils in rheumatoid arthritis, inflammation of the digestive system or meningitis of bacterial origin. In addition, it inhibits the proliferation of hematopoietic precursors.
  • MlPl ⁇ whose nucleic acid and peptide sequences are described in Brown et al. 1989, J. Immunol. 142, 679-68, the content of which is incorporated by reference in the present application, is also produced by T lymphocytes and monocytes. It exerts its chemo-attractive properties on monocytes and neutrophils in cases of bone arthritis and bacterial meningitis. Like MlPl ⁇ , it inhibits the proliferation of hematopoietic precursors.
  • Act-2 Lipes et al., 1988, PNAS, 85, 9704-9708, the content of which is incorporated herein by reference.
  • polypeptide having at least one cytotoxic activity is intended to denote any peptide substance capable of inducing or activating an immune response directed specifically against a tumor cell (the cytotoxic activity is then called anti-tumor activity) or a cell infected with a virus (cytotoxic activity is then called antiviral activity) or to inhibit the growth and / or division of such a cell, in particular tumor or infected. According to a preferred case, said cytotoxic activity results in the death of said cell. According to a particular case, it would also be possible to use compositions according to the present invention in pathological cases associated with cell proliferation, such as for example the phenomena of restenosis.
  • chemo-attraction activity of a given polypeptide in particular derived from the chemokine MIP
  • cells involved in immune reactions such as for example eosinophils, T lymphocytes, monocytes or neutrophils
  • a chemotaxis test Magnhazachi, 1993, Nature Immunity, 12, 57.
  • this type of chemokine inhibiting the proliferation of hematopoietic precursors it is possible to evaluate such a property in vitro according to Graham et al., 1992, Growth Factors, 7, 151.
  • the cytotoxic activity of a given polypeptide in particular an antitumor activity, can be evaluated in vitro by measuring cell survival either by short-term viability tests (such as for example the tryptan blue test or MTT), either by clonogenic survival tests (colony formation) (Brown and Wouters, 1999, Cancer Research, 59, 1391-1399) or in vivo by measuring tumor growth (size and / or volume) in an animal model (Ovejera and Houchens, 1981, Semin. Oncol., 8, 386-393).
  • short-term viability tests such as for example the tryptan blue test or MTT
  • clonogenic survival tests colony formation
  • tumor growth size and / or volume
  • the invention relates to a composition characterized in that said polypeptide having cytotoxic activity is chosen from cytokines, proteins encoded by a gene called “suicide gene” and antiangiogenic protein factors. More particularly, when said polypeptide in (ii) is a cytokine, it is preferably a cytokine chosen from interferons ⁇ , ⁇ and ⁇ , interleukins, and in particular IL-2, 1TL-4 IL -6, IL-10 or IL-12, tumor necrotizing factors (TNF) and colony stimulating factors (GM-CSF, C-CSF, M-CSF ).
  • cytokine chosen from interferons ⁇ , ⁇ and ⁇ , interleukins, and in particular IL-2, 1TL-4 IL -6, IL-10 or IL-12, tumor necrotizing factors (TNF) and colony stimulating factors (GM-CSF, C-CSF, M-CSF .
  • said cytokine is selected from interleukin-2 (IL-2) and interferon gamma (IFN- ⁇ ).
  • Interleukin-2 is in particular responsible for the proliferation of activated T lymphocytes, for the multiplication and activation of cells of the immune system (for the nucleic acid sequence see in particular FR 85 09480).
  • LTFN- ⁇ activates phagocytic cells and increases the expression of class I and II surface antigens of the major histocompatibility complex (for the nucleic acid sequence see in particular FR 85 09225). Said nucleic acid sequences are incorporated by reference into the present application.
  • composition according to the invention is characterized in that it comprises in (ii) at least two nucleic acid sequences coding for all or part of interleukin-2 (IL-2) and all or part of gamma interferon (IFN- ⁇ ).
  • IL-2 interleukin-2
  • IFN- ⁇ gamma interferon
  • the invention also relates to such a composition characterized in that said polypeptide in ( ⁇ ) exhibits at least one enzymatic activity selected from thymidine kinase activity, purine nucleoside phosphorylase activity, guanine or uracil activity or orotate phosphoribosyl transferase and cytosine deaminase activity.
  • polypeptides which are not toxic as such but which exhibit catalytic enzymatic properties capable of transforming an inactive substance (predrogue), for example a nucleoside or a nucleoside analog, into a substance which is highly toxic for the cell, for example a modified nucleoside which can be incorporated into elongated DNA or RNA chains, with the consequence, in particular, of the inhibition of cell division or of cellular dysfunctions leading to the death of the cell containing such polypeptides.
  • the genes coding for such polypeptides are called "suicide genes". Many suicide / predrug gene pairs are currently available. We can cite more particularly, the couples:
  • TK HSV-1 herpes simplex virus type 1 thymidine kinase
  • GCV acyclovir or ganciclovir
  • CDase is an enzyme which intervenes in the metabolic pathway of pyrimidines by which the exogenous cytosine is transformed by means of hydrolytic deamination into uracil.
  • CDase activities have been demonstrated in prokaryotes and lower eukaryotes (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615; Beck et al., 1972, J. Bacteriol. 110, 219-228; De Haan et al., 1972, Antonie van Leeuwenhoek 38, 257-263; Hoeprich et al., 1974, J. Inf. Dis. 130, 112-118; Esders and Lynn, 1985, J. Biol. Chem.
  • FCY1 genes of Saccharomyces cerevisiae (S. cerevisiae) and cod ⁇ of E. coli coding respectively for the CDase of these two organisms are known and their sequences published (EP 402 108; Erbs et al., 1997, Curr. Genêt. 31, 1-6; WO93 / 01281).
  • CDase also deaminates a cytosine analog, 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU) which is a highly cytotoxic compound especially when it is converted into 5-fluoro-UMP (5-FUMP ).
  • Cells lacking CDase activity either because of an inactivating mutation of the gene coding for the enzyme, or because of their natural deficiency for this enzyme (for example mammalian cells) are resistant to 5-FC (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615; Kilstrup et al., 1989, J. Bacteriol. 1989 171, 2124-2127).
  • This phenomenon is due to the excretion by cells expressing CDase activity, of 5-FU which intoxicates neighboring cells by simple diffusion through the cell membrane.
  • This passive diffusion property of 5-FU constitutes an advantage over the rfc / GCV reference system for which the neighborhood effect requires contact with the cells which express tk (Mesnil et al., 1996, Proc. Natl. Acad Sci. USA 93,
  • sensitivity to 5-FC varies widely across cell lines. Low sensitivity is observed, for example, in human tumor lines PANC-1 (pancreatic carcinoma) and SK-BR-3 (breast adenocarcinoma) transduced by a retrovirus expressing the E codA gene. Coli (Harris et al., 1994, Gene Therapy 1, 170-175). This undesirable phenomenon could be explained by the absence or the weak endogenous conversion of 5-FU formed by the enzymatic action of CDase into cytotoxic 5-FUMP.
  • This step normally performed in mammalian cells by the orotate phosphorybosyl transferase (Peters et al., 1991, Cancer 68, 1903-1909), can be absent in certain tumors and thus render gene therapy, based on CDase, ineffective.
  • uracil In prokaryotes and lower eukaryotes, uracil is transformed into UMP by the action of uracil phosphoribosyl transferase (consequently exhibiting UPRTase activity). This enzyme also converts 5-FU to 5-FUMP.
  • furl mutants of the yeast S. cerevisiae are resistant to high concentrations of 5-FU (10 mM) and 5-FC (10 mM) because in the absence of UPRTase activity, 5-FU, originating from deamination of 5-FC by CDase, is not transformed into cytotoxic 5-FUMP (Jund and Lacroute, 1970, J. Bacteriol. 102, 607-615).
  • the upp and FUR1 genes encoding lTJPRTase respectively from E. coli and S. cerevisiae have been cloned and sequenced (Andersen et al., 1992, ⁇ ur. J. Biochem. 204, 51-56; Kern et al., 1990, Gene 88, 149-157).
  • a polypeptide having UPRTase activity denotes a polypeptide capable of converting uracil or one of its derivatives into a monophosphated analogue and, in particular 5-FU into 5-FUMP.
  • mutant is meant the addition, deletion and / or substitution of one or more residues at any location of said polypeptide.
  • the native LTJPRTase in question in the present invention can be of any origin, in particular prokaryotic, fungal or yeast.
  • Lactococcus lactis (Martinussen and Hammer, 1994, J. Bacteriol. 176, 6457-
  • yeast UPRTase and in particular that encoded by the FUR1 gene from S. cerevisiae, the sequence of which is disclosed in
  • Kern et al. (1990, Gene 88, 149-157) is introduced here by reference.
  • the gene sequences and those of the corresponding UPRTases can be found in the literature and specialized databases (SWISSPROT, ⁇ MBL, Genbank, Medline ).
  • application PCT / FR99 / 00904 describes a FUR1 gene devoid of 105 nucleotides 5 ′ of the coding part allowing the synthesis of a UPRTase deleted from the first 35 residues in the N-terminal position and starting with methionine in position 36 in native protein.
  • the expression product of the mutant gene, designated FUR1 ⁇ 105 is capable of complementing a furl mutant of S. cerevisiae.
  • the polypeptide encoded according to the invention is a deletion mutant of a native UPRTase.
  • the deletion is preferably located in the N-terminal region of the original UPRTase. It can be total (concern all the residues of said N-terminal region) or partial (concern one or more residues, whether continuous or not, in the primary structure).
  • a polypeptide is made up of N-terminal, central and C-terminal parts, each representing approximately one third of the molecule. For example, the UPRTase of S.
  • N-terminal part consists of the first 83 residues starting with the so-called initiator methionine located in the first position of the native form.
  • initiator methionine located in the first position of the native form.
  • N-terminal part covers positions 1 to 69.
  • patent applications WO96 / 16183 and PCT / FR99 / 00904 describe the use of a fusion protein coding for a two-domain enzyme having the CDase and UPRTase activities and demonstrate that the transfer of a hybrid gene codA :: upp or FCYlr.FURl or FCYl :: FUR1 ⁇ 105 carried by an expression plasmid increases the sensitivity to 5-FC of transfected B16 cells.
  • the protein and nucleic acid sequences described in these two applications are incorporated into the description of the present application.
  • the polypeptide is a polypeptide fused in phase with at least one second polypeptide.
  • the fusion can take place at any location of the first polypeptide, the N or C-terminal ends are preferred and in particular the N-terminal end.
  • a fusion of CDase and UPRTase activities improves the sensitivity of target cells to 5-FC and 5-FU.
  • a person skilled in the art is able to clone the CDase or UPRTase sequences from the published data, to carry out possible mutations, to test the enzymatic activities of the mutant forms in an acellular or cellular system according to l technology. art or by following the protocol indicated in application PCT / FR99 / 00904 and to merge, in particular in phase, the polypeptides of CDase and UPRTase activity, and consequently all or part of the corresponding genes.
  • the composition of the invention is characterized in that the nucleic acid sequence (ii) is selected from the nucleic sequences of the CodA, upp, FUR1 genes; FCYl and FUR1 ⁇ 105, or by a combination of all or part of said sequences.
  • the invention relates more particularly to a said composition characterized in that said polypeptide in (ii) has at least one CDase activity and one UPRTase activity.
  • nucleic acid sequences are intended to denote both distinct sequences which code for at least two distinct polypeptides as well as fused sequences which code for fusion polypeptides, it being understood that the production of such polypeptides can be carried out under the control of the same regulatory elements (polycistronic cassette) or of independent, identical or different elements, homologous or heterologous with respect to the vector containing them, constitutive or inducible.
  • the composition of the invention comprises at least one nucleic acid sequence (ii) coding for a fusion polypeptide in which a first polypeptide exhibiting UPRTase or CDase activity is fused in phase with at least one second polypeptide, said second polypeptide exhibiting CDase or UPRTase activity, respectively.
  • a polypeptide is characterized in that the fusion with the second polypeptide is carried out at the N-terminal end of said first polypeptide.
  • said composition is characterized in that the nucleic acid sequence coding for said fusion polypeptide is a hybrid sequence comprising:
  • hybrid nucleic acid sequence coding for said fusion polypeptide may also contain an IRES type sequence.
  • the invention relates in particular to such a composition for which the first nucleic acid sequence is selected from upp, FUR1 and FUR1 ⁇ 105, and in that the second nucleic acid sequence is selected from CodA and FCYl, and vice versa.
  • a hybrid nucleic acid sequence is chosen from the hybrid sequences described in patent applications WO96 / 16183 and PCT / FR99 / 00904.
  • the composition according to the present invention is characterized in that said polypeptide having cytotoxic activity (ii) is an anti-angiogenic protein factor.
  • Angiogenesis is the process responsible for the formation of new capillaries from the already existing vascular network. This complex process is finely regulated in healthy tissue by the balance of the effects of many angiogenic and anti-angiogenic factors. However, in certain pathologies, and in particular during the formation of a tumor, this process is deregulated: the angiogenic factors take precedence over the anti-angiogenic factors which allows an important vascularization of the tumors and consequently their rapid development and / or the appearance of metastases.
  • an anti-angiogenic factor is considered to be a cytotoxic agent, in particular an anti-tumor agent.
  • angiostatin for angiostatin, endostatin, the factor platelet PF4, thrombospondin-1, PRP (for Proliferin Related Protein), VEGI (for Vascular Endothelial Growth Inhibitor) metalloproteases and urokinase.
  • the nucleic acid sequences (i) or ( ⁇ ) can be easily obtained by cloning, by PCR or by chemical synthesis according to the conventional techniques in use. They may be native genes or derivatives thereof by mutation, deletion, substitution and / or addition of one or more nucleotides. Furthermore, their sequences are widely described in the literature available to those skilled in the art.
  • the present invention also relates to a composition as presented above, characterized in that said nucleic acid sequences (i) and (ii) are inserted into a recombinant vector of plasmid or viral origin, as well as to a such a recombinant vector carrying such nucleotide sequences placed under the control of the elements necessary for their expression in a host cell.
  • the nucleic acid sequences (i) and (ii) may be present in one or more copies on the same vector.
  • compositions of the invention can comprise said nucleic acid sequences (i) and (ii) inserted in the same recombinant vector or in distinct recombinant vectors.
  • recombinant vector according to the invention is intended to denote a
  • Vector of plasmid or viral origin and optionally such a vector associated with one or more substances improving the transfection efficiency and / or the stability of said vector and / or the protection of said vector in vivo with respect to the immune system of the host organism.
  • substances are widely documented in the literature accessible to those skilled in the art (see for example Felgner et al., 1987, Proc. West. Pharmacol. Soc. 32, 115-121; Hodgson and Solaiman, 1996, Nature Biotechnology 14, 339-342; Remy et al., 1994, Bioconjugate Chemistry 5, 647-654).
  • they may be polymers, lipids in particular cationic, liposomes, nuclear or viral proteins or even neutral lipids. These substances can be used alone or in combination. Examples of such compounds are available in particular in WO patent applications 98/08489, WO 98/17693, WO 98/34910, WO 98/37916, WO 98/53853, EP 890362 or WO 99/05183.
  • a possible combination is a recombinant plasmid vector associated with cationic lipids (DOGS, DC-CHOL, spermine-chol, spermidine-chol etc.) and neutral lipids (DOPE).
  • plasmids which can be used in the context of the present invention are vast. They may be cloning and / or expression vectors. In general, they are known to those skilled in the art and many of them are commercially available, but it is also possible to construct or modify them by genetic manipulation techniques. Mention may be made, as examples, of the plasmids derived from pBR322 (Gibco BRL), pUC (Gibco BRL), pBluescript (Stratagene), pREP4, pCEP4 (Invitrogene) or also p Poly (Lathe et al., 1987, Gene 57, 193-201).
  • a plasmid used in the context of the present invention contains an origin of replication ensuring the initiation of replication in a producer cell and / or a host cell (for example, the ColEl origin will be retained for a plasmid intended to be produced in E. coli and the oriP / EBNAl system if it is desired to be self-replicating in a mammalian host cell, Lupton and Levine, 1985, Mol. Cell. Biol. 5, 2533-2542; Yates and al., Nature 313, 812-815). It can also comprise a selection gene making it possible to select or identify the transfected cells (complementation of an auxotrophy mutation, gene coding for resistance to an antibiotic, etc.).
  • cer sequence which promotes the monomeric maintenance of a plasmid (Summers and Sherrat, 1984, Cell 36, 1097-1103, sequences of integration into the cell genome).
  • a viral vector Being a viral vector, one can envisage a vector deriving from a poxvirus (vaccinia virus, in particular MVA, canaripox, etc.), from an adenovirus, from a retrovirus, from a virus herpes, an alphavirus, a foamyvirus or a virus associated with the adenovirus.
  • a non-replicative and non-integrative vector will be used.
  • the adenoviral vectors are very particularly suitable for the implementation of the present invention. However, it should be noted here that as part of the implementation of the present invention, the nature of the vector is of little importance.
  • Retroviruses have the property of infecting and integrating mainly in dividing cells and in this respect are particularly suitable for cancer application.
  • a recombinant retrovirus according to the invention generally comprises the LTR sequences, an encapsidation region and the nucleotide sequence according to the invention placed under the control of the retroviral LTR or of an internal promoter such as those described below. It can be derived from a retrovirus of any origin (murine, primate, feline, human, etc.) and in particular from MoMuLV (Moloney murine leukemia virus), MVS (Murine sarcoma virus) or Friend murine retrovirus (Fb29).
  • MoMuLV Moloney murine leukemia virus
  • MVS Murine sarcoma virus
  • Fb29 Friend murine retrovirus
  • the retroviral vector according to the invention may include modifications in particular at the level of the LTRs (replacement of the promoter region with a eukaryotic promoter) or of the packaging region (replacement with a heterologous packaging region, for example of the VL30 type). (see French applications 94 08300 and 97 05203).
  • a defective adenoviral vector for replication that is to say devoid of all or part of at least one region essential for replication selected from regions E1, E2, E4 and.
  • a deletion from the El region is preferred.
  • it can be combined with other modification (s) / deletion (s) affecting in particular all or part of the regions E2, E4 and / or L1-L5, insofar as the defective essential functions are complemented in trans by means of 'a line of complementation and / or an auxiliary virus in order to ensure the production of the viral particles of interest.
  • use may be made of second generation vectors of the state of the art (see for example international applications WO94 / 28152 and WO97 / 04119).
  • the deletion of the majority of the region E1 and of the transcription unit E4 is very particularly advantageous.
  • the adenoviral vector can also be devoid of all or part of the non-essential E3 region.
  • the origin of the adenoviral vector according to the invention can be varied both from the point of view of the species and of the serotype.
  • adenovirus of human or animal origin canine, avian, bovine, murine, ovine, porcine, simian .
  • Mention may more particularly be made of the adenoviruses CAV-1 or CAV-2 of canine origin, DAV of avian origin or else Bad of type 3 of bovine origin (Zakharchuk et al., Arch. Virol., 1993, 128: 171 -176; Spibey and Cavanagh, J. Gen.
  • an adenoviral vector of human origin preferably deriving from a serotype C adenovirus, in particular of type 2 or 5, will be preferred.
  • An adenoviral vector according to the present invention can be generated in vitro in Escherichia coli (E. coli) by homologous ligation or recombination (see for example international application WO96 / 17070) or alternatively by recombination in a complementation line.
  • the different adenoviral vectors and their preparation techniques are known (see for example Graham and Prevect, t
  • the elements necessary for expression consist of all of the elements allowing the transcription of the nucleotide sequence into AR ⁇ and the translation of AR ⁇ m into polypeptide, in particular the promoter sequences and / or efficient regulatory sequences in said cell, and optionally the sequences required to allow excretion or expression on the surface of target cells of said polypeptide.
  • These elements can be regulable or constitutive.
  • the promoter is adapted to the vector selected and to the host cell. Mention may be made, by way of examples, of the eukaryotic promoters of the PGK (Phospho Glycerate Kinase), MT (metallothioneine; Me Ivor et al., 1987, Mol . Cell Biol.
  • ⁇ -1 antitrypsin CFTR
  • promoters of the gene coding for muscle creatine kinase, for actin, for the pulmonary surfactant immunoglobulin, ⁇ -actin (Tabin et al., 1982, Mol. Cell Biol. 2, 426-436), SR ⁇ (Takebe et al., 1988, Mol. Cell. Biol.
  • the early promoter of the SV40 virus (Simian Virus), the RSV LTR (Rous Sarcoma Virus), the promoter of MPSV, the promoter TK-HSV-1, the early promoter of the CMV virus (Cytomegalovirus), the promoters of the vaccinia virus p7.5K pH5R, pKlL, p28, pl i and the adenoviral promoters El A and MLP or a combination of said promoters. It can also be a promoter stimulating expression in a tumor or cancer cell.
  • the promoters of the MUC-1 genes overexpressed in breast and prostate cancers (Chen et al., 1995, J. Clin. Invest.
  • the necessary elements may, in addition, include additional elements improving the expression of the nucleotide sequence according to the invention or its maintenance in the host cell. Mention may in particular be made of the intronic sequences (WO 94/29471), secretion signal sequences, nuclear localization sequences, internal sites of translation initiation of IRES type, poly A sequences for transcription termination.
  • the invention relates more particularly to a recombinant vector, in particular a viral vector, and more specifically to an adenoviral vector defective for replication, comprising: (i) a nucleic acid sequence coding for all or part of a MIP chemokine,
  • nucleic acid sequence coding for all or part of a polypeptide having at least one cytotoxic activity, said nucleic acid sequences being placed under the control of the elements necessary for their expression in a host cell and being defined as indicated above.
  • the present invention also relates to a viral particle, in particular adenoviral, comprising a recombinant viral vector according to the invention.
  • a viral particle can be generated from a viral vector according to any conventional technique in the art. Its propagation is carried out in particular in a complementation cell adapted to the deficiencies of said vector.
  • an adenoviral vector use will be made, for example, of a complementation line as described in application WO 94/28152, to line 293 established from human embryonic kidney cells, which effectively complements the El function (Graham et al., 1977, J. Gen. Virol.
  • complementation cell is meant a cell capable of providing in trans the early and / or late factors necessary for the packaging of the genome.
  • the invention also relates to a process for preparing a viral particle, according to which: (i) a recombinant vector according to the invention is introduced into a cell, in particular a complementation cell capable of complementing said vector in trans, so as to obtain a said transfected cell, (ii) culturing said transfected cell under appropriate conditions to allow the production of said viral particle, and
  • the viral particle can be recovered from the culture supernatant but also from the cells.
  • One of the commonly used methods is to lyse the cells by consecutive freeze / thaw cycles to collect the virions in the lysis supernatant. These can be amplified and purified according to the techniques of the art (chromatographic process, ultracentrifugation in particular through a gradient of cesium chloride ).
  • the invention also relates to a eukaryotic host cell comprising the DNA fragments present in the composition according to the invention.
  • Said host cell is advantageously a mammalian cell and, preferably, a human cell. It will preferably be a 293 cell, LCA4 or PERC6. Such a cell is particularly useful for producing viral particles at high titer, without generating particles competent for replication.
  • the invention also relates to a host cell comprising a nucleotide sequence, a recombinant vector according to the invention or infected with a viral particle according to the invention.
  • a host cell consists of any cell transfectable by a recombinant vector or infectable by a viral particle, as defined above.
  • a mammalian and in particular human cell is particularly suitable. It can comprise said vector in a form integrated into the genome or not (episome). It may be a primary or tumor cell of any origin, in particular hematopoietic (totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage ...) (satellite cell, myocyte, myoblast, smooth muscle ...), cardiac, pulmonary, tracheal, hepatic, epithelial or fibroblast.
  • hematopoietic totipotent stem cell, leukocyte, lymphocyte, monocyte or macrophage
  • satellite cell myocyte, myoblast, smooth muscle
  • cardiac pulmonary, tracheal, hepatic, epithelial or fibroblast.
  • the invention also relates to a composition intended for the implementation of an antitumor or antiviral treatment, or any applications requiring cell death, in a mammal comprising:
  • Another object according to the invention consists of a formulation intended for the implementation of a cytotoxic treatment, in particular antitumor or antiviral, in a mammal characterized in that it comprises a composition (based on nucleic acids or polypeptides as described above), an adenoviral vector or a viral particle according to the invention, as well as a support which is acceptable from a pharmaceutical point of view.
  • a support is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength, such as for example a sucrose solution.
  • such a support can contain any solvent, or aqueous or partially aqueous liquid such as sterile non-pyrogenic water.
  • the pH of the formulation is also adjusted and buffered in order to meet the requirements for use in vivo.
  • the formulation may also include a pharmaceutically acceptable diluent, adjuvant or excipient, as well as solubilizers, stabilizers, preservatives.
  • a pharmaceutically acceptable diluent for injectable administration, an aqueous, non-aqueous or isotonic solution formulation is preferred. It can be presented as a single dose or in multidose in liquid or dry form (powder, lyophilisate, etc.) capable of being reconstituted extemporaneously with an appropriate diluent.
  • said formulation also comprises pharmaceutically acceptable amounts of a prodrug capable of being transformed into cytotoxic molecule by a polypeptide having at least one cytotoxic activity.
  • Such a prodrug will be selected in particular from the group consisting of acyclovir or ganciclovir (GCV), cyclophosphophamide, 6-methylpurine deoxyribonucleoside, 6-thioxanthine, cytosine or one of its derivatives or uracil or one of its derivatives.
  • said prodrug is 5-fluorocytosine (5FC) or 5-fluorouracil (5-FU).
  • said formulation may also comprise one or more substances potentiating the cytotoxic effect of 5-FU.
  • drugs which inhibit the enzymes of the de novo biosynthesis pathway for pyrimidines (for example those cited below), drugs such as Leucovorin (Waxman et al., 1982, Eur. J.
  • a formulation according to the invention is more particularly intended for the preventive or curative treatment of diseases by gene therapy and is more particularly intended for proliferative diseases (cancers, tumors, restenosis, etc.) and for diseases of infectious origin, in particular viral for which it is necessary to limit the proliferation of infected cells (induced by hepatitis B or C viruses, HIV, herpes, retroviruses, etc.).
  • a formulation according to the invention can be manufactured in a conventional manner for administration by the local, parenteral or digestive route.
  • routes of administration We can cite for example the intragastric, subcutaneous, intracardiac, intramuscular, intravenous, intraperitoneal, intratumoral, intranasal, intrapulmonary or intratracheal route.
  • administration by aerosol or instillation is advantageous.
  • the administration can take place in single dose or repeated one or more times after a certain interval of interval.
  • the appropriate route of administration and dosage vary according to various parameters, for example, the individual, the disease to be treated or the gene (s) of interest to be transferred.
  • the preparations based on viral particles according to the invention can be formulated in doses of between 10 4 and 10 14 pfu (units forming plaques), advantageously 10 5 and 10 13 pfu and, preferably, 10 6 and 10 12 ufp.
  • doses comprising from 0.01 to 100 mg of DNA, preferably 0.05 to 10 mg and, most preferably, 0.5 to 5 mg may be considered.
  • a composition based on polypeptides preferably comprises from 0.05 to 10 g and, most preferably, from 0.5 to 5 g of said polypeptide.
  • the doses can be adjusted by the clinician.
  • the present invention also relates to the therapeutic or prophylactic use of a composition, of a recombinant vector or of a viral particle according to the invention for the preparation of a medicament intended for the treatment of the human or animal body by therapy gene, in particular for the preparation of a cytotoxic drug, in particular an antitumor or antiviral drug, intended to inhibit the growth or cause the rejection of a tumor or the death of an infected cell.
  • the drug can be administered directly in vivo (for example by intravenous injection, in an accessible tumor or at its periphery, in the lungs by aerosol, in the vascular system by means of an appropriate probe ...) .
  • a preferred use is to treat or prevent cancers, tumors and diseases resulting from unwanted cell proliferation.
  • possible applications include breast, uterine (in particular those induced by papillomas virus), prostate, lung, bladder, liver, colon, pancreas, stomach cancers , esophagus, larynx of the central nervous system and blood (lymphomas, leukemia etc.). It is also useful in the context of cardiovascular diseases, for example to inhibit or delay the proliferation of smooth muscle cells of the vascular wall (restenosis).
  • infectious diseases application to AIDS can be considered.
  • the invention also extends to a method for the treatment of diseases by gene therapy, characterized in that a nucleotide sequence, a recombinant vector, is administered to an organism or to a host cell in need of such treatment.
  • a viral particle or a host cell according to the invention.
  • the treatment method uses a nucleotide sequence, a recombinant vector or a viral particle allowing the expression of a polypeptide according to the invention having a UPRTase activity
  • the administration of the UPRTase and CDase sequences can be simultaneous or consecutive, the order of administration being unimportant.
  • the therapeutic use or the method of treatment also comprises an additional stage according to which the organism or the host cell is administered pharmaceutically acceptable amounts of a pre-drug, advantageously an analog of cytosine and, in particular of 5-FC.
  • a dose of 50 to 500 mg / kg / day can be used with a preference for 200 mg / kg / day.
  • the predrogue is administered according to standard practices and this in a prior, concomitant or even after that of the therapeutic agent according to the invention.
  • the oral route is preferred.
  • Either a single dose of the pre-drug or repeated doses may be administered long enough to allow the production of the toxic metabolite within the host organism or cell.
  • the therapeutic use or the method of treatment is associated with a second treatment of the patient by surgery (in particular by removal of the tumor partially or totally), by radiotherapy or chemotherapy.
  • the treatment according to the invention is applied beforehand, concomitantly or following said second treatment.
  • this treatment will be applied following said second treatment.
  • FIG. 1 represents the evolution of the tumor volume in B6D2 mice implanted with B16F0 tumor cells.
  • FIG. 2 represents the survival rate of these same mice.
  • Groups of 15 mice are treated using compositions comprising adenoviruses expressing the following genes: huMIP ⁇ , huIL2, huMIPl ⁇ + huIL2, empty Ad.
  • FIG. 3 represents the evolution of the tumor volume in mice
  • FIG. 4 represents the survival rate of these same mice.
  • Groups of 15 mice are treated using compositions comprising adenoviruses expressing the following genes: huMIP ⁇ , huIL2, huMIP ⁇ + huIL2, empty Ad.
  • FIG. 5 represents the evolution of the tumor volume in B6D2 mice implanted with P815 tumor cells.
  • FIG. 6 represents the survival rate of these same mice. Groups of 15 mice are treated using compositions comprising adenoviruses expressing the following genes: Tris buffer, huMIP ⁇ + huIL2, huMIPl ⁇ + muIFN ⁇ , huIL2 + muIFN ⁇ .
  • FIG. 7 represents the evolution of the tumor volume in mice
  • mice implanted with RENCA tumor cells. These mice are treated with the aid of compositions comprising adenoviruses expressing the human MlPl ⁇ gene (huMIPl ⁇ ) in combination with adenoviruses expressing the murine IL12 gene (muIL12), or adenoviruses expressing the muIL12 gene, or adenoviruses containing no transgene (Ad empty).
  • adenoviruses expressing the human MlPl ⁇ gene huMIPl ⁇
  • muIL12 murine IL12 gene
  • muIL12 muIL12 gene
  • Ad empty adenoviruses containing no transgene
  • the constructions described below are carried out according to the general techniques of genetic engineering and molecular cloning, detailed in Maniatis et al., (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Spring Harbor, NY) or according to the recommendations from the manufacturer when using a commercial kit.
  • the homologous recombination steps are preferably carried out in the E. coli BJ 5183 strain (Hanahan, 1983, J. Mol. Biol. 166, 557-580).
  • the technique used consists of filling the protruding 5 ′ ends with the large fragment of DNA polymerase I from E. coli (Klenow).
  • the adenoviral genome fragments used in the various constructions described below are indicated precisely according to their position in the nucleotide sequence of the Ad5 genome as disclosed in the Genbank database under the reference M73260.
  • the cells are transfected or transduced and cultured according to standard techniques well known to those skilled in the art.
  • Tumor models Three tumor cell models were chosen in order to evaluate the activity of the composition of the invention: P815 (mastocytoma H-2d, described in Dunn et al, 1957, J. Natl. Cancer Inst., 18 , 587-590), B16FO (melanoma H-2b, described in Wu et al, 1996, Cancer Res., 56, 21-26) and RENCA (renal cell carcinoma H-2d, described in Murphy et al, 1973, J. Natl. Cancer Inst., 50 (4 ), 1023-1025).
  • the cells (3E + 5 for each tumor model) are implanted on D-7 / Dl 1 subcutaneously in the right flank of B6D2 mice aged 6 to 8 weeks.
  • cytotoxic compositions of the invention A volume of 100 ⁇ l of adenoviral vectors (5 ⁇ 10 8 infectious units) is injected directly into the tumors when their volume is close to 4 to 10 mm 3 (D0). This injection is repeated under the same conditions on D1 and D2.
  • composition of the invention administered is controlled by measuring the size of the tumors as well as by measuring the survival time of the mice treated with, if necessary, a control of the animal's immunological status by ELISPOT, test CTL, ...
  • the animals can also be then subjected to a counter-lateral challenge during which a lethal dose of tumor cells is administered to the pre-treated animal.
  • the human MIP cDNA (Accession number with GenBank: X03754; sequence incorporated on request by reference) was assembled by synthetic oligonucleotides according to the sequence described by Obaru, K. & al. 1986, J. Biochem. 99 (3), 885-894. This cDNA was introduced into a vector derived from pBluescript to give the vector pTG 13006.
  • pTG8347 is a p polyll vector (Lathe et al., 1987, Gene 57, 193-201) into which are inserted the Ad5 sequences 1 to 458, the RSV promoter, the rabbit beta-globin 1 intron 2 splicing sequences, the beta polyadenylation sequences - rabbit globin 1 and the Ad5 sequences 3328-5788.
  • Ad5 sequences 1 to 458 the Ad5 sequences 1 to 458
  • the RSV promoter the rabbit beta-globin 1 intron 2 splicing sequences
  • beta polyadenylation sequences - rabbit globin 1 and the Ad5 sequences 3328-5788.
  • the adènoviral vector pTG13010 is reconstituted by recombination in the E. coli BJ 5183 strain between the P ⁇ cI-BsiEII fragment of pTG 13008 and the vector pTG6624 (described in French application 97 06757) linearized by CZ ⁇ l.
  • pTG6624 corresponds to the plasmid p poly II carrying the genome Ad5 deleted from the regions El (nt 459 to 3327) and E3 (nt 28592 to 30470), the MIP expression cassette being inserted in place of El.
  • the final construction pTG13010 contains the genome Ad5 deleted from most of the regions El (nt 459 to 3328) and E3 (nt 28249 to 30758) and, in place of El, a cassette for the expression of the MlPl ⁇ gene placed under the control of the RSV promoter and of the rabbit beta-globin 1 intron 2 splicing sequences.
  • the adenoviral particles are generated by transfection into a line for complementing the El function, for example line 293 (ATCC CRL1573) according to the techniques of the art (Graham and Prevec, 1991, Methods in Molecular Biology Vol7, Gene Transfer and
  • the cDNA of human MIP1 ⁇ (GenBank accession number: J04130; sequence incorporated on request by reference) was assembled by synthetic oligonucleotides according to the sequence described by Lipes, M.A. et al.
  • the Nofl-Asp718 fragment of M13TG13013 containing the MIP1 ⁇ gene is isolated and introduced into the vector pTG8347 cleaved by these same enzymes, to give the transfer vector pTG13015.
  • the adenoviral vector pTG13023 is reconstituted by recombination in the E. coli BJ 5183 strain between the P ⁇ cI-BstEII fragment of pTG13015 and the vector pTG6624 (described in French application 97 06757) linearized by CZ ⁇ l.
  • the final construction pTG 13023 contains the genome Ad5 deleted from most of the regions El (nt 459 to 3328) and E3 (nt 28249 to 30758) and instead and place of E1, a cassette for the expression of the MIP1 ⁇ gene placed under the control of the RSV promoter and of the splice sequences of intron 2 of rabbit ⁇ globin 1.
  • the adenoviral particles are generated by transfection into a line for complementing the El function, for example line 293 (ATCC CRL1573) according to the techniques of the art (Graham and Prevec, 1991, Methods in Molecular Biology Vol7, Gene Transfer and Expression Protocols; Ed EJ Murray, The Human Press Inc, Clinton, NJ).
  • mice thus treated are then subjected to a counter lateral challenge consisting in the administration under the conditions described above of a lethal dose (3.10 5 cells) of tumor cells on D80 / D100. It was thus found that the results described above are also accompanied by an immune state of the mouse such that no tumor is capable of developing after this challenge step.
  • huMIPl ⁇ 2.10 8 infectious units
  • muIL12 murine gene IL12
  • empty Ad empty
  • the adenoviruses are in a solution containing 100 mM Tris and 10 mM MgCl 2 . It was moreover verified that the mice treated under the same conditions with a composition comprising only the adenoviruses expressing the MlP1 gene exhibited tumors of identical or even greater volume than those observed in mice treated with a composition comprising empty Ad.
  • the results obtained according to this example demonstrate a drop in tumor volumes in mice treated with the compositions of the invention comprising an adenovirus expressing MlP1 associated with an adenovirus expressing 1TL12, very particularly by comparison with the results observed during treatment of mice with muIL12 alone.

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