EP0906441A1 - Antitumorzellzusammensetzungen, die auf mindestens drei transgene exprimieren - Google Patents

Antitumorzellzusammensetzungen, die auf mindestens drei transgene exprimieren

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Publication number
EP0906441A1
EP0906441A1 EP97916490A EP97916490A EP0906441A1 EP 0906441 A1 EP0906441 A1 EP 0906441A1 EP 97916490 A EP97916490 A EP 97916490A EP 97916490 A EP97916490 A EP 97916490A EP 0906441 A1 EP0906441 A1 EP 0906441A1
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EP
European Patent Office
Prior art keywords
cells
expression
composition according
gene
virus
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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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EP97916490A
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English (en)
French (fr)
Inventor
Majid Mehtali
Horst Homann
Yves Poitevin
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Transgene SA
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Transgene SA
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Publication date
Priority claimed from PCT/IB1996/000439 external-priority patent/WO1997035996A1/en
Application filed by Transgene SA filed Critical Transgene SA
Publication of EP0906441A1 publication Critical patent/EP0906441A1/de
Withdrawn legal-status Critical Current

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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • 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
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/55—IL-2
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13061—Methods of inactivation or attenuation
    • C12N2740/13062—Methods of inactivation or attenuation by genetic engineering
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
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    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the subject of the present invention is a cell composition for the treatment or prevention of tumors in humans or animals. More particularly, it comprises a population of cells capable of expressing a combination of at least three therapeutic genes having an effect. The invention also relates to the therapeutic use of such a composition in the field of oncology 20
  • Immunotherapy is based on the transfer of genes coding for cytokines and co-stimulatory molecules with the aim of making tumor cells more immunogenic and strengthening the host's anti-tumor immune response.
  • the cytokines thus evaluated in mu ⁇ ns models and, for which have shown an anti-tumor property, are interleukin (IL) 2, 1TL-4, IL-6, IL-7, IL-12, tumor necrosis factor (TNF) of alpha type, GM-CSF (for Granulocyte Macrophage Colony Sttmulating Factor) and interferons (IFN) Xenogenic cells producing IL-2 have thus demonstrated an anti-tumor effect (see European application EP 0 579 791)
  • the cytotoxic approach consists in specifically increasing the sensitivity of tumor cells to chemotherapy by transfer of a so-called suicide gene, the product of which is capable of transforming an inactive precursor into a highly cytotoxic product.
  • suicide gene the product of which is capable of transforming an inactive precursor into a highly cytotoxic product.
  • the most widely used to date is the tk gene of the virus.
  • Herpes Simplex type 1 (HSV-1) coding for the enzyme thymidine kinase (TK) which has the property of converting acyclovir and ganciclovir into phosphorylated analogs of nucleosides
  • TK thymidine kinase
  • the anti-oncogene strategy is based on the introduction into tumor cells of a functional copy of a tumor suppressor gene (for example the gene associated with retinoblastoma or p53) or the inhibition of the expression of oncogenes by the transfer of genes coding for antisense polynucleotides or ⁇ bozymes capable of degrading the messenger RNAs of oncogenes with the aim of reducing or abolishing the proliferation of cancer cells
  • an anti-tumor composition based on cells genetically modified to secrete IL-2 and produce retroviral particles expressing the tk genes of the HSV-1 virus and LFN ⁇ .
  • Such a composition makes it possible to inhibit or delay the Cell proliferation by inducing specific death of tumor cells, better presentation of antigens and stimulation of host immune cells
  • the present invention offers an effective alternative to the techniques of the prior art for treating cancer in humans or 'animal This is why the subject of the present invention is an anti-tumor composition comprising a population of cells allowing the expression of at least three therapeutic anti-tumor genes.
  • therapeutic antitumor genes genes whose expression products have an antitumor effect, in particular for increasing immunity directed specifically against the tumor and / or at least partially inhibiting cell division.
  • genes coding for immunostimulatory polypeptides of the immune response and / or cytotoxic are meant any polypepude capable of amplifying the production of antibodies directed against tumor cells and antigens or of stimulating an immune response mediated by cells, activating T cells to trigger a significant delayed hypersensitivity or cytotoxic response against tumor cells.
  • cytotoxic is meant any polypepude capable of inducing cell death either directly or indirectly via a drug capable of being administered independently
  • immunostimulatory genes which can be used in the context of the present invention, it is possible to envisage genes coding for cytokines and in particular mterleukins (IL), colony stimulating factors (G-CSF, M-CSF and GM-CSF), interferons (LFN), rumor necrosis factors (TNF), co-stimulation factors such as polypeptides B7 1 and B7 2 and factors activating the expression of class II histocompatibthte antigens
  • IL mterleukins
  • G-CSF colony stimulating factors
  • M-CSF and GM-CSF interferons
  • TNF rumor necrosis factors
  • co-stimulation factors such as polypeptides B7 1 and B7 2 and factors activating the expression of class II histocompatibthte antigens
  • IL-2 is particularly preferred It is responsible for the proliferation of activated T lymphocytes, and, in association with IFN ⁇ , stimulates macrophages, natural killer cells NK (for Natural Killer in English) and T cells. In addition, certain studies tend to show that it has a chemotactic role for lymphocytes when it is produced at the level of a rumor.
  • Interferons have antiviral and immunomodulatory properties. They can activate phagocytic cells and increase the expression of class I and II surface antigens of the major histocompatibility complex (MHC) and also stimulate the cytotoxicity of NK cells and those activated by lymphokines (LAK for Lymphokine Activated Killer in English) meet tumor cells.
  • MHC major histocompatibility complex
  • LAK lymphokine Activated Killer in English
  • Colon stimulating factors are involved in the maturation of hematopoietic stem cells and their differentiation into mature cells in the bloodstream.
  • GM-CSF, G-CSF and M-CSF for Granulocyte-Macrophage, Granuiocyte and Macrophage respectively.
  • TNF ⁇ produced by macrophages are responsible for the anti-tumor cytotoxic activity of macrophages and lymphocytes and for local tissue damage. observed in the inflammatory reaction.
  • cytotoxic genes include those encoding a thymidine kinase and, in particular the thymidine kinase (TK) of the Herpes Simplex virus type 1 (HS V- 1), cytosine deaminase, cytochrome P450 2B 1, pu ⁇ ne nucleoside phosphorylase encoded by the DeoD ⁇ 'E.coli gene, nitroreductase and ⁇ -glucoronidase
  • TK thymidine kinase
  • H V-1 Herpes Simplex virus type 1
  • these toxic genes are described in the literature ( see for example Moolten, 1994, Cancer Gène Therapy /, 279-287)
  • the three therapeutic genes used in the context of the present invention code for the TK-HSV1, IL-2 and IFN ⁇ polypeptides.
  • the origin of the immunostimulatory genes is chosen so that they are functional in the host for which the anti-tumor composition according to the invention is intended.
  • the coding sequences for human IL-2 and human LFN ⁇ are preferably retained.
  • the therapeutic genes can be obtained by cloning, by PCR or by chemical synthesis according to the conventional techniques commonly used. They may be native genes or derivatives thereof by mutation, deletion, substitution and / or addition of one or more nucleotides. Of course, they can include the appropriate elements for regulating transcription as well as translation initiation and termination signals allowing their expression.
  • a promoter region which is functional in the cells of the host which we want to treat will be used, preferably in human cells. It may be the promoter region naturally governing the expression of said gene or a promoter region of different origin, for example from eukaryotic or viral genes. On the other hand, the promoter region can be modified so as to contain regulatory sequences, for example a transcription activating element (enhancer).
  • the promoter region chosen may be constitutive or regulable, and in the latter case, in response to certain cellular signals. It will be advantageous to use a tissue-specific promoter region, when the tumor to be treated originates from a particular cell type. Alternatively, the use of a promoter responding to tumor specific signals (e.g. having overexpressed factors by tumor cells) may prove advantageous because the expression is restricted to tumor cells.
  • promoters are generally known to those skilled in the art. Mention may in particular be made of the promoters SV40 (Virus Simian 40), PGK (Adra et al., 1987, Gene 60, 65-74), HMG (Hydroxy-Methyl-Glutaryl-coenzyme A), TK (Thymidine Kinase), LTRs (Long Terminal Repeat) of RSV (Rous Sarcoma Virus), Mo-MLV (Moloney Murine Leukemia Virus) and promoters of genes coding for class 1 MHC antigens which are activated by IFN ⁇ . These examples are not limitative.
  • the therapeutic genes can be placed under the control of elements allowing their expression independently or in common. In other words, they can be expressed in a monocistronic or polycistronic manner. In the latter case, an element allowing the reinitiation of translation will be implemented at the level of the second cistron, for example an internal ribosome entry site (IRES) well known to those skilled in the art.
  • IRES internal ribosome entry site
  • a certain number of IRES have been identified in the 5 ′ region of viral mRNAs and, in particular, picornaviruses such as the poliomyelitis virus (Pelletier et al., 1988, Mol. Cell. Biol.
  • the immunosumulatory polypeptides may be secreted outside the cells making up the antitumor composition according to the invention.
  • the corresponding genes can also include a signal sequence. It can be the natural signal sequence or a heterologous sequence as long as it is functional in the cell host
  • said cell population can derive from a primary or tumor cell, from a cell line, from an organ or can comprise a mixture of cells allowing the expression of the various therapeutic genes.
  • a mixture of cells for example derived from the Véro line (available at ATCC), a part expressing IL2 and another part expressing IFN ⁇ , possibly combined with a retroviral packaging line allowing production of virions or a population of viruses expressing the TK cytotoxic gene.
  • Véro line available at ATCC
  • a part expressing IL2 and another part expressing IFN ⁇ possibly combined with a retroviral packaging line allowing production of virions or a population of viruses expressing the TK cytotoxic gene.
  • a retroviral packaging line allowing the production infectious particles comprising a retroviral vector according to the invention as defined below
  • the cell population composing the antitumor composition according to the invention comprises or consists of retroviral packaging cells allowing the production of infectious particles comprising a retroviral vector
  • the retroviral vector is defective by deletion or mutation of the gag, pol and env viral genes and cannot, therefore, replicate autonomously Its propagation requires the supply of viral polypeptides for which it is deficient
  • An packaging cell is capable of providing in trans all the polypeptides which the retroviral vector cannot synthesize and which are necessary for the constitution of the infectious particles
  • the packaging cells in use in the present invention are derived from a line of human origin and, in particular, of line 293 This can be generated by transfusion ction of vectors allowing expression of the gag / pol genes of the FMuLV virus (F ⁇ end Mu ⁇ ne Leukemia Virus) of the strain FB29 and the env gene of the amphotropic virus 4070A
  • a preferred antitumor composition according to the invention comprises a population of cells comprising a vector allowing the expression of a gene coding for PIL-2 and allowing the production of infectious particles having incorporated a retroviral vector expressing a gene coding for thymidine kinase of the HSV-1 virus and a gene coding for lTFN ⁇
  • the population of cells in use in the context of the present invention is sensitive to a drug allowing its elimination.
  • a drug derived from acyclovir and, in particular, ganciclovir
  • the population of cells can also express a positive selection marker, for example a gene for resistance to an antibiotic facilitating its selection Mention may be made of the genes ne ⁇ (neomycin) conferring resistance to antibiotic G4 I 8, dhjr (dihydrofolate reductase), pac (puroacetyl transferase) (Morgenstern and Land, 1990, Nucleic Acids Res 18, 3587-3596), hygromycin B and gpt (xanthine phospho ⁇ bosyl)
  • the present invention also relates to an packaging cell derived from line 293 and comprising - the gag / pol genes of the FMuLV virus (F ⁇ end Mu ⁇ ne Leukemia Virus) of the strain FB29 and the env gene of the amphotropic virus 4070A, and a vector allowing the of a gene encoding IL-2
  • a suitable vector can be constituted by the vector pTG5324 described below in which the expression of riL-2 is directed by the early promoter of the virus.
  • the present invention also relates to a retroviral vector characterized in that it comprises from 5 'to 3' - a 5 'LTR, an encapsidation region, a gene coding for gamma interferon, a constitutive internal promoter, a gene coding for thymidine kinase of the HS V-1 virus, - an internal ⁇ bosome entry site (IRES), a gene coding for a positive selection marker, and a 3 'LTR
  • a vector according to the invention can derive from any retrovirus.
  • retroviruses such as avian erythroblastosis virus (AEV), avian leukemia virus (AVL), avian sarcoma virus (ASV), necrosis virus of spleen (SNV) and Rous sarcoma virus (RSV), bovine retroviruses, feline retroviruses, mu ⁇ ns retroviruses such as mu ⁇ ne leukemia virus (MuLV), Friend virus (F-MLV) and murine sarcoma virus (MSV) and primate retroviruses.
  • MoMuLV Moloney mu ⁇ ne leukemia virus
  • the numerous retroviral vectors derived from the latter which are described in the literature, in particular the vector
  • the present invention also relates to the use of an anti-tumor composition of an packaging cell or of a retroviral vector according to
  • cancers which could be thus treated are, advantageously, solid tumors such as renal, breast, lung and colon cancers and melanomas.
  • the medicament resulting from the present invention can be administered according to any general route commonly in use, in particular by parenteral route such as the systemic route, subcutaneous intramuscular or intrapetoneal route.
  • parenteral route such as the systemic route, subcutaneous intramuscular or intrapetoneal route.
  • the intra-tumor route is indicated as being particularly advantageous.
  • administration can take place in a single or repeated dose, one or more times after a certain time interval.
  • the medicament will also comprise a support which is acceptable from a pharmaceutical point of view. It may also include a pharmaceutically acceptable carrier, diluent or adjuvant.
  • the appropriate dosage varies according to different parameters, for example the route of administration, the individual to be treated, the nature and severity of the tumor condition, the type of therapeutic genes used.
  • the present invention also relates to a method of treatment of cancer in mammals, according to which an individual in need of such treatment is injected with a pharmaceutically effective amount of an antitumor composition, an encapsidation cell or a retroviral vector according to the invention
  • Figure 1 illustrates the vector pTG5324 allowing expression of human IL-2 from the CMV early promoter. It includes the pac selection gene directed by the SV40 promoter.
  • Figure 2 illustrates the retroviral vector pTG9344 comprising a 5 'LTR. an encapsidation region, a bicistronic expression cassette "tk gene of the HSV-1 virus followed by TIRES EMCVet of the selection gene neo" directed by the murine PGK promoter.
  • Figure 3 illustrates the retroviral vector pTG9326 deriving from the vector pTG9344 by insertion of the gene coding for canine IFN ⁇ .
  • the constructions described below are carried out according to general techniques of genetic engineering and molecular cloning, detailed in Maniatis et al, (1989, Laboratory Manual, Cold Spring Harbor, Laboratory Press, Cold Sp ⁇ ng Harbor, NY) or according to the recommendations of the manufacturer when using a commercial kit
  • the cloning steps using bacterial plasmids are preferably carried out in the coitus XLl-BIue or DH5a strain (Gibco BRL)
  • the M13 vectors are amplified in the E.
  • the procedure is by filling the protruding 5 ′ ends with the large fragment of DNA polymerase I ⁇ E coli (Klenow) or by digestion with nuclease SI followed by a Klenow treatment PCR (Polymerase Chain Reaction) amplification techniques are known to those skilled in the art (see for example PCR Protocols - A guide to methods and applications, 1990, edited by Innis, Gelfand, Sninsky and White, Académie Press Inc)
  • the line E l 7 is a retroviral packaging line derived from the human line 293 (Graham et al, 1977, J Gen Virol 36, 59-72) by transfection of vectors expressing respectively the gag / pol genes of the FMuLV virus (Friend Mu ⁇ ne Leukemia Virus) of the strain FB29 and the env gene of the amphotropic virus 4070A
  • the cells are transfected according to standard techniques well known to those skilled in the art.
  • the DMEM medium Dulbecco's Modified Eagle's Medium, Gibco BRL
  • FCS fetal calf serum
  • EXAMPLE 1 Construction of a retroviral packaging line producing human interleukin-2 fhrL-2 (17-TG5324)
  • the sequences coding for I'hIL-2 are isolated from the vector pTG36 (described in French patent 85 09480) in the form of a PstI fragment, sub-cloned in the vector M 13TG130 (Kieny et al, 1983, Gene 26, 91 -99) and subjected to site-directed mutagenesis so as to introduce a Sa / l site 12 nucleotides downstream of the stop codon (Amersham mutagenesis kit, RPN 1523)
  • the hIL-2 cDNA is pu ⁇ fie of the mutated vector by digestion Sali and inserts into the ⁇ ' / iol site of pBCMGneo (Karasuyama and Melchers, 1988, Eur J Immunol 18, 97-104) located in 3' and 5 'respectively of splicing and polyadenylation signals of the rabbit ⁇ -globin gene
  • pTG5320 The sequences coding for I'hIL-2 are isolated from the vector
  • a BamHI-HindIII fragment carrying the early promoter of the CMV virus (Cytomegalovirus) purified from pLNCX is introduced into the vector p polyIII-I * (Lathe et al, 1987, Gene 57, 193-201) treated with the same enzymes.
  • the Saf [-BamHl purifies fragment of pTG5320 carrying the intron ⁇ -globin, the cDNA hIL -2 and the ⁇ -globin polyadenylation signal
  • the resulting vector pTG5321 is linearized by the enzyme BamHI and a BamHI-Bglll fragment containing the selection gene pac is inserted under the control of the early promoter and of the polyadenylation signal of the SV40 virus
  • the vector thus obtained is designated pTG5322
  • the vector pTG5324 (FIG. 1) is generated by insertion into the preceding vector linearized by BamHI of a BamHI fragment comprising the mitochond ⁇ ale 12 S mu ⁇ ne sequences (Luftalla et al, 1985, Som Cell Mol Genêt 11, 223-238)
  • plasmid pTG5324 20 ⁇ g of plasmid pTG5324 are used to transfect E 17 cells at a density of 40 to 50% according to the standard calcium phosphate technique. The next day, the transfected cells are placed in the presence of puromycin After two weeks in selective medium, the resistant clones are subcultured, propagated and frozen in liquid nitrogen while waiting to check their capacity to secrete hIL-2.
  • 6-well culture plates are used in which 4em] 0 5 cells to be tested are tested.
  • the day next, the medium is changed and harvested 24 h later The amount of hLL-2 present in cell supernatants is estimated by ELISA (R&D Systems Minneapolis, D2050) About a quarter of the clones tested secrete amounts of IL-2 exceeding 2 ⁇ g / ml / 10 6 cells / 24h.
  • the most clone producer designates E17-5324-clone 2 and secretes 3 ⁇ g / ml / 10 6 cells / 24h of hIL-2 is retained for subsequent studies
  • E l cells 7-5324 are intended for human therapeutic use, it is advantageous to test their capacity for resistance to inactivation by the human complement. To do this, 5 ⁇ 10 cells are cultured in an appropriate support. The next day the medium is eliminated and the cells are exposed to 0.5 ml of fresh or inactive human serum by heating (negative control) taken from two different individuals or from FCS (negative control). After 150 min, the culture is continued.
  • the retroviral vector pTG9344 allows the expression in a bicistronic manner of a cytotoxic gene, in this case the tk gene of HSV-1 and of the positive selection gene neo
  • the basic vector is pLXSP which derives from pLXSN (Miller and Rosman, 1989, supra)
  • the PGK promoter obtained from the plasmid PKJ-1 (Adra et ai, 1987 Gene 60, 65-74) is introduced downstream of the packaging region in the form of an EcoRI-Psll fragment (positions - 517 a -20 with respect to the transcriptional dimming site)
  • the tk gene is isolated from the vector pTK- 1 (Spandidos et al, 1982, Exp Cell Res 141, 149-15S, Wagner, 1981, Proc Natl Acad Sci USA 78, 1441-1445) and subclone in the BamHI site of ⁇ BR32S (Covarrubias and Bolivar, 1982 Gene /
  • the resistance to the selection agent G418 depends on the efficiency of reinitiation from TIRES EMCV Consequently, when the culture is carried out in a selective medium, only the cells producing large quantities of bicistronic mRNA are brought to survive thereby ensuring a level of expression high tk gene
  • the vector pTG9344 includes a unique EcoRl restriction site which allows the insertion of an additional gene under the dependence of the 5 'LTR retroviral promoter.
  • Canine LFN ⁇ is cloned from cellular RNA isolated from canine T lymphocytes stimulated by concanavalin
  • a cellular RNA is reverse transc ⁇ t by implementing the degenerate primer oTG4031 (SEQ LD NO 3)
  • the specific fragment is amplified in two stages First of all an internal fragment is produced using primers OTG4 I 69 and OTG4 I 70 (SEQ ID NO 4 and 5) Then, two specific ohgonucleotides oTG4321 and oTG43 19 (SEQ ID NO 6 and 7) are used in combination with two oligo d (T) and primer adapters to generate the 5 'and 3' fragments according to the RACE method (Frohmann et al, 1988, Proc Natl Acad Sa USA 85, 8998-9002) The two PCR fragments are cleaved with Sphl and A ⁇ ll before being inserted into the Sphl site of M13TG131 ( Kieny et al,
  • the murine IFN ⁇ is isolated by PCR on the basis of the sequence data (Gray and Goeddel, 1983, Proc Natl Acad Sci USA 80, 5842-5846) using the primers 0TG7295 and oTG7296 (SEQ ID NO 10 and 1 1 )
  • the retroviral vector pTG9337 resulting from the insertion of the amplification fragment cleaved by EcoKl within the Ec ⁇ Rl site of pTG9344
  • Human LFN ⁇ is isolated by PCR from the vector M13TG2437 which results from subcloning into the vector M 13TG131 of the coding sequence IFN isolated from pTG23 (Tessier et al, 19S4 Nucleic Acids Res 12, 7663-7676)
  • the primers 0TG6 I47 and oTG4983 (SEQ ID NO 12 and 13) are used.
  • the amplified fragment is treated with SI nuclease and then with Klenow before being cloned in an intermediate vector from which it can be isolated to be inserted into the LcoRl site of pTG9344
  • the E17 cells are transfected with 10 ⁇ g of each of the plasmids pTG9326 and pTG5324 by the calcium phosphate method and cultured in a selective medium (1 ⁇ g / ml of puromycin and 1 mg / ml of G418) 24 h after transfection
  • a selective medium (1 ⁇ g / ml of puromycin and 1 mg / ml of G418)
  • the resistant cells are subcloned by limiting dilution (cultured in a 96-well plate of 200 ⁇ l / well of a dilution at a density of 1.5 cells per ml)
  • the cell clones are recovered after two weeks culture and amplified in a conventional manner
  • the clones are cultured at the rate of 4.
  • ⁇ 10 cells After a change of medium, the 24 ha culture supernatant is collected from which the amounts of hIL-2 and of IFN ⁇ secreted
  • the ELISA method (R&D Systems Minneapolis, D2050) previously applied LTFN ⁇ is applied by the method of inhibiting the cytopathic effect of the VSV virus (Vesicular Stomatitis Virus strain lndiana, ATCC VR 158) on the dog kidney epithelial cell line MDCK (Steward II, in The Interferon System, pp 17-19, Sp ⁇ nger-Verlag, NY, Familletti et al, 1981, Methods Enzymology 78, 387) Briefly, 3x l 0 4 MDCK cells / well are cultured in a 96-well microtiter plate and then serial dilutions of the supernatants obtained from the clones resistant to puromycin and to G418 are added.
  • retroviral particles by these clones is evaluated by infection of 1 to 2x 10 permissive 3T3 cells After 24 h of culture, they are exposed for 90 min to 200 ⁇ l of 10 in 10 dilutions of cell supernatant to be tested and 200 ⁇ l of medium containing 16 ⁇ g / ml of polybrene (Sigma) The culture is continued in a medium which is first of all conventional and then selective (5 mg / ml of G418) 24 h after infection The resistant colonies after two weeks are stained with crystal violet (0.05% in an ethanol mixture 10% water 90%) The number of retroviral particles present in the supernatant of the producing clones can be calculated from the number of 3T3 colonies resistant to G41 8 The clone designated below is selected after E 17-TG5324 & TG9326 # 28 which produces 1 ⁇ g / ml / 10 6 cells / 24h of hIL-2, 32 U / ml of TFN ⁇ and 4.5 ⁇ 10 6
  • EXAMPLE 7 Construction of an packaging line secreting hIL-2 and producing the retoviral vector pTG9337
  • the El 7-5324 cells are transfected with 20 ⁇ g of plasmid pTG9337 and the resistant clones are selected in G418 medium (1 mg / ml) After subcloning, the most producing clones are evaluated in terms of hIL-2 secretion , murine IFN ⁇ and of titre in viral particles The methods used are described in the previous example with the exception of the assay technique of murine IFN ⁇ which is quantified by ELISA test (PerSeptive Diagnostics, Cambridge, MA , N ° S-6716) The clone retained E17-TG5324 & TG9337 # 33 produces 1 ⁇ g / ml / 10 6 cells / 24h of hIL-2, 60 ng / ml / 10 6 cells / 24h of IFN ⁇ and has a viral titer of I, 3 ⁇ I 0 "cfu / ml
  • EXAMPLE S Transduction of target cells It is verified that the viral particles produced from the producing lines E 17-TG5324 & TG9326 and E 17-TG5324 & TG9337 are capable of transducing target cells and that the expression of the therapeutic genes is not altered in the cellular context
  • This study is carried out on an established mu ⁇ ne line (3T3 cells) and on two primary lines (P3D6M and P3D4M cells) The latter are derived from dog melanomas and subjected to passage in immunodeficient SCID mice in order to generate homogeneous primary lines 2 ⁇ 10 7 cells at stage 5 , are injected subcutaneously into animals Tumors are removed 3 weeks later and the melanoma cells are kept in culture under standard conditions
  • the target cells are cultured at a rate of 1 to 2 ⁇ 10 4 cells per well and, the following day, infected with 0.5 ml of culture cell culture supernatant previously filtered through a 0.45 ⁇ m membrane. infection continue for 1 to 2 hours then the cells are returned to fresh medium Usually, two transduction cycles are carried out on the same day and the transduced cells are cultured in the presence of G418 (5 mg / ml) The medium is changed every three days until the appearance of resistant colonies whose sensitivity to ganciclovir is evaluated The tests use 5 ⁇ 10 ° cells which are placed the day following their cultivation, in the presence of ganciclovir at concentrations of between 0 and 1000 uM The viability of the cells is determined after one week by the trypane blue test The number of cells counted in the wells in which the culture was carried out in abse ganciclovir represents 100% The results indicate that the three types of target cells transduced by particles from the retoviral vectors pTG9326 and p
  • the capacity of the viral particles of vector pTG9344 is verified to induce a neighboring cytotoxic effect in the transduced cells.
  • the primary cells P3D6M are transduced by the viral particles pTG9344 and the infected cells are selected in the presence of G418 (1 mg. / ml)
  • a co-culture is carried out containing non-transduced P3D6M cells and a certain percentage of the transduced culture (respectively 0, 10, 30, 50, 80 and 100%)
  • the co-culture is maintained for 7 days in the presence of Ganciclovir (1 ⁇ M) and the viability of the cells is estimated by staining with trypane blue.
  • this concentration is chosen so as to be toxic for the transduced cells which express thymidine kinase (3% of surviving cells for the test 100%) while it has little or no effect on the viability of cells not expressing the suicide gene (86% of surviving cells for the 0% test) know of co-culture, ganciclovir exerts a notable toxic effect even on a small percentage of transduced cells A drastic reduction in viability is already observed when the cell mixture contains only 10% of transduced cells (18% of surviving cells for the test 10% and 5% for test 30%) These results indicate that the vector pTG9344 expresses a sufficient quantity of TK to induce cytotoxicity of the infected cell and to propagate this effect to the neighboring non-transduced cells.
  • PS 15 (murine mastocvtome) and B 16 (murine melanoma) cells are transduced by the vector pTG9344 (tk-neo) as a negative control or pTG9337 (IFN ⁇ mu ⁇ n-tk-neo) and the presence of antigens is determined.
  • class 1 and II on the surface of cells infected by conventional immunofluorescence and flow cytometry (FACS) techniques The MHC class II antigens present on the surface of P815 and B 16 cells are demonstrated by an anti- sou ⁇ s coupled to FITC (for Fluorescein isothiocyanate) (Pharmingene, San Diego, CA).
  • FITC for Fluorescein isothiocyanate
  • the cells After infection and maintenance in a selective medium (G418 5 mg / ml) for approximately two weeks, the cells are detached by the action of 10 mM EDTA in a phosphate buffered saline (PBS) and washed twice in the following buffer (PBS, 1% bovine serum albumin, 0.1% human gamma globulin, 10 mM EDTA and 0.02% sodium azide). 1x10 6 cells are incubated for 45 min, at 4 ° C.
  • PBS phosphate buffered saline
  • the cells are fixed (2% formaldehyde in PBS buffer) and analyzed by FACS (Becton Dickinson cytometer, San Jose, CA)
  • the B 16 mu ⁇ nes cells are transduced by the vectors pTG9344 and pTG9337 or as a negative control by a retroviral vector expressing the marker gene.
  • LacZ encoding ⁇ -galactosidase (pTG5391)
  • the infected cells are selected in the presence of G41 S for 14 days trypsinized and resuspended at a density of 2x 10 cells / ml in PBS buffer 100 ⁇ l of this suspension are injected subcutaneously into immunocompetent B6 / D2 generation FI mice Two days later and this during the 6 days which follow, the animals receive an intraperitoneal injection of ganciclovir (100 mg / kg / day) and the number of tumors and their size are examined up to 44 days after implantation.
  • ganciclovir 100 mg / kg / day
  • mice in which were implanted the cells transduced by a vector expressing therapeutic genes do not develop tumors before the 25th day (groups 3 and 4) whereas the animals having received no vector (group 1) or a non-therapeutic vector ( group 2) are decimated well before the 21st day
  • group 1 or a non-therapeutic vector group 2
  • group 3 and 4 the mice having received no vector
  • group 2 a non-therapeutic vector
  • group 3 and 4 the expression of the tk gene alone slows tumor development (group 3) while the vast majority (9 / 10) animals having received B cells 16-9337 (concomitant expression of the tk and LFN ⁇ genes) show no tumor more than 44 days post-implantation
  • NAME Transgene S.A.

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EP97916490A 1996-03-25 1997-03-25 Antitumorzellzusammensetzungen, die auf mindestens drei transgene exprimieren Withdrawn EP0906441A1 (de)

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PCT/IB1996/000439 WO1997035996A1 (en) 1996-03-25 1996-03-25 Packaging cell line based on human 293 cells
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FR9610927A FR2746317B1 (fr) 1996-03-25 1996-09-06 Nouvelles compositions antitumorales
PCT/FR1997/000521 WO1997035995A1 (fr) 1996-03-25 1997-03-25 Compositions cellulaires antitumorales exprimant au moins trois transgenes

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