WO1995009655A1 - Traitement de tumeurs par la transformation genetique des cellules tumorales a l'aide de genes codants des marqueurs selectifs negatifs et des cytokines - Google Patents

Traitement de tumeurs par la transformation genetique des cellules tumorales a l'aide de genes codants des marqueurs selectifs negatifs et des cytokines Download PDF

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WO1995009655A1
WO1995009655A1 PCT/US1994/011251 US9411251W WO9509655A1 WO 1995009655 A1 WO1995009655 A1 WO 1995009655A1 US 9411251 W US9411251 W US 9411251W WO 9509655 A1 WO9509655 A1 WO 9509655A1
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interleukin
agent
tumor
cells
nucleic acid
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PCT/US1994/011251
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English (en)
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Kenneth W. Culver
Charles J. Link, Jr.
Michael R. Blaese
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The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services
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Application filed by The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services filed Critical The Government Of The United States Of America, Represented By The Secretary Of The Department Of Health And Human Services
Priority to JP7510988A priority Critical patent/JPH09504518A/ja
Priority to EP94930031A priority patent/EP0722343A4/fr
Publication of WO1995009655A1 publication Critical patent/WO1995009655A1/fr

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    • 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
    • C07K14/435—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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to the treatment of tumors. More particularly, this invention relates to the treatment of tumors (such as, for example, ovarian cancer) with DNA (RNA) encoding an agent which renders tumor cells sensitive to an interaction agent and DNA (RNA) encoding a cyto ine.
  • tumors such as, for example, ovarian cancer
  • RNA DNA
  • RNA DNA
  • RNA DNA
  • Ovarian carcinoma is the most common cause of death from a gynecological malignancy in the United States with approximately 19,000 newly diagnosed cases per year and a 70% overall mortality rate. Over two-thirds of patients have an advanced stage of the disease at presentation for which systemic chemotherapy is indicated after staging and debulking laparotomy. (Young, et al., "Cancer of the Ovary,” in DeVita, et al., eds. , Cancer Principles and Practice of Oncology, J.B. Lippincott, Philadelphia, pgs. 1226-1254 (1993)). Although about 80% of patients respond to initial treatment with cisplatin-based chemotherapy, i only about 10% to 20% experience durable complete
  • Ovarian cancer has a fairly unigue natural history. Even patients with advanced stages of the disease often have their diseases confined to their abdomens for extended periods of time. The cancer often stays localized to the abdomen and presents great difficulty for the patient by obstruction of the intestines or ureters. As a result, intraperitoneal therapies have been developed for the local administration of chemotherapeutic agents into the peritoneal cavity. (Meyers, Semin. Oncol. , Vol. II, pgs. 275-284 (1984)). These therapies have met with only moderate success because they did not provide the reduced toxicity profiles initially hoped for.
  • Gene transfer has been recognized for some time as a promising avenue to therapies for cancers, among other diseases.
  • the earliest applications of gene transfer for cancer treatment have been indirect approaches focusing on enhancing anti-tumor immune responses. Thus, for instance, attempts have been made to increase the cytotoxicity of immune cells, or to enhance their proliferation.
  • tumor cells have been modified in vitro with cytokine genes and reintroduced into patients in an attempt to immunize the patient to their own cancer.
  • the IL-4 gene was introduced to tumors by Tepper, et al., Cell 57; 503 (1989); the IL-2 gene by Fearon, et al., Cell 60 :397 (1990), and by Gansbacher, et al., J. EXD. Med. 172: 1217 (1990); the interferon-gamma gene by Gansbacher, et al. , Cancer Res. 50; 7820 (1990); and TNF gene by Asher, et ' al., J. Immunol. 146: 3227 (1991).
  • Each of the animal studies demonstrated rejection of genetically altered tumors upon rei plantation, and the mice in these studies were immune to subsequent rechallenge with the same tumor.
  • a murine retroviral vector was employed to introduce a thymidine kinase gene from herpes simplex virus 1 ("HSV-1 tk gene") into C6 rat glioma-derived cell lines in vitro.
  • HSV-1 tk gene herpes simplex virus 1
  • Cells which had taken up the retroviral vector were sensitized to the anti-viral agent ganciclovir, and were preferentially killed when exposed to ganciclovir in the medium.
  • Ezzeddine, et al. were able to use the method to define conditions in vitro for killing essentially all infected cells but not uninfected cells.
  • C6 cells were introduced subcutaneously into nude mice to form tumors and the tumor-bearing mice were treated with ganciclovir.
  • Ganciclovir inhibited the growth of tumors formed by HSV-1 tk expressing C6 cells, but did not affect tumors formed by HSV-1 tk-negative C6 cells.
  • Ezzeddine, et al. thus showed that in vitro retroviral gene transfer can be used to sensitize cells to a cytotoxic agent, which can then be used to kill the cells when they are propagated as tumors in nude mice.
  • the authors did not demonstrate any practical way to introduce an HSV-1 tk gene into tumor cells in situ, however.
  • Ezzeddine, et al. also did not show how to eradicate all neoplastic cells, a prerequisite for tumor remission, when less than all cells in the tumor would take up a tk gene, express the gene at a level sufficient to assure toxicity and, as a consequence, be killed by exposure to ganciclovir.
  • Gene transfer is achieved by infection or tumor cells with urine retroviral vectors carrying the Herpes Simplex thymidine kinase gene and integration of this gene into the genome of the host cell. These vectors are produced continuously by murine vector producer cells that are injected into the tumor mass. Because retroviruses can infect only cells that are synthesizing DNA actively (i.e., replicating cells), a preferential transduction of tumor cells is achieved. This approach now is being evaluated in a clinical trial. (Oldfield, et al. , Human Gene Therapy. Vol. 4, pgs. 39-69 (1993)).
  • PCT Application No. W093/04167 discloses a purported method for transferring therapeutic genes to brain tumor cells in order to kill the cells.
  • a retrovirus containing a selectable marker and at least one gene required for its replication is introduced into producer cells such that integration of the proviral DNA corresponding to the retrovirus into the genome of the producer cell results in the generation of a modified retrovirus wherein at least one of the genes required for replication of the retrovirus is replaced by the therapeutic gene or genes.
  • Producer cells then are selected in which the modified retrovirus is incorporated as part of the genome of the producer cells.
  • the producer cells then are grafted in proximity to the dividing tumor cells in order to infect the tumor cell with the modified retrovirus, thereby transferring the therapeutic gene or genes to the tumor cells.
  • the cells then are killed by administering a substance that is metabolized by the therapeutic gene transferred to the tumor cells into a metabolite that kills the cells.
  • the therapeutic gene may be the Herpes Simplex thymidine kinase gene, and the substance which is metabolized by Herpes Simplex thymidine kinase to kill the tumor cells may be gancyclovir or acyclovir.
  • the cited PCT application shows only (i) that a replication-defective retrovirus which carried an HSV tk gene and a G418 resistance gene could be transduced stably, via G418 selection, into a glioma cell line in vitro; (ii) that the viral tk gene in the transformed cells rendered them about 20-fold more sensitive to ganciclovir than control glioma cells; and (iii) that some glioma tumor cells which formed tumors when implanted in rat brains also expressed a /3-galactosidase marker when the tumors were injected with a producer cell line which produced a retroviral vector with the marker gene.
  • the vector in the described experiments did not carry a tk gene, and there was no systemic administration of a chemotherapeutic agent.
  • the PCT application in question does not show that tumor cells can be rendered sensitive in vivo to any such agent.
  • a viral tk sensitizing gene administered by microinjecting producer cells into a tumor in situ renders sensitive to ganciclovir tumor cells which are not transduced to express the viral tk.
  • the '591 application teaches that this effect radically augments the therapeutic- efficacy of the sensitizing gene in a way that could not have been predicted.
  • anti-tumor therapies may be potentiated by administering, in addition to the sensitizing gene, a gene that stimulates or activates the immune system, thereby increasing the overall percentage of killed neoplastic cells in the tumor.
  • immune response enhancing genes disclosed in the '591 application are cytokines, including but not limited to IL-1 through IL-12, and immune co-activating signal molecules, such as certain MHC determinants.
  • the '591 application discloses IL-2 as a particularly preferred cytokine in this regard.
  • the '591 application is not limited to any particular mechanism by which IL-2 activity might augment an anti-tumor effect of a sensitizing gene. Nonetheless, it is hypothesized there that IL-2 and other immune response enhancing genes would improve therapeutic efficacy by stimulating or activating the immune system.
  • an IL-2 gene augments the action of a sensitizing gene without immune system activity, using ovarian cancer cells as a model system.
  • an IL-2 gene in in vitro experiments potentiated the ganciclovir sensitivity of cells of an ovarian tumor cell line upon transduction with an HSVtk expression vector.
  • the synergistic effect of IL-2 and the sensitizing gene was seen in vivo in the absence of an immune system in implanted ovarian cell tumors in nude mice.
  • the potentiating effect of Interleukin-2 on an HSVtk/ganciclovir-mediated gene therapy for ovarian cell tumors is indicative of an unusual and unrecognized mechanism of anti-tumor activity of a cytokine.
  • a method of treating a tumor in a host comprising administering to the tumor a first nucleic acid sequence, and a second nucleic acid sequence.
  • the first nucleic acid sequence encodes an agent such that the tumor cells are rendered sensitive to an interaction agent; i.e., growth of the tumor cells is inhibited, prevented, or destroyed upon administration of the interaction agent.
  • the second nucleic acid sequence encodes an agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells, but is not an agent which renders tumor cells sensitive to an interaction agent.
  • the tumor Upon administration of the first nucleic acid sequence and the second nucleic acid sequence to the tumor, the tumor then is treated with an interaction agent.
  • the therapeutic effect of the interaction agent is enhanced by the agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells (but is not an interaction agent), and such effect is an effect independent of the immune system of the host.
  • nucleic acid sequence means a DNA or RNA molecule, and includes complete and partial gene sequences, and includes polynucleotides as well. Such term also includes a linear series of deoxyribonucleotides or ribonucleotides connected one to the other by phosphodiester bonds between the 3' and 5' carbons of the adjacent pentoses.
  • the first and second nucleic acid sequences are contained in at least one expression vehicle.
  • expression vehicle means any genetic construct including the first and/or second nucleic acid sequences, and is capable of providing for expression of such sequence(s).
  • the first nucleic acid sequence is contained in a first expression vehicle
  • the second nucleic acid sequence is contained in a second expression vehicle.
  • the expression vehicle may be any expression vehicle which is capable of transfecting cells and expressing the first and/or second nucleic acid sequence(s) in vivo.
  • Such expression vehicles include, but are not limited to, eukaryotic vectors, prokaryotic vectors (such as, for example, bacterial plasmids), and viral vectors.
  • the vector also may be contained within a liposome.
  • the first and second expression vehicles are viral vectors.
  • Viral vectors which may be employed include, but are not limited to, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, and Herpes Virus vectors.
  • the viral vector is a retroviral vector.
  • a first packaging cell line is transduced with a first viral vector, which includes the first nucleic acid sequence which encodes an agent such that the tumor cells are rendered sensitive to an interaction agent, to form a first producer cell line including the first viral vector.
  • a second packaging cell line also is transduced with a second viral vector, which includes the second nucleic acid sequence which encodes an agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells, wherein the agent is not an agent which renders the tumor cells sensitive to an interaction agent, to form a second producer cell line including the second viral vector.
  • the producer cell lines then are administered to the tumor, whereby the producer cell lines generate viral vector particles capable of transducing the tumor cells.
  • each of the first and second viral vectors is a retroviral vector.
  • retroviral vectors which may be employed include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus.
  • the retroviral vector is an infectious but non-replication competent retrovirus.
  • replication competent retroviruses may also be used.
  • Retroviral vectors are useful as agents to mediate retroviral-mediated gene transfer into eukaryotic cells.
  • Retroviral vectors generally are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by the gene(s) of interest. Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • a packaging-defective helper virus is necessary to provide the structural genes of a retrovirus, which have been deleted from the vector itself.
  • the retroviral vector may be one of a series of vectors described in Bender, et al. , J. Virol. , 61:1639-1649 (1987), based on the N2 vector (Armentano, et al., J. Virol. , 61:1647-1650) containing a series of deletions and substitutions to reduce to an absolute minimum the homology between the vector and packaging systems. These changes have also reduced the likelihood that viral proteins would be expressed. In the first of these vectors, LNL-XHC, there was altered, by site-directed mutagenesis, the natural ATG start codon of gag to TAG, thereby eliminating unintended protein synthesis from that point.
  • MoMuLV Moloney murine leukemia virus
  • pPr80 BOg another glycosylated protein
  • MoMuSV Moloney murine sarcoma virus
  • the vector LNL6 was made, which incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV.
  • the 5' structure of the LN vector series thus eliminates the possibility of expression of retroviral reading frames, with the subsequent production of viral antigens in genetically transduced target cells.
  • Miller has eliminated extra env sequences immediately preceding the 3' LTR in the LN vector (Miller, et al. , Biotechniques, 7:980-990, 1989).
  • Safety is derived from the combination of vector genome structure together with the packaging system that is utilized for production of the infectious vector.
  • Miller, et al. have developed the combination of the PPAM3 plasmid (the packaging-defective helper genome) for expression of retroviral structural proteins together with the LN vector series to make a vector packaging system where the generation of recombinant wild-type retrovirus is reduced to a minimum through the elimination of nearly all sites of recombination between the vector genome and the packaging- defective helper genome (i.e. LN with pPAM3).
  • the retroviral vector may be a Moloney Murine Leukemia Virus of the LN series of vectors, such as those hereinabove mentioned, and described further in Bender, et al. (1987) and Miller, et al. (1989).
  • Such vectors have a portion of the packaging signal derived from a mouse sarcoma virus, and a mutated gag initiation codon.
  • the term "mutated” as used herein means that the gag initiation codon has been deleted or altered such that the gag protein or fragments or truncations thereof, are not expressed.
  • the retroviral vector may include at least four cloning, or restriction enzyme recognition sites, wherein at least two of the sites have an average frequency of appearance in eukaryotic genes of less than once in 10,000 base pairs; i.e., the restriction product has an average DNA size of at least 10,000 base pairs.
  • Preferred cloning sites are selected from the group consisting of Notl, SnaBI, Sail, and Xhol.
  • the retroviral vector includes each of these cloning sites. Such vectors are further described in U.S. Patent Application Serial No. 919,062, filed July 23, 1992, and incorporated herein by reference in its entirety.
  • a shuttle cloning vector which includes at least two cloning sites which are compatible with at least two cloning sites selected from the group consisting of Notl, SnaBI, Sail, and Xhol located on the retroviral vector.
  • the shuttle cloning vector also includes at least one desired gene which is capable of being transferred from the shuttle cloning vector to the retroviral vector.
  • the shuttle cloning vector may be constructed from a basic "backbone" vector or fragment to which are ligated one or more linkers which include cloning or restriction enzyme recognition sites. Included in the cloning sites are the compatible, or complementary cloning sites hereinabove described. Genes and/or promoters having ends corresponding to the restriction sites of the shuttle vector may be ligated into the shuttle vector through techniques known in the art.
  • the shuttle cloning vector can be employed to amplify DNA sequences in prokaryotic systems.
  • the shuttle cloning vector may be prepared from plasmids generally used in prokaryotic systems and in particular in bacteria.
  • the shuttle cloning vector may be derived from plasmids such as pBR322; pUC 18; etc.
  • the vectors include one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the hi ⁇ tone, pol III, and ⁇ -actin promoters).
  • Other viral promoters which may be employed include, but are not limited to, adenovirus promoters, TK promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to those skilled in the art from the teachings contained herein.
  • the vectors then are employed to transduce packaging cell lines to form producer cell lines.
  • packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2, -AM, PA12, T19-14X, VT-19-17-H2, CRE, CRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the first and second vectors containing the first and second nucleic acid sequences encoding an agent such that the tumor cells are rendered sensitive to an interaction agent, and an agent capable of providing for the inhibition, prevention, or destruction of the growth of the tumor cells upon expression of the nucleic acid sequence encoding the agent, wherein such agent is not an agent which renders tumor cells sensitive to an interaction agent, may transduce the packaging cells through any means known in the art. Such means include, but are not limited to, electroporation, the use of liposomes, and CaP0 4 precipitation.
  • the first and second producer cell lines then are administered directly to or adjacent to the tumor in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
  • the first and second producer cell lines are administered in as large a volume as will be tolerated by the host.
  • the first and second producer cell lines are administered in an amount of from about 1 x 10 6 cells/kg to about 1 x 10 8 cells/kg of host weight.
  • the exact amount of producer cells to be administered is dependent upon various factors, including but not limited to, the type of the tumor, the location of the tumor, and the size of the tumor. In some cases, repeat administration of the producer cells may be required.
  • the first and second producer cell lines are administered to the tumor such that the vector particles generated by the first and second producer cell lines are able to transduce the tumor cells.
  • the first and second producer cell lines may be administered directly to or adjacent to the tumor, or to a systemic pathway which enables vector particles generated by the first and second producer cell lines to travel to and transduce tumor cells.
  • the producer cell lines may be injected into the blood stream (i.e., intravenous administration), or into the cerebrospinal fluid in order to treat tumors of the central nervous system.
  • the producer cells also may be administered intraperitoneally, subcutaneously, or intramuscularly. The exact mode of administration is dependent upon the type of tumor which is treated.
  • the producer cells may be administered in combination with a pharmaceutically acceptable carrier suitable for administration to a patient.
  • the carrier may be a liquid carrier such as, for example, a saline solution or a buffer solution or other isomolar aqueous solution.
  • the producer cells Upon administration of the first and second producer cell lines to the tumor, the producer cells generate viral vector particles.
  • the viral vector particles then transduce the surrounding tumor cells. Because tumor cells, and in particular cancerous tumor cells, in general are actively replicating cells, the retroviral vector particle would be integrated into and expressed preferentially or exclusively in the tumor cells as opposed to normal cells.
  • first nucleic acid sequence being contained in a first expression vector (such as a retroviral vector)
  • second nucleic acid sequence being contained in a second expression vector (which also may be a retroviral vector)
  • first and second producer cell lines which generate first and second viral vector particles containing the first and second nucleic acid sequences
  • first nucleic acid sequence and the second nucleic acid sequence may be contained in one expression vehicle (such as a retroviral vector).
  • the vector then is transduced into a packaging cell line to form a producer cell line which generates viral vector particles containing the first and second nucleic acid sequences.
  • Such viral vector particles are generated by the producer cell line upon administration of the producer cells to the tumor, whereby such viral vector particles containing the first and second nucleic acid sequences transduce the tumor cells, and the tumor cells express the proteins encoded by the first and second nucleic acid sequences.
  • first nucleic acid sequence and the second nucleic acid sequence may be contained in first and second expression vehicles, respectively.
  • the first and second expression vehicles (which may be first and second retroviral vectors, then may be transduced into a single packaging cell line to form a producer cell line which generates first and second viral vector particles containing the first and second nucleic acid sequence, respectively.
  • the first and second viral vector particles upon administration of the producer cells to the tumor, transduce the tumor cells, and the tumor cells express the proteins encoded by the first and second nucleic acid sequences.
  • Tumors which may be treated in accordance with the present invention include malignant and non-malignant tumors.
  • Malignant (including primary and metastatic) tumors which may be treated include, but are not limited to, those occurring in the adrenal glands; bladder; bone; breast; cervix; endocrine glands (including thyroid glands, the pituitary gland, and the pancreas); colon; rectum; heart; hematopoietic tissue; kidney; liver; lung; muscle; nervous system; brain; eye; oral cavity; pharynx; larynx; ovaries; penis; prostate; skin (including melanoma); testicles; thymu ⁇ ; and uterus.
  • the agent which renders the tumor cells sensitive to an interaction agent is a negative selective marker; i.e., a material which in combination with a chemotherapeutic or interaction agent inhibits, prevents, or destroys the growth of the tumor cells.
  • an interaction agent is administered to the human host.
  • the interaction agent interacts with the negative selective marker in order to prevent, inhibit, or destroy the growth of the tumor cells.
  • Negative selective markers which may be employed include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase; and cytosine deaminase.
  • thymidine kinase such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase
  • cytosine deaminase include, but are not limited to, thymidine kinase, such as Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the negative selective marker is a viral thymidine kinase selected from the group consisting of Herpes Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase, and varicella-zoster virus thymidine kinase.
  • the interaction or chemotherapeutic agent preferably is a nucleo ⁇ ide analogue, for example, one selected from the group consisting of ganciclovir and acyclovir.
  • Such interaction agents are utilized efficiently by the viral thymidine kinases as substrates, and such interaction agents thus are incorporated lethally into the DNA of the tumor cells expressing the viral .thymidine kinases, thereby resulting in the death of the tumor cells.
  • the negative selective marker is cytosine deaminase.
  • cytosine deaminase is the negative selective marker
  • a preferred interaction agent is 5-fluorocytosine. Cytosine deaminase converts 5- fluorocytosine to 5-fluorouracil, which is highly cytotoxic. Thus, the tumor cells which express the cytosine deaminase gene convert the 5-fluorocytosine to 5- fluorouracil and are killed.
  • Another interaction agent which may be employed is l-2-deoxy-2-fluoro- ⁇ -D- arabinofuranosil-5-iodouracil (FIAU) .
  • the interaction agent is administered in an amount effective to inhibit, prevent, or destroy the growth of the transduced tumor cells.
  • the interaction agent may be administered in an amount from 5 mg to 10 mg/kg of host weight per day, depending on overall toxicity to a patient.
  • the interaction agent preferably is administered systemically, such as, for example, by intravenous administration, by parenteral administration, by intraperitoneal administration, or by intramuscular administration.
  • a "bystander effect” may result, i.e., tumor cells which were not originally transduced with the nucleic acid sequence encoding the negative selective marker may be killed upon administration of the interaction agent.
  • the "bystander effect” is disclosed in U.S. Patent Application Serial No. 07/877,519, filed May 1, 1992, which is incorporated herein by reference.
  • the transformed tumor cells may be producing a diffusible form of the negative selective marker that either acts extracellularly upon the interaction agent, or is taken up by adjacent, non- transformed tumor cells, which then become susceptible to the action of the interaction agent. It also is possible that one or both of the negative selective marker and the interaction agent are communicated between tumor cells.
  • the agent which provides for the inhibition, prevention, or destruction of the growth of the tumor cells, wherein the agent is not an agent which renders tumor cells sensitive to an interaction agent, and such agent is encoded by the second nucleic acid sequence is a cytokine.
  • the cytokine is an interleukin.
  • Other cytokines which may be employed include interferons and colony-stimulating factors, such as GM-CSF.
  • Interleukins which may be encoded by the second nucleic acid sequence include, but are not limited to, Interleukin- 1; Interleukin-l ⁇ ; Interleukin-2; Interleukin-3; Interleukin-4; Interleukin-5; Interleukin-6; Interleukin-7; Interleukin-8; Interleukin-9; Interleukin-10; Interleukin- 11; and Interleukin-12.
  • the interleukin is Interleukin-2.
  • an expression vehicle such as a retroviral vector particle
  • a cytokine such as Interleukin-2, for example
  • the expression of the cytokine may activate the immune system against the tumor and aids in eradicating residual tumor cells not killed by the bystander effect of the negative selective marker.
  • cytokine such as Interleukin-2
  • negative selective marker such as Herpes Simplex thymidine kinase
  • a first packaging cell line is transduced with a first retroviral vector, such as those hereinabove described, which includes the Herpes Simplex thymidine kinase gene.
  • a second packaging cell line is transduced with a retroviral vector, such as those hereinabove described, which includes the Interleukin-2 gene.
  • the transduced packaging cell lines are administered in vivo to the tumor in an acceptable pharmaceutical carrier and in an amount effective to inhibit, prevent, or destroy the growth of the tumor.
  • the producer cells Upon administration of the producer cells to the tumor, the producer cells generate a first group of viral particles including a gene encoding the negative selective marker, and a second group of viral particles including a gene encoding a cytokine.
  • the two groups of viral particles transduce the tumor cells.
  • the host then is given an agent such as ganciclovir, or l-2-deoxy-2-fluoro-B-D- arabinofuranosil-5-iodouracil (FIAU), which interacts with the Herpes Simplex Virus thymidine kinase to kill the transduced tumor cells.
  • agent such as ganciclovir, or l-2-deoxy-2-fluoro-B-D- arabinofuranosil-5-iodouracil (FIAU)
  • FIAU Herpes Simplex Virus thymidine kinase
  • Interleukin-2 by transduced tumor cells will stimulate an immune response against the tumor and help to kill those tumor cells which were not transduced with the vector particles and not killed as a result of the Herpes Simplex thymidine kinase bystander effect.
  • the method of the present invention is particularly useful when the targeted tumor is localized in a particular region of the body for extended periods of time, such as, for example, ovarian cancer, which tends to remain localized in the abdomen for extended periods of time; melanoma; renal carcinoma; brain tumors; liver tumors; and head and neck cancer.
  • ovarian cancer cells may be administered intraperitoneally. Such injection of the producer cells also minimizes undesirable propagation of the virus in the body, especially when replication- competent retroviral vectors are used because such vectors are produced continuously.
  • any vector particle which escapes from the local environment of the tumor should immediately bind to another cell. Most cells are not in cycle, however, and therefore will not integrate the genes carried by the vector and will not express any genes which it contains. Thus, the proportion of potential target cells which are in cycle at the time of exposure will be small, and systemic toxic effects on normal tissues will be minimized.
  • a method of treating a tumor in a host comprising administering to the tumor a nucleic acid sequence which encodes a first agent which renders the tumor cells sensitive to an interaction agent.
  • a second agent then is administered to the tumor.
  • the second agent provides for the inhibition, prevention, or destruction of the growth of the tumor cells.
  • the second agent is not an agent which renders tumor cells sensitive to an interaction agent.
  • the second agent enhances the therapeutic effect of the interaction agent by an effect independent of the immune system of the host.
  • the tumor then is treated with the interaction agent.
  • the nucleic acid sequence encoding the first agent is contained in an expression vehicle, which may be a viral vector such as those hereinabove described.
  • the viral vector is contained in a producer cell line, which is administered to the tumor to produce a virus in an amount effective to transform cells of the tumor.
  • the viral vector may be a retroviral vector as hereinabove described and may be administered in amounts hereinabove described.
  • the first agent is a negative selective marker, which may be selected from those hereinabove described, and may be administered in amounts hereinabove described.
  • the second agent is a cytokine, which also may be selected from those hereinabove described.
  • Example 1 Construction of pGlTkSvNa
  • This vector contains the Thymidine Kinase (hTK) gene from herpes simplex virus I regulated by the retroviral promoter and the bacterial gene, neomycin phosphotransferase (Neo R ) driven by an SV40 promoter.
  • hTK Thymidine Kinase
  • the hTK gene confers sensitivity to the DNA analogs acyclovir and ganciclovir, while the Neo R gene product confer resistance to the neomycin analogue, G418.
  • pGlTkSvNa To make pGlTkSvNa, a three step cloning strategy was used. First, the herpes simplex thymidine kinase gene (Tk) was cloned into the Gl plasmid backbone to produce pGlTk. Second, the Neo R gene (Na) was cloned into the plasmid pSvBg to make pSvNa. Finally, SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa.
  • Tk herpes simplex thymidine kinase gene
  • Na Neo R gene
  • SvNa was excised from pSvNa and ligated into pGlTk to produce pGlTkSvNa.
  • Plasmid pGlTkSvNa was derived from plasmid PGl ( Figure 3). Plasmid pGl was constructed from pLNSX (Palmer, et al., Blood, Vol. 73, pgs. 438-445), and incorporated herein by reference. The construction strategy for plasmid pGl is shown in Figure 1. The 1.6kb EcoRI fragment, containing the 5' Moloney Murine Sarcoma Virus (MoMuSV) LTR, and the 3.0kb EcoRl/Clal fragment, containing the 3' LTR, the bacterial origin of replication and the ampicillin resistance gene, were isolated separately.
  • MoMuSV Moloney Murine Sarcoma Virus
  • pGl Figure 3
  • pGl Figure 3
  • MCS 54 base pair multiple cloning site
  • the MCS was designed to generate a maximum number of unique insertion sites, based on a screen of non-cutting restriction enzymes of the pGl plasmid, the neo r gene, the ⁇ -galactosidase gene, the hygromycin r gene, and the SV40 promoter.
  • the structure of the 5' linker was as follows: 5' - 1/2 Ndel - Sphl - Notl - SnaBI - Sail - SacII - Accl - Nrul - Bglll - III 27 bp ribosomal binding signal - Kozak consensus sequence/Ncol - first 21 bp of the lacZ open reading frame - 1/2 BamHI - 3' .
  • the structure of the 3' linker was as follows: 5' - 1/2 mutated EcoRI - last 55 bp of the lacZ open reading frame - Xhol
  • the Kozak consensus sequence (5'-GCCGCCACCATGG-3' ) has been shown to signal initiation of mRNA translation (Kozak, Nucl.Acids Res. 12:857-872, 1984), incorporated herein by reference.
  • the Kozak consensus sequence includes the Ncol site that marks the ATG translation initiation codon..
  • pBR322 (Bolivar et al. , Gene. 2:95, 1977), incorporated herein by reference was digested with Ndel and EcoRI and the 2.1 kb fragment that contains the ampicillin resistance gene and the bacterial origin of replication was isolated.
  • pBg has utility as a shuttle plasmid because the lacZ gene can be excised and another gene inserted into any of the restriction sites that are present at the 5' and 3' ends of the lacZ gene. Because these restriction sites are reiterated in the pGl plasmid, the lacZ gene or genes that replace it in the shuttle plasmid construct can easily be moved into pGl.
  • a 1.74 kB Bglll/PvuII fragment containing the Herpes Simplex Virus Type I thymidine kinase gene (GenBank accession no. V00467, incorporated herein by reference) was excised from the pXl plasmid (Huberman, et al., Exptl. Cell Res. Vol. 153, pgs 347-362 (1984) incorporated herein by reference), blunted with the large (Klenow) fragment of DNA polymerase I, and inserted into the unique SnaBI site in the pGl multiple cloning site, to form plasmid pGlTK. ( Figure 5) .
  • a producer cell line was made from vector plasmid and packaging cells.
  • the PA317/GlTkSvNa producer cell was made by the same general techniques used to make previous clinically relevant retroviral vector producer cell lines.
  • the vector plasmid pGlTkSvNa DNA was transfected into a ecotropic packaging cell line, PE501. Supernatant from the PE501 transfected cells was then used to transinfect the amphotropic packaging cell line (PA317).
  • Clones of transinfected producer cells were then grown in G418 containing medium to select clones that contain the Neo R gene. The clones were then titered for retroviral vector production. Several clones were then selected for further testing and finally a clone was selected for clinical use.
  • PE501 cells 5 x 10 5 PE501 cells (Miller, et al. , Biotechniques, Vol. 7, pgs. 980-990 (1989), incorporated herein by reference) were plated in 100 mm dishes with 10 ml high glucose Dulbecco's Modified Essential Medium (DMEM) growth medium supplemented with 10% fetal bovine serum (HGD10) per dish. The cells were incubated at 37°C, in 5% C0 2 /air overnight. The plasmid pGlTKSvNa then was transfected into PE501 cells by CaP0 4 precipitation using 50 ⁇ g of DNA by the following procedure.
  • DMEM Dulbecco's Modified Essential Medium
  • HFD10 fetal bovine serum
  • a culture dish(es) with optimum precipitate following the overnight incubation then was (were) selected.
  • the dish(es) then was (were) washed again with PBS to remove the salt and the salt solution.
  • 10 ml of HGD10 medium then was added to the dish(es), and the dish(es) incubated at 37°C in a 5% C0 2 atmosphere for about 48 hrs.
  • PE501 ecotropic containing supernatants from such colonies of PE501 cells were collected in volumes of from about 5 to 10 ml, placed in cryotubes, and frozen in liquid nitrogen at -70°C.
  • PA317 cells (Miller et al. Mol. Cell. Biol. 6:2895- 2902 (1986)) then were plated at a density of 5 x 10 4 cells per 100 mm plate on Dulbecco's Modified Essential Medium (DMEM) including 4.5 g/1 glucose, glutamine supplement, and 10% fetal bovine serum (FBS).
  • DMEM Dulbecco's Modified Essential Medium
  • FBS fetal bovine serum
  • the PE501 supernatant then was thawed, and 8 ⁇ g/ml of polybrene was added to the supernatant.
  • the medium was aspirated from the plates of PA317 cells, and 7 to 8 ml of viral supernatant was added and incubated overnight.
  • the PE501 supernatant then was removed and the cells refed approximately 18-20 hours with fresh 10% FBS.
  • the medium was changed to 10% FBS and G418 (800 ⁇ g/ml).
  • the plate then was monitored, and the medium was changed to fresh 10% FBS and G418 to eliminate dying or dead cells as necessary.
  • the plate was monitored for at least 10 to 14 days for the appearance of G418 resistant colonies .
  • Cloning rings were placed around all selected colonies.
  • the cells were tyrpsinized and incubated into wells in a six well dish in 5 ml of HGD10 plus lx hypoxanthine aminopterin thymidine (HAT) .
  • HGD10 lx hypoxanthine aminopterin thymidine
  • clones grew to confluency, they were trypsinized and incubated in a 100 ml dish. As a clone in the 100 ml dish approached confluency, its amphotropic vector-containing supernatant was removed and centrifuged at 1,200 to 1,500 rpm for 5 minutes to pellet out cells.
  • DNA from the plasmid pGlNaSvTk was digested with restriction enzymes Bglll and Smal and the 1163 base pair (bp) Herpes thymidine kinase (TK) fragment was fractionated by agarose gel electrophoresis and isolated. This fragment contains 56 bp of the TK 5'-untranslated region and 1107 bp of the TK translation open reading frame.
  • the 1163 bp TK fragment was ligated to the plasmid vector pSP73 (Promega Corporation, Madison, WI) that had been digested with restriction enzymes Bglll and Smal.
  • pSPTK5' contains the 5 ' portion of the TK open reading frame but lacks the last 21 bp of the open reading frame and the translation termination codon.
  • the linearized pGlNaSvTK was used as a template for polymerase chain reaction (PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence .for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence .for an Xhol site, the TK translation termination codon, and the last 19 bp of the TK open reading frame (5'-PCR) using a forward primer that contains the first 17 bases of the TK open reading frame (5'-GCACCATGGCTTCGTACCCCTGC-3' ) and a reverse primer that contains complementary sequence .for an Xhol site, the TK translation termination codon, and the last
  • PCR products were fractionated on an agarose gel and the expected 1215 bp fragment that includes the full-length TK open reading frame was isolated. The isolated fragment was digested with restriction enzymes Pstl and Xhol, digestion products were fractionated on an agarose gel, and the 420 bp fragment was isolated.
  • pSPTKl pSPTK5 ' was digested with Pstl and the 3993 bp fragment that contains the pSP73 vector and the 5' portion of the TK open reading frame was isolated following agarose gel electophoresis. This 3993 bp fragment was ligated to the PCR-generated 420 bp Pstl/Xhol fragment that contains the 3' end of the TK open reading frame (above). Ligated plasmid DNA was transformed into E.
  • coli DK5 ⁇ competent cells Gibco/BRL, Gaithersburg, MD
  • DNA from ampicillin-resistant colonies was screened by restriction enzyme digestion. Plasmids that appeared to contain the full-length TK open reading frame were termed pSPTKl.
  • the DNA from several pSPTKl clones was dideoxy sequenced in the region from the Pstl site through the Xhol site (the region that was generated by PCR) .
  • pSPTKl clone #4 was found to match the expected TK sequence in this region and was used for construction of pGlTKlSvNa.
  • pGlTKlSvNa pSPTKl DNA was digested with Bglll and the 5' overhanging ends were repaired by incubation of the digested DNA with deoxy nucleotides and Klenow fragment of E. coli DNA polymerase I. The DNA was then digested with Xhol to generate a 1225 bp fragment that contains 56 bp of TK 5'-untranslated region and the full-length TK open reading frame. This blunt/XhoI fragment was ligated to pGlXSvNa DNA that had been digested with SnaBI and Sail.
  • pGlXSvNa the 1.2 kb SvNa fragment was excised from pSvNa (Part A above) with Sail and Hindlll. This fragment was ligated to pGl that had been digested with Sail and Hindlll.
  • the ligated plasmid was termed pGlXSvNa where the "X" denotes a multiple cloning region.
  • pGlTKlSvNa Plasmids that appeared to contain the TK fragment by diagnostic restriction enzyme digestion were termed pGlTKlSvNa.
  • Figure 8. Clone #2 was dideoxy sequenced from the beginning of the 5'-LTR through the end of the 3'-LTR and was found to contain the intact TK open reading frame.
  • pGlTKlSvNa was used to produce a producer cell by combination with PA317 by the hereinabove described method • (Part B above). Such producer cell line was designated as producer cell line PA317/GlSvNa.7.
  • pGl was cut with HindiII and Sail.
  • pSvNa ( Figure 9), which contains the SV40 promoter from pLNSX and the neo r gene from pN2, was also cut with HindiII and Sail, and a Hindlll-Sall fragment containing an SV40 promoter and a ⁇ - galactosidase gene was ligated into Hindlll/Sall digested pGl to form pGlXSvNa ( Figure 10).
  • pGlXSvNa was cut at the SnaBI site and a Bglll-Clal restriction fragment containing the Interleukin-2 leader sequence, and Kozak region, Interleukin-2 secretion signal added by oligomers; and the mature Interleukin-2 coding sequence from ATCC with the 3' untranslated region removed, was ligated into the cut pGlXSvNa to form pGH2SvNa. ( Figure 11) .
  • pGH2SvNa was used to produce a producer cell line by combination with PA317 by the method described in Part B hereinabove.
  • the producer cell line is sometimes hereinafter referred to as PA317/GlI2SvNa.5.
  • pGH2GSvNa was cut at the SnaBI site, and a Bglll- Hindlll restriction fragment containing the Interleukin-2 leader sequence, Kozak region, and a PCR-generated full coding sequence using the Roche Interleukin-2 gene as a template was ligated into the cut pGlXSvNa to form pGlI2GSvNa.ll.
  • Figure 12. pGH2GSvNa was used to produce a producer cell line by combination with PA317 by the method described in Part B hereinabove. The producer cell line is sometimes hereinafter referred to as PA317/GlI2GSvNa.
  • mice 68 athymic nude female mice were injected with 15-30 x 10 6 OVCAR-3 ovarian cancer cells in 1 or 1.5 ml of Hanks BSS solution into the peritoneal cavity. The mice then were divided into four groups. Group A included 19 mice and each mouse received 10 x 10 6 PA317/GlTKSvNa.53 producer cells intraperitoneally 7 days after the injection of the OVCAR-3 cells. Group B included 15 mice and each mouse received 10 x 10 6 PA317/GlI2SvNa.5 producer cells intraperitoneally 7 days after injection of the OVCAR-3 cells.
  • Group C included 18 mice and each mouse received intraperitoneal injections of 10 x 10 6 PA317/GlTKSvNa.53 producer cells and 10 x 10 6 PA317/GlI2SvNa.5 producer cells 7 days after the injection of the OVCAR-3 cells.
  • Group D included 16 mice and each mouse received intraperitoneal injections of 2 x 10 6 PA317/GlTKSvNa.53 producer cells and 2 x 10 6 PA317/GlI2SvNa producer cells 7 days after the injection of the OVCAR-3 cells.
  • mice in Group A received ganciclovir (GCV) in an amount of 5 mg/kg intraperitoneally and twice daily for 14 days. The other mice received no treatment. All the mice then were evaluated for tumor growth. The number of tumor-free mice in each group is given in Table I below. TABLE I Group Mice free of tumors
  • mice Thirty C57BL black 6-8 week female mice each received a subcutaneous injection of 2 x 10 5 transduced MCA205 fibrosarcoma cells. The mice were divided into 3 groups with 10 mice in each group.
  • Group I received MCA205 cells transduced with vector particles generated from pGlTKSvNa.
  • Group II received MCA205 cells transduced with vector particles generated from pGH2SvNa.
  • Group III received 1 x 10 5 MCA205 cells transduced with vector particles generated from pGlTKSvNa and 1 x 10 s MCA205 cells transduced with pGH2SvNa.
  • the average size of the tumors transduced with the Interleukin-2 gene were 32% smaller than the tumors transduced with the Herpes Simplex thymidine kinase gene. All animals that received ganciclovir therapy had complete resolution of their tumors . The animals that received both Herpes Simplex thymidine kinase and Interleukin-2 transduced tumors had significantly decreased times to complete tumor resolution. Thus, the two genes in combination induced more rapid tumor destruction with ganciclovir therapy.
  • cytokines such as Interleukin-2 may activate expression of endogenous thymidine kinase genes in the tumor cells.
  • the induction would have to be surprisingly large because the endogenous enzyme is unable to use ganciclovir as a substrate significantly, if at all.
  • Human OVCAR-3 cells were transduced with vector particles generated from either pGlTKSvNa or pGH2SvNa, and selected in 1.0 mg/ml G418 for 7-14 days. Cells surviving the G418 selection were used as the transduced tumor cells in this example.
  • OVCAR-3 cells were added to the wells of a microtiter plate at 10,000 cells/well in a total volume of 100 ⁇ l of RPMI1640 with 10% fetal calf serum. Each well had a different proportion (in percent) of wild type OVCAR- 3 cells; OVCAR-3 cells transduced with vector particles generated from pGlTKSvNa; and OVCAR-3 cells transduced with vector particles generated from pGH2SvNa.
  • 100 ⁇ l of ganciclovir was added to each well at a concentration of 2.5 ⁇ g/ml, 5.1 ⁇ g/ml or 12.5 ⁇ g/ml.
  • the cells were cultured for 44 to 48 hours in an incubator at 37°C with 5% C0 2 .
  • 0.5 ⁇ Ci of 3 H-thymidine then was added to each well in a volume of 20 ⁇ l into each well of the plate.
  • the cells were harvested and radioactivity was measured as counts per minute (cpm).
  • the cpm is directly proportional to the proliferation rate in this assay.
  • the percent decrease in proliferation was measured for each well. The results are given in Table III below.
  • Interleukin-2 and ganciclovir induce a bystander effect in this assay that is nearly comparable to the Herpes Simplex thymidine kinase effect alone.
  • the bystander effect is potentiated.
  • the in vivo synergy between the Herpes Simplex thymidine kinase and Interleukin-2 may not be simply an enhancement of the immune response, but may include an unexplained mechanism whereby Interleukin-2 directly or indirectly affects the anti-tumor response to ganciclovir. Such mechanism does not include an activity of the immune system.
  • Example 4 The procedure of Example 3 was repeated with respect to A375 melanoma cells. The percent decrease in proliferation of cells in each well containing various proportions of wild-type and transduced cells is given in Table IV below.
  • Example 5 The procedure of Example 3 was repeated with respect to 786-0 renal carcinoma cells. The percent decrease in proliferation of cells in each well containing various proportions of wild-type and transduced cells is given in Table V below.
  • each patient begins the treatment cycle.
  • the cycle is begun by administering to each patient PA317/GlTKlSvNa.7 producer cells and PA317/GlI2GSvNa.11 producer cells through the Tenckhoff catheter over a period of 4 hours.
  • the total volume of fluid administered is between 1 and 4 liters and the producer cells are administered as a 50:50 mixture of cells at a concentration of from about 2x10 6 cells/ml to about lOxlO 6 cells/ml.
  • the total number of producer cells administered per treatment cycle is about lxlO 10 cells.
  • each patient receives an intravenous dose of 5 mg/kg of ganciclovir daily for 14 days. After the 14-day period of ganciclovir treatment, the patients receive no treatment for 7 days to end the treatment cycle. At the end of the 7-day period without ganciclovir treatment, the treatment cycle is repeated.

Abstract

L'invention concerne un procédé pour traiter une tumeur dans un organisme hôte. Ce procédé consiste à administrer à l'organisme hôte au moins un véhicule d'expression comprenant une première séquence d'acides nucléiques codant un agent qui rend les cellules tumorales sensibles à un agent d'interaction, et une deuxième séquence d'acides nucléiques qui code un agent qui assure l'inhibition, la prévention ou la destruction de la croissance des cellules tumorales, ce dernier agent ne rendant pas les cellules tumorales sensibles à un agent d'interaction. La tumeur est ensuite traitée par l'agent d'interaction. De préférence, la première séquence d'acides nucléiques code un marqueur sélectif négatif (comme la thymidine kinase du virus Herpes simplex), et la deuxième séquence d'acides nucléiques code une cytokine (comme l'Interleukine-2), et les première et deuxième séquences d'acides nucléiques sont contenues dans des premier et deuxième vecteurs viraux, et sont eux-mêmes contenus dans une première lignée cellulaire productrice et une deuxième ligne cellulaire productrice, respectivement. Les lignées cellulaires productrices sont administrées à la tumeur, et, de ce fait, les particules virales générées par les lignées cellulaires productrices infectent les cellules tumorales. Les cellules tumorales sont tuées lors de l'administration de l'agent d'interaction, tel que le ganciclovir, à l'organisme hôte.
PCT/US1994/011251 1993-10-06 1994-10-04 Traitement de tumeurs par la transformation genetique des cellules tumorales a l'aide de genes codants des marqueurs selectifs negatifs et des cytokines WO1995009655A1 (fr)

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EP94930031A EP0722343A4 (fr) 1993-10-06 1994-10-04 Traitement de tumeurs par la transformation genetique des cellules tumorales a l'aide de genes codants des marqueurs selectifs negatifs et des cytokines

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0719147A1 (fr) * 1993-08-26 1996-07-03 Baylor College Of Medicine Therapie genique pour tumeurs solides, papillomes et verrues
WO1997032991A2 (fr) * 1996-03-06 1997-09-12 Avigen, Inc. Therapie genique pour le traitement de tumeurs solides, utilisant des vecteurs de virus adeno-associes recombines
GB2355460A (en) * 1999-10-21 2001-04-25 Univ Manchester Gene Therapy
WO2001040503A2 (fr) * 1999-12-06 2001-06-07 Axxima Pharmaceuticals Ag Methode d"identification et de quantification des inhibiteurs de kinase
WO2002041922A1 (fr) * 2000-11-24 2002-05-30 Chugai Seiyaku Kabushiki Kaisha Procede servant a reguler l'activite de l'expression d'un produit genetique transfere dans un organisme vivant
US7354592B2 (en) 1997-09-10 2008-04-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US7452531B2 (en) 1999-10-04 2008-11-18 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
US7514089B2 (en) 1997-09-10 2009-04-07 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
EP2045275A2 (fr) 1998-02-23 2009-04-08 Sumitomo Bakelite Co., Ltd. Compositions de réserve polycyclique dotées d'une résistance à la gravure améliorée
US8420611B2 (en) 2004-08-12 2013-04-16 Cedars-Sinai Medical Center Combined gene therapy for the treatment of macroscopic gliomas
US8883493B2 (en) 2007-01-30 2014-11-11 Cedars-Sinai Medical Center Adenoviral vector comprising herpes simplex virus type 1 thymidine kinase and a transgene for increasing the expression of the transgene
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0476953A2 (fr) * 1990-09-14 1992-03-25 The General Hospital Corporation Destruction butée des cellules néoplastiques à l'acide de virus
WO1993004167A1 (fr) * 1991-08-13 1993-03-04 The Regents Of The University Of California Transfert de gene in vivo a l'aide de cellules implantees produisant un retrovirus

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2091346C (fr) * 1990-09-14 2007-04-24 Philip Frost Methodes et compositions pour la therapie genetique et la potentialisation de l'immunite antitumorale
CA2113990A1 (fr) * 1991-07-26 1993-02-18 Frederick L. Moolten Traitement anticancereux utilisant des cellules malignes
WO1993010218A1 (fr) * 1991-11-14 1993-05-27 The United States Government As Represented By The Secretary Of The Department Of Health And Human Services Vecteurs comprenant des genes etrangers et des marqueurs selectifs negatifs
JPH07506370A (ja) * 1992-05-01 1995-07-13 アメリカ合衆国 バイスタンダー作用による殺腫瘍療法

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0476953A2 (fr) * 1990-09-14 1992-03-25 The General Hospital Corporation Destruction butée des cellules néoplastiques à l'acide de virus
WO1993004167A1 (fr) * 1991-08-13 1993-03-04 The Regents Of The University Of California Transfert de gene in vivo a l'aide de cellules implantees produisant un retrovirus

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 53, issued 01 January 1993, RAM et al., "In Situ Retroviral-Mediated Gene Transfer for the Treatment of Brain Tumors in Rats", pages 83-88. *
J. EXP. MED., Volume 172, issued October 1990, GANSBACHER et al., "Interleukin 2 Gene Transfer into Tumor Cells Abrogates Tumorigenicity and Induces Protective Immunity", pages 1217-1224. *
J. EXP. MED., Volume 177, issued May 1993, MINASI et al., "The Selective Ablation of Interleukin 2-producing Cells Isolated from Transgenic Mice", pages 1451-1459. *
PROC. NATL. ACAD. SCI. USA., Volume 85, issued October 1988, BORRELLI et al., "Targeting of an Inducible Toxic Phenotype in Animal Cells", pages 7572-7576. *
SCIENCE, Volume 254, issued 01 November 1991, GOLUMBEK et al., "Treatment of Established Renal Cancer by Tumor Cells Engineered to Secrete Interleukin-4", pages 713-716. *
SCIENCE, Volume 256, issued 12 June 1992, CULVER et al., "In Vivo Gene Transfer with Retroviral Vector-Producer Cells for Treatment of Experimental Brain Tumors", pages 1550-1552. *
See also references of EP0722343A4 *

Cited By (21)

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EP0719147A4 (fr) * 1993-08-26 1998-09-16 Baylor College Medicine Therapie genique pour tumeurs solides, papillomes et verrues
US6217860B1 (en) 1993-08-26 2001-04-17 Baylor College Of Medicine Gene therapy for solid tumors, papillomas and warts
EP0719147A1 (fr) * 1993-08-26 1996-07-03 Baylor College Of Medicine Therapie genique pour tumeurs solides, papillomes et verrues
US6531456B1 (en) 1996-03-06 2003-03-11 Avigen, Inc. Gene therapy for the treatment of solid tumors using recombinant adeno-associated virus vectors
WO1997032991A2 (fr) * 1996-03-06 1997-09-12 Avigen, Inc. Therapie genique pour le traitement de tumeurs solides, utilisant des vecteurs de virus adeno-associes recombines
WO1997032991A3 (fr) * 1996-03-06 1998-02-05 Avigen Inc Therapie genique pour le traitement de tumeurs solides, utilisant des vecteurs de virus adeno-associes recombines
US5952221A (en) * 1996-03-06 1999-09-14 Avigen, Inc. Adeno-associated virus vectors comprising a first and second nucleic acid sequence
US6218180B1 (en) 1996-03-06 2001-04-17 Avigen, Inc. Gene therapy for the treatment of solid tumors using recombinant adeno-associated virus vectors
US7514089B2 (en) 1997-09-10 2009-04-07 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
US7354592B2 (en) 1997-09-10 2008-04-08 Vion Pharmaceuticals, Inc. Genetically modified tumor-targeted bacteria with reduced virulence
EP2045275A2 (fr) 1998-02-23 2009-04-08 Sumitomo Bakelite Co., Ltd. Compositions de réserve polycyclique dotées d'une résistance à la gravure améliorée
US7452531B2 (en) 1999-10-04 2008-11-18 Vion Pharmaceuticals, Inc. Compositions and methods for tumor-targeted delivery of effector molecules
GB2355460A (en) * 1999-10-21 2001-04-25 Univ Manchester Gene Therapy
US8460932B2 (en) 1999-10-21 2013-06-11 Cedars-Sinai Medical Center Method of treating a disorder by suicide gene therapy
WO2001040503A3 (fr) * 1999-12-06 2002-07-18 Axxima Pharmaceuticals Ag Methode d"identification et de quantification des inhibiteurs de kinase
WO2001040503A2 (fr) * 1999-12-06 2001-06-07 Axxima Pharmaceuticals Ag Methode d"identification et de quantification des inhibiteurs de kinase
WO2002041922A1 (fr) * 2000-11-24 2002-05-30 Chugai Seiyaku Kabushiki Kaisha Procede servant a reguler l'activite de l'expression d'un produit genetique transfere dans un organisme vivant
US8420611B2 (en) 2004-08-12 2013-04-16 Cedars-Sinai Medical Center Combined gene therapy for the treatment of macroscopic gliomas
US8883493B2 (en) 2007-01-30 2014-11-11 Cedars-Sinai Medical Center Adenoviral vector comprising herpes simplex virus type 1 thymidine kinase and a transgene for increasing the expression of the transgene
US10857233B1 (en) 2010-02-09 2020-12-08 David Gordon Bermudes Protease inhibitor combination with therapeutic proteins including antibodies
US11180535B1 (en) 2016-12-07 2021-11-23 David Gordon Bermudes Saccharide binding, tumor penetration, and cytotoxic antitumor chimeric peptides from therapeutic bacteria

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EP0722343A1 (fr) 1996-07-24
CA2173495A1 (fr) 1995-04-13
JPH09504518A (ja) 1997-05-06
EP0722343A4 (fr) 1999-12-29

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