EP0689604A1 - Procede de preparation de vaccins anticancereux - Google Patents

Procede de preparation de vaccins anticancereux

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Publication number
EP0689604A1
EP0689604A1 EP94911921A EP94911921A EP0689604A1 EP 0689604 A1 EP0689604 A1 EP 0689604A1 EP 94911921 A EP94911921 A EP 94911921A EP 94911921 A EP94911921 A EP 94911921A EP 0689604 A1 EP0689604 A1 EP 0689604A1
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EP
European Patent Office
Prior art keywords
dna
cells
adenovirus
transfected
virus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP94911921A
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German (de)
English (en)
Inventor
Max L. Birnstiel
Michael Buschle
Matthew Cotten
Gerhard Maas
Christian Plank
Gotthold Schaffner
Walter Schmidt
Ernst Wagner
Kurt Zatloukal
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Boehringer Ingelheim International GmbH
Original Assignee
Boehringer Ingelheim International GmbH
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Priority claimed from AT0055693A external-priority patent/AT399656B/de
Priority claimed from DE4326821A external-priority patent/DE4326821A1/de
Application filed by Boehringer Ingelheim International GmbH filed Critical Boehringer Ingelheim International GmbH
Publication of EP0689604A1 publication Critical patent/EP0689604A1/fr
Withdrawn legal-status Critical Current

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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • C07—ORGANIC CHEMISTRY
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    • 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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    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • A—HUMAN NECESSITIES
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10361—Methods of inactivation or attenuation
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/20011—Rhabdoviridae
    • C12N2760/20111—Lyssavirus, e.g. rabies virus
    • C12N2760/20122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention relates to gene therapy
  • the immune system has a big one
  • Cancer cells can be observed in early stages of the tumor. In advanced stages one often finds the situation that the cancer cells of the tumor.
  • Cancer vaccines contain cancer cells that are genetically modified or cancer cells in combination with immunostimulating adjuvants that induce a cancer-specific immune response in the patient or
  • MHC-I or MHC-II antigens or by expression of viral proteins such as hemagglutinin (Itaya et al., 1987; Fearon et al., 1988; Plaksin et al., 1988 and Ostrand-Rosenberg et al., 1990) .
  • Interferons are used to activate the immune system so that it recognizes the tumor cells as foreign and rejects them.
  • Stimulation of T cells requires both activation of the T cell receptor and activation of a second receptor on the T cell by a molecule on the surface of the antigen presenting cell (Jenkins and Johnson, 1993).
  • the B7 / CD28 pair represents such a unit of co-stimulating molecules
  • HSA heat stable antigen
  • the aim of the cancer-specific immune response brought about by the transfection of the tumor cells with a gene from one of these groups is that, after removal of the tumor, micrometastases, which are still clinically undetectable and cannot be surgically removed, are destroyed in order to prevent recurrence To prevent cancer.
  • therapy based on cancer vaccines represents active-specific immunotherapy with vaccines from inactivated tumor cells or parts of them, if possible the patient's own, thereby reducing the immune system of the
  • Patients are specifically mobilized against antigens of the individual tumor or at least the same tumor type.
  • cancer vaccines consisting of viable tumor cells have recently been developed (Rosenberg et al., 1992). However, for safety reasons, a vaccine based on cells that are no longer divisible and have a limited lifespan is desirable.
  • One of the critical steps in the manufacture of cancer vaccines is gene transfer to the cells that make up the vaccine or a component thereof.
  • retroviruses can only transduce dividing cells.
  • vectors as are the alternative to the retroviral system
  • adenoviruses for the gene transfer with DNA complexes by means of receptor-mediated endocytosis has been proposed in several works due to the ability of these viruses to release the content of endosomes.
  • the use of adenoviruses increases the use of adenoviruses.
  • adenovirus-polylysine conjugates can be complexed with DNA together with conjugates of transferrin-polylysine, whereby ternary transferrin-polylysine / adenovirus-polylysine / DNA complexes arise (Wagner et al., 1992).
  • the complexes bind
  • the adenovirus breaks open endosomes, which results in the release of the material from the endosome into the cytoplasm.
  • the DNA can then enter the nucleus where the gene is expressed, mostly from episomally localized DNA.
  • This technique has compared to conventional viral and non- Viral gene transfer methods have the following advantages: In this context, since the adenovirus only acts as a means for releasing the transfection complexes from the endosome, the virus can be inactivated with the aid of genetic and / or chemical, possibly in combination with physical methods, which increases safety compared to conventional ones viral techniques increased (Cotten et al., 1992). Furthermore, that
  • Transferrin and adenovirus conjugates can thus be taken up via transferrin and / or adenovirus receptors.
  • transferrin other ligands, specific for certain cell populations, can also be used, e.g. melanoma cells LDL proved to be very suitable. It can be done with the help of this
  • Such peptides which are also referred to as "endosomolytic” or “fusogenic” peptides, have also been used to bring about an increase in gene expression during gene transfer by means of receptor-mediated endocytosis.
  • endosomolytic or “fusogenic” peptides
  • Replication defect of the virus which can be attributed to a defect in the E1A region, could of the
  • transfected cells are partially bypassed, so the defect is "leaky". Furthermore, the yield of virus in the packaging cell lines available to complement the defects of Ad5 dl312 was not satisfactory.
  • the object of the present invention was to develop a method for producing cancer vaccines
  • the invention thus relates to a method for
  • the method is characterized in that tumor cells or fibroblasts are cultivated and the cultivated cells are transfected ex vivo with a composition which contains the following components: ai) a DNA molecule which contains one or more sequences which can be expressed in the cell and which are suitable for one or several, the same or different,
  • Encoded polypeptide b) a conjugate between a DNA-binding molecule and an endosomolytic agent,
  • adenovirus which has a mutation at least in the E4 region
  • adenovirus which, in addition to an effect in the E1A region, has one or more further genetic defects, or
  • Cells binds and is internalized into these, components b) and c) forming an essentially electroneutral complex with the DNA defined in a) in such a way that the transfected cells are so
  • Transfection of fibroblasts mixes these with non-transfected and inactivated tumor cells, and that the cell population may be mixed with
  • the cancer vaccines obtainable by the method according to the invention contain inactivated tumor cells which, surprisingly, despite the
  • Autologous tumor cells possibly in a mixture with fibroblasts, serve as the starting material for tumor vaccines, which are produced individually for each individual patient.
  • the fibroblasts can also be autologous cells, but it is also possible to use cells from a fibroblast cell line, whereby the
  • tumor cells and / or fibroblasts are isolated from tissue samples of the individual to be treated.
  • the methods for this are known to the person skilled in the art.
  • Primary melanoma cells can e.g. B. the easiest
  • Lymph node metastases can be isolated by the
  • Conditions are dissociated mechanically and optionally additionally enzymatically.
  • Isolation from primary tumors is generally more difficult, especially in the case of smaller tumors.
  • the procedure is such that the tumors are surgically removed, mechanically crushed and additionally enzymatically dissociated.
  • Dissociation which can be followed, apply collagenase, DNAse and hyaluronidase.
  • the isolation from other tumors can be done according to the same principle, depending on the surrounding tissue, the methods for isolation and dissociation are varied.
  • methods known from the literature can be used, e.g. the specialist book "Human Cancer in Primary Culture", ed. John R.W. Masters, 1991.
  • the gene constructs are primary instead of in cancer cells
  • Fibroblasts are introduced, which are then mixed with the non-transfected irradiated cancer cells that carry tumor antigens (Fakhrai et al., 1992).
  • the advantage of this embodiment is that
  • Fibroblasts can be easily and in large quantities obtained from the patient, which is particularly important when there are smaller tumors and thus a smaller number of tumor cells, and that the
  • Gene transfer into autologous primary fibroblasts can be standardized more easily than transfection into primary cancer cell isolates from different patients.
  • Another advantage of this embodiment is that the tumor cells do not have to be cultivated over a long period of time, so that their antigenic spectrum, which can be lost in part due to the cultivation, is retained. This also avoids the disadvantage associated with the cultivation of tumor cells that individual populations, for example fibroblasts, that are present in the tumor isolate are contained in a small proportion, or tumor cell clones overgrow the culture.
  • Fibroblasts are made using known methods
  • Transfection medium containing the complexes with components a) to c) treated.
  • simultaneous administration of conjugate b) and the complex between DNA and internalizing factor conjugate is preferred.
  • the complexes can also be used repeatedly.
  • the inactivation of the tumor cells or the fibroblasts can be carried out using methods known per se, e.g. physical methods, such as treatment by means of X-ray or gamma radiation, and / or by means of chemical treatment with mitotic inhibitors, e.g. mitomycin C.
  • mitotic inhibitors e.g. mitomycin C.
  • the suitable inactivation dose can be determined, for example, by incorporating 3 H-thymidine into the cells or their cells at different radiation dosages or concentrations of the chemical inactivating agent and / or different treatment times Proliferation rate and on the other hand the expression of the foreign gene is determined.
  • the transfected fibroblasts are preferably also inactivated. With this measure, one acquired during transfection and / or culture
  • cryoprotectants e.g. Dimethyl sulfoxide (DMSO), switched on as an intermediate step.
  • DMSO Dimethyl sulfoxide
  • the freezing step can be carried out at any point in the process
  • the cancer vaccines produced in vitro by the method according to the invention are administered to the patient
  • Trigger or reactivate immune response Trigger or reactivate immune response.
  • the amount of tumor cells per immunization is in the order of 10 5 - 10 7 cells.
  • the number of added fibroblasts is approximately in the same range, but can optionally be reduced by up to approximately 1/100 of the cell number.
  • cancer vaccines these cells are detectable at the injection site. It was also examined whether these cells were distributed in the blood or in different organs. The detection was carried out using polymerase chain reactions.
  • the cancer vaccine was processed, the cells were transfected with interleukin-2 plasmid DNA. With specific primers, IL-2 and adenovirus DNA fragments were used as markers for the
  • the invention relates in a further aspect
  • Transfection complexes consisting of DNA, containing one or more sequences coding for an immunostimulating polypeptide, a DNA-binding molecule, which preferably has an internalization factor for
  • Tumor cells and / or fibroblasts is conjugated, in particular polylysine, and an endosomolytic conjugate of a DNA-binding molecule and the above-defined adenovirus or a peptide, wherein the DNA-binding molecule c) and the DNA-binding part of the conjugate b) to the DNA are bound.
  • complexes according to the invention an efficient gene transfer into primary human melanoma cells and primary human
  • Fibroblasts can be transfected
  • the DNA molecule is a plasmid which contains a sequence coding for an immunostimulating polypeptide, for example a cytokine, in expressible form.
  • immunostimulating also includes the property of the so-called co-stimulatory molecules, such as B7 (Schwartz, 1992; Townsend and Allison, 1993) or adhesion molecules, for example HSAs, which reinforce the immune response ("heat stable antigens", Kay et al., 1990) or ICAM (Springer et al., 1987); further include immunostimulating
  • Substances also foreign antigens (so-called "neo-antigens"), e.g. viral antigens.
  • the sequence coding for the immunostimulating polypeptide is related to regulatory sequences that are as high as possible
  • Enable expression of the immunomodulatory polypeptide in the target cells Preferably be strong
  • Promoters such as the CMV promoter (Boshart et al., 1985) or the ⁇ -actin promoter (Gunning et al., 1987)
  • the suitable construct can be determined in preliminary tests by comparing the expression values.
  • IL-2 interleukin 2
  • DNA sequences encoding other cytokines such as IL-4, IL-12, IFN- ⁇ , TNF- ⁇ , GM-CSF can also be used (Pardoll, 1992). It can too
  • Combinations of cytokine sequences can be used to enhance the immunostimulating effect, e.g. IL-2 + IFN- ⁇ , IL-2 + IL-4, IL-2 + TNF- ⁇ or TNF- ⁇ + IFN- ⁇ .
  • sequences coding for two different cytokines are preferably present on separate plasmids. This can be done, for example, in the manner according to the invention Experiments based on IL-2 and IFN- ⁇ have been shown to fine-tune cytokine expression by varying the proportions of the two plasmids.
  • a DNA molecule which contains one or more sequence (s) coding for a stimulatory molecule.
  • the co-stimulatory molecule is the heat-stable antigen (Heat Stahle Antigen HSA).
  • HSA Heat-stable antigen
  • IL-2 coding DNA
  • IL-2 coding DNA
  • a DNA molecule which contains one or more sequence (s) coding for a neoantigen.
  • the neoantigen is one
  • Virus protein or a fragment thereof for example the Rabies glycoprotein.
  • a DNA containing the sequence coding for the virus protein can be used either alone or in combination with one for a cytokine,
  • IL-2 encoding DNA preferably IL-2 encoding DNA can be used.
  • the gene transfer system used has proven to be suitable for constructs with a size of 48 kb (Cotten et al., 1992).
  • the DNA coding for an immunostimulating polypeptide may be therapeutic
  • DNA Purity requirements with regard to which they are therapeutically effective DNA must only meet the condition that it contains no sequence that codes for a functionally active in the cell polypeptide.
  • the size of this DNA is also not critical, in general it is expedient that it is in the range of the order of magnitude of the gene therapy-active DNA molecule or is smaller than this.
  • this filler DNA can replace portions of the therapeutically active DNA of different sizes. This has the
  • the therapeutic DNA dose and thus the amount of cytokine expressed in the cell can be varied without having to change the other parameters, which is within the scope of a standardized
  • Turmorvakzin manufacturing process is of great interest.
  • the plasmid DNA used is free of lipopolysaccharides. It was surprisingly found that the expression values are increased considerably if the plasmid DNA is largely free of lipopolysaccharides. These represent a common one
  • the DNA can either be purified by suitable methods, e.g. by a combination of chromatographic methods including polymyxin chromatography, or it can, too
  • the transfection medium Lipopolysaccharide binding reagents such as polymyxin can be added.
  • the target cell is receptors for the adenovirus
  • adenovirus conjugates may suffice as component c) a DNA conjugate which is not conjugated with another internalization factor binding
  • Insert molecule in general, it is the same molecule as that contained in conjugate b), polylysine is preferably used.
  • the adenovirus itself has the function of the internalization factor, in this case it is not necessary to add another internalization factor to the DNA-binding substance
  • component c) primarily has the function of a
  • a non-conjugated DNA binding molecule as component c) can be omitted entirely.
  • the DNA-binding substance defined as component c) is conjugated with an internalization factor.
  • Embodiment of the present invention is used above all if, in the case of using an adenovirus conjugate, the target cell has no or only a few receptors for the adenovirus, for example in Use of a virus of a distant species, or if a peptide conjugate is used as the endosomolytic conjugate b). In the presence of another
  • Virus receptor or by binding to both receptors via the pathway of receptor-mediated endocytosis.
  • the viruses are released from the endosomes, the DNA contained in the complexes is also released into the cytoplasm and thereby escapes lysosomal degradation.
  • the conjugates preferably contained as c) between an internalization factor and a DNA-binding substance are known per se.
  • ligands or fragments thereof which are internalized after binding to a tumor cell or a fibroblast via endocytosis, preferably receptor-mediated endocytosis, or
  • internalization factors are the ligands transferrin (Klausner et al., 1983), asialoglycoproteins (such as asialotransferrin, asialorosomucoid or
  • Asialofetuin Asialofetuin
  • Lectine Goldstein et al., 1980; Sharon, 1987
  • mannosylated glycoproteins (Stahl et al., 1978), lysosomal enzymes (Sly et al., 1982), LDL (Goldstein et al., 1982), modified LDL (Goldstein et al., 1979), lipoproteins that are receptors in the Cell are taken up (apo B100 / LDL); viral proteins;
  • EGF Epidermal Growth Factor
  • TGF Factor Heldin et al., 1982
  • TGF ⁇ Massague et al., 1986
  • TGFß Transforming Growth Factors " ⁇ , ⁇ ”
  • HGF Hepatocyte Growth Factor (Nakamura et al., 1989)
  • nerve growth factor Hosang et al., 1987
  • Insulin-like growth factor I Insulin-like Growth factor
  • Transferrin and EGF are preferred as internalization factors in the context of the present invention.
  • Suitable DNA-binding substances as component c) are e.g. homologous organic
  • Polycations such as polylysine, polyarginine, polyornithine or heterologous polycations with two or more
  • these polycations can have different chain lengths, furthermore non-peptide synthetic
  • DNA-binding substances are also natural DNA-binding proteins of a polycationic character, such as histones or protamines or analogs or fragments thereof, and also spermine or spermidines.
  • the length of the polycation is not critical as long as the complexes are essentially electroneutral. If the DNA consists of 6,000 bp and 12,000 negative charges, the amount of polycation per mole of DNA can e.g. his:
  • the average specialist can use simple
  • Routine experiments select other combinations of polycation length and molar amount.
  • Polylysine is preferably used in the context of the present invention, in particular with a chain length of approximately 200 to 300 lysines.
  • the DNA-binding molecule modified in this way can be used as component c) for the sake of simplicity.
  • Component c) takes over.
  • the internalizing factor-polycation conjugates can be produced chemically or, if the polycation is a polypeptide, by a recombinant route, with regard to the production methods reference is made to the disclosure of EP 388 758.
  • the conjugates can also be prepared according to the method described by Wagner et al., 1991b, by using a glycoprotein, e.g. Transferrin, and the DNA-binding molecule, in particular polylysine, via one or more carbohydrate chains of the glycoprotein with one another
  • a glycoprotein e.g. Transferrin
  • the DNA-binding molecule in particular polylysine
  • An adenovirus conjugate preferably contains b)
  • Adenovirus with a defect in the E4 region was developed by Bridge and Ketner, 1989,
  • the gene product of the open reading frame ORF 6/7 has a very important function.
  • This protein probably binds the cellular transcription factor E2F, making it a highly specific adenoviral transcription factor. It was
  • Gene transfer system based on receptor-mediated endocytosis in which the adenovirus acts as an agent that disrupts the endosomes, has low toxicity when applied to tumor cells, and at the same time has high gene transport efficiency.
  • mutant d11014 which has an intact ORF 4, but in which ORF 3 and ORF 6 and ORF 6/7 are defective, was selected on the basis of its property that it is strongest in the synthesis of viral proteins is impaired. Since the functions of the virus proteins encoded in E4 have not yet been fully elucidated, it is currently not possible to define in which reading frame the mutations must be set in order to achieve the desired effect. Other mutants besides mutant d11014 can be determined empirically by producing E4 mutants as described by Bridge and Ketner and testing them in standardized transfection and cytotoxicity experiments as described in the examples. For the orienting transfection tests, a reporter gene can first be used instead of the cytokine gene.
  • Mutants which have comparable gene expression and cytotoxicity values as d11014 or d11014 are even superior are suitable in the context of the present invention as constituents of the transfection complex, provided they also meet the requirement that the viruses, such as d11014 in the cell line W162, in one the E4 defect
  • complementing cell line can be grown to high titers.
  • an adenovirus with an E4 defect it is also possible to use an adenovirus with a defect in the E1a region which, in addition to the Ela defect, has one or more further gene defects which are set using chemical, chemical / physical or genetic methods was (n).
  • the defects set with the help of chemical or physical / chemical methods are not targeted defects, but can be scattered over the entire virus genome.
  • dl312 an adenovirus mutant called dl312 (Jones and Shenk, 1979), which had been inactivated with 8-methoxypsoralen / UV, was shown to be suitable for the production of cancer vaccines.
  • Psoralen derivatives are used, e.g. 4'-aminomethyl-4, 5 ', 8-trimethylpsoralen, which has been found to be suitable for inactivating adenoviruses in view of their use in gene transfer.
  • a virus is advantageously used that has been inactivated if possible by a standardizable method. The requirement for the
  • Standardizability is mainly due to chemical
  • Inactivation methods fulfilled, which are superior to the chemical / physical methods in this regard.
  • An example of a chemical inactivation method is inactivation with ⁇ -propiolactone (Morgeaux et al., 1993; De Shu et al., 1986; Budowsky and Zalesskaya, 1991).
  • Standardizability is also advantageous. It has been found within the scope of the present invention that the treatment of adenovirus with two aliquots
  • ß-propiolactone causes a decrease in virus titer by 5 log for 4 h at room temperature, which is due to the inactivation achieved with 8-methoxypsoralen is comparable. DNA transport activity is maintained at medium doses of ß-propiolactone (0.3%), while treatment with 1% ß-propiolactone causes this ability to decrease significantly.
  • Analysis of the gene expression from the inactivated virus showed that both the psoralen derivatives and ⁇ -propiolactone block the virus gene expression (E1a and E3) to the same extent.
  • the more sensitive plaque assay showed that psoralen treatment inactivated the virus by more than 7 log, with no plaque observed at the highest virus doses.
  • ß-propiolactone inactivation only called for a drop in
  • Virus titer by 5 log with plaques being observed at higher virus doses.
  • a virus is therefore used which is inactivated using exclusively chemical methods, preferably using ⁇ -propiolactone.
  • ⁇ -propiolactone are illustrated can be determined.
  • the efficiency of virus inactivation by determining the virus titer and / or by means of the
  • Inactivation method takes effect, i.e. which sections can be destroyed can be done with DNA probes
  • Inactivation methods are tested to see if they can block virus replication and transcription functions.
  • Inactivation method when it delivers adenovirus particles that have the endosomolytic activity useful for the DNA transport function that a function of
  • CPE cytopathic endpoint assay
  • Psoralene derivatives completely replicated the virus, so that no plaques were detectable by means of this test.
  • RNA analysis for the detection of the gene expression was carried out either with an adenovirus 5 Ela probe, which recognizes a part of the Ela gene, which is deleted in the adenovirus d1312, or an adenovirus 5 E3 probe, which recognizes a part of the E3 region that codes for the abundant E3 19K glycoprotein (E1a serves as a control; an RNA signal should be missing for the virus dl312, but should be detectable for d11014, since this has a wild type E1 region).
  • E1a serves as a control; an RNA signal should be missing for the virus dl312, but should be detectable for d11014, since this has a wild type E1 region).
  • transfected cells could play because at least two of the E3 genes modulate the surface expression of MHC class I molecules and TNF receptor molecules on the surface of the infected cells.
  • a defect in the E3 region is therefore desirable for the use of the virus dll014 in gene therapy applications in which immunogenic tumor cells are produced, since the expression of this region in this context could interfere with the immune response to transfected cells.
  • polylysine-coupled (or ionically bound) endosomolytic agent is preferably used as part of a ternary or combination complex.
  • Adenovirus can be coupled to polylysine
  • the coupling of virus by chemical means can be carried out in a manner known per se for the coupling of peptides, wherein, if necessary, the individual components are provided with linker substances before the coupling reaction (this measure is necessary if by
  • linker substances are bifunctional compounds that start with
  • a virus has suitable carbohydrate chains, it can contain the DNA-binding substance via one or more
  • Carbohydrate chains of the glycoprotein are connected (Wagner et al., 1991b).
  • virus-polylysine conjugates Another method for producing the virus-polylysine conjugates is the enzymatic coupling of the virus to a DNA-binding substance, in particular a polyamine, by means of a transglutaminase (Zatloukal et al., 1992).
  • Another method for producing the adenovirus-polylysine conjugates is to couple the virus to the polycation via a biotin-protein bridge, preferably a biotin-streptavidin bridge
  • binding to biotin can also take place via avidin.
  • polyclonal or monoclonal antibodies against biotin can be used.
  • the link between the virus and polylysine can also be made by adding polylysine to a lectin is coupled, which has affinity for a virus surface glycoprotein, wherein the binding in such a conjugate takes place via the binding between the lectin and the glycoprotein. If the virus does not have suitable carbohydrate side chains, it can be modified accordingly.
  • the virus can, in case it is on its
  • Surface proteins has regions that are acidic and can therefore bind to a polycation, also ionically bound to the DNA-binding molecule.
  • Conjugates can also be a peptide that is covalently or by ionic binding to a DNA-binding molecule. Regarding the requirements of such
  • Peptides are made and their coupling to the DNA-binding molecule is referred to WO 93/07283.
  • Expression levels for IL-2 can be achieved in melanoma cells. Melanoma cells present in the presence of this
  • Peptide conjugates transfected with transferrin-polylysine conjugates with IL-2 plasmid DNA proved to be cancer vaccines with a prophylactic protective effect against tumor formation.
  • the DNA-binding substance is determined according to the type and amount that the Complexation of the DNA guaranteed, the complexes obtained are preferably essentially
  • Components a), b) and c) must be taken into account that complexation of the DNA takes place and it is ensured that the complex formed is bound to the cell, transported into the cell and that it is released from the endosomes.
  • the chosen internalization factor-conjugate / DNA ratio depends primarily on the size of the polycation molecules and on the number and distribution of the positively charged groups, criteria based on the size and structure of the DNA to be transported
  • the molar ratio of internalization factor: polylysine is preferably approximately 10: 1 to approximately 1:10.
  • the amount of the conjugate portion which is determined by DNA can be determined by titrations after determining the conjugate: DNA ratio which is optimal for the efficiency of transfection and expression -binding substance is replaceable.
  • Polylysine is preferred both as component c), preferably conjugated to a
  • a more suitable method for determining the ratio of the components contained in the complexes is to first import the one into the cell
  • the complexes can be prepared by the
  • Components a), b) and c), which are each in the form of dilute solutions, are mixed.
  • the optimal ratio of DNA to conjugate and non-conjugated DNA-binding substance is determined by
  • the DNA complexes can be produced at physiological salt concentrations. Another possibility is the use of high salt concentrations (about 2 M NaCl) and subsequent adjustment to physiological conditions by slow dilution or dialysis.
  • the amount of conjugate is matched to the respective cell type, especially the
  • a further criterion is the respective internalization factor conjugate, in particular with regard to the internalization factor for which the target cell has a defined number of receptors.
  • the amount of endosomolytic conjugate depends on the amount of DNA to be imported.
  • the optimal concentration of endosomolytic conjugate is determined by titration in preliminary tests with the target cells intended for the transfection and the vector system intended for the transfection, whereby a gene construct is expediently used as DNA, the size of which corresponds to that for the specific one Application largely matches, and for the purpose of simpler
  • the invention relates to those transfected with the complexes according to the invention
  • Tumor cells or fibroblasts Tumor cells or fibroblasts.
  • Fibroblasts mixed with tumor cells in a pharmaceutically acceptable formulation Fibroblasts mixed with tumor cells in a pharmaceutically acceptable formulation.
  • the ready-to-use cancer vaccines are preferably in the form of a suspension, which if necessary
  • Trypsinization is obtained.
  • the cells are suspended in a physiological medium
  • physiological saline or buffer solution which may contain those nutrients, in particular
  • FCS fetal calf serum
  • cell culture media with additives such as calf serum, human serum, human serum albumin and / or hydroxyethyl cellulose ensure the viability of a sufficient number of cells for the vaccine.
  • the tumor vaccines are expediently in serum of blood group AB, and autologous serum can also be used as the medium.
  • the medium may contain additions of growth factors or cytokines such as IFN-gamma or GM- CSF to favorably affect cell antigen presentation.
  • the type of cancer vaccine which can be provided with the aid of the present invention represents a new development in the field of cytokine therapy. It has the advantage that the serious side effects
  • Cancers are treated with the aid of the cancer vaccine according to the invention, e.g. Kidney cancer or breast cancer.
  • Fig. 1 gene transport in primary human
  • Fig. 2 Determination of the ligands involved in the uptake of gene transfer complexes.
  • Fig. 5 Effect of irradiation of tumor cells on the
  • FIG. 7 Influence of the plasmid concentration on the
  • Fig. 8 Loss of tumorigenicity of transfected
  • EGF as a ligand for gene transfer in cells of a human epidermoid carcinoma cell line (A: plus 2% FCS, B: without FCS)
  • Fig. 11 IL-2 expression in mouse melanoma cells under
  • Fig. 12 IL-2 expression in fibroblasts below
  • Fig. 13 Stimulation of an immune response by
  • Fig. 16 Titration of 4'-aminomethyl-4, 5 ', 8-trimethylpsoralen
  • Figure 18 Gene transport activity of adenovirus d11014
  • Methoxypsoralen in the virus genome Fig. 24 Growth of human melanoma cells as a function of the gamma radiation dose
  • Fig. 28 Interleukin-2 expression in human
  • Fig. 29 Influence of the endotoxin content of the DNA on the
  • Plasmid constructs i) DNA plasmid constructs containing the sequence coding for human or mouse IL-2
  • pWS2m a plasmid called pWS2m which contains the mouse IL-2 gene under the control of the cytomegalovirus enhancer / promoter:
  • the plasmid pH ⁇ APr-1 (Gunning et al., 1987) was cut with BamHI and EcoRI.
  • BamHI and EcoRI By means of agarose gel cleaning, a 2.5 kb fragment isolated which contains the ampicillin resistance gene and the origin of replication of pBR322 and the SV40 polyadenylation signal. This fragment was ligated to the CMV promoter / enhancer, which is a 0.7 kb PCR fragment from the vector pAD-CMVI
  • the plasmid obtained was called pWS.
  • the cDNA coding for mouse IL-2 was generated as a PCR fragment from the plasmid BMGneo-mIL-2
  • the vector pWS2 is used, which is obtained in a similar manner to the vector pWS2m, with the difference that, as a template for the PCR amplification, instead of the plasmid BMGneo-mIL-2, the plasmid pIL2-50A (Taniguchi et al., 1983), which contains the cDNA coding for human IL-2.
  • the vector pGShIL-2tet was produced, which contains the tetracycline resistance gene, cut out from pBR327.
  • mice IL-2 sequence For the plasmid pCM2, the mouse IL-2 sequence together with the 5 'and 3' flanking regions (cleavage and poly (A) signals from rabbit-ß-globingen) were used as
  • DNA plasmid construct containing the sequence coding for mouse IFN- ⁇ The plasmid pSVEmuIFN- ⁇ described by Gray and Goeddel, 1983 was used.
  • DNA-plasmid construct pHSA containing the sequence coding for HSA
  • the plasmid pHSA which contains the HSA sequence driven by the CMV promoter, was obtained by cloning the HSA cDNA (Kay et al., 1990) into the BstXI site of the plasmid pCDM8 (Seed, 1987).
  • DNA plasmid construct pWS-RABIES containing the sequence coding for the Rabies glycoprotein
  • the sequence coding for the Rabies glycoprotein was cloned from the vector pKSV-10 (Pharmacia), which contains the Rabies glycoprotein cDNA (Anilionis et al., 1982) into the Bgl II site down from the SV40 promoter as Bgl II Fragment isolated.
  • the vector pWS2m was cut with Bgl II and BamHI.
  • the fragment carrying the murine IL-2 cDNA was separated by agarose gel electrophoresis and the fragment corresponding to the vector pWS was isolated and ligated to the Rabies fragment.
  • the correct Rabies orientation was confirmed by sequencing.
  • the Rabies glycoprotein sequence is as
  • the vector pWS2 described in i) was cut with SalI and BamHI and the fragment thus released, coding for IL-2, was separated by agarose gel electrophoresis.
  • the DNA coding for murine GM-CSF was synthesized from 12 mutually overlapping Oligonucleotides produced.
  • the sequence used corresponded to the sequence described by Miyatake et al., 1985 (coding region from position 32 to position 457), with the difference between silent exchanges in position 178 (A instead of G), position 274 (C instead of G) and position 355 (C instead of G).
  • the AGC triplet was replaced by GTC.
  • the 5 'area was provided with a Sall-compatible overhang, the 3' area with a BamHI-compatible overhang.
  • the 5 'end was provided with a GCCGCC sequence analogous to the IL-2 sequence in pWS2.
  • the synthetic GM-CSF gene was cloned into the vector pWS2 and sequenced using the "shot-gun" method (Sambrook, 1989). The means
  • An IL-2 cassette containing the CMV enhancer / promoter, the sequence coding for IL-2 and the SV40-PolyA sequence was obtained by means of PCR using the vector pWS2 described in i).
  • the PCR product was subjected to restriction enzyme digestion with EcoRI and into the EcoRI / Smal site of the plasmid pUC19
  • the plasmid obtained was named pGShIL-2.
  • the plasmid pBR327 (Soberon et al., 1980), which had been digested with Sspl and Aval, served as the source for the tetracycline resistance gene and parts of the "upstream" region of the ⁇ -lactanase gene (ampicillin resistance gene) .
  • EcoRI / Aval adapter the isolated tet sequence was inserted into the
  • the plasmid pCMV was prepared by removing the BamHI insert of the plasmid pSTCX556 (Severne et al., 1988), treating the plasmid with Klenow fragment and the HindiII / Sspl and Klenow-treated fragment from the plasmid pRSVL (containing the Photinus pyralis
  • Luciferase gene under the control of the Rous Sarcoma Virus LTR enhancer / promoter (Uchida et al., 1977,
  • EGF Extracellular Growth Factor
  • HBS Hemtrachlorose
  • etrachlorose Hemtrachlorose
  • HBS Hetrachlorose
  • elution buffer 1 mg EGF (Epidermal Growth Factor, Sigma, St. Louis, Cat. No. E-4127) was purified by gel filtration (Sephadex G-10) with HBS as the elution buffer.
  • the modified protein was gel filtered at room temperature through a Sephadex G-10 column, whereby 70 nmol EGF, modified with 50 nmol dithiopyridine linker, was obtained.
  • the modified protein was treated with 3-mercaptopropionate-modified polylysine (50 nmol, average chain length 290 lysine monomers,
  • Dithiopyridine linker was obtained.
  • the modified protein was modified with 3-mercaptopropionate
  • Virion concentration was extracted by UV spectrophotometric analysis
  • genomic virus DNA performed (formula: a
  • optical density unit (OD, A260) corresponds to 10 12
  • the adenovirus d11014 described by Bridge and Ketner, 1989, was in the cell line W162, which is derived from the cell line Vero (ATCC No. CCL81) and their
  • Total volume 3.2 ml (Total volume 3.2 ml) and stored at -25oC.
  • biotinylation of the virus could be demonstrated in a qualitative detection after drops of different dilutions on cellulose nitrate membrane: after
  • the biotinylation of adenovirus d11014 was based on 15 ml adenovirus preparation (1.2 ⁇ 10 12 particles per ml), which were treated with 150 ⁇ l, 1 mM NHS-LC-biotin. After 3 hours at room temperature, dialysis was carried out against 1 1 HBS plus 40% glycerol at 4 ° C overnight, then the buffer was replaced with fresh one and a second dialysis was carried out. A virus preparation with a titer of approximately 1.2 ⁇ 10 12 particles per ml was obtained. iv) Inactivation of biotinylated adenovirus with 8-methoxypsoralen
  • the first step was biotinylated
  • Plasmid DNA in 150 ⁇ l HBS was added, mixed well and incubated for a further 30 min.
  • polylysine-modified human transferrin (TfpL) in 150 ⁇ l HBS was added, mixed thoroughly and incubated for 30 min.
  • TfpL polylysine-modified human transferrin
  • mouse melanoma cell line Cloudman S91 (clone M3) was purchased from ATCC (No. CCL 53.1). The cells were grown in 6 cm plastic dishes or T25 culture bottles coated with 0.1% gelatin in Ham's F10 medium containing 12.5% horse serum, 2.5% FCS, 2 mM glutamine and antibiotics. ii) mouse fibroblasts
  • Tissue fragments were allowed to settle for 5 minutes and the supernatant containing the released cells was mixed with 5 ml of DMEM containing 20% FCS. The undissociated material was digested for an additional 30 minutes and the supernatant collected as described above.
  • the combined supernatants were 15 min Centrifuged at 500 xg, the cell pellet in DMEM, containing 20% FCS, resuspended and the rows sown in culture bottles. After 30 minutes, the non-adhered cells were aspirated and fresh medium
  • tissue fragments were carefully pushed through a metal sieve with the plunger of a syringe. The material was then run through several times
  • Antibiotics penicillin / streptamycin or, in the case of human fibroblasts, gentamycin are given.
  • the biopsies were extensively crushed in a tissue culture facility using tweezers and a surgical knife in a laminar air flow in sterile 6 cm plastic dishes. Then were
  • Antibiotics were added and the culture was placed in a 37oC incubator. After 10 days, the medium was replaced with DMEM containing 10% FCS. Then the medium was changed twice a week. 4 weeks after the start of the culture, the cells which were from the Tissue fragments had grown out, trypsinized and plated out in new culture dishes for transfection.
  • An alternative, preferred method was to transfer the pieces of skin into fresh medium after comminution and to wash them with medium once or twice as required. 5 to 10 pieces of tissue were placed in a T25 tissue culture bottle, the surface of which had been wetted with DMEM plus 10% FCS, and distributed evenly, whereupon the bottle was turned over. This caused the biopsies to hang ("hanging drop
  • the KB human epidermoid carcinoma cell line was purchased from ATCC (No. CCL 17). The cells were 6 cm
  • Luciferase activity was performed as by Zenke et al., 1990, Cotten et al., 1990, or in EP 388 758. ii) IL-2 assay
  • interleukin-2 The expression of interleukin-2 was determined using a bioassay as described by Karasuyama and Melchers, 1988. In addition, IL-2 production was carried out using the Becton Dickinson IL-2 ELISA kit (catalog No. 30032) according to the manufacturer's instructions. iii) IFN- ⁇ assay
  • Virus preparations carried out. These experiments have shown that the results obtained with d1312 in this regard can be transferred to psoralen / UV-inactivated adenovirus d1312 and to (psoralen / UV-inactivated) adenovirus d11014. In some experiments, d1312 was used as a representative. example 1
  • HMM-1 and HMM-2 were transfected at different time intervals from the start of culture with complexes which contained the following components: 1.7 x 10 10 adenovirus particles d1312, 100 ng StreptpL, 6 ⁇ g pCMVL-DNA, 7 ⁇ g TfpL .
  • the expression of luciferase was determined 24 hours after the transfection. Expression was based on the protein content of the cell lysates on 1 x 10 6 cells
  • HMM-1 Primary human melanoma cell isolates of the designation HMM-1 were treated with 1.7 x 10 10 adenovirus particles d1312,
  • Transfection complexes were prepared in which the TfpL was replaced by non-conjugated pL (pL). The result of these tests is shown in Fig. 2. It was found that the addition of free transferrin reduced the amount of luciferase expression, which was indicated indicates that the complexes are taken up, at least in part, by binding to the transferrin receptor. On the other hand, however, the still significant gene expression seen in the presence of free transferrin suggests that binding to the adenovirus receptors is also greater
  • HMM-1 primary human melanoma cells per 6 cm petri dish were transfected with complexes consisting of 1.2 x 10 10 adenovirus particles d1312, 1,200 ng streptpL, 6 ⁇ g pCMVL and 6.6 ⁇ g TfpL or
  • Adenovirus particles d1312 (d1312 PI), 600 ng StreptpL, 6 ⁇ g pCMVL and 6.6 ⁇ g TfpL (the optimal amount of
  • Luciferase expression measured per 3 x 10 5 cells.
  • the effect of 8-methoxypsoralen / UV inactivation on gene expression is shown in FIG. 3. 7 days after the transfection there was a sharp decrease in
  • Fibroblasts derived from malignant melanoma were transfected with combination complexes containing either adenovirus d1312, 8-methoxypsoralen / UV-inactivated adenovirus d1312 or adenovirus d11014.
  • the cells were transfected with complexes which contained the following components: 6 ⁇ g pCMVL-DNA,
  • Virus particles per ml 100 ⁇ l aliquots of this mixture or, in order to obtain the virus: cell ratios shown in FIG. 4, a 1: 3 serial dilution of this mixture were applied to 3 ⁇ 10 4 cells per well of a cell culture plate (24 wells per plate) in 500 ⁇ l RPMI + 2% FCS applied. After 2 hours at 37 ° C the medium was replaced with 2 ml RPMI + 10% FCS. After 8 days, the medium was removed and the cells were fixed with 4% formaldehyde and 150 mM NaCl for 5 minutes and then stained with 0.1% crystal violet in 2% ethanol for 10 minutes. The cells were then washed twice with PBS, once with water and photographed. The result of the cytotoxicity test is shown in FIG. 4: With a standard virus: cell ratio of 10,000: 1, adenovirus dl312 is cytotoxic; this cytotoxicity is mitigated by psoralen / UV treatment of the virus.
  • Luciferase expression in light units per ⁇ g protein in the cell lysate was assessed for Luciferase expression in light units per ⁇ g protein in the cell lysate.
  • M-3 mouse melanoma cells (3 x 10 5 cells per 6 cm
  • Petri dish were transfected with 2 x 10 9 adenovirus particles d1312, 250 ng StreptpL, 6 ⁇ g plasmid DNA (containing the mouse IL-2 or the mouse IFN- ⁇ sequence) and 7.5 ⁇ g TfpL. 4 h after the transfection, the cells were washed twice with Ham 's F10 culture medium without serum.
  • proliferradiated tumor cells (“prophylactic mouse model”) a) M-3 melanoma cells (3 x 10 5 cells per T25
  • mice were immunized with 1 x 10 5 cells administered subcutaneously in the back. In parallel, a group of 6 mice were immunized with non-transfected M-3 cells, which were irradiated and further
  • mice had been treated like the transfected cells.
  • the mice received booster immunizations with cell preparations as used for the first immunizations.
  • the animals were exposed to the highly tumorigenic dose of 1 x 10 5 M-3 cells, which was applied at a location which was remote from the previous immunization sites.
  • 4 mice that had not been immunized were exposed to the tumorigenic cells in the same way. 8 weeks after tumor cell implantation, all (4/4) non-immunized animals developed tumors, while all mice immunized with the irradiated, IL-2 transfected M-3 cells were free of tumors (0 tumors in 5 animals) .
  • mice irradiated, non-transfected M-3 cells and irradiated M-3 cells that had been transfected with an "empty" plasmid (pSP) were only partially protected (4 out of 6 mice developed tumors).
  • the development of the tumors in the animals is summarized in Table II and in FIG. 9. The results obtained show that the immunization of mice with IL-2 transfected, irradiated tumor cells induces a systemic immune response that the animals before
  • mice were treated according to the same immunization protocol with irradiated M-3 cells that had been transfected with complexes containing 3 ⁇ 10 9 8-methoxypsoralen / UV-inactivated adenovirus particles d11014, 600 ng streptpL, a) 6 ⁇ g IL-2 plasmid pBCMGneo-mIL-2 (IL-2 100%), b) 5.4 ⁇ g pSP plasmid and 0.6 ⁇ g IL-2 plasmid (IL-2 10%), c) 5.4 ⁇ g IL-2 plasmid and 0.6 ⁇ g IFN - ⁇ plasmid
  • mice (IL-2 90% + IFN- ⁇ 10%) or d) 5.4 ⁇ g pSP plasmid and 0.6 ⁇ g IFN- ⁇ plasmid (IFN- ⁇ 10%), and 4.7 ⁇ g TfpL contained.
  • a group of mice was immunized with irradiated M-3 cells that had been transfected with 3.6 ⁇ 10 9 inactivated
  • Adenovirus particles d1312, 600 ng StreptpL, 6 ug IL-2 plasmid and 4.7 ug TfpL (IL-2 100% d1312).
  • 1 ⁇ 10 5 M-3 cells were implanted in the immunized animals and, as a control, also in the non-immunized animals.
  • the development of the tumors is summarized in Table III.
  • M-3 cells transfected with 100% IL-2 plasmid produced 33,000 units of IL-2 per 1 x 10 6 cells per 24 hours, while M-3 cells transfected with 4% IL-2 plasmid produced 396 units IL-2 produced per 1 x 10 6 cells and 24 h. A week after the
  • Booster injections were made in the immunized animals and, 1 x 10 ⁇ M-3 cells were also implanted in the non-immunized animals for control purposes.
  • further groups of mice were implanted with 3 ⁇ 10 5 M-3 cells and 1 ⁇ 10 6 M-3 cells . To show that the immune response
  • mice which had been immunized with 100% IL-2 transfected, irradiated M-3 cells were given 1 x 10 5 syngeneic
  • Squamous cell carcinoma cells KLN 205 (ATCC No. CRL 1453) implanted. The development of the tumors is shown in Table IV.
  • KB cells were grown at a density of 400,000 cells per 6 cm dish and transfected with various complexes containing 2.5 x 10 9 adenovirus particles d1312, 200 ng streptpL, 6 ⁇ g pCMVL-DNA and optionally 3.8 ⁇ g polylysine, 3.8 ⁇ EGF-pL ( Amount based on polylysine content), or 6 ⁇ g TfpL contained.
  • the complexes containing 2.5 x 10 9 adenovirus particles d1312, 200 ng streptpL, 6 ⁇ g pCMVL-DNA and optionally 3.8 ⁇ g polylysine, 3.8 ⁇ EGF-pL ( Amount based on polylysine content), or 6 ⁇ g TfpL contained.
  • the complexes containing 2.5 x 10 9 adenovirus particles d1312, 200 ng streptpL, 6 ⁇ g pCMVL-DNA and optionally 3.8 ⁇ g polylysine, 3.8 ⁇ E
  • FIG. 10A A value of 20,845,000 light units was obtained with EGFpL; this Value was significantly higher than with TfpL (3970000) or polylysine (plys 10550000).
  • TfpL TfpL + EGF 12350000 light units
  • polylysine pLys + EGF 14055000
  • the expression values refer to 50% of the cells.
  • KB cells were transfected with complexes which inactivated 5 ⁇ 10 9 8-methoxypsoralen / UV
  • Plasmid constructs which were present as a component of ternary complexes, were transfected. 3 ul adenovirus d1312
  • transfection complexes were used for the experiments with 8-methoxypsoralen / UV-inactivated adenovirus d11014 used: 9 ⁇ l virus preparation, 600 ng StreptpL, 6 ⁇ g TfpL, 6 ⁇ g BCMGneo-mIL-2.
  • the IL-2 activity was determined after the time intervals indicated in FIG. BCMGneo-mIL-2 also became 64,000 units after 48 hours and after
  • IL-2 constructs were transfected with IL-2 constructs as in a) and the IL-2 expression was determined on the days after the transfection shown in FIG. 12.
  • the composition of the transfection medium was the same for BCMGneo-mIL-2 as for M-3 cells; an identical transfection complex was made for BMGneo-mIL-2.
  • M3 cells were in Ham's F12 medium plus
  • Vaccine used within 60 min for injection into recipient mice.
  • mice were injected as described in the previous examples. There were two separate vaccine injections with each one week apart
  • mice 100,000 cells carried out in two different places on the back.
  • the mice were injected with 300,000 unmodified, non-irradiated M3 cells in 300 ⁇ l HBSS at a third location on the back.
  • the development of the tumors was checked at weekly intervals.
  • Fig. 13 shows the result of these experiments (the number of days is shown on the abscissa, the tumor size in mmX on the ordinate. The numbers next to the endpoints mean the number of mice with tumors compared to the total number of mice in each group.
  • pSP 6 ⁇ g pSP; 80 IL-2/20 pSP: 4.8 ⁇ g pWS2m plus
  • Tumor growth rate Considerable tumor masses were formed in 4 out of 6 mice.
  • the IL-2 producing cells caused only a moderate decrease in tumors in all mice in the group (3/3) that developed tumors.
  • mice that had been vaccinated with HSA-expressing M3 cells showed a strongly suppressed tumor growth.
  • the mice vaccinated with both HSA and IL-2 were administered at a reduced dose of IL-2 DNA.
  • expressing M3 cells developed tumors at a significantly reduced frequency (2 out of 6), and the tumors formed were small compared to the control tumors (vaccination with pSP-transfected cells); the average size was 18 mm 3 compared to 1149 mm 3 .
  • Example 11 M3 cells were treated and transfected as described in Example 11. Only the exposure time of the transfection complexes, which were produced in the same way as in Example 11 (6 ⁇ g plasmid DNA) on the cells, was four instead of two hours. The injections into the mice were also analogous to Example 11
  • FIG. 14 shows the result of the tests, the structure of the graphic is kept analogous to FIG. 13.
  • pSP negative control shows the expected, significant tumor growth.
  • Psoralen was removed by gel filtration: for this, the virus / psoralen sample (2 ml) was applied to a pharmacia
  • Inactivation performance is achieved when psoralens are used at concentrations close to saturation, while much better performances are achieved at lower concentrations. This could either be due to a crystallization phenomenon that made up the compound removed the solution, or on a filter effect
  • the CPE assay ("Cytopathic Endpoint Assay" or
  • Fig. 15 The numbers on the left in the figure refer to the relative virus titer (filled triangles), the numbers on the right in the figure refer to luciferase light units (open
  • the concentration of 8-methoxypsoralen in mg / ml is plotted on the abscissa. It was shown that treatment of the virus with 0.11 mg / ml 8-methoxypsoralen causes a reduction in the titer which is no different from that caused in preliminary tests with a concentration of 0.33 mg / ml.
  • the DNA transport activity is at this concentration
  • RNA viruses as well as DNA viruses and is 5 mg / ml soluble in aqueous solutions. The used
  • Luciferase light units (open squares). The concentration of 4'-aminomethyl-4,5 ', 8-trimethylpsoralen in ⁇ g / ml is plotted on the abscissa.
  • the virus samples were adjusted to 0.3 M HEPES, pH 7.9
  • ⁇ -propiolactone solutions were added. Concentrated ⁇ -propiolactone solutions were prepared by
  • ⁇ -propiolactone (Sigma, Catalog No. P5648) was diluted with HBS immediately before use. There were
  • virus samples were incubated for 4 hours at room temperature before either being stored at -70 ° C or for the
  • the adenoviruses were exposed to various concentrations of ⁇ -propiolactone at room temperature for 4 hours
  • ß-propiolactone suggested that a modification of viral nucleic acids occurs preferentially at the lower concentration, whereas the agent admits the capsid proteins at the higher concentrations modify and begin to damage the endosomolytic activity of the virus.
  • the adenoviruses were treated with several aliquots of lower ( ⁇ 0.3%) ß-propiolactone concentrations in the hope that one would be preferred
  • Sample 3 Inactivation with 0.3% ⁇ -propiolactone.
  • Sample 4 Inactivation with 2 x 0.3% ß-propiolactone.
  • Sample 5 Inactivation with 3 x 0.2% ⁇ -propiolactone.
  • Sample 6 Inactivation with 4 x 0.15% ß-propiolactone.
  • Sample 7 Inactivation with 1%
  • Luciferase light units D) Determination of the replication ability of inactivated adenoviruses by means of a plaque assay i) Comparison of the plaque assays of adenovirus d11014, inactivated with 8-methoxypsoralen, ⁇ -propiolactone or 4'-aminomethyl-4, 5 ', 8-trimethylpsoralen The plaque assay is used for a more sensitive determination of the replication ability of the virus: Adenovirus 5 requires the penetration of 10 to 50 virus particles in order to generate an infected cell.
  • the CPE assay shown below measures the ability of chemically inactivated viruses to trigger a cytopathic viral infection, but this requires infection of at least 10% of the target population to be detected during the four days that the assay lasts.
  • This assay thus allows the detection of
  • the plaque assay can be used to detect a single plaque that has arisen as a result of the penetration of 10 to 50 viruses; the test is therefore approximately 1,000 times more sensitive than the CPE assay.
  • Virus preparations by means of a plaque assay showed that the ⁇ -propiolactone treatment (2 x 0.3%) caused a decrease in the virus titer by about 5 log (FIG. 19: sample 1: non-activated virus, sample 2: inactivation with 0.33 ⁇ g / ml 8-methoxypsoralen, sample 3: inactivation with 0.11 ⁇ g / ml 8-methoxypsoralen, sample 4: inactivation with 2 x 0.3% ß-propiolactone, sample 5: inactivation with 0.28 mg / ml 4'-aminomethyl-4,5 ' , 8-trimethylpsoralen, sample 6:
  • Psoralen inactivated samples show that less than 10 2 plaque forming units (pfu's) are present in the psoralen inactivated samples.
  • the An important observation is that ß-propiolactone-inactivated viruses form plaques with a demonstrable frequency. So if the ß-propiolactone inactivation in the CPE assay appears to be equivalent to the inactivation of psoralen - in the plaque assay there is a clear one
  • the adenovirus d11014 preparations were made with
  • Example 1 non-activated virus
  • Sample 2 inactivation with 0.11 ⁇ g / ml 8-methoxypsoralen
  • Sample 3 inactivation with 0.3% ß-propiolactone
  • Sample 4 inactivation with 2 x 0.3% ß-propiolactone
  • Sample 5 inactivation with 3 x 0.2% ß-propiolactone
  • sample 6 inactivation with 4 x 0.15% ß-propiolactone
  • sample 7 inactivation with 1%
  • the complexes contained approx. 1 x 10 10 virus particles, 800 ng streptavidin-polylysine, 6 ⁇ g pCMV-DNA and 5.2 ⁇ g
  • the complexes were applied to K562 cells (24 h pre-grown in 50 ⁇ M deferrioxamine / RPMI / 10% FCS, plated at 250,000 cells / ml) at 2 ml per well of a 24-well plate. After two hours
  • RNA Northern analysis was carried out according to the method described by Paeratakul et al., 1988: 48 hours after the transfection, the cells were washed 3 times in HBS and serial dilutions corresponding to 30,000, 10,000 or 3,000 cells were applied to a nitrocellulose filter using a 96 Samples of dot blot device
  • the filters were then washed twice in 2X SSC / 0.1% SDS for 30 minutes at 65 ° C, each followed by 30 minutes in 0.1X SSC, 0.1% SDS.
  • the radioactive pattern was visualized by phosphor imaging.
  • the radioactively labeled ( 32 P) probes were prepared from PCR products from adenovirus d11014 sequences.
  • An Ela probe (383 bp) was prepared using PCR primers for Ad5, bp 736-751 (Ela.l) and for bp 1119-1101 (E1a.2). (This sequence is part of the region deleted in d1312. It serves as
  • RNA signal should be missing in the d1312 samples, but should be contained in the d11014 samples because the latter virus has a wild-type El region.
  • An E3 probe (436 bp) was derived from the most expressed E3 gene, the 19 K glycoprotein gene (Gooding, 1992) using primers made to Ad5, bp 28722-28737 (E3.a) and 29157-29142 (E3.b) (Die
  • E3 should play an important role in the immune response to transfected cells, because at least two of the E3 genes modulate the surface expression of MHC class I molecules and TNF receptor molecules on the surface of infected cells.
  • the gel fragment was then heated to 95 ° C for 10 minutes and 10 ⁇ l of the resulting solution was added for 12 hours
  • Fig. 21 shows the autoradiogram: it was found that there was no Ela expression in the d1312 samples, both in the non-inactivated and in the 8-methoxypsoralen inactivated.
  • d11014 viruses strong, but completely absent both in the 8-methoxypsoralen-treated and in the viruses inactivated twice with 0.3% ß-propiolactone.
  • E3 is less expressed in non-inactivated d1312 adenoviruses than in d11014. This finding is in line with the knowledge of Ela's role as positive
  • RNA sample washed once with 80% ethanol and dissolved in 20 ⁇ l RNase-free water. Contaminating DNA contained in the RNA sample was removed by digestion with RNase-free DNase (Boehringer Mannheim; 60 min at 37 ° C). The DNase was then inactivated by further incubation at 95 ° C for 5 minutes.
  • RNase-free DNase Boehringer Mannheim
  • the reverse transcription reaction mixtures contained 10 ⁇ l RNA solution, 2 ⁇ l 10 mM dNTPs, 4 ⁇ l 25 mM MgCl 2 , 2 ⁇ l 10 X RT buffer (100 mM Tris-HCl, 900 mM KCl, pH 8.3), 1 ⁇ l 50 ⁇ M oligo d (D) 16 .
  • Reverse transcriptase reactions were carried out at 25 ° C for 10 minutes and at 42 ° C for 15 minutes, followed by 5 minutes at 95 ° C to inactivate the reverse transcriptase.
  • 3H 8-methoxypsoralen was determined by counting aliquots of the purified virus in scintillation fluid. Calculations based on the measured
  • Radioactivity built into the DNA showed that there was one psoralen molecule per 800 base pairs of the virus.
  • the virus DNA was purified and cleaved using restriction enzymes in order to obtain ten or eleven DNA fragments: from the 3 H 8-methoxypsoralen-labeled virus the DNA was purified by incubating the virus with 0.4% SDS / 0.4 mg / ml Proteinase K at 56 ° C for 45 min. The sample was taken twice
  • the DNA from the aqueous phase was precipitated with 0.54 volumes of isopropanol after addition of 1/10 volume of 3M sodium acetate, pH 5.
  • the precipitated DNA was centrifuged, washed twice with ice-cold 80% ethanol, air-dried and dissolved in TE.
  • DNA aliquots of non-inactivated adenovirus or of 3 H 8-methoxypsoralen-labeled adenovirus were with the restriction enzymes Hindill or Asp718 according to the manufacturer's instructions (Boehringer Mannheim), but with 10fafh digested higher amounts of enzyme, purified by phenol / chloroform and chloroform extraction, with ethanol
  • the cells were trypsinized, one bottle from each group, centrifuged, the pellet recorded, 100 ⁇ l of cell suspension mixed with trypan blue and counted in the counting chamber. The remaining cells were mixed with new medium and placed in a new culture bottle (1st passage). The process was repeated a week later; One bottle from each group was trypsinized, counted and the 1st passage was counted as a control. The non-irradiated and 5 Gy irradiated cells showed a clear one
  • Melanoma cell cultures were chosen as the safe radiation dose for all experiments with a dose of 100 Gy.
  • PCR polymerase chain reaction
  • M3 melanoma cells were transfected with recombinant mouse interleukin-2 plasmid using the adenovirus transferrin method as described in Example 8 c). After 24 h the cytokine production was determined (20,000-50,000 units of IL-2 in 24 h per
  • the cells were trypsinized in washed serum-free medium and adjusted to a concentration of 3 x 10 5 or 1 x 10 6 cells per 100 ⁇ l isotonic saline solution. 100 ⁇ l of cell suspension were injected subcutaneously into the right flank of DBA / 2 mice using a cannula (see also Examples 7 and 8).
  • PMBZs peripheral mononuclear blood cells
  • the blood mixed with citrate buffer (10 mM) was then separated by a Ficoll gradient and the PMBZs were digested with Proteinase K buffer. DNA purification was done as described above.
  • the reaction mixture was composed of 1 ⁇ g DNA, 1 ⁇ PCR buffer (Boehringer Mannheim), 1 mM
  • PCR reaction times were: 5 minutes at 95 ° C for denaturation, then 40 cycles of 30 seconds 94 ° C, 30 seconds 60 ° C, 2 minutes 72 ° C. After that, the Amplification products mixed with sample buffer and separated in a 2% agarose gel.
  • the specific primers had the following sequences:
  • IL-2 up position 99-122 (SEQ ID NO: 3)
  • Adeno up position 1228-1248 (SEQ ID NO: 5)
  • the detection limit of the amplification products is between 200 and 1,000 copies of DNA.
  • nine DBA / 2 mice were each immunized with 3 ⁇ 10 5 IL-2 plasmid-transfected M3 melanoma cells. After 1, 2 and 5 days, 3 mice were sacrificed and the immunization sites were cut out. The PCR analysis showed that after 2 days the IL-2 DNA began to degrade strongly and after 5 days this DNA was no longer detectable. In an analog experiment, IL-2 plasmid DNA could be detected from the injection site of an animal on day 5 after the injection. In PCR reactions with the adenovirus-specific
  • Injection site the draining lymph nodes, spleen, kidney, liver, colon and the gonads (ovaries)
  • Example 16 a Testing the effectiveness of the cancer vaccine for its protective action against metastasis
  • Therapeutic Mouse Model "Therapeutic Mouse Model"
  • the transfection complexes used, the cultivation of the cells and the implementation of the transfections were carried out as described in Example 8.
  • C57BL / 6J strain mice were used as test animals, 8 animals per group being used.
  • the melanoma cells used were the B16-F10 cells syngeneic for the mouse strain used (NIH DCT tumor depository; Fidler et al., 1975)
  • B16-F10 cells injected intravenously to cause metastasis.
  • the cancer vaccine was administered subcutaneously to immunize against the metastases.
  • transfected cells in the following means cells that have been subjected to transfection treatment; the expression values for the cytokines are given per mouse in 24 hours; the names correspond to those in Fig. 25):
  • the animals of the second group were each immunized with 1 ⁇ 10 5 M3 cells which had been transfected and irradiated with the vector pWE-Gm.
  • the first control group was treated with 1 x 10 5 irradiated but not transfected M3 cells.
  • the second control group received as
  • Tumor development control 5 x 10 3 M3 cells. The animals were observed over a period of more than 4 months. It was found that in the two groups which had received cells expressing a cytokine as tumor vaccine, 80% of the animals before one
  • Control group all tumors developed within 8 weeks. In the second control group, all but one of the animals developed.
  • Example 17 Efficacy of tumor vaccines depending on the cytokine dose in the prophylactic mouse model
  • M3 melanoma cells used were the same as in Example 16.
  • the cytokine plasmids used were the same as in Example 16. The
  • Example 8 Immunization was carried out as described in Example 8, for setting of the tumor ( "Challenge") was in contrast to Example 8 instead of 1 x 10 5 cells 3 x 10 5 cells.
  • the peptide with the designation INF5 (SEQ ID NO: 7) was synthesized using the HBTU activation method (Knorr et al., 1989; 1 mmol scale), 230 mg of TentaGel S-PHB resin (Rapp polymers; 0.27 mmol / g) as the solid phase was used.
  • the first amino acid that was coupled was N- ⁇ -N- ⁇ -di-Fmoc-lysine. This resulted in a head-to-head dimer, with a C-terminal lysine as
  • the stirring mixture is pipetted dropwise into 40 ml of ether and the mixture is left to stand for 1 h.
  • the crude peptide was obtained by centrifugation
  • the peptide was complexed ionically to polylysine.
  • the DNA complexes were mixed with 0.5 ml RPMI 1640 (Gibco), containing 10% FCS, and applied to M3 melanoma cells (1 ⁇ 10 5 cells in 6-well plates). After 4 hours the medium was replaced by fresh medium. The cells were harvested 24 h after the transfection and examined for luciferase activity. Expression corresponding to 12,866,000 light units was detected.
  • Transfection complexes consisting of 3 ⁇ g pGShIL-2tet, 1.5 ⁇ g TfpL290, 5 ⁇ g pL290 and 40 ⁇ g INF5 were, as in i) described, applied to melanoma cells.
  • the amounts of IL-2 secreted into the culture medium within 24 h were measured by means of an ELISA assay (Biokine IL-2 test kit, T Cell Diagnostics). The values were 6,500 BRMP units on the first day and 11,500 BRMP units on the second day, one unit corresponding to 40 pg IL-2.
  • C57BL / 6J strain and B16-F10 cells were used in this experiment. There were two immunizations, each with 1 x 10 5 cells at an interval of 7 days
  • the challenge (1 ⁇ 10 5 cells) was set 7 days after the last immunization.
  • the cancer vaccines were prepared using a transfection complex consisting of 6 ⁇ g pWS-Gm-DNA, 3 ⁇ g TfpL, 10 ⁇ g pL and 40 ⁇ g INF5. There were tumor vaccines
  • the starting material was plasmid pGShIL-2tet and MM3 melanoma cells irradiated with 100 Gy; after 20 h incubation at 37 ° C, the cells were trypsinized and cell number as well
  • Viability determined with trypan blue The cells were then divided into four equal groups to test four different freezing media. The four
  • Sample groups were centrifuged in RPMI 1640 at 800 rpm (120 g) before adding freezing medium, and the cell pellets were placed in 1 ml freezing medium. After this
  • the plasmid pGShIL-2tet was obtained from E. coli overnight cultures (grown in the presence of 5 ⁇ g / ml tetracycline in LB medium). b) Purification of the plasmid DNA from endotoxin
  • Triton X-114 (Sigma) was subjected to three 0 ° C / 30 ° C temperature cycles as described by Bordier, 1981.
  • the extraction of the lipopolysaccharides from the DNA sample was carried out as follows, using modified methods (Aida and Pabst, 1990; Manthorpe et al., 1993): The DNA sample (0.5-1.5 mg / ml in 10 mM Tris, 0.1 mM EDTA, pH 7.4 (TE)) was on
  • a volume of polymyxin resin sludge (Affi-Prep-Polymyxin, Biorad), which corresponded to the volume of the DNA sample, was briefly mixed with three volumes of 0.1 N NaOH, then it was washed three times with five resin volumes of TE. The pelleted resin was taken up again with the DNA samples (in TE 0.8-1.2 mg / ml) and the mixture was stirred at 4 ° C. overnight. Then the sample was put on one
  • Lipopolysaccharide content was determined using the Limulus chromogenic assay based on the Limulus coagulation reaction of Amoebocyte-based (Iwanaga, 1993; available from
  • Particles / ml) and plasmid DNA (6 ⁇ g, diluted in 100 ⁇ l with the lipopolysaccharide content indicated in each case) were prepared as described in the preceding examples.
  • H225 Primary human melanoma cells
  • transfection complexes used the culturing of the cells and the implementation of the transfections were carried out as described in Example 8, with regard to the
  • Colon carcinoma cell line called CT 26 used, the establishment of which was described by Brattain et al., 1988.
  • BALB / c strain mice were used as test animals. 1 ⁇ 10 5 cells were used for each of the two immunizations, the challenge was set with 3 ⁇ 10 5 cells, n Table VII shows the expression values in 24 h for IL-2 (units / mouse), IFN-gamma and GM-CSF (each ng / mouse), secreted between

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Abstract

On prépare des vaccins anticancéreux en traitant des cellules tumorales ou des fibroblastes avec un complexe d'ADN de codage d'un polypeptide immunostimulant et d'une substance de liaison de l'ADN, par exemple la polylysine, de préférence conjuguée avec la transferrine. Le complexe contient en outre un conjugué d'une substance de liaison de l'ADN et d'un peptide endosomolytique ou d'un adénovirus ayant au moins un défaut en E4 ou en E1a associé à d'autres défauts génétiques.
EP94911921A 1993-03-19 1994-03-18 Procede de preparation de vaccins anticancereux Withdrawn EP0689604A1 (fr)

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AT556/93 1993-03-19
AT0055693A AT399656B (de) 1993-03-19 1993-03-19 Verfahren zur herstellung von krebsvakzinen
DE4326821 1993-08-10
DE4326821A DE4326821A1 (de) 1993-08-10 1993-08-10 Verfahren zur Herstellung von Krebsvakzinen
PCT/EP1994/000859 WO1994021808A1 (fr) 1993-03-19 1994-03-18 Procede de preparation de vaccins anticancereux

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CA2190290C (fr) * 1994-05-13 2011-07-05 Michael J. Mastrangelo Procede permettant d'induire une reponse immunitaire au moyen de virus vaccinaux recombines
US6093700A (en) 1995-05-11 2000-07-25 Thomas Jefferson University Method of inducing an immune response using vaccinia virus recombinants encoding GM-CSF
DE4426429A1 (de) * 1994-07-26 1996-02-01 Boehringer Ingelheim Int Verfahren zum Einführen von DNA in höhere eukaryotische Zellen
DE19510344C1 (de) * 1995-03-22 1996-11-07 Boehringer Ingelheim Int Verwendung einer Tumorvakzine
WO1997000085A1 (fr) * 1995-06-19 1997-01-03 University Of Medicine & Dentistry Of New Jersey Therapie genique de tumeurs solides au moyen d'interferons seuls ou associes a d'autres proteines immuno-effectrices
DE19608753C1 (de) * 1996-03-06 1997-06-26 Medigene Gmbh Transduktionssystem und seine Verwendung
US7001765B2 (en) 1996-03-06 2006-02-21 Medigene Ag Adeno-associated virus vector for boosting immunogenicity of cells
DE19608751B4 (de) 1996-03-06 2006-05-18 Medigene Ag Verwendung eines Adeno-assoziierten Virus-Vektors zur Steigerung der Immunogenität von Zellen
DE19631357A1 (de) * 1996-08-02 1998-02-05 Deutsches Krebsforsch Vektor zur Aktivierung des Immunsystems gegen mit Papillomviren bzw. Sequenzen davon assoziierten Zellen
AU737717B2 (en) * 1997-08-13 2001-08-30 Uab Research Foundation, The Vaccination by topical application of genetic vectors
EP1003711B1 (fr) 1997-08-13 2001-11-07 Biontex Laboratories GmbH Nouvelles lipopolyamines, leur representation et leur utilisation
WO1999036433A2 (fr) 1998-01-14 1999-07-22 Morphogenesis, Inc. Substances et methodes pour le traitement de maladies cancereuses
US7348015B2 (en) 1998-01-14 2008-03-25 Morphogenesis, Inc. Antigen modified cancer cell vaccines for cancer therapy
US7795020B2 (en) 1998-01-14 2010-09-14 Morphogenesis, Inc. Tumor cell vaccines
EP2295065B1 (fr) * 1998-02-20 2013-10-09 The University of Miami Complexe de peptide antigènes à protéines de choc thermique modifiées
CA2362578A1 (fr) 1999-02-09 2000-08-17 Kam Leong Vaccins anti-tumeurs
BR0001029A (pt) * 2000-04-10 2001-11-20 Fk Biotecnologia Ltda Processo de transformação de células tumorais
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DE10131148A1 (de) * 2001-06-28 2003-01-16 I P L Internat Pharmaceutics L Xenogene Oligo- oder/und Polyribonukleotide als Mittel zur Behandlung von malignen Tumoren
US7176022B2 (en) * 2002-12-20 2007-02-13 Cell Genesys, Inc. Directly injectable formulations which provide enhanced cryoprotection of cell products
DK2257301T3 (da) 2008-03-03 2014-04-28 Univ Miami Immunterapi baseret på allogene cancerceller.
WO2009117116A2 (fr) 2008-03-20 2009-09-24 University Of Miami Vaccination par protéine de choc thermique gp96 et procédés d'utilisation
AU2011336019B2 (en) * 2010-12-02 2016-07-07 Oncotherapy Science, Inc. TOMM34 peptides and vaccines including the same
WO2016127015A1 (fr) 2015-02-06 2016-08-11 Heat Biologics, Inc. Vecteur co-exprimant un vaccin et des molécules co-stimulantes
EP3487999A4 (fr) * 2016-07-25 2020-05-20 Ascend Biopharmaceuticals Ltd Procédés de traitement d'un cancer
WO2018071405A1 (fr) 2016-10-11 2018-04-19 University Of Miami Vecteurs et cellules de vaccin pour immunité contre le virus zika
US11548930B2 (en) 2017-04-04 2023-01-10 Heat Biologics, Inc. Intratumoral vaccination
AU2018395010B2 (en) * 2017-12-29 2022-07-28 Genemedicine Co., Ltd. Cell sheet for gene delivery

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Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9421808A1 *

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