WO2007057368A1 - Vecteur viral, son utilisation pour la therapie de carcinomes de cellules hepatiques et agent pharmaceutique comprenant ce vecteur - Google Patents

Vecteur viral, son utilisation pour la therapie de carcinomes de cellules hepatiques et agent pharmaceutique comprenant ce vecteur Download PDF

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Publication number
WO2007057368A1
WO2007057368A1 PCT/EP2006/068374 EP2006068374W WO2007057368A1 WO 2007057368 A1 WO2007057368 A1 WO 2007057368A1 EP 2006068374 W EP2006068374 W EP 2006068374W WO 2007057368 A1 WO2007057368 A1 WO 2007057368A1
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vector
nucleic acid
acid sequences
promoter
sequences
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PCT/EP2006/068374
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German (de)
English (en)
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Brigitte PÜTZER
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Universität Rostock
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    • 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]
    • C07K14/5434—IL-12
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • Viral vector its use for the treatment of hepatocellular carcinoma and pharmaceutical agents comprising the vector
  • the invention relates to a viral vector comprising nucleic acid sequences encoding IL-12 and a costimulator protein, and further comprising an AFP promoter and MER-I enhancer.
  • the invention relates to a tricistronic adenoviral vector co-expressing IL-12p40, IL-12p35 and B7.1.
  • the invention also relates to the use of the vector for the therapy of hepatocellular carcinomas and for the production of a pharmaceutical agent for the treatment of hepatocellular carcinomas, preferably of hepatocellular carcinomas.
  • the invention also relates to pharmaceutical agents comprising the vector or virus particles.
  • HCC hepatocellular carcinoma
  • Current therapeutic approaches including, for example, surgery, radiofrequency ablation, chemotherapy, and / or percutaneous ethanol injection, have achieved some success only in smaller tumors while being insufficient in controlling large tumors.
  • a gene therapy for cancer treatment has been proposed in the prior art. Genetic therapeutic methods are based on the administration of a nucleic acid that is incorporated into the tumor cell and has sequences that destroy the tumor cell or contribute to their destruction.
  • the treatment strategy distinguishes between the transmission of tumor suppressor genes, the transmission of oncolytic viruses, immunotherapy and suicide gene therapy.
  • the immune system be stimulated to recognize and remove neoplastic cells from the patient.
  • the immunotherapeutic approach to providing these proteins involves vector-mediated transfer of genes whose products activate the immune system and trigger an immune response directed against the tumor cells.
  • the immune system also recognizes tumor-specific structures on the tumor cells. The activation of the immune system can therefore lead to destruction of the tumor by the components of the immune system.
  • IL-12 has a pronounced antitumor effect.
  • IL-12 activates NK cells as well as cytotoxic T lymphocytes and induces their IFN- ⁇ production.
  • IL-12 also promotes an immune response via the proliferation and activation of Th1 T helper cells and causes inhibition of tumor angiogenesis in vivo.
  • the administration of the recombinant IL-12 as an antitumor agent failed due to the fact that this cytokine has toxic side effects at therapeutic doses.
  • IL-12-encoding nucleic acid It was also investigated whether an IL-12-encoding nucleic acid is introduced into the tumor and allows local activation of the immune system.
  • vectors which code for the different subunits of the heterodimeric IL-12.
  • the protein consists of two subunits with relative molecular weights of 40 kDa and 35 kDa linked by disulfide bridges.
  • Sangro et al. (2004) J Oncology 22 (8), 1389-1397 intratumorally injected an adenoviral vector expressing IL-12 under the control of the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • WO 2004/035799 A2 discloses adenoviral vectors comprising nucleic acid sequences coding for IL-12 and at least one costimulator protein, wherein the costimulator protein is in particular the 4-1 BB ligand. Although this reduced the dosage of the vector, the treatment was associated with fewer side effects. However, the vector has not been successful in clinical trials. The complexity of the multiple costimulatory protein construct and the non-specific control of protein expression over the preferred CMV promoter are considered to be detrimental.
  • tumor-specific promoters under the control of which some types of tumors overexpress oncofetal genes that are not active in normal tissue (Robson & Hirst (2003) J Biomedicine and Biotech., 2, 110-137).
  • Known tumor-specific genes encode the carcinoembryonic antigen (CEA) and the fetoprotein (AFP).
  • AFP is normally expressed in the fetal liver and also occurs in hepatocellular carcinomas.
  • the AFP promoter has been used, for example, for targeted expression of the transgenic HSV-tk, CD and diphtheria toxin A in HCC tumors. Expression of immunostimulatory proteins such. B. IL-12, under AFP promoter control, however, is not described.
  • the activity of the AFP promoter is considered weak.
  • a particular disadvantage is the dependence of the transgene expression on an AFP overexpression of the tumor cells, so that low AFP concentrations lead to a low efficiency of expression in the tumors.
  • the object of the present invention was therefore to provide vectors or pharmaceutical agents which ensure a selective and effective immunotherapy of hepatocellular carcinomas.
  • a viral vector comprising nucleic acid sequences encoding IL-12 and a costimulator protein and nucleic acid sequences comprising at least one promoter, wherein the promoter is the AFP promoter and the nucleic acid sequences further comprise a MER I-enhancer include.
  • the viral vector brings about a particularly advantageous co-expression of the combination of therapeutically efficient genes for IL-12 and a costimulator protein which occurs exclusively in AFP-positive liver cell carcinomas.
  • IL-12 may be present as single chain IL-12, i. H. consist of an amino acid sequence comprising the two subunits of natural IL-12 as a fusion protein; or the two subunits IL-12p35 and IL-12p40 are separated and are translated independently.
  • the subunits IL-12p35 and IL-12p40 are a 1.1 kb fragment and a 2.3 kb fragment, respectively.
  • the cDNA of IL-12 or the subunits is isolated from a genomic library with routine experiments, optionally amplified and subcloned.
  • the cDNA for IL-12p35 may be introduced into the pED (EMC) vector at the PstI restriction site while the cDNA for IL-12p40 is contained in the same pED (EMC) vector at the XbaI restriction site.
  • EMC pED
  • EMC pED
  • nucleic acid sequences which encode a costimulator protein are understood to mean those sequences which, when expressed in cells, produce proteins which are present as cell surface proteins and are bound specifically by receptors of the immune cells, in particular T cells.
  • the cDNA of the costimulator protein is also preferably provided by appropriate techniques in a plasmid, such as pCA 13. Costimulator sequences are known to those of skill in the art cDNA sequences are obtained from appropriate databases or other literature selected, in vivo or in vitro synthesized and inserted into the appropriate plasmid.
  • the nucleic acids for IL-12 or its subunits and the costimulator protein can be contained in a single expression cassette, so that a polycistronic transcript is formed, or each protein-encoding nucleic acid is contained in a separate expression cassette, so that there are several expression cassettes.
  • the order of the nucleic acid sequences within the expression cassette or the arrangement of several expression cassettes in the vector is arbitrary.
  • the individual cistrons within the expression cassette may be separated by non-coding sequence segments, preferably sequences for internal ribosome entry sites (IRES sequences), so that in particular polycistronic transcripts are effectively translated.
  • the IRES sequences originate, for example, from the commercially available pCITE-2 vector (Novagen).
  • an expression cassette is created in which IL-12p40 is upstream of IL-12p35 and downstream of which is the costimulator protein, all protein coding sequences being separated by IRES sequences.
  • the 5 'flanking regions of the AFP gene contain DNA sequences for the AFP enhancer domain and the AFP promoter. Their orientation, designation and nucleotide numbering, as also used in the present specification, corresponds to the AFP genome (Zhang et al. (1992) JBC 267 (15), 10676-10682).
  • the enhancer domain is divided into three fragments, referred to as Enhancer I, Enhancer II, and Enhancer III. Within these fragments are located the MER I enhancers, MER I I enhancers and MER III enhancers, termed minimal enhancer regions (MER).
  • the vector according to the invention may comprise the nucleic acid sequences for the AFP enhancer domain or parts thereof.
  • parts of the AFP enhancer domain are understood to be the abovementioned enhancer fragments, minimal enhancer regions and / or nucleic acid sequences which enhance the gene expression of the expression cassette under their control.
  • the MER I enhancer is preferred as a component of the viral vector.
  • the AFP promoter (about 900 bp fragment) and MER-I enhancer (about 400 bp fragment) are amplified individually or contiguously by PCR and, for example, subcloned into the pCR2.1TOPO vector via EcoRI.
  • the murine AFP promoter contains the base pairs of -1009 to +37 and the human AFP promoter the base pairs of -230 to +29.
  • a preferred murine MER I enhancer according to the invention contains the base pairs from -2497 to-2193 or up to a further 100 base pairs at the 5 'and / or 3' end of the abovementioned sequence region, particularly preferably the base pairs of 2574 to -2190.
  • the person skilled in the art is able, for example by sequence overlays and homology comparisons, to find the nucleic acid sequences corresponding to the preferred murine sequences in other species, such as in humans.
  • the human AFP enhancer comprises the two domains A and B.
  • the enhancer domain A preferably contains the base pairs of -4900 to -3300 and the enhancer domain B the base pairs of -3700 to -3300. It is also possible to use only the enhancer domain B, which alone is sufficient in the meaning of the invention.
  • the vector of the invention will comprise further elements, the z. B. are essential for vector replication or protein expression. They are generated by methods known to those skilled in the art.
  • AdEasy adenovirus shuttle plasmid pShuttle (AdEasy), after co-transformation with the plasmid pAdEasyl, which contains the remaining adenovirus sequences, recombined in E.
  • adenoviral vector is additionally deleted to one Vector of the first generation or the second generation.
  • Corresponding vectors are well known in the art.
  • the nucleic acid sequences encoding IL-12 and the costimulator protein and the nucleic acid sequences comprising the AFP promoter and the MER-I enhancer are arranged in an expression cassette.
  • an expression cassette is to be understood as meaning the entirety of the nucleic acid sequences which is necessary for the expression of one or more genes and which contains at least the gene sequences to be expressed as well as transcriptional and translational regulatory elements which, for example, initiate, enhance, inhibiting, terminating etc. act, wherein the nucleic acid sequences form a functional and possibly also a structural unit, preferably at the level of the primary structure.
  • the expression cassette is under the control of only one promoter and only one enhancer element.
  • several promoters are used in an expression cassette whose number is e.g. corresponds to that of the Cistrone.
  • the costimulator protein is B7.1, B7.2, IL-2 or 4-1 BBL.
  • B7.1 (CD80) which substantially enhances the immune response to the tumor and reduces the toxicity of IL-12 so that the vector dose can be reduced with equal efficiency.
  • the interaction of IL-12 and B7.1 shows a synergistic effect. Their combination is particularly efficient in inducing tumor suppression and a specific, long-lasting anti-tumor immunity.
  • the costimulator protein may also be a cytokine or protein with cytokine activity, i. H. a cytokine agonist.
  • the transgenes are species-specific.
  • the human genes are required and preferred for the purposes of the invention.
  • human B7.1 is the costimulator protein.
  • IL-12 includes IL-12p40 and IL-12p35, preferably human IL-12p40 and human IL-12p35.
  • AFP promoter and the MER-I enhancer in Depending on the species to be treated are to choose.
  • Preferred are a human AFP promoter and a human MER-I enhancer.
  • the nucleic acid sequences may also be derived from other species or modified by methods known in the art, provided that the activity of the proteins remains in the range of at least 50%, preferably at least 70%, of the corresponding activity of the human gene product. Changes in the gene sequence leading to an increase in the activity of the proteins are of course included herein.
  • the nucleic acid sequences further comprise IRES sequences located in the expression cassette between IL-12 and the costimulator protein, or between IL-12p40, IL-12p35 and the costimulator protein.
  • IRES sequences are derived from the 5 'untranslated regions of picornaviruses, which allow for cap-independent initiation of translation on internal AUG triplets.
  • the IRES sequences are derived from Encephalomyo carditis virus (EMCV). With vectors that have an IRES sequence for each cistron that is not immediately behind the promoter, high expression of the immunostimulatory genes is achieved.
  • IRES sequences ensure a uniform co-expression, in particular of the IL-12 subunits IL-12p35 and IL-12p40, which is necessary for the full function of the transgenes.
  • IL-12p35 and IL-12p40 which is necessary for the full function of the transgenes.
  • IRES sequences can be used in a vector. In the latter case, the recombination frequency among these sequences is advantageously minimized, but the expression rate of the proteins may be lowered, so that identical IRES sequences are preferred.
  • the nucleic acid sequences further comprise a polyadenylation signal from simian virus 40 which terminates the expression cassette.
  • the simian virus 40 polyadenylation signal, short Called SV40 polyA signal, is considered part of the expression cassette.
  • nucleic acid sequences comprising hMERI enhancers, hAFP promoter, hLL-12p40, EMCV IRES, hLL-12p35, EMCV IRES, hB7.1 and SV40 polyA signal are described in U.S. Patent Nos. 4,646,731; order in 5'-3 'orientation in the expression cassette.
  • a vector is referred to as a viral vector, if it is a nucleic acid sequence comprising sequences of viral origin, which allow the packaging of the nucleic acid in viral envelopes.
  • the vectors may be an adenoviral vector, adeno-associated vector, lentiviral vector, HSV vector, retroviral vector, baculoviral vector or Semliki-Forrest vector.
  • an adenoviral vector is preferred.
  • the adenoviral vectors may be those of the first generation (with deletions in the regions E1 and E3), the second generation (with deletions in E1, E2, E3, E4 etc.), replication-competent (oncolytic adenoviruses) or helper - Dependent adenoviral vectors act.
  • Corresponding adenoviral vectors are z.T. approved for gene therapy in humans and thus classified as safe.
  • the adenoviral vector has at least one deletion of the region E1.
  • E1 contains the viral genes E1A, E1B and for the protein IX, whose deletions lead to the replication defect of the vector.
  • the E3 region is deleted, so that there is a classic Wargenerationsvektor.
  • the E1 region is substituted by the expression cassette. This means that the expression cassette is located at the site of the E1 region. It does not matter whether the exchange of the nucleic acid sequences takes place simultaneously or with a time offset.
  • the expression cassette is in the deleted E1 region of the adenovirus Type 5 (Ad5), which is a serotype of the adenovirus subgroup C.
  • Ad5 DNA sequences are included, for example, in the shuttle plasmid pShuttle (AdEasy), in particular from map unit (mu) 0 to 1, where 1 mu corresponds to approximately 360 bp, and from mu 9.8 to 15.8. Between the indicated regions are the promoter, a polylinker with unique restriction sites, and an SV40 polyA signal.
  • the remaining DNA sequences of Ad5 are contained in another plasmid, such as pAdEasyl, and are recombined in the course of a co-transformation with the abovementioned Ad5 sequences of the pShuttle plasmid into a functional viral vector according to the invention.
  • the invention relates to a recombinant adenoviral vector (rAdAFPpehlL12B7.1) carrying the expression cassette hlL-12p40-EMCV IRES-hlL-12p35-EMCV IRES-hB7.1-SV40 polyA signal under the control of the upstream MER I enhancer / AFP promoter in the deleted E1 region of Ad5 carries.
  • a vector which comprises a nucleic acid sequence of SEQ ID NO: 1 or variants, mutants, parts of the sequence or 80% homologous sequences which have the same properties.
  • the nucleic acid sequence of SEQ ID NO: 1 represents the non-codogenic strand of the expression cassette.
  • the vector may also comprise the complementary (codogenic) strand.
  • the vector comprises a double-stranded nucleic acid, more preferably a dsDNA comprising the DNA sequence of SEQ ID NO: 1 and the DNA sequence complementary thereto base-paired.
  • Mutants can be obtained, for example, by substitution, deletion, insertion, translocation, inversion and / or addition of at least one nucleotide.
  • Variants of the DNA sequence according to the invention can be modified by modifications such. As alkylation, arylation or acetylation of at least one nucleotide, incorporation of enantiomers and / or by fusion of the sequences with one or more nucleotides or a nucleic acid sequence arise.
  • the vector can also be composed of RNA which represents a variant in the sense of the invention. Parts of the sequences are limited to those sections that are responsible for a particular function of the Vector, in particular the selectivity and expression ability, are sufficient. Any changes in the sequence are necessarily limited by the requirement of functional integrity.
  • a nucleic acid sequence that is functionally analogous to the DNA sequence of SEQ ID NO: 1 has a homology of at least 60%, more preferably at least 70%, most preferably at least 80%.
  • Homologous sequences can also be derived from other species and have the same function. Functionally analogous sequences of different species may have a lower degree of homology, but preferably at least 30%.
  • the present nucleic acid sequence of SEQ ID NO: 1 contains the murine AFP promoter and the murine MER-I enhancer. It is understood that the human AFP promoter and the human MER-I enhancer are included within the scope of the invention SEQ ID NO: 1. They can take the place of the murine constituents. For the indicated changes of such nucleic acid sequences, reference is made to the corresponding standard works of biochemistry and molecular biology.
  • the present invention also relates to virus particles comprising the vector of the invention.
  • virus particles nucleic acids are referred to, which are enclosed by the envelope proteins of a virus.
  • the invention further relates to a cell comprising the vector according to the invention and / or the virus particle according to the invention.
  • the cell in particular a eukaryotic cell, is transfected with the vector or virus particle, resulting in a genetically modified cell.
  • the cells are also called transgenic cells.
  • the invention also relates to cell cultures, tissues, organs and the like, the cells with the o.g. constructs according to the invention. Furthermore, the invention relates to organisms comprising the said cells according to the invention.
  • the present invention also provides a pharmaceutical composition which comprises the vector according to the invention or the virus particle according to the invention, preferably with pharmaceutically acceptable excipients.
  • One Pharmaceutical agent within the meaning of the invention is any agent which can be used in the therapy, follow-up or after-treatment of patients who show, at least temporarily, a pathogenic modification of the overall condition or condition of individual parts of the patient organism, in particular due to hepatocellular carcinoma.
  • the particular dose or dose range for the administration of the pharmaceutical agent according to the invention is large enough to achieve the desired therapeutic effect of inducing an immune response. In general, the dose will vary with the age, constitution and gender of the patient, as well as the severity of the disease.
  • the specific dose, frequency and duration of administration will depend on a variety of factors, such as: Structure of the vector used, expression of the immunostimulatory proteins, dietary habits of the individual to be treated, route of administration, excretion rate and combination with other drugs.
  • the individual dose can be adjusted both in relation to the primary illness and in relation to the occurrence of possible complications. The exact dose can be determined by a person skilled in the art by known means and methods.
  • the virus particles are administered in a concentration of at most 1 ⁇ 10 11 infectious virus particles (PFU) per dosage unit, preferably 1 ⁇ 10 9 PFU per dosage unit.
  • PFU infectious virus particles
  • the vector at a dose of 1x10 9 PFU led to a strong specific expression of the therapeutic genes IL-12 and B7.1 in hepatoma cells, which was not associated with toxic effects.
  • the dosage may also remain well below the values mentioned and amount to not more than 1 ⁇ 10 8 PFU per dosing unit, preferably 1 ⁇ 10 7 PFU per dosing unit, more preferably 1 ⁇ 10 6 PFU per dosing unit.
  • the experiments were also carried out in immunocompetent coat mice (mouse tumor model).
  • the vectors or virus particles according to the invention cause destruction or significant reduction of the tumor already at particularly low dosage.
  • the pharmaceutical agent in a preferred embodiment of the invention may also comprise further active ingredients.
  • the therapeutic effect of the pharmaceutical compositions according to the invention can then be, for example, that by inducing the expression of the immunologically active proteins as a desired side effect, certain anti-tumor drugs work better or by reducing the dose, the number of side effects of these drugs is reduced.
  • the present pharmaceutical agent is suitable for the therapeutic treatment of hepatocellular carcinomas, preferably of hepatocellular carcinomas.
  • hepatocellular carcinomas preferably of hepatocellular carcinomas.
  • the prior teaching of the invention and its embodiments are valid and applicable without limitation to pharmaceutical agents, as appropriate. It is understood that also the host of the pharmaceutical agent is included within the scope of the present invention.
  • the introduction of the pharmaceutical agent into a cell or an organism can be effected in any manner which makes it possible for the tumor cells to be brought into contact with the vectors or virus particles contained in the medium and delivered to the tumor, and for expression of the immunostimulating agents Proteins is induced.
  • the pharmaceutical agent of the present invention may be administered orally, intradermally, subcutaneously, intramuscularly, intravenously and / or intratumorally.
  • the chosen mode of administration will depend on the indication, dose to be administered, individual-specific parameters, etc. In particular, the different modes of administration allow for site-specific therapy that minimizes side effects and reduces the vector dose.
  • the intratumoral injection is preferred.
  • the application can z. B.
  • the pharmaceutically acceptable excipients such as.
  • adjuvants are added.
  • any substance which makes possible, amplifies or modifies an effect with the vectors or virus particles according to the invention is an adjuvant.
  • Known adjuvants are, for example, aluminum compounds such as aluminum hydroxide or aluminum phosphate, saponins such as QS 21, muramyl dipeptide or muramyl tripeptide, proteins such.
  • DNA which has an immuno-stimulatory property or codes for a protein with adjuvant effect can be administered in parallel or in a construct.
  • the pharmaceutical agent may be in the form of a tablet, capsule, powder, solution, dispersion or suspension.
  • the dosage forms of the pharmaceutical agent are prepared with the usual solid or liquid carriers and / or diluents and the commonly used excipients according to the desired mode of administration in a suitable dosage and in a conventional manner.
  • the vectors or virus particles of the present invention in the pharmaceutical agent can be incorporated, for example, in liposomes or liposomes with replication-competent adenoviruses (RCA), as polyglycol-coated adenoviruses, antibody-bridged adenoviruses (which are bound to an antibody specific for the virus and a cell marker ), Cassette in an RCA, or as a tumor-conditionable RCA (with the E1 function under control of a tumor-specific promoter), or mixed with RCAs.
  • adenoviruses RCA
  • pharmaceutically acceptable excipients known to those skilled in the art can also form part of the agents or formulations according to the invention. These excipients also include, for example, salts, various fillers, additional buffering agents, chelating agents, antioxidants, cosolvents, and the like.
  • the pharmaceutical agent is present as an injection solution.
  • aqueous media such as B. distilled Water or physiological saline solutions are used.
  • the pharmaceutical agent can also be present as a solid composition, for example in the lyophilized state, and then by addition of a resolving agent, such as. B. distilled water, prior to use.
  • the concentration of the therapeutically active vectors or virus particles in the formulation may vary between 0.1 to 100% by weight.
  • the initial immunogenicity of the tumor cells can be enhanced by multiple injections.
  • the pharmaceutical agent may be used as a carrier material, such as. B. based on cellulose sulfate, which releases the vector after implantation into the tumor over a period of time.
  • Another object of the invention relates to the use of the vector according to the invention, the virus particle according to the invention or the cell according to the invention for the treatment of hepatocellular carcinomas, preferably of hepatocellular carcinomas.
  • Yet another subject of the invention relates to the use of the vector according to the invention, of the virus particle according to the invention or of the cell according to the invention for the production of a medicament for the treatment of hepatocellular carcinomas, preferably of hepatocellular carcinomas.
  • the invention further teaches a method of treating hepatocellular carcinomas, preferably hepatocellular carcinomas, wherein an effective dose of the vector or virus particle of the invention is administered to a subject to be treated.
  • the subject to be treated is in particular a mammal, preferably a human.
  • the therapeutic principle is to induce an anti-tumor immune response by direct intratumoral injection.
  • tumor cells can also be taken from the patient and these cells treated in vitro with an effective dose of the vector or the virus particle according to the invention.
  • the treated cells can be immediately re-implanted into the organism so that the body's immune system destroys the antigen-presenting tumor cells and the vectors or virus particles - if genetically enabled and present in sufficient concentration - propagate.
  • the treated cells are contacted in vitro with immune cells that attack the tumor cells and their regression, so that subsequently the remaining healthy liver cells are re-implanted.
  • a viral vector which allows the selective and therapeutically efficient expression of IL-12 and a costimulator protein.
  • AFP promoter which is inactive in adult normal liver cells and non-hepatoma cells
  • selective expression of the transgenes in hepatocellular carcinomas preferably in AFP-positive hepatocellular carcinomas
  • Tumor-specific expression spares healthy liver tissue and other tissue.
  • IL-12 is cytotoxic in high concentrations, such as those that occur due to high vector concentrations. This provides increased safety of the vector according to the invention.
  • the coupling of the AFP promoter with the MER-I enhancer ensures high, stable and uniform co-expression of all proteins.
  • the expression is comparable to or even better than that under the control of the CMV promoter and moreover has the decisive advantage of targeted expression in HCC tumors.
  • Co-expression, high concentration level and selectivity of protein expression in the vector according to the invention are accompanied by a considerable improvement in its application and the therapeutic effect.
  • the synergistic effect of the proteins encoded by the vectors of the invention allows the use of low vector dosages, which in turn produce less side effects, such as a reduced risk of autoimmune disease to the patient.
  • the reduction in the vector dose also improves safety in clinical use by reducing the likelihood of spreading the vector in the organism to be treated.
  • Immunotherapy not only eliminates the primary tumor but also the metastases. In addition to the complete tumor regression, the formation of a permanent anti-tumor immunity reached. Furthermore, larger tumor masses or multiple tumor foci can be treated simultaneously without producing side effects.
  • liver toxicity after adenoviral gene transfer of IL12 with the vector according to the invention with AFP promoter / enhancer is substantially lower than that of a vector with IL12 under the control of the CMV promoter, as described, for example, as Ad-2 in DE 102 48 141 A1 (WO2004 / 035799 A2) is described.
  • Fig. 1 shows the vector map of the recombinant adenoviral vector rAdmAFPpehlL12B7.1.
  • Fig. 2 shows the base pair sequence of the recombinant adenoviral vector rAdmAFPpehlL12B7.1.
  • Fig. 3 shows the activity of different promoters in different cell lines.
  • the IL-12p35 and IL-12p40 cDNAs of human IL-12 and the cDNA of costimulatory molecule B7.1 (CD80) were provided by the Genetics Institute (Andover, MA, USA).
  • the IL-12p35 subunit is a 1.1 kb fragment contained in the pED (EMC) vector at the PstI site.
  • the IL-12p40 subunit is a 2.3kb in size Fragment contained in the same vector at the XbaI interface.
  • the hB7.1 cDNA was used as a plasmid (pCA13huB7) by Dr. med. S. Dessureault (Toronto, Canada).
  • the IL-12p40 subunit is upstream of the IL-12p35 cDNA and the CD80 cDNA ( Figure 1). Between the individual cDNAs are each an IRES sequence of encephalomyocarditis virus (EMCV). The IRES sequences are from the pCITE-2 vector from Novagen. The plasmid with the complete expression cassette for human IL-12B7.1 (phlL12B7) was prepared by Dr. med. Stewart provided.
  • EMCV encephalomyocarditis virus
  • rAdmAFPpehll_12B7.1 The gene construct rAdmAFPpehll_12B7.1 was generated with the AdEasy system provided by B. Vogelstein, Memphis, TN, USA (He et al. (1998) PNAS USA 95, 2509-2514).
  • the vector was made replication-defective by elimination of the E1 region containing the viral genes E1A, E1B and protein IX.
  • the E3 region was deleted so that it is a recombinant adenoviral first generation vector. Then, the following steps were performed:
  • the SV40 polyadenylation signal (160 bp fragment) was excised from the pMH4 plasmid via BglII / Sall restriction, blunt ended, and inserted (and sequenced) into the NotI interface of the pShuttle plasmid.
  • the murine ⁇ -fetoprotein (AFP1) promoter (-1009 to +37) was amplified by PCR (899 bp fragment), subcloned into the pCR2.1TOPO vector via EcoRI and then into the polylinker after EcoRI restriction (blunt ended). Integrated region (Sall interface, blunt ended) of the AdEasy pShuttle plasmid (and sequenced).
  • the resulting vector (pShuttle / polyA signal / AFP promoter) was cut with Hindi II and filled in with Klenow (blunt ended).
  • PCR amplified MER-I 2574 to -2190
  • MER-I 2574 to -2190
  • EcoRI digest / blunt ended from pCR2.1TOPO and sequenced.
  • the IL12B7.1 cDNA (3950 bp fragment, Notl / Xhol was excised from the plasmid phlL12B7 and blunt ended) was inserted into the EcoRV site of the resulting plasmid (and sequenced).
  • the plasmid thus generated contains the human IL-12B7.1 human interleukin-12 expression cassette (hlL-12)
  • Gene consisting of the two subunits IL-12p35 and IL-12p40, and the human B7.1 (CD80) gene (hB7.1), under the control of the murine AFP promoter / MER-I enhancer and terminated by the polyadenylation signal from SV40.
  • the recombination was carried out after co-transformation with the plasmid AdEasyl, which contains the remaining adenovirus sequences, in E. coli BJ 5183 bacteria.
  • the resulting virus vector was then propagated in 293 cells.
  • the final vector rAdmAFPpehlL12B7.1 carries the hIL-12p40 ECMV IRES-hIL-12p35 -EMCV IRES-hB7.1 expression cassette in the deleted E1 region of Ad5 ( Figure 1, 2).
  • a reporter gene in the plasmid pGL3 was placed under the control of a different promoter.
  • pGL3 Basic, pGL3 SV40, pGL3 CMV, pGL3 TERT, pGL3 AFP P + E and pGL3 AFP were the human hepatocellular carcinoma cell lines Hep3B and HepG2, human embryonic kidney cells 293, human osteosarcoma cells Saos-2, human foreskin fibroblasts VH6 and the human Cercix cancer cell line HeLa transfected.
  • the results in FIG. 3 show that both the AFP promoter and the AFP promoter Promoter / MER I enhancers resulted in expression of the reporter gene only in the HCC cell lines while they were not active in the non-HCC cell lines. A selectivity of the other promoters could not be observed.
  • the AFP promoter / MER-I enhancer caused significantly greater gene activation than the AFP promoter alone.
  • Example 3 Determination of the liver toxicity after adenoviral gene transfer of IL12 with the vector according to the invention in comparison with the vector Ad-2 from DE 102 48 141 A1
  • the animals were sacrificed 7 days after injection (peak of IL12-related toxicity) and the activity of the liver enzymes GPT / ALAT (glutamate pyruvate transaminase), GOT / ASAT (glutamate-oxaloacetate transaminase), GLDH (glutamate dehydrogenase) and LDH (lactate hydrogenase) analyzed in serum.
  • GPT / ALAT gluta pyruvate transaminase
  • GOT / ASAT gluta-oxaloacetate transaminase
  • GLDH glutlutamate dehydrogenase
  • LDH lactate hydrogenase
  • the LDH levels which are elevated only after severe liver damage and are not liver-specific, also increase slightly after Ad2 injection compared to the AdILI 2B7 vector.
  • the results indicate a lower systemic toxicity of the AdILI 2B7 vector compared to the Ad2 vector, ie liver toxicity can be markedly reduced by combining the hepatocyte-specific AFP promoter / enchancer with IL12. U / I ⁇ SD, p ⁇ 0.05.

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Abstract

L’invention concerne un vecteur viral comprenant des séquences d’acide nucléique codant pour IL-12 et une protéine costimulatrice, ainsi qu’un promoteur AFP et un amplificateur MER-I. L’invention concerne notamment un vecteur adénoviral tricistronique coexprimant IL-12p40, IL-12p35 et B7.1. L’invention a également pour objet l’utilisation du vecteur pour la thérapie de carcinomes de cellules du foie et pour la fabrication d’un agent pharmaceutique pour la thérapie de carcinomes de cellules du foie, de préférence de carcinomes hépatocellulaires. L’invention concerne également des agents pharmaceutiques comprenant le vecteur ou une particule de virus.
PCT/EP2006/068374 2005-11-15 2006-11-13 Vecteur viral, son utilisation pour la therapie de carcinomes de cellules hepatiques et agent pharmaceutique comprenant ce vecteur WO2007057368A1 (fr)

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EP2682459A1 (fr) * 2011-03-02 2014-01-08 Beijing Bio-Targeting Therapeutics Technology Inc. Adénovirus oncolytique pour une thérapie ciblée d'une tumeur humaine, et utilisation associée
JP2014509197A (ja) * 2011-03-02 2014-04-17 北京▲錘▼特生物科技有限公司 標的性治療人腫瘍治療の腫瘍溶解性アデノウイルス及びその応用
EP2682459A4 (fr) * 2011-03-02 2014-12-10 Beijing Bio Targeting Therapeutics Technology Inc Adénovirus oncolytique pour une thérapie ciblée d'une tumeur humaine, et utilisation associée
WO2020239964A1 (fr) * 2019-05-28 2020-12-03 Ospedale San Raffaele S.R.L. Agents et procédés pour le traitement d'infections virales
CN111850041A (zh) * 2020-07-30 2020-10-30 药鼎(北京)国际细胞医学技术有限公司 一种治疗肝癌的含il12双顺反子的病毒构建体及其用途和构建方法
US12071633B2 (en) 2020-10-13 2024-08-27 Kriya Therapeutics, Inc. Viral vector constructs for delivery of nucleic acids encoding cytokines and uses thereof for treating cancer

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