WO2007068772A1 - Nouveaux adenovirus recombinants de replications conditionnee (crad) - Google Patents

Nouveaux adenovirus recombinants de replications conditionnee (crad) Download PDF

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WO2007068772A1
WO2007068772A1 PCT/ES2006/000676 ES2006000676W WO2007068772A1 WO 2007068772 A1 WO2007068772 A1 WO 2007068772A1 ES 2006000676 W ES2006000676 W ES 2006000676W WO 2007068772 A1 WO2007068772 A1 WO 2007068772A1
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gene
recombinant adenovirus
promoter
cells
adenovirus according
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Rubén HERNÁNDEZ ALCOCEBA
Sergia Bortolanza
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Proyecto De Biomedicina Cima, S.L.
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/208—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/76—Viruses; Subviral particles; Bacteriophages
    • A61K35/761—Adenovirus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
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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
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    • 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
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10332—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
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    • 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
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor

Definitions

  • the present invention relates to new recombinant adenoviruses for the treatment of solid tumors, and in particular to new selectively replicative adenoviruses in tumor cells that have altered the pathway of retinoblastoma protein (pRB) and that develop in hypoxia conditions
  • SUBSTITUTE SHEET (RULE 26) advances Probably due to an inefficient transduction of the entire mass of solid tumors.
  • adenoviruses possess to penetrate cells and, using cellular machinery, replicate and package their own viral genome, ultimately causing cell lysis and the propagation of their progeny.
  • adenoviruses are genetically modified so that this replicative and cytopathic activity is attenuated or nullified in normal cells, but not diminished in the target tumor cells.
  • These adenoviruses are commonly known as conditioned replication adenoviruses.
  • the first CRAds incorporate genomic mutations that cause a functional loss only compensated by some specific alterations of the target tumor cells.
  • the incorporation of deletions in the ElA or ElB genes results in the production of the mutated proteins, unable to bind with cellular proteins and block control elements that possess normal cells, but that are already intrinsically blocked in tumor cells.
  • the E1B-55 Kda protein which normally binds and inactivates p53 by inducing entry into S phase (synthesis phase of the cell cycle), is prevented from binding to p53 and consequently will not activate the phase. S and replication, except in tumor cells with an altered p53 pathway.
  • Delta24 adenovirus which incorporates a 24 base pair deletion in the constant region 2 (CR2) of the ElA viral gene.
  • CR2 constant region 2
  • the mutated ElA protein is unable to bind to the pRB retinoblastoma protein, a binding that in normal cells is necessary for induction of the S phase and viral replication.
  • CRAds would only replicate in cells in which the ElA / pRB interaction is not necessary, for example in tumor cells with an altered RB-pl ⁇ pathway.
  • a second type of CRAds is those in which one or more tumor-specific promoters are introduced instead of the endogenous promoters of some of their viral genes (eg ElA, ElB, E4).
  • the endogenous promoters of some of their viral genes eg ElA, ElB, E4.
  • viral replication would be restricted to those tumors in which the specific tumor promoter is re-activated or over-activated. Since many of these promoters are not activated in all tumors, the most recent developments seek to design promoters whose activation is universal in all types of tumors.
  • CRAds have been developed where the transcription of the ElA gene is controlled by the promoter of the E2F-1 gene, by a promoter derived from the gene
  • SUBSTITUTE SHEET (RULE 26) of telomerase reverse transcriptase (TERT), or also by a hypoxia inducible promoter.
  • US6900049, US2005 / 0074430 and WO2004 / 031357 have oncolytic adenoviruses whose replication is dependent on the hypoxia-inducible factor (hypoxia-inducible factor, HIF).
  • the replicative and cytolytic activity of these viruses would be limited to cells whose HIF activity is increased, and in particular to cells that grow under hypoxic conditions.
  • viruses carry one or more genes (ElA, ElB, E4) whose transcription is controlled by a promoter, usually artificial, that incorporates one or more hypoxia-responsive hypoxia-responsive elements.
  • Ad9xHRElA hypoxia-inducible oncolytic adenovirus
  • E2F for example tumor.
  • SUBSTITUTE SHEET (RULE 26) of new generations of oncolytic adenoviruses to maximize their replicative and oncolytic efficiency, maintaining their selectivity for tumor cells, and ultimately, presenting maximum anti-tumor activity and safety. This may be possible by developing new CRAds that "exploit" new regulatory mechanisms, and by selecting CRAds that, by combining different control mechanisms, have better performance. In view of the pre-clinical and clinical trials conducted to date, some specialists have ventured that oncolytic virotherapy alone will hardly be effective in the complete eradication of tumors. However, the clinical data obtained incline them to think that the use of oncolytic adenovirus in combination with radiotherapy, chemotherapy or gene therapy will allow greater anti-tumor efficacy.
  • a first object of the invention relates to a recombinant conditioned replication adenovirus, which for simplicity we will refer to as adenovirus
  • SUBSTITUTE SHEET (RULE 26) recombinant of the invention characterized in that it comprises: a) a promoter operatively linked to the ElA adenoviral gene comprising at least one HRE hypoxia response element; b) a promoter operatively linked to the adenoviral region E4 comprising at least one element of response to the E2F factor. c) an adenoviral ElA gene deleted in the constant region CR2; and d) an exogenous gene of interest.
  • any human adenoviral variety or serotype (which has been isolated in humans) can be used, for example serotypes 2 (Ad2), 5 (Ad5), 11 (AdIl) or 3 (Ad3).
  • Ad5 serotype whose complete sequence is available in GeneBank (Human adenovirus type 5, complete genome; Accession number: AC_000008; 35938 bp linear DNA VRL 26 -JAN-2005).
  • the viral promoter that regulates and controls the transcription of the gene encoding the ElA protein has been replaced by a promoter comprising at least one hypoxia response element (HRE) inducible by the HIF-I factor, preferably more than 3.
  • said promoter operatively linked to the ElA adenoviral gene comprises 9 tandem copies of a hypoxia response element (HRE).
  • HIF-I-inducible hypoxia responses can be obtained from promoters of other genes that include such elements.
  • HREs can be obtained, for example, from the promoter of the VEGF vascular endothelium growth factor gene, the erythropoietin gene, or some glycolytic enzyme genes, for example enolasa-1.
  • a promoter operably linked to a gene means a DNA fragment functionally associated with the coding sequence of said gene, such that said fragment is sufficient to regulate and control the transcription of said sequence.
  • gene coding is the minimum promoter region that includes regulatory (minimum) sequences that allow efficient control of gene transcription.
  • the promoter that regulates and controls transcription of the ElA gene is an artificial promoter that includes several tandem copies of the HRE derived from the human VEGF gene promoter.
  • said promoter comprises the sequence SEQ. ID. NO: 1, which includes 9 tandem copies of the hypoxia response element of the VEGF-A gene, linked in a 3 'position to a TATA sequence from the rat prolactin gene promoter.
  • This minimal artificial promoter (9xHRE promoter) has already been described by Cuevas y cois. (Cancer Research 2003; cited above).
  • the natural promoter of the genes encoding the E4 proteins has been replaced by a promoter that
  • SUBSTITUTE SHEET (RULE 26) it comprises at least one element of response to the E2F factor, preferably more than one.
  • said promoter is a fragment of the human E2F-1 factor promoter comprising the regulatory sequences of the E2F-1 gene, for example a minimal promoter.
  • said promoter comprises the sequence SEQ. ID. NO: 2. This minimal artificial promoter has already been described by Hernández-Alcoceba and cois ("New oncolytic adenoviruses with hypoxia- and estrogen receptor-regulated replication"; Human Gene Therapy, 2002; 13: 1737-1750).
  • the use of the E2F-1 promoter to direct the expression of the ElA, ElB and E4 viral genes has been described in WO01 / 36650.
  • the gene encoding the ElA protein of the recombinant virus of the invention has a deletion in the constant region CR2. More specifically, said deletion affects, totally or partially, the region of CR2 necessary for protein binding of pRB retinoblastoma, so that the mutated ElA protein is unable to bind to pRB.
  • the deletion comprises 8 "LTCHEAGF” amino acids (amino acids 122-129 of the ElA protein, corresponding to nucleotides 923-946 of the aforementioned sequence AC_000008). This deletion corresponds to the Delta24 deletion (Fueyo J. et al. Oncogene; 2000/19: 2-12).
  • the recombinant adenovirus of the invention comprises an ElA viral gene deleted with the Delta24 deletion and, operatively
  • SUBSTITUTE SHEET (RULE 26) linked to said gene, a 9xHRE promoter with SEQ sequence. ID. NO: 1; and an E4 viral gene operably linked to the human E2F-1 promoter of sequence SEQ ID NO: 2.
  • the recombinant virus of the invention comprises an exogenous gene or transgene.
  • said exogenous gene is introduced in the place occupied by the viral genes encoding the gpl9k and 6.1k proteins, which are deleted
  • the exogenous gene introduced into the recombinant adenovirus of the invention may be a reporter gene.
  • luciferase eg luciferase
  • a therapeutic gene such as a tumor suppressor gene, an apoptosis inducing gene, an anti-angiogenic gene, a suicide gene that encodes a pro-drug activating enzyme, or an immunostimulatory gene.
  • the exogenous gene is the luciferase reporter gene.
  • said exogenous gene is the thymidine kinase (TK) suicide gene.
  • the exogenous gene is the interleukin 12 gene (IL-12).
  • WO2004 / 031357 describes a hypoxic-regulated oncolytic virus with the IL-12 gene as a transgene.
  • SUBSTITUTE SHEET (RULE 26)
  • the design of the new CRAd has advantages for its use as an oncolytic virus compared to previous versions, since several elements that determine its efficacy and specificity have been optimized: 1.-
  • the use of a promoter that responds to hypoxia to control the ElA gene allows that the replication and cytopathic effect of the virus be stimulated under conditions of low oxygen tension. These conditions are present in solid tumors, and it has been described that they can decrease the activity of different oncolytic adenoviruses regulated by other mechanisms.
  • an advantage of the recombinant adenovirus of the invention over ONYX-411 is the control of . the ElA and E4 regions by different promoters, and specifically the use of the E2F-1 promoter in the E4 region. This allows a double replication control mechanism, while ONYX-411 depends exclusively on the pRB path in the cells. On the other hand, avoiding the repetition of sequences reduces the
  • SUBSTITUTE SHEET (RULE 26) possibility of recombination, increasing the stability of the viral genome.
  • Interleukin 12 This cytokine has several mechanisms of action that contribute to its antitumor effect. On the one hand, it stimulates the reaction of the immune system against tumors through the production of other cytokines and the activation of effector cells. On the other hand, it has an antiangiogenic effect, which hinders the vascularization of tumors and prevents their growth. These actions make the expression of IL-12 in the context of the CRAd described above present important advantages.
  • SUBSTITUTE SHEET (RULE 26) causes hypoxia, which stimulates the replication of CRAd.
  • HSV-TK Thymidine kinase from Herpes virus type I
  • This enzyme has the function of converting a relatively harmless drug (ganciclovir) into a potent cytotoxic agent.
  • ganciclovir a relatively harmless drug
  • the cells that express HSV-TK in the presence of exogenously administered ganciclovir, die and cause the death of the cells that are in their vicinity. Since the administration of ganciclovir can be done days after administering the CRAd, this can achieve an increase in the destruction of tumor cells once the virus has been amplified, and thus increase its oncolytic capacity. At the same time, this stops viral replication, due to the death of infected cells, and can be considered a safety mechanism in the case of replication in healthy tissues.
  • HSV-TK can be used as a reporter gene in humans using the technique of
  • PET Positron Emission Tomography
  • the CRAd genome has been modified so that exogenous genes can be included in the E3 region. These genes take the place of the viral genes that code for the gpl9k and ⁇ .7k proteins. (Hawkins et al., Gene Therapy (2001) 8, 1123-1131). The deletion of these genes
  • SUBSTITUTE SHEET (RULE 26) adenovirus) does not affect the replication of the virus in tumor cells, since the gpl9k and ⁇ .7k proteins have the function of inhibiting the immune response against infected cells. Since tumor cells have their own mechanisms to inhibit their recognition by the immune system, these deletions can favor the elimination of the virus in normal cells, but not in tumor cells, and therefore contribute to the specificity of viral replication.
  • the inclusion of exogenous genes in this location has additional advantages. On the one hand, the endogenous promoter and polyphenylation sequences of the virus are used, so it is not necessary to include these sequences exogenously. This saves space in the viral genome and larger genes can be included.
  • DNA constructs for the preparation of the recombinant adenovirus of the invention can be obtained by conventional methods of molecular biology, many of them collected in general laboratory manuals (for example, "Molecular Cloning: a
  • SUBSTITUTE SHEET (RULE 26) specifically oriented to the preparation of adenovirus, for example:
  • any cell line that is permissive for the replication and formation of selectively replicative recombinant virions of the recombinant adenovirus of the invention can be used for the propagation of the recombinant adenovirus of the invention.
  • Almost any conventional human tumor cell line could be used.
  • the packaging cells transfected with the adenovirus genome have increased HIF expression and activity, except for HEK293 or PER.C6 cells, which constitutively contribute the El viral genes.
  • SUBSTITUTE SHEET (RULE 26) cells can be subjected to an inducing treatment of HIF expression, such as culture under hypoxia conditions (eg in a hypoxic chamber adjusted to a gaseous composition 93% N 2 / ⁇ % CÜ 2 /1% O 2 ) , culture in the presence of cobalt chloride (eg 10OOM), or any other mimetic treatment of hypoxia conditions that is an inducer of HIF.
  • hypoxia conditions eg in a hypoxic chamber adjusted to a gaseous composition 93% N 2 / ⁇ % CÜ 2 /1% O 2
  • cobalt chloride eg 10OOM
  • the present invention relates to a host cell comprising a recombinant adenovirus previously described and object of the present invention.
  • the invention also relates to a process for the in vitro propagation of said adenovirus object of the present invention which comprises culturing a host cell containing a recombinant adenovirus of the invention, under conditions that allow the expression of said adenovirus.
  • the conditions for optimizing the culture of the host cell will depend on the type of host cell used.
  • the method of producing the recombinant adenovirus of the invention will include isolation and purification thereof.
  • a further object of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a recombinant adenovirus of the invention and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition of the invention can be formulated with a variety of conventional excipients that improve adenovirus stability during the manufacturing, handling, storage and distribution processes of the product, or that are appropriate for therapeutic administration.
  • cryoprotectant sugars, polyols, surfactants, amino acids, polymers, buffers and salts can be used to adjust the pH of the composition, antioxidants and other chelating agents, as well as bacteriostatics and bactericides (Parkins et al. "The formulation of biopharmaceutical products "; Pharmaceutical Science and Technology Today, 2000; 3: 129-137).
  • a sterile aqueous or non-aqueous suspension could also be used, which may contain suspending agents or thickening agents. Concrete excipients and formulations useful for the preparation of the pharmaceutical composition of the invention can be found for example in the Journal of Pharmaceutical Sciences (Evans RK et al.
  • the pharmaceutical composition will contain from 10 3 to 10 15 or more adenoviral particles in aqueous solution.
  • Figure 1 Schematic representation of the generic plasmid for the production of CRAds. The sequence of
  • Type 5 adenovirus is in the form of a plasmid with
  • SUBSTITUTE SHEET (RULE 26) Kanamycin resistance, and can be released by digestion with the restriction enzyme Pac ⁇ .
  • ElAp ElA promoter, which is flanked by recognition sequences for BstBI.
  • E4p promoter of the E4 region, flanked by sequences for SwaI and
  • E3 for the insertion of exogenous genes.
  • LP late promoter of the virus.
  • Figure 2. Schematic representation of the AdHLuc, AdDHLuc, AdDHTK, AdDHIL-12 and AdWTLuc viruses.
  • ITR Inverted Terminal Repeat; HRE, ' Hypoxia Response Element; E2F-lp, promoter of the transcription factor E2F-1.
  • D24 deletion of the CR2 domain of ElA.
  • Figure 3. Cytopathic effect of AdHLuc, AdDHLuc and AdWT in Huh-7 human hepatocarcinoma cells ( Figure A) and in normal IMR-90 fibroblasts ( Figure B).
  • Relative survival is represented with respect to uninfected cells cultured under the same conditions, comparing survival when the cells were maintained in hypoxia conditions compared to that observed in normoxia conditions.
  • Huh-7 cells were infected with 5 viruses / cell, and IMR-90 fibroblasts with 135 viruses / cell to compensate for their lower adenovirus infectivity. The cytopathic effect was evaluated at 5 days after infection for Huh-7 cells and at 10 days for IMR-90 fibroblasts. The asterisk indicates significant differences (p ⁇ 0.05)
  • SUBSTITUTE SHEET (RULE 26) The cells were grown under normoxia or hypoxia. The cultures were photographed (20Ox) 5 days after infection. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology.
  • FIG. 1 Cytopathic effect of the AdHLuc, AdDHLuc and AdWT viruses on BJ cells (infected with 500 viruses / cell) and IMR-90 (125 viruses / cell), for cells maintained under normoxia or hypoxia conditions. Photographed (20Ox) also at 5 days. Cells that undergo cytopathic effect by viric replication are distinguished by loss of adhesion, with increased refringence and rounded morphology. Figure 6. Cell viability assay in Huh-7 and IMR-90 cells infected with AdDHLuc virus
  • AdDHLuc or AdWT when they remained in hypoxia or normoxia conditions for 4 days. units
  • FIG. 8 Graphical comparison of in vitro expression of luciferase (represented as RLU / ⁇ g of total protein; RLU, relative luciferase units) in Huh-7 cells infected by the AdDHLuc, AdHLuc viruses,
  • AdWTLuc or AdCMVLuc when they were kept in hypoxia or normoxia conditions. Infection: MOI of 10 viruses / cell.
  • FIG. 9 Cytotoxicity of the AdDHTK virus in response to hypoxia and GCV treatment. Graphic comparison of the viability of A549 cells infected with an MOI of 0.12 virus / cell under normoxia and hypoxia conditions, after 5 days of incubation with a normal culture medium or supplemented with 100 ⁇ M GCV. The asterisk indicates significant differences (p ⁇ 0.05).
  • FIG. 10 Control of the expression of IL12 in the AdDHIL12 virus. Graphical comparison of the increase in IL12 production measured in the supernatant of HeLa cells infected with the AdDHIL12 virus under conditions of normoxia or hypoxia. The asterisk indicates significant differences (p ⁇ 0.05).
  • FIG. 11 In vivo expression of luciferase in athymic mice with human tumor xenografts (Huh-7 cells), after infection (by intratumoral injection) with the AdDHLuc, AdDHWT and Ad-CMV-Luc viruses.
  • A Luciferase activity in subcutaneous tumors at different times after infection with 2xlO 8 iu of the virus to be tested.
  • B Increase in luciferase activity compared to day 1 in the same mice.
  • C Tumors induced by intrahepatic injection of Huh-7 cells;
  • Example 1 Construction and propagation of recombinant adenoviruses
  • AdDHTK which incorporates the Herpes simplex virus thymidine kinase gene (HSV-TK, suicide gene); and - AdDHIL-12, to which the interleukin-12 gene (IL-12, therapeutic gene) has been incorporated.
  • HSV-TK Herpes simplex virus thymidine kinase gene
  • IL-12 interleukin-12 gene
  • the ElA gene promoter has been replaced by a synthetic promoter (SEQ. ID. NO: 1) that responds to hypoxia; ii) the E4 region promoter has been replaced by the E2F-1 transcription factor promoter (SEQ. ID. NO: 2); iii) a deletion (Delta24 deletion) has been made in the CR2 domain of the ElA gene; and iv) the genes of interest have been introduced to replace the gpl9k / ⁇ .7k genes of the E3 region.
  • SEQ. ID. NO: 1 that responds to hypoxia
  • the E4 region promoter has been replaced by the E2F-1 transcription factor promoter (SEQ. ID. NO: 2);
  • iii) a deletion (Delta24 deletion) has been made in the CR2 domain of the ElA gene; and iv) the genes of interest have been introduced to replace the gpl9k / ⁇ .7k genes of the E3 region.
  • AdWT wild type 5 adenovirus
  • AdWTLuc which differs from AdWT in the inclusion of the luciferase gene in place of the gpl9k / ⁇ .7k genes of the E3 region
  • Ad-CMV-Luc a defective adenovirus that expresses the luciferase gene under the control of the CMV promoter (Vector Biolabs, Philadelphia, Ref. 1000); Y
  • AdHLuc which differs from AdDHLuc because it has not been deleted (Delta24 deletion) in the CR2 domain of the ElA gene.
  • the plasmid pSEHE2F
  • This plasmid is based on the adenovirus type 5 genome, modified to facilitate the replacement of the ElA and E4 promoters with tumor-specific promoters (Hernández Alcoceba R. et al. Human Gene Therapy (2002) 13: 1737-1750).
  • recognition sequences have been introduced for restriction enzymes in specific regions and exogenous promoters that can be easily substituted:
  • ElAp ElA promoter region flanked by recognition sequences for the BstBI enzyme (pSEHE2F contains in this region the artificial promoter 5XEH3, which will be replaced by the 9XHRE promoter in the viruses object of the invention);
  • SUBSTITUTE SHEET (RULE 26) - E4 promoter region (E4p) flanked by recognition sequences for the I-Ceul and SwaI enzymes (pSEHE2F contains in this region the promoter for factor E2F-1, which will be maintained in the viruses object of the invention).
  • This last promoter was obtained by Polymerase Chain Reaction (PCR) from human genomic DNA using the following oligonucleotides: SEQ ID NO 3: 5 'TACTGTAACTATAACGGTCCTAAGGTAGCGTGGTACCATCCGGACAAAGCC-S' and, SEQ ID NO 4: 5 'TAAGTATTTAGG .
  • An insulating sequence (referred to herein as "Ins") is present in pSEHE2F between the adenovirus packaging sequence and the ElA promoter region.
  • This sequence transcription stop sequence of the bovine growth hormone gene
  • Plasmid pSHE2F was modified in successive stages to obtain the plasmids necessary to produce the viruses of the invention and their controls: .- Introduction of the 9XHRE promoter in the ElA region.
  • Plasmid PSEHE2F was digested with BstBI, and the 5XEH3 promoter was replaced by the 9XHRE promoter.
  • an adapter consisting of the following pair of oligonucleotides was used:
  • SUBSTITUTE SHEET (RULE 26) The correct orientation of the promoter was verified by digestion with the restriction enzyme BamHI and sequencing using the following oligonucleotides: SEQ ID NO 7: 5Ad5St: 5 'TAGTGTGGCGGAAGTGTGATGTTG 3' and SEQ ID NO 8: 3Ad5St: 5 'TCTTCGGTAATAACACCTCCG.
  • This new plasmid was called pSHIFE2F. .- Partial deletion of the E3 region.
  • Plasmid pSHIFE2F was modified to delete the genes encoding gpl9k / 6.7k (Dgpl9k / ⁇ .7K) and flanking this area with recognition sequences for the Pl-Scel enzyme, which will allow the incorporation of exogenous genes. To obtain the deletion, successive stages of subcloning were carried out. First, a 4.7 kb fragment was obtained by digestion of plasmid pSHIFE2F with the AgeI enzyme, and subcloned into the Agel site of commercial plasmid pMIB / V5-HisC
  • pMIB-AgeC This new plasmid (called pMIB-AgeC) was digested simultaneously with the Spel and Xbal enzymes and a fragment obtained by PCR was introduced in this position into which a restriction site for the Xmnl enzyme has been introduced at position 28555 relative to the genome of adenovirus type 5.
  • This PCR fragment was obtained with the following pair of oligonucleotides:
  • 3XmnI 5 'CCGATTCTAGAGAAACCTGAATTAGAATAGCCCGTAGAGTTGCTTGA AATTGTTCTAAACCCCAC 3', using plasmid pSEHE2F as a template.
  • the new plasmid was called pMIB-E3Xmn.
  • 5PI-Sce 5 'ACGTAATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCA 3' and, SEQ ID NO 12:
  • 3PI-Sce 5 'TGCCATTTCATTACCTCTTTCTCCGCACCCGACATAGATTACGT 3'.
  • plasmid pMIB-PIScel The resulting plasmid is called pMIB-PIScel, and it has the Pl-Scel site where the adenoviral genes that code for To incorporate this deletion into the genome of adenoviral vectors, plasmid pMIB-PIScel was digested with Agel and Seal enzymes simultaneously, and the 4.1 kb fragment obtained was introduced into plasmid pSHIFE2F digested with the same enzymes. In this way, a plasmid is generated that contains the desired deletion, but is incomplete because it has lost the initial regions of the adenoviral genome. However, from this plasmid the 7.7 kb fragment comprised between the SwaI and Spel enzymes was obtained, which was introduced into the plasmid pSHIFE2F instead of the homonymous fragment. In this way, the genes that
  • SUBSTITUTE SHEET (RULE 26) coding for gpl9K / 6.7k viral proteins were deleted and instead the Pl-Scel restriction site was introduced that can be used for the incorporation of exogenous genes in this area, since it is not present in any other region of the virus.
  • the first exogenous gene that was introduced in the E3 region was the luciferase reporter gene, obtained from plasmid pGL3-Basic (Promega).
  • the 1.6 kb fragment between the HindIII and Xbal sites encoding luciferase was treated with the enzyme T4 polymerase to achieve blunt ends, and was introduced into the Pl-Scel site that had been treated in the same way in the modified adenoviral plasmid previously.
  • the resulting plasmid is called pSHE2F-Luc.
  • Pl-Scel recognition sites for the CIaI enzyme instead of Pl-Scel, because the latter is very large and its symmetrical repetition can cause plasmid recombinations.
  • sequence that is intended to be introduced can be flanked by CIaI sites by ligation of these adapters or by PCR reaction using oligonucleotides that include the recognition sequence for CIaI. If the exogenous gene of interest has internal CIaI sites, as with the
  • SUBSTITUTE SHEET (RULE 26) interleukin 12, NarI sites, which are compatible with CIaI, can be used.
  • SEQ ID NO 16 BElA: 5 'ATCGATCACCTCCGGTACAAGGTTTGG 3'.
  • the AB and BC fragments have a homology region, so that if they are mixed they can partially hybridize. This region has been designed to exclude the bases between position 922 and 947. If the hybridization of these two fragments is used as a template for a PCR reaction with oligonucleotides AElA and
  • SUBSTITUTE SHEET (RULE 26) incorporated the PCR fragment.
  • This intermediate plasmid was named pSdlElA, which has incorporated the modified ElA gene but has lost the 9XHRE promoter and the sequence between bases 9197 and 33756.
  • the 9XHRE promoter was reintroduced using the BstBI site.
  • the 14.8 kb fragment comprised between the Sdal sites was initially subcloned. A triple ligation was then carried out to fuse 3 fragments between RsrII sites that restore the modified viral genome.
  • the 14 kb fragment comes from the plasmid that contains the deletion in the CR2 domain of ElA and the 9XHRE promoter, while the 17 kb fragment (from plasmid pSHE2F-Luc) contains the luciferase gene in the E3 region. Finally, the genome is completed with the 7.7 kb fragment also from pSHE2F-Luc. The plasmid resulting from this fusion was called pSDHE2F-Luc. .- Construction of pAdLuc. For this, the 12.5 kb fragment between the Ndel sites of plasmid pSHE2F-Luc was obtained and introduced into the homonymous sites of plasmid pTG3602.
  • SUBSTITUTE SHEET (RULE 26) CIaI recognition.
  • the oligonucleotides used were: SEQ ID NO 20:
  • 5CIaTK 5 'GTACTATCGATGCTAGCCACCATGGCTTCGTACC 3' and SEQ ID NO 21:
  • 3CIaTK 5 'GTACTATCGATAAGCTTAAGTCAGTTAGCCTCC 3' This fragment was digested with CIaI and subcloned into a plasmid based on pSDHE2F-Luc, in which the E3 deletion is flanked by CIaI sites, as described in the previous section. In this way the luciferase gene is replaced by the TK gene. .- Construction of pSDHE2F-IL12.
  • 5NARIL12 5 'GTACTGGCGCCACCATGGGTCCTCAGAAGCTAACC 3'
  • 3NarIL12 5 'GTACTGGCGCCTAATCCGGATCAATTCTCAGG 3' This fragment was digested with NarI and subcloned into the CIaI site of the pro-viral plasmid described above. In this way the luciferase gene is replaced by the interleukin 12 gene.
  • SUBSTITUTE SHEET (RULE 26) The general structure of the modified plasmids is depicted in Figure 1, and a genome skeleton of the recombinant viruses is shown in Figure 2.
  • the plasmids described above were digested with the enzyme Pac ⁇ , which releases the viral genome from the rest of the bacterial sequences, and transfected into 293 cells (ATCC CRL-1573) by the calcium phosphate precipitation method. Once the cytopathic effect was observed (typically 7-10 days after transfection), the cells were used by 3 consecutive cycles of freezing and thawing. Serial dilutions of the lysate were made and with them A549 cells (ATCC CCL-185; from human lung cancer) were infected under hypoxic conditions (1% oxygen). In this way several clones of the viruses were obtained, which were verified by PCR.
  • the new viruses obtained from plasmids pSHE2F-Luc, pSDHHE2F-Luc, pSDHE2F-TK, pSDHE2F-IL12 and pAdLuc were named AdHLuc, AdDHLuc, AdDHTK, AdDHIL12 and AdWTLuc, respectively.
  • viruses were amplified in A549 cells maintained under hypoxia in the case of AdHLuc, AdDHLuc,
  • AdDHTK AdDHTK, AdDHIL12, and in 293 cells in the case of AdWTLuc. Purification was carried out by standard techniques using a Cesium gradient and subsequent passage through an exclusion column. The virus obtained was stored in a medium buffered with 10% glycerol at -80 ° C
  • AdDHLuc virus The activity of the AdDHLuc virus against control viruses in different cell types was analyzed, both in normal conditions and in hypoxia.
  • the main feature sought in CRAds is their ability to selectively induce the death of tumor cells.
  • SUBSTITUTE SHEET (RULE 26) 2. At 24 hours, infection with the virus to be tested (one type of virus per well, eg AdWT, AdHLuc or AdDHLuc); The viral concentration was adjusted to each cell line based on the infectivity for that line. 3. Incubation at 37 ° C under hypoxic conditions (chamber adjusted to a gaseous composition of 93% N2 / 6% CO2 / 1% O 2 ) or normoxia (environmental gaseous composition).
  • Huh-7 tumor cells (Nakabayashi et al. 1982 were infected. Cancer Res., 42: 3858-3863; from human hepatocarcinoma) and A549 (ATTC CCL-185; from human lung cancer), and normal IMR cells -90 (ATCC CCL-186; from primary human lung fibroblasts) with the various viruses.
  • AdHLuc has a cytotoxic potency equal to
  • AdHLuc and close to AdWT on tumor cells are advantageously it is strongly attenuated in normal cells.
  • SUBSTITUTE SHEET (RULE 26) (ATCC CCL-75.1), which have been maligned by the expression of SV40 T antigen, which blocks the pRB path. WI38-VA13 cells have impaired cell cycle control, and therefore it is expected that the AdDHLuc virus is not attenuated with respect to AdHLuc. Likewise, tests were carried out with normal BJ cells (ATCC CRL-2522; from primary human skin fibroblasts).
  • FIG 4 shows photomicrographs of normal WI-38 and malignant WI38-VA13 fibroblasts infected with the different viruses under hypoxia or normoxia conditions.
  • the cells that are suffering from the cytopathic effect of the virus are distinguished because they lose adhesion to the culture plate and acquire a rounded morphology.
  • AdDHLuc is the virus that is most attenuated in normal cells, while retaining its ability to kill malignant cells.
  • AdHLuc which confirms greater attenuation and the operation of the double control system for its replication. It is important to note that in this type of experiments the decrease in cell viability at low doses is dependent on viral amplification, so this test also indirectly reflects the control of virus replication.
  • AdDHLuc replication In order to directly analyze the control of AdDHLuc replication, A549 cells were infected with the virus under normoxia or hypoxia conditions, and the production of new infective particles was quantified after days.
  • the cells were seeded, infected and grown in the same manner as in the tests to evaluate the cytopathic effect. In this case, 4 days after infection, the cells were used by 3 freeze-thaw cycles and the infective particles were quantified by limit dilution in 293 cells or by immunohistochemistry with anti-adenovirus antibodies (Adeno-X Rapad Titer Kit , BD Biosciences Clontech Cat. No. K1653-1) following the instructions
  • hypoxia activated the amplification of the AdDHLuc virus, while the same does not happen with AdWT, whose replication did not undergo changes in these experimental conditions.
  • luciferase reporter gene in the E3 region of the AdDHLuc virus allows monitoring of the expression of exogenous genes introduced into the virus.
  • luciferase expression is increased in response to viral replication, due to the activation of late promoters and the increase in the number of copies of the viral genome in the cell. Therefore, measuring luciferase activity is an indicator of viral replication.
  • Huh-7 cells were seeded and infected with the AdDHLuc, AdHLuc, AdWTLuc replicative viruses, and with the Ad-CMV-Luc defective virus (Vector Biolabs Cat. No. 1000).
  • the dose of virus used (MOI) was 10 viruses / cell, and the cells were cultured in the same manner as in the tests for cytopathic effect evaluation.
  • Two days after infection, the cells were used to measure Luciferase activity using a commercial kit (Luciferase Assay System, Promega Cat. No. E4030), following the manufacturer's instructions. The activity was expressed as RLü / ⁇ g of total protein (RLU: Relative Luciferase Units).
  • Example 3 Characterization of the AdDHTK virus In v ⁇ tro
  • the cytopathic effect obtained after infection of the A549 cells, in the presence or absence of the Ganciclovir pro-drug (GCV) was analyzed.
  • the test was performed as described in example 2, with the following modifications.
  • the cells were infected with different AdDHTK MOIs under conditions of normoxia or hypoxia, and 24 hours later the infectious medium was removed and two types of wells were differentiated. In one of them standard culture medium with 2% fetal bovine serum was added, and in the other GCV (100 ⁇ M) was included. Cell survival was quantified 5 days later.
  • Figure 9 shows the data obtained when A549 cells were infected with an MOI of 0.12 virus / cell. As can be seen, this small amount of virus did not significantly decrease the survival of tumor cells under conditions
  • HeLa cells (ATTC CCL-2) were seeded in 24-well plates (2xlO 4 cells / well), and infected with the AdDHIL12 virus under normoxia or hypoxia conditions. Since IL12 is a secretable protein, its production in the culture medium was quantified at different times after infection. The detection of murine IL12 was carried out by a commercial ELISA kit (BD
  • IL12 practically does not increase under normoxia conditions, but there is a significant increase at 3 days if the cells are maintained in hypoxia conditions.
  • human tumor xenotransplants were performed in atomic mice (immunosuppressed) by subcutaneous injection (IxIO 7 cells in 150 ⁇ l saline serum) or intrahepatic (1.5 x 6 cells in 50 ⁇ l serum) of Huh-7 cells.
  • the virus to be tested AdDHLuc, Ad-CMV-Luc and AdWTLuc
  • the measurement of luciferase activity was started, performed daily for 10 days.
  • the luciferin substrate was administered intraperitoneally.
  • the expression kinetics and biodistribution of viruses can be monitored in live animals by measuring the emission of light using a high-sensitivity luminometric camera (In vivo imaging system, Xenogen). Under these conditions, the light emission
  • a defective adenovirus such as Ad-CMV-Luc maintains the initial expression levels during the first week and then initiates a slow decrease.
  • AdDHLuc showed a significant increase in luciferase activity during the first 4 days.
  • Figure HB illustrates the luciferase activity relative to the first day post-infection and shows how only in the case of AdDHLuc there is a significant increase in successive days. This is compatible with the achievement of several replication cycles in the
  • Huh-7 cells were injected into the liver of the mice and subsequently the virus was administered (10 9 i.).
  • the virus was administered (10 9 i.).
  • figure HC it can be observed how very high levels of expression are achieved and the increase in the first days is confirmed and then stabilized and a slow descent begins.
  • AdDHLuc is a CRAd capable of serving as an expression vector for exogenous genes with efficacy superior to non-replicative adenoviruses.

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Abstract

L'invention concerne un adénovirus recombinant de réplication conditionnée, caractérisé en ce qu'il comprend un promoteur uni au gène E1A qui comprend des éléments de réponse à l'hypoxie, un promoteur uni à la région E4 qui comprend au moins un élément de réponse au facteur E2F, une délétion dans le domaine CR2 du gène ElA et un gène exogène d'intérêt. L'invention concerne également un procédé permettant d'obtenir ledit adénovirus et une composition comprenant ce dernier.
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US9345787B2 (en) 2008-12-22 2016-05-24 Targovax Oy Adenoviral vectors and methods and uses related thereto
CN102264760A (zh) * 2008-12-22 2011-11-30 昂克斯治疗有限公司 溶瘤性腺病毒载体及与其相关的方法和用途
RU2520823C2 (ru) * 2008-12-22 2014-06-27 Онкос Терапьютикс Ой Аденовирусные векторы и способы и применения, связанные с ними
CN102264760B (zh) * 2008-12-22 2017-03-22 昂克斯治疗有限公司 溶瘤性腺病毒载体及与其相关的方法和用途
AU2009332883B2 (en) * 2008-12-22 2015-05-21 Oncos Therapeutics Oy Oncolytic adenoviral vectors and methods and uses related thereto
WO2010072900A1 (fr) * 2008-12-22 2010-07-01 Oncos Therapeutics Vecteurs adénoviraux oncolytiques, leurs procédés et leurs utilisations
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
CN105307671A (zh) * 2013-04-18 2016-02-03 蒂尔坦生物制药有限公司 增强过继细胞疗法
WO2014170389A1 (fr) * 2013-04-18 2014-10-23 Tilt Biotherapeutics Oy Thérapie cellulaire adoptive améliorée
AU2014255733B2 (en) * 2013-04-18 2019-05-16 Tilt Biotherapeutics Oy Enhanced adoptive cell therapy
US10647963B2 (en) 2013-04-18 2020-05-12 Tilt Biotherapeutics Oy Enhanced adoptive cell therapy
CN105307671B (zh) * 2013-04-18 2020-09-04 蒂尔坦生物制药有限公司 增强过继细胞疗法
CN111658670A (zh) * 2013-04-18 2020-09-15 蒂尔坦生物制药有限公司 溶瘤腺病毒载体与过继t细胞治疗组合物及其用途
US10787645B2 (en) 2013-04-18 2020-09-29 Tilt Biotherapeutics Oy Enhanced adoptive cell therapy
CN111658670B (zh) * 2013-04-18 2024-10-01 蒂尔坦生物制药有限公司 溶瘤腺病毒载体与过继t细胞治疗组合物及其用途

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