WO2000029599A1 - Vecteurs viraux a expression tardive de transgenes - Google Patents

Vecteurs viraux a expression tardive de transgenes Download PDF

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WO2000029599A1
WO2000029599A1 PCT/US1999/026004 US9926004W WO0029599A1 WO 2000029599 A1 WO2000029599 A1 WO 2000029599A1 US 9926004 W US9926004 W US 9926004W WO 0029599 A1 WO0029599 A1 WO 0029599A1
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virus
cell
gene
cells
replication
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Kenneth N. Wills
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Canji, Inc.
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Priority to AU23441/00A priority Critical patent/AU2344100A/en
Priority to CA002351587A priority patent/CA2351587A1/fr
Priority to JP2000582580A priority patent/JP2002530085A/ja
Priority to EP99967094A priority patent/EP1131458A1/fr
Publication of WO2000029599A1 publication Critical patent/WO2000029599A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • C12N2830/60—Vector systems having a special element relevant for transcription from viruses
    • 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
    • C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
    • C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian

Definitions

  • the genome of the adenovirus has been well characterized. This information has been used to design recombinant adenoviruses capable of acting as vectors for the introduction of exogenous DNA into target cells. Many of these viral vectors contain modifications to the early gene products to endow the vectors with specific activities. For example, the early genes El and E2, are transcribed early after infection. The products of these genes are responsible for the suppressing the ability of the infected cell to respond to the infection and to promote viral replication in the host.
  • the immediate early gene El a is transcribed rapidly following infection.
  • the Ela product can immortalize primary cells in vivo.
  • Ela has also been show to bind a cellular protein pl05 RB, the product of the retinoblastoma gene.
  • the Elb gene is not capable of transforming cells on its own, but cooperates with Ela to stably transform cells. Both Ela and Elb are necessary for the full transformation and tumor formation in animals.
  • the Ela gene has a variety of functions. The full scope of activity of the adenovirus Ela region is described in Bayley, S. and Mymryk, J. (1994) Intl. J. of Oncology 5:425-444.
  • the Elb genes are known to interact with host cell proteins. In particular two proteins are produced by differential splicing of the Elb sequence, pl9 and p55.
  • the p55 protein has been well characterized as interacting with the p53 gene product.
  • p53 is a well characterized protein and has been shown to activate programmed cell death (PCD) apoptotic pathways when present in sufficient intracellular concentration.
  • PCD programmed cell death
  • p53 induced apoptosis has been shown to take place in response to a wide variety of cell injury including radiation, DNA damaging agents, etc. By binding to p53, p55 prevents the formation of the active p53 phosphorylated tetramer.
  • Ela and Elb cooperate in their activities. In particular Ela has been shown to induce p53 dependent and p53 independent apoptosis. In addition to inducing other early viral genes, Ela drives quiescent cell into S-phase which is required for viral replication. It is believed that this early pressure to enter S-phase by the virus on the quiescent cell induces a apoptotic response from the cell.
  • the adenovirus responds by the expression of the Elb 55K protein to bind to p53 and delay the induction of apoptosis so that the virus is able to replicate prior to the death of its host.
  • Adenovirus also produced the E1B19K protein which prevents or delays Ela mediated apoptosis as well.
  • Alternative to this type of selectively replicating vector is the employment of a replication deficient adenoviral vector containing extensive elimination of El function.
  • vectors containing elimination of El, E3 and partial E4 deletions have been employed to delivery exogenous transgenes. Such vectors have been employed to deliver the p53 gene to target cells.
  • Replication deficient and selectively replicating vectors have, at least in theory, design drawbacks which are of concern to clinicians. Because the replication deficient vectors will not propagate uncontrollably in the patient, they have a more theoretically appealing safety profile. However, as effective tumor elimination requires the infection of the substantial majority of the tumor cells being infected, a substantial molar excess of vector is commonly used to insure therapeutic effectiveness. Selectively replicating vectors are viewed as being more of an issue from a safety perspective because of their ability to replicate and potentially mutate to form fully replication competent vectors in the patient. However, by exploiting the natural ability to the virus to propagate under particular conditions enables these vectors to spread to surrounding tumor cells. Since the vectors themselves are able to replicate, a lower initial dose of such vectors is required. This is favorable from an immunological perspective as well as for economic reasons in the manufacture of such agents.
  • the adenoviral replicative cycle in human cells can be divided into the early and late phase which are punctuated by the onset of viral DNA replication.
  • the early phase beings when viral particles attach to cells through interaction between the virion fiber domain and cell surface receptors.
  • the virion moves into the cell by either endocytosis or direct penetration of the cytoplasmic membrane and is transported to the nucleus where most of the capsid is shed. In the nucleus, the virion core proteins are removed yielding viral chromosomes that are almost entirely devoid of virion proteins.
  • Expression of the viral genome is temporally coordinated and begins with the Ela region about one hour after infection.
  • the other early genes Elb, E2, E3 and E4 are first express soon after Ela at the 1.5-2.0 hours post infection, A number of the protein products encoded by the early genes are required for viral DNA replication, while other prepare the DNA synthesis machinery of the infected cell for efficient viral DNA replication. Some early virally encoded proteins have been associated with protecting infected cells from immune surveillance.
  • the messenger RNAs for all late gene products are spliced from a primary RNA which is transcribed from the major late promoter (MLP).
  • MLP major late promoter
  • the MLP is located at position 16.5 on the r-strand.
  • the major late promoter is active to a limited extent in the early phase of infection, the transcription does not proceed past map position 39.
  • the MLP is fully activated and continues to map position 99.
  • Each late primary RNA transcript is processed into one of five different mRNAs, L1-L5. These mRNAis all contain a common tripartite leader sequence of 203 nucleotides.
  • Late mRNAs encode capsid components and proteins required for assembly of virions and packaging of the viral chromosome.
  • Viral DNA replication requires the terminal protein for initiation and proceed by a semi-conservative mechanism. With the onset of replication, efficient transcription of the late gene families from the major late promoter begins and attains a maximal level approximately 18 hours post infection. During the late phase viral proteins block cellular DNA and protein synthesis, presumably so that maximum viral macromolecular synthesis can occur. Intermediate gene expression, which actually begins during the early phase, reaches a maximum between 8 -12 hour post infection. Assembly of the virion and packaging of the viral genome begins at about 24 hours after infection. Infected cells are killed because of attrition and lyse yielding approximately 10,000 virions per cell.
  • the present invention provides a conditionally replicating recombinant virus containing a therapeutic transgene under control of a late regulatory element.
  • the invention further provides pharmaceutical formulations and methods of use of same.
  • the present invention further provides recombinant producer cells capable of complementing the packaging genes defective or deleted in the vector.
  • the present invention also provides method of making such vectors and formulations.
  • FIGURES Figure 1 is an autoradiogram of a time course study comparing the levels of p53 expression of a replication competent MLP-p53 (Ad5dElbMLPp53) virus in comparison to the replication deficient p53 vector (ACN53) at two different concentrations (1.8 x 10 8 particles/ml (upper panel) and 1.8 x 10 9 particles/ml (lower panel) in MRC9 cells according to the procedures of Example 2.
  • ACN53 replication deficient MLP-p53
  • the replication competent MLP-p53 construct resulted in expression later in time than the replication deficient CMV-p53 virus (ACN53).
  • FIG. 2 presents the results of an experiment substantially similar to that presented in Figure 1 except that the experiment was conducted in SK-HEP1 hepatocellular carcinoma cells according to the procedures of Example 2.b. herein. Again, the time course experiment demonstrates the temporal and greater expression of p53 in the replication competent MLP-p53 construct.
  • Figure 3 presents the results of an experiment substantially identical to that presented in Figure 1 except that the experiment was conducted in NCI H358 breast cancer cells according to the procedures of Example 2.c. herein. Again, the time course experiment demonstrates the temporal expression of p53 in the replication competent MLP-p53 construct.
  • Figure 4 presents the results of an experiment similar to that presented in Figure 3 except that a replication competent Elbdl55k-CMV-p53 virus was included for comparison and only a single dose was administered according to the procedures of Example 2.d. herein.
  • This time course experiment demonstrates the temporal expression of p53 in the replication competent MLP-p53 construct as compared to the substantially similar construct wherein the p53 gene was under control of the constituitive CMV promoter. This data confirms that the MLP-p53 construct is indeed expressing the p53 in a temporal manner late in the viral replication cycle.
  • Figure 5 is a digest of viral DNA from SK-BR3 cells infected with a one hour pulse of the indicated viruses at a concentration of 1.8 x 10 9 particles/ml and harvested approximately 48 hours later according to the procedures of Example 2.d. herein.
  • the results presented demonstrate that replication competent wild-type Ad5 (Ad5WT), replication competent ElBdl55K (ZAZA) virus and replication competent ElBdl55K- MLP-p53 (55K/MLP53) virus all replicate their viral DNA well while the replication deficient adenovirus control (rAdcon) and the replication deficient vector encoding p53 (FTCB) does not.
  • Figure 6 is a graphical representation of the data obtained in vivo in a PC-3 mouse tumor model.
  • Tumor volume is plotted on the vertical axis and days following administration is plotted on the horizontal axis.
  • the replicating virus ElBdl55K-MLP-p53 (cFAMA) was able to produce tumor regression in an in vivo mouse model of human cancer.
  • the replication competent CMV driven p53 virus also replicates its viral DNA, but to a lesser extent.
  • the present invention provides a replication competent recombinant virus containing a therapeutic transgene operably linked to a late regulatory element.
  • Replication Competent Recombinant Virus The term "replication competent" is made in reference to a virus which is capable of replicating its genome and packaging the replicated viral genome into infectious particles in mammalian cells. It should be noted that the term replication competent does not generally apply to virus that can only be grown in cells which have been modified to provide deleted viral functions in trans.
  • recombinant virus refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism capable of infecting a mammalian cell whose genomes have been modified by conventional recombinant DNA techniques.
  • the viral genome may be RNA or DNA contained with a coated structure of protein of a lipid membrane.
  • virus(es) and viral vector(s) are used interchangeably herein.
  • viruses useful in the practice of the present invention include recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxviridae, adenoviridiae, or picornaviridiae.
  • Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et al.( ⁇ 991) Nature Biotechnology 15:866-870) may also be useful in the practice of the present invention.
  • Minimal vector systems in which the viral backbone contains only the sequences need for packaging of the viral vector and may optionally include a transgene expression cassette may also be produced according to the practice of the present invention.
  • equine herpes virus vectors for human gene therapy are described in WO98/27216 published August 5, 1998.
  • the vectors are described as useful for the treatment of humans as the equine virus is not pathogenic to humans.
  • ovine adenoviral vectors may be used in human gene therapy as they are claimed to avoid the antibodies against the human adenoviral vectors. Such vectors are described in WO 97/06826 published April 10, 1997.
  • the virus is an adenovirus.
  • adenovirus is synonomous with the term “adenoviral vector” and refers to viruses of the genus adenoviridiae.
  • adenoviridiae refers collectively to animal adenoviruses of the genus mastadenovirus including but no limited to human, bovine, ovine, equine, canine, porcine, murine and simian adenovirus subgenera.
  • human adenoviruses includes the A-F sugenera as well as the individual serotypes thereof the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adl lA and Ad I IP), 12, 13,14,15,16,17,18,19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91.
  • bovine adenoviruses includes but is not limited to bovine adenovirus types
  • canine adenoviruses includes but is not limited to canine types 1 (strains CLL, Glaxo, RI261, Utrect, Toronto 26-61) and 2.
  • equine adenoviruses includes but is not limited to equine types 1 and 2.
  • porcine adenoviruses includes but is not limited to porcine types 3 and 4.
  • recombinant adenovirus also includes chimeric (or even multimeric) vectors, i.e. vectors constructed using complementary coding sequences from more than one viral subtype. See, e.g. Feng, et al. Nature Biotechnology 15:866-870.
  • the recombinant adenoviral vector is derived from genus adenoviridiae.
  • Particularly preferred viruses are derived from the human adenovirus type 2 or type 5.
  • the preferred vector is derived from the human adenoviridiae. More preferred are vectors derived from human adenovirus subgroup C. Most preferred are adenoviral vectors derived from the human adenovirus serotypes 2 and 5.
  • the virus is derived human adenovirus Type 5 dl309. d!327, dl520 or wild-type adenovirus.
  • late regulatory element refers to regulatory element which drives transcription of the therapeutic transgene a point later in time than the element which induces initial viral replication. Characteristically, these promoter elements are found driving expression of packaging proteins and other proteins late in the viral life cycle.
  • An example of such late regulatory element when the parent vector is adenovirus is the adenovirus major late promoter (MLP).
  • MLP adenovirus major late promoter
  • Other viral vector systems also possess late temporally regulated promoters.
  • the AcNPV basic gene promoter and the polyhedrin gene promoters may be employed (Sridhar, et al. (1993) FEBS Lett. 315:282-286.
  • the Latent Activated Promoters may be employed. See, e.g.
  • the HPV31b promoter may be employed (Ozbun and Meyers (1998) J. Virol. 72:2715-22).
  • the P39 promoter For parvoviruses, the P39 promoter.
  • the pi 1 promoter For vaccinia virus, the pi 1 promoter.
  • the virus is derived from the genus adenoviridiae and the late regulatory element is the adenoviral Major Late Promoter.
  • the Major Late Promoter is well characterized in the art and resides at approximately map position 16.5 of the adenoviral Type 2 genome.
  • operably linked refers to a linkage of polynucleotide elements in a functional relationship.
  • a nucleic acid sequence is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • Operably linked means that the nucleotide sequences being linked are typically contiguous. However, as enhancers generally function when seperated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.
  • therapeutic transgene refers to a nucleotide sequence the expression of which in the target cell produces a therapeutic effect.
  • therapeutic transgene includes but is not limited to tumor suppressor genes, antigenic genes, cytotoxic genes, cytostatic genes, pro-drug activating genes, apoptotic genes, pharmaceutical genes or anti-angiogenic genes.
  • the vectors of the present invention may be used to produce one or more therapeutic transgenes, either in tandem through the use of IRES elements or through independently regulated promoters.
  • tumor suppressor gene refers to a nucleotide sequence, the expression of which in the target cell is capable of suppressing the neoplastic phenotype and/or inducing apoptosis.
  • tumor suppressor genes useful in the practice of the present invention include the p53 gene, the APC gene, the DPC-4 gene, the
  • BRCA-1 gene the BRCA-2 gene, the WT-1 gene, the retinoblastoma gene (Lee, et al . (1987) Nature 329:642), the MMAC-1 gene, the adenomatous polyposis coli protein (Albertsen, et al ., United States Patent 5,783,666 issued July 21, 1998), the deleted in colon carcinoma (DCC) gene, the MMSC-2 gene, the NF-1 gene, nasopharyngeal carcinoma tumor suppressor gene that maps at chromosome 3p21.3. (Cheng, et al . 1998. Proc. Nat. Acad. Sci. 95:3042-3047), the MTS 1 gene, the CDK4 gene, the NF- 1 gene, the NF2 gene, and the VHL gene.
  • DCC colon carcinoma
  • antigenic genes refers to a nucleotide sequence, the expression of which in the target cells results in the production of a cell surface antigenic protein capable of recognition by the immune system.
  • antigenic genes include carcinoembryonic antigen (CEA), p53 (as described in Levine, A. PCT International Publication No. WO94/02167 published February 3, 1994).
  • CEA carcinoembryonic antigen
  • p53 as described in Levine, A. PCT International Publication No. WO94/02167 published February 3, 1994.
  • the antigenic gene may be fused to the MHC class I antigen.
  • Cytotoxic gene refers to nucleotide sequence, the expression of which in a cell produces a toxic effect. Examples of such cytotoxic genes include nucleotide sequences encoding pseudomonas exotoxin, ricin toxin, diptheria toxin, and the like.
  • Cytostatic gene refers to nucleotide sequence, the expression of which in a cell produces an arrest in the cell cycle.
  • examples of such cytostatic genes include p21, the retinoblastoma gene, the E2F-Rb gene, genes encoding cyclin dependent kinase inhibitors such as P16, pl5, pl8 and pl9, the growth arrest specific homeobox (GAX) gene as described in Branellec, et al . (PCT Publication WO97/16459 published May 9, 1997 and PCT Publication WO96/30385 published October 3, 1996) .
  • cytokine gene refers to a nucleotide sequence, the expression of which in a cell produces a cytokine.
  • cytokines include GM-CSF, the interleukins, especially LL-1, IL-2, LL-4, LL-12, IL-10, IL-19, LL-20, interferons of the alpha, beta and gamma subtypes especially interferon ⁇ -2b and fusions such as interferon ⁇ -2 -l.
  • chemokine gene refers to a nucleotide sequence, the expression of which in a cell produces a cytokine.
  • chemokine refers to a group of structurally related low-molecular cytokines weight factors secreted by cells are structurally related having mitogenic, chemotactic or inflammatory activities. They are primarily cationic proteins of 70 to 100 amino acid residues that share four conserved cysteine residues. These proteins can be sorted into two groups based on the spacing of the two amino-terminal cysteines. In the first group, the two cysteines are separated by a single residue (C-x-C), while in the second group, they are adjacent (C-C).
  • Examples of member of the 'C-x-C chemokines include but are not limited to platelet factor 4 (PF4), platelet basic protein (PBP), interleukin-8 (LL-8), melanoma growth stimulatory activity protein (MGSA), macrophage inflammatory protein 2 (MIP-2), mouse Mig (ml 19), chicken 9E3 (or pCEF-4), pig alveolar macrophage chemotactic factors I and I (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF), and IP 10.
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • LL-8 interleukin-8
  • MGSA melanoma growth stimulatory activity protein
  • MIP-2 macrophage inflammatory protein 2
  • mouse Mig mouse 19
  • chicken 9E3 or pCEF-4
  • pig alveolar macrophage chemotactic factors I and I AMCF-I and -II
  • PBSF pre-B cell growth stimulating factor
  • Examples of members of the 'C-C group include but are not limited to monocyte chemotactic protein 1 (MCP-1), monocyte chemotactic protein 2 (MCP-2), monocyte chemotactic protein 3 (MCP-3), monocyte chemotactic protein 4 (MCP-4), macrophage inflammatory protein 1 ⁇ (MIP-l- ⁇ ), macrophage inflammatory protein 1 ⁇ (MTP-l- ⁇ ), macrophage inflammatory protein 1 ⁇ (MLP- 1- ⁇ ), macrophage inflammatory protein 3- (MIP-3- ⁇ , macrophage inflammatory protein 3 ⁇ (MIP-3- ⁇ ), chemokine (ELC), macrophage inflammatory protein 4 (MLP-4), macrophage inflammatory protein 5 (MIP-5), LD78 ⁇ , RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, 1-309, human protein HCC-1/NCC- 2, human protein HCC-3, mouse protein CIO
  • pharmaceutical protein gene refers to nucleotide sequence, the expression of which results in the production of protein have pharmaceutically effect in the target cell.
  • examples of such pharmceutical genes include the proinsulin gene and analogs (as described in PCT International Patent Application No. WO98/31397, growth hormone gene, dopamine, serotonin, epidermal growth factor, GABA, ACTH, NGF, VEGF (to increase blood perfusion to target tissue, induce angiogenesis, PCT publication WO98/32859 published July 30, 1998), thrombospondin etc.
  • pro-apoptotic gene refers to a nucleotide sequence, the expression thereof results in the programmed cell death of the cell.
  • pro-apoptotic genes include p53, adenovirus E3-11.6K, the adenovirus E4orf4 gene, p53 pathway genes, and genes encoding the caspases.
  • the pl6 gene is also apoptotic in Rb positive, pl6 negative, p53 wild type tumors. (Frizelle, et al . (1998) Oncogene 16:3087-95 and Sandig, et al. (1997) Nature Medicine 3:313) L Pro-Drug Activating Genes:
  • pro-drug activating genes refers to nucleotide sequences, the expression of which, results in the production of protein capable of converting a non- therapeutic compound into a therapeutic compound, which renders the cell susceptible to killing by external factors or causes a toxic condition in the cell.
  • An example of a prodrug activating gene is the cytosine deaminase gene. Cytosine deaminase converts 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU), a potent antitumor agent.
  • the lysis of the tumor cell provides a localized burst of cytosine deaminase capable of converting 5FC to 5FU at the localized point of the tumor resulting in the killing of many surrounding tumor cells.
  • the thymidine kinase (TK) gene in which the cells expressing the TK gene product are susceptible to selective killing by the administration of gancyclovir may be employed.
  • anti-angiogenic genes refers to a nucleotide sequence, the expression of which results in the extracellular secretion of anti-angiogenic factors.
  • Anti- angiogenesis factors include angiostatin, inhibitors of vascular endothelial growth factor (VEGF) such as Tie 2 (as described in PNAS(USA)(1998) 95:8795-8800), and endostatin.
  • VEGF vascular endothelial growth factor
  • modifications and or deletions to the above referenced genes so as to encode functional subfragments of the wild type protein may be readily adapted for use in the practice of the present invention.
  • modifications refers to changes in the genomic structure of the recombinant adenoviral vector. Such modifications include deletions and/or changes in amino acid coding sequence so as to produce a protein deficient in binding to its substrate.
  • the reference to the p53 gene includes not only the wild type protein but also modified p53 proteins, allelic variations thereof, or proteins derived from other mammalian species.
  • the wild-type p53 sequence is well known in the art.
  • Other mammalian p53 molecules are also known in the art and may be incorporated into the practice of the present invention such as murine p53, porcine p53, equine p53, bovine p53, canine p53, etc.
  • modified p53 proteins refers to modifications to the primary, secondary, tertiary, or quaternary structure of the p53 protein which retains the function of p53 proteins.
  • modified p53 proteins include modifications to p53 to increase nuclear retention, deletions such as the ⁇ 13-19 amino acids to eliminate the calpain consensus cleavage site, modifications to the oligomerization domains (as described in Bracco, et al. PCT published application WO97/0492 or United States Patent No. 5,573,925).
  • the p53 sequence may be modified to replace the endogenous tetramerization domains with a leucine zipper oligermization domain.
  • therapeutic transgene may be secreted into the media or localized to particular intracellular locations by inclusion of a targeting moiety such as a signal peptide or nuclear localization signal (NLS).
  • a targeting moiety such as a signal peptide or nuclear localization signal (NLS).
  • transciptionally active proteins e.g. p53
  • the present invention also provides recombinant adenoviruses containing additional modifications to the viral genome such as targeting modifications, modifications to make the vectors replicate selectively in particular cell types or phenotypic states, controlled expression characteristics, suicide genes or additional modifications to enhance cytotoxicity.
  • additional modifications to the viral genome such as targeting modifications, modifications to make the vectors replicate selectively in particular cell types or phenotypic states, controlled expression characteristics, suicide genes or additional modifications to enhance cytotoxicity.
  • additional modifications to the viral genome such as targeting modifications, modifications to make the vectors replicate selectively in particular cell types or phenotypic states, controlled expression characteristics, suicide genes or additional modifications to enhance cytotoxicity.
  • replication control elements refers to DNA sequences inserted into the viral genome or modifications to the viral genome in order to produce recombinant viral vectors which selectively replicate in one cell type versus another cell type, in a cell in one phenotypic state relative to another phenotypic state (cell state specific), or in a given cell type in response to an external stimuli (inducible). Examples of such replication control elements include cell-type specific promoter, cell state specific promoters, and inducible promoters.
  • Cell type specific replication may be achieved by the linkage of a cell type specific promoter to an early viral gene such as the El, Ela, E2 or E4 gene when the virus is selected from the adenovirus genome.
  • the term "cell type specific promoter” refers to promoters which are differentially activated in as a result of cell cycle progression or in different cell types. Examples of cell-type specific promoters includes cell cycle regulatory gene promoters, tissue specific promoters or pathway responsive promoters.
  • the cell type specific promoter is linked to the E4 gene rather than the El gene because factors such as NF-LL6 can substitute for Ela in regulating Ela responsive promoters in the adenovirus in the absence of Ela function (Spergel, et al. (1992) J. Virol. 66:1021-1030).
  • cell cycle regulatory gene promoters describe promoters for genes which are activated substantially upon entry into S-phase. Examples of such promoters include the E2F regulated promoters (e.g. DHFR, DNA polymerase alpha, thymidylate synthase, c-myc and b-myb promoters).
  • E2F regulated promoters e.g. DHFR, DNA polymerase alpha, thymidylate synthase, c-myc and b-myb promoters.
  • Tissue specific promoters are well known in the art and include promoters active preferentially in smooth muscle ( -actin promoter), pancreas specific (Palmiter, et ⁇ Z.(1987) Cell 50:435), liver specific Rovet, et.al. (1992) J. Biol. Chem.. 267:20765; Lemaigne, et al. (1993) J. Biol. Chem.. 268:19896; Nitsch, et al. (1993) Mol. Cell. Biol. 13:4494), stomach specific (Kovarik, et al. (1993) J. Biol. Chem.. 268:9917, pituitary specific (Rhodes, et al. (1993) Genes Dev. 7:913, prostate specific (United States Patent 5,698,443, Henderson, et al. issued December 16, 1997), etc. c. Pathway Responsive Promoters
  • pathway-responsive promoter refers to DNA sequences that bind a certain protein and cause nearby genes to respond transcriptionally to the binding of the protein in normal cells. Such promoters may be generated by incorporating response elements which are sequences to which transcription factors bind. Such responses are generally inductive, though there are several cases where increasing protein levels decrease transcription. Pathway-responsive promoters may be naturally occurring or synthetic. Pathway-responsive promoters are typically constructed in reference to the pathway or a functional protein which is targeted. For example, a naturally occurring p53 pathway-responsive promoter would include transcriptional control elements activated by the presence of functional p53 such as the p21 or bax promoter. Alternatively, synthetic promoters containing p53 binding sites upstream of a minimal promoter (e.g.
  • Synthetic pathway-responsive promoters are generally constructed from one or more copies of a sequence that matches a consensus binding motif. Such consensus DNA binding motifs can readily be determined. Such consensus sequences are generally arranged as a direct or head-to-tail repeat separated by a few base pairs. Elements that include head-to-head repeats (e.g. AGGTCATGACCT) are called palindromes or inverted repeats and those with tail-to- tail repeats are called everted repeats.
  • T3 pathway-responsive promoters thyroid hormone pathway-responsive promoters containing the consensus motif: 5' AGGTCA 3.', the TPA pathway- responsive promoters (TREs), TGF- ⁇ pathway-responsive promoters (as described in Grotendorst, et al (1996) Cell Growth and Differentiation 7: 469-480).
  • TPA pathway- responsive promoters TREs
  • TGF- ⁇ pathway-responsive promoters as described in Grotendorst, et al (1996) Cell Growth and Differentiation 7: 469-480.
  • natural or synthetic E2F pathway responsive promoters may be used.
  • An example of an E2F pathway responsive promoter is described in Parr, et al. (1997), Nature Medicine 3 : 1145- 1149 which describes an E2F- 1 promoter containing 4 E2F binding sites and is reportedly active in tumor cells with rapid cycling.
  • pathway-responsive promoters are well known in the art and can be identified in the Database of Transcription Regulatory Regions on Eukaryotic Genomes accessible through the internet at http://www.eimb.rssi.ru/TRRD. ii. Preferred Pathway Promoters:
  • the vector comprises a synthetic TGF- ⁇ pathway-responsive promoter active in the presence of a functional TGF- ⁇ pathway such as the promoter containing SRE and PAI pathway responsive promoters.
  • PAI refers to a synthetic TGF- ⁇ pathway-responsive promoter comprising sequences responsive to TGF- ⁇ signally isolated from the plasminogen activator-I promoter region.
  • the PAI-pathway-responsive promoter may be isolated as a 749 base pair fragment isolatable from the plasmid p ⁇ OOluc (as described in Zonneveld, et al. (1988) PNAS 85:5525-5529 and available from GenBank under accession number J03836).
  • SRE refers to a synthetic TGF- ⁇ response element comprising a repeat of 4 of the Smad-4 DNA binding sequences (GTCTAGAC as described in Zawel, et al. (1988) Mol. Cell 1:611-617.
  • the SRE response element may be generated by annealing complimentary oligonucleotides encoding the Smad-4 binding sequences and cloning in plasmid pGL#3 - promoter luciferase vector (commercially available from ProMega).
  • a "p53 pathway-responsive promoter” refers to a transcriptional control element active in the presence of a functional p53 pathway.
  • the p53 pathway- responsive promoter may be a naturally occurring transcriptional control region active in the presence of a functional p53 pathway such as the p21 or mdm2 promoter.
  • the p53 pathway-responsive promoter may be a synthetic transcriptional control region active in the presence of a functional p53 pathway such as the SRE and PAI-RE pathway-responsive promoters.
  • p53-CON describes a p53 pathway- responsive promoter containing a synthetic p53 response element constructed by insertion of two synthetic p53 consensus DNA binding sequences (as described in Funk, et al.
  • RGC refers to a synthetic p53 pathway-responsive promoter using a tandem of the p53 binding domains identified in the ribosomal gene cluster.
  • p53CON and RGC response elements can be constructed by annealing complementary oligonucleotides and p53 responsive promoters can be constructed by cloning in plasmid pGL3-promoter luciferase vector (commercially available from ProMega).
  • d Tissue Specific Promoters operably linked to an early gene.
  • Tissue specific promoters are well known in the art and include promoters active preferentially in smooth muscle (alpha-actin promoter), pancreas specific (Palmiter, et al. (1987) Cell 50:435), liver specific Rovet, et.al. (1992) J. Biol. Chem.. 267:20765; Lemaigne, et al . (1993) J. Biol. Chem. 268:19896; Nitsch, et al . (1993) Mol. Cell. Biol. 13:4494), stomach specific (Kovarik, et al . (1993) J. Biol. Chem.. 268:9917, pituitary specific (Rhodes, et al . (1993) Genes Dev. 7:913 and prostate specific antigen promoter. 2. Cell State Specific:
  • phenotypic states examples include the neoplastic phenotype versus a normal phenotype in a given cell type.
  • Selective replication is achieved by the use of viral replication control elements.
  • the term viral replication control element refers to a DNA sequence engineered into the vector of the present invention such that the virus is preferentially enabled to replicate the viral genome in a particular type of target cell.
  • these cell state specific promoters may be linked to an early gene such as El, E2, or preferably E4 to achieve selective replication in response to specific phenotypic states.
  • tumor specific promoters refers to promoters which are active in tumor cells and inactive in cells which are not transformed. Examples of tumor specific promoters include the alpha-fetoprotein promoter, the tyrosinase promoter.
  • the use of tumor specific promoters to achieve conditional replication of adenoviral vectors is described in co- pending United States Patent Application 08/433,798 filed May 3, 1995 and International Patent Application No. PCT/US96/06199 published as International Publication No. WO 96/34969 on November 7, 1996 the entire teaching of which is herein incorporated by reference.
  • the alpha-fetoprotein promoter could be used to replace the endogenous E4 promoter and achieve greater selectivity in conditional replication .
  • Other factors such as NF-LL6 can substitute for Ela in regulating Ela responsive promoters in the adenovirus in the absence of Ela function (Spergel, et al. (1992) J. Viroll 66: 1021-1030) and this can be avoided by substitution of the E4 promoter with a tumor specific promoter.
  • repressor of viral replication refers to a protein, if expressed in a given cell, substantially represses viral replication.
  • the repressor of viral replication will be dependent on the nature of the parent adenoviral vector from which the recombinant vector of the present invention is derived. For example, in the case of adenoviral vectors or other DNA tumor viruses, the E2F-Rb fusion construct as described in European Patent Application No.
  • E2F-Rb fusion protein consists of the DNA binding and DPI heterodimerizations domains of the human E2F transcription factor protein (amino acids 95-286 of wild type E2F) fused to the Rb growth suppression domain (amino acids 379-928 of the wild type Rb protein).
  • the E2F-Rb fusion protein is a potent repressor of E2F-dependent transcription and arrests cells in Gl.
  • the DNA binding domain is located at amino acids 128-193 and the dimerization domain is located at 194-289.
  • the sequence of the human E2F-1 protein is available from GenBank under accession number M96577 deposited August 10, 1992.
  • E2F from other E2F family members of E2F from other species may be employed when constructing a vector for use in other species.
  • the rep protein and its derivates is an effective repressor of viral replication in the absence of adenovirus infection.
  • the virus is derived from herpes simplex virus, the ICPO-NX, a deleted form of the immediate early protein ICPO (Liun, et al. (1998) J. Virol. 72:7785-7795), protein may be used as an effective repressor of viral replication.
  • any protein with dominant negative activity can be used as a repressor of viral replication.
  • an E2F pathway responsive promoter may be used to drive expression of the modified Ela coding sequence.
  • E2F-Rb fusion protein Using a p53 pathway responsive promoter driving expression of E2F-Rb fusion protein, one achieves repression of both El function and E2 function because the E2F-Rb fusion protein will suppress both the E2 and E2F response elements.
  • the p53 response element In p53 deficient tumor cells, the p53 response element is inactive and E2F-Rb is not expressed.
  • inducible promoter refers to promoters which facilitate transcription of the therapeutic transgene preferable (or solely) under certain conditions and/or in response to external chemical or other stimuli.
  • inducible promoters are known in the scientific literature (See, e.g. Yoshida and Hamada (1997) Biochem. Biophys. Res. Comm. 230:426-430; Iida, et al . (1996) J. Virol. 70(9):6054-6059; Hwang, et al . (1997) J. Virol 71(9):7128-7131; Lee, et al . (1997) Mol. Cell. Biol. 17(9):5097-5105; and Dreher, et al . (1997) J.
  • Examples of radiation inducible promoters include those induced by ionizing radiation such as the Egr-1 promoter (as described in Manome, et al. (1998) Human Gene Therapy 9:1409-17; Takahashi, et al . (1997) Human Gene Therapy 8:827-833; Joki. et al. Human Gene Therapy (1995) 6:1507-1513; Boothman, et al.
  • ElBdl55K deletion As previously indicated the Elb 55K protein binds to p53. Consequently, in order to enhance the effect of the p53 introduced by the viral vector it is preferred to introduce Descriptions of ElBdl55K mutations to eliminate p53 binding described in McCormick, United States Patent No. 5,677,178 issued October 14, 1997, the entire teaching of which is herein incorporated by reference.
  • the virus is a recombinant adenoviral vector encoding p53 under control of the MLP promoter containing a deletion of nucleotides 2247-3272 of the adenoviral genome to eliminate the function of the Elb 55K protein.
  • E4 Modifications to increase the potency of the vectors of the present invention include but are not limited to alterations within Elb.
  • the vectors of the present invention may be modified to introduce mutations in E4 to increase the cytotoxicity (Muller, et al. (1992) J. Virol. 66:5867-5878) or contain upregulation of viral cytopathic proteins such as E4orf4 or E3 11.6K proteins.
  • E4 region of the adenovirus genome has been implicated in viral DNA replication, host protein synthesis shut off and viral assembly.
  • E4orf6 is sufficient for DNA replication and late protein synthesis in immortalized cells. However, E4orf6/7 appears to be required for replication in non-dividing cells.
  • E4orf6/7 assists in restricting the replication of the virus in immortalized (i.e. tumor cells) and may be incorporated into the vectors of the present invention.
  • E4 deletions have been shown to reduce the immongenicity of the vectors (Wang, et al. (1997) Gene Therapy 4:393-400; Dedieu, et al. (1997) J. Virol 71 :4626-37), but can affect the persistence of transgene expression depending on the open reading frames of E4 retained and the promoter used to drive expresion of the transgene (Armentano, et al. (1997) J. Virol 71 :2408-2416).
  • the E4 region also encodes a protein (E4orf6) capable of binding to and inactivating the transciptional activity of p53 (Dubner, et al. (1996) Science 272:1470-73). Therefore, it may be desirable to modify the E4 region to delete those open reading frames with undesirable properties for the particular virus construct while retaining those with desired properties.
  • the E4 orf6 region may be deleted to reduce inactivation of p53 while retaining E4orf3 to allow continued expression of p53 and replication of the virus. In order to preserve replication competency of the virus, either E4orf6 or E4orf3 should be retained.
  • the adenovirus may contain modifications to the Ela coding sequence so as to produce Ela gene products which are deficient in binding to one or more p300 protein family members and one or more Rb protein family member protein but retain the transactivating function of the Ela CR3 domain and a deletion of the amino acids from approximately 219 to approximately 289 of the Ela 289R protein (or approximately amino acids 173 to approximately amino acid 243 of the Ela 243R protein.
  • deletion of the binding to the p300 family members is achieved by introducing a deletion corresponding to amino acids 4-25 of the Ela 243R and 289R proteins or amino acids 38-60 of the Ela 243R and 289R proteins.
  • deletion of the binding to the pRb family members is achieved amino acids 111-123 of the Ela 243R and 289R proteins.
  • deletion of the binding to the pRb family members may be achieved by eliminate of amino acids 124-127 of the Ela 243R and 289R proteins.
  • the E3 region of the adenovirus encodes proteins which help adenovirally infected cells avoid clearance by the immune system (Wold, et al. (1995) Curr. Top. Microbiol. Immunol. 199:237-274). Upregulation of this region and subfragments thereof has been shown to prevent or decrease the immune response to virally infected cells, leading to longer term gene expression. (Ilan, et al. (1997) PNAS 94:2587:2592, Bunder, et al. (1997) J. Virol. 71 :7623-28). Therefore, modifications to the E3 region (or sub-components thereof) to overexpress their proteins (e.g. by upregulating the E3 region using a strong constituitive promoter such as CMV) may be desirable to allow for a greater degree of viral replication due to its ability to avoid or delay the immune mediated clearance of infected cells.
  • a strong constituitive promoter such as CMV
  • the present invention provides recombinant viruses which contain "targeting modifications" in order to achieve preferential targeting of the virus to a particular cell type.
  • targeting modification refers to modifications to the viral genome designed to result in preferential infectivity of a particular cell type.
  • Cell type specificity or cell type targeting may also be achieved in vectors derived from viruses having characteristically broad infectivities such as adenovirus by the modification of the viral envelope proteins.
  • cell targeting has been achieved with adenovirus vectors by selective modification of the viral genome knob and fiber coding sequences to achieve expression of modified knob and fiber domains having specific interaction with unique cell surface receptors. Examples of such modifications are described in Wickham, et al. (1997) J.
  • Virol 71(ll):8221-8229 incorporas of RGD peptides into adenoviral fiber proteins
  • Arnberg, et al. (1997) Virology 227:239-244 (modification of adenoviral fiber genes to achieve tropism to the eye and genital tract); Harris and Lemoine (1996) TIG 12(10): 400-405; Stevenson, et al (1997) J. Virol. 71(6):4782- 4790; Michael, et ⁇ /.(1995) gene therapy 2:660-668 (incorporation of gastrin releasing peptide fragment into adenovirus fiber protein); and Ohno, et al.
  • the ElBdl55K-MLP-p53 (FAMA) vector was prepared in substantial accordance with the teaching of Example 1 herein. Briefly, the adenovirus genome was modified by introducing a deletion of base pairs 2247 to 3272 of the adenovirus genome so that the ability of the 55K protein is unable to bind to an inhibit the transcriptional activity of p53. In substitution of this deletion was placed a polyA signal from adenovirus protein IX for the Elbl9K upstream of the deletion and the cDNA encoding the Ad2 major late promoter and tripartite leader sequence driving expression of p53 from the plasmid pA M/53 as describe in Wills, et al . (1994) Human Gene Therapy 5:1079-88. The remainder of the virus sequence is wild-type Ad5.
  • B In vitro Experiments:
  • the vectors of the present invention were evaluated in a mouse human prostate cancer model.
  • the model and experiments were peformed in substantial accordance with the teaching of Example 3 herein.
  • the data is presented in Table 1 below and in Figure 6 of the attached drawings.
  • the vectors of the present invention demonstrated antitumor efficacy in vivo. It should be noted that no only did the tumors fail to grow, but actually regressed in size such that 5 of 6 animals were tumor free 27 days post administration. This is particularly surprising in light of the non-replicating p53 vector FTCB in which p53 is also expressed from the CMV promoter. Also, the effect is not due to merely to the Elb55k deletion (as represented by cZAZA) which showed lower efficacy at 27 days post administration.
  • the present invention further provides a pharmaceutically acceptable formulation of the recombinant adenoviruses in combination with a carrier.
  • the vectors of the present invention may be formulated for dose administration in accordance with conventional pharmaceutical practice with the addition of carriers and excipients. Dosage formulations may include intravenous, intratumoral, intramuscular, intraperitoneal, topical, matrix or aerosol delivery.
  • Carriers may include intravenous, intratumoral, intramuscular, intraperitoneal, topical, matrix or aerosol delivery.
  • carrier refers to compounds commonly used on the formulation of pharmaceutical compounds used to enhance stability, sterility and deliverability of the therapeutic compound.
  • the delivery system is in an acceptable carrier, preferably an aqueous carrier.
  • aqueous carriers may be used, e.g., water, buffered water, 0.8% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorption monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorption monolaurate, triethanolamine oleate, etc.
  • the present invention further provides pharmaceutical formulations of the vectors of recombinant adenoviruses of the present invention with a carrier and a delivery enhancing agent(s).
  • delivery enhancers or “delivery enhancing agents” are used interchangeably herein and includes one or more agents which facilitate uptake of the virus into the target cell. Examples of delivery enhancers are described in co-pending United States Patent Application Serial No. filed July 7, 1998.
  • Examples of such delivery enhancing agents include detergents, alcohols, glycols, surfactants, bile salts, heparin antagonists, cyclooxygenase inhibitors, hypertonic salt solutions, and acetates.
  • Alcohols include for example the aliphatic alcohols such as ethanol, N-propanol, isopropanol, butyl alcohol, acetyl alcohol.
  • Glycols include glycerine, propyleneglycol, polyethyleneglycol and other low molecular weight glycols such as glycerol and thioglycerol.
  • Acetates such as acetic acid, gluconic acid, and sodium acetate are further examples of delivery-enhancing agents.
  • Hypertonic salt solutions like IM NaCl are also examples of delivery-enhancing agents.
  • surfactants are sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethyleneglycol 400, polysorbate 80, polyoxyethylene ethers, polyglycol ether surfactants and DMSO.
  • Bile salts such as taurocholate, sodium tauro-deoxycholate, deoxycholate, chenodesoxycholate, glycocholic acid, glycochenodeoxycholic acid and other astringents such as silver nitrate may be used.
  • Heparin-antagonists like quaternary amines such as protamine sulfate may also be used.
  • Cyclooxygenase inhibitors such as sodium salicylate, salicylic acid, and non-steroidal antiinflammatory drug (NSALDS) like indomethacin, naproxen, diclofenac may be used.
  • Delivery-enhancing agents includes compounds of the Formula I:
  • XI is a cholic acid group or deoxycholic acid group
  • X2 and X3 are each independently selected from the group consisting of a cholic acid group, a deoxycholic acid group, and a saccharide group. At least one of X2 and X3 is a saccharide group.
  • the saccharide group may be selected from the group consisting of pentose monosaccharide groups, hexose monosaccharide groups, pentose-pentose disaccharide groups, hexose-hexose disaccharide groups, pentose- hexose disaccharide groups, and hexose-pentose disaccharide groups.
  • detergent includes anionic, cationic, zwitterionic, and nonionic detergents.
  • Exemplary detergents include but are not limited to taurocholate, deoxycholate, taurodeoxycholate, cetylpyridium, benalkonium chloride, Zwittergent3- 14 detergent, CHAPS (3-[(3-Cholamidopropyl) dimethylammoniol]-l- propanesulfonate hydrate), Big CHAP, Deoxy Big CHAP, Triton-X-100 detergent, C12E8, Octyl-B-D-Glucopyranoside, PLURONIC- F68 detergent, Tween 20 detergent, and TWEEN 80 detergent (CalBiochem Biochemicals).
  • Unit dosage formulations of the present invention may be included in a kit of products containing the recombinant adenovirus of claim 1 in lyophilized form and a solution for reconstitution of the lyophilized product along with instructions for use.
  • Recombinant adenoviruses of the present invention may be lyophilized by conventional procedures and reconstituted.
  • calpain inhibitors refers to a compound which inhibits the proteolytic action of calpain- I, e.g. ⁇ -calpains.
  • calpain inhibitors as used herein includes those compounds having calpain I inhibitory activity in addition to or independent of their other biological activities. A wide variety of compounds have been demonstrated to have activity in inhibiting the proteolytic action of calpains.
  • calpain inhibitors are useful in the practice of the present invention include N-acetyl-leu-leu- norleucinal also known as "calpain inhibitor 1." Additional calpain inhibitors are described in the following United States Patents, herein incorporated by reference, United States Patent No. 5,716,980 entitled Alcohol or aldehyde derivatives and their use; United States Patent No. 5,714,471 entitled Peptide and peptide analog protease inhibitors; United States Patent No. 5,693,617 entitled Inhibitors of the 26s proteolytic complex and the 20s proteasome contained therein; United States Patent No.
  • the calpain inhibitor is N-acetyl- leu-leu-norcinal (calpain inhibitor-1) commercially available from Boehringer- Mannheim (Indianapolis, IN).
  • the present invention provides a method of eliminating neoplastic cells in a mammalian organism by the administration of a recombinant viral vector of the present invention to a neoplastic cell.
  • neoplastic cell is a cell displaying an aberrant growth phenotype characterized by independence of normal cellular growth controls.
  • the term neoplastic cells comprise cells which may be actively replicating or in a temporary non- replicative resting state (Gl or GO). Localized populations of neoplastic cells are referred to as neoplasms.
  • Neoplasms may be malignant or benign. Malignant neoplasms are also referred to as cancers.
  • Neoplastic transformation refers the conversion of a normal cell into a neoplastic cell, often a tumor cell.
  • mammalian organism includes, but is not limited to, humans, pigs, horses, cattle, dogs, cats.
  • the present invention provides a method of ablating neoplastic cells in a mammalian organism in vivo by the administration of a pharmaceutically acceptable formulation of the recombinant adenovirus described above.
  • ablating means the substantial reduction of the population of viable neoplastic cells so as to alleviate the physiological maladictions of the presence of the neoplastic cells.
  • substantially means a reduction in the population of viable neoplastic cells in the mammalian organism by greater than approximately 20% of the pretreatment population.
  • viable means having the uncontrolled growth and cell cycle regulatory characteristics of a neoplastic cell.
  • neoplastic cell is used hereing to distinguish said cells from neoplastic cells which are no longer capable of replication.
  • a tumor mass may remain following treatment, however the population of cells comprising the tumor mass may be dead. These dead cells have been ablated and lack the ability to replicate, even though some tumor mass may remain.
  • a recombinant adenovirus containing a deletion of the E1B-55K gene function and expressing a tumor suppressor gene from the adenoviral major late promoter is formulated with a pharmaceutically acceptable carrier for administration by intravenous, intraperitoneal, or intratumor injection.
  • the appropriate dose and method of administration of the vector to be administered to the mammalian organism in need of treatment will be determined by the skilled artisan taking into account the extent of metastatis of the primary tumor, the delivery enhancer(s) included in the formulation, the extent to which the immunological response is suppressed, etc. Each of these latter factors will decrease the dosage of the vector provided to the mammalian organism in need of treatment.
  • a dosage of approximately 1 x 10 5 to 1 x 10 13 particles preferably 1 x 10 ⁇ to 1 x 10" particles, most preferably x 10 7 to 1 x 10 10 particles
  • the typical course of treatment will be the daily administration of a pharmaceutically acceptable formulation of the vector of the present invention over a period of three to ten days, preferably five to eight days.
  • the tumor suppressor gene is the wild-type p53 gene.
  • the tumor suppressor gene encodes is a VP22-p53 fusion protein.
  • the pharmaceutically acceptable carrier contains a delivery enhancing agent.
  • the delivery enhancing agent is a calpain inhibitor.
  • the recombinant adenoviral vector E1B- dl55K-MLP-p53 is formulated in a carrier solution further comprising the calpain inhibitor n-acetyl-leu-leu-norcinal (calpain inhibitor 1) at a concentration of from approximately 1 to 50 micromolar.
  • the daily dosage may be reduced as compared to a formulation absent such delivery enhancing agents by a factor of one to two logs.
  • adenoviral vector endogenous to the mammalian type being treated.
  • virus from the species to be treated
  • vectors derived from different species which possess favorable pathogenic features For example, it is reported (WO 97/06826 published April 10, 1997) that ovine adenoviral vectors may be used in human gene therapy to minimize the immune response characteristic of human adenoviral vectors. By minimizing the immune response, rapid systemic clearance of the vector is avoided resulting in a greater duration of action of the vector.
  • the present invention provides a method of use of the recombinant adenoviruses alone
  • the recombinant adenoviruses of the present invention and formulations thereof may be employed in combination with conventional chemotherapeutic agents or treatment regimens.
  • chemotherapeutic agents include inhibitors of purine synthesis (e.g., pentostatin, 6-mercaptopurine, ⁇ thioguanine, methotrexate) or pyrimidine synthesis (e.g. Pala, azarbine), the conversion of ribonucleotides to deoxyribonucleotides (e.g. hydroxyurea), inhibitors of dTMP synthesis (5-fluorouracil), DNA damaging agents (e.g.
  • Chemotherapeutic treatment regimens refers primarily to non- chemical procedures designed to ablate neoplastic cells such as radiation therapy. In such instances, the daily dosage in the course of treatment is reduced in comparison to those dosages provided absent such chemotherapeutic agents.
  • the immune system is capable of recognizing and eliminating recombinant viral vectors. As this would effectively reduce the amount of adenovirus reaching the target cell, it is preferable in many instances to administer the compounds of the present invention in combination with immunosuppressive agents such as etoposide.
  • the immunosuppressive agent is administered in advance, preferably for about a week in advance of the introduction of the recombinant viral vector of the present invention to eliminate the humoral immune response to the viral particles.
  • a pharmaceutically acceptable formulation of the vector of the present invention is administered intratumorally following the administration of a immunosuppressive agent for a period of from about one day to about two weeks in advance of administration of the vector.
  • the vector is preferably an adenoviral vector and further contains a deletion of the E1B-55K protein and contains an expression cassette expressing the p53 tumor suppressor gene from an adenoviral major late promoter.
  • the immunosuppressive agent is etoposide and is administered daily for a period of from about 1 to 7 days (preferably 3- 7 days) prior to administration of the vector. In such instances, the daily dosage in the course of treatment is reduced in comparison to those dosages provided absent such immunosuppressive agents.
  • the present invention also provides a method of ablating neoplastic cells in a population of normal cells contaminated by said neoplastic cells ex vivo by the administration of a recombinant adenovirus of the present invention to said population.
  • An example of the application of such a method is currently employed in ex vivo applications such as the purging of autologous stem cell products commonly known as bone marrow purging.
  • stem cell product refers to a population of hematopoietic, progenitor and stem cells capable of reconstituting the long term hematpoietic functionvof a patient who has received myoablative therapy.
  • Stem cell products are conventionally obtained by apheresis of mobilized or non-mobilized peripheral blood.
  • Apheresis is conventionally achieved through the use of known procedures using commercially available apheresis apparatus such as the COBE Spectra Apheresis System, commercially available from COBE International, 1185 Oak Street, Lakewood, CO. It is preferred that treatment conditions be optimized to achieve a "3- log purge” (i.e. removal of approximately 99.9% of the tumor cells from the stem cell produce) and most preferably a "5-log purge” (removal of approximatley 99.999% of tumor cells from the stem cell product).
  • a stem cell product of 100 ml volume would be treated at a concentration of from about lxlO 6 to lxlO 10 particles/ml of the recombinant adenovirus of the present invention for a period of approximately 4 hours at 37°C.
  • the present invention provides a recombinant viral vectors capable of recruiting immature dendritic cells to a tumor site and exposing the dendritic cells to a localized high concentration of tumor antigens characteristic of the tumor present in the patient.
  • the vectors of the present invention are specifically engineered to induce killing of tumor cells.
  • the lysed tumor cell or the apoptotic bodies produced by an apoptosed tumor cell) provides a rich localized concentration of tumor specific proteins.
  • dendritic cell chemoattractants By introducing a gene encoding a dendritic cell chemoattractant, immature dendritic cells capable engulfing tumor antigens areinstallited to the site of the lysed tumor cells thereby engulfing tumor antigents and presenting these antigens to the immune system.
  • dendritic cell chemoattractants refers to chemotactic chemokines capable of attracting and/or directing the migration of dendritic cells to a particular location.
  • Xu, et al suggest that all C-C chemokines, including MCP-1, MCP-2, MCP-3, MIPl alpha, MIP-1 beta, and RANTES, induced migration of DC-enriched cells cultured with or without LL-4.
  • Xu, et al. (1996) J. Leukoc. Biol. 60(3):365-71.
  • the dendritic cell chemoattractant is MLP-3-alpha.
  • the dendritic cell chemoattractant may be expressed intracellular form where it is released upon cell lysis or in secreted form by the use of a signal peptide. Upon expression of the dendritic cell chemoattractant, the dendritic cells then engulf the tumor antigens or apoptotic bodies, mature and migrate through existing pathways to the lymph and present the tumor antigens to the T-cells. The resulting T-cells are then capable of recognizing and killing tumor cells.
  • the appropriate dosage regimen in such instances is in accordance with the typical anti-neoplastic dosage formulation and regimen as described above.
  • the vectors of the present invention are also useful for diagnostic purposes.
  • the vectors of the present invention may incorporate a reporter gene in place of the therapeutic gene which is expressed upon viral replication.
  • reporter gene refers to a gene whose product is capable of producing a detectable signal alone or in combination with additional elements.
  • reporter genes includes the beta-galactosidase gene, the luciferase gene, the green fluorescent protein gene, nucleotide sequences encoding proteins detectable by imaging systems such as X-rays or magnetic field imaging systems (MRI).
  • MRI magnetic field imaging systems
  • such vectors may also be employed to express a cell surface protein capable of recognition by a binding molecule such as a fluorescently labelled antibody.
  • in vivo applications include imaging applications such as X-ray, CT scans or Magnetic Resonance Imaging (MRI).
  • X Method of Makin g The Compositions :
  • the present invention further provides a method of producing the recombinant adenovirus comprising the modifications to packaging domains described above, said method comprising the steps of: a. infecting a producer cell with a recombinant virus; b . culturing said infected producer cell under conditions so as to permit replication of the viral genome in the producer cell; c . harvesting the producer cells, and d. purifying the recombinant adenovirus.
  • infectious means exposing the recombinant adenovirus to the producer cell under conditions so as to facilitate the infection of the producer cell with the recombinant virus.
  • the activities necessary for viral replication and virion packaging are cooperative.
  • conditions be adjusted such that there is a significant probability that the producer cells are multiply infected with the virus.
  • An example of a condition which enhances the production of virus in the producer cell is an increased virus concentration in the infection phase.
  • the total number of viral infections per producer cell can be overdone, resulting in toxic effects to the cell.
  • the present invention provides a method to increase the infectivity of producer cell lines for viral infectivity by the inclusion of a calpain inhibitor.
  • calpain inhibitors useful in the practice of the present invention include calpain inhibitor 1 (also known as N-acetyl-leucyl-leucyl-norleucinal, commercially available from Boehringer Mannheim).
  • producer cell means a cell capable of facilitating the replication of the viral genome of the recombinant adenovirus to be produced and capable of complementing the packaging defects of the recombinant adenovirus.
  • mammalian cell lines are publicly available for the culture of recombinant adenoviruses.
  • the 293 cell line (Graham and Smiley (1977) J. Gen. Virol. 36:59-72) has been engineered to complement the deficiencies in El function and is a preferred cell line for the production of the current vectors.
  • cell lines may be developed incorporating viral sequences stably integrated into the viral genome.
  • Examples of other producer cells parent cell lines which may be employed include HeLa cells, PERC.6 cells (as described in publication WO/97/00326, application serial No. PCT/NL96/00244 and the A549-E1 cell line (as described in International Patent Application No. PCT/US97/810039 published February 23, 1998 as International Publication No. WO98/US3473.
  • the term "culturing under conditions to permit replication of the viral genome” means maintaining the conditions for the infected producer cell so as to permit the virus to propagate in the producer cell. It is desirable to control conditions so as to maximize the number of viral particles produced by each cell. Consequently it will be necessary to monitor and control reaction conditions such as temperature, dissolved oxygen, pH, etc.
  • Commercially available bioreactors such as the CelliGen Plus Bioreactor (commercially available from New Brunswick Scientific, Inc. 44 Talmadge Road, Edison, NJ) have provisions for monitoring and maintaining such parameters. Optimization of infection and culture conditions will vary somewhat, however, conditions for the efficient replication and production of virus may be achieved by those of skill in the art taking into considerations the known properties of the producer cell line, properties of the virus, type of bioreactor, etc.
  • oxygen concentration is preferably maintained from approximately 50% to approximately 120% dissolved oxygen, preferably 100% dissolved oxygen.
  • concentration of viral particles as determined by conventional methods such as HPLC using a Resource Q column
  • the reactor is harvested.
  • harvested means the collection of the cells containing the recombinant adenovirus from the media. This may be achieved by conventional methods such as diffential centrifugation or chromatographic means. At this stage, the harvested cells may be stored or further processed by lysis and purification to isolate the recombinant virus. For storage, the harvested cells should be buffered at or about physiological pH and frozen at -70C.
  • lysis refers to the rupture of the producer cells. Lysis may be achieved by a variety of means well known in the art. When it is desired to isolate the viral particles from the producer cells, the cells are lysed, using a variety of means well known in the art. For example, mammalian cells may be lysed under low pressure (100-200 psi differential pressure) conditions or conventional freeze thaw methods. Exogenous free DNA/RNA is removed by degradation with DNAse/RNAse.
  • purifying means the isolation of a substanially pure population of recombinant virus particles from the lysed producer cells.
  • Conventional purification techniques such as chromatographic or differential density gradient centrifugation methods may be employed.
  • the virus is purified by column chromatography in substantial accordance with the process of Huyghe et al. (1995) Human Gene Therapy 6: 1403-1416 as described in co-pending United States Patent application Serial No. 08/400,793 filed March 7, 1995. Additional methods and procedures to optimize production of the recombinant adenoviruses of the present invention are described in co-pending United States Patent Application Serial No. 09/073,076, filed May 4, 1998.
  • the purified virus is then admixed with appropriate excipients and carriers or delivery enhancing agents.
  • the solution is sterilized for individual packaging and vialed for storage.
  • the virus may be lyphophilized for storage and reconsituted in a solution containing delivery enhancing agents, buffers, preservatives, cryoprotectants and/or carriers.
  • the lyophilized virus and the reconsitution solution may be packaged together as a kit for consumption by the end user along with instructions for appropriate handling and administration.
  • the mutagenic oligonucleotides were first phosphorylated at the 5' end, and then annealed to denatured pXB-ElB template DNA.
  • the annealed primer/template reactions were incubated with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP, dGTP and dTTP) to synthesize a complementary mutant strand.
  • the complementary strand synthesis reaction was then transfomed into the bacterial strain, BMH 71-18 mutS. This bacterial strain is defective for mis-match repair, preventing undesired repair of the mutant strand.
  • the transformants were digested with HindlJI to cut any parental plasmid strands, and retransformed into DH5 ⁇ for amplification. Potential EIB mutants were then screened by restriction enzyme analysis to confirm the desired mutations.
  • This procedure was used to introduce restriction enzyme nuclease cleavage sites in the EIB 55K coding region.
  • the first site was introduced by modifying positions 2247 and 2248 of the wild type Ad5 genome wherein a guanine 2247 was replaced with a thymidine and thymidine 2248 replaced with cytosine (respectively) to introduce a EcoRl cleavage site.
  • a second resriction site was introduced at position 3272 wherein thymidine 3272 was replaced with cytosine site (silent mutation) to introduce an Xhol site.
  • the new restriction enzyme sites were used in a restriction enzyme digest with EcoRI and Xhol.
  • This plasmid is based on the pBR322 derivative pML2 (pBR322 deleted for base pairs 1140-2490) and contains an adenovirus type 5 sequences extending from base pair 1 to 5788 except that it is deleted for adenovirus type 5 base paris 357-3327.
  • a transciption unit is inserted iwhich is comprised of the adenovirus type 2 major late promoter, the adenovirus type 2 tripartite leader DNA and human p53 cDNA.
  • This EcoRI/Xhol fragment is inserted into the EcoRl and Xhol sites introduced into the ElB55k coding region.
  • the polyA sequence that follows wtAd5 pIX was amplified by PCR (Ad5 sequence 4001-4368).
  • the primers used for amplification included sequences to introduce an EcoRl site at the 5' end of the Ad5 sequence, and a SacII site at the 3' end of the Ad5 sequence.
  • This fragment was then inserted into the EcoRl- SacII sites immediately following the EIB 19k coding sequence.
  • the SacII site was included in the MLP-p53 cassette inserted previously, and was upstream of the MLP promoter sequence.
  • the resulting EIB mutation results in a sequence encoding the first 76 amino acids of the E1B55K protein followed by 11 missense amino acids resulting in a non-functional deleted EIB protein.
  • adenovirus cFAMA For recombination to produce adenovirus cFAMA, the viral large fragment and pXCl-ElB53-2A were cotransfected into 293 cells by calcium phosphate mediated transfection. After 5 hours the precipitate was rinsed from the cells and normal media replaced. At 15 days after the initial transfection, viral "comets" were isolated, plaque purified two times, and subsequently viral DNA was screened using restriction enzyme analysis and DNA sequencing. Viral stocks were purified by double cesium chloride gradients and quantitated by column chromatography as described in Huyghe, et al. (1995) Human Gene Therapy 6:1403-1416.
  • the cells were resuspended in lysis buffer (50mM Tris, 250 mM NaCl, 0.1% NP40, 50 mM NaF, 5 mM EDTA) and lysed by the freeze thaw method (repeated three times).
  • the cells were stained with the BioRad dye reagent concentrate provided with the kit and the absorbance was determined at a wavelength of 595 nm.
  • the primary antibody was a murine anti-p53 antibody (commercially available from Novacastra as catalog number 1801 diluted
  • Example 2 This evaluation was performed in substantial accordance with the teachining of Example 2. a above except that the experiment was conducted in SK-HEP1 hepatocellular carcinoma cells and the six well plate was seeded with 7.5 x 10 5 cells per well. The results are presented in Figure 2 of the attached drawings.
  • Example 2 This evaluation was performed in substantial accordance with the teachining of Example 2. a above except that the experiment was conducted in NCI H358 cells except that the six well plate was seeded with 1.5 x 10 5 cells per well. The cells were infected as in Example 2.a. above using 1.8 x 10 9 particles/ml concentration of the indicated viruses for Figure 3 and 1.8 x 10 8 for Figure 4. The results are presented in Figure 3 and 4 of the attached drawings.
  • SK-BR3 cells were infected with a one hour pulse of the following recombinant adenoviral ectors at a concentration of 1.8 x 10 9 particles/ml:
  • rAdcon a recombinant adenovirus lacking El and protein IX function without a p53 coding sequence (Wills, et al.)
  • ElBdl55K A recombinant adenovirus containing the E1B-55K deletion described in Example 1 above with no exogenous transgene cassette.
  • 55K/CMVp53 A recombinant adenovirus cFAIC containing the E1B-55K deletion described above further comprising an expression cassette encoding p53 under control of the CMV promoter
  • Ad5WT Wild type adenovirus type 5.
  • the cells were harvested at approximately 48 hours post infection.
  • the DNA was applied to a agarose gel and stained with ethidium bromide according to techniques well known in the art. The results are presented in Figure 5 of the attached drawings.
  • PC-3 cells prostate carcinoma, p53 null
  • PC-3 cells prostate carcinoma, p53 null
  • virus was intratumorally injected for 5 consecutive days at a dose of 1 x 10 10 particles per injection on days 11-15 post PC-3 cell injection.
  • the six different viral constructs cFAMA, cFAIC, FTCB, ZZCB, cZAZA and wildtype Ad5 were compared.
  • V-PBS refers to phosphate buffered saline containing 3% sucrose and 2mM magnesium chloride. Tumor volume was evaluated on days 1, 4, 7, 11, 15, 19, 23, and 27. The results of the evaluations are presented in Figure 5 of the accompanying drawings and presented in the Table 1 above.
  • the ElBd]55K-MLP-Cytosine Deaminase vector is prepared in substantial accordance with the teaching of Example 1 herein. However, the transfer plasmid containing the p53 coding sequence is replaced with a DNA sequence encoding the cytosine deaminase gene.
  • the AFP-E4-ElBdl55K-MLP-p53 is a recombinant adenovirus which is similar to the ElBdl55K-MLP-p53 vector is prepared in substantial accordance with the teaching of Example 1 herein.
  • the E4 gene is operably linked to the alpha-fetoprotein tumor specific promoter sequence (AFP) facilitating the replication of this virus in hepatocellular carcinoma cells.
  • AFP alpha-fetoprotein tumor specific promoter sequence
  • Other factors such as NF-LL6 can substitute for Ela in regulating Ela responsive promoters in the adenovirus in the absence of Ela function (Spergel, et al (1992) J. Viroll 66:1021-1030) and this can be avoided by substitution of the E4 promoter with a tumor specific promoter.
  • the AFP-E4-ElBcU55K-MLP-IFN2a describes a recombinant adenovirus which is similar to the AFP-E4-ElBdl55K-MLP-p53vector is prepared in substantial accordance with the teaching of Example 5 herein. However, in this construction the p53 gene is replaced with the DNA sequence encoding interferon alpha-2b.
  • the ElAdK)l/07-ElBdi55K-MLP-p53 is a recombinant adenovirus which is substantially similar to the ElBdl55K-MLP-p53 vector is prepared in substantial accordance with the teaching of Example 1 herein.
  • the Ela gene is modified to contain the in-frame deletion mutations dU 101 and dil 107, as described in Jelsma et al, (1998) Virology 163. 494-502 and are constructed using the oligonucleotide site directed technique of Zoeller and Smith (1984) DNA 3, 479-488, as modifed by Kunkel (1985) PNAS 82, 488-492.
  • the M13 template DNA useful for mutagenesis of the EIA region, contains Ad5 sequences from nucleotide positions 22-1339 inserted between the BamHl and Xbal restriction enzyme sites in the multiple cloning sequence of M13mpl9.
  • the resulting bacteriophage construct, M13mpl9ElA is then propagated in dut ung E. coli bacterial strain CJ236 which results in an occasional incorporation of uracil in place of thymidine in the newly synthesized DNA.
  • the oligonucleotides, for construction of the El mutants, are synthesized to consist of sequences of either 11 or 12 nucleotides of Ad5 sense DNA on either side of the sequence that was to be removed.
  • the mutagencic oligonucleotides are first phosphorylated at the 5' end, and then annealed to uracil containing M13mpllElA single- stranded template DNA.
  • the annealed primer/template reactions are incubated with T4 DNA polymerase, T4 DNA ligase and deoxyribonucleotides (dATP, dCTP, dGTP and dTTP) to synthesize a complementary strand containing the El A mutation of interest.
  • the complementary strand synthesis reaction are then transfomed into the ung + wild type host bacterial strain, MV1190.
  • M13mpl9ElA DNA strand which contains uracil, cannot be replicated efficiently in MV1190. Therefore the replicative form double strand DNA containing the EIA mutation of interest is enriched.
  • M13mpl9ElA phage DNA from potential EIA mutants is first screened by restriction enzyme analysis and then by DNA-sequencing, in both strands, to confirm the desired El A-mutations. Construction of an adenovirus comprising the ElAdlOl/deletions is carried out by using homologous recombination in the adenovirus El-region containing 293 cell line by the method of McGory, et al, (1988) Virology 163, 614-617.
  • This method requires two plasmids, one a viral plasmid containing the entire wtAd5 genome modified as in Example 1 to contain the Elbdl55K deletion and the MLP promoter (pXCl-ElB55-2A), and the other a transfer plasmid containing an EIA gene with the dl01/07 double ElA-mutant .
  • the transfer plasmid, pLE2 contains wtAd5 sequences from 22-1774 cloned in the tetracycline gene of pBR322 Jelsma et al, (1988)Virology 163. 494-502.

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Abstract

La présente invention concerne des virus recombinés contenant un transgène thérapeutique lié fonctionnellement à un élément régulateur tardif. Les vecteurs de la présente invention sont capables d'une réplication et d'une lyse de cellules néoplasiques. Les vecteurs peuvent comprendre facultativement des modifications apportées au génome de manière à conférer des fonctions de réplication ou de ciblage thérapeutique conditionnelles supplémentaires. La présente invention concerne également des formulations pharmaceutiques de ces vecteurs. En outre, la présente invention concerne des méthodes d'utilisation de ces vecteurs et des méthodes de préparation des vecteurs.
PCT/US1999/026004 1998-11-18 1999-11-17 Vecteurs viraux a expression tardive de transgenes WO2000029599A1 (fr)

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WO2000029573A2 (fr) * 1998-11-18 2000-05-25 Canji, Inc. Vecteurs adenoviraux
WO2001096602A2 (fr) * 2000-06-16 2001-12-20 Medical Research Council Procedes et materiels relatifs a des vecteurs plasmidiques
EP1180932A1 (fr) * 1999-05-12 2002-02-27 The Uab Research Foundation Adenovirus a pouvoir infectant renforce et a replication conditionnelle, et applications de celui-ci
WO2002048377A2 (fr) * 2000-12-14 2002-06-20 Genvec, Inc. Polynucleotide codant une proteine chimere et vecteurs, cellules afferents, et methodes d'expression correspondantes
US7109029B2 (en) 2001-02-23 2006-09-19 Cell Genesys, Inc. Vector constructs
US8168168B2 (en) 1999-05-12 2012-05-01 Juan Fueyo Infectivity-enhanced conditionally-replicative adenovirus and uses thereof
US9943568B2 (en) 2013-04-18 2018-04-17 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating cancer
US10238698B2 (en) 2012-01-25 2019-03-26 Dnatrix, Inc. Biomarkers and combination therapies using oncolytic virus and immunomodulation
US10316065B2 (en) 2009-05-06 2019-06-11 Fundacio Institut D Investigacio Biomedica De Bellvitge (Idibell) Oncolytic adenoviruses for treating cancer
US10398761B2 (en) 2015-08-25 2019-09-03 Armo Biosciences, Inc. Methods of using combinations of PEG-IL-10 and IL-15 for treating cancers
US11155599B2 (en) 2012-02-02 2021-10-26 Board Of Regents, The University Of Texas System Adenoviruses expressing heterologous tumor-associated antigens

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029573A3 (fr) * 1998-11-18 2000-10-05 Canji Inc Vecteurs adenoviraux
WO2000029573A2 (fr) * 1998-11-18 2000-05-25 Canji, Inc. Vecteurs adenoviraux
EP1180932A4 (fr) * 1999-05-12 2007-12-05 Uab Research Foundation Adenovirus a pouvoir infectant renforce et a replication conditionnelle, et applications de celui-ci
EP1180932A1 (fr) * 1999-05-12 2002-02-27 The Uab Research Foundation Adenovirus a pouvoir infectant renforce et a replication conditionnelle, et applications de celui-ci
US10391183B2 (en) 1999-05-12 2019-08-27 The Uab Research Foundation Infectivity-enhanced conditionally-replicative adenovirus and uses thereof
US8168168B2 (en) 1999-05-12 2012-05-01 Juan Fueyo Infectivity-enhanced conditionally-replicative adenovirus and uses thereof
WO2001096602A2 (fr) * 2000-06-16 2001-12-20 Medical Research Council Procedes et materiels relatifs a des vecteurs plasmidiques
WO2001096602A3 (fr) * 2000-06-16 2002-09-06 Medical Res Council Procedes et materiels relatifs a des vecteurs plasmidiques
WO2002048377A2 (fr) * 2000-12-14 2002-06-20 Genvec, Inc. Polynucleotide codant une proteine chimere et vecteurs, cellules afferents, et methodes d'expression correspondantes
WO2002048377A3 (fr) * 2000-12-14 2003-08-14 Genvec Inc Polynucleotide codant une proteine chimere et vecteurs, cellules afferents, et methodes d'expression correspondantes
US7109029B2 (en) 2001-02-23 2006-09-19 Cell Genesys, Inc. Vector constructs
US10316065B2 (en) 2009-05-06 2019-06-11 Fundacio Institut D Investigacio Biomedica De Bellvitge (Idibell) Oncolytic adenoviruses for treating cancer
US10238698B2 (en) 2012-01-25 2019-03-26 Dnatrix, Inc. Biomarkers and combination therapies using oncolytic virus and immunomodulation
US11065285B2 (en) 2012-01-25 2021-07-20 Dnatrix, Inc. Biomarkers and combination therapies using oncolytic virus and immunomodulation
US11155599B2 (en) 2012-02-02 2021-10-26 Board Of Regents, The University Of Texas System Adenoviruses expressing heterologous tumor-associated antigens
US9943568B2 (en) 2013-04-18 2018-04-17 Armo Biosciences, Inc. Methods of using pegylated interleukin-10 for treating cancer
US10357545B2 (en) 2013-04-18 2019-07-23 Armo Biosciences, Inc. Methods of using interleukin-10 for treating solid tumors
US10398761B2 (en) 2015-08-25 2019-09-03 Armo Biosciences, Inc. Methods of using combinations of PEG-IL-10 and IL-15 for treating cancers

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