WO1999066946A1 - Compositions et procedes servant a provoquer une apoptose chez des cellules exprimant e6 - Google Patents

Compositions et procedes servant a provoquer une apoptose chez des cellules exprimant e6 Download PDF

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WO1999066946A1
WO1999066946A1 PCT/US1999/014057 US9914057W WO9966946A1 WO 1999066946 A1 WO1999066946 A1 WO 1999066946A1 US 9914057 W US9914057 W US 9914057W WO 9966946 A1 WO9966946 A1 WO 9966946A1
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cell
protein
vectors
cells
cancer
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Wafik S. El-Deiry
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Trustees Of The University Of Pennsylvania
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • This invention relates to the field of methods of treatment of cancer.
  • this invention provides a method of treatment of cancers associated with human papillomavirus infection or other tumors in which the E6 oncogene is expressed, and a pharmaceutical preparation and kit to practice the method.
  • HPV human papillomavirus
  • HPV human papillomavirus
  • the E6-oncoprotein encoded by HPV has been shown to target the tumor suppressor protein p53 for degradation via ubiquitin conjugation and subsequent proteolysis (Scheffner et al . , 1990, Cell 63: 1129-1136) .
  • HPV-E6-expressing cancer cells are resistant to the tumor suppressive effects of exogenous wild-type p53 delivered by an adenovirus (Ad) vector (Prabhu et al., 1996, Clin. Cancer Res . 2: 1221-1229).
  • Ad adenovirus
  • Ad-p21 p21 -expressing adenovirus inhibits the growth of E6-over-expressing cells, although the primary effect of p21 over-expression is a growth arrest associated with a large cell phenotype and little, if any, apoptosis
  • p53 tumor suppressor Therapy based on the p53 tumor suppressor is unavailable for cancers associated with expression of the E6 oncogene because the E6 protein targets p53 for degradation by ubiquitin-mediated proteolysis. It has been discovered in accordance with the present invention that the p53 homolog, p73, is not targeted for degradation by E6 and, moreover, is a potent inhibitor of cancer colony growth and inducer of apoptosis, even in cells that over-express E6. Thus, p73 is a superior tumor suppressor protein for treatment of cancers in which the E6 oncogene is expressed, such as those associated with HPV infection.
  • method for inducing apoptosis in an E6-expressing cell.
  • the method comprises administering to the cell an amount of p73 protein effective to induce the apoptosis.
  • the p53 protein is administered as a DNA construct comprising an expressible sequence that encodes the protein.
  • the DNA construct is operably inserted into a viral vector for transforming cells.
  • the method is typically utilized for arresting growth of cancerous cells, particularly cancers associated with infection with E6-expressing viruses, such as HPV.
  • the cell is a cultured cell
  • the cell is obtained from the body of a living organism, the administering is performed ex vivo, and the cell is returned to the living organism.
  • the cell is disposed within a living organism and the administering is performed in vivo.
  • the p73 protein utilized in the method is preferred to be p73 ⁇ or p73 ⁇ , most preferably the latter.
  • the protein comprises a sequence selected from the group consisting of SEQ ID NO:l and SEQ ID NO : 2. If a DNA construct is used, the DNA construct preferably comprises more than 50 nucleotides of SEQ ID NO : 3.
  • an apoptotic, E6-expressing transgenic cell which comprises a heterologous, expressible DNA construct encoding p73.
  • the cell is obtained from a cultured cell line.
  • the cell is a primary cell obtained from a living organism. In yet another embodiment, it is disposed within a living organism.
  • a pharmaceutical preparation for treatment of cancers associated with E6 over-expression comprises a p73 protein associated with a delivery vehicle for delivering proteins to cancer cells.
  • the preparation comprises an expressible DNA construct encoding p73, associated with a delivery vehicle for delivering DNA to cancer cells.
  • the pharmaceutical preparation also may comprise at least one additional active ingredient for treatment of cancer.
  • kits that contains the pharmaceutical preparation and other optional components.
  • the kit may include a second pharmaceutical agent useful for treating cancer.
  • U373 cell line R273H (Kaghad et al . , 1997, Cell 90: 809-819); S 480 cell line: R273H, P309S (Kaghad et al . , 1997, Cell 90: 809-819); SKBr3 cell line: R175H (Kovach et al . , 1991, J. Natl. Cancer Inst . , 83:1004-1009); H80 cell line (also known as U-373 MG) : R273H (Gomez-Manzano et al . , 1996, Cancer Res. 56:694-699).
  • p73 unlike p53 , is not specifically targeted for degradation in Ad-E6 infected cancer cells.
  • S 480 cells were transfected by p73 (lanes 1,2), p73 ⁇ m (lanes 3,4), p73 ⁇ (lanes 5,6), or p73 ⁇ m (lanes 7,8).
  • cells were infected by either Ad-LacZ or Ad-E6 (as indicated) .
  • expression of p73 ⁇ (upper left) or p73 ⁇ (upper right) was detected by immunoblotting using anti-HA antibody and for p53 (lower panels) expression using anti-p53 antibody, as described in Example 1.
  • the band just above p73 ⁇ is a non-specific anti-HA cross-reactive band.
  • isolated nucleic acid refers to a DNA molecule that is separated from sequences with which it is immediately contiguous (in the 5' and 3' directions) in the naturally occurring genome of the organism from which it was derived.
  • the "isolated nucleic acid” may comprise a DNA molecule inserted into a vector, such as a plasmid or virus vector, or integrated into the genomic DNA of a procaryote or eucaryote .
  • An "isolated nucleic acid molecule” may also comprise a cDNA molecule.
  • isolated nucleic acid primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above.
  • the term may refer to an RNA molecule that has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a “substantially pure” form (the term “substantially pure” is defined below) .
  • isolated protein or polypeptide
  • isolated and purified protein or polypeptide
  • This term refers primarily to a protein produced by expression of an isolated nucleic acid molecule of the invention. Alternatively, this term may refer to a protein which has been sufficiently separated from other proteins with which it would naturally be associated, so as to exist in “substantially pure” form.
  • substantially pure refers to a preparation comprising at least 50-60% by weight the compound of interest (e.g., nucleic acid, oligonucleotide, protein, etc.) . More preferably, the preparation comprises at least 75% by weight, and most preferably 90-99% by weight, the compound of interest. Purity is measured by methods appropriate for the compound of interest (e.g. chromatographic methods, agarose or polyacrylamide gel electrophoresis , HPLC analysis, and the like) .
  • Nucleic acid sequences and amino acid sequences can be compared using computer programs that align the similar sequences of the nucleic or amino acids thus define the differences.
  • standard BLAST query parameters utilized by public databases such as GenBank, are utilized herein.
  • the term "substantially the same” refers to nucleic acid or amino acid sequences having sequence variation that do not materially affect the nature of the protein (i.e. the structure, thermostability characteristics and/or biological activity of the protein) .
  • the term “substantially the same” is intended to refer to the coding region and to conserved sequences governing expression, and refers primarily to degenerate codons encoding the same amino acid, or alternate codons encoding conservative substitute amino acids in the encoded polypeptide.
  • the term “substantially the same” refers generally to conservative substitutions and/or variations in regions of the polypeptide not involved in determination of structure or function.
  • percent identical refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical amino acids in the compared amino acid sequence by a sequence analysis program.
  • Percent similar refers to the percent of the amino acids of the subject amino acid sequence that have been matched to identical or conserved amino acids. conserved amino acids are those which differ in structure but are similar in physical properties such that the exchange of one for another would not appreciably change the tertiary structure of the resulting protein.
  • percent identical refers to the percent of the nucleotides of the subject nucleic acid sequence that have been matched to identical nucleotides by a sequence analysis program.
  • Transcriptional and translational control sequences are DNA regulatory elements such as promoters, enhancers, ribosome binding sites, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • expression control sequences or elements are DNA regulatory elements such as promoters, enhancers, ribosome binding sites, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • expression is intended to include transcription of DNA and translation of the mRNA transcript .
  • promoter refers generally to transcriptional regulatory regions of a gene, which may be found at the 5 ' or 3 ' side of the coding region, or within the coding region, or within introns.
  • a promoter is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the typical 5' promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5 1 direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI) , as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • selectable marker gene refers to a gene encoding a product that, when expressed, confers a selectable phenotype such as antibiotic resistance on a transformed cell.
  • operably linked means that the regulatory sequences necessary for expression of a particular coding sequence are placed in the DNA molecule in the appropriate positions relative to the coding sequence so as to enable expression of the coding sequence. This same definition is sometimes applied to the arrangement of transcription units and other regulatory elements (e.g., enhancers or translation regulatory sequences) in an expression vector.
  • a “vector” is a replicon, such as plasmid, phage, cosmid, or virus to which another nucleic acid segment may be operably inserted so as to bring about the replication or expression of the segment.
  • nucleic acid construct or "DNA construct” is sometimes used to refer to a coding sequence or sequences operably linked to appropriate regulatory sequences and inserted into a vector for transforming a cell. This term may be used interchangeably with the term “transforming DNA” .
  • Such a nucleic acid construct may contain a coding sequence for a gene product of interest, along with a selectable marker gene and/or a reporter gene.
  • a "heterologous" region of a nucleic acid construct is an identifiable segment (or segments) of the nucleic acid molecule within a larger molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the native gene) . Allelic variations or naturally-occurring mutational events do not give rise to a heterologous region of DNA as defined herein.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • the transforming DNA may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming DNA may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming DNA has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming DNA.
  • a “clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a “cell line” is a clone of a primary cell that is capable of stable growth in vi tro for many generations.
  • "Killing”, "programmed cell death” and “apoptosis” are used interchangeably in this text to describe a series of cellular events that culminates in the death of the target cell.
  • Apoptosis is a characteristic morphological change in which the cell and its nucleus shrink, condense and fragment. Frequently accompanying this morphological change are the activation of intracellular proteases and nucleases that lead to, for example, cell nucleus involution and nuclear DNA fragmentatio . II . Description
  • HPV-E6 protein targets the p53 tumor suppressor protein for degradation by ubiquitin-mediated proteolysis, making such cancers resistant to p53 -mediated therapy.
  • HPV-E6 targets both endogenous wild-type and mutant p53 for degradation (Fig. 1) . Possibly because p53 mutations are rare in cervical cancer (Busby-Earle et al . , 1994, Br. J. Cancer 69: 732-737) the hypothesis that HPV-E6 could target endogenous mutant p53 for degradation has not been previously directly tested. While several studies have reported low levels of p53 expression and an inverse correlation between the presence of HPV and p53 expression (Scheffner et al . , 1991, Proc . Natl . Acad. Sci. USA 88: 5523-5527; Srivastava et al . , 1992,
  • p73 is a potent inducer of apoptosis and is an effective inhibitor of cancer cell growth.
  • p73 is an excellent substitute for p53 in gene replacement because of its resistance to E6-mediated proteolysis.
  • Other differences of effect of viral oncoproteins have been noted (Marin et al . , 1998, Mol . Cell. Biol. 18:6316-6324; Steengenga et al . , 1999, Mol. Cell. Biol.
  • kits that utilize p73 for arresting the growth of E6 -expressing cells, particularly HPV- infected cancer cells.
  • the treatment of the target cells may be in vivo, within the patient; or ex vivo, removed from the patient, treated, and reintroduced into the patient. It is contemplated that the methods, pharmaceutical preparations and kits of the invention can be used alone or in conjunction with chemotherapy or radiation therapy to treat cancers in vivo . Additionally, the methods, pharmaceutical preparations and kit of the invention can be used for experimental purposes in vi tro with standard cell cultures.
  • the treatment of cancers associated with the over-expression of E6 protein is of particular interest. Several circumstances may result in mammalian cells that over-express E6 protein.
  • HPV infection is well-known to result in cancers of the uterine cervix.
  • HPV infection may also result in esophageal squamous cell cancer, laryngeal papilloma, bronchiolo-alveolar carcinoma, penile carcinoma and bladder carcinoma, among others.
  • E6 -over-expression may also result from a mutation in the mammalian cell genome such that the endogenous E6 gene is over-expressed. All mammalian cells that over-express the E6 protein, regardless of the origin of the phenotype, are contemplated for treatment with the method of the invention.
  • the amino acid sequence of p73 protein on which to base the nucleic acid construct is ideally from the gene that is endogenous to the species which is being treated.
  • Homo sapiens is being treated and the nucleic acid construct encodes SEQ ID NO:l or SEQ ID NO : 2.
  • the nucleic acid sequence is SEQ ID NO : 3.
  • variants of p73 protein also exist in Homo sapiens and the sequences of these variants are also contemplated for use with the invention (DeLaurenzi et al . , 1999, Cell Death Differ. 6:389-390 incorporated by reference herein;
  • amino acid sequence information such as the full length sequence in SEQ ID NO:l and SEQ ID NO : 2 enables the preparation of a synthetic gene that can be used to synthesize the Homo sapiens p73 protein in standard in vivo expression systems or to make viral vectors expressing the p73 protein.
  • the sequence encoding Homo sapiens p73 from isolated native nucleic acid molecules such as SEQ ID NO: 3 can be utilized.
  • the amino acid and nucleic acid sequences found in Genbank Accession Nos . AF138873, Y11419 and AF043641 can be used to prepare the p73 protein endogenous to Mus musculus, Chlorocebus aethiops and Barbus barbus, respectively.
  • an isolated nucleic acid that encodes the amino acid sequence of the invention can be prepared by oligonucleotide synthesis.
  • Codon usage tables can be used to design a synthetic sequence that encodes the protein of the invention.
  • the codon usage table has been derived from the organism in which the synthetic nucleic acid will be expressed.
  • the codon usage for E. coli would be used to design an expression DNA construct to produce the Homo sapiens p73 in E. coli .
  • Synthetic oligonucleotides may be prepared by the phosphoramadite method employed in the Applied Biosystems 38A DNA Synthesizer or similar devices.
  • the resultant oligonucleotide may be purified according to methods known in the art, such as high performance liquid chromatography (HPLC) .
  • HPLC high performance liquid chromatography
  • Nucleic acid molecules encoding p73 also may be isolated from appropriate species using methods well known in the art.
  • Native nucleic acid sequences may be isolated by screening mammalian or other cDNA or genomic libraries with oligonucleotides preferably designed to match the Homo sapiens coding sequence of p73 (SEQ ID NO: 3) .
  • SEQ ID NO: 3 Several other p73 amino acid sequences are now known: Mus musculus , Genbank Accession No. AF138873; Chlorocebus aethiops (Green Monkey) , Genbank Accession No. Y11419; and Barbus barbus , Genbank Accession No. AF043641; each of these sequences is incorporated by reference herein.
  • Oligonucleotides designed to match any of these sequences or to match regions of high homology between these sequences may also be used to screen for mammalian p73 -encoding nucleotides. In positions of degeneracy where more than one nucleic acid residue could be used to encode the appropriate amino acid residue, all the appropriate nucleic acids residues may be incorporated to create a mixed oligonucleotide population, or a neutral base such as inosine may be used.
  • the strategy of oligonucleotide design is well known in the art (see also Sambrook et al . , Molecular Cloning, 1989, Cold Spring Harbor Press, Cold Spring Harbor NY) .
  • PCR polymerase chain reaction primers may be designed by the above method to match a known coding sequence of p73, and these primers used to amplify the native nucleic acids from isolated mammalian cDNA or genomic DNA.
  • Nucleic acids having the appropriate sequence homology with a Homo sapiens p73 synthetic nucleic acid molecule may be identified by using hybridization and washing conditions of appropriate stringency.
  • One common formula for calculating the stringency conditions required to achieve hybridization between nucleic acid molecules of a specified sequence homology (Sambrook et al. , 1989, supra) :
  • T m 81.5°C + 16.6Log [Na+] + 0.41(% G+C) - 0.63 (% formamide) - 600/#bp in duplex
  • the T m is 57 °C.
  • the T m of a DNA duplex decreases by 1 - 1.5°C with every 1% decrease in homology.
  • targets with greater than about 75% sequence identity would be observed using a hybridization temperature of 42 °C.
  • Nucleic acids of the present invention may be maintained as DNA in any convenient cloning vector.
  • clones are maintained in plasmid cloning/expression vector, such as pBluescript
  • P73 protein can be produced by using in vi tro expression methods known in the art.
  • a DNA molecule such as a DNA encoding the amino acid sequence SEQ ID NO : 1 or SEQ ID NO : 2
  • a plasmid vector adapted for expression in a bacterial cell, such as E . coli , or a eukaryotic cell, such as Saccharomyces cerevisiae or other yeast.
  • a commercially available expression/secretion system can be used, whereby the recombinant protein is expressed and thereafter secreted from the host cell, to be easily purified from the surrounding medium.
  • expression/secretion vectors are not used, an alternative approach involves purifying the recombinant protein by affinity separation, such as by immunological interaction with antibodies that bind specifically to the recombinant protein or fusion proteins such as His tags. Such methods are commonly used by skilled practitioners.
  • the method of the invention for treating mammalian cells that over-express E6 comprises administering a therapeutically effective amount of p73 protein to the target cells.
  • the administration of the p73 protein can be accomplished via several methods, including the exposing the target cell, i.e., the E6- over-expressing cell, to p73 protein, or exposing the target cell to a nucleic acid construct that expresses an appropriate p73 coding sequence.
  • p73 e.g, as a protein or as a nucleic acid encoding the protein
  • the choice of method of administration will depend largely on the position of the target cells and the length of time the treatment is needed.
  • Target cells may be removed from the patient and treated ex vivo, and then reintroduced to the patient. Additionally, the treatment may be used in cell cultures for experimental purposes.
  • the target cells comprise E6-over-expressing carcinomas.
  • the target cells are papilloma-virus positive cancers.
  • the target cells are HPV-positive carcinomas of the uterine cervix.
  • the administration of p73 protein to target cells can be accomplished by exposing the target cell to p73 protein.
  • the target cell are tumor cells within an animal, it is preferred that the protein is administered in a protected form to increase their stability cells
  • liposomes are water-filled vesicles composed of several phospholipids layers surrounding an aqueous core with an outer shell capable of providing direction to specific target cells.
  • liposomes are composed of some combination of phosphatidylcholine, cholesterol, phosphatidylglycerol or other glycolipids or phospholipids (Hudson and Black, 1993, American Pharmacy NS33 (5) :23-24) .
  • Insoluble polymers composed of polyethylene may also be used to form a protective layer around the protein, inhibiting degradation while traveling to the target cell (Hudson and Black, 1993, American Pharmacy NS33 (5) :23-24) .
  • Another way to deliver p73 protein to target cells is to couple the protein to a target cell -specific monoclonal antibody. This approach allows the protein to be specifically delivered to the target cell and minimizes toxic effects on non-target cells (Houston, 1993, Current Opinion in Biotechnology 4:739-744) .
  • the p73 protein is administered to the target cell through the use of heterologous nucleic acids that will cause the protein to be synthesized within the target cell.
  • nucleic acids can be temporary residents in the target cell, such as expression plasmids, or they can be stably integrated into the genome of the target cell .
  • Expression plasmids are particularly appropriate for experimental work with cell cultures, such as illustrated in Example 1. The construction of such plasmids and the transformation of target cells with them in vi tro is well known to those of skill in the art of cell biology.
  • Expression vectors suitable for p73 expression in mammalian cells are commercially available (Gene Therapy Systems, San Diego) .
  • Naked DNA and plasmids may be delivered to the target cells by several known means. The naked DNA may be transferred directly into the genetic material of the cells (Wolff et al .
  • the p73 -encoding DNA may be delivered in liposomes (Ledley, 1987, J. Pediatrics 110:1) or proteoliposomes that contain viral envelope receptor proteins (Nicolau et al , 1983, Proc. Natl . Acad. Sci . U.S.A. 80:1068), or the p73- encoding DNA may be coupled to a polylysine-glycoprotein carrier complex.
  • viral vectors are preferred.
  • a variety of viral vector may be used in this invention, included retroviral vectors such as the herpes simplex virus (U.S.
  • Patent 5,288,641 incorporated herein by reference
  • Cytomegalovirus murine leukemia virus
  • Boese et al . 1995, Science 270:475-4719
  • Miller 1992, Curr. Top. Microbiol . Immunol. 158:1
  • Recombinant adeno-associated virus such as those described by U.S. Patent No. 5,139,941 (which is incorporated herein by reference) and recombinant adenoviral vectors (He et al . , 1998, PNAS 95:2509-2514, incorporated by reference herein) are particularly preferred.
  • recombinant lentivirus vectors such as a recombinant Human Immunodeficiency Virus (U.S. Patent No. 5,885,805; Blaese et al . , 1995, Science 270:475-479; Onodera et al . , 1998, J. of Virology 72:1769-1774) and Feline Immunodeficiency Virus.
  • a recombinant Human Immunodeficiency Virus U.S. Patent No. 5,885,805; Blaese et al . , 1995, Science 270:475-479; Onodera et al . , 1998, J. of Virology 72:1769-1774
  • Feline Immunodeficiency Virus Often these vectors have been designed so that they are replication-defective, and the techniques to prepare such vectors are well known in the art (Ghosh-Choudhury and Graham, 1987, Biochem. Biophys . Res. Comm
  • the recombinant vector of the invention comprises a nucleic acid construct comprising a sequence encoding a p73 protein operably linked to an appropriate promoter and other expression-regulatory sequences.
  • a strong constitutive promoter such as a cytomegalovirus promoter, a viral LTR, RSV or SV40 promoter
  • a cytomegalovirus promoter is used.
  • promoters associated with genes that are expressed at high levels in mammalian cells such as elongation factor- 1 and actin, are also contemplated. It is particularly advantageous to use a viral -specific and -regulated promoter to direct expression specifically in affected cancer cells.
  • the HPV-E6 promoter is used.
  • a recombinant adenoviral vector is used to deliver the p73- expressing construct to the target cell.
  • adenoviral vectors for gene therapy is well known in the art (El-Deiry et al . , 1993, Cell 75:817; Blogosklonny and El-Deiry, 1996, Int. J. Cancer 67:386-395; Prabhu et al . , 1996, Clin Cancer Res. 2:1221-1230; Zeng et al . , 1997, Int. J. Oncol. 11:221-226; Mitchell and El-Deiry, 1999, Cell Growth and Diff. 10:223-230; Meng et al . , 1998, Clin.
  • an adenovirus vector has been used successfully to deliver p53 to target cells to treat lung cancer in human patients (Roth et al . , 1996, Nature Med. 2:974 incorporated herein by reference; and U.S. Patent 5,747,469 incorporated herein by reference) . It is contemplated that these protocols with simple variation that will be well known to those in the art can be used to administer the p73 protein to target cells in the invention. In a most preferred embodiment, therapeutically effective amounts of the viral vector are delivered to the cancers by direct injection.
  • p53 and p73 have significant amino acid sequence similarities (Kaghad et al , 1997, Cell 90:809-818, incorporated by reference herein) , particularly in the most conserved regions of p53 : the transactivation, DNA binding and p53 oligomerization domains.
  • a sequence similar to the MDM2- binding domain is also present in p73.
  • the residues in p53 often found mutated in tumors and shown to be required for DNA recognition are conserved and occupy identical positions in p73.
  • the C-terminal domain of p73 shows homology to invertebrate p53 homologs .
  • p73 regulates p53 target genes when p73 is over-expressed in cells (Zhu et al . , 1998, Cancer Research 58:5061-5065; Jost et al., 1997, Nature 389:181-184). Finally, as a result of activation of p53 -responsive genes, p73 can inhibit cell growth and induce apoptosis in a manner similar to p53.
  • compositions that can be used to treat mammalian cells with p73 in vi tro, in vivo and ex vivo .
  • the compositions comprise either p73 protein or nucleic acids encoding p73 protein.
  • the pharmaceutical compositions of the invention are formulated in an appropriate "biologically acceptable medium" .
  • biologically acceptable medium includes any and all solvents, dispersion media and the like which may be appropriate for the desired route of administration of the pharmaceutical preparation, as exemplified in the preceding paragraph.
  • the use of such media for pharmaceutically active substances is known in the art. Except insofar as any conventional media or agent is incompatible with the nucleic acid molecules or proteins to be administered, its use in the pharmaceutical preparation is contemplated.
  • Dosage unit form refers to a physically discrete unit of the pharmaceutical preparation appropriate for the patient undergoing treatment .
  • Each dosage should contain a quantity of active ingredient calculated to produce the desired effect in association with the selected pharmaceutical carrier. Procedures for determining the appropriate dosage unit are well known to those skilled in the art .
  • the pharmaceutical composition also can include various other components as additives or adjuncts.
  • Exemplary pharmaceutically acceptable components or adjuncts which are employed in relevant circumstances include antioxidants, free radical scavenging agents, peptides, growth factors, antibiotics, bacteriostatic agents, immunosuppressives, anticoagulants, buffering agents, anti-inflammatory agents, anti-pyretics, time release binders, anaesthetics, steroids and corticosteroids .
  • Such components can provide additional therapeutic benefit, act to effect the therapeutic action of the pharmaceutical composition, or act towards preventing any potential side effects which may be posed as a result of administration of the pharmaceutical composition.
  • the p73 protein or nucleic acid molecule can be employed as part of a pharmaceutical composition with other compounds (e.g., chemotherapeutic agents) intended to prevent or treat cancer or a related disorder.
  • compositions can vary. They can be administered by inhalation (e.g., in the form of an aerosol either nasally or using delivery articles of the type set forth in U.S. Patent No. 4,922,901 to Brooks et al .
  • topically e.g., in lotion form or as a suppository
  • orally e.g., in liquid form within a solvent such as an aqueous or non-aqueous liquid, or within a solid carrier
  • intravenously e.g., within a dextrose or saline solution
  • infusion or injection e.g., as a suspension or as an emulsion in a pharmaceutically acceptable liquid or mixture of liquids
  • intrathecally e.g., intracerebro- ventricularly
  • transdermally e.g., using a transdermal patch
  • the administration of the pharmaceutical compositions of the present invention can be intermittent, or at a gradual, continuous, constant or controlled rate to a warm-blooded animal, (e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey); but advantageously is preferably administered to a human being.
  • a warm-blooded animal e.g., a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey
  • the time interval between administrations can vary.
  • Administration preferably is such that the active ingredients of the pharmaceutical formulation contact the target cells, whether within or outside the body of a mammalian subject.
  • the appropriate dose of the compound is that amount effective to result in increased levels of p73 protein within the target cell.
  • effective amount therapeutic amount or “effective dose” is meant that amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder.
  • Prevention of the disorder is manifested by delaying the onset of the symptoms of the disorder.
  • Treatment of the disorder is manifested by a decrease in the symptoms associated with the disorder or an amelioration of the recurrence of the symptoms of the disorder.
  • the effective dose can vary, depending upon factors such as the condition of the patient, the severity of the symptoms of the disorder, and the manner in which the pharmaceutical composition is administered.
  • the effective dose of compounds will of course differ from patient to patient but in general includes amounts starting where target cell growth is halted to where the target cell is killed.
  • Dosages contemplated for use with the retroviral vector embodiment of the invention are those suggested in U.S. Patent 5,747,469 (incorporated herein by reference) .
  • One of ordinary skill in the art will know how to determine such doses without undue experimentation .
  • Kits with the components necessary to treat E6- over-expressing target cells by the method of the invention are also provided.
  • the kit contains therapeutically effective amounts of the pharmaceutical preparation of the invention in a container.
  • the pharmaceutical preparation in the kit may be comprised of p73 protein or a DNA construct encoding p73, preferably inserted into a vector for transforming cells.
  • the p73 protein or p73 encoding viral vector may be in the form of a pharmaceutically acceptable sterile solution such as sterile saline, dextrose solution or buffered solution.
  • the p73 protein or p73 encoding viral vector can be lyophilized or desiccated.
  • the kit may optionally further comprise a container of a pharmaceutically acceptable solution, (e.g., saline, dextrose solution, etc.), preferably sterile, to reconstitute the pharmaceutical preparation to form a solution for injection purposes.
  • a pharmaceutically acceptable solution e.g., saline, dextrose solution, etc.
  • instructions may be included in the kit.
  • the kit may additionally comprise pharmaceutical preparations in containers for other therapies related to cancer treatment, such as chemotherapy.
  • Plasmids The mammalian expression vector pCMV-neo-Bam (Baker et al . , 1990, Science 249: 912-915) and the wild-type p53 expression vector SN3 (Baker et al . , 1990, Science 249: 912-915) were obtained from Bert Vogelstein (Johns Hopkins University) . Wild-type and mutant p73 ⁇ and p73 ⁇ plasmids (Jost et al.,1997, Nature 389: 191-194; incorporated herein by reference) were obtained from William G. Kaelin, Jr. (Dana Farber Cancer
  • HPV-E6 expression plasmid (Prabhu et al . , 1996, Clin. Cancer Res. 2: 1221-1229; incorporated herein by reference) was obtained from Kathleen Cho (Johns Hopkins University) . Cell culture and transfection conditions.
  • the mutant p53 -expressing human colon adenocarcinoma cell line SW480 was maintained in culture as previously described (Prabhu et al . , 1996, Clin. Cancer Res. 2: 1221-1229) .
  • the mutant p53 -expressing human glioma cell lines U373 and H80 were obtained from Peter C.
  • Ad-LacZ reagent Panet al . , 1996, Clin. Cancer Res. 2: 1221-1229
  • the HPV type 16 E6-expressing replication deficient adenovirus was prepared and titered as previously described (Satyamoorthy et al . , 1997, Cancer Res. 57: 1873-1876; Prabhu et al . , 1996, Clin. Cancer Res. 2: 1221-1229).
  • the CMV promoter-driven HPV type 16 E6 cDNA was inserted into an E3 -deleted adenovirus by homologous recombination to generate El and E3 deleted replication defective Ad-E6 adenovirus (Satyamoorthy et al . , 1997, Cancer Res. 57: 1873-1876; incorporated herein by reference) .
  • the cloned HPV-E6 DNA sequence was verified and expression of HPV-E6 was verified by Northern blotting of total RNA derived from Ad-E6 versus Ad-LacZ infected cells. Cells were infected using an MOI of 50 as previously described (Prabhu et al . , 1996, Clin. Cancer Res. 2: 1221-1229). Infection of SW480 cells using Ad-LacZ at an MOI of 50 followed by X-gal staining revealed greater than 99% infectivity.
  • TUNEL assays At 48 hrs following transfection, cells were formalin fixed and the extent of apoptosis was assessed by nicked-end labeling using the Apotag kit (Oncor) followed by analysis using fluorescence microscopy.
  • HPV-E6 targets both wild-type and mutant p53 protein for degradation.
  • Ad-E6 E6-expressing adenovirus
  • Fig. 1 a panel of human cancer cells derived from different tissues and containing either endogenous wild-type or mutant p53 were infected.
  • Fig. 1 E6-expressing adenovirus
  • Fig. 1 E6-expressing adenovirus
  • p73 ⁇ The resistance of p73 to E6-dependent proteolysis was observed with p73 ⁇ , p73 ⁇ m, p73 ⁇ , or p73 ⁇ m. This observation suggested that p73 is a candidate for gene replacement in E6-expressing cancer cells. p73 ⁇ induces apoptosis and suppresses growth in HPV E6-expressing human cancer cells. p73 ⁇ was previously found to be a potent activator of p53-dependent gene expression (Kaghad et al . , 1997, Cell 90: 809-819; Jost et al.,1997, Nature 389: 191-194). p73 ⁇ in colony suppression assays in the absence or presence of E6-expression.
  • p73 ⁇ was found to be a potent growth suppressor. Transfection studies revealed that p73 ⁇ was a less potent suppressor of growth of SW480 cancer cells either in the absence or presence of HPV-E6. This could not be explained by dominant negative inhibition of p73 ⁇ by the endogenous p53 mutant in SW480 cells because it was previously shown that p73 shows negligible interaction with p53 (Kaghad et al . , 1997, Cell 90: 809-819) . p73 ⁇ has been previously shown to be an inducer of apoptosis, similar to p53 (Jost et al.,1997, Nature 389: 191-194).

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Abstract

L'invention concerne des procédés, des compositions pharmaceutiques et des trousses servant à provoquer une mort cellulaire programmée chez des cellules exprimant l'oncogène E6. Les procédés et les compositions sont particulièrement appropriées pour le traitement de cancers impliquant des infections à un virus exprimant E6, tel que le virus du papillome humain (HPV). Les procédés et les compositions utilisent l'homologue de p53, p73. A la différence de p53, p73 n'est pas ciblé par l'oncoprotéine E6 en vue d'une dégradation induite par l'ubiquitine, et constitue de ce fait une alternative viable à une thérapie à base de p53 pour traiter des cancers exprimant E6.
PCT/US1999/014057 1998-06-24 1999-06-23 Compositions et procedes servant a provoquer une apoptose chez des cellules exprimant e6 WO1999066946A1 (fr)

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WO2003047613A1 (fr) * 2001-12-03 2003-06-12 Biotech Research Ventures Pte Limited Utilisation de c-jun ou d'agents d'activation de c-jun tels que les uv ou les c-jun n-terminal kinases (jnk) pour traiter le cancer
WO2004108142A2 (fr) * 2003-06-06 2004-12-16 The University Of Manchester Traitement d'etats lies au vieillissement
WO2006054138A1 (fr) * 2004-11-19 2006-05-26 Istituti Fisioterapici Ospitalieri Peptide capable de briser un complexes proteiques m-p53/p63, m-p53/73 et m-p53/proteines isoformes respectives dans des cellules tumorales, ainsi qu'utilisation de ce peptide dans le champ pharmacologique
WO2008093083A2 (fr) * 2007-01-31 2008-08-07 Vibio Limited Marqueur pronostique pour le cancer
US7557187B2 (en) 1996-02-02 2009-07-07 Sanofi-Aventis Purified SR-p70 protein
EP2370582A2 (fr) * 2008-12-04 2011-10-05 Opko Curna, LLC Traitement de maladies liées à un gène suppresseur de tumeur par inhibition d'un transcrit antisens naturel du gène

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Cited By (16)

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US7838648B2 (en) 1996-02-02 2010-11-23 Sanofi Aventis Purified SR-p70 protein
US7557187B2 (en) 1996-02-02 2009-07-07 Sanofi-Aventis Purified SR-p70 protein
US7459282B2 (en) 2001-12-03 2008-12-02 Biotech Research Ventures Pte Limited Use of c-Jun or c-Jun activating agents such as UV or c-Jun N-terminal kinases (JNKs) for treating cancer
WO2003047613A1 (fr) * 2001-12-03 2003-06-12 Biotech Research Ventures Pte Limited Utilisation de c-jun ou d'agents d'activation de c-jun tels que les uv ou les c-jun n-terminal kinases (jnk) pour traiter le cancer
WO2004108142A2 (fr) * 2003-06-06 2004-12-16 The University Of Manchester Traitement d'etats lies au vieillissement
WO2004108142A3 (fr) * 2003-06-06 2005-04-21 Univ Manchester Traitement d'etats lies au vieillissement
JP2008520641A (ja) * 2004-11-19 2008-06-19 イスティトゥート フィシオテラピシ オスピタリエリ 腫瘍細胞で形成されるｍ−ｐ５３／ｐ６３、ｍ−ｐ５３／ｐ７３およびｍ−ｐ５３／各イソ型タンパク質複合体を分解可能なペプチドおよび薬理学分野におけるその使用
WO2006054138A1 (fr) * 2004-11-19 2006-05-26 Istituti Fisioterapici Ospitalieri Peptide capable de briser un complexes proteiques m-p53/p63, m-p53/73 et m-p53/proteines isoformes respectives dans des cellules tumorales, ainsi qu'utilisation de ce peptide dans le champ pharmacologique
JP4664984B2 (ja) * 2004-11-19 2011-04-06 イスティトゥート フィシオテラピシ オスピタリエリ 腫瘍細胞で形成されるｍ−ｐ５３／ｐ６３、ｍ−ｐ５３／ｐ７３およびｍ−ｐ５３／各イソ型タンパク質複合体を分解可能なペプチドおよび薬理学分野におけるその使用
US7939624B2 (en) 2004-11-19 2011-05-10 Instituti Fisioterapici Ospitalieri Peptide able to break the m-p53/p63, m-p53/p73 and m-p53/respective isoform proteins complex formed in the tumor cells and uses thereof in the pharmacological field
WO2008093083A3 (fr) * 2007-01-31 2008-09-25 Univ Manchester Marqueur pronostique pour le cancer
WO2008093083A2 (fr) * 2007-01-31 2008-08-07 Vibio Limited Marqueur pronostique pour le cancer
EP2370582A2 (fr) * 2008-12-04 2011-10-05 Opko Curna, LLC Traitement de maladies liées à un gène suppresseur de tumeur par inhibition d'un transcrit antisens naturel du gène
EP2370582A4 (fr) * 2008-12-04 2013-05-15 Curna Inc Traitement de maladies liées à un gène suppresseur de tumeur par inhibition d'un transcrit antisens naturel du gène
RU2746478C2 (ru) * 2008-12-04 2021-04-14 КьюРНА, Инк. Лечение связанных с геном-супрессором опухолей заболеваний посредством ингибирования природного транскрипта в антисмысловой ориентации относительно этого гена
US11697814B2 (en) 2008-12-04 2023-07-11 Curna, Inc. Treatment of tumor suppressor gene related diseases by inhibition of natural antisense transcript to the gene

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