WO2000021575A2 - Inhibiteurs de calpaine et applications de ces inhibiteurs - Google Patents

Inhibiteurs de calpaine et applications de ces inhibiteurs Download PDF

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WO2000021575A2
WO2000021575A2 PCT/US1999/021453 US9921453W WO0021575A2 WO 2000021575 A2 WO2000021575 A2 WO 2000021575A2 US 9921453 W US9921453 W US 9921453W WO 0021575 A2 WO0021575 A2 WO 0021575A2
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cells
cell
calpain inhibitor
calpain
administration
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PCT/US1999/021453
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WO2000021575A3 (fr
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Isabella A. Atencio
Drake M. Laface
Muralidhara Ramachandra
Paul W. Shabram
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Canji, Inc.
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Priority to AU10926/00A priority Critical patent/AU1092600A/en
Publication of WO2000021575A2 publication Critical patent/WO2000021575A2/fr
Publication of WO2000021575A3 publication Critical patent/WO2000021575A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K38/1758—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals p53
    • 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
    • A61K38/55—Protease inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • Programmed cell death is a natural process by which an organism eliminates particular cells in a regulated process.
  • Programmed cell death pathways are initiated by a variety of stimuli, some internal to the cell (e.g. DNA damage) while others are activated as a result of external factors (e.g. fas ligand).
  • a single mediator of apoptosis has not been elucidated. Rather, a variety of apoptotic mechanisms are in a potential state for response at all times.
  • the fact that many molecules are identified as possessing both pro- and anti-apoptotic activity e.g. caspases,NF-kB, API, and p53), there appears to be a fine balance of factors which permit survival or terminate the cell.
  • p53 While no single mediator of apoptosis has been identified, mutations in p53 are recognized as the most common genetic alterations associated with human cancer, diseases characterized by abberent regulation of cell cycle control and apoptosis. Although p53 was first characterized as a dominant cooperating oncogene, it was later determined that the form of p53 under investigation at that time was a mutant, inactive and long-lived form of the wild-type protein. This mutant protein displaced the short-lived wild-type native form from its enzymatic substrate binding sites and was mistakenly deemed to act dominantly. p53 is now thought of as a tumor suppressor gene.
  • p53 The sequence specific DNA binding properties of p53 regulate the transcription of a continually expanding number of genes involved in regulation of the cell cycle and apoptosis.
  • p53 has been implicated in the transcriptional downregulation of the bcl2 gene and upregulation of the bax gene, precipitating a cascade of events leading to apoptosis.
  • the complete mechanism of action of p53 is not fully elucidated.
  • p53 binds to many important cellular proteins and is involved in the control of gene expression, cell cycle regulation and apoptosis, it is observed that some tumors produce wildtype p53, yet do not undergo apoptosis.
  • the intracellular cytosolic concentration of p53 appears to be maintained by a balance of continual synthesis and degradation.
  • p53 exists as an inactive cytosolic monomeric protein with a very short half-life. Increasing data support the view that, in normal cells, stability of p53 is controlled through mdm-2 binding and ubiquination, through an, as yet, incompletely understood mechanism. Binding of p53 to mdm-2 has been shown to lead to ubiquitination of p53 protein. Following ubiquitination, the p53 is believed to be degraded through a common proteasome mediated degradative pathway. p53 exerts its function as a tetramer of phosphorylated p53 subunits. The phosphorylation step leading to activation of p53 function is mediated by DNA-dependent protein kinase ("DNA-PK"). DNA- PK appears to be activated in response to DNA damage. Woo, et al. (1998) Nature 394:700-704; Lane, D. (1998) Nature 394:616-617.
  • DNA-PK DNA-dependent protein kinase
  • the p53 apoptotic pathway is clearly an important pathway employed by the organism to eliminate aberrant cell types.
  • p53 has been demonstrated to play a role in many biochemical pathways related to human carcinogenesis. In approximately 50% of human cancers, the p53 gene is inactivated through mutational, viral, or other cellular components. Somatic mutations in the DNA binding domain of p53 are found in the majority of human tumors bearing p53 alterations. It has been demonstrated that restoration of p53 function in p53 deficient tumor cells will induce apoptosis of tumor cells.
  • rAd-p53 replication defective recombinant adenovirus vectors expressing wildtype p53
  • Calpain a calcium dependent cysteine protease
  • Calpain has two primary isoforms (m-calpain and ⁇ - calpain) distinguished by the calcium concentration required for their activation in vitro.
  • Calpain consists of a heavy and light chain.
  • the heavy chain consists of a cysteine proteinase domain.
  • the light chain is a calcium binding domain and possesses four EF-hands characteristic of calcium binding proteins.
  • the native form of calpain is inactive except at relatively high Ca+2 concentrations.
  • ⁇ -calpain also known as calpain I
  • ⁇ M calcium levels for activation In vitro experiments indicate that ⁇ -calpain (also known as calpain I) requires ⁇ M calcium levels for activation.
  • m-calpain also known as calpain II
  • calpain II requires a much higher (mM) calcium concentration for activation.
  • Calpain is a cytosolic protease whose activity is modulated by the cytosolic inhibitor protein, calpastatin. Upon activation, calpain has been shown to translocate to the cell membrane where it is sequestered from its inhibitors (Lane et al, 1992; Molinari and Carafoli, 1997). Additionally, calpain has been shown to possess in vitro proteolytic action against a broad range of substrates including components of receptor signaling pathways, interleukin receptors where the common cytokine receptor ⁇ chain is cleaved by calpains (Noguchi, et al.
  • calpain inhibitors demonstrate a broad range of substrates in vitro, its role in vivo remains unclear.
  • the therapeutic applications suggested for calpain inhibitors include the prevention of neurodegradation and cellular damage caused by excessive activation of glutamate receptors in neuronal cells, retarding cataract formation , minimization of degeneration of neuronal tissues following injury, mycoardial infaction, angioplastic restenosis, prevention of cartilage damage and subsequent inflammation, muscular dystrophy and platelet aggregation associated with thrombosis.
  • In vivo experiments in mice and rats have shown that prior infusion of calpain inhibitors prevents neuronal and hepatocyte cell death due to hypoxia, and increases survival after orthotopic liver transplants.
  • calpain inhibitors In vitro , calpain inhibitors have been observed to attenuate apoptosis in primary rat hepatocytes in response to a variety of apoptotic stimuli. Additionally, calpain inhibitors have been shown to inhibit activation-induced programmed cell death of T-cells from HIV positive donors (Sarin et al, 1994). Calpain has been shown to cleave the precursor form of IL-la into a 17kD C-terminal fragment referred to as "mature" IL-la and a 16kD N-terminal fragment which migrates to the nucleus and induces apoptosis in vitro. In each of these instances, the primary application of the calpain inhibitor is to prevent cell death.
  • p53 gene therapy has focused on the use of recombinant viral, particularly adeno viral, vectors to result in the intracellular expression of p53.
  • a significant excess of the recombinant virus must be administered to the patient because not every viral particle administered will infect the target cell. This is due at least in part to the effects of diffusion, clearance and/or neutralization by the organism, and that not every interaction between the viral particle and a target cell results in infection and productive expression.
  • the systemic administration of excess recombinant virus poses potential safety concerns to the individual undergoing treatment. Consequently, methods which can be employed to lower the total dose of the recombinant viral vector while maintaining a therapeutically effective dose are desired.
  • the present invention provides a method of inducing apoptosis in p53 positive tumor cells by the administration of a calpain inhibitor alone or in combination with rAd-p53 or in combination with rAd-p53 and in tumors with mutated or null p53 status.
  • the present invention also provides a decreased CTL response to the administration of recombinant adenoviral vectors by the concomitant administration of calpain inhibitors. The combined effect being increased p53 expression.
  • the present invention provides a method to enhance p53-mediated apoptosis in a cell by the administration of p53 in combination with a calpain inhibitor.
  • the present invention further provides a method of inducing cell death in p53 positive tumor cells by the administration of a therapeutically effective dose of a calpain inhibitor.
  • the present invention further provides a method of increasing the infectivity of a cell to a viral vector by treatment of the cell with a calpain inhibitor.
  • the present invention further provides a method of enhancing transciption of a therapeutic transgene from the CMV promoter.
  • the present invention further provides a method of suppressing the in vivo CTL response to viral vectors by the use of calpain inhibitors.
  • the present invention further provides pharmaceutical formulations of p53 and a calpain inhibitor in a pharmaceutically acceptable carrier.
  • the present invention further provides a method of ablating neoplastic cells in a mammalian organism in vivo by the co-administration of a calpain inhibitor and p53.
  • the present invention further 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 adeno virus in combination with a calpain inhibitor.
  • Figure 1 is a graphical representation percentage of cells undergoing apoptosis (as determined by annexin V-FITC positive staining) in response to increasing concentrations of CI-1 in the SK-Hepl( «) RKO( ⁇ ), HLF ( ⁇ ), DLD- 1(D) and the HEP3B( ) cell lines.
  • the vertical axis represents the change in the percentage annexin V-FTTC positive staining cells as determined by flow cytometry.
  • the horizontal axis represents the ⁇ M concentration of CI-1.
  • Figure 2 are the results of flow cytometry experiments comparing the amount of BrdU labelling of cells to detect a G0/G1 arrest in response to calpain inhibitor 1 treatment in cell lines which differ in endogeneous p53 status.
  • Figure 3 is a histogram representing the percentage of cells undergoing apoptosis (vertical axis) measured at 17 hours post treatment in response to the adminstration of various concentrations of rAd-p53 and/or CI-1.
  • histogram D5 DLD1 cells in response to 10 ⁇ M CI-1 plus lxlO 9 rAd-p53;
  • Figure 4 is a graphical representation of the percent annexin V postive HLF cells (apoptotic cells) as a function of increasing dosage of rAd-p53 alone ( ⁇ ), and rAd-p53 in combination with 10 ⁇ M CI-1 (•).
  • the horizontal axis represents the concentration in units of [number of particles of rAd-p53/milliliter of solution].
  • the vertical axis represents the percent of annexin V positive (apoptotic) cells as determined by flow cytometry.
  • Figure 5 contains the results of Western blots measuring the levels of p53 and p21 in rAd-p53 infected cells in the presence of varying concentrations of CI-1.
  • each lane is a "+" of "-" which indicates if the cells were pulsed with lxlO 9 rAd-p53 for one hour (+) or not (-).
  • the row of numbers below indicates the ⁇ M concentration of CI-1 to which the cells were exposed at 17 hours post-infection.
  • the upper row of chemiluminescent signals provides the levels of p53 while the lower row of chemiluminescent signals provides the levels of p21.
  • HLF cells showed the increases in p53 and p21 protein levels.
  • 5 ⁇ M CI-1 was added with rAd-p53, the levels of p53 increased two fold, as quantitated on NTH imaging.
  • lO ⁇ M CI-1 was added, the levels of p53 and p21 increased approximately three fold. Similar results were seen with the cell lines SK-Hepl and DLD1.
  • the cell line RKO showed a two fold increase in p53 and p21 levels in response to 5 ⁇ M CI-1, while a higher increase in p53 levels at lO ⁇ M CI-1 was seen, about a five fold increase over rAd-p53 alone.
  • Figure 6 are microscopic photographs (5 days post treatment) of 10 micron X-gal stained cross sections from livers of C57 BIJ6 mice sacrificed 3 days post infection with a recombinant adenoviral vector expressing the ⁇ -galactosidase marker gene (BGCG) in combination with 120 mg/kg of CI-1 (Panel A) and BGCG alone (Panel B). CI-1 was intraperitoneally administered on days 1, 2, and 3. BGCG (rAd-b-gal) was admistered by tail vein injection on day 2.
  • BGCG rAd-b-gal
  • Figure 7 A and B are histogram graphical representations respectively of the number of viral copies of DNA and transgene RNA per 6.8 x 10 5 HLF cells (as determined by PCR and RT-PCR respectively) in response to treatment in the presence and absence of CI-1.
  • the vertical axis represents the number of copies of p53 DNA detected by PCR (figure 7A) and p53 mRNA (figure 7B)per 6.8xl0 5 HLF cells.
  • Column A represents treatment with lxlO 7 ACN53 alone.
  • Column B represents treatment with lxlO 7 ACN53 in the presence of 10 ⁇ M CI-1.
  • Column C represents treatment with 3xl0 7 ACN53.
  • Figure 8 is a graphical representation of the data obtained by the administration of calpain inhibitor 2 in combination with a recombinant adenvirus encoding p53 to the SK-Hep-1 hepatocellular carcinoma cell line.
  • the cells were treated with the combinations indicated below each histogram and measured for apoptosis by annexin V staining at 17 hours post treatment. The percentage of cells positive for annexin V staining is indicated on the vertical axis.
  • the induction of apoptosis is selective to inhibitors of microcalpains.
  • Figure 9 is a graphical representation of the data obtained by the administration of calpain inhibitor 1 in combination with a recombinant adenovirus encoding p53 to primary ocular fibroblast cells.
  • the percentage of cells positive for annexin V staining is indicated on the vertical axis.
  • calpain inhibitor 1 incombination of ACN53 failed to induce apoptosis in primary ocular fibroblasts.
  • Figure 10 presents the results of a gel shift assay demonstrating the activation of NF-kB and AP-1 in the HLF hepatocellular carcinoma cell line measured at 24 hours post treatment as more fully described in Example 8.
  • Panel A illustrates the induction of NF-kb upon administration of calpain inhibitor 1.
  • Panel B illustrates the induction of AP-1 upon administration of calpain inhibitor 1.
  • Figure 11 is a graphical results of the cytotoxicity assay of CTL against ACN53 transduced EL-4 targets in C57/B16 mice injected with 60 mg/Kg ( ⁇ ) or 120 mg/Kg ( ⁇ ) calpain inhibitor 1 in combination with ACN53 as more fully described in Example 7 herein. Percent lysis is plotted on the vertical axis and time on the horizontal axis. The ACN53 alone is represented by the circles (•) and the naive control level is represented by the squares ( ⁇ ). As can be seen from the data presented, the CTL response to ACN53 was substantially reduced in the presence of calpain inhibitor 1.
  • Figures 12 A,B and C present the results of an in vivo mouse model following adminsitration of recombinant adenovirus encoding p53 alone or in combination with calpain inhibitors. Tumor growth regression was evaluated at Day 4, Day 8 and Day 11 post-treatment ( Figures 12 A,B and C respectively).
  • the present invention provides a method to enhance apoptosis in a cell by the administration of p53 in combination with a calpain inhibitor.
  • apoptosis means a form of programmed cell death that is characterized by specific morphologic and biochemical properties. Morphologically, apoptosis is characterized by a series of structural changes in dying cells: blebbing of the plasma membrane, condensation of the cytoplasm and nucleus, and cellular fragmentation into membrane apoptotic bodies.
  • apoptosis is characterized by the degradation of chromatin, initially into large fragments of 50-300 kilobases and subsequently into smaller fragments that are monomers and multimers of 200.
  • the execution of apoptosis minimizes the leakage of cellular constituents from dying cells. This distinguishes apoptosis from necrosis, which usually results from trauma that causes injured cells to swell and lyse, releasing the cytoplasmic material that stimulates an inflammatory response.
  • p53 refers to the product of the p53 tumor suppressor gene.
  • the p53 protein is well known in the art. Nucleotide sequences encoding p53 may be isolated from libraries or synthesized using known techniques.
  • the term “p53” refers to the the wild-type p53 protein as well as mutations or truncations thereof, which display essentially the same function as the wild-type protein. It will be readily apparent to those of skill in the art that 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.
  • the reference to the p53 gene includes not only the wild type protein but also modified 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 term p53 includes p53 molecules derived from human as well as other mammalian sources such as porcine p53, equine p53, bovine p53, canine p53, etc.
  • p53 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).
  • a similar targeting moiety derived from the HIV Tat protein is also described in Vives, et al. (1997) J. Biol. Chem. 272:16010-16017.
  • the term "administration of p53” refers to p53 gene therapy as well as p53 protein therapy.
  • the term"p53 gene therapy refers to the introduction to a cell of a nucleotide sequence encoding p53 operably linked to expression control sequences so as to effect expression (transcription and translation) of the the p53 gene in the cell.
  • the term "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. For instance, 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 seqences 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.
  • p53 gene therapy includes both in vivo and ex vivo gene therapy. Ex vivo gene therapy refers to the administration of an expression vector comprising a nucleotide sequence encoding p53 to a population cells obtained from a living organism with the expectation of those cells being reimplanted into the organism. Ex vivo gene therapy is described in Anderson, et al.
  • ex vivo gene therapy examples include the purging of tumor cells from stem cell products.
  • In vivo gene therapy refers to the administration of an expression vector comprising a nucleotide sequence encoding p53 to a living organism.
  • Examples of in vivo gene therapy administration include intratumoral, regional (intraperitoneal) or subcutaneous (IM or IV) administration.
  • expression vector refers to viral an non- viral vectors comprising a p53 expression cassette.
  • expression cassette is used herein to define a nucleotide sequence containing regulatory elements and a transgene coding sequence so as to result in the transcription and translation of a transgene in a transduced cell.
  • regulatory element refers to promoters, enhancers, transcription terminators, polyadenylation sites, and the like.
  • the expression cassette may also contain other sequences aiding expression and/or secretion of the therapeutic gene
  • the regulatory elements may be arranged so as to allow, enhance or facilitate expression of the transgene only in a particular cell type.
  • the expression cassette may be designed so that the transgene is under control of an inducible promoter, tissue specific or tumor specific promoter, or temporal promoter.
  • 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. Examples of 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.
  • temporal promoters refers to promoters which drive transcription or the therapeutic transgene at a point later in the viral cycle relative to the promoter controlling expression of the response element and are used in conjunction with viral vector systems.
  • temporally regulated promoters include the adenovirus major late promoter (MLP), other late promoters.
  • MLP adenovirus major late promoter
  • E3 promoter which is strictly dependent on E1A and viral replication.
  • examples of temporal promoter include the latent activated promoters.
  • non-viral vector refers to an autonomously replicating, extrachromosomal circular DNA molecule, distinct from the normal genome and nonessential for cell survival under nonselective conditions capable of effecting the expression of a DNA sequence in the target cell.
  • non-viral vectors include plasmids. Plasmids autonomously replicate in bacteria to facilitate bacterial production, but it is not necessary that such plasmids replicate in the target cell in order to achieve the therapeutic effect. Additional genes, such as those encoding drug resistance, can be included to allow selection or screening for the presence of the recombinant vector.
  • Such additional genes can include, for example, genes encoding neomycin resistance, multi-drug resistance, thymidine kinase, ⁇ -galactosidase, dihydrofolate reductase (DHFR), and chloramphenicol acetyl transferase.
  • the non-viral vector is provided in a non viral delivery system.
  • Examples of non-viral delivery systems used to introduce an exogenous nucleotide sequence to a target cell include expression plasmids capable of directing the expression of the therapeutic gene of interest in the target cell.
  • the expression plasmid containing the therapeutic gene may be encapsulated in liposomes.
  • liposomes includes emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like.
  • the delivery of nucleotide sequences to target cells using liposome carriers is well known in the art.
  • a variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al. Ann. Rev. Biophys. Bioeng. 9:467 (1980), Szoka, et al. United States Patent No 4,394,448 issued July 19, 1983, as well as U.S. Patent Nos. 4,235,871, 4,501,728,
  • Liposomes useful in the practice of the present invention may be formed from one or more standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol.
  • vesicle forming lipids include DC-chol, DOGS, DOTMA, DOPE, DOSPA, DMRIE, DOPC, DOTAP, DORIE, DMRIE-HP, n-spermidine cholesterol carbamate and other cationic lipids as disclosed in United States Patent No. 5,650,096.
  • lipids are generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. Additional components may be added to the liposome formulation to increase serum half-life such as polyethylene glycol coating (so called "PEG-ylation") as described in United States Patent Nos. 5,013,556 issued May 7, 1991 and 5,213,804 issued May 25, 1993.
  • PEG-ylation polyethylene glycol coating
  • a lipid enacpulated expression plasmid may incorporate modified surface cell receptor ligands to facilitate targeting.
  • the liposomes either filled or decorated with a desired composition of the invention of the invention can delivered systemically, or can be directed to a tissue of interest, where the liposomes then deliver the selected therapeutic/immunogenic peptide compositions.
  • ligands includes antibodies, monoclonal antibodies, humanized antibodies, single chain antibodies, chimeric antibodies or functional fragments (Fv, Fab, Fab') thereof.
  • the DNA constructs of the invention can be linked through a polylysine moiety to a targeting moiety as described in Wu, et al. United States Patent No. 5,166,320 issued November 24, 1992 and Wu, et al., United States Patent No. 5,635,383 issued June 3, 1997, the entire teachings of which are herein incorporated by reference.
  • viral vector refers to a virus comprising a p53 expression cassette.
  • virus refers to any of the obligate intracellular parasites having no protein-synthesizing or energy-generating mechanism.
  • 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.
  • the 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 picornnaviridiae.
  • the viruses may be naturally occurring viruses or their viral genomes may be modified by recombinant DNA techniques to include expression of exogenous transgenes and may be engineered to be replication deficient, conditionally replicating or replication competent.
  • Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See e.g., Feng, et ⁇ /.(1997) 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 viral vector 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 subgenera 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 1,2,3,4,7, and 10.
  • 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.
  • viral vector includes replication deficient, replication competent and conditionally replicating viral vectors.
  • replication deficient refers to vectors which are incapable of replication in a wild type mammalian cell.
  • the producer cell line In order to produce such vectors in quantity, the producer cell line must be cotransfected with a helper virus or modified to complement the missing functions.
  • 293 cells have been engineered to complement adenoviral El deletions allowing propagation of the El deleted replication deficient adenoviral vectors in 293 cells. See Graham, et al. (1977) J. Gen. Virol. 38:59.
  • replication competetnt viral vectors refers to a viral vector which is capable of infection, DNA replication, assembly and packaging within an infected cell.
  • conditionally replicating viral vectors is used herein to refer to replication competent vectors which are designed to achieve selective expression in particular cell types while avoiding untoward broad spectrum infection. Examples of conditionally replicating vectors are described in Bischoff, et /.(1996) Science 274:373-376; Pennisi, E. (1996) Science 274:342-343; Russell, S.J. (1994) Eur. J. of Cancer 30A(8):1165-1171.
  • Cell type specificity or cell type targeting may also be achieved in viral vectors derived from viruses having characteristically broad infectivities 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 /.(1997) J.
  • Virol 71(11):8221- 8229 incorporas into adenoviral fiber proteins
  • Arnberg, et /.(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 ⁇ /.(1997) J. Virol.
  • particular moieties may be conjugated to the viral surface to achieve targeting (See, e.g. Nilson, et al. (1996) gene therapy 3:280-286 (conjugation of EGF to retroviral proteins).
  • targeting modifications may be introduced into the viral vectors of the present invention in addition to or in combination with other modifications to the viral genome.
  • Targeting modifications may be used with replication deficient, replication competent or conditionally replicating viruses.
  • the preferred vector is derived from the genus adenoviridae. More preferred are vectors derived from human adenovirus types 2 and 5. These vectors may incorporate particular modifications to enhance their therapeutic potential. For example they may include deletions of Ela and Elb genes.
  • Certain other regions may be enhanced or deleted to provide specific features.
  • upregulation of the E3 region is described to reduce the immunogenicity associated with human adenoviral vectors administered to human subjects.
  • the E4 region has been implicated as important to expression of transgenes from the CMV promoter, however the E4orf 6 protein has been described as leading to the degradation of p53 in target cells in the presence of Elb large protein.
  • Steegenga, et al. (1998) Oncogene 16:345-347. Consequently, the elimination of such sequences from p53 gene therapy using adenoviral vectors.
  • p53 is administered in a viral vector delivery system in the ACN53 vector (described in Wills, et al. (1994) Human Gene Therapy 5:1079-1088).
  • p53 protein therapy refers to the administration of p53 protein sequence to a cell in such pharmaceutical formulations to effect the intracellular delivery of an active form of the protein to the cell and to exert its cell regulatory effects therein.
  • protein delivery system refers to 53 protein formulation for the intracellular delivery of the p53 protein to the target cell. Protein delivery systems comprise the therapeutic protein admixed with conventional carriers and excipients which stabilize and/or facilitate the uptake of the protein in the target cell.
  • calpain inhibitor 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. Examples of 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.
  • P53 was administered to the cells by the use of the replication defective adenoviral vector ACN53 described in Wills, et al, supra.
  • the effect of varying concentrations of rAd-p53 was investigated in combination with CI-1 in a range of 5-20 ⁇ M.
  • the effects of individual and combination treatment was determined in the in DLD1, RKO, HLF and SK-Hepl cells was investigated in substantial accordance with the teaching of Examples 2 and 3 herein.
  • the percentage of apoptotic cells was determined at 17 hours post treatment (with the exception of U87 cells (24 hours) and HeLa cells (68 hours)) with annexin V positive staining.
  • the results are presented in graphical format in Figure 3 of the attached drawings. In brief, the results were as follows:
  • ZZCB empty cassette control virus
  • Calpains have been shown to play a role in p53 stability. (Gonen et al, 1997; Kubbutat and Vousden, 1997). In targeting p53 for degradation, ⁇ -calpains, and to a much lesser extent milli-calpains, reportedly recognize the tertiary structure of the protein and preferentially cleave wildtype p53 at a single cleavage site located at the N-terminal region overlapping the mdm-2 binding site (Kubbutat and Vousden, 1997). Deletion of the calpain consensus cleavage site in p53 has been shown to inhibit the ability of mdm-2 to bind to p53 resulting in an increase in p53 half-life.
  • the p53 null cell line Hep3B was infected with IxlO 9 particle/ml and assayed for p53 protein levels by western blots 17 hours post-infection. The results are shown in figure 5. No p53 protein was detected in untreated Hep3B. p53 protein was detected in cells infected with rAd53 alone, and icreased greater then 10 fold when infected with rAd-p53 in the presence of lO ⁇ M calpain inhibitor 1. The results presented in figure 5 demonstrate a 2-5X increase in p53 levels in response to calpain inhibitor 1 in cell lines with endogenous wildtype and mutated p53 status.
  • CI-1 may not, therefore, be the sole mechanism by which enhanced cell death has been achieved. This result also suggests that CI-1 stabilizes exogenous as well as endogenous p53, and was confirmed using a p53 null cell line, Hep3B. Thus, a new mechanism of action is involved other than p53 stability in the increased induction of apoptosis by the combination of CI-1 and p53.
  • HLF cells were infected with increasing concentrations of rAd-p53 with or without lO ⁇ M CI-1, and assayed for percentage apoptotic cells by annexin V-FITC staining 42 hours post-treatment in substantial accordance with the teaching of Example 3 herein.
  • a dosage response curve was plotted and is shown in Figure 4. Untreated cells and cells treated with lO ⁇ M CI-1 alone showed low background annexin V staining (2-3% respectively).
  • the present invention provides a method to enhance apoptosis in a cell by the administration of p53 in combination with a calpain inhibitor.
  • p53 positive refers to the genotype of a cell which possesses at least one copy of a gene encoding a functional p53 molecule. p53 positive is distinguished from p53 negative which refers to the geneotype of a cell which does not possess at least one copy of a gene encoding a functional p53 molecule.
  • the present invention provides a method of inducing p53 mediated apoptosis in a p53 positive cell by the adminsitration of a calpain inhibitor.
  • CI-1 N-acetyl-leu-leu-norleucinal
  • the data presented above demonstrates that at 26 hours post-treatment with CI-1, p53 wildtype tumor cells demonstrated a significant increase in the percentage of cells undergoing apoptosis in contrast to the p53 mutated and null tumor cells.
  • the data presented demonstrates an increasing dose response with increasing concentrations of CI-1.
  • concentrations of CI-1 At 20 ⁇ M concentration, the increase in the percent of cells annexin V positive over background staining became more significant m cells with p53 wildtype status.
  • SK-Hepl cells increased the percentage of annexin V positive cells from background staining of 20% to 66%. In RKO cells, an increase from 10% to 52% cells annexin V positive cells was seen.
  • HLF increased from a background staining of 12% to 27% and DLD-1 increased from 8% to 11%.
  • SK-Hepl increased annexin V positive cell to 73%, RKO to 61%, while DLD1 increased to 23%, and HLF to 21%.
  • the hepatocellular carcinoma cell line with null p53 status, Hep3B, (Fig 1) showed no increase in the percentage of apoptotic cells in response to even the highest concentration of inhibitor (50 ⁇ M).
  • Other cell lines treated with CI-1 included the p53 wild type glioblastoma cell line U87, which showed an increase in the percentage of apoptotic cells from 5% to 72% m response to 20 ⁇ M CI-1 treatment.
  • a colorectal cell line RKO, with p53 wildtype status showed 47% of the control cells incorporating BrdU in response to DMF, while 16% of cells incorporated BrdU label in response to 5 ⁇ M CI-1.
  • DLD1 cells showed 35% of cells incorporating BrdU without calpain treatment, and upon 5 ⁇ M CI-1 treatment for 17 hours, the percentage of cells incorporating BrdU decreased only to about 25%.
  • Micro-calpain Inhibitors Increase the Infectivity of a Target Cell to Viral Vectors:
  • the present invention provides a method of increasing the infectivity of a cell to a viral vector by treatment of the cell with a calpain inhibitor.
  • ITE Infection Transduction Efficiency
  • ITE -In (1 - F) / I ⁇ V t° 5
  • F represents the fraction of cells in a given population which are "positive" for the parameter under evaluation (e.g. expression of the virally encoded gene);
  • ⁇ represents the average surface are of the cell;
  • I is a constant proportional to the diffusion coefficient and will be affected by the virus, the viscosity of the solution, etc. and is determined empirically,
  • t is the time of exposure and V is the concentration of particles per unit volume.
  • viral vector is described above with the exception that the expression cassette is not limited to the p53 gene rather includes a therapeutic transgene.
  • 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 supressing the neoplastic phenotype and/or inducing apoptosis.
  • 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). In order to facilatate immune recognition, 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 PI 6, pl5, pl8 and pi 9, 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 IL-1, IL-2, IL-4, IL-12, IL-10, IL-19, IL-20, interferons of the ⁇ , ⁇ and ⁇ subtypes especially interferon -2b and fusions such as interferon ⁇ -2 - 1.
  • 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 a ino acid residues that share four conserved cysteine 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 (TL- 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 II (AMCF-I and -II), pre-B cell growth stimulating factor (PBSF),and IP10.
  • PF4 platelet factor 4
  • PBP platelet basic protein
  • TL- 8 interleukin-8
  • MGSA melanoma growth stimulatory activity protein
  • MIP-2 macrophage inflammatory protein 2
  • mouse Mig mouse Mig
  • chicken 9E3 or pCEF-4
  • pig alveolar macrophage chemotactic factors I and II 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-1- ⁇ ), macrophage inflammatory protein 1 ⁇ (MlP-l- ⁇ ), macrophage inflammatory protein 1 ⁇ (MTP-l- ⁇ ), macrophage inflammatory protein 3 (MLP-3- , macrophage inflammatory protein 3 ⁇ (MIP-3- ⁇ ), chemokine (ELC), macrophage inflammatory protein 4 (MTP-4), macrophage inflammatory protein 5 (MIP-5), LD78 ⁇ , RANTES, SIS-epsilon (p500), thymus and activation-regulated chemokine (TARC), eotaxin, 1-309, human protein HCC-l/NCC-2, human protein HCC-3, mouse
  • 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, GAB A, ACTH, NGF, VEGF (to increase blood perfusion to target tissue, induce angiogenesis, PCT publication WO98/32859 publsihed 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.
  • pro-drug activing 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 to 5 fluorouracil, 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.
  • TK thymidine kinase
  • 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
  • therapeutic genes 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).
  • the effect of pretreatment of the cells with CI-1 increased the infectivity of the cells to the virus to approximately 130 times that of the untreated cells, as compared without pre-treatment, where the effect was 50 fold higher.
  • the cells be pretreated with the calpain inhibitor prior to exposure to the viral vector to maximize the infectivity of the cell line to the vector.
  • mice were injected with 1 x 10 10 particles of rAd-p53 per injection intratumorally in combination with intraperitoneally injected CI-1 at a concentration of 60 mg/kg for a period of 5 days, 2 days off and then for 5 more days.
  • Tumor growth/regression was evaluated at Day 4, Day 8 and Day 11 post-treatment (figures 12 A,B and C respectively).
  • the rate of decrease in tumor growth was at least 40% reduction in fold increase in tumor volume in mice treated with calpain inhibitor 1 in addition to FTCB, in comparison with the mice treated with the FTCB vector alone. It is particularly noteworthy in this instance that calpain inhibitor alone provided significant antitumor effects.
  • NFkB Nuclear Factor Kappa B
  • GGGGACTTTCC Nuclear Factor Kappa B
  • NFkB Nuclear Factor Kappa B
  • GGGGACTTTCC DNA sequence within the immunoglobulin light chain kappa enhancer region in mice and humans.
  • NFkB is an inducible factor which is present in a wide variety of cell types. This factor regulates the transcription of a wide variety of cellular and viral genes including c-myc, the interleukins, receptors, adhesion molecules, p53 and the CMV early promoter. This factor is induced in response to a variety of primarily pathogenic stimuli including IL-1, TNF- ⁇ , adhesion, bacterial lipopolysaccharides (LPS), and oxidative stress. Because induction of NFkB is blocked by antioxidants, it is believed that activation of NFkB employs reactive oxide intermediates (ROIs) as intracellular second messengers in response to the above stimuli.
  • ROIs reactive oxide intermediates
  • Calpain inhibitor I has been shown to be an inhibitor of the proteolysis of IkB and hence of an inhibitor of the activation of NFkB. Ruetten and Thiemmermann (1997) Br. J. Pharmacol 121 :695-704. Similar studies indicating that the subsequent degradation of IkB involves calpains and that calpain inhibitors decrease NFkB activity exist. For example, Milligan, et al indicate that CI-1 decreases the nuclear translocation of NFkB and that CI-1 inhibited the degradation of IkB, Arch Biochem Biophys (1996) 335:388-395. See also, Claudio, et al.
  • the present invention further provides a method of enhancing transcription of a therapeutic transgene from the CMV promoter.
  • enhancing transcription refers to an increase in the transcriptional activity of those sequences operably linked to the CMV promter.
  • CMV promoter means the immediate early promoter of the cytomegalovirus.
  • the promoter may be isolated as a restriction endonuclease fragment from the commercially available vector pCMV ⁇ (GenBank Accession Number U02451). NFkB has been conclusively demonstrated to enhance transcription from the CMV promoter.
  • the CMV promoter is a strong constituitive promoter widely utilized in the expression of exogenous transgenes from viral vectors. Recent reports indicated an upregulation of the CMV promoter driven genes in response to recombinant adenoviral infection. Loser, et al. (1998) J. Virol. 72:180-190. As previously indicated, the addition of agents which raise intracellular p53 concentration, regardless of mechanism, are desired to induce p53 mediated apoptosis.
  • CI-1 demonstrably increases the level of p53 produced by ACN53 in a cell may involve upregulation of the p53 transgene driven by the CMV promoter by the in vivo stabilization of NFkB.
  • the transcriptional factor API is activated in response to calpain inhibitor 1 treatment, and there are API binding sites on the CMV promoter for activation of promoter.
  • mice were sacrificed three days and nine days later (four-and ten days post-infection respectively), and livers were harvested for quantitative PCR and RT-PCR to detect viral DNA and viral transcript (b-galactosidase) respectively.
  • the present invention also provides a method to suppress the in vivo CTL response to viral vectors by the use of calpain inhibitors.
  • N-acetyl-leucinyl- leucinyl-norleucinal (Calpain Inhibitor I) has proteosome as well as calpain inhibitor activity.
  • CI-1 is primarily an inhibitor of calpain.
  • CI-1 has been shown to inhibit proteasome function. This inhibition effects antigenic peptide presentation to T cells as the proteosome is the primary enzymatic complex that degrades proteins into peptides prior to insertion into the ER through the TAP complex where loading onto MHC class I occurs (Rock, et al. ).
  • the calpain inhibitors may also effect antigen presentation by a mechanism that is not generally obvious. Namely, the calpain inhibitor may inhibit apoptosis of non-transduced cells such as antigen presenting cells (e.g., macrophages, dendritic cells) which may affect the capacity for antigen transfer to dendritic cells through phagocytosis of the apoptotic cells, thus effecting maturation of the dendritic cells and antigen presentation function following migration to regional lymphnodes where the dendritic cells activate T cells.
  • antigen presenting cells e.g., macrophages, dendritic cells
  • the present invention further provides a pharmaceutical formulations of p53 and a calpain inhibitor in a pharmaceutically acceptable carrier.
  • formulation refers to pharmaceutical formulations comprising a protein, viral or non-viral delivery system for administration in vivo or ex vivo to an individual in need of treatment.
  • In vivo administration will typically employ the compositions of the present invention formulated for intramuscular, intravenous, intrarumoral, intrahepatic artery, intraperitoneal or intrvesicular, injectable depot- type devices or topical routes of administration.
  • the compositions of the present invention include polymer matrices, hydrogel matrices, polymer implants, or encapsulated formulations to allow slow or sustained release of the compositions.
  • formulations for the delivery of transgenes via non-viral delivery systems to the airway epithelia is described in Debs and Zhu, United States Patent No 5,641,622 issued June 24, 1997.
  • p53 refers to the p53 protein therapy and gene therapy systems for the delivery of a nucleotide sequence encoding p53. While the combined effect of p53 and the calpain inhibitor are not limited to a given vehicle for the introduction of the p53 molecule, in the preferred practice of the invention, the p53 molecule is introduced by gene therapy methods using a viral vector derived from genus adenoviridiae. Particularly preferred viruses are derived from the human adenovirus type 2 or type 5. Such viruses are preferably replication deficient by modifications or deletions in the Ela and/or Elb coding regions. Other modifications to the viral genome to achieve particular expression characteristics or permit repeat administration or lower immune response are preferred.
  • recombinant adenoviruses having complete or partial deletions of the E4 coding region, optionally retaining E4orf6 and E4orf6/7.
  • the E3 coding sequence may be deleted but is preferably retained.
  • the promoter operator region of E3 be modified to increase expression of E3 to achieve a more favorable immunological profile for the therapeutic vectors.
  • human adenoviral type 5 vectors containing a DNA sequence encoding p53 under control of the cytomegalovirus promoter region and the tripartite leader sequence having E3 under control of the CMV promoter and deletion of E4 coding regions while retaining E4orf6 and E4orf6/7.
  • the vector is ACN53, as described in Wills, et al. (1994) Human gene therapy 5:1079-1088.
  • carriers 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, sorbitan 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, sorbitan monolaurate, triethanolamine oleate, etc.
  • delivery enhancers or “delivery enhancing agents” are used interchangeably herein and includes agents which facilitate the transfer of the nucleic acid or protein molecule to the target cell.
  • delivery enhancing agents 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 1M NaCl are also examples of delivery-enhancing agents.
  • surfactants are sodium dodecyl sulfate (SDS) and lysolecithin, polysorbate 80, nonylphenoxypolyoxyethylene, lysophosphatidylcholine, polyethylenglycol 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 (NSAIDS) like indomethacin, naproxen, diclofenac may be used.
  • Delivery- enhancing agents includes compounds of the formula I:
  • XI and X2 are selected from the group consisting of a cholic acid group, a deoxcholic acid group and a saccharide group, m is an integer from 2 to 8 and preferably 2 or 3, n is an integer from 2 to 8 and preferably 2 or 3, and R is a cationic group, a saccharide group or a structure -CO-X3 wherein X3 is a sachharide 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, Zwittergent®3-14 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).
  • the pharmaceutical formulation may be provided in a single premixed formulation or provided in a kit of parts for mixing by the end user.
  • kit refers to a unit packaged combination of the elements of a formulated calpain inhibitor and a protein, viral or non-viral p53 formulation. Such kits promote the proper use and formulation of the materials when used in combination and avoid improper dosing.
  • the formulated elements of the kit may be in ready to use or precursor form (e.g. lyophilized form) requiring reconsitution in a solution.
  • the kit prefereably contains the appropriate carriers and solvents to reconstitute the precursor form of the p53 and calpain inhibitor elements.
  • the present invention provides a method of ablating neoplastic cells in a mammalian organism in vivo by the co-administration of a calpain inhibitor and p53.
  • 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.
  • viable neoplastic cell is used hereing to distinguish said cells from neoplastic cells which are no longer capable of replication. For example, 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.
  • 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).
  • Gl or GO temporary non-replicative resting state
  • neoplasms may be malignant or benign.
  • Malignant neoplasms are also referred to as cancers.
  • cancer is used interchangeably herein with the term tumor.
  • 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 methods and compositions of the present invention may be used for the treatment of a variety of organisms suffering from of diseases associated with p53 dysfunction and may be used to eliminate p53 negative cells from a population of cells.
  • the formulations and methods of the present invention may be used for the treatment of a variety of mammalian species suffering from such maladies including humans, pigs, horses, cattle, dogs, cats, preferably by employing vectors encoding, for example, human p53, porcine p53, equine p53, bovine p53, canine p53 (Velhoen & Milner (1998) Oncogene 16:1077- 1084), feline p53, etc. respectively.
  • vectors encoding, for example, human p53, porcine p53, equine p53, bovine p53, canine p53 (Velhoen & Milner (1998) Oncogene 16:1077- 1084), feline p53, etc. respectively.
  • Preferably one employs an adenoviral vector endogenous to the mammalian type being treated.
  • 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.
  • co-administration refers to the administration within a sufficient time such that the therapeutic effect of one element has not been eliminated prior to the administration of the second agent. For example, it has been observed that the prior treatment of cells with calpain inhibitor 1 will increase the infectivity of those cells to viral vectors. Consequently, although not administered at the same time, the calpain inhibitor is deemed to be co-administered with the virus when the infectivity enhancing effect of the calpain inhibitor persists.
  • Examples of diseases currently demonstrated as candidates for treatment with p53 therapy include a broad variety of cancers commonly associated with p53 mutations. Approximately 65% of all cancer cell types studied to date implicate that p53 dysfunction is assocated with the neoplastic phenotype of these cells.
  • the dosage of a recombinant adenoviral vector encoding p53 may be decreased by approximately a factor of 2 logs when the concentration of CI-1 is approximately lO ⁇ M ⁇ M to achieve intracellular p53 dosage substantially equivalent to the administration of the rAd-p53 vector alone.
  • the total dosage of the p53 to be administered to an organism in need of treatment is based on a variety of factors.
  • such factors include the infectivity of the particular vector with respect to the target cell, the total number of viral copies of the virus produced in the target cell, the relative strength of the control regions used to express the p53 molecule (i.e the intracellular and particularly intranuclear p53 concentration), etc.
  • p53 protein therapy as well as p53 gene therapy
  • these factors may be readily determined by those of skill in the art.
  • the optimal human dose of has been determined in human clinical trials to be approximately 2xl0 13 PN. Consequently, one may readily determine based on the above data that a substantially equivalent effective dose of ACN53 may be decreased by a factor of 2 log in combination with a 10 ⁇ M concentration of CI-1.
  • the formulations of the present invention also provide a means to increase p53 dose or increase p53 expression without the need to increase the vector dose which is associated with toxicity in some systems.
  • the method of the present invention 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. Examples of combination therapy when the therapeutic gene is p53 are described in Nielsen, et al. WO/9835554A2 published August 20, 1998. Examples of the utility of the combination of p53 and DNA damaging compounds are described in United States Patent No. 5,747,469 issued May 5, 1998.
  • the method of the present invention may include the co-administration of immunosuppressive agents.
  • immunosuppressive agents include cyclosporine, azathioprine, methotrexate, cyclophosphamide, lymphocyte immune globulin, antibodies against the CD3 complex, adrenocorticosteroids, sulfasalzaine, FK-506, methoxsalen, and thalidomide.
  • selectively replication or conditionally replication adenoviral vectors are employed, the elimination of E3-11.6K protein to minimize cell lysis induced by adenovirus until apoptosis is achieved. This would minimize the immune response until after the initial round of localized spreading occurs. At this later time, once apotosis of the initially infected cells is achieved and localized virus spreading is permitted, the immune response would be advantageous.
  • 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 o reconstitutin gthe long term hematpoietic functionof a patient who has received myoablative therpay.
  • Stem cell products are conventionally obtained by apheresis or 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 approximately 99.999% of tumor cells from the stem cell product).
  • a stem cell product of 100 ml volume would be treated with up to 5 x 10 ⁇ particles per milliter (5xl0 13 particles) of the recombinant adenovirus of the present invention for a period of approximately 4 hours at 37C.
  • P53 gene therapy particularly the use of adenoviral vectors encoding p53 have been used to effectively purge tumor cells from a population of stem cells. Results to date indicate that approximately 8xl0 9 PN/ml of stem cell product achieve the 5-log purge considered optimal for autologous implantation.
  • the use of calpain inhibitors can enhance the effect of rAd-p53 vectors by reducing the dosage by approximately 2 logs.
  • the data presented indicates that treatment of the cells with CI-1 increases the infectivity of the cells to rAd vectors by an additional 1-3 logs depending on the timing of the calpain inhibitor treatment.
  • the combination of pretreatment with CI-1 in combination with a rAd-53 gene therapy would be expected to reduce the dosage required to achieve a 5-log purge by approximately 3-4 logs.
  • an effective dose of ACN53 for a 5 log purge of a stem cell product would be approximately 1 x 10 7 to approximately 5 x 10 8 PN/ml in the presence of 10 ⁇ M CI-1.
  • Example 1 Construction of Viral Vectors
  • a viral vector backbone was created based on a human adenovirus type 5 genome comprising deletions of the Ela and Elb and protein IX gene functions and partial deletion of the E3 coding region. Specifically, the deletions of base pairs 355 to 3325 was used to eliminate Ela and Elb functions, deletion of base pairs 3325 to 4021 was used to eliminate protein IX function and deletions of 28592 to 30470 were used to eliminate E3 functions. See Wills, et al. (1994) Human Gene Therapy 5:1079-1088. The DNA sequence encoding the cytomegalovirus immediate early promoter without the presence of the CMV promoter intron was inserted into the rAd viral genome.
  • This vector without an exogenouse transgene was used as control vector and was designated ZZCB.
  • the wildtype p53 coding sequence was inserted into this vector backbone so as to achieve expression of the p53 sequence under control of the CMV promoter.
  • This vector was denoted ACN53 and is also referred to interchangeably as FTCB.
  • NCI-H596 cells were obtained from the American Type Culture Collection, Peoria, IL under accession number ATCC # HTB 178). NCI-H596 cells were grown in Ham's F- 12 DMEM (commercially available from Irvine Scientific) supplemented with sodium pyruvate and 10% fetal bovine serum. HLF cells were obtained from the Japanese Cancer Research Resource Bank, National Institute of Health, Tokyo, Japan). SK-Hepl cells were obtained from the American Type Culture Collection, Peoria, JL under accession number ATCC #HTB 52). RKO cells were obtained from M. Brattain, Medical College Hospital, Toledo, Ohio.
  • DLD-1 cells were obtained from the American Type Culture Collection, Peoria, IL under accession number ATCC # CCL 221). Hep 3B 2.1-7 cells were obtained from the American Type Culture Collection, Peoria, IL under accession number ATCC # HB 8064).
  • NCI-H596 cells were obtained from the American Type Culture Collection, Peoria, IL under accession number ATCC # HTB 178). All cell lines with the exception of NCI-H596 were grown in Delbecco' s Modified Eagles media (DME) supplemented with sodium pyruvate and 10% fetal bovine serum. NCI-H596 cells were grown in Ham's F-12 DME supplemented with glutamine and 10% fetal bovine serum.
  • DME Delbecco' s Modified Eagles media
  • Example 3 Apoptotic Effect Of Virus and/or CI-1 on Tumor Cells An aliquot containing approximately I x lO 6 cells of each type was placed in a T-225 flask. The effect of varying concentrations of rAd-p53 (from IxlO 7 to 2xl0 9 ACN53 particles/ml of solution) was investigated in combination with CI-1 in a range of 5-20 ⁇ M. In each instance, the cell line was infected with IxlO 7 -IxlO 9 particles/ml of cell culture with the ACN53 using the empty cassette virus (ZZCB) as a control. In each instance, the cells were exposed to the virus foy a period of one hour. At the end of this time, the virus was washed off the from the cells and fresh media containing an appropriate concentration of CI-1 was added.
  • ZZCB empty cassette virus
  • HLF cells were infected at increasing log concentrations of rAd-p53 ranging from lx 10 5 to lx 10 9 for one hour at which time virus was washed off and replaced with fresh media (viral infection with no treatment) or with lO ⁇ M calpain inhibitor 1.
  • Cells were assayed for apoptosis by annexin V-FTTC staining at 24 and 42 hours post-infection.
  • CI-1 was obtained from Boehringer-Mannheim Biochemicals (Indianapolis, IN). The CI-1 containing media was prepared as follows. CI-1 was diluted in dimethylformamide (DMF) to a concentration of 33.3 ⁇ M/ ⁇ l. The CI-1 solution in DMF was added to achieve a final concentration in the media of 5 or 10 ⁇ M final concentration of CI-1. An equivalent volume of DMF alone was added to the control cells. Calpain inhibitor 2, N-acetyl-leu-leu-methioninal (Boehringer Mannheim) was added to cell lines at 5-50 ⁇ M final concentration with or without rAd-p53 for 26 hours.
  • DMF dimethylformamide
  • the percentage of cells undergoing apoptosis was determined by convential apoptotic markers. Apoptosis was monitored visually by observing blebbed nuclei characteristic of apoptosis, by propidium iodide staining (using propidium iodide commercially available from Molecular Probes, Inc.) followed by flow cytometric analysis to look for subgenomic populations of cells, and by labeling cells with annexin V-HTC (commercially available from Boehringer Mannheim) in substantial accordance with the instructions provided by the manufacturer, followed by flow cytometric analysis on a FACScan Flow Cytometer (commercially available from Becton Dickinson).
  • CI-1 In order to determine any increases in p53 function in response to calpain inhibitor 1, the quantity of cells undergoing G0/G1 cell cycle arrest in response to CI-1 administration, was assayed by bromodeoxyuridine labeling of cells .
  • Each of the cell lines described in Example 2 above were exposed to a concentration of 0 or 5 ⁇ M CI-1 (in DMF) as described in Example 3 above.
  • Cell lines treated with DMF alone or 5 ⁇ M final concentration of calpain inhibitor 1 for 17 hours were pulse labeled with a final concentration of lO ⁇ M bromodeoxyuridine (Boehringer Mannheim) for two hours.
  • Cells were harvested for bivariate BrdU/DNA flow cytometric analysis by fixation in 70% ethanol, followed digestion with .08% pepsin for 30 minutes at 37 degrees. Cells were centrifuged at 1500RPM, and 2N HCL was added. Cells were incubated at 37 degrees for 20 minutes, followed by addition of 1M sodium borate. Cells were washed in IFA/tween 20 (.01M HEPES, .005% sodium azide, 0.5% tween 20, 5% FBS, 3.75M NaCl), and anti- BrdU antibody, diluted 1:10 in IFA without tween 20, was added for 30 minutes. Cells were washed in IFA/tween 20, and incubated in IFA tween 20/RNase for 15 minutes at 37 degrees.
  • Example 5 Western blotting At 17 hours post treatment, cells were lysed in protein lysis buffer (50mM tris, 250mM NaCl, 50mM NaF, 5mM EDTA and 0.1% NP-40 with ImM PMSF). Cell lysates were subjected to electrophoresis. lO ⁇ g of protein was added per lane on a 12% polyacrylamide gel and transfered onto nitrocellulose membranes. The membranes were subjected to western blot analysis using antibodies specific for p53 or p21 (Calbiochem, p53 antibody Ab-6, and p21 antibody Ab-7 ). HRP- conjugated secondary antibody (Amersham) was added for one hour at which time the blots were washed. Blots were developed using enhanced chemiluminescence detection system (Amersham) and quantitated using NIH imaging analysis.
  • protein lysis buffer 50mM tris, 250mM NaCl, 50mM NaF, 5mM EDTA and 0.1% NP-40 with ImM
  • Taqman® EZ RT-PCR core reagents (rTth DNA polymerase, AmpErase UNG, deoxy ATP, deoxy CTP, dexoy GTP, deoxy UTP, 5x Taqman® EZ buffer, and manganese acetate solution) were obtained from Perkin-Elmer, as Part No. N808-0236.
  • Oligonucleotide Primer "A” " (5' Taqman® p53 5'- AACGGTACTCCGCCACC) and Primer “B” (3' Taqman® p53; 5'- CGTGTCACCGTCGTGGA) and 10 ⁇ M Taqman® Probe (5'-FAM- CAGCTGCTCGAGAGGTTTTCCGATCC-TAMRA) were obtained from Perkin Elmer. Diethyl pyrocarbonate (DEPC) treated water was obtained from United States Biochemical Cat. No. 70783 or equivalent.). tRNA was obtained from Sigma Cat. No. R5636, 10 ⁇ g/ml.
  • DEPC Diethyl pyrocarbonate
  • RT Master Mix A quantity of reverse transcription master mix (RT Master Mix ) was prepared proportional to the number of samples to be amplififed.
  • RT Master Mix for one sample comprises: 7 ⁇ L Mn(OAC) 2 , 10 ⁇ L Gene Amp 5X Taqman® Buffer, 3 ⁇ L Oligonucleotide Primer "A”, 10 ⁇ m, 3 ⁇ L Oligonucleotide Primer "B”, 10 ⁇ m, 6 ⁇ L deoxynucleotide driphosphates, 0.5 ⁇ L of rTth polymerase, 0.5 ⁇ L UNG, and 1.5 ⁇ L Taqman® Probe, 10 ⁇ m and Q.S.
  • thermocycler Perkin Elmer ABI Prism 7700 Sequence Detector
  • PCR ASSAY The following materials were obtained from Perkin Elmer Corporation under Part No. N808-0028: Gene Amp 10X Taqman Buffer A, 25 mM MgCl2, 5 ⁇ M Oligonucleotide Primer "A”, 5 ⁇ M Oligonucleotide Primer “B”, 5 units/ ⁇ L AmpliTaq® Gold DNA Polymerase, 10 ⁇ M Taqman Probe. Deoxynucleotide Triphosphates , dATP, dCTP, and dGTP are 10 mM. dUTP is 20 mM.
  • primers and probe were used p53 PCR Primer "A” (5' Taqman p53) from 5'-3' is AAC GGT ACT CCG CCA CC; Primer "B” (3' Taqman p53) from 5'-3' is CGT GTC ACC GTC GTG GAA; Taqman probe (p53 Taqman Probe) from 5 '-3' is FAM-CAG CTG CTC GAG AGG TTT TCC GAT CC-TAMRA.
  • primers and probe were used for B-Actin PCR.; Sequence of Primer "A” (5' Taqman B-Actin) from 5'-3' is TCA CCC ACA CTG TGC CCA TCT ACG A; Primer "B” (3' Taqman B- Actin) from 5'-3' is CAG CGG AAC CGC TCA TTG CCA ATG G; Taqman probe (B-Actin Taqman Probe) from 5 '-3' is FAM-ATG CCC CCC CCA TGC CAT CCT GCG T-TAMRA.
  • thermocycler Perkin Elmer, ABI Prism 7700 Sequence Detector
  • Example 7 Evaluation of In Vivo CTL Response
  • C57BL/6 mice (2 mice per dose) were injected intravenously with 9xl0 9 particles/animal (iv/200 ⁇ l) of a recombinant adenoviral vector encoding the ⁇ gal reporter gene under control of the CMV promoter.
  • Naive mice were employed as controls.
  • the spleens were harvested at day 10 for immunological analysis. Livers were harvested for analysis of ⁇ -gal transgene expression. Cells were be dispersed to a single cell suspension and washed 3 times. The cells were be resuspended complete RPMI-10 (RPMI + 10% FCS, 2ME (5 ⁇ M), glutamine, Na pyruvate, Pen/Strep).
  • Responder CTL cells were seeded in 24-well plates at 8xl0 6 cells per well in 1.5 ml RPMI-5%.
  • the responder CTL cells were restimulated by adding syngeneic (C57BL/6) spleen cells that were transduced for 6 hours with 2xl0 10 BGCG particles/ml and then mitomycin treated to inhibit proliferation and/or activation of stimulator cell populations.
  • the responder CTL were co-cultured with the stimulator cells at 37°C in a humidified incubator for seven days. Conconavilin A supernate (10%) was added to the CTL restimulation cultures on day 2 to promote CTL expansion.
  • the Effector CTL cells were harvested, washed 2 times and counted to enumerate the CTL effector cells.
  • the CTL blast cells were plated in 96-well plates (round-bottom) at various cell densities with IxlO 4 51 Cr -labeled P815 target cells expressing antigen (ELA target cells were transduced overnight with IxlO 10 PN/ml BGCG to induce antigen expression and then labeled 2hr with 51 -chromium) to give various Effector to target ratios in a total volume of 200ul RPMI-10.
  • the Effector CTL cells were co-cultivated with the target cells for 6 hours and the specific 51 Cr release were determined by counting harvested supernates (lOO ⁇ l) in the ⁇ counter.
  • NF-kB Promega E329
  • AP-1 Promega E320
  • Nuclear extracts (l ⁇ g protein) were incubated in a 10 ⁇ l (final volume) reaction mixture containing 10 mM Tris-HCl (pH 7.5), 0.5 mM DTT, 0.5 mM EDTA, 1 mM MgC12, 4% glycerol and 50 ng/ml poly(dl-dC) at room temperature for 10 min.
  • oligonucleotides (-100,000 cpm) were then added and the reaction mixtures were incubated for another 20 min at room temperature. After 20 min incubation, 5 ⁇ l of 60% glycerol was added to each reaction and the samples were subjected to native polyacrylamide gel electrophoresis. After electrophoresis gels were dried and exposed to an X-ray film at -70°C.

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Abstract

Cette invention concerne un procédé qui permet d'accroître l'apoptose dans une cellule et qui consiste à administrer du p53 combiné à un inhibiteur de calpaïne. Cette invention concerne également un procédé qui permet d'accroître l'infectivité d'une cellule par un vecteur viral et qui consiste à traiter la cellule à l'aide d'un inhibiteur de calpaïne. Cette invention concerne en outre un procédé permettant d'accroître la transcription d'un transgène thérapeutique à partir du promoteur CMV. Cette invention concerne aussi un procédé permettant de supprimer la réponse CTL in vivo à des vecteurs viraux à l'aide d'inhibiteurs de calpaïne. Cette invention concerne également des formulations pharmaceutiques de p53 et d'un inhibiteur de calpaïne dans un excipient acceptable sur le plan pharmaceutique. Cette invention concerne aussi un procédé d'ablation in vivo de cellules néoplastiques dans l'organisme d'un mammifère, lequel consiste à effectuer l'administration conjuguée d'un inhibiteur de calpaïne et de p53. Cette invention concerne enfin un procédé d'ablation de cellules néoplastiques ex vivo dans une population de cellules normales qui a été contaminée par lesdites cellules néoplastiques, lequel procédé consiste à administrer une combinaison d'un adénovirus recombinant et d'un inhibiteur de calpaïne à ladite population.
PCT/US1999/021453 1998-10-15 1999-10-14 Inhibiteurs de calpaine et applications de ces inhibiteurs WO2000021575A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6803233B2 (en) 2000-07-31 2004-10-12 The Regents Of The University Of California Model for Alzheimer's disease and other neurodegenerative diseases
US7837991B2 (en) 1994-05-31 2010-11-23 Aventis Pharma, S.A. Method of cancer treatment by p53 protein control

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
EASTHAM J A ET AL: "IN VIVO GENE THERAPY WITH P53 OR P21 ADENOVIRUS FOR PROSTATE CANCER" CANCER RESEARCH,US,AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, vol. 55, no. 22, 15 November 1995 (1995-11-15), pages 5151-5155, XP000606345 ISSN: 0008-5472 *
HAMADA KATSUYUKI ET AL: "Adenovirus-mediated transfer of a wild-type p53 gene and induction of apoptosis in cervical cancer." CANCER RESEARCH 1996, vol. 56, no. 13, 1996, pages 3047-3054, XP002137540 ISSN: 0008-5472 *
KUBBUTAT M H ET AL: "Proteolytic cleavage of human p53 by calpain: a potential regulator of protein stability." MOLECULAR AND CELLULAR BIOLOGY, (1997 JAN) 17 (1) 460-8., XP000906747 *
LIU T -J ET AL: "APOPTOSIS INDUCTION MEDIATED BY WILD-TYPE P53 ADENOVIRAL GENE TRANSFER IN SQUAMOUS CELL CARCINOMA OF THE HEAD AND NECK" CANCER RESEARCH,US,AMERICAN ASSOCIATION FOR CANCER RESEARCH, BALTIMORE, MD, vol. 55, 15 July 1995 (1995-07-15), pages 3117-3122, XP002027381 ISSN: 0008-5472 *
PARIAT M ET AL: "Proteolysis by calpains: a possible contribution to degradation of p53" MOLECULAR AND CELLULAR BIOLOGY,US,WASHINGTON, DC, vol. 17, no. 5, May 1997 (1997-05), pages 2806-2815, XP002110841 ISSN: 0270-7306 *
SHINOHARA K ET AL: "Apoptosis induction resulting from proteasome inhibition." BIOCHEMICAL JOURNAL, (1996 JUL 15) 317 ( PT 2) 385-8., XP000906742 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7837991B2 (en) 1994-05-31 2010-11-23 Aventis Pharma, S.A. Method of cancer treatment by p53 protein control
US6803233B2 (en) 2000-07-31 2004-10-12 The Regents Of The University Of California Model for Alzheimer's disease and other neurodegenerative diseases

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