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US20030059400A1 - Light emitting microorganisms and cells for diagnosis and therapy of tumors - Google Patents

Light emitting microorganisms and cells for diagnosis and therapy of tumors Download PDF

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US20030059400A1
US20030059400A1 US10/189,918 US18991802A US2003059400A1 US 20030059400 A1 US20030059400 A1 US 20030059400A1 US 18991802 A US18991802 A US 18991802A US 2003059400 A1 US2003059400 A1 US 2003059400A1
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tumor
tumors
protein
cell
pharmaceutical composition
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Aladar Szalay
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Genelux Corp
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Priority claimed from EP01118417A external-priority patent/EP1281772A1/fr
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Priority to US10/866,606 priority Critical patent/US8568707B2/en
Assigned to GENELUX CORPORATION reassignment GENELUX CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: SZALAY, ALADAR A.
Assigned to GENELUX CORPORATION reassignment GENELUX CORPORATION TO CORRECT COVER SHEET Assignors: SZALAY, ALADAR A.
Priority to US11/981,976 priority patent/US8586022B2/en
Priority to US11/982,102 priority patent/US20090117049A1/en
Priority to US11/982,035 priority patent/US20090123382A1/en
Priority to US11/982,040 priority patent/US20090117048A1/en
Abandoned legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • 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
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0045—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent agent being a peptide or protein used for imaging or diagnosis in vivo
    • A61K49/0047—Green fluorescent protein [GFP]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/495—Transforming growth factor [TGF]
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent

Definitions

  • the present invention relates to diagnostic and pharmaceutical compositions comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, e.g. a luminescent or fluorescent protein.
  • the present invention also relates to the use of said microorganism or cell for tumor-targeting or tumor-imaging.
  • said microorganism or cell additionally contain an expressible DNA sequence encoding a protein suitable for tumor therapy, e.g. a cytotoxic or cytostatic protein.
  • the concentration of virus particles in the tumors shows variations among patients.
  • the presence of human papillomavirus in squamous cell carcinomas of the esophagus ranges from 0 to 72% (10-15).
  • no virus particles have been found in tumor-free areas of the esophageal epithelium of the same patient suggesting that the virus particles are located only in the tumor tissues.
  • the presence and amplification of the virus-encoded fusion proteins in the same tumor were monitored in live animals by observing GFP fluorescence under a stereomicroscope and by collecting luciferase-catalyzed light emission under a low-light video-imaging camera.
  • Tumor-specific light emission was detected 4 days after viral injection in nude mice carrying subcutaneous C6 glioma implants ranging in size from 25 to 2500 mm 3 . The signal became more intense after the 4th postinjection day and lasted for 30 to 45 days, indicating continued viral replication.
  • Tumor accumulation of rVV-ruc-gfp virus particles was also seen in nude mice carrying subcutaneous tumors developed from implanted PC-3 human prostate cells, and in mice with orthotopically implanted MCF-7 human breast tumors. Further, intracranial C6 rat glioma cell implants in immunocompetent rats and MB-49 human bladder tumor cell implants in C57 mice were also targeted by the Vaccinia virus. Cross sections of a C6 glioma revealed that light emission was clustered in “patches” at the periphery of the tumor where the fast-dividing cells reside. In contrast, cross sections of breast tumors revealed that fluorescent “islands” were distributed throughout the tumors.
  • light-emitting cells or microorganims e.g. Vaccinia virus can be used to detect and treat primary and metastatic tumors.
  • the light-emitting bacteria were not observed to be released into the circulation and to re-colonize subsequently implanted tumors in the same animal. Further, mammalian cells expressing the Ruc-GFP fusion protein, upon injection into the bloodstream, were also found to home into and propagate in glioma tumors.
  • the present invention relates to a diagnostic or pharmaceutical composition
  • a diagnostic or pharmaceutical composition comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal.
  • Any microorganism or cell is useful for the diagnostic method of the present invention, provided that they replicate in the organism, are not pathogenic for the organism e.g. attenuated and, are recognized by the immune system of the organism, etc.
  • the diagnostic or pharmaceutical composition comprises a microorganism or cell containing a DNA sequence encoding a luminescent and/or fluorescent protein.
  • DNA sequence encoding a luminescent and/or fluorescent protein also comprises a DNA sequence encoding a luminescent and fluorescent protein as fusion protein.
  • the diagnostic or pharmaceutical composition of the present invention comprises a microorganism or cell containing a DNA sequence encoding a protein capable of inducing a signal detectable by magnetic resonance imaging (MRI), e.g. metall binding proteins.
  • MRI magnetic resonance imaging
  • the protein can bind contrast agents, chromophores, ligands or compounds required for visualization of tissues.
  • the DNA sequences encoding a luminescent and/or fluorescent protein are present in a vector or an expression vector.
  • the DNA sequences can also be contained in a recombinant virus containing appropriate expression cassettes.
  • Suitable viruses that may be used in the diagnostic or pharmaceutical composition of the present invention include baculovirus, vaccinia, Sindbis virus, Sendai virus, adenovirus, an AAV virus or a parvovirus, such as MVM or H-1.
  • the vector may also be a retrovirus, such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV or GaLV.
  • a suitable promoter is e.g. human cytomegalovirus “immediate early promoter” (pCMV).
  • pCMV immediate early promoter
  • tissue and/or organ specific promoters are useful.
  • the DNA sequences encoding a luminescent and/or fluorescent protein are operatively linked with a promoter allowing high expression.
  • promoters e.g. inducible promoters are well-known to the person skilled in the art.
  • DNA sequences encoding luminescent or fluorescent proteins that can be used in the diagnostic or pharmaceutical of the present invention.
  • Renilla luciferase from Renilla reniformis (Lorenz et al., PNAS USA 88 (1991), 4438-4442) and green fluorescent protein from Aequorea victoria (Prasher et al., Gene 111 (1987), 229-233) have been described that allow the tracing of bacteria or viruses based on light emission.
  • Transformation and expression of these genes in bacteria allows detection of bacterial colonies with the aid of the low light imaging camera or individual bacteria under the fluorescent microscope (Engebrecht et al., Science 227 (1985), 1345-1347; Legocki et al., PNAS 83 (1986), 9080-9084; Chalfie et al., Science 263 (1994), 802-805).
  • Luciferase genes have been expressed in a variety of organisms. Promoter activation based on light emission, using lux AB fused to the nitrogenase promoter, was demonstrated in Rhizobia residing within the cytoplasm of cells of infected root nodules by low light imaging (Legocki et al., PNAS 83 (1986), 9080-9084; O'Kane et al., J. Plant Mol. Biol. 10 (1988), 387-399). Fusion of the lux A and lux B genes resulted in a fully functional luciferase protein (Escher et al., PNAS 86 (1989), 6528-6532).
  • This fusion gene (Fab2) was introduced into Bacillus subtilis and Bacillus megatherium under the xylose promoter and then fed into insect larvae and was injected into the hemolymph of worms. Imaging of light emission was conducted using a low light video camera. The movement and localization of pathogenic bacteria in transgenic arabidopsis plants, which carry the pathogen-activated PAL promoter-bacterial luciferase fusion gene construct, was demonstrated by localizing Pseudomonas or Ervinia spp. infection under the low light imager as well as in tomato plant and stacks of potatoes (Giacomin and Szalay, Plant Sci. 116 (1996), 59-72).
  • Bacteria containing the complete lux operon sequence when injected intraperitoneally, intramuscularly, or intravenously, allowed the visualization and localization of bacteria in live mice indicating that the luciferase light emission can penetrate the tissues and can be detected externally (Contag et al., Mol. Microbiol. 18 (1995), 593-603).
  • the microorganism is a bacterium, e.g. attenuated Particularly preferred is attenuated Salmonella thyphimurium, attenuated Vibrio cholerae or attenuated Listeria monocytogenes or E. coli.
  • viruses such as Vaccinia virus, AAV, a retrovirus etc. are also useful for the diagnostic and therapeutic compositions of the present invention.
  • the virus is Vaccinia virus.
  • the cell of the diagnostic or therapeutic composition of the present invention is a mammalian cell such as stem cells which can be autologous or heterologous concerning the organism.
  • the luminescent or fluorescent protein is a luciferase, green fluorescent protein (GFP) or red fluorescent protein (RFP).
  • the microorganism or cell of the diagnostic or pharmaceutical composition of the present invention additionally contains a gene encoding a substrate for the luciferase.
  • the microorganism or cell of the diagnostic or pharmaceutical composition of the present invention contains a ruc-gfp expression cassette which contains the Renilla luciferase (ruc) and Aequorea gfp cDNA sequences under the control of a strong synthetic early/late (PE/L) promoter of Vaccinia or the luxCDABE cassette.
  • a preferred use of the microorganisms and cells described above is the preparation of a diagnostic composition for tumor-imaging.
  • the diagnostic composition of the present invention can be used e.g. during surgery, to identify tumors and metastasis.
  • the diagnostic composition of the present invention is useful for monitoring a therapeutic tumor treatment.
  • Suitable devices for analysing the localization or distribution of luminescent and/or fluorescent proteins in an organism, organ or tissue are well known to the person skilled in the art and, furthermore described in the literature cited above as well as the Examples, below.
  • the microorganisms and cells can be modified in such a way that they bind metalls and consequently are useful in MRI technology to make this more specific.
  • the present invention also relates to a pharmaceutical composition containing a microorganism or cell as described above, wherein said microorganism or cell furthermore contains one or more expressible DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors, such as a cytotoxic protein, a cytostatic protein, a protein inhibiting angiogenesis, or a protein stimulating apoptosis.
  • a protein(s) suitable for tumor therapy and/or elimination of metastatic tumors such as a cytotoxic protein, a cytostatic protein, a protein inhibiting angiogenesis, or a protein stimulating apoptosis.
  • proteins are well-known to the person skilled in the art.
  • the protein can be an enzyme converting an inactive substance (pro-drug) administered to the organism into an active substance, i.e. toxin, which is killing the tumor or metastasis.
  • the enzyme can be glucuronidase converting the less toxic form of the chemoterapeutic agent glucuronyldoxorubicin into a more toxic form.
  • the gene encoding such an enzyme is directed by a promoter which is inducible additionally ensuring that the conversion of the pro-drug into the toxin only occurs in the target tissue, i.e. tumor.
  • promoters are e.g. IPTG-, antibiotic-, heat-, pH-, light-, metall-, aerobic-, host cell-, drug-, cell cycle- or tissue specific-inducible promoters.
  • suitable proteins are human endostatin and the chimeric PE37/TGF-alpha fusion protein.
  • Endostatin is a carboxyterminal peptide of collagen XVIII which has been characterized (Ding et al., PNAS USA 95 (1998), 10443). It has been shown that endostatin inhibits endothelial cell proliferation and migration, induces G1 arrest and apoptosis of endothelial cells in vitro, and has antitumor effect in a variety of tumor models. Intravenous or intramuscular injection of viral DNA and cationic liposome-complexed plasmid DNA encoding endostatin result in limited expression levels of endostatin in tumors. However intratumoral injection of purified endostatin shows remarkable inhibition of tumor growth.
  • Pseudomonas exotoxin is a bacterial toxin secreted by Pseudomonas aeruginosa.
  • PE elicits its cytotoxic effect by inactivating elongation factor 2 (EF-2), which results in blocking of protein synthesis in mammalian cells.
  • EF-2 elongation factor 2
  • Single chain PE is functionally divided into three domains: domain Ia is required for binding to cell surface receptor, domain II is required for translocating the toxin into the target cell cytosol, and domain III is responsible for cytotoxicity by inactivating EF-2.
  • PE40 is derived from wild type Pseudomonas exotoxin that lacks the binding domain Ia.
  • TGF alpha/PE40 fusion protein where the C-terminus of TGF alpha polypeptide has been fused in frame with the N-terminus of the PE40 protein.
  • TGF alpha is one of the ligands of epidermal growth factor receptor (EGFR), which has been shown to be preferentially expressed on the surface of a variety of tumor cells.
  • EGFR epidermal growth factor receptor
  • TGF alpha-PE40 chimeric protein The toxicity of TGF alpha-PE40 chimeric protein is dependent on a proteolytic processing step to convert the chimeric protein into its active form, which is carried out by the target.
  • a new chimeric toxin protein that does not require processing has been constructed by Theuer and coworkers (J.Biol.Chem. 267 (1992), 16872).
  • the novel fusion protein is termed PE37/TGF alpha, which exhibited higher toxicity to tumor cells than the TGF alpha-PE40 fusion protein.
  • the protein suitable for tumor therapy is endostatin (for inhibition of tumor growth) or recombinant chimeric toxin PE37/transforming growth factor alpha (TGF-alpha) (for cytotoxicity to tumor cells).
  • endostatin for inhibition of tumor growth
  • TGF-alpha transforming growth factor alpha
  • the delivery system of the present application even allows the application of compounds which could so far not be used for tumor therapy due to their high toxicity when systemicly applied.
  • Such compounds include proteins inhibiting elongation factors, proteins binding to ribosomal subunits, proteins modifying nucleotides, nucleases, proteases or cytokines (e.g. IL-2, IL-12 etc.), since experimental data suggest that the local release of cytokines might have a positive effect on the immunosuppressive status of the tumor.
  • the microorganism or cell can contain a BAC (Bacterial Artificial Chromosome) or MAC (Mammalian Artificial Chromosome) encoding several or all proteins of a specific pathway, e.g. anti-angionesis, apoptosis, woundhealing-pathway or anti-tumor growth. Additionally the cell can be cyber cell or cyber virus endocing these proteins.
  • BAC Bacterial Artificial Chromosome
  • MAC Mammalian Artificial Chromosome
  • the microorganisms or cells of the present invention are preferably combined with suitable pharmaceutical carriers.
  • suitable pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions, such as oil/water emuslions, various types of wetting agents, sterile solutions etc..
  • Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose.
  • Administration of the microorganisms or cells may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the preferred route of administration is intravenous injection
  • the route of administration depends on the nature of the tumor and the kind of microorganisms or cells contained in the pharmaceutical composition.
  • the dosage regimen will be determined by the attending physian and other clinical factors.
  • dosages for any one patient depends on many factors, including the patient's size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind, size and localization of the tumor, general health and other drugs being adminstered concurrently.
  • Preferred tumors that can be treated with the microorganisms or cells of the present invention are bladder tumors, breast tumors, prostate tumors, glioma tumors, adenocarcinomas, ovarial carcinomas, and pancreatic carcinomas; liver tumors, skin tumors.
  • FIG. 1 External Imaging of GFP Expression in Subcutaneous C6 Glioma Tumors in Nude Mice
  • C6 glioma cells (5 ⁇ 10 5 ) were implanted subcutaneously into the right lateral thigh. At designated days after tumor cell implantation, the animals were infected intravenously with 1 ⁇ 10 8 pfu of rVV-ruc-gfp virus particles. GFP expression was monitored under a fluorescence stereomicroscope. Bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of subcutaneous glioma tumor are shown. GFP signal can be observed in tumors as small as 22 mm 3 in size (B-B′′), or as old as 18 days (about 2500 mm 3 in size) (A-A′′).
  • FIG. 2 Visualization of Tumor Angiogenesis
  • FIG. 3 Expression of GFP in Subcutaneous Glioma Tumor of the Same Animal
  • FIG. 4 Bright Field (A) and Fluorescence (B) Images of Tumor Cells Expressing GFP
  • FIG. 5 Low Light Image of the Anesthetized Nude Mouse to Indicate the Location of Renilla luciferase-Triggered Light Emission in the Presence of Intravenously Injected Substrate Coelenterazine (5 ⁇ g Ethanol Solution)
  • FIG. 6 Monitoring Tumor-Specific Viral Infection Based on GFP Gene Expression in a Variety of Tumor Models
  • FIG. 7 Monitoring Vaccinia-Mediated GFP Expression in a Breast Tumor Model
  • FIG. 8 Visualization of the Clearance of Light Emitting Bacteria From Nude Mice Based on the Detection of Light Emission Under the Low Light Imager
  • Nude mice were intravenously injected with 10 7 cells of attenuated S. typhimurium (A, B) and V. cholera (C, D). Both strains were transformed with pLITE201 carrying the lux operon. Photon collection was done 20 min (A, C) and 2 days (B, D) after bacterial injections.
  • FIG. 9 Homing of Glioma Tumors by Attenuated Bacteria
  • FIG. 10 Homing in of Bacteria onto Breast Tumors
  • FIG. 11 Homing in of Bacteria Onto Bladder Tumors in C57 Mice
  • C57 mice were intravenously injected with 10 7 attenuated V. cholera transformed with pLITE201 encoding the lux operon.
  • luminescence was noted in the bladder region of the whole animal (A). The animal was sacrificed and an abdominal incision was made to expose the bladder. The light emission was limited to the bladder region (B). With the removal of the bladder (C) from the mouse, the entire source of light emission was removed (D) as demonstrated by the overlay of the low light photon emission image over the photographic image of the excised bladder.
  • FIG. 12 Homing in of Bacteria onto Brain Glioma Tumors in Lewis Rats
  • Lewis rats were intravenously injected with 10 8 cells of attenuated V. cholera transformed with pLITE201 encoding the lux operon. Twenty-four hours after bacterial injection, faint luminescence was noted in the head region of the whole animal during visualization under the low light imager. The animals were sacrificed and their brain removed. Photon collection was carried out for one minute from rats with (A) and without (B) brain tumors. Strong luminescence was confirmed in regions of the brain of the rats with the brain tumor (marked with arrows in A). Luminescence was completely absent in the control brain tissues (B).
  • FIG. 13 Transformed Human Fibrosarcoma Cells Home in on Subcutaneous Glioma Tumors in Nude Mice
  • FIG. 14 Homing of Attenuated Listeria monocytogenes into Subcutaneous Prostate Tumors
  • the bacterial strains used were attenuated Salmonella typhimurium (SL7207 hisG46, DEL407[aroA544::Tn10]), attenuated Vibrio cholerae (Bengal 2 Serotyp 0139, M010 DattRS1), and attenuated Listeria monocytogenes (D2 mpl, actA, plcB).
  • the bacterial strains were kindly provided by Prof. W. Göbel (University of Würzburg, Germany).
  • the plasmid pLITE201 containing the luxCDABE cassette was obtained from (Voisey and Marincs, Biotech 24, 1998, 56-58).
  • the plasmid pXylA-dual with the operon sequence of gfp-cDNA, lux AB, lux CD, and lux E under the control of the Xylose promoter was kindly provided by Dr. Phil Hill (University of Nottingham, UK).
  • the bacteria were transformed by electroporation.
  • the rat C6 nitrosourea-induced glioma cell line (ATCC, Rockille, Md.) was cultured in RPMI-1640 medium (Cellgro, Mediatech, Inc., Herndon, Va.) supplemented with 10% (v/v) FBS and 1 ⁇ penicillin/streptomycin.
  • the human PC3 prostate carcinoma cell line (ATCC, Rockville, Md.) and the Human MB-49 bladder tumor cells and rat 9L glioma cells were maintained in DMEM medium (Cellgro, Mediatech, Inc., Herndon, Va.) supplemented with L-glutamine and 10% (v/v) FBS.
  • HT1080 fibrosarcoma cells (ATCC, Manassas, Va.) were cultured in F12 minimal essential media (Cellgro, Mediatech, Inc., Herndon, Va.) supplemented with 10% FBS and 1 ⁇ penicillin/streptomycin.
  • F12 minimal essential media Cellgro, Mediatech, Inc., Herndon, Va.
  • the MCF-7 human mammary carcinoma cell line (ATCC, Rockville, Md.), permanently transformed with a plasmid carrying pro-IGF-II cDNA (obtained from Dr. Daisy De Leon, Loma Linda Iuniversity, Loma Linda, Calif.) was cultured in DMEM/F12 medium supplemented with 5% FBS and 560 ⁇ g/ml of G418 (Life Technologies, Grand Island, N.Y.).
  • PT67 packing cells (Clontech, Palo Alto, Calif.) were cultured in DMEM medium supplemented with 10% (v/v) FBS. At 70% confluence, PT67 cells were transformed with pLEIN (Clontech, Palo Alto, Calif.) using calcium phosphate precipitation method (Profection Mammalian Transfection Systems, Promega, Madison, Wis.) for 12 hours. Fresh medium was replenished at this time.
  • Retroviral supernatant collected from PT67 cells 48 hours post transformation were filtered through a 0.45 ⁇ m filter and was added to target HT1080 cells along with polybrene to a final concentration of 4 ⁇ g/ml.
  • the medium was replaced after 24 hours and the cells were treated with G418 selection at 400 ⁇ g/ml and stepwise increased to 1200 ⁇ g/ml.
  • C57BL/6J Min/+ mice were obtained from Jackson Laboratories (Bar Harbor, Me.)
  • Min multiple intestinal neoplasia is an autosomal dominant trait involving a nonsense mutation in codon 850 of the murine Apc gene, which renders these animals susceptible to spontaneous intestinal adenoma formation.
  • Female BALB/c athymic nu/nu mice baring MCF-7 human breast tumor implants were generated and kindly provided by Dr. Daisy DeLeon and Dr.
  • Vaccinia virus Lister strain (LIVP) was used as a wild type virus.
  • Recombinant Vaccinia virus rVV-ruc-gfp was constructed by inserting, via homologous recombination, the ruc-gfp-cassette into the Vaccinia virus genome (Wang et al., Proc. Biolumin. Chemilumin. 9, 1996, 419-422).
  • the virus was amplified in CV-1 cells by addition of virus particles at a multiplicity of infection (MOI) of 0. 1 pfu/cell to CV-1 cell monolayers followed by incubation at 37° C.
  • MOI multiplicity of infection
  • C6 glioma cells were grown, harvested and the cell number was determined by the Trypan Blue exclusion method.
  • Disinfectant was applied to the skin surface, then approximately 5 ⁇ 10 5 cells were suspended in 100 ⁇ l of phosphate buffered saline (PBS) and injected subcutaneously into the right lateral thigh of each mouse.
  • Tumor growth was monitored by recording the size of the tumor with a digital caliper.
  • Tumor volume (mm 3 ) was estimated by the formula (L ⁇ H ⁇ W)/2, where L is the length, W is the width, and H is the height of the tumor in mm.
  • Intracerebral glioma tumors were generated by injecting C6 glioma cells into the head of rats. Prior to injection, rats were anesthetized with sodium pentobarbital (Nembutal® Sodium solution, Abbot Laboratories, North Chicago, Ill.; 60 mg/kg body weight). A midline scalp incision (0.5-1 cm) was made, the skin was retracted, and a 1 mm burr hole was made in the skull at a location 2 mm to the left and 2.5 mm posterior to the brigma. Tumor cells were pipetted into an insulin syringe, which was fitted with a 29-gauge needle and mounted in a stereotactic holder.
  • the needle was inserted vertically through the burr hole to a depth of 3 mm. After injection into the brain of 5 ⁇ 10 5 C6 cells in a 10 ⁇ l volume, the needle was kept in place for 15 sec and then withdrawn. The skin incision was closed with surgical clips. Mice bearing subcutaneous prostate tumors were generated over a period of one month following subcutaneous implantation of 3 ⁇ 10 6 PC3 human prostate cells.
  • MB-49 human bladder tumor cells were implanted in the C57 mouse bladder to produce animals with bladder tumors.
  • female nude mice were first implanted with 0.72 mg/90 day-release 17 ⁇ -estradiol pellets (Innovative Research, Rockville, Md.) in the skin to facilitate breast tumor development and metastasis.
  • 1 ⁇ 10 6 MCF-7 human breast carcinoma cells transformed with pro-IGF-II Dull et al., Nature 310 (1984), 777-781 were implanted in the mammary fat pad.
  • tumors developed from implanted cells were resected and minced into 1-mm 3 cubes for tissue transplantation into the mammary fat pad.
  • coelenterazine 0.5 ⁇ g/ ⁇ l diluted ethanol solution
  • luciferase assay buffer 0.5 M NaCl; 1 mM EDTA; and 0.1 M potassium phosphate, pH 7.4
  • ARGUS 100 Hamamatsu, Hamamatsu, Japan
  • Imaging of GFP expression in tumors of live animals was performed using a Leica MZ8 stereo fluorescence microscope equipped with a mercury lamp power supply and a GFP filter (excitation at 470 run). Images were captured using a SONY DKC-5000 3CCD digital photo camera.
  • Vaccinia virus (1 ⁇ 10 8 pfu) carrying the Renilla luciferase—GFP fusion expression cassette (rVV-ruc-gfp) was introduced intravenously into nude mice with no tumors. The animals were observed once every 3 days over a two-week time period under the low-light imager to monitor luciferase catalyzed light emission immediately after intravenous injection of coelenterazine, and under a fluorescence microscope to visualize GFP expression. Neither apparent luminescence nor green fluorescence was detected in the animals when imaged externally, except at certain locations that had small skin lesions. Such luminescence and fluorescence signals disappeared after a few days once the lesions had healed.
  • nude mice were intravenously injected with 1 ⁇ 10 8 rVV-ruc-gfp Vaccinia virus particles one day after subcutaneous C6 cell implantation. Surprisingly, 4 days after viral injection GFP expression was seen in 5-day-old C6 tumors that had a volume of about 25 mm 3 (FIG. 1B-B′′). Examination of labeled Vaccinia virus tumor targeting by visualization of GFP expression in implanted tumors younger than 5 days was not feasible in live mice, since sufficient levels of marker gene expression required approximately 4 days to allow detection under a fluorescence microscope.
  • FIG. 1A-A′′, D-D′′, E-E′′ and FIG. 2 show that the location of blood vessels and the neovascularization within the periphery of the enlarging tumor were readily visible and confirmed by external visualization against a bright green fluorescent background (FIG. 1A-A′′, D-D′′, E-E′′ and FIG. 2).
  • the recombinant rVV-ruc-gfp virus carried a second marker gene, which encoded the Renilla luciferase in the form of a fusion protein with GFP. Therefore we were able to directly superimpose the site of GFP fluorescence with light emission from Renilla luciferase in the tumors. Immediately after coelenterazine (substrate for Renilla luciferase) was delivered by intravenous injection, a very strong luciferase activity was recorded only in the tumor region under a low light video camera (FIG. 5).
  • Vaccinia virus was recombinantly introduced into mice that carried different types of implanted tumors.
  • One of these tumor models was a nude mouse with implanted subcutaneous PC-3 human prostate carcinoma.
  • the PC3 implants from which tumors developed grew at a much slower rate than the implanted subcutaneous glioma tumors, these tumors showed the same dynamics with regards to Vaccinia virus infection when identical titers (1 ⁇ 10 8 ) were injected intravenously (FIG. 6A-A′′). Similar to our findings with glioma tumors, GFP expression was initially detected 4 days after virus injection, and the fluorescence lasted throughout the 3-week observation period.
  • mice with established breast tumors were also used for labeled Vaccinia injections. These breast tumors were allowed to grow for 6 months after the animals received implants of MCF-7 human breast carcinoma cells transformed with pro-IGF-11 cDNA. At the time of Vaccinia virus injection, the tumors had reached maximum growth and the tumor volume (about 400-500 mm 3 ) did not change significantly during the experimental period. Similar to previous experiments, 6 days after intravenous delivery of 1 ⁇ 10 8 rVV-ruc-gfp virus particles, strong GFP expression was observed in the breast tumor region (FIG. 6B-B′′, FIG. 7A-A′′ and B-B′′) and nowhere else in the body.
  • FIG. 7C-C′′ Examination of cross sections of virus-infected breast tumors revealed luminescent “islands” throughout the tumors without any indication of central or peripheral preference of infection.
  • the MCF-7 tumor cells used in these breast tumor models are known to metastasize and in addition to the primary solid tumor, a smaller metastasized tumor found on the left lateral side of the body showed GFP fluorescence (FIG. 7D-D′′, E-E′′, and F-F′′).
  • Excised lung tissues were also examined for detection of metastases. Metastasized tumors as small as 0.5 mm in diameter on the surface of the lung were positive for GFP fluorescence (FIG. 7G-G′′).
  • rVV-ruc-gfp virus (1 ⁇ 10 8 pfu) was intravenously injected. Five days later, tumor-specific GFP expression was detected in the newly formed glioma tumor in addition to GFP expression seen in the original breast tumor.
  • Two additional tumor models including Lewis rats with intracranial C6 rat glioma tumors and C57 mice with MB-49 human bladder tumors in the bladder, were used for Vaccinia injections.
  • Tumor-affinity of virus particles is a phenomenon limited to tumors in nude mice with a diminished T-lymphocyte function or whether it is a general protective property of tumors that may be demonstrated also in immunocompetent animals.
  • Lewis rats with intracranial C6 rat glioma tumors and C57 mice with MB-49 human bladder tumors in the bladder were used.
  • a total of 5 ⁇ 10 5 C6 glioma cells in a 100 ⁇ l volume were stereotactically implanted in the brains of 2 of 4 immunocompetent Lewis rats, and the tumors were allowed to grow for 5 days.
  • the other 2 rats were injected intracranially with phosphate-buffered saline to serve as controls.
  • all 4 rats were intravenously injected with rVV-ruc-gfp virus particles via the femoral vein.
  • GFP expression was detected in the brains with implanted intracranial tumors (FIG. 6C-C′′) while no GFP expression was seen in the control brains.
  • C57 mice with or without bladder tumors, were divided into two groups.
  • One group was injected intravenously with rVV-ruc-gfp Vaccinia virus (1 ⁇ 10 8 pfu) and the other with saline solution as control.
  • Five days after virus injection the animals were sacrificed and examined under the fluorescence microscope. GFP expression was observed in the bladder tumor region in C 57 mice but not in control mice (FIG. 6D-D′′).
  • nude mice with ten-day-old tumors (about 500 mm 3 ) in the tight hind leg were injected intravenously via the femoral vein with 10 7 S. typhimurium or 10 7 V. cholera in a 50 ⁇ l volume of bacterial suspension.
  • the incision wounds were sutured and the animals were monitored for six days under the low light imager. At each observation time point, photons were collected for exactly one minute.
  • luminescent bacteria were disseminated throughout the whole body of the animal similar to the findings in the non-tumorous mice (FIG. 9A).
  • the purpose of this experiment was to determine whether the size of the tumor has any influence on its ability to be colonized by bacteria.
  • Tumors were induced in the right hind leg of nude mice by subcutaneous injection of glioma cells as described.
  • On days 0, 2, 4, 6, 8, and 10 of tumor induction attenuated S. typhimurium and V. cholera with the pLITE201 plasmid were injected intravenously through the femoral vein.
  • Presence of luminescent bacteria in the tumor was determined by photon collection for exactly one minute under the low light imager two and four days post-infection.
  • the tumor volume was also determined by measuring the dimensions with a digital caliper. The earliest time-point when luminescent activity was noted in the tumors was on day eight after tumor induction. Corresponding tumor volumes were approximately 200 mm 3 .
  • FIG. 11D shows the continued colonization and propagation of the bacteria in the main tumor, while the metastatic, smaller tumor had become cleared. Luminescent activity continued for over 45 days in the right breast tumor. Similar experiments were conducted using E. coli to demonstrate that homing in of tumors by bacteria is not strain dependent (FIGS. 11E and 11F).
  • Nude mice with human breast tumors were injected intravenously with 5 ⁇ 10 5 human fibrosarcoma cells, which were permanently transformed with retrovirus derived from pLEIN. Seven days post-injection, the animals were anesthetized Nembutal, and monitored under a fluorescent stereomicroscope. Fluorescent cells were noted only in the tumor region of the whole mice through the skin (FIG. 14A 1 - 3 ). Upon exposure of the tumor tissues by reflection of the overlying skin (FIG. 14B 1 - 3 ), and in cross sections of the tumors (FIG. 14C 1 - 3 ), fluorescent patches were visible in distinct regions. Close examination of the organs of the mice showed the presence of small clusters of fluorescent cells in the lungs of the animals, demonstrating the affinity of the fibrosarcoma cells for the lungs in addition to the tumorous tissue.
  • the therapeutic proteins, endostatin and Pseudomonas exotoxin/TGF alpha fusion protein are required to be-secreted from the bacteria into the medium or into the cytosol of tumor cells for inhibition of tumor growth.
  • the ompF signal sequence can be placed upstream of the coding sequence of endostatin, which facilitates the secretion into the periplasmic space.
  • an additional protein, the PAS protein needs to be coexpressed with endostatin.
  • PAS has been shown to cause membrane leakiness and the release of secreted proteins into the medium (Tokugawa et al., J.Biotechnol. 37 (1994), 33; Tokugawa et al., J.Biotechnol. 35 (1994), 69).
  • the second construct for the secretion of Pseudomonas exotoxin/TGF alpha fusion protein from E. coli has the OmpA signal sequence upstream of the fusion gene and the release from the periplasmic space into the medium is facilitated by sequences present in domain II of the exotoxin (Chaudhary et al., PNAS 85 (1988), 2939; Kondo et al., J.Biol.Chem.
  • LLO listeriolysin
  • vectors can be generated where the therapeutic protein encoding genes are under the control of the T7 promoter or the P spac synthetic promoter (Freitag and Jacobs, Infect.Immun. 67 (1999), 1844). Without exogenous induction, the levels of the therapeutic proteins are low in E. coli and in L. monocytogenes. The minimal levels of therapeutic proteins in bacteria provide greater safety following intravenous injection of the engineered bacteria.
  • the construction of the endostatin secretion vector to be used in E. coli is as follows.
  • the coding sequence of human endostatin (591 bp) will be amplified by PCR from the plasmid pES3 with the introduction of the required restriction sites on both ends, followed by ligation into a pBluescript (Clontech Corp., USA) cloning vector to generate pBlue-ES.
  • the ompF signal sequence (Nagahari et al., EMBO J. 4 (1985), 3589) is amplified with Taq polymerase and inserted upstream in frame with the endostatin sequence to generate pBlue-ompF/ES.
  • the expression cassette driven by the T7 promoter will be excised, and inserted into the pLITE201 vector described in Example 1(B), above, carrying the luxCDABE cassette, to produce the plasmid pLITE-ompF/ES.
  • the sequence encoding the PAS factor (a 76 amino acid polypeptide) will be amplified from the chromosomal DNA of Vibrio alginolyticus (formerly named Achromobacter iophagus ) (NCIB 11038) with Taq polymerase using the primers 5′-GGGAAAGACATGAAACGCTTA3-′ and 5′-AAACAACGAGTGAATTAGCGCT-3′, and inserted into the multiple cloning sites of pCR-Blunt (Clontech Corp., USA) to create the expression cassette under the control of the lac promoter.
  • the resulting plasmid will be named pCR-PAS.
  • the lac promoter linked to the pas gene will be excised from pCR-PAS and inserted into pLITEompF/ES to yield the final plasmid BSPT#1-ESI.
  • Plasmid pVC85 (Pastan, see above) contains a T7 promoter, followed by an ompA signal sequence, and a sequence encoding domain II and III of Pseudomonas exotoxin (PE40).
  • the DNA sequence encoding PE40 will be excised with restriction enzymes and replaced with a fragment of PE37/TGF alpha (Pseudomonas exotoxin A 280-613/TGF alpha) obtained from the plasmid CT4 (Pastan, see above) to create the plasmid pVC85-PE37/TGF alpha.
  • the expression cassette of ompAPE37/TGF alpha linked to the T7 promoter will be excised and inserted into pLITE201 to yield the final plasmid BSPT#2-PTI.
  • PNAS in press. will be inserted into pCHHI-ES to generate BSPT#3-ESc, and into pCCHI-PE37/TGF alpha to generate BSPT#4-PTc.
  • the listeriolysin promoter in BSPT#3-ESc and BSPT#4-PTc will be replaced with the P spac promoter from the plasmid pSPAC (Yansura and Henner, PNAS USA 81 (1984), 439) to generate BSPT#5-ESi and BSPT#6-PTi.
  • P spac is a hybrid promoter consisting of the Bacillus subtilis bacteriophage SPO-1 promoter and the lac operator. IPTG-induced GFP expression from the P spac promoter has been documented in L. monocytogenes in the cytosol of mammalian cells.
  • endostatin and Pseudomonas exotoxin/TGF alpha fusion protein synthesized within E. coli and L. monocytogenes will be determined by extracting these proteins from the cell pellet.
  • the secreted proteins in the medium will be concentrated and analyzed by gel separation and the quantity will be determined by Western blotting. It is imperative to determine the percentage of the newly synthesized proteins expressed from each plasmid construct in either E. coli or L. monocytogenes that is present in the medium. It is also essential to confirm, in addition to constitutive expression of endostatin and Pseudomonas exotoxin/TGF alpha fusion protein, that expression can be induced in E. coli and in L.
  • E. coli strains (DH5 ⁇ and BL21( ⁇ DE3) will be transformed with BSPT#1-ESi and BSPT#2-PTi plasmid DNA.
  • L. monocytogenes strain EGDA2 will be transformed with plasmids BSPT#3-ESc, BSPT#4-PTc, BSPT#5-ESi, and BSPT#6-PTi individually. After plating on appropriate antibiotic-containing plates, individual colonies will be selected from each transformation mixture. These colonies will be screened under a low light imager and fluorescence microscope for luciferase and GFP expression, respectively. Three colonies with the most intense light emission from each transformation batch will be chosen for further studies.
  • the cells will be grown in minimal medium to log phase. After centrifuging down the bacteria, the supernatants will be passed through a 0.45- ⁇ m-pore-size filter, and the bacterium-free medium will be used for precipitation of the secreted proteins. The precipitates will be collected by centrifugation. Pellets will be washed, dried, and re-suspended in sample buffer for protein gel separation. Proteins from aliquots corresponding to 10 ⁇ l of bacterial culture will be compared to proteins from 200 ⁇ l of culture supernatant after separation in a 10% SDS-polyacrylamide gel.
  • VEGF vascular endothelial growth factor
  • HUVEC human umbilical vein endothelial cell
  • HUVECs After incubation, the HUVECs will be placed in the upper chamber.
  • the migration of HUVECs into the lower chamber induced by VEGF 165 (R&D Systems, Minneapolis, Minn.) will be quantified by microscopic analysis.
  • the concentration of functional endostatin in the medium will be directly proportional to the degree of inhibition of HUVEC migration.
  • the inhibitory activity of the chimeric toxin in mammalian cells will be measured based on inhibition of de novo protein synthesis by inactivating EF-2 (Carroll and Collier, J.Biol.Chem. 262 (1987), 8707). Aliquots of bacterium-free supernatants obtained from the expression of various recombinant PE secretion constructs in E. coli and in L. monocytogenes will be added to the C6 glioma cells or to HCTI 16 colon carcinoma cells. Following treatment with medium, the mammalian cells will be pulsed with [ 3 H]-leucine, and the incorporation will be determined in the protein fraction. To determine the presence of secreted chimeric toxin proteins in L.
  • the bacteria will be eliminated from the medium by gentamicin treatment.
  • the mammalian cells containing L. monocytogenes in the cytosol will be lysed, and the released bacteria removed from the lysate by filtration.
  • the mammalian cell lysate containing the secreted chimeric toxins will be assayed in protein synthesis inhibition experiments.
  • the inhibition of [ 3 H]-leucine incorporation in tumor cell culture will be directly proportional to the amount of the biologically active chimeric toxin protein in the medium and cell lysate.
  • E. coli (DH5 ⁇ ) carrying the DsRed (Matz et al., Nat.Biotech. 17 (1999), 969) expression cassette under the control of a constitutive promoter are used in this experiment.
  • L. monocytogenes EGD strain derivatives with in-frame deletion in each of the virulence genes were individually labeled with the green fluorescent protein cassette driven by the constitutive SOD promoter.
  • C6 glioma or HCT116 colon carcinoma tumors The localization and intratumoral distribution of bacteria will first be studied in nude mice with implanted C6 glioma or HCT116 colon carcinoma tumors.
  • C6 glioma or HCT116 colon carcinoma cells (5 ⁇ 10 5 in 100 ⁇ l) will be subcutaneously injected into the right hind leg of the animals. Twelve days after tumor cell injection, the animals will be anesthetized, and the left femoral vein surgically exposed.
  • Light-emitting bacteria (1 ⁇ 10 6 cells re-suspended in 50 ⁇ l of saline) will be intravenously injected, and the wound incision will be closed with sutures.
  • Tumors will be measured three times a week using a caliper. Tumor volume will be calculated as follows: small diameter ⁇ large diameter ⁇ height/2.
  • Intracerebral glioma tumors will be generated by injecting C6 glioma cells into the head of Wistar rats. Rats will be anesthetized with Ketamine (70-100 mg/kg body weight) and Xylazine (8-10 mg/kg body weight). A midline scalp incision (0.5-1 cm) will be made, skin will be reflected, and a 1 mm burr hole will be made in the skull located 2 mm to the left and 2.5 mm posterior to the brigma. Tumor cells will be pipetted into an insulin syringe fitted with a 29-gauge needle and mounted in a stereotactic holder. The needle will be inserted vertically through the burr hole to a depth of 3 mm.
  • the localization of bacteria in the tumor will also be analyzed using cryosectioned tumor tissues.
  • a reliable morphological and histological preservation, and reproducible GFP or RFP detection may be obtained using frozen sections after a slow tissue freezing protocol (Shariatmadari et al., Biotechniques 30 (2001), 1282). Briefly, tumor tissues will be removed from the sacrificed animals to a Petri dish containing PBS and dissected into the desired size. The samples will be mixed for 2 h in 4% paraformaldehyde (PFA) in PBS at room temperature. They will be washed once with PBS, and embedded in Tissue-Tek at room temperature, and then kept in the dark at 4° C.
  • PFA paraformaldehyde
  • the tissue will be kept at ⁇ 20° C. for 30 min. Then, 10- to 50- ⁇ m-thick sections will be cut with a Reichert-Jung Cryocut 1800 cryostat and collected on poly-L-lysine (1%)-treated microscope slides. During sectioning, the material will be kept at room temperature to avoid several freezing and thawing cycles. Finally, the sections will be rinsed in PBS and mounted in PBS and kept in the dark at 4° C.
  • each tumor will be cut into two halves.
  • One half of the tumor will be used for preparing thick sections (60-75 ⁇ m), which will be analyzed under a fluorescence stereomicroscope to observe the distribution of bacteria in the sections of tumors obtained from each time point of the experiment.
  • the regions of interest will be identified, thin sectioned, prepared, and analyzed with laser scanning cytometry and under the confocal microscope followed by image reconstruction.
  • mice with spontaneous tumors will be obtained and used in intravenous injection experiments with E. coli carrying the bacterial lux operon. Two animals of each tumor model will be used, and the luciferase light emission monitored dally under the low light imager. It is expected that the spontaneously occurring tumors can be imaged similarly to the implanted tumors based on bacterial luciferase expression. Two of the spontaneous tumor models, mice with adenocarcinoma of the large intestine and mice with adenocarcinoma of the mammary tissue, will be used for bacterial localization experiments following intravenous injection of E. coli expressing RFP and L. monocytogenes expressing GFP as described above.
  • the endostatin and chimeric toxin gene cassettes are linked to signal peptide encoding sequences, which facilitate the secretion of these proteins into the extracellular space in the tumor or into the cytosol of infected tumor cells.
  • Both proteins secreted from bacteria into the extracellular space of the tumor are expected to function similarly to directly injected purified proteins.
  • Both proteins secreted from L. monocytogenes into the cytosol of the infected tumor cells will resemble the viral delivery system reported earlier for endostatin.
  • the bacterial systems can be used as a constitutive secretion system or as an exogenously added IPTG-activatable secretion system in the tumor.
  • the secreted amount of proteins inhibiting tumor growth can be determined.
  • IPTG IP-linked glycoprotein
  • the inhibitory protein secretion from the intravenously injected bacteria will be kept at minimum while in blood circulation. This will provide an added safety to the recipient tumorous animals during delivery of bacteria.
  • the onset and duration of the therapy can be controlled by the addition of IPTG.
  • the bacterial delivery system can be eliminated by administration of antibiotics, similar to treating a bacterial infection.
  • the inhibitory effect of endostatin and the cytotoxicity of the chimeric toxin secreted by E. coli and L. monocytogenes in tumors will be determined as follows. Thirty-five nude mice bearing 10-day-old C6 tumors will be injected with bacterial constructs as follows: (a) Five mice with E. coli engineered to secrete endostatin; (b) Five mice with E. coli engineered to secrete chimeric toxin; (c) Five mice with L. monocytogenes engineered to secrete endostatin; (d) Five mice with L. nionocytogeties engineered to secrete chimeric toxin; (e) Five mice with E.
  • control group five mice injected with E. coli expressing bacterial luciferase alone, and five mice with L. monocytogenes expressing GFP.
  • each tumor volume will be determined. Three days after injection, the replication of bacteria in the tumors will be monitored under a low light imager or under a fluorescence stereomicroscope. The light emission and the tumor volume will be measured daily up to 20 days after bacterial injection. Ten days after injection, one animal from each group will be sacrificed and the levels of the secreted proteins present in the tumor tissue will be analyzed using Western blot analysis. These experiments will result in inhibition of tumor growth in endostatin treated animals or a more dramatic tumor regression in animals treated with chimeric toxin proteins. The tumor growth in control animals is not expected to be affected by the bacteria alone.
  • mice with spontaneous adenocarcinoma of mammary tissue will be used to study the effect of secreted proteins on tumor growth.
  • An experimental scheme identical to that described for the C6 tumor analysis will be used.
  • the presence of endostatin or chimeric toxin in the tumor tissue will be determined by Western blot analysis.
  • An identical experimental design will be used to assay the effect of IPTG-induction of endostatin and chimeric toxin production in bacteria in C6 tumors as well as in the spontaneously occurring breast tumor mouse model. It is expected that multiple IPTG induction of protein expression in bacteria might be required for successful tumor therapy.
  • mice with 12-day-old C6 tumors will be intravenously injected with E. coli expressing the bacterial luciferase.
  • antibiotic therapy will be initiated by intraperitoneal administration of gentamicin (5 mg/kg body weight) twice daily, or the newly discovered clinafloxacin (CL960) (Nichterlein et al., Monbl.Bakteriol. 286 (1997), 401). This treatment will be performed for 5 days, and the effect of antibiotics on the bacteria will be monitored by imaging light emission from the animals daily.

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US20090117049A1 (en) 2009-05-07
ATE452205T1 (de) 2010-01-15
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KR20100063143A (ko) 2010-06-10
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NZ530818A (en) 2007-07-27
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