WO2001025399A2 - Imagerie non invasive de tumeurs par des bacteries ciblees sur des tumeurs - Google Patents

Imagerie non invasive de tumeurs par des bacteries ciblees sur des tumeurs Download PDF

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WO2001025399A2
WO2001025399A2 PCT/US2000/027397 US0027397W WO0125399A2 WO 2001025399 A2 WO2001025399 A2 WO 2001025399A2 US 0027397 W US0027397 W US 0027397W WO 0125399 A2 WO0125399 A2 WO 0125399A2
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Prior art keywords
tumor
labeled
subject
marker
bacteria
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PCT/US2000/027397
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English (en)
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WO2001025399A3 (fr
Inventor
David G. Bermudes
Ivan Cheung-Lam King
Ronald G. Blasberg
Juri G. Tjuvajev
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Vion Pharmaceuticals, Inc.
Sloan-Kettering Institute For Cancer Research
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Priority to CA002386806A priority Critical patent/CA2386806A1/fr
Priority to AU79936/00A priority patent/AU7993600A/en
Priority to EP00970577A priority patent/EP1414499A4/fr
Publication of WO2001025399A2 publication Critical patent/WO2001025399A2/fr
Publication of WO2001025399A3 publication Critical patent/WO2001025399A3/fr

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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
    • 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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is concerned with non-invasive imaging methods to detect solid tumors in vivo.
  • the invention is also concerned with non-invasive methods to monitor and/or treat solid tumors.
  • mice injected with bacillus Calmette-Guerin have increased serum levels of TNF and that TNF-positive serum caused necrosis of the sarcoma Meth A and other transplanted tumors in mice.
  • BCG Bacillus Calmette-Guerin
  • TNF ⁇ -mediated septic shock is among the primary concerns associated with bacteria, and can have toxic or lethal consequences for the host (Bone, 1992, JAMA 268: 3452-3455; Dinarello et al, 1993, JAMA 269: 1829-1835). Further, dose-limiting, systemic toxicity of TNF ⁇ has been the major barrier to effective clinical use. Modifications which reduce this form of an immune response would be useful because TNF ⁇ levels would not be toxic, and a more effective concentration and/or duration of the therapeutic vector could be used.
  • Salmonella have been demonstrated to be capable of being tumor-targeted, possess anti-tumor activity and are useful in delivering genes such as the herpes simplex virus thymidine kinase (HSV TK) to solid tumors (Pawelek et al, WO 96/40238).
  • HSV TK herpes simplex virus thymidine kinase
  • Pawelek et al. (1997, Cancer Res. 57:4537-4544) employed a gene fusion consisting of the ⁇ -lactamase secretion signal followed by the entire coding sequence of HSVl-tk.
  • the gene product of this construct was secreted into the periplasmic space of the bacterium and was shown to be functionally active.
  • Antitumor studies with a strain carrying this construct demonstrated that treatment with ganciclovir resulted in an enhancement of antitumor activity only when the HSVl-tk plasmid was present, thus establishing the therapeutic potential of Salmonella expressing functionally active HSVl-tk.
  • Pawelek et al. Modifications to the lipid composition of tumor-targeted bacteria which alter the immune response as a result of decreased induction of TNF ⁇ production were suggested by Pawelek et al. (Pawelek et al, WO 96/40238). Pawelek et al. provided methods for isolation of genes from Rhodobacter responsible for monophosphoryl lipid A (MLA) production. MLA acts as an antagonist to septic shock. Pawelek et al.
  • MLA monophosphoryl lipid A
  • Hone and Powell disclose methods to produce gram-negative bacteria having non-pyrogenic Lipid A or LPS. Hone and Powell propose using non-pyrogenic bacteria only for vaccine purposes.
  • Maskell describes a mutant strain of Salmonella having a mutation in the msbB gene which induces TNF ⁇ at a lower level as compared to a wild type strain.
  • Bermudes et al, WO 99/13053 teach compositions and methods for the genetic disruption of the msbB gene in tumor-targeted Salmonella, which result in Salmonella possessing a lesser ability to elicit TNF ⁇ and reduced virulence compared to the wild type.
  • some such mutant Salmonella have increased sensitivity to chelating agents as compared to wild type Salmonella.
  • the mutant tumor-targeted Salmonella deliver a gene product such as a pro-drug converting enzyme useful as an anti-tumor agent.
  • Blasberg and Tjuvajev developed a non-invasive imaging system to detect gene transfer and expression in target tissues which is useful for monitoring and evaluating in vivo gene therapy (Tjuvajev et al, 1995, Cancer Res. 55:6126-6132; Tjuvajev et al, 1996, Cancer Res. 56:4087-4095; Tjuvajev et al, 1998, Cancer Res. 58:4333-4341.
  • the system uses a transfer vector containing HSV1-TK and a labeled marker substrate to noninvasively image target tissue or cells which have taken up the marker gene.
  • the present invention provides non-invasive methods to detect solid tumors in vivo by delivery of a marker gene to a solid tumor.
  • the vector for delivery of the marker gene to the solid tumor is a heterogeneous or homogenous population of tumor-targeted bacteria.
  • the present invention also provides other non-invasive methods to detect solid tumors using tumor targeted bacterial vectors. Such methods comprise, in vivo, detecting a compound incorporated into the tumor-targeted bacterial vectors, detecting an infection caused by the bacterial vectors, or detecting an antigen present on the surface of the bacterial vectors after the bacterial vectors have been administered to a subject.
  • the marker gene When a marker gene is delivered to a solid tumor, the marker gene can be detected directly in the tumor, by the use of a labeled moiety that interacts with the marker gene product, or by the use of a labeled marker substrate. More particularly, in one embodiment, the invention encompasses use of attenuated tumor-targeted bacteria or bacterial vectors, such as, e.g., Salmonella, as a vector for the delivery of a marker gene to an appropriate site of action, e.g., the site of a solid tumor. If the marker gene product itself is detectable by non- invasive methods, the marker gene product itself can be detected directly.
  • attenuated tumor-targeted bacteria or bacterial vectors such as, e.g., Salmonella
  • a labeled moiety that interacts with the marker gene product is detected, or a labeled marker substrate is used and a labeled marker metabolite is detected.
  • a tumor can thus be localized or detected by scanning a subject to detect the marker gene product, a labeled complex comprising the marker gene product and its interacting moiety, or a labeled marker metabolite, respectively, thereby imaging the tumor.
  • the attenuated tumor-targeted bacteria of the invention are facultative aerobes or facultative anaerobes which are modified to encode the marker gene.
  • the marker gene encodes a fluorescent protein and the marker gene product is detected directly.
  • the marker gene encodes strepatividin and is detected by administering labeled biotin, in which case the marker gene is indirectly detected by detection of the labeled biotin.
  • the marker gene product is a heterologous protein that is immunologically detectable, and the marker gene product is detected by administration ofa labeled antibody.
  • the marker gene is HSVl-tk or VZV-tk and the marker substrate is a labeled 2'-fluoro-nucleoside analogue.
  • the labeled marker substrate is a labeled 2'-fluoro-5-iodo-l-beta-D-arabinofuranosyl-uracil (FIAU).
  • the labeled marker substrate is a radiolabeled
  • 2'-fluoro-nucleoside analogues were chosen for use in the subject invention because they are selectively phosphorylated by HSV1-TK or VZV-TK, can be radiolabeled with appropriate radionuclides for imaging e.g., with SPECT or PET and are resistant to metabolic degradation in vivo (see, for example, Abrams et al, 1985, Int. J. Appl. Radiat. Isot. 36:
  • tumor imaging methods of the invention do not require the use of a marker gene.
  • tumor imaging comprises administering a labeled compound to the subject, which labeled compound is preferentially inco ⁇ orated into the bacteria, and detecting the labeled
  • the tumor is imaged by scanning to image an infection caused by the bacteria at tumor site.
  • the present invention also provides a non-invasive method to monitor the course of disease or effectiveness of tumor treatment. Repeated imaging of tumor tissue during the course of treatment allows direct observation 0 of changes in size. In addition to detection and monitoring, the present invention also provides methods for therapy to inhibit tumor growth or reduce the tumor size.
  • HSV1-TK and VZV-TK metabolize marker substrates as well as pro-drug substrates, and may be genetically engineered to prefer a specific substrate.
  • the present invention is based, in part, on the surprising discovery that the 5 metabolic activities of tumor-targeted bacteria are of sufficient magnitude and duration while in tumor cells or the tumor environment to be able to express, incorporate, trap, bind or otherwise hold within the tumor region, sufficient amounts of a detectably labeled agent to allow non-invasive detection of the tumor.
  • a detectably labeled agent for example, expression of a ⁇ - lactamase:HSVl-TK fusion protein with a periplasmic localization signal has been found to 0 cause the trapping effect of phosphorylation of a marker substrate (e.g., FIAU) and permits signal localization for monitoring.
  • a marker substrate e.g., FIAU
  • the marker gene product of a tumor-targeted bacterial vector is retained in the cytosol and periplasm of the vector, where such vector is used for imaging or monitoring a tumor.
  • the marker gene product of a tumor-targeted 5 bacterial vector is retained in the cytosol of the vector, where such vector is used for imaging or monitoring a tumor.
  • the present method employing genetically engineered tumor-targeted bacteria, such as Salmonella can be used to detect solid tumors which are at least 2 times, more preferably 4-5 times, and most preferably 10 times smaller than can be detected using current methods including but not limited to x-ray and CT scanning.
  • the method can be used to detect and/or monitor tumor metastases as well as primary solid tumors.
  • bacteria such as Salmonella
  • bacteria can accept multiple genes either by insertion into the bacterial chromosome or by transformation with multiple plasmids.
  • This invention provides a method of detecting a tumor by imaging in a subject comprising: (a) administering to the subject a tumor-targeted bacteria containing a marker gene, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment and the marker gene is expressed in the tumor-targeted bacteria, thereby generating a marker gene product; and (b) scanning the subject to detect the marker gene product, thereby imaging the tumor in the subject.
  • the marker gene is detected directly. In other embodiment, the marker gene is detected indirectly.
  • indirect detection further comprises administering to the subject a labeled marker-binding moiety after step (a), wherein the marker gene product of step (a) binds to the labeled marker-binding moiety; and wherein said scanning is after clearance of residual labeled marker-binding moiety not bound to the marker gene product, thereby detecting the labeled marker-binding moiety localized to the tumor and imaging the tumor in the subject.
  • indirect detection further comprises administering to the subject a labeled marker substrate after step (a), wherein the tumor-targeted bacteria expressing the marker gene product of step (a) metabolizes the labeled marker substrate to produce a labeled marker metabolite; and wherein said scanning is after clearance of residual marker substrate not metabolized by the marker gene product, thereby detecting the labeled marker metabolite localized to the tumor and imaging the tumor in the subject.
  • This invention provides a method of detecting a tumor by imaging in a subject comprising: (a) administering to the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor cells and or the tumor environment; (b) administering to the subject a labeled compound that is preferentially incorporated into the tumor-targeted bacteria; and (c) scanning the subject to detect the labeled compound, thereby detecting the tumor-targeted bacteria and imaging the tumor in the subject.
  • the tumor-targeted bacteria is a mutant having an enhanced preference to incorporate the labeled compound.
  • the bacteria is a mutant at the asd locus and the marker compound is diaminopimelic acid.
  • the present invention further provides methods of imaging a tumor in a subject comprising (a) administering to the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment, and (b) scanning to image an infection caused by the bacteria, thereby detecting and imaging the tumor in the subject.
  • imaging the infection comprises detecting sequestered polymo ⁇ honuclear neutrophils at the site of infection, for example by using a labeled antibody that detects an antigen, such as CD 15, present on the polymo ⁇ honuclear neutrophils, or by using a labeled chemotactic peptide analog that binds to a receptor present on the polymo ⁇ honuclear neutrophils.
  • the present invention provides methods of imaging a tumor in a subject comprising: (a) administering to the subject a tumor-targeted bacteria, wherein the tumor- targeted bacteria targets the tumor cells and/or the tumor; (b) administering a labeled antibody to the subject, wherein the antibody binds to an antigen present on the surface of the tumor-targeted bacteria; and (c) scanning the subject to detect the labeled antibody, thereby imaging the tumor in the subject.
  • the antigen present on the surface of the tumor-targeted bacteria is an O-antigen, an H-antigen, or an outer membrane protein.
  • the present invention provides a method of monitoring a tumor during the course ofa disease and/or treatment in a subject comprising: (a) administering to the subject a tumor-targeted microorganism containing a marker gene, wherein the tumor- targeted microorganism targets the tumor cells and/or the tumor environment and the marker gene is expressed in the tumor-targeted microorganism, thereby generating a marker gene product; (b) scanning the subject to detect the marker gene product, thereby imaging the tumor in the subject; and (c) repeating steps (a) and (b) as needed during the course of the disease in the subject.
  • the present invention provides a method of monitoring a tumor during the course of a disease and/or treatment in a subject comprising: (a) administering to the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment; (b) administering to the subject a labeled compound that is preferentially inco ⁇ orated into the tumor-targeted bacteria;(c) scanning the subject to detect the labeled compound, thereby detecting the tumor-targeted bacteria and imaging the tumor in the subject; and (d) repeating steps (a) through (c) as needed during the course of the disease in the subject.
  • the present invention provides a method of monitoring a tumor during the course ofa disease in a subject comprising (a) administering to the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment; (b) scanning to image an infection caused by the bacteria, thereby detecting and imaging the tumor in the subject; and (c) repeating steps (a) and (b) as needed during the course of the disease in the subject.
  • the present invention provides a method of monitoring a tumor during the course of a disease in a subject comprising (a) administering to the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor; (b) administering a labeled antibody to the subject, wherein the antibody binds to an antigen present on the surface of the tumor-targeted bacteria; (c) scanning the subject to detect the labeled antibody, thereby imaging the tumor in the subject; and (d) repeating steps (a) through (c) as needed during the course of the disease in the subject.
  • this invention provides methods of simultaneously imaging and treating a tumor in a subject.
  • simultaneous imaging and treatment comprises administering tumor-targeted bacteria expressing a marker gene and a suicide gene capable of converting a prodrug into a cytotoxic drug, together with a labeled marker substrate.
  • a single tumor-targeted bacterial vector may contain more than one bacterial expression construct, wherein at least one construct is used for imaging the tumor, as above, and at least one construct is used for treating the tumor.
  • a population of at least two tumor-targeted bacterial vectors is administered, wherein one vector is for imaging as above, and another vector is for therapeutic treatment.
  • the invention provides a non-invasive, clinically applicable method for imaging tumors which can be implemented using existing imaging techniques to monitor and evaluate in vivo cancer treatments in human subjects.
  • compositions for carrying out the noninvasive imaging (and optionally treatment) methods of the invention comprising a population of the tumor-targeted bacteria of the invention suitable for noninvasive tumor imaging.
  • a composition of the invention comprises a population of tumor-targeted bacteria which harbor a recombinant streptavidin gene operably linked to a promoter.
  • the promoter is preferentially active at the tumor site or in the environment.
  • the tumor- targeted bacteria is a partial asd mutant with an enhanced preference to inco ⁇ orate labeled DAP.
  • the invention provides pharmaceutical compositions comprising a population of the tumor-targeted bacteria of the invention suitable for non-invasive tumor imaging, and a pharmaceutically acceptable carrier.
  • the invention provides kits comprising compositions of the invention for carrying out the non-invasive detection methods of the invention, as well as for carrying out the non-invasive detection and treatment methods of the invention.
  • the invention provides kits comprising in one or more containers (a) a purified population of tumor-targeted bacteria and (b) a detectably labeled molecule.
  • the bacteria contain a marker gene operably linked to a promoter.
  • the labeled molecule is a labeled moiety which binds to the marker gene product.
  • the labeled molecule is labeled biotin and the marker gene is streptavidin.
  • the labeled molecule is a labeled substrate of the marker gene product.
  • the labeled molecule is a labeled compound which is preferentially inco ⁇ orated into the tumor-targeted bacteria.
  • the tumor- targeted bacteria is a mutant having an enhanced preference to inco ⁇ orate the labeled compound.
  • the bacteria is mutant at the asd locus and the labeled compound is DAP.
  • the labeled molecule is a labeled antibody that detects an antigen present on polymo ⁇ honuclear neutrophils or on the surface of the tumor-targeted bacteria, or a labeled chemotactic peptide analog that binds to a receptor present on polymo ⁇ honuclear neutrophils.
  • Facultative aerobic and facultative anaerobic bacteria useful for the methods and compositions of the present invention include but are not limited to Escherichia coli (including but not limited to pathogenic (e.g., entero-invasive or uropathogenic) Escherichia coli), Salmonella spp., Shigella spp., Streptococcus spp., Yersinia enterocolitica, Listeria monocytogenies, and Mycoplasma hominis.
  • Escherichia coli including but not limited to pathogenic (e.g., entero-invasive or uropathogenic) Escherichia coli), Salmonella spp., Shigella spp., Streptococcus spp., Yersinia enterocolitica, Listeria monocytogenies, and Mycoplasma hominis.
  • the bacterial vector is an attenuated strain of Salmonella genetically engineered to express an altered Lipid A which reduces the virulence of the strain, and genetically engineered to express a gene product which aids in preventing the growth of a solid tumor, which gene product is under the control of an irradiation-inducible promoter.
  • solid tumors include, but are not limited to, sarcomas, carcinomas, lymphomas and other solid tumor cancers, such as renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors and tumors of the central nervous system, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
  • sarcomas such as renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors and tumors of the central nervous system, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
  • Salmonella spp. encompasses all Salmonella species, including: Salmonella typhi, Salmonella choleraesuis, and Salmonella enteritidis. Serotypes of Salmonella are also encompassed herein, for example, typhimurium, a subgroup of Salmonella enteritidis, commonly referred to as Salmonella typhimurium.
  • Attenuation Attenuation is a modification so that a bacterium or bacterial vector is less pathogenic. The end result of attenuation is that the risk of toxicity as well as other side-effects is decreased, when the bacterium or bacterial vector is administered to the patient.
  • Virulence Virulence is a relative term describing the general ability to cause disease, including the ability to kill normal cells or the ability to elicit septic shock (see specific definition below).
  • Septic shock is a state of internal organ failure due to a complex cytokine cascade, initiated by TNF ⁇ .
  • the relative ability of a bacterium or bacterial vector to elicit TNF ⁇ is used as one measure to indicate its relative ability to induce septic shock.
  • Gene product refers to any molecule capable of being encoded by a nucleic acid, including but not limited to, a protein or another nucleic acid, e.g., DNA, RNA dsRNAi, ribozyme, DNAzyme, etc.
  • the nucleic acid which encodes for the gene product of interest is not limited to a naturally occurring full-length "gene" having non-coding regulatory elements.
  • Tumor-targeted is defined as the ability to distinguish between a cancerous target cell or tissue and the non-cancerous counte ⁇ art cell or tissue so that tumor-targeted bacteria, such as Salmonella preferentially attach to, infect and/or remain viable in the cancerous target cell or the tumor environment.
  • Chelating agent sensitivity is defined as the effective concentration at which bacteria proliferation is affected, or the concentration at which the viability of bacteria, as determined by recoverable colony forming units (c.f.u.), is reduced.
  • an Omp-like protein includes any bacterial outer membrane protein, or portion thereof (e.g., signal sequence, leader sequence, periplasmic region, transmembrane domain, multiple transmembrane domains, or combinations thereof).
  • the Omp-like protein is at least a portion of OmpA, OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, a porin-like protein, PhoA, PhoE, lamB, ⁇ -lactamase, an enterotoxin, protein A, endoglucanase, peptidoglycan-associated lipoprotein (PAL), FepA, FhuA, NmpA, NmpB, NmpC, or a major outer membrane lipoprotein (such as LPP), etc.
  • PAL peptidoglycan-associated lipoprotein
  • a release factor includes any protein, or functional portion thereof which enhances release of bacterial components.
  • a release factor is a bacteriocin release protein.
  • Release factors include, but are not limited to, the bacteriocin release protein (BRP) encoded by the cloacin D13 plasmid, the BRPs encoded by the colicin E1-E9 plasmids, or BRPs encoded by the colicin A, N or D plasmids.
  • BRP bacteriocin release protein
  • Direct detection of a marker gene product: As used herein, direct detection of a marker gene product indicates that the signal detected is a function of the gene product itself.
  • Indirect detection of a marker gene product:
  • indirect detection of a marker gene products entails that detection ofa label moiety that co-localizes with the marker gene product, for example by binding to the marker gene product or as a result of being a metabolite of a reaction catalyzed by the marker gene product.
  • composition of the invention minimally comprises a population of tumor-targeted bacteria suitable for use in the non- invasive tumor imaging or tumor imaging and treatment methods described herein.
  • a composition of the invention optionally further comprises a population of tumor-targeted bacteria suitable for tumor therapy.
  • a pharmaceutical of the invention or a pharmaceutical composition of the invention refers to a composition of the invention that comprises a pharmaceutically acceptable carrier.
  • FIG. 1 demonstrates the ability of the imaging system to detect [ 14 C]-FIAU in tumor-bearing mice pretreated with Salmonella expressing HSV-TK .
  • a tumor- implanted mouse was treated with VNP20009 expressing the plasmid p5-3 from Pawelek et al (1997, Cancer Res., 57:4537-4599) four days prior to i.v. injection of labeled marker substrate ([ 14 C]-FIAU).
  • tumor tissue samples were removed from sacrificed animals and subjected to QAR. Histology of the tumor and surrounding tissue is shown on the left, the digital autoradiogram on the right and the merged image in the center.
  • [ 14 C]- FIAU can be clearly detected within the tumor, with little or no accumulation in the surrounding tissue. See text in Section 6 for details.
  • FIG. 2 demonstrates the tumor-targeting selectivity of VNP20009 expressing the plasmid p5-3.
  • Liver tissue was collected from mice treated as described in the text in Section 6 and subjected to QAR. Histology of the tissue is shown on the left and the digital autoradiogram on the right. Little or no labeled marker substrate can be detected.
  • FIG. 3 A shows fluorescence of an untreated tumor and figure 3B shows fluorescence observed in tumors following treatment with GFP-containing Salmonella strain VNP20009.
  • FIG. 4 shows the effect of administering non-Salmonella tumor-targeted bacteria on tumor growth, as a measure of tumor volume versus time following administration of the bacteria.
  • the present invention provides novel compositions and non-invasive methods of detecting tumors which exploit the tumor-specificity of tumor- targeting bacteria, as described below. Because the tumor targeting bacteria do not discriminate between different tumor types, the described compositions and methods can be used to simultaneously image multiple tumor types, in contrast to the tumor imaging reagents currently in use, which are largely specific to a single tumor type or tumor subtype.
  • the present invention provides a method of detecting a tumor by imaging in a subject comprising: (a) administering to the subject a tumor-targeted bacteria containing a marker gene, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment and the marker gene is expressed in the tumor-targeted bacteria, thereby generating a marker gene product; and (b) scanning the subject to detect the marker gene product, thereby imaging the tumor in the subject.
  • the marker gene is detected directly. In other embodiment, the marker gene is detected indirectly.
  • indirect detection further comprises administering to the subject a labeled marker-binding moiety after step (a), wherein the marker gene product of step (a) binds to the labeled marker-binding moiety; and wherein said scanning is after clearance of residual labeled marker-binding moiety not bound to the marker gene product, thereby detecting the labeled marker-binding moiety localized to the tumor and imaging the tumor in the subject.
  • indirect detection further comprises administering to the subject a labeled marker substrate after step (a), wherein the tumor-targeted bacteria expressing the marker gene product of step (a) metabolizes the labeled marker substrate to produce a labeled marker metabolite; and wherein said scanning is after clearance of residual marker substrate not metabolized by the marker gene product, thereby detecting the labeled marker metabolite localized to the tumor and imaging the tumor in the subject.
  • the present invention provides a method of monitoring a tumor during the course ofa disease and/or treatment in a subject comprising (a) administering to the subject a tumor-targeted microorganism containing a marker gene, wherein the tumor- targeted microorganism targets the tumor cells and/or the tumor environment and the marker gene is expressed in the tumor-targeted microorganism, thereby generating a marker gene product; (b) scanning the subject to detect the marker gene product, thereby imaging the tumor in the subject; and (c) repeating steps (a) and (b) as needed during the course of the disease in the subject.
  • the invention provides a non-invasive, clinically applicable method for imaging tumors which can be implemented using existing imaging techniques to monitor and evaluate in vivo cancer treatments in human subjects.
  • the imaging and monitoring methods of the present invention can use tumor- targeted bacterial vectors as previously described in WO 96/40238 and WO 99/13053, which are inco ⁇ orated-by-reference herein in their entirety, for imaging solid tumor cancers, such as sarcomas, carcinomas, lymphomas or other solid tumor cancers, for example, renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors and tumors of the central nervous system, including, but not limited to, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, melanoma.
  • solid tumor cancers such as sarcomas, carcinomas, lymphomas or other solid tumor cancers, for example, renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors and tumors of the central nervous system, including, but not limited to
  • novel imaging, monitoring, detecting and treatment methods of the present invention can be used with existing nuclear medicine instrumentation as previously described in United States Patent No. 5,703,056 entitled “Non-Invasive Imaging of Gene Transfer” issued December 30, 1997, which is inco ⁇ orated-by-reference herein in its entirety.
  • the present invention provides a means of imaging or monitoring tumors by delivering high levels of the products of marker gene(s), either alone or together with therapeutic gene(s), using various tumor-targeted strains of bacteria (preferably modified and/or attenuated) which selectively accumulate at or within tumors while expressing the marker gene(s) and/or therapeutic genes.
  • the vectors When administered to a subject, e.g., an animal for veterinary use or to a human for clinical use, the vectors can be used alone or may be combined with any physiological carrier such as water, an aqueous solution, normal saline, or other physiologically acceptable excipient. In general, the dosage would range from about 1 to lxlO 9 c.f.u./kg, preferably about 1 to lxlO 2 c.f.u/kg.
  • the vectors of the present invention can be administered by a number of routes, including but not limited to: orally, topically, injection including, but limited to intravenously, intraperitoneally, subcutaneously, intramuscularly, intrathecally, intratumorally, i.e., direct injection into the tumor, etc.
  • the imaging methods of the invention are used to monitor the efficiency of vector targeting prior to administration of a suicide substrate.
  • visualization of a bacterial vector at a tumor site serves the pu ⁇ ose of identifying the tumor site and provides an indication of whether sufficient vector targeting to a tumor has indeed taken place.
  • a single tumor-targeted bacterial vector contains both a marker gene and a suicide gene.
  • the bacterial vector harbors a mutation that increases inco ⁇ oration of the labeled compound into the bacteria and contains a suicide gene.
  • two tumor-targeted bacterial vectors are administered, each vector containing either a marker gene or a suicide gene.
  • administration of a labeled marker substrate, compound or binding moiety for imaging is performed. This permits assessment of the specificity of the vector for the tumor by the practitioner. Further, the amount of vector targeted to the tumor can be determined by correlating the intensity of the image with the amount administered. If it is determined that sufficient amount of vector has been delivered with sufficient specificity, suicide substrate can then be administered to treat the tumor. For further details relating to this embodiment, see Section 5.4., infra.
  • the invention provides methods for imaging or monitoring a tumor while simultaneously treating the tumor using one or more of the tumor- targeting bacterial vectors described and referenced herein.
  • a subject may be diagnosed with a solid tumor cancer by any method known in the art.
  • the vector, i.e., the tumor-targeted bacteria, used in simultaneous imaging or monitoring and treatment may be isolated using the methods of the present invention with target cell lines or using model tumors in mice.
  • a biopsy of tumor cells is used in a selection assay for isolating a vector which is super-infective and tumor-specific for the tumor of the subject.
  • the vector used is genetically modified to express, for example, a suicide gene or to delete virulence factors, or both, as described herein.
  • the isolated vector may be analyzed for sensitivity to antibiotics to insure the eradication of the vector from the patient's body after successful treatment or if the patient experiences complications due to the administration of the vector.
  • the methods of in vivo imaging and treatment are combined, wherein the imaging and treatment vectors are separate tumor- targeted bacterial vectors.
  • each vector is capable of expressing a mutant gene selected for substrate specificity, wherein the imaging mutant gene is retained in the cytosol of a first tumor-targeted bacterial vector and the treatment mutant gene is in the periplasm of a second tumor-targeted bacterial vector.
  • a single vector contains both a suicide gene and an imaging gene, where the suicide gene and imaging gene are carried on different plasmids or are inco ⁇ orated into bacterial DNA.
  • different mutants of HSVl-TK or VZV-TK serve as both the suicide gene and the imaging gene in the same tumor-targeted bacterial vector.
  • the suicide gene and the imaging gene are carried in two different vectors administered simultaneously to a subject, preferably the two different vectors are produced from the same bacterial strain and exhibit the same tumor- specificity.
  • the two different vectors may also be administered sequentially within a short time of one another, and in any order.
  • the imaging instrumentation such as ⁇ camera or single-photon emission computed tomography (SPECT), PET, MRI, etc.
  • SPECT single-photon emission computed tomography
  • MRI magnetic resonance imaging
  • relevant labeled marker substrates as described in United States Patent No. 5,703,056 entitled “Non-Invasive Imaging of Gene Transfer” issued December 30, 1997 to Blasberg and Tjuvajev, which patent is inco ⁇ orated-by-reference herein in its entirety, can be used in the novel imaging, monitoring, detecting and treatment methods of the present invention.
  • tumor-targeted bacteria are engineered to become bacterial vectors of the invention to express one or more marker genes (optionally, including one or more therapeutic genes) suitable for imaging or monitoring (or optionally, including treating) a tumor.
  • marker genes optionally, including one or more therapeutic genes
  • tumor-targeted bacteria are administered in conjunction with a labeled compound that is inco ⁇ orated into the bacteria for tumor imaging, monitoring, and, optionally, treatment. It will be understood by one skilled in the art that imaging a tumor using the methods of the invention is equivalent to tumor detection.
  • a marker gene is a gene coding for an enzyme that may be expressed in the tumor-targeted bacterial vector and that catalyzes a reaction with a labeled marker substrate, resulting in the accumulation of a detectable marker within the vector.
  • the he ⁇ es simplex virus thymidine kinase gene (HSVl-tk) and the varicella zoster virus thymidine kinase gene (VZV-tk) are preferred "marker genes" and a labeled 2- 'fluoro-nucleoside analogue is their preferred marker substrate.
  • FIAU 2'-fluoro-5-iodo-l- ⁇ -D-arabinofuranosyl- uracil
  • HSVl-TK HSV1 thymidine kinase
  • VZV-TK varicella zoster virus thymidine kinase
  • a preferred marker substrate is
  • the HSVl-TK- or VZV-TK-expressing vector is administered to the subject concu ⁇ ently with a pro-drug (e.g. ganciclovir or acyclovir).
  • the pro-drug is phosphorylated in the periplasm of the microorganism which is freely permeable to nucleotide triphosphates.
  • the phosphorylated ganciclovir or acycolvir toxic false DNA precursors, readily pass out of the periplasm of the microorganism and into the cytoplasm and nucleus of the host cell where they inco ⁇ orate into host cell DNA, thereby causing the death of the host cell.
  • the marker gene product of a tumor- targeted bacterial vector is retained in the cytosol of the vector, where such vector is used for imaging or monitoring a tumor.
  • Marker gene product retention in the cytosol ofa tumor-targeted bacterial vector aids in the imaging or monitoring of particularly small, difficult-to-detect tumors (i.e. less than one gram) by maximizing marker gene product accumulation at the tumor site.
  • tumor-targeted bacterial vector expression constructs are preferably designed such that a marker gene product (e.g., a thymidine kinase enzyme, a glucokinase enzyme, or a cytochrome P-450 enzyme) is not secreted from the bacterial vector.
  • the marker gene product of a tumor-targeted bacterial vector is retained in the cytosol and periplasm of the vector, where such vector is used for imaging or monitoring a tumor. In another embodiment, the marker gene product of a tumor-targeted bacterial vector is retained in the periplasm of the vector, where such vector is used for imaging or monitoring a tumor.
  • a vector containing a marker gene for imaging a tumor is under the specific regulatory control of certain types of promoters.
  • These promoters may be either constitutive, in which the genes are continually expressed, inducible, in which the genes are expressed only upon the presence of an inducer molecule(s) or cell-type or tumor-environment specific control, in which genes are expressed only or preferentially in certain cell types or only in the tumor-environment, respectively.
  • the expression of the gene is controlled by a bacterial promoter which may be activated by secretions or other molecules specific to a given tumor.
  • the bacterial promoter is activated only by secretions or other molecules specific to a given tumor.
  • the detecting and monitoring compositions and/or methods of the present invention allow for the localization and identification of tumors for which whole body detection methods are unavailable or impractical.
  • the detecting and monitoring compositions and/or methods of the present invention further allow for the localization and identification of tumors smaller than those diagnosable by methods available prior to the invention.
  • prior methods are limited to detection of a tumor having at least one gram in mass. Accordingly, in one embodiment, a tumor having less than one gram mass is detected or monitored. In another embodiment, a tumor from 0.99 grams to 0.01 grams mass is detected or monitored. In yet another embodiment, a tumor from 0.9 grams to 0.1 grams mass is detected or monitored. In yet still another embodiment, a tumor from 0.8 grams to 0.2 grams mass is detected or monitored. In a preferred embodiment, a tumor of from about 0.05 to about 0.1 grams mass is detected or monitored (plus or minus twenty percent).
  • the imaging methods of the present invention are used to monitor the progression of a tumor over time.
  • one or more tumors are visualized according to the methods of the invention over time, thereby allowing detection of any changes in size, shape and/or location of tumors being monitored.
  • the efficacy of an anti-cancer treatment, such as chemotherapy is monitored by visualizing the tumor before, during and/or after treatment. Suppression of tumor growth or shrinkage or disappearance of tumors during monitoring is indicative ofa successful course of treatment.
  • the imaging methods of the invention are used to monitor the efficiency of vector targeting.
  • visualization serves the pu ⁇ ose of identifying the location of the vector to determine whether sufficient vector targeting to a tumor has taken place.
  • a single tumor-targeted bacterial vector contains both a marker gene and a therapeutic gene.
  • separate vectors each containing either a marker gene or a therapeutic gene are used.
  • administration of marker substrate and imaging is performed to assess the specificity of the vector for the tumor and the amount of vector targeted to the tumor. If it is determined that sufficient amount of vector has been delivered with sufficient specificity, suicide substrate can be administered to treat the tumor.
  • the marker gene and the suicide gene are the same gene, e.g., HSVl-TK or VZV-TK.
  • the suicide gene and the marker gene are mutants of the same gene selected for marker substrate specificity and suicide substrate specificity, respectively.
  • the different vectors, each containing either a suicide gene or a marker gene are produced from the same strain of bacteria so that they have similar or identical targeting characteristics.
  • a tumor-targeted bacterial vector used for in vivo imaging according to the methods of the invention may be attenuated such that, when administered to a subject, the vector is less toxic to the subject and easier to eradicate from the subject's system.
  • a vector is super-infective and specific for a target tumor.
  • such a vector is also sensitive to a broad range of antibiotics.
  • a tumor-targeted bacterial vector carrying a gene for imaging also carries a gene, such as a gene encoding a pro-drug converting enzyme, which is expressed and secreted by the vector in or near the target tumor.
  • a gene can be under the control of either constitutive, inducible or tumor cell-type or tumor-environment specific promoters.
  • such a gene is expressed and secreted only when a vector has invaded the cytoplasm of a target tumor cell, thereby limiting the effects due to expression of the gene to the target site of the tumor.
  • the marker gene expressed by the bacterial vector encodes a directly detectable marker gene product.
  • a marker gene can be a gene encoding a fluorescent protein, a bioluminescent protein, or a chemiluminescent protein.
  • the marker gene encodes a fluorescent molecule.
  • the fluorescent molecule is firefly luciferase.
  • the protein encoded by the marker gene is GFP from Aequorea victoria or a mutant thereof.
  • the GFP expressed in a tumor-targeted bacteria for tumor imaging according to the methods of the invention can be encoded by its naturally occurring coding sequence or by a coding sequence that has been modified for optimal bacterial codon usage. Mutations can be introduced into the coding sequence to produce GFP mutants with altered fluorescence wavelength or intensity or both.
  • the GFP mutant is a blue GFP.
  • the fluorescent protein is a yellow or red-orange emitter recently discovered in reef corals (Matz et al, 1999, Nature Biotechnol. 17:969-973). Whole-body imaging of GFP-expressing tumors and metastases has been reported by Yang et al (2000, Proc. Nat'l Acad. Sci. U S A 97:1206-11).
  • a marker gene product can be detected indirectly.
  • a marker gene product can be detected by administration of a labeled moiety that binds to the marker gene product.
  • binding of a labeled moiety to a marker gene product encompasses protein-protein interactions, protein-compound interactions, protein-peptide interactions, enzyme-substrate interactions, and chelating activity.
  • the labeled moiety can be a proteinaceous or non- proteinaceous molecule, e.g., a carbohydrate.
  • a proteinaceous marker-binding moiety can be a ligand, an antibody, or any other peptide or polypeptide that interacts specifically with, and has a high affinity to, the marker gene product.
  • the invention provides indirect methods of imaging a tumor in a subject comprising administering to the subject a tumor-targeted bacteria containing a marker gene, wherein the tumor-targeted bacteria targets the tumor cells and/or the tumor environment and the marker gene is expressed in the tumor-targeted bacteria, thereby generating a marker gene product; administering to the subject a labeled marker-binding moiety, wherein the marker gene product binds to the labeled marker- binding moiety; and scanning the subject to detect the marker gene product, wherein said scanning is after clearance of residual labeled marker-binding moiety not bound to the marker gene product, thereby detecting the labeled marker-binding moiety localized to the tumor and imaging the tumor in the subject.
  • the marker gene expressed by the bacterial vector is avidin or streptavidin. These proteins bind biotin with high affinity (Kendrew , ed, The Encyclopedia of Molecular Biology, 1994, Blackwell Science Ltd., pps. 80 and 1037). Streptavidin, a protein from Streptomyces avidinii, can chelate biotin as well as other compounds (e.g., boron, see Sano, 1999, Bioconjug Chem. 10:905- 911). The accumulation of chelated detectable agents, such as labeled biotin, can be detected by various methods and thus be used to facilitate the localization and monitoring of a tumor.
  • the marker gene expressed by the bacterial vector encodes an enzyme (e.g., bacterial lacZ protein or chloramphenicol acetyl transferase (CAT)).
  • An enzyme encoded by a marker can be detected by the administration of a labeled substrate analog.
  • the marker gene expressed by the bacterial vector encodes a receptor, or a protein comprising the ligand binding domain of a receptor, for example a fusion protein comprising the ligand-binding domain of the receptor and a protein which targets the ligand-binding domain to the outer bacterial membrane.
  • the receptor is a nicotinic acetylcholine receptor, which can be imaged by PET and SPECT using radiolabeled nicotine analogs.
  • halogenated 3-pyridyl ether compounds can be used for imaging the alpha 4 beta 2 nicotinic acetylcholine receptor (Gundisch, 2000, Cu ⁇ Pharm Des 6(11):1143-57).
  • the receptor is a dopamine or serotonin receptor.
  • the marker gene expressed by the bacterial vector is, or a protein comprising an immunologically detectable epitope or other binding moiety (e.g., myc, glutathione-S-transferase (GST), or hexahistidine).
  • an immunologically detectable epitope or other binding moiety e.g., myc, glutathione-S-transferase (GST), or hexahistidine.
  • GST glutathione-S-transferase
  • hexahistidine hexahistidine
  • the marker gene product When the marker gene product possesses catalytic activity, the gene product can be detected indirectly by virtue of its activity, for example by production of a detectable metabolite following a chemical modification of a substrate molecule. Accordingly, preferred marker genes to practice the present embodiments are enzymes.
  • the invention provides an indirect method of tumor imaging comprising administering to the subject a tumor-targeted bacterial vector containing a marker gene, wherein the tumor-targeted bacterial vector targets to the tumor and/or infects cells of the tumor, under conditions in which the marker gene is expressed in the tumor-targeted bacterial vector, thereby generating a marker gene product; administering to the subject a labeled marker substrate under conditions in which the labeled marker substrate is metabolized by the marker gene product to produce a labeled marker metabolite which is substantially retained in the tumor-targeted bacterial vector throughout a time-period sufficient for imaging the labeled marker metabolite; and scanning the subject to detect the labeled marker metabolite, thereby imaging and detecting a tumor in the subject.
  • the generation of the marker metabolite from the marker substrate entails the release of a quenched label from the substrate (see, e.g., Bell and Taylor-Robinson,
  • Suitable enzymes that can produce detectably labeled metabolites include but are not limited to malate dehydrogenase, staphylococcal nuclease, ⁇ -5-steroid isomerase, yeast alcohol dehydrogenase, ⁇ -glycerophosphate, dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, ⁇ -galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.
  • the tumor-targeted bacteria also express a permease or transporter which allows for enhanced uptake of the substrate of the marker gene product enzyme.
  • the tumor- targeted bacteria that express lacZ would also express lacY to increase lactose or galactose uptake.
  • suitable enzymes that can produce detectably labeled metabolites are proteases.
  • Suitable protease substrates for imaging are polymerized protease recognition sites bearing a quenched label, which have been called “repeating graft co-polymers" (Weissleder et al, 1999, Nature Biotechnology 17:375-378). Cleavage of the substrate by the recombinant protease expressed by the tumor-targeted vector at the tumor site releases the quenched label, i.e., generates a labeled metabolite, allowing imaging of the tumor. Synthesis of quenched substrates can be done according to the principles described in Weissleder et al. (1999, Nature Biotechnology 17:375-378).
  • the marker gene expressed by the vector is a wild type, natural mutant or genetically-engineered HSVl-TK or VZV-TK gene, and its substrate is a labeled nucleoside analog such as FIAU, ACV or GCV.
  • the nucleoside analog is a 2'-fluoro-nucleoside analogue such as FIAU, .
  • FIAU This accumulated, labeled FIAU is then imaged by scanning the subject using, for example, positron emission tomography, ⁇ camera or single-photon emission computed tomography.
  • the FIAU is phosphorylated in the cytosol of the microorganism instead of the periplasm to enhance accumulation and subsequent imaging. This may be accomplished using a vector genetically engineered to express TK in the cytosol of the microorganism.
  • the labeled 2'-fluoro-nucleoside analogue is 5-[ 123 I]-, 5-[ 124 I]- or 5-[ 131 I]-2'-fluoro-5-iodo-l- ⁇ -D- arabinofuranosyl- uracil; 5-[ 18 F]-2'-fluoro-5-fluoro-l- ⁇ -D- arabinofuranosyl- uracil; 2-[ ⁇ C]- or 5-([ ⁇ C]-methy)-2'-fluoro-5-methyl-l- ⁇ -D-arabinofuranosyl- uracil; 2-[ u C]- or 5-([ n C]- ethyl)-2'-fluoro-5-ethyl-l- ⁇ -D-arabinofuranosyl- uracil; 5-(2-[ 18 F]-ethyl)-2'-fluoro-5-(2- fluoro-
  • the marker gene expressed by the vector is wild type, natural mutant or genetically-engineered yeast glucokinase gene, and its substrate is a labeled 3-O-methyl glucose.
  • the labeled 3-O-methyl glucose is [ U C]- or [ 18 F]-3-O-methyl glucose.
  • the marker gene expressed by the vector is wild type, natural mutant or genetically-engineered cytochrome P-450 Bl gene, and its substrate is a labeled imidazole substrate.
  • the labeled imidazole substrate is 2-[ ⁇ C]-misonidazole, 2-[ n C]-metronidazole or 3-[ 18 F]- fluoromisonidazole.
  • the invention also encompasses the detection of a tumor by administering to the subject in which tumor detection is desired tumor-targeted bacteria.
  • the tumor targeted bacteria is then detected by administering to the subject a labeled compound which is inco ⁇ orated or localized to the bacteria itself.
  • the labeled compound can be chosen to be inco ⁇ orated into the bacterial cell wall.
  • the labeled compound is labeled with one of the labels described in Section 5.3, infra and detected as described.
  • the labeled compound is labeled diaminopimelic acid (DAP), as described below.
  • the labeled compound is a labeled glucose or glucose analog which, because of the higher proliferation rate of the bacteria (as well as the tumor cells) relative to normal mammalian cells, will be preferentially inco ⁇ orated into the dividing bacteria and tumor cells. This improves on the current imaging systems in which labeled glucose analogs are used to image tumors by enhancing the specific signal and reducing the non-specific signal, allowing the detection of smaller tumors, including metastases.
  • the labeled glucose analog is 18 F- fluorodeoxyglucose ( I8 F-FDG), which can be detected by PET scanning.
  • the bacteria's metabolism is manipulated to increase its inco ⁇ oration of the labeled compound.
  • the bacteria has an auxotrophic mutation which enhances its uptake of the labeled compound.
  • such a mutation further compromises its ability to live independently of the compound, such that its viability is largely dependent on the administration of the compound. Following administration of the compound and imaging the bacteria (and, indirectly, the tumor(s)), such bacteria are gradually cleared from the system.
  • the tumor-targeted bacteria is mutant for the asd locus, which makes the bacteria more susceptible to lysis due to compromised cell wall integrity. Rescue of this mutant can be achieved by exogenously providing DAP(Galan et al, 1990, Gene 94:24-35).
  • DAP labeled DAP
  • the DAP becomes inco ⁇ orated into the cell wall. Therefore, administering labeled DAP to a subject to whom asd tumor-targeting bacteria have been administered enhances the inco ⁇ oration of DAP into the cells walls of the bacteria, resulting in an enhanced signal for imaging relative to the use of non-asd tumor-targeting bacteria.
  • the asd mutation is a hypomo ⁇ hic but not a null mutation, so that the bacteria can survive administration and targeting to the tumor prior to administration of the labeled DAP.
  • a tumor is detected by detecting an infection (e.g., the immune response) caused by administering a composition of the invention to a subject.
  • an infection e.g., the immune response
  • tumor-targeted bacteria are administered to a subject and the subject scanned to detect an infection caused by the bacteria at the tumor site.
  • the infection, and therefore the tumor is detected indirectly by detecting the presence of an antigen that is highly or specifically expressed at the infection site.
  • the antigen detected is a neutrophil-specific antigen.
  • Polymo ⁇ honuclear neutrophils are sequestered at infection sites and labeled monoclonal neutrophil-specific antigens have been successful to diagnose infections.
  • the neutrophil-specific antigen is CD15.
  • Tc-labeled anti-CD 15 monoclonal antibody is commercially available under the trade name LeuTech (Palatin Technologies, Inc.), which has been used to detect appendicitis (Kipper et al, 2000, J Nucl Med 41(3):449-55).
  • the infection, and therefore the tumor is detected indirectly by using a labeled chemotactic peptide analogs which bind to leukocyte receptors (see, e.g., Fischman et al, 1994, Semin Nucl Med).
  • the labeled chemotactic peptide analog is labeled N-formyl-methionyl-leucyl-phenylalanine (ForMLF).
  • the labeled chemotactic peptide analog is labeled neutrophil peptide- 1. "Tc-labeled neutrophil peptide- 1 has been used to detect bacterial infections (Welling et al, 1999, J Nucl Med 40(12):2073-80).
  • a tumor is detected by detecting a surface antigen expressed by the tumor-targeted bacteria.
  • tumor-targeted bacteria are administered to a subject.
  • a labeled antibody which binds to an antigen present on the surface of the tumor-targeted bacteria is then administered, and the subject scanned to 10 detect the label. Therefore the tumor is detected indirectly by detecting the presence of an antigen that is expressed on the bacterial surface.
  • the surface antigen detected by the antibody is an O- antigen (an abbreviation for O-polysaccharide antigen).
  • the surface antigen detected by the antibody is an H-antigen (H-antigens are flagellar antigens).
  • the surface antigen detected by the antibody is an outer membrane protein.
  • the present invention provides non-invasive methods of imaging tumors in
  • labels that can be used are radioisotopes (such as 3 Hydrogen, "Carbon, '"Carbon, 13 Nitrogen, 18 Flourine, 12 Iodine, 12 Iodine, 125 Iodine, 131 Iodine, 11 'indium, 64 Copper, 67 Copper, 43 Scandium, 44 Scandium, 46 Scandium, 47 Scandium,
  • radioisotopes such as 3 Hydrogen, "Carbon, '"Carbon, 13 Nitrogen, 18 Flourine, 12 Iodine, 12 Iodine, 125 Iodine, 131 Iodine, 11 'indium, 64 Copper, 67 Copper, 43 Scandium, 44 Scandium, 46 Scandium, 47 Scandium,
  • Fluorophores can also be used as labels for marker detection.
  • examples of such compounds are rhodamine, fluorescein, Cy5, Cy3, succinimidyl 6-(N-(7-nitrobenz-2-oxa-l, 3-diazol-4-yl)amino)hexanoate (also known as NBT), R- phycoerythrin, allophycocyanin, 6-((7-amino-4-methylcoumarin- 3-acetyl)amino)hexanoic
  • Label detection can be performed with a whole-body optical fluorescence imaging system (Yang et al, 2000, Proc. Nat'l Acad. Sci. U S A 97:1206-11).
  • the label is chelated or quenched until it reaches the tumor site, where the label is released.
  • fluorophores Weissleder et al
  • the present invention further provides methods by which the signal of the label or the marker gene product to be imaged can be enhanced. Such methods encompass enhancing the specific signal, reducing the background or non-specific signal, or both.
  • the invention provides a method further comprising 0 waiting a time-period after administration of the labeled compound, substrate or binding moiety sufficient to allow at least 67% of non-specific label to clear from the subject.
  • the invention provides a method further comprising waiting a time-period after administration of the labeled compound, substrate or binding moiety sufficient to allow at least 80% of non-specific label to clear from the subject.
  • the invention provides a method further comprising waiting a time-period after administration of the labeled compound, substrate or binding moiety sufficient to allow at least 90% of non-specific label to clear from the subject.
  • non-specific label refers to labeled (i) compound, (ii) substrate or (iii) binding moiety that (i) has not been inco ⁇ orated into the bacteria, (ii) has not been metabolized by the marker 0 gene product, or (iii) is not bound to the marker gene product, respectively.
  • the label in a labeled substrate is quenched or chelated for release at a tumor site upon production of a labeled metabolite.
  • the labeled substrate is undetectable or generates a reduced signal until the label is activated, thereby reducing the non-specific signal that could be generated from non- 5 specific binding or poor clearance of the substrate.
  • the specific signal is enhanced by maximizing the interaction of the marker gene product with the labeled compound, substrate or binding moiety.
  • the marker gene product is brought into the vicinity of the labeled compound, substrate or binding moiety, for example by promoting its release into the extracellular environment.
  • the labeled compound, substrate or binding moiety is brought into the vicinity of the marker gene product, for example by promoting its uptake into or retention in the bacterial vector.
  • the attenuated tumor-targeted bacterial vectors of the invention which express at least one marker gene product, also express at least molecule which functions to permeabilize the bacteria cell membrane(s) or enhance the release of intracellular components into the extracellular environment, e.g. at the tumor site, thereby enhancing the delivery of the marker gene product(s).
  • release factors Such molecules which permeabilize the bacterial cell or enhance release are designated as "release factors”.
  • the release factor expressed by the bacterial vector of the invention may be endogenous to the modified attenuated tumor-targeted bacteria or it may exogenous (e.g., encoded by a nucleic acid that is not native to the attenuated tumor-targeted bacteria).
  • a release factor may be encoded by a nucleic acid comprising a plasmid, or by a nucleic acid which is integrated into the genome of the attenuated tumor-targeted bacteria.
  • a release factor may be encoded by the same nucleic acid or plasmid that encodes the marker gene of interest, or by a separate nucleic acid or plasmid..
  • such a factor is one of the Bacteriocin Release Proteins, or BRPs (herein refe ⁇ ed to in the generic as BRP).
  • BRP Bacteriocin Release Proteins
  • the BRP employed in the invention can originate from any source known in the art.
  • BRP proteins include, but are not limited to, the BRP from the cloacin D13 plasmid, the BRP from one of the colicin E1-E9 plasmids, and BRP from colicin A, N or D plasmids.
  • the BRP is of cloacin D13 (pCloDF13 BRP).
  • the enhanced release system comprises overexpression of a porin protein; see e.g., Sugawara and Nikaido, 1992, J. Biol. Chem.267:2507-l l).
  • release factors and porins should be optimized to enhance release of cellular contents but not result in bacterial lysis.
  • such expression is under the control of a promoter that is preferentially active in the tumor environment.
  • the release factor or porin coding sequence can be expressed under the control the pepT promoter, which is activated in response to the anaerobic nature of the tumor environment (see, e.g., Lang et al, 1996, Gene, 168:169-171).
  • the release factor or porin coding sequence is expressed under the control of a tet promoter, and expression of the release factor or porin is induced upon administration of tetracycline to the subject.
  • the present invention also provides methods for local delivery of one or more marker gene products to the tumor environment through secretion machinery.
  • tumor-targeted bacteria are engineered to express one or more nucleic acid molecules encoding one or more fusion proteins comprising an Omp-like protein, or portion thereof (e.g., signal sequence, leader sequence, periplasmic region, transmembrane domain, multiple transmembrane domains, or combinations thereof; see Section 3.1 for definition of "Omp-like protein”) and marker gene product.
  • the Omp-like protein is at least a portion of OmpA
  • the signal sequence is constructed to be more hydrophobic (e.g., by the insertion or replacement of amino acids within the signal sequence to hydrophobic amino acids, e.g., Leucine).
  • the marker gene product is expressed as a fusion protein to an Omp-like protein, or portion thereof.
  • Bacterial outer membrane proteins are integral membrane proteins of the bacterial outer membrane, possess multiple membrane- spanning domains and are often attached to one or more lipid moieties. Outer membrane proteins are initially expressed in precursor form (the pro-Omp) with an amino terminal signal peptide that directs the protein to the membrane, upon which the signal peptide is cleaved by a signal peptidase to produce the mature protein.
  • a marker gene product is constructed as a fusion protein with an Omp-like protein. In this embodiment, the gene product has enhanced delivery to the outer membrane of the bacteria.
  • the fusion of a marker gene product to an Omp-like protein is used to enhance localization of marker gene product to the periplasm.
  • the fusion of a marker gene product to an Omp-like protein is used to enhance release of the gene product.
  • a release factor e.g., BRP, supra
  • BRP BRP
  • a fusion protein of the invention comprises a proteolytic cleavage site.
  • the proteolytic cleavage site may be endogenous to the gene product or endogenous to the Omp-like protein, or the proteolytic cleavage site may be constructed into the fusion protein.
  • the Omp-like protein of the invention is a hybrid Omp comprising structural elements that originate from separate proteins.
  • the Omp-like protein is OmpA; the same principles used in the construction of OmpA-like fusion proteins are applied to other Omp fusion proteins, keeping in mind the structural configuration of the specific Omp-like protein.
  • the native OmpA protein contains eight anti-parallel transmembrane ⁇ -strands within the 170 amino acid N-terminal domain of the protein. Between each pair of transmembrane domains is an extracellular or intracellular loop, depending on the direction of insertion of the transmembrane domain.
  • the C-terminal domain consists of 155 amino acids which are located intracellularly and presumably contact the peptidoglycan occupying the periplasmic space.
  • Expression vectors have been generated that facilitate the generation of OmpA fusion proteins. For example, Hobol et al. (1995, Dev. Biol. Strand. 84:255-262) have developed vectors containing the OmpA open reading frame with linkers inserted within the sequences encoding the third or fourth extracellular loops that allow the in- frame insertion of the heterologous protein of choice.
  • the portion of the OmpA fusion protein containing the marker gene product is at the extracellular bacterial surface.
  • the fusion protein comprises an even number or odd number of membrane-spanning domains of OmpA located N-terminal to the gene product.
  • the gene product is situated between two extracellular loops of OmpA for presentation to the tumor cell by the bacterial cell.
  • the invention provides expression plasmids of gene product fusion proteins at the bacterial extracellular surface.
  • the plasmid denoted T ⁇ c(Xpp)ompA comprises a trc promoter-driven lipopolyprotein (lpp) anchor sequence fused to a truncated omp A transmembrane sequence.
  • the plasmid is denoted TrcompA comprises a trc promoter-driven ompA gene signal sequence.
  • Such plasmids may be constructed to comprise a nucleic acid comprising or encoding one or more gene product(s) of the invention.
  • a marker gene product is preceded or flanked by consensus cleavage sites for a metalloprotease or serine protease that is abundant in tumors, for release of the gene product into the tumor environment. Whether the marker gene product is preceded or flanked by protease cleavage sites depends on whether it is located terminally or internally in the fusion protein, respectively.
  • Similar fusion proteins may be constructed with any of the Omp-like proteins using the strategies described above in terms of OmpA.
  • the selection of the portion of the Omp-like protein to be fused to a marker gene product will depend upon the location that is desired for the expression of the gene product (e.g. periplasmic, extracellular, membrane bound, etc.).
  • o Construction of fusion proteins for expression in bacteria are well known in the art and such methods are within the scope of the invention. (See, e.g., Makrides, S., 1996, Microbiol. Revs 60:512-538 which is inco ⁇ orated herein by reference in its entirety).
  • the invention encompasses an embodiment where the marker gene product is an enzyme.
  • This class of marker gene product is detected by the addition of its substrate attached to a labeled moiety.
  • the enzyme and substrate must be brought into close proximity.
  • the tumor- targeted bacteria can be modified to increase the retention of the enzyme substrate in its 0 cytoplasm and to facilitate greater contact with the enzyme.
  • the enzyme is a pro-drug converting enzyme.
  • This class of enzymes modulate the chemical nature of a benign drug to produce a cytotoxic agent and, as such, can also be used for treatment of a tumor as well as its detection and localization (see Section 5.6 and Table 2 below).
  • 5 Enhanced cytoplasmic retention of the labeled enzyme substrate can be accomplished through the manipulation of the transport systems normally responsible for pumping the compounds out of the cell. Such transporters are called drug efflux systems.
  • One prefe ⁇ ed modification of the host organism is to disable or severely compromise one or more drug efflux systems in the host organism.
  • Membrane-associated 0 energy driven efflux plays a major role in drug resistance in most organisms, including bacteria, yeasts, and mammalian cells (Nikaido, 1994, Science 264:382-388; Balzi et al, 1994, Biochim Biophys Acta 1187:152-162; Gottesman et al, 1993, Ann Rev Biochem 62:385).
  • Normally, a wild type efflux system actively secretes potentially toxic compounds, thus reducing their accumulation inside the host organism.
  • the efflux systems that can be expressed in a tumor-targeting vector of the invention can also be of yeast or plant origin. Efflux systems in animals, plants, yeast, and bacteria are mechanistically related, often involving ATP binding cassette transporters (see, e.g., Thomas et al, 2000, Plant Cell 12(4):519-33). Recent data suggests that certain efflux systems are structurally conserved among yeast, plants and bacteria (Harley and Saier, 2000, J. Mol. Microbiol. Biotechnol.2(2): 195-8).
  • a yeast gene which can be used as an efflux system is the bfrl+ gene, which confers brefeldin A resistance to Schizosaccharomyces pombe (Nagao et al, 1995, J. Bacteriol. 177(6): 1536-43).
  • a yeast gene which can be used as an efflux system is the CDR1 gene of Candida albicans, which confers resistance to cyclohexamide and chloramphenicol (Prasad et al, 1995, Cu ⁇ Genet 27:320-329).
  • Bacteria useful in the present invention are those facultative aerobic and facultative anaerobic bacteria which are able to differentiate between cancerous cells and non-cancerous counte ⁇ art cells.
  • the bacteria are able to differentiate between melanoma cells and melanocytes or between colon cancer cells and normal colon epithelial cells.
  • Illustrative examples of bacteria useful in the present invention as tumor-targeted bacteria and/or for isolation of tumor-targeted bacteria include, but are not limited to the following facultative aerobes and anaerobes: Escherichia coli, including but not limited to pathogenic Escherichia coli (e.g., entero-invasive or uropathogenic Escherichia coli), Salmonella spp., Shigella spp., Streptococcus spp., Yersinia enterocolitica, Listeria monocytogenies, and Mycoplasma hominis.
  • Escherichia coli including but not limited to pathogenic Escherichia coli (e.g., entero-invasive or uropathogenic Escherichia coli), Salmonella spp., Shigella spp., Streptococcus spp., Yersinia enterocolitica, Listeria monocytogenies, and Mycoplasma hominis.
  • Salmonella spp. are particularly useful strains for the present invention, since they show natural preference for attachment to and penetration into certain solid tumor cancer cells in tissue culture, as opposed to non-cancerous counte ⁇ art cells.
  • the term "Salmonella” is used generically herein to refer to any Salmonella species). Since Salmonella have a natural ability to distinguish between cancerous cells and their non- cancerous counte ⁇ art cells, and preferentially replicate in tumors (in mice), they are directly applicable to the methods for treatment according to the present invention. Bacteria such as Salmonella are causative agents of disease in humans and animals.
  • the bacterial vectors including but not limited to Salmonella, are attenuated in their virulence for causing disease.
  • Attenuation in addition to its traditional definition in which a bacterium is modified so that the bacterium is less pathogenic, is intended to include also the modification of a bacterial strain so that a lower titer of that derived bacterial strain can be administered to a patient and still achieve comparable results as if one had administered a higher titer of the parental bacterial strain.
  • the end result serves to reduce the risk of toxic shock or other side effects due to administration of the strain to the patient.
  • Such attenuated bacteria are isolated by means of a number of techniques. For example, attenuation can be achieved by the deletion or disruption of DNA sequences which encode for virulence factors that insure survival of the bacteria in the host cell, especially macrophages and neutrophils.
  • deletion or disruption techniques include, for example, homologous recombination, chemical mutagenesis, radiation mutagenesis, or transposon mutagenesis.
  • Those virulence factors that are associated with survival in macrophages are usually specifically expressed within the macrophages in response to stress signals, for example, acidification, or in response to host cell defensive mechanisms such macropinocytosis (Fields et al, 1986, Proc. Natl. Acad. Sci. USA 83:5189-5193).
  • Table 4 of International Publication WO 96/40238 is an illustrative list of Salmonella virulence factors whose deletion results in attenuation.
  • LPS lipopolysaccharide
  • LA lipid A
  • Altering the LA content of bacteria, such as Salmonella can be achieved by the introduction of mutations in the LPS biosynthetic pathway.
  • LPS biosynthetic pathway Several enzymatic steps in LPS biosynthesis and the genetic loci controlling them in Salmonella have been identified (Raetz, 1993, J. Bacteriol. 175:5745-5753 and references therein), as well as co ⁇ esponding mutants.
  • One such illustrative mutant is firA, a mutation within the gene that encodes the enzyme UDP-3-O(R-30 hydroxymyristyl)-glycocyamine N-acyltransferase, which regulates the third step in endotoxin biosynthesis (Kelley et al, 1993, J. Biol. Chem.
  • Bacterial strains bearing this type of mutation produce a lipid A that differs from wild-type lipid A in that it contains a seventh fatty acid, a hexadecanoic acid (Roy and Coleman, 1994, J. Bacteriol. 176:1639-1646). Roy and Coleman demonstrated that in addition to blocking the third step in endotoxin biosynthesis, the firA ' mutation also decreases enzymatic activity of lipid A 4' kinase that regulates the sixth step of lipid A biosynthesis.
  • the bacteria of the invention are tumor- targeted, i.e., the bacteria preferentially attaches to, infect, and/or remain viable in a tumor or tumor cell versus a normal tissue, non-tumor or non-tumor cell.
  • Suitable methods for obtaining attenuated tumor-targeted bacteria are described in Sections 6.1 and 6.2 of International Publication WO 96/40238, which sections are inco ⁇ orated by reference herein in their entirety.
  • the resulting bacteria are highly specific and, in one embodiment super- infective, the difference between the number of infecting bacteria found at the target tumor or tumor cell as compared to the non-cancerous counte ⁇ arts becomes larger and larger as the dilution of the bacterial culture is increased such that lower titers of bacteria can be used with positive results.
  • the techniques described in International Publication WO 96/40238 can also be used to produce attenuated tumor-targeted Salmonella or non-Salmonella bacterial strains.
  • an illustrative example of an attenuated tumor-targeted bacterium having an LPS pathway mutant is the msbB ' Salmonella mutant described in International Publication WO 99/13053, which is inco ⁇ orated herein by reference in its entirety; see especially Section 6.1.2 which describes the characteristic of the msbB- Salmonella mutant.
  • One characteristic of the msbB ' Salmonella is decreased ability to induce a TNF- ⁇ response compared to the wild-type bacterial strain.
  • the msbB ' Salmonella induce TNF- ⁇ expression at levels of about 5 percent to about 40 percent compared to the levels induced by wild-type Salmonella.
  • the TNF- ⁇ response induced by whole bacteria or isolated or purified LPS can be assessed in vitro or in vivo using commercially available assay systems such as by enzyme linked immunoassay (ELISA). Comparison of TNF- ⁇ production on a per colony forming unit ("c.f.u.") or on a ⁇ g/kg basis, is used to determine relative activity. Lower TNF- ⁇ levels on a per unit basis indicate decreased induction of TNF- ⁇ production.
  • the msbB ' Salmonella strain is modified to express a marker gene product which aids in reducing the volume of, or inhibiting the growth of, the solid tumor cancer.
  • the present invention also encompasses the use of derivatives of msbB ' attenuated tumor-targeted Salmonella mutants.
  • the stability of the attenuated phenotype is important such that the strain does not revert to a more virulent phenotype during the course of treatment of a patient.
  • Such stability can be obtained, for example, by providing that the virulence gene is disrupted by deletion or other non-reverting mutations on the chromosomal level rather than epistatically.
  • Another method of insuring the attenuated phenotype is to engineer the bacteria such that it is attenuated in more than one manner.
  • bacteria can be made much more susceptible to lysis by manipulating the cell wall integrity.
  • mutations or deletions in the asd locus are used to severely compromise cell wall integrity.
  • This type of mutant bacteria relies on exogenously provided diaminopimelic acid to survive (Galan et al, 1990, Gene 94:24-35).
  • this supplement can be withdrawn from the patient to accelerate the clearance of tumor-targeted bacteria.
  • different pathways can be manipulated to attenuate the bacteria, such as, e.g.
  • a mutation in the pathway for lipid A production such as the msbB ' mutation (International Publication WO 99/13053) and one or more mutations to auxotrophy for one or more nutrients or metabolites, such as uracil biosynthesis, purine biosynthesis, and arginine biosynthesis as described by Bochner, 1980, J. Bacteriol. 143:926-933.
  • the tumor-targeted msbB ' Salmonella is also auxotrophic for purine.
  • the attenuated tumor-targeted bacteria are attenuated by the presence of a mutation in AroA, msbB, Purl or SerC.
  • the attenuated tumor-targeted bacteria are attenuated by the presence ofa deletion in AroA, msbB, Purl or SerC.
  • any attenuated tumor-targeted bacteria may be used in the imaging and monitoring methods of the invention.
  • the detection and monitoring methods can be combined with administration of a bacterial strain described in Section 5.5 which has been genetically modified to express one or more gene products of interest and serve as a bacterial vector which aids in reducing the volume of, or inhibiting the growth of, the solid tumor cancer.
  • these embodiments encompass detection (and/or monitoring) and anti-tumor treatment, i.e., inhibition of tumor growth or reduction of tumor volume.
  • the same bacteria which express a marker gene can express an antitumor gene product.
  • the marker gene itself is utilized as an anti-tumor gene.
  • At least two different bacteria can be used, expressing a marker gene and one expressing an anti -tumor gene product (referred to herein as a "gene product of interest").
  • the gene product of interest is selected from the group consisting of proteinaceous and nucleic acid molecules.
  • the nucleic acid molecule can be double- stranded or single-stranded DNA or double-stranded or single-stranded RNA, as well as triplex nucleic acid molecules.
  • the nucleic acid molecule can function as a ribozyme, or antisense nucleic acid, etc. Release of the gene product of interest into the tumor environment may be enhanced in various ways.
  • the gene product of interest is co-expressed with a release factor (e.g., BRP, see Section 5.4.1).
  • a release factor e.g., BRP, see Section 5.4.1
  • the gene product of interest is expressed as a fusion protein with an Omp-like protein, or portion thereof (see Section 5.4.2).
  • Antisense nucleotides are oligonucleotides that bind in a sequence- specific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that has complementary sequences, antisense prevents translation of the mRNA (see, e.g., U.S. Patent Nos. 5,168,053; 5,190,931; 5,135,917; 5,087,617).
  • Triplex molecules refer to single DNA strands that bind duplex DNA forming a collinear triplex molecule, thereby preventing transcription (see, e.g., U.S. Patent No. 5,176,996).
  • a ribozyme is an RNA molecule that specifically cleaves RNA substrates, such as mRNA, resulting in inhibition or interference with cell growth or expression.
  • RNA substrates such as mRNA
  • Ribozymes can be targeted to any RNA transcript and can catalytically cleave that transcript (see, e.g., U.S. Patent No. 5,272,262; U.S. Patent No. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246).
  • the proteinaceous molecule is a cellular toxin, e.g., saporin, a ribosome inactivating protein, or a porin protein, such as gonococcal PI porin protein.
  • the proteinaceous molecule is an anti-angiogenesis protein, an antibody or an antigen.
  • the proteinaceous molecule is a cytokine, e.g., IL-2, or an anti-angiogenic factor, e.g., endostatin, or a pro-drug converting enzyme, e.g. , He ⁇ es Simplex Virus ("HSV") thymidine kinase or cytosine deaminase.
  • HSV He ⁇ es Simplex Virus
  • the nucleic acid molecule encoding the gene product of interest is from about 6 base pairs to about 100,000 base pairs in length.
  • the nucleic acid molecule is from about 20 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid molecule is from about 100 base pairs to about 10,000 base pairs in length. Even more preferably, it is a nucleic acid molecule from about 500 pairs to about 2,000 base pairs in length.
  • the nucleic acid encoding a gene product of interest is provided in an expression vector in operative linkage with a selected promoter, and optionally in operative linkage with other elements that participate in transcription, translation, localization, stability and the like.
  • the gene product of interest is cytotoxic or cytostatic to a cell by inhibiting cell growth through interference with protein synthesis or through disruption of the cell cycle.
  • a product may act, for example, by cleaving rRNA or ribonucleoprotein, inhibiting an elongation factor, cleaving mRNA, or other mechanism that reduced protein synthesis to a level such that the cell cannot survive.
  • cytotoxic or cytostatic molecules include but are not limited to saporin, the ricins, abrin, and other ribosome inactivating proteins (RIPs).
  • the gene product of interest is a pro-drug converting enzyme or nucleic acid encoding the same, i.e. an enzyme that
  • pro-drug converting enzymes include, HSV-TK, bacterial cytosine deaminase, cytochrome P-450 NADPH oxidoreductase, and those listed on page 33 and in Table 2 of WO 96/40238 by Pawelek et al, which is inco ⁇ orated herein in its entirety. Further examples of pro-drug converting enzymes can be seen in Table 2 below.
  • a pro-drug converting enzyme need not be a secreted protein if co-expressed with a release factor such as BRP (see Section 5.4.1).
  • the pro-drug converting enzyme is cytochrome p450 NADPH oxidoreductase which acts upon mitomycin C and porfiromycin (Murray et al,
  • a gene product of interest is an inhibitor of inducible nitric oxide synthase (NOS) or of endothelial nitric oxide synthase.
  • NOS inducible nitric oxide synthase
  • NO endothelial nitric oxide synthase.
  • NO is implicated to be involved in the regulation of vascular growth and in arteriosclerosis. NO is formed from L-arginine by nitric oxide synthase (NOS) and 0 modulates immune, inflammatory and cardiovascular responses.
  • the gene product of interest is cytotoxic or cytostatic to a cell by inhibiting the production or activity of a protein involved in cell proliferation, such as an oncogene or growth factor, (e.g., bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8) or cellular receptor or ligand.
  • a protein involved in cell proliferation such as an oncogene or growth factor, (e.g., bFGF, int-2, hst-1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8) or cellular receptor or ligand.
  • the inhibition can be at the 5 level of transcription or translation (mediated by a gene product that is a ribozyme or triplex DNA), or at the level of protein activity (mediated by a gene product that is an inhibitor of a growth factor pathway, such as a dominant negative mutant).
  • a gene product of interest is a cytokine, chemokine, or an immunomodulating protein or a nucleic acid encoding the same, 0 such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-10 (IL-10), interleukin-15 (IL-15), interleukin-18 (IL-18), endothelial monocyte activating protein-2 (EMAP2) GM-CSF, IFN- ⁇ , IFN- ⁇ , MIP-3 ⁇ , SLC, MIP-3 ⁇ , or an MHC gene, such as HLA-B7.
  • IL-1 interleukin-1
  • IL-2 interleukin-2
  • IL-4 interleukin-4
  • IL-5 interleukin-5
  • IL-10 interleukin-10
  • IL-15 interleukin-15
  • IL-18 interleukin-18
  • EEMAP2 endothelial monocyte activating protein-2
  • the cytokine may also be a member of the TNF family, including but not limited to, tumor necrosis factor- ⁇ (TNF- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), TNF- 5 ⁇ -related apoptosis-inducing ligand (TRAIL), TNF- ⁇ -related activation-induced cytokine (TRANCE), TNF- ⁇ -related weak inducer of apoptosis (TWEAK), CD40 ligand (CD40L), LT- ⁇ (lymphotoxin alpha), LT- ⁇ (lymphotoxin beta), OX40L (OX40 ligand), FasL, CD27L (CD27 ligand), CD30L (CD30 ligand), 4-1BBL, APRIL (a proliferation-inducing ligand), LIGHT (a 29 kDa type II transmembrane protein produced by activated T cells), TL1 (a tumor necrosis factor-like cytokine), TNFSF16, TNFSF17,
  • the gene product of interest is tumor necrosis factor- ⁇ (TNF- ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), TNF- ⁇ - related apoptosis-inducing ligand (TRAIL), TNF- ⁇ -related activation-induced cytokine (TRANCE), TNF- ⁇ -related weak inducer of apoptosis (TWEAK), and CD40 ligand (CD40L), or a functional fragment thereof.
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TNF- ⁇ tumor necrosis factor- ⁇
  • TRANCE TNF- ⁇ -related activation-induced cytokine
  • TWEAK TNF- ⁇ -related weak inducer of apoptosis
  • CD40L CD40 ligand
  • nucleic acid molecules encoding costimulatory molecules such as B7.1 and B7.2, ligands for CD28 and CTLA-4 respectively, can also be delivered to enhance T cell mediated immunity.
  • costimulatory molecules such as B7.1 and B7.2, ligands for CD28 and CTLA-4 respectively
  • an immunomodulating agent is, ⁇ -l,3-galactosyl transferase, whose expression on tumor cells allows complement-mediated cell killing.
  • the immunomodulating agent is a tumor-associated antigen, i.e.
  • tumor-associated antigens are described in Kuby, 1992,
  • a gene product of interest is an anti-angiogenic molecule or protein associated with angiogenesis, such as, e.g., endostatin, angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor antagonist, the 16 kDa proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of platelet factor-4, the anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-angiogenic factor designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of thrombospondin I, the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR containing peptides, the small anti-angiogenic peptides of laminin, fibronectin, procollagen and EGF, and peptide antagonists of integrin
  • angiogenesis such
  • a gene product of interest is a Flt-3 ligand or nucleic acid encoding the same.
  • the gene product of interest is a cytotoxic polypeptide or peptide, or a functional fragment thereof.
  • cytotoxic polypeptides or peptides include, but are not limited to, members of the bacteriocin family
  • bacteriocin family members include,
  • bacteriocin release protein BRP
  • ColEl bacteriocin release protein
  • ColEla ColElb ColE2, ColE3, ColE4, ColE5, ColE6, ColE7, ColE8, ColE9
  • Colicins A Colicin K
  • Colicin L Colicin M
  • cloacin DF13 pesticin Al 122
  • staphylococcin 1580 butyricin 7423
  • pyocin RI or AP41 megacin A-216, and vibriocin.
  • the gene product of interest is colicin E3.
  • the gene product of interest is BRP.
  • Colicin E3 has been shown to have a profoundly cytotoxic effect on mammalian cells (see, Smarda et al, 1978, Folia Microbiol. 23:272-277), including a leukemia cell model system (see, Fiska et al, 1978, Experimentia 35: 406-407).
  • ColE3 cytotoxicity is a function of protein synthesis arrest, mediated by inhibition of 80S ribosomes (Turnowsky et al, 1973, Biochem. Biophys. Res. Comm. 52:327-334). More
  • ColE3 has ribonuclease activity (Saunders, 1978, Nature 274: 113-114).
  • ColE3 is a 60kDa protein complex consisting of a 50kDa and a 1 OkDa protein in a 1 : 1 ratio, the larger subunit having the nuclease activity and the smaller subunit having inhibitory function of the 5 OkDa subunit.
  • the 5 OkDa protein acts as a cytotoxic protein (or toxin)
  • the lOkDa protein acts as an anti-toxin. Accordingly, in
  • ColE3 when ColE3 is used as a secondary marker gene product, the larger ColE3 subunit or an active fragment thereof is expressed alone or at higher levels than the smaller subunit. In a prefe ⁇ ed mode of the embodiment, ColE3 expression is accompanied by BRP expression to enhance release into the tumor environment. In yet another embodiment of the invention, the ColE3 50kDa toxin and lOkDa anti-toxin are encoded on a single
  • the toxin/anti-toxin can act as a selection system for the Salmonella which carry the plasmid, such that Salmonella which lose the plasmid are killed by the toxin.
  • the lOkDa anti-toxin is on the chromosome, separate from the colE3 toxin on the plasmid, resulting in a barrier to transmission of other bacteria (see, Diaz et al,
  • the bacteriocin is cloacin DF13.
  • Cloacin DF13 functions in an analogous manner to ColE3.
  • the protein complex is of 67KDa molecular weight.
  • the individual components are 57kDa and 9kDa in size.
  • DF13 can cause the leakage of cellular potassium.
  • the bacteriocin is colicin V (See, e.g., Pugsley, A.P. and Oudega, B. "Methods for Studing Colicins and their Plasmids" Plasmids a Practical Approach, 1987, ed. by K.G. Hardy; Gilson, L. et al, 1990, The EMBO Journal vol. 9 pp3875-3884).
  • the bacteriocin is selected from the group consisting of colicin E2 (a dual subunit colicin similar to ColE3 in structure but with endonuclease rather than ribonuclease activity); Colicins A, El, la, lb, or K, which form ion-permeable channels, causing a collapse of the proton motive force of the cell and leading to cell death; colicin L which inhibits protein, DNA & RNA synthesis; colicin M which causes cell sepsis by altering the osmotic environment of the cell; pesticin Al 122 which functions in a manner similar to colicin B function; staphycoccin 1580, a pore- forming bacteriocin; butyricin 7423 which indirectly inhibits RNA, DNA and protein synthesis through an unknown target; Pyocin PI, or protein resembling a bacteriophage tail protein that kills cells by uncoupling respiration from solute transport; Pyocin
  • the bacteriocin is BRP.
  • the cytotoxic activity of BRP is mediated by the release of cellular components.
  • the gene products of interest are tumor inhibitory enzymes or functional fragments thereof.
  • tumor inhibitory enzymes include, but are not limited to, methionase, asparaginase, lipase, phospholipase, protease, ribonuclease (excluding colE3), DNAse, and glycosidase.
  • the primary marker gene product is methionase.
  • the gene product of interest comprises a number of viral gene products.
  • the gene product of interest comprises all the viral proteins encoded by an adeno virus or he ⁇ es virus genome.
  • the gene product of interest is all the viral proteins encoded by an adenovirus genome except for the E IB viral protein such that this particular adenovirus can only replicate in a mammalian cell lacking p53 activity.
  • Additional types of cellular toxins that may be delivered according to the methods of the present invention are antibody molecules that are preferably expressed within the target cell; hence, these antibody molecules have been given the name "intrabodies.”
  • Conventional methods of antibody preparation and sequencing are useful in the preparation of intrabodies and the nucleic acid sequences encoding same; it is the site of action of intrabodies that confers particular novelty on such molecules. (For a review of various methods and compositions useful in the modulation of protein function in cells via the use of intrabodies, see International Application WO 96/07321).
  • Intrabodies are antibodies and antibody derivatives (including single-chain antibodies or "SCA") introduced into cells as transgenes that bind to and incapacitate an intracellular protein in the cell that expresses the intrabodies or derivatives.
  • intrabodies encompass monoclonals, single chain antibodies, V regions, and the like, as long as they bind to the target protein.
  • Intrabodies to proteins involved in cell replication, tumorigenesis, and the like e.g., HER2/neu, VEGF, VEGF receptor, FGF receptor, FGF
  • the intrabody can also be a bispecific intrabody.
  • Such a bispecific intrabody is engineered to recognize both (1) the desired epitope and (2) one of a variety of "trigger" molecules, e.g., Fc receptors on myeloid cells, and CD3 and CD2 on T cells, that have been identified as being able to cause a cytotoxic T cell to destroy a particular target.
  • "trigger" molecules e.g., Fc receptors on myeloid cells, and CD3 and CD2 on T cells
  • antibodies to HER2/neu also called erbB-2
  • HER2/neu has a pivotal role in the progression of certain tumors, human breast, ovarian and non-small lung carcinoma. Thus, inhibiting the function of HER2/neu may result in slowing or halting tumor growth (see, e.g. U.S. Patent No. 5,587,458).
  • Nucleic acid molecules and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g., International Publication WO 93/01286, U.S. Patent Nos. 5,218,088; 5,175,269; 5,109,124). Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
  • Antisense oligonucleotides may be designed to resist degradation by endogenous nucleolytic enzymes using linkages such as phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphoramidate, phosphate esters, and the like (see, e.g., Stein in: Oligodeoxynucleotides, Antisense
  • Particularly useful antisense nucleotides and triplex molecules are molecules that are complementary to bind to the sense strand of DNA or mRNA that encodes a protein involved in cell proliferation, such as an oncogene or growth factor, (e.g., bFGF, int-2, hst- 1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8).
  • an oncogene or growth factor e.g., bFGF, int-2, hst- 1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8.
  • Other useful antisense oligonucleotides include those that are specific for IL-8 (see, e.g., U.S. Patent No.
  • c-src c-fos H-ras (lung cancer), K-ras (breast cancer), urokinase (melanoma), BCL2 (T-cell lymphoma), IGF- 1 (glioblastoma), IGF-1 (glioblastoma), IGF-1 receptor (glioblastoma), TGF- ⁇ l, and CRIPTO EGF receptor (colon cancer).
  • These particular antisense plasmids reduce tumorigenicity in athymic and syngenic mice.
  • nucleotide sequences of the genes encoding these gene products are well known (see GenBank).
  • a nucleic acid molecule encoding one of the may be isolated by standard methods, such as amplification (e.g., PCR), probe hybridization of genomic or cDNA libraries, antibody screening of expression libraries, chemically synthesized or obtained from commercial or other sources.
  • Nucleic acid molecules and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g. , International Publication WO 93/01286, U.S. Patent Nos. 5,218,088; 5,175,269; 5,109,124). Identification of oligonucleotides and ribozymes for use as antisense agents involve methods well known in the art.
  • the gene product of interest is a fragment, analog, or variant of the wild-type full-length gene product, or a nucleic acid encoding the same.
  • the derivative, analog or variant is functionally active, e.g. , capable of exhibiting one or more functional activities associated with a full-length, wild-type marker gene product.
  • such derivatives, analogs or variants which have the desired therapeutic properties can be used to inhibit tumor growth.
  • Derivatives or analogs of an marker gene product can be tested for the desired activity by procedures known in the art, including those described herein.
  • variants can be made by altering gene sequences by substitutions, additions (e.g., insertions) or deletions that provide molecules having the same or increased anti-tumor function relative to the wild-type marker gene product.
  • the variants of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of the gene product, including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change, i.e., the altered sequence has at least one conservative substitution.
  • any of the gene product of interest-encoding nucleic acids that are of mammalian origin can be altered to employ bacterial codon usage by methods known in the art. Prefe ⁇ ed codon usage is exemplified in Cu ⁇ ent Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc. New York, and Zhang et al, 1991, Gene 105: 61.
  • the gene product of interest is expressed as a fusion protein.
  • a gene product is constructed as a chimeric or fusion protein comprising the gene product or a fragment thereof joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein.
  • such a chimeric protein is produced by recombinant expression of a nucleic acid comprising or encoding the gene product of interest, e.g., comprising a TNF encoding sequence, joined in-frame to a coding sequence for a different protein.
  • a chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric product into the expression vehicle of choice by methods commonly known in the art.
  • Chimeric nucleic acids comprising portions ofa nucleic acid comprising or encoding a gene product of interest fused to any heterologous protein-encoding sequence may be constructed.
  • the fusion protein comprises an affinity tag such as a hexahistidine tag, or other affinity tag that may be used in purification, isolation, identification, or assay of expression.
  • the fusion protein comprises a protease cleavage site such as a metal protease or serine cleavage site.
  • a protease site co ⁇ esponding to a protease which is active at the site of a tumor is constructed into a fusion protein of the invention.
  • an gene product of interest is constructed as a fusion protein to an Omp-like protein, or portion thereof (e.g., signal sequence, leader sequence, periplasmic region, transmembrane domain, multiple transmembrane domains, or combinations thereof; see supra, Section 3.1 for definition of "Omp-like protein").
  • the diagnostic imaging system including but not limited to streptavidin, also has antitumor activity.
  • the diagnostic imaging system is combined with BRP, such as BRP and streptavidin, where the marker gene product streptavidin has increased antitumor activity when secreted.
  • the agent chelated by streptavidin has antitumor activity, such as would be mediated by high specific localization of gamma-emitting radioactive biotin.
  • the agent chelated by streptavidin has antitumor activity when activated by another agent, such as when boron is activated by neutrons (Sano, 1999, Bioconjug Chem. 10:905-911).
  • the present invention provides methods for local delivery of one or more fusion proteins comprising a ferry peptide and a marker gene product to a solid tumor by tumor-targeted bacteria.
  • Ferry peptides used in fusion proteins have been shown to facilitate the delivery of a polypeptide or peptide of interest to virtually any cell within diffusion limits of its production or introduction (see., e.g., Bayley, 1999, Nature Biotechnology 17:1066-1067; Fernandez et al, 1998, Nature Biotechnology 16:418- 420; and Derossi et al, 1998, Trends Cell Biol. 8:84-87).
  • tumor-targeted bacteria are engineered to express a nucleic acid molecule encoding a fusion protein comprising a ferry peptide and a marker gene product.
  • tumor-targeted bacteria are engineered to express one or more nucleic acid molecules encoding one or more fusion proteins comprising a ferry peptide and a marker gene product.
  • ferry peptides include, but are not limited to, peptides derived from the HIV TAT protein, the antennapedia homeodomain (penetratin), Kaposi fibroblast growth factor (FGF) membrane-translocating sequence (MTS), and he ⁇ es simplex virus VP22.
  • penetratin the antennapedia homeodomain
  • FGF Kaposi fibroblast growth factor
  • MTS membrane-translocating sequence
  • VP22 he ⁇ es simplex virus
  • the present invention also provides methods for local delivery of one or more fusion proteins comprising a signal peptide, ferry peptide and an marker gene product to a solid tumor by tumor-targeted bacteria.
  • tumor-targeted bacteria are engineered to express one or more nucleic acid molecules encoding one or more fusion proteins comprising a signal sequence, a ferry peptide and an marker gene product.
  • the present invention also provides methods for local delivery of one or more fusion proteins comprising a signal peptide, a proteolytic cleavage site, a ferry peptide and an marker gene product to a solid tumor by tumor-targeted bacteria.
  • tumor-targeted bacteria are engineered to express one or more nucleic acid molecules encoding one or more fusion proteins comprising a signal sequence, a proteolytic cleavage site, a ferry peptide and an marker gene product.
  • the present invention also provides methods for local delivery of one or more fusion proteins of the invention and one or more marker gene products of the invention to the site of a solid tumor by tumor-targeted bacteria.
  • the expression of both the fusion protein(s) and marker gene product(s) at the site of the solid tumor by an tumor-targeting bacteria improves the level of tumor or tumor cell growth inhibited compared to when either fusion protein(s) alone or the marker gene product(s) alone is expressed.
  • the marker gene product can be a pro-drug converting enzyme or a pro-drug.
  • the pro-drug converting enzyme can be fused or conjugated to a polypeptide therapeutic of the invention.
  • Exemplary pro-drug converting enzymes are provided in Table 2 below.
  • Carboxylesterase CPT-1 1 (I ⁇ notecan) Kojima ef ⁇ t, 1998 Cytosine deaminase 5-FC (5-fluorocytos ⁇ ne) Hamstra e ⁇ ⁇ t, 1999; Carboxypeptidase A methotrexate-alanine Haenseler ef ⁇ t, 1992
  • imaging and treatment are 0 combined in the same vector.
  • the same recombinant gene expressed by the vector is both a marker gene and a therapeutic.
  • the marker gene substrate is radiolabeled.
  • the substrate can be radiolabeled FIAU or GCV (e.g., 131 I-FIAU or 131 I- GCV), the co ⁇ esponding marker metabolite, by virtue of its radioactivity, will provide a 5 therapeutic benefit on the patient as well as permitting tumor imaging.
  • the marker gene encodes an antigen and a labeled antibody administered for imaging
  • the antibody can be radiolabeled and the radiolabel would serve both as an imaging tool and a therapeutic agent.
  • the same principle can be applied to other embodiments, for example those in which a labeled compound is administered that is inco ⁇ orated into the vector (such 30 as labeled DAP in asd mutant vectors as described above), the labeled compound can be labeled with a radionuclide that is used for both imaging and therapy.
  • the marker gene product and/or the anti-tumor gene product must be 35 expressed at the tumor site. Because the tumor can be in, but is not limited to, a mammalian or avian cell, the promoter controlling the expression of the marker gene product and/or the anti-tumor gene product must be compatible with such cells.
  • the mammalian cell can be, but is not limited to, human, canine, feline, equine, bovine, porcine, rodent, etc.
  • the choice of promoter will depend on the type of target cell and the degree or type of expression control desired. Promoters that are suitable for use in the present invention include, but are
  • promoter 5 not limited to, constitutive, inducible, tissue-specific, cell type-specific and temporal- specific and need not necessarily function in a mammalian cell.
  • Another type of promoter useful in the present invention is an event-specific promoter which is active or up-regulated in response to the occurrence of an event, such as viral infection.
  • an event such as viral infection.
  • the HIV LTR is an event specific promoter.
  • the promoter is inactive unless the tat gene product is
  • Exemplary promoters useful in the present invention include, but are not limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304- 310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22:787-797), the he ⁇ es thymidine kinase promoter (Wagner
  • CMV cytomegalovirus
  • RNA promoter 20 cauliflower mosaic virus 35S RNA promoter (Gardner, et al, 1981, Nucl. Acids Res. 9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate carboxylase (Herrera-Estrella et al, 1984, Nature 310:115-120); promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the following
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115-
  • immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-658; Adames et al, 1985, Nature 318:533-538; Alexander et al, 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert et al, 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46:89-94; myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-712); myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), prostate specific antigen gene control region which is active in prostate cells, and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234:1372-1378).
  • Another exemplary promoter is one that has enhanced activity in the tumor environment; for example, a promoter that is activated by the anaerobic environment of the tumor such as the PI promoter of the /?ep7 gene. Activation of the PI promoter is dependent on the FNR transcriptional activator (Strauch et al, 1985, J. Bacteriol. 156:743- 751).
  • the PI promoter is a mutant promoter that is induced at higher levels under anaerobic conditions than the native PI promoter, such as the pepT200 promoter whose activity in response to anaerobic conditions is induced by CRP-cAMP instead of FNR (Lombardo et al, 1997, J. Bacteriol.
  • potABCD an anaerobically-induced promoter
  • potABCD is an operon that is divergently expressed from pepT under anaerobic conditions.
  • the promoter in the pepT gene responsible for this expression has been isolated (Lombardo et al, 1997, J. Bacteriol. 179:1909-1917).
  • the promoter is an antibiotic-induced promoter, such as the tet promoter of the TnlO transposon.
  • the promoter can be an antibiotic- induced promoter, such as the tet promoter of the TnlO transposon.
  • the tet promoter is a single-mer, which single-mer responds in an all-or- nothing manner to the presence of tetracycline and provides a genetically stable on-off switch.
  • the tet promoter is multimerized, for example three- fold. Such a multimer responds in a graded manner to the presence of tetracycline and provides a more manipulable system for control of gene expression.
  • Promoter activity would then be induced by administering to a subject who has been treated with the attenuated tumor- targeted bacteria of the invention an appropriate dose of tetracycline.
  • tet inducible expression system was initially described for eukaryotic systems such as Schizosaccharomyces pombe (Faryar and Gatz, 1992, Cu ⁇ ent Genetics 21:345-349) and mammalian cells (Lang and Feingold, 1996, Gene 168:169-171)
  • recent studies extend its applicability to bacterial cells. For example, Stieger et al.
  • repressor sequences In addition to the promoter, repressor sequences, negative regulators, or tissue-specific silencers can be inserted to reduce non-specific expression of the gene product. Moreover, multiple repressor elements may be inserted in the promoter region.
  • One type of repressor sequence is an insulator sequence. Illustrative examples of repressor sequences which silence background transcription are found in Dunaway et al, 1997, Mol. Cell Biol. 17:182-129; Gdula et al, 1996, Proc. Natl. Acad. Sci. USA 93:9378-9383; Chan et al, 1996, J. Virol. 70:5312-5328.
  • sequences which increase the expression of the gene product can be inserted in the expression vector, e.g., ribosome binding sites.
  • Expression levels of the transcript or translated product can be assayed by any method known in the art to ascertain which promoter/repressor sequences affect expression.
  • a first tumor-targeted bacterial vector which expresses a first gene product is used for imaging or monitoring a tumor.
  • Such first tumor-targeted bacterial vector for imaging or monitoring may be used alone or together with a second tumor-targeted bacterial vector which expresses a second gene product used to produce a tumor growth inhibitory response or a reduction of tumor volume in a subject (including but not limited to a human patient) having a solid tumor cancer.
  • the method comprises administering to a subject, a pharmaceutical composition comprising an effective amount of facultative aerobic or facultative anaerobic tumor-targeted bacteria suitable for imaging or monitoring a tumor.
  • the method comprises administering to a subject, a pharmaceutical composition comprising an effective amount ofa first facultative aerobic or facultative anaerobic, attenuated, tumor-targeted bacteria suitable for imaging or monitoring a tumor and a second facultative aerobic or facultative anaerobic, attenuated, tumor- targeted bacteria suitable for producing a tumor growth inhibitory response or a reduction of tumor volume in the subject.
  • the method comprises administering, to a subject, a pharmaceutical composition comprising an effective amount of a facultative aerobic or facultative anaerobic, attenuated, tumor-targeted bacterial vector which has been genetically modified to express a gene product of interest, which aids in reducing the volume, or inhibiting the growth of the tumor, in combination with one or more a pharmaceutical compositions comprising an effective amount of facultative aerobic or facultative anaerobic tumor-targeted bacteria suitable for imaging or monitoring a tumor.
  • the method comprises administering, to a subject, a pharmaceutical composition comprising an effective amount of a facultative aerobic or facultative anaerobic, attenuated, tumor-targeted Salmonella spp.
  • a gene product of interest which aids in reducing the volume or inhibiting the growth of a solid tumor together with a pharmaceutical composition comprising an effective amount of facultative aerobic or facultative anaerobic tumor-targeted bacteria suitable for imaging or monitoring a tumor.
  • Solid tumors include, but are not limited to, sarcomas, carcinomas or other solid tumor cancers, such as renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors and tumors of the central nervous system, including, but not limited to, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer, colon cancer, and melanoma.
  • the subject is preferably an animal, including but not limited to animals such as cows, pigs, chickens, etc., and is preferably a mammal, and most preferably human.
  • Effective treatment of a solid tumor includes but is not limited to, inhibiting tumor growth, reducing tumor volume, etc.
  • the amount of the pharmaceutical composition of the invention which is effective in imaging or monitoring or the treatment of a solid tumor cancer will depend on the nature of the solid tumor, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the solid tumor, and should be decided according to the judgment of the practitioner and each patient's circumstances.
  • suitable dosage ranges are generally from about 1.0 c.f.u./kg to about 1 x 10 10 c.f.u./kg; optionally from about 1.0 c.f.u./kg to about 1 x 10 9 c.f.u./kg; optionally from about 1.0 c.f.u./kg to about 1 x 10 8 c.f.u./kg; optionally from about 1 x 10 2 c.f.u./kg to about 1 x 10 9 c.f.u./kg; optionally from about 1 x 10 4 c.f.u./kg to about 1 x 10 9 c.f.u./kg; optionally from about 1 x 10 2 c.f.u./kg to about 1 x 10 8 c.f.u./kg; optionally from about 1 x 10 4 c.f.u./kg to about 1 x 10 8 c.f.u./kg. Effective doses may be extrapolated from dose-res,
  • Various delivery systems are known and can be used to administer a pharmaceutical composition of the present invention.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds may be administered by any convenient route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions of the invention may be desirable to administer locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, by injection, by means of a catheter, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.
  • administration can be by direct injection at the site (or former site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
  • the bacteria or bacterial vector can be delivered in a controlled release system.
  • a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al, 1980, Surgery 88:507; Saudek et al, 1989, N. Engl. J. Med. 321 :574).
  • polymeric materials can be used (see Medical Applications of Controlled Release, 1974, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability, Drug Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York; Ranger and Peppas, 1983, J.
  • a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138).
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
  • an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
  • composition of the invention should be administered in a carrier that is pharmaceutically acceptable.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia or receiving specific or individual approval from one or more generally recognized regulatory agencies for use in animals, and more particularly in humans.
  • carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • Buffered saline is a prefe ⁇ ed ca ⁇ ier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion and the like. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
  • Such compositions will contain a therapeutically effective amount of the Therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
  • the formulation should suit the mode of administration.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the mass of radiopharmaceutical produced from a no carrier-added synthesis can be calculated using Avogadro's number, the half- life of the radionuclide (in sec), the molecular weight of the radiopharmaceutical and the administered dose (in Bq).
  • a dose of 32 ng of FIAU represents only 1/110,000 to 1/3,720,000 of the daily dose (and 3.1xl0"5 to 1.6xl0" of the total dose) that was administered to patients in the 14 to 28 day hepatitis B clinical trials, where no FIAU-related toxicity was observed. Only in the 168 day clinical trial (H3X-PPPC) did severe liver, pancreatic and neurotoxicity appear.
  • a 32 ⁇ g dose of FIAU represents less than 0.03 to 1.0 percent of the daily dose that was administered to patients in the 14 to 28 day hepatitis B clinical trials, where no FIAU-related toxicity was observed.
  • Various delivery systems are known and can be used to administer a pharmaceutical composition of the present invention.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, subcutaneous and intravenous.
  • Administration can be systemic or local.
  • administration can be by direct injection at the site (or former site) of a tumor.
  • the tumor-targeted bacteria or bacterial vectors which express a gene product of interest and the pro-drug treatment may be advantageously used in a combination method with one or more doses of i ⁇ adiation to produce a tumor growth inhibitory response or a reduction of tumor volume, in a subject, including a human patient, having a solid tumor cancer.
  • kits for carrying out the non-invasive detection methods of the invention and kits for carrying out the non-invasive detection and treatment methods of the invention.
  • kits comprise in one or more containers a purified population of tumor-targeted bacteria and a labeled moiety (including but not limited to biotin, an antibody or a chemotactic peptide), substrate, or compound which is useful for the non- invasive imaging in conjunction with said population of tumor- targeted bacteria.
  • the label can be a radionuclide, paramagnetic ion or fluorophore.
  • the label is sequestered.
  • the tumor-targeted bacteria harbor a recombinant marker gene operably linked to a promoter.
  • the marker gene is streptavidin.
  • the tumor-targeted bacteria is a mutant with an enhanced preference to inco ⁇ orate a labeled compound.
  • bacteria is mutant for the asd locus, most preferably only a partial mutant for said locus.
  • the kit further comprises a second population of tumor-targeted bacteria suitable for tumor therapy in the container in which the tumor- targeted bacteria useful for imaging is present or in a separate container.
  • the tumor-targeted bacteria suitable for imaging is also suitable for therapy.
  • the kit further comprises a pharmaceutically acceptable carrier in the container in which the tumor-targeted bacteria is present or in a separate container. Instructions are optionally included for using the bacteria to carry out the non-invasive imaging methods of the present invention.
  • kits can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the invention provides indirect methods of tumor imaging comprising administering to the subject a tumor- targeted bacterial vector containing a marker gene, wherein the tumor-targeted bacterial vector targets to the tumor and/or infects cells of the tumor, under conditions in which the marker gene is expressed in the tumor-targeted bacterial vector, thereby generating a marker gene product; administering to the subject a labeled marker substrate under conditions in which the labeled marker substrate is metabolized by the marker gene product to produce a labeled marker metabolite which is substantially retained in the tumor-targeted bacterial vector throughout a time-period sufficient for imaging the labeled marker metabolite; and scanning the subject to detect the labeled marker metabolite, thereby imaging and detecting a tumor in the subject.
  • the gene and a labeled marker substrate are selected as a self-complementary pair from the group consisting of: wild-type, natural mutant, or genetically-engineered he ⁇ es simplex virus-thymidine kinase or varicella zoster virus-thymidine kinase gene and a labeled nucleoside analog such as FIAU, ACV or GCV.
  • the nucleoside analog is a 2'-fluoro-nucleoside such as FIAU.
  • the labeled 2'-fluoro-nucleoside analogue can be 5-[ 123 I]-, 5-[ 124 I]- or 5-[ 131 I]-2'-fluoro-5-iodo-l- ⁇ -D-arabinofuranosyl- uracil; 5-[ I8 F]-2'-fluoro-5-fluoro-l- ⁇ -D- arabinofuranosyl- uracil; 2-[ u C]- or 5-([ u C]-methy)-2'-fluoro-5-methyl-l- ⁇ -D- arabinofuranosyl- uracil; 2-[ u C]- or 5-([ ⁇ C]-ethyl)-2'-fluoro-5-ethyl-l- ⁇ -D- arabinofuranosyl- uracil; 5-(2-[ l8 F]-ethyl)-2'-fluoro-5-(2- fluoro-ethyl)-l- ⁇ -D
  • D-arabinofuranosyl- uracil or 5-[ 123 I]-, 5-[ 124 1]- or 5-[ 131 I]-2'-fluoro-5-iodovinyl-l- ⁇ -D- aribofuranosyl- uracil.
  • the TK can have a therapeutic as well as imaging pu ⁇ ose.
  • a labeled pro-drug e.g. a labeled ganciclovir or acyclovir
  • the pro-drug is phosphorylated in the periplasm of the microorganism which is freely permeable to nucleotide triphosphates.
  • the labeled metabolites, phosphorylated ganciclovir or acycolvir which are toxic false DNA precursors, readily pass out of the periplasm of the microorganism and into the cytoplasm and nucleus of the host cell.
  • the phosphorylated ganciclovir or acycolvir inco ⁇ orate into host cell DNA, thereby causing the death of the host cell.
  • two recombinant tk genes are administered, either by way of two different bacterial vectors or a single bacteria vector comprising two recombinant tk genes.
  • the two recombinant tk genes are selected for different substrate specificity and subcellular localization.
  • the imaging mutant tk gene is retained in the cytosol of a first tumor-targeted bacterial vector and the treatment mutant tk gene is in the periplasm of a second tumor-targeted bacterial vector.
  • mice C57B/6 were subcutaneously implanted with C38 colon tumor cells and staged until the tumors were palpable (20 days).
  • Salmonella expressing HSVl-TK from plasmid p5-3 Salmonella expressing HSVl-TK from plasmid p5-3 (Pawelek et al, 1997, Cancer Res.
  • [ 14 C]-FIAU was injected i.v. After 23 hours, the animals were sacrificed and muscle, liver and tumor tissue samples removed and homogenized for radioactivity (DPM) and bacterial cfu determinations per gram of tissue. The accumulation of [ 14 C]-FIAU is presented in
  • FIG. 1 and 2 A sample of tumor and liver from mouse 3 were subjected to QAR. Cryosections were cut and exposed for quantitative analysis. The printouts of tumor and liver sections are shown in FIG. 1 and 2. In each of these figures, histology is on the left, digital autoradiogram is on the right and the merged image is in the center.
  • Cytosolic HSVl-TK Expression Expression of HSVl-TK in the Salmonella cytoplasm increases signal localization and detection over periplamic expression.
  • a vector coding for cytosolic HSVl- TK is made with PCR primers [forward 5'-GATCCCATGGCTTCGTACCCCGGCC-3 * (SEQ ID NO: 1) and reverse 5'-CTAGAAGCTTCAGTGGCTATGGCAGGGC-3' (SEQ ID NO:2)] to generate a product.
  • the template for the PCR reaction is a plasmid containing the HSVl-TK (McKnight, 1980, Nucleic Acids Research 8: 5949-5964).
  • PCR is performed for example as 1 cycle of 95 °C for 5 min: 35 cycles of 95 °C for 1 min; 55 °C for lmin; 72 °C for 2 min: and 1 cycle of 72 °C for 10 min using Ready-to-go PCR beads (Pharmacia) or equivalent.
  • the approximately 1.5 kilobase PCR product is then gel purified, followed by restriction digestion with Ncol and HindXXX and the product ligated into the Ncol and HindXXX sites of pTrc99a (Pharmacia).
  • TK activity is assayed using a modification of the method of Summers and Summers (1977, J. Virol. 24:314-318).
  • kinase substrates [ 125 IdC (Dupont/ ⁇ ew England Nuclear) or 3 H-ganciclovir (Moravek Biochemicals)] are incubated with bacterial cell lysate at 37 °C for 1 hour and then bound to DE81 paper (Whatman). After washing, the associated radioactivity is determined in a scintilation counter.
  • HSVl-tk mutants that are able to bind FIAU preferentially over thymidine can be selected.
  • Six codons hypothesized as playing a role in substrate binding (Balasubramanian et. al., 1990, J. Gen. Virol. 71:2979-2987; Leu 159, He 160, Phe 161, Ala 168, Leu 169, and Leu 170) in wild type HSVl-tk are targeted for random mutagenesis.
  • the construction of the random sequence inserts is essentially as described in Black et al. (1996, PNAS 9:3525-3529). Briefly, two random oligonucleotides are constructed: MB126 5'-TGGGAGCTCACATGCCCCGCCCCCGGCCCTCACCNNNNNNNGACCG
  • oligos 10 with Klenow fragment.
  • the annealed oligos are then amplified by PCR using the primers: PI : 5'-TGGGAGCTCACATGCCCCGCC-3' (SEQ ID NO:5) and P2: 5'- ATGAGGTACCG-3' (SEQ ID NO:6). This amplification generates the random inserts with the restriction sites SacX and Kp X.
  • the random sequence inserts from above are ligated into the wild type HSVl-tk open reading frame to replace the wild type segment.
  • a dummy vector is created.
  • This vector (pET23d backbone for high overexpression in the BL21DE3 strain) contains the wild type HSVl-tk open reading frame 0 with a piece of non-functional DNA at the insertion site. This vector has no HSVl-TK activity unless there is successful replacement of the dummy fragment with a fragment from the random inserts that results in the production of an active enzyme.
  • the dummy and PCR amplified random inserts are cut with KpnX and Sac I and gel isolated.
  • the vector and gel purified PCR amplified random inserts are ligated with Kp ⁇ X and SacX.
  • E. coli strain 5 BL21(DE3) tdk " [F ompT hsdSB( ⁇ ' mB )g ⁇ / dcm tdk (DE3)] Black et al, 1996, PNAS 9:3525-3529) is transformed with the ligated mixture.
  • the endogenous HSVl-tk in this strain has been deleted so only those bacteria that receive a HSVl-tk construct that is active will survive the initial positive selection screen.
  • the enzyme expressed must be an active enzyme. Any clone harboring a HSVl-tk construct that does not yield active HSVl-TK enzyme is eliminated.
  • the first selection of the library is based on HSVl-TK activity, by the method of Black and Loeb (1993, Biochem. 32:11618-11626). After transformation and recovery, the transformants are plated onto LB + carbenicillin (carb 50 ) to score total transformants.
  • HSVl-TK selection media 2% BBL Trypticase peptone, 0.5% NaCl, 0.8% Gel-Rite, 0.2% glucose, 50mg/mL carb, lOmg/mL 5'- fluorodeoxyuridine (FUdR), 2mg/mL thymidine, 12.5mg/mL uridine
  • LB+carb 50 LB+carb 50 in parallel.
  • the basis of the selection is that FUdR is phosphorylated by HSVl-TK to form FdUMP which is an inhibitor of thymidiylate synthase and thus the inhibition of dTMP production.
  • the requirement for dTMP can therefore only be filled by an active HSVl-TK enzyme.
  • HSVl-tk + clones are scored after incubation at 37 °C for 24 hours. Selection of FIAU sensitive clones is performed essentially as in the method of Black et al. (1996, PNAS 9:3525-3529) with minor modifications. All positive clones from above are picked and inoculated into HSVl-TK selection media in a 96 well plate.
  • All clones are serial diluted into saline and streaked onto HSVl-TK selection plates that contain 2mg/ml thymidine or HSVl-TK selection plates that contain lmg/ml of thymidine plus decreasing concentrations of FIAU (2mg/ml to Omg/ml).
  • HSVl-TK selection plates that contain 2mg/ml thymidine or HSVl-TK selection plates that contain lmg/ml of thymidine plus decreasing concentrations of FIAU (2mg/ml to Omg/ml).
  • This example illustrates a genetically engineered Salmonella expressing a mutant TK enzyme having an enhanced binding affinity for a pro-drug such as ganciclovir.
  • Salmonella are useful in one embodiment of the invention, i.e., in a method to detect and treat a solid tumor.
  • the enhanced-ganciclovir-affmity TK advantageously provides a therapeutic effect and is used in combination with Salmonella expressing TK having an affinity for radiolabeled FIAU which permits in vivo detection of a solid tumor upon administration of the bacteria.
  • Salmonella HSVl-TK where the TK enzyme is mutant and capable of binding ganciclovir at a higher affinity than wild type, is generated according the method published by Black et al(X996, PNAS 9:3525-3529). These authors demonstrated the isolation of an HSVl-TK mutant with a 40-fold increase in specificity for ganciclovir.
  • HSVl-TK 13y LIFDRHPIAALLCYP 1 /J (SEQ ID NO: 7) Mutant #75 159 LLLDRHPIACMLCYP 173 (SEQ ID NO:8)
  • the mutant sequence is introduced into the coding sequence of HSVl-tk by PCR-mediated site directed mutagenesis (Ausubel et al, 1995, in Short Protocols in Molecular Biology. third edition, NY: 18.6-18.22).
  • the mutations are introduced using two paired sets of primers, each of which generates approximately half of the complete sequence with a slight overlap and can be spliced together using a shared Sau3 AX site engineered to occur in the overlap:
  • Set 1 PCR primers (forward) 5'-GATCCCATGGCTTCGTACCCCGGCC-3' (SEQ ID NO:9) and (reverse) 5'-CGGCGATCGG ATGGCGGTCGAGGAGGAGGG-3' (SEQ ID NO:10);
  • Set 2 PCR primers (forward) 5'- CCATCCGATC GCCGTCATGC TGTGC-3' (SEQ ID NO:l 1) and (reverse) 5'-CTAGAAGCTTCAGTGGCTATGGCAGGGC-3' (SEQ ID NO: 12)
  • the HSVl-tk mutant described above (mutant #75; Black et al, 1996, P ⁇ AS 9:3525-3529) is a "super" mutant in that it has a higher affinity for ganciclovir and a lower affinity for thymidine than wild type HSVl-TK. Because this enzyme has these properties it is more toxic to cells primarily because it is less able to bind thymidine so there is less competition in the cells for thymidine and thus more enzyme available to bind and metabolize the ganciclovir pro-drug.
  • the optimal imaging times for in vivo tumor detection can be determined as described below.
  • mice (C57B/6) are implanted with C38 colon tumor cells and staged until the tumors are palpable. Salmonella expressing HSVl-TK or HSV1 -"super" TK are injected into the tumor and after five days [ l31 I]- FIAU is injected intravenously. Gamma camera imaging is performed at various times, for example 1 hr, 3 hr, 9 hr, 24 hr and 48 hr after FIAU administration. The accumulation of radiolabeled FIAU present in tumors and selected organs can also be determined in animals sacrificed for example at 24 and 48 hours post FIAU administration. Tissue is processed for determination of radioactive FIAU content inco ⁇ orated into DNA by acid precipitation.
  • Total tissue radioactivity is determined and the percent acid precipitated (% inco ⁇ orated into DNA) versus the percent acid soluble (% as uninco ⁇ orated FIAU, % uninco ⁇ orated phosphorylated FIAU, and % radiolabeled metabolites) is determined. Tissue radioactivity ( % dose/gram) is plotted versus time after FIAU injection.
  • Salmonella have been demonstrated to target a wide variety of solid tumors following systemic administration. However, some tumor types, including glioma and prostate cancers may benefit from local-regional administration. The utility of tumor- targeted genetically engineered HSVl-TK expressing Salmonella injected either i.v. or by direct intra-tumoral administration for detection, monitoring and/or therapy can be assessed. Although i.v. administration of genetically altered Salmonella with selective "biological" targeting of the tumor in vivo is a prefe ⁇ ed mode of application, it is clinically feasible to directly administer these bacteria into the tumor bed at time of surgery.
  • mice (C57B/6) are implanted with C38 colon tumor cells and staged until the tumors are palpable. Salmonella expressing HSVl-TK or HSV1 -"super" TK is injected directly into the tumor when tumors are palpable.
  • Salmonella expressing HSVl-TK or HSV1 -"super" TK is injected directly into the tumor when tumors are palpable.
  • [ 123 I]-FIAU is administered 1, 2, 4, 6, 10 or 14 days after Salmonella injection.
  • Gamma camera imaging is done at 1 hr, 3 hr, 9 hr, 24 hr and 48 hr after FIAU administration.
  • the accumulation of radiolabeled FIAU present in tumors and selected organs is also determined in animals sacrificed at 24 and 48 hours. Tissue is processed for determination of radioactive FIAU content inco ⁇ orated into DNA by acid precipitation.
  • tissue radioactivity is determined and the percent acid precipitated (% inco ⁇ orated into DNA) versus the percent acid soluble (% as uninco ⁇ orated FIAU, % uninco ⁇ orated phosphorylated FIAU, and % radiolabeled metabolites) is determined. Data is plotted as tissue radioactivity (% dose/gram) versus time after Salmonella administration, bacterial count (number of bacteria/gram) versus time after Salmonella administration and bacterial count/tissue radioactivity ratio (number of bacteria/%) dose) vs. time after Salmonella administration. 6.7. Other Tumor Models
  • tumor-targeted genetically engineered Salmonella for imaging can be assessed in a spectrum of solid tumors and tumor growth environments.
  • Orthotopic models include liver tumors arising from murine C38 colon carcinoma, lung tumors arising from B 16F 10 melanoma and breast tumors arising either from MD A-MB-231 or spontaneously in C-neu transgenic mice.
  • Bacteria targeting and imaging are compared in orthotopically placed tumors (liver, lung and breast) with s.c. tumors as a reference.
  • the spontaneous breast tumor model also provides metastases to multiple sites for assay in this system.
  • Salmonella bacteria expressing a labeled marker gene are administered intravenously in combination, e.g., simultaneously or separately within a specific time period, with labeled marker substrate and tumor detection follows the protocol described above. The animal is scanned and images of tumor are obtained. This imaging/treatment technique is broadly applicable to different orthotopic tumor sites and different tumor cell lines.
  • the gene containing streptavidin was obtained from the plasmid BBG9 R & D Systems, Minneapolis, MN).
  • the plasmid was first transformed to Salmonella strain YS501 and re analyzed for restriction digestion pattern, and then subsequently transformed to the tumor specific strain YS1646 with and without the Bacteriocin Release Protein (BRP) plasmid pSWl (Bio 101, Vista, CA). Streptavidin production in the supernatant was detected by inducing a streptavidin BRP-containing strain with 1.0 ⁇ g/ml mitomycin for 4 hrs followed by centrifugation.
  • BRP Bacteriocin Release Protein
  • Green fluorescent protein has been used to demonstrate the localization of tumors (Yang et al, 2000, Whole-body optical imaging of green fluorescent protein- expressing tumors and metastases. Proc Natl Acad Sci U S A. 97:1206-11), however, efficient means of delivery of GFP by systemically administered vectors has not been shown.
  • Green fluorescence protein was obtained from Clonetech (Palo Alto, CA) and subcloned into the ptrc99a expression vector (Pharmacia, Piscataway, NJ). The resulting trc-GFP plasmid was transformed into tumor-specific Salmonella strain 5 VNP20009. GFP-containing Salmonella strain VNP20009 were injected i.v. (2 x 10 6 ) into mice bearing HTB117 human lung carcinoma. After 5 days, the mice were sacrificed and tumors were examined for the presence of fluorescent bacteria using standard cryomicrotomy and fluorescent microscopy techniques (FIG. 3).
  • Figure 3A shows fluorescence of an untreated tumor and figure 3B shows fluorescence observed in tumors 10 following treatment with GFP-containing Salmonella strain VNP20009.
  • LacZY was mobilized into Salmonella YS1646 as a F' factor containing the 0 entire lacZ operon, including the lacY gene, which is responsible for lactose transport, as follows: Salmonella strain YS1646 was engineered to express the F' as follows such that the strain is able to be infected by phage. Salmonella strain YS501 (recD ' , chloramphenicol resistant) was mated with E. coli strain NH4104, which carries the F' plasmid containing the lactose operon, and Salmonella colonies were selected for chloramphenicol resistance, lac + 5 on minimal media containing lactose and chloramphenicol.
  • This strain designated YS501- F' (which is also met) was mated with Salmonella strain YS1646 (which is also pur) and Salmonella colonies were selected on minimal media containing lactose and purine but lacking methionine. This strain was designated YS1646-F'. The presence of the F' lac results in lac+ Salmonella. 0
  • the plasmid pSPLuc+ containing the gene for firefly luciferase was obtained from Promega (Madison, Wl).
  • pSPLuc ⁇ was digested with Ncol and Xbal and ligated into pTrc99a (Pharmacia, NJ).
  • This clone was then amplified by PCR using primers forward 5 5'-GTGTGCGGCCGCAATATTACTGAAATGAGCTGTTGACAATTAATCATCC-3' (SEQ ID NO: 13) and reverse 5'- GTGTGATATCGCATGCCTGCAGGTCGACTCTAGAATTAC-3' (SEQ ID NO: 14) which add Notl and EcoRV sites to the ends of the luciferase open reading frame.
  • the resulting product was subjected to restriction digestion with Notl and EcoRV and cloned into a promoterless ⁇ -galactosidase vector (Genbank # AF 192277 ) also cut with Notl and EcoRV to remove most of the ⁇ -galactosidase coding region.
  • the plasmid was transfe ⁇ ed to VNP20009.
  • the resulting clone has the partially constitutive trc promoter driving expression of the luciferase gene expression and results in functional expression of the luciferase protein. This demonstrates that tumor-specific bacteria can express luciferase.
  • Shigella flexneri (“ 'Shigella”), a gram-negative Shigella species which functions under both aerobic and anaerobic conditions, and Listeria monocytogenes (“Listeria”; ATCC strain 43251), a gram-positive species which functions under aerobic and anaerobic conditions, were grown at 37 °C either in Luria broth (LB) liquid media with shaking at approximately 225 revolutions per minute, or on LB solid media containing 1.5% agar.
  • LB consisted of 10 g tryptone, 5 g yeast extract and 10 g NaCl per liter. The pH of LB was adjusted to 7 using a IN solution of NaOH.
  • Streptococcus agalactiae (“Streptococcus”; ATCC strain 13813), a Streptococcus species which functions under both aerobic and anaerobic conditions, was grown in heart brain infusion ("BHI”; Difco) liquid media or on BHI solid media according to the methods described for Shigella and Listeria. Streptococcus was grown to an OD600 of 0.85, co ⁇ esponding to an estimated 2.5 x 10 8 c.f.u./ml. The bacterial culture was used without freezing for tumor-targeting experiments (Section 6.12.2 below).
  • Streptococcus colonies were inoculated into liquid culture as described above, grown to an OD600 of 1.2, and frozen at -80°C in 15% glycerol, co ⁇ esponding to 1.2 x 10 8 c.f.u./ml after thawing.
  • the bacteria were administered to mice having established melanomas, and the ratio of the concentration of bacteria in the tumor to the concentration of bacteria in the liver in each mouse was determined as follows.
  • mice were implanted with B16F10 murine melanoma tumor cells and the bacteria administered when the tumors weighed approximately lg, or approximately after 14-16 days post tumor cell implantation.
  • frozen Shigella and 5 Listeria cultures were thawed at room temperature and diluted into phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • Streptococcus administration a fresh culture was diluted into PBS.
  • the bacteria were administered intravenously into the mice in the following amounts: 2 x 10 6 c.f.u. o ⁇ Shigella; 1 x 10 4 or 1 x 10 5 c.f u. o ⁇ Listeria; and 1 x 10 5 or 1 x 10 6 c.f.u. of
  • mice were sacrificed and tumors and livers harvested and homogenized. Serial dilutions of the homogenates were then plated to the appropriate media for each species.
  • Counts are based on colony forming units (c.f.u.) given as the mean ⁇ standard error (SE).
  • mice were implanted subcutaneously with B16F10 murine melanoma tumor • ⁇ cells (5 x 10 5 cells per animal).
  • the Shigella, Listeria or Streptococcus were administered intravenously when the tumors weighed approximately 0.3 g.. Frozen stock of the bacteria (see Section 6.12.1) were thawed at room temperature and diluted into PBS.
  • the bacteria were administered intravenously into the mice in the following amounts: 2 x 10 6 c.f.u. of Shigella; X x 10 5 c.f.u. o ⁇ Listeria; and 1 x 10 6 c.f.u. o ⁇ Streptococcus.
  • Tumor volume was monitored approximately every 5 days. Tumor growth is graphically depicted as tumor volume versus time in FIG. 4. Tumor growth in mice receiving the tumor-targeted Listeria, Shigella or Streptococcus was inhibited by at least approximately 40% relative to tumor growth in control animals, and up to approximately 65% in the case o ⁇ Listeria. These data indicate that these facultative, gram positive and gram negative bacteria reduce tumor volume or inhibit tumor growth.

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Abstract

L'invention concerne des compositions et des procédés non invasifs permettant de détecter des tumeurs solides au moyen de bactéries ciblées sur des tumeurs. Plus précisément, l'invention concerne des compositions et des procédés permettant de détecter des tumeurs in vivo par administration d'un gène marqueur à une tumeur solide par le biais d'une population de bactéries ciblées sur des tumeurs. Elle concerne des procédés permettant de détecter des tumeurs solides par détection d'un composé incorporé aux bactéries, d'une infection provoquée par les bactéries sur le(s) site(s) de la tumeur, ou d'un antigène présent sur la surface des vecteurs bactériens. La détection d'une tumeur implique la détection d'un produit génique marqueur exprimé par les bactéries ciblées sur des tumeur. Ledit produit peut être détecté directement dans la tumeur à l'aide d'un fragment étiqueté qui interagit avec ce produit, ou d'un substrat marqueur étiqueté. Ainsi, si un produit génique marqueur est directement détectable, il peut l'être directement. Dans le cas contraire, un fragment étiqueté interagissant avec le produit génique marqueur est détecté, ou on utilise un substrat marqueur étiqueté, ce qui permet de détecter un métabolite marqueur étiqueté. On peut donc localiser ou détecter une tumeur par balayage d'un sujet afin de détecter le produit génique marqueur, un complexe étiqueté comprenant ledit produit et son fragment interactif, ou un métabolite marqueur étiqueté, respectivement, mettant ainsi en image la tumeur. Plus précisément, les bactéries ciblées sur les tumeurs atténuées sont des aérobies ou des anaérobies facultatifs modifiés pour coder le gène marqueur. En outre, l'invention concerne des compositions et des procédés de mise en image et de traitement simultanés une tumeur chez un sujet au moyen d'au moins une population d'organismes ciblés sur les tumeurs. Elle concerne également des procédés non invasifs, cliniquement applicables de mise en image de tumeurs, que l'on peut mettre en oeuvre par des techniques d'imagerie existantes pour surveiller et évaluer in vivo des traitements de cancer chez des sujets humains. Elle concerne enfin des trousses associées.
PCT/US2000/027397 1999-10-04 2000-10-04 Imagerie non invasive de tumeurs par des bacteries ciblees sur des tumeurs WO2001025399A2 (fr)

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AU79936/00A AU7993600A (en) 1999-10-04 2000-10-04 Non-invasive tumor imaging by tumor-targeted bacteria
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EP1369491A1 (fr) * 2002-06-05 2003-12-10 Aladar A. Szalay Microorganismes et cellules luminescents pour le diagnostic et la thérapie de maladies asociées avec du tissu blessé ou inflammé
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