JP4634710B2 - Microorganisms and cells for tumor diagnosis and treatment - Google Patents

Description

Detailed Description of the Invention

  The present invention relates to a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, such as a luminescent or fluorescent protein, and in certain embodiments, further for tumor therapy and / or elimination of metastatic tumors It relates to diagnostic and pharmaceutical compositions comprising DNA sequences encoding suitable proteins, such as cytotoxic or cytostatic proteins.

  The presence of bacteria in the tumor was reported about 50 years ago. Several publications have demonstrated early clinical findings that an unexpected large number of bacteria were found in tumors excised from human patients. Researchers argue that chronic infection can make cells susceptible to malignant growth. Chronic infection of various strains of Chlamydia is associated with lung and cervical cancer and malignant lymphoma. Another well-described link between the presence of certain bacterial species and cancer development is Helicobacter pylori in patients with gastric ulcers. Increased levels of H. pylori-related antibodies are found in patients with duodenal ulcers and adenocarcinoma of the stomach. These observations indicate the concomitant presence of bacteria at the tumor site; however, it was not clear whether the microorganism was responsible for tumor formation or whether the neoplastic tissue was prone to bacterial colonization. A strictly anaerobic bacterium, Clostridiumpasteurianum, injected intravenously into mice, replicated selectively in tumors, suggesting a hypoxic microenvironment at the necrotic center. Intravenous injection of attenuated Salmonella typhimurium mutants resulted in increased bacterial titers in tumor tissue compared to other organs in mice in histological and bacteriological analyses.

  Similarly, the presence of viral particles has been reported in human breast tumors excised as early as 1965. More recently, based on polymerase chain reaction (PCR) data, human papillomavirus has been claimed to be associated with anogenital tumors and esophageal cancer, breast cancer, and the most common cervical cancer. Presence of hepatitis C virus in human hepatocellular carcinoma, presence of Epstein-Barr virus in squamous cell carcinoma of Kirnura disease, presence of mouse mammary tumor virus-like particles (MMTV) in human breast cancer, macaque astrocytoma The presence of the SV40 virus in, and the presence of herpesvirus in turtle fibropapilloma. Surprisingly, the concentration of viral particles in the tumor varies between patients. The presence of human papillomavirus in squamous cell carcinoma of the esophagus ranges from 0 to 72% (10 to 15). In contrast to tumor tissue, no virus particles were found in the same patient's esophageal epithelial area without tumor, suggesting that the virus particles are located only in the tumor tissue.

  However, so far it has been clearly shown whether the above mentioned microorganisms are responsible for the development of tumor-like disorders (other than papillomavirus) or whether, for example, tumors or bacteria can attract and / or protect viruses. There wasn't. Thus, there was no basis for the use of such microorganisms for tumor diagnosis or treatment. Conventional tumor diagnostic methods such as MRI (magnetic resonance imaging) lack sensitivity and specificity, and treatment methods such as surgery are invasive and not very sensitive.

  Therefore, it is an object of the present invention to provide an efficient and reliable means for tumor diagnosis and treatment that overcomes the disadvantages of currently used diagnostic and therapeutic approaches.

According to the invention, this is achieved by the subject matter defined in the claims. When a vaccinia virus carrying the luminescent fusion gene construct rVV-ruc-gfp (LIVP strain) is injected intravenously into nude mice, it must be cleared from all organs within 4 days as determined by the disappearance of luminescence. Was found. In contrast, if the fate of injected vaccinia virus is similarly followed in nude mice with tumors grown from subcutaneously implanted C6 rat glioma cells, the viral particles are retained for a long time in the tumor tissue Was found and produced long-term luminescence. The presence and amplification of virus-encoded fusion proteins in the same tumor is monitored in living animals by observing GFP fluorescence under a stereomicroscope and by collecting luciferase-catalyzed luminescence under a low light video imaging camera It was done. Tumor-specific luminescence was detected in nude mice carrying subcutaneous C6 glioma implants with a size of 25-2500 mm ³ 4 days after virus injection. The signal became strong 4 days after injection and persisted for 30-45 days, indicating continued viral replication. Tumor accumulation of rVV-ruc-gfp virions is also seen in nude mice bearing subcutaneous tumors developed from transplanted PC-3 human prostate cells and mice with orthotopic transplanted MCF-7 human breast tumors It was. Furthermore, intracranial C6 rat glioma cell transplants in immunocompetent rats and MB-49 mouse bladder tumor cell transplants in C57 mice were also targeted by vaccinia virus. A cross section of C6 glioma showed that the luminescence was clustered in “patches” around the periphery of the tumor where there were rapidly dividing cells. In contrast, the cross section of the breast tumor showed fluorescent “islands” distributed throughout the tumor. In addition to primary breast tumors, small metastatic tumors are also detected outside the contralateral breast region and in nodules on the exposed lung surface, suggesting metastasis to the contralateral breast and lung. In summary, luminescent cells or microorganisms, such as vaccinia virus, can be used to detect and treat primary and metastatic tumors.

  Similar results were obtained with luminescent bacteria (Salmonella, Vibrio, Listeria, E. coli) that were injected intravenously into mice and immediately visible in all animals under a low light imager. Luminescence was not detected in both athymic (nu / nu) and immunocompetent C57 mice 36 hours after bacterial injection as a result of clearing by the immune system. In the skin wounds of animals injected intravenously, bacterial luminescence increases and remains detectable until 6 days after injection. In nude mice with tumors developed from transplanted C6 glioma cells, luminescence disappeared completely from the animals 36 hours after delivery of the bacteria, similar to mice without tumors. However, 48 hours after injection, unexpectedly, a strong and rapidly increasing luminescence originating only from the tumor area was observed. This observation indicates continuous bacterial replication in the tumor tissue. The degree of luminescence depends on the bacterial strain used. A homing-inprocess with persistent luminescence has also been shown in nude mice bearing prostate, bladder, and breast tumors. In addition to primary tumors, metastatic tumors could also be visualized as illustrated in the breast tumor model. Tumor-specific luminescence was also observed in immunocompetent C57 mice with bladder tumors and Lewis rats with brain glioma implants. Once in the tumor, it was observed that the luminescent bacteria were released into the circulation without being observed and recolonized following the transplanted tumor in the same animal. Furthermore, it has also been found that mammalian cells expressing the Ruc-gfp fusion protein are induced in glioma and proliferate upon injection into the bloodstream.

  These findings include: (a) multifunctional viral vector design useful for detection of tumors based on signals such as luminescence, and / or suppression of tumor development and / or angiogenesis, eg, signaled by quenching and (B) Opens the way for the development of bacterial and mammalian cell-based tumor targeting systems in combination with therapeutic gene constructs for the treatment of cancer. These systems have the following advantages: (a) they specifically target the tumor without affecting normal tissues; (b) expression and secretion of the therapeutic gene construct is preferably controlled by an inducible promoter Under, can switch secretion on or off; and (c) the location of the delivery system within the tumor can be confirmed by direct visualization prior to activating gene expression and protein delivery .

  Accordingly, the present invention relates to diagnostic and / or pharmaceutical compositions containing microorganisms or cells comprising a DNA sequence that encodes a detectable protein or a protein capable of inducing a detectable signal.

  In a preferred embodiment, the microorganism or cell of said diagnostic and / or pharmaceutical composition further comprises a tumor treatment and / or metastatic tumor, such as a cytotoxic protein, a cytostatic protein, a protein that inhibits angiogenesis or a protein that stimulates apoptosis. A DNA sequence that encodes a protein suitable for elimination. Such proteins are well known to those skilled in the art and further examples of suitable proteins are given below.

  Any microorganism or cell is useful in the compositions of the present invention if they replicate in the organism, are not pathogenic to the organism (eg, attenuated), are recognized by the organism's immune system, etc. is there. Examples of microorganisms useful in the present invention are bacteria and viruses. The term “bacteria” as used herein refers to bacteria that are not themselves tumor-targeted (ie, they cannot distinguish between cancerous cells or tissues and non-cancerous corresponding cells or tissues). . This is because the results of experiments leading to the present invention show that they accumulate in the tumor due to the fact that bacteria and the like are not exposed to attack by the host cell's immune system in this environment. A list of candidate bacteria that are useful for the purposes of the present invention and are not tumor targeted is shown in Table 1 below. One skilled in the art can readily identify such bacteria that are not tumor targeted by commonly available methods, such as those described in Section 6.1 of WO96 / 40238. Preferably, the bacterium is an intercellular bacterium such as E. coli, E. faecalis, Vibrio cholerae, Vibrio fischeri, Vibrio Harveyi, Lactobacillus spp., Pseudomonas spp. In the method of the invention, viruses and cells that are not tumor-targeted, in particular mammalian cells, are preferred. Particularly preferred are cytoplasmic viruses.

  In a particularly preferred embodiment, the diagnostic and / or pharmaceutical composition contains a microorganism or cell comprising a DNA sequence encoding a photoprotein and / or a fluorescent protein.

  The term “DNA sequence encoding photoprotein and / or fluorescent protein” as used herein also includes DNA sequences encoding photoprotein and fluorescent protein as fusion proteins.

  In an alternative preferred embodiment of the diagnostic and / or pharmaceutical composition, the protein encoded by the DNA sequence is a cellular receptor capable of binding to a ligand that can be a diagnostic or therapeutic ligand. The ligand may be a protein (including a large or small peptide, an antibody, etc.), a synthetic compound (such as a synthetic steroid analog), and the like. Therefore, the in vivo location of the labeled ligand in living animals and human patients can be visualized in real time, for example by SPECT or PET. Most any known protein ligand-receptor pair for tumor labeling is useful in the methods of the invention. Preferably, to increase specificity, the mutant protein ligand (or analog if it is a compound) or mutant ligand receptor can be genetically or chemically manipulated so that they are Does not bind to sex molecules. In addition to increased specificity, these variants / analogs also limit deleterious effects on normal host physiology.

In a more preferred embodiment of the diagnostic and / or pharmaceutical composition of the present invention, the ligand is a radionuclide labeled ligand. The ligand is, for example, a radionuclide-labeled ligand that is specifically expressed on the surface of a tumor cell, eg, carrying a receptor protein gene construct, eg, following intravenous delivery of engineered bacterial, viral, or mammalian cells It is useful for tumor visualization by single photon emission computed tomography (SPECT) or positron-computed tomography (PET) resulting from the binding of radionuclide-labeled ligands to their receptors. Radionuclides can be useful for conventional tumor scintigraphy, PET and possibly internal radiation therapy of tumors. Examples of radionuclides useful in the present invention are (a) β ⁺ -emitters such as ¹¹ C, ¹³ N, ¹⁵ O or ⁶⁴ Cu or (b) γ-emitters such as ¹²³ I. For example, other radionuclides that can be used as tracers for PET are ⁵⁵ Co, ⁶⁷ Ga, ⁶⁸ Ga, ⁶⁰ Cu (II), ⁶⁷ Cu (II), ⁵⁷ Ni, ⁵⁵ Co, ⁵² Fe, ¹⁸ F, etc. Is included.

SPECT and PET are sensitive techniques that may be useful for tumor imaging according to the present invention. Both SPECT and PET can detect trace amounts of β ⁺ and γ emissions from radionuclides. PET is even more sensitive than SPECT. In experiments with small experimental animals, tumor imaging is performed using a microPET apparatus, which is commercially available, for example, by Concorde Microsystems (Knoxville, TN).

Examples of useful radionuclide-labeled drugs include ⁶⁴ Cu-labeled engineered antibody fragments (Wu et al., PNASUSA 97 (2002), 8495-8500), ⁶⁴ Cu-labeled somatostatin (Lewis et al., J. Med. Chem. 42 ( 1999), 1341-1347, ⁶⁴ Cu- pyruvaldehyde - bis (N4-methyl ^{thiosemicarbazone) (64 Cu-PTSM) (} Adonai et al, PNASUSA 99 (2002), 3030-3035 , 52 Fe- citric acid (Leenders al, J.Neural Transm.suppl.43 (1994), 123-132 , 52 Fe / 52m Mn- citric acid) (Calonder et al, J.Neurochem.73 (1999), 2047-2055) and ⁵² Fe- labeled water Iron oxide (III) -sucrose complex (Beshara et al., Br. J. Haematol. 104 (1999), 288-295, 296-302).

  To apply a radionuclide-labeled ligand for tumor detection, a gene encoding a receptor protein to which the ligand can bind is delivered by an intravenously injected bacterial, viral or mammalian cell of the invention. Since specific intravenously injected bacteria, viruses and mammalian cells specifically replicate in tumors are shown in the examples below, receptor protein expression in tumors is targeted for targeting by radionuclide labeled ligands. Label the tumor.

For example, in the case of vaccinia virus, the virus can be used to transport a genetic construct that encodes a receptor protein that can specifically bind to a ligand to allow efficient tumor detection. Intravenous injection of recombinant vaccinia virus allows receptor gene delivery and surface expression of the receptor protein in tumor tissue. The radionuclide labeled ligand is then injected intravenously into the host. Specific binding between the radionuclide-labeled ligand and its receptor expressed on the tumor cell surface allows detection of tumors based on β ⁺ -or γ-luminescence derived only from the tumor. Labeling the tumor with a radionuclide allows easy discrimination between the tumor and normal tissue. Therefore, a cue β ⁺ -or γ-detector attached to a surgical blade holder can be designed. Based on β ⁺ -or γ-luminescence signals, labeled tumors can be excised cleanly with minimal removal of normal tissue.

Gallium-67 is particularly preferred for the purposes of the present invention. Gallium-67 ( ⁶⁷ GA) can be used for diagnostic imaging using PET, SPECT, or scintigraphy. It is known for its ability to accumulate in inflammatory lesions and tumors, particularly lymphomas, and many other types of tumors such as pancreatic tumors, lung tumors and the like. ^The mechanism of ⁶⁷ Ga uptake has been proposed through both transferrin-dependent and transferrin-independent pathways. In the transferrin-dependent pathway, transferrin receptor overexpression has been shown to significantly increase ⁶⁷ Ga uptake by tumor cells. Furthermore, anti-transferrin receptor antibodies significantly block ⁶⁷ Ga by tumor cells. For the imaging of small tumors, very high concentrations of ⁶⁷ Ga are required to overcome the background signal. Thus, in this case, following intravenous injection of the virus, the use of a recombinant vaccinia virus carrying the transferrin receptor gene construct to specifically overexpress the transferrin receptor on the surface of tumor cells of a surviving animal or human patient. preferable. ⁶⁷ Ga is also delivered intravenously. The high level of ⁶⁷ Ga accumulation in tumor cells with help from overexpressed transferrin receptor serves to significantly improve tumor detection capabilities in surviving animals and human patients.

  In another preferred embodiment, the diagnostic and / or pharmaceutical composition of the present invention comprises a DNA sequence encoding a protein capable of inducing a signal that is detectable by magnetic resonance imaging (MRI), eg, a metal binding protein. Contains containing microorganisms or cells. In addition, the protein may bind to contrast agents, chromophores, ligands or compounds that are required for tissue visualization. Preferably, the protein is a cellular receptor capable of binding to a paramagnetic- or superparamagnetic-metal-labeled ligand. For example, a paramagnetic- or superparamagnetic-metal-labeled ligand specific on the surface of a tumor cell following intravenous delivery of an engineered bacterial, viral, or mammalian cell of the invention carrying the receptor protein gene construct Tumor visualization by MRI resulting from binding to their receptors expressed in has several advantages. The high level of accumulation of these metals in the tumor facilitates tumor detection. Virtually any paramagnetic or superparamagnetic metal can be used for this purpose. Preferably, due to the systemic toxicity of bare heavy metals, paramagnetic or superparamagnetic is carefully chosen to keep harmful effects to a minimum. In most currently available contrast agents, the metal particles are either linked to a chelate or coated with a polymer and are used much more safely than bare particles. Therefore, the use of chelated or polymer-coated metal tagged ligands is preferred.

  Methods for making protein-linked contrast agents are well known to those skilled in the art, for example, protein ligands (or other compound ligands) are first chemically attached to a chelate (eg, diethylenetriaminepentaacetic acid (DTPA)). The The chelate-protein complex is then labeled with a metal. Similar labeling methods are used in making gallium-labeled somatostatin analogs, and are used to produce indium-111-labeled low density lipoproteins using DTPA-bis (stearylamide). Details of protein modification with chelates used in contrast agents have been previously described. For example, modification of a protein with the bifunctional chelate 2- (4-isothiocyanotobenzyl-6-methyl) diethylenetriamine-pentaacetic acid (MX-DTPA) (Mirzadeh et al., Bioconjug. Chem. 1 (1990), 59-65 ) And the modification of proteins with diethylenetriamine-pentaacetic acid cyclic anhydride (cDTPAA) (Duncan and Welch, J. Nucl. Med. 34 (1993), 1728-1738).

  Alternatively, MRI can be performed by the following approach.

(A) Modified gadolinium activation by enzyme / protein delivery This approach is based on the principle of Gene-Directed Enzyme Prodrug Therapy (GDEPT), where the enzyme / protein is The non-toxic prodrug delivered to is activated into an active toxic drug. Specific activation of weak relaxed MRI contrast agents to strong relaxed states by enzymes / proteins in tumors enables tumor detection. Enzyme / protein delivery to the tumor is achieved by engineered bacteria, viruses, and mammalian cells injected intravenously, which includes a gene encoding β-galactosidase (or any other related enzyme). Possess. β-galactosidase can be either extracellularly secreted or expressed on the surface of bacteria or mammalian cells. An example of an MRI agent that can be cleaved by β-galactosidase and used in MRI imaging for tumor detection is (4,7,10-tri (acetic acid) -1- (2-β-galactopyranosylethoxy ) -1,4,7,10-tetraazacyclododecane) gadolinium (Egad) (Moats et al., 1997). In this compound, the galactopyranose residue is located at the 9th coordination site of the Gd ³⁺ ion. Because of this obstacle, water protons were excluded from interaction with Gd ³⁺ ions, and therefore the effect on T1 relaxation time was reduced. In the presence of β-galactosidase, the enzyme cleaves galactopyranose from the chelate, releasing the coordination site, allowing an irreversible transition of the contrast agent from a weak relaxed state to a strong relaxed state. Since bacterial-, viral-, or mammalian cell-based enzyme expression occurs only in tumors, enzyme-mediated relaxation state transitions also occur only in tumors. Therefore, this enzyme-mediated activation of MRI contrast agents can be used for tumor detection.

In addition to the MRI contrast agents described above, similar new contrast agents can be developed in which other types of residues can be attached to the chelate. These residues can be removed by its corresponding enzyme (similar to the removal of galactopyranose by galactosidase) (eg, α-mannosidase, α- and β-glucosidase, β-glucuronidase), the number of Gd ³⁺ ions Release 9 coordination sites and change T1 relaxation time for tumor detection. Genes encoding these enzymes can all be delivered by the engineered bacteria, viruses, or mammalian cells of the present invention. In addition, new contrast agents using metals other than gadolinium can be developed for enzyme-activated MRI tumor imaging.

  This approach can be combined with treatment as shown in the following section.

(B) First, manipulation of a delivery vector system for activation of a contrast agent (eg, modified gadolinium), followed by the injection of a prodrug injected with the same constitutively expressed enzyme (eg, β-galactosidase) Activation-1 gene product based detection and treatment system (OGPBDTS)
For example, a gadolinium-based contrast agent can be activated by an enzyme (eg, β-galactosidase) as described above, which can be used for tumor detection. The intravenously delivered prodrug (CBI, TMI, PCI, etc.) (described in US Pat. No. 5,646,298) is then cleaved by the same β-galactosidase within the tumor, and the tumor for cancer treatment Can produce active cytotoxic drugs against cells.

(C) Delivery vector system manipulation in two gene product-based detection and therapy systems (TGPBDTS) Gene 1 is linked to a constitutive promoter and senses contrast agents (eg, metal binding proteins, modified gadolinium, or other agents) Gene 2 is linked to an exogenously active promoter, which is silenced without an activating drug, and only after the detection vector system is found to be positive by MRI, Switch on. Activation of the vector-based promoter gene construct is accomplished by injection or by oral administration of an activator drug. Repeated injection of contrast agent allows real-time monitoring of tumor size and metastatic location during the treatment procedure.

  Preferably, in order to transfect cells, DNA sequences encoding the above-mentioned (diagnostic or therapeutic) proteins, for example photoproteins and / or fluorescent proteins, are present in vectors or expression vectors. Those skilled in the art are familiar with the examples. The DNA sequence can also be included in a recombinant virus that contains an appropriate expression cassette. Suitable viruses that can be used in the diagnostic or pharmaceutical composition of the present invention include baculovirus, vaccinia, Sindbis virus, Sendai virus, adenovirus, AAV virus or parvovirus such as MVM or H-1. . The vector can also be a retrovirus such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV or GaLV. For expression in mammals, a suitable promoter is, for example, the human cytomegalovirus “early promoter” (pCMV). In addition, tissue and / or organ specific promoters are useful. Preferably, for example, a DNA sequence encoding a photoprotein and / or a fluorescent protein is operably linked to a promoter that allows high expression. Such promoters, for example inducible promoters, are well known to those skilled in the art. Preferably, the construct is inserted into the bacterial genome by appropriate integration. When such constructs are produced in mammalian cells using mammalian vectors, cell lines that are stably transformed with single copy insertions are produced for long-term expression in tumor cells.

  General methods known to those skilled in the art can be used to generate the above DNA sequences and to construct expression vectors or viruses containing the DNA sequences. These methods include, for example, in vitro recombination techniques, synthesis methods, as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. And in vivo recombination methods. Methods for transfection of cells using the above vectors, methods for selecting transfectants by phenotype and methods for expressing DNA sequences are known to those skilled in the art.

  One skilled in the art knows DNA sequences that encode proteins, such as photoproteins or fluorescent proteins that can be used in the diagnostic and / or pharmaceutical compositions of the invention. From Vibrio harveyi (Belas et al., Science 218 (1982), 791-793) and Vibrio fischerii (Foran and Brown, Nucleic acids Res.) That allow tracking of bacteria, viruses or mammalian cells based on luminescence over the past decade. 16 (1988), 177) bacterial luciferase, firefly luciferase (de Wet et al., Mol. Cell. Biol. 7 (1987), 725-737), aequorin from Aequoreavictoria (Prasher et al., Biochem. 26 (1987), 1326 -1332), light emission from Renilla luciferase from Renilla reniformis (Lorenz et al., PNASUSA 88 (1991), 4438-4442) and green fluorescent protein from Aequorea victoria (Prasher et al., Gene 111 (1987), 229-233) The identification and isolation of structural genes encoding proteins has been described. Transformation and expression of these genes in bacteria allows detection of bacterial colonies with the aid of a low light imaging camera or individual bacteria under a fluorescent microscope (Engebrecht et al., Science 227 (1985), 1345- 1347; Legocki et al., PNAS 83 (1986), 9080-9084; Chalfie et al., Science 263 (1994), 802-805).

  The luciferase gene is expressed in various organisms. Luminescence-based promoter activation using lux AB fused to a nitrogenase promoter has been shown in Rhizobia present in the cytoplasm of infected nodule cells by low light imaging (Legocki et al., PNAS83 (1986), 9080 -9084; O'Kane et al., J. Plant Mol. Biol. 10 (1988), 387-399). Fusion of the luxA and luxB genes resulted in a fully functional luciferase protein (Escher et al., PNAS86 (1989), 6528-6532). This fusion gene (Fab2) was introduced into Bacillus subtilis and Bacillus megatherium under the xylose promoter, then fed to insect larvae and injected into the insect hemolymph. Luminescence imaging was performed using a low light video camera. Under low light imager by localizing Pseudomonas or Ervininiaspp. Infection, pathogenic bacterial migration and localization in transformed Arabidopsis plants (which carry the pathogenic activation PAL promoter-bacterial luciferase fusion gene construct) As well as in tomato plants and many potatoes (Giacomin and Szalay, Plant Sci. 116 (1996), 59-72).

  All luciferases expressed in bacteria require exogenous addition of a substrate such as decanal or coelenterazine for luminescence. In contrast, visualization of GFP fluorescence does not require a substrate, but an excitation light source is required. More recently, gene clusters (including luxCDABE) encoding bacterial luciferases and proteins for providing intracellular decanals have been developed by Xenorhabdusluminescens (Meighen and Szittner, J. Bacteriol. 174 (1992), 5371-5381) and Photobacterium leiognathi ( Lee et al., Eur. J. Biochem. 201 (1991), 161-167), transmitted to bacteria, and produces continuous luminescence independent of exogenously added substrate (Fernandez-Pinas And Wolk, Gene 150 (1994), 169-174). Bacteria containing the complete lux operon sequence allow visualization and localization of bacteria in living mice when injected intraperitoneally, intramuscularly or intravenously, and luciferase luminescence can penetrate the tissue and externally It can be detected (Contag et al., Mol. Microbiol. 18 (1995), 593-603).

  Preferably, the microorganism of the present invention is a bacterium that is not tumor-targeted, for example an attenuated bacterium. A list of candidate bacterial strains that may be useful for the present invention (ie, BMPT (bacterial mediated protein therapy) and tumor imaging) and that cannot be tumor targeted is listed in Table 1. One skilled in the art can readily identify such bacteria that are not tumor-targeted by commonly available methods, such as those described in Section 6.1 of WO96 / 40238.

  Bacteria such as microorganisms associated with milk and cheese that are naturally consumed by most individuals and have intrinsic anti-tumor activity when injected directly into solid tumors for safe and direct applicability to humans Cell lines are preferred. Such bacteria also include bacteria naturally isolated from various types of tumors or from human tumors that have developed coexistence (symbiosis) with certain types of tumors. Extracellular secretion of therapeutic proteins by bacteria is mediated by signal peptides or endogenous protein secretion pathways. To provide additional safety to BMPT, bacterial inducible promoters, such as the IPTG-inducible lac promoter, can be used to exogenously regulate therapeutic protein production by bacteria. IPTG-regulated expression of mammalian proteins in bacteria is well documented. IPTG has also been shown to be functional for promoter activation in animals. In addition to the lac promoter, other examples of promoters that allow regulation of gene expression in bacteria include the ara promoter (activated by arabinose) and the PLtetO-1 promoter (activated by anhydrotetracycline (aTc)) (Lutz And Bujard, Nucleic Acids Res. 25 (1997), 1203-1210).

  Attenuated Vibrio cholerae is particularly preferred.

A further example of a bacterium useful for the preparation of a diagnostic composition for monitoring tumor imaging or therapeutic tumor treatment is a magnetic bacterium (metal-bound bacterial mediated tumor detection (MBBMTD)). Such bacteria allow tumor detection by accumulation of iron-based contrast agents. Magnetic bacteria can be isolated from fresh marine sediments. These can produce magnetic particles (Fe ₃ O ₄ ) (Blakemore, Anuu. Rev. Microbiol. 36 (1982), 217-238). To do so, they have an efficient iron uptake system that makes available both insoluble and soluble forms of iron. Magnetospirillummagneticum AMB-1 is an example of such a magnetic bacterium that is isolated and cultured for magnetic particle production (Yang et al., Enzyme Microb. Technol. 29 (2001), 13-19). These magnetic bacteria (naturally occurring or genetically modified), when injected intravenously, have the same tumor accumulation ability as Vibriocholera, for example, these bacteria have iron-based imaging in the tumor Can be used to accumulate agents and then allow tumor detection by MRI. Similarly, other naturally isolated bacterial metal-accumulating strains can be used for tumor targeting, absorption of metal from contrast agents, and eventually tumor imaging.

  Alternatively, viruses such as vaccinia virus, AAV, retrovirus are also useful in the diagnostic and therapeutic compositions of the present invention; see Table 2 listing examples of viruses useful in the present invention.

  Preferably, the virus is a vaccinia virus.

  Preferably, the cells of the diagnostic or therapeutic composition of the present invention are mammalian cells such as stem cells that can be autologous or xenogeneic to the organism. Examples of suitable cell types are shown in Table 3.

  In a further preferred embodiment of the diagnostic and / or therapeutic composition of the invention, the luminescent or fluorescent protein is luciferase, green fluorescent protein (GFP) or red fluorescent protein (RFP).

  In particularly preferred embodiments, the microorganism or cell of the diagnostic and / or pharmaceutical composition of the invention further comprises a gene encoding a substrate for luciferase. In an even more preferred embodiment, the microorganism or cell of the diagnostic and / or pharmaceutical composition of the invention comprises Renilla luciferase (ruc) and Aequorea gfp cDNA under the control of a strong synthetic early / late (RE / L) promoter of vaccinia. Contains a ruc-gfp expression cassette or luxCDABE cassette containing the sequence.

  A preferred use of the microorganisms and cells is the preparation of a diagnostic composition for tumor imaging. The diagnostic composition of the present invention can be used, for example, during surgery to identify tumors and metastases. Furthermore, the diagnostic compositions of the present invention are useful for monitoring therapeutic tumor treatment. For example, suitable devices for analyzing the localization or distribution of luminescence and / or fluorescent proteins in an organism, organ or tissue are well known to those skilled in the art and are further described in the references cited above and in the examples below. The In addition, microorganisms and cells can be modified to bind metals, which is useful for MRI techniques and makes tumor localization more clear.

  The invention also relates to antibacterial agents (eg, antibacterial or antiviral compounds) for monitoring tumor imaging or therapeutic tumor treatment, such as peptides or proteins fused to a detectable protein. This diagnostic composition is useful for tumor detection (peptide-bound tumor targeting vector detection (PLTTVD)) via the binding of bacteria localized to tumors and antimicrobial compounds (eg, luminescent or radiolabeled antimicrobial peptides / proteins) It is.

  The present invention also relates to the use of antibacterial agents (eg antibacterial or antiviral compounds) for the preparation of tumor therapeutic pharmaceutical compositions, eg peptides or proteins fused to therapeutic proteins.

Examples of naturally occurring antibacterial proteins include ubiquicidin (UBI, 6.7 kDa) and lactoferrin (hLF, 77 kDa). UBI was initially isolated from mouse macrophages and later from human airway epithelial cells. hLF is found in mucosa and neutrophils and is known to bind to the surface structure of both gram negative and gram positive bacteria. Small synthetic peptides containing bacterial binding domains of UBI or hLF are designed for imaging bacterial infections that can be distinguished from sterile inflammation (Nibbering et al., Nucl. Med. Commun. 19 (1998), 1117- 1121; Welling et al., Nucl. Med. Biol. 29 (2000), 413-422; Welling et al., Eur. J. Nucl. Med. 27 (2002), 2929-301). These peptides are radiolabeled with ^99m Tc and allow real-time visualization in live animals using two-dimensional scintigraphy. For example, a two-dimensional gamma camera (eg, Toshiba GCA 7100 / UI, Tokyo, Japan) equipped with a low energy universal parallel hole collimator can be used to visualize ^99m Tc labeled antimicrobial peptides in live animals or humans. In order to utilize these synthetic peptides for tumor detection, a tumorous individual is first infected with a specific strain of extracellular bacteria. Following bacterial colonization of the tumor, a radiolabeled compound, such as a peptide, is delivered intravenously. Specific binding of labeled peptides to bacteria in the tumor can be visualized in real time by scintigraphy, thus allowing for localization of the tumor in humans and animals. ^In addition to labeling with ^99m Tc, compounds such as synthetic peptides can also be labeled with paramagnetic or superparamagnetic metal particles for visualization using MRI, or compounds can be non-invasive visualization with SPECT or PET Can be labeled with a radionuclide (eg, ⁵⁵ CO, ⁶⁸ Ga, ⁶⁰ CU (II), ¹²³ I, etc.). In addition, compounds such as synthetic peptides can also be used with fluorescent probes, fluorescent proteins (eg, green fluorescent protein (GFP) or DsRed), or luminescent proteins (eg, Renilla luciferase, firefly luciferase) for real-time visualization in an individual. Can be tagged.

  In addition to UBI and hLF, there are many other examples of antimicrobial peptides / proteins produced by bacteria, plants, invertebrates, and vertebrates including humans that are useful for the purposes of the present invention (Schroder, Cell. Mol. Life Sci. 56 (1999), 32-46; Cole and Ganz, Biotechniques 29 (2000), 822-826, 828, 830-831), for example, pediocin PA-1, derived from lactic acid bacteria, Bacillus Graviscidin S from Brevis, protegrin-1 from porcine leukocytes, SMAP-29 from sheep myeloid cells, buforin I and buforin II from Asian toads, β-defensin, LL-37, human cathelicidin (cathelicidin) ) Fragments derived from hCAP-18, alasine I derived from catfish, granulysin, PAMP derived from Propionibaeterium jensenii, and the like. Based on these naturally occurring antibacterial proteins, small peptides can be designed and synthesized to eliminate the bacterial killing effect of the original protein while retaining the ability to bind bacteria. These synthetic peptides can then be used for bacteria-mediated tumor detection.

  Similar to the detection of bacteria with antibacterial compounds (eg, peptides / proteins), antiviral compounds (eg, peptides / proteins) can be used to detect viral particles in tumors. Lactoferricin is one example of an antiviral peptide / protein that can be used to design peptides for detecting viral particles. In addition to the antimicrobial peptides / proteins discussed, radiolabeled or photoprotein / probe labeled antibody fragments can also be used to target bacteria or viruses that are localized to the tumor for tumor detection.

  In addition, the antimicrobial compound can be fused to a metal binding protein (fusion peptide binding tumor targeting vector detection (PLTTVD)) for detecting bacteria, viruses, etc. of the invention in tumors. After allowing the fusion construct to bind to the engineered bacteria or the like in the tumor, a metal agent (which can be used for detection by MRI, PET or SPECT) is injected intravenously. Indirect tumor detection is enabled by the binding and absorption of metal agents by bacteria and the like.

  The invention also relates to a pharmaceutical composition comprising a microorganism or cell comprising a DNA sequence encoding a cellular receptor that allows binding of the ligand and further a ligand that binds to a toxin (= chimeric toxin). Suitable toxins that can be conjugated to a ligand are well known to those skilled in the art. Some chimeric toxins have been previously described. First, a Tfn-CRM107 chimeric toxin was prepared by conjugating transferrin to mutant diphtheria toxin CRM107 (Johnson et al., J. Neurisurg. 70 (1989), 240-248), which targets the transferrin receptor. To do. Second, 425.3-PE antitoxin was prepared by conjugating anti-EGF receptor antibody to total Pseudomonas exotoxin A (Myklebust et al., Cancer Res. 53 (1993), 3784-3788). Third, Tfn-PE was prepared by conjugating transferrin to Pseudomonas exotoxin (Hall et al., J. Neurosurg. 76 (1992), 838-844; Hall et al., Neurosurgery 34 (1994), 649-656). It has been shown in animal models and human patients that these chimeric toxins have antitumor activity against medulloblastoma, small cell lung cancer, glioma xenografts and intracranial tumors. Thus, for example, recombinant vaccinia viruses can be designed to specifically express transferrin receptors on the surface of tumor cells to allow the use of these receptor-targeted chimeric toxins for anti-tumor treatment.

  The present invention also relates to a pharmaceutical composition comprising (a) a microorganism or cell comprising a DNA sequence encoding a cellular receptor that allows binding of the ligand and (b) the ligand bound to a therapeutic protein. Suitable therapeutic proteins and methods for generating therapeutic ligand fusion proteins by binding therapeutic proteins to ligand proteins are well known to those skilled in the art. Examples of suitable therapeutic proteins are shown in Tables 4 and 5.

  The binding of therapeutic ligand fusion proteins to the ligand receptor is targeted by the therapeutic protein to the tumor tissue, since the tumor tissue can be labeled with the ligand receptor using genetically engineered bacteria, viruses or cells injected intravenously Make it possible. Intracellular bacteria and viruses are particularly useful for labeling tumor cell surfaces with engineered receptor proteins. However, due to the “rolling effect” of therapeutic proteins on neighboring cells, extracellular bacteria and mammalian cells can also be used to deliver therapeutic proteins.

  In the case of gene-directed enzyme prodrug therapy (GDEPT) (see below), for example, extracellular bacteria can be used to secrete enzyme ligand fusion proteins. The tumor cell surface can be labeled with a receptor protein by a virus. Following intravenous delivery of the prodrug, cells expressing the receptor are not only bathed and killed with the active drug, but the surrounding tumor cells (which may not be labeled with the receptor) are also killed.

  Viruses such as vaccinia virus can also be used to label tumor cell surfaces with receptor proteins. For example, tumor tissue is specifically infected by intravenously injected genetically engineered vaccinia virus (eg, carrying a transferrin receptor gene) (which also encodes a single peptide for cell surface expression) . Transferrin receptor expression on the tumor cell surface marks these cells for targeting by the therapeutic ligand fusion protein. In this case, the ligand can be a transferrin protein and the therapeutic protein can be Pseudomonas exotoxin (or any other cytotoxic therapeutic protein). Tumor cell internalization of the transferrin / transferrin receptor pair allows for internalization of the therapeutic protein, which then specifically confers cytotoxicity to the tumor cells. The transferrin / transferrin receptor pair is just one of many examples of ligand receptor pairs that can be used. Furthermore, mutant ligands and mutant receptors with very specific affinity for each other can be used to avoid binding to endogenous proteins.

  Further examples of suitable proteins are human endostatin and chimeric PE37 / TGF-α fusion proteins. Endostatin is the carboxy-terminal peptide of collagen XVIII, which has been characterized (Ding et al., PNAS USA 95 (1998), 10443). Endostatin has been shown to inhibit endothelial cell proliferation and migration, induce endothelial cell G1 arrest and apoptosis in vitro, and has antitumor effects in various tumor models. Intravenous or intramuscular injection of viral DNA and cationic liposome-complexed plasmid DNA encoding endostatin resulted in limited endostatin expression levels in tumors. However, intratumoral injection of purified endostatin shows significant inhibition of tumor growth. Pseudomonas exotoxin is a bacterial toxin secreted by Pseudomonasaeruginosa. PE manifests its cytotoxic effect by inactivating elongation factor 2 (EF-2), thereby producing a block of protein synthesis in mammalian cells. Single chain PE is functionally divided into three domains: domain Ia is required for binding to cell surface receptors, domain II is required to transfer toxins to the target cell cytosol, and domain III is EF. Manages cytotoxicity by inactivating -2. PE40 is derived from a wild type Pseudomonas exotoxin that lacks the binding domain Ia. Other proteins such as antibody fragments or protein ligands can be inserted in place of the binding domain. This makes the PE40 ligand fusion protein specific for its receptor. One of the chimeric toxins produced by highly specific genetic engineering is the TGFα / PE40 fusion protein, in which the C-terminus of the TGFα polypeptide is fused in-frame with the N-terminus of the PE40 protein. TGFα is one of the epidermal growth factor receptor (EGFR) ligands and has been shown to be selectively expressed on the surface of various tumor cells. TGFα-PE40 fusion protein has been shown to be very toxic to tumor cells with elevated EGFR on the cell surface, while being hardly toxic to nearby cells that present little surface EGFR ing. The toxicity of TGFα-PE40 chimeric protein depends on the proteolytic processing step to convert the chimeric protein to its active form, which step is performed by the target. In order to overcome the need for proteolysis, new chimeric toxic proteins have been constructed by Theuer and colleagues (J. biol. Chem. 267 (1992), 16872). The new fusion protein is called PE37 / TGFα, which is more toxic to tumor cells than the TGFα-PE40 fusion protein.

  In particularly preferred embodiments of the diagnostic and / or pharmaceutical compositions of the invention, the ligand is not a naturally occurring ligand, but any compound (eg, antibody) that can specifically bind to the receptor. The term “antibody” preferably relates to antibodies consisting essentially of pooled monoclonal antibodies with different epitope specificities as well as separate monoclonal antibody preparations. Monoclonal antibodies are produced from antigens containing fragments of specific receptors by methods well known to those skilled in the art (see, for example, Koehler et al., Nature 256 (1975), 495). As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab) refers to intact molecules and antibody fragments (eg, Fab and F (ab ′)) that can specifically bind to a receptor. 2 fragments). Fab and F (ab ′) 2 fragments lack the intact antibody Fc fragment, disappear more rapidly from the circulation, and have less nonspecific tissue binding than intact antibodies (Wahl et al., J. Nucl. Med 24: 316-325 (1983)). Accordingly, these fragments, and the products of FAB or other immunoglobulin expression libraries are preferred. Furthermore, the antibodies of the present invention include chimeric, single chain, and humanized antibodies.

  Antibody ligands are useful, for example, for anti-tumor therapy using a fusion protein between an antibody and a therapeutic protein (eg, an immunotherapeutic protein): First, a gene encoding a cell surface receptor is injected intravenously. Delivered to tumor cells of living organisms by genetically engineered bacteria and viruses. Such receptors can be, for example, transferrin receptor, EGF receptor, somatostatin receptor, and the like. Receptor overexpression on the cell surface after bacterial or viral infection can be used to mark tumor cells. Next, a fusion protein (immunotherapy protein) between the antibody, preferably an antibody fragment (specific for overexpressed surface receptors) and a therapeutic protein (eg any type of toxin, see Table 5) Prepared. Intravenously injected fusion proteins bind to the marked tumor cells and are cytotoxic to these cells in the tumor tissue.

  The present invention also provides a microorganism or cell as described above and further (a) an antimicrobial compound fused to a protein suitable for tumor therapy and / or metastasis and / or (b) a detectable protein or detectable The present invention relates to diagnostic and / or pharmaceutical compositions containing antimicrobial compounds fused to proteins capable of inducing signals. Anti-tumor treatments using such fusion proteins between antibacterial compounds (eg, peptides / proteins) and therapeutic proteins encode hybrid proteins between antibacterial peptides / proteins and therapeutic proteins (see Table 5 above) It can be performed by first preparing a fusion gene construct. After protein expression, the hybrid protein is purified and ready for intravenous injection. Next, for example, luminescent bacteria (which can be recognized and bound by antimicrobial peptides / proteins) are injected intravenously into the subject for tumor-specific accumulation. Specific binding of antimicrobial peptides / proteins to the bacteria in the tumor helps to concentrate the therapeutic protein specifically in the tumor and thus can reveal tumor-specific cytotoxicity.

  In addition, proteins suitable for tumor therapy and / or elimination of metastatic tumors are enzymes that convert inactive substances (prodrugs) administered to an organism into active substances that kill tumors or metastases (ie, toxins) ( Gene-directed enzyme prodrug therapy (GDEPT)). The principle of GDEPT is to activate non-toxic prodrugs with systemically delivered enzymes / proteins into active toxic drugs that are cytotoxic to tumors. Specifically, GDEPT is a two-step procedure. In the first step, a gene encoding a foreign enzyme is administered, directed to the tumor, and can be expressed using specific transcription elements. In the second step, a prodrug is administered and activated by a foreign enzyme expressed in the tumor. If the enzyme / protein is present only in the tumor, the active drug is also produced only in the tumor and is therefore cytotoxic only to the tumor cells while at the same time having minimal systemic toxicity. The gene encoding the enzyme / protein is specifically delivered to the tumor using intravenously injected genetically engineered bacteria, viruses or (mammalian) cells of the invention. These genes can be under the control of either a constitutive promoter or an exogenously regulated inducible promoter that ensures that the conversion of the prodrug to a toxin occurs only in the target tissue, ie the tumor. Such promoters are, for example, IPTG-, antibiotic-, heat-, pH-, light-, metal-, aerobic-, host cell-, drug-, cell cycle- or tissue-specific-inducible promoters.

  For example, the enzyme can be a glucuronidase that converts the less toxic form of the chemotherapeutic agent, glucuronyl doxorubicin, to a more toxic form. Preferably, the gene encoding such an enzyme is directed by a promoter that is a constitutive or inducible promoter. Additional examples of enzyme / prodrug pairs useful in the pharmaceutical compositions of the invention are listed in Table 4 above.

In addition, the delivery system of the present application can even apply compounds that have not previously been used for tumor therapy due to their high toxicity when applied systemically. Such compounds include proteins inhibiting elongation factors, proteins binding to ribosomal subunits, proteins modifying nucleotides, nucleases, proteinase A chromatography peptidase or cytokines (e.g., IL-2, IL-12) ( the experimental data , Because it suggests that local release of cytokines has a positive effect on the immunosuppressive state of the tumor).

  In addition, the microorganism or cell can be a BAC (bacterial artificial chromosome) or MAC (mammalian artificial chromosome) that encodes some or all of the proteins of a particular pathway (eg, anti-angiogenesis, apoptosis, wound healing or anti-tumor growth). Can be included. Furthermore, the cells can be computer designed cyber cells or cyber viruses that encode these proteins in multiple combinations.

  For administration, the microorganism or cell of the invention is preferably referred to as a suitable pharmaceutical carrier. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions and the like. Such carriers can be formulated according to the usual methods and can be administered to the subject at a suitable dose. Administration of the microorganism or cell can be performed in various ways, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. A preferred route of administration is intravenous injection. The route of administration will, of course, depend on the nature of the tumor and the type of microorganism or cell contained in the pharmaceutical composition. The dosage regimen is determined by the physician based on various clinical factors. As is well known in the medical field, medication for any one patient can be determined by patient size, body surface area, age, sex, specific compound being administered, time and route of administration, tumor type, size and It depends on many factors, including localization, general health and other drugs administered at the same time.

  Suitable tumors that can be treated with the microorganisms or cells of the present invention are bladder tumors, breast tumors, prostate tumors, brain tumors, colon tumors, lung tumors, ovarian cancers, and pancreatic cancers; liver cancers, skin tumors.

  The present invention is illustrated by examples.

Example 1: Materials and Methods
(A) Bacterial strain. The bacterial strains used were attenuated Salmonellatyphimurium (SL7207 hisG46, DEL407 [aroA544 :: Ynl0]), attenuated Vibrio cholerae (Bengal 2Serotyp 0139, M010 DattRS1) and attenuated Listeria monocytogenes (D2 mpl, actA, plcB) Met. Bacterial strains were kindly provided by Prof. W. Goebel (University of Wuerzburg, Germany).

(B) Plasmid construct. Plasmid pLITE201 containing the luxCDABE cassette was obtained from Dr. Marines, Biotech 24, 1998, 56-58). The plasmid pXylA-dual with gfp-cDNA operon sequences, luxAB, luxCD and luxE under the control of the xylose promoter was kindly provided by Dr. Phil Hill (University of Nottingham, UK).

(C) Bacterial transformation. Bacteria were transformed by electroporation.

(D) Tumor cell line. Rat C6 nitrosourea-induced glioma cell line (ATCC, Rockille, Inc.) in RPMI-1640 medium (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with 10% (v / v) FBS and 1 × penicillin / streptomycin. MID) was cultured. Human PC3 prostate cancer cell line (ATCC, Rockville, MD) and MB-49 mouse bladder in DMEM medium (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with L-glutamine and 10% (v / v) FBS Tumor cells and rat 9L glioma cells were maintained. HT1080 fibrosarcoma cells (ATCC, Manassas, VA) were cultured in F12 minimal essential medium (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with 10% FBS and 1 × penicillin / streptomycin. In DMEM / F12 medium supplemented with 5% FBS and 560 μg / ml G418 (Life Technologies, Grand Island, NY), a plasmid carrying pro-IGF-II cDNA (Dr. Daisy De Leon, Loma Linda University, Loma Linda, MCF-7 human breast cancer cell line (ATCC, Rockville, MD) permanently transformed with (obtained from CA) was cultured.

(E) Production and propagation of retroviruses to produce luminescent stable transformed cell lines. PT67 packed cells (Clontech, Palo Alto, Calif.) Were cultured in DMEM medium supplemented with 10% (v / v) FBS. At 67% confluence, PT67 cells were transformed with pLEIN (Clontech, Palo Alto, Calif.) Using the calcium phosphate precipitation method (Profection Mammalian Transfection Systems, Promega, Madison, Wis.) For 12 hours. At this time, fresh medium was replenished. Retroviral supernatant recovered from PT67 cells 48 hours after transformation was filtered through a 0.45 μm filter and added to target HT1080 cells with polybrene to a final concentration of 4 μg / ml. After 24 hours, the medium was replaced and the cells were treated with G418 selection at 400 μg / ml and gradually increased to 1200 μg / ml.

(F) Growth of recombinant vaccinia virus. Vaccinia virus Lister strain (LIVP) was used as wild type virus. Recombinant vaccinia virus rVV-ruc-gfp is constructed by inserting the ruc-gfp-cassette (Wang et al., Proc. Biolumin. Chemilumin. 9, 1996, 419-422) into the vaccinia virus genome via homologous recombination did. Viral in CV-1 cells by addition of viral particles at a multiplicity of infection (MOI) of 0.1 pfu / cell to CV-1 cell monolayer followed by 1 hour incubation at 37 ° C. with simple agitation every 10 minutes Was amplified. At this time, the supernatant liquid with virus particles was removed, and the cell monolayer was washed once with serum-free medium. Fully grown media was then added and the cells were incubated at 37 ° C. RVV-ruc-gfp virions grown in CV-1 cells were purified via a sucrose gradient. The plaque assay was used 72 hours after infection and the titer of the recombinant virus was determined by staining the cells with ethanol containing 50% crystal violet solution.

(G) Production of mice bearing tumor grafts. Five to six week old male BALB / c athymic nu / nu mice (25-30 g body weight) and Lewis rats (250-300 g body weight) were purchased from Harlan (Frederick, MD). C57BL / 6J Min / + mice were obtained from Jackson Laboratories (Bar Harbor, ME) and Min (multiple intestinal tumorigenesis) is an autosomal dominant feature containing a nonsense mutation at codon 850 of the murine Apc gene. Makes it susceptible to sudden intestinal adenoma formation.

To obtain tumors in nude mice, C6 glioma cells were grown and harvested and the cell number was determined by trypan blue exclusion. Disinfectant was applied to the skin surface, then about 5 × 10 ⁵ cells were suspended in 100 μl phosphate buffered saline (PBS) and injected subcutaneously into the right thigh of each mouse. Tumor growth was monitored by recording tumor size using a digital caliper. Tumor volume (mm ³ ) with formula (L × H × W) / 2, where L is the length of the tumor, W is the width of the tumor, and H is the height of the tumor (mm) Estimated by

Intracerebral gliomas were prepared by injecting C6 glioma cells into the rat's head. Prior to injection, rats were anesthetized with sodium pentobarbital (Nembutal® sodium solution, Abbot Laboratories, North Chicago, IL; 60 mg / kg body weight). A midline scalp incision (0.5-1 cm) was made, the skin was contracted, and a 1 mm barhole was made 2 mm to the left of the brigma and 2.5 mm posterior to the skull. Tumor cells were pipetted into an insulin syringe equipped with a 29 gauge needle and secured to a stereotaxic holder. A needle was inserted vertically through the bar hole to a depth of 3 mm. After injection of 5 × 10 ⁵ C6 cells in a 10 μl volume into the brain, the needle was held in place for 15 seconds and then withdrawn. The skin incision was closed with a surgical clip. After subcutaneous implantation of 3 × 10 ⁶ PC3 human prostate cells, mice bearing subcutaneous prostate tumors were produced over a period of one month.

MB-49 mouse bladder tumor cells were transplanted into C57 mouse bladders to produce animals with bladder tumors. To create animals with breast cancer (Tian and DeLeon, submitted as publication), the skin of female nude mice was first transplanted with 0.72 mg / 90-day released 17β-estradiol pellet (Innovative Research, Rockville, MD) and the chest Promoted tumor development and metastasis. One day after estrogen pellet implantation, 1 × 10 ⁶ MCF-7 human breast cancer cells transformed with pro-IGF-II (Dull et al., Nature 310 (1984), 777-781) were implanted into the mammary fat pad. For the orthotopic graft, a tumor developed from the transplanted cells was partially excised and cut into 1 mm ³ cubes for tissue transfer to the mammary fat pad.

(H) Assay of Renilla luciferase in surviving animals. Each mouse was anesthetized with Nembutal (60 mg / kg body weight) before each renilla luciferase assay. Measure Renilla luciferase activity after intravenous injection of a mixture of 5 μl coelenterazine (0.5 μg / μl diluted ethanol solution) and 95 μl luciferase assay buffer (0.5 M NaCl, 1 mM EDTA; and 0.1 M potassium phosphate, pH 7.4) did. The entire surviving animal was then imaged using a Hamamatsu ARGUS 100 low light video camera in a dark box and the images were recorded using Image Pro Plus 3.1 software (Media Cybernetics, SilverSpring, MD). In order to accurately locate the site of luminescence, a false colored photon generation image was overlaid on the animal grayscale image.

(I) Detection of luminescence and fluorescence. Immediately prior to imaging, mice and rats were anesthetized using Nembutal® (60 mg / kg body weight). Animals were placed in a dark box for photon counting and recording of superimposed images (ARGUS100 Hamamatsu, Hamamatsu, Japan). Photon recovery was 1 minute from the animal's ventral and dorsal visual fields. The light image was then recorded, then the low light image was overlaid on the light image, and the position of luminescence activity was recorded.

  Imaging of GFP expression in tumors of surviving animals was performed using a Leica MZ8 stereofluorescence microscope equipped with a mercury lamp power supply and a GFP filter (excitation at 470 nm). Images were recorded using a SONYDKC-5000 3CCD digital photo camera.

(J) Histology of tumor tissue. Under anesthesia, the animals were euthanized with an overdose Nembutal®. The tissue of interest was removed, embedded in a Tissue-TekOCT compound (Miles Scientific, Naperville, IL) and immediately frozen in liquid nitrogen without fixation. The frozen sections were cut at -20 ° C. using Reichert-JungCryocut 1800 cryostat. Tissue GFP fluorescence was monitored under a Leica fluorescence microscope and images were recorded using Photoshop software.

Example 2: Results obtained by intravenous injection of recombinant vaccinia virus rVV-ruc-gfp into mice
(A) Monitoring of virus-mediated marker gene expression in immunodeficient mice Vaccinia virus (1 × 10 ⁸ pfu) carrying the Renilla luciferase-GFP fusion expression cassette (rVV-ruc-gfp) was transferred to nude mice without tumor Intravenously introduced. Animals were observed under a low light imager once every 3 days for a period of 2 weeks, monitored for luciferase-catalyzed luminescence immediately after intravenous injection of coelenterazine, and visualized GFP expression under a fluorescence microscope. When imaged externally, no obvious luminescence or green fluorescence was detected in the animals except at certain locations with small skin lesions. Such luminescent and fluorescent signals disappeared after a few days once the lesion had healed. Animals were sacrificed 1 and 2 weeks after viral infection and their organs were removed and tested for the presence of luminescent and GFP fluorescent signals. One week after virus injection, neither luminescence nor green fluorescence could be detected in the brain, liver, lung, spleen, kidney or testis. These results indicated that the rVV-ruc-gfp virus did not show organ specificity after injection and the virus appears to be cleared from the animal by the immune system immediately after systemic delivery via the bloodstream.

(B) Visualization of vaccinia virus-mediated marker gene expression in glioma of surviving nude mice The distribution of injected vaccinia virus in nude mice bearing C6 glioma implanted subcutaneously was investigated. Nude mice with tumors approximately 500 mm ³ in size were injected intravenously with 1 × 10 ⁸ pfu of rVV-ruc-gfp virus. Seven days after virus injection, animals were monitored for GFP expression under a fluorescence microscope to determine the presence and multiplicity of virus infection in tumors that grew to a size of approximately 2500 mm ³ . Surprisingly, green fluorescence was detected only in the tumor area of surviving animals. Seven days after virus injection, GFP fluorescence was very intensely localized in a patch-like pattern confined to the tumor area (FIGS. 1A-A ″). These patches, often found at the ends of vascular branches, can indicate local viral infection of tumor cells surrounding the leaky ends of the capillaries. During real-time observation of the same tumor, the GFP signal from the center of these patches begins to disappear and a new green fluorescent center appears in the form of a ring around the extinguisher patch. New sites of strong GFP fluorescence can arise from the progression of viral infection to nearby cells inside the tumor during the growth and expansion of the tumor. After careful examination of the mice, there was no detectable green fluorescence anywhere on the body surface or in the dissected organ except for the tumor area. This experiment clearly showed that mature solid tumors could be easily localized by labeled vaccinia virus based on luminescence, and also showed the affinity of virus particles for tumor tissue.

To determine if tumor size and angiogenesis are critical factors for virus retention in tumors, nude mice were given 1 x 10 ⁸ rVV-ruc-gfp vaccinia one day after subcutaneous C6 cell transplantation. Intravenous injection using virus particles. Surprisingly, 1 day after virus injection, GFP expression was seen in 5 day old C6 tumors with a volume of about 25 mm ³ (FIG. 1B-B ″). Tests of labeled vaccinia virus tumor targeting by visualization of GFP expression in transplanted tumors younger than 5 days could not be performed in surviving mice. This is because about 4 days were required for marker gene expression at a sufficient level to allow detection under a fluorescence microscope.

  The finding that injection of rVV-ruc-gfp vaccinia virus into the host's bloodstream results in GFP expression and accumulation in tumors suitable for non-invasive tumor detection suggests that we have entered this virus in the same animal in real time And allowed to follow the replication process (FIGS. 1C-C '', D-D '', E-E ''). A continuously increasing level of GFP fluorescence was observed in the same animals through 20 days after virus injection, which is the scheduled time before the animals were sacrificed. Such an increase in detectable fluorescence indicated very strong viral replication in tumor tissue, indicating that the latter functions as a protective immunological tolerance environment for viral replication. Virus replication in tumors was demonstrated by determining the virus titer and luminescence of isolated virus particles in cell culture. Interestingly, vascular localization and neovascularization within the periphery of the expanding tumor was readily visible and confirmed by externally visualizing a bright green fluorescent background (Figure 1A). ~ A '', D ~ D '', E ~ E '' and FIG. 2).

  To determine the location of viral infection within the tumor, the animals were sacrificed and the skin over the tumor was carefully folded to expose the tumor. In exposed tumors, GFP fluorescence was seen to be concentrated exclusively in the tumor tissue (FIGS. 3B-B ″ and D-D ″). Non-neoplastic femoral muscle did not show any fluorescence of viral infection as indicated by the arrows in FIGS. Tumor-covered skin was also non-fluorescent (indicated by asterisks in FIGS. 3B-B ″ and D-D ″). However, a cross-sectional view of the tumor revealed that a strong green fluorescent region was almost found as a patch in the vicinity of the tumor where the actively dividing tumor cells were probably located (in FIGS. 3C-C ''). Double arrow).

  To further investigate the pattern of viral infection in C6 glioma based on GFP expression, tumor tissue was cut for microscopic analysis under a fluorescence microscope. Comparative analysis of various tissue sections revealed that GFP fluorescence was present in large clusters of cells inside the tumor (Figure 4), but in normal tissues such as heart, lung, liver, spleen, and kidney The fluorescence was not visible.

  In addition to GFP, the recombinant rVV-ruc-gfp virus carried a second marker gene encoding Renilla luciferase in the form of a fusion protein with GFP. Therefore, the present inventors were able to directly superimpose the luminescence derived from Renilla luciferase and the site of GFP fluorescence in the tumor. Immediately after coelenterazine (substrate for Renilla luciferase) was delivered by intravenous injection, very strong luciferase activity was recorded only in the tumor area under a low light video camera (FIG. 5). By reducing the sensitivity of the low light video camera to avoid saturation of light detection, we were able to identify Renilla luciferase gene expression in localized patches around the tumor. These patch-like patterns correlated exactly with the GFP signal.

(C) Activity of vaccinia virus delivered to the bloodstream on various tumors implanted in animals Determine whether vaccinia virus attraction is limited to glioma or whether this attraction can be observed in other tumors To do so, recombinant vaccinia virus was recombinantly introduced into mice with various types of transplanted tumors. One of these tumor models was nude mice with transplanted subcutaneous PC-3 human prostate cancer. PC3 grafts from which tumor development originated grew at a much slower rate compared to transplanted subcutaneous gliomas, but these tumors were vaccinia virus when injected intravenously with the same titer (1 x 10 ⁸ ) The same kinetics were shown for the infection (Fig. 6A-A "). Similar to our findings on glioma, GFP expression was first detected 4 days after virus injection, and fluorescence continued throughout the 3 week observation period.

Female nude mice with established breast tumors were also used for labeled vaccinia injections. These breast tumors were grown for 6 months after the animals received a graft of MCF-7 human breast cancer cells transformed with pro-IGF-11 cDNA. At the time of vaccinia virus injection, the tumor reached maximum growth and the tumor volume (approximately 400-500 mm ³ ) did not change significantly during the experimental period. Similar to the previous experiment, 6 days after intravenous delivery of 1 × 10 ⁸ rVV-ruc-gfp virions, strong GFP expression was observed in the breast tumor area (FIGS. 6B-B '', 7A-A ′). 'And BB''), not observed elsewhere in the body.

  Investigation of a cross-section of a virally infected breast tumor revealed luminescent “islands” throughout the tumor without showing preference for the center or periphery of the infection (FIGS. 7C-C ″). The MCF-7 tumor cells used in these breast tumor models are known to metastasize, and the smaller metastatic tumor found on the left side of the body in addition to the primary solid tumor showed GFP fluorescence (Figure 7D- D ″, E to E ″ and F to F ″). The removed lung tissue was also examined for detection of metastases. Metastatic tumors about 0.5 mm in diameter on the surface of the lung were positive for GFP fluorescence (FIGS. 7G-G ″). The presence of strong Renilla luciferase-mediated luminescence confirmed the expression of the luciferase-GFP fusion protein in these breast tumors when the substrate coelenterazine was injected intravenously into living animals, but not elsewhere in the body. These experiments showed that intravenously delivered vaccinia virus particles were selectively accumulated and replicated in primary and metastatic breast tumors in nude mice, presumably as a result of immunocompromised state of the tumor microenvironment.

In order to determine whether the virus particles can move out of the tumor and re-enter the circulation, we injected C6 glioma cells into the thighs of mice and were infected with labeled vaccinia virus. A second tumor was formed in an animal that already had a breast tumor. If viral particles are released from the tumor and reenter the circulation in a significant number, they can colonize newly transplanted gliomas. Monitoring of these second tumors showed that GFP signal was not visible in new gliomas 7 and 14 days after transplantation of glioma cells. To illustrate that the newly transplanted glioma can be targeted by labeled vaccinia virus, a second dose of rVV-ruc-gfp virus (1 × 10 ⁸ pfu) was injected intravenously. Five days later, tumor-specific GFP expression was detected in newly formed gliomas in addition to the GFP expression found in the original breast tumor. These findings suggested that the virus particles in the infected tumor were not re-released into the circulation or released in sufficient numbers to infect and replicate in the second tumor.

Two additional tumor models were used for vaccinia infection, including Lewis rats with intracranial C6 rat glioma and C57 mice with MB-49 mouse bladder tumor in the bladder. Whether virion tumor affinity is a tumor-limited phenomenon in nude mice with reduced T-lymphocyte function, or is it a general protective property of tumors that can also be shown in immunocompetent animals To determine whether Lewis rats with intracranial C6 rat glioma and C57 mice with MB-49 mouse bladder tumor in the bladder were used. A total of 5 × 10 ⁵ C6 glioma cells in a 100 μl volume were implanted stereotaxically into 2 brains of 4 immunocompetent Lewis rats and tumors were allowed to grow for 5 days. Two other rats were injected intracranial with phosphate buffered saline to serve as a control. On day 6, all four rats were injected intravenously with rVV-ruc-gfp virus particles via the femoral blood vessels. Five days after virus injection, all four mice were sacrificed and their brains were carefully removed for analysis by fluorescence microscopy. GFP expression was detected in brains with transplanted intracranial tumors (FIGS. 6C-C ″), whereas no GFP expression was seen in control brains. In parallel experiments, C57 mice with or without bladder tumors were divided into two groups. One group was injected intravenously with rVV-ruc-gfp vaccinia virus (1 × 10 ⁸ pfu) and the other intravenously with saline as a control. Five days after virus injection, animals were sacrificed and examined under a fluorescence microscope. GFP expression was observed in the bladder tumor area of C57 mice, but not in control mice (FIGS. 6D-D ″).

  Taken together, these experiments show that vaccinia virus particles are selectively accumulated and retained in various tumors, possibly protected by the tumor microenvironment, and they survive in immunocompromised and non-neoplastic tissues of immunocompetent animals Indicates not obtained. The tumor targeting process by intravenous injection of vaccinia virus carrying a luminescent double marker gene has demonstrated the ability to detect primary and metastatic tumors in surviving animals of the vaccinia virus line.

Example 3 Results of Intravenous Injection of Bacteria and Mammalian Luminescent Cells into Mice (A) Visualization of Luminescent Bacteria Present in Whole Animals After Intravenous Injection Final results of intravenously injected luminescent bacteria in animals To determine, 10 ⁷ bacteria carrying pLITE201 plasmid in 50 μl were injected into the left femoral vessel of mice under anesthesia. After closing the incision with sutures, mice were monitored in real time under a low light imager (ARGUS100 Camera System, Hamamatsu, Hamamatsu, Japan) and photons were collected for 1 minute. Imaging was repeated at 2 day time intervals to determine the presence of luminescence from a given animal. The distribution pattern of luminescence after intravenous injection of bacteria into mice was found to be unique to the bacterial strain used. Injection of attenuated V. cholera into the bloodstream immediately produced localized luminescence in the liver. However, S. typhimurium injections spread throughout the animal's body, suggesting differences in interaction with the host cell system (FIGS. 8A-8D). Imaging of the same animals 24 hours and 48 hours after infection showed that all of the detectable luminescence rapidly decreased from the earlier time and was completely removed from the injected animals. These findings suggest that luminescent bacteria injected into the bloodstream via the femoral blood vessels are removed. This process was confirmed by photon generation analysis of isolated organs and found to lack luminescence. Similar data was obtained in immunocompetent mice and rats, suggesting that removal of bacteria from the blood is effective in both systems.

(B) Bacteria home to glioma in nude mice To examine whether bacteria preferentially colonize in neoplastic tissue, tumor on day 10 (approximately 500 mm ³ ) in right hind limb Nude mice having a 50 μl volume of bacterial suspension containing 10 ⁷ S. typhimurium or 10 ⁷ V. cholera were injected intravenously via the femoral vein. After injection, the incised wound was sutured and the animals were monitored for 6 days under a low light imager. At each observation time, photons were collected for exactly 1 minute. In mice injected with S. typhimurium, fluorescent bacteria were scattered throughout the animal, similar to the findings in tumor-free mice (FIG. 9A). Nude mice injected with V. cholera exhibited fluorescence activity only in the liver region during the initial observation (FIG. 9E). Regardless of the species of bacteria injected, fluorescence activity was observed only in the tumor area 2 days after injection (FIGS. 9B and 9F). Monitoring of mice under dark imaging devices on days 4 and 6 after injection showed a decrease in the amount of detectable fluorescence in animals injected with S. typhimurium (FIGS. 9C and 9D). This finding is in marked contrast to the findings in the tumors of mice injected with V. cholera, indicating not only bacterial survival in the tumor mass, but also bacterial growth and a dramatic increase in luminescence. (FIGS. 9G and 9H).

Nude mice with PC3 human prostate tumor subcutaneously in the right hind limb were injected intravenously with 10 ⁷ attenuated L. monocytogenes transformed with psod-gfp plasmid DNA with gfp cDNA. GFP fluorescence was observed under a fluorescence stereomicroscope. 27 hours after bacterial injection, GFP signal was detected only in the tumor area (FIG. 14 ). No GFP signal was observed in other parts of the animal.

(C) Determination of minimum size and age of glioma required for bacterial infection The purpose of this experiment is to determine whether the size of the tumor has any effect on the ability to be colonized by the bacteria To find out. Tumors were induced in the right hind limb of nude mice by subcutaneous injection of glioma cells as described. On days 0, 2, 4, 6, 8, and 10 of tumor induction, attenuated S. typhimurium and V. cholera with pLITE201 plasmid were injected intravenously via the femoral vein. On days 2 and 4 after injection, the presence of fluorescent bacteria in the tumor was examined by collecting photons for exactly 1 minute under a low light imaging device. Tumor volume was measured by measuring the size using a digital caliper. The earliest time point at which fluorescence activity was observed in the tumor was 8 days after tumor induction. The corresponding tumor volume was about 200 mm ³ .

(D) Bacteria home to breast tumors in nude mice To investigate whether bacterial colonization of tumors is restricted to glioma cells or this is a common phenomenon observed for all tumors To determine whether or not, female nude mice with tumors in the right breast pad were intravenously injected with a 50 μl volume of bacterial suspension containing 10 ⁷ V. cholera. Within the first 10 minutes after seeding, 1 min under low light image forming apparatus, when monitoring the animals, a typical fluorescence pattern in the liver region was shown (Figure 10 A). Two days later, the liver disappeared obvious fluorescent bacteria, but the breast tumor was colonized by the labeled V. cholera. In addition to the main tumor, metastatic tumors showed fluorescence activity in the left breast (Fig. 10 B). Day 5, the animals showed a loss of colony forming bacteria in incisional wounds, tumors remained fluoresced (Figure 10 C). Figure 10 D showed continued colonization and proliferation in the main tumor, disappeared in small tumors metastatic. Fluorescence activity continued for 45 days in right breast tumors. Similar experiments, performed using E. coli, homing to the tumor by bacteria was shown not to be species-dependent (Fig. 10 E and 10 F).

A second tumor (C6 glioma) was induced in the right hind limb of these animals to see if bacteria from the tumor entered the bloodstream in significant amounts and colonized elsewhere. Tumors were allowed to grow for 10 days. No fluorescence activity is observed in glioma, indicating the absence of significant bacteria that can cause colony formation in this tumor. However, when the animals were intravenously challenged with 10 ⁷ attenuated V. cholera, the leg tumors showed strong fluorescent activity.

  The findings of these experiments indicate that larger tumors retain bacteria more effectively over time. Furthermore, bacteria within the tumor do not leak into the blood in amounts sufficient to infect sensitive sites such as other tumors.

(E) Bacteria home to bladder tumors in immunocompetent mice C57 mice were injected intravenously with 10 ⁷ attenuated V.cholera transformed with pLITE201 encoding the lux operon. On day 9 after bacterial delivery, fluorescence activity was recorded by photon collection for 1 minute under a low light imaging device. Luminescence was observed in the bladder region of the whole animal (FIG. 11A ). The animals were sacrificed and an abdominal incision was made to expose the bladder. Fluorescence activity was confirmed clearly be limited to the bladder (Figure 11 B). When taking out the bladder from mice, where the fluorescence activity disappeared even in animals, excised bladders continued to show light emission (FIG. 11 C). Based on the results of this experiment, bacteria can target tumors in immunocompetent and nude mice. In addition, bacteria can target smaller tumors.

(F) Bacteria home to glioma in rat brain In immunocompetent animals, to have a glioma in the brain to see if the bacterium can cross the blood brain barrier and target the tumor Lewis rats were injected intravenously via the left femoral vein with 10 ⁸ attenuated V. cholera carrying the pLITE201 plasmid. The next day, all animals were monitored for 1 minute under a low light imaging device and a low level of fluorescent activity was observed through the skull. Rats were sacrificed and a piece of brain tissue was removed to further assess the exact location of fluorescent bacteria. Visualization of ablation brain under the image forming apparatus showed a strong fluorescence activity in certain regions of the brain (Figure 12 A). The labeled bacteria were injected intravenously, the same image forming control rats without tumors, it was shown that the fluorescent activity is not present at all (Figure 12 B).

(G) Transformed human fibrosarcoma cells home into subcutaneous gliomas in nude mice 5 × 10 ⁵ humans permanently transformed with pLEIN-derived retrovirus into nude mice with human breast tumors Fibrosarcoma cells were injected intravenously. Seven days after injection, the animals were anesthetized with Nembutal and monitored under a fluorescent stereomicroscope. Fluorescent cells were observed only in the tumor area in the whole body of the mouse through the skin (FIG. 13 A1 to 3). When exposing the tumor tissue by inversion-layer skin (FIG. 13 B1~3), in tumors of the cross-section (Fig. 13 C1 -3), fluorescent patches were visible in distinct regions. A close examination of the mouse organ shows the presence of a small population of fluorescent cells in the animal's leg, which indicates the affinity of the fibrosarcoma cells for the lung in addition to the neoplastic tissue.

  Example 4: Construction of a bacterial plasmid vector having a photoprotein encoding coding cassette and a therapeutic gene expression construct in cis configuration

(A) Principle Using the above-described luminescent expression system, tumors could be imaged based on luminescence for up to 45 days in animals. These findings suggest significant DNA stability in bacteria in the absence of selection. Thus, by placing the therapeutic gene cassette in the cis configuration with the photoprotein expression cassette on the same replicon, luminescence can be used as an indicator of the presence and stability of the therapeutic construct. In contrast to photoproteins, therapeutic proteins, endostatin and Pseudomonas exotoxin / TGFα fusion protein must be secreted from bacteria into the culture medium or into the cytosol of tumor cells to inhibit tumor growth It is. Two constructs with different signal sequences can be designed to achieve secretion of the protein from the extracellularly replicated E. coli into the tumor. For secretion of endostatin, the ompF signal sequence can be placed upstream of the coding region of endostatin, which facilitates secretion into the periplasmic space. In order to release endostatin into the medium, it is necessary to co-express an additional protein, PAS protein, with endostatin. PAS has been shown to cause membrane leakage and release of secreted proteins into the medium (Tokugawa et al., J. Biotechnol. 37 (1994), 33; Tokugawa et al., J. Biotechnol. 35 (1994), 69 ). A second construct for secretion of Pseudomonas exotoxin / TGFα fusion protein from E. coli has an OmpA signal sequence upstream of the fusion gene, and release from the periplasmic space into the medium is the second exogenous exotoxin II. Promoted by sequences present in the domain (Chaudhary et al., PNAS 85 (1988), 2939: Kondo et al., J. Biol. Chem. 263 (1988), 9470; Kihara and Pastan, Bioconj. Chem. 5 (1994), 532). Coding sequences of listeriolysin (LLO) (Mengaud et al., Infect. Immun. 56 (1988), 766) to promote secretion of endostatin and Pseudomonas exotoxin / TGFα fusion protein from L. monocytogenes It may be arranged upstream of

For regulation of endostatin and Pseudomonas exotoxin / TGFα fusion protein expression levels in bacteria, vectors can be made in which the gene encoding the therapeutic protein is under the control of a T7 promoter or a P _spac synthetic promoter (Freitag and Jacobs, Infect Immun. 67 (1999), 1844). Without exogenous induction, therapeutic protein levels are low in E. coli and L. monocytogenes. The minimal level of therapeutic protein in bacteria provides greater safety after intravenous injection of engineered bacteria. The following describes the construction of six plasmid DNAs for constitutive and regulated expression of endostatin and Pseudomonas exotoxin / TGFα fusion protein in E. coli and L. monocytogenes. All plasmids that are transferred into E. coli have a constitutively expressed bacterial Lux operon, and all plasmids that are transferred into L. monocytogenes have a constitutively expressed sod-gfp cassette. Plasmids BSPT # 1-ESi and BSPT # 2-Pti are replicable only in E. coli and plasmids BSPT # 3, # 4, # 5 and # 6 replicate in E. coli and L. monocytogenes.

(B) Construction of plasmid vector for protein expression and secretion from E. coli The construction of endostatin secretion vector used in E. coli is as follows. The human endostatin coding sequence (591 bp) is amplified by PCR from plasmid pES3 with the necessary restriction sites at both ends and then ligated into the pBluescript (Clontech, Corp., USA) cloning vector to create pBlue-ES. The ompF signal sequence (Nagahari et al., EMBO J. 4 (1985), 3589) is amplified with Taq polymerase and inserted in-frame upstream of the endostatin sequence to create pBlue-ompF / ES. An expression cassette driven by the T7 promoter is excised and inserted into the pLITE201 vector carrying the luxCDABE cassette described in Example 1 (B) above to produce the plasmid pLITE-ompF / ES. The sequence encoding the PAS factor (76 amino acid polypeptide) was expressed by Vibrioalginolyticus (formerly Achromobacter iophagus) with Taq polymerase using primers 5'-GGGAAAGACATGAAACGCTTA3'- and 5'-AAACAACGAGTGAATTAGCGCT-3 '(NCIB 11038 ) And inserted into the multiple cloning site of pCR-Blunt (Clontech, Corp., USA) to prepare an expression cassette under the control of the lac promoter. The resulting plasmid is named pCR-PAS. The lac promoter linked to the pas gene is excised from pCR-PAS and inserted into pLITEompF / ES to obtain the final plasmid BSPT # 1-ESI.

  Plasmid pVC85 (Kondo et al., 1998, J. Biol. Chem. 263: 9470-9475) contains a T7 promoter followed by an ompA signal sequence and a sequence (PE40) encoding the second and third domains of Pseudomonas exotoxin. contains. A DNA sequence encoding PE40 was excised with a restriction enzyme, and a fragment of PE37 / TGFα (Pseudomonas exotoxin A280-613 / TGFα) obtained from plasmid CT4 (Kihara and Pastan, 1994, Bioconjug. Chem. 5; 532-538) and Replace with the plasmid pVC85-PE37 / TGFα. The ompAPE37 / TGFα expression cassette linked to the T7 promoter is excised and inserted into pLITE201 to obtain the final plasmid BSPT # 2-PTI.

(C) Construction of a plasmid vector for protein expression and secretion from L. monocytogenes A gene encoding endostatin or PE37 / TGFα is inserted downstream of the listeriolysin (LLO) signal sequence in plasmid pCHHI, and pCHHI- ES and pCHHI-PE37 / TGFα are prepared. By ligating the above-described secretion cassette to a listeriolysin promoter obtained from the pCHHI vector, constitutive expression of the therapeutic protein is obtained. The sod-gfp expression cassette excised from the plasmid psod-gfp (Goeta et al. PNAS during printing) is inserted into pCHHI-ES to produce BSPT # 3-ESc, and inserted into pCHHI-PE37 / TGFα to obtain BSPT # 4. -Make PTc. For expression of therapeutic proteins under the control of an IPTG-inducible promoter, the listeriolysin promoter in BSPT # 3-ESc and BSPT # 4-PTc was transformed into plasmid pSPAC (Yansura and Henner, PNAS USA 81 (1984), 439) is replaced with the P _spac promoter and BSPT # 5-ESi and BSPT # 6-PTi are produced. P _spac is a hybrid promoter consisting of the bacteriophage SPO-1 promoter and the lac operator. IPTG-inducible GFP expression from the P _spac promoter has been demonstrated in L. monocytogenes in the cytosol of mammalian cells.

Example 5: Demonstration of luciferase and GFP expression in bacteria and confirmation of their function in endostatin and recombinant toxin / TGFα fusion protein secretion and culture assays E. coli and L. monocytogenes in tumor tissue of living animals To be able to detect the presence of luciferase and GFP levels constitutively expressed in bacteria needs to be sufficient. Therefore, after transformation with the recipient Escherichia coli or L. monocytogenes construct described in Example 4, the clone with the highest luciferase luminescence or GFP fluorescence is selected. In addition to characterizing the luminescence from each selected colony prior to intravenous injection, it is necessary to confirm the ability of the selected transformants to secrete endostatin and Pseudomonas exotoxin / TGFα fusion protein into the medium. The presence of endostatin and Pseudomonas exotoxin / TGFα fusion proteins synthesized in E. coli and L. monocytogenes is examined by extracting these proteins from cell pellets. The protein secreted into the medium is concentrated, analyzed by gel separation, and the amount is measured by Western blot. It is important to examine the proportion of newly synthesized protein expressed from each plasmid construct in either E. coli or L. monocytogenes present in the medium. In addition to constitutive expression of endostatin and Pseudomonas exotoxin / TGFα fusion protein, it is also essential to confirm that expression can be induced in E. coli and L. monocytogenes when IPTG is added to the bacterial culture medium. In order to design future tumor treatment protocols, it is first necessary to compare the relative amount of protein secreted by the constitutive expression system in bacterial cultures with the expression level induced in a defined period of time. When produced from the proposed construct, it is equally essential to test that both proteins are biologically active when synthesized in E. coli and L. monocytogenes. Both proteins were previously synthesized in E. coli and shown to be active.

  The results of the experiments described below should confirm that endostatin is secreted well from E. coli using the OmpF signal peptide in combination with PAS pore-forming protein expression. These experiments also use the OmpA signal peptide in combination with PE domain II to show whether PE40 / TGFα and PE37 / TGFα fusion proteins are secreted from bacteria. In addition, a listeriolysin signal peptide may also promote the secretion of endostatin and chimeric toxin / TGFα fusion protein into the medium and into the cytosol of infected tumor cells. It can be expected that both proteins secreted into the medium will be biologically active using an endothelial cell migration inhibition assay and a protein synthesis inhibition assay. The presence and amount of these proteins can be regulated by replacing the constitutive promoter with a promoter that can be induced by IPTG.

  In addition to the secretion systems described below, alternative secretion systems such as the E. coli HlyBD-dependent secretion pathway (Schlor et al., Mol. Gen. Genet. 256 (1997), 306) can also be used. Alternative to other Gram-positive bacteria such as Bacillus.sp. Endoxylanase signal peptide (Choi et al., Appl. Microbiol. Biotechnol. 53 (2000), 640; Jeong and Lee, Biotechnol. Bioeng. 67 (2000), 398) A secretion signal can be introduced.

(A) Confirmation of secretion of endostatin and Pseudomonas exotoxin / TGFα fusion protein from bacteria to growth medium Transformation of E. coli strains (DH5α and BL21 (λDE3)) with BSPT # 1-ESi and BSPT # 2-PTi plasmid DNA To do. L. monocytogenes strain EGDA2 is individually transformed with plasmids BSPT # 3-ESc, BSPT # 4-PTc, BSPT # 5-ESi and BSPT # 6-PTi. After plating on appropriate antibiotic-containing plates, individual colonies are selected from each transformation mixture. These colonies are screened for luciferase and GFP expression, respectively, under a weak light imaging device and a fluorescence microscope. Three colonies with the highest luminescence intensity from each transformation batch are selected for further study. To confirm the secretion of endostatin and Pseudomonas exotoxin / TGFα fusion protein from each of the selected transformants, cells are grown to log phase in minimal essential medium. After centrifugation to sediment the bacteria, the supernatant is passed through a 0.45 μm pore size filter and a bacteria-free medium is used for the precipitation of secreted proteins. The precipitate is collected by centrifugation. The pellet is washed, dried, and resuspended in sample buffer for protein gel separation. After separation on a 10% SDS-polyacrylamide gel, the aliquot-derived protein corresponding to 10 μl of bacterial culture is compared to 200 μl of protein from the culture supernatant. Western blot analysis is performed using a polyclonal antibody against endostatin (following the antibody production protocol described by Timpl, Methods Enzymol. 82 (1982), 472) and a monoclonal antibody against TGFα (Oncogene Research Products, Cambridge, MA, USA). The optimal anti-conditions for secretion are established by sampling the growth medium at different times during growth. Similar methods have previously been used to analyze secreted proteins in Salmonellatyphimurium culture supernatants (Kaniga et al., J. Bacteriol. 177 (1995), 3965). By using these methods, the amount of secreted protein in the bacterial culture medium made by each of the constructs without induction is established. To estimate the increase in the amount of secreted protein in the medium, IPTG-dependent promoter activation experiments were performed by adding IPTG to the log phase bacterial culture for 3-6 hours, and the secreted protein was Assay.

(B) Confirmation of biological activity of endostatin secreted by E. coli and L. monocytogenes using migration inhibition assay Endostatin is a vascular endothelial growth factor (VEGF) -induced human umbilical vein endothelial cell (HUVEC)- It has been shown to inhibit migration. Thus, the biological activity of endostatin secreted by bacteria can be tested using the HUVEC migration assay provided by CascadeBiologics, Portland, OR. Inhibition of cell migration is assessed in a 48-well chemotaxis chamber (NeuroProbe, Galthersburgs, MD) (Polverine et al., Methods Enzymol. 198 (1991), 440). Bacteria-free supernatant from each secreted construct is added to HUVEC for a 30 minute preincubation. After incubation, HUVEC is placed in the upper chamber. Migration to the lower chamber of HUVEC induced by VEGF ₁₆₅ (R & D Systems, Minneapolis, NIN) is quantified by microscopic analysis. The functional endostatin concentration in the medium is directly proportional to the degree of inhibition of HUVEC migration.

(C) Cytotoxicity test of secreted recombinant PE toxin in tumor cell cultures The inhibitory activity of chimeric toxins in mammalian cells is measured based on the inhibition of novel protein synthesis by inactivation of EF-2 (Carroll and Collier, J. Biol. Chem. 262 (1987), 8707). Aliquots of bacterial-free supernatants obtained from expression of various recombinant PE secretion constructs in E. coli and L. monocytogenes are added to C6 glioma cells or HCTI16 colon carcinoma cells. After treatment with media, mammalian cells are pulsed with [ ³ H] -leucine and uptake is measured in the protein fraction. To examine the presence of secreted chimeric toxin protein in L. monocytogenes-infected mammalian cells, bacteria are removed from the medium by gentamicin treatment. Mammalian cells containing L. monocytogenes in the cytosol are lysed and the released bacteria are removed from the lysate by filtration. Mammalian cell lysates containing secreted chimeric toxins are assayed in protein synthesis inhibition experiments. Inhibition of [ ³ H] -leucine uptake in tumor cell culture is directly proportional to the amount of biologically active chimeric toxin protein in the medium and cell lysate.

Example 6: Invasion, localization and distribution of intravenous bacteria in the tumor of a live animal (A) Principle A live animal because only a few intravenous bacteria escape from the immune system by entering the tumor Its direct localization in is impossible due to emission limitations. Their localization can only be confirmed by delimiting the tumor to confirm the initial center of luminescence. Looking at sections at later time points, bacteria can be seen throughout the tumor due to rapid replication. To examine whether one or many bacteria have entered the same site, red fluorescent protein can be used to label extracellularly replicating E. coli, and green fluorescent protein can be used for intracellularly replicating L. monocytogenes. By visualizing the distribution of red and green fluorescence in the tissue sections, the entry site and replication and localization of E. coli and L. monocytogenes can be measured individually and simultaneously in the center and peripheral regions of the tumor. The pattern of entry and distribution obtained in transplanted tumors can be expected to be similar to that of spontaneous tumors, thus bacterial-based diagnostics and protein therapies are effective approaches.

  Through the experiments described in section (B), in the following, the entry, replication and distribution of luminescent bacteria in spontaneous tumors may be compared with the distribution pattern in transplanted tumors. Furthermore, double labeling experiments allow the operator to confirm the exact location of extracellularly replicated E. coli and intracellularly replicated L. monocytogenes in the same tumor section. Finally, it can be ascertained (after 5 days of bacterial colonization) whether the bacteria are evenly distributed within the tumor or whether preferential localization occurs around the tumor or in the necrotic center. The potential for reduced bacterial entry within naturally occurring tumors due to the immunity of these animals can be resolved by increasing the number of intravenously injected bacteria.

(B) Intravenous injection of red fluorescent protein expressing E. coli and green fluorescent protein expressing L. monocytogenes into nude mice and rodents with transplanted and spontaneous tumors DsRed (Matz et al., Nat. Biotech under the control of a constitutive promoter 17 (1999), 969) E. coli (DH5α) with an expression cassette is used for this experiment. L. monocytogenes EGD strain derivatives with in-frame deletions in each of the toxic genes were individually labeled with a green fluorescent protein cassette driven by a constitutive SOD promoter.

First, bacterial localization and intratumoral distribution are studied in nude mice with transplanted C6 glioma or HCT116 colon cancer tumors. C6 glioma or HCT116 colon cancer tumor cells (5 × 10 ^{5 in} 100 μl) are injected subcutaneously into the animal's right hind limb. Twelve days after tumor cell injection, the animals are anesthetized and the left femoral vein is surgically exposed. Luminescent bacteria (1 × 10 ⁶ suspended in 50 μl saline) are injected intravenously and the incision is closed with suture. Tumors are measured 3 times a week using calipers. Tumor volume is calculated as follows: small diameter × large diameter × height / 2.

  Based on GFP or RFP, bacterial localization within the tumor is also analyzed using frozen section tumor tissue. Reliable morphological and histological preservation and reproducible GFP or RFP detection can be obtained using the slow tissue freezing protocol (Shariatmadari et al., Biotechniques 30 (2001), 1282). Briefly, tumor tissue is removed from a sacrificed animal into a PBS-containing petri dish and cut to the desired size. Samples are mixed in PBS containing 4% paraformaldehyde (PFA) for 2 hours at room temperature. These are washed once with PBS, embedded in Tissue-Tek at room temperature, then kept in the dark at 4 ° C for 24 hours and slowly frozen at -70 ° C. Tissues are maintained at -20 ° C for 30 minutes prior to subdivision. Sections 10-50 μm thick are then cut with a Reichert-JungCryocut 1800 cryostat and collected on poly-L-lysine (1%) treated microscope slides. During fragmentation, the material is maintained at room temperature to avoid several freeze / thaw cycles. Finally, the sections are rinsed in PBS, placed in PBS and kept in the dark at 4 ° C.

To monitor the entry of luminescent E. coli and L. monocytogenes from the bloodstream into the tumor, 27 nude mice are injected with C6 tumor cells and 27 nude mice are injected with HCT116 colon cancer tumor cells. Twelve days after tumor development, 9 animals in group C6 and 9 animals in group HCT116 are given intravenous injections of E. coli with RFP constructs. Another 9 animals in each group are injected intravenously with L. monocytogenes transformed with the GFP cDNA construct. Nine animals from each tumor model in the third group receive both E. coli and L. monocytogenes (1 x 10 ⁶ cells each). At 5 hours, 25 hours, and 5 days after injection, 3 animals in each treatment group are sacrificed and their tumors excised and treated individually as described in the freeze fragmentation protocol described above. After freezing, each tumor is cut in half. One tumor is used for the preparation of thick sections (60-75 μm), which are analyzed under a fluorescence stereomicroscope to observe the bacterial distribution in the tumor sections obtained at each time point of the experiment. The region of interest is identified, sliced, prepared, and analyzed under a confocal microscope with laser scanning cytometry before the image is reconstructed.

  In parallel experiments, animals with spontaneous tumors listed in Table 6 are obtained and used for intravenous injection experiments using E. coli with the bacterial lux operon. Two animals of each tumor model are used and luciferase luminescence is monitored daily under a low light imaging device. Spontaneous tumors are expected to be able to image similar to transplanted tumors based on bacterial luciferase expression. Two naturally occurring tumor models, mice with adenocarcinoma of the large intestine and mice with adenocarcinoma of the breast tissue, are used for experiments under bacterial localization after intravenous injection of RFP-expressing E. coli and GFP-expressing L. monocytogenes as described above To do. These experiments are expected to highlight the importance of bacterial-based diagnostic and protein therapy systems.

Example 7: Bacteria-mediated tumor targeting and bacterial secretory protein therapy (A) principle in rodents with transplanted or spontaneous tumors As shown in the previous examples, intravenous injection of luminescent bacteria is , Resulting in invasion, replication and accumulation only in the tumor area of the animal. This process can be monitored by imaging of luminescence in the tumor. Placing endostatin and chimeric toxin-expressing gene cassettes in cis position relative to the luminescent gene cassette provides an indirect in vivo detection system for these primary and special delivery through bacteria.

  Ligation of endostatin and chimeric toxin-expressing gene cassettes with a signal peptide encoding the sequence facilitates secretion of these proteins into the extracellular space of the tumor or into the cytosol of infected tumor cells. Both proteins secreted from bacteria into the extracellular space of the tumor are expected to function in the same way as purified proteins injected directly. Both proteins secreted into the cytosol of infected tumor cells from L. monocytogenes are similar to the viral delivery systems previously reported for endostatin. The bacterial system may be used in tumors as a constitutive secretion system or as an exogenously added IPTG-activatable secretion system. By regulating the expression level of the therapeutic protein in bacteria that colonize the tumor, the amount of protein secreted that inhibits tumor growth can be determined. Without the addition of IPTG, inhibitory protein secretion from intravenously injected bacteria is kept to a minimum in the blood circulation. This provides additional safety for recipient animals with tumors during bacterial delivery. The BSPT system can be used to control the onset and duration of treatment by the addition of IPTG. Upon completion of the treatment, the bacterial delivery system can be removed by administration of antibiotics, similar to the treatment of bacterial infections.

(B) Measurement of the effect of endostatin and Pseudomonas exotoxin / TGFα fusion protein secreted by E. coli and L. monocytogenes on tumor growth in animals with transplanted tumors Secreted by E. coli and L. monocytogenes in tumors The inhibitory effect of endostatin and the cytotoxicity of the chimeric toxin are examined as follows. 35 nude mice with C6 tumors on day 10: (a) E. coli engineered to secrete endostatin into 5 mice; (b) Chimeric toxin secreted into 5 mice E. coli engineered to: (c) L. monocytogenes engineered to secrete endostatin in 5 mice; (d) L. monocytogenes engineered to secrete chimeric toxins in 5 mice. (E) E. coli secreting endostatin and chimeric toxin in 5 mice; (f) Control group: E. coli expressing only bacterial luciferase in 5 mice and GFP expressed in 5 mice Inject bacterial constructs like L. monocytogenes. Each tumor volume is measured at the time of bacterial injection. Three days after injection, bacterial replication in the tumor is monitored under a low light imaging device or under a fluorescence stereo microscope. Luminescence and tumor volume are measured daily up to 20 days after bacterial injection. Ten days after injection, one animal in each group is sacrificed and the level of secreted protein present in the tumor tissue is analyzed by Western blot analysis. These experiments result in inhibition of tumor growth in endostatin treated animals or more dramatic tumor regression in animals treated with chimeric toxin protein. In control animals, tumor growth is not expected to be affected by bacteria alone.

  In a follow-up experiment, a naturally occurring adenocarcinoma type of breast tissue (FVB-neuN (N # 202) strain, Table 6) is used to study the effect of secreted proteins on tumor growth. The same experimental scheme as described in C6 tumor analysis is used. At the end of tumor treatment, the presence of endostatin and chimeric toxins in the tumor tissue is examined by Western blot analysis. The same experimental design is used to assay the IPTG-induced effects of endostatin and chimeric toxin production in bacteria in C6 tumors and in naturally occurring breast tumor mouse models. It is expected that multi-IPTG induction of protein expression in bacteria may be required for good tumor therapy.

  At any stage of tumor treatment, it may be necessary to remove the luminescent therapeutic gene-containing bacteria from the animal. To perform this experiment, mice with day 12 C6 tumors are intravenously injected with bacterial luciferase-expressing E. coli. Three days after injection, antibiotic treatment is administered by intraperitoneal administration of gentamicin (5 mg / kg body weight) twice a day or newly discovered clinafloxacin (CL960) (Nichterlein et al., Zentralbl. Bakteriol. 286 (1997), 401) Start. This treatment is performed for 5 days and the effect of antibiotics on the bacteria is monitored by daily luminescence imaging from animals.

  By completing the above experiment, endostatin and chimeric toxin proteins secreted into the tumor are expected to cause inhibition of tumor growth and measurable tumor regression. Tumor regression is expected to be achieved in both groups of rodents with transplanted tumors and rodents with spontaneous tumors. Simultaneous application of secreted endostatin and chimeric toxin protein in tumor treatment may give the most promising results. Removal of engineered bacteria from the tumor by administration of antibiotics is a means to add to the safety of the bacterial secretory protein therapy (BSPT) of the present invention.

FIG. 1 shows external imaging of GFP expression in subcutaneous C6 glioma of nude mice. C6 glioma cells (5 × 10 ⁵ ) were implanted subcutaneously in the right thigh. On designated days after tumor cell transplantation, animals were intravenously infected with 1 × 10 ⁸ pfu rVV-ruc-gfp virus particles. GFP expression was monitored under a fluorescent stereomicroscope. Shown are bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of subcutaneous glioma. A GFP signal can be observed in tumors as small as 22 mm ³ (B-B ″) or as large as 18 days of age (approximately 2500 mm ³ ) (A-A ″). In older tumors, GFP expression was seen in a “patch” -like pattern (indicated by arrows at A ′). Marker gene expression in tumors of the same animal is monitored continuously 4 days (C-C ''), 7 days (D-D ''), and 14 days (E-E '') after intravenous virus infection Can be done. (Bar = 5mm). FIG. 2 is a visualization of tumor angiogenesis. C6 glioma cells (5 × 10 ⁵ ) were implanted subcutaneously into the right thigh of nude mice. Ten days after tumor cell transplantation, animals were intravenously infected with 1 × 10 ⁹ pfu of rVV-ruc-gfp. GFP expression was monitored 7 days after virus injection. Angiogenesis at the surface of subcutaneous C6 glioma is shown against a bright green fluorescent background in tumors following vaccinia-mediated gene expression. Bright field (A), fluorescence (B), and bright field, fluorescence overlay (C) images of subcutaneous glioma are revealed. (Bar = 5mm). FIG. 3 shows the expression of GFP in subcutaneous glioma of the same animal. Five days after subcutaneous implantation of 5 × 10 ⁵ C6 glioma cells into the right lateral thigh, 10 ⁸ rVV-ruc-gfp virus particles were injected intravenously. Five days after virus injection, animals were anesthetized and sacrificed for analysis of GFP expression under a fluorescence microscope. The covered skin was folded (B-B ''), in a cross-sectional view (C-C ''), and in a cut leg (D-D ''), the tumor was visualized externally (A-- A '' ). Bright field (A), fluorescence (B), and bright field, fluorescence overlay (C) images of subcutaneous glioma are revealed. The strongest GFP expression is seen as a patch located along the outer surface of the right tumor (double arrow in CC). The sharp difference in GFP expression in tumor tissue and normal muscle tissue (arrows in DD) is clearly recognizable. Mark the folded skin with an asterisk (B-B '' and D-D ''). (Bar = 5mm). FIG. 4 shows bright field (A) and fluorescence (B) images of tumor cells expressing GFP. Frozen sections (30 μ thick) of glioma tissue were prepared from nude mice intravenously injected with 1 × 10 ⁸ rVV-ruc-gfp virus particles. (Bar = 50 μm). FIG. 5 is a low light image of anesthetized nude mice, showing the position of renilla luciferase-induced luminescence in the presence of the intravenously injected substrate coelenterazine (5 μg ethanol solution). FIG. 6 shows subcutaneous PC-3 human prostate tumor (A to A ″) and MCF-7 human breast tumor (B to B ″) in nude mice, intracranial C6 rat glioma (C to C in Lewis rats). '', Arrows indicate tumor location), and monitoring of tumor-specific viral infection based on GFP gene expression in various tumor models including MB-49 mouse bladder tumor (D-D '') in C-57 mice Indicates. Animals were monitored 7 days after intravenous injection of 1 × 10 ⁸ rVV-ruc-gfp virus particles. Tumor bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images are shown. (Bar = 5mm). FIG. 7 shows monitoring of vaccinia-mediated GFP expression in a breast tumor model. Nude mice bearing breast tumors were injected intravenously with 1 × 10 ⁸ rVV-ruc-gfp virus particles. Both primary tumors (A-A '', B-B '' and C-C '') and metastatic tumors (D-D '', E-E '' and F-F '') are external ( A to A '' and D to D ''), removing the covering skin (B to B '' and E to E '') and tearing them open (C to C '' and F-F ''), brightfield, fluorescence (') and brightfield, fluorescence overlay (") set of images visualized. GFP expression in lung metastases in the same animal was also visualized (GG). (Bar = 5 mm (A to A ″ to F to F ″), and Bar = 1 mm (G to G ″). FIG. 8 shows the visualization of the clearance of luminescent bacteria from nude mice based on detection of luminescence under a low light imager. Nude mice were injected intravenously with 10 ⁷ cells of attenuated S. typhimurium (A, B) and V.cholera (C, D). Both strains were transformed with pLITE201 carrying the lux operon. Photon collection was performed 20 minutes (A, C) and 2 days (B, D) after bacterial injection. FIG. 9 shows the homing of glioma by attenuated bacteria. Nude mice with C6 glioma in the right hind limb were subjected to 10 ⁷ attenuated S. typhimurium (AD) and V. cholesterol (EH) co-transformed with pLITE201 plasmid DNA encoding the lux operon. ) Was injected intravenously. Photon recovery was performed for 1 minute under a low light imager. Mice injected with S. typhimurium immediately showed luminescence throughout the animal (A). In contrast, luminescence in mice injected with V.cholera was visible in the liver area (E). Two days after bacterial injection, both groups of mice showed luminescence only in the tumor area (B, F). Luminescence in tumors infected with S. typhimurium decreased slowly at 4 days (C) and 6 days (D) after bacterial injection. Tumors infected with V. cholera show greatly increased luminescence at 4 days (G) and 6 days (H) after injection, suggesting sustained replication of bacteria in the tumor tissue. FIG. 10 shows bacterial homing to breast tumors. Nude mice with breast tumors in the right chest pad are given 10 ⁷ attenuated V.cholera (AD) or 10 ⁷ E. coli (EH) transformed with pLITE201 plasmid DNA encoding the lux operon. Used for intravenous injection. Photon recovery was performed for 1 minute under a low light imager. After 20 minutes of bacterial delivery, luminescent V. cholera was observed in the liver (A). Forty-eight hours after injection, luminescence was shown in the primary breast tumor in the right breast region and the metastatic tumor in the left breast region (arrow) and in the cut wound (B). On day 5, luminescence was only visible in the tumor area and not in the wound (C). Eight days after bacterial injection, luminescence activity was abolished from smaller tumor areas, but remained strongly in the primary breast tumor (D). Two days after intravenous bacterial injection, E. coli homing to breast tumors in nude mice was also observed (E: side view, F: ventral view). FIG. 11 shows bacterial homing to bladder tumors in C57 mice. C57 mice were injected intravenously with 10 ⁷ attenuated V.cholera transformed with pLITE201 encoding the lux operon. Nine days after bacterial delivery, it showed luminescence in the bladder region of the entire animal (A). The animals were sacrificed, an abdominal incision was made, and the bladder was exposed. Luminescence was limited to the bladder region (B). With the removal of the bladder from the mouse (C), all sources of luminescence were removed (D) as illustrated by the overlay of the weak photon generation image over the photographic image of the excised bladder. FIG. 12 shows bacterial homing to brain glioma in Lewis rats. Lewis rats were injected intravenously with attenuated V. cholera of 10 ⁸ cells transformed with pLITE201 encoding the lux operon. Twenty-four hours after bacterial injection, the whole animal showed weak luminescence in the head region during visualization under a low light imager. The animals were sacrificed and their brains removed. Photon recovery was performed for 1 minute from rats with brain tumors (A) and rats without brain tumors (B). Strong luminescence was confirmed in the brain region of rats with brain tumors (arrowed in A). There was no luminescence in the control brain tissue (B). FIG. 13 shows that transformed human fibrosarcoma cells home on subcutaneous gliomas in nude mice. Nude mice with subcutaneous gliomas were injected intravenously with 5 × 10 ⁵ human fibrosarcoma cells permanently transformed with a retrovirus derived from pLEIN. Seven days after injection, the animals were anesthetized and monitored under a fluorescent stereomicroscope. Fluorescent cells were shown only in the tumor area of the entire mouse through the skin (A1-3). Upon exposure of the tumor tissue by wrapping the covered skin (B1-3), in the cross-sectional view of the tumor (C1-3), fluorescent patches could be recognized in separate areas. Precise experiments with mouse organs showed the presence of small clusters of fluorescent cells in the lungs of animals, indicating the affinity of fibrosarcoma cells for the lung in addition to tumor tissue (D1-3). (Bar = 5 mm (A1 to C3), = 1 mm (D1 to D3)). FIG. 14 shows homing of attenuated Listeria monocytogenes to subcutaneous prostate tumor. Nude mice with subcutaneous human PC3 prostate tumor in the right hind leg were injected intravenously with 10 ⁷ attenuated L. monocytogenes transformed with psod-gfp plasmid DNA carrying gfpcDNA, under fluorescence stereomicroscope GFP fluorescence was observed. GFP signal was detected only in the tumor area 27 hours after bacterial injection. Tumors are shown with a set of visible light (a), fluorescence (b), and visible light and fluorescence overlay (C) images. (Bar = 5mm).

Claims (20)

Tumor in a subject, tumor tissue, cancer or for detecting or imaging a metastasis, or tumor in a subject, tumor tissue, cancer or for monitoring therapeutic treatment by imaging a metastasis, systemic use of a vaccinia virus Lister strain for the preparation of a composition for administration, the virus, see contains a DNA encoding a protein that induces a detectable protein or detectable signal, said composition, systemically Use, formulated for administration . Virus The use according to claim 1, wherein the The Institute of Viral Preparations Reshonzu Lister strain (LIVP). Composition Use according to claim 1 or 2 that is formulated for intravenous administration. Composition, intraperitoneal, subcutaneous, intratumoral, using intramuscular, or formulated are that claims 1 to 3, wherein any one for intradermal administration. Use according to any of claims 1 to 4 , wherein the virus allows detection of tumor, tumor tissue, cancer or metastasis by detection of light. Proteins that induce detectable signals include magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron-computed tomography (PET), scintigraphy, gamma camera, β ⁺ detector, Or use according to any of claims 1 to 5 , which induces a signal detectable by a gamma detector. Use according to any of claims 1 to 6 , wherein the detectable protein or the protein that induces a detectable signal emits light or induces the emission of light. 8. Use according to claim 7 , wherein the detectable protein or the protein that induces a detectable signal is a fluorescent protein or a photoprotein. 8. Use according to claim 7 , wherein the protein capable of inducing a detectable signal is luciferase, green fluorescent protein or red fluorescent protein. Use according to any of claims 1 to 6 , wherein the detectable protein or the protein that induces a detectable signal binds to a contrast agent, chromophore or ligand for tissue visualization. The use according to any of claims 1 to 6 , wherein the protein that induces a detectable signal is a metal binding protein. The use according to any of claims 1 to 6 , wherein the protein that induces a detectable signal is a cellular receptor that binds to a detectable ligand for detection of a tumor, tumor tissue, cancer, or metastasis in a subject. . Use according to claim 12 , wherein the cellular receptor is a transferrin receptor. 13. Use according to claim 12 , wherein the detectable ligand is a metal-labeled ligand or a ligand that binds to a metal. 15. Use according to any of claims 1 to 14 , wherein the tumor is a breast, prostate, bladder, brain, colon, lung, ovary, pancreas, liver or skin tumor. Use according to any of claims 1 to 15 , wherein the virus comprises DNA encoding a therapeutic product. The therapeutic product is cytotoxic protein, cytostatic protein, protein that inhibits angiogenesis, protein that stimulates apoptosis, protein that inhibits elongation factor, protein that binds to ribosomal subunit, protein that modifies nucleotide, nuclease, 17. Use according to claim 16 , selected from among proteases, cytokines, toxins, enzymes and receptors. The therapeutic products are rsCD40L, Fas-ligand, TRAIL, TNF, anti-GA733-2, microcin E492, diphtheria toxin, Pseudomonas exotoxin, Shiga toxin, endostatin, glucuronidase, β-galactosidase, thymidine kinase, western 17. Use according to claim 16 , selected from horseradish peroxidase, carboxypeptidase G2, cytochrome P450, nitroreductase, cytosine deaminase, carboxylesterase, tyrosinase and verotoxin. 17. Use according to claim 16 , wherein the therapeutic product is a cellular receptor that binds to a therapeutic ligand for tumor therapy. 20. Use according to claim 19 , wherein the therapeutic ligand comprises a ligand bound to a toxin.

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