Indian Patents
Title of Invention

A COMPOSITION COMPRISING A VACCINIA VIRUS
Abstract Described are diagnostic and pharmaceutical compositions comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, e.g. a luminescent or fluorescent protein, or a protein inducing a signal detectable by magnetic resonance imaging (MRI) or a cell receptor capable of binding a ligand and, particular embodiment, furthermore (a) DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors, e.g. a cytotoxic or cytostatic protein.
Full Text FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
&
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10, rule 13)
THERAPY FOR TUMORS
GENELUX CORPORATION, a corporation organized under the laws of Delaware at 3030 Bunker Hill Street, Suite 310, San Diego, California 92109, United States of America
The following specification particularly describes the invention and the manner in which it is to be performed.

2-
THERAPY FOR TUMORS
The present invention relates to diagnostic and pharmaceutical
compositions comprising a microorganism or cell containing a
DNA sequence encoding a detectable protein or a protein.

capable of inducing a detectable signal, e.g. a luminescent or
fluorescent protein and in a particular embodiment,
furthermore (a) DNA sequence(s) encoding (a) protein(s)
suitable for tumor therapy and/or elimination of metastatic
tumors, e.g. a cytotoxic or cytostatic protein.
Presence of bacteria in tumors was reported approximately fifty years ago Several publications substantiated the earlier clinical findings that unexpectedly large numbers of bacteria were discovered in excised tumors from human patients. Investigators argue that chronic infections may predispose cells to malignant growth. Chronic infections of various strains of Chlamydia have been associated with lung and cervical cancer as well as malignant lymphoma. Another well described association between the presence of a specific bacterial species and cancer development is Helicobocter pylori in patients with gastric ulcers. Elevated levels of II pylori-associated antibodies have been found in patients with duodenal ulcer and gastric adenocarcinoma. These observations demonstrate a concomitant presence of bacteria at tumor sites however, it was not yet clear whether the microorganisms were the cause of tumor formation or whether the. tumorous tissues were more susceptible to bacterial colonization. Intravenously injected strict anaerobic bacteria, Clostridium pesteurianvm, into mice replicated selectively in the tumor suggesting a hypoxic microenvironment in the necrotic center. Intravenous injection of attenuated SaJmonella typhimurium mutants resulted in elevated bacterial titers in the tumor tissues in comparison to the other organs of mice upon histologic and bacteriologic analyses.

3,
Similarly, the presence of virus particles was reported in excised human breast turners as early as 1965. More recently, based on polymerase chain reaction (PCR) data, the human papillomavirus has been claimed to be associated with anogenital tumors and esophageal cancers, breast cancers, and most commonly, cervical cancers- In addition, the presence of hepatitis C virus in human hepatocellular carcinoma, Epstein-Barr virus in squamous cell carcinoma in Kirnura’s disease, mouse mammary tumor virus-like paiticles (MMTV) in human breast cancer, SV40 virus in macaque astrocytoma, and herpesvirus in turtle fibropapilloma ha6 been reported. Surprisingly, the concentration of virus particles in the tumors shows variations among patients. The presence of human papillomavirus in squamous cell carcinomas j of the esophagus ranges from 0 to 72% {10- 15). In contrast to tumor tissues, no virus particles have been found in tumor-free areas of the esophageal epithelium of the same patient suggesting that the virus particles are located only in the tumor tissues.
However, so far it could not be shown definitely whether the above discussed microorganisms are responsible for the development of disorders like tumors (except for papillomaviruses) or whether e.g. tumors can attract and/or protect viruses or bacteria. Accordingly, there was no basis for the use of such microorganisms for the diagnosis or therapy of tumors. Conventional tumor diagnostic methods, such as IviR.1 {Magnetic Resonance Imaging) lack sensitivity and specificity and therapeutic methods, e.g. surgery, are invasive and not very sensitive.
Therefore, it is the object of the present invention to provide a means for the efficient and reliable diagnosis as well as the therapy of tumors which overcomes the disadvantages of the diagnostic and therapeutic approaches presently used.

-3-
According to the present invention this is achieved by the subject matters defined in the claims. When Vaccinia virus-{LIVP strain) carrying the light emitting fusion gene construct rW-ruc-gfp was injected intravenously into nude mice, the virus particles were found to be cleared from all internal organs within 4 days as determined by extinction of light emission. In contrast, when the fate of the injected Vaccinia virus was similarly followed in nude mice bearing tumors grown from subcutaneously implanted C6 rat glioma cells, virus particles were found to be retained over time in the tumor tissues, resulting in lasting light emission. The presence and amplification of the virus-encoded fusion proteins in the same tumor were monitored in live animals by observing GEP fluorescence under a stereomicroscope and by collecting luciferase-catalyzed light emission under a low-light video-imaging camera. Tumor-specific light emission was detected 4 days after viral injection in nude mice carrying subcutaneous C6 glioma implants ranging in size from
25 to 2500 (mm3. The signal became more intense after the 4th
postinjection day and lasted for 30 to 45 days, indicating continued viral replication. Tumor accumulation of rW-ruc-gfp virus particles was also seen in nude mice carrying subcutaneous tumors developed from implanted PC-3 human prostate cells and in mice with orthotopically implanted MCF-7 human breast tumors. Further, intracranial C6 rat glioma cell implants in immunocompetent rats and MB-49 mouse 'bladder tumor cell implants in C57 mice were also targeted by the Vaccinia virus. Cross sections of a C6 glioma revealed that light emission was clustered in „patches„ at the periphery of the tumor where the fast-dividing cells reside. In contrast, cross sections of breast tumors revealed that fluorescent ,,islands,, were distributed throughout the tumors. In addition to primary breast tumors, small metastatic tumors were also detected externally in the contralateral breast region, as

faifi as in nodules on the exposed lung surface, suggesting metastasis to the contralateral breast and lung. In summary, light-emitting cells or microorganims, e.g. Vaccinia virus can be used to detect and treat primary and metastatic tumors.
Similar results were obtained with light-emitting bacteria
(Salmonella, Vibrior Listeria, E. coli) which were injected
intravenously into mice and which could be visualized in whole
animals under a low light imager immediately. No light
emission was detected thirty-six hours after bacterial
injection in both athymic (nu/nu) mice and immunocompetent C57
mice as a result of clearing by the immune system.; In the
cutaneous wound of an intravenously injected animal, the
bacterial light emission increases and remains detectable up
to six days post-injection. In nude mice baring tumors

deyeloped from implanted C6 glioma ceil light emission was
abolished from the animal entirely thirty-six hours after
delivery of bacteria, similar to mice without tumors. However,
forty-eight hours post-injection, unexpectedly, a strong
rapidly increasing light emission originating only from the
tumor regions was observed. This observation indicates a
continuous bacterial replication in the tumor tissue. The
extent of light emission is dependent on the bacterial strain
used. The homing-in process together with the sustained light
emission was also demonstrated in nude mice carrying prostate,
bladder, and breast tumors. In addition to primary tumors
metastatic tumors could also be visualized as exemplified in
the breast tumor model. Tumor-specific light emission was also
observed in immunocompetent C57 mice with bladder tumors as
well as in Lewis rats with brain glioma implants. Once in the
tumor the light-emitting bacterid wet not observed to be
released into the circulation and to re-colonize subsequently
implanted tumors in the same animal. Further, mammalian cells
expressing the Ruc-gfp fusion protein, upon injection into the
bloodstream were also found to home into and propagate in
glioma tumors.

These findings open the way for (a) designing multifunctional viral vectors useful for the detection of tumors based on signals like light emission and/or for suppression of tumor development and/or angiogenesis signaled by e.g., light extinction and (b) the development of bacterium- and mammalian cell-baaed tumor. targeting systems in combination with therapeutic gene constructs for the treatment of cancer. These systems have the following advantages: (a) They target the tumor specifically without affecting normal tissue (b) the expression and secretion of the therapeutic gene constructs are, preferably, under1 the control of an inducible .promoter, enabling secretion to be switched on or off and (c) the location of the delivery system inside the tumor can be verified by direct visualization before activating gone expression and protein delivery.
Accordingly, the present invention relates to a diagnostic and/or pharmaceutical composition comprising a microorganism or cell containing a DNA sequence encoding a (detectable protein or a protein capable of inducing a detectable signal.
In a preferred embodiment, the microorganism or cell of said diagnostic and/or pharmaceutical composition furthermore contains (a) DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors, such as a cytotoxic protein, a cytostatic protein, a protein inhibiting angiogenesis or a protein stimulating apoptosis. Such proteins are well-known to the person skilled in the art and further examples of suitable proteins are given below.
Any microorganism or cell is useful for the compositions of the present invention, provided that they replicate in the organism, are not pathogenic for the organism, e.g. attenuated, and are recognized by the immune system of the organism, etc. Examples of microorganisms useful for the

7

present invention are bacteria and viruses. The term bacteria" as used herein refers to bacteria which are per -se not tumor-targeted (i.e. they can not differentiate between. cancerous cell or tissue and the non-cancerous-- counterpart cell or tissue) since the results of the experiments leading to the present invention show that bacteria etc. accumulate in the tumor due to the fact that in this environment they are not exposed to attack by the immune system of the host. A list of candidate bacteria which might be useful for the purposes of the present invention and which might not be tumor-targeted are given in Table 1, below. The person skilled in the art can easily identify such bacteria which are not tumor-targeted by
commonly available methods, eg. the methods described in

section 6.1 of WO 96/40238. Preferably, said bacteria are
intercellular bacteria such as E. coli, E. faecalisr Vibrio
cholerae, Vibrio fischeri. Vibrio harveyi, Lactobacillus spp. ,
Pseudomonas spp. In the method of the present invention,
viruses and cells, particularly mammalian cells, are preferred

which are not tumor-targeted. Particularly preferred are
cytoplasmic viruses.
In a particularly preferred embodiment, the diagnostic and/or pharmaceutical composition comprises a microorganism or cell containing a DNA sequence encoding a luminescent and/or fluorescent protein.
As used herein, the term „DNA sequence encoding a luminescent and/or fluorescent protein,, also comprises a DNA sequence-encoding a luminescent and fluorescent protein as fusion protein.
In an alternative preferred embodiment of the diagnostic and/or pharmaceutical composition, the protein encoded by the. DNA sequence is a ceil receptor capable of binding a ligand which can be a diagnostic or therapeutic ligand. The ligand

s
Man be a protein (including large or small peptides,
antibodies, etc.), a synthetic compound such as a synthetic steroid analog), etc. Therefore, the in vivo location of labeled ligands in live animals and human patients can be visualized, e.g, in real time by SPECT or PET. Almost any known protein ligand-receptor pair for tumor labeling Is useful in the method of the present invention. Preferably, to increase specificity, mutant protein ligands (or analogs if it is a chemical compound) or mutant ligand receptors can be genetically or chemically engineered so that they will not bind to any endogenous molecules. In addition to increasing specificity, these mutants/analogs will also limit adverse effect to the normal host physiology.

In a more preferred embodiment of the diagnostic and/or pharmaceutical composition of the present invention, the ligand is a radionuclide-labelled ligand. Said ligand is, e.g., useful for tumor visualization by single-photon emission computed tomography (SPECT) or positron-emission tomography (PET) resulting from the binding of radionuclide-labelled ligand to its receptors expressed specifically on the surface of tumor cells following intravenous delivery of, e.g., engineered bacteria,, viruses, or mammalian cells carrying the receptor protein gene constructs.. Radionuclides may be used for conventional tumor scintigraphy, PET and possibly internal radiotherapy of tumors. Examples of radionuclides useful in the present invention are (a) B+-emitters such as 11C, 13N, 150 or 64Cu or (b) -emitters such as U'3I. Other radionuclides that can, e.g., be used as tracers for PET include 55Co, 67Ga, 63Ga, 60Cu(II), 63Cu(II), 51Ni, __59Co, 57Fe, l6F, etc.
SPECT and PET are sensitive techniques that may be used for tumor imaging according to the present invention. Both SPECT and PET are capable of detecting trace amounts of B+ and y

9
emission from radionuclides. PET is even more sensitive than
SPECT. In experiments using small laboratory animals, tumor
imaging is .performed using a microPET instrument, which 'is
commercially available through, e.g., Concorde Microsystems
(Knoxville, TN).
Examples of useful radionuclide-labeled agents are ^Cu-labeled engineered antibody fragment (Wu et al., PNAS USA 91_ (2002), 8495-8500), 64Cu-labeled somatostatin (Lev/is et al.., J.Med.Chem. 42 (1999), 1341-1347, 64Cu~pyruvaldehyde- bis(N4-methylthio.semicarbazone) (64Cu-PTSM) (Adonai et al., PNAS USA 99 (2002),; 3030-3035, :S2Fe-citrate (Leenders et al., J.Neural Transm. Suppl- 43 (1994), 123-132, 52Fe/52mMn--citrate

9), 2047-2055) and
(Calonder | et al., J.Neurochem. 73 (199J
Fe-labeled iron(III) hydroxide-sucrose {complex (Breshara et al./ Br J. Haematol. 104 (1999), 268-295, 296-302).
In order to apply the radionuclide-labelled ligand in tumor detection, the genes encoding the receptor proteins that the ligands may bind are delivered by intravenously injected bacteria, viruses, or mammalian cells according to the present--invention-. Since it could be shown in the examples, below, that certain intravenously injected bacteria, viruses and mammalian cells replicate specifically in the tumors, expression of the receptor proteins in the tumors, will mark the tumors for targeting by the radionuclide-labeled ligands.
For example, in the case of Vaccinia virus, to allow efficient tumor detection, the virus can be used to carry gene constructs encoding receptor proteins that can bind specifically to the ligands. Intravenously injection of recombinant Vaccinia virus allows the delivery of the receptor gene and surface expression of the receptor protein in the tumor tissues. Then, the radionuclide-labeled ligands are injected intravenously into the host. The specific binding between radionuolide-labelled ligands and their receptors

10
-9-
expressed on the tumor cell surface allows the detection of tumors baaed on β+ or γ-emissions originating only from the tumors. Labeling the tumors with radionuclides will allow easy distinction between tumors and- normal tissues. Therefore, handhold β+- or γ-detectors that are attached to a surgical blade holder can be designed. Based on the signal of β*- or y-emission, the labelled tumors can be cleanly excised while the removal of normal tissues is kept at a minimum.
Particularly preferred is gallium-67 for the purposes of the
present invention. Galliuiti-67 (67Ga) can be used for
diagnostic, imaging using PET, SPECT, or scintigraphy. It is
known for its ability to I accumulate in inflammatory lesions
and tumors, especially in lymphomas, but also in many other
types of. tumors, such as in pancreatic tumors, lung tumors
etc. i The mechanism of 67Ga uptake has been proposed to be
through both transferrin-dependent route and transferrin
independent | route. In the transferrin-dependent route,, it
has been shown that over-expression of transferrin receptor
significantly increases 67Ga uptake- by cumor ceils.
Furthermore, anti-transferrin receptor antibody
significantly blocks 67Ga by tumor cells. For small tumor
imaging, very high concentrations of G1Ga are required to

overcome background signal. Thus, in this case, the use of
recombinant Vaccinia virus carrying a transferrin receptor
gene construct for over-expressing transferrin receptors
specifically on the surface of tumor cells in live animals
or human patients following intravenous injection of the
viruses is preferred. 67Ga will be also- -delivered
intravenously. High level accumulation of 6'Ga in tumor cells
with the help from the over-expressed transferrin receptors
helps to significantly improve tumor detection abilities in
live animals and human patients.

11
10
In an alternative preferred embodiment, the diagnostic and/or pharmaceutical composition of the present invention comprises a microorganism or cell containing a DNA sequence encoding a protein capable of inducing a signal detectable by magnetic resonance imaging (MRI), e.g. metal binding proteins. Furthermore, the protein can bind contrast agents, chromophores, Uganda or compounds required for visualisation of tissues. Preferably, said protein is a cell receptor capable of binding a paramagnetic- or superparamagnetic metal-labelled ligand- Tumor visualization by MRI, resulting from the binding of paramagnetic- or superparamagnetic-metal-labelled ligands to their receptors expressed specifically on the surface of tumor cells following, e.g., intravenous delivery of engineered bacteria, viruses, or mammalian cells according to the present invention carrying the I receptor protein gene constructs has several advantages. The high level of accumulation of these metals in the tumors will facilitate tumor detection, virtually any paramagnetic or superparamagnetic metals can be used for this purpose.' Preferably, duo. to the systemic toxicity of naked heavy metals, the paramagnetic or superparamagnetic are carefully selected to keep adverse effects at minimal. In most currently available contrast agents, the metal particles are either linked to chelates or are coated with polymers, which are much safer to use than naked particlas. Therefore, the use of ligands tagged with chelated or polymer-coated metals is preferred.
Methods for generating contrast agents linked with proteins are well known to the person skilled in the art, e.g., the protein ligand (or other chemical compound ligand) is first chemically attached to the chelates (e.g. diethylene triamine pentaacetic acid (DTPA)). Then, the chelate-protein complex is labeled with metals. A similar labeling method has been used in generating gallium-labeled somatostatin

12
11
analogs and used in producing indium-lll-labeled Mow density" lipoprotein using DTPA-bis(stearylamide). Details of protein modifications with chelates used in contrast agents have been previously described. For example, modifying proteins with the bifunctional chelate 2- (isothiocyanotdbenzyl-e-methyl) diethylenetriamine-pentaacetic acid (MX-DTPA)) (Mirzadeh et al., Bioconjug. Chem. 1 (1990), 59-65) and modifying proteins with the cyclic anhydride : of diethylenetriamine-pentaacetic acid (cDTPAA) (Duncan and Welch, J. Nucl. Med. 34 (1993), 1728-1738) have been described.
Alternatively, MRI can be carried out! by the following approaches;
(a) Modified gadolinium activation duel to enzyme/protein delivery
This approach Is based on the principles of Gene-Directed Enzyme Prodrug Therapy (GDEPT)/ in which case the enzymes/proteins activate a systemically delivered nontoxic prodrug into active toxic drug. Specific activation of the MRI contrast agent in weak relaxivity state into strong relaxivity state by the enzyme/protein in the tumors allows tumor detection. The enzyme/protein delivery to tumors is achieved through intravenously injected engineered bacteria, viruses, and mammalian cells, which carry the gene encoding β-galactosidase (or any other related -enzyme). The β- galactosidase can be in either extracellular secreted form or expressed an the bacteria or mammalian cell surface. 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-galactopyranosyiethoxy)-1,4,7,10-

tetraazacyclododecane) gadolinium (Egad) (Moats et al., 1997,). In this compound, a galactopyranose residue is positioned at the 9th coordination site of the Gd3+ ion. Due to this blockage, water protons are excluded from interacting with Gd3+ ion, and therefore diminished effect on Tl relaxation time. In the presence cf 13-gal act o 51 close, the enzyme cleaves the galactopyranose from the chelate, freeing the coordination site, and allows irreversible transition of the contrast agent from a weak to a strong relaxivity state. Since the bacterium-
virus-, or mammalian cell-based enzyme expression occurs only in tumors, the enzyme-mediated relaxivity state transition will also occur only in the tumors. Therefore, this enzyme-mediated. activation of MRI contrast agents can be used for tumor detection

In addition to the above MRI contrast agent, similar new contrast agents may be. developed in which other types of residues may be attached to the chelates. These residues can be removed by their corresponding enzymes (similar to the removal of galactopyranose by galactosidase) (e.g. α-
mannosidases, a- and (β-glucosidases, β-glucuronidases) to free
the 9th coordination site of the Gd3+ ion and modify the Tl
relaxation time for tumor detections. The genes encoding these
enzymes may all be delivered by the engineered bacteria,

viruses, or mammalian cells according to the present
invention. Moreover, new contrast agents using metals other
than gadolinium may be developed for enzyme-activated MRI
tumor imaging.
This approach can be combined with therapy as shown, in the following sections.

14
13
Engineering of delivery vector systems for activation of the contrast agents (e.g. modified gadolinium) first and subsequently, the activation of an injected prodrug by the same constitutively expressed enzyme (e.g. β-galactosidase) -One-Gene-Product-Based Detection and Therapy System (OGPBDTS)
For example, the gadolinium-based contrast agent can be activated by an enzyme (e.g. β-galactosidase) / as described above, which can be used for tumor detection. Subsequently, the intravenously delivered prodrugs, such as CBI, TMI, PCI (described in US-patent 5,646,298), may be cleaved by the same β-galactosidase in the tumors, to yield active cytotoxic drugs against tumor cells for cancer treatment.
(c) Engineering of delivery vector systems with the Two-Gene-Product-Based Detection and Therapeutic Systems (TGPBDTS)
Gene 1 is linked to a. constitutive promoter and produces the enzymes/proteins for sensing the contrast agents (e.g. metal-binding proteins, modified gadolinium, or other agents) and gene 2 is linked to an exogenously activatable promoter, which is silent without the activator drug, and only turned on after the detection vector system is found positive by MRI. The activation of the vector-based promoter gene construct is achieved by injection or by oral administration, of the activator drug. The repeated injection of the contrast agent allows the real-time monitoring of tumor size and location of metastases during the treatment procedure.

15
14
Preferably, for transfecting the cells the DNA sequences encoding] the (diagnostic or therapeutic) proteins described above/ e.g., a luminescent and/or fluorescent protein are present in a vector or an expression vector. A person skilled in the art is familiar with examples thereof. The DNA sequences can also be contained in a recombinant virus containing- appropriate expression cassettes. Suitable viruses that may be used in the diagnostic or pharmaceutical composition of the present invention include baculovirus, vaccinia/ sindbis virus, Sendai virus, adenovirus, an AAV virus or a parvovirus, such as MVM orH-1. The vector may also be a retrovirus, such as MoMULV, MoMuLV, HaMuSV, MuMTV, RSV or GaLW For expression in mammals; a suitable promoter is e.g. human cytomegalovirus ,,immediate early promoter,, (pCMv) . i Furthermore, tissue and/or organ) specific-. promoters are useful. Preferably, the DNA sequences encoding, e.g., a luminescent and/or fluorescent protein are operatively linked with a promoter allowing high expression. Such promoters, e.g. inducible promoters are; well-known to the person skilled In the art. Preferably, the above constructs are inserted into the bacterial genome by stable integration. If such constructs are made in mammalian cells using mammalian vectors, stably transformed cell lines with a single-copy insertion will be generated for long-term expression in tumor cells.
For generating the above described DMA sequences and i:uc constructing expression vectors or viruses which contain said DNA sequences, it is possible to use general methods known in the art. These methods include e.g. in vitro -recombination techniques, synthetic methods and in vivo recombination methods as described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2rtJ edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, for example. Method.'? of transfecting cells, of phenotypically selecting

transfectants and of expressing the DNA sequences by using the above described vectors are known in the art.
The person skilled in the art knows DNA sequences encoding
proteins, e.g., luminescent or fluorescent .proteins that can
be used in the diagnostic and/or pharmaceutical composition of
the present invention. During the past decade, the
identification and isolation of structural genes encoding
light-emitting proteins from bacterial luciferase from Vibrio
harveyi (Belas et al., Science 2_1_8 (1982), 791-793) and from
Vibrio' fischerii (Foran and Brown, Nucleic acids Res. 16
1998) 177), firefly luciferase (de Wet et al. , Mol. Cell.
Biol 7 (1987), 72b-737), aequorin from AequGiea victoria
Prasher et al.., Biochem. 26 (1987), 1326-1332), Renilla.
'luciferase from Renilla reniformis (Lorenz et al., PNAS USA 6 8
(1991), 4438-4442) and green fluorescent protein from Aequorea,
victoria (Prasher et al., Gene 1JLL (1987), 229-233) have-been
described that allow the tracing of ; bacteria, viruses or
mammalian cells based on light emission. Transformation and
expression of these genes in bacteria allows detection of
bacterial colonies with the aid of the low light imaging
camera or individual bacteria under the fluorescent microscope
(Engebrecht et all., Science 227 (1985), 1345-1347; Legocki et
al., PNAS 83_ (1986), 9080-9084; Chalfie et al., Science 263
(1994), 8G2-805)
Luciferase genes have been expressed in a variety of organisms. Promoter activation based on light; emission, using lux AB fused to the nitrogenase promoter, was demonstrated in Rhizobia residing within the cytoplasm of cells, of infected root nodules by low light imaging (Legocki et at., PNAS 83 (1986)., 908-0-9084; O’Kane et al. , J. Plant Mol. Biol. 10 (1988), 387-3991. Fusion of the lux A and lux B genes resulted

17
16
in a fully functional luciferase protein (Eschar et al., FNAS 85 (1989) , 6528-6532). This fusion gene (Fab2) was , introduced into Bacillus .subtilis and Bacillus megatherium under the xylose promoter and then fed into insect larvae and was injected into the hemolymph of worms. Imaging of light emission was conducted using a low light video camera. The movement and localization of pathogenic bacteria in transgenic arabidopsis plants, which carry the pathogen-activated PAL promoter-bacterial luciferase fusion gene construct, was demonstrated by localizing Pseudomonas or Ervinia spp: infection under the low light imager as well as in tomato plant and stacks of potatoes (Giacomin and Szalay, Plant Sci. 116 (1996), 59-72).
All of the Luciferases expressed in bacteria require exogenously added substrates such as decanal or coelenterazine for light emission. In contrast, while visualization of GFP fluorescence does not require a substrate, an excitation light source is needed. More recently, the gene cluster encoding the bacterial luciferase and the proteins for providing decanal within the cell, which includes luxCDABE was isolated from Xenorhabdus luminescens (Meighen and Szittner, J. Bacterid 174 (1992), 5371-5381) and Photobacterium leiognathx (Lee et al., Eur. J. Biochem. 201. (1991), 161-167) and transferred into bacteria resulting in continuous light emission independent of exogenously added substrate (Fernandez-Pinas and WoIk, Gene 150 (1994), 169-174). Bacteria containing the complete lux operon sequence, when injected intraperitoneally, intramuscularly, or intravenously, allowed the visualization and localization of bacteria in live mice indicating that the luciferase- light emission can penetrate the tissues and can be detected externally (Contag et al., Mol. Microbiol. 18 (1995), 593-603).
18

Preferably, the microorganism of the present invention -is a
bacterium which is not tumor- -targeted, e.g. an attenuated bacterium. A list of candidate bacterial strains that might be useful for the present invention (i.e. BMPT (bacterium-mediated protein therapy) and tumor imaging) and which might not be tumor-targeted are listed in Table 1. The person skilled in the art can easily identify such bacteria which are not tumor-targeted by commonly available methods, e.g. the methods described in section 6.1 of WO 96/4 0238,
For safety and direct applicability to humans, bacterial cell lines such as milk and cheese associated microorganisms are preferred which are naturally consumed by most individuals and have intrinsic anti-tumor activity when injected directly to solid tumors, Such bacteria also include bacteria that were naturally, isolated from human tumors which developed co-existence (symbiosis) with a variety of types of tumors or with a specific type of tumor. Extracellular secretion of the therapeutic proteins by1 bacteria is -mediated- through either signal peptides or endogenous protein secretion pathways. To provide additional safety to the BMPT/ bacterial inducible promoters, such as IPTG-induced lac promoter may be used to' exogenously regulate therapeutic protein production by bacteria. IPTG- regulated expression of mammalian proteins in bacteria has been well documented. IPTG has also been shown' to be functional in promoter activation in animals. In addition to lac promoter, other examples of promoters that allow regulation of gene expression in bacteria include the ara promoter (activated by arabinose) and PLtetO-1 promoter (activated by anhydrotetracycline (aTc)) (Lutz and Bujard Nucleic Acids Pes. 25 (1997), 1203-1210).

19
Table 1. Examples of candidate bacterial strains which might be useful in the present invention.
Bacterial
Strain Brief description References

Aerobes, grant
positive
Lactobacillus bulgaricus
Lactobacillus
casei
Lctiobacillus acidophilus

Yogurl bacteria; nonpathogenic (part of normal flora beneficial organism needed by human body); blastolysin fraction isolated from L. bulgaricus shows antitumor effect (Bogdanov et al. 1975; Bogdanov et al. 1977; Ket-linskii et al. 1987); may reduce the risk of developing colon tumors in humans (Wollowski et. al. 2001); ATCC#11842.
Nonpathogenic (part of normal flora beneficial organism needed by human body)'); i.v. injected LC9018 strain of L. easel shows markedly inhibition of the growth of subcuta-neously inoculated sarcoma-180 in mice (Kato et al. 1981); shows antitumor activity fro in i.p. injected L. casei (LC9018 strain) (Kato et al. 1988); ATCC// 393.
Nonpathogenic (part of normal flora beneficial organism needed by human body); inhibitory effect on liver tumori-genesis (Mizutani and Mitsuoka 1980); reduces 1,2-di-methylhydxazbe (DMH)-induced intestinal tumors in male Sprague-Dawley rats (Mcintosh e( al 1999); shows strong antiproliferative effect of milk fermented by L. acidophilus on the growth of a human, breast cancer cell line (Biffi et al. 1997):

S intends et al. 1971, JBacte-riol 107:382-384;
Bogdanov et al. 197.5, FEES Lett 57:259-261,
Bogdanov et al. 1977, Biull Eksp Biol Med 84:709-712;
Kethinskii et al. 1987, Vopr Onkol 33:51-56;
Wollowski el. al. 2001, Am 3 Clin Nutr 73:451S-455S Kato et al. 1981, Garm 72:517-523;
Kato et al 1988, Cancer Immunol immunooelher 26j2l5-221__ Mizutani and Mitsuoka 1980, Cancer Lett 11:89-95;
Biffi et al. 1997, Nutr Cancer 28:93-99;
Mcintosh et al. 1999. Nutr

20

'SL^-s'-" Cancer 35:153-159
Lactobacillus brevis Nonpathogenic (part of normal flora beneficial organism needed by human body);
Lactobacillus paracasei Nonpathogenic (part of normal flora beneficial organism needed by human body); shows antiproliferative effect of milk fermented by L. paracasei on the growth of a human breast cancer cell line (Biffi et al. 1997); Biffi et a! 1997, Nutr Cancer 28:93-99
Lactobacillus plantarum Nonpathogenic (part of normal flora beneficial organism needed by human body); MuiosaM et al. (2000) described that "that daily administration of I. plantarum L-137 is required to exert an antitumor effect at the late stages of tumor development when 1L-12 production is considerably impaired"; Murosaki et al. 2000, Cancer Immunol Immunother 49:157-164
Lactobacillus rhamnpsus Nonpathogenic (part of normal flora beneficial organism needed by human body); -
Lactobaci salivarius llus Nonpathogenic (part of normal flora beneficial organism needed by human body); modification of enteric flora in EL-10 lenockout mice by probiotic lactobacilli (through milk feeding) was associated with reduced prevalence of colon cancer and. mucosal inflammatory activity (O'Mahony et al. 2001); O'Mahony et al. 2001, Ali-ment Pharmacol Ther 15:1219-1225
Lactobaci sporogene Urns Nonpathogenic (part of normal flora beneficial organism needed by human body);
Lactobacillus lactis Nonpathogenic (part of normal flora beneficial organism needed by human body); —
Lactobacillus fermentum ATCC#933S
Streptococcus thermophilus Yogurt bacteria; nonpathogenic (part of normal flora beneficial organism needed, by human body); reported tumor-specific transplantation resistance in mice after treatment of initial tumors with S. thermophilus (Kaklij and Kelkar 1996); T-lymphocytes are involved in antitumor activity exhibited by S. thermophilus (Kaklij et al. 1991); intraperitoneal administration of iS1. thermopllilus resulted in complete core in a very significant proportion of ascitic form of sarcoma-180 or Ehrlich ascites carcinoma tumor-bearing mice (Kelkar et al. 1988): ATCC# BAA-;'.50D. Kelkar et al. 1988, Cancer Lett 42:73-77;Kaldij et al. 1991, Cancer Lett 56:37-43:Kaklij and Kelkar 1996, Microbiol Immunol _IO:55-58_ Banerjee et al. 1988,'Arch Intern Med 148:1769-;_1774_ Nauciel and Goguel 1977, J Natl Cancer Inst 59:1723-177.6
Bacillus sub-tilts Nonpathogenic; B. subtilis bacteremia lias been significantly linked with cancer patients (Banerjee et al. 1988);
Bacillus megaterium Nonpathogenic, peplidoglycan from B. megaterium inhi-bits tumor growth (Nauciel and Goguel 1977);

21
•. ■ J-.
Welllus polymyxa Nonpathogenic; Lauieusaus et al. '1975, Proc Natl Acad Sci' USA 72:3656-3660;Saito and Watanabe 1981, Microbiol Immunol 25:13-22
Mycobacterium smeg-matis Nonpathogenic; fast-growing; Saito and Walanabc (1981) showed that "the bacteriocin from M smegmaris produced morphological alterations and inhibition of synthesis of ribonucleic acid, deoxyribonucleic acid and protein in the transformed but not in the non transformed cells";
Mycobacterium vaccae Nonpathogenic;
Mycobacterium micrott Nonpathogenic; ATCC# 35782
Mycobacte-tium habana Nonpathogenic; slow-growing, photochromogen originially isolated from monkeys;
Listeria monocytoge-ms Intracellular pathogen
Enterococcus faecalis Have been isolated from tumors and infection endocarditis; ATCC# 29212, 51299 Milbrandt 1998, Clin Cardiol 21:123-126


Aerobes, gram negative -a
Escherichia coli Intravenously injected E. coli shows tumor-specific localization. Shabahang et al.unpublished data

22

Almonella typhitmirium
Vibrio cholera
Vibrio harveyi
Pseudomo-nas fluores-cens
Pseudomorus putida

Bermudes and associates have used intravenous) y injected Salmonella as a protein delivery vector (Pawelck ct al. 1997; Bermudes ct al. 2000, Cluitmonl et al. 2000; Zheng et al. 2000;)Bermudes and associates showed a Salmonella (TK)-dependent |(1.4)C]FIAU accumulation of at least 30-fold higher in tumor tissue compared to muscle tissue (Tjuvajev et al; 2001); Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma showed NO ANTITUMOR effect (Toso et al. 2002)
Intravenously injected V. cholera shows tumor-specific localization; nonpathogenic strains of V. cholera are available;
Nonpathogenic; luminescent bacterial ATCC# 700106. Nonpathogenic; motile by means of multiple polar flagella; P. fluorescens bacteremia has bean reported in P. putida bacteremia has, been reported in cancer patients (Martino et al. 1996); L-Methiobinase from P. putida depletes methionine and inhibits tumor growth (Kokkina-kis ct al! 1997); normally grow at 26' C, but ATCC# 43142, 47054 can grow at 37°C.

Pawelek et at. 1997, Cancer ; Kes 57:4537-* 4544; ,' .. ' "
Bermudes ct al. 2000, Adv Exp Med Bial 465:57-63;
Clairmont et al. 2000, J Meet Dis 181:1996-
2002;
Zheng el al.
2000, Oncol
Res 12:127-
135;
Tjuvajev et al.
2001, J
Control Re
lease 74:313-
315;
Toso et id,
2002, j Clia
Oncol 20:142-
152
Shabahang et. al.
unpublished data
Hsueh et al. 1998, J Clin Microbiol 36:2914-29 i 7;
Picot et al. 2001, Microbes infect 3:985-995
Pekhover. al 19S3, Biull Eksp Biol Med 9:5:87-88;
Anaissie et al

23
"1 1987,"AmJ j Med 82:1191-I 1194;
Martino et al. 1996, Eur J Clin Microbiol Infect Dis 15:610-615;
Kokkinakis et al. 1997, Nutr Cancer 29:195-204;
Miki et al. 2000, Cancer Res 60:2696-2702

Anaerobes, \ gram positive
Bifidobacte-
rium bifidum
Bifidobacterium lorigum
Bifidobacterium infants

Nonpathogenic (part of normal flora beneficial organism
needed by human body); shewed by Kimura et al. (1980)
that "selectively, localized and proliferated in several types
of mouse tumors following administration" and "None
of the same bacilli could be detected in the tissues of
healthy organs such as the liver, spleen, kidney, lung,
blood, bone marrow, and muscle 48 or 96 hr after i.v.
administration into "tumor-bearing mice"; shows antiproli
ferative, effect of milk: fermented by B. bifidum on the
growth of a human breast cancer cell line (Biffi et al.
1997); ATCC # 11863, 15696
Nonpathogenic (part of normal flora beneficial organism needed by human body), inhibitory effect on liver tumori-genesis (Mizutani and Mitsuoka 1980); B. longum was shown to "selectively localized to and proliferated in 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors after systemic application." (Yazawa et al. 2001); was shown to selectively localize and grow in hypoxia tumors (Yazawa et al. 2000); ATCO'/l 5707
Nonpathogenic (part of normal flora beneficial organism needed by human body); shows strong antiproliferative effect of milk fermented by B. infantis on the growth of a_

Kimura et al. 1980, Cancer Res 40:2061-2068;
Biffi et al 1997, Nutr Cancer 28:93-99
Mizutani and Mitsuoka 1980, Cancer Lett 11:89-95;
Yazawa et al.
2000, Cancer
Gene Ther
7:269-274;
Yazawa et al.'
2001, Breast
Cancer Res
Treat 66:165-
170
Kohwi et al. 1978. Gann 69:613-618:

24

human breast cancer cell line (Biffi et al. 1997).and other ] tumor cells or tumors (Kohwi et al. 1978; Sekine ct al. 1985; Sekine et al. 1995); A!CC# 15697 Sekine ct al. 1985, Cancer Res 45:1300-1307;Sekine et al. 1995, Biol Pharm Bull 18:148-153;Biffi et al. 1997, Nutr Cancer 28:93-99
Bifidobacterium latero-sporus Nonpathogenic (part of normal flora beneficial organism needed by human body):
Bifidobacterium animalis Shows .antiproliferative effect of milk fermented by B. animals on the growth of a. human breast cancer cell line (Bill et al 1997); ATCC# 25527 Biffi et al. 1997; Nutr \Cancer 28:93-99
Actinomyces israelii Actinimycetes are fungus-like bacteria that form filamentous branches. They are known to reside in the mouth and in the intestinal tract. Pathogenic proliferation of the organisms, which is usually a result of trauma to the region of infection, can lead to actinomycosis.
Eubacterium lentum Eubacterium species are normal flora of the intestinal tract. However, they may cause opportunistic infections. E lentum, the most often isolated species, has been linked to endocarditis and some wound infectious.
Peptostrepto-coccus an-aerobius One of the most common bacteria found in non-sparing anaerobic (NSA) infections in certain surgical group of patient:; ATCC# 27337 Chatterjee and Chakraborti 1995, J Indian Med Assoc 93:333-5, 339
PeptocQccus prevotii Found in wound infections; Chatterjee and Chakraborti 1995, J Indian Med Assoc 93:333-5,339
Clostridium novyi Strict anaerobe; wildly Lu soil; motility is inhibited in the presence of oxygen; Vogelstein lab (Dang et al. 2091) showed that "intravenously injected C. novyl-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells"', ""Large numbers (up to 108 in a volume of 500 p.1) of C. novyl and C. sordellti spores could be injected intraveuously into normal mice without causing any noticeable side effects."; ATCC # 19402 Dang et al.2001, ProcNatl Acad SciUSA98:1.5155-15160;Nuyts et al.2002, Aoli-cancerDrugs

25
13:115-125
Clostridium sordellii ATCC.V97I4
Clostridium absortum ATCC// 27555
Clostridium acetobutyli-cum Theys et al. (2001) demonstrated "Specific targeting of cytosine deaminase to solid tumors by engineered Clostridium acetobutylicum"; Other related papers (Theys et al. 1999); ATCC# 824 Theys et al, 1999, Appl Environ Microbiol 65:4295-4300,Theys et al. 2001, Cancer Gene Titer 8:294-297
Clostridium bifermentans ATCC # 17836
Clostridium difficile The link between C. difficile and cancer patients has been documented (Anand and Glatt .1993; Simon et. al. 2000)!; ATCC# 700057 Anand and Glatt 1993, Clin Infect Dis17:109-113Scheller et al. 1995, Arch Dis Child72:219-222;Wehl et al.1999, MedPediatr Oncol32:336-343;Simon etal.2000, InfectControl HospEpidemiol21:592-596
Clostridium histotyticum ATCC# 19401 '
Clostridium perfringens \ Pathogenic; C. perfringens bacteremia has been often reported in cancer patients, C. perfringens enterotoxin kills tumor cells and reduce tumor growth (Michl et al. 2001); ATCC# 3624, 13124 Bodey etal. 1991, Cancer 67:1928-1942;Michl etal. 2001,Gastroenterology 121:678-684'Minton etal. 1995, FEMS Microbiol Rev
Clostridium beijerinckii Brown and associates at Stanford described that "To demonstrate {be specificity of this approach for tumor targe-tin/5, we ;uf;rayf;.Hf7iiigly injected the inactive spore form of

26
~~b C. beijerinckii, which upon transition to a reproductive state v\ ill express the IL coli nitroreductase gone Nitroreductase activity was detectable in 10 of 10 tumors during the first 5 days after intravenous injection of inactive clostridial spores, indicating a rapid transition from spore to reproductive state" (Lemmon et al. 1997); other related papers on C. beijetinclai tumor targeting (Minton et al. 1995, FEMS Microbiol Rev 17:357-364; Fox et al. 1996); ATCC# 25752 17:357-364;Fox. et al.1996, GeneTher 3:173-178;Lemmon -et al.1997, GeneTher 4:791-796Liu ct al 2002, Gene Ther 9:291-296
Clostridium sporogenes Harmless saprophyte; Brown and associates at Stanford described that "systemic delivery of 5-FC into mice previously injected with CD-transformed spores of C. sporogenes produced greater antitumor effect than maximally tolerated doses of 5-FU"; ATCC# 19404
Staphylococcus aureus Non-motile, non-sporing and facultatively anaerobic; a halotolerant (salt tolerant;) organism associated with the nasal mucosa of mammals which has both benign and pathogenic strains, 5. aureus bacteremia is frequently seen in cancer patients; ATCC #25923
Staphyioqpcj-cus epidermi-dis Nonpathogenic normal microflora component of the skin;
■

Anaerobes, gram negative Chatterjee and Chaluaborti 1995, J Indian Med Assoc 93:333-5, 339
Acidamino-coccusfer-mentcns Found- in wound infections;


Plant bacteria, gram positive
Clavibacter michiganesis subsp. michi-gamnsis Pathogen of tomato


Plant bacter ria, gram negative
Agrobacte-rium numefa-tiens. A. (tumefaciens in plant tumors (Ullrich and Aloni 2C00); Kunk et al. (2001, Proc Natl Acad Sci U S A 98:1871-1876) described "Agrobaeturlium attaches to and geneti- Aksac 1974, Turk Hij Teer Biyoi Derg

27

Erwinia her-bicola
Azorhizobium caulinodans
Xanthomonas campestris pv. vesicatoria
Xanthomorias campestris pv. campestris

cally transforms types of human cells"; it has been reported that antibodies against A tumecfociens were found in patients with various cancers (Aksac 1974, 'link Hij Teer Biyol Derg 34:48-51); grow at 26-30°C; ATCC# 15955
Non-capsulated, non-spore forming, short, rods with a single monotrichons polar flagellum, and are harmless to humans; purified tyrosine phenol-lyase from E. herbicola significantly inhibited growth of established B-16 melanoma tumors;
Symbiotic, colonize plants, fix nitrogen;
Pathogen of pepper and tomato
Pathogen of beat and cabbage; E. coil lac promoter was shown to be functional in. this plant pathogen;

34.48-51;
Zambryski. et al. 1982, J Mol Appl Genet 1:361-370;
•Rezmer et al. 1999, Planta 209:399^105,
Ullrich and Aloni 2000, J Exp Bot 51:1951-1960;
Azmi et at. 2001, Planta
May;213(l):2 9-36,
Kunik et at. 2001, Proe. Natl Acad Sci USA
98:1871-1876

Meadows et al. 1976, Cancer Res 36:167-167'
Soby and Darnels 1996, Appl Micro-, biol Biotech-nol 46:559-561

...J
Note: The content of this table is by no-means to be exhaustive. Any other similar bacterial strains, which are not listed in this table, arc also considered to he included.
Particularly preferred is attennated Vibrio Cholarae
28
A further example of a bacterium useful for the preparation of a diagnostic composition for tumor-imaging or monitoring a therapeutic tumor treatment is a magnetic bacterium (Metal-Binding-Bacteria-Mediated Tumor Detection (MBBMTD)) . Such bacteria allow tumor detection through the accumulation, of iron-based contrast agents. Magnetic bacteria can be isolated from fresh and marine sediments. They are capable of producing magnetic particles (Fe3O4) (Blakemore, Annu. Rev.
Microbiol. 3_6 (1982), 217-238). To do so, they have efficient iron uptake systems, which allow them to utilize both insoluble and soluble forms of iron. Magnetospirilluma magneticum AMB--1 is an example of such magnetic bacteria that has been isolated and cultured for magnetic particle production (Yang et al., Enzyme Microb. Technol. 29 (2001), 13-19)., Since it can be expected that these magnetic bacteria .(naturally occuring or genetically modified)/ when injected intravenously, possess the similar tumor accumulation ability as that of Vibrio cholera, for example, these bacteria can be used for accumulating iron-based contrast agents in the tumors, which in turn allows tumor detection by MRI. Similarly, other naturally isolated metal-accumulating strains of bacteria can be used for tumor targeting/ absorption of metals from contrast agents, and eventually tumor imaging.
Alternatively, viruses such as Vaccinia virus, AAV,, a retrovirus etc. are also useful for the diagnostic and therapeutic compositions of the present invention; see Table -2 listing examples of viruses useful in the present invention.

29
Table 2 Example of viruses useful in the present invention

Viral strains Brief description ReferencesYu et a!. 2002, unpublished data
Vaccinia virus We have shown that viral thymidine kinase mutation is not necessary for tumor accumulation by intravenously injected Vaccinia virus.
Epstein-Barr virus
Human papillomavirus zur Hausen 2002, Nat Rev Cancel 2:342-350
Retrovirus
Adenovirus Lindsey 2002, Lancet Oncol 3:264
Adenoassoci-ated virus
SV4Q virus i
Cytomegalovirus
Newcastle Disease Virus safe, replicates in and kill tumor cells; local, injection is more effective than systemic injection Schirrmacher et al.2001, Int J Oncol18:y45-y52
Bovine enterovirus Bovine enterovirus has been shown to exhibit wide tissue tropism for cell types in vitro. It also shows oncolytic activity towards human cells. Smyth et al 2002, Int JMol Med 10:49-53
high stability, low toxicity, and broad host range Beyer etal. 2002, J Virol 76: 1488-1495
LymphocyticChoriomeningitisVirus (LCMV) High stability, low toxicity, and broad host range Beyer etal. 2002,JVirol76: 1488-1495
Derivatives of the Edmon-ston-B strain of measles virus (M'-Ed) safe, live attenuated, has been shown to induce regression of human lymphoma xenografts in immunodeficient mice Grote et al 2(001,Blood 97:3746-3754
Herpes simplex virus type 1 Lachmann and Efstathiou 1999, Curr Opin Mol Ther 1:622-632;Wu et al. 200), Cancer Res 61:3009-3015
Attenuated yellow fever (YF) virus powerful vaccine,, safe Barrett 1997, Biologi-cals 25:17-25;McAllister et al. 2000, J Viiol 74:9197-205
Note: The content of this table is by no-means to be exhaustive. Any other similar viral strains, which are not listed in this table, are. also considered to be included.

30
Preferably, the virus is Vaccinia virus.
Preferably, the cell of the diagnostic or therapeutic composition of the present invention is a mammalian cell such as stem cells which can be autologous or heterologous to the organism. Examples of suitable cell types are shown in Table 3.
Table 3. Examples of mammalian cells useful in the present invention

Mammalian cells Brief description Reference
Stem cells It has been shown that neutral stem cells implanted intravenously outside the central nervous system target to an intracanial tumor. Additionally, when implanted intracanially at distant sites from the tumor, such as into the cerebral ventricles, the donor neural stem cells migrate through normal brain tissues to target the human glioblastoma cells. A boody et al. 2000, ProcNatl Acad Sci U S A 97:12846-12851;Herrlinger et al. 2000, Mol Ther 1:347-257
Have been shown to infiltrate the pleural space of patients to target malignant pleural mesotheliomas.(Harrison et al. 2000, Ann Thorac Surg 70:407-411). We have shown that intravenously injected fibrosarcoma cells accumulate in breast tumors and subcutaneous glioma tumors (Yu et al. unpublished data)
Note: The contents of this table is by no means to be exhaustive. Any other similar types of mammalian cells, which are not listed in this table, are also considered to be included.

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

31
In a particularly preferred embodiment, the microorganism or cell of the diagnostic and/or pharmaceutical composition f the present invention additionally contains his a gene encoding a substrate for the luciferase. In an. even more pre tier red embodiment, the microorganism or cell of the diagnostic and/or pharmaceutical composition of the present invention contains a ruc-gfp expression cassette which contains the Renilla luciferase (aruc) and Aeguorea gfp cDNA sequences under the control of a strong synthetic early/late (PE/L) promoter of Vaccinia or the luxCDABE cassette.
A preferred use of the microorganisms and cells described above is the preparation of a diagnostic composition for tumor-imaging. The diagnostic composition of the present invention, can be used e.g. during surgery, to identify tumors and metastasis. Furthermore, the diagnostic composition of the. present invention is useful for monitoring a therapeutic tumor treatment. Suitable devices for analysing, e.g., the localization or distribution of luminescent and/or fluorescent proteins in an organism, organ or tissue are well known to the person skilled in the art and, furthermore described in the literature cited above as well as the examples, below. Additionally, the microorganisms and cells can be modified .in such a Way that they bind metals and consequently are useful in MRI technology to make tumor localization more specific.
The present, invention also relates to the use of an antimicrobial, e.g. antibacterial or antiviral compound, e.g., peptide or protein fused to a detectable protein, for the preparation of a diagnostic composition for tumor imaging or: monitoring a therapeutic tumor treatment. This ; diagnostic composition is useful for tumor detection through the binding of the antimicrobial compound, e.g., a light-emitting or radiolabeled antimicrobial peptide/protein with bacteria localized in tumors (Peptide- linked - Tumor- Targeting vector

32
Defection (PLTTVD)
The present invention also relates to the use of an antimicrobial, e.g. antibacterial or antiviral compound, e.g., peptide or protein fused to a therapeutic protein for the preparation of a pharmaceutical composition for tumor therapy.
Examples of naturally occurring antimicrobial proteins include
ubiquicidin (UBI, 6.7 kDa) and lactoferrin (hLF, 77 kDa). UBI
was originally isolated from murine macrophages and later from human airway epithelial cells. hLF is found in mucosa] and neutrophils- and is known, to bind to surface structures 1 of both gram-negative and grain-positive bacteria. Small synthetic peptides containing the bacteria binding domains of UBI of hLF have been designed for imaging of bacterial infections, which can be discriminated from sterile inflammations (Nibbering et al., Nucl. Med. Commun.19 (1998), 1117-1121; Welling et al., Nucl. Med. Biol 2_9 (2000), 413-422; Welling et al., Eur. J. Nucl. Med. 27_ (2002), 2929-301). These peptides are radiolabeled with 99mTc, which allows real-time visualisation in live animal using planar scintigraphy. For example, a planar gamma camera (e.g. Toshiba GCA 7100/UI, Tokyo, Japan) equipped with a low-energy general-purpose parallel-hole collimator can be used to visualize the distribution of 9'n,Tc-labelled antimicrobials peptides in live animals or humans. To apply these synthetic peptides for tumor detection, - first the tumorous individuals are infected with a particular strain of extracellular bacteria. After bacterial colonization of the tumors, the radiolabelled compounds, e.g., peptides are delivered intravenously. Specific binding of the labeled peptides to the bacteria in the tumors can be visualized in real time by scintigraphy, which therefore allows the localization of tumors in humans

33
and animals. In addition to labeling with 99mTc, the compounds, e.g., synthetic peptides can also be labeled with paramagnetic- or superparamagnetic-metal particles for visualization using MRI or the compounds can be labeled with radionuclides, Such as 55CO, 66Ga, C0CtT (II), 123l etc. for noninvasive visualization by SPECT or PET. Furthermore, the
compounds, e.g,, synthetic peptides can also be tagged with
fluorescent probes, fluorescent proteins (such as green fluorescent protein (GFP) or DsRed), or luminescent proteins (such as Renilla luciferase, firefly luciferase) for real-time visualization in an individual.
In addition to UBI and BLF, there are many other examples of antimicrobial peptides/proteins produced by bacteria, plants, invertebrates, and vertebrates including humans (Schroder, Cell. Mol. Life Sci. 56 (1999), 32-46; Cole and Ganz, Biotechniques 29 (2000), 822-826, 828, 830-831) .which are useful for the purposes of the present invention, e.g. pediocin PA-1 from lactic acid bacteria, gramicidin S from Bacillus brevis), protegrin-1 from porcine leukocytes, SMAP-29 from sheep myeloid cells, buforin I & buforin II from Asian toad, beta-defensins, LL-37, a fragment of human cathelicidin hCAP-18, arasin I from catfish, granulysin, PAMP from Popionibaeterium jensenii etc. based on these naturally occurring antimicrobial proteins, small peptides can be designed and synthesized to retain the bacteria-binding capability while eliminating the bacteria-killing effect by the original proteins. These synthetic peptides could then be used in bacterium-mediated tumor detection.
Similar to detection of bacteria by antibacterial compounds
(e.g. peptides/proteins), antiviral compounds (e.g.
peptides/proteins) may be used for detecting viral particle.
in the tumors. Lactoferricin is one of the example of

34
antiviral peptide/protein that may be used to design peptides for detecting viral particles. In addition to the antimicrobial peptides/proteins discussed, radiolabeled- or light-emitting-protein/probe-labeled antibody fragments can also be used to target bacteria or viruses localized in tumors for tumor detection.
Moreover, the antimicrobial compound can be fused to a metal-binding protein (Fusion-Peptide-Linked Tumor Targeting Vector Detection (PLTTVD)) for detecting the accumulation of bacteria, viruses etc, according to the present invention in tumors. After allowing the binding of the fusion construct to the engineered bacteria etc. in tumor, metal agents (which can be used for detection by MRI, PET or SPECT) are injected intravenously. Binding and absorption of the metal agents by bacteria etc will allow indirect tumor detection.
The present invention also relates to a pharmaceutical composition comprising a microorganism or cell containing TMK sequence(s) encoding a cell receptor capable of binding a ligand and furthermore a ligand which is coupled to a toxin ( chimeric toxin). Suitable toxins which can be coupled to the ligand are well known to the person skilled in the art. Several chimeric toxins have been described previously. First, • the Tfn-CRM 107 chimeric toxin was prepared by conjugating transferrin to the mutated diphtheria toxin CRM 107 (Johnson et al.., J. Neurosurg. 70 (1989), 240-248), which targets the transferrin receptor. Second, the 42S.3--PE immunotoxin was prepared by conjugating anti-EGF receptor antibody to the whole Pseudomonas exotoxin A {Myklebust et
al., Cancer Res- 53 (1993), 3784-3788). Third, the Tfn-PE
was prepared by conjugating transferrin to the-i Pseudomonas exotoxin (Hall et al., J. Neurosurg. 76 (1992), 838-844; hall et al., Neurosurgery 34 (1994), 649-656). It could be shown in animal models and human patients that these

35
chimeric toxins have anti-tumor activities against medulloblastoma, small cell lung cancer, glioma xenografts and intracranial tumors. Thus, e.g. recombinant Vaccinia. virus can be designed to specifically express transferrin receptors on the surface of tumor cells allowing the use of these receptor-targeting chimeric toxins for antitumor treatment.
Th present invention also relates to a pharmaceutical composition comprising (a) a microorganism or cell containing a DNA sequence encoding a cell receptor capable, of binding a ligand and (b) said ligand, which is coupled to a therapeutic protein. Suitable therapeutic proteins as well as methods for generating therapeutic ligand fusion proteins by linking therapeutic proteins to the ligand proteins are well known to the person skilled in the art. Examples of suitable therapeutic proteins are shown in. Tables 4 and 5.









36
Table 4. Examples of enzyme/prodrug pairs in which the delivery of the enzyme gene is facilitated
by intravenously injected microorganisms and cells:

Therapeutic proteins (enzymes), drug, prodrug Description References
Herpes simplex virus thymidine kinase (HSVTK)+ Ganciclovir (GCV) Most well-knownEnzyme/prodrug combination. Moolten Cancer Res. 1386;46:5276-5281
Herpes simplex virus thymidine kinase (HSV-TK)+ A-5021 (I ’S,2’R)- 9{[1’,2’- bis (hydroxymethyl)cycloprop-1-yl]methyl}guanine A-5021 has highly selective anti-hepatic activity and was selectively phosphorylated by viral TK in herpes virus infected cells. The anti herpetic activity of A-5021 was not potent in comparison with ACV and penciclovir. Hasegawa et al. 2000, Cancer Gene Ther 7:557-562
Horseradish peroxidase (HRP)+ Indole-3-acetic acid (IAA) When activated by purified HRP, IAA was not shown to inhibit colony information in mammalian cells, whereas neither enzyme nor prodrug alone was cytotoxic at the same concentration or times The HRP/IAA-inducted cell kill was effective in normoxic and anoxic condition Greco et al. 2000, Cancer Gene Ther 7:1414-1420
Bacterial enzyme carboxy-peptidase G2 (COG2)+4-([2-chloroethyl][2-mesyloxyethyl]amino)benoyl-L-glutamic acid (CDMA)or + 4-{N,N-bis (2-iodoethyl) amino] phenoxycarbonyl L glutamic acid (ZD2767P) CPG2 can be expressed both intracellularly or tethered to the outer surface of mammalian cells, where it is able to activate mustard prodrug for use in suicide gene therapy. Spooner et al. 2000, Cancer Gene Ther 7:1348-1356
Human cytochrome P450 CYP1A2+ acetaminophen Acetaminophen is cytotoxic through the cytochrome P450 mediated generation of a chemically reactive metabolite, N-acetylbezoquinoneimine Thatcher et al. 2000, Cancer Gene Ther 7:521-525



37




(NABQI).



Rabbit cytochrome P450 4B1 (CYP4B1)
+ 4-ipomeanol (4-1M)

CYP4B1 is able to induce tumor cell death at low micro-molar concentrations in glioblastoma cells; after treatment with the prodrug 4-IM.

Mohr et al. 2000, Cancer Gene Ther. 7:1008-1014-
Heuser et al. 2000, Cancer Gone Ther 7:806-12



Rat cytochrome P450 4B1 (CifP2Bl)
+ oxaphosporines, such as ifoslfemidd (TFO)

The CYP2B1 gene product activates oxaphosphorines to the hydroxy form, giving rise to the toxic products phosphatide mustard and acrolein, which alkylate DNA and proteins, respectively.

Kammertoens et al. 2000, Cancer Gene Ther 7:629-636




Djeha et al 240
E. coli nitroreductase (NTR)
+ CB1954

CB1954 is a weak
monofunctional alkylating
agent that is converted by NTR into 2- and 4-hydroxylainino derivatives. Cellular thioesters suck as acetyl coenzyme A subsequently convert the latter into a powerful bifunctional alkylating - agent that can kill both proliferating and nonproliferating cells.


Cancer Gene
Djeha et al. 2000, Ther. 7:721-731:
2001, Mol Ther 3:233-



PTX0147 expressing human (CMV)
is the plasmid
NTR. from the
cytomegalovirus
early pro-
moter/enhancer and also carries the b-globin second intron and poly (A) sequences and a G413 selectable marker.

Westphal et al. 2000, Cancer Gene Ther 7:97-106

Weedon et al. 2000, hit J Cancer 86:848-854

E. coli cytosine deaminase (CJD), E. coli uracil phosphoribosyltransferase (UPRT)

Despite CD expression, a number of tumor cells were 5-FC-resistant, which may be attributable to tire lack of an active cytosine transport

Koyama et al. 2000, Cancer Gene liter. 7:101.5-1022;
Thcys et al. 2001, Cancer Gene Ther 8:294-297;

38



5-fluorocytosine (5-FC)

system in mammalian cells and
to the degradation of the
formed 50FU by
dihydropyrimidine dehydrogenase (DPD). In the gene transfer strategy, to improve the effect of the CD/5-FC system, it might be possible to transduce the enzyme gene that converts 5-FU to its active forms. One of the candidates is E. coli UPRT. It is a pyrmidine salvage enzyme and is characteristic to bacteria. It directly converts 5-FU to 5-fluorouridine monophosphate (FUMP) at the first step of 5-FU Activation and has the potential "to enhance the activating pathway against DPD.

Kammertoens et a(. 2000, Cancel Gene Ther 7:629-636;
Block et al. 2000, Cancer Gene Ther 7:438-445;
Bentires-Alj et al. 2000 Cancer Gene Ther 7:20-26;
Kawamura et al. 2000, Cancer Gene Ther 7:637-643;
Li et al. 1997, Cancer Gene Ther 4:113-117



Cytochrome P450 enzymes
+ cyclophosphamide (CPA)

Liver tissue has a high content of P45Q enzymes active toward CPA and is the major organ responsible for CPA activation. Activated CPA generated in the liver circulates through the blood and gains entry to tumor tissue to exert its therapeutic effects.

Huang et al 2000, Cancer Gene Ther, 7:1034-1042; '*'.'
Kan et al. 2001, Cancer Gene Ther 8:473-482

Intratumorai CPA activation
can result in a high, localized
concentration of active drug
metabolite at its site of action,
which may maximize
therapeutic effects while at the same time minimizing the host toxicities associated with hepatic drug activation.

rabbit carboxylesterase
+ 7-ethyl-10-[4-(1-piper]-dino)-l-piperi-

Exposure of neuroblastoma cell lines or of mixtures of these cell lines with CD34 (+) cells at a ratio of 10:90 to

Meek et al. 2001, Cancer Res 61:5083-5089

39

duip]carhoiiyloxyoamp- Vpthecin(CPT-ll)

replication-deficient AdRSVrCE for 24 b and subsequent exposure of cells to 1-5 microM CPT-11 for 4 h increased the toxicity of CPT-11 to three

Neuroblastoma ceiJ linc3 (SJ'NB-1, NB-1691, and SK-N-SH) from approximately 20-50-fold and eradicated their clonogenic potential.

Mushroom tyrosinase

+' bis^2-«hloroethyi)ami uo-
4-hydroxyplienylamino-
methanone28

A sterically undemanding
prodrug bis-(2-chloro-
ethyl)aniino-4-hy-droxyphenylaniiriometbanone 2S was kynthesised and founcl to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the caxboxyHc acid of which is the natural substrate for tyrosinase.

Jordan et all 2001, Bioorg Med Chem 9:1549-1558



E. coli fi-galactpsidase
+ l-chlorornethyl-5-hydroxy-l32-dilaydro-3H-berjz[e]kidole (CC-1065)
or+l-(l'-chloroethyl)-5-
hydroxy-l ^-dihydro-SB -
baizfejipdole
A mutant of carboxypep ti-dase G2 (CPG2, glutainate carboxypeptidase
+ 4-[bis(2-iodoetbyl)aminoj-phenyloxycarbonyl-L-glu-tamio acid
or + 3-fluoro-4-[bi5(2-
cMorefliyl)amino] benzo} ri -
jUfilutamic acid

Prodrug cleaved ; by
galactosidase shows high cytotoxicity towards human bronchial carcinoma cells of line A549.
Activation of all three of the prodrugs not only kills the cells expressing the mutant CPG2 on the surface but also the neighboring cells through bystander effect.

Tietze Res
Friedlos el al. 2002, Cancer 62:1724-1729



or + 3,5-difluoro-4 [bis(.2-
iodoethyl)amino)benzoyl-
L-glutamic acid
Note: The content of this table is by no-means to be exhaustive Any other similar enzyme-prodrug pairs, which are not listed in this table, are also considered to be included.


Further examples of-therapeutic proteins that can be used for BMPT (bacterium-

mediated protein therapy), VMP'I' (virus-mediated protein )rotein therapy), e.g. inCMPT (mammaiiaii-cell-mediated therapy) or CMFT (cell-mediated, jroteui therapy), against tumors;
Therapeutic proteins, drugs Description References
rsCD40L Ligand of CD40, sensitizing tumor cells to apoptosis induced by a variety of agents, including TNF-alpha, anti-Fas, and cytotoxic drugs. Eliopoulos et al. 2000, Mole. Cell. Biol. 20:5503-5515
Fas-ligand Sharma et al. 2000, Pharmacol Ther. 88:333-347
TRAIL Ligand for death receptors such as DcR2, DcRl, DR5, DR4. Golstein 1997, Curr. Biol. 7:R750-753.j, i.:
TNF TNF is the ligand for TNFR1, which mediates cell-death signaling. Baker and Reddy 1996, Oncogene 12:1-9;Theys et al. 1999, Appl Environ Microbiol 65:4295-4300;Lammertyn etal. 1997, Appl Environ Microbiol 63:1808-1813 '

Recombinant antibodies
Anti-GA733-2i Secreted or membrane-anchored form of monoclonal antibody (mAb) .(CO 17-1 A) specific for Ag (GA733-2) that is present on the surface of most gastrointestinal carcinomas. Paul et al. 2000, Cancer Gene Ther 7:615-623
Anticancer drugs adria-mycin (ADM), cytosine Some tumor cells express Fas antigen on their surface, and Nakmura el al. 1997, Anticancer Res. .17:173-179;

42


arabinoside (Ara-C), cisplatin, doxorubicin, mitomycin C, fluorouracil, campto-thecin, cis-diammine-dichloroplatinum (H), CDDP, etc.
+anti-Fas. antibody

apoptosis is induced in those cells by 1gM-anti-Fas MAb
Clinically relevant
concentrations of anticancer drugs enhance Fas receptor expression on the plasma membrane of tumor cells.
encoding anti-Fas
By the combination of ADM or Ara-C with IgM-anti-Fas MoAb, the induction of apoptosis in HL60 leukemic cells -was enhanced significantly we may use bacteria or 'viruses to deliver
antibody. Expression of the anti-Fas antibody on the surface of tumor cells may sensitizes these cells to chemotherapy agents, which therefore may improve the efficiency of chemotherapy.

Micheau et al. 1997, J. Nail. Cancer Inst. 89:783-7S9;
Chang et al. 1998, Osaka City Med. J. 44:173-180;
Mizutani et al. 1997, Cancer 79:1180-1189;
Jiang et al. 1999, Hematology 29:101-110;
Mizutani et al. 1998, J. Urol. 160:561-
570 !

Bacterial toxins

Microcin E492

A low-molecular mass channel-
forming bacteriocin (7,887 Da.)
produced by Klebsiella
pneumoniae. It has been shown
that microcin E492 induces;
apoptosis in certain human cell
lines. Treatment with zVAD-
fmk, a general caspase in
hibitor, completely blocked the
cytotoxic effect of this bacte
riocin, which may provide a
safety mechanism when micro
cin is used in -anti-tumor
treatment. Microcin-E492-
insensitive mutants of E. coli K12 have been isolated., which may be used as a carrier to

de-oieuzo ).9'J'\, Arch Microbiol 139:72-75;
Hetz et al. 2002, Proc Natl Acad Sci U S A 99:2696-2701

43

deliver microcin (Pugsley et al 1986, J Gen Microbiol 132:3253-3259).
diphtheria toxin (DT)■ Toxin-labeled monoclonal antibodies have been used to target tumor ceil surface receptors for-cell killing-(Kreit-maii 2001, Curr Pharm Biotechnol 2:313-325; Thomas et al. 2002, J Pediatr Surg 37:539-544). Toxin-label protein ligands have also been used to target cell surface receptors (Olson et al. 1997, Int J Cancer 73:865-870; Arora et al. 1999, Cancer Res 59:183-188; Wild et al. 2000, Br jCancer83:1077-1083
Pseudomonas exotoxin
Escherichia coli Shiga toxins Shiga toxins, Shiga-like toxin I (SLT-I) and Shiga-like toxin U (SLT-II) are cell-associated cytotoxins, which have been shown to be able to kill tumor cells. The toxins kill target cells by inducing apoptosis. O'Brien etal. 1992, Curr Top Microbiol Immunol 180:65-94;Nakao and Takeda 2000, J Nat Toxins 9:299-313
Escherichia coli Vero-toxin 1 (VTI) VT1 is an E. coli elaborated subunit toxin active only against (tumor) cell lines limit express the VT1 receptor, globotriaosyl ceramide-Gb3. In an example of VTl action, it has been shown that VTl eliminates human astrocytoma, xenograft in nude mice Farkas-Himsley et al. 1995, Proc Natl Acad Sci USA 92:6996-7000;Arab et al. 1PS9, Oncol Res 11:33-39


44

Plant toxins
Hyperforin Hyperforin is a plant derived antibiotic. It was shown that hyperforin can inhibit tumor growth by activating mitochondria-mediated apoptosis pathway. Hostanska et al. 2002, Pharmazie 57:323-331;Schempp et al. 2002, Oncogene 21:1242-1250
Note: The content of this table is by no-means to be exhaustive. Any other similar therapeutic
proteins, -which are not listed in this table, are also considered to be included.
Since the tumor tissues can be labeled with ligand receptors using intravenously injected engineered bacteria, viruses or cells, the binding of therapeutic-ligand fusion proteins to the ligand receptors will enable the targeting of the therapeutic, proteins to the turner tissues. Intracellular bacteria and viruses are particularly useful in labeling the tumor cell surface with designated receptor proteins. However, due to "bystander effect" of therapeutic proteins on neighboring cells, extracellular bacteria and mammalian cells can also be used to deliver the therapeutic proteins.
In the case of Gene-Directed Enzyme Prodrug Therapy (GDEPT) (see below) , for example, extracellular bacteria may be used to secrete the enzyme-ligand fusion protein. The tumor ceil surface may be labeled with receptor proteins by viruses. After the intravenous delivery of prodrug, not only the cells expressing the receptors will be bathed with and killed by the active drug, the surrounding tumor cells (which may not be labeled with .receptors] will also be killed.
Viruses (such as Vaccinia virus) may also be used to label the tumor cell surface with receptor proteins. For example, the

45
tumor tissues can be specifically infected by intravenously injected engineered Vaccinia virus carrying, e.g., a transferrin receptor gene (also encoding a single peptide for cell surface expression). Expression of the transferrin receptor on the tumor cell surface will mark these cells for targeting by therapeutic-ligand fusion proteins. In this case, the ligand is the transferrin protein, and the therapeutic protein could be a Pseudomonas exotoxin (or any other cytotoxic therapeutic proteins). Tumor cell internalization of the transferrin/transferrin receptor pair allows the internalization of the therapeutic protein, which in turn delivers the cytotoxicity specifically to the tumor cells. Transferrin/transferrin receptor pair is only one of many examples of ligand-receptor pairs that can be used. In addition, mutant ligands and mutant receptors with highly Specific affinity 'toward each other may be used to avoid the binding to endogenous proteins.
Additional examples of suitable proteins are human endostatin and the chimeric PE37/TGF-ajlpha fusion protein. Endostatin is a carboxyterminal peptide of collagen XJVIII which has been characterized (Ding et al., PNAS USA 95 (1998), 10443). It has been shown that endostatin inhibits endothelial cell proliferation and migration, induces Gl arrest and apoptosis of endothelial cells in vitro, and has antitumor effect in a variety of tumor models. Intravenous or intramuscular injection of viral DNA and cationic liposome-complexed plasmid DNA encoding endostatin result in limited expression levels of endostatin in tumors. However intratumoral injection of purified endostatin shows remarkable inhibition of tumor growth. Pseudomonas exotoxin is a bacterial toxin secreted by Pseudomonas aeruginosa. PE elicits its cytotoxic effect by inactivating elongation factor 2 (EF-2), which results in blocking of protein synthesis in mammalian cells. Single chain PE is functionally divided into three domains: domain la is

46
required for binding to cell surface receptor/ domain II is required for translocating the toxin into the target cell. cytosol, and domain III is responsible for cytotoxicity by inactivating EF-2. PE40 is derived from wild type Pseudomonas exotoxin that lacks the binding domain la. Other proteins such as antibody fragments or protein ligands can be inserted in place of the binding domain. This will render the PE40-ligand fusion protein specific to its receptor. One of the highly, specific engineered chimeric toxins is the TGF alpha/PE40 fusion protein, where the C-terminus of TGF alpha polypeptide has been fused in frame with the C-terminus of the PE40 protein. TGF alpha is one of the ligands of epidermal growth factor receptor (EGFR), which has been shown to be preferentially expressed on the surface of a variety of tumor cells. TGF alpha-PE40 fusion protein has been shown to be highly toxic to tumor cells with elevated (EGFR) on the cell surface and while it is less toxic to nearby cells displaying fewer numbers of surface EGFR. The toxicity of TGF alpha-PE40 chimeric protein is dependent on a proteolytic processing step to convert the chimeric protein into its active form, which is carried out by the target. - To overcome the requirement for proteolysis, a new chimeric toxin protein that does not require processing has been constructed by Theuer and-coworkers (J. Biol. Chem. 267 (1992), 16872). The novel fusion protein is termed PE37/TGF alpha, which exhibited higher toxicity to tumor cells than the TGF alpha-PE40 fusion protein.
In a particular preferred embodiment of the diagnostic and/or. pharmaceutical composition of the present invention the ligand is not the naturally—occurring ligand but any compound which is capable, of specifically binding to the receptor, e.g. an antibody. The term "antibody", preferably, relates to antibodies which consist essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations. Monoclonal antibodies are made from an.

47
antigen containing fragments of the particular receptor by methods well known to those skilled in the art (see, e.g., Kohler et al., Nature 256 (1975), 495), As used herein, the terra "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab)2 fragments) which are capable of specifically binding to a receptor. Fab and F(ab)2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). Thus, these fragments are preferred, as well as the Products of an FAB or other immunoglobulin expression library. Moreover, antibodies of the present invention include chimeric, single chain, and humanized antibodies.
Antibody-ligands are, e.g., useful in anti-tumor therapy, using fusion proteins (i.e. immuno therapeutic proteins) between antibodies and therapeutic proteins: Firstly, the genes encoding cell surface receptors are delivered to tumors cells in live organisms via intravenously injected engineered bacteria and viruses. Such receptors can be e.g., transferrin receptor EGF receptor, somatostatin receptor etc. Over-expression of receptors on the cell surface following bacterial or viral infections will be used to mark the tumor cells. Secondly, a fusion protein (an immuno therapeutic protein) between an antibody, preferably an antibody fragment, (specific to the overexpressed surface receptors) and a therapeutic protein (e.g. any type of toxins, see Table 5) is prepared. The intravenously injected fusion protein may bind to the marked tumor cells and exhibit cytotoxicity toward these ceils in the tumor tissues.
The present invention also relates to a diagnostic and/or pharmaceutical composition containing a microorganism or cell as described above and furthermore (a) an antimicrobial compound fused to a protein suitable for tumor therapy and/or

48
alimination of. Metastatic tumors and/or (b) an antimicrobial compound fused to a detectable protein or a protein capable of inducing a detectable signal. Anti-tumor therapy using such fusion proteins between antimicrobial compounds (e.g. peptides/proteins) and therapeutic proteins can be carried by firstly preparing fusion gene constructs encoding hybrid proteins between antimicrobial peptides/proteins and therapeutic proteins (see Table 5, above) . After protein expression, the hybrid proteins are purified and ready for intravenous injection. Secondly, e.g. light-emitting bacteria, which can be recognized and bound by the antimicrobial peptides/proteins, are injected intravenously into the subject for tumor-specific accumulation. The specific binding of antimicrobial peptides/proteins to bacteria in the tumors helps to concentrate therapeutic proteins specifically in the tumors, which therefore may elicit tumor specific cytotoxicity.
Furthermore, the protein suitable for tumor therapy and/or elimination of metastatic tumors can be an enzyme converting an inactive substance (prodrug) administered to the organism into an active substance, i.e. toxin, which kills the tumor or metastasis (Gene-Directed Enzyme Prodrug Therapy (GDEPT). The principle of GDEPT is that an enzyme/protein activates a systemically delivered nontoxic prodrug into active toxic drug, which is cytotoxic to tumors. To be specific, GDEPT is a two-step treatment. In the first step, the gene encoding a foreign enzyme is administered and directed to the tumor where it may be expressed using .specific transcriptional elements. In the second step, prodrugs are administered and activated by the foreign enzyme expressed at the tumor. If the enzyme/proteins are present only in the tumors, the active drugs will also be produced only in the tumors, and therefore exhibit cytotoxicity only toward the tumor cells, while at the same time, systemic toxicity is kept to a minimum. The gene

encoding enzyme/protein is delivered specifically to the tumors using Intravenously injected engineered bacteria, viruses, or (mammalian) cells according to the present invention. These genes can be under the control of either constitutive promoters or exogenously regulated inducible promoters additionally ensuring that the conversion of the prodrug into the toxin only occurs in the target tissue, i.e. tumor. Such promoters are e.g. IPTG-, antibiotic-, heat-, pH-, light-, metal-, aerobic-, host cell-, drug-, cell cycle- or tissue specific-inducible promoters.
For example, the enzyme can be glucuronidase converting the less toxic form of the chemoterapeutic agent glucuronyldoxorubicin into a more toxic form. Preferably, the
gene encoding such an enzyme is directed by a promoter which is a constitutive or inducible promoter. Further examples of enzyme/prodrug pairs which are useful in the pharmaceutical composition of the present invention are listed in Table 4, above.
Moreover, the delivery system-of the present application even allows the application of compounds which could so far not be used for tumor therapy due to their high toxicity when systemicly applied. Such compounds include proteins inhibiting elongation factors, proteins binding to ribosomal subunits. proteins modifying nucleotides, nucleases, proteases or cytokines (e.g. IL-2, IL-12 etc.), since experimental data suggest that the local release of cytokines might have a positive effect on the immunosuppressive status of the tumor.
Furthermore, the microorganism or cell can contain BAC (Bacterial Artificial Chromosome) or MAC (Mammalian Artificial Chromosome) encoding several or all proteins of a specific pathway, e.g. antiangiogenesis, apoptosis, wound-healing or anti-tumor growth. Additionally the cell can be a computer-

50
designed cyber cell or cyber virus endocing these proteins,in multiple combinations.
For administration, the microorganisms or cells of the present invention are preferably combined with suitable pharmaceutical carriers. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emuslions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Administration of the microorganisms or cells may be effected by different ways e.g., by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The preferred route of administration is intravenous injection. The route of administration of course depends on the nature of the tumor and the kind of microorganisms or cells contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician based on various clinical factors. As is well known in the medical arts, dosages for any one patient depends on many factors, including the patients size, body surface area, age, sex, the particular compound to be administered, time and route of administration, the kind, size and localization of the tumor, general health and other drugs being adminstered concurrently.
Preferred tumors that can be treated with the microorganisms or cells of the present invention are bladder1 tumors, breast tumors, prostate tumors, brain tumors, colon tumors, lung tumors, ovarial carcinomas, and pancreatic carcinomas; liver tumors, skin tumors.
Brief description of the drawings
Figure 1: External imaging of GFP expression in subcutaneous C6 glioma tumors in nude mice

51
5-e-
C6 glioma cells (5 x lG5) were implanted subcutaneously into -the right lateral thigh. At designated days after tumor cell implantation, the animals were infected intravenously with 1 x-109 pfu of rW-ruc-gfp virus particles GFP expression was monitored under a fluorescence stereomicroscope. Bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of subcutaneous glioma tumor are shown. GFP signal can be observed in tumors as small as 22 mm3 in size (B-B”), or as old as 18 days (about 2500 mm3 in size) (A-A" X. In older tumors, GFP expression was seen in "patch”-like patterns (indicated by arrows in Ar). Marker gene expression in the tumor of the same animal can be monitored continuously 4 (C-C"), 7 (D-D"), and 14 (E-E”) days after intravenous viral injection. (Bars = 5 mm.)
Figure 2 Visualization of tumor angiogenesis
C6 glioma cells (5 x 105) were implanted subcutaneously into the right lateral thigh of nude mice. Ten days after tumor cell -implantation, the animals were infected intravenously with 1 x 108 pfu of rW-ruc-gfp. GFP expression was monitored 7 days post-viral injection. Vascularization at the surface of the subcutaneous C6 glioma tumor is shown against the bright green fluorescent background in the tumor following Vaccinia-mediated gene expressions. Bright field (A), fluorescence (B), and bright field, fluorescence overlay. (C) images of subcutaneous glioma tumor are illustrated. (Bars = 5 mm.)
Figure 3: Expression of GFP in subcutaneous glioma tumor of the same animal
Five days after the subcutaneous implantation of 5 x 10s C6 glioma cells into the right lateral thigh, 10b of rW-ruc-gfp virus particles were injected intravenously. Five days after viral injection the animal was anesthetized and sacrificed for analysis of GFP expression under fluorescence microscope. The tumor was visualized externally (A-K), with the overlying

skin reflected (B-B”), in cross section (C-C”), and in the amputated leg (D-D”). Bright field (A), fluorescence (B), and bright field, fluorescence overlay (C) images of subcutaneous glioma tumor are illustrated. The strongest GFP expressions
are seen as patches located along the outer surface of the tumor on the right (double arrows in C-C”) . Sharp difference of GFP expression in tumor tissue and in the normal muscle tissue (arrows in D-D”) is clearly visible. Asterisks mark the reflected skin (B-B" and D-D”)- (Bars = 5 mm.)
Figure 4: Bright field (A) and fluorescence (B) images of tumor Cells expressing GFP
Frozen sections (30 um thick) of the glioma tumor tissues were prepared from a nude mouse that has been intravenously
injected with 1 x 108 of rW-ruc-gfp virus
particles. (Bars =
50 p.m
Figure 5: Low light image of the anesthetized nude mouse to
indicate the location of Renilla luciferase-triggered light emission in the presence of intravenously injected substrate 1 coelenterazine (5 p.g ethanol solution)
Figure 6: Monitoring tumor-specific viral infection based on GFP gene expression in a variety of tumor models
including subcutaneous PC-3 human prostate tumor (A-A”) and MCF-7 human breast tumor (B-B”) in nude mice, intracranial C6 rat glioma tumor {C-C', arrows indicate the location of the tumor) in Lev/is rats, and MB-49 mouse bladder tumor (D-D”) in C57 mice. Animals were monitored 7 days after intravenous injections of" 1 n 108 of rW-ruc-gfp virus particles. Bright field (top), fluorescence (middle), and bright field, fluorescence overlay (bottom) images of the tumor are illustrated. (Bars = 5 nun.)

53

Figure 7: Monitoring Vaccinia-mediated GFP expression la a breast tumor model
Nude mouse carrying breast tumor was injected intravenously with 1 x 106 of rW-ruc-gfp virus particles. Both the primary tumor (A-Ar', B-B”, and C-C”) and the metastasized tumor (D-D", E-E", and F-F") were visualized externally (A-A” and-D-D”), with overlying skin removed (B-B” and E-E,”). and when they were split open (C-C” and F-F") in a set of bright field, fluorescence (x) and bright field, fluorescence overlay (V) images. GFP expression in lung metastases in the same animal was also visualized (G-G") (Bars = 5 mm (A-A" to F-F”), and Bars - 1 mm. (G-G" ) .
Figure 8: Visualization of the clearance of light emitting bacteria from nude mice based on the detection of light emission under the low light imager
Nude mice were intravenously injected with 107 cells of attenuated S. typhimurium (A, B) and V. cholera (c, D). Both strains were transformed with pLITE201 carrying the lux operon. Photon collection was (done 20 rain (A, C) and 2 days (El, D) after bacterial injections.
Figure 9: Homing of glioma tumors by attenuated bacteria Nude mice with a C6 glioma tumor in the right hind leg were intravenously injected with 107 attenuated S. typhimurium (A-D) and with . V. cholera (E-H) both transformed with pLITE201 plasmid DNA encoding the lux operon. Photon collection was carried out for one minute under the low light imager. Mice injected with S. typhimurium exhibited luminescence immediately through the whole animal (A) . In contrast, luminescence in the mice injected with V. cholera was visible in the liver area (E) . Two days after bacterial injection, both groups of mice demonstrated luminescence only in the tumor region (B, F). The light emission in the tumors infected with S. typhimurium slowly diminished four (C) and six (D)

54
days after bacterial injection. Tumors infected with V. cholera showed enormously increased light emission four (G) and six (H) days after injection suggesting continued replication of the bacteria in the tumor tissues.
Figure 10: Homing in of bacteria to breast tumors
Nude mice with breast tumors in the right breast pad were intravenously injected with 107 attenuated V., cholera (A-D) or with 107 E. coli (E-F) transformed with pLITE201 plasmid DNA encoding the lux operon. Photon collection was carried out for one minute under the low light imager. Twenty minutes after bacterial delivery, luminescent V. cholera were observed in the liver (A). Forty-eight hours after injection, light emission was noted in the primary breast tumor in the right breast area and a metastatic turner (arrow) in the left breast area, and in the incision wound (B) , At five days, the light
!: '• !-■
emission was visible only in the tumor regions, and not at the wound (C) . Eight days after bacterial injection, the luminescent activity was abolished from the smaller tumor region but remained strong in the primary breast tumor (D). Homing in of E. coli to breast tumors in nude mice was also observed two days after intravenous bacterial injection (E: side view, F: ventral view).
Figure 11; Homing in of bacteria to bladder tumors in C57 mice C57 mice were intravenously injected with 107 attenuated V. cholera transformed with pLITE201 encoding the lux operon. Nine days after bacterial delivery, luminescence was noted in the bladder region of the whole animal (A). The animal was sacrificed and an abdominal incision was made to expose the bladder. The light emission was limited to the bladder region (B) With the removal of the bladder (c) from the mouse, the, entire source of light emission was removed (D) as demonstrated, by the overlay of the low light photon emission image over the photographic image of the excised bladder.


Figure 12: Homing in of bacteria to brain glioma tumors in
Lewis rats
Lewis rats were intravenously injected with 10B cells of attenuated V. cholera transformed with pLITE20l encoding the lux operon. Twenty-four hours after bacterial injection, faint luminescence was noted in the head region of the whole animal during visualization under the low light imager. The animals were sacrificed and their brain removed. Photon collection was carried out for one minute from rats with (A) and without (B) brain tumors. Strong luminescence was confirmed in regions of the brain of the rats with the brain tumor (marked with arrows in A) - Luminescence was completely absent in the control brain tissues (B).
Figure 13: Transformed human fibrosarcoma cells home in on subcutaneous glioma tumors in nude mice
Nude mice with subcutaneous glioma tumors were injected intravenously with 5 x 105 human fibrosarcoma cells, which were permanently transformed with retrovirus derived from pLEIN. Seven days post:- injection, the animals were anesthetized and monitored under a fluorescent stereomicroscope. Fluorescent cells were noted only in the tumor region of the v/hole mice through the skin (Al-3). Upon exposure of the tumor tissues by reflection of the overlying skin (Bl -3), and in cross sections of the tumors (CI -3), fluorescent patches were visible in distinct regions. Close examination of the organs of the mice showed the presence of small clusters of fluorescent cells in the lungs of the animals, demonstrating the affinity of the fibrosarcoma cells for the lungs in addition to the tumorous tissues (Dl-3). (Bars - 5 mm (A1-C3), = 1 mm (D1-D3)).
Figure 14: Homing of attenuated Listeria monocytogenes into subcutaneous prostate tumors

56
Nude mice with subcutaneous human PC3 prostate tumor in : the light hind leg were intravenously injected, with 107 attenuated L. monocytogenes transformed with psod-gfp plasmid " DNA carrying the gfp cDNA, GFP fluorescence was observed under a fluorescence stereo microscope. Twenty-seven hours after bacterial injection, GFP signal was detected only in the tumor region. The tumor is shown in a set of visible light (a), fluorescent— (b), and visible and fluorescent light overlay (C) images. (Bars = 5 mm.)
The present invention is explained by the examples.
Example 1: Materials and Methods
(A) Bacterium strains. The bacterial strains used were attenuated Salmonella typhimurium (SL7207 hisG46, DEL407faroA544::TnlO]), attenuated. Vibrio cholerae
(Bengal 2 Serotyp 0139, MO 10 DattRSl), and attenuated Listeria monocytogenes (D2 mpl, actA, plcB) .The bacterial strains were kindly provided by Prof. w. GObel
(University of Wurzburg, Germany) .
(B) Plasmid constructs. The plasmid pLITE201 containing the
lux CDABB cassette was obtained from Dr. Marines, Biotech
24,1998, 56-58). The plasmid pXylA-dual with the operon
sequence of gfp~cDNA, lux AB, LUX CD, and lux E under the
control of the Xylose promoter was Kindly provided by Dr.
Phil Hill (University of Nottingham, UK).
(C) Transformation of bacteria
The bacteria were transformed by electroporation.
(D) Tumor Cell linos. The rat C6 nitrosourea-induced ylioma
cell line (ATCG, Rockille, MID) was cultured in RPMI-1640

57
medium (Cellgro, Mediatech, Inc, Herndon, VA) supplemented with 10% (v/v) FBS and 1 x penicillin/streptomycin. The human PC3 prostate carcinoma cell line (ATCC, Rockville, MD) and the MB-49 mouse bladder tumor cells and rat 9L glioma cells were maintained in DMEM medium (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with L-glutamine and 10% (v/v) FBS..HT1080 fibrosarcoma, cells (ATCC, Manassas, VA) were cultured in F12 minimal essential media (Cellgro, Mediatech, Inc., Herndon, VA) supplemented with 10% FBS and 1 x penicillin/streptomycin. The MCF-7 human mammary carcinoma cell line (ATCC, Rockville,-MD), permanently transformed with a plasmid carrying pro-IGF-II cDNA (obtained from Dr, Daisy De Leon, Loma Linda

University, LJ->ma Linda, CA) was cultured in DMEM/F12

medium supplemented with 5% FBS and 560 ug/ml of G418

(Life Technologies, Grand Island, NY)

(E) Production and propagation of retrovirus to generate a
light-emitting stably transformed cell line. PT67 packing
cells (Clontech, Palo Alto, CA) were cultured in DMEM medium
supplemented with 10% (v/v) FBS. At 70% confluence, PT67 cells
wer'4 transformed with pLEIN (Clontech, Palo Alto, CA) using
calcium phosphate precipitation method (Protection Mammalian
Transfection Systems,. Promega, Madison, WI) for 12 hours.
Fresh medium was replenished at this time. Retroviral
supernatant collected from FT67 cells 48 hours post
transformation was filtered through a 0.45 pm filter and was
added to target HT1080 cells along with polybrene to a final
concentration of 4 ug/ml. The medium was replaced after 24
hours and the cells were treated with G418 selection at 400 |.i
g/ml and stepwise increased to 1200 ug/ml.
(F) Propagation of recombinant Vaccinia virus. Vaccinia virus
Lister strain (LIVP) was used Recombinant Vaccinia virus rW-ruc-gfp was constructed by
inserting, via homologous recombination, the ruc-gfp-cassette

(Wang
et al., Proc Bioluiuin. Chemilumin. 9, 1996, 419-422;

into the Vaccinia virus genome. The virus was amplified in CV-1 cells by addition of virus particles at a multiplicity of infection (MOD of 0. 1 pfu/cell to CV-1 cell monolayers followed by incubation at 37°C for 1 h with brief agitation every 10 min. At this time, the supernatant fluid with virus particles, was removed, and the cell monolayers were washed once with, serum free medium. Complete growth medium was then
added, and the cells were incubated at 37°C rW-ruc-gfp virions propagated in CV-1 cells were purified through a sucrose gradient. A plaque assay was used 72 h after infection to determine the titer of recombinant virus by staining the cells with 50% crystal Violet solution in ethanol.
(G) Generation of mice carrying tumor implants. Five- to six-wee^-old male BALB/e athymic nu/nu mice (25-30 g in body weight) and . Lewis rats (250-300 g in body weight) . were purchased front Harlan {Frederick, MD) . C573L/6J Min/+ mice were obtained from Jackson Laboratories (Bar Harbor, ME), Min
(multiple intestinal neoplasia) is an autosomal dominant trait involving a nonsense mutation in codon 850 of the murine Ape gene, which renders these animals susceptible to spontaneous intestinal adenoma formation.
To obtain tumors in nude mice, Co glioma cells were grown, harvested and the cell number was determined by the Trypan Blue exclusion method. Disinfectant was applied to the skin surface, then approximately 5 x 105 cells were suspended in 100 ul of phosphate buffered saline (PBS1 and, injected subcutaneously into the right lateral thigh of each mouse-Tumor growth was monitored by recording the size of the tumor with digital caliper. Tumor volume (mm3 ) was estimated by the formula (L x H x W), 2, where L is the length, W is the width, and H is the height of the tumor in mm.

57
Intracerebral glioma tumors were generated by injecting C6 glioma cells into the head of rats. Prior to injection, rats
were anesthetized with sodium pentobarbital (Nembutal® Sodium solution, Abbot Laboratories, North Chicago, lL; 60 mg/kg body weight). A midline scalp incision (0-5 -1 cm!) was made, the skin was retracted, and a 1 mm burr hole was made in the skull at a location 1 mm to the left and 2.5 mm posterior to (the brigma. Tumor cells were pipetted into an insulin syringe, which was fitted with a 29-gauge needle and mounted in a stereotactic holder. The needle was inserted vertically through the burr hole to a depth of 3 mm. After injection into
the brain of 5 x 10 C6 cells in a 10 |al volume, the needle was
kept1 in place for 15 second then withdrawn. The skin incision
was closed with surgical clips. Mice bearing subcutaneous
prostate tumors were generated over a period of one month
following subcutaneous implantation of 3 x 106 PC3 human
prostate cells.
MB- 49 mouse bladder tumor cells were implanted -in the C57 mouse bladder to produce animals with bladder I tumors. To generate animals with breast cancer (Tian and DeLeon, submitted for publication), female nude mice were first implanted with 0.72 mg/90 day-release 17β-estradiol pellets (Innovative Research, Rockville, MD) in the skin to facilitate breast tumor development and metastasis. One day after estrogen pellet implantation, 1 x 106 MCF-7 human breast carcinoma cell transformed with pro-IGF-II (Dull et al., Nature 310 (1984), 777-781) were implanted in the mammary fat pad. For orthotopic transplants, tumors developed from implanted cells were resected and minced into 1-mm3 cubes for tissue transplantation into the mammary fat pad.
(H) Assay of Renilla luciferase in live animals. Mice were anesthetized with Nembutal (60 mg/kg body weight) before every Renilla luciferase assay. Renilla luciferase activities were

60
determined after intravenous injection of a mixture of 5 pl of coelenterazine (0,5 pg/pl diluted ethanol solution) and 95 ul of luciferase assay buffer (0. 5 M NaCl; 1 mM EDTA; and 0. 1 M potassium phosphate, pH 7.4), Whole live animals were then imaged in a dark box using the Hamamatsu ARGUS100 low light Video camera, and the images were recorded using image Pro Plus 3.1 software (Media Cybernetics, silver Spring, MD). The pseudocolored photon emission image was superimposed onto the gray scale image of the animal in order to precisely locate the site of light emission.
(I) Detection of luminescence and fluorescence. Immediately
before imaging, mice and rats were anesthetized with Nembutal® (60 mg/kg body weight). The animals were placed inside the dark box for photon counting and recording superimposed images (ARGUS 100, Hamamatsu, Hamamatsu, Japan), Photon collection was for one minute from ventral and dorsal views of the animals. & light image was then recorded and the low light image was then superimposed over the light image to record the location of luminescent activity.
Imaging of GFP expression in tumors of live animals was performed using a Leica M28 stereo fluorescence microscope equipped with a mercury lamp power supply and a GFP filter (excitation at 470 nm) . Images were captured using a SONY DKC-5000 3CCD digital photo camera.
(J) Histology of tumor tissues. Under anesthesia, the animals
were euthanized with an overdose of Nembutal®. The tissues of interest were removed, embedded in Tissue-Tek OCT compound (Miles Scientific, Naperville, IL) and immediately frozen in liquid nitrogen without fixation. Frozen sections were cut at
-20°C using a Reichert-Jung Cryocut 1800 cryostat. CFP fluorescence of the tissues was monitored under a Leica

61

fluorescence microscope and the. images were recorded using Photoshop software.
Example 2: Results obtained by intravenous injection of recombinant Vaccinia virus rW-ruc-gfp into mice
(A) Monitoring of virus-mediated marker gene expression in"
immunodeficient mice
Vaccinia virus (1 x 10e pfu) carrying the Renilla luciferase GFP fusion expression cassette (rW-ruc-gfp) was introduced intravenously into nude mice with no tumors. The animals were observed once every 3 days over a two-week time period under the low-light imager to monitor luciferase catalyzed light emission immediately after intravenous injection of coelenterazine, and under a fluorescence microscope to visualize GFP expression. Neither apparent luminescence nor green fluorescence was detected in the animals when imaged externally, except at certain locations that had small skin lesions. Such luminescence and fluorescence signals disappeared after a few days once the lesions had healed. Animals were sacrificed one week and two weeks after viral infection, and their organs were removed and examined for the presence of luminescence and GFP fluorescence signals. One week after viral injection, no luminescence or green fluorescence could be detected in brain, liver, lung, spleen, kidney or testis. These results indicated that the rW-ruc-gfp virus did not show organ, specificity after injection and that the virus seemed to be cleared from the animal by the immune system soon after systemic, delivery via the bloodstream.
(B)Visualization of Vaccinia virus-mediated marker gene expression in glioma tumors of live nude mice

62
the distribution of injected Vaccinia virus in nude mice bearing subcutaneously implanted C6 glioma tumors was examined. Nude mice with tumors approximately 500 mm3 in size were injected intravenously with 1 x 106 pfu of the rW-ruc-gfp virus. Seven days after virus injection, the animals were monitored for GFP expression under a fluorescence microscope to determine the presence of viral infection and multiplication in the tumors, which had grown to approximately 2500 mm3 in size. Surprisingly, green fluorescence was detected only in the tumor regions in live animals. Seven days after viral injection, the GFP fluorescence was very intensely localized in a patch-like pattern restricted to the tumor region (Fig. lA-A”) . These patches, often seen at the end of blood vessel branches, may have indicated local viral infection of tumor cells that surround the leaky terminals of capillary vessels. During real-time, observation I of the same tumors, the GFP signal from the center of these patches started to disappear, and new green fluorescent centers appeared in the form of rings at the periphery of the fading patches. The new sites of intense GFP fluorescence may have resulted from progression of the viral infection to nearby cells within the tumor during tumor growth and expansion. After careful examination of the mice, with the exception of the tumor region, no detectable green fluorescence was seen elsewhere on the body surface or in the dissected organs. This experiment clearly showed that a mature solid tumor could be easily localized by the labeled Vaccinia virus, based on light-emission/ and it also demonstrated the affinity of virus particles for the tumor tissue.
To determine whether tumor size and vascularization are decisive factors for viral retention in tumors, nude mice were intravenously injected with 1 x 10a rW-ruc-gfp Vaccinia virus particles one day after subcutaneous C6 cell implantation. Surprisingly, 4 days after viral injection GFP expression was

seen in 5-day-old C6 tumors that had a volume of about 25 mm3 “Fig. 1B-B"). Examination of labeled Vaccinia virus tumor targeting by visualization of GFP expression in implanted tumors younger than 5 days was not feasible in live mice, since sufficient levels of marker gene expression required approximately 4 days to allow detection under a fluorescence microscope.
The finding that injection of the rW-ruc-gfp Vaccinia virus into the bloodstream of the host resulted-1 in GFP expression and accumulation in tumors suitable for non-invasive tumor detection I allowed us to follow the entry and replication process of this virus in the same animal irk real time (Fig. 1 C-C”, D-D" and E-E”). A continuously increasing level of GFP fluorescence was observed in the same animal throughout 20 days following viral injection, which was the time scheduled before sacrificing the animals. Such an increase in detectable
fluorescence was indicative of a very strong viral replication in the tumor tissue, the latter appearing to function as a protective immunoprivileged environment for viral replication. The viral replication in the tumors was verified by determining the viral titer and light emission of the isolated viral particles in cell cultures. Interestingly, the location of blood vessels and the neovascularization within the periphery of the enlarging tumor were readily visible and confirmed by external visualization against a bright green fluorescent background (Fig. 1A-A", D-D", E-E." and Fig. 2).
To determine the location of viral infection within the tumors, the animals were sacrificed and the skin over the tumor was carefully reflected to expose the tumor. In the exposed tumor, GFP fluorescence was found to be concentrated exclusively in the tumor tissue (Fig. 3B-B” and D-D”). The non-tumorous thigh muscles did not show any fluorescence of viral infection, as indicated by arrows in Fig. 3D-D”. The skin overlying the tumor was also non-fluorescent (indicated

64
by asterisks in Fig. 3B-B" and D-D”). Cross sections of the
tumor, however, revealed that strong green fluorescent regions
were mostly found as patches in the periphery of the tumor
(double. arrows in Fig. 3 C-C”) where the actively dividing
tumor cells are presumably located.
To further examine the pattern of viral infection in C6 glioma tumors based on GFP expression, the tumor tissues were sectioned for microscopic analysis under the fluorescence microscope. Comparative analysis of various tissue sections revealed that GFP fluorescence was present in large clusters of cells within the tumor (Fig, 4), but no fluorescence was visible in normal tissues such as the heart, lung, liver, spleen, and kidney.
In addition to GFP, the recombinant rW-ruc-gfp virus carried a second marker gene, which encoded the Renilla luciferase in the form of a fusion protein with GFP. Therefore we were able to directly superimpose the site of GFP fluorescence with light emission from Renilla luciferase in the tumors. Immediately after coelenterazine (substrate for Renilla luciferase) was delivered by intravenous injection, a very strong luciferase activity was recorded only in the tumor region under a low light video camera (Fig. 5). By lowering the sensitivity of the low light video camera to avoid saturation of light detection, we were able to identify the Renilla luciferase gene expression in localized patches in the periphery of the tumor. These patch-like patterns precisely correlated with the GFP signals.
(C) Affinity of Vaccinia virus delivered to the bloodstream
for different tumors implanted into animals
To determine whether the at traction of the Vaccinia virus was limited to glioma tumors or whether this attraction could be

64-
observed in ether turner u, recombinant vaccinia virus was -recombinantly introduced into mice that carried different types of implanted tumors. One of these tumor models was a nude mouse with implanted subcutaneous PC-3 human. prostate carcinoma. Although the PC3 implants from which tumors developed grew at a much slower rate than the implanted subcutaneous glioma tumors, these tumors showed the same dynamics with regards to Vaccinia virus infection when identical titers (1 x 108) were injected intravenously (Fig. 6A-A"). Similar to our findings with glioma tumors, GFP expression was 3 initially detected 4 days after virus injection, and the fluorescence lasted throughout the 3-week observation period.
■'■'■■.■"': '■'> '■';'■ ; .
Female nude mice with established breast tumors were also used
for labeled Vaccinia injections. These breast tumors were
allowed to grow for 6 months after the animals received
implants of MCF-7 human breast carcinoma cells transformed
with pro-IGF-11 cDNA- At the time of Vaccinia virus injection,
the tumors had reached maximum growth and the tumor volume
(about 400-500 am3) did not change significantly during the
experimental period- Similar to previous experiments, 6 days
after intravenous delivery of 1 x 10e rW-ruc-gfp virus
particles, strong GFP expression was observed in the breast
armor region (Fig. 6B-B', Fig. 7A-A” and B-B”) and nowhere
else in the body.
Examination of cross sections of virus-infected breast tumors revealed luminescent „islands„ throughout the tumors without any indication of central or peripheral preference of. infection (Fig. 7C-C”) . The MCF-7 tumor cells used in these breast tumor models are known to metastasize and in addition for the primary solid tumor, a smaller metastasized tumor found on the left side of the body showed GFP fluorescence (Fig. 7 D-D”, E-E”, and F-F”). Excised lung tissues were also

66
examined for detection of metastases. Metastasized tumors as
small as 0.5 mm in diameter on the surface of the lung were
positive for GFP fluorescence (Fig. 7G-G"). The presence of a
strong Renilla luciferase-mediated light emission confirmed
the expression of the luciferase GFP fusion protein in these
breast tumors but nowhere else in the body when the substrata
coelenterazine was injected intravenously -into the live
animals. These experiments showed that intravenously delivered
Vaccinia virus particles were selectively accumulated to and
replicated in primary and metastasized breast tumors in nude
mice, likely as a result of the immunocompromised state of the
tumor microenvironment.

To determine whether virus particles could move out of tumors
And reenter the circulation, we injected C6 glioma cells into thigh of mice to form a second tumor in animals already carrying a breast tumor infected with labeled Vaccinia virus. If' the virus particles Were released from the tumor to re-enter the circulation in significant numbers, they would be able; to colonize the newly implanted glioma tumor. Monitoring o>f these second tumors showed that no GFP signal was visible in the new glioma tumor 7 and 14 days after implantation of the glioma cells. To demonstrate that the newly implanted glioma tumors could be targeted by labeled Vaccinia virus, a second dose of rW-ruc-gfp virus (1 x 108 pfu) was intravenously injected. Five days later, tumor-specific GFP expression was detected in the newly formed glioma tumor in addition to GFP expression seen in the original breast tumor. These findings suggested that the virus particles in infected. tumors were either not released back into the circulation at
all, or were not released in sufficient numbers to infect and
replicate in a second tumor.
Two additional tumor models, including Lewis rats with intracranial C6 rat glioma tumors and C57 mice with MB-4 9

67
mouse bladder tumors in the bladder, were used for Vaccinia injections. To determine whether tumor-affinity of virus particles is a phenomenon limited to tumors in nude mice with a diminished T-lymphocyte function or whether it is a general protective property of tumors that may be demonstrated also in immunocompetent animals, Lewis rats with intracranial Co rat glioma tumors and C57 mice with MB-49 mouse bladder tumors in" the bladder were used. A total of 5 x 105 C6 glioma cells in a 100 p.1 volume were stereotactically implanted in the brains of 2 of 4 immunocompetent Lewis rats, and the tumors were allowed to grow for 5 days. The other 2 rats were Injected intracranially with phosphate-buffered saline to serve as controls. On day six, all 4 rats were intravenously injected with rW-ruc-gfp virus particles via the femoral vein. Five days after virus injection, all 4 animals were sacrificed, and their brains were carefully excised for analysis by fluorescence microscopy. GFP expression was detected in the brains with implanted intracranial tumors (Fig. 6C-C”) while no GFP expression was seen in the control brains. In parallel experiments, C57 mice, with or without bladder tumors, were divided into two groups. One group was injected intravenously with rW-ruc-gfp Vaccinia virus (1 x 108 pfu.) and the other with saline solution as control. Five days after virus injection, the animals were sacrificed and examined under the fluorescence microscope. GFP expression was observed in the bladder tumor region in C 57 mice but not in control mice (Fig. 6D-D”).
Taken together, these experiments show that Vaccinia virus particles of tumors, probably protected by the tumor microenvironment, and that they were not able to survive in the non-tumorous tissues of immunocompromised as well as immunocompetent animals. The tumor-targeting process by intravenously injected Vaccinia virus carrying the

68
light-emitting dual marker gene demonstrated the ability of the Vaccinia virus system to detect primary and metastatic tumors in live animals.
Example 3: Results of intravenous injection of bacterial and mammalian light-emitting cells into mice
(A) Visualization of light emitting bacteria present in whole animals after intravenous injection
To determine the fate of intravenously injected luminescent bacteria in the animals, lb7 bacteria carrying the pLITE201 plasmid in 50 pl were injected into the left femoral vein of
mice under anesthesia- Following closure of the incision with sutures, the mice were monitored under the low light imager (ARGUS 100 Camera System, Hamamatsu, Hamamatsu, Japan) in real time land photons were collected for one minute. The imaging was repeated in two-day time intervals to determine the presence of light emission from a given animal. It was found that the distribution pattern of light emission following an intravenous injection of bacteria into mice was characteristic of the bacterial strains used. Injection of the attenuated V. cholera into the bloodstream resulted in light emission localized in the liver immediately. Injection of s typhimurium, however, was widely disseminated throughout the body of the animal suggesting a difference in the interaction with host cell system (Figure 8A-3D). Imaging the same animals 24 and 48 hours post-infection showed that all of the detectable light emission from the earlier time diminished rapidly and was eliminated completely from the injected animal. These findings suggest that light emitting bacteria injected into the bloodstream via the femoral vein are cleared. This process was confirmed by photon emission analysis of excised organs, which were found to lack light

69
emission. Similar data were obtained in immunocompetent mice arid rats suggesting that the removal of bacteria from the blood is efficient in both systems.
(B) Bacteria home in to glioma tumors in nude mice
To determine, if bacteria preferentially colonize tumorous tissues, nude mice with ten-day-old tumors (about 500 mm3) in the right hind leg were injected intravenously via the femoral
vein with 107 S. typhimurium or 107 V. cholera in a 50 ul
volume of bacterial: suspension. Following injection, the
incision wounds were sutured and the animals were monitored
for; six days under the low light imager. At each observation
time point, photons were collected for exactly one minute. In
{mice injected with 5. typhimurium, luminescent bacteria were
disseminated throughout the whole body of the animal similar
to the findings in the non-tumorous mice {Figure 9A) . Nude
mice injected with V. cholera, demonstrated luminescent
activity only in the liver region during the early observation
period (Figure 9E). Regardless of the bacterial strain
injected, two days after injection, luminescent activity was
observed only in the tumor region (Figures 9B and 9F) .
Monitoring of the mice under the low light imager on days four
and six post-injection showed decreased amounts of detectable luminescence in the tumors of animals injected with s.
typhimurium (Figures 9C and 9D. This finding was in marked
contrast with the findings In the tumor of mice injected with or mice injected with V. cholera, which demonstrated net only survival but also propagation of the bacteria in the tumor mass with a dramatic increase in light, emission (Figures 9G and 9H) .
Nude mice bearing subcutaneous human PC3 human prostate tumor in the right hind leg were intravenously injected with 107 attenuated L. monocytogenes transformed with psod-gfp plasmid DNA carrying the gfp cDNA. GFP fluorescence was observed under-

69

a fluorescence stereomicroscope. Twenty-seven hours after bacterial injection, GFP signal was detected only in the tumor region (Figure 10). No GFP signal was observed in the rest of the animal.
(C) Determination of minimum size and age of glioma tumors necessary for bacterial infection.
The purpose of this experiment was to determine whether the
-size of the tumor has .any influence on its ability to be
colonized by bacteria. Tumors Were induced in the right hind
[leg of nude mice by subcutaneous injection of glioma cells as
described. On days 0„; 2, 4, 6, 8 and 10 of tumor induction attenuated S. typhimurium and V. cholera with the PLITE201 plasmid were injected; intravenously through the femoral vein. Presence of luminescent bacteria in the tumor was determined by photon collection for exactly one minute under the low light. imager two and four days post-infection. The tumor
volume was also determined by measuring the dimensions with a
digital caliper. Th£ earliest time-point when luminescent

activity was noted in the tumors was on day eight after tumor
induction. Corresponding tumor volumes were approximately 200
mm3.
(D) Bacteria home in to breast tumors of nude mice
In order to determine whether bacterial colonization of tumors is limited to glioma cells or whether this is a general phenomenon observed with all tumors, female nude mice bearing tumors in the right breast pad were intravenously injected with 107 V. cholera in a 50 ul volume of bacterial suspension. The animals were monitored within the first 10 minutes after inoculation under the low light imager for one minute and demonstrated the typical luminescent pattern in the liver region (Figure 11A). Two days later, while the liver had

71
become clear of luminescent bacteria, the breast tumor was
colonized by. the labeled V. cholera. In addition to the main.
tumor, a metastatic tumor in the left breast demonstrated
luminescent activity (Figure 11B). On day five, the animals
had cleared the bacteria that colonized the incision wound,
however, the tumors remained luminescent (Figure 1-1C). Figure
11D shows the continued colonization and propagation of the
bacteria in the main tumor, while the metastatic, smaller
tumor had become cleared- Luminescent activity continued for
over 45 days in the right breast tumor. Similar experiments
were conducted using E. coli to demonstrate that homing in of
tumors by bacteria is not strain dependent (Figures .1 IE and
To determine whether the bacteria from the tumor enter the blood circulation in significant quantities to colonize other sites, a second tumor (C6 glioma) was induced in. these animals in the right hind leg. The tumor was allowed to grow for 10 days. No luminescent activity was observed in the glioma tumor demonstrating the absence of a significant bacteria that would cause colonization of this tumor. However, when the animal was rechallenged with 107 attenuated V. cholera intravenously, the leg tumor showed strong luminescent activity.
The findings of these experiments demonstrate that larger tumors retain bacteria more effectively over time. Furthermore, the bacteria within the tumors do not escape into

the blood in sufficient quantities to infect susceptible sites

such as other tumors.
(E) Bacteria home in to bladder tumors in immunocompetent mice
C57 mice were intravenously injected with 107 attenuated V.
cholera transformed with pLITE201 encoding the lux operon. On
day nine after bacterial delivery, luminescent activity was

72
recorded by photon collection for one minute under the low light imager. Light emission was noted in the bladder region of the whole animal (Figure 12A), The animals were sacrificed and an abdominal incision was made to expose the bladder. Luminescent activity was positively confirmed to be limited to the bladder (Figure 12B). Upon removal of the bladder from the mice luminescent activity was no longer visible anywhere in the animals, however, the excised bladders continued to demonstrate light emission (Figure 12C). Based on the results of this experiment, bacteria can target tumors in immunocompetent, as well as nude mice. Furthermore, the bacteria can also target smaller tumors.
(F) Bacteria home in to glioma tumors in the brain of' rats
Lewis rats with glioma tumors in the brain were intravenously injected) with 108 attenuated V. cholera with the pLITE20l plasmid through the left femoral vein to determine if bacteria can cross the blood-brain barrier and target tumors in immunocompetent animals- The whole animals were monitored for one minute under the low light imager the following day and low levels of luminescent activity was observed through the skull. The rats were sacrificed and the brain tissue was removed in one piece in order to further evaluate the exact location of the luminescent bacteria. Visualization of the excised brain under the imager demonstrated strong luminescent activity in specific regions of the brain (Figure 13A). Similar imaging of control rats without brains tumors, which were intravenously injected with the labeled bacteria, iemonstrated absence of any luminescent activity (Figure 13B).
(G) Transformed human fibrosarcoma cells home in to subcutaneous glioma tumors in nude mice

"73
Nude mice with human breast tumors were injected intravenously With 5 x 105 human fibrosarcoma cells, which were permanently transformed with retrovirus derived from pLEIN. Seven days post-injection, the animals were anesthetized with Nembutal, and monitored under a fluorescent stereomicroscdpe. Fluorescent cells were noted only in the tumor region of the whole mice through the skin (Figure 14A1-3). Upon exposure of the tumor tissues by reflection of the overlying skin, (Figure 14B1-3), and in cross sections of the tumors (Figure 1'4C1 -3), fluorescent patches were visible in distinct, regions. Close examination of the organs of the mice showed the presence of small clusters of fluorescent cells in the lungs of the animals, demonstrating the affinity of the fibrosarcoma cells "for the lungs in addition to the tumorous tissue.

Example 4: Construction of bacterial plasmid vectors that
carry the light-emitting protein encoding expression cassettes
and the therapeutic gene expression constructs in cis
configuration
(A) Rationale
Using the light-emitting expression systems described above, tumors could be imaged based on light emission for up to 45 days in animals. These findings suggest a remarkable plasmid DNA stability in bacteria in the absence of selection. Therefore, by placing the therapeutic gene cassette in cis configuration with the light-emitting protein expression cassette on the same replicon, light emission can be used as an indicator of therapeutic construct presence and stability. In contrast to light-emitting proteins, the therapeutic proteins, endostatin and Pseudomonas exotoxin/TGF alpha fusion protein, are required to be secreted from the bacteria into the medium or into the cytosol of tumor cells for inhibition

74
of tumor growth. To achieve protein secretion from the -tracellularly replicating E. coli cells into the tumor, two constructs with different signal sequences can be designed. For secretion of endostatin, the ompF signal sequence can be placed upstream of the coding sequence of endostatin which facilitates the secretion into the periplasmic space. To release the endostatin into the medium, an additional protein, the PAS protein, needs to be coexpressed with endostatin. PAS has been shown to cause membrane leakiness and the release of secreted proteins into the medium (Tokugawa et al., J.Biotechnol. 37 (1994), 33; Tokugawa et al., J.Biotechnol. 35 (1994), 69). The second construct for the secretion of Pseudomonas exotoxin/TGF alpha fusion protein from E. coli has the OmpA signal sequence upstream of the fusion gene and the release from the periplasmic space into the medium is facilitated by frequences present in domain II of the exotoxin (Chaudhary et al., PNAS 85 (1988), 2939; Kondo et al., J.Biol.Chem. 263 (1988), 9470; Kihara and Pastan, Bioconj.Chem. 11 (1994), 532). To promote secretion of endostatin and Pseudomonas exotoxin/TGF alpha fusion protein from L. monocytogenes, the signal sequence of listeriolysin (LLO) (Mengaud et all, Infect. Immun. S6 (1988), 766) can be placed upstream of each coding sequence.
For regulation of endostatin and Pseudomonas exotoxin/TGF alpha fusion protein expression levels in bacteria, vectors can be generated where the therapeutic protein encoding genes are under the control of the T7 promoter or the P.,pac synthetic promoter (Freitag and Jacobs, Infect .Immun. 67 (1999), 1844). Without exogenous induction, the levels of the therapeutic proteins are low in E. coli and in L. monocytogenes. The minimal levels of therapeutic protein in bacteria, provide greater safety following intravenous injection of the engineered bacteria. In the following, tie construction of six plasmid DNAs for constitutive and regulated expression of

75
endostatin and Pseudomonas exotoxin/TGF alpha fusion protein in E. coli and L. monocytogenes is described. All plasmids to be transferred into E, coli will carry the constitutively expressed bacterial lux operon, and all the plasmids to be transferred into L. monocytogenes will carry the constitutively expressed sod-gfp cassette. Plasmids BSPT#l-ESi and BSPT#2-Pti are able to replicate in E. coli only/- and plasmids BSPT#3, #4, #5, and #6 replicate in E. coli and L. monocytogenes.
(B) Construction of plasmid vectors for protein expression and
secretion from E. coli
The construction of the endostatin secretion vector to be used in E. coli is as follows. The coding sequence cf human endostatin (591 bp) will be amplified by PCR from the plasmid pES3 with the introduction of the required restriction sites on both ends, followed by ligation into a pBluescript {Clontech Corp., USA) cloning vector to generate pBlue-ES. The ompF signal sequence (Nagahari et al., EHBO J. 4/(1985), 3589) is amplified with Tag polymerase and inserted upstream in frame with the endostatin sequence to generate pBlue-ompF/ES. The expression cassette driven by the T7 promoter will be excised, and inserted into the pLITE201 vector described in Example 1(B), above, carrying the luxCDABE cassette, to produce the plasmid pLITE-ompF/ES. The sequence encoding the PAS factor (a 76 amino acid polypeptide) will be amplified from the chromosomal DNA of Vibrio alginolyticus (formerly named Achromobacter iophagus) (NCIB 11038) with Taq polymerase using the primers 5’-GGGAAAGACATGAAACGCTTA3-‘ and –5’- AAACAACGAGTGAATTAGCGCT-3’, and inserted into the multiple cloning sites of pCR-Blunt (Clontech Corp., USA) to create the expression cassette under the control of the lac promoter.- The resulting plasmid will be named pCR-PAS. The lac promoter linked to the pas gene will be excised from pCR-PAS and

76
inserted into pLITEompF/ES to yield the final •plasmid BSPT#l-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 encoding domain II and III of
Pseudomonas exotoxin (PE40). The DNA sequence encoding PE40
will be excised with restriction enzymes and replaced with a
fragment of PE37/TGF alpha (Pseudomonas exotoxin A 280-613/TGF
alpha) obtained from the plasmid CT4 (Kihara & Pastan, 1994,
Bioconjug. Chem. 5: 532-538) j to create the plasmid
pt€85-PE37/TGF alpha. The expression cassette of ompAPE3T/TGF
alpha linked to the T7 promoter will be excised and inserted
into pLM201 to yield the final plasmid BSPT#2-PTI.

(C) Construction of plasmid vectors for protein expression and secretion from L. monocytogenes
Genes encoding endostatin; or PE37/TGF alpha will be inserted downstream of the listeriolysin (LLC) signal sequence in the plasmid pCHHI to generate pCHHI-ES and pCCHI-PE37/TGF alpha. Constitutive expression of the therapeutic proteins will be obtained by linking the above secretion cassettes to the listeriolysin promoter obtained from the pCHHI vector. The sod-gfp expression cassette, excised from the plasmid psod-gfp (Gotz et al. PNAS in press.) will be inserted into pCHHl-ES to generate BSPTO-ESc, and into pCCHI-PE37/TGF alpha to generate BSPT#4-PTC. For the expression of the therapeutic proteins under the control of an IPTG inducible promoter, the listeriolysin promoter in BSPT#3-ESc and BSPT#4-PTc will be replaced with the Pspac promoter from the plasmid pSPAC (Yansura and Henner, FNAS USA £1 (1984), 439) to generate BSPT#5-E31 and BSPT#5-PTi. Pspac is a hybrid promoter consisting , if the Bacillus subtilis bacteriophage SPO-1 promoter and the lac operator. IPTG-induced GFP expression from the Pspac. promoter

77
has been documented in L. monocytogenes in the cytosol of mammalian cells.
Example 5: Demonstration of the expression of luciferase and GFP in bacteria and verification of the secretion of
endostatin and recombinant toxin/TGF alpha fusion protein and
their function in cell culture assays
To be able to detect the presence of E. coli and L. monocytogenes in tumor tissues in live animals, the levels of the constitutively expressed luciferase and GFP in bacteria need to be adequate. Therefore, after transformation of recipient E. coli or L. monocytogenes with the constructs described in Example 4, the colonies with the highest luciferase light emission or GFP fluorescence will be selected. In addition to characterizing light emission from each selected colony before intravenous injection, the ability of the selected transformants to secret endostatin and Pseudomonas exotoxin. TGF alpha fusion protein into the medium-needs to be confirmed. The presence of endostatin and Pseudomonas exotoxin, TGF alpha fusion protein synthesized within E. coli and L. monocytogenes will be determined by extracting those proteins from the cell pellet. The secreted proteins in the medium will be concentrated and analyzed by gel separation and the quantity will be determined by Western blotting. It is imperative to determine the percentage of the newly synthesized proteins expressed from each plasmid construct in either E. coli or L. monocytogenes that is present in the medium. It is also essential to confirm, in addition to constitutive expression of endostatin and Pseudomonas exotoxin/TGF alpha fusion protein, that expression can be induced in E. coli and in L. monocytogenes upon the addition of IPTG to the bacterial culture medium. For the design of future tumor therapy protocols, the relative amounts

78
of protein secreted by the constitutive expression system needs to be compared to the induced expression levels for a defined time period first in bacterial cultures. It is equally essential to determine that both proteins when synthesized in E. coli and L. monocytogenes are biologically active if generated from the proposed constructs. Both proteins were synthesized previously in E. coli and were shown to be active,
The-results of the experiments described below should confirm whether endostatin is successfully secreted from E. coli using the OmpF signal peptide in combination with PAS pore forming
protein expression. These experiments will also show if the PE40/TGF alpha and PE37/TGF alpha fusion proteins are secreted
from bacteria using the GmpA signal peptide in combination
with domain II of PE. Further, the listeriolysin signal peptide may also facilitate the secretion lot endostatin and
the chimeric toxin/TGF alpha fusion protein into the medium as well as into the-, eytosol of infected tumor cells. Using the
endothelial cell migration inhibition assay and the protein synthesis inhibition assay, it can be expected to determine that both proteins secreted into the medium are biologically active. The presence and quantities of these proteins may be regulated by replacing the constitutive promoters with promoters that can be induced by IPTG.
In addition to the secretion system described below, alternative secretion systems such as the E. coli HlyBD-dependent secretion pathway (Senior et al., MolvGen.Genet, 256 (1997), 306, may be useful. Alternative secretion signals from ether gram positive bacteria, such as the Bacillus .sp. endoxylanase signal peptide (Choi et al., Appl.Microbiol. Biotechnol. 53 (2000), 640; Jeorg and Lee, Biotechnol.Bioeng. 67. (2000), 398) can be introduced.
(A) Confirmation of endostatin and Pseudomonas exotoxin/TGF

alpha fusion protein secretion from bacteria into growth
medium
E. coli strains (DH5a and BL21 (XDE3) will be transformed with
BSPT#l-£Si and BSPT#2-PTi plasmid DNA, L. monocytogenes strain
E6DA2 will be transformed with plasmids '. BSPT#3-ESc,
BSPT#4-PTc, BSPT#5-ES1 and BSPT#6-PTi individually. After
plating on appropriate antibiotic-containing plates,
individual colonies will be selected from each transformation
mixture. These colonies will be screened under a low light
imager and fluorescence microscope for luciferase and GFP
expression, respectively. Three colonies with the most intense
J light emission from each transformation batch will be chosen
for further studies. To verify the secretion of endostatin and
pseudomas exotoxin/TGF alpha fusion protein from each selected
transformant, the cells will be grown in minimal medium to log
phase. After centrifuging. down the bacteria, the supernatants
will be passed through a 0.45-um-pore-size filter, and the I bacterium-free medium will be used for precipitation of the secreted proteins- the i precipitates will be collected by center if legation. Peliets will be washed,, dried, and re-suspended in. sample buffer for protein gel separation. Proteins from aliquots corresponding to 10 |il of bacterial
culture will be compared to proteins from 200 u.1 of culture supernatant after separation in a 10% SDS-polyacrylamide gel. Western blot analysis will be performed using polyclonal antibody against endostatin (following the antibody production protocol described by Timpl, Methods Enzymol. 82 (1982), 472) and monoclonal antibody against TGF alpha (Oncogene Research Products, Cambridge, MA, USA). The optimal growth conditions will be . established for secretion by sampling the growth medium at different times during growth. A similar method has been used previously to analyze secreted proteins in Salmonella typhimurium culture supernatant (Kaniga et al., J.Bacteriol. 177 (1995), 3965). By use of these methods, the

amount of secreted proteins in the bacterial culture medium generated by each of the constructs without induction will be established. To estimate the increase in the amount of secreted proteins in the medium, IPTG-dependent promoter activation experiments will be carried out by adding IPTG to the bacterial culture in log phase for 3 to 6 hours, and the secreted proteins will be assayed as above.
(B) Verification of the biological activity of endostatin secreted by E. coli and L. monocytogenes using a migration
inhibition assay
It has been shown that endostatin inhibits vascular.
endothelial growth factor (VEGF)-induced human umbilical vein
endothelial cell (HUVEC) migration. Thus, the biological
activity of endostatin secreted by bacteria can be tasted
using the HUVEC migration assay provided by Cascade Biologies/
Portland, OR. The inhibition of cell migration will be
assessed in 48-weli chemotaxis chambers (Neuro Probe,
Galthersburgs, MD (Polverine et al., Methods Enzymol. 198
(1991), 440), Bacterium-free supernatant from each secretion
construct will be added to HUVECs for preincubation for 30
min. After incubation, the HUVECs will be placed in the upper
chamber. The migration of HUVECs into the lower chamber
induced by Ve(iF165 (R&D Systems, Minneapolis, NIN) will be
quantified by microscopic analysis. The concentration of
functional endostatin in the medium will be directly
proportional to the degree of inhibition of HUVEC migration.
(C) Testing the cytotoxic activity of secreted recombinant PE
toxin in tumor cell cultures
The inhibitory activity of the chimeric toxin in mammalian cells will c-e measured based on inhibition of de novo protein synthesis by inactivating EF-2 (Carroll and Collier,

JTMol.Chem. 262 (1987), 8707). Aliquots of bacterium-free
supernatants obtained front the expression of various
recombinant PE secretion constructs in E, coli and in X.
monocytogenes will be added to the C6 glioma cells or to HCTI
Wi6 colon carcinoma cells. Following treatment with medium, the
mammalian cells will be pulsed with [3H]-leucine, and the
incorporation, will be determined in the protein fraction. To
determine the presence of secreted chimeric toxin proteins in
L. monocytogenes-infected mammalian cells, the bacteria will
be eliminated from the medium by gentamicin treatment. The
mammalian cells containing L. monocytogenes in the cytosol
will be lysed, and the released bacteria I removed from the
lysate by filtration. The mammalian cell lysate containing the
secreted chimeric toxins will be assayed in protein synthesis
inhibition experiments. The inhibition of [3H] -leucine
incorporation in tumor cell culture fill be directly
proportional to the amount of the biologically active chimeric
toxin protein in the medium and cell lysate.
Example 6: Determination of the entry, localization and
distribution of intravenously injected bacteria in tumors of
live animals
(A) Rationale
Since only a small number of intravenously injected bacteria escape the immune system by entering the tumor, their immediate localization is not possible due to limited light emission in live animals. Their location can only be verified by sectioning the tumor to identify the early centers of Light emission. Looking at sections at a later time point, bacteria - can be seen throughout the entire tumor due to rapid replication- To determine whether one or multiple bacteria enter through the same site, red fluorescent protein can be

used to label the extracellularly replicating E. coli and green fluorescent protein for the intracellularly replicating L. monocytogenes. By visualizing the distribution of the red and green fluorescence in tissue sections, the entry sites as well as replication and localization of E- coli and L. monocytogenes can be determined individually and simultaneously -in the central or peripheral regions of the tumor. It can be expected that the patterns of entry and distribution obtained in implanted tumors mimic those of spontaneous tumors, accordingly, the bacterium-based diagnosis and protein therapy will become a valid approach.

With the experiments described in section (B), below, the entry, replication, and distribution of light-emitting bacteria in spontaneous tumors can be compared to the distribution patterns in implanted tumors. Further, double-labeling experiments will allow the operator to precisely locate the extracellularly replicating E. coli and the intracellularly replicating L. monocytogenes in the same tumor sections. Lastly, it can be determined (subsequent to a five-day I bacterial colonization) whether bacteria are distributed evenly in the tumors or preferential localization occurs in the periphery of the tumor or in the necrotic center. A possible reduction in bacterial entry into spontaneously occurring tumors due to the immunocompetence of these animals can be overcome by increasing the number of intravenously injected bacteria.
(B) Intravenous injection cf E. coli expressing red
fluorescent protein and L. monocytogenes expressing green
fluorescent protein into nude mice and into rodents with
implanted and spontaneous tumors
E. coli (DH5a) carrying the DsRed (Matz et al., Nat.Biotech. 17 (1999) , 969) expression cassette under the control of a

83
constitutive promoter are used in this experiment. L. monocytogenes EGD strain derivatives with in-frame deletion in each of the virulence genes were individually labeled with the green fluorescent protein cassette driven by the constitutive SOD promoter.
The localization and intratumoral distribution of bacteria
will first be studied in nude mice with implanted C6 glioma or
HCT116 colon carcinoma tumors. C6 glioma or HCT116 colon
{carcinoma cells (5 x 10s in 100 ul) will be subcutaneously
injected into the right hind leg of the animals. Twelve days
after tumor cell injection- the animals will be anesthetized,
and the left femoral vein surgically exposed. Light-emitting
bacteria (1 x 3 06 cells re-suspended in 50 pL-of saline) will
be intravenously injected, and the wound incision will be
closed with sutures. Tumors will be measured three times a

I week using a caliper. Tumor volume will be calculated as
Follows: small diameter x large diameter x height / 2.
The localization of bacteria in the tumor, based on GFP or RFP will also be analyzed using cryosectioned tumor tissues. A reliable morphological and histological preservation, and reproducible GFP or RFP detection may be obtained using frozen sections after a slow tissue freezing protocol (Shariatmadari et al., Biotechniques 30 (2001), 1282). Briefly, tumor tissues will be removed from the sacrificed animals to a Petri dish containing PBS and dissected into the desired size. The samples will be mixed for 2 h in 4% paraformaldehyde (PFA) in PBS at room temperature. They will be washed once with PBS, and embedded in Tissue-Tek at room temperature, and then kept in the dark at 4°C for 24 h and slowly frozen at -70°C. Before sectioning, the tissue will be kept at ~20°C for 30 min. Then,
10- to 50-jim-thick sections will be cut with a Reichert-Juno
Cryocut 1800 cryostat and collected on poly-L-lysine (1%)-treated microscope slides. During sectioning, the

84
material will be kept at room temperatures to avoid several freezing and thawing cycles. Finally, the sections will be
rinsed in PBS and mounted in PBS and kept in the dark at 4°C.
To monitor the entry of light emitting E, coli and L. monocytogenes from the blood stream into the tumor, 27 nude mice will be injected with C6 tumor cells, and 27 nude mice
with HCT116 colon carcinoma cells. Twelve days after - tumor, development, 9 animals from the C6 group and 9 from the HCT116
group will receive an intravenous injection of E, coli with the RFP construct. Another 9 animals from each group will
receive an intravenous injection of L, monocytogenes transformed with the GFP^ cDNA construct. The !third group of 9 animals from each tumor model will receive both E. coli and X.
monocytogenes (1 x 10s cells of each). Five hours, 25 hours,
and 5 days after direction, three animals of each treatment
group will be sacrificed/ their tumors excised,, and processes individually as described in the above cryosectioning protocol. After freezing, each tumor will be cut into two halves. One half of the tumor will be used for preparing thick sections (60-75 tm.) r which will be analyzed under a fluorescence stereomicroscope to observe the distribution of bacteria in the sections of tumors obtained from each time point of the experiment. The regions of interest will be identified, thin sectioned, prepared, and analysed with laser scanning cytometry and under the confocal microscope followed by image reconstruction.
In parallel experiments, animals with spontaneous tumors, as listed in Table 6, will be obtained and used in intravenous injection experiments with E, coli carrying the bacterial lux operon. Two animals of each tumor model will be used, and the luciferase light emission monitored daily under the low light: imager. It is expected that the spontaneously occurring tumors can he imaged similarly to the implanted tumors based en

85
bacterial luciferase expression-. Two of the spontaneous tumor models mice with adenocarcinoma of the large in testing-arid mice with adenocarcinoma of the mammary tissue, will" "be -used for bacterial localization experiments following intravenous' injection of E. coli expressing P.FP and L. monocytogenes expressing GFP as described above. It can be expected that these experiments will emphasize the significance of the bacterium-
based-diagnosis and protein therapy system.

Table6
Spontaneous tumor animal models

Animal species
Mouse
Mouse
Mouse
Rat

Strain name
129/Sv-Madh3TMI par
FVB/N-T(fN(UPII-
SV40T)29Xrw
FVB-N neuN(N#202)
F344/CrCrlB R

Tumor
description
Spontaneous adenocarcinoma of
large
intestine
Spontaneous
I carcinoma
of bladder
with metastasis to the liver Spontaneous adenocarcinoma of mammary tissue
Spontaneous carcinoma
of pituitary

Source
Jackson Laboratories Bar Harbor, ME
Jackson Laboratories Bar Harbor, ME
Jackson Laboratories Bar Harbor,
ME
Charles River Laboratories,

Reference
Zhu et al.,
Cell 94 (1989), 703
Zhang et
al., Cancer
Res. 59
(1999),
3512

Guy et al.,
PNAS USA 99
(1992),
10579 . ,
Hosokawa et
al
Toxicol.Pat hol. 21

Wilmington, (1993), 203 MA
Example 7: Verification of bacterium-mediated tumor targeting
and bacterium-secreted protein therapy in rodents with
implanted or spontaneous tumors
(A) Rationale
As shown in the previous examples, intravenous injection of light-emitting bacteria results in entry, replication, and accumulation only in the tumor regions in animals. This process can be monitored by imaging of light emission in tumors. Placing the endostatin and chimeric toxin expressing gene cassettes in cis configuration with a light-emitting gene cassette provides an indirect detection system, in vivo for their temporal and spacial delivery via bacteria.
the endostatin and chimeric toxin gene cassettes are linked to signal peptide encoding sequences, which facilitate the secretion of these proteins into the extracellular space in the tumor or into the cytosol of infected tumor cells. Both proteins secreted from bacteria into the extracellular space of the tumor are expected to function similarly to directly injected purified proteins. Both proteins secreted from L. monocytogenes into the cytosol of the infected tumor cells will resemble the viral delivery system reported earlier for endostatin. The bacterial systems can be used as a-constitutive secretion system or as an exogenously added IPIb-activatable secretion system in the tumor. By regulating the expression levels of the therapeutic proteins in bacteria that colonize the tumor, the secreted amount of proteins inhibiting tumor growth can be determined. Without the

addition of IPTG/ the inhibitory protein secretion from the. intravenously injected bacteria will be kept at minimum while in blood circulation. This will provide added safety to the recipient tumorous animals during delivery of bacteria. Using the BSPT system, the onset and duration of the therapy can .be controlled by the addition of IPTG. Upon completion of the treatment, the bacterial delivery system can be eliminated by administration of antibiotics, similar to treating a bacterial infection.
(B)Determination of the effect of endostatin and Pseudomonas
exotoxin/TGF alpha fusion protein secreted by E. coli and L.
monocytogenes on tumor growth in animals with implanted tumors
',.- ■'■:■.■ ■ j
itie xmiujicwi-^ CL JLtsuu <_>JL ci:uysLai ui cuiu. Lilt; cyLULUAiui;Ly \JL.
M
the chimeric toxin secreted by E. coli and L; monocytogenes in
tumors will be determined as follows. Thirty-five nude mice
bearing 10-day-old C6 tumors will be injected with bacterial
constructs as follows: (a) Five mice with E. coli engineered
to secrete endostatin; {b} Five mice with E. coli engineered

to secrete chimeric toxin; (c) Five mice with L. monocytogenes
engineered to secrete endostatin; (d) Five mice with L.
monocytogenes engineered to secrete chimeric toxin; (e) Five
mice with E. coli secreting endostatin and chimeric toxin; (f)
control group: five mice injected with E. coli expressing
bacterial luciferase alone, and five mice with L.
monocytogenes expressing GFP. At the time of. bacteria
injection, each tumor volume will be determined. Three days
after injection, the replication of bacteria in the tumors
will be monitored under a low light imager or under- a
fluorescence stereomicroscope. The light emission and the
tumor volume will be measured daily up to 20 days after
bacterial injection. Ten days after injection, one animal from
each group will be sacrificed and the levels of the secreted
proteins present in the tumor tissue, will be analyzed using

88

Western blot analysis. These experiments will result in inhibition of tumor growth in endostatin treated animals or a more dramatic tumor regression in animals treated with chimeric toxin proteins. The tumor growth in control animals is not expected to be affected by the bacteria alone.
In a follow-up experiment, mice with spontaneous
adenocarcinoma of mammary tissue (strain FVB-neuN(N#2Q2),
Table 6) i will be used to study the; effect of secreted proteins
on tumor growth. An experimental scheme identical to that
described for the C6 tumor analysis will be used. At the
completion of tumor therapy, the presence of endostatin or
chimeric toxin in the tumor tissue will be determined by
Western blot analysis. An identical experimental design will
be used to1 assay the effect of IPTG-induction of endostatin
and chimeric toxin production in bacteria in C6 tumors as well
as in the spontaneously occurring breast tumor mouse model. It
is expected that multiple IPTG induction of protein expression
in bacteria might be required for successful tumor therapy.
At any stage of tumor treatment, it may be required to remove
the light emitting and therapeutic gene containing bacteria
from the animal. To carry out this experiment, mice with
12-day-old C6 tumors will be intravenously injected with /•;.
coli expressing the bacterial luciferase. Three days after
injection, antibiotic, therapy will be initiated by

intraperitoneal administration of gentamicin (5 mg/kg body
weight) twice daily, or the newly discovered clinafloxacin
(CL960) (Nichterlein et al., Zentralbl .Bakteriol. 286 (1997),
401) . This treatment will be performed for 5 days, and the
effect of antibiotics on the bacteria will be monitored by
imaging light emission from the animals daily.
By completing the above experiments, it is expected that endostatin and chimeric toxin proteins secreted into the tumors will cause the inhibition of tumor growth and

89

measurable tumor regression. It is anticipated that tumor regression will be achieved in both groups of rodents with implanted tumors and with spontaneously occurring tumors. Experiments with simultaneous application of secreted endostatin and chimeric toxin proteins in tumor treatment may give the most promising results. The removal of the engineered bacteria from the tumor by administration of antibiotics is an added safety measure of the bacterium-secreted protein therapy (BSPT) of the present invention.

90
I Claim:
A diagnostic and/or pharmaceutical composition comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal.
2. The diagnostic and/or pharmaceutical composition of claim 1, wherein said microorganism or cell furthermore contains (a) DNA sequence (s) encoding (a) protein (s) suitable for tumor therapy and/or elimination of metastatic tumors.
3. The diagnostic and/or pharmaceutical composition of claim 1 or 2, wherein said protein capable of inducing a detectable signal is a luminescent and/or fluorescent protein.
4. The diagnostic and/or pharmaceutical composition of anyone of claims 1 to 3, wherein said protein is a cell receptor capable of binding a ligand.
5. The diagnostic and/or pharmaceutical composition of claim 4, wherein said composition further comprises a ligand which is a radionuclide-labelled ligand.
6. The diagnostic and/or pharmaceutical composition of claim 4, wherein said composition further comprises a ligand which is a paramagnetic-or superparamagnetic-metal-labelled ligand.
7. The diagnostic and/or pharmaceutical composition of claim 4, wherein said composition further comprises a ligand which is coupled to a toxin.
8. The diagnostic and/or pharmaceutical composition of claim 4, wherein said composition further comprises a ligand which is coupled to a therapeutic protein.
9. The diagnostic and/or pharmaceutical composition of any one of claims 4 to 8, wherein said ligand is an antibody.

91
10. The diagnostic and/or pharmaceutical composition of
Claiml, wherein said protein capable of inducing a detectable
signal is a protein inducing a signal detectable by magnetic resonance imaging (MRI) or capable of binding a contrasting agent, chromophore or a ligand required for visualization of tissues.
11. The diagnostic and/or pharmaceutical composition of any
one of claims 1 to 10, wherein said migroorganism is a
bacterium or a virus.
12. The diagnostic and/or pharmaceutical composition of claim 11, wherein said bacterium is a magnetic bacterium.
13. The diagnostic and/or pharmaceutical composition of claim 11, wherein said virus is Vaccinia virus.
14. The diagnostic and/or pharmaceutical composition of claim 11, wherein said bacterium is attenuated Vibrio cholera.
15. The diagnostic and/or pharmaceutical composition of any one of claims 1 to 10, wherein said cell is a mammalian cell.
16. The diagnostic and/or pharmaceutical composition of claim 3, wherein said luminescent or fluorescent protein is luciferase, RFP or GFP.
17. The diagnostic and/or pharmaceutical composition of claim 16, wherein said microorganism or cell additionally contains a gene encoding a substrate for a luciferase.
18. the diagnostic and/or pharmaceutical composition of any one of claims 1 to 17 further comprising (a) an antimicrobial compound fused to a protein suitable for tumor therapy and/or elimination of metastatic tumors and/or (b) an antimicrobial compound fused to a detectable protein or a protein capable of inducing a detectable signal.

92
19. The diagnostic and pharmaceutical composition of any one of claims 1,2, 3 and 10 to 17, wherein said protein suitable
for tumor therapy and/or elimination of metastatic tumors is endostatin or recombinant chimeric toxin PE37/transforming growth factor alpha (TGF-alpha).
20. The diagnostic and pharmaceutical composition of any one of claims 1,2 and 10 to 17, wherein said protein suitable for tumor therapy and/or elimination of metastatic tumors is a protein capable of converting a nontoxic prodrug into a toxic drug which is cytotoxic to a tumor.
21. The diagnostic and pharmaceutical composition according to any one of claims 1 to 17, wherein said DNA sequence (s) is (are) on a BAC, MAC, cyber cell or cyber virus.
22. the diagnostic and pharmaceutical composition substantially as herein described with reference to the foregoing examples and accompanying drawings.

Dated this 30th day of October 2006


RAJESHWARI .H
OF K & S PARTNERS
AGENT FOR THE APPLICANTS

93
ABSTRACT
Summary
Microorganisms and cells for diagnosis and therapy of tumors
Described are diagnostic and pharmaceutical, compositions comprising a microorganism or cell containing a DNA sequence encoding a detectable protein or a protein capable of inducing a detectable signal, e.g. a luminescent or fluorescent protein, and, in a particular embodiment, furthermore (a) DNA sequence(s) encoding (a) protein(s) suitable for tumor therapy and/or elimination of metastatic tumors, e.g. a cytotoxic or cytostatic protein.




To,
The Controller of Patents,
The Patent Office,
Mumbai.

Documents:

1282-MUMNP-2006-ABSTRACT 27-11-2007.pdf

1282-mumnp-2006-abstract.doc

1282-mumnp-2006-abstract.pdf

1282-MUMNP-2006-CLAIMS 27-11-2007.pdf

1282-mumnp-2006-claims.doc

1282-mumnp-2006-claims.pdf

1282-MUMNP-2006-CORRESPONDENCE 3-7-2008.pdf

1282-MUMNP-2006-CORRESPONDENCE(20-10-2008).pdf

1282-mumnp-2006-correspondence-received.pdf

1282-mumnp-2006-description (complete).pdf

1282-MUMNP-2006-DESCRIPTION(COMPLETE) 27-11-2007.pdf

1282-MUMNP-2006-DRAWING 27-11-2007.pdf

1282-mumnp-2006-drawing.pdf

1282-MUMNP-2006-FORM 1 27-11-2007.pdf

1282-mumnp-2006-form 2 27-11-2007.pdf

1282-MUMNP-2006-FORM 2(TITLE PAGE) 27-11-2007.pdf

1282-MUMNP-2006-FORM 3 3-7-2008.pdf

1282-mumnp-2006-form-1.pdf

1282-mumnp-2006-form-2.doc

1282-mumnp-2006-form-2.pdf

1282-mumnp-2006-form-26.pdf

1282-mumnp-2006-form-3.pdf

1282-mumnp-2006-form-5.pdf

1282-MUMNP-2006-PETITION UNDER RULE 137(20-10-2008).pdf

abstract1.jpg


« Previous Patent
Next Patent »
Patent Number 235608
Indian Patent Application Number 1282/MUMNP/2006
PG Journal Number 30/2009
Publication Date 24-Jul-2009
Grant Date 08-Jul-2009
Date of Filing 01-Nov-2006
Name of Patentee GENELUX CORPORATION
Applicant Address DELAWARE AT 3030 BUNKER HILL STREET SUITE 310 SAN DIEGO CALIFORNIA 92109,
Inventors:
# Inventor's Name Inventor's Address
1 ALADAR A. SZALAY HESSENSTRASSE 64 97078 WURZBURG GERMANY
PCT International Classification Number A61K35/66
PCT International Application Number PCT/IB2002/04767
PCT International Filing date 2002-07-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 01118417.3 2001-07-31 EUROPEAN UNION