Note: Descriptions are shown in the official language in which they were submitted.
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NON-INVASIVE TUMOR IMAGING BY TUMOR-TARGETED BACTERIA
This application claims priority to U.S. provisional patent application No.
60/157,620, filed on October 4, 1999, the content of which is incorporated
herein by
reference its entirety.
1. Field of the Invention
The present invention is concerned with non-invasive imaging methods to
detect solid tumors in vivo. The invention is also concerned with non-invasive
methods to
monitor and/or treat solid tumors.
2. Background of the Invention
2.1. Cancer Chemotherapy
A major problem in the chemotherapy of solid tumor cancers is delivery of
therapeutic agents, such as drugs, in sufficient concentrations to eradicate
tumor cells while
at the same time minimizing damage to normal cells. Thus, studies in many
laboratories are
directed toward the design of biological delivery systems, such as antibodies,
cytokines, and
viruses for targeted delivery of drugs, pro-drug converting enzymes, and/or
genes into
tumor cells. See e.g., Crystal, 1995, Science 270: 404-410.
2.2. Bacterial Infections and Cancer
Early clinical observations reported cases in which certain cancers were
reported to regress in patients with bacterial infections, See Nauts et al.,
1953, Acta Medica
Scandinavica 145:1-102, (Suppl. 276); Shear, 1950; J.A.M.A. 142:383-390. Since
these
observations, Lee et al., 1992, Proc. Natl. Acad. Sci. USA 89:1847-1851 (Lee
et al.) and
Jones et al., 1992, Infect. Immun. 60:2475-2480 (Jones et al.) isolated
mutants of
Salmonella typhimurium that were able to invade HEp-2 (human epidermoid
carcinoma)
cells in vitro in significantly greater numbers than the wild type strain. The
"hyperinvasive"
mutants were isolated under conditions of aerobic grov~rth of the bacteria
that normally
repress the ability of wild type strains to invade HEp-2 animal cells.
However, such
hyperinvasive Swlmonella typhimurium as described by Lee et al. and Jones et
al. carry the
risk of pan-invasive infection and could lead to wide-spread bacterial
infection in the cancer
patient.
Carswell et al., 19 7 S, Proc. Natl. Acad. Sci. USA 72:3666-3669,
demonstrated that mice injected with bacillus Calmette-Guerin (BCG) have
increased serum
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levels of TNF and that TNF-positive serum caused necrosis of the sarcoma Meth
A and
other transplanted tumors in mice. As a result of such observations,
immunization of cancer
patients with BCG injections is currently utilized in some cancer therapy
protocols. See
Sosnowski, 1994, Compr. Ther. 20:695-701; Barth and Morton, 1995, Cancer 75
(Suppl.
2):726-734; Friberg, 1993, Med. Oncol. Tumor. Pharmacother. 10:31-36 for
reviews of
BCG therapy.
However, TNFa-mediated septic shock is among the primary concerns
associated with bacteria, and can have toxic or lethal consequences for the
host (Bone,
1992, JAMA 268: 3452-3455; Dinarello et al., 1993, JAMA 269: 1829-1835).
Further,
dose-limiting, systemic toxicity of TNFa has been the major barrier to
effective clinical use.
Modifications which reduce this form of an immune response would be useful
because
TNFa levels would not be toxic, and a more effective concentration and/or
duration of the
therapeutic vector could be used.
Further, resistance to antibiotics can complicate eliminating the presence of
bacteria within the human body (Tschape, 1996, D T W Dtsch Tierarztl
Wochenschr 1996
103:273-7; Ramos et al., 1996, Enferm Infec. Microbiol. Clin. 14: 345-51).
2.3. Tumor-targeted Bacteria
Genetically engineered Salmonella have been demonstrated to be capable of
being tumor-targeted, possess anti-tumor activity and are useful in delivering
genes such as
the herpes simplex virus thymidine kinase (HSV TK) to solid tumors (Pawelek et
al., WO
96/4023 8).
Pawelek et al. (1997, Cancer Res. 57:4537-4544) employed a gene fusion
consisting of the (3-lactamase secretion signal followed by the entire coding
sequence of
HSV 1-tk. The gene product of this construct was secreted into the periplasmic
space of the
bacterium and was shown to be functionally active. Antitumor studies with a
strain
carrying this construct demonstrated that treatment with ganciclovir resulted
in an
enhancement of antitumor activity only when the HSV1-tk plasmid was present,
thus
establishing the therapeutic potential of Salmonella expressing functionally
active HSV1-tk.
2.4. Decreased Induction of TNFa by Modified Bacterial Lipid A
Modifications to the lipid composition of tumor-targeted bacteria which alter
the immune response as a result of decreased induction of TNFa production were
suggested
by Pawelek et al. (Pawelek et al., WO 96/40238). Pawelek et al. provided
methods for
isolation of genes from Rhodobacter responsible for monophosphoryl lipid A
(MLA)
production. MLA acts as an antagonist to septic shock. Pawelek et al. also
suggested the
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use of genetic modifications in the lipid A biosynthetic pathway, including
the mutation
firA, which codes for the third enzyme UDP-3-O (R-30 hydroxylmyristoly)-
glucosamine N-
acyltransferase in lipid A biosynthesis (Kelley et al., 1993, J. Biol. Chem.
268: 19866-
19874). Pawelek et al. showed that mutations in the firA gene induce lower
levels of
TNFa. In Escherichia coli, the gene msbB (mlt) which is responsible for the
terminal
myristylation of lipid A has been identified (Engel, et al., 1992, J.
Bacteriol. 174:6394-
6403; Karow and Georgopoulos 1992, J. Bacteriol. 174: 702-710; Somerville et
al., 1996, J.
Clin. Invest. 97: 359-365). Genetic disruption of this gene results in a
stable non-
conditional mutation which lowers TNFa induction (Somerville et al., 1996, J.
Clin. Invest.
97: 359-365; Somerville, WO 97/25061).
Hone and Powell, W097/18837 ("Hone and Powell"), disclose methods to
produce gram-negative bacteria having non-pyrogenic Lipid A or LPS. Hone and
Powell
propose using non-pyrogenic bacteria only for vaccine purposes.
Maskell, W098/33923, describes a mutant strain of Salmonella having a
1 S mutation in the msbB gene which induces TNFa at a lower level as compared
to a wild type
strain.
Bermudes et al., WO 99/13053, teach compositions and methods for the
genetic disruption of the msbB gene in tumor-targeted Salmonella, which result
in
Salmonella possessing a lesser ability to elicit TNFa and reduced virulence
compared to the
wild type. In certain embodiments, some such mutant Salmonella have increased
sensitivity
to chelating agents as compared to wild type Salmonella. In other embodiments,
the mutant
tumor-targeted Salmonella deliver a gene product such as a pro-drug converting
enzyme
useful as an anti-tumor agent.
2.5. Imaging Gene Therapy
Blasberg and Tjuvajev developed a non-invasive imaging system to detect
gene transfer and expression in target tissues which is useful for monitoring
and evaluating
in vivo gene therapy (Tjuvajev et al., 1995, Cancer Res. 55:6126-6132;
Tjuvajev et al.,
1996, Cancer Res. 56:4087-4095; Tjuvajev et al., 1998, Cancer Res. 58:4333-
4341. The
system uses a transfer vector containing HSV 1-TK and a labeled marker
substrate to non-
invasively image target tissue or cells which have taken up the marker gene.
Imaging methods have been described for monitoring gene therapy in United
States Patent No. 5,703,056 entitled "Non-Invasive Imaging of Gene Transfer"
issued
December 30, 1997 to Blasberg and Tjuvajev. Although a variety of vectors are
used to
image gene therapy, these references do not discuss the use of tumor-targeted,
facultative
aerobic or facultative anaerobic bacteria or bacterial vectors, alone or in
combination with
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other agents, to detect tumors by imaging or to treat solid tumors. Further,
these references
do not discuss the use of facultative aerobic or facultative anaerobic
bacterial vectors for
delivery of any substance therapeutic to solid tumor sites.
Citation or identification of any reference in Section 2, or any section of
this
application shall not be construed as an admission that such reference is
available as prior
art to the present invention.
3. Summar~of the Invention
The present invention provides non-invasive methods to detect solid tumors
in vivo by delivery of a marker gene to a solid tumor. The vector for delivery
of the marker
gene to the solid tumor is a heterogeneous or homogenous population of tumor-
targeted
bacteria. The present invention also provides other non-invasive methods to
detect solid
tumors using tumor targeted bacterial vectors. Such methods comprise, in vivo,
detecting a
compound incorporated into the tumor-targeted bacterial vectors, detecting an
infection
caused by the bacterial vectors, or detecting an antigen present on the
surface of the
bacterial vectors after the bacterial vectors have been administered to a
subject.
When a marker gene is delivered to a solid tumor, the marker gene can be
detected directly in the tumor, by the use of a labeled moiety that interacts
with the marker
gene product, or by the use of a labeled marker substrate. More particularly,
in one
embodiment, the invention encompasses use of attenuated tumor-targeted
bacteria or
bacterial vectors, such as, e.g., Salmonella, as a vector for the delivery of
a marker gene to
an appropriate site of action, e.g., the site of a solid tumor. If the marker
gene product itself
is detectable by non-invasive methods, the marker gene product itself can be
detected
directly. Alternatively, a labeled moiety that interacts with the marker gene
product is
detected, or a labeled marker substrate is used and a labeled marker
metabolite is detected.
A tumor can thus be localized or detected by scanning a subject to detect the
marker gene
product, a labeled complex comprising the marker gene product and its
interacting moiety,
or a labeled marker metabolite, respectively, thereby imaging the tumor.
Specifically, the
attenuated tumor-targeted bacteria of the invention are facultative aerobes or
facultative
anaerobes which are modified to encode the marker gene.
In one embodiment, the marker gene encodes a fluorescent protein and the
marker gene product is detected directly. In another embodiment, the marker
gene encodes
strepatividin and is detected by administering labeled biotin, in which case
the marker gene
is indirectly detected by detection of the labeled biotin. In another
embodiment, the marker
gene product is a heterologous protein that is immunologically detectable, and
the marker
gene product is detected by administration of a labeled antibody.
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In a specific embodiment, the marker gene is HSV 1-tk or VZV-tk and the
marker substrate is a labeled 2'-fluoro-nucleoside analogue. In a preferred
embodiment, the
labeled marker substrate is a labeled 2'-fluoro-5-iodo-1-beta-D-
arabinofuranosyl-uracil
(FIAU). In other specific embodiments, the labeled marker substrate is a
radiolabeled
acyclovir (ACV) or a radiolabeled ganciclovir (GCV). As disclosed herein, the
2'-fluoro-nucleoside analogues were chosen for use in the subject invention
because they are
selectively phosphorylated by HSV1-TK or VZV-TK, can be radiolabeled with
appropriate
radionuclides for imaging e.g., with SPECT or PET and are resistant to
metabolic
degradation in vivo (see, for example, Abrams et al., 1985, Int. J. Appl.
Radiat. Isot. 36:
233-238) thereby facilitating interpretation of the resultant images and
measurements.
In other embodiments of the invention, the tumor-imaging methods of the
invention do not require the use of a marker gene. In certain specific
embodiments, tumor
imaging comprises administering a labeled compound to the subject, which
labeled
compound is preferentially incorporated into the bacteria, and detecting the
labeled
compound. In other specific embodiments, the tumor is imaged by scanning to
image an
infection caused by the bacteria at tumor site.
In addition to initial tumor detection, the present invention also provides a
non-invasive method to monitor the course of disease or effectiveness of tumor
treatment.
Repeated imaging of tumor tissue during the course of treatment allows direct
observation
of changes in size. In addition to detection and monitoring, the present
invention also
provides methods for therapy to inhibit tumor growth or reduce the tumor size.
HSV 1-TK and VZV-TK metabolize marker substrates as well as pro-drug
substrates, and
may be genetically engineered to prefer a specific substrate.
The present invention is based, in part, on the surprising discovery that the
metabolic activities of tumor-targeted bacteria are of sufficient magnitude
and duration
while in tumor cells or the tumor environment to be able to express,
incorporate, trap, bind
or otherwise hold within the tumor region, sufficient amounts of a detectably
labeled agent
to allow non-invasive detection of the tumor. For example, expression of a (3-
lactamase:HSVl-TK fusion protein with a periplasmic localization signal has
been found to
cause the trapping effect of phosphorylation of a marker substrate (e.g.,
FIAU) and permits
signal localization for monitoring. Accordingly, according to the present
invention, in
another embodiment, the marker gene product of a tumor-targeted bacterial
vector is
retained in the cytosol and periplasm of the vector, where such vector is used
for imaging or
monitoring a tumor. In another embodiment, the marker gene product of a tumor-
targeted
bacterial vector is retained in the cytosol of the vector, where such vector
is used for
imaging or monitoring a tumor.
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The present method employing genetically engineered tumor-targeted
bacteria, such as Salmonella, can be used to detect solid tumors which are at
least 2 times,
more preferably 4-5 times, and most preferably 10 times smaller than can be
detected using
current methods including but not limited to x-ray and CT scanning.
Advantageously, the
method can be used to detect and/or monitor tumor metastases as well as
primary solid
tumors. Unlike viral vectors which can only accommodate a small segment of
exogenous
DNA, bacteria, such as Salmonella, can accept multiple genes either by
insertion into the
bacterial chromosome or by transformation with multiple plasmids.
This invention provides a method of detecting a tumor by imaging in a
subject comprising: (a) administering to the subject a tumor-targeted bacteria
containing a
marker gene, wherein the tumor-targeted bacteria targets the tumor cells
and/or the tumor
environment and the marker gene is expressed in the tumor-targeted bacteria,
thereby
generating a marker gene product; and (b) scanning the subject to detect the
marker gene
product, thereby imaging the tumor in the subject. In certain embodiments of
the
invention, the marker gene is detected directly. In other embodiment, the
marker gene is
detected indirectly. In one embodiment, indirect detection further comprises
administering
to the subject a labeled marker-binding moiety after step (a), wherein the
marker gene
product of step (a) binds to the labeled marker-binding moiety; and wherein
said scanning is
after clearance of residual labeled marker-binding moiety not bound to the
marker gene
product, thereby detecting the labeled marker-binding moiety localized to the
tumor and
imaging the tumor in the subject. In another embodiment, indirect detection
further
comprises administering to the subject a labeled marker substrate after step
(a), wherein the
tumor-targeted bacteria expressing the marker gene product of step (a)
metabolizes the
labeled marker substrate to produce a labeled marker metabolite; and wherein
said scanning
is after clearance of residual marker substrate not metabolized by the marker
gene product,
thereby detecting the labeled marker metabolite localized to the tumor and
imaging the
tumor in the subj ect.
This invention provides a method of detecting a tumor by imaging in a
subject comprising: (a) administering to the subject a tumor-targeted
bacteria, wherein the
t~or-targeted bacteria targets the tumor cells and/or the tumor environment;
(b)
administering to the subject a labeled compound that is preferentially
incorporated into the
tumor-targeted bacteria; and (c) scanning the subject to detect the labeled
compound,
thereby detecting the tumor-targeted bacteria and imaging the tumor in the
subject. In a
preferred embodiment, the tumor-targeted bacteria is a mutant having an
enhanced
preference to incorporate the labeled compound. In a preferred mode of the
embodiment,
the bacteria is a mutant at the asd locus and the marker compound is
diaminopimelic acid.
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The present invention further provides methods of imaging a tumor in a
subject comprising (a) administering to the subject a tumor-targeted bacteria,
wherein the
tumor-targeted bacteria targets the tumor cells and/or the tumor environment,
and (b)
scanning to image an infection caused by the bacteria, thereby detecting and
imaging the
tumor in the subject. In a preferred embodiment, imaging the infection
comprises detecting
sequestered polymorphonuclear neutrophils at the site of infection, for
example by using a
labeled antibody that detects an antigen, such as CD15, present on the
polymorphonuclear
neutrophils, or by using a labeled chemotactic peptide analog that binds to a
receptor
present on the polymorphonuclear neutrophils.
The present invention provides methods of imaging a tumor in a subject
comprising: (a) administering to the subject a tumor-targeted bacteria,
wherein the tumor-
targeted bacteria targets the tumor cells and/or the tumor; (b) administering
a labeled
antibody to the subject, wherein the antibody binds to an antigen present on
the surface of
the tumor-targeted bacteria; and (c) scanning the subject to detect the
labeled antibody,
thereby imaging the tumor in the subject. In a preferred embodiment, the
antigen present on
the surface of the tumor-targeted bacteria is an O-antigen, an H-antigen, or
an outer
membrane protein.
Further, the present invention provides a method of monitoring a tumor
during the course of a disease and/or treatment in a subject comprising: (a)
administering to
the subject a tumor-targeted microorganism containing a marker gene, wherein
the tumor-
targeted microorganism targets the tumor cells and/or the tumor environment
and the
marker gene is expressed in the tumor-targeted microorganism, thereby
generating a marker
gene product; (b) scanning the subject to detect the marker gene product,
thereby imaging
the tumor in the subject; and (c) repeating steps (a) and (b) as needed during
the course of
the disease in the subject.
Further, the present invention provides a method of monitoring a tumor
during the course of a disease and/or treatment in a subject comprising: (a)
administering to
the subject a tumor-targeted bacteria, wherein the tumor-targeted bacteria
targets the tumor
cells and/or the tumor environment; (b) administering to the subject a labeled
compound
that is preferentially incorporated into the tumor-targeted bacteria;(c)
scanning the subject
to detect the labeled compound, thereby detecting the tumor-targeted bacteria
and imaging
the tumor in the subject; and (d) repeating steps (a) through (c) as needed
during the course
of the disease in the subject.
Further, the present invention provides a method of monitoring a tumor
during the course of a disease in a subject comprising (a) administering to
the subject a
tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor
cells and/or
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the tumor environment; (b) scanning to image an infection caused by the
bacteria, thereby
detecting and imaging the tumor in the subject; and (c) repeating steps (a)
and (b) as needed
during the course of the disease in the subject.
Yet further, the present invention provides a method of monitoring a tumor
during the course of a disease in a subject comprising (a) administering to
the subject a
tumor-targeted bacteria, wherein the tumor-targeted bacteria targets the tumor
cells and/or
the tumor; (b) administering a labeled antibody to the subject, wherein the
antibody binds to
an antigen present on the surface of the tumor-targeted bacteria; (c) scanning
the subject to
detect the labeled antibody, thereby imaging the tumor in the subject; and (d)
repeating
steps (a) through (c) as needed during the course of the disease in the
subject.
Still further, this invention provides methods of simultaneously imaging and
treating a tumor in a subj ect. In one embodiment, simultaneous imaging and
treatment
comprises administering tumor-targeted bacteria expressing a marker gene and a
suicide
gene capable of converting a prodrug into a cytotoxic drug, together with a
labeled marker
substrate. A single tumor-targeted bacterial vector may contain more than one
bacterial
expression construct, wherein at least one construct is used for imaging the
tumor, as above,
and at least one construct is used for treating the tumor. Alternatively, a
population of at
least two tumor-targeted bacterial vectors is administered, wherein one vector
is for imaging
as above, and another vector is for therapeutic treatment.
The invention provides a non-invasive, clinically applicable method for
imaging tumors which can be implemented using existing imaging techniques to
monitor
and evaluate in vivo cancer treatments in human subjects.
Yet further, the invention provides compositions for carrying out the non-
invasive imaging (and optionally treatment) methods of the invention, said
compositions
comprising a population of the tumor-targeted bacteria of the invention
suitable for non-
invasive tumor imaging. In one embodiment, a composition of the invention
comprises a
population of tumor-targeted bacteria which harbor a recombinant streptavidin
gene
operably linked to a promoter. In a preferred mode of the embodiment, the
promoter is
preferentially active at the tumor site or in the environment. In another
embodiment, the
tumor-targeted bacteria is a partial asd mutant with an enhanced preference to
incorporate
labeled DAP.
Yet further, the invention provides pharmaceutical compositions comprising
a population of the tumor-targeted bacteria of the invention suitable for non-
invasive tumor
imaging, and a pharmaceutically acceptable Garner.
Yet further, the invention provides kits comprising compositions of the
invention for carrying out the non-invasive detection methods of the
invention, as well as
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for carrying out the non-invasive detection and treatment methods of the
invention.
Specifically, the invention provides kits comprising in one or more containers
(a) a purified
population of tumor-targeted bacteria and (b) a detectably labeled molecule.
In certain
embodiments, the bacteria contain a marker gene operably linked to a promoter.
In one
embodiment, the labeled molecule is a labeled moiety which binds to the marker
gene
product. In a preferred mode of the embodiment, the labeled molecule is
labeled biotin and
the marker gene is streptavidin. In another embodiment, the labeled molecule
is a labeled
substrate of the marker gene product. In other embodiments, the labeled
molecule is a
labeled compound which is preferentially incorporated into the tumor-targeted
bacteria. In
one embodiment, the tumor-targeted bacteria is a mutant having an enhanced
preference to
incorporate the labeled compound. In one mode of the embodiment, the bacteria
is mutant
at the asd locus and the labeled compound is DAP. In yet other embodiments,
the labeled
molecule is a labeled antibody that detects an antigen present on
polymorphonuclear
neutrophils or on the surface of the tumor-targeted bacteria, or a labeled
chemotactic
peptide analog that binds to a receptor present on polymorphonuclear
neutrophils.
Facultative aerobic and facultative anaerobic bacteria useful for the methods
and compositions of the present invention include but are not limited to
Escherichia coli
(including but not limited to pathogenic (e.g., entero-invasive or
uropathogenic)
Escherichia coli), Salmonella spp., Shigella spp., Streptococcus spp.,
Yersinia
enterocolitica, Listeria monocytogenies, and Mycoplasma hominis. In a
preferred
embodiment, the bacterial vector is an attenuated strain of Salmonella
genetically
engineered to express an altered Lipid A which reduces the virulence of the
strain, and
genetically engineered to express a gene product which aids in preventing the
growth of a
solid tumor, which gene product is under the control of an irradiation-
inducible promoter.
Illustrative examples of solid tumors include, but are not limited to,
sarcomas, carcinomas, lymphomas and other solid tumor cancers, such as renal
carcinoma,
mesoendothelioma, bladder cancer, germ line tumors and tumors of the central
nervous
system, breast cancer, prostate cancer, cervical cancer, uterine cancer, lung
cancer, ovarian
cancer, testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic
cancer, stomach
cancer, liver cancer, colon cancer, and melanoma.
3.1. Definitions
As used herein, Salmonella spp. encompasses all Salmonella species,
including: Salmonella typhi, Salmonella choleraesuis, and Salmonella
enteritidis.
Serotypes of Salmonella are also encompassed herein, for example, typhimurium,
a
subgroup of Salmonella enteritidis, commonly referred to as Salmonella
typhimurium.
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Attenuation: Attenuation is a modification so that a bacterium or bacterial
vector is less pathogenic. The end result of attenuation is that the risk of
toxicity as well as
other side-effects is decreased, when the bacterium or bacterial vector is
administered to the
patient.
Virulence: Virulence is a relative term describing the general ability to
cause
disease, including the ability to kill normal cells or the ability to elicit
septic shock (see
specific definition below).
Septic shock: Septic shock is a state of internal organ failure due to a
complex cytokine cascade,, initiated by TNFa. The relative ability of a
bacterium or
bacterial vector to elicit TNFa is used as one measure to indicate its
relative ability to
induce septic shock.
Gene product: Gene product refers to any molecule capable of being
encoded by a nucleic acid, including but not limited to, a protein or another
nucleic acid,
e.g., DNA, RNA dsRNAi, ribozyme, DNAzyme, etc. The nucleic acid which encodes
for
the gene product of interest is not limited to a naturally occurring full-
length "gene" having
non-coding regulatory elements.
Treatment: In addition to its ordinary meaning, the term treatment
encompasses inhibition of progression of symptoms or amelioration of symptoms
of a
disease or disorder such as a solid tumor cancer.
Tumor-targeted: Tumor-targeted is defined as the ability to distinguish
between a cancerous target cell or tissue and the non-cancerous counterpart
cell or tissue so
that tumor-targeted bacteria, such as Salmonella preferentially attach to,
infect and/or
remain viable in the cancerous target cell or the tumor environment.
Chelating agent sensitivity: Chelating agent sensitivity is defined as the
effective concentration at which bacteria proliferation is affected, or the
concentration at
which the viability of bacteria, as determined by recoverable colony forming
units (c.f.u.), is
reduced.
Omp-like protein: As used herein, an Omp-like protein includes any
bacterial outer membrane protein, or portion thereof (e.g., signal sequence,
leader sequence,
periplasmic region, transmembrane domain, multiple transmembrane domains, or
combinations thereof). In specific embodiments, the Omp-like protein is at
least a portion
of OmpA, OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, a porin-like protein, PhoA, PhoE,
lama, (3-lactamase, an enterotoxin, protein A, endoglucanase, peptidoglycan-
associated
lipoprotein (PAL), FepA, FhuA, NmpA, NmpB, NmpC, or a major outer membrane
lipoprotein (such as LPP), etc.
Release factor: As used herein, a release factor includes any protein, or
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functional portion thereof which enhances release of bacterial components. In
one
embodiment a release factor is a bacteriocin release protein. Release factors
include, but are
not limited to, the bacteriocin release protein (BRP) encoded by the cloacin D
13 plasmid,
the BRPs encoded by the colicin E1-E9 plasmids, or BRPs encoded by the colicin
A, N or
D plasmids.
Direct detection (of a marker gene product): As used herein, direct detection
of a marker gene product indicates that the signal detected is a function of
the gene product
itself.
Indirect detection (of a marker gene product): As used herein, indirect
detection of a marker gene products entails that detection of a label moiety
that co-localizes
with the marker gene product, for example by binding to the marker gene
product or as a
result of being a metabolite of a reaction catalyzed by the marker gene
product.
Composition of the invention: As used herein, a composition of the invention
minimally comprises a population of tumor-targeted bacteria suitable for use
in the non-
invasive tumor imaging or tumor imaging and treatment methods described
herein. A
composition of the invention optionally further comprises a population of
tumor-targeted
bacteria suitable for tumor therapy.
Pharmaceutical of the invention or pharmaceutical composition of the
invention: As used herein, a pharmaceutical of the invention or a
pharmaceutical
composition of the invention refers to a composition of the invention that
comprises a
pharmaceutically acceptable carrier.
4. Brief Description of the Figures
The present invention may be understood more fully by reference to the
following detailed description, illustrative examples of specific embodiments
and the
appended figures.
FIG. 1 demonstrates the ability of the imaging system to detect ['4C]-FIAU
in tumor-bearing mice pretreated with Salmonella expressing HSV-TK . A tumor-
implanted mouse was treated with VNP20009 expressing the plasmid p5-3 from
Pawelek et
al. (1997, Cancer Res., 57:4537-4599) four days prior to i.v. injection of
labeled marker
substrate (['4C]-FIAU). After 23 hours, tumor tissue samples were removed from
sacrificed
animals and subjected to QAR. Histology of the tumor and surrounding tissue is
shown on
the left, the digital autoradiogram on the right and the merged image in the
center. ['4C]-
FIAU can be clearly detected within the tumor, with little or no accumulation
in the
surrounding tissue. See text in Section 6 for details.
FIG. 2 demonstrates the tumor-targeting selectivity of VNP20009 expressing
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the plasmid p5-3. Liver tissue was collected from mice treated as described in
the text in
Section 6 and subjected to QAR. Histology of the tissue is shown on the left
and the digital
autoradiogram on the right. Little or no labeled marker substrate can be
detected.
FIG. 3A shows fluorescence of an untreated tumor and figure 3B shows
fluorescence observed in tumors following treatment with GFP-containing
Salmonella
strain VNP20009.
FIG. 4 shows the effect of administering non-Salmonella tumor-targeted
bacteria on tumor growth, as a measure of tumor volume versus time following
administration of the bacteria.
5. Detailed Description of the Invention
The present invention provides novel compositions and non-invasive
methods of detecting tumors which exploit the tumor-specificity of tumor-
targeting
bacteria, as described below. Because the tumor targeting bacteria do not
discriminate
between different tumor types, the described compositions and methods can be
used to
simultaneously image multiple tumor types, in contrast to the tumor imaging
reagents
currently in use, which are largely specific to a single tumor type or tumor
subtype.
The present invention provides a method of detecting a tumor by imaging in
a subject comprising: (a) administering to the subject a tumor-targeted
bacteria containing a
marker gene, wherein the tumor-targeted bacteria targets the tumor cells
and/or the tumor
environment and the marker gene is expressed in the tumor-targeted bacteria,
thereby
generating a marker gene product; and (b) scanning the subject to detect the
marker gene
product, thereby imaging the tumor in the subject. In certain embodiments of
the
invention, the marker gene is detected directly. In other embodiment, the
marker gene is
detected indirectly. In one embodiment, indirect detection further comprises
administering
to the subject a labeled marker-binding moiety after step (a), wherein the
marker gene
product of step (a) binds to the labeled marker-binding moiety; and wherein
said scanning is
after clearance of residual labeled marker-binding moiety not bound to the
marker gene
product, thereby detecting the labeled marker-binding moiety localized to the
tumor and
imaging the tumor in the subject. In another embodiment, indirect detection
further
comprises administering to the subject a labeled marker substrate after step
(a), wherein the
tumor-targeted bacteria expressing the marker gene product of step (a)
metabolizes the
labeled marker substrate to produce a labeled marker metabolite; and wherein
said scanning
is after clearance of residual marker substrate not metabolized by the marker
gene product,
thereby detecting the labeled marker metabolite localized to the tumor and
imaging the
tumor in the subj ect.
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Further, the present invention provides a method of monitoring a tumor
during the course of a disease and/or treatment in a subject comprising (a)
administering to
the subject a tumor-targeted microorganism containing a marker gene, wherein
the tumor-
targeted microorganism targets the tumor cells and/or the tumor environment
and the
marker gene is expressed in the tumor-targeted microorganism, thereby
generating a marker
gene product; (b) scanning the subject to detect the marker gene product,
thereby imaging
the tumor in the subject; and (c) repeating steps (a) and (b) as needed during
the course of
the disease in the subject. The invention provides a non-invasive, clinically
applicable
method for imaging tumors which can be implemented using existing imaging
techniques to
monitor and evaluate in vivo cancer treatments in human subjects.
The imaging and monitoring methods of the present invention can use
tumor-targeted bacterial vectors as previously described in WO 96/40238 and WO
99/13053, which are incorporated-by-reference herein in their entirety, for
imaging solid
tumor cancers, such as sarcomas, carcinomas, lymphomas or other solid tumor
cancers, for
example, renal carcinoma, mesoendothelioma, bladder cancer, germ line tumors
and tumors
of the central nervous system, including, but not limited to, breast cancer,
prostate cancer,
cervical cancer, uterine cancer, lung cancer, ovarian cancer, testicular
cancer, thyroid
cancer, astrocytoma, glioma, pancreatic cancer, stomach cancer, liver cancer,
colon cancer,
melanoma. The novel imaging, monitoring, detecting and treatment methods of
the present
invention can be used with existing nuclear medicine instrumentation as
previously
described in United States Patent No. 5,703,056 entitled "Non-Invasive Imaging
of Gene
Transfer" issued December 30, 1997, which is incorporated-by-reference herein
in its
entirety.
In various embodiments, the present invention provides a means of imaging
or monitoring tumors by delivering high levels of the products of marker
gene(s), either
alone or together with therapeutic gene(s), using various tumor-targeted
strains of bacteria
(preferably modified and/or attenuated) which selectively accumulate at or
within tumors
while expressing the marker genes) and/or therapeutic genes.
When administered to a subject, e.g., an animal for veterinary use or to a
human for clinical use, the vectors can be used alone or may be combined with
any
physiological Garner such as water, an aqueous solution, normal saline, or
other
physiologically acceptable excipient. In general, the dosage would range from
about 1 to
1x109 c.f.u./kg, preferably about 1 to 1x102 c.f.u/kg.
The vectors of the present invention can be administered by a number of
routes, including but not limited to: orally, topically, injection including,
but limited to
intravenously, intraperitoneally, subcutaneously, intramuscularly,
intrathecally,
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intratumorally, i. e., direct inj ection into the tumor, etc.
In certain embodiments, the imaging methods of the invention are used to
monitor the efficiency of vector targeting prior to administration of a
suicide substrate. In
these embodiments, visualization of a bacterial vector at a tumor site serves
the purpose of
identifying the tumor site and provides an indication of whether sufficient
vector targeting
to a tumor has indeed taken place. In a preferred embodiment, a single tumor-
targeted
bacterial vector contains both a marker gene and a suicide gene. In another
preferred
embodiment where imaging is achieved by administering a labeled compound that
is
incorporated into the bacteria, the bacterial vector harbors a mutation that
increases
incorporation of the labeled compound into the bacteria and contains a suicide
gene. In
another preferred embodiment, two tumor-targeted bacterial vectors are
administered, each
vector containing either a marker gene or a suicide gene. After administration
of one or
more vectors, administration of a labeled marker substrate, compound or
binding moiety for
imaging is performed. This permits assessment of the specificity of the vector
for the tumor
by the practitioner. Further, the amount of vector targeted to the tumor can
be determined
by correlating the intensity of the image with the amount administered. If it
is determined
that sufficient amount of vector has been delivered with sufficient
specificity, suicide
substrate can then be administered to treat the tumor. For further details
relating to this
embodiment, see Section 5.4., infra.
In other embodiments, the invention provides methods for imaging or
monitoring a tumor while simultaneously treating the tumor using one or more
of the
tumor-targeting bacterial vectors described and referenced herein. For
example, a subject
may be diagnosed with a solid tumor cancer by any method known in the art. The
vector,
i.e., the tumor-targeted bacteria, used in simultaneous imaging or monitoring
and treatment
may be isolated using the methods of the present invention with target cell
lines or using
model tumors in mice. In another embodiment, a biopsy of tumor cells is used
in a
selection assay for isolating a vector which is super-infective and tumor-
specific for the
tumor of the subject. In a preferred embodiment, the vector used is
genetically modified to
express, for example, a suicide gene or to delete virulence factors, or both,
as described
herein. In addition, the isolated vector may be analyzed for sensitivity to
antibiotics to
insure the eradication of the vector from the patient's body after successful
treatment or if
the patient experiences complications due to the administration of the vector.
In a highly preferred embodiment, the methods of in vivo imaging and
treatment are combined, wherein the imaging and treatment vectors are separate
tumor-
targeted bacterial vectors. In one embodiment, each vector is capable of
expressing a
mutant gene selected for substrate specificity, wherein the imaging mutant
gene is retained
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in the cytosol of a first tumor-targeted bacterial vector and the treatment
mutant gene is in
the periplasm of a second tumor-targeted bacterial vector. Iri another
embodiment, a single
vector contains both a suicide gene and an imaging gene, where the suicide
gene and
imaging gene are carned on different plasmids or are incorporated into
bacterial DNA. In
another embodiment, different mutants of HSV1-TK or VZV-TK serve as both the
suicide
gene and the imaging gene in the same tumor-targeted bacterial vector.
In an embodiment where the suicide gene and the imaging gene are carried
in two different vectors administered simultaneously to a subject, preferably
the two
different vectors are produced from the same bacterial strain and exhibit the
same tumor-
specificity. The two different vectors may also be administered sequentially
within a short
time of one another, and in any order.
It will be understood by one of ordinary skill in the art that the imaging
instrumentation, such as 'y camera or single-photon emission computed
tomography
(SPELT), PET, MRI, etc., and the corresponding relevant labeled marker
substrates as
described in United States Patent No. 5,703,056 entitled "Non-Invasive Imaging
of Gene
Transfer" issued December 30, 1997 to Blasberg and Tjuvajev, which patent is
incorporated-by-reference herein in its entirety, can be used in the novel
imaging,
monitoring, detecting and treatment methods of the present invention.
Subjects are scanned using such technologies by methods well known to
those skilled in the art. Standard nuclear medicine imaging equipment is
employed.
5.1. Vectors for Imaging or Monitoring a Tumor
In a preferred embodiment of the invention, tumor-targeted bacteria (see
Section 5.5 infra) are engineered to become bacterial vectors of the invention
to express one
or more marker genes (optionally, including one or more therapeutic genes)
suitable for
imaging or monitoring (or optionally, including treating) a tumor.
In another preferred embodiment of the invention, tumor-targeted bacteria
are administered in conjunction with a labeled compound that is incorporated
into the
bacteria for tumor imaging, monitoring, and, optionally, treatment.
It will be understood by one skilled in the art that imaging a tumor using the
methods of the invention is equivalent to tumor detection.
In an exemplary embodiment, a marker gene is a gene coding for an enzyme
that may be expressed in the tumor-targeted bacterial vector and that
catalyzes a reaction
with a labeled marker substrate, resulting in the accumulation of a detectable
marker within
the vector. The herpes simplex virus thymidine kinase gene (HSV1-tk) and the
varicella
zoster virus thymidine kinase gene (VZV-tk) are preferred "marker genes" and a
labeled 2-
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'fluoro-nucleoside analogue is their preferred marker substrate. More
specifically,
2'-fluoro-5-iodo-1-(3-D-arabinofuranosyl- uracil (FIALn is a preferred "marker
substrate" for
the enzyme HSV1 thymidine kinase (HSV1-TK), the gene product of HSV1-tk and
for the
enzyme the varicella zoster virus thymidine kinase (VZV-TK), the gene product
of VSV-tk.
In another embodiment, a preferred marker substrate is
2'-fluoro-S-iodo-1-(3-D-ribofuranosyl- uracil. For additional labeled marker
substrates
useful when the marker gene is tk, see infra at Section 5.2. Other self
complementary pairs
of marker genes and labeled marker substrates are also discussed infra at
Section 5.2. In a
preferred mode of the exemplary embodiment, the HSV1-TK- or VZV-TK-expressing
vector is administered to the subject concurrently with a pro-drug (e.g.
ganciclovir or
acyclovir). The pro-drug is phosphorylated in the periplasm of the
microorganism which is
freely permeable to nucleotide triphosphates. The phosphorylated ganciclovir
or acycolvir,
toxic false DNA precursors, readily pass out of the periplasm of the
microorganism and into
the cytoplasm and nucleus of the host cell where they incorporate into host
cell DNA,
thereby causing the death of the host cell.
In a highly preferred embodiment, the marker gene product of a tumor-
targeted bacterial vector is retained in the cytosol of the vector, where such
vector is used
for imaging or monitoring a tumor. Marker gene product retention in the
cytosol of a
tumor-targeted bacterial vector aids in the imaging or monitoring of
particularly small,
difficult-to-detect tumors (i.e. less than one gram) by maximizing marker gene
product
accumulation at the tumor site. Accordingly, tumor-targeted bacterial vector
expression
constructs are preferably designed such that a marker gene product (e.g., a
thymidine kinase
enzyme, a glucokinase enzyme, or a cytochrome P-450 enzyme) is not secreted
from the
bacterial vector. In another embodiment, the marker gene product of a tumor-
targeted
bacterial vector is retained in the cytosol and periplasm of the vector, where
such vector is
used for imaging or monitoring a tumor. In another embodiment, the marker gene
product
of a tumor-targeted bacterial vector is retained in the periplasm of the
vector, where such
vector is used for imaging or monitoring a tumor.
In another embodiment of the invention, a vector containing a marker gene
for imaging a tumor is under the specific regulatory control of certain types
of promoters.
These promoters may be either constitutive, in which the genes are continually
expressed,
inducible, in which the genes are expressed only upon the presence of an
inducer
molecules) or cell-type or tumor-environment specific control, in which genes
are
expressed only or preferentially in certain cell types or only in the tumor-
environment,
respectively. In one embodiment, for example, the expression of the gene is
controlled by a
bacterial promoter which may be activated by secretions or other molecules
specific to a
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given tumor. In a preferred embodiment, the bacterial promoter is activated
only by
secretions or other molecules specific to a given tumor.
5.2. Methods for Detecting or Monitoring b~ging
The detecting and monitoring compositions and/or methods of the present
invention allow for the localization and identification of tumors for which
whole body
detection methods are unavailable or impractical. The detecting and monitoring
compositions and/or methods of the present invention further allow for the
localization and
identification of tumors smaller than those diagnosable by methods available
prior to the
invention. Generally, prior methods are limited to detection of a tumor having
at least one
gram in mass. Accordingly, in one embodiment, a tumor having less than one
gram mass is
detected or monitored. In another embodiment, a tumor from 0.99 grams to 0.01
grams
mass is detected or monitored. In yet another embodiment, a tumor from 0.9
grams to 0.1
grams mass is detected or monitored. In yet still another embodiment, a tumor
from 0.8
grams to 0.2 grams mass is detected or monitored. In a preferred embodiment, a
tumor of
from about 0.05 to about 0.1 grams mass is detected or monitored (plus or
minus twenty
percent).
In another embodiment, the imaging methods of the present invention are
used to monitor the progression of a tumor over time. In this embodiment, one
or more
tumors are visualized according to the methods of the invention over time,
thereby allowing
detection of any changes in size, shape and/or location of tumors being
monitored. In
another embodiment, the efficacy of an anti-cancer treatment, such as
chemotherapy, is
monitored by visualizing the tumor before, during and/or after treatment.
Suppression of
tumor growth or shrinkage or disappearance of tumors during monitoring is
indicative of a
successful course of treatment.
In yet another embodiment, the imaging methods of the invention are used to
monitor the efficiency of vector targeting. In this embodiment, visualization
serves the
purpose of identifying the location of the vector to determine whether
sufficient vector
targeting to a tumor has taken place. In a particular embodiment, a single
tumor-targeted
bacterial vector contains both a marker gene and a therapeutic gene. In
another particular
embodiment, separate vectors each containing either a marker gene or a
therapeutic gene are
used. In one embodiment, after administration of the vector(s), administration
of marker
substrate and imaging is performed to assess the specificity of the vector for
the tumor and
the amount of vector targeted to the tumor. If it is determined that
sufficient amount of
vector has been delivered with sufficient specificity, suicide substrate can
be administered
to treat the tumor. In a preferred embodiment, the marker gene and the suicide
gene are the
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same gene, e.g., HSV1-TK or VZV-TK. In another preferred embodiment, the
suicide gene
and the marker gene are mutants of the same gene selected for marker substrate
specificity
and suicide substrate specificity, respectively. In a preferred embodiment,
when two
vectors are used, the different vectors, each containing either a suicide gene
or a marker
gene, are produced from the same strain of bacteria so that they have similar
or identical
targeting characteristics.
In another embodiment, a tumor-targeted bacterial vector used for in vivo
imaging according to the methods of the invention may be attenuated such that,
when
administered to a subject, the vector is less toxic to the subject and easier
to eradicate from
the subject's system. In a specific embodiment, such a vector is super-
infective and specific
for a target tumor. In a more preferred embodiment, such a vector is also
sensitive to a
broad range of antibiotics.
In another embodiment, a tumor-targeted bacterial vector carrying a gene for
imaging also carnes a gene, such as a gene encoding a pro-drug converting
enzyme, which
is expressed and secreted by the vector in or near the target tumor. Such a
gene can be
under the control of either constitutive, inducible or tumor cell-type or
tumor-environment
specific promoters. In a preferred embodiment, such a gene is expressed and
secreted only
when a vector has invaded the cytoplasm of a target tumor cell, thereby
limiting the effects
due to expression of the gene to the target site of the tumor.
5.2.1 Direct Detection of Marker Gene Products
In certain embodiments of the present invention, the marker gene expressed
by the bacterial vector encodes a directly detectable marker gene product.
Such a marker
gene can be a gene encoding a fluorescent protein, a bioluminescent protein,
or a
chemiluminescent protein.
In a preferred embodiment, the marker gene encodes a fluorescent molecule.
In a preferred mode of the embodiment, the fluorescent molecule is firefly
luciferase. In
another preferred mode of the embodiment, the protein encoded by the marker
gene is GFP
from Aequorea victoria or a mutant thereof.
The GFP expressed in a tumor-targeted bacteria for tumor imaging according
to the methods of the invention can be encoded by its naturally occurring
coding sequence
or by a coding sequence that has been modified for optimal bacterial codon
usage.
Mutations can be introduced into the coding sequence to produce GFP mutants
with altered
fluorescence wavelength or intensity or both. Such mutations are largely in
the vicinity of
residues 65-67, which form the chromophore of the protein. Examples of useful
GFP
mutations for use as marker genes according to the methods of the present
invention can be
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found in U.S. Patent Nos. 5,777,079 and 5,804,387 and International
Publication
W097/11094. In another preferred mode of the embodiment, the GFP mutant is a
blue
GFP. Examples of blue GFPs are described by Heim and Tsien (1996, Curr. Biol.
6:178-82). In yet another preferred mode of the embodiment, the fluorescent
protein is a
yellow or red-orange emitter recently discovered in reef corals (Matz et al.,
1999, Nature
Biotechnol. 17:969-973). Whole-body imaging of GFP-expressing tumors and
metastases
has been reported by Yang et al. (2000, Proc. Nat'1 Acad. Sci. U S A 97:1206-
11).
5.2.2 Detection of Marker Gene Products
via a Labeled Marker-binding Moiety
In certain embodiments of the present invention, a marker gene product can
be detected indirectly. For example, a marker gene product can be detected by
administration of a labeled moiety that binds to the marker gene product. As
used herein,
binding of a labeled moiety to a marker gene product encompasses protein-
protein
interactions, protein-compound interactions, protein-peptide interactions,
enzyme-substrate
interactions, and chelating activity. The labeled moiety can be a
proteinaceous or non-
proteinaceous molecule, e.g:, a carbohydrate. A proteinaceous marker-binding
moiety can
be a ligand, an antibody, or any other peptide or polypeptide that interacts
specifically with,
and has a high affinity to, the marker gene product.
Accordingly, as described above, the invention provides indirect methods of
imaging a tumor in a subject comprising administering to the subject a tumor-
targeted
bacteria containing a marker gene, wherein the tumor-targeted bacteria targets
the tumor
cells and/or the tumor environment and the marker gene is expressed in the
tumor-targeted
bacteria, thereby generating a marker gene product; administering to the
subject a labeled
maker-binding moiety, wherein the marker gene product binds to the labeled
marker-
binding moiety; and scanning the subject to detect the marker gene product,
wherein said
scanning is after clearance of residual labeled marker-binding moiety not
bound to the
marker gene product, thereby detecting the labeled marker-binding moiety
localized to the
tumor and imaging the tumor in the subj ect.
In certain embodiments of the present invention, the marker gene expressed
by the bacterial vector is avidin or streptavidin. These proteins bind biotin
with high
affinity (Kendrew , ed, The Encyclopedia of Molecular Biology, 1994, Blackwell
Science
Ltd., pps. 80 and 1037). Streptavidin, a protein from Streptomyces avidinii,
can chelate
biotin as well as other compounds (e.g., boron, see Sano, 1999, Bioconjug
Chem. 10:905-
911). The accumulation of chelated detectable agents, such as labeled biotin,
can be
detected by various methods and thus be used to facilitate the localization
and monitoring of
a tumor.
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In other embodiments of the present invention, the marker gene expressed by
the bacterial vector encodes an enzyme (e.g., bacterial lacZ protein or
chloramphenicol
acetyl transferase (CAT)). An enzyme encoded by a marker can be detected by
the
administration of a labeled substrate analog.
In yet other embodiments of the present invention, the marker gene
expressed by the bacterial vector encodes a receptor, or a protein comprising
the ligand
binding domain of a receptor, for example a fusion protein comprising the
ligand-binding
domain of the receptor and a protein which targets the ligand-binding domain
to the outer
bacterial membrane. In one embodiment, the receptor is a nicotinic
acetylcholine receptor,
which can be imaged by PET and SPECT using radiolabeled nicotine analogs. For
example, halogenated 3-pyridyl ether compounds can be used for imaging the
alpha 4 beta 2
nicotinic acetylcholine receptor (Gundisch, 2000, Curr Pharm Des 6(11):1143-
57). In other
embodiments, the receptor is a dopamine or serotonin receptor.
In yet other embodiments of the present invention, the marker gene
expressed by the bacterial vector is, or a protein comprising an
immunologically detectable
epitope or other binding moiety (e.g., myc, glutathione-S-transferase (GST),
or
hexahistidine). To facilitate easier detection of the marker gene product with
reduced
background signal levels, the marker gene encodes a protein that is either
expressed at very
low levels in the subject or, most preferably, an exogenous protein.
5.2.3 Detection of Marker Gene Products via
Production of a Labeled Marker Metabolite
When the marker gene product possesses catalytic activity, the gene product
can be detected indirectly by virtue of its activity, for example by
production of a detectable
metabolite following a chemical modification of a substrate molecule.
Accordingly,
preferred marker genes to practice the present embodiments are enzymes.
Accordingly, as described above, the invention provides an indirect method
of tumor imaging comprising administering to the subject a tumor-targeted
bacterial vector
containing a marker gene, wherein the tumor-targeted bacterial vector targets
to the tumor
~~°r infects cells of the tumor, under conditions in which the marker
gene is expressed in
the tumor-targeted bacterial vector, thereby generating a marker gene product;
administering to the subject a labeled marker substrate under conditions in
which the
labeled marker substrate is metabolized by the marker gene product to produce
a labeled
marker metabolite which is substantially retained in the tumor-targeted
bacterial vector
t~oughout a time-period sufficient for imaging the labeled marker metabolite;
and scanning
the subject to detect the labeled marker metabolite, thereby imaging and
detecting a tumor
in the subject.
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In certain specific embodiments, the generation of the marker metabolite
from the marker substrate entails the release of a quenched label from the
substrate (see,
e.g., Bell and Taylor-Robinson, 2000, Gene Therapy 7:1259-1264).
Suitable enzymes that can produce detectably labeled metabolites include but
are not limited to malate dehydrogenase, staphylococcal nuclease, 8-5-steroid
isomerase,
yeast alcohol dehydrogenase, a-glycerophosphate, dehydrogenase, triose
phosphate
isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose
oxidase,
[3-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase,
glucoamylase and acetylcholinesterase. In one embodiment, the tumor-targeted
bacteria
also express a permease or transporter which allows for enhanced uptake of the
substrate of
the marker gene product enzyme. In an exemplary mode of the embodiment, the
tumor-
targeted bacteria that express lacZ would also express lacy to increase
lactose or galactose
uptake.
In other embodiments, suitable enzymes that can produce detectably labeled
metabolites are proteases. Suitable protease substrates for imaging are
polymerized
protease recognition sites bearing a quenched label, which have been called
"repeating graft
co-polymers" (Weissleder et al., 1999, Nature Biotechnology 17:375-378).
Cleavage of the
substrate by the recombinant protease expressed by the tumor-targeted vector
at the tumor
site releases the quenched label, i.e., generates a labeled metabolite,
allowing imaging of the
tumor. Synthesis of quenched substrates can be done according to the
principles described
in Weissleder et al. (1999, Nature Biotechnology 17:375-378).
In a preferred embodiment, the marker gene expressed by the vector is a wild
type, natural mutant or genetically-engineered HSV1-TK or VZV-TK gene, and its
substrate is a labeled nucleoside analog such as FIAU, ACV or GCV. In a
particular mode
of the embodiment, the nucleoside analog is a 2'-fluoro-nucleoside analogue
such as FIAU,
. Upon concurrent expression of the tk gene and administration of
radioactively labeled
FIAU to the subject, the FIAU is phosphorylated in the microorganism. The
phosphorylated FIAU accumulates in the microorganism. This accumulated,
labeled FIAU
is then imaged by scanning the subject using, for example, positron emission
tomography,
'y camera or single-photon emission computed tomography. In a highly-preferred
embodiment, the FIAU is phosphorylated in the cytosol of the microorganism
instead of the
periplasm to enhance accumulation and subsequent imaging. This may be
accomplished
using a vector genetically engineered to express TK in the cytosol of the
microorganism.
In certain modes of the embodiment in which the marker gene is TK, the
labeled 2'-fluoro-nucleoside analogue is S-['z3I]-, S-['z4I]- or 5-['3'I]-2'-
fluoro-5-iodo-1-13-D-
arabinofuranosyl- uracil; S-['8F]-2'-fluoro-5-fluoro-1-13-D- arabinofuranosyl-
uracil; 2-["C]-
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or 5-(["C]-methy)-2'-fluoro-S-methyl-1-13-D-arabinofuranosyl- uracil; 2-["C]-
or 5-(["C]-
ethyl)-2'-fluoro-5-ethyl-1-13-D-arabinofuranosyl- uracil; S-(2-['$F]-ethyl)-2'-
fluoro-5-(2-
fluoro-ethyl)-1-13-D-arabinofuranosyl- uracil; 5-['z3I]-, 5-['z4I]- or 5-
['3'I]-2'-fluoro-5-
iodovinyl-113-D-arabinofuranosyl- uracil; 5-['z3I]-, 5-['z4I]- or 5-['3'I]-2'-
fluoro-5-iodo-1-13-
D-arabinofuranosyl- uracil; or S-['z3I]-, S-[lza I]- or 5-['3'I]-2'-fluoro-S-
iodovinyl-1-13-D-
aribofuranosyl- uracil.
In another preferred embodiment, the marker gene expressed by the vector is
wild type, natural mutant or genetically-engineered yeast glucokinase gene,
and its substrate
is a labeled 3-O-methyl glucose. In certain modes of the embodiment, the
labeled
3-O-methyl glucose is ["C]- or ['gF]-3-O-methyl glucose.
In yet another preferred embodiment, the marker gene expressed by the
vector is wild type, natural mutant or genetically-engineered cytochrome P-450
B 1 gene,
and its substrate is a labeled imidazole substrate. In certain modes of the
embodiment, the
labeled imidazole substrate is 2-["C]-misonidazole, 2-["C]-metronidazole or 3-
['8F]-
fluoromisonidazole.
5.2.4 Detection of Labeled Compounds Incorporated Into Bacteria
The invention also encompasses the detection of a tumor by administering to
the subject in which tumor detection is desired tumor-targeted bacteria. The
tumor targeted
bacteria is then detected by administering to the subject a labeled compound
which is
incorporated or localized to the bacteria itself. For example, the labeled
compound can be
chosen to be incorporated into the bacterial cell wall. The labeled compound
is labeled with
one of the labels described in Section 5.3, infra and detected as described.
In one embodiment, the labeled compound is labeled diaminopimelic acid
(D~), as described below. In another embodiment, the labeled compound is a
labeled
glucose or glucose analog which, because of the higher proliferation rate of
the bacteria (as
well as the tumor cells) relative to normal mammalian cells, will be
preferentially
incorporated into the dividing bacteria and tumor cells. This improves on the
current
imaging systems in which labeled glucose analogs are used to image tumors by
enhancing
the specific signal and reducing the non-specific signal, allowing the
detection of smaller
tumors, including metastases. In a preferred embodiment, the labeled glucose
analog is'8F-
fluorodeoxyglucose ('gF-FDG), which can be detected by PET scanning.
In certain embodiments, the bacteria's metabolism is manipulated to increase
its incorporation of the labeled compound. In one embodiment, the bacteria has
an
auxotrophic mutation which enhances its uptake of the labeled compound.
Preferably, such
a mutation further compromises its ability to live independently of the
compound, such that
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its viability is largely dependent on the administration of the compound.
Following
administration of the compound and imaging the bacteria (and, indirectly, the
tumor(s)),
such bacteria are gradually cleared from the system.
In one mode of the embodiment, the tumor-targeted bacteria is mutant for the
asd locus, which makes the bacteria more susceptible to lysis due to
compromised cell wall
integrity. Rescue of this mutant can be achieved by exogenously providing
DAP(Galan et
al., 1990, Gene 94:24-35). When labeled DAP is provided to bacteria with an
asd mutation,
the DAP becomes incorporated into the cell wall. Therefore, administering
labeled DAP to
a subject to whom asc~ tumor-targeting bacteria have been administered
enhances the
incorporation of DAP into the cells walls of the bacteria, resulting in an
enhanced signal for
imaging relative to the use of non-asd- tumor-targeting bacteria.
Additionally, depletion of
DAP from the subject eventually results in bacterial death, thereby clearing
the bacteria
from the subject after imaging. In a preferred mode of the embodiment, the
asa' mutation is
a hypomorphic but not a null mutation, so that the bacteria can survive
administration and
targeting to the tumor prior to administration of the labeled DAP.
5.2.5 Detection of Bacterial Infections
In certain embodiments of the invention, a tumor is detected by detecting an
infection (e.g., the immune response) caused by administering a composition of
the
invention to a subject.
In one embodiment, tumor-targeted bacteria are administered to a subject
and the subject scanned to detect an infection caused by the bacteria at the
tumor site. The
infection, and therefore the tumor, is detected indirectly by detecting the
presence of an
antigen that is highly or specifically expressed at the infection site. In a
preferred mode of
the embodiment, the antigen detected is a neutrophil-specific antigen.
Polymorphonuclear
neutrophils are sequestered at infection sites and labeled monoclonal
neutrophil-specific
antigens have been successful to diagnose infections. In a preferred mode of
the
embodiment, the neutrophil-specific antigen is CD15. A 99Tc-labeled anti-CD15
monoclonal antibody is commercially available under the trade name LeuTech
(Palatin
Technologies, Inc.), which has been used to detect appendicitis (Kipper et
al., 2000, J Nucl
Med 41(3):449-55).
In another embodiment, the infection, and therefore the tumor, is detected
indirectly by using a labeled chemotactic peptide analogs which bind to
leukocyte receptors
(see, e.g., Fischman et al., 1994, Semin Nucl Med). In one mode of the
embodiment, the
labeled chemotactic peptide analog is labeled N-formyl-methionyl-leucyl-
phenylalanine
(ForMLF). In another mode of the embodiment, the labeled chemotactic peptide
analog is
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labeled neutrophil peptide-1. 99Tc-labeled neutrophil peptide-1 has been used
to detect
bacterial infections (Welling et al., 1999, J Nucl Med 40(12):2073-80).
5.2.6 Detection of Bacterial Surface Antigens
In certain embodiments of the invention, a tumor is detected by detecting a
surface antigen expressed by the tumor-targeted bacteria.
According to the present embodiment, tumor-targeted bacteria are
administered to a subject. A labeled antibody which binds to an antigen
present on the
surface of the tumor-targeted bacteria is then administered, and the subject
scanned to
detect the label. Therefore the tumor is detected indirectly by detecting the
presence of an
antigen that is expressed on the bacterial surface.
In one embodiment, the surface antigen detected by the antibody is an O-
antigen (an abbreviation for O-polysaccharide antigen). In another embodiment,
the surface
antigen detected by the antibody is an H-antigen (H-antigens are flagellar
antigens). In yet
another embodiment, the surface antigen detected by the antibody is an outer
membrane
protein.
5.3. Labels and Methods of Detection Thereof
The present invention provides .non-invasive methods of imaging tumors in
vivo by detecting tumor-targeted bacteria or a marker gene product expressed
by a tumor-
targeted bacteria, said detection being by way of a labeled moiety,
metabolite, or
compound. Some examples of labels that can be used are radioisotopes (such as
3Hydrogen,
"Carbon, '4Carbon, '3Nitrogen, 'BFlourine, 'z3Iodine, 'z4lodine, 'zSIodine,
'3'Iodine,
"'Indium, 64Copper, 6'Copper, 43Scandium, 44Scandium, 46Scandium, 4'Scandium,
48Scandium,'zGallium,'3Gallium, zo3Bismuth, zosBismuth, z'zBismuth,
z'3Bismuth,
z'4Bismuth, SzIron, SSCobalt, 68Gallium.); paramagnetic metal ions (such as
54Iron, 56Iron,
5'Iron, SBIron,'S'Gadolinium, SSManganese); and ferromagnetic metals (such as
magnetite
(Fe304), y-fernc oxide (y-Fez03), cobalt fernte, nickel ferrite, manganese
fernte). Label
detection can be performed with y camera or single-photon emission computed
tomography
(SPELT), PET, and MRI.
Fluorophores can also be used as labels for marker detection. Examples of
such compounds are rhodamine, fluorescein, CyS, Cy3, succinimidyl
6-(N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoate (also known as NBT),
R-
phycoerythrin, allophycocyanin, 6-((7-amino-4-methylcoumarin- 3-
acetyl)amino)hexanoic
acid (also known as AMCA), 7-dimethylaminocoumarin-4- acetic acid (also known
as
DMACA), and 5-carboxytetramethylrhodamine. Label detection can be performed
with a
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whole-body optical fluorescence imaging system (Yang et al., 2000, Proc. Nat'1
Acad. Sci.
U S A 97:1206-11).
The particular label, optimal label position and method of labeling for each
labeled moiety, substrate or compound that is administered to a subject for
imaging or a
combination of imaging and treatment will depend on the chemical nature of the
moiety,
substrate or compound and its intended use (imaging only or imaging and
therapeutic use)
and can be determined by one of skill in the art.
In certain specific embodiments, the label is chelated or quenched until it
reaches the tumor site, where the label is released. Both fluorophores
(Weissleder et al.,
1999, Nature Biotechnology 17:375-378) and paramagnetic ions (Bell and Taylor-
Robinson, 2000, Gene Therapy 7:1259-1264), which have been successfully used
as
quenched labels that are eventually released to produce a signal, can be used
according to
the present invention.
5.4. Improving the Signal to Noise Ratio
The present invention further provides methods by which the signal of the
label or the marker gene product to be imaged can be enhanced. Such methods
encompass
enhancing the specific signal, reducing the background or non-specific signal,
or both.
In one embodiment, the invention provides a method further comprising
waiting a time-period after administration of the labeled compound, substrate
or binding
moiety sufficient to allow at least 67% of non-specific label to clear from
the subject. In
another embodiment, the invention provides a method further comprising waiting
a
time-period after administration of the labeled compound, substrate or binding
moiety
sufficient to allow at least 80% of non-specific label to clear from the
subject. In yet another
embodiment, the invention provides a method further comprising waiting a time-
period
after administration of the labeled compound, substrate or binding moiety
sufficient to
allow at least 90% of non-specific label to clear from the subject. As used
herein, the term
non-specific label refers to labeled (i) compound, (ii) substrate or (iii)
binding moiety that
(i) has not been incorporated into the bacteria, (ii) has not been metabolized
by the marker
gene product, or (iii) is not bound to the marker gene product, respectively.
In certain other embodiments of the invention, the label in a labeled
substrate
is quenched or chelated for release at a tumor site upon production of a
labeled metabolite.
The labeled substrate is undetectable or generates a reduced signal until the
label is
activated, thereby reducing the non-specific signal that could be generated
from non-
specific binding or poor clearance of the substrate.
In yet other embodiments of the invention, the specific signal is enhanced by
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maximizing the interaction of the marker gene product with the labeled
compound,
substrate or binding moiety. In certain specific embodiments, as described
below, the
marker gene product is brought into the vicinity of the labeled compound,
substrate or
binding moiety, for example by promoting its release into the extracellular
environment. In
$ other specific embodiments, as described below, the labeled compound,
substrate or binding
moiety is brought into the vicinity of the marker gene product, for example by
promoting its
uptake into or retention in the bacterial vector.
5.4.1 Non-Selective Increase in Bacterial Release of Intracellular Compounds
In certain embodiments of the invention, the attenuated tumor-targeted
bacterial vectors of the invention, which express at least one marker gene
product, also
express at least molecule which functions to permeabilize the bacteria cell
membranes) or
enhance the release of intracellular components into the extracellular
environment, e.g. at
the tumor site, thereby enhancing the delivery of the marker gene product(s).
Such
1 S molecules which permeabilize the bacterial cell or enhance release are
designated as
"release factors".
The release factor expressed by the bacterial vector of the invention may be
endogenous to the modified attenuated tumor-targeted bacteria or it may
exogenous (e.g.,
encoded by a nucleic acid that is not native to the attenuated tumor-targeted
bacteria). A
release factor may be encoded by a nucleic acid comprising a plasmid, or by a
nucleic acid
which is integrated into the genome of the attenuated tumor-targeted bacteria.
A release
factor may be encoded by the same nucleic acid or plasmid that encodes the
marker gene of
interest, or by a separate nucleic acid or plasmid..
In a preferred embodiment, such a factor is one of the Bacteriocin Release
Proteins, or BRPs (herein referred to in the generic as BRP). The BRP employed
in the
invention can originate from any source known in the art. Examples of BRP
proteins
include, but are not limited to, the BRP from the cloacin D13 plasmid, the BRP
from one of
the colicin E1-E9 plasmids, and BRP from colicin A, N or D plasmids. In a
preferred
embodiment, the BRP is of cloacin D 13 (pCIoDF 13 BRP).
In another embodiment of the invention, the enhanced release system
comprises overexpression of a porin protein; see e.g., Sugawara and Nikaido,
1992, J. Biol.
Chem.267:2507-11 ).
Expression levels of release factors and porins should be optimized to
enhance release of cellular contents but not result in bacterial lysis.
Preferably, such
expression is under the control of a promoter that is preferentially active in
the tumor
environment. For example, the release factor or porin coding sequence can be
expressed
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under the control the pepT promoter, which is activated in response to the
anaerobic nature
of the tumor environment (see, e.g., Lang et al., 1996, Gene, 168:169-171). In
another
specific embodiment of the invention, the release factor or porin coding
sequence is
expressed under the control of a tet promoter, and expression of the release
factor or porin is
induced upon administration of tetracycline to the subject.
5.4.2 Targeted Marker Gene Release
The present invention also provides methods for local delivery of one or
more marker gene products to the tumor environment through secretion
machinery. In a
preferred embodiment, tumor-targeted bacteria are engineered to express one or
more
nucleic acid molecules encoding one or more fusion proteins comprising an Omp-
like
protein, or portion thereof (e.g., signal sequence, leader sequence,
periplasmic region,
transmembrane domain, multiple transmembrane domains, or combinations thereof;
see
Section 3.1 for definition of "Omp-like protein") and marker gene product.
In specific embodiments, the Omp-like protein is at least a portion of OmpA,
OmpB, OmpC, OmpD, OmpE, OmpF, OmpT, a porin-like protein, PhoA, PhoE, lama, (3-
lactamase, an enterotoxin, protein A, endoglucanase, peptidoglycan-associated
lipoprotein
(PAL), FepA, FhuA, NmpA, NmpB, NmpC, or a major outer membrane lipoprotein
(such
as LPP), etc. In certain embodiments of the invention, the signal sequence is
constructed to
be more hydrophobic (e.g., by the insertion or replacement of amino acids
within the signal
sequence to hydrophobic amino acids, e.g., Leucine).
In several embodiments, the marker gene product is expressed as a fusion
protein to an Omp-like protein, or portion thereof. Bacterial outer membrane
proteins are
integral membrane proteins of the bacterial outer membrane, possess multiple
membrane-
spanning domains and are often attached to one or more lipid moieties. Outer
membrane
proteins are initially expressed in precursor form (the pro-Omp) with an amino
terminal
signal peptide that directs the protein to the membrane, upon which the signal
peptide is
cleaved by a signal peptidase to produce the mature protein. In one
embodiment, a marker
gene product is constructed as a fusion protein with an Omp-like protein. In
this
embodiment, the gene product has enhanced delivery to the outer membrane of
the bacteria.
In one embodiment, the fusion of a marker gene product to an Omp-like
protein is used to enhance localization of marker gene product to the
periplasm. In another
embodiment, the fusion of a marker gene product to an Omp-like protein is used
to enhance
release of the gene product. In another embodiment, a release factor (e.g.,
BRP, supra) is
expressed in a cell which also expresses a fusion protein comprising a marker
gene product
fused to an Omp-like protein. In this embodiment, the co-expression of the
release factor
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allows for enhanced release of the fusion protein from the periplasmic space.
In other embodiments of the invention, a fusion protein of the invention
comprises a proteolytic cleavage site. The proteolytic cleavage site may be
endogenous to
the gene product or endogenous to the Omp-like protein, or the proteolytic
cleavage site
may be constructed into the fusion protein. In certain specific embodiments,
the Omp-like
protein of the invention is a hybrid Omp comprising structural elements that
originate from
separate proteins.
In an exemplary mode of the embodiment, the Omp-like protein is OmpA;
the same principles used in the construction of OmpA-like fusion proteins are
applied to
other Omp fusion proteins, keeping in mind the structural configuration of the
specific
Omp-like protein.
For example, the native OmpA protein contains eight anti-parallel
transmembrane ~3-strands within the 170 amino acid N-terminal domain of the
protein.
Between each pair of transmembrane domains is an extracellular or
intracellular loop,
depending on the direction of insertion of the transmembrane domain. The C-
terminal
domain consists of 155 amino acids which are located intracellularly and
presumably
contact the peptidoglycan occupying the periplasmic space. Expression vectors
have been
generated that facilitate the generation of OmpA fusion proteins. For example,
Hobol et al.
(1995, Dev. Biol. Strand. 84:255-262) have developed vectors containing the
OmpA open
reading frame with linkers inserted within the sequences encoding the third or
fourth
extracellular loops that allow the in-frame insertion of the heterologous
protein of choice.
In another embodiment of the invention, the portion of the OmpA fusion
protein containing the marker gene product is at the extracellular bacterial
surface. In one
aspect of the embodiment, the fusion protein comprises an even number or odd
number of
membrane-spanning domains of OmpA located N-terminal to the gene product. In
another
aspect of the embodiment, the gene product is situated between two
extracellular loops of
OmpA for presentation to the tumor cell by the bacterial cell. In specific
embodiments, the
invention provides expression plasmids of gene product fusion proteins at the
bacterial
extracellular surface. For example, the plasmid denoted Trc(lpp)ompA,
comprises a trc
promoter-driven lipopolyprotein (lpp) anchor sequence fused to a truncated
ompA
transmembrane sequence. As another example, the plasmid is denoted TrcompA
comprises
a trc promoter-driven ompA gene signal sequence. Such plasmids may be
constructed to
comprise a nucleic acid comprising or encoding one or more gene products) of
the
invention.
Optionally, a marker gene product is preceded or flanked by consensus
cleavage sites for a metalloprotease or serine protease that is abundant in
tumors, for release
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of the gene product into the tumor environment. Whether the marker gene
product is
preceded or flanked by protease cleavage sites depends on whether it is
located terminally
or internally in the fusion protein, respectively.
Similar fusion proteins may be constructed with any of the Omp-like
proteins using the strategies described above in terms of OmpA. In the
construction of such
fusion proteins, as will be apparent to one of ordinary skill in the art, the
selection of the
portion of the Omp-like protein to be fused to a marker gene product will
depend upon the
location that is desired for the expression of the gene product (e.g.
periplasmic,
extracellular, membrane bound, etc. ).
Construction of fusion proteins for expression in bacteria are well known in
the art and such methods are within the scope of the invention. (See, e.g.,
Makrides, S.,
1996, Microbiol. Revs 60:512-538 which is incorporated herein by reference in
its entirety).
5.4.3 Increased Cytoplasmic Retention of Enzyme Substrates
1 S The invention encompasses an embodiment where the marker gene product
is an enzyme. This class of marker gene product is detected by the addition of
its substrate
attached to a labeled moiety. To successfully detect the tumor-targeted
bacteria, the enzyme
and substrate must be brought into close proximity. To aid in this process,
the tumor-
targeted bacteria can be modified to increase the retention of the enzyme
substrate in its
cytoplasm and to facilitate greater contact with the enzyme.
In a preferred mode of this embodiment, the enzyme is a pro-drug converting
enzyme. This class of enzymes modulate the chemical nature of a benign drug to
produce a
cytotoxic agent and, as such, can also be used for treatment of a tumor as
well as its
detection and localization (see Section 5.6 and Table 2 below).
Enhanced cytoplasmic retention of the labeled enzyme substrate can be
accomplished through the manipulation of the transport systems normally
responsible for
pumping the compounds out of the cell. Such transporters are called drug
efflux systems.
One preferred modification of the host organism is to disable or severely
compromise one or more drug efflux systems in the host organism. Membrane-
associated
energy driven efflux plays a major role in drug resistance in most organisms,
including
bacteria, yeasts, and mammalian cells (Nikaido, 1994, Science 264:382-388;
Balzi et al.,
1994, Biochim Biophys Acta 1187:152-162; Gottesman et al., 1993, Ann Rev
Biochem
62:385). Normally, a wild type efflux system actively secretes potentially
toxic
compounds, thus reducing their accumulation inside the host organism.
In bacteria, a large number of efflux systems have been studied which can
pump out a wide variety of structurally unrelated molecules ranging from, for
example,
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polyketide antibiotics (acrAE genes of E. coli, Ma et al. 1993, J Bacteriol
17$:6299-6313),
fluroquinolines and ethidium bromide (bmr of Bacillus subtilis and nor A of
Staphylococcus
aureus, Neyfakh et al. 1993, Antimicrob Agents Chemother 37:128-129),
doxorubicin (drr
of Streptomyces peucetius, to quaternary amines (qacE of Klebsiella aerogenes
and mvrC of
$ E. coli). See Table 1 for a list of non-limiting examples of efflux systems.
Any such efflux
systems may be used in a tumor-targeted vector of the invention.
The efflux systems that can be expressed in a tumor-targeting vector of the
invention can also be of yeast or plant origin. Efflux systems in animals,
plants, yeast, and
bacteria are mechanistically related, often involving ATP binding cassette
transporters (see,
e.g., Thomas et al., 2000, Plant Cell 12(4):$19-33). Recent data suggests that
certain efflux
systems are structurally conserved among yeast, plants and bacteria (Harley
and Saier,
2000, J. Mol. Microbiol. Biotechnol.2(2):19$-8). In one embodiment, a yeast
gene which
can be used as an efflux system is the bfrl+ gene, which confers brefeldin A
resistance to
Schizosaccharomyces pombe (Nagao et al., 199$, J. Bacteriol. 177(6):1$36-43).
In another
1$ embodiment, a yeast gene which can be used as an efflux system is the CDRl
gene of
Candida albicans, which confers resistance to cyclohexamide and
chloramphenicol (Prasad
et al., 199$, Curr Genet 27:320-329).
Table 1. List of compounds that are secreted by active drug efflux systems
Chemical class Specific name Efflux systems
cationic dyes rhomadamine-6G bmr
ethidium bromide
acriflavine acrAE
basic antibiotics puromycin bmr
doxorubicin drr, mdr
2$ hydrophilic antibiotics novobiocin acrAE
macrolide
hydrophobic antibiotics beta-lactams
organic cation tetraphenyl
phosphonium bmr
uncharged taxol mdr
chloramphenicol bmr
weak acid nalidixic acid emr
mithramycin mdr
zwitterions fluoroquinolines bmr
detergent SDS acrAE
3$
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5.5. Useful Bacteria
Bacteria useful in the present invention are those facultative aerobic and
facultative anaerobic bacteria which are able to differentiate between
cancerous cells and
non-cancerous counterpart cells. For example, the bacteria are able to
differentiate between
$ melanoma cells and melanocytes or between colon cancer cells and normal
colon epithelial
cells. Illustrative examples of bacteria useful in the present invention as
tumor-targeted
bacteria and/or for isolation of tumor-targeted bacteria include, but are not
limited to the
following facultative aerobes and anaerobes: Escherichia coli, including but
not limited to
pathogenic Escherichia coli (e.g., entero-invasive or uropathogenic
Escherichia coli),
Salmonella spp., Shigella spp., Streptococcus spp., Yersinia enterocolitica,
Listeria
monocytogenies, and Mycoplasma hominis. While, for ease of explanation, the
description
below refers specifically to Salmonella, the methods of and compositions used
in the
invention are in no way intended to be restricted to Salmonella but rather
encompass any
and all of the bacteria taught herein as useful.
1$ Factors contributing to attenuation and tumor-targeting are described
herein and may be used to construct or select an appropriate strain of
bacteria for use in the
methods of the invention. For example, methods to select and isolate tumor-
targeted
bacteria are described in Section 6.1, and methods to attenuate bacteria are
described in
Section 6.2 of International publication W096/40238, which sections are
incorporated
herein by reference in their entirety. Examples of attenuated tumor-targeted
bacteria are
also described in International Application W099/13053, which is incorporated
herein by
reference in its entirety.
Salmonella spp. are particularly useful strains for the present invention,
since they show natural preference for attachment to and penetration into
certain solid
2$ tumor cancer cells in tissue culture, as opposed to non-cancerous
counterpart cells. (The
term "Salmonella" is used generically herein to refer to any Salmonella
species). Since
Salmonella have a natural ability to distinguish between cancerous cells and
their non-
cancerous counterpart cells, and preferentially replicate in tumors (in mice),
they are
directly applicable to the methods for treatment according to the present
invention.
Bacteria such as Salmonella are causative agents of disease in humans
and animals. One such disease that can be caused by Salmonella is sepsis,
which is a
serious problem because of the high mortality rate associated with the onset
of septic shock
(Bone, 1993, Clinical Microbiol. Revs. 6:57-68). Therefore, to allow the safe
use of
Salmonella in the present invention, the bacterial vectors, including but not
limited to
Salmonella, are attenuated in their virulence for causing disease. In the
present application,
attenuation, in addition to its traditional definition in which a bacterium is
modified so that
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the bacterium is less pathogenic, is intended to include also the modification
of a bacterial
strain so that a lower titer of that derived bacterial strain can be
administered to a patient
and still achieve comparable results as if one had administered a higher titer
of the parental
bacterial strain. The end result serves to reduce the risk of toxic shock or
other side effects
due to administration of the strain to the patient. Such attenuated bacteria
are isolated by
means of a number of techniques. For example, attenuation can be achieved by
the deletion
or disruption of DNA sequences which encode for virulence factors that insure
survival of
the bacteria in the host cell, especially macrophages and neutrophils. Such
deletion or
disruption techniques are well known in the art and include, for example,
homologous
recombination, chemical mutagenesis, radiation mutagenesis, or transposon
mutagenesis.
Those virulence factors that are associated with survival in macrophages are
usually
specifically expressed within the macrophages in response to stress signals,
for example,
acidification, or in response to host cell defensive mechanisms such
macropinocytosis
(Fields et al., 1986, Proc. Natl. Acad. Sci. USA 83:5189-5193). Table 4 of
International
Publication WO 96/40238 is an illustrative list of Salmonella virulence
factors whose
deletion results in attenuation.
Yet another method for the attenuation of the bacteria, such as
Salmonella, is to modify substituents of the bacteria which are responsible
for the toxicity
of that bacteria. For example, lipopolysaccharide (LPS) or endotoxin is
primarily
responsible for the pathological effects of bacterial sepsis. The component of
LPS which
results in this response is lipid A ("LA"). Elimination or mitigation of the
toxic effects of
LA results in an attenuated bacteria since 1) the risk of septic shock in the
patient is reduced
and 2) higher levels of the bacterial strain can be tolerated.
Altering the LA content of bacteria, such as Salmonella, can be achieved
by the introduction of mutations in the LPS biosynthetic pathway. Several
enzymatic steps
in LPS biosynthesis and the genetic loci controlling them in Salmonella have
been
identified (Raetz, 1993, J. Bacteriol. 175:5745-5753 and references therein),
as well as
corresponding mutants. One such illustrative mutant is firA, a mutation within
the gene that
encodes the enzyme UDP-3-O(R-30 hydroxymyristyl)-glycocyamine N-
acyltransferase,
which regulates the third step in endotoxin biosynthesis (Kelley et al., 1993,
J. Biol. Chem.
268:19866-19874). Bacterial strains bearing this type of mutation produce a
lipid A that
differs from wild-type lipid A in that it contains a seventh fatty acid, a
hexadecanoic acid
(Roy and Coleman, 1994, J. Bacteriol. 176:1639-1646). Roy and Coleman
demonstrated
that in addition to blocking the third step in endotoxin biosynthesis, the
firA- mutation also
decreases enzymatic activity of lipid A 4' kinase that regulates the sixth
step of lipid A
biosynthesis.
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In addition to being attenuated, the bacteria of the invention are tumor-
targeted, i.e., the bacteria preferentially attaches to, infect, and/or remain
viable in a tumor
or tumor cell versus a normal tissue, non-tumor or non-tumor cell. Suitable
methods for
obtaining attenuated tumor-targeted bacteria are described in Sections 6.1 and
6.2 of
International Publication WO 96/40238, which sections are incorporated by
reference herein
in their entirety. As the resulting bacteria are highly specific and, in one
embodiment super-
infective, the difference between the number of infecting bacteria found at
the target tumor
or tumor cell as compared to the non-cancerous counterparts becomes larger and
larger as
the dilution of the bacterial culture is increased such that lower titers of
bacteria can be used
with positive results. The techniques described in International Publication
WO 96/40238
can also be used to produce attenuated tumor-targeted Salmonella or non-
Salmonella
bacterial strains.
An illustrative example of an attenuated tumor-targeted bacterium
having an LPS pathway mutant is the msbB- Salmonella mutant described in
International
Publication WO 99/13053, which is incorporated herein by reference in its
entirety; see
especially Section 6.1.2 which describes the characteristic of the msbB-
Salmonella mutant.
One characteristic of the msbB- Salmonella is decreased ability to induce a
TNF-a response
compared to the wild-type bacterial strain. The msbB- Salmonella induce TNF-a
expression
at levels of about 5 percent to about 40 percent compared to the levels
induced by wild-type
Salmonella.
The TNF-a response induced by whole bacteria or isolated or purified
LPS can be assessed in vitro or in vivo using commercially available assay
systems such as
by enzyme linked immunoassay (ELISA). Comparison of TNF-a production on a per
colony forming unit ("c.f.u.") or on a ~g/kg basis, is used to determine
relative activity.
Lower TNF-a levels on a per unit basis indicate decreased induction of TNF-a
production.
In a preferred embodiment, the msbB~ Salmonella strain is modified to express
a marker
gene product which aids in reducing the volume of, or inhibiting the growth
of, the solid
tumor cancer.
The present invention also encompasses the use of derivatives of msbB-
attenuated tumor-targeted Salmonella mutants.
The stability of the attenuated phenotype is important such that the
strain does not revert to a more virulent phenotype during the course of
treatment of a
patient. Such stability can be obtained, for example, by providing that the
virulence gene is
disrupted by deletion or other non-reverting mutations on the chromosomal
level rather than
epistatically.
Another method of insuring the attenuated phenotype is to engineer the
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bacteria such that it is attenuated in more than one manner. For example,
bacteria can be
made much more susceptible to lysis by manipulating the cell wall integrity.
In one
embodiment, mutations or deletions in the asd locus are used to severely
compromise cell
wall integrity. This type of mutant bacteria relies on exogenously provided
diaminopimelic
acid to survive (Galan et al., 1990, Gene 94:24-35). At the termination of
therapy, this
supplement can be withdrawn from the patient to accelerate the clearance of
tumor-targeted
bacteria. In other embodiments different pathways can be manipulated to
attenuate the
bacteria, such as, e.g., a mutation in the pathway for lipid A production,
such as the msbB-
mutation (International Publication WO 99/13053) and one or more mutations to
auxotrophy for one or more nutrients or metabolites, such as uracil
biosynthesis, purine
biosynthesis, and arginine biosynthesis as described by Bochner, 1980, J.
Bacteriol.
143:926-933. In a preferred embodiment, the tumor-targeted msbB- Salmonella is
also
auxotrophic for purine. In certain embodiments, the attenuated tumor-targeted
bacteria are
attenuated by the presence of a mutation in AroA, msbB, Purl or Serf. In
another
embodiment the attenuated tumor-targeted bacteria are attenuated by the
presence of a
deletion in AroA, msbB, Purl or Serf.
Accordingly, any attenuated tumor-targeted bacteria may be used in the
imaging and monitoring methods of the invention.
5.6. Vectors For Combining Treatment With Imaging
In certain embodiments of the present invention, the detection and
monitoring methods can be combined with administration of a bacterial strain
described in
Section 5.5 which has been genetically modified to express one or more gene
products of
interest and serve as a bacterial vector which aids in reducing the volume of,
or inhibiting
the growth of, the solid tumor cancer. Hence, these embodiments encompass
detection
(and/or monitoring) and anti-tumor treatment, i.e., inhibition of tumor growth
or reduction
of tumor volume. The same bacteria which express a marker gene can express an
anti-
tumor gene product. In specific modes of the embodiment, the marker gene
itself is utilized
as an anti-tumor gene. Alternatively, at least two different bacteria can be
used, expressing
a marker gene and one expressing an anti-tumor gene product (referred to
herein as a "gene
product of interest"). The gene product of interest is selected from the group
consisting of
proteinaceous and nucleic acid molecules. The nucleic acid molecule can be
double-
stranded or single-stranded DNA or double-stranded or single-stranded RNA, as
well as
triplex nucleic acid molecules. The nucleic acid molecule can function as a
ribozyme, or
antisense nucleic acid, etc. Release of the gene product of interest into the
tumor
environment may be enhanced in various ways. In one embodiment the gene
product of
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interest is co-expressed with a release factor (e.g., BRP, see Section 5.4.1).
In another
embodiment, the gene product of interest is expressed as a fusion protein with
an Omp-like
protein, or portion thereof (see Section 5.4.2).
Antisense nucleotides are oligonucleotides that bind in a sequence-
specific manner to nucleic acids, such as mRNA or DNA. When bound to mRNA that
has
complementary sequences, antisense prevents translation of the mRNA (see,
e.g., U.S.
Patent Nos. 5,168,053; 5,190,931; 5,135,917; 5,087,617). Triplex molecules
refer to single
DNA strands that bind duplex DNA forming a collinear triplex molecule, thereby
preventing transcription (see, e.g., U.S. Patent No. 5,176,996).
A ribozyme is an RNA molecule that specifically cleaves RNA
substrates, such as mRNA, resulting in inhibition or interference with cell
growth or
expression. There are at least five known classes of ribozymes involved in the
cleavage
and/or ligation of RNA chains. Ribozymes can be targeted to any RNA transcript
and can
catalytically cleave that transcript (see, e.g., U.S. Patent No. 5,272,262;
U.S. Patent No.
1 S 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and
5,093,246).
In various embodiments, the proteinaceous molecule is a cellular toxin,
e.g., saporin, a ribosome inactivating protein, or a porin protein, such as
gonococcal PI
porin protein. In other embodiments, the proteinaceous molecule is an anti-
angiogenesis
protein, an antibody or an antigen. In yet other embodiments, the
proteinaceous molecule is
a cytokine, e.g., IL-2, or an anti-angiogenic factor, e.g., endostatin, or a
pro-drug converting
enzyme, e.g., Herpes Simplex Virus ("HSV") thymidine kinase or cytosine
deaminase.
The nucleic acid molecule encoding the gene product of interest is from
about 6 base pairs to about 100,000 base pairs in length. Preferably the
nucleic acid
molecule is from about 20 base pairs to about 50,000 base pairs in length.
More preferably
the nucleic acid molecule is from about 100 base pairs to about 10,000 base
pairs in length.
Even more preferably, it is a nucleic acid molecule from about 500 pairs to
about 2,000 base
pairs in length.
The nucleic acid encoding a gene product of interest is provided in an
expression vector in operative linkage with a selected promoter, and
optionally in operative
linkage with other elements that participate in transcription, translation,
localization,
stability and the like.
In certain embodiments, the gene product of interest is cytotoxic or
cytostatic to a cell by inhibiting cell growth through interference with
protein synthesis or
through disruption of the cell cycle. Such a product may act, for example, by
cleaving
rRNA or ribonucleoprotein, inhibiting an elongation factor, cleaving mRNA, or
other
mechanism that reduced protein synthesis to a level such that the cell cannot
survive.
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Examples of such cytotoxic or cytostatic molecules include but are not limited
to saporin,
the ricins, abrin, and other ribosome inactivating proteins (RIPS).
In another mode of this embodiment, the gene product of interest is a
pro-drug converting enzyme or nucleic acid encoding the same, i.e. an enzyme
that
modulates the chemical nature of a drug to produce a cytotoxic agent.
Illustrative examples
of pro-drug converting enzymes include, HSV-TK, bacterial cytosine deaminase,
cytochrome P-450 NADPH oxidoreductase, and those listed on page 33 and in
Table 2 of
WO 96/40238 by Pawelek et al., which is incorporated herein in its entirety.
Further
examples of pro-drug converting enzymes can be seen in Table 2 below. WO
96/40238
also teaches methods for production of secreted fusion proteins comprising
such pro-drug
converting enzymes. According the present invention, a pro-drug converting
enzyme need
not be a secreted protein if co-expressed with a release factor such as BRP
(see Section
5.4.1). In a specific embodiment, the pro-drug converting enzyme is cytochrome
p450
NADPH oxidoreductase which acts upon mitomycin C and porfiromycin (Murray et
al.,
1994, J. Pharmacol. Exp. Therapeut. 270:645-649).
In another mode of the embodiment, a gene product of interest is an
inhibitor of inducible nitric oxide synthase (NOS) or of endothelial nitric
oxide synthase.
Nitric oxide (NO) is implicated to be involved in the regulation of vascular
growth and in
arteriosclerosis. NO is formed from L-arginine by nitric oxide synthase (NOS)
and
modulates immune, inflammatory and cardiovascular responses.
In another mode of the embodiment, the gene product of interest is
cytotoxic or cytostatic to a cell by inhibiting the production or activity of
a protein involved
in cell proliferation, such as an oncogene or growth factor, (e.g., bFGF, int-
2, hst-1/K-FGF,
FGF-S, hst-2/FGF-6, FGF-8) or cellular receptor or ligand. The inhibition can
be at the
level of transcription or translation (mediated by a gene product that is a
ribozyme or triplex
DNA), or at the level of protein activity (mediated by a gene product that is
an inhibitor of a
growth factor pathway, such as a dominant negative mutant).
In another mode of the embodiment, a gene product of interest is a
cytokine, chemokine, or an immunomodulating protein or a nucleic acid encoding
the same,
such as interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-4 (IL-4),
interleukin-5 (IL-5),
interleukin-10 (IL-10), interleukin-15 (IL-15), interleukin-18 (IL-18),
endothelial monocyte
activating protein-2 (EMAP2) GM-CSF, IFN-y, IFN-a, MIP-3a, SLC, MIP-3~3, or an
MHC
gene, such as HLA-B7. The cytokine may also be a member of the TNF family,
including
but not limited to, tumor necrosis factor-a (TNF-a), tumor necrosis factor-~3
(TNF-Vii), TNF-
a-related apoptosis-inducing ligand (TRAIL), TNF-a-related activation-induced
cytokine
(TRANCE), TNF-a-related weak inducer of apoptosis (TWEAK), CD40 ligand
(CD40L),
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LT-a (lymphotoxin alpha), LT-~i (lymphotoxin beta), OX40L (0X40 ligand), Fast,
CD27L
(CD27 ligand), CD30L (CD30 ligand), 4-1BBL, APRIL (a proliferation-inducing
ligand),
LIGHT (a 29 kDa type II transmembrane protein produced by activated T cells),
TL1 (a
tumor necrosis factor-like cytokine), TNFSF 16, TNFSF 17, and AITR-L (ligand
of the
activation-inducible TNFR family member). In other embodiment, the gene
product of
interest is tumor necrosis factor-a (TNF-a), tumor necrosis factor-(3 (TNF-
~3), TNF-a-
related apoptosis-inducing ligand (TRAIL), TNF-a-related activation-induced
cytokine
(TRANCE), TNF-a-related weak inducer of apoptosis (TWEAK), and CD40 ligand
(CD40L), or a functional fragment thereof.. For a review, see, e.g., Kwon et
al., 1999, Curr.
Opin. Immunol. 11:340-345, which describes members of the TNF family. Delivery
of
such immunomodulating these marker gene products will modulate the immune
system,
increasing the potential for host antitumor immunity. Alternatively, nucleic
acid molecules
encoding costimulatory molecules, such as B7.1 and B7.2, ligands for CD28 and
CTLA-4
respectively, can also be delivered to enhance T cell mediated immunity. Yet
another
immunomodulating agent is, a-1,3-galactosyl transferase, whose expression on
tumor cells
allows complement-mediated cell killing. In yet another alternative mode of
the
embodiment, the immunomodulating agent is a tumor-associated antigen, i.e. a
molecule
specifically that is expressed by a tumor cell and not in the non-cancerous
counterpart cell,
or is expressed in the tumor cell at a higher level than in the non-cancerous
counterpart cell.
Illustrative examples of tumor-associated antigens are described in Kuby,
1992,
Immunology, W.H. Freeman and Company, New York, NY, 15' Edition, pp. 515-520
which
is incorporated by reference herein. Other examples of tumor-associated
antigens are
known to those of skill in the art.
In another mode of the embodiment, a gene product of interest is an
anti-angiogenic molecule or protein associated with angiogenesis, such as,
e.g., endostatin,
angiostatin, apomigren, anti-angiogenic antithrombin III, the 29 kDa N-
terminal and a 40
kDa C-terminal proteolytic fragments of fibronectin, a uPA receptor
antagonist, the 16 kDa
proteolytic fragment of prolactin, the 7.8 kDa proteolytic fragment of
platelet factor-4, the
anti-angiogenic 24 amino acid fragment of platelet factor-4, the anti-
angiogenic factor
designated 13.40, the anti-angiogenic 22 amino acid peptide fragment of
thrombospondin I,
the anti-angiogenic 20 amino acid peptide fragment of SPARC, RGD and NGR
containing
peptides, the small anti-angiogenic peptides of laminin, fibronectin,
procollagen and EGF,
and peptide antagonists of integrin a~~i3 and the VEGF receptor. In a
preferred embodiment
of the invention, the anti-angiogenic molecule is a functional fragment of
endostatin,
apomigren or thrombospondin I.
In another mode of the embodiment, a gene product of interest is a Flt-3
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ligand or nucleic acid encoding the same.
In another specific embodiment, the gene product of interest is a
cytotoxic polypeptide or peptide, or a functional fragment thereof. Examples
of cytotoxic
polypeptides or peptides include, but are not limited to, members of the
bacteriocin family
(see e.g., Konisky, 1982, Ann. Rev. Microbiol. 36:125-144), verotoxin,
cytotoxic necrotic
factor 1 (CNF1), cytotoxic necrotic factor 2 (CNF2), Pasteurella multiocida
toxin (PMT),
Pseudomonas endotoxin, hemolysin, CAAX tetrapeptides which are potent
competitive
inhibitors of farnesyltransferase, cyclin inhibitors, Raf kinase inhibitors,
CDC kinase
inhibitors, caspases, p53, p16, and p21. Examples of bacteriocin family
members, include,
but are not limited to, bacteriocin release protein (BRP), ColEl, ColEla,
ColElb ColE2,
ColE3, ColE4, ColES, ColE6, ColE7, ColEB, ColE9, Colicins A, Colicin K,
Colicin L,
Colicin M, cloacin DF13, pesticin Al 122, staphylococcin 1580, butyricin 7423,
pyocin R1
or AP41, megacin A-216, and vibriocin. In a preferred embodiment, the gene
product of
interest is colicin E3. In another preferred embodiment, the gene product of
interest is BRP.
For example, Colicin E3 (ColE3) has been shown to have a profoundly
cytotoxic effect on mammalian cells (see, Smarda et al, 1978, Folia Microbiol.
23:272-277),
including a leukemia cell model system (see, Fiska et al, 1978, Experimentia
35: 406-407).
ColE3 cytotoxicity is a function of protein synthesis arrest, mediated by
inhibition of 805
ribosomes (Turnowsky et al., 1973, Biochem. Biophys. Res. Comm. 52:327-334).
More
specifically, ColE3 has ribonuclease activity (Saunders, 1978, Nature 274:113-
114). In its
naturally occurring form, ColE3 is a 60kDa protein complex consisting of a
SOkDa and a
l OkDa protein in a 1:1 ratio, the larger subunit having the nuclease activity
and the smaller
subunit having inhibitory function of the SOkDa subunit. Thus, the SOkDa
protein acts as a
cytotoxic protein (or toxin), and the lOkDa protein acts as an anti-toxin.
Accordingly, in
one embodiment, when ColE3 is used as a secondary marker gene product, the
larger ColE3
subunit or an active fragment thereof is expressed alone or at higher levels
than the smaller
subunit. In a preferred mode of the embodiment, ColE3 expression is
accompanied by
BRP expression to enhance release into the tumor environment. In yet another
embodiment
of the invention, the ColE3 SOkDa toxin and l OkDa anti-toxin are encoded on a
single
plasmid within an attenuated tumor-targeted bacteria, such as Salmonella. In
this
embodiment, the toxin/anti-toxin can act as a selection system for the
Salmonella which
carry the plasmid, such that Salmonella which lose the plasmid are killed by
the toxin. In
another embodiment, the l OkDa anti-toxin is on the chromosome, separate from
the colE3
toxin on the plasmid, resulting in a barner to transmission of other bacteria
(see, Diaz et al.,
1994, Mol. Microbio. 13:855-861).
In another preferred mode of this embodiment, the bacteriocin is cloacin
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DF13. Cloacin DF13 functions in an analogous manner to ColE3. The protein
complex is
of 67KDa molecular weight. The individual components are 57kDa and 9kDa in
size. In
addition to its ribonuclease activity, DF 13 can cause the leakage of cellular
potassium.
In yet another preferred mode of this embodiment, the bacteriocin is
colicin V (See, e.g., Pugsley, A.P. and Oudega, B. "Methods for Studing
Colicins and their
Plasmids" Plasmids a Practical Approach, 1987, ed. by K.G. Hardy; Gilson, L.
et al., 1990,
The EMBO Journal vol. 9 pp3875-3884).
In another embodiment, the bacteriocin is selected from the group consisting
of colicin E2 (a dual subunit colicin similar to ColE3 in structure but with
endonuclease
rather than ribonuclease activity); Colicins A, E1, Ia, Ib, or K, which form
ion-permeable
channels, causing a collapse of the proton motive force of the cell and
leading to cell death;
colicin L which inhibits protein, DNA & RNA synthesis; colicin M which causes
cell sepsis
by altering the osmotic environment of the cell; pesticin Al 122 which
functions in a
manner similar to colicin B function; staphycoccin 1580, a pore-forming
bacteriocin;
butyricin 7423 which indirectly inhibits RNA, DNA and protein synthesis
through an
unknown target; Pyocin P1, or protein resembling a bacteriophage tail protein
that kills cells
by uncoupling respiration from solute transport; Pyocin AP41 which has a
colicin E2-like
mode of action; and megacin A-216 which is a phospholipase that causes leakage
of
intracellular material (for a general review of bacteriocins, see Konisky,
1982, Ann. Rev.
Microbio1.36:125-144).
In yet another mode of this embodiment, the bacteriocin is BRP. The
cytotoxic activity of BRP is mediated by the release of cellular components.
In another specific embodiment, the gene products of interest are tumor
inhibitory enzymes or functional fragments thereof. Examples of tumor
inhibitory enzymes
include, but are not limited to, methionase, asparaginase, lipase,
phospholipase, protease,
ribonuclease (excluding colE3), DNAse, and glycosidase. In a preferred
embodiment, the
primary marker gene product is methionase.
In a particular embodiment, the gene product of interest comprises a number
of viral gene products. For example, the gene product of interest comprises
all the viral
proteins encoded by an adenovirus or herpes virus genome. In a particular
example, the
gene product of interest is all the viral proteins encoded by an adenovirus
genome except
for the E1B viral protein such that this particular adenovirus can only
replicate in a
mammalian cell lacking p53 activity.
Additional types of cellular toxins that may be delivered according to the
methods of the present invention are antibody molecules that are preferably
expressed
within the target cell; hence, these antibody molecules have been given the
name
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"intrabodies." Conventional methods of antibody preparation and sequencing are
useful in
the preparation of intrabodies and the nucleic acid sequences encoding same;
it is the site of
action of intrabodies that confers particular novelty on such molecules. (For
a review of
various methods and compositions useful in the modulation of protein function
in cells via
the use of intrabodies, see International Application WO 96/07321).
Intrabodies are antibodies and antibody derivatives (including single-chain
antibodies or "SCA") introduced into cells as transgenes that bind to and
incapacitate an
intracellular protein in the cell that expresses the intrabodies or
derivatives. As used herein,
intrabodies encompass monoclonals, single chain antibodies, V regions, and the
like, as
long as they bind to the target protein. Intrabodies to proteins involved in
cell replication,
tumorigenesis, and the like (e.g., HERZ/neu, VEGF, VEGF receptor, FGF
receptor, FGF)
are especially useful. The intrabody can also be a bispecific intrabody. Such
a bispecific
intrabody is engineered to recognize both (1) the desired epitope and (2) one
of a variety of
"trigger" molecules, e.g., Fc receptors on myeloid cells, and CD3 and CD2 on T
cells, that
have been identified as being able to cause a cytotoxic T cell to destroy a
particular target.
For example, antibodies to HER2/neu (also called erbB-2) may be used to
inhibit the function of this protein. HER2/neu has a pivotal role in the
progression of
certain tumors, human breast, ovarian and non-small lung carcinoma. Thus,
inhibiting the
function of HER2/neu may result in slowing or halting tumor growth (see, e.g.
U.5. Patent
No.5,587,458).
Nucleic acid molecules and oligonucleotides for use as described herein can
be synthesized by any method known to those of skill in this art (see, e.g.,
International
Publication WO 93/01286, U.S. Patent Nos. 5,218,088; 5,175,269; 5,109,124).
Identification of oligonucleotides and ribozymes for use as antisense agents
and DNA
encoding genes for targeted delivery for genetic therapy involve methods well
known in the
art. For example, the desirable properties, lengths and other characteristics
of such
oligonucleotides are well known. Antisense oligonucleotides may be designed to
resist
degradation by endogenous nucleolytic enzymes using linkages such as
phosphorothioate,
methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate,
phosphoramidate,
phosphate esters, and the like (see, e.g., Stein in: Oligodeoxynucleotides,
Antisense
Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp. 97-117,
1989);
Jager et al., 1988, Biochemistry 27:7237).
Particularly useful antisense nucleotides and triplex molecules are molecules
that are complementary to bind to the sense strand of DNA or mRNA that encodes
a protein
involved in cell proliferation, such as an oncogene or growth factor, (e.g.,
bFGF, int-2, hst-
1/K-FGF, FGF-5, hst-2/FGF-6, FGF-8). Other useful antisense oligonucleotides
include
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those that are specific for IL-8 (see, e.g., U.S. Patent No. 5,241,049), c-
src, c fos H-ras
(lung cancer), K-ras (breast cancer), urokinase (melanoma), BCL2 (T-cell
lymphoma), IGF-
1 (glioblastoma), IGF-1 (glioblastoma), IGF-1 receptor (glioblastoma), TGF-
ail, and
CRIPTO EGF receptor (colon cancer). These particular antisense plasmids reduce
tumorigenicity in athymic and syngenic mice.
The nucleotide sequences of the genes encoding these gene products are well
known (see GenBank). A nucleic acid molecule encoding one of the may be
isolated by
standard methods, such as amplification (e.g., PCR), probe hybridization of
genomic or
cDNA libraries, antibody screening of expression libraries, chemically
synthesized or
obtained from commercial or other sources.
Nucleic acid molecules and oligonucleotides for use as described herein can
be synthesized by any method known to those of skill in this art (see, e.g.,
International
Publication WO 93/01286, U.S. Patent Nos. 5,218,088; 5,175,269; 5,109,124).
Identification of oligonucleotides and ribozymes for use as antisense agents
involve
methods well known in the art.
In certain embodiments, the gene product of interest is a fragment, analog, or
variant of the wild-type full-length gene product, or a nucleic acid encoding
the same. The
derivative, analog or variant is functionally active, e.g., capable of
exhibiting one or more
functional activities associated with a full-length, wild-type marker gene
product. As one
example, such derivatives, analogs or variants which have the desired
therapeutic properties
can be used to inhibit tumor growth. Derivatives or analogs of an marker gene
product can
be tested for the desired activity by procedures known in the art, including
those described
herein.
In particular, variants can be made by altering gene sequences by
substitutions, additions (e.g., insertions) or deletions that provide
molecules having the
same or increased anti-tumor function relative to the wild-type marker gene
product. For
example, the variants of the invention include, but are not limited to, those
containing, as a
primary amino acid sequence, all or part of the amino acid sequence of the
gene product,
including altered sequences in which functionally equivalent amino acid
residues are
substituted for residues within the sequence resulting in a silent change,
i.e., the altered
sequence has at least one conservative substitution.
Any of the gene product of interest-encoding nucleic acids that are of
mammalian origin can be altered to employ bacterial codon usage by methods
known in the
art. Preferred codon usage is exemplified in Current Protocols in Molecular
Biology, Green
Publishing Associates, Inc., and John Wiley & Sons, Inc. New York, and Zhang
et al.,
1991, Gene 105: 61.
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In certain embodiments, the gene product of interest is expressed as a fusion
protein. In a specific embodiment, a gene product is constructed as a chimeric
or fusion
protein comprising the gene product or a fragment thereof joined at its amino-
or
carboxy-terminus via a peptide bond to an amino acid sequence of a different
protein. In
$ one mode of this embodiment, such a chimeric protein is produced by
recombinant
expression of a nucleic acid comprising or encoding the gene product of
interest, e.g.,
comprising a TNF encoding sequence, joined in-frame to a coding sequence for a
different
protein. Such a chimeric product can be made by ligating the appropriate
nucleic acid
sequences encoding the desired amino acid sequences to each other by methods
known in
the art, in the proper coding frame, and expressing the chimeric product into
the expression
vehicle of choice by methods commonly known in the art. Chimeric nucleic acids
comprising portions of a nucleic acid comprising or encoding a gene product of
interest
fused to any heterologous protein-encoding sequence may be constructed. In a
specific
mode, the fusion protein comprises an affinity tag such as a hexahistidine
tag, or other
affinity tag that may be used in purification, isolation, identification, or
assay of expression.
In another specific mode of this embodiment, the fusion protein comprises a
protease
cleavage site such as a metal protease or serine cleavage site. In this
particular mode, it is in
some cases preferred that a protease site corresponding to a protease which is
active at the
site of a tumor is constructed into a fusion protein of the invention. In
certain embodiments,
an gene product of interest is constructed as a fusion protein to an Omp-like
protein, or
portion thereof (e.g., signal sequence, leader sequence, periplasmic region,
transmembrane
domain, multiple transmembrane domains, or combinations thereof; see supra,
Section 3.1
for definition of "Omp-like protein").
In another embodiment, the diagnostic imaging system, including but not
limited to streptavidin, also has antitumor activity. In one embodiment, the
diagnostic
imaging system is combined with BRP, such as BRP and streptavidin, where the
marker
gene product streptavidin has increased antitumor activity when secreted. In
another
embodiment, the agent chelated by streptavidin has antitumor activity, such as
would be
mediated by high specific localization of gamma-emitting radioactive biotin.
In another
embodiment, the agent chelated by streptavidin has antitumor activity when
activated by
another agent, such as when boron is activated by neutrons (Sano, 1999,
Bioconjug Chem.
10:905-911 ).
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In another embodiment, the present invention provides methods for local
delivery of one or more fusion proteins comprising a ferry peptide and a
marker gene
product to a solid tumor by tumor-targeted bacteria. Ferry peptides used in
fusion proteins
have been shown to facilitate the delivery of a polypeptide or peptide of
interest to virtually
any cell within diffusion limits of its production or introduction (see.,
e.g., Bayley, 1999,
Nature Biotechnology 17:1066-1067; Fernandez et al., 1998, Nature
Biotechnology 16:418-
420; and Derossi et al., 1998, Trends Cell Biol. 8:84-87). Accordingly,
engineering tumor-
targeted bacteria to express fusion proteins comprising a ferry peptide and a
marker gene
product enhances the ability of a marker gene product to be internalized by
tumor cells. In a
specific embodiment, tumor-targeted bacteria are engineered to express a
nucleic acid
molecule encoding a fusion protein comprising a ferry peptide and a marker
gene product.
In another embodiment, tumor-targeted bacteria are engineered to express one
or more
nucleic acid molecules encoding one or more fusion proteins comprising a ferry
peptide and
a marker gene product. Examples of ferry peptides include, but are not limited
to, peptides
derived from the HIV TAT protein, the antennapedia homeodomain (penetratin),
Kaposi
fibroblast growth factor (FGF) membrane-translocating sequence (MTS), and
herpes
simplex virus VP22.
The present invention also provides methods for local delivery of one or
more fusion proteins comprising a signal peptide, ferry peptide and an marker
gene product
to a solid tumor by tumor-targeted bacteria. In a specific embodiment, tumor-
targeted
bacteria are engineered to express one or more nucleic acid molecules encoding
one or more
fusion proteins comprising a signal sequence, a ferry peptide and an marker
gene product.
The present invention also provides methods for local delivery of one or
more fusion proteins comprising a signal peptide, a proteolytic cleavage site,
a ferry peptide
and an marker gene product to a solid tumor by tumor-targeted bacteria. In a
specific
embodiment, tumor-targeted bacteria are engineered to express one or more
nucleic acid
molecules encoding one or more fusion proteins comprising a signal sequence, a
proteolytic
cleavage site, a ferry peptide and an marker gene product.
The present invention also provides methods for local delivery of one or
more fusion proteins of the invention and one or more marker gene products of
the
invention to the site of a solid tumor by tumor-targeted bacteria. Preferably,
the expression
of both the fusion proteins) and marker gene products) at the site of the
solid tumor by an
tumor-targeting bacteria improves the level of tumor or tumor cell growth
inhibited
compared to when either fusion proteins) alone or the marker gene products)
alone is
expressed.
As discussed above, in certain embodiments of the invention, the marker
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gene product can be a pro-drug converting enzyme or a pro-drug. The pro-drug
converting
enzyme can be fused or conjugated to a polypeptide therapeutic of the
invention.
Exemplary pro-drug converting enzymes are provided in Table 2 below.
TABLE 2
REPRESENTATIVE PRO-DRUG CONVERTING ENZYMES FOR USE IN VECTOR THERAPY
Enzyme Pro-drue Reference
Carboxypeptidasebenzoic acid mustards Bashawe et al., 1988;
G2 Springer et al.,
1990
aniline mustards Davies et al., 1994
phenol mustards Springer et al., 1990
Beta-glucuronidasep-hydroxyaniline mustard-Roffer et al., 1991
glucuronide
epirubicin-glucuronide Halsma et al., 1992
Mitaku et al., 1994
Penicillin-V-amidaseadriamycin-N phenoxyacerylKen et al., 1990
Penicillin-G-amidaseN-(4'-hydroxyphenyl acetyl)-Bignami et al., 1992
palytoxin doxorubicin
melphalan Vrudhula et al., 1993
R-lactamase nitrogen mustard-cephalosporinAlexander et al., 1991
~3-phenylenediamine
vinblastine derivative-cephalosporin
cephalosporin mustard Meyer et al., 1993
Svensson et al., 1993
p-glucosidase cyanophenylmethyl-(3-D-gluco-Rowlandson-Busza et
al., 1991
2$
pyranosiduronic acid
(i-galactosidasep-galactose-drug conjugatesFarquhar et al., 1997
(e.g.,
alkylating agents, purine
and
pyridmide antimetabolites"
anthracyclines, campothecin,
and
taxol)
3~
Nitroreductase 5-(adaridin-1-yl-)2, Knox et al., 1988; Somani
4- and
dinitrobenzamide Wilman, 1994
CarboxylesteraseCPT-11 (Irinotecan) Kojima et al., 1998
Cytosine deaminase5-FC (5-fluorocytosine) Hamstra et al., 1999;
Carboxypeptidasemethotrexate-alanine Haenseler et al., 1992
A
Bagshawe et al., 1988, Br. J. Cancer 58:700-703.
Springer et al., 1990, J. Med. Chem. 33:677-681.
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Davies et al., 1994, Ann. Oncol. 5 (Suppl 5):73(abstr).
Springer et al., 1994, A novel bisiodo-phenol mustard in antibody-directed
enzyme pro-drug therapy (ADEPT). In:
Programme of Eleventh Hammersmith Conference. Advances in the Application of
Monoclonal Antibodies.
London: Hammersmith Hospital (abstr).
Haisma et al., 1992(a), Cancer Immunol. Immunother. 34:343-348.
Roffler et al., 1991, Biochem. Pharmacol. 42:2062-2065.
Haisma et al., 1992(b), Br. J. Cancer 88:474-478.
Mitaku et al., 1994, Ann. Oncol. 5 (Suppl 5):76 (abstr).
Kerr et al., 1990, Cancer Immunol. Immunother. 31:202-206.
Bignami et al., 1992, Cancer Res. 52:5759-5764.
Vrudhula et al., 1993, J. Med. Chem. 38:919-923.
Alexander et al., 1991, Tetrahedron Lett. 32:3296-3272.
Meyer et al., 1993, Cancer Res. 53:3956-3963.
Svensson et al., 1992, Bioconj. Chem. 3:176-181.
Rowlandson-Busza et al., 1991, Cytotoxicity following specific activation of
amygladin. In: Monoclonal
Antibodies, Epenetos AA (ed), London: Chapman & Hall, pp. 179-183.
Farquhar et al., 1997, Proceedings of the American Association for Cancer
Research 38, p. 613-14, abstract no.
4121.
Knox et al., 1988, Biochem. Pharmacol. 41:4661-4669.
Somani et al., 1994, Ann. Oncol. 5 (Suppl 5):73 (abstr).
Kojima et al., 1998, J. Clin. Invest. 101(8):1789-96
Hamstra et al., 1999, Hum. Gene Ther. 10(12):1993-2003
Haenseler, E., Esswein, A., Vitols, K.S., Montejano, V., Mueller et al., 1992,
Biochemistry 31:214-220.
As discussed above, in certain embodiments, imaging and treatment are
combined in the same vector. In certain embodiments, the same recombinant gene
expressed by the vector is both a marker gene and a therapeutic. In one non-
limiting mode
of the embodiment, the marker gene substrate is radiolabeled. For example,
when the
marker gene is TK, the substrate can be radiolabeled FIAU or GCV (e.g.,'3'I-
FIAU or'3'I-
GCV), the corresponding marker metabolite, by virtue of its radioactivity,
will provide a
therapeutic benefit on the patient as well as permitting tumor imaging.
Similarly, wherein
the marker gene encodes an antigen and a labeled antibody administered for
imaging, the
antibody can be radiolabeled and the radiolabel would serve both as an imaging
tool and a
therapeutic agent. The same principle can be applied to other embodiments, for
example
those in which a labeled compound is administered that is incorporated into
the vector (such
as labeled DAP in asd mutant vectors as described above), the labeled compound
can be
labeled with a radionuclide that is used for both imaging and therapy.
5.7. Promoters for Control of Both Marker and Therapeutic Genes
The marker gene product and/or the anti-tumor gene product must be
expressed at the tumor site. Because the tumor can be in, but is not limited
to, a mammalian
or avian cell, the promoter controlling the expression of the marker gene
product and/or the
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anti-tumor gene product must be compatible with such cells. The mammalian cell
can be,
but is not limited to, human, canine, feline, equine, bovine, porcine, rodent,
etc. The choice
of promoter will depend on the type of target cell and the degree or type of
expression
control desired. Promoters that are suitable for use in the present invention
include, but are
not limited to, constitutive, inducible, tissue-specific, cell type-specific
and temporal-
specific and need not necessarily function in a mammalian cell. Another type
of promoter
useful in the present invention is an event-specific promoter which is active
or up-regulated
in response to the occurrence of an event, such as viral infection. For
example, the HIV
LTR is an event specific promoter. The promoter is inactive unless the tat
gene product is
present, which occurs upon HIV infection.
Exemplary promoters useful in the present invention include, but are not
limited to, the SV40 early promoter region (Bernoist and Chambon, 1981, Nature
290:304-
310), the promoter contained in the 3' long terminal repeat of Rous sarcoma
virus
(Yamamoto, et al., 1980, Cell 22:787-797), the herpes thymidine kinase
promoter (Wagner
et al., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the cytomegalovirus
("CMV")
promoter, the regulatory sequences of the tyrosinase gene which is active in
melanoma cells
(Siders et al., 1998, Gen. Ther. 5:281-291), the regulatory sequences of the
metallothionein
gene (Brinster et al., 1982, Nature 296:39-42); plant expression vectors
comprising the
nopaline synthetase promoter region (Herrera-Estrella et al., Nature 303:209-
213) or the
cauliflower mosaic virus 355 RNA promoter (Gardner, et al., 1981, Nucl. Acids
Res.
9:2871), and the promoter of the photosynthetic enzyme ribulose biphosphate
carboxylase
(Herrera-Estrella et al., 1984, Nature 310:11 S-120); promoter elements from
yeast or other
fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter,
PGK
(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, and the
following
animal transcriptional control regions, which exhibit tissue specificity and
have been
utilized in transgenic animals: elastase I gene control region which is active
in pancreatic
acinar cells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold
Spring Harbor
Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-S 15);
insulin gene
control region which is active in pancreatic beta cells (Hanahan, 1985, Nature
315:115-
122), immunoglobulin gene control region which is active in lymphoid cells
(Grosschedl et
al., 1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538; Alexander
et al.,
1987, Mol. Cell. Biol. 7:1436-1444), mouse mammary tumor virus control region
which is
active in testicular, breast, lymphoid and mast cells (Leder et al., 1986,
Cell 45:485-495),
albumin gene control region which is active in liver (Pinkert et al., 1987,
Genes and Devel.
1:268-276), alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al.,
1985, Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;
alpha 1
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antitrypsin gene control region which is active in the liver (Kelsey et al.,
1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active in myeloid
cells
(Mogram et al., 1985, Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94;
myelin basic
protein gene control region which is active in oligodendrocyte cells in the
brain (Readhead
et al., 1987, Cell 48:703-712); myosin light chain-2 gene control region which
is active in
skeletal muscle (Sani, 1985, Nature 314:283-286), prostate specific antigen
gene control
region which is active in prostate cells, and gonadotropic releasing hormone
gene control
region which is active in the hypothalamus (Mason et al., 1986, Science
234:1372-1378).
Another exemplary promoter is one that has enhanced activity in the tumor
environment; for example, a promoter that is activated by the anaerobic
environment of the
tumor such as the P1 promoter of the pepT gene. Activation of the P1 promoter
is
dependent on the FNR transcriptional activator (Strauch et al., 1985, J.
Bacteriol. 156:743-
751). In a specific embodiment, the P1 promoter is a mutant promoter that is
induced at
higher levels under anaerobic conditions than the native P1 promoter, such as
the pepT200
1 S promoter whose activity in response to anaerobic conditions is induced by
CRP-cAMP
instead of FNR (Lombardo et al., 1997, J. Bacteriol. 179:1909-1917). In
another
embodiment, an anaerobically-induced promoter is used, e.g., the potABCD
promoter.
potABCD is an operon that is divergently expressed from pepT under anaerobic
conditions.
The promoter in the pepT gene responsible for this expression has been
isolated (Lombardo
et al., 1997, J. Bacteriol. 179:1909-1917).
Yet another exemplary promoter is an antibiotic-induced promoter, such as
the tet promoter of the TnlO transposon. Alternatively, the promoter can be an
antibiotic-
induced promoter, such as the tet promoter of the TnlO transposon. In a
preferred
embodiment, the tet promoter is a single-mer, which single-mer responds in an
all-or-
nothing manner to the presence of tetracycline and provides a genetically
stable on-off
switch. In another embodiment, the tet promoter is multimerized, for example
three-fold.
Such a multimer responds in a graded manner to the presence of tetracycline
and provides a
more manipulable system for control of gene expression. Promoter activity
would then be
induced by administering to a subject who has been treated with the attenuated
tumor-
targeted bacteria of the invention an appropriate dose of tetracycline.
Although the tet
inducible expression system was initially described for eukaryotic systems
such as
Schizosaccharomyces pombe (Faryar and Gatz, 1992, Current Genetics 21:345-349)
and
mammalian cells (Lang and Feingold, 1996, Gene 168:169-171), recent studies
extend its
applicability to bacterial cells. For example, Stieger et al. (1999, Gene
226:243-252) have
shown 80-fold induction of the firefly luciferase gene upon tet induction when
operably
linked to the tet promoter. Optimization of the tet expression system for use
according to
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the methods of the invention is described in Section 6 below. An advantage of
this
promoter is that it is induced at very low levels of tetracycline,
approximately 1/100th of the
dosage required for antibiotic activity.
In addition to the promoter, repressor sequences, negative regulators, or
S tissue-specific silencers can be inserted to reduce non-specific expression
of the gene
product. Moreover, multiple repressor elements may be inserted in the promoter
region.
One type of repressor sequence is an insulator sequence. Illustrative examples
of repressor
sequences which silence background transcription are found in Dunaway et al.,
1997, Mol.
Cell Biol. 17:182-129; Gdula et al., 1996, Proc. Natl. Acad. Sci. USA 93:9378-
9383; Chan
et al., 1996, J. Virol. 70:5312-5328. In certain embodiments, sequences which
increase the
expression of the gene product can be inserted in the expression vector, e.g.,
ribosome
binding sites. Expression levels of the transcript or translated product can
be assayed by
any method known in the art to ascertain which promoter/repressor sequences
affect
expression.
5.8. Methods And Compositions For Treatment
According to the present invention, a first tumor-targeted bacterial vector
which expresses a first gene product is used for imaging or monitoring a
tumor. Such first
tumor-targeted bacterial vector for imaging or monitoring may be used alone or
together
with a second tumor-targeted bacterial vector which expresses a second gene
product used
to produce a tumor growth inhibitory response or a reduction of tumor volume
in a subject
(including but not limited to a human patient) having a solid tumor cancer. In
one
embodiment of the present invention, the method comprises administering to a
subject, a
pharmaceutical composition comprising an effective amount of facultative
aerobic or
facultative anaerobic tumor-targeted bacteria suitable for imaging or
monitoring a tumor. In
another embodiment of the present invention, the method comprises
administering to a
subject, a pharmaceutical composition comprising an effective amount of a
first facultative
aerobic or facultative anaerobic, attenuated, tumor-targeted bacteria suitable
for imaging or
monitoring a tumor and a second facultative aerobic or facultative anaerobic,
attenuated,
tumor-targeted bacteria suitable for producing a tumor growth inhibitory
response or a
reduction of tumor volume in the subject. In yet another embodiment, the
method
comprises administering, to a subject, a pharmaceutical composition comprising
an
effective amount of a facultative aerobic or facultative anaerobic,
attenuated, tumor-targeted
bacterial vector which has been genetically modified to express a gene product
of interest,
which aids in reducing the volume, or inhibiting the growth of the tumor, in
combination
with one or more a pharmaceutical compositions comprising an effective amount
of
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facultative aerobic or facultative anaerobic tumor-targeted bacteria suitable
for imaging or
monitoring a tumor. In a preferred embodiment, the method comprises
administering, to a
subject, a pharmaceutical composition comprising an effective amount of a
facultative
aerobic or facultative anaerobic, attenuated, tumor-targeted Salmonella spp.
which
expresses a gene product of interest which aids in reducing the volume or
inhibiting the
growth of a solid tumor together with a pharmaceutical composition comprising
an effective
amount of facultative aerobic or facultative anaerobic tumor-targeted bacteria
suitable for
imaging or monitoring a tumor.
Solid tumors include, but are not limited to, sarcomas, carcinomas or other
solid tumor cancers, such as renal carcinoma, mesoendothelioma, bladder
cancer, germ line
tumors and tumors of the central nervous system, including, but not limited
to, breast
cancer, prostate cancer, cervical cancer, uterine cancer, lung cancer, ovarian
cancer,
testicular cancer, thyroid cancer, astrocytoma, glioma, pancreatic cancer,
stomach cancer,
liver cancer, colon cancer, and melanoma. The subject is preferably an animal,
including
but not limited to animals such as cows, pigs, chickens, etc., and is
preferably a mammal,
and most preferably human. Effective treatment of a solid tumor, includes but
is not limited
to, inhibiting tumor growth, reducing tumor volume, etc.
The amount of the pharmaceutical composition of the invention which is
effective in imaging or monitoring or the treatment of a solid tumor cancer
will depend on
the nature of the solid tumor, and can be determined by standard clinical
techniques. In
addition, in vitro assays may optionally be employed to help identify optimal
dosage
ranges. The precise dose to be employed will also depend on the route of
administration,
and the seriousness of the solid tumor, and should be decided according to the
judgment of
the practitioner and each patient's circumstances. However, suitable dosage
ranges are
generally from about 1.0 c.f.u./kg to about 1 x 10'° c.f.u./kg;
optionally from about 1.0
c.f.u./kg to about 1 x 109 c.f.u.lkg; optionally from about 1.0 c.f.u./kg to
about 1 x 108
c.f.u./kg; optionally from about 1 x 10z c.f.u./kg to about 1 x 109 c.f.u./kg;
optionally from
about 1 x 104 c.f.u./kg to about 1 x 109 c.f.u./kg; optionally from about 1 x
102 c.f.u./kg to
about 1 x 10g c.f.u./kg; optionally from about 1 x 104 c.f.u.lkg to about 1 x
10$ c.f.u./kg.
Effective doses may be extrapolated from dose-response curves derived from in
vitro or
animal model test systems.
Various delivery systems are known and can be used to administer a
pharmaceutical composition of the present invention. Methods of introduction
include but
are not limited to intradermal, intramuscular, intraperitoneal, intravenous,
subcutaneous,
intranasal, epidural, and oral routes. The compounds may be administered by
any
convenient route, for example by infusion or bolus injection, by absorption
through
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epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal
mucosa, etc.)
and may be administered together with other biologically active agents.
Administration can
be systemic or local. In addition, it may be desirable to introduce the
pharmaceutical
compositions of the invention into the central nervous system by any suitable
route,
including intraventricular and intrathecal injection; intraventricular
injection may be
facilitated by an intraventricular catheter, for example, attached to a
reservoir, such as an
Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use
of an
inhaler or nebulizer, and formulation with an aerosolizing agent.
In a specific embodiment, it may be desirable to administer the
pharmaceutical compositions of the invention locally to the area in need of
treatment; this
may be achieved by, for example, and not by way of limitation, local infusion
during
surgery, by injection, by means of a catheter, or by means of an implant, said
implant being
of a porous, non-porous, or gelatinous material, including membranes, such as
sialastic
membranes, or fibers. In one embodiment, administration can be by direct
injection at the
1 S site (or former site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
In another embodiment, the bacteria or bacterial vector can be delivered in a
controlled release system. In one embodiment, a pump may be used (see Langer,
supra;
Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980,
Surgery 88:507;
Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment,
polymeric
materials can be used (see Medical Applications of Controlled Release, 1974,
Langer and
Wise (eds.), CRC Pres., Boca Raton, Florida; Controlled Drug Bioavailability,
Drug
Product Design and Performance, 1984, Smolen and Ball (eds.), Wiley, New York;
Ranger
and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy
et al.,
1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et
al., 1989,
J. Neurosurg. 71:105). In yet another embodiment, a controlled release system
can be
placed in proximity of the therapeutic target, i.e., the brain, thus requiring
only a fraction of
the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of
Controlled Release,
supra, vol. 2, pp. 115-138).
Other controlled release systems are discussed in the review by Langer
(1990, Science 249:1527-1533).
In a preferred embodiment, the composition is formulated in accordance with
routine procedures as a pharmaceutical composition adapted for intravenous
administration
to human beings. Typically, compositions for intravenous administration are
solutions in
sterile isotonic aqueous buffer. Where necessary, the composition may also
include a
solubilizing agent and a local anesthetic such as lidocaine to ease pain at
the site of the
injection. Generally, the ingredients are supplied either separately or mixed
together in unit
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dosage form, for example, as a dry lyophilized powder or water free
concentrate in a
hermetically sealed container such as an ampoule or sachette indicating the
quantity of
active agent. Where the composition is to be administered by infusion, it can
be dispensed
with an infusion bottle containing sterile pharmaceutical grade water or
saline. Where the
composition is administered by injection, an ampoule of sterile water for
injection or saline
can be provided so that the ingredients may be mixed prior to administration.
5.9. Considerations For Pharmaceutical Compositions
A composition of the invention should be administered in a carrier that is
phaceutically acceptable. The term "pharmaceutically acceptable" means
approved by
a regulatory agency of the Federal or a state government or listed in the U.S.
Pharmacopeia
or other generally recognized pharmacopeia or receiving specific or individual
approval
from one or more generally recognized regulatory agencies for use in animals,
and more
particularly in humans. The term "carner" refers to a diluent, adjuvant,
excipient, or vehicle
with which the therapeutic is administered. Such pharmaceutical Garners can be
sterile
liquids, such as water and oils, including those of petroleum, animal,
vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
Buffered saline
is a preferred carrier when the pharmaceutical composition is administered
intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can also be
employed as liquid
carriers, particularly for injectable solutions. The composition, if desired,
can also contain
minor amounts of wetting or emulsifying agents, or pH buffering agents. These
compositions can take the form of solutions, suspensions, emulsion and the
like. Examples
of suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences"
by E.W. Martin. Such compositions will contain a therapeutically effective
amount of the
Therapeutic, preferably in purified form, together with a suitable amount of
Garner so as to
provide the form for proper administration to the patient. The formulation
should suit the
mode of administration. In a preferred embodiment, the composition is
formulated in
accordance with routine procedures as a pharmaceutical composition adapted for
intravenous administration to human beings. Typically, compositions for
intravenous
administration are solutions in sterile isotonic aqueous buffer.
The reported serious complications and deaths following administration of
FIAU in patients raise concerns regarding the administration of FIAU in
clinical trials,
including radiolabeled imaging trials. It should be noted, however, that
serious FIAU-
related toxicities are dependent on total dose and length of administration.
In the FIAU
imaging methods, FIAU is given as a single dose and at doses considerably
lower than that
used in prior clinical trials (32 ng as compared to 1176 - 2940 mg). The
calculations for the
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actual dose (mg) of FIAU used in imaging are given below. The radiosynthesis
of several
2'-fluoro nucleosides has been described including carrier-added and no
carrier-added
syntheses. The mode of synthesis, % yield, specific activity and the mass-dose
equivalent is
presented in Table 3.
S
Table 3. Radiosynthesis fluoronucleosides
of
2'-
2'- StudyMode of SynthesisYield Specific Dose Dose Equiv.
Activity
fluoro- (ref. (%) (GBq/mmol) (MBq) (mg)
#)
nucleo-
side
FIAU [1] ['z3I], ['3'I]*~90% 110 - 600 148 0.092 -
0.49
[2] ["'I]* - 100 - 200 148 0.28 - 0.56
f no carrier-added~45% - 148 0.000032
11
*carrier-added: 25 mg NaI
[1] Misra et al., 1986, Appl. Radiat. Isot. 37:901-905.
[2] Tovell et al., 1987, J. Med. Virol. 22:183-188.
The mass of radiopharmaceutical produced from a no Garner-added synthesis
can be calculated using Avogadro's number, the half life of the radionuclide
(in sec), the
molecular weight of the radiopharmaceutical and the administered dose (in Bq).
The
calculation of the mass-dose equivalent for a no carrier-added synthesis of
[1241]-FIAU is
presented:
Avogadro's Number (Na) - 6.023 x 1023 atoms/mole
[124I~ half life (tyz) - 3.46 x 105 sec
[124I] _ FIAU dose (Bq) - 148 MBq (4mCi)
[124I] _ FIAU molecular weight (MW) - 376 g/mole
Number of radiolabeled molecules (Nm) - Bq (dps) x tl~z (sec)
- ( 1.48 x 106) x (3.46 x 105)
- 5.12 x 1013 molecules
Moles - Nm/Na
- 5.12 x 1013 molecules / 6.023 x 1023
molecules/mole
- 8.50 x 10-11 moles
Mass (for a no carrier-added synthesis) - moles x MW
- 8.50 x 10-11 moles x 376 g/mole
- 3.2 x 10-8 g
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- 32 n~
Review of all published phase 1 and phase 2 clinical studies involving FIAU
administration, the published radiosynthesis procedures for labeling FIAU, and
the
calculation of the mass-dose equivalent for a no carrier-added synthesis of
[1241]_FIAU
clearly demonstrates that the potential for toxicity associated with the
projected diagnostic
studies is very verb low. For example, the equivalent mass for a 148 MBq (4
mCi) dose of
no Garner-added [1241]_FIAU proposed for PET imaging studies in patients is
only 32 ng.
A dose of 32 ng of FIAU represents only 1/110,000 to 1/3,720,000 of the daily
dose (and
3.1x10-5 to 1.6x10-7 of the total dose) that was administered to patients in
the 14 to 28 day
hepatitis B clinical trials, where no FIAU-related toxicity was observed. Only
in the 168
day clinical trial (H3X-PPPC) did severe liver, pancreatic and neurotoxicity
appear.
Even assuming the synthesis results in a 1000-fold lower specific activity
compared to a no carrier-added synthesis, this would amount to a pharmacologic
dose of 32
pg of FIAU. A 32 pg dose of FIAU represents less than 0.03 to 1.0 percent of
the daily
dose that was administered to patients in the 14 to 28 day hepatitis B
clinical trials, where
no FIAU-related toxicity was observed.
5.10. Methods of Delivery
Various delivery systems are known and can be used to administer a
pharmaceutical composition of the present invention. Methods of introduction
include but
are not limited to intradermal, intramuscular, intraperitoneal, subcutaneous
and intravenous.
Administration can be systemic or local. In a specific embodiment, it may be
desirable to
administer the pharmaceutical compositions of the invention locally to the
area in need of
treatment; this may be achieved by, for example, and not by way of limitation,
local
infusion during surgery, by injection or by means of a catheter. In one
embodiment,
administration can be by direct injection at the site (or former site) of a
tumor. The present
invention, the tumor-targeted bacteria or bacterial vectors which express a
gene product of
interest and the pro-drug treatment, may be advantageously used in a
combination method
with one or more doses of irradiation to produce a tumor growth inhibitory
response or a
reduction of tumor volume, in a subject, including a human patient, having a
solid tumor
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cancer.
5.11. Kits
The invention also provides kits for carrying out the non-invasive detection
methods of the invention and kits for carrying out the non-invasive detection
and treatment
methods of the invention. Such kits comprise in one or more containers a
purified
population of tumor-targeted bacteria and a labeled moiety (including but not
limited to
biotin, an antibody or a chemotactic peptide), substrate, or compound which is
useful for the
non-invasive imaging in conjunction with said population of tumor-targeted
bacteria. The
label can be a radionuclide, paramagnetic ion or fluorophore. In one
embodiment, the label
is sequestered.
In one embodiment, the tumor-targeted bacteria harbor a recombinant marker
gene operably linked to a promoter. In a preferred mode of the embodiment, the
marker
gene is streptavidin. In another embodiment, the tumor-targeted bacteria is a
mutant with
an enhanced preference to incorporate a labeled compound. In a preferred mode
of the
embodiment bacteria is mutant for the asd locus, most preferably only a
partial mutant for
said locus.
In other embodiments, the kit further comprises a second population of
tumor-targeted bacteria suitable for tumor therapy in the container in which
the tumor-
targeted bacteria useful for imaging is present or in a separate container. In
yet other
embodiments, the tumor-targeted bacteria suitable for imaging is also suitable
for therapy.
In other embodiments, the kit further comprises a pharmaceutically
acceptable carrier in the container in which the tumor-targeted bacteria is
present or in a
separate container.
Instructions are optionally included for using the bacteria to carry out the
non-invasive imaging methods of the present invention.
Optionally associated with such kits can be a notice in the form prescribed
by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or
biological products, which notice reflects approval by the agency of
manufacture, use or
sale for human administration.
5.12. Exemplary Embodiment: Thymidine Kinase as a Marker Gene
As described above, the invention provides indirect methods of tumor
imaging comprising administering to the subject a tumor-targeted bacterial
vector
containing a marker gene, wherein the tumor-targeted bacterial vector targets
to the tumor
and/or infects cells of the tumor, under conditions in which the marker gene
is expressed in
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the tumor-targeted bacterial vector, thereby generating a marker gene product;
administering to the subject a labeled marker substrate under conditions in
which the
labeled marker substrate is metabolized by the marker gene product to produce
a labeled
marker metabolite which is substantially retained in the tumor-targeted
bacterial vector
$ throughout a time-period sufficient for imaging the labeled marker
metabolite; and scanning
the subject to detect the labeled marker metabolite, thereby imaging and
detecting a tumor
in the subj ect.
Merely for purposes of illustration and not limitation, the gene and a labeled
marker substrate are selected as a self complementary pair from the group
consisting o~
wild-type, natural mutant, or genetically-engineered herpes simplex virus-
thymidine kinase
or varicella zoster virus-thymidine kinase gene and a labeled nucleoside
analog such as
FIAU, ACV or GCV. In a particular mode of the embodiment, the nucleoside
analog is a
2'-fluoro-nucleoside such as FIAU.
The labeled 2'-fluoro-nucleoside analogue can be $-['z3I]-, $-[lzal]- or
1$ $-['3'I]-2'-fluoro-$-iodo-1-13-D-arabinofuranosyl- uracil; $-['8F]-2'-
fluoro-$-fluoro-1-13-D-
arabinofuranosyl- uracil; 2-["C]- or $-(["C]-methy)-2'-fluoro-$-methyl-1-13-D-
arabinofuranosyl- uracil; 2-["C]- or $-(["C]-ethyl)-2'-fluoro-$-ethyl-1-13-D-
arabinofuranosyl- uracil; $-(2-['8F]-ethyl)-2'-fluoro-$-(2- fluoro-ethyl)-1-13-
D-
arabinofuranosyl- uracil; $-['z3I]-, $-['z4I]- or $-['3'I]-2'-fluoro-$-
iodovinyl-113-D-
ar'abinofuranosyl- uracil; $-['z3I]-, $-[lzal]- or $-['3'I]-2'- fluoro-$-iodo-
1-13-
D-arabinofuranosyl- uracil; or $-['z3I]-, $-[lza I]- or 5-['3'I]-2'-fluoro-$-
iodovinyl-1-13-D-
aribofuranosyl- uracil.
In certain embodiments, the TK can have a therapeutic as well as imaging
purpose. Upon concurrent expression of the TK and administration of a labeled
pro-drug
2$ (e.g. a labeled ganciclovir or acyclovir) to the subject, the pro-drug is
phosphorylated in the
periplasm of the microorganism which is freely permeable to nucleotide
triphosphates. The
labeled metabolites, phosphorylated ganciclovir or acycolvir, which are toxic
false DNA
precursors, readily pass out of the periplasm of the microorganism and into
the cytoplasm
and nucleus of the host cell. In addition to providing the signal for imaging,
the
phosphorylated ganciclovir or acycolvir incorporate into host cell DNA,
thereby causing the
death of the host cell.
In another embodiment, two recombinant tk genes are administered, either
by way of two different bacterial vectors or a single bacteria vector
comprising two
recombinant tk genes. The two recombinant tk genes are selected for different
substrate
3$ specificity and subcellular localization. For example, the imaging mutant
tk gene is
retained in the cytosol of a first tumor-targeted bacterial vector and the
treatment mutant tk
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gene is in the periplasm of a second tumor-targeted bacterial vector.
6. Examples
The following series of examples are presented by way of illustration and not
by way of limitation on the scope of the invention. Instead, the examples are
meant to
illustrate certain variations of genetically engineered tumor-targeted
Salmonella useful for
the methods of the invention to detect, monitor and/or treat solid tumors.
6.1. Example: Tumor Detection Using Tumor-targeted
Salmonella Expressing HSVl-TK
The initial study was conducted using s.c. implanted tumors to provide a
standard easily reproduced tumor model. Mice (C57B/6) were subcutaneously
implanted
with C38 colon tumor cells and staged until the tumors were palpable (20
days).
Salmonella expressing HSV 1-TK from plasmid p5-3 (Pawelek et al., 1997, Cancer
Res.
57:4537-4544) in VNP20009 [strain YS1645 purl , msbB-, xyl, tets, amps] (ATCC#
202165; WO 96/40238) were injected i.v. (2x106 cfu/animal) and after four
days, 10 pCi
['4C]-FIAU was injected i.v. After 23 hours, the animals were sacrificed and
muscle, liver
and tumor tissue samples removed and homogenized for radioactivity (DPM) and
bacterial
cfu determinations per gram of tissue. The accumulation of ['4C]-FIAU is
presented in
Table 4.
Table 4
Sample mouse # cfu/gram tissue DPM/gram tissue
-bacteria
muscle 1 - 474
muscle 2 - 949
liver 1 - 9800
liver 2 - 8394
tumor 1 - 9761
tumor 2 - 5 818
+bacteria
muscle 3 5.0x103 474
muscle 4 4.6x 103 949
liver 3 7.1x105 9800
liver 4 1.3x106 8394
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tumor 3 1.2x109 9761
tumor 4 1.0x109 5818
A sample of tumor and liver from mouse 3 were subjected to QAR.
Cryosections were cut and exposed for quantitative analysis. The printouts of
tumor and
liver sections are shown in FIG. 1 and 2. In each of these figures, histology
is on the left,
digital autoradiogram is on the right and the merged image is in the center.
These figures clearly document the ability of Salmonella expressing HSV1-
TK to specifically accumulate ['4C]-FIAU within the tumor, with little or no
accumulation
in the surrounding skin, and little or no relative accumulation in the liver.
Hence the tumor-
targeted Salmonella expressing HSV1-TK are useful when administered in
combination
with radiolabeled FIAU for tumor detection via in vivo imaging.
6.2. Example: Cytosolic HSVl-TK Expression
Expression of HSV 1-TK in the Salmonella cytoplasm increases signal
localization and detection over periplamic expression. A vector coding for
cytosolic HSV 1-
TK is made with PCR primers [forward 5'-GATCCCATGGCTTCGTACCCCGGCC-3'
(SEQ ID NO:1) and reverse 5'-CTAGAAGCTTCAGTGGCTATGGCAGGGC-3' (SEQ ID
N0:2)] to generate a product. The template for the PCR reaction is a plasmid
containing
the HSV 1-TK (McKnight, 1980, Nucleic Acids Research 8: 5949-5964). PCR is
performed
for example as 1 cycle of 95 °C for 5 min: 35 cycles of 95 °C
for 1 min; 55 °C for lmin;
72°C for 2 min: and 1 cycle of 72°C for 10 min using Ready-to-go
PCR beads (Pharmacia)
or equivalent. The approximately 1.5 kilobase PCR product is then gel
purified, followed
by restriction digestion with NcoI and HindIII and the product ligated into
the NcoI and
HindIII sites of pTrc99a (Pharmacia). TK activity is assayed using a
modification of the
method of Summers and Summers (1977, J. Virol. 24:314-318). The kinase
substrates
['ZSIdC (Dupont/New England Nuclear) or 3H-ganciclovir (Moravek Biochemicals)]
are
incubated with bacterial cell lysate at 37°C for 1 hour and then bound
to DE81 paper
(Whatman). After washing, the associated radioactivity is determined in a
scintilation
counter.
6.3. Example: Enhanced Specificity of HSVl-TK For FIAU
Using a combination of positive and negative selection (Black et al., 1996,
PNAS 9:3525-3529), active HSV1-tk mutants that are able to bind FIAU
preferentially over
thymidine can be selected. Six codons hypothesized as playing a role in
substrate binding
(Balasubramanian et. al., 1990, J. Gen. Virol. 71:2979-2987; Leu 159, Ile 160,
Phe 161, Ala
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168, Leu 169, and Leu 170) in wild type HSV 1-tk are targeted for random
mutagenesis.
The construction of the random sequence inserts is essentially as described in
Black et al.
(1996, PNAS 9:3$2$-3$29). Briefly, two random oligonucleotides are
constructed: MB126
$'-TGGGAGCTCACATGCCCCGCCCCCGGCCCTCACC GACCG
$ CCATCCCATC-3' (SEQ ID N0:3) and MB127 $'-ATAAGGTACCGCGCGGCCGG
GTAGC GGCGATGGGATGGCGG-3' (SEQ ID N0:4) where N
designates all 4 nucleotides in a 100% random equimolar mixture. The oligos
are annealed,
extended and amplified by PCR essentially as described by Black and Loeb
(1993,
Biochem. 32:11618-11626). The oligos are purified a$er synthesis, annealed and
extended
with Klenow fragment. The annealed oligos are then amplified by PCR using the
primers:
P1 : $'-TGGGAGCTCACATGCCCCGCC-3' (SEQ ID NO:$) and P2: $'
ATGAGGTACCG-3' (SEQ ID N0:6). This amplification generates the random inserts
with the restriction sites SacI and KpnI.
In order to create the random library (Black and Hruby, 1990, J. Biol. Chem.
1$ 26$:17$84-17$94; Black et al., 1996, PNAS 9:3$2$-3$29), the random sequence
inserts
from above are ligated into the wild type HSV1-tk open reading frame to
replace the wild
type segment. However, to avoid background activity due simply to uncut wild
type HSV1-
tk vector, a dummy vector is created. This vector (pET23d backbone for high
overexpression in the BL21DE3 strain) contains the wild type HSV1-tk open
reading frame
with a piece of non-functional DNA at the insertion site. This vector has no
HSV 1-TK
activity unless there is successful replacement of the dummy fragment with a
fragment from
the random inserts that results in the production of an active enzyme. The
dummy and PCR
amplified random inserts are cut with KpnI and Sac I and gel isolated. The
vector and gel
purified PCR amplified random inserts are ligated with KpnI and SacI. E. coli
strain
2$ BL21(DE3) tdk- [F- ompT hsdSB(rB- mB-)gal dcm tdk (DE3)] ( Black et al.,
1996, PNAS
9:3$2$-3$29) is transformed with the ligated mixture. The endogenous HSV1-tk
in this
strain has been deleted so only those bacteria that receive a HSV 1-tk
construct that is active
will survive the initial positive selection screen.
In order to create a useful HSVl-TK that is able to bind FIAU with a high
affinity, the enzyme expressed must be an active enzyme. Any clone harboring a
HSV1-tk
construct that does not yield active HSV1-TK enzyme is eliminated. In order to
accomplish
this, the first selection of the library is based on HSV 1-TK activity, by the
method of Black
and Loeb (1993, Biochem. 32:11618-11626). After transformation and recovery,
the
transformants are plated onto LB + carbenicillin (carb 5°) to score
total transformants.
3$ Individual colonies are then picked and streaked onto HSV1-TK selection
media (2% BBL
Trypticase peptone, 0.$% NaCI, 0.8% Gel-Rite, 0.2% glucose, $Omg/mL carb,
lOmg/mL $'-
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fluorodeoxyuridine (FUdR), 2mg/mL thymidine, 12.5mg/mL uridine) and LB+carb
5° in
parallel. The basis of the selection is that FUdR is phosphorylated by HSV 1-
TK to form
FdUMP which is an inhibitor of thymidiylate synthase and thus the inhibition
of dTMP
production. The requirement for dTMP can therefore only be filled by an active
HSV 1-TK
enzyme. Since the endogenous HSV1-TK enzyme in strain KY895 has been
disrupted, the
only active HSV 1-TK must come from the random insert library. Only those
clones that
have received an active form of HSV1-TK will be able to grow on this selection
media.
Uridine is added to inhibit the thymidine phosphatase. HSV1-tk + clones are
scored after
incubation at 37°C for 24 hours.
Selection of FIAU sensitive clones is performed essentially as in the method
of Black et al. (1996, PNAS 9:3525-3529) with minor modifications. All
positive clones
from above are picked and inoculated into HSV1-TK selection media in a 96 well
plate. All
clones are serial diluted into saline and streaked onto HSV1-TK selection
plates that contain
2mg/ml thymidine or HSV1-TK selection plates that contain lmg/ml of thymidine
plus
decreasing concentrations of FIAU (2mg/ml to Omg/ml). Those clones that are
able to bind
and thus phosphorylate FIAU over thymidine are not able to grow on plates
containing
FIAU, since the HSV1-TK enzyme is bound to the FIAU and thus not available to
phosphorylate the thymidine needed for cell survival.
6.4. Example: Enhanced Activation of Ganciclovir by HSVl-TK
This example illustrates a genetically engineered Salmonella expressing a
mutant TK enzyme having an enhanced binding affinity for a pro-drug such as
ganciclovir.
Such Salmonella are useful in one embodiment of the invention, i.e., in a
method to detect
and treat a solid tumor. The enhanced-ganciclovir-affinity TK advantageously
provides a
therapeutic effect and is used in combination with Salmonella expressing TK
having an
affinity for radiolabeled FIAU which permits in vivo detection of a solid
tumor upon
administration of the bacteria.
Salmonella HSVl-TK, where the TK enzyme is mutant and capable of
binding ganciclovir at a higher affinity than wild type, is generated
according the method
published by Black et a1.(1996, PNAS 9:3525-3529). These authors demonstrated
the
isolation of an HSV 1-TK mutant with a 40-fold increase in specificity for
ganciclovir.
When this gene was expressed in tumor cells, a dramatic increase in
sensitivity to
ganciclovir was observed. The protein sequence of the naturally occurring form
and the
mutant are shown:
HSVl-TK 'S9LIFDRHPIAALLCYP"3 (SEQ ID N0:7)
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Mutant #75 'S9LLLDRHPIACMLCYP"3 (SEQ ID N0:8)
The mutant sequence is introduced into the coding sequence of HSV1-tk by PCR-
mediated
site directed mutagenesis (Ausubel et al., 1995, in Short Protocols in
Molecular Biolo~y,
third edition, NY:18.6-18.22). The mutations are introduced using two paired
sets of
primers, each of which generates approximately half of the complete sequence
with a slight
overlap and can be spliced together using a shared Sau3AI site engineered to
occur in the
overlap: Set 1 PCR primers (forward) 5'-GATCCCATGGCTTCGTACCCCGGCC-3' (SEQ
ID N0:9) and (reverse) 5'-CGGCGATCGG ATGGCGGTCGAGGAGGAGGG-3' (SEQ ID
NO:10); Set 2 PCR primers (forward) 5'- CCATCCGATC GCCGTCATGC TGTGC-3'
(SEQ ID NO:11) and (reverse) 5'-CTAGAAGCTTCAGTGGCTATGGCAGGGC-3' (SEQ
ID N0:12) are used to generate a product using wild type tk as a template,
followed by
restriction digestion with 1) PCR set 1, NcoI and Sau3AI, and 2) PCR set 2,
Sau3AI and
HindIII, and the two products simultaneously ligated into the NcoI and HindIII
sites of
pTrc99a (Pharmacia). Clones derived are tested for ganciclovir and FIAU
activation
profiles using the HSV1-TK enzyme assay based on Summers and Summers (1977, J.
Virol. 24:314-318) and described above.
The HSVl-tk mutant described above (mutant #75; Black et al., 1996,
PNAS 9:3525-3529) is a "super" mutant in that it has a higher affinity for
ganciclovir and a
lower affinity for thymidine than wild type HSV 1-TK. Because this enzyme has
these
properties it is more toxic to cells primarily because it is less able to bind
thymidine so there
is less competition in the cells for thymidine and thus more enzyme available
to bind and
metabolize the ganciclovir pro-drug.
6.5. Example: Optimal Time For Imaging After
Labeled FIAU Intravenous Administration
It has been repeatedly observed that Salmonella targeting of tumors
progresses over the first 3 to 5 days, with day 5 exhibiting the full
targeting potential in all
cases (Pawelek et al., 1997, Cancer Res. 57:4537-454; Zheng et al., 1997,
Annual Meeting
of the American Association for Cancer Research, abstract 60; King et al.,
1998, Annual
Meeting of the American Association for Cancer Research, abstract 3484; Low et
al., 1999,
Nature Biotech. 17:37-41). It is advantageous to determine the optimal time
for imaging
after radiolabeled FIAU administration in order to achieve a maximum "signal
to
background".
The optimal imaging times for in vivo tumor detection can be determined as
described below.
Mice (C57B/6) are implanted with C38 colon tumor cells and staged until
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the tumors are palpable. Salmonella expressing HSV1-TK or HSV1-"super" TK are
injected into the tumor and after five days ['3'I]- FIAU is injected
intravenously. Gamma
camera imaging is performed at various times, for examplel hr, 3 hr, 9 hr, 24
hr and 48 hr
after FIAU administration. The accumulation of radiolabeled FIAU present in
tumors and
selected organs can also be determined in animals sacrificed for example at 24
and 48 hours
post FIAU administration. Tissue is processed for determination of radioactive
FIAU
content incorporated into DNA by acid precipitation. Total tissue
radioactivity is
determined and the percent acid precipitated (% incorporated into DNA) versus
the percent
acid soluble (% as unincorporated FIAU, % unincorporated phosphorylated FIAU,
and
radiolabeled metabolites) is determined. Tissue radioactivity ( % dose/gram)
is plotted
versus time after FIAU injection.
6.6. Example: Time Course of Bacterial And Radioactivity Localization
After Direct Intra-tumoral Administration of Salmonella
Salmonella have been demonstrated to target a wide variety of solid tumors
following systemic administration. However, some tumor types, including glioma
and
prostate cancers may benefit from local-regional administration. The utility
of tumor-
targeted genetically engineered HSV 1-TK expressing Salmonella injected either
i.v. or by
direct intra-tumoral administration for detection, monitoring and/or therapy
can be assessed.
Although i.v. administration of genetically altered Salmonella with selective
"biological"
targeting of the tumor in vivo is a preferred mode of application, it is
clinically feasible to
directly administer these bacteria into the tumor bed at time of surgery.
Mice (C57B/6) are implanted with C38 colon tumor cells and staged until
the tumors are palpable. Salmonella expressing HSV1-TK or HSV1-"super" TK is
injected
directly into the tumor when tumors are palpable. ['z3I]-FIAU is administered
1, 2, 4, 6, 10
or 14 days after Salmonella injection. Gamma camera imaging is done at 1 hr, 3
hr, 9 hr, 24
hr and 48 hr after FIAU administration. The accumulation of radiolabeled FIAU
present in
tumors and selected organs is also determined in animals sacrificed at 24 and
48 hours.
Tissue is processed for determination of radioactive FIAU content incorporated
into DNA
by acid precipitation. Total tissue radioactivity is determined and the
percent acid
precipitated (% incorporated into DNA) versus the percent acid soluble (% as
unincorporated FIAU, % unincorporated phosphorylated FIAU, and % radiolabeled
metabolites) is determined. Data is plotted as tissue radioactivity (%
dose/gram) versus
time after Salmonella administration, bacterial count (number of
bacteria/gram) versus time
after Salmonella administration and bacterial count/tissue radioactivity ratio
(number of
bacteria/% dose) vs. time after Salmonella administration.
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6.7. Other Tumor Models
The use of tumor-targeted genetically engineered Salmonella for imaging
can be assessed in a spectrum of solid tumors and tumor growth environments.
Orthotopic
models include liver tumors arising from murine C38 colon carcinoma, lung
tumors arising
from B 16F 10 melanoma and breast tumors arising either from MDA-MB-231 or
spontaneously in C-neu transgenic mice. Bacteria targeting and imaging are
compared in
orthotopically placed tumors (liver, lung and breast) with s.c. tumors as a
reference. The
spontaneous breast tumor model also provides metastases to multiple sites for
assay in this
system. Salmonella bacteria expressing a labeled marker gene are administered
intravenously in combination, e.g., simultaneously or separately within a
specific time
period, with labeled marker substrate and tumor detection follows the protocol
described
above. The animal is scanned and images of tumor are obtained. This
imaging/treatment
technique is broadly applicable to different orthotopic tumor sites and
different tumor cell
lines.
6.8. Example: Detection of Tumor-specific Bacteria Mediated by Biotin Binding
The gene containing streptavidin was obtained from the plasmid BBG9 R &
D Systems, Minneapolis, MN). The plasmid was first transformed to Salmonella
strain
YS501 and re analyzed for restriction digestion pattern, and then subsequently
transformed
to the tumor specific strain YS1646 with and without the Bacteriocin Release
Protein (BRP)
plasmid pSWl (Bio 101, Vista, CA). Streptavidin production in the supernatant
was
detected by inducing a streptavidinBRP-containing strain with 1.0 pg/ml
mitomycin for 4
hrs followed by centrifugation. Supernatant samples were spotted to
nitrocellulose to allow
binding, followed by a blocking step containing 3% bovine serum albumin and
0.05%
Tween 20 in phosphate buffered saline. The presence of streptavidin was
detected by
overlaying the nitrocellulose strip with a biotin-conjugated alkaline
phosphatase (Sigma, St.
Louis, MO), followed by washing and then a color detection using nitro blue
tetrazolium
and 5-bromo-1-chloro-3-indol phosphate (NBTBCIP). By comparison with a serial
dilution of a streptavidin standard using the same assay, the tumor-specific
bacterial
supernatant was determined to contain 1 p,g/ml or greater.
6.9. Example: Detection of Tumor-specific Bacteria Mediated Fluorescence
Green fluorescent protein has been used to demonstrate the localization of
tumors (Yang et al., 2000, Whole-body optical imaging of green fluorescent
protein-
expressing tumors and metastases. Proc Natl Acad Sci U S A. 97:1206-11),
however,
efficient means of delivery of GFP by systemically administered vectors has
not been
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shown.
Green fluorescence protein (GFP) was obtained from Clonetech (Palo Alto,
CA) and subcloned into the ptrc99a expression vector (Pharmacia, Piscataway,
NJ). The
resulting trc-GFP plasmid was transformed into tumor-specific Salmonella
strain
VNP20009. GFP-containing Salmonella strain VNP20009 were injected i.v. (2 x
106) into
mice bearing HTB 117 human lung carcinoma. After 5 days, the mice were
sacrificed and
tumors were examined for the presence of fluorescent bacteria using standard
cryomicrotomy and fluorescent microscopy techniques (FIG. 3). Figure 3A shows
fluorescence of an untreated tumor and figure 3B shows fluorescence observed
in tumors
following treatment with GFP-containing Salmonella strain VNP20009.
These results demonstrate that a tumor specific bacterium can express GFP,
and that GFP can be localized to tumors using tumor-specific bacteria.
6.10. Example: Cloning~3-galactosidase Into Tumor-specific Salmonella
The LacZ open reading frame was cloned into the expression vector trc99a
(Pharmacia) as a BspHI/StyI fragment from a promoterless (3-gal vector
(Genbank
Accession #AF 192277) and transformed into Salmonella YS 1646. Positive clones
were
detected as blue colonies on X-gal plates.
LacZY was mobilized into Salmonella YS 1646 as a F' factor containing the
entire lacZ operon, including the lacy gene, which is responsible for lactose
transport, as
follows: Salmonella strain YS 1646 was engineered to express the F' as follows
such that the
strain is able to be infected by phage. Salmonella strain YS501 (recD-,
chloramphenicol
resistant) was mated with E. coli strain NH4104, which carries the F' plasmid
containing the
lactose operon, and Salmonella colonies were selected for chloramphenicol
resistance, lac+
on minimal media containing lactose and chloramphenicol. This strain,
designated YS501-
F' (which is also met-) was mated with Salmonella strain YS 1646 (which is
also pur ) and
Salmonella colonies were selected on minimal media containing lactose and
purine but
lacking methionine. This strain was designated YS 1646-F'. The presence of the
F' lac
results in lac+ Salmonella.
6.11. Example: Cloning Firefly Luciferase Into Tumor-specific Salmonella
The plasmid pSPLuc+ containing the gene for firefly luciferase was obtained
from Promega (Madison, WI). pSPLuc+ was digested with NcoI and XbaI and
ligated into
pTrc99a (Pharmacia, NJ). This clone was then amplified by PCR using primers
forward
5'-GTGTGCGGCCGCAATATTACTGAAATGAGCTGTTGACAATTAATCATCC-3'
(SEQ ID N0:13) and reverse 5'-
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GTGTGATATCGCATGCCTGCAGGTCGACTCTAGAATTAC-3' (SEQ ID N0:14)
which add NotI and EcoRV sites to the ends of the luciferase open reading
frame. The
resulting product was subjected to restriction digestion with NotI and EcoRV
and cloned
into a promoterless (3-galactosidase vector (Genbank # AF192277 ) also cut
with NotI and
EcoRV to remove most of the ~i-galactosidase coding region. The plasmid was
transferred
to VNP20009. The resulting clone has the partially constitutive trc promoter
driving
expression of the luciferase gene expression and results in functional
expression of the
luciferase protein. This demonstrates that tumor-specific bacteria can express
luciferase.
6.12. Example: Non-Salmonella Bacteria Target Tumors and Inhibit Tumor Growth
6.12.1 Materials and Methods
Shigella flexneri ("Shigella"), a gram-negative Shigella species which
functions under both aerobic and anaerobic conditions, and Listeria
monocytogenes
("Listeria"; ATCC strain 43251), a gram-positive species which functions under
aerobic and
1 S aerobic conditions, were grown at 37 ° C either in Luria broth (LB)
liquid media with
shaking at approximately 225 revolutions per minute, or on LB solid media
containing
1.5% agar. LB consisted of 10 g tryptone, S g yeast extract and 10 g NaCI per
liter. The pH
of LB was adjusted to 7 using a 1N solution of NaOH. Generally, cultures were
streaked
out on solid media and incubated until visible growth was observed, and then
introduced
into liquid media and grown to an appropriate density before freezing in 15%
glycerol at
-80°C. Shigella was grown to an ODboo of 1.9, corresponding to 1.03 x
109 c.f.u./ml.
Listeria was grown to an OD6oo of 0.6, corresponding to 0.98 x 10g c.f.u./ml.
Streptococcus agalactiae ("Streptococcus"; ATCC strain 13813), a
Streptococcus species which functions under both aerobic and anaerobic
conditions, was
~'o~'n in heart brain infusion ("BHI"; Difco) liquid media or on BHI solid
media according
to the methods described for Shigella and Listeria. Streptococcus was grown to
an OD600
of 0.85, corresponding to an estimated 2.5 x 10g c.f.u./ml. The bacterial
culture was used
without freezing for tumor-targeting experiments (Section 6.12.2 below). For
tumor
inhibition experiments (Section 6.12.3 below), Streptococcus colonies were
inoculated into
liquid culture as described above, grown to an OD600 of 1.2, and frozen at -
80°C in 15%
glycerol, corresponding to 1.2 x 108 c.f.u./ml after thawing.
6.12.2 Non-Salmonella Genera Target Selectively to Solid Tumors
To determine the tumor-targeting capabilities of Shigella, Listeria and
Streptococcus, the bacteria were administered to mice having established
melanomas, and
the ratio of the concentration of bacteria in the tumor to the concentration
of bacteria in the
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liver in each mouse was determined as follows.
The mice were implanted with B 16F 10 murine melanoma tumor cells and
the bacteria administered when the tumors weighed approximately 1 g, or
approximately
after 14-16 days post tumor cell implantation. Prior to administration, frozen
Shigella and
Listeria cultures were thawed at room temperature and diluted into phosphate
buffered
saline (PBS). For Streptococcus administration, a fresh culture was diluted
into PBS. The
bacteria were administered intravenously into the mice in the following
amounts: 2 x 106
c.f.u. of Shigella; 1 x 104 or 1 x 105 c.f.u. of Listeria; and 1 x 105 or 1 x
106 c.f.u. of
Streptococcus.
At day 5 following administration of the bacteria, the mice were sacrificed
and tumors and livers harvested and homogenized. Serial dilutions of the
homogenates
were then plated to the appropriate media for each species.
The results of these experiments are shown in Table S. These data indicate
that Shigella, Listeria, and Streptococcus, which function under both aerobic
and anaerobic
conditions, have highly significant targeting ratios for tumors as compared to
normal tissues
(liver) when administered in vivo.
Table 5.
Strain Dose Tumor c.f.u./g Liver c.f.u./ Ratio Strain
(mean ~ SE) (mean ~ SE)
2 x 106 6.7 ~ 5.8 x 10' S.5 ~ 5.8 x 102 120,000:1 Shigella
1 x 104 2.7 ~ 2.4 x 10g 7.3 ~ 6.9 x 105 370:1 Listeria
1 x 105 2.3 ~ 0.8 x 10g 3.1 ~ 1.9 x 106 70:1 Listeria
1 x 105 6.1 ~ 2.9 x 108 2.4 ~ 2.4 x 103 250,000:1 Streptococcus
1 x 106 3.0 ~ 1.6 x 109 2.5 ~ 2.5 x 102 12,000,000:1 Streptococcus
Tumor to normal tissue (liver) relative accumulations. Counts are based on
colony forming units (c.~u.)
given as the mean ~ standard error (SE).
6.12.3 Administration of Non-Salmonella Genera Reduces
Volume or Inhibits Growth of Solid Tumors
To determine the ability of the tumor-targeting Shigella, Listeria and
Streptococcus to inhibit tumor growth, the effect of administration of the
tumor-specific
bacteria on the growth of established melanomas was determined as follows:
Mice were implanted subcutaneously with B16F10 murine melanoma tumor
cells (5 x 105 cells per animal). The Shigella, Listeria or Streptococcus were
administered
intravenously when the tumors weighed approximately 0.3 g.. Frozen stock of
the bacteria
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(see Section 6.12.1) were thawed at room temperature and diluted into PBS. The
bacteria
were administered intravenously into the mice in the following amounts: 2 x
106 c.f.u. of
Shigella; 1 x 105 c.f.u. of Listeria; and 1 x 106 c.f.u. of Streptococcus.
Following the first week of administration of the tumor-specific bacteria, two
doses (one on day 5, one on day 8 post-bacteria) of 500 mg ampicillin/kg body
weight were
administered to the mice that had received Listeria, for the alleviation of
systemic effects of
the bacteria.
Tumor volume was monitored approximately every 5 days. Tumor growth is
graphically depicted as tumor volume versus time in FIG. 4. Tumor growth in
mice
receiving the tumor-targeted Listeria, Shigella or Streptococcus was inhibited
by at least
approximately 40% relative to tumor growth in control animals, and up to
approximately
65% in the case of Listeria. These data indicate that these facultative, gram
positive and
gram negative bacteria reduce tumor volume or inhibit tumor growth.
7. Microorganism Deposits
As described in W099/13053 published March 18, 1999, the following
microorganisms were deposited with the American Type Culture Collection
(ATCC), 10801
University Blvd., Manassas, Virginia 20110-2209, on 25 August, 1998, and have
been
assigned the indicated Accession numbers:
Microor;_ a~ ATCC Accession No.
YS1646 202165
YS1456 202164
The invention claimed and described herein is not to be limited in scope by
the specific embodiments, including but not limited to the deposited
microorganism
embodiments, herein disclosed since these embodiments are intended as
illustrations of
several aspects of the invention. Indeed, various modifications of the
invention in addition
to those shown and described herein will become apparent to those skilled in
the art from
the foregoing description. Such modifications are also intended to fall within
the scope of
the appended claims.
A number of references are cited herein, the entire disclosures of which are
incorporated herein, in their entirety, by reference.
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SEQUENCE LISTING
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SLOAN-KETTERING INSTITUTE FOR CANCER RESEARCH
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3
