Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS FOR TUMOR-TARGETED
DELIVERY OF EFFECTOR MOLECULES
This application claims priority to U.S. provisional patent applications Nos.
60/157,500, 60/157,581, and 60/157,637, filed on October 4, 1999, the contents
of each of
which is incorporated herein by reference its entirety.
1. FIELD OF THE INVENTION
The present invention relates to the delivery of one or more primary effector
molecules) to a solid tumor for the treatment or inhibition of the tumor. More
particularly,
the invention is related to the preparation and use of attenuated tumor-
targeted bacteria,
such as, e.g., Salmonella, as a vector for the delivery of one or more primary
effector
molecules) to an appropriate site of action, e.g., the site of a solid tumor.
Specifically, the
attenuated tumor-targeted bacteria of the invention is a facultative aerobe or
facultative
anaerobe which is modified to encode one or more primary effector molecule(s).
The
primary effector molecules) of the invention include members of the TNF
cyokine family,
anti-angiogenic factors, and cytotoxic polypeptides or peptides. The primary
effector
molecules of the invention are useful, for example, to treat a sUlld tumor
cancer such as a
carcinoma, melanoma, lymphoma, sarcoma, or metastases derived from these
tumors. The
Invention further relates to the surprising discovery that primary effector
molecules) such
as TNF family members, anti-angiogenic factors, and cytotoxic polypeptides or
peptides
can be delivered locally to tumors by attenuated tumor-targeted bacteria with
reduced
toxicity and reduced immunological complications to the host. The invention
also relates to
the delivery of one or more optional effector molecules) (termed "secondary
effector
molecules") which may be delivered by the attenuated tumor-targeted bacteria
in
conjunction with the primary effector molecule(s). The secondary effector
molecules)
provide additional anti-tumor therapeutic activity, enhance release of the
primary effector
molecules) from the attenuated tumor-targeted bacteria, and/or enhance uptake
of the
primary effector molecules) at the appropriate site of action, e.g., at the
site of a solid
~mor.
2. BACKGROUND OF THE INVENTION
A neoplasm, or tumor, is a neoplastic mass resulting from abnormal cell
growth,
which can be benign or malignant. Benign tumors generally remain localized.
Malignant
~mors generally have the potential to invade and destroy neighboring body
tissue and
spread to distant sites and cause death (for review, see Robins and Angell,
1976, Basic
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Pathology, 2d Ed., W.B. Saunders Co., Philadelphia, pp. 68-122). A tumor is
said to have
metastatized when it has spread from one organ or tissue to another.
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, R.G., 1995, Science 270:404-410).
2.1. CELLULAR IMMUNITY AND CYTOKINES
One strategy for the treatment of cancer involves enhancing or activating a
cellular
immune response. Successful induction of a cellular immune response directed
toward
autologous tumors offers several advantages over conventional chemotherapy: 1
) immune
recognition is highly specific, being directed exclusively toward tumors; 2)
growth at
metastatic sites can be suppressed through immune surveillance; 3) the
diversity of immune
response and recognition can compensate for different resistance mechanisms
employed by
tumor cells; 4) clonal expansion of cytotoxic T cells can occur more rapidly
than the
expanding tumor, resulting in antitumor mechanisms which ultimately overwhelm
the
tumor; and 5) a memory response can suppress disease recurrence in its
earliest stages,
prior to physical detection. Clinical studies of responding patients have
borne out results
from animal models demonstrating that successful immunotherapy involves the
activation
of CD8+ T cells (class I response), although evidence exists for participation
of CD4+ T
cells, macrophages, and NK cells. See, e.g., Chapoval et al., 1998, J.
Immunol. 161:6977-
6984; Gollub et al., 1998, J. Clin. Invest. 102:561-575; Kikuchi et al., 1999,
Int. J. Cancer
80:425-430; Pan et al., 1995, Int. J. Cancer 80:425-430; Saffran et al., 1998,
Cancer Gene
Ther. 5:321-330; and Zimmermann et a1.,1999, Eur. J. Immunol. 29:284-290.
2.2. - TUMOR NECROSIS FACTOR (TNF)
FAMILY OF CYTOHINES
The best characterized member of the TNF family is TNF-a. TNF-a is known to
exert pleiotropic effects on the immune system. TNF-a is a cytokine which can
exert
potent cytotoxic effects directly on tumor cells. TNF-a is generally thought
to exert its
anti-tumor effects via other mechanisms such as stimulation of proliferation
and
differentiation, and prevention of apoptosis in monocytes (see, e.g., Mangan
et al., 1991, J.
Immunol. 146:1541-1546; and Ostensen et a1.,1987, J. Immunol. 138:4185-4191),
promotion of tissue factor-like procoagulant activity and suppression of
endothelial cell
surface anticoagulant activity, ultimately leading to clot formation within
the tumor
(reviewed in Beutler and Cerami, 1989, Ann. Rev. Immunol. 7:625-655; and
Vassalli, P.,
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1992, Ann. Rev. Immunol. 10:411-452). However, as a result of these
properties, systemic
administration of TNF-a results in lethal consequences in the host due to
disseminated
intravascular coagulation.
Other cytokines have also been implicated in anti-tumor responses. IL-2 is a
class I
cytokine and is also thought to play a role in anti-tumor response. For
example,
spontaneously regressing melanomas have been associated with elevated
intratumoral
levels of TNF-a and IL-2. See, e.g., Beutler and Cerami, 1989; Annu. Rev.
Iinmunol.
7:625-655; Lowes et al., 1997, J. Invest. Dermatol. 108:914-919; Mangan et
al., 1991, J.
Immunol. 146:1541-1546; Scheruich et al., 1987, J. Immunol. 138: 1786-1790.
Both TNF-a and IL-2 aid in lymphocyte homing, and IL-2 has been shown to
induce tumor infiltration of natural killer (NIA) cells, T-cells, and
lymphokine activated
killer (LAK) cells (see, e.g., Etter et al., 1998, Cytokine 10:395-403;
Reinhardt et al., 1997,
Blood 89:3837-46; Chen et al., 1997, J. Neuropathol. Exp. Neurol. 56:541-50;
Vora et al.,
1996, Clin. Exp. Immunol. 105:155-62; Luscinskas et al., 1996, J. Immunol.
157:326-35;
Kjaergaard et al., 1998, Scand. J. Immunol. 47, 532-540; Johansson et al.,
1996, Nat.
Immun. 15:87-97; and Watanabe et al., 1997, Am. J. Pathol. 150:1869-80). In
the presence
of both TNF-a and IL-2, the cytolytic activity of NK and LAK cells is
increased, even
when directed against TNF-insensitive cell lines (see, e.g, Ostensen et al.,
1987, J.
Immunol. 138:4185-4191). However, therapeutic levels of IL-2 have also been
shown to be
toxic to the host.
Clearly, dose-limiting toxicity from systemic cytokine administration poses a
significant barrier to realizing the potential of cytokines in cancer therapy.
Moreover,
systemic cytokine delivery can result in decreased homing of syngeneic T
cells, thus
opposing targeted immunotherapy, in addition to resulting in unwanted clinical
side effects.
See Addison et al., 1998, Gene Ther. 5:1400-1409; Albertini et al., 1997,
Clin. Cancer Res.
3~ 1277-1288; Becker et al., 1996, Proc. Natl. Acad. Sci. USA 93:7826-7831;
Book et al.,
1998, J. Neuroimmunol. 92:50-59; Cao et al., 1998, J. Cancer Res. Clin. Oncol.
124:88-92;
D'Angelica et al., 1999, Cancer Immunol. Immunother. 47:265-271; Deszo et al.,
1996,
Clin. Cancer Res. 2:1543-1552; Kjaergaard et al., 1998, Scand. J. Immunol.
47:532-540;
Ostensen et al., 1987, J. Immunol. 138:4185-4191; and Schimnacher et al.,
1998, Clin.
Cancer Res. 4:2635-2645.
2.3. DELIVERY OF CYTOHINES
Recent experimental animal and clinical studies have attempted to bypass
systemic
toxicity of cytokines and administer higher doses, through sub-systemic or
alternative
methods of delivery of cytokines. In murine models, sarcoma-180 tumors have
been
treated with administration of a fusogenic liposome-encapsulated TNF-a gene,
and
systemic administration of polyethylene glycol-encapsulated TNF-a, which could
localize
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to the tumor vasculature (see Tsutsumi et al., 1996, Jpn. J. Cancer Res.
87:1078-1085).
Sensitization of tumors to TNF-a by endothelial-monocyte-activating
polypeptide II has
also been reported (see, Marvin et al., 1999, J. Surg. Res. 63:248-255; Wu et
al., 1996,
Cancer Res.59:205-212).
In clinical studies, complete tumor eradication has been observed following
high-
dose TNF-a administration to patients via isolated limb perfusion, in
combination with
interferon-a or melphalan. However, this technique presents severe risks to
the patient if
the cytokines are not completely removed following treatment. Further, these
treatments
require limb isolation, which, in itself presents risks to the patient. See
Eggermont et al.,
1997, Semin. Oncol. 24:547-555 Fraker et al., 1995, Cancer J. Sci. Am. 1:122-
130;
Lejeune et a1.,1998, Curr. Opin. Immunol. 10:573-580; Marvin et al., 1996, J.
Surg. Res.
63:248-255; Mizuguchi et al., 1998, Cancer Res. 58:5725-5730; Tsutsumi et al.,
1996, Jpn.
J. Cancer Res. 87:1078-1085; and Wu et a1.,1996, Cancer Res. 59, 205-212.
Previous studies by Carrier et al, 1992, J. Immunol. 148:1176-81, Saltzman et
al.,
1997, Cancer Biother. Radiopharm. 12:37-45, SaItzman et al., 1997, J. Pediat.
Surgery
32:301-306 have reported the use of attenuated Salmonella strains to deliver
IL-113
(Carrier) and IL-2 (Saltzman) directly to livers and spleens, the natural
sites of Salmonella
infection, to serve as vaccine strains or affect hepatic metastases.
Saltzman's studies used
oral administration of Salmonella in which bacteria ac-e taken up by GALT (gut
associated
lymphoid tissue) and transported to liver and spleen. However, these
infections are limited
to the natural sites of infection.
2.4. ANGIOGENESIS AND TUMORIGENESIS
Another strategy for the treatment of cancer involves the inhibition of
angiogenesis.
Angiogenesis is the process of growth of new capillaries from preexisting
blood vessels.
New capillaries are formed by a process in which the endothelial cells of the
preexisting
blood vessel, using proteolytic enzymes such as matrix metalloproteases,
degrade the
basement membranes in their vicinity, proliferate, migrate into surrounding
stromal tissue
and form microtubes . The process of angiogenesis is very tightly regulated by
an interplay
between negative and positive factors, and in adults is normally restricted to
the female
reproductive cycle and wound repair (Malonne et al., 1999, Clin. Exp.
Metastasis 17:1-14).
Aberrant or abnormal regulation of angiogenesis has been implicated in many
human
disorders, including diabetic retinopathy, psoriasis, rheumatoid arthritis,
cardiovascular
disease, and tumorigenesis (Folkman, 1995, Nat. Med. 1:27-31 ).
Angiogenesis is a critical process for tumor growth and metastasis. Tumor
formation is divided into two stages, the prevascular and vascular stages.
Studies have
shown that cells of prevascular tumors proliferate as rapidly as do cells from
vascularized
tumors. However, prevascular tumors rarely grow to more than 2-3 mm3 because
of the
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existence of an equilibrium between cell proliferation and cell death, the
latter resulting
from the hypoxic nature of the prevascular tumor (Folkman, 1995, Nat. Med.
1:27-31). The
switch from the prevascular to vascular stage requires a shift in the balance
of the
regulatory factors of angiogenesis from a net balance favoring negative
factors to one in
which the positive factors, such as fibroblast growth factor (FGF) and
vascular endothelial
growth factor (VEGF), predominate (Cao, 1998, Prog. Mol. Subcell. Biol. 20:161-
176).
The shift in balance between regulatory factors is a result of the up-
regulation of the
angiogenic factors and the simultaneous down-regulation of anti-angiogenic
factors
(Folkman, 199$, N. Eng. J. Med. 333:1757-1763).
2~5~ ANTI-ANGIOGENIC FACTORS
Anti-angiogenic factors were postulated to exist on the basis of several
related
phenomena that led to the conclusion that primary tumors often inhibited the
growth of
their metastases (Cao, 1998, Prog. Mol. Subcell. Biol. 20:161-176). The first
of these
factors to be isolated was mouse angiostatin, a 38 kDa proteolytic fragment of
plasminogen
1$ that is released into the circulation by primary Lewis lung carcinoma
tumors and prevents
the growth of secondary metastases (O'Reilly et al., 1994, Cell 79:315-328).
In humans,
peptides of 40, 42 and 4$ kDa produced by the limited proteolysis of
plasminogen with
metalloelastase have anti-angiogenic activity comparable to mouse angiostatin
(O'Reilly et
al., 1994, Cell 79:315-328). Plasminogen itself has no such activity. It is
also thought that
tumor-associated macrophages are responsible for the production of
angiostatin, since
tumor cells themselves have no detectable angiostatin mRNA. Macrophage
metalloelastase
expression is induced by granulocyte colony stimulating factor (GM-CSF)
secreted by the
tumor cells (Dong et al., 1997, Cell 88:801-810). In certain tumors,
angiostatin production
is catalyzed by serine proteases rather than metalloelastase, where serine
proteases are
2$ produced directly by the tumor cells (Gately et al., 1997, Cancer Res.
56:4887-4890).
Administration of angiostatin at a concentration of 100mg/kg/day to
experimental mice
with primary tumors resulted in a strong inhibition of tumor growth without
toxic side
effects. The tumors regrew within 2 weeks of cessation of the angiostatin
treatment,
indicating that the tumors regress into a dormant state rather than completely
die as a result
of the treatment (O'Reilly et al., 1996, Nat. Med. 2:689-692).
After the discovery of angiostatin, other angiogenesis inhibitors, including
several
angiogenesis-inhibiting peptides, were discovered and isolated. A more potent
inhibitor of
angiogenesis than angiostatin is kringle 5, a peptide comprising the fifth
kringle domain of
plasminogen (angiostatin comprises kringle domains 1-4). Kringle 5 can be
produced by
the proteolysis of plasminogen, and recombinant forms are also active (Cao et
al., 1997, J.
Biol. Chem. 272:22924-22928).
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Endostatin was isolated in a manner similar to the isolation of angiostatin
(O'Reilly
et al., 1997, Cell 88:1-20), the source being a murine hemangioendothelioma
rather than a
Lewis lung carcinoma. The peptide has an apparent molecular mass of 20 kDa
whose
sequence corresponds to the C-terminal of collagen XVIII (O'Reilly et al.,
1997, Cell 88:1-
20), a region called NC 1 that is divergent among various collagen molecules
(Oh et al.,
1994, Proc. Natl. Acad. Sci. USA 91:4229-4233; and Rehn et al., 1994, Proc.
Natl. Acad.
Sci. USA 91:4234-4238). In mice, the growth of Lewis lung carcinoma metastases
is
suppressed by the administration 0.3 mg/kg/day of recombinant endostatin, and
the primary
tumor regresses to a dormant state when the peptide is administered at 20
mg/kg/day.
Functional recombinant endostatin can be produced from inclusion bodies,
either in vitro by
denaturation and refolding, or in vivo by the~sustained release of
subcutaneously
administered endostatin inclusion body preparations (O'Reilly et al., 1997,
Cell 88:1-20).
An alternative method of endostatin delivery consisting of intramuscular
administration of
an endostatin expression plasmid results in only the partial inhibition of
tumor growth in a
mouse model system (Blezinger et al., 1999, Nat. Biotech. 17:343-348).
Similarly,
endostatin or angiotensin-encoding plasmids complexed to liposomes that were
delivered
intravenously resulted in a partial inhibition of tumor growth in a nude mouse
model of
breast cancer (Chen et al., 1999, Cancer Res. 59:3308-3312).
Recently, a novel anti-angiogenic activity has been attributed to a C-terminal
truncation peptide of the Serpiri (Serine Protease Inhibitor) anti-thrombin
(O'Reilly et al.,
1999, Science 285:1926-1928). Full length anti-thrombin has no inherent anti-
angiogenic
activity, but upon cleavage of the C-terminal reactive loop of the protein by
thrombin, anti-
thrombin acquires potent angiogenic activity. The proteolytic fragment is
referred to
hereinafter as anti-angiogenic anti-thrombin.
Other angiogenesis-inhibiting peptides known in the art include the 29 kDa
N-terminal and a 40 kDa C-terminal proteolytic fragments of fibronectin
(Homandberg et
al., 1985, J. Am. Pathol. 120:327-332); the 16 kDa proteolytic fragment of
prolactin (Clapp
et al., 1993, Endocrinology 133:1292-1299); and the 7.8 kDa proteolytic
fragment of
platelet factor-4 (Gupta et al., 1995, Proc. Natl. Acad. Sci. USA 92:7799-
7803).
In addition to those naturally produced proteolytic fragments that have
demonstrated anti-angiogenic effects, several synthetic peptides that
correspond to regions
of known extracellular matrix proteins have been assessed for activity in
inhibiting
angiogenesis. Synthetic peptides which have been demonstrated to be functional
endothelial inhibitors, i.e. angiogenesis inhibitors, include a 13 amino acid
peptide
corresponding to a fragment of platelet factor-4 (Maione et al., 1990, Cancer
Res. 51:2077-
2083); a l4 amino acid peptide corresponding to a fragment of collagen I
(Tolma et al.,
1993, J. Cell Biol. 122:497-511); a 19 amino acid peptide corresponding to a
fragment of
Thrombospondin I (Tolsma et al., 1993, J. Cell Biol. 122:497-511); and a 20
amino acid
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peptide corresponding to a fragment of SPARC (Sage et al., 1995, J. Cell.
Biochem.
57:1329-1334), a secreted cysteine-rich extracellular matrix glycoprotein
whose expression
in human melanoma cells leads to reduced cellular invasion in vitro and
reduced
tumorigenicity in an in vivo nude mouse model (Ledda et al., 1996, Nature Med.
3:171-176)
. Other peptides of less than 10 amino acids that inhibit angiogenesis and
correspond to
fragments of laminin, .fibronectin, procollagen, and EGF have also been
described (see the
review by Cao, 1998, Prog. Mol. Subcell. Biol. 20:161-176).
The small fibronectin peptides that inhibit angiogenesis generally comprise
the
motif RGD. RGD is a peptide motif (amino acids Arg-Gly-Asp) used by proteins
for
recognition and binding to integrin molecules. The expression of integrin
a~(33 is associated
with angiogenic blood vessels and inhibition of its activity by monoclonal
antibodies blocks
vascularization (Brooks et al., 1994, Science 264:569-571). This has been
confirmed by a
study showing that the administration of cyclic pentapeptides containing the
RGD motif
inhibits the activity of vitronectin receptor-type integrins and block retinal
neovascularization (Hammes et al., 1996, Nature Medicine 2:529-533). The anti-
1 S angiogenic effect of integrin Mockers such as cyclic pentapeptides and
monoclonal
antibodies has been shown to promote tumor regression by inducing the
apoptosis of
angiogenic blood vessels (Brooks et al., 1994, Cell 79:1157-1164). Peptides
comprising
the RGD motif, and another integrin binding motif, NGR (amino acids Asn-Gln-
Arg),
showed markedly enhanced anti-tumor activity
The inhibition of the activity of another type of cell surface receptor,
namely the
urokinase plasminogen activator (uPA) receptor, also results in the inhibition
of
angiogenesis. The uPA receptor, upon ligand binding, initiates a proteolytic
cascade that is
necessary for the basement membrane invasion step of angiogenesis. Inhibition
of the uPA
receptor by receptor antagonists inhibits angiogenesis, tumor growth (Min et
al., 1996,
Cancer Res. 56: 2428-2433) and metastasis (Crowley et al., 1993, Proc. Natl.
Acad. Sci.
USA 90:5021-5025). Such antagonists have been identified by bacteriophage
peptide
display of random peptides (Goodson et al., Proc. Natl. Acad. Sci. USA 91:7129-
7133).
Dominant negative forms of the receptor's ligand, uPA, have also been
identified (Min et
al., 1996, Cancer Res. 56: 2428-2433).
file the discovery of angiostatin, endostatin and other anti-angiogenic
peptides
provided an exciting new approach for cancer therapy, the reality of a course
of treatment
involving one or more of these peptides is the impracticality of the
production of immense
amounts of peptides (stemming from the cost and/or labor of having to produce,
for an
average person of 65 kg or 143 lbs, approximately 1.3 or 6.5 grams of protein
per day,
depending on the peptide) and the duration of the treatment (which has to be
sustained if
the tumor is to stay in regression). It is thought that the two main reasons
that these
peptides have to be administered in such large quantities are that, first, a
majority are
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degraded in the blood stream and, second, of the molecules that do survive
degradation
only a very limited proportion make their way to the tumor. Thus, it would be
a great
advantage to the field of tumor therapy if anti-angiogenic proteins or
peptides could be
delivered more efficiently to the tumor and in a more cost-effective and
patient-friendly
manner.
2.6. BACTERIOCIN FAMILY
Colicin E3 (referred to hereinafter as ColE3) is a bacteriocin, i.e., a
bacterial
proteinaceous toxin with selective activity, in that its host is immune to the
toxin.
Bacteriocins may be encoded by the host genome or by a plasmid, may have a
broad or
narrow range of hosts, and may have a simple structure comprising one or two
subunits or
may be a multi-subunit structure (Konisky, 1982, Ann. Rev. Microbiol. 36:125-
144). In
addition, a bacteriocin host has an immunity against the bacteriocin. The
immunity is
found in all cells of a given host population, even those that do not express
the bacteriocin.
The cytotoxicity of ColE3 results from its inhibition of protein synthesis
(Nomura,
1963, Cold Spring Harbor Symp. Quant. Biol. 28:315-324). The target of ColE3
activity is
the 16S component of bacterial ribosomes, which is common to the 30S and 70S
ribosomes
(Bowman et al., 1971, Proc. Natl. Acad. Sci. USA. 68:964-968), and the
activity results in
the degradation of the ribosome (Meyhack, 1970, Proc. Natl. Acad. Sci. USA).
ColE3
activity is unique among RNAses, in that it does not cause the overall
degradation of RNA,
but cleaves mRNA molecules 49 nucleotides from the end, resulting in the
separation of
the rRNA from the mRNA and thereby inhibiting translation. The ribonuclease
activity of
ColE3 resides in the molecule itself, rather than being mediated by another
protein
(Saunders, 1978, Nature 274:113-114). ColE3 is also able to penetrate the
inner and outer
membranes of the target cell.
In its naturally occurring form, ColE3 is a 60kDa protein complex consisting
of a
SOkDa and a lOkDa 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.
The 50 kDa subunit comprises at least two functional domains, an N-terminal
region
required for translocation across target cell membranes, and a C-terminal
region with
catalytic (RNAse) activity. Within the host organism, the activity of the
large subunit is
inhibited by the small subunit. The subunits are thought to dissociate upon
entry of the
toxin into the target cell as a result of interaction with the target cell's
outer membrane
(reviewed by Konisky, 1982, Ann. Rev. Microbiol. 36:125-144).
The toxicity of the large subunit of ColE3 has been utilized to prevent the
lateral
spread of cloned genes among microorganisms. Diaz et al. (1994, Mol.
Microbiol. 13:855-
861) separated the two components of ColE3 such that the small (anti-toxic)
subunit was
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expressed as a chromosomally integrated coding sequence and the large subunit
was
expressed from a plasmid. Bacteria with the chromosomally integrated small
subunit are
immune to plasmids that express the ColE3 large subunit, but if the plasmid
were to be
laterally transferred to another recipient that lacked the small subunit, that
cell would be
killed.
Colicin E3 (ColE3) has also 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., 1979, Experimentia 35:406-407).
ColE3
activity targets the 405 subunit of the 805 mammalian ribosome (Turnowsky et
al., 1973,
Biochem. Biophys. Res. Comm. 52:327-334).
2.7. 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-I02, (Suppl. 276); and 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 typhimuriatm that were able to invade HEp-2 (human epidermoid
carcinoma)
veils in vitro in significantly greater numbers than the wild-type strain. The
"hyperinvasive" mutants were isolated under conditions of aerobic growth of
the bacteria
that normally repress the ability of wild-type strains to invade HEp-2 animal
cells.
However, such hyperinvasive Salmonella typhimuYium 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., 1975, Proc. Natl. Acad. Sci. USA 72:3666-3669, demonstrated
that
mice injected with bacillus Calmette-Guerin (BCG) have increased serum 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. 20:695-70I; Barth and Morton, 1995, Cancer 75 (Suppl.
2):726-734;
Friberg, 1993, Med. Oncol. Tumor. Pharmacother. 10:3I-36 for reviews of BCG
therapy.
However, TNF-a-mediated septic shock is among the primary concerns associated
with bacteria, and can have toxic or lethal consequences for the host (Bone,
1992, JAMA
268:3452-3455; Dinarello et al., 1993, JAMA 269:1829-1835). Further, dose-
limiting,
systemic toxicity of TNF-a has been the major barrier to effective clinical
use.
Modifications which reduce this form of an immune response would be useful
because
TNF-a levels would not be toxic, and a more effective concentration and/or
duration of the
therapeutic vector could be used.
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2.8. TUMOR-TARGETED BACTERIA
Genetically engineered Salmonella have been demonstrated to be capable of
tumor
targeting, possess anti-tumor activity and are useful in delivering effector
genes such as the
herpes simplex thymidine kinase (HSV TK) to solid tumors (Pawelek et al., WO
96/40238).
S
2.9. DECREASED INDUCTION OF TNF-a
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
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 hydroxylmyristoyl)-
glucosamine
-acyltransferase in lipid A biosynthesis (Kelley et al., 1993, J. Biol. Chem.
268:19866-
I 9874). Pawelek et al. showed that mutations in the firA gene induce lower
levels of
TNFa.
In Escherichia coli, the gene rnsbB (mlt) which is responsible for the
terminal
myristalization 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). These references, however, do
not
suggest that disruption of the msbB gene in tumor-targeted Salmonella vectors
would result
in bacteria which are less virulent and more sensitive to chelating agents.
The problems associated with the use of bacteria as gene delivery vectors
center on
the general ability of bacteria to directly kill normal mammalian cells as
well as their
ability to overstimulate the immune system via TNFa which can have toxic
consequences
for the host (Bone, 1992, JAMA 268:3452-3455; and Dinarello et al., 1993, JAMA
269:1829-1835). In addition to these factors, resistance to antibiotics can
severely
complicate coping with the presence of bacteria within the human body
(Tschape, 1996, D
T W Dtsch Tierarztl Wochenschr 1996 103:273-7; Ramos et al., 1996, Enferin
Infec.
Microbiol. Clin. 14: 345-51).
Hone and Powell, W097/18837 ("Hone and Powell"), disclose methods to produce
gram-negative bacteria having non-pyrogenic Lipid A or LPS.
Maskell, W098/33923, describes a mutant strain of Salmonella having a mutation
in the msbB gene which induces TNFa at a lower level as compared to a wild
type strain.
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Bermudes et al., WO 99/13053, teach compositions and methods for the genetic
disruption of the msbB gene in Salmonella, which results 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. See also, Low et al., 1999, Nature
Biotech. 17:37-
47.
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. SUMMARY OF THE TNVENTION
The present invention provides methods for delivering one or more primary
effector
molecules) to a solid tumor. In an embodiment, the methods provide for
delivery of a
high level of one or more primary effector molecules. In particular, the
invention provides
methods by which a primary effector molecule(s), which may be toxic or induce
unwanted
1 S effects (e.g., unwanted immunological effects) when delivered systemically
to a host, can
be delivered locally to tumor by an attenuated tumor-targeted bacteria, such
as Salmonella
with reduced toxicity to the host. The present invention encompasses the
preparation and
the use of attenuated tumor-targeted bacteria, such as, e.g., Salmonella, as a
vector for the
delivery of one or more primary effector molecules) and optionally, one or
more
secondary effector molecule(s), to an appropriate site of action, e.g., the
site of a solid
tumor. Specifically, the attenuated tumor-targeted bacteria of the invention
are facultative
aerobes or facultative anaerobes which are engineered to encode one or more
primary
effector molecules) and optionally, one or more secondary effector
molecule(s).
The present invention provides attenuated tumor-targeted bacteria engineered
to
express nucleic acid molecules encoding primary effector molecules at the site
of a solid
tumor. In a specific embodiment, attenuated tumor-targeted bacteria are
engineered,to
express a nucleic acid molecule encoding a primary effector molecule. In
another
embodiment, attenuated tumor-targeted bacteria are engineered to express one
or more
nucleic acid molecules encoding one or more primary effector molecules. In
accordance
with this embodiment, a single bacterial strain is engineered to express one
or more nucleic
acid molecules encoding one or more primary effector molecules at the site of
a solid
tumor. In another embodiment, more than one attenuated tumor-targeted
bacterial strain is
engineered to express one or more nucleic acid molecules encoding one or more
primary
effector molecules. In a mode of this embodiment, the attenuated tumor-
targeted bacterial
strains are of the same species. In another mode of this embodiment, the
attenuated tumor-
targeted bacterial strains are of different species (e.g., Listeria and
Salmonella).
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The primary effector molecules of the invention are useful for the treatment
of a
solid tumor cancer such as a carcinoma, melanoma, lymphoma, or sarcoma. As
used
herein, "treatment of a solid tumor" or "treat a solid tumor" encompasses
inhibiting the
growth of a tumor or tumor cells, reducing the volume of a tumor, killing
tumor cells, or
spreading of tumor cells (metastasis). In a specific embodiment, the primary
effector
molecules of the invention induce a local immune response at the site of the
tumor that
results in the inhibition of growth of a tumor or tumor cells, the killing of
tumor cells, or
the prevention of the spread of tumor cells to other parts of the body.
Accordingly, the
primary effector molecules provide a therapeutic effect for treatment of a
tumor.
The primary effector molecules can be derived from any known organism,
including, but not limited to, animals, plants, bacteria, fungi, and protista,
or viruses. In a
preferred mode of one embodiment of the invention, the primary effector
molecules) is
derived from a mammal. In a more preferred mode of this embodiment, the
primary
effector molecules) is derived from a human. The primary effector molecules of
the
invention include members of the TNF family, anti-angiogenic factors,
cytotoxic
p°lypeptides or peptides, tumor inhibitory enzymes, and functional
fragments thereof.
In a specific embodiment, the primary effector molecules of the invention are
members of the TNF family or functional fragments thereof. Examples of TNF
family
members, include, but are not limited to, 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), CD40 ligand (CD40L), 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),
TNFSF16,
TNFSF 17, and AITR-L (ligand of the activation-inducible TNFR family member).
In a
preferred embodiment, a primary effector molecule of the invention is tumor
necrosis
factor-a (TNF-a), tumor necrosis factor-~i (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.
In another specific embodiment, the primary effector molecules of the
invention are
anti-angiogenic factors or functional fragments thereof. Examples of anti-
angiogenic
factors, include, but are not limited to, endostatin, angiostatin, apomigren,
anti-angiogenic
antithrombin III, the 29 kDa N-terminal and a 40 kDa C-terminal proteolytic
fragments of
Bbronectin, 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-
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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~(33 and the VEGF receptor. In a preferred embodiment
of the
invention, a primary effector molecule of the invention is a functional
fragment of
endostatin, apomigren or thrombospondin I.
In another specific embodiment, the primary effector molecules of the
invention are
cytotoxic polypeptides or peptides, or functional fragments thereof. Examples
of cytotoxic
polypeptides or peptides include, but are not limited to, members of the
bacteriocin family,
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. In a
preferred embodiment, the primary effector molecule is a member of the
bacteroicin
family, with the proviso that said bacteriocin family member is not a
bacteriocin release
protein (BRP). Examples of bacteriocin family members, include, but are not
limited to,
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
specific
embodiment, the primary effector molecule is colicin E3.
In another specific embodiment, the primary effector molecules of the
invention 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), DNAase, and glycosidase. In a
preferred
embodiment, the primary effector molecule is methionase.
The present invention also provides methods for local, combinatorial delivery
of
one or more primary effector molecules) and one or more secondary effector
molecules)
to solid tumors by attenuated tumor-targeted bacteria, such as Salmonella. In
a specific
embodiment, attenuated tumor-targeted bacteria are engineered to express a
nucleic acid
molecule encoding a primary effector molecule and a secondary effector
molecule. In
another embodiment, attenuated tumor-targeted bacteria are engineered to
express one or
more nucleic acid molecules encoding one or more primary effector molecules
and one or
more secondary effector molecules. In accordance with this embodiment, a
single bacterial
strain is engineered to express one or more nucleic acid molecules encoding
one or more
primary effector molecules and one or more secondary effector molecules at the
site of a
solid tumor. In another embodiment, more than one attenuated tumor-targeted
bacterial
strain is engineered to express one or more nucleic acid molecules encoding
one or more
primary effector molecules and one or more secondary effector molecules at the
site of a
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solid tumor. In a mode of this embodiment, the attenuated tumor-targeted
bacterial strains
are of the same species. In another mode of this embodiment, the attenuated
tumor-
targeted bacterial strains are of different species (e.g., Listeria and
Salmonella).
The secondary effector molecules) of the invention provide additional anti-
tumor
therapeutic activity, enhance release of the primary effector molecules) from
the
attenuated tumor-targeted bacteria, and/or enhance internalization at the site
of action, e.g.,
at the site of a solid tumor. The secondary effector molecules) of the
invention comprise a
molecule (such as an anti-tumor protein, including but not limited to a
cytotoxins, an
enzyme abd a bacteriocin; a pro-drug converting enzyme; an antisense molecule;
a
ribozyme; an antigen; etc.) which is delivered in addition to the primary
effector
molecules) by the methods of the invention'to treat a solid tumor cancer such
as a
carcinoma, melanoma, lymphoma, or sarcoma.
The secondary effector molecules can be derived from any known organism,
including, but not limited to, animals, plants, bacteria, fungi, and protista,
or viruses. In
certain embodiments, the secondary effector molecule is derived from a
bacteria or virus.
In certain preferred embodiments of the invention, the secondary effector
molecules) is
derived from a bacterium (e.g. BRP). In other preferred embodiments of the
invention, the
secondary effector molecules) is derived from a virus (e.g., TAT). In yet
other preferred
embodiments of the invention, the secondary effector molecult:(~) is derived
from a
mammal. In certain preferred embodiments, the secondary effector molecules) is
derived
from a human.
The invention provides attenuated tumor-targeted bacteria comprising effector
molecules) which are encoded by a plasmid or transfectable nucleic acid. In a
preferred
embodiment of the invention, the attenuated tumor-targeted bacteria is
Salmonella. When
more than one effector molecule (e.g., primary or secondary) is expressed in
an attenuated
~mor-targeted bacteria, such as Salmonella, the effector molecules may be
encoded by the
same plasmid or nucleic acid, or by more than one plasmid or nucleic acid. The
invention
also provides attenuated tumor-targeted bacteria comprising effector
molecules) which are
encoded by a nucleic acid which is integrated into the bacterial genome.
Integrated
effector molecules) may be endogenous to an attenuated tumor-targeted
bacteria, such as
Salmonella, or may be introduced into the attenuated tumor-targeted bacteria
(e.g., by
introduction of a nucleic acid which encodes the effector molecule, such as a
plasmid,
transfectable nucleic acid, transposon, etc.) such that the nucleic acid
encoding the effector
molecule becomes integrated into the genome of the attenuated tumor-targeted
bacteria.
The invention provides a nucleic acid molecule encoding an effector molecule
which
nucleic acid is operably linked to an appropriate promoter. A promoter
operably linked to
a nucleic acid encoding an effector molecule may be homologous (i.e., native)
or
heterologous (i.e., not native to the nucleic acid encoding the effector
molecule).
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Examples of suitable promoters include but are not limited to the Tet
promoter, trc, pepT,
lac, sulA, pol II (dinA), ruv, recA, uvrA, uvrB, uvrD, umuDC, IexA, cea, caa,
recN and
pagC.
The present invention also provides methods for local delivery of one or more
fusion proteins comprising a signal sequence and an effector molecule by
attenuated
tumor-targeted bacteria. In a preferred embodiment, attenuated 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 infra, Section 3.1 for definition of "Omp-like
protein") and an
effector molecule. Without intending to be limited as to mechanism, the
present inventors
believe that the Omp-like protein acts as an anchor or tether for the effector
molecule to the
outer membrane, or serves to localize the effector molecule to the bacterial
outer
membrane. In certain embodiments, the effector molecule has enhanced delivery
to the
outer membrane of the bacteria. In one embodiment, the fusion of an effector
molecule to
an Omp-like protein is used to enhance localization of an effector molecule to
the
periplasm. In certain other embodiments, the fusion of an effector molecule to
an Omp-
like protein is used to enhance release of the effector molecule. Examples of
Omp-like
proteins include, but are not limited to, at least a portion of each of the
following: 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, and a major outer membrane lipoprotein
(such
as LPP). 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 effector molecule or endogenous to the Omp-like protein, or the
proteolytic cleavage
site may be constructed into the fusion protein.
The present invention also provides methods for local delivery of one or more
fusion proteins comprising a ferry peptide and an effector molecule to a solid
tumor by
attenuated 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
attenuated
tumor-targeted bacteria to express fusion proteins comprising a ferry peptide
and an
effector molecule enhances the ability of an effector molecule to be
internalized by tumor
cells. In a specific embodiment, attenuated tumor-targeted bacteria are
engineered to
express a nucleic acid molecule encoding a fusion protein comprising a ferry
peptide and
an effector molecule. In another embodiment, attenuated tumor-targeted
bacteria are
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engineered to express one or more nucleic acid molecules encoding one or more
fusion
proteins comprising a ferry peptide and an effector molecule. In accordance
with these
embodiments, the effector molecule may be a primary or secondary effector
molecule.
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 effector
molecule to a
solid tumor by attenuated tumor-targeted bacteria. In a specific embodiment,
attenuated
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
effector molecule. In accordance with this embodiment, the effector molecule
may be a
primary or secondary effector molecule.
The present invention also provides methods for local delivery of one or more
fusion proteins comprising a signal peptide, a protolytic cleavage site, a
ferry peptide and
an effector molecule to a solid tumor by attenuated tumor-targeted bacteria.
In a specific
embodiment, attenuated tumor-targeted bacteria are engineered to express one
or more
nucleic acid molecules encoding one or more fusion proteins comprising a
signal sequence,
a protolytic cleavage site, a ferry peptide and an effector molecule. In
accordance with this
embodiment, the effector molecule may be a primary or secondary effector
molecule.
In certain embodiments, a single bacterial strain is engineered to express one
or
more nucleic acid molecules encoding a fusion protein of the invention at the
site of a solid
tumor. In certain other embodiments, more than one attenuated tumor-targeted
bacterial
strain is engineered to express one or more nucleic acid molecules encoding
one or more
fusion proteins of the invention at the site of a solid tumor. In modes of
these
embodiments, the attenuated tumor-targeted bacterial strains are of the same
species. In
another modes of these embodiments, the attenuated tumor-targeted bacterial
strains are of
different species (e.g., Listeria and Salmonella).
The present invention also provides methods for local delivery of one or more
fusion proteins of the invention and one or more effector molecules of the
invention to the
site of a solid tumor by attenuated tumor-targeted bacteria. Preferably, the
expression of
both the fusion proteins) and effector molecules) at the site of the solid
tumor by an
attenuated tumor-targeting bacteria improves the level of tumor or tumor cell
growth
inhibited compared to when either fusion proteins) alone or the effector
molecules) alone
is expressed.
The present invention also provides expression of a primary effector molecule
and
optionally, a secondary effector molecule in an attenuated tumor-targeted
bacteria, such as
Salmonella, which bacteria has an enhanced release system. In a preferred
embodiment of
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the invention, the enhanced release is associated with expression of a release
factor by the
attenuated tumor-targeted bacteria. In one embodiment, the release allows
enhanced
release of effector molecules from the cytoplasmic or periplasmic space. A
release factor
may be endogenous to the attenuated tumor-targeted bacteria or it may
exogenous (i.e.,
encoded by a nucleic acid molecule 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 geriome of the attenuated tumor-
targeted bacteria.
A release factor may be encoded by the same nucleic acid or plasmid that
encodes a
primary effector molecule, or by a separate nucleic acid or plasmid. A release
factor may
be encoded by the same nucleic acid or plasmid that encodes a secondary
effector
molecule, or by a separate nucleic acid or plasmid. In a preferred embodiment,
the release
factor is a Bacteriocin Release Protein (BRP). In a specific embodiment, the
BRP is that of
the cloacin DF 13 plasmid, one of colicin E 1-E9 plasmids, or the colicin A, N
or D
plasmids. In a preferred embodiment, the BRP is of cloacin DF 13 (pCIoDF 13
BRP). In
another embodiment of the invention, the enhanced release system comprises
overexpression of a porin protein.
The present invention also provides expression of a fusion protein of the
invention
in an attenuated tumor-targeted bacteria, such as Salmonella, which bacteria
has an
enhanced release system. In a specific embodiment, the release factor is
expressed in a cell
which also expresses a fusion protein comprising a primary effector molecule
fused to an
imp-like protein. In this embodiment, the co-expression of the release factor
allows for
enhanced release of the fusion protein from the periplasmic space.
In one embodiment, the present invention provides methods of delivering high
levels of effector molecules or fusion proteins using modified, attenuated
tumor-targeted
strains of bacteria, which selectively accumulate within tumors while
expressing the
effector molecules or fusion proteins. In a specific mode, a modified,
attenuated tumor-
targeted strain of bacteria selectively amplifies effector molecules within
tumors. While
the teachings of the following sections are discussed, for simplicity, with
reference
specifically to Salmonella, the compositions and methods of the invention are
in no way
meant to be restricted to Salmonella but encompass any other bacteria to which
the
teachings apply. Specifically, the invention provides an attenuated tumor-
targeted
bacterium which is a facultative aerobe or facultative anaerobe. Examples of
attenuated
tumor-targeted bacteria include, but are not limited to, Escherichia coli,
including
enteroinvasive Escherichia coli, Salmonella spp., Shigella spp., Yersinia
enterocohtica,
Listeria monocytogenies, Mycoplasma hominis, and Streptococcus spp.
The present invention also provides pharmaceutical compositions comprising a
pharmaceutically acceptable Garner and an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
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molecules. The present invention also provides pharmaceutical compositions
comprising a
pharmaceutically acceptable carrier and an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
molecules and one or more secondary effector molecules. The present invention
also
provides pharmaceutical compositions comprising a pharmaceutically acceptable
carrier
and an attenuated tumor-targeted bacteria engineered to contain one or more
nucleic acid
molecules encoding one or more fusion proteins of the invention. Further, the
present
invention provides pharmaceutical compositions comprising a pharmaceutically
acceptable
carrier and an attenuated tumor-targeted bacteria engineered to contain one or
more nucleic
acid molecules encoding one or more fusion proteins of the invention and one
or more
effector molecules (i.e., primary or/and secondary molecules). In a preferred
embodiment,
the attenuated tumor-targeted bacteria is Salmonella.
The pharmaceutical compositions of the invention are useful for the treatment
of
solid tumors. Solid tumors include, but are not limited to, sarcomas,
carcinomas,
lymphomas, and other solid tumor cancers, including, but not limited to germ
line tumors,
~mors 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,
melanoma, renal
cancer, bladder cancer, and mesothelioma.
The present invention provides methods for delivering a primary effector
molecule
for the treatment of a solid tumor cancer comprising administering, to an
animal,
preferably a mammal and most preferably a human, in need of such treatment, a
pharmaceutical composition comprising an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
molecules. The present invention also provides methods for delivering a
primary effector
molecule for the treatment of a solid tumor cancer comprising administering,
to an animal,
preferably a mammal and most preferably a human, in need of such treatment, a
pharmaceutical composition comprising an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
molecules and one or more secondary effector molecules. The present invention
also
provides methods for delivering a primary effector molecule for the treatment
of a solid
tumor cancer comprising administering, to an animal, preferably a mammal and
most
preferably a human, in need of such treatment, a pharmaceutical composition
comprising
an attenuated tumor-targeted bacteria engineered to contain one or more
nucleic acid
molecules encoding one or more fusion proteins of the invention. Further, the
present
~ invention provides methods for delivering a primary effector molecule for
the treatment of
a solid tumor cancer comprising administering, to an animal, preferably a
mammal and
most preferably a human, in need of such treatment, a pharmaceutical
composition
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comprising an attenuated tumor-targeted bacteria engineered to contain one or
more
nucleic acid molecules encoding one or more fusion proteins of the invention
and one or
more effector molecules (i.e., primary or/and secondary molecules). In a
preferred
embodiment, the attenuated tumor-targeted bacteria is Salmonella. In a
specific mode, the
attenuated tumor-targeted bacteria comprises an enhanced release system.
In certain embodiments, attenuated tumor-targeted bacteria engineered to
express
one or more nucleic acid molecules encoding one or more effector molecules
and/or,fusion
proteins can be used in conjunction with other known cancer therapies. For
example,
attenuated tumor-targeted bacteria engineered to express one or more nucleic
acid
molecules encoding one or more effector molecules and/or fusion proteins can
be used in
conjunction with a chemotherapeutic agent. Examples of chemotherapeutic agents
include,
but are not limited to, cisplatin, ifosfamide, paclitaxol, taxanes,
topoisomerase I inhibitors
(e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine,
oxaliplatin, S-
fluorouracil (5-FU), leucovorin, vinorelbine, temodal, taxol, cytochalasin B,
gramicidin D,
emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine,
colchicin, doxorubicin,
1 S daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,
actinomycin D,
1-dehydrotestosterone, melphalan, glucocorticoids, procaine, tetracaine,
lidocaine,
propranolol, and puromycin homologs, and cytoxan. Alternatively, attenuated
tumor-
targeted bacteria engineered to express one or more nucleic acid molecules
encoding one
or more effector molecules and/or fusion proteins can be used in conjunction
with radiation
therapy.
The present invention includes the sequential or concomitant administration of
anti-
cancer agents and attenuated tumor-targeted bacteria engineered to express one
or more
nucleic acid molecules encoding one or more effector molecules and/or fusion
proteins.
The invention encompasses combinations of anti-cancer agents and attenuated
tumor-
targeted bacteria engineered to express one or more nucleic acid molecules
encoding one
or more effector molecules and/or fusion proteins that are additive or
synergistic.
The invention also encompasses combinations of anti-cancer agents and
attenuated
tumor-targeted bacteria engineered to express one or more nucleic acid
molecules encoding
one or more effector molecules and/or fusion proteins that have different
sites of action.
Such a combination provides an improved therapy based on the dual action of
these'
therapeutics whether the combination is synergistic or additive. Thus, the
novel
combinational therapy of the present invention yields improved efficacy over
either agent
used as a single-agent therapy.
3.1. DEFINITIONS AND ABBREVIATIONS
As used herein, Salmonella encompasses all Salmonella species, including:
Salmonella typhi, Salmonella choleraesuis, and Salmonella enteritidis.
Serotypes of
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Salmonella are also encompassed herein, for example, typhimurium, a subgroup
of
Salmonella enteritidis, commonly referred to as Salmonella typhimurium.
Analog: As used herein, the term "analog" refers to a polypeptide that
possesses a
similar or identical function as a primary or secondary effector molecule but
does not
necessarily comprise a similar or identical amino acid sequence of a primary
or secondary
effector molecule, or possess a similar or identical structure of a primary or
secondary ,
effector molecule. A polypeptide that has a similar amino acid sequence refers
to a
polypeptide that satisfies at least one of the following: (a) a polypeptide
having an amino
acid sequence that is at least 30%, at least 35%, at least 40%, at least 45%,
at least 50%, at
least SS%, at least 60%, at least 65%, at least 70%, at least 75%, at least
80%, at least 85%,
at least 90%, at least 95% or at least 99% identical to the amino acid
sequence of a primary
or secondary effector molecule described herein; (b) a polypeptide encoded by
a nucleotide
sequence that hybridizes under stringent conditions to a nucleotide sequence
encoding a
primary or secondary effector molecule described herein of at least S
contiguous amino
acid residues, at least 10 contiguous amino acid residues, at least 15
contiguous amino acid
residues, at least 20 contiguous amino acid residues, at least 25 contiguous
amino acid
residues, at least 40 contiguous amino acid residues, at least 50 contiguous
amino acid
residues, at least 60 contiguous amino residues, at least 70 contiguous amino
acid residues,
at least 80 contiguous amino acid residues, at least 90 contiguous amino acid
residues, at
least 100 contiguous amino acid residues, at least 125 contiguous amino acid
residues, or at
least 150 contiguous amino acid residues; and (c) a polypeptide encoded by a
nucleotide
sequence that is at least 30%, at least 35%, at least 40%, at least 45%, at
least 50%, at least
SS%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at
least 85%, at
least 90%, at least 95% or at least 99% identical to the nucleotide sequence
encoding a
primary or secondary effector molecule described herein. A polypeptide with
similar
structure to a primary or secondary effector molecule described herein refers
to a
polypeptide that has a similar secondary, tertiary or quaternary structure of
primary or
secondary effector molecule described herein. The structure of a polypeptide
can be
determined by methods known to those skilled in the art, including but not
limited to,
peptide sequencing, X-ray crystallography, nuclear magnetic resonance,
circular dichroism,
and crystallographic electron microscopy.
Anti-angiogenic factor: An anti-angiogenic factor is any proteinaceous
molecule
which has anti-angiogenic activity, or a nucleic acid encoding such a
proteinaceous
molecule. In a preferred embodiment, the anti-angiogenic factor is a peptide
fragment or
cleavage fragment of a larger protein.
Attenuation: Attenuation is a modification so that a microorganism or 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 microorganism or vector is administered to the
patient.
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Bacteriocin: A bacteriocin is a bacterial proteinaceous toxin with selective
activity,
in that the bacterial host is immune to the toxin. Bacteriocins may be encoded
by the
bacterial host genome or by a plasmid, may be toxic to a broad or narrow range
of other
bacteria, and may have a simple structure comprising one or two subunits or
may be a
multi-subunit structure. In addition, a host expressing a bacteriocin has
immunity against
the bacteriocin.
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.
Derivative: As used herein, the term "derivative" in the context of a
"derivative of
a polypeptide" refers to a polypeptide that comprises an amino acid sequence
of a
polypeptide, such as a primary or secondary effector molecule, which has been
altered by
the introduction of amino acid residue substitutions, deletions or additions,
or by the
covalent attachment of any type of molecule to the polypeptide. The term
"derivative" as
used herein in the context of a "derivative of a primary or a secondary
effector molecule"
refers to a primary or secondary effector molecule which has been so modified,
e.g., by the
covalent attachment of any type of molecule to the primary or secondary
molecule. For
example, but not by way of limitation, a primary or secondary effector
molecule may be
modified, e.g., by proteolytic cleavage, linkage to a cellular ligand or other
protein, etc. A
derivative of a primary or secondary effector molecule may be modified by
chemical
modifications using techniques known to those of skill in the art (e.g., by
acylation,
phosphorylation, carboxylation, glycosylation, selenium modification and
sulfation).
Further, a derivative of a primary or secondary effector molecule may contain
one or more
non-classical amino acids. A polypeptide derivative possesses a similar or
identical
function as a primary or secondary effector molecule described herein. The
term
"derivative" in the context of a "derivative of an msbB' attenuated tumor-
targeted
Salmonella mutant" refers to a modified msbB- Salmonella mutant as defined in
International Publication No. WO 99/13053 at page 17, incorporated herein by
reference in
its entirety.
Fragment: As used herein, the term "fragment" refers to a peptide or
polypeptide
comprising an amino acid sequence of at least 2 contiguous amino acid
residues, at least 5
contiguous amino acid residues, at least 10 contiguous amino acid residues, at
least 15
contiguous amino acid residues, at least 20 contiguous amino acid residues, at
least 25
contiguous amino acid residues, at least 40 contiguous amino acid residues, at
least 50
contiguous amino acid residues, at least 60 contiguous amino residues, at
least 70
contiguous amino acid residues, at least contiguous 80 amino acid residues, at
least
contiguous 90 amino acid residues, at least contiguous 100 amino acid
residues, at least
contiguous 125 amino acid residues, at least 150 contiguous amino acid
residues, at least
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contiguous 175 amino acid residues, at least contiguous 200 amino acid
residues, or at least
contiguous 250 amino acid residues of the amino acid sequence of a primary or
secondary
effector molecule.
Functional fragment: As used herein, the term "functional fragment" refers to
a
fragment of a primary or secondary effector molecule that retains at least one
functiomof
the primary or secondary effector molecule (e.g., enzymatic activity, anti-
angiogenic
activity, or anti-tumor activity of the effector molecule).
Fusion protein: As used herein, the term "fusion protein" refers to a
polypeptide
that comprises an amino acid sequence of primary or secondary effector
molecule, or
functional fragment or derivative thereof, and an amino acid sequence of a
heterologous
p°lypeptide (e.g., a non-primary or non-secondary effector molecule).
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,
1 S 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.
Purified: As used herein, "purified" attenuated tumor-targeted bacterial
strain is
substantially free of contaminating proteins or amino acids ( e.g., debris
from dead
bacteria), or media. An attenuated tumor-targeted bacterial strain that is
substantially free
of contaminating proteins or amino acids includes preparations of attenuated
tumor-
targeted bacteria having less than about 30%, 20%, 10%, or 5% (by dry weight)
of
contaminating protein or amino acid.
Release factor: As used herein, a release factor includes any protein, or
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.
Septic shock: Septic shock is a state of internal organ failure due to a
complex
cytokine cascade, initiated by TNF-a. The relative ability of a microorganism
or vector to
elicit TNF-a is used as one measure to indicate its relative ability to induce
septic shock.
Tumor-targeted: Tumor-targeted is defined as the ability to preferentially
localize
to a cancerous target cell or tissue relative to a non-cancerous counterpart
cell or tissue and
replicate. Thus, a tumor-targeted bacteria such as Salmonella preferentially
attaches to,
infects and/or remains viable in the cancerous target cell or the tumor
environment.
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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).
As used herein, the strain designations VNP20009 (International Publication
No.
WO 99/13053), YS 1646 and 41.2.9 are used interchangeably and each refer to
the strain
deposited with the American Type Culture Collection and assigned Accession No.
202165.
As used herein, the strain designations YS1456 and 8.7 are used
interchangeably and each
refer to the strain deposited with the American Type Culture Collection and
assigned
Accession No. 202164.
The present invention may be understood more fully by reference to the
following
detailed description, illustrative examples of specific embodiments and the
appended
figures.
4. BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Coding sequence for the mature human TNF-a. Both DNA (SEQ ID
.
N0:3) and protein (SEQ ID N0:4) sequences are indicated.
FIG. 2. Derivation of the Salmonella VNP20009 serf-strain.
FIG. 3. TNF-a, expression from a chromosomally-integrated trc promoter driven
TNF-a, gene in Salmonella fyphimurium.
FIG. 4. Coding sequence for the synthetic OmpA signal sequence (nucleotides 1-
63) fusion to the mature human TNF-a (nucleotides 67-543). Both DNA (SEQ ID
N0:7)
and protein (SEQ ID N0:8) sequences are indicated for the fusion construct.
FIG. 5. Periplasmic localization and processing of an OmpA/TNF-a, fusion
protein
in E-coli (JM 109 strain).
FIG. 6. Coding sequence for the OmpA signal sequence (nucleotides 1-63) fusion
to the mature human TRAIL (nucleotides 67-801). Both DNA (SEQ >D N0:9) and
protein
(SEQ ID NO:10) sequences are indicated for the fusion construct.
FIG. 7. Expression and processing of an OmpA TRAIL fusion protein in E-coli
0109 strain).
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FIG. 8. Coding sequence for the modified OmpA signal sequence (nucleotides 1-
63) fusion to the mature (C125A) human IL-2 (nucleotides 64-462). Both DNA
(SEQ ID
NO:11) and protein (SEQ ID N0:12) sequences are indicated for the fusion
construct.
FIG. 9. Expression and processing of mature human IL-2 fused to the phoA(8L)
or
ompA (8L) synthetic signal peptides.
FIG. I0. Coding sequence for the modif ed phoA signal sequence (nucleotides 1-
63) fusion to the mature (C125A) human IL-2 (nucleotides 64-462). Both DNA
(SEQ ID
N0:13) and protein (SEQ ID N0:14) sequences are indicated for the fusion
construct.
FIG. 11. In vivo anti-tumor efficacy of an attenuated strain of Salmonella
typhimurium expressing the mature form of human TNF-a.
FIG. 12. Effect of BRP expression on anti-tumor efficacy in vivo. The figure
shows
1 S a graphic representation of mean tumor size over time of a C57BL/6 mouse
population
with B 16 melanoma tumors treated with ( 1 ) a PBS control; (2) VI~TP20009;
and (3)
VNP20009 harboring the pSW 1 plasmid, which comprises the BRP gene.
FIG. 13. Anaerobic induction of ~3-gal gene expression under the control of
the
pepT promoter in Salmonella. FIG. 13A demonstrates the in vitro induction of
~i-gal
expression in response to anaerobic conditions of two strains of Salmonella,
YS 1456 and
VNP20009. FIG. 13B demonstrates the in vivo induction of ~i-gal in tumor v.
liver cells of
VNP20009 Salmonella expressing BRP, ~3-gal, or BRP and ~i-gal.
FIG. 14. Tetracycline induction of (3-gal gene expression under the control of
the
Tet promoter in Salmonella. The dose-response indicates a linear response to
Tetracycline
up to a concentration of approximately 0.15 pg/ml, after which there response
declines,
presumably as a result of the antibiotic function of Tetracycline.
FIG. 15. Hexahistidine-endostatin (HexaHIS-endostatin) expression from the
pTrc99a vector. FIG. 15A shows the expression of HexaHIS-endostatin from three
independent clones transformed into Salmonella (VNP20009). FIG. 15B shows the
expression of HexaHIS-endostatin from five independent clones transformed into
E.coli
(DHSa). Even numbered lanes indicate extracts from uninduced cultures, whereas
odd
numbered lanes indicate the corresponding IPTG-induced cultures.
FIG. 16. Expression of HexaHIS-endostatin from the plasmid YA3334:
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HexaHIS-endostatin in the asd system (utilizing the trc promoter) is able to
express a band
of the correct size for HexaHIS-endostatin (~25kD) by Western analysis with a
anti-
histidine antibody (lanes 1-8 correspond to eight independent clones).
FIG. 17. Efficacy of VNP20009 cells expressing endostatin on C38 murine colon
carcinoma. The figure shows a graphic representation of mean tumor size over
time of a
mouse population with established C38 tumors treated with (1) a PBS control;
(2) asd -
VNP20009 carrying an empty YA3334 vector; (3) asd -VNP20009 which expresses
hexahistidine-endostatin; (4) and VNP20009 which expresses hexahistidine-
endostatin and
BRP.
l0
FIG. 18. Efficacy of VNP20009 cells expressing endostatin on DLD I human colon
carcinoma. The figure shows a graphic representation of mean tumor size over
time of a
nude mouse population with established DLD1 tumors treated with (1) a PBS
control; (2)
asd- VNP20009 carrying an empty YA3334 vector; and (3) VNP20009 which
expresses
15 hexahistidine-endostatin and BRP.
FIG. 19. Anti-proliferative activity of lysates from attenuated tumor-targeted
Salmonella expressing human endostatin on endothelial cells. This figure shows
the
inhibition of human vein endothelial cell (HUVEC) proliferation in response to
bFGF and
20 lysates corresponding to 8 x 10$ bacteria. As a control Salmonella
containing the empty
pTrc vector was used. Each data point is a mean of quadruplicate values from a
representative experiment. Samples were normalized by the number of bacteria.
FIG. 20. Anti-proliferative activity of lysates from attenuated tumor-targeted
25 Salmonella expressing platelet factor-4 peptide (amino acids 47-70 of
platelet factor-4) and
thrombospondin peptide(13.40) on endothelial cells. This figure shows the
inhibition of
human vein endothelial cell (HUVEC) proliferation in response to bFGF and
lysates
corresponding to 3.2 x 10g bacteria. As a control Salmonella containing the
empty pTrc
vector was used. Each data point is a mean of quadruplicate values from a
representative
30 experiment. Samples were normalized by the number of bacteria.
FIG. 21. Construction of the pE3.shuttle -1 Vector.
FIG. 22. Construction of the Col E3-CA38 Vector (GenBank Accession Number
35 AF129270). The nucleotide sequence of the Col E3-CA38 Vector is as depicted
in SEQ
ID NO: 1. The Col E3-CA38 Vector contains 5 open reading frames as depicted in
SEQ ID
Nos: 2-5, respectively.
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FIG. 23. Construction of the Col E3-CA38/BRP-1 vector.
FIG. 24. Bar Graph showing the amount of lethal units of colicin E3 produced
by
each strain.
FIG. 25. Halo assay for various strains exposed to ultraviolet light or x-
rays.
FIG. 26. Efficacy of 41.2.9/Col E3 on C38 murine colon carcinoma.
FIG. 27. Anti-tumor activity of 41.2.9/Col/E3 on DLD1 human colon carcinoma in
~~u mice.
FIG. 28. Efficacy of 41.2.9/Col E3 on B 16 murine melanoma.
FIG. 29. Cytotoxicity of Salmonella expressing cloned E. coli CNFI .
FIG. 30. Hela cells exposed to CNF1 (A) show enlargement and multinucleation
relative to normal Hela cells (B).
FIG. 31. The msbB portion of the pCVD442-msbB vector in the 3' to 5'
orientation
(as viewed in th FIG. 32 map), with a deletion in the middle of msbB and
containing
internal Notl, PacI, SpliI, SfiI, SwaI and DraI polylinker in its place (SEQ
ID N0:61). See
FIG. 32.
FIG. 32. Restriction map and schematic of the pCVD442-msbB vector for cloning
DNA in the DmsbB region and subsequent insertion on the chromosome. msbBdel,
the 5'
and 3' regions of DmsbB; mob RP4, the mobilization element in order for the
plasmid to be
transferred from one strain to another. bla; the beta-lactamase gene which
confers
sensitivity to b-lactam antibiotic such as carbenicillin and ampicillin. sacB,
the gene which
confers sensitivity to sucrose.
FIG. 33. 1) pCVD442-Tet-BRP-AB vector, 2) homologous recombination with the
DmsbB chromosomal copy in Salmonella YS50102, 3) chromosomal integration in
Salmonella YS50102, and following phage transduction to strain VNP20009, 4)
sucrose
resolution resulting in strain 41.2.9-Tet-BRP-AB. oriR6K, the plasmid origin
of
replication; mobRP4, the mobilization element in order for the plasmid to be
transferred
from one strain to another. amp; the beta-lactamase gene which confers
sensitivity to b-
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lactam antibiotic such as carbenicillin and ampicillin. sacB, the gene which
confers
sensitivity to sucrose. Note: not drawn to scale.
FIG. 34. Percent cytotoxicity of tetBRPAB clone #26 and clone #31 compared to
positive and negative controls (HSC10 and 41.2.9) following 72 hours of
exposure to
SKOV3 cells (Ave N=8). Expression of verotoxin was induced by tetracycline,
(see clones
26 and 31). Tetracycline treatment (+); and no tetracycline treatment (-). The
E. coli strain
HSC10 was used as a positive control for percent cytotoxicity.
FIG. 35. Halo formation on blood agar for attenuated tumor-targeted Salmonella
in
the absence of tetracycline (1A) and the presence of tetracycline (1B). Halo
formation for
attenuated tumor-targeted Salmonella engineered to constitutively express
SheA. in the
absence of tetracycline (2A) and the presence of tetracycline (2B). Halo
formation for
attenuated tumor-targeted Salmonella engineered to express tetracycline
inducible SheA in
the absence of tetracycline (3A) and the presence of tetracycline (3B).
FIG. 36. (A) An illustration of the TAT-apoptin fusion protein without the
hexahistadine tag. (B) An illustration of the TAT-apoptin fusion protein with
the
hexahistadine tag. (C) A) An illustration of the TAT-apoptin fusion protein
with an
OmpA-8L signal sequence.
FIG. 37. Coding sequence for TAT-apoptin fusion protein. Both DNA (SEQ ID
N0:57) and protein (SEQ ID N0:58) sequences are indicated.
FIG. 38. Coding sequence for hexahistidine-TAT-apoptin fusion protein. Both
DNA (SEQ ID N0:59) and protein (SEQ ID N0:60) sequences are indicated.
FIG. 39. Efficacy of VNP20009/cytoxan combination therapy on M27 lung
carcinoma growth in C57BL/6 mice.
FIG. 40. Efficacy of VNP20009/mitomycin combination therapy on M27 lung
carcinoma growth in C57BL/6 mice.
FIG. 41. Efficacy of VNP20009/cisplatin combination therapy on M27 lung
carcinoma growth in C57BL/6 mice.
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5. DETAILED DESCRIPTION OF THE INVENTION
The present invention utilizes attenuated tumor-targeted strains of bacteria
to
deliver high levels of therapeutic primary effector molecules) to tumors. The
present
invention provides the advantage of bypassing potential systemic toxicity of
certain
primary effector molecules (e.g., septic shock caused by TNF-a). The present
invention
provides delivery of one or more primary effector molecules) and optionally,
one or more
secondary effector molecules) to a solid tumor. More particularly, the
invention
encompasses the preparation and the use of attenuated tumor-targeted bacteria,
such as,
e.g., Salmonella, as a vector for the delivery of one or more primary effector
molecules)
and optionally, one or more secondary effector molecule(s), to an appropriate
site of action,
e.g., the site of a solid tumor. Specifically, the attenuated tumor-targeted
bacteria of the
invention are facultative aerobes or facultative anaerobes, which are
engineered to encode
one or more primary effector molecules) and optionally, one or more secondary
effector
molecule(s).
The attenuated tumor-targeted bacterial-based delivery system presently
described
provides local delivery of one or more effector molecules) to the site of
solid tumors. The
invention provides safe and effective methods by which a primary effector
molecule(s),
which may be toxic or induce an unwanted side effect (e.g., an unwanted
immunological
effect) when delivered systemically to a host, can be delivered locally to
tumors by an
attenuated tumor-targeted bacteria, such as Salmonella with reduced toxicity
to the host.
The invention also provides combinatorial delivery of one or more primary
effector
molecules) and optionally, one or more secondary effector molecules) which are
delivered by an attenuated tumor-targeted bacteria, such as Salmonella. The
invention also
provides combinatorial delivery of different attenuated tumor-targeted
bacteria carrying
one or more different primary effector molecules) and/or optionally, one or
more different
secondary effector molecule(s).
The present invention also provides methods for local delivery of one or more
fusion proteins comprising an effector molecule by attenuated tumor-targeted
bacteria
engineered to express said fusion proteins at the site of the solid tumor(s).
In one
embodiment, attenuated tumor-targeted bacteria are engineered to express a
fusion protein
comprising a signal peptide and an effector molecule. In another embodiment,
attenuated
tumor-targeted bacteria are engineered to express a fusion protein comprising
a signal
peptide, a proteolytic cleavage site, and an effector molecule. In another
embodiment,
attenuated tumor-targeted bacteria are engineered to express a fusion protein
comprising a
ferry peptide and an effector molecule. In another embodiment, attenuated
tumor-targeted
bacteria are engineered to express a fusion protein comprising a signal
peptide, a ferry
peptide and an effector molecule. In yet another embodiment, attenuated tumor-
targeted
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bacteria are engineered to express a fusion protein comprising a signal
peptide, a
proteolytic cleavage site, a ferry peptide and an effector molecule.
Attenuated tumor-
targeted bacteria are engineered to express one or more fusion proteins of the
invention and
one or more effector molecules of the invention.
The present invention also provides pharmaceutical compositions comprising a
pharmaceutically acceptable carrier and an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
molecules. The present invention also provides pharmaceutical compositions
comprising a
pharmaceutically acceptable carrier and an attenuated tumor-targeted bacteria
engineered
to contain one or more nucleic acid molecules encoding one or more primary
effector
molecules and one or more secondary effector molecules. Further, the present
invention
provides pharmaceutical compositions comprising a pharmaceutically acceptable
Garner
and an attenuated tumor-targeted bacteria engineered to contain one or more
nucleic acid
molecules encoding one or more fusion proteins and one or more effector
molecules.
The present invention provides methods of treating solid tumor cancers in an
animal, said methods comprising administering to an animal in need thereof an
attenuated
tumor-targeted bacteria engineered to express one or more nucleic acid
molecules encoding
one or more primary effector molecules. The present invention also provides
methods of
treating solid tumor cancers in an animal, said methods comprising
administering to an
animal in need thereof an attenuated tumor-targeted bacteria engineered to
express one or
more nucleic acid molecules encoding one or more primary effector molecules
and one or
more secondary effector molecules. Further, the present invention provides
methods of
treating solid tumor cancers in an animal, said methods comprising
administering to an
animal in need thereof an attenuated tumor-targeted bacteria engineered to
contain one or
more nucleic acid molecules encoding one or more fusion proteins and one or
more
effector molecules. Preferably, the animal is a mammal (e.g., a dog, a cat, a
horse, a cow, a
monkey, or a pig) and more preferably the animal is a human. Examples of solid
tumor
cancers include, but are not limited to, sarcomas, carcinomas, lymphomas, and
other solid
tumor cancers, 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, central
nervous
system cancer, germ cell line cancer, melanoma, renal cancer, bladder cancer,
and
mesothelioma.
Although not intending to be limited to any one mechanism, the inventors
believe
that the present invention results in the targeted expression of the effector
molecules) at
the site of a tumor by delivery of the attenuated tumor-targeted bacterial
vector containing
the effector molecule(s).
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For reasons of clarity, the detailed description is divided into the following
subsections: Bacterial Vectors; Primary Effector Molecules for Tumor Therapy;
Secondary
Effector Molecules for Co-expression With Primary Effector Molecules;
Derivatives and
Analogs; Fusion Proteins; Expression Vehicles; and Methods and Compositions
for
Delivery.
5.1. BACTERIAL VECTORS
Any attenuated tumor-targeted bacteria may be used in the methods of the
invention. More specifically, the attenuated tumor-targeted bacteria used in
the methods of
the invention are facultative aerobes or facultative anaerobes. Examples of
attenuated
~mor-targeted bacteria that are facultative aerobes or facultative anaerobes
which may be
used in the methods of the invention include, but are not limited to,
Escherichia coli
including enteroinvasive Escherichia coli, Salmonella spp., Shigella spp.,
Yersinia
enterocohtica, Listeria monocytogenies, Mycoplasma hominis, and Streptococcus
spp..
Factors contributing to attenuation and tumor-targeting are described herein
and
1 S may be used to construct or select an appropriate bacterial strain 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 are incorporated herein by
reference.
Examples of attenuated tumor-targeted bacteria are also described in
International
Application W099/13053, which is incorporated herein by reference in its
entirety. In
certain embodiments of the invention, a bacteria may be modified by methods
known in
the art to be attenuated or highly attenuated.
The present invention provides attenuated tumor-targeted bacteria as a vector
for
the delivery of one or more primary effector molecules (e.g., a TNF family
member, a
c~otoxic peptide or polypeptide, a tumor inhibitory enzyme, or an anti-
angiogenic factor)
alone or in combination with a one or more secondary effector molecule(s). The
present
invention also provides attenuated tumor-targeted bacteria as a vector for the
delivery of
one or more fusion proteins of the invention alone or in combination with one
or more
effector molecules. In a preferred embodiment of the invention, the attenuated
tumor-
targeted bacteria which is engineered to express one or more nucleic acid
molecule
encoding effector molecules and/or fusion proteins is Salmonella.
While the teachings of the following section refers specifically to
Salmonella, the
compositions and methods of the invention are in no way meant to be restricted
to
Salmonella but encompass any other bacterium to which the teachings apply.
Suitable
bacterial species include, but are not limited to, Escherichia coli including
enteroinvasive
Escherichia coli, Salmonella spp., Shigella spp., Yersinia enterocohtica,
Listeria
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monocytogenies, Mycoplasma hominis, Streptococcus spp., wherein the bacterium
is a
facultative aerobe or facultative anaerobe.
5.1.1 SALMONELLA VECTORS
Any attenuated tumor-targeted bacteria can be modified using the teaching of
the
invention to encode one or more primary effector molecules and optionally, one
or more
secondary effector molecules to produce a novel attenuated tumor-targeted
bacteria useful
for the delivery of one or more effector molecules of the invention to a solid
tumor.
Further, any attenuated tumor-targeted bacteria can be modified using the
teaching of the
invention to encode one or more fusion proteins of the invention and
optionally, one or
more effector molecules to produce a novel attenuated tumor-targeted bacteria
useful for
the delivery of fusion proteins and effector molecules of the invention to a
solid tumor.
Bacteria such as Salmonella is a causative agent 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,
1 S Clinical Microbiol. Revs. 6:57-68). Therefore, to allow the safe use of
Salmonella vectors
in the present invention, the bacterial vectors such as Salmonella are
attenuated in their
virulence for causing disease. In the present application, attenuation, in
addition to its
traditional definition in which a microorganism vector is modified so that the
microorganism vector is less pathogenic, is intended to include also the
modification of a
microorganism vector so that a lower titer of that derived microorganism
vector can be
administered to a patient and still achieve comparable results as if one had
administered a
higher titer of the parental microorganism vector. The end result serves to
reduce the risk
of toxic shock or other side effects due to administration of the vector 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-S 193). Table 4 of International Publication WO 96/40238 is
an
illustrative list of Salmonella virulence factors whose deletion results in
attenuation.
1'et another method for the attenuation of the bacterial vectors, 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
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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 vector 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 hydroxymyristoyl)-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.
In addition to being attenuated, the bacterial vectors of the invention are
tumor-
targeted, i.e. the bacteria preferentially attaches to, infects, and/or
remains 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 Section 6.1
(pages 25-32;
tumor-targeting) and Section 6.2.2 (pages 43-S l;attenuation) of International
Publication
WO 96/40238, which are incorporated herein by reference. As the resulting
vectors are
highly specific and 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 microorganism culture is
increased such
that lower titers of microorganism vectors 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 vectors.
An illustrative example of an attenuated tumor-targeted bacterium having an
LPS
pathway mutant is the msbB~ Salmonella mutant described in International
Publication
W099/13053, 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 vector. The msbB- Salmonella induce TNF-a
expression at levels of about S percent to about 40 percent compared to the
levels induced
by wild-type Salmonella.
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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.~u.") or on a pglkg 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 vector is modified to contain one or more
primary
effector molecules) and optionally, one or more secondary effector molecules)
of the
invention.
The present invention also encompasses the use of derivatives of msbB-
attenuated
tumor-targeted Salmonella mutants. Derivatives of msbB- attenuated tumor-
targeted
Salmonella mutants can be modified using the teaching of the invention to
encode one or
more primary effector molecules) and optionally, one or more secondary
effector
molecules) to produce a novel attenuated tumor-targeted bacteria useful for
the delivery of
one or more effector molecules) of the invention to a solid tumor.
The stability of the attenuated phenotype is important such that the strain
does not
revert to a more virulent phenotype during the course of treatment of a
patient. Such
stability can be obtained, for example, by providing that the virulence gene
is disrupted by
deletion or other non-reverting mutations on the chromosomal level rather than
epistatically.
Another method of insuring the attenuated phenotype is to engineer the
bacteria
such that it is attenuated in more than one manner, 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 encoding or expressing at least one primary effector molecule
is also
auxotrophic for purine. In certain embodiments, the attenuated tumor-targeted
bacteria
encoding or expressing at least one primary effector molecule are attenuated
by the
presence of a mutation in AroA, msbB, Purl or Serf. In other embodiments, the
attenuated
tumor targeted bacteria encoding at least one primary effector molecule 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 methods
of
the invention to express and deliver one or more primary effector molecules)
and.
optionally, one or more secondary effector molecules) to a solid tumor cancer.
In
preferred embodiments, the attenuated tumor-targeted bacteria are constructed
to express
one or more primary effector molecules) and optionally, one or more secondary
effector
molecule(s). Further, any attenuated tumor-targeted bacteria may be used in
the methods
of the invention to express and deliver one or more fusion proteins and
optionally, one or
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more effector molecules to a solid tumor cancer. In preferred embodiments, the
attenuated
tumor-targeted bacteria are constructed to express one or more fusion proteins
and
optionally, one or more effector molecules.
5.2. PRIMARY EFFECTOR MOLECULES
FOR TUMOR THERAPY
The invention provides for delivery of primary (and optionally secondary)
effector
molecules) by attenuated tumor-targeted bacteria, such as Salmonella. The
effector
molecules of the invention are proteinaceous molecules, (e.g., protein
(including but not
limited to peptide, polypeptide, protein, post-translationally modified
protein, etc.). The
invention further provides nucleic acid molecules which encode the primary
effector
molecules of the invention.
The primary effector molecules can be derived from any known organism,
including, but not limited to, animals, plants, bacteria, fungi, and protista,
or viruses. In a
preferred embodiment of the invention, the primary effector molecules) is
derived from a
mammal. In a more preferred embodiment, the primary effector molecules) is
derived
from a human. The primary effector molecules of the invention comprise members
of the
TNF family, anti-angiogenic factors, cytotoxic polypeptides or peptides, tumor
inhibitory
enzymes, and functional fragments thereof.
In a specific embodiment, the primary effector molecules of the invention are
members of the TNF family or functional fragments thereof. Examples of TNF
family
members, include, but are not limited to, tumor necrosis factor-a (TNF-a),
tumor necrosis
factor-~i (TNF-(3), 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), LT-a, LT-(3, OX40L, CD40L, Fast, CD27L, CD30L,
4-1 BBL, APRIL, LIGHT, TL 1, TNFSF 16, TNFSF 17, and AITR-L. In a preferred
embodiment, a primary effector molecule of the invention 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
review see, e.g., Kwon, B. et al., 1999, Curr. Opin. Immunol. 11:340-345,
which describes
members of the TNF family. Also, Table 1 herein below, lists classic and
standardized
nomenclature of exemplary members of the TNF family. In a preferred embodiment
of
the invention the primary effector molecule of the invention is 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), or CD40 ligand (CD40L).
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TABLE 1
TNF FAMILY MEMBERS
Classic Nomencalture Standardized Nomenclature
LT-a TNFSF 1
TNF-a TNFSF2
LT-(3 TNFSF3
OX40L TNESF4
CD40L TNFSFS
FaSL ' TNFSF6
CD27L TNFSF7
CD30L TNFSF8
4-1BBL TNFSF9
TRAIL TNFSF 10
TRANCE TNFSF 11
TWEAK TNFSF12
APRIL TNFSF 13
LIGHT TNFSF 14
TL 1 TNFSF 15
--- TNFSF 16
--- TNFSF 17
AITR-L TNFSF 18
In another specific embodiment, the primary effector molecules of the
invention are
anti-angiogenic factors or functional fragments thereof. Examples of anti-
angiogenic
factors, include, but are not limited to, endostatin, angiostatin, apomigren,
anti-angiogenic
antithrombin III, the 29 lcDa N-terminal and a 40 lcDa 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~~33 and the VEGF receptor.
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In a preferred embodiment of the invention, a primary effector molecule of the
invention is endostatin. Naturally occurring endostatin consists of the C-
terminal 180
amino acids of collagen XVIII (cDNAs encoding two splice forms of collagen
XVIII have
Genbank Accession No. AF18081 and AF18082).
In another preferred embodiment of the invention, a primary effector molecule
of
the invention is plasminogen fragments (the coding sequence for plasminogen
can be found
in Genbank Accession No. NM 000301 and A33096). Angiostatin peptides naturally
include the four kringle domains of plasminogen, kringle 1 through kringle 4.
It has been
demonstrated that recombinant kringle 1, 2 and 3 possess the anti-angiogenic
properties of
the native peptide, whereas kringle 4 has no such activity (Cao et al., 1996,
J. Biol. Chem.
271:29461-29467). Accordingly, the angiostatin effector molecule of the
invention
comprises at least one and preferably more than one kringle domain selected
from the
group consisting of kringle 1, kringle 2 and kringle 3. In a specific
embodiment, the
primary effector molecule of the invention is a human angiostatin molecule
selected from
the group consisting of 40 kDa isoform, the 42 kDa isoform, the 45 kDa
isofbrm, or a
combination thereof. In another embodiment, the primary effector molecule is
the kringle
5 domain of plasminogen, which is a more potent inhibitor of angiogenesis than
angiostatin
(angiostatin comprises kringle domains 1-4).
In another preferred embcdiment of the invention, a primary effector molecule
of
the invention is antithrombin III. Antithrombin III, which is referred to
hereinafter as
antithrobin, comprises a heparin binding domain that tethers the protein to
the vasculature
walls, and an active site loop which interacts with thrombin. When
antithrombin is
tethered to heparin, the protein elicits a conformational change that allows
the active loop
to interact with thrombin, resulting in the proteolytic cleavage of said loop
by thrombin.
The proteolytic cleavage event results in another change of conformation of
antithrombin,
which (i) alters the interaction interface between thrombin and antithrombin
and (ii)
releases the complex from heparin (Carrell, 1999, Science 285:1861-1862, and
references
therein). O'Reilly et al. (1999, Science 285:1926-1928) have discovered that
the cleaved
antithrombin has potent anti-angiogenic activity. Accordingly, in one
embodiment, the
anti-angiogenic factor of the invention is the anti-angiogenic form of
antithrombin. For the
delivery of said protein to a solid tumor according to the methods of the
invention, the
bacterial vector is modified to express full length antithrombin Genbank
Accession No.
NM 000488 and a proteolytic enzyme that catalyzes the cleavage of antithrombin
to
produce the anti-angiogenic form of the protein. The proteolytic enzyme is
selected from
the group comprising thrombin, pancreatic elastases, and human neutrophil
elastase. In a
preferred embodiment, the proteolytic enzyme is pancreatic elastase. Methods
for the
recombinant expression of functional pancreatic elastase are taught by Shirasu
(Shirasu et
al., 1987, J. Biochem. 102:1555-1563).
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In another preferred embodiment of the invention, a primary effector molecule
of
the invention is the 40 kDa and/or 29 kDa proteolytic fragment of fibronectin.
The
expression vehicles for these fragments can be generated by standard methods
using the
full length nucleic acid sequence encoding the fibronectin precursor protein
(Genbank
Accession No. X02761 ), and a description of the maturation of the encoded
protein. In a
preferred embodiment the 40 kDa and/or the 29 kDa fragment of fibronectin is
expressed
as a cytoplasmic protein under the control of the trc promoter, for example by
insertion
into the pTrc99A plasmid.
In another preferred embodiment of the invention, a primary effector molecule
of
the invention is a urokinase plasminogen activator (uPA) receptor antagonist.
In one mode
of the embodiment, the antagonist is a dominant negative mutant of uPA (see,
e.g.,
Crowley et al., 1993, Proc. Natl. Acad. Sci. USA 90:5021-5025). In another
mode of the
embodiment, the antagonist is a peptide antagonist or a fusion protein thereof
(Goodson et
al., 1994, Proc. Natl. Acad. Sci. USA 91:7129-7133). In yet another mode of
the
embodiment, the antagonist is a dominant negative soluble uPA receptor (Min et
al., 1996,
Cancer Res. 56:2428-2433).
In another preferred embodiment of the invention, a primary effector molecule
of
the invention is the 16 kDa N-terminal fragment of prolactin, comprising
approximately
120 amino acids, or a biologically active fragment thereof (the coding
sequence for
prolactin can be found in Genbank Accession No. NM 000948). In a specific
embodiment, said prolactin fragment has a Cys58 ~Ser58 mutation to circumvent
undesired cross-linking of the protein by disulfide bonds.
In another preferred embodiment of the invention, a primary effector molecule
of
the invention is the 7.8 kDa platelet factor-4 fragment. In a specific
embodiment, the 7.8
kDa platelet factor-4 fragment is expressed as a fusion protein wherein the
amino terminal
comprises the first 35 amino acids of E. coli ~i-glucoronidase. In another
embodiment, the
heparin binding lysines of platelet factor-4 are mutated to glutamic acid
residues, which
results in a variant protein having potent anti-angiogenic activity (Maione et
al., 1991,
Cancer Res. 51:2077-2083). The coding sequence for platelet factor-4 has the
Genbank
Accession No. NM 002619.
In another preferred embodiment of the invention, a primary effector molecule
of
the invention is a small peptide corresponding to the anti-angiogenic 13 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, the small anti-angiogenic peptides of
laminin,
f bronectin, procollagen, or EGF, or small peptide antagonists of integrin
a~~33 or the VEGF
receptor. In a specific embodiment, the small peptides are expressed in tandem
to increase
protein stability. The sequences of the small peptides are provided by Cao
(1998, Prog.
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Mol. Subcell. Biol. 20:161-176), with the exception of VEGF receptor
antagonists (Soker
et al., 1993, J. Biol. Chem. 272:31582-31588). In a highly preferred
embodiment, the
small peptide comprises an RGD or NGR motif. In certain modes of the
embodiment, the
RGD or NGR containing peptide is presented on the cell surface of the host
bacteria, for
example by fusing the nucleic acid encoding the peptide in frame with a
nucleic acid
encoding one or more extracellular loops of OmpA.
In another specific embodiment, the primary effector molecules of the
invention are
cytotoxic polypeptides or peptides, or functional fragments thereof. A
cytotoxic
polypeptide or peptide is cytotoxic or cytostatic to a cell, for example, by
inhibiting cell
growth through interference with protein synthesis or through disruption of
the cell cycle.
Such a product may act 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.
Examples of cytotoxic polypeptides or peptides include, but are not limited
to,
members of the bacteriocin family, verotoxin, cytotoxic necrotic factor 1 (CNF
1; e.g., E.
~oli CNF 1 and Vibrio fischeri CNF 1 ), cytotoxic necrotic factor 2 (CNF2),
Pasteurella
multiocida toxin (PMT), hemolysin, CAAX tetrapeptides which are potent
competitive
inhibitors of farnesyltransferase, saporin, the ricins, abrin, other ribosome
inactivating
proteins (RIPS), Pseudomonas exotoxin, inhibitors of DNA, RNA or protein
synthesis,
antisense nucleic acids, other metabolic inhibitors (e.g., DNA or RNA cleaving
molecules
such as DNase and ribonuclease, protease, lipase, phospholipase), prodrug
converting
enzymes (e.g., thymidine kinase from HSV and bacterial cytosine deaminase),
light-activated porphyrin, ricin, ricin A chain, maize RIP, gelonin,
cytolethal distending
toxin, diphtheria toxin, diphtheria toxin A chain, trichosanthin, tritin,
pokeweed antiviral
protein (PAP), mirabilis antiviral protein (MAP), Dianthins 32 and 30, abrin,
monodrin,
bryodin, shiga, a catalytic inhibitor of protein biosynthesis from cucumber
seeds (see, e.g.,
International Publication WO 93/24620), Pseudomonas exotoxin, E. cola heat-
labile toxin,
E. cola heat-stable toxin, EaggEC stable toxin-1 (EAST), biologically active
fragments of
cytotoxins and others known to those of skill in the art. See, e.g., O'Brian
and Holmes,
Protein Toxins of Escherichia cola and Salmonella in Escherichia and
Salmonella. Cellular
and Molecular BioloQV, Neidhardt et al. (eds.), pp. 2788-2802, ASM Press,
Washington,
D.C. for a review of E. cola and Salmonella toxins.
In a preferred embodiment, the primary effector molecule is a member of the
bacteroicin family (see e.g., Konisky, 1982, Ann. Rev. Microbiol. 36:125-144),
with the
proviso that said bacteriocin family member is not a bacteriocin release
protein (BRP).
3 5 Examples of bacteriocin family members, include, but are not limited to,
CoIE 1, CoIE 1 a,
ColElb ColE2, ColE3, ColE4, ColES, ColE6,.ColE7, ColEB, ColE9, Colicin A,
Colicin K,
Colicin L, Colicin M, cloacin DF13, pesticin Al 122, staphylococcin 1580,
butyricin 7423,
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pyocin R1 or AP41, megacin A-216, microcin M15, and vibriocin (Jayawardene and
Farkas-Himsley,1970, J. Bacteriology vol. 102 pp 382-388). Most preferably the
primary
effector molecules) is colicin E3 or V, although colicins A, E1, E2, Ia, Ib,
K, L, and M
(see, Konisky, 1982, Ann. Rev. Microbiol. 36:125-144) are also suitable as a
primary
effector molecule(s). In another preferred mode of this embodiment, the
bacteriocin is a
cloacin, most preferably cloacin DF13.
In a preferred embodiment, the primary effector molecules) is ColEl, ColE2,
ColE3, ColE4, ColES, ColE6, ColE7, ColEB, or ColE9. Colicin E3 (ColE3) has
been
shown to have a profoundly cytotoxic effect on mammalian cells (Smarda et al.,
1978,
Folia Microbiol. 23:272-277), including a leukemia cell model system (Fiska et
al., 1978,
Experientia 35:406-40. 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 lOkDa 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
effector molecule, the larger ColE3 subunit or an active fragment thereof is
expressed
alone or at higher levels than the smaller subunit. In another embodiment of
the invention,
the~ColE3 SOkDa toxin and lOkDa 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 10
kDa anti-toxin is on the chromosome, separate form the colE3 toxin on the
plasmid,
resulting in a barrier to transmission to other bacteria. (See Section 5.6,
infra).
In another preferred embodiment, the primary effector molecules) is cloacin DF
13.
Cloacin DF 13 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, DF13 can cause the leakage of cellular
potassium.
In another preferred embodiment, the primary effector molecules) is colicin V
(Pugsley, A.P. and Oudega, B. "Methods for Studying Colicins and Their
Plasmids" in
Plasmids, a Practical Approach 1987, ed. By K.G. Hardy; Gilson, L. et al. EMBO
J. 9:
3875-3884).
In another embodiment, the primary effector molecules) is colicin E2 (a dual
subunit colicin similar to ColE3 in structure but with endonuclease rather
than ribonuclease
activity); colicins A, El, 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
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inhibits protein, DNA & RNA synthesis; colicin M which causes cell sepsis by
altering the
osmotic environment of the cell; pesticin A1122 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;
or megacin A-216 which is a phospholipase that causes leakage of intracellular
material
(for a general review of bacteriocins, see Konisky, 1982, Ann. Rev. Microbiol.
36:125-
144); colicin A (Pugsley, A.P. and Oudega, B. "Methods for Studying Colicins
and Their
Plasmids" in Plasmids, a Practical Approach 1987, ed. By K.G. Hardy).
Accordingly, a primary effector molecule may comprise any bacteriocin
described
herein or known in the art, with the proviso that said bacteriocin is not a
bacteriocin release
protein.
In another specific embodiment, the primary effector molecules of the
invention are
tumor inhibitory enzymes or functional fragments thereof. Examples of tumor
inhibitory
1 S enzymes include, but are not limited, methionase, asparaginase, lipase,
phospholipase,
protease, ribonuclease, DNAase, and glycosidase. In a preferred embodiment,
the primary
effector molecule is methionase.
The primary effector molecules of the invention are useful, for example, to
treat, or
prevent a solid tumor cancer such as a carcinoma, melanoma, lymphoma, or
sarcoma.
The invention provides nucleic acid molecules encoding a primary effector
molecule. The invention also provides nucleic acid molecules encoding one or
more
primary effector molecules) and optionally, one or more secondary effector
molecule(s).
The invention provides nucleic acids encoding effector molecules) of the
invention which
is operably linked to an appropriate promoter. Optionally, the nucleic acids
encoding an
effector molecules) may be operably linked to other elements that participate
in
transcription, translation, localization, stability and the like.
The nucleic acid molecule encoding a primary effector molecule is from about 6
to
about 100,000 base pairs in length. Preferably, the nucleic acid is from about
20 base pairs
to about 50,000 base pairs in length. More preferably, the nucleic acid
molecule is from
about 20 base pairs to about 10,000 base pairs in length. Even more
preferably, the nucleic
acid molecule is about 20 base pairs to about 4000 base pairs in length.
5.3. SECONDARY EFFECTOR MOLECULES FOR
CO-EXPRESSION WITH PRIMARY EFFECTOR
MOLECULES
In certain embodiments of the invention, the primary effector molecule (e.g.,
a TNF
family member, a cytotoxic peptide or polypeptide, an anti-angiogenic factor,
or a tumor
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inhibitory enzyme) is optionally co-expressed in a bacterial vector with
another molecule,
i.e. a secondary effector molecule. The secondary effector molecule provides
additional
therapeutic value and/or facilitates the release of the contents of the
modified bacterial
vector (which expresses at least one primary effector molecule and optionally
one or more
secondary effector molecules) into the surrounding environment. As used
herein, the term
"additional therapeutic value" indicates that the secondary effector molecule
provides an
additive or synergistic, cytostatic, or cytotoxic effect on a tumor, e.g., iri
addition to that
provided by the primary effector molecule(s). Thus, a secondary effector
molecule
functions as an additional therapeutic factor and/or a release factor.
Preferably, the
secondary effector molecule, whether a therapeutic or release factor (or
both), is
preferentially or specifically activated or expressed at the desired site,
i.e. at the site of the
tumor. In certain embodiments, the secondary effector molecule can serve two
functions,
i.e. promote the release of the bacterial cell contents (e.g., by promoting
bacterial cell lysis
or quasi lysis) and provide therapeutic value (e.g., by cytotoxicity to the
tumor cells). In
certain non-limiting embodiments, the cytotoxicity of the secondary effector
molecule can
be mediated by the patient's immune system; accordingly such a secondary
effector
molecule can function as an immunomodulator.
In certain embodiments of the invention, the attenuated tumor-targeted
bacterial
vector of the invention is engineered to express at least one secondary
effector molecule
which has anti-tumor activity, i.e. expression of the secondary effector
molecule results in
killing or inhibition of the growth of a tumor or tumor cells.
The secondary effector molecule is proteinaceous or a nucleic acid molecule.
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.
~tisense 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; 5,190,93 l; 5,135,917; and 5,087,617). Triplex molecules refer to
single DNA
strands that bind duplex DNA forming a colinear 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. 5,144,019;
and U.S. Patent
Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246).
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As described above for the primary effector molecule, a nucleic acid encoding
or
comprising a secondary effector molecule is provided in operative linkage with
a selected
promoter, and optionally in operative linkage with other elements that
participate in
transcription, translation, localization, stability and/or the like. Further,
the secondary
effector molecule can be expressed using the same promoter as the primary
effector
molecule and an internal ribosome binding site, or using a different promoter
than the
primary effector molecule.
The nucleic acid molecule encoding the secondary effector molecule 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 20 base pairs to about 10,000 base pairs in
length. Even more
preferably, it is a nucleic acid molecule from about 20 pairs to about 4,000
base pairs in
length.
The nucleotide sequences of the effector molecules encoding the secondary
effector
molecules described below are well known (see GenBank). A nucleic acid
molecule
encoding a secondary effector molecule, which secondary effector molecule is a
cytotoxic
or cytostatic factor or a biologically active fragment, variant or derivative
thereof, 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; and
5,109,124).
Identification of oligonucleotides and ribozymes for use as antisense agents
involve
methods well known in the art.
5.3.1. FACTORS PROVIDING ADDITIONAL
THERAPEUTIC VALUE
In certain embodiments of the invention, the attenuated tumor-targeted
bacterial
vector of the invention, which expresses at least one primary effector
molecule and is
preferably a Salmonella vector, expresses at least one secondary effector
molecule which
has anti-tumor activity, i.e. expression of the secondary effector molecule
results in killing
or inhibition of the growth of a tumor or tumor cells or spread of tumor
cells, thereby
augmenting the cytotoxic or cytostatic action of the primary effector
molecule. In one
embodiment, the effects on the tumor of the secondary effector molecule are
additive to
those of the primary effector molecule. In a preferred embodiment, the effects
are supra-
additive or synergistic, i.e. greater than the sum of the effects of the
primary and secondary
effector molecules if administered separately.
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In certain embodiments, the secondary effector molecule 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.
Examples of
such secondary effector molecules include but are not limited to saporin, the
ricins, abrin,
and other ribosome inactivating proteins (RIPS).
In another embodiment, the secondary effector molecule 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 are listed on page 33 and in Table 2 of WO 96/40238 by Pawelek et al.,
which is
incorporated herein by reference. WO 96/40238 also teaches methods for
production of
secreted fusion proteins comprising such pro-drug converting enzymes.
According to 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, infra, Section 5.3.2). 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 embodiment, the secondary effector
molecules) is
co-expressed with a release factor such as BRP, and cause the release of co-
factors (e.g.,
NADH, NADPH, ATP, etc.) which enhance pro-drug converting enzyme activity. In
another mode of the embodiment, a secondary effector molecule is co-expressed
with a
release factor such as BRP, leading to the release of an activated drug (e.g.,
a drug which is
activated within the bacterial cytoplasm or periplasm, and then released from
the bacterial
vector).
In another embodiment, a secondary effector molecule is an inhibitor of
inducible
nitric oxide synthase (NOS) or of endothelial nitic oxide synthase. Nitric
oxide (NO) is
implicated to be involved in the regulation of vascular growth and in
arterosclerosis. NO is
formed from L-arginine by nitric oxide synthase (NOS) and modulates immune,
inflammatory and cardiovascular responses.
In another embodiment, the secondary effector molecule is cytotoxic or
cytostatic
to a cell by inhibiting the production or activity of a protein involved in
cell proliferation,
such as an oncogene or growth factor, (e.g., bFGF, int-2, hst-1/K-FGF, FGF-5,
hst-2/FGF-
6, FGF-8) or cellular receptor or ligand. The inhibition can be at the level
of transcription
or translation (mediated by a secondary effector molecule that is a ribozyme
or triplex
DNA), or at the level of protein activity (mediated by a secondary effector
molecule that is
an inhibitor of a growth factor pathway, such as a dominant negative mutant).
In another embodiment, a secondary effector molecule is a cytokine, chemokine,
or
an immunomodulating protein or a nucleic acid encoding the same, such as
interleukin-1
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(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. Delivery of such immunomodulating effector molecules 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 both CD28 and CTLA-4, 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. Further, another
immunomodulating
agent is a tumor-associated antigen, i.e. a molecule specifically that is
expressed by a
~mor 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, Immunology, W.H. Freeman and
Company,
New York, NY, 1s' Edition (1992), 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 embodiment, a secondary effector molecule is a Flt-3 ligand or
nucleic
acid encoding the same. In another embodiment, a secondary molecule is BRP.
In a specific embodiment, a secondary effector molecule is not a TNF family
member when the primary effector molecule is a TNF family member. In another
specific
embodiment, a secondary effector molecule is not an anti-angiogenic factor
when the
primary effector molecule is an anti-angiogenic factor. In another specific
embodiment, a
secondary molecule is not a cytotoxic peptide or polypeptide when the
secondary molecule
is a cytotoxic peptide or polypeptide. In another specific embodiment, a
secondary
molecule is not a tumor inhibiting enzyme when the primary effector molecule
is a a tumor
inhibiting enzyme.
5.3.2. FACTORS THAT PROMOTE THE RELEASE OF
ANTI-TUMOR EFFECTOR MOLECULES INTO
THE TUMOR ENVIRONMENT
In certain other embodiments of the invention, the attenuated tumor-targeted
bacterial vector of the invention, which expresses at least one primary
effector molecule
and is preferably a Salmonella vector, expresses at least one secondary
effector 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 primary and/or secondary effector molecule(s).
Such
secondary effector molecule which permeabilizes the bacterial cell or enhances
release is
designated "a release factor". In certain embodiments, the release factor also
advantageously has anti-tumor activity.
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The release factor expressed by the bacterial vector of the invention may be
endogenous to the modified attenuated tumor-targeted bacteria or it may be
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
a primary
effector molecule, or by a separate nucleic acid or plasmid. A release factor
may be
encoded by the same nucleic acid or plasmid that encodes a secondary effector
molecule,
or by a separate nucleic acid or plasmid. In one embodiment, the release
factor is
expressed in a cell which also expresses a fusion protein comprising a primary
effector
molecule fused to an Omp-like protein. In this embodiment, the co-expression
of the
release factor allows for enhanced release of the fusion protein from the
periplasmic space.
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 including but not limited to
the cloacin
DF13 plasmid, one of colicin E1-E9 plasmids, or from colicin A, N or D
plasmids. In a
preferred embodiment, the BRP is of cloacin DF 13 (pCIoDF 13 BRP).
Generally, BRPs are 45-52 amino acid peptides that are initially synthesized
as
precursor molecules (PreBRP) with signal sequences that are not cleaved by
signal
endopeptidases. BRP activity is thought to be mediated, at least in part, by
the detergent-
resistant outer membrane phopholipase A (PIdA) and is usually associated with
an increase
in the degradation of outer membrane phospholipid (for a general review on
BRPs, see van
der Wal et al., 1995, FEMS Microbiology Review 17:381-399). Without limitation
as to
mechanism, BRP promotes the preferential release of periplasmic components,
although
the release of cytoplasmic components is also detected to a lesser extent.
When moderately
overexpressed, BRP may cause the bacterial membrane to become fragile,
inducing quasi-
lysis and high release of cytoplasmic components. Additionally, it is thought
that when
BRP is expressed at superhigh levels, the protein can cause bacterial cell
lysis, thus
delivering cellular contents by lytic release. In this embodiment, BRP
expression may be
correlated with BRP activity (e.g., release of bacterial contents). For
example, superhigh
B~ activity results in bacterial cell lysis of substantially all bacteria.
Thus, as used
herein, "superhigh expression" is defined as the expression level of BRP which
results in
bacterial cell lysis of substantially all bacteria. Moderate BRP activity, is
associated with
partial or enhanced release of bacterial contents as compared to a control
bacteria which is
not expressing BRP, without obligate lysis of the bacteria. Thus, in this
embodiment,
moderate overexpression of BRP is defined as the expression level at which
release of
cytoplasmic components is enhanced, without bacterial lysis of substantially
all of the
bacteria. Substantially all of the bacteria, as used herein, is more than 60%
of the bacteria,
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preferably more than 70%, more preferably 80%, still more preferably more than
90% and
most preferably 90-100% of bacteria.
In a specific embodiment of the invention, the BRP protein is a pCIoDFI3 BRP
mutant whose lytic function has been uncoupled from its protein release
function, thereby
enhancing protein release without bacterial lysis (van der Wal et al., 1998,
App. Env.
Microbiol. 64:392-398). This embodiment allows for prolonged protein release
from the
5.
bacterial vector, while reducing the need for frequent administration of the
vector. In
another specific embodiment, the BRP of the invention is a pCIoDF 13 BRP with
a
shortened C-terminus, which in addition to protein release causes cell lysis
(Luirink et al.,
1989, J. Bacteriol. 171:2673-2679).
In another embodiment of the invention, the enhanced release system comprises
overexpression of a porin protein; see e.g., Sugawara, E. and Nikaido, H.,
1992, J. Biol.
Chem.267:2507-11.
In certain embodiments when a BRP is expressed by the bacterial vector of the
invention, the BRP may be endogenous to the modified attenuated tumor-targeted
bacteria
or it may be exogenous (e.g., encoded by a nucleic acid that is not native to
the attenuated
tumor-targeted bacteria). A BRP 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 BRP may be encoded by the same nucleic acid or plasmid that
encodes a
primary effector molecule, or by a separate nucleic acid or plasmid. A BRP may
be
encoded by the same nucleic acid or plasmid that encodes a secondary effector
molecule,
or by a separate nucleic acid or plasmid. In one embodiment, the BRP-like
protein is
expressed in a cell which also expresses a fusion protein comprising an
effector molecule
fused to an Omp-like protein. In this embodiment, the co-expression of the BRP
allows for
enhanced release of the fusion protein.
In a preferred specific embodiment of the invention a BRP encoding nucleic
acid is
encoded by a colicin plasmid. In another specific embodiment of the invention,
the BRP
encoding nucleic acid is expressed under the control of the native BRP
promoter, which is
an SOS promoter that responds to stress (e.g., conditions that lead to DNA
damage such as
UV light) in its normal host (for BRP, Enterococcus cloacae), yet is partially
constitutive
in Salmonella. In a preferred embodiment, the BRP encoding nucleic acid is
expressed
under the control of the pepT promoter, which is activated in response to the
anaerobic
nature of the tumor environment (see e.g., Lombardo et al., 1997, J.
Bacteriol. 179:1909-
17).
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
singlemer, which singlemer responds in an all-or-nothing manner to the
presence of
tetracycline or analogs thereof such as doxicycline and anhydrotetracycline
and provides a
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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 effector
molecule
levels. 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.
An advantage
of this promoter is that it is induced at very low levels of tetracycline,
approximately 1/lOth
of the dosage required for antibiotic activity.
5.4. DERIVATIVES AND ANALOGS
The invention further encompasses bacterial vectors that are modified to
encode or
deliver a derivative, including but not limited to a fragment, analog, or
variant of a primary
and/or secondary effector molecule, 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 effector molecule. As one
example, such
derivatives, analogs or variants which have the desired therapeutic properties
can be used
to inhibit tumor growth or the spread of tumor cells (metastasis). Derivatives
or analogs of
an effector molecule 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 effector molecule encoding
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
effector
molecule. 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 an
effector molecule, 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 primary or secondary effector-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 105:61-72.
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In a specific embodiment, a derivative, analog or variant of a primary or
secondary
molecule comprises a nucleotide sequence that hybridizes to the nucleotide
sequence
encoding the primary or secondary molecule, or fragment thereof under
stringent
conditions, e.g., hybridization to filter-bound DNA in 6x sodium
chloride/sodium citrate
(SSC) at about 45 °C followed by one or more washes iri 0.2xSSC/0.1%
SDS at about
S SO-65 ° C, under highly stringent conditions, e.g., hybridization to
filter-bound nucleic acid
in 6xSSC at about 45 °C followed by one or more washes in O.IxSSC/0.2%
SDS at about
68 °C, or under other stringent hybridization conditions which are
known to those of skill
in the art (see, for example, Ausubel, F.M. et al., eds. , 1989, Current
Protocols in
Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley &
Sons, Inc.,
New York at pages 6.3.1-6.3.6 and 2.10.3). '
Derivatives or analogs of a primary or secondary effector molecule include but
are
not limited to those molecules comprising regions that are substantially
homologous to the
primary or secondary effector molecule or fragment thereof (e.g., in various
embodiments,
at least 60% or 70% or 80% or 90% or 95% identity over an amino acid sequence
of
identical size without any insertions or deletions or when compared to an
aligned sequence
in which the alignment is done by a computer homology program known in the
art) or
whose encoding nucleic acid is capable of hybridizing to an effector molecule
protein
effector molecule encoding sequence, under high stringency, moderate
stringency, or low
stringency conditions.
To determine the percent identity of two amino acid sequences or of two
nucleic
acids, e.g. between the sequences of a primary effector molecule and other
known
sequences, the sequences are aligned for optimal comparison purposes (e.g.,
gaps can be
introduced in the sequence of a first amino acid or nucleic acid sequence for
optimal
alignment with a second amino or nucleic acid sequence). The amino acid
residues or
nucleotides at corresponding amino acid positions or nucleotide positions are
then
compared. When a position in the first sequence is occupied by the same amino
acid
residue or nucleotide as the corresponding position in the second sequence,
then the
molecules are identical at that position. The percent identity between the two
sequences is
a function of the number of identical positions shared by the sequences (i.e.,
% identity = #
of identical positions/total # of positions (e.g., overlapping positions) x
100). In one
embodiment, the two sequences are the same length.
The determination of percent identity between two sequences can be
accomplished
using a mathematical algorithm. A preferred, non-limiting example of a
mathematical
algorithm utilized for the comparison of two sequences is the algorithm of
Karlin and
Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2264-2268, modified as in Karlin
and
Altschul, 1993, Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul, et al., 1990, J.
Mol.
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Biol. 215:403-410. BLAST nucleotide searches can be performed with the NBLAST
program, score = 100, wordlength = 12 to obtain nucleotide sequences
homologous to a
nucleic acid molecules of the invention. BLAST protein searches can be
performed with
the XBLAST program, score= 50, wordlength = 3 to obtain amino acid sequences
homologous to a protein molecules of the invention. To obtain gapped
alignments for
comparison purposes, Gapped BLAST can be utilized as described in Altschul et
al., 1997,
Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can be used to
perform an
iterated search which detects distant relationships between molecules (Id.).
When utilizing
BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the
respective
programs (e.g., XBLAST and NBLAST) can be used. See
http://www.ncbi.nlm.nih.gov.
Another preferred, non-limiting example ofa mathematical algorithm utilized
for the
comparison of sequences is the algorithm of Myers and Miller, CABIOS (1989).
Such an
algorithm is incorporated into the ALIGN program (version 2.0) which is part
of the GCG
sequence alignment software package. When utilizing the ALIGN program for
comparing
amino acid sequences, a PAM120 weight residue table, a gap length penalty of
12, and a
1 S gap penalty of 4 can be used. Additional algorithms for sequence analysis
are known in the
art and include ADVANCE and ADAM as described in Torellis and Robotti, 1994,
Comput. Appl. Biosci., 10:3-5; and FASTA described in Pearson and Lipman,
1988, Proc.
Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option that sets
the sensitivity
and speed of the search. If ktup=2, similar regions in the two sequences being
compared
are found by looking at pairs of aligned residues; if ktup=1, single aligned
amino acids are
examined. ktup can be set to 2 or 1 for protein sequences, or from 1 to 6 for
DNA
sequences. The default if ktup is not specified is 2 for proteins and 6 for
DNA. For a
further description of FASTA parameters, see
http://bioweb.pasteur.fr/docs/man/man/fasta.l.html#sect2, the contents of
which are
incorporated herein by reference.
Alternatively, protein sequence alignment may be carried out using the
CLUSTAL W algorithm, as described by Higgins et al., 1996, Methods Enzymol.
266:383-
402.
The percent identity between two sequences can be determined using techniques
similar to those described above, with or without allowing gaps. In
calculating percent
identity, only exact matches are counted.
A primary effector molecule or a secondary effector molecule, or derivatives,
or
analogs thereof can be produced by various methods known in the art. The
manipulations
which result in their production can occur at the nucleic acid or protein
level. For example,
a cloned effector molecule encoding sequence encoding, for example, an
effector
molecule can be modified by any of numerous strategies known in the art
(Sambrook et al.,
1989, Molecular Cloning, A Laboratory Manual, 2d ed., Cold Spring Harbor
Laboratory
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Press, Cold Spring Harbor, New York). The sequence can be cleaved at
appropriate sites
with restriction endonuclease(s), followed by further enzymatic modification
if desired,
isolated, and ligated in vitro. In the production of a modified effector
molecule encoding a
derivative or analog of a primary or secondary effector molecule, care should
be taken to
ensure that the modified effector molecule encoding sequence remains within
the same
translational reading frame as the native protein, uninterrupted by
translational stop signals,
in the effector molecule encoding sequence region where the desired primary or
secondary
effector molecule activity is encoded.
Additionally, a nucleic acid sequence encoding an effector molecule can be
mutated
in vitro or in vivo, to create and/or destroy translation, initiation, and/or
termination
sequences, or to create variations in coding regions and/or to form new
restriction
endonuclease sites or destroy preexisting ones, to facilitate further in vitro
modification. In
a preferred specific embodiment, an effector molecule-encoding nucleic acid
sequence is
mutated, for example, to produce a more potent variant. Any technique for
mutagenesis
known in the art can be used, including but not limited to, chemical
mutagenesis, in vitro
site-directed mutagenesis (Hutchinson et al., 1978, J. Biol. Chem. 253:6551),
use of TAB~
linkers (Pharmacia), PCR with primers containing a mutation, etc. In a
preferred
embodiment, conservative amino acid substitutions are made at one or more
predicted non-
essential amino acid residues of an effector molecule. A "conservative amino
acid
substitution" is one in which the amino acid residue is replaced with an amino
acid residue
having a side chain with a similar charge. Families of amino acid residues
having side
chains with similar charges have been defined in the art. These families
include amino
acids with basic side chains (e.g., lysine, arginine, histidine), acidic side
chains (e.g.,
aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine,
asparagine,
glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine,
leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-
branched side
chains ( e.g., threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine,
phenylalanine, tryptophan, histidine). Alternatively, mutations can be
introduced randomly
along all or part of the coding sequence, such as by saturation mutagenesis,
and the
resultant mutants can be screened for biological activity to identify mutants
that retain
activity. Following mutagenesis, the encoded protein can be expressed and the
activity of
the protein can be determined.
In other embodiments, the effector molecules or fusion proteins of the
invention are
constructed to contain a protease cleavage site.
5.5. FUSION PROTEINS
In certain embodiments, the invention provides a primary or secondary effector
molecule which is constructed as a fusion protein (e.g., covalently bonded to
a different
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protein). The invention provides nucleic acids encoding such fusion proteins.
In certain
other embodiments of this invention, the nucleic acid encoding a fusion
protein of the
invention is operably linked to an appropriate promoter.
In a specific embodiment, an effector molecule is constructed as a chimeric or
fusion protein comprising an effector molecule or fragment thereof (preferably
consisting
of at least a domain or motif of the effector molecule, or at least 5, at
least 10, at least 25, at
least 50, at least 75, or at least .100 amino acids of the effector molecule)
joined at its
amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a
different
protein. In specific embodiments, fusion comprises at least 2, at least 6, at
least 10, at least
20, at least 30, at least 50, at least 75, or at least 100 contiguous amino
acids of a
heterologous polypeptide or fragment thereof that is functionally active. In
one
embodiment, such a fusion protein or chimeric protein is produced by
recombinant
expression of a nucleic acid encoding the primary effector molecule (e.g., a
TNR-coding
sequence, an anti-angiogenic factor-coding sequence, a tumor inhibitory enzyme-
coding
sequence, or a cytotoxic polypeptide-coding 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 encoding an
effector molecule
used to any heterologous polypeptide-encoding sequences may be constructed. A
specific
embodiment relates to a chimeric protein comprising a fragment of a primary or
secondary
effector molecule of at least 5, at least 10, at least 25, at least 50, or at
least 100 amino
acids, or a fragment that displays one or more functional activities of the
full-length
primary or secondary effector molecule.
In a specific embodiment, a 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 embodiment, a
fusion protein
comprises a protease cleavage site such as a metal protease or serine cleavage
site. In this
embodiment, 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 several embodiments, an effector molecule is constructed as a fusion
protein to an Omp-
like protein, or fragment thereof (e.g., signal sequence, leader sequence,
periplasmic
region, transmembrane domain, multiple transmembrane domains, or combinations
thereof; see infra, Section 3.1 for definition of "Omp-like protein").
In a preferred embodiment, an effector molecule (primary or secondary) of the
invention is expressed as a fusion protein with an outer membrane protein (Omp-
like
protein). Bacterial outer membrane proteins are integral membrane proteins of
the
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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, an effector molecule is constructed as
a fusion
protein with an Omp-like protein. In this embodiment, the primary effector
molecule has
enhanced delivery to the outer membrane of the bacteria. Without intending to
be limiting
as to mechanism, the Omp-like protein is believed by the inventors to act as
an anchor or
tether for the effector molecule to the outer membrane, or serves to localize
the protein to
the bacterial outer membrane. In one embodiment, the fusion of an effector
molecule to an
Omp-like protein is used to enhance localization of an effector molecule to
the periplasm.
In another embodiment, the fusion of an effector molecule to an Omp-like
proteins is used
to enhance release of an effector molecule. 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, ~i-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). As illustrative examples, see Sections 7.1-7.4, infra.
In other embodiments of the invention, a fusion protein of the invention
comprises
a proteolytic cleavage site. The protolytic cleavage site may be endogenous to
the effector
molecule 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, Hobom et
al. (1995,
Dev. Biol. Strand. 84:255-262) have developed vectors containing the OmpA open
reading
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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 one embodiment of the invention, the portion of the OmpA fusion protein
containing the primary- effector molecule has enhanced expression in the
periplasm. In one
aspect of the embodiment, the fusion protein comprises prior to maturation
either the signal
sequence or the signal sequence followed by at least one membrane-spanning.
domain of
OmpA, located N-terminal to the primary effector molecule. The signal sequence
is
cleaved and absent from the mature protein. In another aspect of the
embodiment, the
primary effector molecule is at the N-terminus of the OmpA fusion, rending
inconsequential to the positioning of the primary effector molecule the number
of
membrane spanning domains of OmpA utilized, as long as the fusion protein is
stable. In
yet another aspect of the embodiment, the primary effector molecule is
situated between
the N - and C-terminal domains of OmpA such that a soluble periplasmic protein
containing the primary effector molecule upon cleavage by a periplasmic
protease within
the periplasm. In certain aspects of this embodiment, it is preferred that a
bacterial vector
which expresses a periplasmic primary effector molecule also coexpresses BRP
to enhance
release of the effector molecule from the bacterial cell.
In another embodiment of the invention, the portion of the OmpA fusion protein
containing the primary effector molecule 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 primary effector
molecule. In another aspect of the embodiment, the primary effector molecule
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
effector
molecule 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 encoding
signal
sequence. Such plasmids may be constructed to comprise a nucleic acid encoding
one or
more effector molecules) of the invention.
Optionally, an effector molecule is preceded or flanked by consensus cleavage
sites
for a metalloprotease or serine protease that is abundant in tumors, for
release of the
effector molecule into the tumor environment. Whether the primary effector
molecule 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
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of the Omp-like protein to be fused to an effector molecule will depend upon
the location
that is desired for the expression of the effector molecule (e.g.,
periplasmic, extracellular,
membrane bound, etc.). Such fusion protein constructions as described herein
for primary
effector molecules are also appropriate .for secondary effector molecules.
In a preferred embodiment, an effector molecule is fused to a ferry peptide.
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 attenuated tumor-targeted bacteria to
express fusion
proteins comprising a ferry peptide and an effector molecule enhances the
ability of an
effector molecule to be internalized by tumor cells. In a specific embodiment,
attenuated
tumor-targeted bacteria are engineered to express a nucleic acid molecule
encoding a
fusion protein comprising a ferry peptide and an effector molecule. In another
embodiment, attenuated tumor-targeted bacteria are engineered to express one
or more
nucleic acid molecules encoding one or more fusion proteins comprising a ferry
peptide
and an effector molecule. In accordance with these embodiments, the effector
molecule
may be a primary or secondary effector molecule. 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), herpes simplex virus VP22, polyhistadine (e.g., hexahistadine;
6H),
polylysine (e.g., hexalysine; 6K), and polyarginine (e.g., hexaarginine; 6R)
(see, e.g.,
Blanke et al., 1996, Proc. Natl. Acad. Sci. USA 93:8437-8442).
In another preferred embodiment, a fusion protein comprises a signal peptide,
ferry
peptide and an effector molecule. In a specific mode of this embodiment,
attenuated
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
effector molecule. In accordance with this mode, the effector molecule is a
primary or
secondary effector molecule.
In another preferred embodiment, a fusion protein comprises a signal peptide,
a
protolytic cleavage site; a ferry peptide and an effector molecule to a solid
tumor by
attenuated tumor-targeted bacteria. In a specific embodiment, attenuated tumor-
targeted
bacteria are engineered to express one or more nucleic acid molecules encoding
one or
more fusion proteins comprising a signal sequence, a protolytic cleavage site,
a ferry
peptide and an effector molecule. In accordance with this embodiment, the
effector
molecule may be a primary or secondary effector molecule.
By way of non-limiting example, colicin activity may be enhanced by addition
of
internalizing peptides derived from HIV TAT, herpes simplex virus VP22,
antennapaedia,
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6H, 6K, and 6R. The fusion can be either C-terminal, N-terminal, or internal.
Internal
fusions are especially preferred where the fusion follows the N-terminal
signal sequence
cleavage peptide. The fusion protein may further comprise an N-terminal signal
sequence
such OmpA or a C-terminal signal sequence such as hlyA.
In a preferred embodiment, an effector molecule is fused to the delivery
portion of
a toxin. Various toxins are known to have self delivery capacity, where one
portion of the
toxin acts as a delivery agent for the second portion of the toxin. For
example, Ballard et
al., 1996, Proc. Natl. Acad. Sci. USA 93:12531-12534 demonstrated that the
anthrax
protective agent (PA) which mediates the entry of lethal factor (LF) and edema
factor into
the cytosolic compartment of mammalian cells, is also capable of mediating
entry of
protein fusions to a truncated form of LF (LFri; 255 amino acid residues).
Thus, effector
molecules of the invention, except those that function outside the cell, can
be fused to the
LFn, or other toxin systems, including, but limited to, diptheria toxin A
chain residues 1-
193 (Blanke et al., 1996, Proc. Natl. Acad. Sci. USA 93:8437-8442), cholera
toxin,
verotoxin, E. coli heat labile toxins (LTs), E. coli heat stable toxins (STs),
entero-
1 S hemolysins, enterotoxins, cytotoxins, EAggEC stable toxin 1 (EAST), CNFs,
cytolethal
distending toxin, a-hemolysins, (3-hemolysins, and SheA hemolysins (for review
see, e.g.,
O'Brien and Holmes, 1996. Protein toxins of Escherichia coli and Salmonella.
Cellular and
Molecular Biology, Neidhardt et al. (eds), ASM Press, Washington, D.C., pp2788-
2802).
In a specific embodiment, a primary effector rizolecule is fused to the
delivery portion of a
toxin. In another specific embodiment, a secondary effector molecule is fused
to the
delivery portion of a toxin.
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).
5.6. EXPRESSION VEHICLES
The present invention provides attenuated tumor-targeted bacteria which have
been
engineered to encode one or more primary effector molecules and optionally,
one or more
secondary effector molecules. The invention provides attenuated tumor-targeted
bacteria
comprising effector molecules) which are encoded by a plasmid or transfectable
nucleic
acid. In a preferred embodiment of the invention, the attenuated tumor-
targeted bacteria is
Salmonella. When more than one effector molecule (e.g., primary or secondary)
is
expressed in an attenuated tumor-targeted bacteria, such as Salmonella, the
effector
molecules may be encoded by the same plasmid or nucleic acid, or by more than
one
plasmid or nucleic acid molecule. The invention also provides attenuated tumor-
targeted
bacteria comprising effector molecules) which are encoded by a nucleic acid
molecule
which is integrated into the bacterial genome. Integrated effector molecules)
may be
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endogenous to an attenuated tumor-targeted bacteria, such as Salmonella, or
may be
introduced into the attenuated tumor-targeted bacteria (e.g., by introduction
of a nucleic
acid which encodes the effector molecule, such as a plasmid, transfectable
nucleic acid,
transposon, etc.) such that the nucleic acid molecule encoding the effector
molecule
becomes integrated into the genome of the attenuated tumor-targeted bac._ ia.
In a
preferred embodiment of the invention, the attenuated tumor-targeted bacteria
is
Salmonella. The invention provides a nucleic acid molecule encoding an
effector molecule
which nucleic acid is operably linked to an appropriate promoter. A promoter
operably
linked to a nucleic acid molecule encoding an effector molecule may be
homologous (i.e.,
native) or heterologous (i.e., not native to the nucleic acid molcule encoding
the effector
molecule).
The nucleotide sequence coding for an effector molecule of the invention or a
functionally active analog or fragment or other derivative thereof, can be
inserted into an
appropriate expression vehicle, e.g., a plasmid which contains the necessary
elements for
the transcription and translation of the inserted protein-coding sequence. The
necessary
transcriptional and translational signals can be supplied by the effector
molecule and/or its
flanking regions. Alternatively, an expression vehicle is constructed by
inserting a
structural DNA sequence encoding a desired protein together with suitable
translation
initiation and termination signals in operable reading phase with a functional
promoter
using one of a variety of methods known in the art for the manipulation of
DNA. See,
generally, Sambrook et al., 1989, Molecular Biology: A Laboratory Manual, Cold
Spring
Harbor Press, Cold Spring Harbor, NY; Ausubel et al., 1995, Current Protocols
in
Molecular Biology, Greene Publishing, New York, NY. These methods may include
in
vitro recombinant DNA and synthetic techniques and in vivo recombinants
(genetic
recombination). The invention provides a nucleic acid molecule encoding an
effector
molecule which nucleic acid is operably linked to an appropriate promoter.
The present invention also provides attenuated tumor-targeted bacteria which
have
been modified to encode one or more fusion proteins and optionally, one or
more effector
molecules. The invention provides attenuated tumor-targeted bacteria
comprising fusion
proteins which are encoded by a plasmid or transfectable nucleic acid. When
more than
one fusion protein and/or effector molecule (e.g., primary or secondary) is
expressed in an
attenuated tumor-targeted bacteria, such as Salmonella, the fusion proteins
and/or effector
molecules may be encoded by the same plasmid or nucleic acid, or by more than
one
plasmid or nucleic acid. The invention also provides attenuated tumor-targeted
bacteria
comprising fusion proteins which are encoded by a nucleic acid which is
integrated into the
bacterial genome. The invention also provides a nucleic acid molecule encoding
an fusion
protein which nucleic acid molecule is operably linked to an appropriate
promoter. The
nucleotide sequence coding for a fusion protein of the invention can be
inserted into an
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appropriate expression vehicle, e.g., a plasmid which contains the necessary
elements for
the transcription and translation of the inserted protein-coding sequence.
In certain specific embodiments of the invention, the expression vehicle of
the
invention is a plasmid. Large numbers of suitable plasmids are known to those
of skill in
the art and are commercially available for generating the recombinant
constructs of the
present invention.
Such commercial plasmids include, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA).
These
pBR322 "backbone" sections are combined with an appropriate promoter and the
structural
sequence to be expressed. pBR322 is considered to be a low copy number
plasmid. If
higher levels of expression are desired, the plasmid can be a high copy number
plasmid, for
example a plasmid with a pUC backbone. pUC plasmids include but are not
limited to
pUCl9 (see e.g., Yanisch-Perron et al. 1985, Gene 33:103-119) and pBluescript
(Stratagene).
The following plasmids are provided by way of example and may be used in
conjunction with the methods of the invention. Bacterial: pBs, phagescript,
phiX174,
pbluescript SK, pBs KS, pNHBa, pNHl6a, pNHl8a, pNH46a (Stratagene); pTrc99A,
pKK223-3, pKK233-3, pDR540, pRITS (Pharmacia). A commercial plasmid with a
pBR322 "backbone" may also be used, for example, pKK223-3 (Pharmacia Fine
Chemicals, Uppsala, Sweden) and GEM 1 (Promega Biotec, Madison, WI, USA).
These
are combined with an appropriate promoter and the structural sequence to be
expressed.
pCET, pTS (as described in Section 6 herein).
In specific embodiments of the invention, a plasmid encoding an effector
molecule
is the pTS-TNF-a plasmid, the pTS-BRP plasmid, or the pTS-BRPTNF-a plasmid as
described in Section 6 herein.
In a specific embodiment of the invention, the fusion protein of the invention
for
secretion into the periplasmic space comprising the OmpA signal sequence and
the primary
effector protein are encoded by the plasmid pIN-III-ompA-Hind , which contains
the DNA
sequence encoding the ompA signal sequence upstream of a linker sequence into
which the
coding sequence for the primary effector molecule can be cloned. In a
preferred specific
embodiment, the lac inducible promoter of pIN-III-ompA-Hind vector is replaced
by a
pepT or tet promoter. (See, Rentier-Delrue et al. ( 1988), Nuc. Acids Res.
16:8726).
The present invention also provides transposon-mediated chromosomal
integration
of effector molecules. Any transposon plasmid known in the art may be used in
the
methods of the invention so long as a nucleic acid encoding an effector
molecule can be
constructed into the transposon cassette. For example, the invention provides
a transposon
plasmid, comprising a transposon or minitransposon, and an MCS.
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In certain embodiments of the invention, the plasmid of the invention is a
transposon plasmid, i.e., comprises a transposon in which the sequence
encoding an
effector molecule of interest is inserted. Transposon plasmids contain
trasposon cassettes
which cassette becomes integrated into the bacterial genome. Accordingly, a
nucleic acid
encoding an effector molecule or fusion protein thereof is inserted into the
transposon
cassette. Thus, a transposon insertion integrates the cassette into the
bacterial genome.
The coding sequence of the effector molecule can be operably linked to a
promoter, or can
be promoterless. In the latter case, expression of the selectable marker is
driven by a
promoter at the site of transposon insertion into the bacterial genome.
Colonies of bacteria
having a transposon insertion are screened for expression levels that meet the
requirements
of the invention, e.g. that express sufficient levels of cytokine to promote
tumor
cytotoxicity, stasis, or regression.
In certain embodiments, in addition to the transposon, the transposon plasmid
comprises, outside the inverted repeats of the transposon, a transposase gene
to catalyse the
insertion of the transposon into the bacterial genome without being carried
along with the
transposon, so that bacterial strains with stable transposon insertions are
generated.
Transposons to be utilized by the present invention include but are not
limited to
Tn7, Tn9, TnlO and TnS. In a preferred embodiment, the transposon plasmid is
pNK2883
(ATCC) having an ampicillin resistance gene located outside the TnlO insertion
elements
and the nucleic acids encoding one or more effector molecules) is inserted
between the
two TnlO insertion elements (e.g., within the transposon cassette).
Preferably, the
construct is made such that additional sequences encoding other elements is
inserted
between the two TnlO insertion elements. In specific embodiments, such
elements may
optionally include (1) a promoterless copy a selectable marker (e.g., SerC,
AroA, etc) for
positive selection of the bacteria containing the plasmid; (2) a BRP gene, (3)
a promoter for
the effector molecule (such as trc) operably linked to the nucleic acid
encoding the one or
more primary effector molecules) (such as TNF-a, or a fusion protein thereof,
e.g., an
OmpA-TNF-a fusion), (4) a terminator for the nucleic acid encoding the one or
more
effector molecule(s).
In one embodiment, after the manipulation of the plasmid as appropriate and
selection of those clones having the desired construct using the ampicillin
resistance
properties encoded by the plasmid, the antibiotic selection is removed through
plasmid loss
and strains having a chromosomal transposon insert are chosen for
administering to human
subjects (e.g., by plating on selective media).
In another specific embodiment, the plasmid pTS is used which comprises an
altered target specificity transposase gene and a minitransposon, containing
the coding
sequences for a promoterless serf gene and an MCS. In another specific
embodiment, the
plasmid pTS-BRP is used which comprises an altered target specificity
transposase gene
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and a minitransposon, containing the coding sequences for a promoterless serf
gene, and
alkylating agent-inducible bacteriocin release factor, and an MCS.
In a preferred embodiment, a transposon plasmid for selection of transposon-
mediated chromosomal integrants, comprises:
a) a transposase gene, for transposon excision and integration, located
outside
of the transposon insertion sequence (e.g., outside of the transposon
cassette);
b) a wild-type coding sequence corresponding to the selection gene deleted in
the bacterial strain (e.g., serC) as well as a ribosomal binding site and
terminator for the wild-type gene, but lacking a promoter. This sequence is
preferably located immediately following the left TN10 transposon insertion
sequence;
c) optionally, between the right and left insertion sequences is a nucleic
acid
sequence encoding a release enhancing nucleic acid (e.g., BRP); and
d) a multiple cloning site (MCS) located between the right and left insertion
sequences, containing unique restriction sites within the plasmid, for the
incorporation of effector molecule. The MCS is preferably located
immediately following the release enhancing nucleic acid (if used) and just
prior to the right TN10 insertion sequence.
In another embodiment, the gene disruption resulting from random integration
of
effector molecules onto the host chromosome, identifies the suitability of the
gene location
for effector insertion.
In yet another embodiment, the expression vehicle is an extrachromosomal
plasmid
that is stable without requiring antibiotic selection, i.e. is self
maintained. In one non-
limiting example, the self maintained expression vehicle is a Salmonella
virulence plasmid.
For example, in one embodiment of the invention, the plasmid selection system
is
maintained by providing a function which the bacteria, such as Salmonella,
lacks and on
the basis of which those Salmonella having the function can be selected for at
the expense
of those that do not. In one embodiment, the Salmonella of the invention is an
auxotrophic.
mutant strain and the expression plasmid provides the mutant or absent
biosynthetic
. enzyme function. The Salmonella which contain the expression plasmid can be
selected
for by growing the cells on growth medium which lacks the nutrient that only
the desired
cells, i.e. those with the expression plasmid, can metabolize. In a highly
preferred aspect
of this embodiment, the Salmonella of the invention has an obligatory
requirement for
DAP (meso-diaminopimelic acid), most preferably by deletion of the asd gene.
DAP is a
component of the peptidoglycan present in the periplasm of Gram-negative
bacteria, which
is required for the integrity of the bacterial outer membrane. Absence of DAP
results in
bacterial cell lysis resulting from the loss of outer membrane integrity. The
asd (~i='
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aspartate semialdehyde dehydrogenase) gene encodes an enzyme in the DAP
biosynthetic
pathway. Gram-negative bacteria which lack asd function can be grown by
supplying
DAP to the culture media. Plasmids, e.g. the expression plasmids of the
invention, that
carry the asd gene sequence operably linked to a homologous or heterologous
promoter can
be selected for by growing Gram-negative bacteria that lack asd activity in
the absence of
DAP (see, e.g., U.S. Patent No. 5,840,483 to Curtiss, III).
Other non-antibiotic selection systems are known in the art and are within the
scope
of the invention. For example, a selection system utilizing a plasmid
comprising a stable
toxin and less stable anti-toxin may be used to select for bacteria which
maintain such a
plasmid.
In another embodiment, the expression vehicle is an extrachromosomal plasmid
that is selectable by non-antibiotic means, for example a colicin plasmid. As
used herein, a
colicin plasmid minimally encodes a colicin toxin and an anti-colicin, the
colicin toxin
being more stable than the anti-colicin, such that any bacteria which loses
the colicin
plasmid is killed as a result of the perdurance of the colicin toxin. In a
preferred
embodiment, the colicin toxin is the large subunit of ColE3 and the anti-
colicin is the small
subunit of ColE3.
In other embodiments of the invention, the expression vehicle is a ~, vector,
more
specifically a lysogenic ~, vector. In a preferred embodiment, the bacterial
host comprising
the ~, vector further comprises a temperature-sensitive ~, repressor which is
functional at
30°C but not 37°C. Consequently, the bacterial host can be grown
and manipulated in vivo
at 30°C without expression of the primary and/or secondary effector
molecule which may
be toxic to the bacterial cell. Upon introduction of the bacterial strain into
the subject, the
~, repressor is inactivated by normal body temperature and expression of the
primary
effector molecule, and optionally a secondary effector molecule, is activated.
Expression of a nucleic acid sequence encoding an effector molecule or fusion
protein may be regulated by a second nucleic acid sequence so that the
effector molecule is
expressed in a bacteria transformed with the recombinant DNA molecule. For
example,
expression of an effector molecule may be controlled by any promoter/enhancer
element
known in the art. A promoter/enhancer may be homologous (i.e., native) or
heterologous
(I~e., not native). Promoters which may be used to control the expression of
an effector
molecule, e.g. a cytokine, or fusion protein in bacteria include, but are not
limited to
prokaryotic promoters such as the (3-lactamase promoter (Villa-Kamaroff et
al., 1978,
Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the lac promoter (DeBoer et
al., 1983,
Proc. Natl. Acad. Sci. U.S.A. 80:21-25; Scientific American, 1980, 242:74-94).
Other
promoters emcompassed by the present invention include, but are not limited
to, lacI, lacZ,
T3, T7, gpt, lambda PR, lambda P~, trc, pagC, sulA, pol II (dinA), ruv, recA,
uvrA, uvrB,
uvrD, umuDC, lexA, cea, caa, and recN (see, e.g., Schnarr et al., 1991.
Biochimie 73:423-
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431). In a preferred embodiment, the promoter is trc, (see, e.g., Amann et
al., 1988, Gene
69:301-15).
In a particular embodiment, in which the primary effector molecule is a
colicin
expressed under the control of a SOS-responsive promoter, the attenuated
bacterial strain
may be treated with x-rays, ultraviolet radiation, an alkylating agent or
another DNA
damaging agent such that expression of the colicin is increased. Exemplary
SOS-responsive promoters include, but are not limited to, recA, sulA, umuC,
dinA, ruv,
uvrA, uvrB, uvrD, lexA, cea, caa, recN, etc.
In another preferred embodiment, the promoter 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
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, the 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) and can be used according
to the
methods of the present invention.
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
multimerized, for example three-fold. 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.
An advantage of this promoter is that it is induced at very lov levels of
tetracycline,
approximately 1/lOth of the dosage required for antibiotic activity.
Once a plasmid is constructed comprising an effector molecule or fusion
protein is
introduced into the attenuated tumor-targeted bacteria, effector molecule
expression or
fusion protein expression can be assayed by any method known in the art
including but not
limited to biological activity, enzyme activity, Northern blot analysis, and
Western blot
analysis. (See Sambrook et al., 1989, Molecular Biology: A Laboratory Manual,
Cold
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Spring Harbor Press, Cold Spring Harbor, NY; Ausubel et al., 1995, Current
Protocols in
Molecular Biology, Greene Publishing, New York, NY).
5.7. COMBINATION THERAPY
In certain embodiments, attenuated tumor-targeted bacteria are used in
conjunction
with other known cancer therapies to treat a solid cancer tumor. In certain
other
i
embodiments, attenuated tumor-targeted bacteria engineered to express one or
more
nucleic acid molecules encoding one or more effector molecules and/or fusion
proteins are
used in conjunction with other known cancer therapies to treat a solid cancer
tumor. For
example, attenuated tumor-targeted bacteria engineered to express one or more
nucleic acid
molecules encoding one or more effector molecules and/or fusion proteins can
be used in
conjunction with a chemotherapeutic agent. Examples of chemotherapeutic agents
include,
but are not limited to, cisplatin, ifosfamide, taxanes such as taxol and
paclitaxol,
topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211),
gemcitabine,
vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine,
temodal,
cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, tenoposide,
vincristine,
vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione,
mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,
glucocorticoids,
procaine, tetracaine, lidocaine, propranolol, and puromycin homologs, and
cytoxan.
Alternatively, attenuated tumor-targeted bacteria engineered to express one or
more nucleic
acid molecules encoding one or more effector molecules and/or fusion proteins
can be used
in conjunction with radiation therapy (e.g., gamma radiation or x-ray
radiation). Any
radiation therapy protocol can be used depending upon the type of cancer to be
treated. For
example, but not by way of limitation,, x-ray radiation can be administered;
in particular,
high-energy megavoltage (radiation of greater that 1 MeV energy) can be used
for deep
~mors, and electron beam and orthovoltage x-ray radiation can be used for skin
cancers.
Gamma ray emitting radioisotopes, such as radioactive isotopes of radium,
cobalt and other
elements may also be administered to expose tissues to radiation.
The present invention includes the sequential or concomitant administration of
an
anti-cancer agent and attenuated tumor-targeted bacteria. The invention
encompasses
combinations of anti-cancer agents and attenuated tumor-targeted bacteria that
are additive
or synergistic.
The invention also encompasses combinations of one or more anti-cancer agents
and attenuated tumor-targeted bacteria that have different sites of action.
Such a
combination provides an improved therapy based on the dual action of these
therapeutics
whether the combination is synergistic or additive. Thus, the novel
combinational therapy
of the present invention yields improved efficacy over either agent used as a
single-agent
therapy.
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The present invention also includes the sequential or concomitant
administration of
an anti-cancer agent and attenuated tumor-targeted bacteria engineered to
express one or
more nucleic acid molecules encoding one or more effector molecules and/or
fusion
proteins. The invention encompasses combinations of anti-cancer agents and
attenuated
tumor-targeted bacteria engineered to express one or more nucleic acid
molecules encoding
one or more effector molecules and/or fusion proteins that are additive or
synergistic.
The invention also encompasses combinations of one or more anti-cancer agents
and attenuated tumor-targeted bacteria engineered to express one or more
nucleic acid
molecules encoding one or more effector molecules and/or fusion proteins that
have
different sites of action. Such a combination provides an improved therapy
based on the
dual action of these therapeutics whether the combination is synergistic or
additive. Thus,
the novel combinational therapy of the present invention yields improved
efficacy over
either agent used as a single-agent therapy.
5.8. METHODS AND COMPOSITIONS FOR DELIVERY
1 S The invention provides methods by which one or more primary effector
molecules
which may be toxic when delivered systemically to a host, can be delivered
locally to
tumors by an attenuated tumor-targeted bacteria with reduced toxicity to the
host. , In one
embodiment, the primary effector molecule is useful to treat sarcomas,
lymphomas,
carcinomas, or other solid tumor cancers. In certain non-limiting embodiments,
the
effector molecule is useful for inducing local immune response at the site of
the tumor.
According to the present invention, the attenuated tumor-targeted bacterial
vectors
containing a nucleic acid molecules encoding one or more primary effector
molecules and
optionally one or more primary effector molecules are advantageously used in
methods to
inhibit the growth of a tumor, reduce the volume of a tumor, or prevent the
spread of tumor
cells in an animal, including a human patient, having a solid tumor cancer.
The present invention provides methods for delivering one or more. primary
effector
molecules for the treatment of a solid tumor cancer comprising administering,
to an animal
in need of such treatment, a pharmaceutical composition comprising an
attenuated tumor-
targeted bacteria comprising one or more nucleic acid molecules encoding one
or more
primary effector molecules operably linked to one or more appropriate
promoters. The
present invention also provides methods for delivering one or more primary
effector
molecules for the treatment of a solid tumor cancer comprising administering,
to an animal
in need of such treatment, a pharmaceutical composition comprising an
attenuated tumor-
targeted bacteria comprising one or more nucleic acid molecules encoding one
or more
pnmary effector molecules and one or more secondary effector molecules
operably linked
to one or more appropriate promoters. In one embodiment, the primary effector
molecule
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is a TNF family member, a cytotoxic peptide or polypeptide, an anti-angiogenic
factor, a
tumor inhibitory enzyme, or a functional fragment thereof.
The present invention provides methods for delivering one or more fusion
proteins
of the invention for the treatment of a. solid tumor cancer comprising
administering, to an
animal in need of such treatment, a pharmaceutical composition comprising an
attenuated
tumor-targeted bacteria comprising one or more nucleic acid molecules encoding
one or
more fusion proteins of the invention operably linked to one or more
appropriate
promoters. The present invention also provides methods for delivering one or
more fusion
proteins of the invention and one or more effector molecules for the treatment
of a solid
tumor cancer comprising administering, to an animal in need of such treatment,
a
pharmaceutical composition comprising an attenuated tumor-targeted bacteria
comprising
one or more nucleic acid molecules encoding one or more fusion proteins of the
invention
and one or more effector molecules operably linked to one or more appropriate
promoters.
In a preferred embodiment, the attenuated tumor-targeted bacteria is
Salmonella.
In another embodiment, the attenuated tumor-targeted bacteria comprises an
enhanced
release system. In a preferred embodiment, the animal is a mammal. In a highly
preferred
embodiment, the animal is a human.
The invention also provides combinatorial delivery of one or more primary
effector
molecules and optionally, one or more secondary effector molecules which are
delivered
by an attenuated tumor-targeted bacteria such as Salmonella. The invention
also provides
combinatorial delivery of different attenuated tumor-targeted bacteria
carrying one or more
different primary effector molecules and/or optionally, one or more different
secondary
effector molecules.
The invention also provides delivery of one or more fusion proteins of the
invention
which are delivered by an attenuated tumor-targeted bacteria such as
Salmonella. The
invention also provides combinatorial delivery of one or more fusion proteins
of the
invention and optionally, one or more effector molecules of the invention,
which are
delivered by an attenuated tumor-targeted bacteria such as Salmonella. The
invention also
provides combinatorial delivery of different attenuated tumor-targeted
bacteria carrying
one or more different fusion proteins and/or optionally, one or more different
effector
molecules.
Solid tumors include, but are not limited to, sarcomas, carcinomas and other
solid
tumor cancers, including, but not limited to germ line tumors, 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, melanoma, renal cancer, bladder cancer,
and
mesothelioma. The subject is preferably an animal, including but not limited
to animals
such as cows, pigs, chickens, dogs, cats, horses, etc., and is preferably a
mammal, and most
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preferably human. As used herein, treatment of a solid tumor, includes but is
not limited
to, inhibiting tumor growth, inhibiting tumor cell proliferation, reducing
tumor volume, or
inhibiting the spread of tumor cells to other parts of the body (metastasis).
The present invention provides a pharmaceutical composition comprising a
pharmaceutically acceptable Garner and an attenuated tumor-targeted bacteria
comprising
one or more nucleic acid molecules encoding one or more primary effector
molecules
operably linked to one or more appropriate promoters. The present invention
provides a
pharmaceutical composition comprising a pharmaceutically acceptable Garner and
an
attenuated tumor-targeted bacteria comprising one or more nucleic acid
molecules
encoding one or more primary effector molecules and one or more secondary
effector
molecules operably linked to one or more appropriate promoters.
The present invention provides a pharmaceutical composition comprising a
pharmaceutically acceptable carrier and an attenuated tumor-targeted bacteria
comprising
one or more nucleic acid molecules encoding one or more fusion proteins of the
invention
operably linked to one or more appropriate promoters. The present invention
provides a
1 S pharmaceutical composition comprising a pharmaceutically acceptable Garner
and an
attenuated tumor-targeted bacteria comprising one or more nucleic acid
molecules
encoding one or more fusion proteins of the invention and one or more effector
molecules
operably linked to one or more appropriate promoters.
The present inventiomalso provides a pharmaceutical composition comprising a
pharmaceutically acceptable Garner and an attenuated tumor-targeted bacteria.
The present
invention also provides a pharmaceutical composition comprising a
pharmaceutically
acceptable carrier and an attenuated tumor-targeted bacteria comprising one or
more
primary effector molecules and optionally, one or more secondary effector
molecules.
Such compositions comprise a therapeutically effective amount of an attenuated
tumor-
targeted Salmonella vector comprising one or more primary effector molecules
and
optionally one or more secondary effector molecules, and a pharmaceutically
acceptable
Garner. The present invention also provides a pharmaceutical composition
comprising a
pharmaceutically acceptable carrier and an attenuated tumor-targeted
Salmonella
comprising one or more fusion proteins of the invention and optionally, one or
more
effector molecules. Such compositions comprise a therapeutically effective
amount of an
attenuated tumor-targeted Salmonella vector comprising one or more fusion
proteins of the
invention and optionally one or more effector molecules, and a
pharmaceutically
acceptable carrier.
In a specific embodiment, 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 for use in animals,
and more
particularly in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or
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vehicle with which the therapeutic is administered. Such pharmaceutical
carriers can be
sterile liquids, such as water and oils, including those of petroleum, animal,
vegetable or
synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil,
olive oil, and the
like. Saline is a preferred Garner 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. Suitable
pharmaceutical
excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice,
flour, chalk, silica
gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk,
glycerol, propylene, glycol, water, ethanol and the like. 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,
tablets, pills,
capsules, powders, sustained-release formulations and the like. Oral
formulation can
include standard carriers such as pharmaceutical grades of mannitol, lactose,
starch,
magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc.
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 attenuated tumor-targeted bacteria, in purified form, together
with a suitable
amount of carrier so as to provide the form for proper administration to the
patient. The
formulation should suit the mode of administration.
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
suspending agent and a local anesthetic such as lignocaine to ease pain at the
site of the
injection. Generally, the ingredients are supplied either separately or mixed
together in
unit dosage form, for example, as a dry lyophilized powder or water free
concentrate in a
hermetically sealed container such as an ampoule or sachette indicating the
quantity of
active agent. 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.
The amount of the pharmaceutical composition of the invention which will be
effective in the treatment or prevention of a solid tumor cancer will depend
on the nature of
the cancer, 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 in the formulation will also depend on the route of
administration, and
the seriousness of the cancer, and should be decided according to the judgment
of the
practitioner and each patient's circumstances. However, suitable dosage ranges
are
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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 10$ 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./kg to about 1 x 108 c.f.u./kg;
and optionally
from about 1 x 104 c.f.u./kg-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,
intrathecal, intranasal, epidural, and oral routes. Methods of introduction
may also be
intra-tumoral (e.g., by direct administration into the area of the tumor).
The compositions may be administered by any convenient route, for example by
infusion or bolus injection, by absorption through epithelial or mucocutaneous
linings (e.g.,
oral mucosa, rectal and intestinal mucosa, etc.) and may be administered
together with
other biologically active agents. Administration can be systemic or local. 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
~lbers. In one embodiment, administration can be by direct injection at the
site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic tissue.
The attenuated tumor-targeted bacteria comprising one or more primary effector
molecules and optionally, one or more secondary effector molecules maybe
delivered in a
controlled release system. The attenuated tumor-targeted bacteria comprising
one or more
Vision proteins of the invention and optionally, one or more effector
molecules may also be
delivered in a controlled release system. In one embodiment, a pump may be
used (see
Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et
al., 1980,
Surgery 88:507; and Saudek et al., 1989, N. Engl. J. Med. 321:574). In another
embodiment, polymeric materials can be used (see Medical Applications of
Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974);
Controlled Drug
Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.),
Wiley,
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol. Chem.
23:61
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(1983); see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann.
Neurol.
25:351; and 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, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
Other controlled release systems are discussed in the review by Langer (1990,
Science 249:1527-1533) and may be used in connection with the administration
of the
attenuated tumor-targeted bacteria comprising one or more primary effector
molecules)
and optionally, one or more secondary effector molecule(s).
The invention also provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions of
the invention. Optionally associated with such containers) can be a notice in
the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration.
The present invention also provides methods for treating a solid tumor
comprising
administering to an animal in need thereof, a pharmaceutical composition of
the invention
and at least one other known cancer therapy. In a specific embodiment, an
animal with a
solid tumor cancer is administered a pharmaceutical composition of the
invention and at
least one chemotherapeutic agent. Examples of chemotherapeutic agents include,
but are
not limited to, cisplatin, ifosfamide, taxanes such as taxol and paclitaxol,
topoisomerase I.
inhibitors (e.g., CPT-l l, topotecan, 9-AC, and GG-211), gemcitabine,
vinorelbine,
oxaliplatin, 5-fluorouracil (S-FU), leucovorin, vinorelbine, temodal,
cytochalasin B,
gramicidin D, emetine, mitomycin, etoposide, tenoposide, vincristine,
vinblastine,
colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,
mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,
tetracaine,
lidocaine, propranolol, and puromycin homologs, and cytoxan.
The present invention includes the sequential or concomitant administration of
pharmaceutical composition of the invention and an anti-cancer agent such as a
chemotherapeutic agent. In a specific embodiment, the pharmaceutical
composition of the
invention is administered prior to (e.g., 2 hours, 6 hours, 12 hours, 1 day, 4
days, 6 days, 12
days, 14 days, 1 month or several months before) the administration of the
anti-cancer
agent. In another specific embodiment, the pharmaceutical composition of the
invention is
administered subsequent to (e.g., 2 hours, 6 hours, 12 hours, 1 day, 4 days, 6
days, 12
days, 14 days, 1 month or several months after) the administration of an anti-
cancer agent.
In a specific embodiment, the pharmaceutical composition of the invention is
administered
concomitantly with an anti-cancer agent. The invention encompasses
combinations of anti-
cancer agents and attenuated tumor-targeted bacteria engineered to express one
or more
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nucleic acid molecules encoding one or more effector molecules and/or fusion
proteins that
are additive or synergistic.
The invention also encompasses combinations of anti-cancer agents and
attenuated
tumor-targeted bacteria engineered to express one or more nucleic acid
molecules encoding
one or more effector molecules and/or fusion proteins that have different
sites of action.
Such a combination. provides an improved therapy based on the dual action of
these
therapeutics whether the combination is synergistic or additive. Thus, the
nove1
combinational therapy of the present invention yields improved efficacy over
either agent
used as a single-agent therapy.
In one embodiment, an animal with a solid tumor cancer is administered a
pharmaceutical composition of the invention and treated with radiation therapy
(e.g.,
gamma radiation or x-ray radiation). In a specific embodiment, the invention
provides a
method to treat or prevent cancer that has shown to be refractory to radiation
therapy. The
pharmaceutical composition may be administered concurrently with radiation
therapy.
Alternatively, radiation therapy may be administered subsequent to
administration of a
pharmaceutical composition of the invention, preferably at least an hour, five
hours, 12
hours, a day, a week, a month, more preferably several months (e.g., up to
three months),
subsequent to administration of a pharmaceutical composition.
The radiation therapy administered prior to, concurrently with, or subsequent
to the
administration of the pharmaceutical composition of the invention can be
administered by
any method known in the art. Any radiation therapy protocol can be used
depending upon
the type of cancer to be treated. For example, but not by way, of limitation,
x-ray radiation
can be administered; in particular, high-energy megavoltage (radiation of
greater that 1
MeV energy) can be used for deep tumors, and electron beam and orthovoltage x-
ray
radiation can be used for skin cancers. Gamma ray emitting radioisotopes, such
as
radioactive isotopes of radium, cobalt and other elements may also be
administered to
expose tissues to radiation.
Additionally, the invention also provides methods of treatment of cancer with
a
Pharmaceutical composition as an alternative to radiation therapy where the
radiation
therapy has proven or may prove too toxic, i.e., results in unacceptable or
unbearable side
effects, for the subject being treated.
5.9. DEMONSTRATION OF THERAPEUTIC OR
PROPHYLACTIC UTILITY OF PHARMACEUTICAL
COMPOSITIONS OF THE INVENTION
The pharmaceutical compositions of the invention are preferably tested in
vitro, and
then in vive for the desired therapeutic or prophylactic activity, prior to
use in humans. For
example, in vitro assays which can be used to determine whether administration
of a
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specific pharmaceutical composition is indicated, include in vitro cell
culture assays in
which a patient tissue sample is grown in culture, and exposed to or otherwise
administered
a pharmaceutical composition, and the effect of such composition upon the
tissue sample is
observed. -
Pharmaceutical compositions of the invention can be tested for their ability
to
augment 'activated immune cells by contacting immune cells with a test
pharmaceutical
composition or a control and determining the ability of the test
pharmaceutical composition
to modulate (e.g., increase) the biological activity of the immune cells. The
ability of a test
composition to modulate the biological activity of immune cells can be
assessed by
detecting the expression of cytokines or antigens, detecting the proliferation
of immune
cells, detecting the activation of signaling molecules, detecting the effector
function of
immune cells, or detecting the differentiation of immune cells. Techniques
known to those
of skill in the art can be used for measuring these activities. For example,
cellular
proliferation can be assayed by 3H-thymidine incorporation assays and trypan
blue cell
counts. Cytokine and antigen expression can be assayed, for example, by
immunoassays
including, but are not limited to, competitive and non-competitive assay
systems using
techniques such as western blots, immunohisto-chemistry radioimmunoassays,
ELISA
(enzyme linked immunosorbent assay), "sandwich" immunoassays,
immunoprecipitation
assays, precipitin reactions, gel diffusion precipitin reactions,
immunodiffusion assays,
agglutination assays, complement-fixation assays, immunoradiometric assays,
fluorescent
immunoassays, protein A immunoassays and FACS analysis. The activation of
signaling
molecules can be assayed, for example, by kinase assays and electromobility
shift assays
(EMSAs). The effector function of T-cells can be measured, for example, by a S
1 Cr-
release assay (see, e.g., Palladino et al., 1987, Cancer Res. 47:5074--5079
and Blachere et
al., 1993, J. Immunotherapy 14:352-356).
Pharmaceutical compositions of the invention can be tested for their ability
to
reduce tumor formation in animals suffering from cancer. Pharmaceutical
compositions of
the invention can also be tested for their ability to alleviate of one or more
symptoms
associated with a solid tumor cancer. Further, pharmaceutical compositions of
the
invention can be tested for their ability to increase the survival period of
patients suffering
from a solid tumor cancer. Techniques known to those of skill in the art can
be used to
analyze the function of the pharmaceutical compostions of the invention in
animals.
In various specific embodiments, in vitro assays can be carned out with
representative cells of cell types involved in a solid tumor cancer, to
determine if a
pharmaceutical composition of the invention has a desired effect upon such
cell types.
Pharmaceutical compositions of the invention for use in therapy can be tested
in
suitable animal model systems prior to testing in humans, including but not
limited to rats,
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mice, chicken, cows, monkeys, pigs, dogs, rabbits, etc. For in vivo testing,
prior to
administration to humans, any animal model system kriown in the art may be
used.
The following series of examples are presented by way of illustration and not
by
way of limitation on the scope of the invention.
6. EXAMPLE: EXPRESSION OF TNF-a BY ATTENUATED
TUMOR-TARGETED SALMONELLA
The following example demonstrates that attenuated tumor-targeted bacteria,
such
as Salmonella, containing a nucleic acid molecule encoding a TNF family member
are
capable of expressing the TNF family member.
6.1. CONSTRUCTION OF TNF-a PLASMIDS
The plasmids described herein serve to illustrate examples of specific
embodiments
of the invention. As will be apparent to one of ordinary skill in the art,
promoter and/or
effector molecule-encoding nucleic acids such as the trc promoter and/or TNF-a
encoding
nucleic acids may be replaced with other appropriate promoter or effector
molecules by
methods known in the art.
For plasmid-based bacterial expression of effector molecule-encoding nucleic
acids
using the trc promoter, the plasmid Trc99A (commercially available from
Pharmacia) or
TrcHisB (commercially available from InVitrogen) were used. Both plasmids
employ an
Nco I site, as the start codon, followed by a multiple cloning site.
6.1.1. THE_pCET PLASMID
For plasmid-based bacterial expression of effector molecule encoding nucleic
acids
using a dual ~,P~, or ~,PR promoter, the pCET plasmid was constructed as
follows. Plasmid
pCE33 (Elvin et al., 1990, Gene 87:123-126) was sequentially cleaved with the
restriction
enzyme Cla I and blunt-ended with mung bean nuclease, followed by cleavage
with the
restriction enzyme BamHI. Next, the resulting 1.4 kb fragment was ligated into
a 2.1 kb
Ssp IlBam HI fragment of pUCl9 (commercially available from GIBCO) to create
plasmid
pCI. Plasmid pCI was cleaved with restriction enzyme BamHI and blunt-ended
with mung
bean nuclease, followed by cleavage with restriction enzyme Afl III. The
resultant 3.1 kb
band was isolated. Plasmid TrcHisB was partially digested with the restriction
enzyme
Clal, blunt-ended with T4 DNA polymerase, followed by cleavage with Afl III.
The
resultant 0.6 kb band, containing the minicistron and terminator, was then
ligated into the
3.1 kb pCI fragment to give plasmid pCET. As with Trc99A or TrcHisB, pCET
employs
an NcoI site as the start codon, followed by the TrcHisB multiple cloning
site. Growth of
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bacteria harboring any plasmid containing the ~,P~, or ~,PR promoter, was
performed at 30°C.
6.1.2. THE STS PLASMID
A plasmid, denoted pTS, employing transposon-mediated chromosomal integration
and serine prototrophic selection of effector molecule-encoding nucleic acids,
was
constructed as follows. The plasmid pNK2883 (commercially available from the
American
Type Culture Collection (ATCC)) was cleaved with restriction enzyme Bam HI and
the 4.8
kb band isolated. The Salmonella typhimurium serf encoding nucleic acid was
isolated
from S. typhimurium strain 14028 (commercially available from the ATCC) by PCR
using
a forward primer of sequence GAAGATCTTCCGGAGGAGGGGAAATG (SEQ ID
NO:1 ), and a reverse primer, of sequence
CGGGATCCGAGCTCGAGGGCCCGGGAAAGGATCTAAGAAGATCC(SEQID
N0:2). The PCR reaction mixture was cleaved with restriction enzymes Bgl II
and Bam
HI, and the 1.1 kb PCR product isolated and ligated into the 4.8 pNK2883
fragment to give
a plasmid, denoted pTS. A cloning sited immediately 3' to the serf encoding
nucleic acid
1 S was present for the insertion of effector molecule-encoding nucleic acids.
6.1.3. THE pTS-TNF-a PLASMID
A plasmici (pTS-TNF-a), for the pTS-mediated chromosomal integration of a trc
promoter-driven human TNF-a encoding nucleic acid, was constructed as follows.
Plasmid PYA3332 is the ASD plasmid PYA272 (see, e.g., U.S. Patent No.
5,840,483 to
Curtiss, III) with the origin of replication replaced by that of the colEl
plasmid (see, e.g.,
Bazaral and Helsinki, 1970, Biochem 9:399-406). Plasmid PYA3332 was cleaved
with
restriction enzyme Nco I and blunt-ended with mung bean nuclease. The blunt-
ended
fragment was then cleaved with restriction enzyme Hind III and the 3.3 kb DNA
fragment
was isolated. An E. coli-optimized human TNF-a encoding nucleic acid (see,
Pennica et
al., 1984 Nature 312:724-729; and Salztman, et al., 1996, Cancer Biotherapy
11:145-153)
as depicted in FIG.1, was then cleaved with restriction enzyme Nde I, blunt-
ended with T4
DNA polymerase, and then cleaved with restriction enzyme with Hind III. The
resulting
0.5 kb fragment was ligated into the 3.3 kb PYA3332 fragment to give plasmid
Asd34TNF-a. Asd34TNF-a was then cleaved with restriction enzyme Bgl II, and
the 1.1
kb fragment, encoding the trc promoter-driven TNF-a encoding nucleic acid, and
ligated
into the Bam HI site of pTS to give plasmid pTS-TNF-a.
6.1.4. THEpTS-BRP PLASMID
A plasmid, denoted pTS-BRP, employing transposon-mediated chromosomal
integration of the BRP encoding nucleic acid and serine prototrophic selection
of effector
molecule-encoding nucleic acids, was constructed as follows. A BRP encoding
nucleic
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acid was isolated from plasmid pSWI (commercially available from Bio101,
Vista, CA) by
PCR using a forward primer, of sequence CCGACGCGTTGACACCTGAAAACTGGAG
(SEQ ID NO:S), and a reverse primer, of sequence
CCGACGCGTGAAAGGATCTCAAGAAGATC (SEQ ID N0:6), and cloned into a
TOPO-TA cloning plasmid (commercially available from InVitrogen, Carlsbad, CA)
to
give a plasmid, denoted pBRP#5. Plasmid pBRP#5 was cleaved with restriction
enzymes
Apa I and Bam HI, and the resultant 0.6 kb band, containing the BRP encoding
nucleic
acid, was ligated into the 5.9 kb Apa IlBam HI proto-pTS fragment to give the
plasmid
pTS-BRP. Cloning sites both 5' and 3' to the BRP encoding nucleic acids were
present for
the insertion of effector molecule-encoding nucleic acids.
6.1.5. THE pTS-BRPTNF-a PLASMID
A plasmid (pTS-BRPTNF-a), for the pTS-mediated chromosomal integration of the
BRP and trc promoter-driven TNF-a encoding nucleic acids, was constructed as
follows.
Plasmid Asd34TNF-a, described above for the construction of pTS-TNF-a, was
cleaved
with restriction enzyme Bgl II, and the 1.1 kb fragment, encoding the trc
promoter-driven
TNF-a encoding nucleic acid, was ligated into the Bam HI site of pTS-BRP to
give
plasmid pTS-BRPTNF-a.
6.2. INTEGRATION OF EFFECTOR MOLECULE-ENCODING
NUCLEIC ACID INTO THE SALMONELLA HOST
CHROMOSOME
The system described here employs OserC- Salmonella strains auxotrophic for
serine or glycine, and plasmids which restore serine/glycine prototrophy upon
chromosomal integration into an actively transcribed region. However, it is
well known in
the art that other selection markers can be used to select for chromosomal
integrants, and
such markers are within the scope of the invention. See, e.g., Kleckner et
al., 1991, Meth.
Enzymol. 204:139-180.
pTS or pTS-BRP plasmids containing effector molecule-encoding nucleic acids
may be introduced into serf-Salmonella strains by a number of means well-known
in the
art, including chemical transformation and electroporation. Following the
introduction of
effector molecule-encoding nucleic acids, Salmonella are grown in ampicillin-
containing
growth medium for a minimum of 2 hours, and more preferably 6 hours or longer.
Bacteria are then placed in medium capable of selecting bacteria prototrophic
for serine,
e.g., M56 medium. Atlas, R.M. "Handbook of Microbiological Media." L.C. Parks,
ed.
CRC Press, Boca Raton, Florida, 1993. Bacteria harboring chromosomal
integrations of
effector molecule-encoding nucleic acids are capable of growth in the
selective media.
Effector molecule-encoding nucleic acid expression is then measured, as
illustrated below.
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Effector molecule-encoding nucleic acid expression may be measured by any of
several
methods known to those skilled in the art, such as by enzymatic activity,
biological
activity, Northern blot analysis, or Western blot analysis.
6.2.1. DELIVERY AND EXPRESSION OF SALMONELLA-
EXPRESSED TNF-a
A trc promoter-driven TNF-a encoding nucleic acid was inserted into the Bam HI
site of pTS-BRP to give a plasmid, denoted pTS-BRPTNF-a, as described above.
Plasmid
pTS-BRPTNF-a was electroporated into an attenuated strain of S. typhimurium,
strain
VNP20009, (see International Publication WO 99/13053) constructed to be serf-
such that
the genotype was OmsbB, Opurl, OserC (FIG. 2), by standard methods known in
the art.
Without limitation as to mechanism, integration of the plasmid into the
bacterial genome
allows for activation of the serf encoding nucleic acid and leads to a serCr
phenotype.
Accordingly, bacteria harboring a chromosomal integration of the TNF-a
encoding nucleic
acid were selected by plating the electroporated bacteria on M56 agar plates
supplemented
with adenine. Bacteria were further characterized for loss of ampicillin
resistance,
indicative of plasmid loss, and concomitant loss of plasmid-based TNF-a
expression.
In order to examine and quantify TNF-a expression by the tumor-targeted
bacteria
of the invention, Salmonella harboring a chromosomal integration of the TNF-a
encoding
nucleic acids were grown overnight, and a measured sample of the culture was
used in
Western blot analysis. Specifically, TNF-a expression from a representative
serf+,
ampicillin-sensitive clone, denoted pTS-BRPTNF-a Clone 2, is shown in FIG. 3.
Western
blot analysis revealed that bacterial protein, corresponding to 3.9x10' cfu of
pTS-
BRPTNF-a Clone 2 bacteria (Lane 1), expressed more than 50 ng TNF-a (Lane 5),
indicating expression of TNF-a at levels greater than 10 ng/10' bacteria.
Therefore, the
human TNF-a was successfully expressed from a chromosomally-integrated, trc
promoter-
driven, TNF-a encoding nucleic acid in Salmonella.
7. EXAMPLE: ATTENUATED TUMOR-TARGETED
BACTERIA EXPRESSING OMPA
FUSION PROTEINS
Periplasmic localization of proteins by fusion to various signal peptides is
dependent on both the signal peptide and the protein. For example, proteins
can be
localized to the periplasmic compartment of bacteria by fusion of a signal
peptide to the
amino terminus of the protein. Without limitation, periplasmic localization is
believed to
facilitate release of bacterial components (such as proteins) by requiring the
component to
traverse only a single membrane in order to be released into the surrounding
environment.
In contrast, cytoplasmic localization requires that the component traverse
both the inner
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and outer membranes of bacteria in order to be released into the surrounding
environment.
Further, periplasmic localization of certain protiens may aid in biological
activity.
A variety of methods known in the art may be used to target an effector
molecule of
the invention to the periplasm. . This example demonstrates that the fusion of
the ompA
signal peptide to the amino terminus of an effector molecule such as a TNF-a,
TRAIL
(TNF-a-related apoptosis-inducing ligand), and interleukin-2 (IL-2) results in
the
periplasmic localization and subsequent processing of proteins.
7.1. PROCESSING OF AN OMPA-TNF-a FUSION PROTEIN
TNF-a expression in four different clones, expressing a plasmid-based trc
promoter-driven ompA-TNF-a fusion protein in JM109 bacteria, was examined by
Western
blot analysis of whole cell lysate. Periplasmic localization was demonstrated
by cleavage
of the precursor fusion proteins to mature TNF-a by signal peptidase, an
enzyme located in
the periplasm. In all four clones, following induction with IPTG,
overexpression of TNF-
a resulted in the appearance of TNF-a as a doublet migrating at approximately
20 kd (FIG.
S~ lanes 4-7), corresponding to both unprocessed and processed forms. For
comparison, a
Salmonella strain harboring a chromosomally-integrated TNF-a encoding nucleic
acids,
expressing the mature (processed) form of TNF-a, was used as a positive
control (FIG. 5,
lane 3). TNF-a expression was not detected in bacteria lacking the TNF-a
encoding
nucleic acids (FIG. S, lane 2).
These results demonstrated that fusion of the mature human TNF-a protein to
the
E. coli ompA signal peptide (as depicted in FIG. 4) resulted in periplasmic
localization and
processing when expressed in E. coli. Further, it was unknown whether
overexpression of
a secreted protein would be toxic to the bacterial host as a result of
overwhelming the
normal secretory apparatus. The present demonstration of expression of a
processed
~mpA-TNF-a fusion protein indicated that the normal secretory apparatus was
capable of
accommodating the high-level expression of secreted proteins.
7.2. PROCESSING OF AN OMPA-TRAIL FUSION PROTEIN
The ability of the ompA signal peptide to periplasmically localize TNF family
members was extended to TRAIL (TNF-a-related apoptosis-inducing ligand),
another
member of the TNF family. For these experiments, a trc promoter-driven TRAIL
encoding nucleic acids, encoding the mature form of human TRAIL (hTRAIL), was
fused
to the coding sequence of the ompA signal peptide (as depicted in FIG.6). Two
different
ompAlTRAIL junctions were examined, one encoding an Ncol site and one encoding
an
Ndel site (See FIG.6 for Ndel containing sequence). Western analysis of both
types of
clones is shown in FIG. 7. Using an anti-hTRAIL antibody, Western blot
analysis revealed
that bacteria over-expressing the ompA-TRAIL with the Nco I junction expressed
both
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processed (28.2 kd) and unprocessed (30.2 kd) forms of hTRAIL (FIG. 7, lanes 2-
4),
whereas bacteria overexpressing the ompA-TRAIL with the Nde I junction
expressed the
processed form exclusively (FIG. 7, lanes 4-7), indicating that the Nde I
junction provided
more efficient processing.
These results demonstrated that fusion of the mature human TRAIL protein to
the
E. coli ompA signal peptide resulted in periplasmic localization and
processing. Further, it
was unknown whether overexpression of the secreted protein would be toxic to
the
bacterial host as a result of overwhelming the normal secretory apparatus. The
present
demonstration of expression of a processed ompA-TRAIL fusion protein indicated
that the
normal secretory apparatus was capable of accommodating the high-level
expression of
secreted proteins.
7.3. PROCESSING OF AN OMPA(8L)-IL-2 FUSION PROTEIN
A secondary effector molecule (IL-2) was expressed as a fusion protein. Fusion
of
mature (C125A) hIL-2 to the wild-type OmpA signal sequence, used above for TNF-
a and
T~'IL, did not permit processing of IL-2. In order to examine the periplasmic
localization and processing of the human IL-2 cytokine, mature human (C125A)
IL-2 was
fused to a modified ompA signal peptide, denoted ompA(8L), as depicted in FIG.
8. The
modified ompA signal peptide was modified by replacing amino acids 6-17 of the
ompA
signal with those depicted in Figure 8. Expression and processing are shown in
FIG. 9
(lanes 6 and 7). Each lane represents a single clone. Results of Western blot
analysis
indicated that virtually complete processing was observed with the ompA(8L)
signal
peptide (FIG. 9, lanes 6 and 7).
7.4. PROCESSING OF AN PHOA(8Ll-IL-2 FUSION PROTEIN
A second fusion protein was examined for periplasmic localization and
processing
of human IL-2, and compared with the fusion protein of Section 7.3. The
expression and
processing of mature human (C 125A) IL-2 fused to a modified phoA signal
peptide,
denoted phoA(8L), as depicted in FIG.10 was examined. Expression and
processing are
shown in FIG. 9. Partial processing was observed with the synthetic phoA(8L)
signal
peptide (FIG. 9, lanes 4 and 5), whereas more complete processing was observed
with the
ompA(8L) signal peptide (FIG. 9, lanes 6 and 7).
These results indicate that localization and processing of IL-2 was provided
by
different signal peptides. The results also demonstrate that periplasmic
localization of
proteins by fusion to various signal peptides is dependent on both the signal
peptide and
the protein.
The results of the fusion protein studies indicate that a secondary effector
protein of
the invention, such as IL-2, can be expressed and localized to the bacterial
per~iplasm by
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fusion with the a protein signal peptide such as OmpA or PhoA. As will be
apparent to one
of ordinary skill in the art, other signal sequences can be used to cause
periplasmic
localization of an effector molecule can be used. As will further be apparent
to one of
ordinary skill in the art, other effector molecules of the invention can be
substituted for the
effector molecules described in the examples herein.
8. EXAMPLE: ANTI-TUMOR EFFICACY OF SALMONELLA
(ONISBB, APUR~ EXPRESSING THE MATURE
FORM OF TNF-a
The following experiment demonstrates that an attenuated tumor-targeted
bacteria
such as Salmonella containing a nucleic acid encoding a primary effector
molecule (e.g., a
TNF family member) can deliver the primary effector to mammalian tumors and
cause a
decrease in tumor volume.
The ability of TNF-a expression to increase the anti-tumor efficacy of
Salmonella
typhimurium was evaluated in a staged murine Colon 38 carcinoma model. For
these
experiments, 1 mm3 tumor fragments, derived from a Colon 38 tumor, were
implanted into
C57BL/6 mice and tumors were allowed to grow to a mean size of approximately
0.3 g, at
which time animals were randomly placed into the following treatment groups
(n=10): 1)
untreated; 2) Salmonella typhimuric~m (~msbB, Opurl, serf) (parental strain);
and.3) pTS-
BRPTNF-a (Clone 2 described above). Mice in each group either remained
untreated or
received a single intravenous injection of 1 x 106 cfu of the appropriate
bacterial strain.
Tumor size was measured weekly, beginning at the time of bacteria inoculation.
In the group receiving attenuated tumor-targeted Salmonella expressing TNF-a,
tumor regression was apparent by the second week following treatment, with
complete
regression observed in 6 of the animals within 4 weeks following treatment
(FIG. 11).
Tumors in the untreated group progressively increased in size, whereas tumors
in the group
treated with the parental Salmonella typhimurium (OmsbB, Opacrl, OserG~ strain
displayed
partial regression between weeks 3-4 following treatment, after which tumors
progressively increased in size (FIG. 11 ).
These results demonstrate that attenuated tumor-targeted Salmonella are able
to
express and deliver an effector molecule such as a TNF family member to a
tumor. Such
Salmonella are useful in the treatment of tumors and provide enhanced tumor
regression
results as compared to parent Salmonella strains which do not express the TNF
family
member.
The demonstration of complete tumor regression, by Salmonella expressing TNF-a
from chromosomally-integrated nucleic acid, indicates that biologically
effective
expression can result from chromosomally integrated-effector molecule encoding
nucleic
acids.
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9. EXAMPLE: ENHANCED DELIVERY OF NUCLEIC
ACID MOLECULES BY
BRP EXPRESSING BACTERIA
In order to demonstrate that BRP activity could enhance the release of a
plasmid
from a tumor-targeted attenuated bacteria such as Salmonella, a tumor-targeted
attenuated
Salmonella strain was constructed that contained BRP on a plasmid as well as a
second
plasmid used as a marker for release (pTrc99a with AMP marker). To assay
activity of
BRP, the Salmonella with or without BRP was grown in culture by standard
methods. The
resulting supernatant was then cleared of any remaining bacteria by
centrifugation and
filtration and the cleared supernatant was then added to competent cells and
underwent a
transformation reaction. These "recipient" cells were then plated onto LB amp
to look for
uptake of the AMP marker plasmid. An increase in the number of AMP resistant
colonies
with BRP would indicate that more plasmid was released into the media from
strains
expressing BRP. Results are summarized in Table 2 below:
Table 2
Plasmid Average # of Amp
Colonies/Transformation
pTrc99a alone 125
pTrc99a+BRP (pSWl) 383 .
These results demonstrate that the presence of BRP increased the amount of amp
plasmid
secreted to the media. Thus, transformation into "recipient cells" with
supernatants from
cells expressing BRP gave higher number of colonies. These results demonstrate
that BRP
enhanced release of a secondary effector molecule, which comprised a nucleic
acid
plasmid. Accordingly, the results show that BRP is useful for plasmid release
or DNA
delivery. In addition, these Salmonella strains that expressed BRP and were
able to deliver
DNA and remained replication competent as a population.
10. EXAMPLE: BRP EXPRESSION DOES NOT IMPAIR
TUMOR-TARGETING OR TUMOR-
INHIBITING ABILITY OF ATTENUATED
TUMOR-TARGETED SALMONELLA
The following example demonstrates that attenuated tumor-targeted bacteria can
be
engineered to express BRP in conjunction with one or more effector molecules
to enhance
the delivery of effector molecules to tumors without inhibiting the ability of
bacteria to
target the tumor.
Solid tumor models were obtained by subcutaneous injection of B16 melanoma
cells in the right hind flank of C57BL/6 mice. For tumor implantation, cells
were
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detached from the flask by trypsinization, washed, and suspended in phosphate
buffered
saline at 2.5x106 cells/ml. An aliquot of 0.2 ml of the cell suspension, for a
total of 5x105
cells/mouse, was injected on Day 0. When tumor volumes reached 150-200 mm3,
approximately 10 days after implantation, the mice were randomized into three
groups of
ten mice and each group received a different treatment. The control group
(curve #1 on
FIG. 12) received 0.2m1s of PBS. Another group received 0.2 ml containing
2x106
c.f.u./mouse of the attenuated tumor-targeted strain of Salmonella VNP20009
(curve #2 on
FIG. 12). The third group received 0.2 ml containing 2x106 c.f.u./mouse of the
attenuated
tumor-targeted strain of Salmonella comprising pSW l, a plasmid comprising the
BRP gene
under the control of its native promoter (curve #3 on FIG. 12). The BRP gene
is SOS
inducible in E. coli, although the inventors Believe, without limitation as to
mechanism,
that it is partially constitutive in Salmonella, producing low to moderate
levels of the BRP
protein, which are further enhanced by the SOS nature of the tumor
environment. Mice
injected with BRP-expressing VNP20009 Salmonella showed nearly identical anti-
tumor
responses to those injected with non-BRP-expressing VNP20009, indicating that
the
survival or tumor-targeting ability of these Salmonella is not altered by BRP
expression,
nor is their ability to inhibit tumor growth. The outcome of BRP-expression on
attenuated
tumor-targeted Salmonella is in direct contrast to the effect of the
expression of secreted
HSV-thymidine kinase (HSV-TK), which HSV-TK expression results in the loss of
VNP20009's tumor-inhibiting abilities (Pavelek et al., 1997, Cancer Res.
57:4537-4544).
Thus, the BRP system can be used to enhance the delivery of primary and/or
secondary
effector molecules to tumors without further modification.
11. EXAMPLE: penT PROMOTER EXPRESSION VEHICLES
This example demonstrates the in vitro and in vivo expression of a nucleic
acid
molecule encoding reporter such as (3-gal under the control of the pepT
promoter in an
attenuated tumor-targeted bacteria such as Salmonella.
11.1. CONSTRUCTION OF penT-BRP- ~3GAL EXPRESSION PLASMIDS
The pepT promoter was cloned by PCR amplification of the region from an
isolated
colony of wild type Salmonella typhimurium (ATCC 14028) using the following
primers:
Forward: S'-AGT CTA GAC AAT CAG GCG AAG AAC GG-3' (SEQ ID NO:15)
Reverse: 5'AGC CAT GGA GTC ACC CTC ACT TTT C-3' (SEQ ID N0:16).
The PCR conditions consisted of 1 cycle of 95°C for 5 minutes, 35
cycles of 95°C
for 1 minute, 65 ° C for 1 minute, 72 ° C for 2 minutes and 1
cycle of 72 ° C for 10 minutes.
The PCR product was cloned into the PCR 2.1 cloning vector (Invitrogen,
Carlsbad,
California), and is referred to as PepT/PCR 2.1.
The PepT/PCR 2.1 vector was digested with NcoI and XbaI. The pepT fragment
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was gel isolated and ligated into the [3-gal Zterm vector digested with the
same enzymes.
Zterm (Temporary Genbank Bankit No. 296495) is a promoterless ~3-gal plasmid
generated
by cloning the ~3Ga1 open reading frame into pUC 19. The resultant plasmid was
called
pepT-~3GAL.
11.2. IN VITRO EXPRESSION OF pepT-~3GAL AND
> MEASUREMENT OF nepT-~iGAL ACTIVITY
Salmonella strains YS 1456 (CC 14 in FIG. 13A; for the genetic make up of the
strain, see WO 96/40238) or VNP20009 (CC16 in FIG. 13A) harboring pepT-~iGAL
were
grown under either anaerobic or aerobic conditions to an OD6oo of ~0.5-0.8. (3-
gal activity
was measured by the method of Birge and Low (1974, J. Mol. Biol. 83:447-457).
The
results are shown in FIG. 13A, and demonstrate approximately 14- to 24- fold
induction of
~i-gal activity upon growth of the bacteria under anaerobic conditions.
11.3. IN VIVO EXPRESSION OF pepT-~iGAL AND
MEASUREMENT OF pepT-GAL ACTIVITY
Cells of the Salmonella strain YS 1456 harboring the pepT-~3ga1 expression
plasmids, a BRP expression plasmid (pSWl from BIO101 (Vista, California),
which
comprises the pCIoDF 13 BRP coding sequence under the control of its native
promoter) or
both expression plasmids were injected intravenously into tumor bearir~l;
mice. Five days
post injection, tumors and livers were homogenized and bacteria were isolated
to show that
the presence of plasmids for pepT-(3ga1 and/or BRP did not interfere with the
ability of
these bacteria to target tumors. In addition, the tumor and liver homogenates
were used to
measure (3ga1 activity to determine whether active (3ga1 could be measured in
vivo and
whether the pepT promoter was induced in an anaerobic tumor environment. The
results,
shown in FIG. 13B, indicate very high levels of pepT promoter activity in the
tumor
environment. There is no significant increase in liver expression of (3ga1
over the
background level, which is thought to arise from the low activity of the pepT
promoter in
the aerobic liver environment and/or the low targeting of the bacterial vector
to the liver.
12. EXAMPLE: TETRACYCLINE INDUCIBLE
EXPRESSION SYSTEM
This example demonstrates the expression of a nucleic acid molecule encoding a
reporter gene such as ~3-gal under the control of the tet promoter in an
attenuated.tumor-
targeted bacteria such as Salmonella.
The tet promoter was cloned from a mini-TN10 transposon by PCR
amplification using the following primers:
Forward: S'-GGA TCC TTA AGA CCC ACT TTC ACA TTT AAG T-3' (SEQ ID N0:17)
Reverse: 5'-GGT TCC ATG GTT CAC TTT TCT CTA TCA C-3' (SEQ ID N0:18).
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The PCR conditions were as follows: one cycle of 95 °C for 5 minutes;
35 cycles of
95°C for 1 minute, 60°C for 1 minute, 72°C for 2 minutes;
and one cycle of 72°C for 10
minutes.
The ~400bp PCR fragment was gel isolated and cloned into the PCR 2.1 vector
(Invitrogen). The PCR2.1/tet promoter vector was digested with NcoI and BamHI.
The
400 by tet promoter fragment was gel isolated and ligated into the
promoterless ~i-gal
vector Zterm that had been digested with the same two enzymes. The ligation
mixture was
transformed and the transformed bacteria were plated to tetracycline/X-gal
plates. Positive
colonies were isolated on the basis of their blue color. Extracts from several
positive clones
were made, and assayed by the method of Birge and Low (1974, J. Mol. Biol.
83:447-457)
for ~3-gal activity in the presence of tetracycline. One clone was isolated
and assayed for ~i-
gal expression over a range of tetracycline concentrations. The results of the
assay, which
demonstrate tlTe induction of (3-gal activity by tetracycline in a dose-
dependent manner, are
shown in FIG. 14.
13. EXAMPLE: INHIBITION OF TUMOR GROWTH BY
ATTENUATED TUMOR-TARGETED
SALMONELLA EXPRESSING ENDOSTATIN
The following example demonstrates the generation of endostatin-expressing
attenuated tumor-targeted Salrnonella, and the in vivo efficacy of tumor
treatment by such
Salmonella.
13.1 CONSTRUCTION OF ENDOSTATIN
EXPRESSION PLASMIDS
Endostatin was PCR amplified from a human placental cDNA library using the
following primers:
Forward: 5'-GTG TCC ATG GGG CAC AGC CAC CGC GAC TTC CAG-3' (SEQ ID
N0:19)
Reverse: S'-ACA CGA GCT CCT ACT TGG AGG CAG TCA TGA AGC T-3' (SEQ ID
N0:20).
The resulting PCR product was cloned into the PCR2.1 vector (Invitrogen).
Hexahistidine-endostatin was PCR amplified using the above constructed plasmid
as a
template with the following primers:
Forward: 5'-GTG TCC ATG GCT CGG CGG GCA AGT GTC GGG ACT GAC CAT
CAT CAT CAT CAT CAT CAC AGC CAC CGC GAC TTC-3' (SEQ ID N0:21)
Reverse: S'-GTG CGG ATC CCT ACT TGG AGG CAG TCA TGA AGC TG-3' (SEQ ID
N0:22).
The conditions for the PCR amplification consisted of 1 cycle of 95 °C
5 min; 30 .
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cycles of 95°C for 1 min, 55°C for 1 minute, and 72°C for
2 minutes; and 1 cycle of 72°C
forl0 minutes.
The resulting product was a DNA fragment with NcoI (S') and BamHI (3')
restriction sites encoding human endostatin having the peptide sequence
MARR.ASVGTDHHHHHH (SEQ ID N0:23) at its amino terminus.
The PCR product was digested with NcoI and BamHI and the 550 by product was
gel isolated and ligated into the pTrc99A vector that had been previously cut
with the same
enzymes. The ligation reaction products were transformed into E. coli DHSa and
the
attenuated tumor-targeted Salmonella strain VNP20009.
The hexahistidine-endostatin coding sequence was also cloned into the
expression
vector YA3334 as a NcoI/BamHI fragment.' YA3334 is the asd plasmid PYA272
(Curtiss III, U.S. Patent No. 5,840,483) with the origin of replication
replaced by that of
the ColEl (Bazaral and Helsinki, 1970, Biochem 9:399-406). Plasmid DNA
prepared from
positive clones was isolated and transformed into the Salmonella strain 8324,
which is
VNP20009 with an asd mutation. This strain was generated according to the
methods
described in Curtiss III (U.S. Patent No. 5,840,483).
13.2. IN VITRO EXPRESSION OF ENDOSTATIN BY
ATTENUATED TUMOR-TARGETED SALMONELLA
Different strains of Salrnonella VNP20009 and E. coli DHSa strains containing
the
pTrc99A-hexahistidine-endostatin plasmid were grown to mid-log phase
(O.D.6oo0.6-
0.8), at which point each culture was split, one half receiving 0.1 mM IPTG
for induction
of trc promoter activity and the other half receiving no IPTG.. After three
further hours of
growth, bacterial extracts were prepared and the expression of hexahistidine-
endostatin
was confirmed by Western blot analysis with an anti-histidine antibody
(Clontech, Palo
Alto, California). FIGS. 15A and 15B show the results of the Western blots
which
demonstrate pTrc99A hexahistidine-endostatin (HexHIS-endostatin) expression in
E. coli
DHSa and Salmonella VNP20009, respectively. While the trc promoter shows no
activity
in E. coli. in the absence of IPTG, the same promoter is constitutively active
in Salmonella
Hexahistidine-endostatin is expressed a single band of approximately 25kD,
which is the
predicted molecular weight for the fizsion protein.
The hexahistidine-endostatin fusion protein was similarly expressed from the
YA3334 plasmid, which utilizes the trc promoter to direct expression. A
protein of the
predicted mass of 25 kDa was detected using the anti-histidine antibody, as
shown in
FIG. 16. In FIG. 16, all bacterial cultures from which the samples were
derived had been
induced with 0.1 mM IPTG for three hours.
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13.3. EFFICACY OF ATTENUATED TUMOR-TARGETED
SALMONELLA EXPRESSING ENDOSTATIN ON
C38 MURINE COLON CARCINOMA
Colon 38 tumor fragments of 2x2x2 mm3 volume were implanted subcutaneously in
9 week old female C57BL/6 mice. When the tumor volumes reached 1000 mm', they
were
removed, cut into fragments of 2x2x2 mm3. The fragments were serially passaged
for
further cycles and the resulting 2x2x2 mm3 fragments were implanted
subcutaneously at
the right flanks of female C57BL/6 mice. When tumor volumes reached 150-200
mm',
approximately 24 days after implantation, the mice were randomized into six
groups of ten
mice and each group received a different treatment. One control group received
0.2m1s of
PBS. Another control group received 0.2 ml containing 1x106 c.f.u. ofthe
attenuated
tumor-targeted strain of Salmonella VNP20009 carrying a control asd plasmid,
i.e. an asd
plasmid that has no insert, as described in Section 5.6, supra. The first
experimental group
received 0.2 ml containing 1x106 c.f.u. of VNP20009 expressing a hexahistidine-
endostatin
fusion protein in an asd plasmid. The second experimental group received
VNP20009 with
the same expression construct as the first group and further expressed BRP.
FIG. 17 shows the results of these experiments, which demonstrate the efficacy
of
tumor inhibition by the VNP20009 strains expressing hexahistine-endostatin.
After 60
days of treatment, the median tumor size in those VNP20009 Salmonella
expressing
endostatin was approximately 13% of the median tumor size in control animals,
and over
30% less than the median tumor size in animals treated with VNP20009
Salmonella
harboring an empty vector. Of the surviving animals, many exhibited static
tumor growth,
as indicated by small changes in net tumor size, and one exhibited a strong
regression of
the tumor. Incomplete penetrance or effectiveness of the treatment most likely
reflects an
imperfect delivery system for endostatin, in concordance with O'Reilly et
al.'s (1997, Cell
88:277-285) finding that endostatin accumulates in inclusion bodies. The
delivery system
for endostatin is enhanced by the expression of BRP. BRP expression is
controlled by its
natural promoter, which normally shows an SOS response in bacteria. BRP
expression was
shown to, decrease mean tumor volume to approximately 6% of the mean tumor
volume of
the control population. Furthermore, within the mouse populations treated with
hexhistidine-endostatin and BRP, several of the mice exhibited striking
reductions in tumor
volume over time, wherein the tumor volume regressed to approximately 10% or
less of
the initial tumor volume. The effect of BRP is likely to be two-fold: first,
BRP itself may
possess anti-tumor activity, and second, BRP promotes the release of
periplasmic contents
and to some extent the release of cytoplasmic contents, including endostatin,
which
prevents the protein from accumulating in inclusion bodies.
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13.4. EFFICACY OF ATTENUATED TUMOR-TARGETED
SALMONELLA EXPRESSING ENDOSTATIN
ON DLD HUMAN COLON CARCINOMA
Cultures of DLD1 cells grown in log phase were trypsinized, washed with PBS
and
the cells reconstituted to a suspension of SxlO'cells/ml in PBS. O.lml
aliquots of single
cell suspensions, each containing 5x106 cells, were injected subcutaneously
into the right
flanks of 9-week old nude female mice (NulNu-CD 1 from Charles River). The
mice were
randomly divided into three groups of ten animals each, then staged at 10-15
days after
injection, or when tumor volume reached 200-400 mm'.
The first group of mice was the control group, and each received an 0.3m1
injection
of PBS. The second group of mice received 0.3m1 containing 1x106 c.fu. ofthe
attenuated
~mor-targeted strain of Salmonella VNP20009 carrying a control asd plasmid.
The third
group of mice received 0.3m1 containing 1x106 c.fu. of the attenuated tumor-
targeted
strain of Salmonella VNP20009 carrying an asd plasmid which expresses a
hexahistidine-
endostatin fusion protein and BRP. The tumors were monitored and measured
twice a
week. FIG. 18 is a graphic representation of tumor volume after administration
of the three
1 S treatments, demonstrating the inhibitory effect of the hexahistidine-
endostatin expressing
attenuated tumor targeted Salmonella on the growth of DLD1 human colon
carcinoma.
VNP20009 carrying the empty vector PYA3332 was not able to significantly
inhibit
tumor growth. However, VNP20009 expressing endostatin and BRP was able to
inhibit
tumor growth. These results demonstrate that the combination of endostatin
plus BRP
increases the anti-tumor effect of either the VNP20009 carrying the PYA3332
vector
(strain 8324).
14. EXAMPLE: EXPRESSION OF ANTI-ANGIOGENIC
FACTORS BY ATTENUATED
TUMOR-TARGETED SALMONELLA
The following example shows the methodology used to engineer attenuated tumor-
targeted bacteria such as Salmonella to express the anti-angiogenic factors
thrombospondin
AHR, platelet factor-4 and apomigren.
14.1. CONSTRUCTION OF A PLASMID CONTAINING THE NUCLEIC
ACID SEQUENCE ENCODING THROMBOSPONDIN AHR
The peptide sequence, TiP 13.40: AYRWRLSHRPKTGFIRWMYEG (SEQ ID
N0:24), corresponding to the anti-angiogenic homology region (AHR) of
thrombospondin (see, e.g., Patent application No. C07K-14/78), was reverse
engineered
and codon optimized for expression in Salmonella, resulting in the DNA
sequence:
GCG TAC CGC TGG CGC CTG TCC CAT CGC CCG AAA ACC GGC TTT ATC
CGC GTG GTG ATG TAC GAA GGC (SEQ ID N0:25). Complementary
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oligonucleotides (Oligo 13:40-1 and Oligo 13:40-2) were produced to synthesize
this
peptide. At the 5' end a sequence coding for the processing region of OMPA and
an
SpeI restriction site were added. At the 3' end, a stop codon was added with a
BarrlHI
restriction site. The two oligos were annealed to generate the double stranded
DNA
fragment. The DNA fragment was cut with SpeIlBamHI and ligated to the
SpeIBamHI
cut vector pTrc801IL2 to produce the plasmid pTrc801-13.40 containing the full
length
modified OmpA leader sequence. When processed, the sequence produces the full
length 13.40 thrombospondin peptide.
Oligo 13.40-1:
5 ~gtgtactagtgtgg_c ca~GCGTACCGCTGGCGCCTGTCCCATCGCCCGAA.AACC
GGCTTTATCCGCGTGGTGATGTACGAAGGCTAAggatccgcgc 3' (SEQ ID N0:26)
Oligo 13.40-2:
5'gcgcggatccTTAGCCTTCGTACATCACCACGCGGATAAAGCCGGTTTTCGGGC
GATGGGACAGGCGCCAGCGGTACGCcgcctg ,c~ccacactagtacac 3' (SEQ ID IvT0:27)
(Restriction sites are italicized and the OmpA processing recognition site is
underlined.)
14.2. CONSTRUCTION OF A PLASMID CONTAINING
THE NUCLEIC ACID SEQUENCE ENCODING
PLATELET FACTOR-4 PEPTIDE (47-701
The peptide consisting of amino acid residues 47-70 of the C-terminus of
platelet factor-4 (PF-4; see, e.g., Maione et al., 1990, Science 247:77- 79
and Jouan et
al., 1999, Blood 94:984-993) was codon-optimized for expression in Salmonella.
The
peptide, which is depicted below, includes a DLQ-motif responsible for
inhibitory
activity of PF-4 on CFU-GM progenitor cells and a clusters of basic amino
acids which
is the major heparin binding domain.
Platelet Factor-4:
MSSAAGFCASRPGLLFLGLLLLPLVVAFASAEAEEDGDLQCLCVKTTSQV
~~ITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES (SEQ
ID N0:28)
Signal peptide = underlined & in bold
Lys 61,62, 65,66 = major heparin binding domain (in bold)
DLQ (7-9, 54-56) = inhibitory activity on CFU-GM progenitor cells (in bold)
Complementary oligonucleotides (oligo PF4-1 and oligo PF4-2) were produced to
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synthesize this peptide. At the 5' end a sequence coding for the processing
region of
OmpA and a SpeI restriction site were added. At the 3' end, a stop codon was
added
with a BamHI restriction site. The two oligos were annealed to generate the
double
stranded DNA fragment. After restriction digest the fragment was ligated into
the
SpeIlBamHI restricted vector pTrc801 to produce the plasmid pTrc801-PF4. When
processed, the sequence produces the full length PF-4 (47-70) peptide.
Oligo PF4-1
5'cttcactagt~tQ~c~ca~QC~AACGGCCGCAAAATCTGCCTGGACCTGCAGGCGCCGCT
GTACA,~~AAAAATCATCAAAAAACTGCTGGAAAGCTAA ggatcc gcg3' (SEQ ID
N0:29)
Oligo PF4-2
5'cgcggatccTTAGCTTTCCAGCAGTTTTTTGATGATTTTTTTGTACAGCGGCGCCTG
CAGGTCCAGGCAGATTTTGCGGCCGTTc cg~ ct~c~cacactagtgaag3' (SEQ ID N0:30)
(Restriction sites are italicized and the ompA processing recognition site is
underlined.)
14.3. CONSTRUCTION OF A PLASMID
CONTAINING THE NUCLEIC ACID
SEQUENCE ENCODING APOMIGREN
The anti-angiogenic peptide apomigren
(IYSFDGRDIMTDPSWPQKVIWHGSSPHGVRLVDNYCEA
WRTADTAVTGLASPLSTGKILDQKAYSCANRLIVLCIENSFMTDARK (SEQ ID
N0:31; see, e.g., International Publication No. W099/29856) corresponds to the
C-
terminus of restin, which is a proteolytic fragment of collagen XV.
Oligonucleotides
(oligo ApomSF and oligo Apom6F) were designed to amplify the DNA fragment from
human cDNA. At the 5' end a sequence coding for the processing region of OmpA
and a
SpeI restriction site were added. At the 3' end, a stop codon was added with a
BamHI
restriction site:
Oligo ApomSF: 5'- ggcttc actagt t c ca c ATATACTCCTTTGATGGTCG -3' (SEQ
ID N0:32)
Oligo Apom6R: 5'- cgc ggatcc TTACTTCCTAGCGTCTGTCATGAAACTG -3' (SEQ ID
N0:33)
(Restriction sites are italicized and the OmpA processing recognition site is
underlined.)
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A fragment of the correct size was obtained by PCR using placental cDNA as
template.
The PCR product was cut with SpeIlBamHI and ligated to the SpeIBamHI
restricted
vector pTrc801 containing the modified ompA signal sequnce to produce the
plasmid
pTrc801-Apom. When processed, the sequence produces the Apomigren peptide.
14.4. ANTI-ANGIOGENIC PEPTIDES PRODUCED BY SALMONELLA
INHIBITING ENDOTHELIAL CELL PROLIFERATION
pTrcOmpA-Endostatin, pTrc801-PF4 and pTrc801-13.40 plasmids were
electroporated into attenuated tumor-targeted Salmonella VNP20009 strains.
Salmonella
strains expressing pTrcOmpA-Endostatin, pTrc801-PF4 and pTrc801-13.40 were
screened
for anti-proliferative activity as described b~c Feldman et al., 2000, Cancer
Res. 60:1503-
1506 and Blezinger et al., 1999, Nature Biotech.17:343-348. Five-rnl cultures
of
individual colonies were grown for 4 hours. Cell lysates were produced by
resuspending
the cell pellet in 1/20 volume HUVEC medium containing 100 mg/ml gentamycin
and
performing 3 consecutive freeze/thaw cycles. The lysates were cleared by
centrifugation
and filter sterilized using a 0.2 mm syringe filter. Ten, twenty-f ve or fifty
ml of the
1 S lysates were added to human vein endothelial cells (HUVECs) in 96 well
plates containing
100 ml basal medium 2% FCS plus 10 ng/ml FGF. As a control Salmonella
containing the
empty pTrc vector were used. Plates were incubated for 72 hours and
proliferation was
measured by MST assay (Mosman et al., 1983, J. Immunol. Methods 65:55-63).
The preliminary results in FIGS. 19 and 20 show that the platelet factor-4
peptide
(pF4_2), the thrombospondin peptide 13.40 (13.40-3) and endostatin produced by
Salmonella seem to have anti-proliferative activity betveen 40-60%.
15. EXAMPLE: EXPRESSION OF A BACTERIOCIN
FAMILY MEMBER BY ATTENUATED
TUMOR-TARGETED SALMONELLA
This example demonstrates that attenuated tumor-targeted bacteria, such as
Salmonella, containing a nucleic acid encoding a bacteriocin family member are
capable of
expressing the bacteriocin family member.
15.1. CONSTRUCTION OF COLE3 PLASMIDS
The plasmids described herein serve to illustrate examples of specific
embodiments
of the invention. As will be apparent to one of ordinary skill in the art,
promoter and/or
effector molecule-encoding nucleic acids such as the trc promoter and/or
bacteriocin
encoding nucleic acids may be replaced with other appropriate promoter or
effector
m°lecules by methods known in the art.
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15.1.1. THE pE3.SHUTTLE-1 INTERMEDIATE
VECTOR PLASMID
pE3.shuttle-1 represents the intermediate vector used to create a cassette
containing
a multiple cloning site and lacZ fragment for cloning/selection into the
plasmid vector
ColE3-CA38 (SEQ ID N0:34). To facilitate the cloning of BRP into E3, BRP was
first
cloned onto an intermediate shuttle vector (FIG. 21 ). This vector contains a
lacZ fragment
which can be used to select clones on lactose in a bacterial strain with a
mutations) in
chromosomal lacZ. The BRP fragment was then cloned into the E3 plasmid SmaI
site
(FIG. 22) as a cassette containing the IacZ alpha complementation fragment.
The lacZ
fragment makes insert selection possible (i.e. Lac+) at this step. Although
the naturally
occurnng E3 plasmid has no antibiotic selection markers (FIG. 23), selection
for the
presence of the plasmid is possible by using a halo assay (Pugsley, A.P. and
Oudega, B.
"Methods for Studying Colicins and Their Plasmids" in Plasmids, a Practical
Approach
1987, ed. By K.G. Hardy; Gilson, L. et al. EMBO J. 9: 3875-3884). This shuttle
vector
should facilitate not only the cloning of BRP onto the E3 plasmid, but any DNA
that could
be combined with E3 or E3BRP. The new E3BRP plasmid was then transformed into
41.2.9 and tested for activity. Preliminary halo forming assays demonstrated
that the
presence of BRP on the plasmid did not interfere with the ability of this
strain to produce
E3. To determine if 41.2.9 E3BRP had enhanced activity over 41.2.9 E3 the
amount of
lethal units of E3 produced by each strain was determined (FIG. 24). 41.2.9
E3BRP
produces 100% more lethal units than 41.2.9 E3 alone, demonstrating that this
strain has an
enhanced activity over 41.2.9 E3 alone.
15.1.2. HALO "STAB" ASSAY FOR E3 ACTIVITY
The sensitive tester strain (SK522) is grown to an ODboo of 0.8. One hundred
p1 of
tester strain is added to 3m1 of warm (~55°C) LB soft agar (for a
100x15mm dish) and
quickly poured onto an LB agar plate. The plate is rocked gently to spread the
overlay
evenly over the plate and the agar allowed to solidify for 10-15 minutes.
Colonies of E.
coli or Salmonella for which E3 activity assay is desired are isolated with a
sterile
toothpick and "stabbed" into the agar. The agar plates are then inverted and
incubated at
37°C overnight. The following day a halo or clearing zone appears
around the E3 stab as
the secreted Colicin E3 kills the sensitive strain. The colonies can be
further induced to
increase E3 production or secretion by treatment with any of a variety of SOS-
inducing
agents such as an alkyalating agent (e.g., mitomycin), ultraviolet light or X-
ray.
The results of one of the halo assays are shown in FIG. 25. When a bacterial
strain
secretes a colicin in the presence of a sensitive strain grown on a bacterial
lawn on a petri
dish, the secreted colicin diffuses out and kills the bacterial cells
contained in the bacterial
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lawn, lysing them thus creating a clear zone or halo. The size of the halo
corresponds to
the amount of colicin secreted. The results shown in FIG. 25 show a number of
strains.
No halos are ever observed around strains not containing the colE3-CA38
plasmid. In the
absence of induction, colicin is produced by the Salmonella strains. Also
evident is that
with various types of induction (i.e., alkylating agents, UV light" X-rays),
all of the halos
increase in size in a dose-dependent manner.
15.1.3. OVERLAY ASSAY FOR SELECTIVE E3 CLONES
Transformants are plated with various dilutions (up to 1:10,000) onto LB and
grown for 2 hours at 37°C. The sensitive tester strain is then prepared
as above in the halo
assay arid an overlay poured with soft agar. After allowing to solidify for 10
minutes, the
plate is then inverted and incubated overnight at 37°C. Small clearing
zones then appear
the following day (which resemble bacteriophage plaques) with a small colony
(or
colonies) in the middle of the clearing zone.
15.1.4. "PLAQUE" OR HALO PURIFICATION ASSAY
The small colony at the center of the clearing zone in the overlay agar
described
above is then isolated using a sterile pasteur pipette. In the case of either
no visible colony
or for the case of multiple colonies in one halo, the entire halo is picked
with a sterile
pasteur pipette. The colony or halo is transferred into SOOpL of LB. Dilutions
(up to
1 ~ 10,000) are made and repIated on LB agar and allowed to grow fox 2 hours
at 37°C. An
overlay is then poured with the sensitive tester strain as outlined above. The
following day,
all or most of the colonies should have halos around them.
16. EXAMPLE: E3 INJECTION IN VIVO, AND
DETERMINATION OF THE PERCENT
RETENTION OF PLASMID IN SALMONELLA
The following example demonstrates the retention of the colE3-CA38 plasmid in
Salmonella in vivo.
Homogenates of tumor and liver from two mice 30 days post injection of either
41.29 (or 41.2.9E3-CA38) were used for the studies. In the description to
follow, L=Liver,
T=Tumor. All four homogenates were plated for CFU and colonies were picked for
analysis by msbB PCR and for colicin production. Almost pure cultures of
colonies similar
to 41.2.9 were obtained from all homogenates. Five colonies were picked from
each for
colicin and PCR analysis. An additional 30 colonies were picked form the
41.2.9 E3 T and
L plates for further analysis as there seemed to be a mixed population of
colicin producers
and non producers in the 41.2.9E3 liver homogenate. Based on these results, an
additional
100 colonies from 41.2.9E3 tumor and liver were picked and tested for colicin
production
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and msbB PCR. Distribution and plasmid retention were calculated from the
combined
date.
The results of the E3 Injection in vivo, Determination of the Percent
retention of
plasmid in Salmonella are shown below in Table 3.
positive
number in msbB
issue positive PCR plasmid
issue FU/ml weight FU/gm for colicin retention
41.29L 1.07E+031.33 4.02E+03 0/5 100% n/a
41.29T 1.26e+070.26 2.42E+08 0/5 100% n/a
41.29E3L 1.15E+042.34 2.46E+04 87/135 100% 64.44
41.29E3T 1.09e+060.35 1.56E+07 134/135 100% 99.26
Table 3
In order for the colE3 plasmid to have an effect in vivo, and in order for it
to carry other
genes to the site of the tumor in vivo, the colE3 plasmid must be effectively
retained in
vivo. The results obtained in this experiment were surprising and also
advantageous since
the target of the effector is the tumor, and therefore there would be less
effect on the liver
itself.
17. EXAMPLE: TUMOR TARGETING OF
VARIOUS 41.2.9. STRAINS IN
THE M27 LUNG TUMOR MODEL
The following experiment demonstrates that the ability of 41.2.9 colE3 and
41.2.9
colE3 BRP and 41.2.9 colE3 BRP-m (modified BRP) Salmonella strains to target
tumors.
The Salmonella strains listed in Table 4 below were injected into M27 lung
tumor-
bearing animals and animals were sacrificed on Day 7. Organ weights were
assayed the
next day for calculation of cfu/g.. Tumors and livers were homogenized and
plated on
msbB to determine the colony forming units (c.f.u.). In groups 1, 2, 4, and 6,
the strains all
accumulated in the tumors to approximately 4x10$ cfu/g with varying
accumulation in the
livers ranging from 6x104 to 4x106 cfu/g. Table 4 summarizes the data for all
groups and is
represented by the average cfu/g. All strains were found to have good tumor
accumulation
(better than 10$ c.fu./gram tissue) and all strains gave positive tumor to
liver ratios. The
BRP colE3 had the best ratio, but was not necessarily better than all other
strains available.
The E3 and E3BRP strains accumulate to fairly high levels in tumors with tumor
to liver
ratios between 100-200:1.
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Table 4
Group Strain Tumor (T) cfu/g tissue Ratio
Liver (L) (Tumor:Liver)
1 41.2.9/E3 T 5.1~1.1x10g I3I:I
1 41.2.9/E3 L 3.9~3.6x 1
O6
2 41.2.9/E3BRPT 4.6~2.7x10g 209:1
2 41.2.9/E3BRPL 2.21.3x106
6' 4I .2.9/E3BRPmT 3.50. I 5x 90:1
1 O$
6' 41.2.9/E3BRPmL 3.93.6x106
BRPm refers to a modified BRP that contains point mutations at position 96 (G
to an A resulting in
an amino acid change of a glycine to an arginine) and at position 114 (T to an
A resulting in an
amino acid change of a serine to a threonine). The mutant BRPm no longer
causes quasi lysis but is
still able to secrete proteins from the bacteria (van der Wal, F.,
Koningstein, G., Ten Hagen, C.M.,
pudega, B. and Luirink, J. (1998) Optimization of Bacteriocin Release Protein
(BRP)-Mediated
Protein Release by Escherichia coli: Random Mutagenesis of the pCIoDF 13-
Derived BRP Gene to
Uncouple Lethality and Quasi-Lysis from Protein Release. Applied and
Environmental
Microbiology vol. 64 pp 392-398).
18. EXAMPLE: EFFICACY OF 41.2.9/COLE3 ON
C38 MURINE COLON CARCINOMA
The following example demonstrates the ability of 41.2.9/ColE3 to inhibit the
growth of C38 murine colon carcinoma.
Colon 38 tumor fragment (2x2x2 mm3) was implanted in C57BL/6 mice (female,
Age: 9 weeks) subcutaneously. After tumor volume reached to 1,000 mm3, the
tumors
«ere removed from the mice under sterile condition and cut into small
fragments (about
2x2x2 mm3 mm3/fragment), and repeated above procedure for 5 cycles. The
fragments
were implanted into mice subcutaneously at the right flank by using a tumor
implantation
needle on Day 0 of tumor implants.
Animals were randomized on Day 0 of Salmonella administration when tumor
volume reached 150-200 mm3. Frozen stocks of 41.2.9 and 41.2.9/ColE3 were
thawed at
room temperature, and diluted in PBS to a final concentration of 7.5 x 106
cfu/ml,
respectively. Aliquots of 0.2 ml bacterial suspension (I.5x106 CFU/mouse) were
administered intravenously into mice as group indicated on Day 0. The bacteria
suspension were diluted to 1x103 CFU, plated on msbB plates and incubated
overnight to
determine the number of bacterial cfu which were administered. The tumors were
measured twice per week up to the end of the experiment. Three tumors of each
group
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(ColE3 ) were dissected and processed for determining cfu and retention of
plasmid.
Groups:
Mice
1. Untreated control g
2. _4_1.2_.9 1.5x10 /mouse 8
~ 3. ~ 41.2.9/ColE3 ( 1.5x 10 /mouse) 8
The results for the efficacy of 41.2.9/ColE3 on C38 marine colon carcinoma are
shown in FIG. 26. The data demonstrate that mice treated by intravenous
injection with
VNP20009 (41.2.9) are able to significantly inhibit the growth of C38 marine
colon
carcinoma. In addition, when mice were treated with VNP20009 containing the
ColE3
plasmid, tumor regression (i.e., tumors were smaller at the end of the
experiment than at
the beginning) was achieved.
19. EXAMPLE: ANTI-TUMOR ACTIVITY OF
VNP20009/COLE3 ON DLD1 HUMAN
COLON CARCINOMA IN NUDE MICE
The following example demonstrates the enhanced ability of Salmonella mutant
VNP20009/ColE3 (41.2.9/ColE3) to inhibit the growth of DLD 1 human colon
carcinoma
relative to Salmonella mutant 41.2.9.
DLD1 cells grown in log phase were removed by trypsinization, washed with PBS,
and reconstituted to 5x10' cell/ml PBS. Single cell suspensions (0.1 ml) were
injected into
Nude mice (Nu/Nu-CD 1 female, Age: 9 weeks; from Charles River) subcutaneously
on
Day 0 (5 x 106 cells/mouse) at right flank. Ten animals were used in each
group,
randomized and staged at about 10-15 days after tumor implantation, when tumor
size
reached 300-400mm3. CFU of Salmonella mutant 41.2.9 and 41.2.9/ColE3 were
counted
one day ahead. Bacteria (41.2.9 and 41.2.9/ColE3) were diluted to 1x10'
CFU/ml. Aliquots
of 0.2 ml bacterial suspensions (2x 106 CFU/mouse) were inj ected
intraveneously into mice
on days indicated. The bacteria suspension was diluted to 1x103 CFU, plated
each
solutions 100u1 on msbB plates and the plates incubated overnight. The
bacteria colonies
were counted next day. The tumors were measured twice per week.
Groups:
Mice
1. Untreated control PBS 10
2. 41.2.9 2x10 /mouse 10
3. 41.2.9/ColE3 2x10 /mouse 10
The results of the anti-tumor activity of 41.2.9/ColE3 on DLD1 human colon
carcinoma in nude mice are shown in FIG. 27. The colicin E3-containing 41.2.9
strain
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shows enhanced activity as compared to strain 41.2.9 alone.
20. EXAMPLE: EFFICACY OF 41.2.9/COLE3 ON B16 MURINE
MELANOMA IN C57BL/G MICE
The following example demonstrates the ability of Salmonella mutant
41.2.9/ColE3
to inhibit the growth B 16-F 10 melanoma.
B 16-F 10 cells grown in log phase were removed by trypsinization, washed with
PBS, and reconstituted to 5 x106 cell/ml PBS. Single cell suspensions (0.1 ml)
were
injected into C57BL/6 mice (female, Age: 9 weeks) subcutaneously on Day 0 (5
x105
cells/mouse) at right flank. Ten animals were used in each group, and
randomized at day
9, when tumor volume reached 150-200 mm3. Frozen stocks of Salmonella clones
41.2.9
and 41.2.9/ColE3 were thawed at room temperature, and diluted in PBS to a
final
concentration of 7.5 x 106 cfu/ml, respectively. Aliquots of 0.2 ml bacterial
suspension
(1.5x106 CFU/mouse) were administered intravenously into mice as group
indicated on
Day 9. The bacteria suspension were diluted to 1x103 CFU, plated on msbB
plates and
incubated overnight to determine the number of bacterial cfu which were
administered.
The tumors were measured twice per week up to the end of the experiment.
Groups:
Mice
1 Untreated control 10
3. 41.2.9 1.5x10 /mouse 10
5. 41.2.9/ColE3 1.5x10 /mouse 10
The results of the efficacy of 41.2.9/ColE3 on B 16 murine melanoma in C57BL/6
mice are shown in FIG. 28. The data demonstrate that mice treated by
intravenous
injection with 41.2.9 (41.2.9) are able to significantly inhibit the growth of
B16 murine
melanoma. In addition, mice treated with 41.2.9/ColE3 showed a significant
decrease in
tumor size at early time points (up to day 37) compared to 41.2.9 alone. This
finding is
very important because smaller tumor sizes are more readily susceptible to
other
therapeutics (e.g., chemotherapeutic agents and radiation such as x-rays).
21. EXAMPLE: ANTI-TUMOR EFFICACY OF
41.2.9/E3 COMBINED WITH BRP
The following example demonstrates that the coexpression of BRP and E3 in
Salmonella mutant 41.2.9 increases the anti-tumor efficacy of mutant.
The coexpression of BItP and E3 in Salmonella mutant 41.2.9 increases the
amount of E3 secreted from the bacteria in vitro. If BRP was able to increase
the amount
of E3 secreted from the Salmonella in vivo then it could be hypothesized that
this
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additional extracellular E3 would be readily available to the tumor cells and
thus increase
the cytotoxicity to these cells. In this experiment 4 groups of animals (10
animals per
group) were tested:
Grou number Treatment
1 Control no treatment
2 41.2.9
3 41.2.9/E3
4 41.2.9/E3/BRP
The model used in this experiment was the human lung carcinoma line HTB 177.
The cells
were implanted into the flank of mice subcutaneously on day 1. When the tumors
reached
to approximately SOOmm3, on day 14 the animals were injected by intravenous
injection
with 1x106 cfu of the strain described in the above table, or with saline in
the case of group
1. The tumor volume was measured weekly up to day 24. The results in Table 5
show that
while 41.2.9 by itself is able to inhibit tumor growth (40% inhibition), the
combination
with E3 is able to increase the anti-tumor efficacy (63%). However, when the
strain
carrying both E3 and B1ZP is used in this model, the anti-tumor efficacy is
further enhanced
(67% inhibition compared to untreated control) and the enhanced inhibition is
quite
significant at the earlier time points (Table 5).
Strain Da 17 Da 20 Da 24
41.2.9 SO 38 40
41.2.9/E3 63 58 63
~ 41.2.9/E3/BRP~ 97 ~ 82 [ 67
In conclusion, treatment with Salmonella carrying both the cytotoxic colicin
E3 and
the enhanced secretion system BItP results in an increase in anti-tumor
efficacy compared
to the untreated control and to treatment with 41.2.9/E3 alone.
22. EXAMPLE: COMBINATION OF COLICIN
E3-CONTAINING SALMONELLA
WITH X-RAY TREATMENT
The following example demonstrates that the combination of 41.2.9 with two
doses
of X-ray significantly increases the survival time of mice above that seen for
X-ray alone.
The schedule was as follows: At day 0, tumors were implanted by the
administration of B 16F 10 melanoma (5 x 105 cells/mouse) s.c. in the right
side, at mid
body of 100 C57B6 female mice (5-7 wks of age). At day 8, colicin E3-
containing
Salmonella 41.2.9 was injected and at days 12, and 26, x-rays were
administered.
Table 5: Percent Tumor Growth Inhibition Compared to Untreated Control
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The results of the combination of colicin E3-containing Salmonella with x-ray
treatment are shown in Table 6.
Table 6
Category n=( Days to 1g mean T/C
)
A sham lSGy (6) 12, 12, 18, 18; 18, 17 1.0
21
J lSGy x-rays (9) 14, 14, 18, 21, 25, 33 1.9
35, 35, 67,
l2dpt, 26dpt 67
K 41.2.9 +lSGy (9) 21, 28, 35, 35, 56, 47 2.8
60, 60, 60,
x-raysl2dpt, 26dpt 67 '
regression #1,2
L 41.2.9/E3+lSGy (9) 28, 39, 53, 56, 56, 57 3.3
60, 67, 74,
x-rays l2dpt, 26dpt 7g
re ression d32
This data demonstrates that the combination of 41.2.9 with two doses of X-ray
significantly increases the survival time of mice above that seen for X-ray
alone. E3
further increased the survival time of mice above that seen for 41.2.9 plus X-
ray.
23. EXAMPLE: EXPRESSION OF CYTOTOXIC NECROTIC
FACTORS BY TUMOR-TARGETED BACTERIA
The following example demonstrates that the expression of E. coli cytotoxic
necrotic factor 1 (CNFI) by tumor-targeted bacteria.
Cytotoxic necrotic factors include, but are not limited to, E. coli cytotoxic
necrotic
factor 1 (CNF1; Falbo et al., 1993, Infect. Immun. 61:4904-4914), Vibrio
fischeri CNFI
(Lin et al., 1998, Biochem. Biophys. Res. Comm. 250:462-465) and E. coli
cytotoxic
necrotic factor 2 (CNF2; Sugai et al., 1999, Infect. Immun. 67:6550-6557). The
CNF-
family also includes Pasteurella multiocida toxin (PMT) which shares 27%
identical
residues and 80% conserved residues of the n-terminal portion of CNF2 (Oswald
et al.,
1994, Proc. Acad, Sci. USA 91:3814-3818).
C~ 1 was cloned from E. coli J96 (ATCC 700336) by PCR using the primers
(forward) 5'- GTGTCATGAAAATGGGTAACCAATGGCAAC -3' (SEQ ID N0:35) and
(reverse) 5'- CACAGAGCTCGCGCTAACAAAACAGCACAAGGGAG -3' (SEQ ID
N0:36) using standard PCR. An approximately 3100 by product was obtained and
cloned
into the NcoI and SacI sites of pTrc99a for expression of the protein as well
as DNA
sequencing using E. coli as the DNA cloning host. DNA sequencing was performed
by
standard methods at the Yale University Keck Biotechnology laboratory. The DNA
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sequencing confirmed that the cloned PCR product was CNF 1 with only minor
sequence
variation of 6 of 3065 base pairs.
The CNF1 plasmid was electroporated into an E. coli DNA cloning host DHSa and
Salmonella strain YS1646 (International Publication No. WO 99/13053). The
expression
of CNF 1 was determined in the E. coli DNA cloning host and Salmonella strain
YS 1649
using a standard LDH assay (Promega, Madison, WI, Cytotox 96~). FIG. 29 shows
that
the presence of the CNF-containing plasmid results in enhanced cytotoxicity.
A,
subsequent assay was used to show that Salmonella carrying the CNF-containing
plasmid
also exhibit other known properties of CNF 1 such as multinucleation (Rycke et
al., 1990, J.
Clin. Microbiol. 28: 694-699). Hela cells exposed to CNF1 were examined for
nuclei by
light microscopy. The results in FIG. 30 clearly show that the presence of
CNF1 in
Salmonella results in the expected multinucleation and cell enlargement.
24. EXAMPLE: EXPRESSION OF VEROTOXIN
BY TUMOR-TARGETED BACTERIA
The following example demonstrates the cytotoxicity of verotoxin AB produced
by
1 S ~mor-targeted bacteria engineered to express verotoxin AB.
Verotoxin (syn. HSC10 toxin, Shiga toxin, shiga-like toxin, Shigella toxin).
This
toxin was isolated from a colicin-producing E. coli strain HSC10, and was
originally
thought to be a colicin (Farkas-Himsley et al., 1995, Proc. Natl. Acad: Sci.
92(15):6996-
7000) . It has a long history of antitumor activity, especially for ovarian
cancer and brain
~mors, however, the antitumor activity is associated with purifed
preparations, not with
whole live bacteria.
Verotoxin was cloned from E. coli HSC10 (ATCC 55227) using primers based
upon the published sequence for verotoxin I and confirmed by DNA-sequencing at
the
Yale Keck Biotechnology Center using standard DNA sequencing techniques. The
expression of verotoxin was accomplished using the BRP gene under control of
the
tetracyclin-inducible promoter polycistronic with the verotoxin A and B
subunits. This
tetracyclin-inducible BRP verotoxin AB was cloned into a vector for
chromosomal
integration using the msbB gene.
24.1. CONSTRUCTION OF VECTORS
24.1.1. AMPLIFICATION AND CLONING OF AB
Verotoxin AB (AB) was generated by PCR using the following primers:
H19B-7: forward: 5'-GTGTCCATGGCTAAAACATTATTAATAGCTGCATCGC-3'
(SEQ ID N0:37); and
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QSTX-R1: reverse 5'-GTGTCTGCAGAACTGACTGAATTGAGATG-3.' (SEQ ID
N0:38).
These primers also contain outer NcoI(5') and PstI(3') restriction
endonuclease sites for
cloning into the NcoI and PstI sites of ptrc99A.
24.1.2. AMPLIFICATION AND CLONING OF TETBRP
TetBRP-AB was constructed in the intermediate vector pSP72-F6/R6. TetBRP was
generated by PCR using the following primers: Tet-5': forward 5'-
GTGTAGATCTTTAAGACCCACTTTCACATTTAAGTTG-3' (SEQ ID N0:39) and
BRP-TET-3': reverse 5'-CACAGGATCCTTACTGAACCGCGATCCCCG-3' (SEQ ID
N0:40). These primers contain BgIII(5') and BamHI(3') restriction endonuclease
sites for
cloning into BgIII and BamHI sites of pSP72-F6/R6 vector.
24.1.3. SUBCLONING OF AB INTO pSP72-F6/R6-TETBRP
ptrc99A-AB was digested with BamHI and AvaI restriction endonucleases to
remove AB for insertion into pSP72F6/R6-TetBRP, also digested with BamHI and
AvaI
restriction endonucleases. The pSP72F6/R6 vector contains multiple restriction
endonuclease sites for cloning in addition to a portion of the ~i-gal gene for
lacZ-alpha
complementation in trans. Both the vector (pSP72F6/R6-TetBRP) and the AB
insert were
resolved on a 0.8% 1XTAE agarose gel and purified using the Qiagen gel
extraction kit.
The vector and insert were ligated using T4 ligase and transformed into DHSa
E. coli cells
using the heat shock method. The cells were plated to LB plates containing 100
pg/ml
Amp and 40 ~g/ml X-gal . Positive colonies were selected based on ampicillin
resistance
and the presence of a functional ~i-gal gene (positive colonies were blue).
24.1.4. SUBCLONING OF TETBRP-AB Il''TO pCDV442
pSP72F6/R6-TetBRP-AB was digested with NotI and SfiI restriction endonucleases
for subcloning into the pCVD442 vector, also digested with NotI and SfiI
restriction
endonucleases.
24.1.5. msbB CHROMOSOMAL VECTOR
A vector capable of undergoing homologous recombination with the DmsbB gene in
the chromosome of strain VNP20009 (a.k.a. YS 1646 in International Publication
No. WO
99/13053) was constructed in the suicide vector pCVD442 (Donnenberg and Kaper,
1991,
Infection and Immunity 59: 4310-4317). Primers for PCR were designed that
would
generate portions of the 5' and 3' sections of the msbB deletion occurring in
VNP20009 as
two separate products (msbB-5': forward 5'-GTG TGA GCT CGA TCA ACC AGC AAG
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CCG TTA ACC CTC TGA C-3' (SEQ ID N0:41) and reverse 5' GTG TGC ATG CGG
GGG GCC ATA TAG GCC GGG GAT TTA AAT GCA AAC GTC CGC CGA AAC GCC
GAC GCA C-3' (SEQ ID N0:42); and msbB-3':forward 5'-GTG TGC ATG CGG GGT
TAA TTA AGG GGG CGG CCG CGT GGT ATT GGT TGA ACC GAC GGT GCT CAT
GAC ATC GC-3' (SEQ ID N0:43) and reverse 5'-GTG TCT CGA GGA TAT CAT TCT
GGC CTC, TGA CGT TGT G-3' (SEQ ID N0:44). These primers also contain outer
SacI
(5') and AvaI (3') restriction endonuclease sites to facilitate cloning into
the SacI and SaII
sites of pCVD442 when these two fragments are joined via a common SphI site
and
generate internal Notl, PacI, SphI, SfiI, SwaI and DraI, in order to
facilitate cloning of DNA
fragments into the DmsbB for stable chromosomal integration without antibiotic
resistance
(FIG. 31). This vector is referred to as pCVD442-msbB (see FIGS. 32 and 33).
In order to clone the Tet-BRP-AB into the pCVD442- msbB, the Tet-BRP-AB
plasmid DNA was restriction digested and the appropriate DNA was purified and
a ligation
reaction containing these two components was performed using T4 ligase. The
ligation
reaction was then transformed to DHS 1 pir and colonies screened for the
presence and
orientation of the Tet-BRP-AB. The Tet-BRP-AB clone was transformed into the
strain
SM101 pir (Donnenberg and Kaper, 1991, supra) and the plasmid designated
pCVD442-
Tet-BRP-AB. Colonies of SM101 pir were screened for Tet-BRP-AB gene by PCR,
and a
SM101 pir clone pCVD442- Tet-BRP-AB was chosen for use as a mating donor to
Salmonella strains. SM 101 pir containing the pCVD442- Tet-BRP-AB was mated to
a
Salmonella strain YS50101 (a spontaneous derivative of the tetracycline-
resistant strain
YS82 (Low et al., 1999, supra) with enhanced resistance to Difco MacConkey
agar) by
standard methods (Davis, R. W., Botstein, D., and Roth, J. R. 1980. Advanced
Bacterial
Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor) and
selected for on
plates containing SO pg/mL carbenicillin (carb) and 300 pg/mL streptomycin
(strep). The
resulting YS50102- pCVD442- Tet-BRP-AB clones were checked for pCVD442- Tet-
BRP-
AB gene by PCR.
24.2. TRANSFER OF THE CHROMOSOMALLY INTEGRATED
pCVD442-Tet-BRP-AB INTO 41.2.9 (YS1646) TO
GENERATE THE STRAIN 41.2.9-Tet-BRP-AB
Using bacteriophage P22 (mutant HT105/1 int-201; Davis et al., 1980), 41.2.9
was
transduced to carbenicillin resistance using strain YS50102- Tet-BRP-AB as
donor. The
presence of the bla and sacB genes from pCVD442 allowed the selection of a
carbr (or
ampr) sucs strain denoted 41.2.9- pCVD-Tet-BRP-AB-1 which contained both the
DmsbB
and DmsbB- Tet-BRP-AB genes (FIG. 33, #3). Strain 41.2.9- Tet-BRP-AB -1 was
plated
on LB sucrose to select a sucr Garbs derivative to remove the DmsbB gene and
leave the
DmsbB- Tet-BRP-AB gene according to the methods of Donnenberg and Kaper, 1991,
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supra (FIG. 33, #4) except that the LB-sucrose agar plates were made without
NaCI, and
the plates were incubated at 30°C. After the growth of colonies on
these plates, they were
gridded to an msbB plate and replica plated to either carbenicillin- or
sucrose-containing
plates in order to detect the presence of a clone which lacked both the
antibiotic and sucrase
markers. The resulting clones were checked for the presence of the Tet-BRP-AB
gene by
PCR. One such derivative containing the chromosomally integrated Tet-BRP-AB
and
lacking sucrose sensitivity and carbenicillin resistance was denoted as 41.2.9-
Tet-BRP-
verotoxin AB.
41.2.9-Tet-BRP-verotoxin AB was tested for cytotoxicity in vitro using a
standard
LDH cytotoxicity assay (Cytotox96~; Promega, Madison, Wisconsin). The results
are
shown in FIG. 34, demonstrating the toxic properties of verotoxin-expressing
clones 26 and
31. Clones 26 and 31 had a significantly higher percentage of cytotoxity when
treated with
tetracycline than when not treated with tetracycline.
25. EXAMPLE: EXPRESSION OF HEMOLYSL~I BY
TUMOR-TARGETED BACTERIA
The following example demonstrates that tumor-targeted bacteria can be
engineered
to express hemolytic proteins such as hemolysin constitutively or under
inducible control.
Hemolysins are well known cytotoxic proteins which have the ability to lyse
red
blood cells (see, e.g., Beutin, 1991, Med. Microbiol. Immunol 180:167-182).
SheA
(Genbank Number EC0238954) is a silent hemolysin found in most wild type
E.coli which
is not normally expressed (Fernandez et al., 1998, FEMS Micriobiol Lett 168:85-
90). SheA
(a.k.a. hlyE; Genbank Number U57430) was cloned by PCR using the following
primers
(forward) S'-TTTTTTCCAT GGCTATTATG ACTGAAATCG TTGCAGATAA AACGG-
3' (SEQ ID N0:45) and (reverse) 5'-TTTTTTAAGC TTCCCGGGTC AGACTTCAGG
TACCTCAAAG AGTGTC-3' (SEQ ID N0:46) from wild type E. coli (strain 2507, Yale
University E. coli Genetic Stock Center) under standard PCR conditions. The
PCR product
of the correct size was cloned into the NcoI and HindIII sites of ptrc99a
(Pharmacia) in
order to place it under the partially constitutive trc promoter. The PCR
product was also
cloned into the tet-bgal-Z-term vector (described supra) cut with NcoI and
EcoRV. E. coli
DHSa (Gibco) were then transformed with the plasmids and plated to blood agar
(trypic soy
agar with 5% sheep blood; BioMerieux, Lombard, IL) with and without the
addition of 0.2
ug/ml tetracycline. Positive colonies were picked as those containing halos of
clearing
around the colony which indicates hemolysis. Positive colonies were subjected
to standard
plasmid purification and transformed to Salmonella YS501 and re-screened for
halos.
Constitutive halo formation is shown in FIG. 35 (2A and 2B) for the trc99a
construct, where a halo is observed with or without added tetracycline.
Tetracycline-
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dependent halo formation is shown in FIG. 35 (3A and 3B) for the tetracycline-
promoter
driven SheA, where no halo is observed without the addition tetracycline.
These results
demonstrate that a tumor-targeted bacterium can express a hemolytic protein,
either
constitutively or under inducible control.
26. EXAMPLE: EXPRESSION OF METHIONASE BY
TUMOR-TARGETED BACTERIA
The following example demonstrates that attenuated tumor-targeted bacteria
such as
Salmonella can be engineered to express methionase.
Methionase is an enzyme that degrades methionine, an essential ammino acid
necessary for tumor growth. Methods have been described for administration of
purified
methionase to inhibit tumor growth or to administer a DNA or viral vector
which codes for
methionase (International Publication No. W000/29589 by Xu and Tan). Xu and
Tan did
not disclose methods for using tumor-specific bacterial vectors for delivery
of methionase,
and, in order to achieve efficacy with purified protein, large amounts of
methionase are
required. A novel method for delivering methionase directly to the tumors it
to express the
enzyme using tumor-targeted bacteria.
The following primers were generated for methionase from Pneudomas putida
based
upon Genbank No. L43133:
Forward: METH-XHOI
5'-CCGCTCGAGATGCACGGCTCCAACAAGCTCCCA-3' (SEQ ID N0:47); and
Reverse: METH-BAM
5'-CGCGGATCCTTAGGCACTCGCCTTGAGTGCCTG-3' (SEQ ID N0:48)
Using the above listed primers (4 mM) and an isolated colony of Pneudomas
putida as the
template, the sequence of methionase was amplified by PCR under the following
conditions:
one cycle of 94°C for 5 minutes, followed by 35 cycles of: 94°C
for 1 minute, 60°C for 1
minute and 72°C for 2 minutes. A final amplification step of
72°C for 10 minutes was
included as the last step of the PCR reaction. PCR products were resolved on
0.8% 1X TAE
agarose gel and a PCR product of the expected size for methionase (~ 1196 bp)
was
identified. The band was excised from the gel and purified using the Qiagen
gel extraction
kit.
Both the pSP72 vector and the isolated gel purifed methionase gene obtained
above
were digested with the restriction enzymes Xho I and Bam HI. The digested
vector and
methionase were resolved on a 0.8% 1XTAE agarose gel. The products of the
digestion
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corresponding to the linearized vector and digested methionase gene were
excised from the
gel and purified using the Qiagen gel extraction kit. The linearized vector
and the insert
(methionase) were ligated together using T4 ligase. The ligation mixture was
transformed
into DhSa E.coli cells by a heat shock method. After recovery, the cells were
plated to LB
media containing 100 mg/mL of ampicillan (Amp) to select for those cells that
contain the
intact pSP72 vector. Amp resistant colonies were identified and the presence
of the pSP72
vector containing the methionase gene were confirmed by plasmid preparation
using a
Qiagen mini-prep kit and restriction digest with the enzymes Eco RI and Bsp
HI.
Clone #9, was sent for sequencing to the Yale sequencing Facility, Yale
University School
of Medicine. Sequence was done using both the SP6 (forward) and T7 (reverse)
sequencing
primers. Results demonstrate 100% sequence match to published methionase
sequence with
the exception of the TGA stop codon which was changed to TAA by PCR.
Methionase activity can be determined using the methionase assay described in
Hori
et al., 1996, Cancer Research 56:2116-2122
27. EXAMPLE: EXPRESSION OF APOPTIN PROTEIN
AS A TAT FUSIONS IN ATTENUATED
TU11IOR-TARGETED BACTERIA
The following example demonstrates that attenuated tumor-targeted bacteria can
be
engineered to express and secrete fusion proteins comprising an effector
molecule and a
ferry peptide such as TAT, antennapedia, VP22, and Kaposi FGF MTS.
27.1. CONSTRUCTION OF TAT-APOPTIN VECTORS
The canary virus (CAV) protein apoptin is known to induce apoptosis in
neoplastic
cells, as when delivered by adenoviral vectors (see, e.g., Noteborn et al.,
1999, Gene
Therapy 6:882-892).
In order to generate a protein which could be transcribed in the cytoplasm of
Salmonella and yet have the ability to be transported to the nucleus of a
tumor cell and
cause apoptosis, the apoptin protein was fused to a peptide derived from the
human
immunodeficiency virus (HIV) TAT protein (see, e.g., Schwartze et al., 1999,
Science
285:1569-1572). Since TAT protein fusions have also been shown to be
functional when
fused to poly-histadine (hexahistadine) amino acids which both increase the
positive charge
and facilitate protein purification (Schwartze et al., 1999, supra), the TAT-
apoptin fusion
was generated with and without the hexahistadine (FIG. 36 A and B). Further,
the TAT-
apoptin fusion can be generated with and without an OmpA-8L signal sequence
(FIG. 36A
and C).
The apoptin and hexahistadine apoptin are assembled using overlapping
oligonucleotides. The nucleic acid sequence encoding apoptin was generated by
PCR using
the following oligonucleotides:
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TAP 1: 5'- GATCCCATGG CTTATGGCAG A,~~AAAA.ACGC CGTCAGCGCC
GTCGCATGAA CGCGCTGCAG GAAGATACCC CGCCGGGCCC GTCCACCGTG
TTTCGCCCGC CG-3' (SEQ ID N0:49)
TAP2: 5'- GGGACAGGGT GATGGTGATG CCCGCGATGC CGATGCGGAT
TTCGCGGCAA TGCGGGGTTT CCAGCGGGCG GGAGGAGGTC GGCGGGCGAA
ACACGGTGGA CGG-3'(SEQ ID N0:50)
TAP3: 5'- GGCATCGCGG GCATCACCAT CACCCTGTCC CTGTGCGGCT
GCGCGAACGC GCGCGCGCCG ACCCTGCGCT CCGCGACCGC GGATAACTCC
G~'CACCG GC-3'(SEQ ID N0:51) '
TAP4: 5'-GCGATATTCG GACGGATCGC AGGAGCGTTT TTTGGACGGC
GGTTTCGGCT GATCGGTGCG CAGATCCGGG ACGTTTTTAA AGCCGGTGTT
TTCGGAGTTA TCCGCGGTCG C-3' (SEQ ID N0:52)
TAPS: 5'-CCTGCGATCC GTCCGAATAT CGCGTCTCCG AACTGAAAGA
ATCCCTGATC ACCACCACCC CGTCCCGCCC GCGCACCGCC CGCCGCTGCA
TCCGCCTCTG AAAGCTTCAT G-3' (SEQ ID N0:53)
TAP6: 5'-CATGAAGCTT TCAGAGGCGG ATGCAGCGGC GGGCGGTGCG C-3'
(SEQ ID N0:54)
The nucleic acid sequence encoding the hexahistadine-containing version of the
TAT-
apoptin fusion protein was generated using TAP 2-TAP6 oligonucleotides and
TAP6H1
oligonucleotide (5'-GATCCCATGG CTCATCACCA TCACCACCAT TATGGCCGCA
' AAAAACGCCG TCAGCGCCGT CGCATGAACG CGCTGCAGGA AGATACCCCG
CCGGGCCC-3'; SEQ ID N0:55). The nucleic acid sequence encoding the OmpABL-
containing version of the TAT-apoptin fusion protein is generated from the PCR
product of
TAP1-TAPE oligonucleotides by PCR using TAP6 oligonucleotide and omp8LF1
oligonucleotide (5'- GATCCCATGG CTAA.AA.AGAC GGCTCTGGCG CTTCTGCTCT
TGCTGTTAGC GCTGACTAGT GTAGCGCAGG CCTATGGCCG CAAAAAACGC
CGTCAGCGCC -3'; SEQ ID N0:56).
Each oligonucleotide is formulated into a stock solution which is 4 pM in
concentration. Using premixed PCR reaction beads (Pharmacia, Ready-to-go
beads), 2 p1
of each oligonucleotide was used. The PCR reaction consisted of one cycle at
95 ° for 5
minutes; thirty-five cycles at 95°C for 1 minute, 60 °C for 1
minute, 72°C for 1 minute; and
one cycle at 72°C for 10 minutes. The PCR reaction was then extracted
with
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phenol/chloroform, precipitated with ethanol, redissolved in water and
subjected to
restriction digestion with Nco I and Hind III. The restriction-digested PCR
product was
resolved by gel electrophoresis and the product of the correct size
(approximately 420 and
450 by for TAT-apoptin and hexahistadine-TAT-apoptin, respectively) were
excised from
the gel and isolated using standard molecular biology techniques. These
products are
ligated into Nco I and Hind III digested ptrc99a (Pharmacia) and result in the
ptrc99a-TAT-
apoptin construct. The correct DNA sequence was obtained for both the TAT-
apoptin (FIG.
37) and the hexahistadine TAT-apoptin (FIG 38).
27.2. DEMONSTRATION OF SECRETION
AND UPTAKE OF TAT-APOPTIN
Attenuated tumor-targeted bacteria are transformed with the ptrc99a-TAT-
apoptin
construct by standard techniques known in the art (e.g., by heat shock or
electroporation)
and cultured in medium. The supernatant from the bacterial culture is tested
for the
presence of TAT-apoptin using techniques known to those of skill in the art
(e.g., Western
Blot analysis or ELISA). Once the presence of the TAT-apoptin in the
supernatant of the
bacterial culture is confirmed, the bacterial culture supernatant is incubated
with
mammalian cells (e.g., NIH3T3, CHO, 293, and 293T cells) and the presence of
the TAT-
apoptin inside the cells is confirmed by apoptin assays known to those of
skill in the art.
27.3. DEMONSTRATION THE UPTAKE OF
TAT-APOPTIN INTRATUMORALLY
Attenuated tumor-targeted bacteria engineered to express TAT-apoptin or
apoptin
are administered intravenously to a B 16 tumor model. The mice are sacrificed
several days
after administration of the bacteria and the organ weights are determined.
Tumors are
assayed for the presence and localization of TAT-aproptin or apoptin using
apoptosis assays
(e.g., DNA laddering and Fluorescein In Situ Cell Death Detection Kit
(Boehringer
Mannheim, Mannheim, Germany)) known to those of skill in the art. Further, the
size of the
tumors are assayed to determine anti-tumor activity of the TAT-apoptin. Tumors
are also
homogenized and plated to determine the colony forming units (c.fu.).
28. EXAMPLE: EFFICACY OF THE COMBINATION OF
VNP20009 AND CHEMOTHERAPEUTIC
AGENTS ON THE GROWTH OF M27
LUNG CARCINOMA IN MICE
The following example demonstrates that the administration of attenuated tumor-
targeted bacteria in combination with a chemotherapeutic agent may act
synergistically or
additively to inhibit the growth of solid tumors such as lung carcinoma.
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28.1. EFFICACY OF THE COMBINATION OF VNP20009
AND CYTOXAN OR VNP20009 AND MITOMYCIN C ON
THE GROWTH OF M27 LUNG CARCINOMA IN MICE
Liquid nitrogen stored M27 murine lung carcinoma cells (1x106/ml x 1m1) were
recovered by rapidly thawing the cells at 37°C and cultured with 10 ml
of DMEM culture
medium containing 10% fetal calf serum (FCS) at 37°C, 5% CO2. After
passing the cells
for two generations, M27 cells in log phase were removed by trypsinization,
washed with 1
x PBS, and reconstituted to 2.5x106 cells/ml with 1 x PBS for tumor
implantation. An M27
cell suspension was implanted into 100 C57BL/6 mice (female, aged 8 weeks, 20
g; 5 x 105
cells/mouse) subcutaneously at the right flank on Day 0. The mice were
randomly divided
into ten groups with each group consisting of 10 mice.
Salmonella strain VNP20009 was diluted to 5x106 CFU/ml with 1 x PBS with our
standard dilution procedures. Each mouse was intravenously administered 0.2 ml
of diluted
Salmonella (1x106 CFU/mouse) on day 12 according to Table 6, infra. In order
to
determine the actual number of injected bacteria, the 5x106 CFU/ml bacterial
suspensions
were further diluted to 1x103 CFU/ml and plated on nutrient agar (MsbB plates;
International Publication No. WO 99/13053). The colonies formed were counted
the next
day.
The mitomycin C (Sigma) and cytoxan (Sigma) were administered to mice
according to Table 7, infra. The second dose of mitomcyin C was given to the
combination
groups on day 22 but not those treated with mitomycin C only due to the large
size of the
~mor. 200 mpk of Cipro (Bayer Inc., West Haven, CT) was administered to each
mouse
treated with VNP20009 alone or VNP20009 + chemotherapeutic drugs since severe
toxic
reactions were observed in groups treated with VNP20009 + cytoxan. The tumor
volume
was measured twice a week until the end of the experiment. The behavior,
appearance and
mortality of the animals was observed daily. The mice were kept in a clean,
temperature
constant laboratory. The bedding was changed twice a week and the mice were
provided
with enough food and drinking water.
Table 7
Group Number of Mice
No treatment control 10
3 mpk mitomycin C, i.v., day 10
15
5 mpk, mitomycin C, i.v., day 10
15
150 mpk cytoxan, i.p., day 15 10
200 mpk cytoxan, i.p., day 1 10
S
VNP20009, 1x106/mouse i.v., 10
day 12
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Group Number of Mice
VNP20009, 1 x 106/mouse i.v., 10 .
day 12 + 3
mpk mitomycin C, i.v., days
15 & 22
VNP20009, 1x106/mouse i.v., 10
day 12 + 5
mpk mitomycin C, i.v., days
15 & 22
VNP20009, 1x106/mouse i.v., 10
day 12 + 150
mpk cytoxan, i.p., day 15 '
VNP20009, 1 x 106/mouse i.v., 10
day 12 + 200
mpk cytoxan, i.p., day 15
As shown to FIG. 39, the combination treatment with VNP20009 + cytoxan
inhibited the growth of the M27 lung carcinoma more than VNP20009 treatment
alone or
cytoxan treatment alone. As shown in FIG. 40, the combination of VNP20009 +
mitomycin
C inhibited the growth of the M27 lung carcinoma more than mitomycin C alone.
However,
the combination of VNP20009 + mitomycin C did not inhibit the growth of the
M27 lung
carcinoma more than VNP20009 treatment alone (FIG. 40). These results suggest
that the
administration of attenuated tumor-targeted bacteria in combination with a .
cheniotherapeutic agent may act synergistically or additively to inhibit the
growth of solid
tumors such as lung carcinoma.
28.2. EFFICACY OF THE COMBINATION OF VNP20009
AND CISPLATIN ON THE GROWTH OF M27
LUNG CARCINOMA IN MICE
Liquid nitrogen stored M27 murine lung carcinoma cells (1x106/ml x 1m1) were
recovered by rapidly thawing the cells at 37°C and cultured with 25 ml
of DMEM culture
medium containing 10% fetal calf serum (FCS) at 37°C, 5% CO2. After
passing the cells
for two generations, M27 cells in log phase (about 90-95% saturation) were
removed by
trypsinization, washed with 1 x PBS, and reconstituted to 2.5x106 cells/ml
with 1 x PBS for
tumor implantation. An M27 cell suspension (0.2 ml) was implanted into 36
C57BL/6 mice
(female, aged 8 weeks, 20 g; 5x105 cells/mouse) subcutaneously at the right
flank on day 0.
The mice were randomly divided into groups with each group consisting of 9
mice.
Salmonella strains VNP20009 was diluted to 5x106 CFU/ml with 1 x PBS with our
standard dilution procedures. Each mouse was administered via the tail vein
0.2 ml of
Salmonella (1x106 CFU/mouse) on day 12 according to Table 8, infra. In order
to
determine the actual number of injected bacteria, the 5x106 CFU/ml bacterial
suspensions
were further diluted to 1 x 103 CFU/ml and plated on MsBb plates. The colonies
formed
were counted the next day.
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The cisplatin was administered to mice on day 14, two days post bacterial
injection
(Table 8, infra). The cisplatin was diluted to 0.5 mg/ml with normal saline
prior to
administration. The tumor volume was measured twice a week until the end of
the
experiment. The behavior, appearance and mortality of the animals was observed
daily.
The mice were kept in a clean, temperature constant laboratory. The bedding
was changed
twice a week and the mice were provided with enough food and drinking water.
Table 8
Group Number of Mice
Control (no treatment) ,
~p20009, 1x106/mouse i.v., on 9
day 12
5 mpk cisplatin, i.p. qw x 2,
on day 14, 19
VNP20009, 1 x 106/mouse i.v., 9
on day 12 + S
mpk cisplatin, i.p. qw x 2,
on day 14, 19, 33
As shown in FIG. 41, the combination treatment with VNP20009 + cisplatin
inhibited the growth of the M27 lung carcinoma more than VNP20009 treatment
alone or
cisplatin treatment alone. These results suggest that the administration of
attenuated tumor-
targeted bacteria in combination with chemotherapeutic agent such as cisplatin
may act
synergistically or additively to inhibit the growth of solid tumors such as
lung carcinoma.
The present invention is not to be limited in scope by the specific
embodiments
described herein. Indeed, various modifications of the invention in addition
to those
described herein will become apparent to those skilled in the art from the
foregoing
description and accompanying figures. Such modifications are intended to fall
within the
scope of the appended claims.
Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
35
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